Sample records for morphogenetic protein receptors

  1. Immunohistochemical localization of bone morphogenetic proteins and the receptors in epiphyseal growth plate.

    PubMed

    Yazaki, Y; Matsunaga, S; Onishi, T; Nagamine, T; Origuchi, N; Yamamoto, T; Ishidou, Y; Imamura, T; Sakou, T

    1998-01-01

    The expression of bone morphogenetic proteins (BMPs) and BMP receptors (BMPRs) in the epiphyseal growth plate has not been clarified. In this study, we studied immunohistochemically the spatial and temporal localization of BMP-2/4, osteogenic protein-1 (OP-1, or BMP-7), and BMP receptors (BMPR-IA, BMPR-IB, and BMPR-II) in the epiphyseal plate of growing rats. The proximal parts of tibia in growing rats were observed. At 12 weeks after birth, BMP-2/4 and OP-1 were expressed markedly in proliferating and maturing chondrocytes. BMPR-IA, IB and II were clearly co-expressed in proliferating and maturing chondrocytes, and the expression was decreased in hypertrophic chondrocytes. At 24 weeks, the expression of BMP-2/4 and OP-1 was decreased, but BMPRs were still well-expressed in proliferating chondrocytes. The temporal and spatial expression of BMP and BMPR suggests that BMP and BMP receptors play roles in the multistep cascade of enchondral ossification in the epiphyseal growth plate.

  2. The Bone Morphogenetic Protein Type Ib Receptor Is a Major Mediator of Glial Differentiation and Cell Survival in Adult Hippocampal Progenitor Cell Culture

    PubMed Central

    Brederlau, A.; Faigle, R.; Elmi, M.; Zarebski, A.; Sjöberg, S.; Fujii, M.; Miyazono, K.; Funa, K.

    2004-01-01

    Bone morphogenetic proteins (BMPs) act as growth regulators and inducers of differentiation. They transduce their signal via three different type I receptors, termed activin receptor-like kinase 2 (Alk2), Alk3, or bone morphogenetic protein receptor Ia (BMPRIa) and Alk6 or BMPRIb. Little is known about functional differences between the three type I receptors. Here, we have investigated consequences of constitutively active (ca) and dominant negative (dn) type I receptor overexpression in adult-derived hippocampal progenitor cells (AHPs). The dn receptors have a nonfunctional intracellular but functional extracellular domain. They thus trap BMPs that are endogenously produced by AHPs. We found that effects obtained by overexpression of dnAlk2 and dnAlk6 were similar, suggesting similar ligand binding patterns for these receptors. Thus, cell survival was decreased, glial fibrillary acidic protein (GFAP) expression was reduced, whereas the number of oligodendrocytes increased. No effect on neuronal differentiation was seen. Whereas the expression of Alk2 and Alk3 mRNA remained unchanged, the Alk6 mRNA was induced after impaired BMP signaling. After dnAlk3 overexpression, cell survival and astroglial differentiation increased in parallel to augmented Alk6 receptor signaling. We conclude that endogenous BMPs mediate cell survival, astroglial differentiation and the suppression of oligodendrocytic cell fate mainly via the Alk6 receptor in AHP culture. PMID:15194807

  3. Bone morphogenetic protein 4 and bone morphogenetic protein receptor expression in the pituitary gland of adult dogs in healthy condition and with ACTH-secreting pituitary adenoma.

    PubMed

    Sato, A; Ochi, H; Harada, Y; Yogo, T; Kanno, N; Hara, Y

    2017-01-01

    The purpose of this study was to investigate the expression of bone morphogenetic protein 4 (BMP4) and its receptors, bone morphogenetic protein receptor I (BMPRI) and BMPRII, in the pituitary gland of healthy adult dogs and in those with ACTH-secreting pituitary adenoma. Quantitative polymerase chain reaction analysis showed that the BMP4 messenger RNA expression level in the ACTH-secreting pituitary adenoma samples was significantly lower than that in the normal pituitary gland samples (P = 0.03). However, there were no statistically significant differences between samples with respect to the messenger RNA expression levels of the receptors BMPRIA, BMPRIB, and BMPRII. Double-immunofluorescence analysis of the normal canine pituitary showed that BMP4 was localized in the thyrotroph (51.3 ± 7.3%) and not the corticotroph cells. By contrast, BMPRII was widely expressed in the thyrotroph (19.9 ± 5.2%) and somatotroph cells (94.7 ± 3.6%) but not in the corticotroph cells (P < 0.001, thyrotroph cells vs somatotroph cells). Similarly, in ACTH-secreting pituitary adenoma, BMP4 and BMPRII were not expressed in the corticotroph cells. Moreover, the percentage of BMP4-positive cells was also significantly reduced in the thyrotroph cells of the surrounding normal pituitary tissue obtained from the resected ACTH-secreting pituitary adenoma (8.3 ± 7.9%) compared with that in normal canine pituitary (P < 0.001). BMP4 has been reported to be expressed in corticotroph cells in the human pituitary gland. Therefore, the results of this study reveal a difference in the cellular pattern of BMP4-positive staining in the pituitary gland between humans and dogs and further revealed the pattern of BMPRII-positive staining in the dog pituitary gland. These species-specific differences regarding BMP4 should be considered when using dogs as an animal model for Cushing's disease. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Uncovering Molecular Bases Underlying Bone Morphogenetic Protein Receptor Inhibitor Selectivity

    PubMed Central

    Alsamarah, Abdelaziz; LaCuran, Alecander E.; Oelschlaeger, Peter; Hao, Jijun; Luo, Yun

    2015-01-01

    Abnormal alteration of bone morphogenetic protein (BMP) signaling is implicated in many types of diseases including cancer and heterotopic ossifications. Hence, small molecules targeting BMP type I receptors (BMPRI) to interrupt BMP signaling are believed to be an effective approach to treat these diseases. However, lack of understanding of the molecular determinants responsible for the binding selectivity of current BMP inhibitors has been a big hindrance to the development of BMP inhibitors for clinical use. To address this issue, we carried out in silico experiments to test whether computational methods can reproduce and explain the high selectivity of a small molecule BMP inhibitor DMH1 on BMPRI kinase ALK2 vs. the closely related TGF-β type I receptor kinase ALK5 and vascular endothelial growth factor receptor type 2 (VEGFR2) tyrosine kinase. We found that, while the rigid docking method used here gave nearly identical binding affinity scores among the three kinases; free energy perturbation coupled with Hamiltonian replica-exchange molecular dynamics (FEP/H-REMD) simulations reproduced the absolute binding free energies in excellent agreement with experimental data. Furthermore, the binding poses identified by FEP/H-REMD led to a quantitative analysis of physical/chemical determinants governing DMH1 selectivity. The current work illustrates that small changes in the binding site residue type (e.g. pre-hinge region in ALK2 vs. ALK5) or side chain orientation (e.g. Tyr219 in caALK2 vs. wtALK2), as well as a subtle structural modification on the ligand (e.g. DMH1 vs. LDN193189) will cause distinct binding profiles and selectivity among BMP inhibitors. Therefore, the current computational approach represents a new way of investigating BMP inhibitors. Our results provide critical information for designing exclusively selective BMP inhibitors for the development of effective pharmacotherapy for diseases caused by aberrant BMP signaling. PMID:26133550

  5. Binding of integrin α1 to bone morphogenetic protein receptor IA suggests a novel role of integrin α1β1 in bone morphogenetic protein 2 signalling.

    PubMed

    Zu, Yan; Liang, Xudong; Du, Jing; Zhou, Shuai; Yang, Chun

    2015-11-05

    Here, we observed that integrin α1β1 and bone morphogenetic protein receptor (BMPR) IA formed a complex and co-localised in several cell types. However, the molecular interaction between these two molecules was not studied in detail to date and the role of the interaction in BMPR signalling remains unknown; thus, these were investigated here. In a steered molecular dynamics (SMD) simulation, the observed development of the rupture force related to the displacement between the A-domain of integrin α1 and the extracellular domain of BMPR IA indicated a strong molecular interaction within the integrin-BMPR complex. Analysis of the intermolecular forces revealed that hydrogen bonds, rather than salt bridges, are the major contributors to these intermolecular interactions. By using Enzyme-linked immunosorbent assay (ELISA) and co-immunoprecipitation (co-IP) experiments with site-directed mutants, we found that residues 85-89 in BMPR IA play the most important role for BMPR IA binding to integrin α1β1. These residues are the same as those responsible for bone morphogenetic protein 2 (BMP-2)/BMPR IA binding. In our experiments, we also found that the interference of integrin α1β1 up regulated the level of phosphorylated Smad1, 5, 8, which is the downstream of BMP/BMPR signalling. Therefore, our results suggest that integrin α1β1/BMPR IA may block BMP-2/BMPR IA complex information and interfere with the BMP-2 signalling pathway in cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Connective tissue growth factor and bone morphogenetic protein 2 are induced following myocardial ischemia in mice and humans.

    PubMed

    Rutkovskiy, Arkady; Sagave, Julia; Czibik, Gabor; Baysa, Anton; Zihlavnikova Enayati, Katarina; Hillestad, Vigdis; Dahl, Christen Peder; Fiane, Arnt; Gullestad, Lars; Gravning, Jørgen; Ahmed, Shakil; Attramadal, Håvard; Valen, Guro; Vaage, Jarle

    2017-09-01

    We aimed to study the cardiac expression of bone morphogenetic protein 2, its receptor 1 b, and connective tissue growth factor, factors implicated in cardiac embryogenesis, following ischemia/hypoxia, heart failure, and in remodeling hearts from humans and mice. Biopsies from the left ventricle of patients with end-stage heart failure due to dilated cardiomyopathy or coronary artery disease were compared with donor hearts and biopsies from patients with normal heart function undergoing coronary artery bypass grafting. Mouse model of post-infarction remodeling was made by permanent ligation of the left coronary artery. Hearts were analyzed by real-time polymerase chain reaction and Western blotting after 24 hours and after 2 and 4 weeks. Patients with dilated cardiomyopathy and mice post-infarction had increased cardiac expression of connective tissue growth factor. Bone morphogenetic protein 2 was increased in human hearts failing due to coronary artery disease and in mice post-infarction. Gene expression of bone morphogenetic protein receptor 1 beta was reduced in hearts of patients with failure, but increased two weeks following permanent ligation of the left coronary artery in mice. In conclusion, connective tissue growth factor is upregulated in hearts of humans with dilated cardiomyopathy, bone morphogenetic protein 2 is upregulated in remodeling due to myocardial infarction while its receptor 1 b in human failing hearts is downregulated. A potential explanation might be an attempt to engage regenerative processes, which should be addressed by further, mechanistic studies.

  7. Downregulation of bone morphogenetic protein receptor 2 promotes the development of neuroblastoma.

    PubMed

    Cui, Ximao; Yang, Yili; Jia, Deshui; Jing, Ying; Zhang, Shouhua; Zheng, Shan; Cui, Long; Dong, Rui; Dong, Kuiran

    2017-01-29

    Neuroblastoma (NB) is the most common extracranial solid tumor of childhood. In this study, we examined the expression of bone morphogenetic protein receptor 2 (BMPR2) in primary NB and adjacent non-tumor samples (adrenal gland). BMPR2 expression was significantly downregulated in NB tissues, particularly in high-grade NB, and was inversely related to the expression of the NB differentiation markers ferritin and enolase. The significance of the downregulation was further explored in cultured NB cells. While enforced expression of BMPR2 decreased cell proliferation and colony-forming activity, shRNA-mediated knockdown of BMPR2 led to increased cell growth and clonogenicity. In mice, NB cells harboring BMPR2 shRNA showed significantly increased tumorigenicity compared with control cells. We also performed a retrospective analysis of NB patients and identified a significant positive correlation between tumor BMPR2 expression and overall survival. These findings suggest that BMPR2 may play an important role in the development of NB. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Bone morphogenetic protein type IA receptor signaling regulates postnatal osteoblast function and bone remodeling.

    PubMed

    Mishina, Yuji; Starbuck, Michael W; Gentile, Michael A; Fukuda, Tomokazu; Kasparcova, Viera; Seedor, J Gregory; Hanks, Mark C; Amling, Michael; Pinero, Gerald J; Harada, Shun-ichi; Behringer, Richard R

    2004-06-25

    Bone morphogenetic proteins (BMPs) function during various aspects of embryonic development including skeletogenesis. However, their biological functions after birth are less understood. To investigate the role of BMPs during bone remodeling, we generated a postnatal osteoblast-specific disruption of Bmpr1a that encodes the type IA receptor for BMPs in mice. Mutant mice were smaller than controls up to 6 months after birth. Irregular calcification and low bone mass were observed, but there were normal numbers of osteoblasts. The ability of the mutant osteoblasts to form mineralized nodules in culture was severely reduced. Interestingly, bone mass was increased in aged mutant mice due to reduced bone resorption evidenced by reduced bone turnover. The mutant mice lost more bone after ovariectomy likely resulting from decreased osteoblast function which could not overcome ovariectomy-induced bone resorption. In organ culture of bones from aged mice, ablation of the Bmpr1a gene by adenoviral Cre recombinase abolished the stimulatory effects of BMP4 on the expression of lysosomal enzymes essential for osteoclastic bone resorption. These results demonstrate essential and age-dependent roles for BMP signaling mediated by BMPRIA (a type IA receptor for BMP) in osteoblasts for bone remodeling.

  9. Role of RGM coreceptors in bone morphogenetic protein signaling

    PubMed Central

    Halbrooks, Peter J; Ding, Ru; Wozney, John M; Bain, Gerard

    2007-01-01

    Background The repulsive guidance molecule (RGM) proteins, originally discovered for their roles in neuronal development, have been recently identified as co-receptors in the bone morphogenetic protein (BMP) signaling pathway. BMPs are members of the TGFβ superfamily of signaling cytokines, and serve to regulate many aspects of cellular growth and differentiation. Results Here, we investigate whether RGMa, RGMb, and RGMc play required roles in BMP and TGFβ signaling in the mouse myoblast C2C12 cell line. These cells are responsive to BMPs and are frequently used to study BMP/TGFβ signaling pathways. Using siRNA reagents to specifically knock down each RGM protein, we show that the RGM co-receptors are required for significant BMP signaling as reported by two cell-based BMP activity assays: endogenous alkaline phosphatase activity and a luciferase-based BMP reporter assay. Similar cell-based assays using a TGFβ-induced luciferase reporter show that the RGM co-receptors are not required for TGFβ signaling. The binding interaction of each RGM co-receptor to each of BMP2 and BMP12 is observed and quantified, and equilibrium dissociation constants in the low nanomolar range are reported. Conclusion Our results demonstrate that the RGMs play a significant role in BMP signaling and reveal that these molecules cannot functionally compensate for one another. PMID:17615080

  10. Defective cellular trafficking of the bone morphogenetic protein receptor type II by mutations underlying familial pulmonary arterial hypertension.

    PubMed

    John, Anne; Kizhakkedath, Praseetha; Al-Gazali, Lihadh; Ali, Bassam R

    2015-04-25

    Familial pulmonary arterial hypertension (FPAH) is a relatively rare but fatal disorder characterized by elevated arterial pressure caused by abnormal proliferation of endothelial cells of the arteries, which eventually leads to heart failure and death. FPAH is inherited as an autosomal dominant trait and is caused by heterozygous mutations in the BMPR2 gene encoding the bone morphogenetic protein type II receptor (BMPR2). BMPR2 belongs to the TGF β/BMP super-family of receptors involved in a signal transduction cascade via the SMAD signaling pathway. The BMPR2 polypeptide is composed of 1038 amino acids and consists of a ligand binding domain, a kinase domain and a cytoplasmic tail. To investigate the cellular and functional consequence of BMPR2 mutations, C-terminally FLAG-tagged constructs of eighteen pathogenic BMPR2 missense mutants were generated by site directed mutagenesis and expressed in HeLa and HEK-293T cell lines. The subcellular localizations of the mutant proteins were investigated using immunostaining and confocal microscopy. Post-translational modifications of the proteins were analyzed by Endoglycosidase H deglycosylation assay. Our results indicated that mutations in the ligand binding domain affecting highly conserved cysteine residues resulted in retention of the mutant proteins in the endoplasmic reticulum (ER), as evident from their co-localization with the ER resident protein calnexin. The kinase domain mutants showed both ER and plasma membrane (PM) distributions, while the cytoplasmic tail domain variants were localized exclusively to the PM. The subcellular localizations of the mutants were further confirmed by their characteristic glycosylation profiles. In conclusion, our results indicate that ER quality control (ERQC) is involved in the pathological mechanism of several BMPR2 receptor missense mutations causing FPAH, which can be explored as a potential therapeutic target in the future. Copyright © 2015. Published by Elsevier B.V.

  11. [Bone morphogenetic proteins (BMP): clinical application for reconstruction of bone defects].

    PubMed

    Sierra-García, Gerardo Daniel; Castro-Ríos, Rocío; Gónzalez-Horta, Azucena; Lara-Arias, Jorge; Chávez-Montes, Abelardo

    2016-01-01

    Since the introduction of bone morphogenetic proteins, their use has become an invaluable ally for the treatment of bone defects. These proteins are potent growth factors, related to angiogenic and osteogenic activity. The osteoinductive capacity of recombinant bone morphogenetic protein (rhBMP) in the formation of bone and cartilage has been confirmed in in vitro studies and evaluated in clinical trials. To obtain a therapeutic effect, administration is systemic, by injection over the physiological dose. Among the disadvantages, ectopic bone formation or high morbidity in cases of spinal fusion is observed. In this review, the roles of bone morphogenetic proteins in bone repair and clinical applications are analyzed. These findings represent advances in the study of bone regeneration and application of growth factors for more predictable results.

  12. A soluble bone morphogenetic protein type IA receptor increases bone mass and bone strength

    PubMed Central

    Baud’huin, Marc; Solban, Nicolas; Cornwall-Brady, Milton; Sako, Dianne; Kawamoto, Yoshimi; Liharska, Katia; Lath, Darren; Bouxsein, Mary L.; Underwood, Kathryn W.; Ucran, Jeffrey; Kumar, Ravindra; Pobre, Eileen; Grinberg, Asya; Seehra, Jasbir; Canalis, Ernesto; Pearsall, R. Scott; Croucher, Peter I.

    2012-01-01

    Diseases such as osteoporosis are associated with reduced bone mass. Therapies to prevent bone loss exist, but there are few that stimulate bone formation and restore bone mass. Bone morphogenetic proteins (BMPs) are members of the TGFβ superfamily, which act as pleiotropic regulators of skeletal organogenesis and bone homeostasis. Ablation of the BMPR1A receptor in osteoblasts increases bone mass, suggesting that inhibition of BMPR1A signaling may have therapeutic benefit. The aim of this study was to determine the skeletal effects of systemic administration of a soluble BMPR1A fusion protein (mBMPR1A–mFc) in vivo. mBMPR1A–mFc was shown to bind BMP2/4 specifically and with high affinity and prevent downstream signaling. mBMPR1A–mFc treatment of immature and mature mice increased bone mineral density, cortical thickness, trabecular bone volume, thickness and number, and decreased trabecular separation. The increase in bone mass was due to an early increase in osteoblast number and bone formation rate, mediated by a suppression of Dickkopf-1 expression. This was followed by a decrease in osteoclast number and eroded surface, which was associated with a decrease in receptor activator of NF-κB ligand (RANKL) production, an increase in osteoprotegerin expression, and a decrease in serum tartrate-resistant acid phosphatase (TRAP5b) concentration. mBMPR1A treatment also increased bone mass and strength in mice with bone loss due to estrogen deficiency. In conclusion, mBMPR1A–mFc stimulates osteoblastic bone formation and decreases bone resorption, which leads to an increase in bone mass, and offers a promising unique alternative for the treatment of bone-related disorders. PMID:22761317

  13. Disruption of the bone morphogenetic protein receptor 2 pathway in nitrofen-induced congenital diaphragmatic hernia.

    PubMed

    Gosemann, Jan-Hendrik; Friedmacher, Florian; Fujiwara, Naho; Alvarez, Luis A J; Corcionivoschi, Nicolae; Puri, Prem

    2013-08-01

    Congenital diaphragmatic hernia (CDH) remains a major therapeutic challenge despite advances in neonatal resuscitation and intensive care. The high mortality and morbidity in CDH has been attributed to pulmonary hypoplasia and persistent pulmonary hypertension (PH). Bone morphogenetic protein receptor 2 (BMPR2) plays a key role in pulmonary vasculogenesis during the late stages of fetal lung development. BMPR2 is essential for control of endothelial and smooth muscle cell proliferation. Dysfunction of BMPR2 and downstream signaling have been shown to disturb the crucial balance of proliferation of smooth muscle cells contributing to the pathogenesis of human and experimental PH. We designed this study to investigate the hypothesis that BMPR2 signaling is disrupted in nitrofen-induced CDH. Pregnant rats were treated with nitrofen or vehicle on gestational day 9 (D9). Fetuses were sacrificed on D21 and divided into CDH and control. Quantitative real-time polymerase chain reaction, Western blotting, and confocal-immunofluorescence were performed to determine pulmonary gene expression levels and protein expression of BMPR2 and related proteins. Pulmonary Bmpr2 gene expression levels were significantly decreased in nitrofen-induced CDH compared to controls. Western blotting and confocal microscopy revealed decreased pulmonary BMPR2 protein expression and increased activation of p38(MAPK) in CDH compared to controls. The observed disruption of the BMPR2 signaling pathway may lead to extensive vascular remodeling and contribute to PH in the nitrofen-induced CDH model. BMPR2 may therefore represent a potential target for the treatment of PH in CDH. © 2013 Wiley Periodicals, Inc.

  14. Repulsive Guidance Molecules (RGMs) and Neogenin in Bone Morphogenetic Protein (BMP) signaling

    PubMed Central

    Tian, Chenxi; Liu, Jun

    2015-01-01

    Summary Bone morphogenetic proteins (BMPs) belong to the transforming growth factor-beta (TGFβ) superfamily. BMPs mediate a highly conserved signal transduction cascade through the type I and type II serine/threonine kinase receptors and intracellular Smad proteins. The BMP pathway regulates multiple developmental and homeostatic processes. Mutations in this pathway can cause various diseases in humans, such as skeletal disorders, cardiovascular diseases and various cancers. Multiple levels of regulation, including extracellular regulation, help to ensure proper spatiotemporal control of BMP signaling in the right cellular context. The family of repulsive guidance molecules (RGMs) and the type I trans-membrane protein neogenin, a paralog of DCC (Deleted in Colorectal Cancer), have been implicated in modulating the BMP pathway. In this review, we discuss the properties and functions of RGM proteins and neogenin, focusing on their roles in the modulation of BMP signal transduction. PMID:23740870

  15. Bone Morphogenetic Proteins, Antagonists and Receptors in Prostate Cancer

    DTIC Science & Technology

    2005-01-01

    expressed in prostate. This work investigates BMP receptors and BMP antagonists to understand the basic mechanisms to inhibit the BMP signaling in...during embryoge- nesis, and prostate cancer metastases to bone. BMP functions can be inhibited by antagonists such as Noggin or DAN. DAN is a protein...protein along with a constant 0-6 -1 10 100 1000 1O0ng/ml of BMP-6, we were able to show a ng/ml BMP-6 dose-dependent inhibition of BMP-6 activity in DU

  16. The ventralizing activity of Radar, a maternally expressed bone morphogenetic protein, reveals complex bone morphogenetic protein interactions controlling dorso-ventral patterning in zebrafish.

    PubMed

    Goutel, C; Kishimoto, Y; Schulte-Merker, S; Rosa, F

    2000-12-01

    In Xenopus and zebrafish, BMP2, 4 and 7 have been implicated, after the onset of zygotic expression, in inducing and maintaining ventro-lateral cell fate during early development. We provide evidence here that a maternally expressed bone morphogenetic protein (BMP), Radar, may control early ventral specification in zebrafish. We show that Radar ventralizes zebrafish embryos and induces the early expression of bmp2b and bmp4. The analysis of Radar overexpression in both swirl/bmp2b mutants and embryos expressing truncated BMP receptors shows that Radar-induced ventralization is dependent on functional BMP2/4 pathways, and may initially rely on an Alk6-related signaling pathway. Finally, we show that while radar-injected swirl embryos still exhibit a strongly dorsalized phenotype, the overexpression of Radar into swirl/bmp2b mutant embryos restores ventral marker expression, including bmp4 expression. Our results suggest that a complex regulation of different BMP pathways controls dorso-ventral (DV) patterning from early cleavage stages until somitogenesis.

  17. Identification of bone morphogenetic protein 9 (BMP9) as a novel profibrotic factor in vitro.

    PubMed

    Muñoz-Félix, José M; Cuesta, Cristina; Perretta-Tejedor, Nuria; Subileau, Mariela; López-Hernández, Francisco J; López-Novoa, José M; Martínez-Salgado, Carlos

    2016-09-01

    Upregulated synthesis of extracellular matrix (ECM) proteins by myofibroblasts is a common phenomenon in the development of fibrosis. Although the role of TGF-β in fibrosis development has been extensively studied, the involvement of other members of this superfamily of cytokines, the bone morphogenetic proteins (BMPs) in organ fibrosis has given contradictory results. BMP9 is the main ligand for activin receptor-like kinase-1 (ALK1) TGF-β1 type I receptor and its effect on fibrosis development is unknown. Our purpose was to study the effect of BMP9 in ECM protein synthesis in fibroblasts, as well as the involved receptors and signaling pathways. In cultured mice fibroblasts, BMP9 induces an increase in collagen, fibronectin and connective tissue growth factor expression, associated with Smad1/5/8, Smad2/3 and Erk1/2 activation. ALK5 inhibition with SB431542 or ALK1/2/3/6 with dorsomorphin-1, inhibition of Smad3 activation with SIS3, and inhibition of the MAPK/Erk1/2 with U0126, demonstrates the involvement of these pathways in BMP9-induced ECM synthesis in MEFs. Whereas BMP9 induced Smad1/5/8 phosphorylation through ALK1, it also induces Smad2/3 phosphorylation through ALK5 but only in the presence of ALK1. Summarizing, this is the first study that accurately identifies BMP9 as a profibrotic factor in fibroblasts that promotes ECM protein expression through ALK1 and ALK5 receptors. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Novel loci interacting epistatically with Bone Morphogenetic Protein Receptor 2 cause familial pulmonary arterial hypertension

    PubMed Central

    Rodriguez-Murillo, Laura; Subaran, Ryan; Stewart, William C. L.; Pramanik, Sreemanta; Marathe, Sudhir; Barst, Robyn J.; Chung, Wendy K.; Greenberg, David A.

    2009-01-01

    Background Familial pulmonary arterial hypertension (FPAH) is a rare, autosomal-dominant inherited disease with low penetrance. Mutations in the Bone Morphogenetic Protein Receptor 2 (BMPR2) have been identified in at least 70% of FPAH patients. However, the lifetime penetrance of these BMPR2 mutations is 10-20%, suggesting that genetic and/or environmental modifiers are required for disease expression. Our goal in this study is to identify genetic loci that may influence FPAH expression in BMPR2-mutation-carriers. Methods We performed a genome-wide linkage scan in 15 FPAH families segregating for BMPR2 mutations. We used a dense SNP array and a novel multi-scan linkage procedure that provides increased power and precision for the localization of linked loci. Results We observed linkage evidence in four regions: 3q22 (median LOD=3.43), 3p12 (median LOD = 2.35), 2p22 (median LOD = 2.21), and 13q21 (median LOD = 2.09). When used in conjunction with the nonparametric bootstrap, our approach yields high-resolution to identify candidate gene regions containing putative BMPR2-interacting genes. Imputation of the disease model by LOD score maximization indicates that the 3q22 locus alone predicts most FPAH cases in BMPR2-mutation carriers, providing strong evidence that BMPR2 and the 3q22 locus interact epistatically. Conclusions Our findings suggest that genotypes at loci in the newly-identified regions, especially at 3q22, could improve FPAH risk prediction in FPAH families and suggest other targets for therapeutic intervention. PMID:19864167

  19. Novel loci interacting epistatically with bone morphogenetic protein receptor 2 cause familial pulmonary arterial hypertension.

    PubMed

    Rodriguez-Murillo, Laura; Subaran, Ryan; Stewart, William C L; Pramanik, Sreemanta; Marathe, Sudhir; Barst, Robyn J; Chung, Wendy K; Greenberg, David A

    2010-02-01

    Familial pulmonary arterial hypertension (FPAH) is a rare, autosomal-dominant, inherited disease with low penetrance. Mutations in the bone morphogenetic protein receptor 2 (BMPR2) have been identified in at least 70% of FPAH patients. However, the lifetime penetrance of these BMPR2 mutations is 10% to 20%, suggesting that genetic and/or environmental modifiers are required for disease expression. Our goal in this study was to identify genetic loci that may influence FPAH expression in BMPR2 mutation carriers. We performed a genome-wide linkage scan in 15 FPAH families segregating for BMPR2 mutations. We used a dense single-nucleotide polymorphism (SNP) array and a novel multi-scan linkage procedure that provides increased power and precision for the localization of linked loci. We observed linkage evidence in four regions: 3q22 ([median log of the odds (LOD) = 3.43]), 3p12 (median LOD) = 2.35), 2p22 (median LOD = 2.21), and 13q21 (median LOD = 2.09). When used in conjunction with the non-parametric bootstrap, our approach yields high-resolution to identify candidate gene regions containing putative BMPR2-interacting genes. Imputation of the disease model by LOD-score maximization indicates that the 3q22 locus alone predicts most FPAH cases in BMPR2 mutation carriers, providing strong evidence that BMPR2 and the 3q22 locus interact epistatically. Our findings suggest that genotypes at loci in the newly identified regions, especially at 3q22, could improve FPAH risk prediction in FPAH families. We also suggest other targets for therapeutic intervention.

  20. Accelerators of Osteogenesis by Recombinant Human Bone Morphogenetic Protein-2

    PubMed Central

    Okubo, Yasunori; Kusumoto, Kenji; Bessho, Kazuhisa

    2007-01-01

    Bone morphogenetic protein (BMP) appears to be one of the most promising cytokine and for clinical use in reconstructive surgery for bony defects and augmentation. To evaluate the effect of basic fibroblast growth factor (bFGF), FK506, elcatonin, and hyperbaric oxygenation (HBO) on osteoinduction by recombinant human bone morphogenetic protein-2 (rhBMP-2), 2 or 5 μg of rhBMP-2 was implanted into intramuscular sites of rats. At 21 days after implantation, the osteoinductive activity in the treatment group and control group was compared radiographically, biochemically, and histologically. The amount of new bone in the treatment group was significantly greater than that in the control group. The alkaline phosphatase activity and calcium content in the treatment group were significantly higher than those in the control group. These results suggest that bFGF, FK506, elcatonin, and HBO accelerated the activity and rate of osteoinduction by rhBMP2. These results may be useful when BMP is applied clinically in near future. PMID:21901062

  1. Regulation of bone morphogenetic proteins in early embryonic development

    NASA Astrophysics Data System (ADS)

    Yamamoto, Yukiyo; Oelgeschläger, Michael

    2004-11-01

    Bone morphogenetic proteins (BMPs), a large subgroup of the TGF-β family of secreted growth factors, control fundamental events in early embryonic development, organogenesis and adult tissue homeostasis. The plethora of dose-dependent cellular processes regulated by BMP signalling demand a tight regulation of BMP activity. Over the last decade, a number of proteins have been identified that bind BMPs in the extracellular space and regulate the interaction of BMPs with their cognate receptors, including the secreted BMP antagonist Chordin. In the early vertebrate embryo, the localized secretion of BMP antagonists from the dorsal blastopore lip establishes a functional BMP signalling gradient that is required for the determination of the dorsoventral or back to belly body axis. In particular, inhibition of BMP activity is essential for the formation of neural tissue in the development of vertebrate and invertebrate embryos. Here we review recent studies that have provided new insight into the regulation of BMP signalling in the extracellular space. In particular, we discuss the recently identified Twisted gastrulation protein that modulates, in concert with metalloproteinases of the Tolloid family, the interaction of Chordin with BMP and a family of proteins that share structural similarities with Chordin in the respective BMP binding domains. In addition, genetic and functional studies in zebrafish and frog provide compelling evidence that the secreted protein Sizzled functionally interacts with the Chd BMP pathway, despite being expressed ventrally in the early gastrula-stage embryo. These intriguing discoveries may have important implications, not only for our current concept of early embryonic patterning, but also for the regulation of BMP activity at later developmental stages and tissue homeostasis in the adult.

  2. Growth differentiation factor 3 is induced by bone morphogenetic protein 6 (BMP-6) and BMP-7 and increases luteinizing hormone receptor messenger RNA expression in human granulosa cells.

    PubMed

    Shi, Jia; Yoshino, Osamu; Osuga, Yutaka; Akiyama, Ikumi; Harada, Miyuki; Koga, Kaori; Fujimoto, Akihisa; Yano, Tetsu; Taketani, Yuji

    2012-04-01

    To examine the relevance of growth differentiation factor 3 (GDF-3) and bone morphogenetic protein (BMP) cytokines in human ovary. Molecular studies. Research laboratory. Eight women undergoing salpingo-oophorectomy and 30 women undergoing ovarian stimulation for in vitro fertilization. Localizing GDF-3 protein in human ovaries; granulosa cells (GC) cultured with GDF-3, BMP-6, or BMP-7 followed by RNA extraction. The localization of GDF-3 protein in normal human ovaries via immunohistochemical analysis, GDF-3 messenger RNA (mRNA) expression evaluation via quantitative real-time reverse transcription and polymerase chain reaction (RT-PCR), and evaluation of the effect of GDF-3 on leuteinizing hormone (LH) receptor mRNA expression via quantitative real-time RT-PCR. In the ovary, BMP cytokines, of the transforming growth factor beta (TGF-β) superfamily, are known as a luteinization inhibitor by suppressing LH receptor expression in GC. Growth differentiation factor 3, a TGF-β superfamily cytokine, is recognized as an inhibitor of BMP cytokines in other cells. Immunohistochemical analysis showed that GDF-3 was strongly detected in the GC of antral follicles. An in vitro assay revealed that BMP-6 or BMP-7 induced GDF-3 mRNA in GC. Also, GDF-3 increased LH receptor mRNA expression and inhibited the effect of BMP-7, which suppressed the LH receptor mRNA expression in GC. GDF-3, induced with BMP-6 and BMP-7, might play a role in folliculogenesis by inhibiting the effect of BMP cytokines. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  3. Mechanisms of Lipid Accumulation in the Bone Morphogenetic Protein Receptor Type 2 Mutant Right Ventricle

    PubMed Central

    Brittain, Evan L.; Fessel, Joshua P.; Penner, Niki; Atkinson, James; Funke, Mitch; Grueter, Carrie; Jerome, W. Gray; Freeman, Michael; Newman, John H.; West, James; Hemnes, Anna R.

    2016-01-01

    Rationale: In heritable pulmonary arterial hypertension with germline mutation in the bone morphogenetic protein receptor type 2 (BMPR2) gene, right ventricle (RV) dysfunction is associated with RV lipotoxicity; however, the underlying mechanism for lipid accumulation is not known. Objectives: We hypothesized that lipid accumulation in cardiomyocytes with BMPR2 mutation occurs owing to alterations in lipid transport and impaired fatty acid oxidation (FAO), which is exacerbated by a high-lipid (Western) diet (WD). Methods: We used a transgenic mouse model of pulmonary arterial hypertension with mutant BMPR2 and generated a cardiomyocyte cell line with BMPR2 mutation. Electron microscopy and metabolomic analysis were performed on mouse RVs. Measurements and Main Results: By metabolomics analysis, we found an increase in long-chain fatty acids in BMPR2 mutant mouse RVs compared with controls, which correlated with cardiac index. BMPR2-mutant cardiomyocytes had increased lipid compared with controls. Direct measurement of FAO in the WD-fed BMPR2-mutant RV showed impaired palmitate-linked oxygen consumption, and metabolomics analysis showed reduced indices of FAO. Using both mutant BMPR2 mouse RVs and cardiomyocytes, we found an increase in the uptake of 14C-palmitate and fatty acid transporter CD36 that was further exacerbated by WD. Conclusions: Taken together, our data suggest that impaired FAO and increased expression of the lipid transporter CD36 are key mechanisms underlying lipid deposition in the BMPR2-mutant RV, which are exacerbated in the presence of dietary lipids. These findings suggest important features leading to RV lipotoxicity in pulmonary arterial hypertension and may point to novel areas of therapeutic intervention. PMID:27077479

  4. Essential Roles of Epithelial Bone Morphogenetic Protein Signaling During Prostatic Development

    PubMed Central

    Omori, Akiko; Miyagawa, Shinichi; Ogino, Yukiko; Harada, Masayo; Ishii, Kenichiro; Sugimura, Yoshiki; Ogino, Hajime; Nakagata, Naomi

    2014-01-01

    Prostate is a male sex-accessory organ. The prostatic epithelia consist primarily of basal and luminal cells that differentiate from embryonic urogenital sinus epithelia. Prostate tumors are believed to originate in the basal and luminal cells. However, factors that promote normal epithelial differentiation have not been well elucidated, particularly for bone morphogenetic protein (Bmp) signaling. This study shows that Bmp signaling prominently increases during prostatic differentiation in the luminal epithelia, which is monitored by the expression of phosphorylated Smad1/5/8. To elucidate the mechanism of epithelial differentiation and the function of Bmp signaling during prostatic development, conditional male mutant mouse analysis for the epithelial-specific Bmp receptor 1a (Bmpr1a) was performed. We demonstrate that Bmp signaling is indispensable for luminal cell maturation, which regulates basal cell proliferation. Expression of the prostatic epithelial regulatory gene Nkx3.1 was significantly reduced in the Bmpr1a mutants. These results indicate that Bmp signaling is a key factor for prostatic epithelial differentiation, possibly by controlling the prostatic regulatory gene Nkx3.1. PMID:24731097

  5. Leiomyoma-derived transforming growth factor-β impairs bone morphogenetic protein-2-mediated endometrial receptivity.

    PubMed

    Doherty, Leo F; Taylor, Hugh S

    2015-03-01

    To determine whether transforming growth factor (TGF)-β3 is a paracrine signal secreted by leiomyoma that inhibits bone morphogenetic protein (BMP)-mediated endometrial receptivity and decidualization. Experimental. Laboratory. Women with symptomatic leiomyomas. Endometrial stromal cells (ESCs) and leiomyoma cells were isolated from surgical specimens. Leiomyoma-conditioned media (LCM) was applied to cultured ESC. The TGF-β was blocked by two approaches: TGF-β pan-specific antibody or transfection with a mutant TGF-β receptor type II. Cells were then treated with recombinant human BMP-2 to assess BMP responsiveness. Expression of BMP receptor types 1A, 1B, 2, as well as endometrial receptivity mediators HOXA10 and leukemia inhibitory factor (LIF). Enzyme-linked immunosorbent assay showed elevated TGF-β levels in LCM. LCM treatment of ESC reduced expression of BMP receptor types 1B and 2 to approximately 60% of pretreatment levels. Preincubation of LCM with TGF-β neutralizing antibody or mutant TGF receptor, but not respective controls, prevented repression of BMP receptors. HOXA10 and LIF expression was repressed in recombinant human BMP-2 treated, LCM exposed ESC. Pretreatment of LCM with TGF-β antibody or transfection with mutant TGF receptor prevented HOXA10 and LIF repression. Leiomyoma-derived TGF-β was necessary and sufficient to alter endometrial BMP-2 responsiveness. Blockade of TGF-β prevents repression of BMP-2 receptors and restores BMP-2-stimulated expression of HOXA10 and LIF. Blockade of TGF signaling is a potential strategy to improve infertility and pregnancy loss associated with uterine leiomyoma. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  6. Promotion of Bone Morphogenetic Protein Signaling by Tetraspanins and Glycosphingolipids

    PubMed Central

    Szymczak, Lindsey C.; Aydin, Taner; Yun, Sijung; Constas, Katharine; Schaeffer, Arielle; Ranjan, Sinthu; Kubba, Saad; Alam, Emad; McMahon, Devin E.; He, Jingpeng; Shwartz, Neta; Tian, Chenxi; Plavskin, Yevgeniy; Lindy, Amanda; Dad, Nimra Amir; Sheth, Sunny; Amin, Nirav M.; Zimmerman, Stephanie; Liu, Dennis; Schwarz, Erich M.; Smith, Harold; Krause, Michael W.; Liu, Jun

    2015-01-01

    Bone morphogenetic proteins (BMPs) belong to the transforming growth factor β (TGFβ) superfamily of secreted molecules. BMPs play essential roles in multiple developmental and homeostatic processes in metazoans. Malfunction of the BMP pathway can cause a variety of diseases in humans, including cancer, skeletal disorders and cardiovascular diseases. Identification of factors that ensure proper spatiotemporal control of BMP signaling is critical for understanding how this pathway is regulated. We have used a unique and sensitive genetic screen to identify the plasma membrane-localized tetraspanin TSP-21 as a key new factor in the C. elegans BMP-like “Sma/Mab” signaling pathway that controls body size and postembryonic M lineage development. We showed that TSP-21 acts in the signal-receiving cells and genetically functions at the ligand-receptor level. We further showed that TSP-21 can associate with itself and with two additional tetraspanins, TSP-12 and TSP-14, which also promote Sma/Mab signaling. TSP-12 and TSP-14 can also associate with SMA-6, the type I receptor of the Sma/Mab pathway. Finally, we found that glycosphingolipids, major components of the tetraspanin-enriched microdomains, are required for Sma/Mab signaling. Our findings suggest that the tetraspanin-enriched membrane microdomains are important for proper BMP signaling. As tetraspanins have emerged as diagnostic and prognostic markers for tumor progression, and TSP-21, TSP-12 and TSP-14 are all conserved in humans, we speculate that abnormal BMP signaling due to altered expression or function of certain tetraspanins may be a contributing factor to cancer development. PMID:25978409

  7. Bone morphogenetic protein type I receptor inhibition induces cleft palate associated with micrognathia and cleft lower lip in mice.

    PubMed

    Lai, Yongzhen; Xie, Changfu; Zhang, Shixian; Gan, Guowu; Wu, Di; Chen, Weihui

    2016-07-01

    Gain-of- and loss-of-function studies have demonstrated that changes in bone morphogenetic protein (BMP) signaling during embryo development cause craniofacial malformations, including cleft palate. It remains uncertain whether BMP signaling could be targeted pharmacologically to affect craniofacial morphogenesis. Pregnant C57Bl/6J mice were treated with the BMP type I receptor inhibitor LDN-193189 at the dose of 3, 6, or 9 mg/kg twice a day by intraperitoneal injection from embryonic day 10.5 (E10.5) to E15.5. At E16.5, embryos were investigated by facial measurement analysis and histology to determine the optimal concentration for malformation. Subsequent embryonic phenotypes were analyzed in detail by histology, whole-mount skeletal staining, micro-computed tomography, and palatal organic culture. We further used immunohistochemistry to analyze protein expression of the BMP-mediated canonical and noncanonical signaling components. The optimal concentration of LDN-193189 was determined to be 6 mg/kg. In utero, LDN-193189 exposures induced partial clefting of the anterior palate or complete cleft palate, which was attributed to a reduced cell proliferation rate in the secondary palate, and delayed palatal elevation caused by micrognathia. Analysis of signal transduction in palatal shelves at E12.5 and E13.5 identified a significant reduction of BMP/Smad signaling (p-Smad1/5/8) and unchanged BMP noncanonical signaling (p-p38, p-Erk1/2) after treatment with LDN-193189. The results of this study indicate that LDN-193189 can be used to manipulate BMP signaling by selectively targeting the BMP/Smad signaling pathway to affect palatal morphogenesis and produce phenotypes mimicking those caused by genetic mutations. This work established a novel mouse model for teratogen-induced cleft palate. Birth Defects Research (Part A) 106:612-623, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  8. Facilitated receptor-recognition and enhanced bioactivity of bone morphogenetic protein-2 on magnesium-substituted hydroxyapatite surface

    PubMed Central

    Huang, Baolin; Yuan, Yuan; Li, Tong; Ding, Sai; Zhang, Wenjing; Gu, Yuantong; Liu, Changsheng

    2016-01-01

    Biomaterial surface functionalized with bone morphogenetic protein-2 (BMP-2) is a promising approach to fabricating successful orthopedic implants/scaffolds. However, the bioactivity of BMP-2 on material surfaces is still far from satisfactory and the mechanism of related protein-surface interaction remains elusive. Based on the most widely used bone-implants/scaffolds material, hydroxyapatite (HAP), we developed a matrix of magnesium-substituted HAP (Mg-HAP, 2.2 at% substitution) to address these issues. Further, we investigated the adsorption dynamics, BMPRs-recruitment, and bioactivity of recombinant human BMP-2 (rhBMP-2) on the HAP and Mg-HAP surfaces. To elucidate the mechanism, molecular dynamic simulations were performed to calculate the preferred orientations, conformation changes, and cysteine-knot stabilities of adsorbed BMP-2 molecules. The results showed that rhBMP-2 on the Mg-HAP surface exhibited greater bioactivity, evidenced by more facilitated BMPRs-recognition and higher ALP activity than on the HAP surface. Moreover, molecular simulations indicated that BMP-2 favoured distinct side-on orientations on the HAP and Mg-HAP surfaces. Intriguingly, BMP-2 on the Mg-HAP surface largely preserved the active protein structure evidenced by more stable cysteine-knots than on the HAP surface. These findings explicitly clarify the mechanism of BMP-2-HAP/Mg-HAP interactions and highlight the promising application of Mg-HAP/BMP-2 matrixes in bone regeneration implants/scaffolds. PMID:27075233

  9. Facilitated receptor-recognition and enhanced bioactivity of bone morphogenetic protein-2 on magnesium-substituted hydroxyapatite surface

    NASA Astrophysics Data System (ADS)

    Huang, Baolin; Yuan, Yuan; Li, Tong; Ding, Sai; Zhang, Wenjing; Gu, Yuantong; Liu, Changsheng

    2016-04-01

    Biomaterial surface functionalized with bone morphogenetic protein-2 (BMP-2) is a promising approach to fabricating successful orthopedic implants/scaffolds. However, the bioactivity of BMP-2 on material surfaces is still far from satisfactory and the mechanism of related protein-surface interaction remains elusive. Based on the most widely used bone-implants/scaffolds material, hydroxyapatite (HAP), we developed a matrix of magnesium-substituted HAP (Mg-HAP, 2.2 at% substitution) to address these issues. Further, we investigated the adsorption dynamics, BMPRs-recruitment, and bioactivity of recombinant human BMP-2 (rhBMP-2) on the HAP and Mg-HAP surfaces. To elucidate the mechanism, molecular dynamic simulations were performed to calculate the preferred orientations, conformation changes, and cysteine-knot stabilities of adsorbed BMP-2 molecules. The results showed that rhBMP-2 on the Mg-HAP surface exhibited greater bioactivity, evidenced by more facilitated BMPRs-recognition and higher ALP activity than on the HAP surface. Moreover, molecular simulations indicated that BMP-2 favoured distinct side-on orientations on the HAP and Mg-HAP surfaces. Intriguingly, BMP-2 on the Mg-HAP surface largely preserved the active protein structure evidenced by more stable cysteine-knots than on the HAP surface. These findings explicitly clarify the mechanism of BMP-2-HAP/Mg-HAP interactions and highlight the promising application of Mg-HAP/BMP-2 matrixes in bone regeneration implants/scaffolds.

  10. Bone morphogenetic proteins in musculoskeletal medicine.

    PubMed

    Giannoudis, Peter V; Einhorn, Thomas A

    2009-12-01

    Ongoing research at the molecular level has expanded our understanding of the physiological processes that regulate the complex phenomena of fracture healing and bone regeneration. A number of key molecules have been identified and shown to facilitate the progression of healing from one stage to another, leading to an uneventful outcome. Among these candidate molecules, bone morphogenetic proteins (BMPs) possess potent osteoinductive properties. They interact with osteoprogenitor cells, regulating both mitogenesis and differentiation potential. Since the discovery of BMPs, a number of experimental and clinical trials have supported their safety and efficacy of their use in therapy. Nonetheless, at times their efficacy falls short of expectations. Several factors have been identified as contributing to this result. It is anticipated that, as our knowledge expands and we understand better the complex pathways and cascades of molecular events attributable to BMPs, the application of these molecules in the clinical setting will continue to increase and to show more favourable outcomes. Copyright 2009 Elsevier Ltd. All rights reserved.

  11. The bone morphogenetic protein axis is a positive regulator of skeletal muscle mass

    PubMed Central

    Chen, Justin L.; Qian, Hongwei; Liu, Yingying; Bernardo, Bianca C.; Beyer, Claudia; Watt, Kevin I.; Thomson, Rachel E.; Connor, Timothy; Turner, Bradley J.; McMullen, Julie R.; Larsson, Lars; McGee, Sean L.; Harrison, Craig A.

    2013-01-01

    Although the canonical transforming growth factor β signaling pathway represses skeletal muscle growth and promotes muscle wasting, a role in muscle for the parallel bone morphogenetic protein (BMP) signaling pathway has not been defined. We report, for the first time, that the BMP pathway is a positive regulator of muscle mass. Increasing the expression of BMP7 or the activity of BMP receptors in muscles induced hypertrophy that was dependent on Smad1/5-mediated activation of mTOR signaling. In agreement, we observed that BMP signaling is augmented in models of muscle growth. Importantly, stimulation of BMP signaling is essential for conservation of muscle mass after disruption of the neuromuscular junction. Inhibiting the phosphorylation of Smad1/5 exacerbated denervation-induced muscle atrophy via an HDAC4-myogenin–dependent process, whereas increased BMP–Smad1/5 activity protected muscles from denervation-induced wasting. Our studies highlight a novel role for the BMP signaling pathway in promoting muscle growth and inhibiting muscle wasting, which may have significant implications for the development of therapeutics for neuromuscular disorders. PMID:24145169

  12. Inhibition of bone morphogenetic protein signaling attenuates anemia associated with inflammation

    PubMed Central

    Steinbicker, Andrea U.; Sachidanandan, Chetana; Vonner, Ashley J.; Yusuf, Rushdia Z.; Deng, Donna Y.; Lai, Carol S.; Rauwerdink, Kristen M.; Winn, Julia C.; Saez, Borja; Cook, Colleen M.; Szekely, Brian A.; Roy, Cindy N.; Seehra, Jasbir S.; Cuny, Gregory D.; Scadden, David T.; Peterson, Randall T.; Bloch, Kenneth D.

    2011-01-01

    Anemia of inflammation develops in settings of chronic inflammatory, infectious, or neoplastic disease. In this highly prevalent form of anemia, inflammatory cytokines, including IL-6, stimulate hepatic expression of hepcidin, which negatively regulates iron bioavailability by inactivating ferroportin. Hepcidin is transcriptionally regulated by IL-6 and bone morphogenetic protein (BMP) signaling. We hypothesized that inhibiting BMP signaling can reduce hepcidin expression and ameliorate hypoferremia and anemia associated with inflammation. In human hepatoma cells, IL-6–induced hepcidin expression, an effect that was inhibited by treatment with a BMP type I receptor inhibitor, LDN-193189, or BMP ligand antagonists noggin and ALK3-Fc. In zebrafish, the induction of hepcidin expression by transgenic expression of IL-6 was also reduced by LDN-193189. In mice, treatment with IL-6 or turpentine increased hepcidin expression and reduced serum iron, effects that were inhibited by LDN-193189 or ALK3-Fc. Chronic turpentine treatment led to microcytic anemia, which was prevented by concurrent administration of LDN-193189 or attenuated when LDN-193189 was administered after anemia was established. Our studies support the concept that BMP and IL-6 act together to regulate iron homeostasis and suggest that inhibition of BMP signaling may be an effective strategy for the treatment of anemia of inflammation. PMID:21393479

  13. LIM mineralization protein-1 potentiates bone morphogenetic protein responsiveness via a novel interaction with Smurf1 resulting in decreased ubiquitination of Smads.

    PubMed

    Sangadala, Sreedhara; Boden, Scott D; Viggeswarapu, Manjula; Liu, Yunshan; Titus, Louisa

    2006-06-23

    Development and repair of the skeletal system and other organs is highly dependent on precise regulation of bone morphogenetic proteins (BMPs), their receptors, and their intracellular signaling proteins known as Smads. The use of BMPs clinically to induce bone formation has been limited in part by the requirement of much higher doses of recombinant proteins in primates than were needed in cell culture or rodents. Therefore, control of cellular responsiveness to BMPs is now a critical area that is poorly understood. We determined that LMP-1, a LIM domain protein capable of inducing de novo bone formation, interacts with Smurf1 (Smad ubiquitin regulatory factor 1) and prevents ubiquitination of Smads. In the region of LMP responsible for bone formation, there is a motif that directly interacts with the Smurf1 WW2 domain and can effectively compete with Smad1 and Smad5 for binding. We have shown that small peptides containing this motif can mimic the ability to block Smurf1 from binding Smads. This novel interaction of LMP-1 with the WW2 domain of Smurf1 to block Smad binding results in increased cellular responsiveness to exogenous BMP and demonstrates a novel regulatory mechanism for the BMP signaling pathway.

  14. Comparative genomic analysis of the eight-membered ring cystine knot-containing bone morphogenetic protein antagonists.

    PubMed

    Avsian-Kretchmer, Orna; Hsueh, Aaron J W

    2004-01-01

    TGF-beta family proteins with a cystine knot motif serve as ligands for diverse families of plasma membrane receptors. Bone morphogenetic protein (BMP) antagonists represent a subgroup of these proteins, some of which bind BMPs and antagonize their actions during development and morphogenesis. Availability of completed genome sequences from diverse organisms allows bioinformatic analysis of the evolution of BMP antagonists and facilitates their classification. Using a regular expression algorithm (http://BioRegEx.stanford.edu), an exhaustive search of the human genome identified all cystine knot-containing BMP antagonists. Based on the size of the cystine ring, these proteins were divided into three subfamilies: CAN (eight-membered ring), twisted gastrulation (nine-membered ring), as well as chordin and noggin (10-membered ring). The CAN family can be divided further into four subgroups based on a conserved arrangement of additional cysteine residues-gremlin and PRDC, cerberus and coco, and DAN, together with USAG-1 and sclerostin. We searched for orthologs of human BMP antagonists in the genomes of model organisms and analyzed their phylogenetic relationship. New human paralogs were identified together with the verification of orthologous relationships of known genes. We also discuss the physiological roles of the CAN subfamily of BMP antagonists and the associated genetic defects. Based on the known three-dimensional structure of key cystine knot proteins, we postulated disulfide bondings for eight-membered ring BMP antagonists to predict their potential folding and dimerization.

  15. Orphan nuclear receptor chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) protein negatively regulates bone morphogenetic protein 2-induced osteoblast differentiation through suppressing runt-related gene 2 (Runx2) activity.

    PubMed

    Lee, Kkot-Nim; Jang, Won-Gu; Kim, Eun-Jung; Oh, Sin-Hye; Son, Hye-Ju; Kim, Sun-Hun; Franceschi, Renny; Zhang, Xiao-Kun; Lee, Shee-Eun; Koh, Jeong-Tae

    2012-06-01

    Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) is an orphan nuclear receptor of the steroid-thyroid hormone receptor superfamily. COUP-TFII is widely expressed in multiple tissues and organs throughout embryonic development and has been shown to regulate cellular growth, differentiation, and organ development. However, the role of COUP-TFII in osteoblast differentiation has not been systematically evaluated. In the present study, COUP-TFII was strongly expressed in multipotential mesenchymal cells, and the endogenous expression level decreased during osteoblast differentiation. Overexpression of COUP-TFII inhibited bone morphogenetic protein 2 (BMP2)-induced osteoblastic gene expression. The results of alkaline phosphatase, Alizarin Red staining, and osteocalcin production assay showed that COUP-TFII overexpression blocks BMP2-induced osteoblast differentiation. In contrast, the down-regulation of COUP-TFII synergistically induced the expression of BMP2-induced osteoblastic genes and osteoblast differentiation. Furthermore, the immunoprecipitation assay showed that COUP-TFII and Runx2 physically interacted and COUP-TFII significantly impaired the Runx2-dependent activation of the osteocalcin promoter. From the ChIP assay, we found that COUP-TFII repressed DNA binding of Runx2 to the osteocalcin gene, whereas Runx2 inhibited COUP-TFII expression via direct binding to the COUP-TFII promoter. Taken together, these findings demonstrate that COUP-TFII negatively regulates osteoblast differentiation via interaction with Runx2, and during the differentiation state, BMP2-induced Runx2 represses COUP-TFII expression and promotes osteoblast differentiation.

  16. Bone Regeneration Using Bone Morphogenetic Proteins and Various Biomaterial Carriers

    PubMed Central

    Sheikh, Zeeshan; Javaid, Mohammad Ahmad; Hamdan, Nader; Hashmi, Raheel

    2015-01-01

    Trauma and disease frequently result in fractures or critical sized bone defects and their management at times necessitates bone grafting. The process of bone healing or regeneration involves intricate network of molecules including bone morphogenetic proteins (BMPs). BMPs belong to a larger superfamily of proteins and are very promising and intensively studied for in the enhancement of bone healing. More than 20 types of BMPs have been identified but only a subset of BMPs can induce de novo bone formation. Many research groups have shown that BMPs can induce differentiation of mesenchymal stem cells and stem cells into osteogenic cells which are capable of producing bone. This review introduces BMPs and discusses current advances in preclinical and clinical application of utilizing various biomaterial carriers for local delivery of BMPs to enhance bone regeneration. PMID:28788032

  17. Bone Morphogenetic Protein (BMP) signaling in development and human diseases

    PubMed Central

    Wang, Richard N.; Green, Jordan; Wang, Zhongliang; Deng, Youlin; Qiao, Min; Peabody, Michael; Zhang, Qian; Ye, Jixing; Yan, Zhengjian; Denduluri, Sahitya; Idowu, Olumuyiwa; Li, Melissa; Shen, Christine; Hu, Alan; Haydon, Rex C.; Kang, Richard; Mok, James; Lee, Michael J.; Luu, Hue L.; Shi, Lewis L.

    2014-01-01

    Bone Morphogenetic Proteins (BMPs) are a group of signaling molecules that belongs to the Transforming Growth Factor-β (TGF-β) superfamily of proteins. Initially discovered for their ability to induce bone formation, BMPs are now known to play crucial roles in all organ systems. BMPs are important in embryogenesis and development, and also in maintenance of adult tissue homeostasis. Mouse knockout models of various components of the BMP signaling pathway result in embryonic lethality or marked defects, highlighting the essential functions of BMPs. In this review, we first outline the basic aspects of BMP signaling and then focus on genetically manipulated mouse knockout models that have helped elucidate the role of BMPs in development. A significant portion of this review is devoted to the prominent human pathologies associated with dysregulated BMP signaling. PMID:25401122

  18. Bone morphogenetic protein 9 (BMP9) controls lymphatic vessel maturation and valve formation

    PubMed Central

    Levet, Sandrine; Ciais, Delphine; Merdzhanova, Galina; Mallet, Christine; Zimmers, Teresa A.; Lee, Se-Jin; Navarro, Fabrice P.; Texier, Isabelle; Feige, Jean-Jacques; Bailly, Sabine

    2013-01-01

    Lymphatic vessels are critical for the maintenance of tissue fluid homeostasis and their dysfunction contributes to several human diseases. The activin receptor-like kinase 1 (ALK1) is a transforming growth factor-β family type 1 receptor that is expressed on both blood and lymphatic endothelial cells (LECs). Its high-affinity ligand, bone morphogenetic protein 9 (BMP9), has been shown to be critical for retinal angiogenesis. The aim of this work was to investigate whether BMP9 could play a role in lymphatic development. We found that Bmp9 deficiency in mice causes abnormal lymphatic development. Bmp9-knockout (KO) pups presented hyperplastic mesenteric collecting vessels that maintained LYVE-1 expression. In accordance with this result, we found that BMP9 inhibited LYVE-1 expression in LECs in an ALK1-dependent manner. Bmp9-KO pups also presented a significant reduction in the number and in the maturation of mesenteric lymphatic valves at embryonic day 18.5 and at postnatal days 0 and 4. Interestingly, the expression of several genes known to be involved in valve formation (Foxc2, Connexin37, EphrinB2, and Neuropilin1) was upregulated by BMP9 in LECS. Finally, we demonstrated that Bmp9-KO neonates and adult mice had decreased lymphatic draining efficiency. These data identify BMP9 as an important extracellular regulator in the maturation of the lymphatic vascular network affecting valve development and lymphatic vessel function. PMID:23741013

  19. Bone morphogenetic protein 9 (BMP9) and BMP10 enhance tumor necrosis factor-α-induced monocyte recruitment to the vascular endothelium mainly via activin receptor-like kinase 2.

    PubMed

    Mitrofan, Claudia-Gabriela; Appleby, Sarah L; Nash, Gerard B; Mallat, Ziad; Chilvers, Edwin R; Upton, Paul D; Morrell, Nicholas W

    2017-08-18

    Bone morphogenetic proteins 9 and 10 (BMP9/BMP10) are circulating cytokines with important roles in endothelial homeostasis. The aim of this study was to investigate the roles of BMP9 and BMP10 in mediating monocyte-endothelial interactions using an in vitro flow adhesion assay. Herein, we report that whereas BMP9/BMP10 alone had no effect on monocyte recruitment, at higher concentrations both cytokines synergized with tumor necrosis factor-α (TNFα) to increase recruitment to the vascular endothelium. The BMP9/BMP10-mediated increase in monocyte recruitment in the presence of TNFα was associated with up-regulated expression levels of E-selectin, vascular cell adhesion molecule (VCAM-1), and intercellular adhesion molecule 1 (ICAM-1) on endothelial cells. Using siRNAs to type I and II BMP receptors and the signaling intermediaries (Smads), we demonstrated a key role for ALK2 in the BMP9/BMP10-induced surface expression of E-selectin, and both ALK1 and ALK2 in the up-regulation of VCAM-1 and ICAM-1. The type II receptors, BMPR-II and ACTR-IIA were both required for this response, as was Smad1/5. The up-regulation of cell surface adhesion molecules by BMP9/10 in the presence of TNFα was inhibited by LDN193189, which inhibits ALK2 but not ALK1. Furthermore, LDN193189 inhibited monocyte recruitment induced by TNFα and BMP9/10. BMP9/10 increased basal IκBα protein expression, but did not alter p65/RelA levels. Our findings suggest that higher concentrations of BMP9/BMP10 synergize with TNFα to induce the up-regulation of endothelial selectins and adhesion molecules, ultimately resulting in increased monocyte recruitment to the vascular endothelium. This process is mediated mainly via the ALK2 type I receptor, BMPR-II/ACTR-IIA type II receptors, and downstream Smad1/5 signaling. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Heterozygous Null Bone Morphogenetic Protein Receptor Type 2 Mutations Promote SRC Kinase-dependent Caveolar Trafficking Defects and Endothelial Dysfunction in Pulmonary Arterial Hypertension*

    PubMed Central

    Prewitt, Allison R.; Ghose, Sampa; Frump, Andrea L.; Datta, Arumima; Austin, Eric D.; Kenworthy, Anne K.; de Caestecker, Mark P.

    2015-01-01

    Hereditary pulmonary arterial hypertension (HPAH) is a rare, fatal disease of the pulmonary vasculature. The majority of HPAH patients inherit mutations in the bone morphogenetic protein type 2 receptor gene (BMPR2), but how these promote pulmonary vascular disease is unclear. HPAH patients have features of pulmonary endothelial cell (PEC) dysfunction including increased vascular permeability and perivascular inflammation associated with decreased PEC barrier function. Recently, frameshift mutations in the caveolar structural protein gene Caveolin-1 (CAV-1) were identified in two patients with non-BMPR2-associated HPAH. Because caveolae regulate endothelial function and vascular permeability, we hypothesized that defects in caveolar function might be a common mechanism by which BMPR2 mutations promote pulmonary vascular disease. To explore this, we isolated PECs from mice carrying heterozygous null Bmpr2 mutations (Bmpr2+/−) similar to those found in the majority of HPAH patients. We show that Bmpr2+/− PECs have increased numbers and intracellular localization of caveolae and caveolar structural proteins CAV-1 and Cavin-1 and that these defects are reversed after blocking endocytosis with dynasore. SRC kinase is also constitutively activated in Bmpr2+/− PECs, and localization of CAV-1 to the plasma membrane is restored after treating Bmpr2+/− PECs with the SRC kinase inhibitor 3-(4-chlorophenyl)-1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP2). Late outgrowth endothelial progenitor cells isolated from HPAH patients show similar increased activation of SRC kinase. Moreover, Bmpr2+/− PECs have impaired endothelial barrier function, and barrier function is restored after treatment with PP2. These data suggest that heterozygous null BMPR2 mutations promote SRC-dependent caveolar trafficking defects in PECs and that this may contribute to pulmonary endothelial barrier dysfunction in HPAH patients. PMID:25411245

  1. Smad7 Regulates the Adult Neural Stem/Progenitor Cell Pool in a Transforming Growth Factor β- and Bone Morphogenetic Protein-Independent Manner▿

    PubMed Central

    Krampert, Monika; Chirasani, Sridhar Reddy; Wachs, Frank-Peter; Aigner, Robert; Bogdahn, Ulrich; Yingling, Jonathan M.; Heldin, Carl-Henrik; Aigner, Ludwig; Heuchel, Rainer

    2010-01-01

    Members of the transforming growth factor β (TGF-β) family of proteins modulate the proliferation, differentiation, and survival of many different cell types. Neural stem and progenitor cells (NPCs) in the adult brain are inhibited in their proliferation by TGF-β and by bone morphogenetic proteins (BMPs). Here, we investigated neurogenesis in a hypomorphic mouse model for the TGF-β and BMP inhibitor Smad7, with the hypothesis that NPC proliferation might be reduced due to increased TGF-β and BMP signaling. Unexpectedly, we found enhanced NPC proliferation as well as an increased number of label-retaining cells in vivo. The enhanced proliferation potential of mutant cells was retained in vitro in neurosphere cultures. We observed a higher sphere-forming capacity as well as faster growth and cell cycle progression. Use of specific inhibitors revealed that these effects were independent of TGF-β and BMP signaling. The enhanced proliferation might be at least partially mediated by elevated signaling via epidermal growth factor (EGF) receptor, as mutant cells showed higher expression and activation levels of the EGF receptor. Conversely, an EGF receptor inhibitor reduced the proliferation of these cells. Our data indicate that endogenous Smad7 regulates neural stem/progenitor cell proliferation in a TGF-β- and BMP-independent manner. PMID:20479122

  2. Intracellular amyloid beta expression leads to dysregulation of the mitogen-activated protein kinase and bone morphogenetic protein-2 signaling axis

    PubMed Central

    Cruz, Eric; Kumar, Sushil; Yuan, Li; Arikkath, Jyothi

    2018-01-01

    Alzheimer’s disease (AD) is a neurodegenerative syndrome classically depicted by the parenchymal accumulation of extracellular amyloid beta plaques. However, recent findings suggest intraneuronal amyloid beta (iAβ1–42) accumulation precedes extracellular deposition. Furthermore, the pathologic increase in iAβ1–42 has been implicated in dysregulation of cellular mechanisms critically important in axonal transport. Owing to neuronal cell polarity, retrograde and anterograde axonal transport are essential trafficking mechanism necessary to convey membrane bound neurotransmitters, neurotrophins, and endosomes between soma and synaptic interfaces. Although iAβ1–42 disruption of axonal transport has been implicated in dysregulation of neuronal synaptic transmission, the role of iAβ1–42 and its influence on signal transduction involving the mitogen-activated protein kinase (MAPK) and morphogenetic signaling axis are unknown. Our biochemical characterization of intracellular amyloid beta accumulation on MAPK and morphogenetic signaling have revealed increased iAβ1–42 expression leads to significant reduction in ERK 1/2 phosphorylation and increased bone morphogenetic protein 2 dependent Smad 1/5/8 phosphorylation. Furthermore, rescue of iAβ1–42 mediated attenuation of MAPK signaling can be accomplished with the small molecule PLX4032 as a downstream enhancer of the MAPK pathway. Consequently, our observations regarding the dysregulation of these gatekeepers of neuronal viability may have important implications in understanding the iAβ1–42 mediated effects observed in AD. PMID:29470488

  3. Positioning cell wall synthetic complexes by the bacterial morphogenetic proteins MreB and MreD.

    PubMed

    White, Courtney L; Kitich, Aleksandar; Gober, James W

    2010-05-01

    In Caulobacter crescentus, intact cables of the actin homologue, MreB, are required for the proper spatial positioning of MurG which catalyses the final step in peptidoglycan precursor synthesis. Similarly, in the periplasm, MreC controls the spatial orientation of the penicillin binding proteins and a lytic transglycosylase. We have now found that MreB cables are required for the organization of several other cytosolic murein biosynthetic enzymes such as MraY, MurB, MurC, MurE and MurF. We also show these proteins adopt a subcellular pattern of localization comparable to MurG, suggesting the existence of cytoskeletal-dependent interactions. Through extensive two-hybrid analyses, we have now generated a comprehensive interaction map of components of the bacterial morphogenetic complex. In the cytosol, this complex contains both murein biosynthetic enzymes and morphogenetic proteins, including RodA, RodZ and MreD. We show that the integral membrane protein, MreD, is essential for lateral peptidoglycan synthesis, interacts with the precursor synthesizing enzymes MurG and MraY, and additionally, determines MreB localization. Our results suggest that the interdependent localization of MreB and MreD functions to spatially organize a complex of peptidoglycan precursor synthesis proteins, which is required for propagation of a uniform cell shape and catalytically efficient peptidoglycan synthesis.

  4. [The role of Smads and related transcription factors in the signal transduction of bone morphogenetic protein inducing bone formation].

    PubMed

    Xu, Xiao-liang; Dai, Ke-rong; Tang, Ting-ting

    2003-09-01

    To clarify the mechanisms of the signal transduction of bone morphogenetic proteins (BMPs) inducing bone formation and to provide theoretical basis for basic and applying research of BMPs. We looked up the literature of the role of Smads and related transcription factors in the signal transduction of BMPs inducing bone formation. The signal transduction processes of BMPs included: 1. BMPs combined with type II and type I receptors; 2. the type I receptor phosphorylated Smads; and 3. Smads entered the cell nucleus, interacted with transcription factors and influenced the transcription of related proteins. Smads could be divided into receptor-regulated Smads (R-Smads: Smad1, Smad2, Smad3, Smad5, Smad8 and Smad9), common-mediator Smad (co-Smad: Smad4), and inhibitory Smads (I-Smads: Smad6 and Smad7). Smad1, Smad5, Smad8, and probable Smad9 were involved in the signal transduction of BMPs. Multiple kinases, such as focal adhesion kinase (FAK), Ras-extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K), and Akt serine/threonine kinase were related to Smads signal transduction. Smad1 and Smad5 related with transcription factors included core binding factor A1 (CBFA1), smad-interacting protein 1 (SIP1), ornithine decarboxylase antizyme (OAZ), activating protein-1 (AP-1), xenopus ventralizing homeobox protein-2 (Xvent-2), sandostatin (Ski), antiproliferative proteins (Tob), and homeodomain-containing transcriptian factor-8 (Hoxc-8), et al. CBFA1 could interact with Smad1, Smad2, Smad3, and Smad5, so it was involved in TGF-beta and BMP-2 signal transduction, and played an important role in the bone formation. Cleidocranial dysplasia (CCD) was thought to be caused by heterozygous mutations in CBFA1. The CBFA1 knockout mice showed no osteogenesis and had maturational disturbance of chondrocytes. Smads and related transcription factors, especially Smad1, Smad5, Smad8 and CBFA1, play an important role in the signal transduction of BMPs inducing bone

  5. Role of bone morphogenetic protein-7 in renal fibrosis

    PubMed Central

    Li, Rui Xi; Yiu, Wai Han; Tang, Sydney C. W.

    2015-01-01

    Renal fibrosis is final common pathway of end stage renal disease. Irrespective of the primary cause, renal fibrogenesis is a dynamic process which involves a large network of cellular and molecular interaction, including pro-inflammatory cell infiltration and activation, matrix-producing cell accumulation and activation, and secretion of profibrogenic factors that modulate extracellular matrix (ECM) formation and cell-cell interaction. Bone morphogenetic protein-7 is a protein of the TGF-β super family and increasingly regarded as a counteracting molecule against TGF-β. A large variety of evidence shows an anti-fibrotic role of BMP-7 in chronic kidney disease, and this effect is largely mediated via counterbalancing the profibrotic effect of TGF-β. Besides, BMP-7 reduced ECM formation by inactivating matrix-producing cells and promoting mesenchymal-to-epithelial transition (MET). BMP-7 also increased ECM degradation. Despite these observations, the anti-fibrotic effect of BMP-7 is still controversial such that fine regulation of BMP-7 expression in vivo might be a great challenge for its ultimate clinical application. PMID:25954203

  6. Inductive specification and axonal orientation of spinal neurons mediated by divergent bone morphogenetic protein signaling pathways

    PubMed Central

    2011-01-01

    Background Bone morphogenetic protein (BMP)7 evokes both inductive and axon orienting responses in dorsal interneurons (dI neurons) in the developing spinal cord. These events occur sequentially during the development of spinal neurons but in these and other cell types such inductive and acute chemotactic responses occur concurrently, highlighting the requirement for divergent intracellular signaling. Both type I and type II BMP receptor subtypes have been implicated selectively in orienting responses but it remains unclear how, in a given cell, divergence occurs. We have examined the mechanisms by which disparate BMP7 activities are generated in dorsal spinal neurons. Results We show that widely different threshold concentrations of BMP7 are required to elicit the divergent inductive and axon orienting responses. Type I BMP receptor kinase activity is required for activation of pSmad signaling and induction of dI character by BMP7, a high threshold response. In contrast, neither type I BMP receptor kinase activity nor Smad1/5/8 phosphorylation is involved in the low threshold orienting responses of dI axons to BMP7. Instead, BMP7-evoked axonal repulsion and growth cone collapse are dependent on phosphoinositide-3-kinase (PI3K) activation, plausibly through type II receptor signaling. BMP7 stimulates PI3K-dependent signaling in dI neurons. BMP6, which evokes neural induction but does not have orienting activity, activates Smad signaling but does not stimulate PI3K. Conclusions Divergent signaling through pSmad-dependent and PI3K-dependent (Smad-independent) mechanisms mediates the inductive and orienting responses of dI neurons to BMP7. A model is proposed whereby selective engagement of BMP receptor subunits underlies choice of signaling pathway. PMID:22085733

  7. Downregulated bone morphogenetic protein signaling in nitrofen-induced congenital diaphragmatic hernia.

    PubMed

    Makanga, Martine; Dewachter, Céline; Maruyama, Hidekazu; Vuckovic, Aline; Rondelet, Benoit; Naeije, Robert; Dewachter, Laurence

    2013-08-01

    Bone morphogenetic proteins (BMP) have been shown to play crucial roles in not only lung and heart development, but also in the pathogenesis of pulmonary vascular remodeling in pulmonary hypertension (PH). We therefore hypothesized that BMP signaling could be altered in nitrofen-induced congenital diaphragmatic hernia (CDH) and associated PH. Pregnant rats were exposed to either 100 mg nitrofen or vehicle on embryonic day (E) 9.5. On E17 and E21, fetuses were delivered by cesarean section, killed and checked for left-sided CDH. The tissue was then harvested for pathobiological evaluation. In nitrofen-induced CDH, pulmonary expressions of BMP4, BMP receptor (BMPR) type 2 and Id1 decreased on E17 and E21. On E17, pulmonary gremlin-1 expression increased, while BMP7 decreased. In the lungs, Id1 expression was correlated to BMP4 and BMPR2 and inversely correlated to gremlin-1 expression. Myocardial expressions of BMPR2, BMPR1A, BMP7 and SERCA-2A decreased, while gremlin-1 and noggin expressions increased on E17. On E21, myocardial expressions of Id1 and SERCA-2A decreased, while gremlin-1 expression increased. Moreover, BMPR2 and BMPR1A expressions were correlated to SERCA-2A expression and inversely correlated to pro-apoptotic Bax/Bcl2 ratio within the myocardium. Downregulation of BMP signaling seems to contribute to pulmonary and myocardial anomalies observed in nitrofen-induced CDH.

  8. Maxillary anterior ridge augmentation with recombinant human bone morphogenetic protein 2.

    PubMed

    Edmunds, Ryan K; Mealey, Brian L; Mills, Michael P; Thoma, Daniel S; Schoolfield, John; Cochran, David L; Mellonig, Jim

    2014-01-01

    No human studies exist on the use of recombinant human bone morphogenetic protein 2 (rhBMP-2) on an absorbable collagen sponge (ACS) as a sole graft material for lateral ridge augmentation in large ridge defect sites. This series evaluates the treatment outcome of maxillary anterior lateral ridge augmentation with rhBMP-2/ACS. Twenty patients were treated with rhBMP-2/ACS and fixation screws for space maintenance. Cone beam volumetric tomography measurements were used to determine gain in ridge width, and a bone core biopsy was obtained. The mean horizontal ridge gain was 1.2 mm across sites, and every site gained width.

  9. Bone morphogenetic protein 4 antagonizes hair cell regeneration in the avian auditory epithelium.

    PubMed

    Lewis, Rebecca M; Keller, Jesse J; Wan, Liangcai; Stone, Jennifer S

    2018-07-01

    Permanent hearing loss is often a result of damage to cochlear hair cells, which mammals are unable to regenerate. Non-mammalian vertebrates such as birds replace damaged hair cells and restore hearing function, but mechanisms controlling regeneration are not understood. The secreted protein bone morphogenetic protein 4 (BMP4) regulates inner ear morphogenesis and hair cell development. To investigate mechanisms controlling hair cell regeneration in birds, we examined expression and function of BMP4 in the auditory epithelia (basilar papillae) of chickens of either sex after hair cell destruction by ototoxic antibiotics. In mature basilar papillae, BMP4 mRNA is highly expressed in hair cells, but not in hair cell progenitors (supporting cells). Supporting cells transcribe genes encoding receptors for BMP4 (BMPR1A, BMPR1B, and BMPR2) and effectors of BMP4 signaling (ID transcription factors). Following hair cell destruction, BMP4 transcripts are lost from the sensory epithelium. Using organotypic cultures, we demonstrate that treatments with BMP4 during hair cell destruction prevent supporting cells from upregulating expression of the pro-hair cell transcription factor ATOH1, entering the cell cycle, and fully transdifferentiating into hair cells, but they do not induce cell death. By contrast, noggin, a BMP4 inhibitor, increases numbers of regenerated hair cells. These findings demonstrate that BMP4 antagonizes hair cell regeneration in the chicken basilar papilla, at least in part by preventing accumulation of ATOH1 in hair cell precursors. Copyright © 2018 Elsevier B.V. All rights reserved.

  10. Bone morphogenetic protein (BMP)1-3 enhances bone repair.

    PubMed

    Grgurevic, Lovorka; Macek, Boris; Mercep, Mladen; Jelic, Mislav; Smoljanovic, Tomislav; Erjavec, Igor; Dumic-Cule, Ivo; Prgomet, Stefan; Durdevic, Dragan; Vnuk, Drazen; Lipar, Marija; Stejskal, Marko; Kufner, Vera; Brkljacic, Jelena; Maticic, Drazen; Vukicevic, Slobodan

    2011-04-29

    Members of the astacin family of metalloproteinases such as human bone morphogenetic protein 1 (BMP-1) regulate morphogenesis by processing precursors to mature functional extracellular matrix (ECM) proteins and several growth factors including TGFβ, BMP2, BMP4 and GFD8. We have recently discovered that BMP1-3 isoform of the Bmp-1 gene circulates in the human plasma and is significantly increased in patients with acute bone fracture. We hypothesized that circulating BMP1-3 might have an important role in bone repair and serve as a novel bone biomarker. When administered systemically to rats with a long bone fracture and locally to rabbits with a critical size defect of the ulna, recombinant human BMP1-3 enhanced bone healing. In contrast, neutralization of the endogenous BMP1-3 by a specific polyclonal antibody delayed the bone union. Invitro BMP1-3 increased the expression of collagen type I and osteocalcin in MC3T3-E(1) osteoblast like cells, and enhanced the formation of mineralized bone nodules from bone marrow mesenchymal stem cells. We suggest that BMP1-3 is a novel systemic regulator of bone repair. Copyright © 2011 Elsevier Inc. All rights reserved.

  11. Effects of recombinant human bone morphogenetic protein 7 (rhBMP-7) on the behaviour of oral squamous cell carcinoma: a preliminary in vitro study.

    PubMed

    Lappin, D F; Abu-Serriah, M; Hunter, K D

    2015-02-01

    We investigated the effects of recombinant human bone morphogenetic protein-7 (rhBMP-7) on the behaviour of oral keratinocytes and head and neck squamous cell carcinoma (SCC) cells in vitro. Expression of all three BMP receptors was high (p<0.01), and rhBMP-7 exhibited significant dose-related inhibitory effects on the doubling time and viability of cancer cells (p<0.01), but not on the proliferation or viability of oral keratinocytes. It elicited no significant effect on the invasion of Matrigel in SCC of the head and neck. Results indicate that in cell culture, rhBMP-7 exerts antineoplastic effects. This should be tested in an orthotopic animal model to more closely replicate in vivo effects. Copyright © 2014. Published by Elsevier Ltd.

  12. Stromal derived factor-1 regulates bone morphogenetic protein 2-induced osteogenic differentiation of primary mesenchymal stem cells

    PubMed Central

    Hosogane, Naobumi; Huang, Zhiping; Rawlins, Bernard A.; Liu, Xia; Boachie-Adjei, Oheneba; Boskey, Adele L.; Zhu, Wei

    2010-01-01

    Stromal derived factor-1 (SDF-1) is a chemokine signaling molecule that binds to its transmembrane receptor CXC chemokine receptor-4 (CXCR4). While we previously detected that SDF-1 was co-required with bone morphogenetic protein 2 (BMP2) for differentiating mesenchymal C2C12 cells into osteoblastic cells, it is unknown whether SDF-1 is similarly involved in the osteogenic differentiation of mesenchymal stem cells (MSCs). Therefore, here we examined the role of SDF-1 signaling during BMP2-induced osteogenic differentiation of primary MSCs that were derived from human and mouse bone marrow. Our data showed that blocking of the SDF-1/CXCR4 signal axis or adding SDF-1 protein to MSCs significantly affected BMP2-induced alkaline phosphatase (ALP) activity and osteocalcin (OCN) synthesis, markers of preosteoblasts and mature osteoblasts, respectively. Moreover, disrupting the SDF-1 signaling impaired bone nodule mineralization during terminal differentiation of MSCs. Furthermore, we detected that blocking of the SDF-1 signaling inhibited the BMP2-induced early expression of Runt-related factor-2 (Runx2) and osterix (Osx), two “master” regulators of osteogenesis, and the SDF-1 effect was mediated via intracellular Smad and Erk activation. In conclusion, our results demonstrated a regulatory role of SDF-1 in BMP2-induced osteogenic differentiation of MSCs, as perturbing the SDF-1 signaling affected the differentiation of MSCs towards osteoblastic cells in response to BMP2 stimulation. These data provide novel insights into molecular mechanisms underlying MSC osteogenesis, and will contribute to the development of MSC therapies for enhancing bone formation and regeneration in broad orthopaedic situations. PMID:20362069

  13. Alk2/ACVR1 and Alk3/BMPR1A Provide Essential Function for Bone Morphogenetic Protein-Induced Retinal Angiogenesis.

    PubMed

    Lee, Heon-Woo; Chong, Diana C; Ola, Roxana; Dunworth, William P; Meadows, Stryder; Ka, Jun; Kaartinen, Vesa M; Qyang, Yibing; Cleaver, Ondine; Bautch, Victoria L; Eichmann, Anne; Jin, Suk-Won

    2017-04-01

    Increasing evidence suggests that bone morphogenetic protein (BMP) signaling regulates angiogenesis. Here, we aimed to define the function of BMP receptors in regulating early postnatal angiogenesis by analysis of inducible, endothelial-specific deletion of the BMP receptor components Bmpr2 (BMP type 2 receptor), Alk1 (activin receptor-like kinase 1), Alk2 , and Alk3 in mouse retinal vessels. Expression analysis of several BMP ligands showed that proangiogenic BMP ligands are highly expressed in postnatal retinas. Consistently, BMP receptors are also strongly expressed in retina with a distinct pattern. To assess the function of BMP signaling in retinal angiogenesis, we first generated mice carrying an endothelial-specific inducible deletion of Bmpr2 . Postnatal deletion of Bmpr2 in endothelial cells substantially decreased the number of angiogenic sprouts at the vascular front and branch points behind the front, leading to attenuated radial expansion. To identify critical BMPR1s (BMP type 1 receptors) associated with BMPR2 in retinal angiogenesis, we generated endothelial-specific inducible deletion of 3 BMPR1s abundantly expressed in endothelial cells and analyzed the respective phenotypes. Among these, endothelial-specific deletion of either Alk2 / acvr1 or Alk3 / Bmpr1a caused a delay in radial expansion, reminiscent of vascular defects associated with postnatal endothelial-specific deletion of BMPR2, suggesting that ALK2/ACVR1 and ALK3/BMPR1A are likely to be the critical BMPR1s necessary for proangiogenic BMP signaling in retinal vessels. Our data identify BMP signaling mediated by coordination of ALK2/ACVR1, ALK3/BMPR1A, and BMPR2 as an essential proangiogenic cue for retinal vessels. © 2017 The Authors.

  14. Recombinant human bone morphogenetic protein 2 in augmentation procedures: case reports.

    PubMed

    Luiz, Jaques; Padovan, Luis Eduardo Marques; Claudino, Marcela

    2014-01-01

    To successfully rehabilitate edentulous patients using endosseous implants, there must be enough available bone. Several techniques have been proposed for augmentation of sites with insufficient bone volume. Although autogenous bone has long been considered the gold standard for such procedures, the limited availability of graft material and a high morbidity rate are potential disadvantages of this type of graft. An alternative is to use recombinant human bone morphogenetic protein 2 (rhBMP-2), which is able to support bone regeneration in the oral environment. These cases demonstrate the applicability of rhBMP-2 in maxillary sinus elevation and augmentation procedures in the maxilla to enable dental implant placement. The use of rhBMP-2 in alveolar augmentation procedures had several clinical benefits for these patients.

  15. Activation of Bone Morphogenetic Protein 4 Signaling Leads to Glomerulosclerosis That Mimics Diabetic Nephropathy*

    PubMed Central

    Tominaga, Tatsuya; Abe, Hideharu; Ueda, Otoya; Goto, Chisato; Nakahara, Kunihiko; Murakami, Taichi; Matsubara, Takeshi; Mima, Akira; Nagai, Kojiro; Araoka, Toshikazu; Kishi, Seiji; Fukushima, Naoshi; Jishage, Kou-ichi; Doi, Toshio

    2011-01-01

    Diabetic nephropathy (DN) is the most common cause of chronic kidney disease. We have previously reported that Smad1 transcriptionally regulates the expression of extracellular matrix (ECM) proteins in DN. However, little is known about the regulatory mechanisms that induce and activate Smad1. Here, bone morphogenetic protein 4 (Bmp4) was found to up-regulate the expression of Smad1 in mesangial cells and subsequently to phosphorylate Smad1 downstream of the advanced glycation end product-receptor for advanced glycation end product signaling pathway. Moreover, Bmp4 utilized Alk3 and affected the activation of Smad1 and Col4 expressions in mesangial cells. In the diabetic mouse, Bmp4 was remarkably activated in the glomeruli, and the mesangial area was expanded. To elucidate the direct function of Bmp4 action in the kidneys, we generated transgenic mice inducible for the expression of Bmp4. Tamoxifen treatment dramatically induced the expression of Bmp4, especially in the glomeruli of the mice. Notably, in the nondiabetic condition, the mice exhibited not only an expansion of the mesangial area and thickening of the basement membrane but also remarkable albuminuria, which are consistent with the distinct glomerular injuries in DN. ECM protein overexpression and activation of Smad1 in the glomeruli were also observed in the mice. The mesangial expansion in the mice was significantly correlated with albuminuria. Furthermore, the heterozygous Bmp4 knock-out mice inhibited the glomerular injuries compared with wild type mice in diabetic conditions. Here, we show that BMP4 may act as an upstream regulatory molecule for the process of ECM accumulation in DN and thereby reveals a new aspect of the molecular mechanisms involved in DN. PMID:21471216

  16. The coat morphogenetic protein SpoVID is necessary for spore encasement in Bacillus subtilis.

    PubMed

    Wang, Katherine H; Isidro, Anabela L; Domingues, Lia; Eskandarian, Haig A; McKenney, Peter T; Drew, Kevin; Grabowski, Paul; Chua, Ming-Hsiu; Barry, Samantha N; Guan, Michelle; Bonneau, Richard; Henriques, Adriano O; Eichenberger, Patrick

    2009-11-01

    Endospores formed by Bacillus subtilis are encased in a tough protein shell known as the coat, which consists of at least 70 different proteins. We investigated the process of spore coat morphogenesis using a library of 40 coat proteins fused to green fluorescent protein and demonstrate that two successive steps can be distinguished in coat assembly. The first step, initial localization of proteins to the spore surface, is dependent on the coat morphogenetic proteins SpoIVA and SpoVM. The second step, spore encasement, requires a third protein, SpoVID. We show that in spoVID mutant cells, most coat proteins assembled into a cap at one side of the developing spore but failed to migrate around and encase it. We also found that SpoIVA directly interacts with SpoVID. A domain analysis revealed that the N-terminus of SpoVID is required for encasement and is a structural homologue of a virion protein, whereas the C-terminus is necessary for the interaction with SpoIVA. Thus, SpoVM, SpoIVA and SpoVID are recruited to the spore surface in a concerted manner and form a tripartite machine that drives coat formation and spore encasement.

  17. Thyroid Hormone-Induced Hypertrophy in Mesenchymal Stem Cell Chondrogenesis Is Mediated by Bone Morphogenetic Protein-4

    PubMed Central

    Karl, Alexandra; Olbrich, Norman; Pfeifer, Christian; Berner, Arne; Zellner, Johannes; Kujat, Richard; Angele, Peter; Nerlich, Michael

    2014-01-01

    Chondrogenic differentiating mesenchymal stem cells (MSCs) express markers of hypertrophic growth plate chondrocytes. As hypertrophic cartilage undergoes ossification, this is a concern for the application of MSCs in articular cartilage tissue engineering. To identify mechanisms that elicit this phenomenon, we used an in vitro hypertrophy model of chondrifying MSCs for differential gene expression analysis and functional experiments with the focus on bone morphogenetic protein (BMP) signaling. Hypertrophy was induced in chondrogenic MSC pellet cultures by transforming growth factor β (TGFβ) and dexamethasone withdrawal and addition of triiodothyronine. Differential gene expression analysis of BMPs and their receptors was performed. Based on these results, the in vitro hypertrophy model was used to investigate the effect of recombinant BMP4 and the BMP inhibitor Noggin. The enhancement of hypertrophy could be shown clearly by an increased cell size, alkaline phosphatase activity, and collagen type X deposition. Upon induction of hypertrophy, BMP4 and the BMP receptor 1B were upregulated. Addition of BMP4 further enhanced hypertrophy in the absence, but not in the presence of TGFβ and dexamethasone. Thyroid hormone induced hypertrophy by upregulation of BMP4 and this induced enhancement of hypertrophy could be blocked by the BMP antagonist Noggin. BMP signaling is an important modulator of the late differentiation stages in MSC chondrogenesis and the thyroid hormone induces this pathway. As cartilage tissue engineering constructs will be exposed to this factor in vivo, this study provides important insight into the biology of MSC-based cartilage. Furthermore, the possibility to engineer hypertrophic cartilage may be helpful for critical bone defect repair. PMID:23937304

  18. The coat morphogenetic protein SpoVID is necessary for spore encasement in Bacillus subtilis

    PubMed Central

    Wang, Katherine H.; Isidro, Anabela L.; Domingues, Lia; Eskandarian, Haig A.; McKenney, Peter T.; Drew, Kevin; Grabowski, Paul; Chua, Ming-Hsiu; Barry, Samantha N.; Guan, Michelle; Bonneau, Richard; Henriques, Adriano O.; Eichenberger, Patrick

    2009-01-01

    SUMMARY Endospores formed by Bacillus subtilis are encased in a tough protein shell known as the coat, which consists of at least 70 different proteins. We investigated the process of spore coat morphogenesis using a library of 40 coat proteins fused to GFP and demonstrate that two successive steps can be distinguished in coat assembly. The first step, initial localization of proteins to the spore surface, is dependent on the coat morphogenetic proteins SpoIVA and SpoVM. The second step, spore encasement, requires a third protein, SpoVID. We show that in spoVID mutant cells, most coat proteins assembled into a cap at one side of the developing spore but failed to migrate around and encase it. We also found that SpoIVA directly interacts with SpoVID. A domain analysis revealed that the N-terminus of SpoVID is required for encasement and is a structural homolog of a virion protein, whereas the C-terminus is necessary for the interaction with SpoIVA. Thus, SpoVM, SpoIVA and SpoVID are recruited to the spore surface in a concerted manner and form a tripartite machine that drives coat formation and spore encasement. PMID:19775244

  19. Turning Bone Morphogenetic Protein 2 (BMP2) On and Off in Mesenchymal Cells†

    PubMed Central

    Rogers, Melissa B.; Shah, Tapan A.; Shaikh, Nadia N.

    2016-01-01

    The concentration, location, and timing of bone morphogenetic protein 2 (BMP2, HGNC:1069, GeneID: 650) gene expression must be precisely regulated. Abnormal BMP2 levels cause congenital anomalies and diseases involving the mesenchymal cells that differentiate into muscle, fat, cartilage, and bone. The molecules and conditions that influence BMP2 synthesis are diverse. Understandably, complex mechanisms control Bmp2 gene expression. This review includes a compilation of agents and conditions that can induce Bmp2. The currently known trans-regulatory factors and cis-regulatory elements that modulate Bmp2 expression are summarized and discussed. This article is protected by copyright. All rights reserved PMID:25776852

  20. Efficient delivery of recombinant human bone morphogenetic protein (rhBMP-2) with dextran sulfate-chitosan microspheres.

    PubMed

    Xia, Yuan-Jun; Xia, Hong; Chen, Ling; Ying, Qing-Shui; Yu, Xiang; Li, Li-Hua; Wang, Jian-Hua; Zhang, Ying

    2018-04-01

    Bone morphogenetic protein-2 (BMP-2) serves an important role in the development of bone and cartilage. However, administration of BMP-2 protein alone by intravenous delivery is not very effective. Sustained delivery of stabilized BMP-2 by carriers has been proven necessary to improve the osteogenesis effect of BMP-2. The present study constructed a novel drug delivery system using dextran sulfate (DS)-chitosan (CS) microspheres and investigated the efficiency of the delivery system on recombinant human bone morphogenetic protein (rhBMP-2). The microsphere morphology, optimal ratio of DS/CS/rhBMP-2, and drug loading rate and entrapment efficiency of rhBMP-2 CS nanoparticles were determined. L929 cells were used to evaluate the cytotoxicity and effect of DS/CS/rhBMP-2 microspheres on cell proliferation. Differentiation study was conducted using bone marrow mesenchymal stem cells (BMSCs-C57) cells treated with DS/CS/rhBMP-2 microspheres or the control microspheres. The DS/CS/rhBMP-2 microspheres delivery system was successfully established. Subsequent complexation of rhBMP-2-bound DS with polycations afforded well defined microspheres with a diameter of ~250 nm. High protein entrapment efficiency (85.6%) and loading ratio (47.245) µg/mg were achieved. Release of rhBMP-2 from resultant microspheres persisted for over 20 days as determined by ELISA assay. The bioactivity of rhBMP-2 encapsulated in the CS/DS microsphere was observed to be well preserved as evidenced by the alkaline phosphatase activity assay and calcium nodule formation of BMSCs-C57 incubated with rhBMP-2-loaded microspheres. The results demonstrated that microspheres based on CS-DS polyion complexes were a highly efficient vehicle for delivery of rhBMP-2 protein. The present study may provide novel orientation for bone tissue engineering for repairing and regenerating bone defects.

  1. Survey of the Effectiveness of Internet Information on Patient Education for Bone Morphogenetic Protein.

    PubMed

    Huang, Meng; Briceño, Valentina; Lam, Sandi K; Luerssen, Thomas G; Jea, Andrew

    2016-03-01

    In light of recent reports of potential short- and long-term complications of bone morphogenetic protein (BMP) and increasing "off-label" use among spine surgeons, we wished to analyze online information on BMP and its controversial uses, as patients frequently search the Internet for medical information, even though the quality and accuracy of available information are highly variable. Between December 2014 and January 2015, we conducted a Google search to identify the 50 most accessed websites providing BMP information using the search phrase "bone morphogenetic protein." Websites were classified based on authorship. Each website was examined for the provision of appropriate patient inclusion and exclusion criteria, surgical and nonsurgical treatment alternatives, purported benefits, disclosure of common and potential complications, peer-reviewed literature citations, and discussion of off-label use. Two percent of websites were authored by private medical groups, 2% by academic medical groups, 10% by insurance companies, 16% by biomedical industries, 4% by news sources, 0% by lawyers, and 66% by others. Sixty-two percent referenced peer-reviewed literature. Benefits and complications were reported in 44% and 26% of websites, respectively. Surgical and nonsurgical treatment alternatives were mentioned in 16% and 4% of websites, respectively. Discussion of off-label BMP use occurred in 18% of websites. Our study showed the ineffectiveness of the Internet in reporting quality information on BMP use. We found that websites authored by insurance companies provide an acceptable foundation for patient education. This, however, cannot replace the need for a thorough dialogue between doctor and patient about risks, benefits, and indications. Copyright © 2016. Published by Elsevier Inc.

  2. The Controversy Surrounding Bone Morphogenetic Proteins in the Spine: A Review of Current Research

    PubMed Central

    Hustedt, Joshua W.; Blizzard, Daniel J.

    2014-01-01

    Bone morphogenetic proteins have been in use in spinal surgery since 2002. These proteins are members of the TGF-beta superfamily and guide mesenchymal stem cells to differentiate into osteoblasts to form bone in targeted tissues. Since the first commercial BMP became available in 2002, a host of research has supported BMPs and they have been rapidly incorporated in spinal surgeries in the United States. However, recent controversy has arisen surrounding the ethical conduct of the research supporting the use of BMPs. Yale University Open Data Access (YODA) recently teamed up with Medtronic to offer a meta-analysis of the effectiveness of BMPs in spinal surgery. This review focuses on the history of BMPs and examines the YODA research to guide spine surgeons in their use of BMP in spinal surgery. PMID:25506287

  3. High-dose bone morphogenetic protein-induced ectopic abdomen bone growth.

    PubMed

    Deutsch, Harel

    2010-02-01

    Infuse [bone morphogenetic protein (BMP)] is increasingly used in spinal fusion surgery. The authors report a rare complication of BMP use. This is a case report. A 55-year-old male underwent a thoracic T8 to the pelvis fusion for degenerative lumbar disc disease and pseudarthrosis at another institution. The procedure involved an anterior and posterior approach with the use of multiple units of BMP. The patient presented to our institution with complaints of weight loss, pain, tenderness, and increasing solid growth in the left lower quadrant several months after his surgery. A computed tomography revealed ectopic bone growth in the retroperitoneal area and pelvis contiguous to the anterior lumbar exposure. The anterior wound was re-explored, and a large sheet of ectopic bone was removed from the retroperitoneal space. We report a rare case of extraspinal ectopic bone growth because of the use of multiple packages of BMP. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  4. Bone Morphogenetic Protein 4 Promotes Vascular Smooth Muscle Contractility by Activating MicroRNA-21 (miR-21), which Down-regulates Expression of Family of Dedicator of Cytokinesis (DOCK) Proteins*

    PubMed Central

    Kang, Hara; Davis-Dusenbery, Brandi N.; Nguyen, Peter H.; Lal, Ashish; Lieberman, Judy; Van Aelst, Linda; Lagna, Giorgio; Hata, Akiko

    2012-01-01

    The bone morphogenetic protein 4 (BMP4) signaling pathway plays a critical role in the promotion and maintenance of the contractile phenotype in vascular smooth muscle cell (vSMC). Misexpression or inactivating mutations of the BMP receptor gene can lead to dedifferentiation of vSMC characterized by increased migration and proliferation that is linked to vascular proliferative disorders. Previously we demonstrated that vSMCs increase microRNA-21 (miR-21) biogenesis upon BMP4 treatment, which induces contractile gene expression by targeting programmed cell death 4 (PDCD4). To identify novel targets of miR-21 that are critical for induction of the contractile phenotype by BMP4, biotinylated miR-21 was expressed in vSMCs followed by an affinity purification of mRNAs associated with miR-21. Nearly all members of the dedicator of cytokinesis (DOCK) 180-related protein superfamily were identified as targets of miR-21. Down-regulation of DOCK4, -5, and -7 by miR-21 inhibited cell migration and promoted cytoskeletal organization by modulating an activity of small GTPase. Thus, this study uncovers a regulatory mechanism of the vSMC phenotype by the BMP4-miR-21 axis through DOCK family proteins. PMID:22158624

  5. Production of Transgenic Pigs with an Introduced Missense Mutation of the Bone Morphogenetic Protein Receptor Type IB Gene Related to Prolificacy.

    PubMed

    Zhao, Xueyan; Yang, Qiang; Zhao, Kewei; Jiang, Chao; Ren, Dongren; Xu, Pan; He, Xiaofang; Liao, Rongrong; Jiang, Kai; Ma, Junwu; Xiao, Shijun; Ren, Jun; Xing, Yuyun

    2016-07-01

    In the last few decades, transgenic animal technology has witnessed an increasingly wide application in animal breeding. Reproductive traits are economically important to the pig industry. It has been shown that the bone morphogenetic protein receptor type IB (BMPR1B) A746G polymorphism is responsible for the fertility in sheep. However, this causal mutation exits exclusively in sheep and goat. In this study, we attempted to create transgenic pigs by introducing this mutation with the aim to improve reproductive traits in pigs. We successfully constructed a vector containing porcine BMPR1B coding sequence (CDS) with the mutant G allele of A746G mutation. In total, we obtained 24 cloned male piglets using handmade cloning (HMC) technique, and 12 individuals survived till maturation. A set of polymerase chain reactions indicated that 11 of 12 matured boars were transgene-positive individuals, and that the transgenic vector was most likely disrupted during cloning. Of 11 positive pigs, one (No. 11) lost a part of the terminator region but had the intact promoter and the CDS regions. cDNA sequencing showed that the introduced allele (746G) was expressed in multiple tissues of transgene-positive offspring of No.11. Western blot analysis revealed that BMPR1B protein expression in multiple tissues of transgene-positive F1 piglets was 0.5 to 2-fold higher than that in the transgene-negative siblings. The No. 11 boar showed normal litter size performance as normal pigs from the same breed. Transgene-positive F1 boars produced by No. 11 had higher semen volume, sperm concentration and total sperm per ejaculate than the negative siblings, although the differences did not reached statistical significance. Transgene-positive F1 sows had similar litter size performance to the negative siblings, and more data are needed to adequately assess the litter size performance. In conclusion, we obtained 24 cloned transgenic pigs with the modified porcine BMPR1B CDS using HMC. c

  6. Alk2/ACVR1 and Alk3/BMPR1A Provide Essential Function for Bone Morphogenetic Protein Induced Retinal Angiogenesis

    PubMed Central

    Lee, Heon-Woo; Chong, Diana C.; Ola, Roxana; Dunworth, William P.; Meadows, Stryder; Ka, Jun; Kaartinen, Vesa M.; Qyang, Yibing; Cleaver, Ondine; Bautch, Victoria L.; Eichmann, Anne; Jin, Suk-Won

    2017-01-01

    Objective Increasing evidence suggests that Bone Morphogenetic Protein (BMP) signaling regulates angiogenesis. Here, we aimed to define the function of BMP receptors in regulating early post-natal angiogenesis by analysis of inducible, endothelial specific deletion of the BMP receptor components Bmpr2, Alk1, Alk2 and Alk3 in mouse retinal vessels. Approach and Results Expression analysis of several BMP ligands showed that pro-angiogenic BMP ligands are highly expressed in postnatal retinas. Consistently, BMP receptors are also strongly expressed in retina with a distinct pattern. To assess the function of BMP signaling in retinal angiogenesis, we first generated mice carrying an endothelial-specific inducible deletion of BMP Type 2 receptor (Bmpr2). Postnatal deletion of Bmpr2 in endothelial cells substantially decreased the number of angiogenic sprouts at the vascular front and branchpoints behind the front, leading to attenuated radial expansion. To identify critical BMPR1s associated with BMPR2 in retinal angiogenesis, we generated endothelial-specific inducible deletion of three BMPR1s abundantly expressed in endothelial cells and analyzed the respective phenotypes. Among these, endothelial specific deletion of either Alk2/acvr1 or Alk3/Bmpr1a caused a delay in radial expansion, reminiscent of vascular defects associated with postnatal endothelial specific deletion of BMPR2, suggesting that ALK2/ACVR1 and ALK3/BMPR1A are likely to be the critical BMPR1s necessary for pro-angiogenic BMP signaling in retinal vessels. Conclusions Our data identify BMP signaling mediated by coordination of ALK2/ACVR1, ALK3/BMPR1A, and BMPR2 as an essential pro-angiogenic cue for retinal vessels. PMID:28232325

  7. [Slow-release recombinant human bone morphogenetic protein-2 suppresses chromium wear particle-induced osteolysis in rats].

    PubMed

    Li, Gan; Li, Qi; Lin, Li-Jun; Duan, Xin; Zhang, Xi-Qi

    2012-03-01

    To observe the effect of a slow-release recombinant human bone morphogenetic protein-2 (rhBMP-2) formulation on the expressions of receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) in a murine air pouch model of bone implantation. A cranial bone allograft was implanted in the air pouch induced on the back of the recipients. The rat models were then randomized into 5 groups, including a blank control group, chromium particle group, and 3 rhBMP-2 groups receiving 50, 100 or 200 µg/L slow-release rhBMP-2 in addition to chromium particles. Three weeks later, the expressions of RANKL and OPG in the air pouch was detected using Western blotting and RT-PCR, and the positively stained area for osteoclasts in the bone graft was determined with TRAP staining for drug effect assessment. RANKL and OPG expressions were found in the air pouches in all the 5 groups. RANKL and OPG protein and mRNA expressions, RANKL/OPG ratio and osteoclast staining area in the bone graft were the highest in chromium particle group (P<0.05), but were significantly decreased by treatment with the slow-release rhBMP-2 formulation (P<0.05); the measurements showed no significant differences between the blank control group and 200 µg/L rhBMP-2 group (P>0.05). Chromium particles can cause osteolysis by increasing the RANKL/OPG ratio in rats, and intervention with slow-release rhBMP-2 can significantly promote bone formation and suppress bone resorption by decreasing RANKL/OPG ratio.

  8. Heterotopic ossification after the use of recombinant human bone morphogenetic protein-7

    PubMed Central

    Papanagiotou, Marianthi; Dailiana, Zoe H; Karachalios, Theophilos; Varitimidis, Sokratis; Hantes, Michael; Dimakopoulos, Georgios; Vlychou, Marianna; Malizos, Konstantinos N

    2017-01-01

    AIM To present the incidence of heterotopic ossification after the use of recombinant human bone morphogenetic protein-7 (rhBMP-7) for the treatment of nonunions. METHODS Bone morphogenetic proteins (BMPs) promote bone formation by auto-induction. Recombinant human BMP-7 in combination with bone grafts was used in 84 patients for the treatment of long bone nonunions. All patients were evaluated radiographicaly for the development of heterotopic ossification during the standard assessment for the nonunion healing. In all patients (80.9%) with radiographic signs of heterotopic ossification, a CT scan was performed. Nonunion site palpation and ROM evaluation of the adjacent joints were also carried out. Factors related to the patient (age, gender), the nonunion (location, size, chronicity, number of previous procedures, infection, surrounding tissues condition) and the surgical procedure (graft and fixation type, amount of rhBMP-7) were correlated with the development of heterotopic ossification and statistical analysis with Pearsons χ2 test was performed. RESULTS Eighty point nine percent of the nonunions treated with rhBMP-7, healed with no need for further procedures. Heterotopic bone formation occurred in 15 of 84 patients (17.8%) and it was apparent in the routine radiological evaluation of the nonunion site, in a mean time of 5.5 mo after the rhBMP-7 application (range 3-12). The heterotopic ossification was located at the femur in 8 cases, at the tibia in 6, and at the humerus in οne patient. In 4 patients a palpable mass was present and only in one patient, with a para-articular knee nonunion treated with rhBMP-7, the size of heterotopic ossification affected the knee range of motion. All the patients with heterotopic ossification were male. Statistical analysis proved that patient’s gender was the only important factor for the development of heterotopic ossification (P = 0.007). CONCLUSION Heterotopic ossification after the use of rhBMP-7 in nonunions was

  9. Clinical application of bone morphogenetic proteins for bone healing: a systematic review.

    PubMed

    Krishnakumar, Gopal Shankar; Roffi, Alice; Reale, Davide; Kon, Elizaveta; Filardo, Giuseppe

    2017-06-01

    This paper documents the existing evidence on bone morphogenetic proteins (BMPs) use for the treatment of bone fractures, non-union, and osteonecrosis, through a review of the clinical literature, underlying potential and limitations in terms of cost effectiveness and risk of complications. A systematic review was performed on the PubMed database using the following string: (bone morphogenetic proteins OR BMPs) and (bone repair OR bone regeneration) including papers from 2000 to 2016. The search focused on clinical trials dealing with BMPs application to favor bone regeneration in bone fractures, non-union, and osteonecrosis, in English language, with level of evidence I, II, III, and IV. Relevant data (type of study, number of patients, BMPs delivery material, dose, site, follow-up, outcome, and adverse events) were extracted and analyzed. Forty-four articles met the inclusion criteria: 10 randomized controlled trials (RCTs), 7 comparative studies, 18 case series, and 9 case reports. rhBMP-2 was documented mainly for the treatment of fractures, and rhBMP-7 mainly for non-unions and osteonecrosis. Mixed results were found among RCTs and comparative papers: 11 reported positive results for BMPs augmentation, 3 obtained no significant effects, and 2 showed negative results. The only study comparing the two BMPs showed a better outcome with rhBMP-2 for non-union treatment. Clinical evidence on BMPs use for the treatment of fractures, non-union, and osteonecrosis is still controversial, with the few available reports being mainly of low quality. While positive findings have been described in many studies, mixed results are still present in the literature in terms of efficacy and adverse events. The difficulties in drawing clear conclusions are also due to the studies heterogeneity, mainly in terms of different BMPs applied, with different concomitant treatments for each bone pathology. Therefore, further research with well-designed studies is needed in order to

  10. Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry.

    PubMed

    Siverino, Claudia; Tabisz, Barbara; Lühmann, Tessa; Meinel, Lorenz; Müller, Thomas; Walles, Heike; Nickel, Joachim

    2018-03-29

    Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present several limitations that have led to the use of biomaterials in combination with osteogenic growth factors, such as bone morphogenetic proteins (BMPs). Commonly used absorption or encapsulation methods require supra-physiological amounts of BMP2, typically resulting in a so-called initial burst release effect that provokes several severe adverse side effects. A possible strategy to overcome these problems would be to covalently couple the protein to the scaffold. Moreover, coupling should be performed in a site-specific manner in order to guarantee a reproducible product outcome. Therefore, we created a BMP2 variant, in which an artificial amino acid (propargyl-L-lysine) was introduced into the mature part of the BMP2 protein by codon usage expansion (BMP2-K3Plk). BMP2-K3Plk was coupled to functionalized beads through copper catalyzed azide-alkyne cycloaddition (CuAAC). The biological activity of the coupled BMP2-K3Plk was proven in vitro and the osteogenic activity of the BMP2-K3Plk-functionalized beads was proven in cell based assays. The functionalized beads in contact with C2C12 cells were able to induce alkaline phosphatase (ALP) expression in locally restricted proximity of the bead. Thus, by this technique, functionalized scaffolds can be produced that can trigger cell differentiation towards an osteogenic lineage. Additionally, lower BMP2 doses are sufficient due to the controlled orientation of site-directed coupled BMP2. With this method, BMPs are always exposed to their receptors on the cell surface in the appropriate orientation, which is not the case if the factors are coupled via non-site-directed coupling techniques. The product outcome is highly controllable and, thus, results in materials with homogeneous properties, improving their applicability for the repair of critical size bone defects.

  11. Collagen I derived recombinant protein microspheres as novel delivery vehicles for bone morphogenetic protein-2.

    PubMed

    Mumcuoglu, Didem; de Miguel, Laura; Jekhmane, Shehrazade; Siverino, Claudia; Nickel, Joachim; Mueller, Thomas D; van Leeuwen, Johannes P; van Osch, Gerjo J; Kluijtmans, Sebastiaan G

    2018-03-01

    Bone morphogenetic protein-2 (BMP-2) is a powerful osteoinductive protein; however, there is a need for the development of a safe and efficient BMP-2 release system for bone regeneration therapies. Recombinant extracellular matrix proteins are promising next generation biomaterials since the proteins are well-defined, reproducible and can be tailored for specific applications. In this study, we have developed a novel and versatile BMP-2 delivery system using microspheres from a recombinant protein based on human collagen I (RCP). In general, a two-phase release pattern was observed while the majority of BMP-2 was retained in the microspheres for at least two weeks. Among different parameters studied, the crosslinking and the size of the RCP microspheres changed the in vitro BMP-2 release kinetics significantly. Increasing the chemical crosslinking (hexamethylene diisocyanide) degree decreased the amount of initial burst release (24h) from 23% to 17%. Crosslinking by dehydrothermal treatment further decreased the burst release to 11%. Interestingly, the 50 and 72μm-sized spheres showed a significant decrease in the burst release compared to 207-μm sized spheres. Very importantly, using a reporter cell line, the released BMP-2 was shown to be bioactive. SPR data showed that N-terminal sequence of BMP-2 was important for the binding and retention of BMP-2 and suggested the presence of a specific binding epitope on RCP (K D : 1.2nM). This study demonstrated that the presented RCP microspheres are promising versatile BMP-2 delivery vehicles. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. MB109 as bioactive human bone morphogenetic protein-9 refolded and purified from E. coli inclusion bodies

    PubMed Central

    2014-01-01

    Background The development of chemical refolding of transforming growth factor-beta (TGF-β) superfamily ligands has been instrumental to produce the recombinant proteins for biochemical studies and exploring the potential of protein therapeutics. The osteogenic human bone morphogenetic protein-2 (hBMP-2) and its Drosophila DPP homolog were the early successful cases of refolding into functional form. Despite the similarity in their three dimensional structure and amino acid sequences, several other TGF-β superfamily ligands could not be refolded readily by the same methods. Results Here, we report a comprehensive study on the variables of a rapid-dilution refolding method, including the concentrations of protein, salt, detergent and redox agents, pH, refolding duration and the presence of aggregation suppressors and host-cell contaminants, in order to identify the optimal condition to refold human BMP-9 (hBMP-9). To produce a recombinant form of hBMP-9 in E. coli cells, a synthetic codon-optimized gene was designed to encode the mature domain of hBMP-9 (Ser320 – Arg429) directly behind the first methionine, which we herein referred to as MB109. An effective purification scheme was also developed to purify the refolded MB109 to homogeneity with a final yield of 7.8 mg from 100 mg of chromatography-purified inclusion bodies as a starting material. The chemically refolded MB109 binds to ALK1, ActRIIb and BMPRII receptors with relatively high affinity as compared to other Type I and Type II receptors based on surface plasmon resonance analysis. Smad1-dependent luciferase assay in C2C12 cells shows that the MB109 has an EC50 of 0.61 ng/mL (25 pM), which is nearly the same as hBMP-9. Conclusion MB109 is prone to be refolded as non-functional dimer and higher order multimers in most of the conditions tested, but bioactive MB109 dimer can be refolded with high efficiency in a narrow window, which is strongly dependent on the pH, refolding duration, the presence of

  13. Regulators and effectors of bone morphogenetic protein signalling in the cardiovascular system.

    PubMed

    Luo, Jiang-Yun; Zhang, Yang; Wang, Li; Huang, Yu

    2015-07-15

    Bone morphogenetic proteins (BMPs) play key roles in the regulation of cell proliferation, differentiation and apoptosis in various tissues and organs, including the cardiovascular system. BMPs signal through both Smad-dependent and -independent cascades to exert a wide spectrum of biological activities. Cardiovascular disorders such as abnormal angiogenesis, atherosclerosis, pulmonary hypertension and cardiac hypertrophy have been linked to aberrant BMP signalling. To correct the dysregulated BMP signalling in cardiovascular pathogenesis, it is essential to get a better understanding of how the regulators and effectors of BMP signalling control cardiovascular function and how the dysregulated BMP signalling contributes to cardiovascular dysfunction. We hence highlight several key regulators of BMP signalling such as extracellular regulators of ligands, mechanical forces, microRNAs and small molecule drugs as well as typical BMP effectors like direct downstream target genes, mitogen-activated protein kinases, reactive oxygen species and microRNAs. The insights into these molecular processes will help target both the regulators and important effectors to reverse BMP-associated cardiovascular pathogenesis. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

  14. Repulsive Guidance Molecule is a structural bridge between Neogenin and Bone Morphogenetic Protein

    PubMed Central

    Healey, Eleanor G.; Bishop, Benjamin; Elegheert, Jonathan; Bell, Christian H.; Padilla-Parra, Sergi; Siebold, Christian

    2015-01-01

    Repulsive guidance molecules (RGMs) control crucial processes spanning cell motility, adhesion, immune cell regulation and systemic iron metabolism. RGMs signal via two fundamental signaling cascades: the Neogenin (NEO1) and the Bone Morphogenetic Protein (BMP) pathways. Here, we report crystal structures of the N-terminal domains of all human RGM family members in complex with the BMP ligand BMP2, revealing a novel protein fold and a conserved BMP-binding mode. Our structural and functional data suggest a pH-linked mechanism for RGM-activated BMP signaling and offer a rationale for RGM mutations causing juvenile hemochromatosis. We also determined the ternary BMP2–RGM–NEO1 complex crystal structure, which combined with solution scattering and live-cell super-resolution fluorescence microscopy, indicates BMP-induced clustering of the RGM–NEO1 complex. Our results show how RGM acts as the central hub linking BMP and NEO1 and physically connecting these fundamental signaling pathways. PMID:25938661

  15. A polymorphism in a conserved posttranscriptional regulatory motif alters bone morphogenetic protein 2 (BMP2) RNA:protein interactions.

    PubMed

    Fritz, David T; Jiang, Shan; Xu, Junwang; Rogers, Melissa B

    2006-07-01

    The bone morphogenetic protein (BMP)2 gene has been genetically linked to osteoporosis and osteoarthritis. We have shown that the 3'-untranslated regions (UTR) of BMP2 genes from mammals to fishes are extraordinarily conserved. This indicates that the BMP2 3'-UTR is under stringent selective pressure. We present evidence that the conserved region is a strong posttranscriptional regulator of BMP2 expression. Polymorphisms in cis-regulatory elements have been proven to influence susceptibility to a growing number of diseases. A common single nucleotide polymorphism (SNP) disrupts a putative posttranscriptional regulatory motif, an AU-rich element, within the BMP2 3'-UTR. The affinity of specific proteins for the rs15705 SNP sequence differs from their affinity for the normal human sequence. More importantly, the in vitro decay rate of RNAs with the SNP is higher than that of RNAs with the normal sequence. Such changes in mRNA:protein interactions may influence the posttranscriptional mechanisms that control BMP2 gene expression. The consequent alterations in BMP2 protein levels may influence the development or physiology of bone or other BMP2-influenced tissues.

  16. Bone Morphogenetic Protein 15 (BMP15) Acts as a BMP and Wnt Inhibitor during Early Embryogenesis*

    PubMed Central

    Di Pasquale, Elisa; Brivanlou, Ali H.

    2009-01-01

    Bone morphogenetic protein 15 (BMP15) belongs to an unusual subgroup of the transforming growth factor β (TGFβ) superfamily of signaling ligands as it lacks a key cysteine residue in the mature region required for proper intermolecular dimerization. Naturally occurring BMP15 mutation leads to early ovarian failure in humans, and BMP15 has been shown to activate the Smad1/5/8 pathway in that context. Despite its important role in germ cell specification, the embryological function of BMP15 remains unknown. Surprisingly, we find that during early Xenopus embryogenesis BMP15 acts solely as an inhibitor of the Smad1/5/8 pathway and the Wnt pathway. BMP15 gain-of-function leads to embryos with secondary ectopic heads and to direct neural induction in intact explants. BMP15 inhibits BMP4-mediated epidermal induction in dissociated explants. BMP15 strongly inhibits BRE response induced by BMP4 and blocks phosphorylation and activation of Smad1/5/8 MH2-domain. Mechanistically, BMP15 protein specifically interacts with BMP4 protein, suggesting inhibition upstream of receptor binding. Loss-of-function experiments using morpholinos or a naturally occurring human BMP15 dominant-negative mutant (BMP15-Y235C) leads to embryos lacking head. BMP15-Y235C also eliminates the inhibitory activity of BMP15 on BRE (BMP-responsive element). Finally, we show that BMP15 inhibits the canonical branch of the Wnt pathway, upstream of β-catenin. We, thus, demonstrate that BMP15 is necessary and sufficient for the specification of dorso-anterior structures and highlight novel mechanisms of BMP15 function that strongly suggest a reinterpretation of its function in ovaries specially for ovarian failure. PMID:19553676

  17. Differential ubiquitination of Smad1 mediated by CHIP: implications in the regulation of the bone morphogenetic protein signaling pathway.

    PubMed

    Li, Ren-Feng; Shang, Yu; Liu, Di; Ren, Ze-Song; Chang, Zhijie; Sui, Sen-Fang

    2007-11-30

    Smad1, a downstream regulator of the bone morphogenetic protein (BMP) receptors, is tightly regulated by the ubiquitin-proteasomal degradation system. To dissect the mechanisms that underlie the regulation of Smad1, it is important to investigate the specific ubiquitination site(s) in Smad1. Here we report that the alpha-NH(2) group of the N terminus and the epsilon-NH(2) groups of internal lysine residues 116, 118 and 269 (K116, K118 and K269) of Smad1 are ubiquitin acceptor sites mediated by the carboxyl terminus of Hsc70-interacting protein (CHIP). The in vitro degradation assay indicates that ubiquitination at the N terminus partially contributes to the degradation of Smad1. Furthermore, we demonstrate that the ubiquitination level of pseudo-phosphorylated Smad1 by CHIP is stronger than that of wild-type Smad1 and can be strongly inhibited by a phosphorylated tail of Smad1, PIS(pS)V(pS). Third, our results indicate that Hsp70 facilitates CHIP-mediated poly-ubiquitination of Smad1 whereas it attenuates CHIP-meditated mono-ubiquitination of Smad1. Finally, consistent with the in vitro observation, we show that CHIP preferentially mediates the degradation of phospho-Smad1/5 in vivo. Taken together, these results provide us a hint that CHIP might preferentially regulate phosphorylated Smad1 and thus the BMP signaling.

  18. Two Variants of Recombinant Human Bone Morphogenetic Protein-2 (rhBMP-2) with Additional Protein Domains: Synthesis in an Escherichia coli Heterologous Expression System.

    PubMed

    Karyagina, A S; Boksha, I S; Grunina, T M; Demidenko, A V; Poponova, M S; Sergienko, O V; Lyashchuk, A M; Galushkina, Z M; Soboleva, L A; Osidak, E O; Bartov, M S; Gromov, A V; Lunin, V G

    2017-05-01

    Two variants of recombinant human bone morphogenetic protein-2 (rhBMP-2) with additional N-terminal protein domains were obtained by expression in E. coli. The N-terminal domains were s-tag (15-a.a. oligopeptide from bovine pancreatic ribonuclease A) and lz (leucine zipper dimerization domain from yeast transcription factor GCN4). The s-tag-BMP-2 and lz-BMP-2 were purified by a procedure that excluded a long refolding stage. The resulting dimeric proteins displayed higher solubility compared to rhBMP-2 without additional protein domains. Biological activity of both proteins was demonstrated in vitro by induction of alkaline phosphatase in C2C12 cells, and the activity of s-tag-BMP-2 in vivo was shown in various experimental animal models.

  19. Bone morphogenetic protein 9 as a key regulator of liver progenitor cells in DDC-induced cholestatic liver injury.

    PubMed

    Addante, Annalisa; Roncero, Cesáreo; Almalé, Laura; Lazcanoiturburu, Nerea; García-Álvaro, María; Fernández, Margarita; Sanz, Julián; Hammad, Seddik; Nwosu, Zeribe C; Lee, Se-Jin; Fabregat, Isabel; Dooley, Steven; Ten Dijke, Peter; Herrera, Blanca; Sánchez, Aránzazu

    2018-05-11

    Bone morphogenetic protein 9 (BMP9) interferes with liver regeneration upon acute injury, while promoting fibrosis upon carbon tetrachloride-induced chronic injury. We have now addressed the role of BMP9 in 3,5 diethoxicarbonyl-1,4 dihydrocollidine (DDC)-induced cholestatic liver injury, a model of liver regeneration mediated by hepatic progenitor cell (known as oval cell), exemplified as ductular reaction and oval cell expansion. WT and BMP9KO mice were submitted to DDC diet. Livers were examined for liver injury, fibrosis, inflammation and oval cell expansion by serum biochemistry, histology, RT-qPCR and western blot. BMP9 signalling and effects in oval cells were studied in vitro using western blot and transcriptional assays, plus functional assays of DNA synthesis, cell viability and apoptosis. Crosslinking assays and short hairpin RNA approaches were used to identify the receptors mediating BMP9 effects. Deletion of BMP9 reduces liver damage and fibrosis, but enhances inflammation upon DDC feeding. Molecularly, absence of BMP9 results in overactivation of PI3K/AKT, ERK-MAPKs and c-Met signalling pathways, which together with an enhanced ductular reaction and oval cell expansion evidence an improved regenerative response and decreased damage in response to DDC feeding. Importantly, BMP9 directly targets oval cells, it activates SMAD1,5,8, decreases cell growth and promotes apoptosis, effects that are mediated by Activin Receptor-Like Kinase 2 (ALK2) type I receptor. We identify BMP9 as a negative regulator of oval cell expansion in cholestatic injury, its deletion enhancing liver regeneration. Likewise, our work further supports BMP9 as an attractive therapeutic target for chronic liver diseases. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  20. Gene Therapy of Bone Morphogenetic Protein for Periodontal Tissue Engineering

    PubMed Central

    Jin, Q-M.; Anusaksathien, O.; Webb, S.A.; Rutherford, R.B.; Giannobile, W.V.

    2009-01-01

    Background The reconstruction of lost periodontal support including bone, ligament, and cementum is a major goal of therapy. Bone morphogenetic proteins (BMPs) have shown much potential in the regeneration of the periodontium. Limitations of BMP administration to periodontal lesions include need for high-dose bolus delivery, BMP transient biological activity, and low bioavailability of factors at the wound site. Gene transfer offers promise as an alternative treatment strategy to deliver BMPs to periodontal tissues. Methods This study utilized ex vivo BMP-7 gene transfer to stimulate tissue engineering of alveolar bone wounds. Syngeneic dermal fibroblasts (SDFs) were transduced ex vivo with adenoviruses encoding either green fluorescent protein (Ad-GFP or control virus), BMP-7 (Ad-BMP-7), or an antagonist of BMP bioactivity, noggin (Ad-noggin). Transduced cells were seeded onto gelatin carriers and then transplanted to large mandibular alveolar bone defects in a rat wound repair model. Results Ad-noggin treatment tended to inhibit osteogenesis as compared to the control-treated and Ad-BMP-7-treated specimens. The osseous lesions treated by Ad-BMP-7 gene delivery demonstrated rapid chrondrogenesis, with subsequent osteogenesis, cementogenesis and predictable bridging of the periodontal bone defects. Conclusion These results demonstrate the first successful evidence of periodontal tissue engineering using ex vivo gene transfer of BMPs and offers a new approach for repairing periodontal defects. PMID:12666709

  1. Research on Navy-Related Combat Casualty Care Issues, Navy Operational-Related Injuries and Illnesses and Approaches to Enhance Navy/Marine Corps Personnel Combat Performance.

    DTIC Science & Technology

    1998-06-01

    role of bone morphogenetic protein (BMP) receptors in bone regeneration in periodontal tissues . Tissue samples for these studies are in the accrual...34 Characterization of Bone Morphogenetic Protein Receptors in Oral Tissues " collection of clinical samples will proceed in preparation for assay. • Relative to the...transcribed. • Relative to the project * Characterization of Bone Morphogenetic Protein Receptors in Oral Tissues ", collection of clinical samples is

  2. Nanostructured hydroxyapatite surfaces-mediated adsorption alters recognition of BMP receptor IA and bioactivity of bone morphogenetic protein-2.

    PubMed

    Huang, Baolin; Yuan, Yuan; Ding, Sai; Li, Jianbo; Ren, Jie; Feng, Bo; Li, Tong; Gu, Yuantong; Liu, Changsheng

    2015-11-01

    Highly efficient loading of bone morphogenetic protein-2 (BMP-2) onto carriers with desirable performance is still a major challenge in the field of bone regeneration. Till now, the nanoscaled surface-induced changes of the structure and bioactivity of BMP-2 remains poorly understood. Here, the effect of nanoscaled surface on the adsorption and bioactivity of BMP-2 was investigated with a series of hydroxyapatite surfaces (HAPs): HAP crystal-coated surface (HAP), HAP crystal-coated polished surface (HAP-Pol), and sintered HAP crystal-coated surface (HAP-Sin). The adsorption dynamics of recombinant human BMP-2 (rhBMP-2) and the accessibility of the binding epitopes of adsorbed rhBMP-2 for BMP receptors (BMPRs) were examined by a quartz crystal microbalance with dissipation. Moreover, the bioactivity of adsorbed rhBMP-2 and the BMP-induced Smad signaling were investigated with C2C12 model cells. A noticeably high mass-uptake of rhBMP-2 and enhanced recognition of BMPR-IA to adsorbed rhBMP-2 were found on the HAP-Pol surface. For the rhBMP-2-adsorbed HAPs, both ALP activity and Smad signaling increased in the order of HAP-Sin

  3. Bone morphogenetic protein and bone metastasis, implication and therapeutic potential.

    PubMed

    Ye, Lin; Mason, Malcolm D; Jiang, Wen G

    2011-01-01

    Bone metastasis is one of the most common and severe complications in advanced malignancies, particularly in the three leading cancers; breast cancer, prostate cancer and lung cancer. It is currently incurable and causes severe morbidities, including bone pain, hypercalcemia, pathological fracture, spinal cord compression and consequent paralysis. However, the mechanisms underlying the development of bone metastasis remain largely unknown. Bone morphogenetic proteins (BMPs) belong to the TGF-beta superfamily and are pluripotent factors involved in the regulation of embryonic development and postnatal homeostasis of various organs and tissues, by controlling cellular differentiation, proliferation and apoptosis. Since they are potent regulators for bone formation, there is an increasing interest to investigate BMPs and their roles in bone metastasis. BMPs have been implicated in various neoplasms, at both primary and secondary tumors, particularly skeletal metastasis. Recently studies have also suggested that BMP signaling and their antagonists play pivotal roles in bone metastasis. In this review, we discuss the current knowledge of aberrations of BMPs which have been indicated in tumor progression, and particularly in the development of bone metastasis.

  4. Bone morphogenetic protein-2 and bone therapy: successes and pitfalls.

    PubMed

    Poon, Bonnie; Kha, Tram; Tran, Sally; Dass, Crispin R

    2016-02-01

    Bone morphogenetic proteins (BMPs), more specifically BMP-2, are being increasingly used in orthopaedic surgery due to advanced research into osteoinductive factors that may enhance and improve bone therapy. There are many areas in therapy that BMP-2 is being applied to, including dental treatment, open tibial fractures, cancer and spinal surgery. Within these areas of treatment, there are many reports of successes and pitfalls. This review explores the use of BMP-2 and its successes, pitfalls and future prospects in bone therapy. The PubMed database was consulted to compile this review. With successes in therapy, there were descriptions of a more rapid healing time with no signs of rejection or infection attributed to BMP-2 treatment. Pitfalls included BMP-2 'off-label' use, which lead to various adverse effects. Our search highlighted that optimising treatment with BMP-2 is a direction that many researchers are exploring, with areas of current research interest including concentration and dose of BMP-2, carrier type and delivery. © 2015 Royal Pharmaceutical Society.

  5. Electrostatics and N-glycan-mediated membrane tethering of SCUBE1 is critical for promoting bone morphogenetic protein signalling.

    PubMed

    Liao, Wei-Ju; Tsao, Ku-Chi; Yang, Ruey-Bing

    2016-03-01

    SCUBE1 (S1), a secreted and membrane-bound glycoprotein, has a modular protein structure composed of an N-terminal signal peptide sequence followed by nine epidermal growth factor (EGF)-like repeats, a spacer region and three cysteine-rich (CR) motifs with multiple potential N-linked glycosylation sites, and one CUB domain at the C-terminus. Soluble S1 is a biomarker of platelet activation but an active participant of thrombosis via its adhesive EGF-like repeats, whereas its membrane-associated form acts as a bone morphogenetic protein (BMP) co-receptor in promoting BMP signal activity. However, the mechanism responsible for the membrane tethering and the biological importance of N-glycosylation of S1 remain largely unknown. In the present study, molecular mapping analysis identified a polycationic segment (amino acids 501-550) in the spacer region required for its membrane tethering via electrostatic interactions possibly with the anionic heparan sulfate proteoglycans. Furthermore, deglycosylation by peptide N-glycosidase F treatment revealed that N-glycans within the CR motif are essential for membrane recruitment through lectin-mediated surface retention. Injection of mRNA encoding zebrafish wild-type but not N-glycan-deficient scube1 restores the expression of haematopoietic and erythroid markers (scl and gata1) in scube1-knockdown embryos. We describe novel mechanisms in targeting S1 to the plasma membrane and demonstrate that N-glycans are required for S1 functions during primitive haematopoiesis in zebrafish. © 2016 Authors; published by Portland Press Limited.

  6. Complications of Anterior Cervical Fusion using a Low-dose Recombinant Human Bone Morphogenetic Protein-2

    PubMed Central

    Kukreja, Sunil; Ahmed, Osama I; Haydel, Justin; Nanda, Anil

    2015-01-01

    Objective There are several reports, which documented a high incidence of complications following the use of recombinant human bone morphogenetic protein-2 (rhBMP-2) in anterior cervical fusions (ACFs). The objective of this study is to share our experience with low-dose rhBMP-2 in anterior cervical spine. Methods We performed a retrospective analysis of 197 patients who underwent anterior cervical fusion (ACF) with the use of recombinant human bone morphogenetic protein-2 (rhBMP-2) during 2007-2012. A low-dose rhBMP-2 (0.7mg/level) sponge was placed exclusively within the cage. In 102 patients demineralized bone matrix (DBM) was filled around the BMP sponge. Incidence and severity of dysphagia was determined by 5 points SWAL-QOL scale. Results Two patients had prolonged hospitalization due to BMP unrelated causes. Following the discharge, 13.2%(n=26) patients developed dysphagia and 8.6%(n=17) patients complained of neck swelling. More than half of the patients (52.9%, n=9) with neck swelling also had associated dysphagia; however, only 2 of these patients necessitated readmission. Both of these patients responded well to the intravenous dexamethasone. The use of DBM did not affect the incidence and severity of complications (p>0.05). Clinico-radiological evidence of fusion was not observed in 2 patients. Conclusion A low-dose rhBMP-2 in ACFs is not without risk. However, the incidence and severity of complications seem to be lower with low-dose BMP placed exclusively inside the cage. Packing DBM putty around the BMP sponge does not affect the safety profile of rhBMP-2 in ACFs. PMID:26217385

  7. Establishment of a cell line producing bone morphogenetic protein from a human osteosarcoma.

    PubMed

    Takaoka, K; Yoshikawa, H; Masuhara, K; Sugamoto, K; Tsuda, T; Aoki, Y; Ono, K; Sakamoto, Y

    1989-07-01

    A human osteosarcoma cell line was established from a biopsy specimen from a 13-year-old girl. The osteosarcoma tissue was maintained in athymic nude mice (Balb C nu/nu) by serial transplantation for three years. The tumor was excised from a host mouse and digested with collagenase. The isolated cells were cultured by 98 passages in 14 months, and clones of osteosarcoma cells were obtained by limiting dilution. A clone named human osteosarcoma cell 6 (H-OS-6) that showed the osteoblastic phenotypes of productions of bone morphogenetic protein (BMP) and alkaline phosphatase and a response to human parathyroid hormone (h-PTH 1-34) was selected. The morphology of its chromosomes indicated its human origin. This human osteosarcoma cell line is unique in producing BMP under in vitro conditions.

  8. Activated protein C (APC) can increase bone anabolism via a protease-activated receptor (PAR)1/2 dependent mechanism.

    PubMed

    Shen, Kaitlin; Murphy, Ciara M; Chan, Ben; Kolind, Mille; Cheng, Tegan L; Mikulec, Kathy; Peacock, Lauren; Xue, Meilang; Park, Sang-Youel; Little, David G; Jackson, Chris J; Schindeler, Aaron

    2014-12-01

    Activated Protein C (APC) is an anticoagulant with strong cytoprotective properties that has been shown to promote wound healing. In this study APC was investigated for its potential orthopedic application using a Bone Morphogenetic Protein 2 (rhBMP-2) induced ectopic bone formation model. Local co-administration of 10 µg rhBMP-2 with 10 µg or 25 µg APC increased bone volume at 3 weeks by 32% (N.S.) and 74% (p<0.01) compared to rhBMP-2 alone. This was associated with a significant increase in CD31+ and TRAP+ cells in tissue sections of ectopic bone, consistent with enhanced vascularity and bone turnover. The actions of APC are largely mediated by its receptors endothelial protein C receptor (EPCR) and protease-activated receptors (PARs). Cultured pre-osteoblasts and bone nodule tissue sections were shown to express PAR1/2 and EPCR. When pre-osteoblasts were treated with APC, cell viability and phosphorylation of ERK1/2, Akt, and p38 were increased. Inhibition with PAR1 and sometimes PAR2 antagonists, but not with EPCR blocking antibodies, ameliorated the effects of APC on cell viability and kinase phosphorylation. These data indicate that APC can affect osteoblast viability and signaling, and may have in vivo applications with rhBMP-2 for bone repair. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  9. Key role of the expression of bone morphogenetic proteins in increasing the osteogenic activity of osteoblast-like cells exposed to shock waves and seeded on bioactive glass-ceramic scaffolds for bone tissue engineering.

    PubMed

    Muzio, Giuliana; Martinasso, Germana; Baino, Francesco; Frairia, Roberto; Vitale-Brovarone, Chiara; Canuto, Rosa A

    2014-11-01

    In this work, the role of shock wave-induced increase of bone morphogenetic proteins in modulating the osteogenic properties of osteoblast-like cells seeded on a bioactive scaffold was investigated using gremlin as a bone morphogenetic protein antagonist. Bone-like glass-ceramic scaffolds, based on a silicate experimental bioactive glass developed at the Politecnico di Torino, were produced by the sponge replication method and used as porous substrates for cell culture. Human MG-63 cells, exposed to shock waves and seeded on the scaffolds, were treated with gremlin every two days and analysed after 20 days for the expression of osteoblast differentiation markers. Shock waves have been shown to induce osteogenic activity mediated by increased expression of alkaline phosphatase, osteocalcin, type I collagen, BMP-4 and BMP-7. Cells exposed to shock waves plus gremlin showed increased growth in comparison with cells treated with shock waves alone and, conversely, mRNA contents of alkaline phosphatase and osteocalcin were significantly lower. Therefore, the shock wave-mediated increased expression of bone morphogenetic protein in MG-63 cells seeded on the scaffolds is essential in improving osteogenic activity; blocking bone morphogenetic protein via gremlin completely prevents the increase of alkaline phosphatase and osteocalcin. The results confirmed that the combination of glass-ceramic scaffolds and shock waves exposure could be used to significantly improve osteogenesis opening new perspectives for bone regenerative medicine. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

  10. Bone morphogenetic protein-mediated interaction of periosteum and diaphysis. Citric acid and other factors influencing the generation of parosteal bone.

    PubMed

    Kübler, N; Urist, M R

    1990-09-01

    In rabbits, after long-bone growth is complete and the cambium layer regresses, mesenchymal-type cells with embryonic potential (competence) for bone development persist in the adventitial layer of periosteum. These cells are not determined osteoprogenitor cells (stem cells) because bone tissue differentiation does not occur when adult periosteum is transplanted into a heterotopic site. In this respect, adventitial cells differ from bone marrow stroma cells. In a parosteal orthotopic site in the space between the adult periosteum and diaphysis, implants of bone morphogenetic protein (BMP) and associated noncollagenous proteins (BMP/NCP) induce adventitia and adjacent muscle connective-tissue-derived cells to switch from a fibrogenetic to a chondroosteoprogenetic pattern of bone development. The quantity of induced bone is proportional to the dose of BMP/NCP in the range from 10 to 50 mg; immature rabbits produced larger deposits than mature rabbits in response to BMP/NCP. Preoperative local intramuscular injections of citric, edetic, or hyaluronic acids in specified concentrations markedly enhanced subperiosteal BMP/NCP-induced bone formation. The quantity of bovine or human BMP/NCP-induced bone formation in rabbits is also increased by very low-dose immunosuppression but not by bone mineral, tricalcium phosphate ceramic, inorganic calcium salts, or various space-occupying, unspecific chemical irritants. Although composities of BMP/NCP and allogeneic rabbit tendon collagen increased the quantity of bone in a parosteal site, in a heterotopic site the composite failed to induce bone formation. In a parosteal site, the conditions permitting BMP/NCP-induced bone formation develop, and the end product of the morphogenetic response is a duplicate diaphysis. How BMP reactivates the morphogenetic process in postfetal mesenchymal-type adventitial cells persisting in adult periosteum (including adjacent muscle attachments) is not known.

  11. Expression of bone morphogenetic proteins and Msx genes during root formation.

    PubMed

    Yamashiro, T; Tummers, M; Thesleff, I

    2003-03-01

    Like crown development, root formation is also regulated by interactions between epithelial and mesenchymml tissues. Bone morphogenetic proteins (BMPs), together with the transcription factors Msx1 and Msx2, play important roles in these interactions during early tooth morphogenesis. To investigate the involvement of this signaling pathway in root development, we analyzed the expression patterns of Bmp2, Bmp3, Bmp4, and Bmp7 as well as Msx1 and Msx2 in the roots of mouse molars. Bmp4 was expressed in the apical mesenchyme and Msx2 in the root sheath. However, Bmps were not detected in the root sheath epithelium, and Msx transcripts were absent from the underlying mesenchyme. These findings indicate that this Bmp signaling pathway, required for tooth initiation, does not regulate root development, but we suggest that root shape may be regulated by a mechanism similar to that regulating crown shape in cap-stage tooth germs. Msx2 expression continued in the epithelial cell rests of Malassez, and the nearby cementoblasts intensely expressed Bmp3, which may regulate some functions of the fragmented epithelium.

  12. Silibinin promotes osteoblast differentiation of human bone marrow stromal cells via bone morphogenetic protein signaling.

    PubMed

    Ying, Xiaozhou; Sun, Liaojun; Chen, Xiaowei; Xu, Huazi; Guo, Xiaoshan; Chen, Hua; Hong, Jianjun; Cheng, Shaowen; Peng, Lei

    2013-12-05

    Silibinin is the major active constituent of the natural compound silymarin; several studies suggest that silibinin possesses antihepatotoxic properties and anticancer effects against carcinoma cells. However, no study has yet investigated the effect of silibinin on osteogenic differentiation of human bone marrow stem cells (hBMSCs). The aim of this study was to evaluate the effect of silibinin on osteogenic differentiation of hBMSCs. In this study, the hBMSCs were cultured in an osteogenic medium with 0, 1, 10 or 20 μmol/l silibinin respectively. hBMSCs viability was analyzed by cell number quantification assay and cells osteogenic differentiation was evaluated by alkaline phosphatas (ALP) activity assay, Von Kossa staining and real time-polymerase chain reaction (RT-PCR). We found that silibinin promoted ALP activity in hBMSCs without affecting their proliferation. The mineralization of hBMSCs was enhanced by treatment with silibinin. Silibinin also increased the mRNA expressions of Collagen type I (COL-I), ALP, Osteocalcin (OCN), Osterix, bone morphogenetic protein-2 (BMP-2) and Runt-related transcription factor 2 (RUNX2). The BMP antagonist noggin and its receptor kinase inhibitors dorsomorphin and LDN-193189 attenuated silibinin-promoted ALP activity. Furthermore, BMP-responsive and Runx2-responsive reporters were activated by silibinin treatment. These results indicate that silibinin enhances osteoblast differentiation probably by inducing the expressions of BMPs and activating BMP and RUNX2 pathways. Thus, silibinin may play an important therapeutic role in osteoporosis patients by improving osteogenic differentiation of BMSCs. © 2013 Elsevier B.V. All rights reserved.

  13. Analysis of Recombinant Human Bone Morphogenetic Protein-2 Use in the Treatment of Lumbar Degenerative Spondylolisthesis

    PubMed Central

    Yao, Qingqiang; Cohen, Jeremiah R.; Buser, Zorica; Park, Jong-Beom; Brodke, Darrel S.; Meisel, Hans-Joerg; Youssef, Jim A.; Wang, Jeffrey C.; Yoon, S. Tim

    2016-01-01

    Study Design Retrospective database review. Objective To identify trends of the recombinant human bone morphogenetic protein-2 (rhBMP-2) use in the treatment of lumbar degenerative spondylolisthesis (LDS). Methods PearlDiver Patient Record Database was used to identify patients who underwent lumbar fusion for LDS between 2005 and 2011. The distribution of bone morphogenetic protein use rate (BR) in various surgical procedures was recorded. Patient numbers, reoperation numbers, BR, and per year BR (PYBR) were stratified by geographic region, gender, and age. Results There were 11,335 fusion surgeries, with 3,461 cases using rhBMP-2. Even though PYRB increased between 2005 and 2008, there was a significant decrease in 2010 for each procedure: 404 (34.5%) for posterior interbody fusion, 1,282 (34.3%) for posterolateral plus posterior interbody fusion (PLPIF), 1,477 (29.2%) for posterolateral fusion, and 335 (22.4%) for anterior lumbar interbody fusion. In patients using rhBMP-2, the reoperation rate was significantly lower than in patients not using rhBMP-2 (0.69% versus 1.07%, p < 0.0001). Male patients had higher PYBR compared with female patients in 2008 and 2009 (p < 0.05). The West region and PLPIF had the highest BR and PYBR. Conclusions Our data shows that the revision rates were significantly lower in patients treated with rhBMP-2 compared with patients not treated with rhBMP-2. Furthermore, rhBMP-2 use in LDS varied by year, region, gender, and type of fusion technique. In the West region, the posterior approach and patients 65 to 69 years of age had the highest rate of rhBMP-2 use. PMID:27853658

  14. Strategies for delivering bone morphogenetic protein for bone healing.

    PubMed

    Begam, Howa; Nandi, Samit Kumar; Kundu, Biswanath; Chanda, Abhijit

    2017-01-01

    Bone morphogenetic proteins (BMPs) are the most significant growth factors that belong to the Transforming Growth Factor Beta (TGF-β) super-family. Though more than twenty members of this family have been identified so far in humans, Food and Drug Administration (FDA) approved two growth factors: BMP-2 and BMP-7 for treatments of spinal fusion and long-bone fractures with collagen carriers. Currently BMPs are clinically used in spinal fusion, oral and maxillofacial surgery and also in the repair of long bone defects. The efficiency of BMPs depends a lot on the selection of suitable carriers. At present, different types of carrier materials are used: natural and synthetic polymers, calcium phosphate and ceramic-polymer composite materials. Number of research articles has been published on the minute intricacies of the loading process and release kinetics of BMPs. Despite the significant evidence of its potential for bone healing demonstrated in animal models, future clinical investigations are needed to define dose, scaffold and route of administration. The efficacy and application of BMPs in various levels with a proper carrier and dose is yet to be established. The present article collates various aspects of success and limitation and identifies the prospects and challenges associated with the use of BMPs in orthopaedic surgery. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Genomewide Analysis of Aryl Hydrocarbon Receptor Binding Targets Reveals an Extensive Array of Gene Clusters that Control Morphogenetic and Developmental Programs

    PubMed Central

    Sartor, Maureen A.; Schnekenburger, Michael; Marlowe, Jennifer L.; Reichard, John F.; Wang, Ying; Fan, Yunxia; Ma, Ci; Karyala, Saikumar; Halbleib, Danielle; Liu, Xiangdong; Medvedovic, Mario; Puga, Alvaro

    2009-01-01

    Background The vertebrate aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that regulates cellular responses to environmental polycyclic and halogenated compounds. The naive receptor is believed to reside in an inactive cytosolic complex that translocates to the nucleus and induces transcription of xenobiotic detoxification genes after activation by ligand. Objectives We conducted an integrative genomewide analysis of AHR gene targets in mouse hepatoma cells and determined whether AHR regulatory functions may take place in the absence of an exogenous ligand. Methods The network of AHR-binding targets in the mouse genome was mapped through a multipronged approach involving chromatin immunoprecipitation/chip and global gene expression signatures. The findings were integrated into a prior functional knowledge base from Gene Ontology, interaction networks, Kyoto Encyclopedia of Genes and Genomes pathways, sequence motif analysis, and literature molecular concepts. Results We found the naive receptor in unstimulated cells bound to an extensive array of gene clusters with functions in regulation of gene expression, differentiation, and pattern specification, connecting multiple morphogenetic and developmental programs. Activation by the ligand displaced the receptor from some of these targets toward sites in the promoters of xenobiotic metabolism genes. Conclusions The vertebrate AHR appears to possess unsuspected regulatory functions that may be potential targets of environmental injury. PMID:19654925

  16. Fine mapping of the human bone morphogenetic protein-4 gene (BMP4) to chromosome 14q22-q23 by in situ hybridization

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wijngaard, A. van den; Boersma, C.J.C.; Olijve, W.

    Bone morphogenetic protein-4 (BMP-4) is a member of the transforming growth factor-{beta} (TGF-{beta}) superfamily and is involved in morphogenesis and bone cell differentiation. Recombinant BMP-4 can induce ectopic cartilage and bone formation when implanted subcutaneously or intramuscularly in rodents. This ectopic bone formation process resembles the process of bone formation during embryogenesis and fracture healing. A cosmid clone containing the complete human bone morphogenetic protein-4 gene (BMP4) was isolated (details to be published elsewhere) and used as a probe to determine the precise chromosomal localization of the human BMP4 gene. This cosmid clone was labeled with biotin-14-dATP and hybridized inmore » situ to chromosomal preparations of metaphase cells as described previously. In 20 metaphase preparations, an intense and specific fluorescence signal (FITC) was detected on the q arm of chromosome 14. The DAPI-counterstained chromosomes were computer-converted into GTG-like banding patterns, allowing the regional localization of BMP4 within 14q22-q23. 10 refs., 1 fig.« less

  17. Purification of bone morphogenetic protein-2 from refolding mixtures using mixed-mode membrane chromatography.

    PubMed

    Gieseler, Gesa; Pepelanova, Iliyana; Stuckenberg, Lena; Villain, Louis; Nölle, Volker; Odenthal, Uwe; Beutel, Sascha; Rinas, Ursula; Scheper, Thomas

    2017-01-01

    In this study, we present the development of a process for the purification of recombinant human bone morphogenetic protein-2 (rhBMP-2) using mixed-mode membrane chromatography. RhBMP-2 was produced as inclusion bodies in Escherichia coli. In vitro refolding using rapid dilution was carried out according to a previously established protocol. Different membrane chromatography phases were analyzed for their ability to purify BMP-2. A membrane phase with salt-tolerant properties resulting from mixed-mode ligand chemistry was able to selectively purify BMP-2 dimer from refolding mixtures. No further purification or polishing steps were necessary and high product purity was obtained. The produced BMP-2 exhibited a biological activity of 7.4 × 10 5  U/mg, comparable to commercial preparations. Mixed-mode membrane chromatography can be a valuable tool for the direct purification of proteins from solutions with high-conductivity, for example refolding buffers. In addition, in this particular case, it allowed us to circumvent the use of heparin-affinity chromatography, thus allowing the design of an animal-component-free process.

  18. In vitro and in vivo protein release and anti-ischemia/reperfusion injury properties of bone morphogenetic protein-2-loaded glycyrrhetinic acid-poly(ethylene glycol)-b-poly(l-lysine) nanoparticles

    PubMed Central

    Shan, Fang; Liu, YuJuan; Jiang, Haiying; Tong, Fei

    2017-01-01

    Here, we describe a bone morphogenetic protein-2 (BMP-2) nanocarrier based on glycyrrhetinic acid (GA)-poly(ethylene glycol) (PEG)-b-poly(l-lysine) (PLL). A protein nanocarrier was synthesized, characterized and evaluated as a BMP-2 delivery system. The designed nanocarrier was synthesized based on the ring-opening polymerization of amino acid N-carboxyanhydride. The final product was measured with 1H nuclear magnetic resonance. GA-PEG-b-PLL nanocarrier could combine with BMP-2 through electrostatic interaction to form polyion complex (PIC) micelles. BMP-2 could be rapidly and efficiently encapsulated through the GA-PEG-b-PLL nanocarrier under physiological conditions, exhibiting efficient encapsulation and sustained release. In addition, the GA-PEG-b-PLL-mediated BMP-2 delivery system could target the liver against hepatic diseases as it has GA-binding receptors. The anti-hepatic ischemia/reperfusion injury (anti-HI/RI) effect of BMP-2/GA-PEG-b-PLL PIC micelles was investigated in rats using free BMP-2 and BMP-2/PEG-b-PLL PIC micelles as controls, and the results showed that BMP-2/GA-PEG-b-PLL PIC micelles indicated significantly enhanced anti-HI/RI property compared to BMP-2 and BMP-2/PEG-b-PLL. All results suggested that GA-PEG-b-PLL could be used as a potential BMP-2 nanocarrier. PMID:29089759

  19. Bone morphogenetic protein use in spine surgery-complications and outcomes: a systematic review.

    PubMed

    Faundez, Antonio; Tournier, Clément; Garcia, Matthieu; Aunoble, Stéphane; Le Huec, Jean-Charles

    2016-06-01

    Because of significant complications related to the use of autologous bone grafts in spinal fusion surgery, bone substitutes and growth factors such as bone morphogenetic protein (BMP) have been developed. One of them, recombinant human (rh) BMP-2, has been approved by the Food and Drug Administration (FDA) for use under precise conditions. However, rhBMP-2-related side effects have been reported, used in FDA-approved procedures, but also in off-label use.A systematic review of clinical data was conducted to analyse the rhBMP-2-related adverse events (AEs), in order to assess their prevalence and the associated surgery practices. Medline search with keywords "bone morphogenetic protein 2", "lumbar spine", "anterolateral interbody fusion" (ALIF) and the filter "clinical trial". FDA published reports were also included. Study assessment was made by authors (experienced spine surgeons), based on quality of study designs and level of evidence. Extensive review of randomised controlled trials (RCTs) and controlled series published up to the present point, reveal no evidence of a significant increase of AEs related to rhBMP-2 use during ALIF surgeries, provided that it is used following FDA guidelines. Two additional RCTs performed with rhBMP-2 in combination with allogenic bone dowels reported increased bone remodelling in BMP-treated patients. This AE was transient and had no consequence on the clinical outcome of the patients. No other BMP-related AEs were reported in these studies. This literature review confirms that the use of rhBMP-2 following FDA-approved recommendations (i.e. one-level ALIF surgery with an LT-cage) is safe. The rate of complications is low and the AEs had been identified by the FDA during the pre-marketing clinical trials. The clinical efficiency of rhBMP-2 is equal or superior to that of allogenic or autologous bone graft in respect to fusion rate, low back pain disability, patient satisfaction and rate of re-operations. For all other off

  20. Heparin Microparticle Effects on Presentation and Bioactivity of Bone Morphogenetic Protein-2

    PubMed Central

    Hettiaratchi, Marian H.; Miller, Tobias; Temenoff, Johnna S.; Guldberg, Robert E.; McDevitt, Todd C.

    2014-01-01

    Biomaterials capable of providing localized and sustained presentation of bioactive proteins are critical for effective therapeutic growth factor delivery. However, current biomaterial delivery vehicles commonly suffer from limitations that can result in low retention of growth factors at the site of interest or adversely affect growth factor bioactivity. Heparin, a highly sulfated glycosaminoglycan, is an attractive growth factor delivery vehicle due to its ability to reversibly bind positively charged proteins, provide sustained delivery, and maintain protein bioactivity. This study describes the fabrication and characterization of heparin methacrylamide (HMAm) microparticles for recombinant growth factor delivery. HMAm microparticles were shown to efficiently bind several heparin-binding growth factors (e.g. bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (FGF-2)), including a wide range of BMP-2 concentrations that exceeds the maximum binding capacity of other common growth factor delivery vehicles, such as gelatin. BMP-2 bioactivity was assessed on the basis of alkaline phosphatase (ALP) activity induced in skeletal myoblasts (C2C12). Microparticles loaded with BMP-2 stimulated comparable C2C12 ALP activity to soluble BMP-2 treatment, indicating that BMP-2-loaded microparticles retain bioactivity and potently elicit a functional cell response. In summary, our results suggest that heparin microparticles stably retain large amounts of bioactive BMP-2 for prolonged periods of time, and that presentation of BMP-2 via heparin microparticles can elicit cell responses comparable to soluble BMP-2 treatment. Consequently, heparin microparticles present an effective method of delivering and spatially retaining growth factors that could be used in a variety of systems to enable directed induction of cell fates and tissue regeneration. PMID:24881028

  1. Analysis of Recombinant Human Bone Morphogenetic Protein-2 Use in the Treatment of Lumbar Degenerative Spondylolisthesis.

    PubMed

    Yao, Qingqiang; Cohen, Jeremiah R; Buser, Zorica; Park, Jong-Beom; Brodke, Darrel S; Meisel, Hans-Joerg; Youssef, Jim A; Wang, Jeffrey C; Yoon, S Tim

    2016-12-01

    Study Design  Retrospective database review. Objective  To identify trends of the recombinant human bone morphogenetic protein-2 (rhBMP-2) use in the treatment of lumbar degenerative spondylolisthesis (LDS). Methods  PearlDiver Patient Record Database was used to identify patients who underwent lumbar fusion for LDS between 2005 and 2011. The distribution of bone morphogenetic protein use rate (BR) in various surgical procedures was recorded. Patient numbers, reoperation numbers, BR, and per year BR (PYBR) were stratified by geographic region, gender, and age. Results  There were 11,335 fusion surgeries, with 3,461 cases using rhBMP-2. Even though PYRB increased between 2005 and 2008, there was a significant decrease in 2010 for each procedure: 404 (34.5%) for posterior interbody fusion, 1,282 (34.3%) for posterolateral plus posterior interbody fusion (PLPIF), 1,477 (29.2%) for posterolateral fusion, and 335 (22.4%) for anterior lumbar interbody fusion. In patients using rhBMP-2, the reoperation rate was significantly lower than in patients not using rhBMP-2 (0.69% versus 1.07%, p  < 0.0001). Male patients had higher PYBR compared with female patients in 2008 and 2009 ( p  < 0.05). The West region and PLPIF had the highest BR and PYBR. Conclusions Our data shows that the revision rates were significantly lower in patients treated with rhBMP-2 compared with patients not treated with rhBMP-2. Furthermore, rhBMP-2 use in LDS varied by year, region, gender, and type of fusion technique. In the West region, the posterior approach and patients 65 to 69 years of age had the highest rate of rhBMP-2 use.

  2. Activation of the PI3K/Akt pathway mediates bone morphogenetic protein 2-induced invasion of pancreatic cancer cells Panc-1.

    PubMed

    Chen, Xiong; Liao, Jie; Lu, YeBin; Duan, XiaoHui; Sun, WeiJia

    2011-06-01

    Bone morphogenetic proteins (BMPs) signaling has an emerging role in pancreatic cancer. However, because of the multiple effects of different BMPs, no final conclusions have been made as to the role of BMPs in pancreatic cancer. In our studies, we have focused on bone morphogenetic protein 2(BMP-2) because it induces an epithelial to mesenchymal transition (EMT) and accelerates invasion in the human pancreatic cancer cell line Panc-1. It has been reported that the phosphatidylinositol 3-kinase (PI3K)/Akt pathway mediates invasion of gastric and colon cancer cells, which is unrevealed in pancreatic cancer cells. The objective of our study was to investigate whether BMP-2 mediated invasion might pass through the PI3K/Akt pathway. Our results show that expression of phosphorylation of Akt was increased by treatment with BMP-2, but not Noggin, a BMP-2 antagonist. Then pretreatment of Panc-1 cells with LY294002, an inhibitor of the PI3K/AKT pathway, significantly inhibited BMP-2-induced EMT and invasiveness. The data suggest that BMP-2 accelerates invasion of panc-1 cells via the PI3K/AKT pathway in panc-1 cells, which gives clues to searching new therapy targets in advanced pancreatic cancer.

  3. Synergistic effects of fibronectin and bone morphogenetic protein on the bioactivity of titanium metal.

    PubMed

    Biao, M N; Chen, Y M; Xiong, S B; Wu, B Y; Yang, B C

    2017-09-01

    To improve the biological properties of bioactive titanium metal, recombinant human bone morphogenetic protein 2(rhBMP-2) and fibronectin (Fn) were adsorbed on its surface solely or contiguously to modify the anodic oxidized titanium (AO-Ti), acid-alkali-treated titanium (AA-Ti), and polished titanium (P-Ti). It is found that the different bioactive titanium surface structures had great influence on protein adsorption. The adsorption amounts of BMP adsorbed solely and Fn/BMP adsorbed contiguously were AA-Ti > P-Ti > AO-Ti, and that for Fn adsorbed solely was AA-Ti ≈ P-Ti > AO-Ti. The conformation of proteins was changed remarkably after the adsorption. For BMP, the α-helix decreased on AA-Ti and stabilized on P-Ti and AO-Ti. For Fn, the β-sheet on PT-Ti and AA-Ti increased significantly. For Fn/BMP, the percentage of β-sheet on AA-Ti increased, and that of α-helix on all samples was stable. MSCs showed greater adhesion and spreading on Fn/BMP groups. MTT and Elisa tests showed that the synergistic effects of proteins made the cells proliferate and differentiate faster. It indicated both the surface structure and the synergistic effects of proteins could influence the biological properties of titanium metals. It provides research foundation for improving the biological properties of bioactive titanium metals by simultaneous application of several proteins. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2485-2498, 2017. © 2017 Wiley Periodicals, Inc.

  4. Molecular dynamics simulations of the adsorption of bone morphogenetic protein-2 on surfaces with medical relevance.

    PubMed

    Utesch, Tillmann; Daminelli, Grazia; Mroginski, Maria Andrea

    2011-11-01

    Bone morphogenetic protein-2 (BMP-2) plays a crucial role in osteoblast differentiation and proliferation. Its effective therapeutic use for ectopic bone and cartilage regeneration depends, among other factors, on the interaction with the carrier at the implant site. In this study, we used classical molecular dynamics (MD) and a hybrid approach of steered molecular dynamics (SMD) combined with MD simulations to investigate the initial stages of the adsorption of BMP-2 when approaching two implant surfaces, hydrophobic graphite and hydrophilic titanium dioxide rutile. Surface adsorption was evaluated for six different orientations of the protein, two end-on and four side-on, in explicit water environment. On graphite, we observed a weak but stable adsorption. Depending on the initial orientation, hydrophobic patches as well as flexible loops of the protein were involved in the interaction with graphite. On the contrary, BMP-2 adsorbed only loosely to hydrophilic titanium dioxide. Despite a favorable interaction energy between protein and the TiO(2) surface, the rapid formation of a two-layer water structure prevented the direct interaction between protein and titanium dioxide. The first water adlayer had a strong repulsive effect on the protein, while the second attracted the protein toward the surface. For both surfaces, hydrophobic graphite and hydrophilic titanium dioxide, denaturation of BMP-2 induced by adsorption was not observed on the nanosecond time scale.

  5. Constitutively Active Akt Induces Ectodermal Defects and Impaired Bone Morphogenetic Protein Signaling

    PubMed Central

    Segrelles, Carmen; Moral, Marta; Lorz, Corina; Santos, Mirentxu; Lu, Jerry; Cascallana, José Luis; Lara, M. Fernanda; Carbajal, Steve; Martínez-Cruz, Ana Belén; García-Escudero, Ramón; Beltran, Linda; Segovia, José C.; Bravo, Ana

    2008-01-01

    Aberrant activation of the Akt pathway has been implicated in several human pathologies including cancer. However, current knowledge on the involvement of Akt signaling in development is limited. Previous data have suggested that Akt-mediated signaling may be an essential mediator of epidermal homeostasis through cell autonomous and noncell autonomous mechanisms. Here we report the developmental consequences of deregulated Akt activity in the basal layer of stratified epithelia, mediated by the expression of a constitutively active Akt1 (myrAkt) in transgenic mice. Contrary to mice overexpressing wild-type Akt1 (Aktwt), these myrAkt mice display, in a dose-dependent manner, altered development of ectodermally derived organs such as hair, teeth, nails, and epidermal glands. To identify the possible molecular mechanisms underlying these alterations, gene profiling approaches were used. We demonstrate that constitutive Akt activity disturbs the bone morphogenetic protein-dependent signaling pathway. In addition, these mice also display alterations in adult epidermal stem cells. Collectively, we show that epithelial tissue development and homeostasis is dependent on proper regulation of Akt expression and activity. PMID:17959825

  6. Probing receptor structure/function with chimeric G-protein-coupled receptors.

    PubMed

    Yin, Dezhong; Gavi, Shai; Wang, Hsien-yu; Malbon, Craig C

    2004-06-01

    Owing its name to an image borrowed from Greek mythology, a chimera is seen to represent a new entity created as a composite from existing creatures or, in this case, molecules. Making use of various combinations of three basic domains of the receptors (i.e., exofacial, transmembrane, and cytoplasmic segments) that couple agonist binding into activation of effectors through heterotrimeric G-proteins, molecular pharmacology has probed the basic organization, structure/function relationships of this superfamily of heptahelical receptors. Chimeric G-protein-coupled receptors obviate the need for a particular agonist ligand when the ligand is resistant to purification or, in the case of orphan receptors, is not known. Chimeric receptors created from distant members of the heptahelical receptors enable new strategies in understanding how these receptors transduce agonist binding into receptor activation and may be able to offer insights into the evolution of G-protein-coupled receptors from yeast to humans.

  7. Functional relevance of G-protein-coupled-receptor-associated proteins, exemplified by receptor-activity-modifying proteins (RAMPs).

    PubMed

    Fischer, J A; Muff, R; Born, W

    2002-08-01

    The calcitonin (CT) receptor (CTR) and the CTR-like receptor (CRLR) are close relatives within the type II family of G-protein-coupled receptors, demonstrating sequence identity of 50%. Unlike the interaction between CT and CTR, receptors for the related hormones and neuropeptides amylin, CT-gene-related peptide (CGRP) and adrenomedullin (AM) require one of three accessory receptor-activity-modifying proteins (RAMPs) for ligand recognition. An amylin/CGRP receptor is revealed when CTR is co-expressed with RAMP1. When complexed with RAMP3, CTR interacts with amylin alone. CRLR, initially classed as an orphan receptor, is a CGRP receptor when co-expressed with RAMP1. The same receptor is specific for AM in the presence of RAMP2. Together with human RAMP3, CRLR defines an AM receptor, and with mouse RAMP3 it is a low-affinity CGRP/AM receptor. CTR-RAMP1, antagonized preferentially by salmon CT-(8-32) and not by CGRP-(8-37), and CRLR-RAMP1, antagonized by CGRP-(8-37), are two CGRP receptor isotypes. Thus amylin and CGRP interact specifically with heterodimeric complexes between CTR and RAMP1 or RAMP3, and CGRP and AM interact with complexes between CRLR and RAMP1, RAMP2 or RAMP3.

  8. Combination of bone morphogenetic protein-2 plasmid DNA with chemokine CXCL12 creates an additive effect on bone formation onset and volume.

    PubMed

    Wegman, F; Poldervaart, M T; van der Helm, Y J; Oner, F C; Dhert, W J; Alblas, J

    2015-07-27

    Bone morphogenetic protein-2 (BMP-2) gene delivery has shown to induce bone formation in vivo in cell-based tissue engineering. In addition, the chemoattractant stromal cell-derived factor-1α (SDF-1α, also known as CXCL12) is known to recruit multipotent stromal cells towards its release site where it enhances vascularisation and possibly contributes to osteogenic differentiation. To investigate potential cooperative behaviour for bone formation, we investigated combined release of BMP-2 and SDF-1α on ectopic bone formation in mice. Multipotent stromal cell-seeded and cell-free constructs with BMP-2 plasmid DNA and /or SDF-1α loaded onto gelatin microparticles, were implanted subcutaneously in mice for a period of 6 weeks. Histological analysis and histomorphometry revealed that the onset of bone formation and the formed bone volume were both enhanced by the combination of BMP-2 and SDF-1α compared to controls in cell-seeded constructs. Samples without seeded multipotent stromal cells failed to induce any bone formation. We conclude that the addition of stromal cell-derived factor-1α to a cell-seeded alginate based bone morphogenetic protein-2 plasmid DNA construct has an additive effect on bone formation and can be considered a promising combination for bone regeneration.

  9. Effectiveness of recombinant human bone morphogenetic protein-7 in the management of congenital pseudoarthrosis of the tibia: a randomised controlled trial.

    PubMed

    Das, Sakti Prasad; Ganesh, Shankar; Pradhan, Sudhakar; Singh, Deepak; Mohanty, Ram Narayan

    2014-09-01

    Despite the popularity and an increased use of bone morphogenetic protein to improve bone healing in patients with congenital pseudoarthrosis of the tibia (CPT), no previous study has compared its efficacy against any other procedure. We randomised 20 consecutive patients (mean age 4.1 years) with CPT (Crawford type IV) associated with neurofibromatosis type 1(NF1) and no previous history of surgery into two groups. Group 1 received recombinant human bone morphogenetic protein-7 (rhBMP-7) along with intramedullary Kirschner (K)-wire fixation and autologous bone grafting; group 2 received only K wire and grafting. Outcome measures were time to achieve union, Johnston grade, tibial length and the American Orthopaedic Foot and Ankle Society (AOFAS) score, which were evaluated preoperatively and at five year follow-up. Study results showed that patients in group 1 achieved primary bone union at a mean of 14.5 months [standard error (SE) 5.2], whereas group 2 took a mean of 17.11 months (SE 5.0). However, the log-rank test showed no difference in healing times between groups at all time points (P = 0.636). There was a statistically significant pre- to post operative improvement (P < 0.05) within groups for the other outcome measures. In a five year follow-up, these results suggest that rh-BMP-7 and autologous bone grafting is no better than autologous grafting alone.

  10. The calcium-sensing receptor and its interacting proteins

    PubMed Central

    Huang, Chunfa; Miller, R Tyler

    2007-01-01

    Abstract Seven membrane-spanning, or G protein-coupled receptors were originally thought to act through het-erotrimeric G proteins that in turn activate intracellular enzymes or ion channels, creating relatively simple, linear signalling pathways. Although this basic model remains true in that this family does act via a relatively small number of G proteins, these signalling systems are considerably more complex because the receptors interact with or are located near additional proteins that are often unique to a receptor or subset of receptors. These additional proteins give receptors their unique signalling ‘personalities’. The extracellular Ca-sensing receptor (CaR) signals via Gαi, Gαq and Gα12/13, but its effects in vivo demonstrate that the signalling pathways controlled by these subunits are not sufficient to explain all its biologic effects. Additional structural or signalling proteins that interact with the CaR may explain its behaviour more fully. Although the CaR is less well studied in this respect than other receptors, several CaR-interacting proteins such as filamin, a potential scaffolding protein, receptor activity modifying proteins (RAMPs) and potassium channels may contribute to the unique characteristics of the CaR. The CaR also appears to interact with additional proteins common to other G protein-coupled receptors such as arrestins, G protein receptor kinases, protein kinase C, caveolin and proteins in the ubiquitination pathway. These proteins probably represent a few initial members of CaR-based signalling complex. These and other proteins may not all be associated with the CaR in all tissues, but they form the basis for understanding the complete nature of CaR signalling. PMID:17979874

  11. Bone morphogenetic protein-4 strongly potentiates growth factor-induced proliferation of mammary epithelial cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Montesano, Roberto; Sarkoezi, Rita; Schramek, Herbert

    2008-09-12

    Bone morphogenetic proteins (BMPs) are multifunctional cytokines that elicit pleiotropic effects on biological processes such as cell proliferation, cell differentiation and tissue morphogenesis. With respect to cell proliferation, BMPs can exert either mitogenic or anti-mitogenic activities, depending on the target cells and their context. Here, we report that in low-density cultures of immortalized mammary epithelial cells, BMP-4 did not stimulate cell proliferation by itself. However, when added in combination with suboptimal concentrations of fibroblast growth factor (FGF)-2, FGF-7, FGF-10, epidermal growth factor (EGF) or hepatocyte growth factor (HGF), BMP-4 potently enhanced growth factor-induced cell proliferation. These results reveal a hithertomore » unsuspected interplay between BMP-4 and growth factors in the regulation of mammary epithelial cell proliferation. We suggest that the ability of BMP-4 to potentiate the mitogenic activity of multiple growth factors may contribute to mammary gland ductal morphogenesis as well as to breast cancer progression.« less

  12. Bone Morphogenetic Protein Usage in Anterior Lumbar Interbody Fusion: What Else Can Go Wrong?

    PubMed

    Elias, Elias; Nasser, Zeina; Winegan, Lona; Verla, Terence; Omeis, Ibrahim

    2018-03-01

    Bone morphogenetic protein (BMP) graft showed promising outcome during early phases of its use. However, unreported adverse events and off-label use shattered its safe profile and raised concerns regarding its indication. In 2008 the U.S. Food and Drug Administration prohibited its use in anterior cervical spine procedures due to the possibility of edema, hematoma, and need to intubate. At the molecular level, BMPs act as multifactorial growth factors playing a role in cartilage, heart, and bone formation. However, its unfavorable effect on bone overgrowth or heterotopic ossification post spine surgeries has been described. Reported cases in the literature were limited to epidural bone formation. We present a rare and interesting case of a 59-year-old female, in whom BMP caused intradural bone growth several years after an anterior lumbar interbody fusion surgery. Caution must be exercised while using BMPs because of inadvertent complications. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Genetic polymorphisms in bone morphogenetic protein receptor type IA gene predisposes individuals to ossification of the posterior longitudinal ligament of the cervical spine via the smad signaling pathway.

    PubMed

    Wang, Hao; Jin, Weitao; Li, Haibin

    2018-02-20

    The present study investigated the molecular mechanisms underlying the 4A > C and -349C > T single nucleotide polymorphisms (SNPs) in bone morphogenetic protein receptor type IA (BMPR-IA) gene, which significantly associated with the occurrence and the extent of ossification of the posterior longitudinal ligament (OPLL) in the cervical spine. The SNPs in BMPR-IA gene were genotyped, and the association with the occurrence and severity of OPLL were evaluated in 356 OPLL patients and 617 non-OPLL controls. In stably transfected mouse embryonic mesenchymal stem cells (C3H10T1/2), the expression levels of the BMPR-IA gene and Smad4 protein as well as phosphorylated Smad1/5/8 were detected by Western blotting. In addition, the alkaline phosphatase (ALP) and osteocalcin (OC) activity of osteogenesis specificity protein was assessed using the ALP quantitation and osteocalcin radioimmunoassay kit, respectively. The 4A > C and the -349C > T polymorphisms of BMPR-IA gene were significantly associated with the development of OPLL in the cervical spine. The C allele type in 4A > C polymorphism significantly increases the occurrence and the extent of OPLL. The T allele type in -349C > T polymorphism significantly increases the susceptibility to OPLL, but not the extent of OPLL. The current results further validate our previous observations. The expression levels of BMPR-IA gene were significantly increased in pcDNA3.1/BMPR-IA (mutation type, MT -349C > T; MT 4A > C; MT -349C > T and 4A > C) vector-transfected C3H10T1/2 cells compared to the wild type (WT) vector-transfected cells. The levels of phosphorylated Smad1/5/8 and ALP activity were significantly increased in pcDNA3.1/BMPR-IA (MT -349C > T) vector-transfected C3H10T1/2 cells compared to the WT vector-transfected cells. However, no significant differences were observed in the protein levels of phosphorylated Smad1/5/8 and the ALP activity between MT A/C and WT vector

  14. Moonlighting Proteins and Protein–Protein Interactions as Neurotherapeutic Targets in the G Protein-Coupled Receptor Field

    PubMed Central

    Fuxe, Kjell; Borroto-Escuela, Dasiel O; Romero-Fernandez, Wilber; Palkovits, Miklós; Tarakanov, Alexander O; Ciruela, Francisco; Agnati, Luigi F

    2014-01-01

    There is serious interest in understanding the dynamics of the receptor–receptor and receptor–protein interactions in space and time and their integration in GPCR heteroreceptor complexes of the CNS. Moonlighting proteins are special multifunctional proteins because they perform multiple autonomous, often unrelated, functions without partitioning into different protein domains. Moonlighting through receptor oligomerization can be operationally defined as an allosteric receptor–receptor interaction, which leads to novel functions of at least one receptor protomer. GPCR-mediated signaling is a more complicated process than previously described as every GPCR and GPCR heteroreceptor complex requires a set of G protein interacting proteins, which interacts with the receptor in an orchestrated spatio-temporal fashion. GPCR heteroreceptor complexes with allosteric receptor–receptor interactions operating through the receptor interface have become major integrative centers at the molecular level and their receptor protomers act as moonlighting proteins. The GPCR heteroreceptor complexes in the CNS have become exciting new targets for neurotherapeutics in Parkinson's disease, schizophrenia, drug addiction, and anxiety and depression opening a new field in neuropsychopharmacology. PMID:24105074

  15. Recombinant human bone morphogenetic protein-2 for grade III open segmental tibial fractures from combat injuries in Iraq.

    PubMed

    Kuklo, T R; Groth, A T; Anderson, R C; Frisch, H M; Islinger, R B

    2008-08-01

    This is a retrospective consecutive case series of 138 Gustillo-Anderson type IIIB and IIIC segmental tibial fractures treated at Walter Reed Army Medical Center in soldiers injured in Iraq between March 2003 and March 2005. Five patients with a head injury and four who were lost to follow-up were excluded. The patients were treated definitively with either a ringed external fixator or a reamed intramedullary nail, evaluated in terms of supplementary bone grafting with either autogenous bone (group 1, 67 patients) or recombinant human bone morphogenetic protein-2 at 1.50 mg/ml applied to an absorbable collagen sponge (group 2, 62 patients). The mechanism of injury, defect size and classification, associated injuries, presence of infection, preliminary treatment/fixation, number of procedures before definitive management, time to and details of definitive management, subsequent infection, re-operation, smoking history and other complications were noted. Radiographs were assessed for union, delayed union or nonunion by an independent investigator. All the patients were male. Their mean age was 26.6 years (20 to 42) and the mean follow-up was for 15.6 months (12 to 32). Group 2 had a slightly higher profile of concomitant injuries and a slightly worse fracture classification, but these were not significant. The rate of union was 76% (51 of 67) for group 1 and 92% for group 2 (57 of 62; p = 0.015). There was also a higher rate of subsequent infection in group 1 (14.9%) compared with group 2 (3.2%; p = 0.001) and a higher rate of re-operation (28%) in group 1 (p = 0.003). There were no observed hypersensitivity reactions to the recombinant human bone morphogenetic protein-2 implant.

  16. Gradual downhill running improves age-related skeletal muscle and bone weakness: implication of autophagy and bone morphogenetic proteins.

    PubMed

    Kim, Jeong-Seok; Lee, Young-Hee; Yi, Ho-Keun

    2016-12-01

    What is the central question of this study? Exercise training by running has an effect on age-related muscle and bone wasting that improves physical activity and quality of life in the elderly. However, the effect of downhill running on age-related muscle and bone wasting, and its mechanisms, are unclear. What is the main finding and its importance? Gradual downhill running can improve skeletal muscle growth and bone formation by enhancing autophagy and bone morphogenetic protein signalling in aged rats. Therefore, downhill running exercise might be a practical intervention to improve skeletal muscle and bone protection in the elderly. Recent evidence suggests that autophagy and the bone morphogenetic protein (BMP) signalling pathway regulate skeletal muscle growth and bone formation in aged rats. However, the effect of downhill running on muscle growth and bone formation is not well understood. Thus, we investigated the effect of downhill and uphill running on age-related muscle and bone weakness. Young and late middle-aged rats were randomly assigned to control groups (young, YC; and late middle-aged, LMC) and two types of running training groups (late middle-aged downhill, LMD; and late middle-aged uphill, LMU). Training was progressively carried out on a treadmill at a speed of 21 m min -1 with a slope of +10 deg for uphill training versus 16 m min -1 with a slope of -16 deg for downhill training, both for 60 min day -1 , 5 days week -1 for 8 weeks. Downhill and uphill training increased autophagy-related protein 5, microtubule-associated protein light chain, Beclin-1 and p62 proteins in aged rats. In addition, superoxide dismutase, haem oxygenase-1 and the BMP signalling pathway were elevated. Phosphorylation of mammalian target of rapamycin and myogenic differentiation were increased significantly in the LMD and LMU groups. Consequently, in the femur, BMP-2, BMP-7 and autophagy molecules were highly expressed in the LMD and LMU groups. These results

  17. The changing world of G protein-coupled receptors: from monomers to dimers and receptor mosaics with allosteric receptor-receptor interactions.

    PubMed

    Fuxe, Kjell; Marcellino, Daniel; Borroto-Escuela, Dasiel Oscar; Frankowska, Malgorzata; Ferraro, Luca; Guidolin, Diego; Ciruela, Francisco; Agnati, Luigi F

    2010-10-01

    Based on indications of direct physical interactions between neuropeptide and monoamine receptors in the early 1980s, the term receptor-receptor interactions was introduced and later on the term receptor heteromerization in the early 1990s. Allosteric mechanisms allow an integrative activity to emerge either intramolecularly in G protein-coupled receptor (GPCR) monomers or intermolecularly via receptor-receptor interactions in GPCR homodimers, heterodimers, and receptor mosaics. Stable heteromers of Class A receptors may be formed that involve strong high energy arginine-phosphate electrostatic interactions. These receptor-receptor interactions markedly increase the repertoire of GPCR recognition, signaling and trafficking in which the minimal signaling unit in the GPCR homomers appears to be one receptor and one G protein. GPCR homomers and GPCR assemblies are not isolated but also directly interact with other proteins to form horizontal molecular networks at the plasma membrane.

  18. Differing impact of the deletion of hemochromatosis-associated molecules HFE and transferrin receptor-2 on the iron phenotype of mice lacking bone morphogenetic protein 6 or hemojuvelin.

    PubMed

    Latour, Chloé; Besson-Fournier, Céline; Meynard, Delphine; Silvestri, Laura; Gourbeyre, Ophélie; Aguilar-Martinez, Patricia; Schmidt, Paul J; Fleming, Mark D; Roth, Marie-Paule; Coppin, Hélène

    2016-01-01

    Hereditary hemochromatosis, which is characterized by inappropriately low levels of hepcidin, increased dietary iron uptake, and systemic iron accumulation, has been associated with mutations in the HFE, transferrin receptor-2 (TfR2), and hemojuvelin (HJV) genes. However, it is still not clear whether these molecules intersect in vivo with bone morphogenetic protein 6 (BMP6)/mothers against decapentaplegic (SMAD) homolog signaling, the main pathway up-regulating hepcidin expression in response to elevated hepatic iron. To answer this question, we produced double knockout mice for Bmp6 and β2-microglobulin (a surrogate for the loss of Hfe) and for Bmp6 and Tfr2, and we compared their phenotype (hepcidin expression, Bmp/Smad signaling, hepatic and extrahepatic tissue iron accumulation) with that of single Bmp6-deficient mice and that of mice deficient for Hjv, alone or in combination with Hfe or Tfr2. Whereas the phenotype of Hjv-deficient females was not affected by loss of Hfe or Tfr2, that of Bmp6-deficient females was considerably worsened, with decreased Smad5 phosphorylation, compared with single Bmp6-deficient mice, further repression of hepcidin gene expression, undetectable serum hepcidin, and massive iron accumulation not only in the liver but also in the pancreas, the heart, and the kidneys. These results show that (1) BMP6 does not require HJV to transduce signal to hepcidin in response to intracellular iron, even if the loss of HJV partly reduces this signal, (2) another BMP ligand can replace BMP6 and significantly induce hepcidin expression in response to extracellular iron, and (3) BMP6 alone is as efficient at inducing hepcidin as the other BMPs in association with the HJV/HFE/TfR2 complex; they provide an explanation for the compensatory effect of BMP6 treatment on the molecular defect underlying Hfe hemochromatosis in mice. © 2015 by the American Association for the Study of Liver Diseases.

  19. Genetic analyses of bone morphogenetic protein 2, 4 and 7 in congenital combined pituitary hormone deficiency.

    PubMed

    Breitfeld, Jana; Martens, Susanne; Klammt, Jürgen; Schlicke, Marina; Pfäffle, Roland; Krause, Kerstin; Weidle, Kerstin; Schleinitz, Dorit; Stumvoll, Michael; Führer, Dagmar; Kovacs, Peter; Tönjes, Anke

    2013-12-01

    The complex process of development of the pituitary gland is regulated by a number of signalling molecules and transcription factors. Mutations in these factors have been identified in rare cases of congenital hypopituitarism but for most subjects with combined pituitary hormone deficiency (CPHD) genetic causes are unknown. Bone morphogenetic proteins (BMPs) affect induction and growth of the pituitary primordium and thus represent plausible candidates for mutational screening of patients with CPHD. We sequenced BMP2, 4 and 7 in 19 subjects with CPHD. For validation purposes, novel genetic variants were genotyped in 1046 healthy subjects. Additionally, potential functional relevance for most promising variants has been assessed by phylogenetic analyses and prediction of effects on protein structure. Sequencing revealed two novel variants and confirmed 30 previously known polymorphisms and mutations in BMP2, 4 and 7. Although phylogenetic analyses indicated that these variants map within strongly conserved gene regions, there was no direct support for their impact on protein structure when applying predictive bioinformatics tools. A mutation in the BMP4 coding region resulting in an amino acid exchange (p.Arg300Pro) appeared most interesting among the identified variants. Further functional analyses are required to ultimately map the relevance of these novel variants in CPHD.

  20. Genetic analyses of bone morphogenetic protein 2, 4 and 7 in congenital combined pituitary hormone deficiency

    PubMed Central

    2013-01-01

    Background The complex process of development of the pituitary gland is regulated by a number of signalling molecules and transcription factors. Mutations in these factors have been identified in rare cases of congenital hypopituitarism but for most subjects with combined pituitary hormone deficiency (CPHD) genetic causes are unknown. Bone morphogenetic proteins (BMPs) affect induction and growth of the pituitary primordium and thus represent plausible candidates for mutational screening of patients with CPHD. Methods We sequenced BMP2, 4 and 7 in 19 subjects with CPHD. For validation purposes, novel genetic variants were genotyped in 1046 healthy subjects. Additionally, potential functional relevance for most promising variants has been assessed by phylogenetic analyses and prediction of effects on protein structure. Results Sequencing revealed two novel variants and confirmed 30 previously known polymorphisms and mutations in BMP2, 4 and 7. Although phylogenetic analyses indicated that these variants map within strongly conserved gene regions, there was no direct support for their impact on protein structure when applying predictive bioinformatics tools. Conclusions A mutation in the BMP4 coding region resulting in an amino acid exchange (p.Arg300Pro) appeared most interesting among the identified variants. Further functional analyses are required to ultimately map the relevance of these novel variants in CPHD. PMID:24289245

  1. Membrane receptor location defines receptor interaction with signaling proteins in a polarized epithelium.

    PubMed

    Amsler, K; Kuwada, S K

    1999-01-01

    Signal transduction from receptors is mediated by the interaction of activated receptors with proximate downstream signaling proteins. In polarized epithelial cells, the membrane is divided into subdomains: the apical and basolateral membranes. Membrane receptors may be present in one or both subdomains. Using a combination of immunoprecipitation and Western blot analyses, we tested the hypothesis that a tyrosine kinase growth factor receptor, epidermal growth factor receptor (EGFR), interacts with distinct signaling proteins when present at the apical vs. basolateral membrane of a polarized renal epithelial cell. We report here that tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma) was induced only when basolateral EGFR was activated. In contrast, tyrosine phosphorylation of several other signaling proteins was increased by activation of receptor at either surface. All signaling proteins were distributed diffusely throughout the cytoplasm; however, PLC-gamma protein also displayed a concentration at lateral cell borders. These results demonstrate that in polarized epithelial cells the array of signaling pathways initiated by activation of a membrane receptor is defined, at least in part, by the membrane location of the receptor.

  2. Increased Retinal Expression of the Pro-Angiogenic Receptor GPR91 via BMP6 in a Mouse Model of Juvenile Hemochromatosis.

    PubMed

    Arjunan, Pachiappan; Gnanaprakasam, Jaya P; Ananth, Sudha; Romej, Michelle A; Rajalakshmi, Veeranan-Karmegam; Prasad, Puttur D; Martin, Pamela M; Gurusamy, Mariappan; Thangaraju, Muthusamy; Bhutia, Yangzom D; Ganapathy, Vadivel

    2016-04-01

    Hemochromatosis, an iron-overload disease, occurs as adult and juvenile types. Mutations in hemojuvelin (HJV), an iron-regulatory protein and a bone morphogenetic protein (BMP) coreceptor, underlie most of the juvenile type. Hjv(-/-) mice accumulate excess iron in retina and exhibit aberrant vascularization and angiomas. A succinate receptor, GPR91, is pro-angiogenic in retina. We hypothesized that Hjv(-/-) retinas have increased BMP signaling and increased GPR91 expression as the basis of angiomas. Expression of GPR91 was examined by qPCR, immunofluorescence, and Western blot in wild-type and Hjv(-/-) mouse retinas and pRPE cells. Influence of excess iron and BMP6 on GPR91 expression was investigated in ARPE-19 cells, and wild-type and Hjv(-/-) pRPE cells. Succinate was used to activate GPR91 and determine the effects of GPR91 signaling on VEGF expression. Signaling of BMP6 was studied by the expression of Smad1/5/8 and pSmad4, and the BMP-target gene Id1. The interaction of pSmad4 with GPR91 promoter was studied by ChIP. Expression of GPR91 was higher in Hjv(-/-) retinas and RPE than in wild-type counterparts. Unexpectedly, BMP signaling was increased, not decreased, in Hjv(-/-) retinas and RPE. Bone morphogenetic protein 6 induced GPR91 in RPE, suggesting that increased BMP signaling in Hjv(-/-) retinas was likely responsible for GPR91 upregulation. Exposure of RPE to excess iron and succinate as well as BMP6 and succinate increased VEGF expression. Bone morphogenetic protein 6 promoted the interaction of pSmad4 with GPR91 promoter in RPE. G-protein-coupled receptor 91 is a BMP6 target and Hjv deletion enhances BMP signaling in retina, thus underscoring a role for excess iron and hemochromatosis in abnormal retinal vascularization.

  3. Influences of Reduced Expression of Maternal Bone Morphogenetic Protein 2 on Embryonic Development

    PubMed Central

    Singh, Ajeet P.; Castranio, Trisha; Scott, Greg; Guo, Dayong; Harris, Marie A.; Ray, Manas; Harris, Stephan E.; Mishina, Yuji

    2009-01-01

    Bone morphogenetic protein 2 (BMP2) was originally found by its osteoinductive ability, and recent genetic analyses have revealed that it plays critical roles during early embryogenesis, cardiogenesis, decidualization as well as skeletogenesis. During a course of evaluation of the conditional allele for Bmp2, we found that the presence of a neo cassette, a selection marker needed for gene targeting events in embryonic stem cells, in the 3’ untranslated region of exon 3 of Bmp2, reduced the expression levels of Bmp2 both in embryonic and maternal tissues. Some of the embryos that were genotyped as transheterozygous for the floxed allele with the neo cassette over the conventional null allele (fn/−) showed a lethal phenotype including defects in cephalic neural tube closure and ventral abdominal wall closure. Embryos exhibiting these abnormalities were increased when genotypes of the pregnant females were different; when expression levels of Bmp2 in maternal tissues were lower, a larger proportion of fn/− embryos exhibit these abnormalities. These results suggest that the expression levels of Bmp2 together in both in embryonic and maternal tissues influence the normal neural tube closure and body wall closure with different thresholds. PMID:18769073

  4. Increased bone morphogenetic protein signaling contributes to age-related declines in neurogenesis and cognition.

    PubMed

    Meyers, Emily A; Gobeske, Kevin T; Bond, Allison M; Jarrett, Jennifer C; Peng, Chian-Yu; Kessler, John A

    2016-02-01

    Aging is associated with decreased neurogenesis in the hippocampus and diminished hippocampus-dependent cognitive functions. Expression of bone morphogenetic protein 4 (BMP4) increases with age by more than 10-fold in the mouse dentate gyrus while levels of the BMP inhibitor, noggin, decrease. This results in a profound 30-fold increase in phosphorylated-SMAD1/5/8, the effector of canonical BMP signaling. Just as observed in mice, a profound increase in expression of BMP4 is observed in the dentate gyrus of humans with no known cognitive abnormalities. Inhibition of BMP signaling either by overexpression of noggin or transgenic manipulation not only increases neurogenesis in aging mice, but remarkably, is associated with a rescue of cognitive deficits to levels comparable to young mice. Additive benefits are observed when combining inhibition of BMP signaling and environmental enrichment. These findings indicate that increased BMP signaling contributes significantly to impairments in neurogenesis and to cognitive decline associated with aging, and identify this pathway as a potential druggable target for reversing age-related changes in cognition. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. The evaluation of lyophilized polymer matrices for administering recombinant human bone morphogenetic protein-2.

    PubMed

    Duggirala, S S; Rodgers, J B; DeLuca, P P

    1996-07-01

    Novel unitary devices, prepared by lyophilization of viscous solutions of sodium carboxymethylcellulose (CMC) and methylcellulose (MC), were evaluated as sustained-release delivery systems for recombinant human bone morphogenetic protein-2 (rhBMP-2). In vitro characterization of the unitary devices, which contained rhBMP-2-loaded poly (d,l lactide-co-glycolide) (PLGA) bioerodible particles (BEPs), was conducted over a 2-month period. Determinations included buffer uptake, mass and molecular weight loss and rhBMP-2 release from the unitary devices. CMC devices imbibed approximately 16 times their weight of buffer, while with MC, equilibrium uptake was approximately 6 times the dry weight of the devices. Overall mass loss percentages were approximately 55 and 35%, respectively, for CMC and MC devices. rhBMP-2 release from the devices was essentially a triphasic process: an initial phase during which "free" protein (rhBMP-2 present on the surface and within the pores of the PLGA BEPs) was released, a lag period during which no release was discerned, and then release of "bound" rhBMP-2 (protein adsorbed to the BEPs). The release of bound protein correlated with the mass loss of the polymer which began after 3 weeks. Release from the unitary devices was lower than that from the BEPs alone, due to a retardation effect of the gelled CMC/MC polymers. In rabbits in which full-thickness cranial bone defects were created, the implants were well tolerated and induced significant new bone growth during an 8-week evaluation period. The CMC devices appear to have induced bone earlier (at 2 weeks), but this did not affect eventual 8-week results. CMC devices without rhBMP-2 appeared to provide some bone conduction, in contrast to the blank MC devices.

  6. Bone morphogenetic protein 9 regulates tumor growth of osteosarcoma cells through the Wnt/β-catenin pathway.

    PubMed

    Lv, Zilan; Wang, Chuan; Yuan, Taixian; Liu, Yuehong; Song, Tao; Liu, Yueliang; Chen, Chu; Yang, Min; Tang, Zuchuan; Shi, Qiong; Weng, Yaguang

    2014-02-01

    Bone morphogenetic protein 9 (BMP9) is a member of the transforming growth factor-β (TGF-β) family, which has been shown to regulate the progression of several tumors. Recent studies indicated that BMP9 affects osteosarcoma (OS) processes, but its specific roles and molecular mechanisms have yet to be fully elucidated. The human OS cell lines 143B and MG63 were used for the present study. We found that BMP9 overexpression suppressed the growth of OS cells, whereas inhibition of BMP9 reversed this effect. Our results also showed that BMP9 overexpression induced G0/G1 phase arrest and apoptosis in OS cells. We further investigated the possible molecular mechanisms mediating the biological role of BMP9. We observed that BMP9 overexpression reduced β-catenin mRNA and protein levels, and also downregulated its downstream proteins c-Myc and osteoprotegerin (OPG) and inhibited the phosphorylation levels of GSK-3β (Ser 9) in OS cells, whereas inhibition of BMP9 reversed these effects. Moreover, the suppressive effects of BMP9 overexpression on OS cells was reversed by exogenous β-catenin expression, but augmented by β-catenin silencing. In conclusion, our results revealed that BMP9 can regulate tumor growth of OS cells through the Wnt/β-catenin pathway. Therefore, BMP9 may be a new therapeutic target in OS.

  7. The Role of Endocytosis during Morphogenetic Signaling

    PubMed Central

    Gonzalez-Gaitan, Marcos; Jülicher, Frank

    2014-01-01

    Morphogens are signaling molecules that are secreted by a localized source and spread in a target tissue where they are involved in the regulation of growth and patterning. Both the activity of morphogenetic signaling and the kinetics of ligand spreading in a tissue depend on endocytosis and intracellular trafficking. Here, we review quantitative approaches to study how large-scale morphogen profiles and signals emerge in a tissue from cellular trafficking processes and endocytic pathways. Starting from the kinetics of endosomal networks, we discuss the role of cellular trafficking and receptor dynamics in the formation of morphogen gradients. These morphogen gradients scale during growth, which implies that overall tissue size influences cellular trafficking kinetics. Finally, we discuss how such morphogen profiles can be used to control tissue growth. We emphasize the role of theory in efforts to bridge between scales. PMID:24984777

  8. G Protein-coupled Receptor Kinases of the GRK4 Protein Subfamily Phosphorylate Inactive G Protein-coupled Receptors (GPCRs).

    PubMed

    Li, Lingyong; Homan, Kristoff T; Vishnivetskiy, Sergey A; Manglik, Aashish; Tesmer, John J G; Gurevich, Vsevolod V; Gurevich, Eugenia V

    2015-04-24

    G protein-coupled receptor (GPCR) kinases (GRKs) play a key role in homologous desensitization of GPCRs. It is widely assumed that most GRKs selectively phosphorylate only active GPCRs. Here, we show that although this seems to be the case for the GRK2/3 subfamily, GRK5/6 effectively phosphorylate inactive forms of several GPCRs, including β2-adrenergic and M2 muscarinic receptors, which are commonly used as representative models for GPCRs. Agonist-independent GPCR phosphorylation cannot be explained by constitutive activity of the receptor or membrane association of the GRK, suggesting that it is an inherent ability of GRK5/6. Importantly, phosphorylation of the inactive β2-adrenergic receptor enhanced its interactions with arrestins. Arrestin-3 was able to discriminate between phosphorylation of the same receptor by GRK2 and GRK5, demonstrating preference for the latter. Arrestin recruitment to inactive phosphorylated GPCRs suggests that not only agonist activation but also the complement of GRKs in the cell regulate formation of the arrestin-receptor complex and thereby G protein-independent signaling. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Bone morphogenetic protein antagonist gene NOG is involved in myeloproliferative disease associated with myelofibrosis.

    PubMed

    Andrieux, Joris; Roche-Lestienne, Catherine; Geffroy, Sandrine; Desterke, Christophe; Grardel, Nathalie; Plantier, Isabelle; Selleslag, Dominik; Demory, Jean-Loup; Laï, Jean-Luc; Leleu, Xavier; Le Bousse-Kerdiles, Caroline; Vandenberghe, Peter

    2007-10-01

    In a case with secondary myelofibrosis occurring after essential thrombocythemia, cytogenetic analysis revealed an isolated translocation t(X;17)(q27;q22) in all cells. We found that a bacterial artificial chromosome (BAC) encompassing the breakpoint on chromosome 17 long arm contained only one gene, NOG. We therefore investigated the occurrence of this rare breakpoint in myeloproliferative disorders (MPDs). We identified three more patients with a 17q abnormality in MPDs: myelofibrosis with myeloid metaplasia (MMM); chronic myeloid leukemia positive for t(9;22)(q34;q11) with additional t(4;17)(p15;q22) at diagnosis; and myelofibrosis complicating polycythemia vera. All three cases exhibited a split of BACs containing NOG. The protein encoded by NOG, noggin, acts as an antagonist to bone morphogenetic secreted protein 2 and 4 (BMP2 and BMP4). A comparative analysis of gene expression on Agilent 22K oligonucleotide microarrays in purified CD34+ cells from the blood of MMM patients showed significant downregulation of BMPR2, BMPR1B, BMP2, and BMP8; upregulation of BMP3 and BMP10; and a trend to lower expression of NOG. Thus, given that expression and release of BMPs are important in the induction of osteosclerosis and angiogenic activity, the observed BMP deregulations could be triggered by potential NOG genetic alterations in the four cases here described, and may contribute to the myelofibrotic process characterized by bone marrow stromal reaction including collagen fibrosis, osteosclerosis, and angiogenesis.

  10. Bone morphogenetic protein 7 (BMP-7) influences tendon-bone integration in vitro.

    PubMed

    Schwarting, Tim; Lechler, Philipp; Struewer, Johannes; Ambrock, Marius; Frangen, Thomas Manfred; Ruchholtz, Steffen; Ziring, Ewgeni; Frink, Michael

    2015-01-01

    Successful graft ingrowth following reconstruction of the anterior cruciate ligament is governed by complex biological processes at the tendon-bone interface. The aim of this study was to investigate in an in vitro study the effects of bone morphogenetic protein 7 (BMP-7) on tendon-bone integration. To study the biological effects of BMP-7 on the process of tendon-bone-integration, two independent in vitro models were used. The first model involved the mono- and coculture of bovine tendon specimens and primary bovine osteoblasts with and without BMP-7 exposure. The second model comprised the mono- and coculture of primary bovine osteoblasts and fibroblasts. Alkaline phosphatase (ALP), lactate dehydrogenase (LDH), lactate and osteocalcin (OCN) were analyzed by ELISA. Histological analysis and electron microscopy of the tendon specimens were performed. In both models, positive effects of BMP-7 on ALP enzyme activity were observed (p<0.001). Additionally, similar results were noted for LDH activity and lactate concentration. BMP-7 stimulation led to a significant increase in OCN expression. Whereas the effects of BMP-7 on tendon monoculture peaked during an early phase of the experiment (p<0.001), the cocultures showed a maximal increase during the later stages (p<0.001). The histological analysis showed a stimulating effect of BMP-7 on extracellular matrix formation. Organized ossification zones and calcium carbonate-like structures were only observed in the BMP-stimulated cell cultures. This study showed the positive effects of BMP-7 on the biological process of tendon-bone integration in vitro. Histological signs of improved mineralization were paralleled by increased rates of osteoblast-specific protein levels in primary bovine osteoblasts and fibroblasts. Our findings indicated a role for BMP-7 as an adjuvant therapeutic agent in the treatment of ligamentous injuries, and they emphasized the importance of the transdifferentiation process of tendinous fibroblasts

  11. Ionotropic glutamate receptors: regulation by G-protein-coupled receptors.

    PubMed

    Rojas, Asheebo; Dingledine, Raymond

    2013-04-01

    The function of many ion channels is under dynamic control by coincident activation of G-protein-coupled receptors (GPCRs), particularly those coupled to the Gαs and Gαq family members. Such regulation is typically dependent on the subunit composition of the ionotropic receptor or channel as well as the GPCR subtype and the cell-specific panoply of signaling pathways available. Because GPCRs and ion channels are so highly represented among targets of U.S. Food and Drug Administration-approved drugs, functional cross-talk between these drug target classes is likely to underlie many therapeutic and adverse effects of marketed drugs. GPCRs engage a myriad of signaling pathways that involve protein kinases A and C (PKC) and, through PKC and interaction with β-arrestin, Src kinase, and hence the mitogen-activated-protein-kinase cascades. We focus here on the control of ionotropic glutamate receptor function by GPCR signaling because this form of regulation can influence the strength of synaptic plasticity. The amino acid residues phosphorylated by specific kinases have been securely identified in many ionotropic glutamate (iGlu) receptor subunits, but which of these sites are GPCR targets is less well known even when the kinase has been identified. N-methyl-d-aspartate, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and heteromeric kainate receptors are all downstream targets of GPCR signaling pathways. The details of GPCR-iGlu receptor cross-talk should inform a better understanding of how synaptic transmission is regulated and lead to new therapeutic strategies for neuropsychiatric disorders.

  12. Exaggerated inflammatory response after use of recombinant bone morphogenetic protein in recurrent unicameral bone cysts.

    PubMed

    MacDonald, Kevin M; Swanstrom, Morgan M; McCarthy, James J; Nemeth, Blaise A; Guliani, Teresa A; Noonan, Kenneth J

    2010-03-01

    Recurrent unicameral bone cysts (UBCs) can result in significant morbidity during a child's physical and emotional development. Multiple treatment options are available and a review of the literature fails to clearly define the optimal treatment for UBCs. Recombinant bone morphogenetic protein (BMP) has been used with success in other disorders of poor bone formation. This manuscript is the first to report on the use of recombinant BMP in the treatment of UBCs. Three patients with recurrent UBCs underwent revision surgery with recombinant BMP. Radiographic and medical review was performed and is reported here. In these patients, the use of BMP failed to fully resolve their UBC; 2 patients had complete recurrence that required further surgery. In addition to poor radiographic results, all patients developed exaggerated inflammatory responses in the acute postoperative period. Each child developed clinically significant limb swelling and pain that mimicked infection. On the basis of our poor radiographic results and a paradoxical clinical result, we no longer recommend the use of recombinant BMP in the manner reported here for the treatment of recurrent UBCs. Level IV, case series.

  13. The morphogenetic MreBCD proteins of Escherichia coli form an essential membrane-bound complex.

    PubMed

    Kruse, Thomas; Bork-Jensen, Jette; Gerdes, Kenn

    2005-01-01

    MreB proteins of Escherichia coli, Bacillus subtilis and Caulobacter crescentus form actin-like cables lying beneath the cell surface. The cables are required to guide longitudinal cell wall synthesis and their absence leads to merodiploid spherical and inflated cells prone to cell lysis. In B. subtilis and C. crescentus, the mreB gene is essential. However, in E. coli, mreB was inferred not to be essential. Using a tight, conditional gene depletion system, we systematically investigated whether the E. coli mreBCD-encoded components were essential. We found that cells depleted of mreBCD became spherical, enlarged and finally lysed. Depletion of each mre gene separately conferred similar gross changes in cell morphology and viability. Thus, the three proteins encoded by mreBCD are all essential and function in the same morphogenetic pathway. Interestingly, the presence of a multicopy plasmid carrying the ftsQAZ genes suppressed the lethality of deletions in the mre operon. Using GFP and cell fractionation methods, we showed that the MreC and MreD proteins were associated with the cell membrane. Using a bacterial two-hybrid system, we found that MreC interacted with both MreB and MreD. In contrast, MreB and MreD did not interact in this assay. Thus, we conclude that the E. coli MreBCD form an essential membrane-bound complex. Curiously, MreB did not form cables in cell depleted for MreC, MreD or RodA, indicating a mutual interdependency between MreB filament morphology and cell shape. Based on these and other observations we propose a model in which the membrane-associated MreBCD complex directs longitudinal cell wall synthesis in a process essential to maintain cell morphology.

  14. Physiological exercise loading suppresses post-traumatic osteoarthritis progression via an increase in bone morphogenetic proteins expression in an experimental rat knee model.

    PubMed

    Iijima, H; Ito, A; Nagai, M; Tajino, J; Yamaguchi, S; Kiyan, W; Nakahata, A; Zhang, J; Wang, T; Aoyama, T; Nishitani, K; Kuroki, H

    2017-06-01

    To evaluate the dose-response relationship of exercise loading in the cartilage-subchondral bone (SB) unit in surgically-induced post-traumatic osteoarthritis (PTOA) of the knee. Destabilized medial meniscus (DMM) surgery was performed on the right knee of 12-week-old male Wistar rats, and sham surgery was performed on the contralateral knee. Four weeks after the surgery, the animals were subjected to moderate (12 m/min) or intense (21 m/min) treadmill exercises for 30 min/day, 5 days/week for 4 weeks. PTOA development in articular cartilage and SB was examined using histological and immunohistochemical analyses, micro-computed tomography (micro-CT) analysis, and biomechanical testing at 8 weeks after surgery. Gremlin-1 was injected to determine the role of bone morphogenetic protein (BMP) signaling on PTOA development following moderate exercise. Moderate exercise increased BMP-2, BMP-4, BMP-6, BMP receptor 2, pSmad-5, and inhibitor of DNA binding protein-1 expression in the superficial zone chondrocytes and suppressed cartilage degeneration, osteophyte growth, SB damage, and osteoclast-mediated SB resorption. However, intense exercise had little effect on BMP expression and even caused progression of these osteoarthritis (OA) changes. Gremlin-1 injection following moderate exercise caused progression of the PTOA development down to the level of the non-exercise DMM-operated knee. Exercise regulated cartilage-SB PTOA development in DMM-operated knees in a dose-dependent manner. Our findings shed light on the important role of BMP expression in superficial zone chondrocytes in attenuation of PTOA development following physiological exercise loading. Further studies to support a mechanism by which BMPs would be beneficial in preventing PTOA progression are warranted. Copyright © 2016 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

  15. Dexamethasone Enhances Osteogenic Differentiation of Bone Marrow- and Muscle-Derived Stromal Cells and Augments Ectopic Bone Formation Induced by Bone Morphogenetic Protein-2

    PubMed Central

    Yuasa, Masato; Yamada, Tsuyoshi; Taniyama, Takashi; Masaoka, Tomokazu; Xuetao, Wei; Yoshii, Toshitaka; Horie, Masaki; Yasuda, Hiroaki; Uemura, Toshimasa; Okawa, Atsushi; Sotome, Shinichi

    2015-01-01

    We evaluated whether dexamethasone augments the osteogenic capability of bone marrow-derived stromal cells (BMSCs) and muscle tissue-derived stromal cells (MuSCs), both of which are thought to contribute to ectopic bone formation induced by bone morphogenetic protein-2 (BMP-2), and determined the underlying mechanisms. Rat BMSCs and MuSCs were cultured in growth media with or without 10-7 M dexamethasone and then differentiated under osteogenic conditions with dexamethasone and BMP-2. The effects of dexamethasone on cell proliferation and osteogenic differentiation, and also on ectopic bone formation induced by BMP-2, were analyzed. Dexamethasone affected not only the proliferation rate but also the subpopulation composition of BMSCs and MuSCs, and subsequently augmented their osteogenic capacity during osteogenic differentiation. During osteogenic induction by BMP-2, dexamethasone also markedly affected cell proliferation in both BMSCs and MuSCs. In an in vivo ectopic bone formation model, bone formation in muscle-implanted scaffolds containing dexamethasone and BMP-2 was more than two fold higher than that in scaffolds containing BMP-2 alone. Our results suggest that dexamethasone potently enhances the osteogenic capability of BMP-2 and may thus decrease the quantity of BMP-2 required for clinical application, thereby reducing the complications caused by excessive doses of BMP-2. Highlights: 1. Dexamethasone induced selective proliferation of bone marrow- and muscle-derived cells with higher differentiation potential. 2. Dexamethasone enhanced the osteogenic capability of bone marrow- and muscle-derived cells by altering the subpopulation composition. 3. Dexamethasone augmented ectopic bone formation induced by bone morphogenetic protein-2. PMID:25659106

  16. Colloid, adhesive and release properties of nanoparticular ternary complexes between cationic and anionic polysaccharides and basic proteins like bone morphogenetic protein BMP-2.

    PubMed

    Petzold, R; Vehlow, D; Urban, B; Grab, A L; Cavalcanti-Adam, E A; Alt, V; Müller, M

    2017-03-01

    Herein we describe an interfacial local drug delivery system for bone morphogenetic protein 2 (BMP-2) based on coatings of polyelectrolyte complex (PEC) nanoparticles (NP). The application horizon is the functionalization of bone substituting materials (BSM) used for the therapy of systemic bone diseases. Nanoparticular ternary complexes of cationic and anionic polysaccharides and BMP-2 or two further model proteins, respectively, were prepared in dependence of the molar mixing ratio, pH value and of the cationic polysaccharide. As further proteins chymotrypsin (CHY) and papain (PAP) were selected, which served as model proteins for BMP-2 due to similar isoelectric points and molecular weights. As charged polysaccharides ethylenediamine modified cellulose (EDAC) and trimethylammonium modified cellulose (PQ10) were combined with cellulose sulphatesulfate (CS). Mixing diluted cationic and anionic polysaccharide and protein solutions according to a slight either anionic or cationic excess charge colloidal ternary dispersions formed, which were cast onto germanium model substrates by water evaporation. Dynamic light scattering (DLS) demonstrated, that these dispersions were colloidally stable for at least one week. Fourier Transform Infrared (FTIR) showed, that the cast protein loaded PEC NP coatings were irreversibly adhesive at the model substrate in contact to HEPES buffer and solely CHY, PAP and BMP-2 were released within long-term time scale. Advantageously, out of the three proteins BMP-2 showed the smallest initial burst and the slowest release kinetics and around 25% of the initial BMP-2 content were released within 14days. Released BMP-2 showed significant activity in the myoblast cells indicating the ability to regulate the formation of new bone. Therefore, BMP-2 loaded PEC NP are suggested as novel promising tool for the functionalization of BSM used for the therapy of systemic bone diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Injectable chitosan microparticles incorporating bone morphogenetic protein-7 for bone tissue regeneration

    PubMed Central

    Mantripragada, Venkata P.; Jayasuriya, Ambalangodage C.

    2014-01-01

    This study investigates the influence of the controlled release of bone morphogenetic protein 7 (BMP-7) from cross-linked chitosan microparticles on pre-osteoblasts (OB-6) in vitro. BMP-7 was incorporated into microparticles by encapsulation during the particle preparation and coating after particle preparation. Chitosan microparticles had an average diameter of 700 μm containing ~100 ng of BMP-7. The release study profile indicates that nearly 98% of the BMP-7 coated on the microparticles was released in a period of 18 days while only 36% of the BMP-7 encapsulated in the microparticles was released in the same time period. Cell attachment study indicated that the BMP-7 coated microparticles have many cells adhered on the microparticles in comparison with microparticles without growth factors on day 10. DNA assay indicated a statistical significant increase (p<0.05) in the amount of DNA obtained from BMP-7 encapsulated and coated microparticles in comparison with microparticles without any growth factors. A real time RT-PCR experiment was performed to determine the expression of a few osteoblast specific genes - Dlx5, runx2, osterix, osteopontin, osteocalcin, and bone sialoprotein. The results thus suggest that chitosan microparticles obtained by coacervation method are biocompatible and helps in improving the encapsulation efficiency of BMP-7. Also BMP-7 incorporated in the microparticles is being released in a controlled fashion to support attachment, proliferation and differentiation of pre-osteoblasts, thus acting as a good scaffold for bone tissue regeneration. PMID:24497318

  18. Bone morphogenetic protein signaling is impaired in an Hfe knockout mouse model of hemochromatosis

    PubMed Central

    Corradini, Elena; Garuti, Cinzia; Montosi, Giuliana; Ventura, Paolo; Andriopoulos, Billy; Lin, Herbert Y.; Pietrangelo, Antonello; Babitt, Jodie L.

    2009-01-01

    Background and Aims Mutations in HFE are the most common cause of the iron-overload disorder hereditary hemochromatosis (HH). Levels of the main iron regulatory hormone, hepcidin, are inappropriately low in HH mouse models and patients with HFE mutations, indicating that HFE regulates hepcidin. The bone morphogenetic protein 6 (BMP6)-SMAD signaling pathway is an important endogenous regulator of hepcidin expression. We investigated whether HFE is involved in BMP6-SMAD regulation of hepcidin expression. Methods The BMP6-SMAD pathway was examined in Hfe knockout (KO) mice and in wild-type (WT) mice as controls. Mice were placed on diets of varying iron content. Hepcidin induction by BMP6 was examined in primary hepatocytes from Hfe KO mice; data were compared with those of WT mice. Results Liver levels of Bmp6 mRNA were higher in Hfe KO mice; these were appropriate for the increased hepatic levels of iron in these mice, compared with WT mice. However, levels of hepatic phosphorylated Smad 1/5/8 protein (an intracellular mediator of Bmp6 signaling) and Id1 mRNA (a target gene of Bmp6) were inappropriately low for the body iron burden and Bmp6 mRNA levels in Hfe KO, compared with WT mice. BMP6 induction of hepcidin expression was reduced in Hfe KO hepatocytes compared with WT hepatocytes. Conclusions HFE is not involved in regulation of BMP6 by iron, but does regulate the downstream signals of BMP6 that are triggered by iron. PMID:19591830

  19. Receptor recruitment: A mechanism for interactions between G protein-coupled receptors

    PubMed Central

    Holtbäck, Ulla; Brismar, Hjalmar; DiBona, Gerald F.; Fu, Michael; Greengard, Paul; Aperia, Anita

    1999-01-01

    There is a great deal of evidence for synergistic interactions between G protein-coupled signal transduction pathways in various tissues. As two specific examples, the potent effects of the biogenic amines norepinephrine and dopamine on sodium transporters and natriuresis can be modulated by neuropeptide Y and atrial natriuretic peptide, respectively. Here, we report, using a renal epithelial cell line, that both types of modulation involve recruitment of receptors from the interior of the cell to the plasma membrane. The results indicate that recruitment of G protein-coupled receptors may be a ubiquitous mechanism for receptor sensitization and may play a role in the modulation of signal transduction comparable to that of the well established phenomenon of receptor endocytosis and desensitization. PMID:10377404

  20. Metabotropic Glutamate Receptors and Interacting Proteins in Epileptogenesis

    PubMed Central

    Qian, Feng; Tang, Feng-Ru

    2016-01-01

    Neurotransmitter and receptor systems are involved in different neurological and neuropsychological disorders such as Parkinson's disease, depression, Alzheimer’s disease and epilepsy. Recent advances in studies of signal transduction pathways or interacting proteins of neurotransmitter receptor systems suggest that different receptor systems may share the common signal transduction pathways or interacting proteins which may be better therapeutic targets for development of drugs to effectively control brain diseases. In this paper, we reviewed metabotropic glutamate receptors (mGluRs) and their related signal transduction pathways or interacting proteins in status epilepticus and temporal lobe epilepsy, and proposed some novel therapeutical drug targets for controlling epilepsy and epileptogenesis. PMID:27030135

  1. Increased Retinal Expression of the Pro-Angiogenic Receptor GPR91 via BMP6 in a Mouse Model of Juvenile Hemochromatosis

    PubMed Central

    Arjunan, Pachiappan; Gnanaprakasam, Jaya P.; Ananth, Sudha; Romej, Michelle A.; Rajalakshmi, Veeranan-Karmegam; Prasad, Puttur D.; Martin, Pamela M.; Gurusamy, Mariappan; Thangaraju, Muthusamy; Bhutia, Yangzom D.; Ganapathy, Vadivel

    2016-01-01

    Purpose Hemochromatosis, an iron-overload disease, occurs as adult and juvenile types. Mutations in hemojuvelin (HJV), an iron-regulatory protein and a bone morphogenetic protein (BMP) coreceptor, underlie most of the juvenile type. Hjv−/− mice accumulate excess iron in retina and exhibit aberrant vascularization and angiomas. A succinate receptor, GPR91, is pro-angiogenic in retina. We hypothesized that Hjv−/− retinas have increased BMP signaling and increased GPR91 expression as the basis of angiomas. Methods Expression of GPR91 was examined by qPCR, immunofluorescence, and Western blot in wild-type and Hjv−/− mouse retinas and pRPE cells. Influence of excess iron and BMP6 on GPR91 expression was investigated in ARPE-19 cells, and wild-type and Hjv−/− pRPE cells. Succinate was used to activate GPR91 and determine the effects of GPR91 signaling on VEGF expression. Signaling of BMP6 was studied by the expression of Smad1/5/8 and pSmad4, and the BMP-target gene Id1. The interaction of pSmad4 with GPR91 promoter was studied by ChIP. Results Expression of GPR91 was higher in Hjv−/− retinas and RPE than in wild-type counterparts. Unexpectedly, BMP signaling was increased, not decreased, in Hjv−/− retinas and RPE. Bone morphogenetic protein 6 induced GPR91 in RPE, suggesting that increased BMP signaling in Hjv−/− retinas was likely responsible for GPR91 upregulation. Exposure of RPE to excess iron and succinate as well as BMP6 and succinate increased VEGF expression. Bone morphogenetic protein 6 promoted the interaction of pSmad4 with GPR91 promoter in RPE. Conclusions G-protein-coupled receptor 91 is a BMP6 target and Hjv deletion enhances BMP signaling in retina, thus underscoring a role for excess iron and hemochromatosis in abnormal retinal vascularization. PMID:27046124

  2. Disruption of Axonal Transport Perturbs Bone Morphogenetic Protein (BMP) - Signaling and Contributes to Synaptic Abnormalities in Two Neurodegenerative Diseases

    PubMed Central

    Kang, Min Jung; Hansen, Timothy J.; Mickiewicz, Monique; Kaczynski, Tadeusz J.; Fye, Samantha; Gunawardena, Shermali

    2014-01-01

    Formation of new synapses or maintenance of existing synapses requires the delivery of synaptic components from the soma to the nerve termini via axonal transport. One pathway that is important in synapse formation, maintenance and function of the Drosophila neuromuscular junction (NMJ) is the bone morphogenetic protein (BMP)-signaling pathway. Here we show that perturbations in axonal transport directly disrupt BMP signaling, as measured by its downstream signal, phospho Mad (p-Mad). We found that components of the BMP pathway genetically interact with both kinesin-1 and dynein motor proteins. Thick vein (TKV) vesicle motility was also perturbed by reductions in kinesin-1 or dynein motors. Interestingly, dynein mutations severely disrupted p-Mad signaling while kinesin-1 mutants showed a mild reduction in p-Mad signal intensity. Similar to mutants in components of the BMP pathway, both kinesin-1 and dynein motor protein mutants also showed synaptic morphological defects. Strikingly TKV motility and p-Mad signaling were disrupted in larvae expressing two human disease proteins; expansions of glutamine repeats (polyQ77) and human amyloid precursor protein (APP) with a familial Alzheimer's disease (AD) mutation (APPswe). Consistent with axonal transport defects, larvae expressing these disease proteins showed accumulations of synaptic proteins along axons and synaptic abnormalities. Taken together our results suggest that similar to the NGF-TrkA signaling endosome, a BMP signaling endosome that directly interacts with molecular motors likely exist. Thus problems in axonal transport occurs early, perturbs BMP signaling, and likely contributes to the synaptic abnormalities observed in these two diseases. PMID:25127478

  3. Increased bone morphogenetic protein 7 signalling in the kidneys of dogs affected with a congenital portosystemic shunt.

    PubMed

    van Dongen, Astrid M; Heuving, Susanne M; Tryfonidou, Marianna A; van Steenbeek, Frank G; Rothuizen, Jan; Penning, Louis C

    2015-05-01

    Dogs with a congenital portosystemic shunt (CPSS) often have enlarged and hyper-filtrating kidneys. Although expression of different growth factors has been well-described in the livers of dogs affected with a CPSS, their expression in the kidneys has yet to be determined. Bone morphogenetic protein 7 (BMP-7), hepatocyte growth factor (HGF) and transforming growth factor (TGF)-β have been implicated in renal development (BMP-7, HGF) or the onset of renal fibrosis (TGF-β). Moreover, BMP-7 and HGF have protective properties in renal fibrosis. In this study, the expression and activity of BMP-7 were investigated in renal biopsies obtained from 13 dogs affected with a CPSS and compared to similar samples from age-matched healthy control dogs. Both quantitative reverse-transcriptase PCR and Western blotting showed up-regulated BMP-7 signalling in kidneys of CPPS-affected dogs. These research findings may help to explain the renal pathology/dysfunction in dogs affected with a CPSS. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. [Effect of chemical air pollution on congenital morphogenetic variants].

    PubMed

    Kotysheva, E N

    2011-01-01

    The paper presents the results of investigating the impact of chemical ambient air pollution on congenital morphogenetic variants (CMVs) in 4-7-year-old children. CMVs have been ascertained to depend on the pattern and level of chemical ambient air pollution. Mild morphogenetic disorders develop in the conceptive, organogenetic, and early fetogenetic periods.

  5. Structural basis for receptor activity-modifying protein-dependent selective peptide recognition by a G protein-coupled receptor

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Booe, Jason M.; Walker, Christopher S.; Barwell, James

    Association of receptor activity-modifying proteins (RAMP1-3) with the G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) enables selective recognition of the peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) that have diverse functions in the cardiovascular and lymphatic systems. How peptides selectively bind GPCR:RAMP complexes is unknown. We report crystal structures of CGRP analog-bound CLR:RAMP1 and AM-bound CLR:RAMP2 extracellular domain heterodimers at 2.5 and 1.8 Å resolutions, respectively. The peptides similarly occupy a shared binding site on CLR with conformations characterized by a β-turn structure near their C termini rather than the α-helical structure common to peptides that bind relatedmore » GPCRs. The RAMPs augment the binding site with distinct contacts to the variable C-terminal peptide residues and elicit subtly different CLR conformations. Lastly, the structures and accompanying pharmacology data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes.« less

  6. Structural basis for receptor activity-modifying protein-dependent selective peptide recognition by a G protein-coupled receptor

    DOE PAGES

    Booe, Jason M.; Walker, Christopher S.; Barwell, James; ...

    2015-05-14

    Association of receptor activity-modifying proteins (RAMP1-3) with the G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) enables selective recognition of the peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) that have diverse functions in the cardiovascular and lymphatic systems. How peptides selectively bind GPCR:RAMP complexes is unknown. We report crystal structures of CGRP analog-bound CLR:RAMP1 and AM-bound CLR:RAMP2 extracellular domain heterodimers at 2.5 and 1.8 Å resolutions, respectively. The peptides similarly occupy a shared binding site on CLR with conformations characterized by a β-turn structure near their C termini rather than the α-helical structure common to peptides that bind relatedmore » GPCRs. The RAMPs augment the binding site with distinct contacts to the variable C-terminal peptide residues and elicit subtly different CLR conformations. Lastly, the structures and accompanying pharmacology data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes.« less

  7. Plant nuclear hormone receptors: a role for small molecules in protein-protein interactions.

    PubMed

    Lumba, Shelley; Cutler, Sean; McCourt, Peter

    2010-01-01

    Plant hormones are a group of chemically diverse small molecules that direct processes ranging from growth and development to biotic and abiotic stress responses. Surprisingly, genome analyses suggest that classic animal nuclear hormone receptor homologs do not exist in plants. It now appears that plants have co-opted several protein families to perceive hormones within the nucleus. In one solution to the problem, the hormones auxin and jasmonate (JA) act as “molecular glue” that promotes protein-protein interactions between receptor F-boxes and downstream corepressor targets. In another solution, gibberellins (GAs) bind and elicit a conformational change in a novel soluble receptor family related to hormone-sensitive lipases. Abscisic acid (ABA), like GA, also acts through an allosteric mechanism involving a START-domain protein. The molecular identification of plant nuclear hormone receptors will allow comparisons with animal nuclear receptors and testing of fundamental questions about hormone function in plant development and evolution.

  8. The G protein-coupled receptors deorphanization landscape.

    PubMed

    Laschet, Céline; Dupuis, Nadine; Hanson, Julien

    2018-07-01

    G protein-coupled receptors (GPCRs) are usually highlighted as being both the largest family of membrane proteins and the most productive source of drug targets. However, most of the GPCRs are understudied and hence cannot be used immediately for innovative therapeutic strategies. Besides, there are still around 100 orphan receptors, with no described endogenous ligand and no clearly defined function. The race to discover new ligands for these elusive receptors seems to be less intense than before. Here, we present an update of the various strategies employed to assign a function to these receptors and to discover new ligands. We focus on the recent advances in the identification of endogenous ligands with a detailed description of newly deorphanized receptors. Replication being a key parameter in these endeavors, we also discuss the latest controversies about problematic ligand-receptor pairings. In this context, we propose several recommendations in order to strengthen the reporting of new ligand-receptor pairs. Copyright © 2018 Elsevier Inc. All rights reserved.

  9. Osteogenic Protein-1 (Bone Morphogenetic Protein-7) in the Treatment of Tibial Nonunions

    PubMed Central

    FRIEDLAENDER, GARY E.; PERRY, CLAYTON R.; DEAN COLE, J.; COOK, STEPHEN D.; CIERNY, GEORGE; MUSCHLER, GEORGE F.; ZYCH, GREGORY A.; CALHOUN, JASON H.; LAFORTE, AMY J.; YIN, SAMUEL

    2005-01-01

    Background: The role of bone morphogenetic proteins (BMPs) in osseous repair has been demonstrated in numerous animal models. Recombinant human osteogenic protein-1 (rhOP-1 or BMP-7) has now been produced and was evaluated in a clinical trial conducted under a Food and Drug Administration approved Investigational Device Exemption to establish both the safety and efficacy of this BMP in the treatment of tibial nonunions. The study also compared the clinical and radiographic results with this osteogenic molecule and those achieved with fresh autogenous bone. Materials and Methods: One hundred and twenty-two patients (with 124 tibial nonunions) were enrolled in a controlled, prospective, randomized, partially blinded, multi-center clinical trial between February, 1992, and August, 1996, and were followed at frequent intervals over 24 months. Each patient was treated by insertion of an intramedullary rod, accompanied by rhOP-1 in a type I collagen carrier or by fresh bone autograft. Assessment criteria included the severity of pain at the fracture site, the ability to walk with full weight-bearing, the need for surgical re-treatment of the nonunion during the course of this study, plain radiographic evaluation of healing, and physician satisfaction with the clinical course. In addition, adverse events were recorded, and sera were screened for antibodies to OP-1 and type-I collagen at each outpatient visit. Results: At 9 months following the operative procedures (the primary end-point of this study), 81% of the OP-1-treated nonunions (n = 63) and 85% of those receiving autogenous bone (n = 61) were judged by clinical criteria to have been treated successfully (p = 0.524). By radiographic criteria, at this same time point, 75% of those in the OP-1-treated group and 84% of the autograft-treated patients had healed fractures (p = 0.218). These clinical results continued at similar levels of success throughout 2 years of observation, and there was no statistically

  10. Endocytosis and membrane receptor internalization: implication of F-BAR protein Carom

    PubMed Central

    Xu, Yanjie; Liu, Suxuan; Xia, Jixiang; Stein, Sam; Ramon, Cueto; Xi, Hang; Wang, Luqiao; Xiong, Xinyu; Zhang, Lixiao; He, Dingwen; Yang, William; Zhao, Xianxian; Cheng, Xiaoshu; Yang, Xiaofeng; Wang, Hong

    2016-01-01

    Endocytosis is a cellular process mostly responsible for membrane receptor internalization. Cell membrane receptors bind to their ligands and form a complex which can be internalized. We previously proposed that F-BAR protein initiates membrane curvature and mediates endocytosis via their binding partners. However, F-BAR protein partners involved in membrane receptor endocytosis and the regulatory mechanism remain unknown. In this study, we established a group of database mining strategies to explore mechanisms underlying receptor-related endocytosis. We identified 34 endocytic membrane receptors and 10 regulating proteins for vesicle formation in clathrin-dependent endocytosis (CDE), a major process of membrane receptor internalization. We found that F-BAR protein FCHSD2 (Carom) may facilitate endocytosis via 9 endocytic partners. Carom is highly expressed, along with highly expressed endocytic membrane receptors and partners, in endothelial cells and macrophages. We established 3 models of Carom-receptor complex and their intracellular trafficking based on protein-protein interaction and subcellular localization. We conclude that Carom may mediate receptor endocytosis and transport endocytic receptors to the cytoplasm for receptor signaling and lysosome/proteasome degradation, or to the nucleus for RNA processing, gene transcription and DNA repair. PMID:28199211

  11. Collagenase-3 binds to a specific receptor and requires the low density lipoprotein receptor-related protein for internalization

    NASA Technical Reports Server (NTRS)

    Barmina, O. Y.; Walling, H. W.; Fiacco, G. J.; Freije, J. M.; Lopez-Otin, C.; Jeffrey, J. J.; Partridge, N. C.

    1999-01-01

    We have previously identified a specific receptor for collagenase-3 that mediates the binding, internalization, and degradation of this ligand in UMR 106-01 rat osteoblastic osteosarcoma cells. In the present study, we show that collagenase-3 binding is calcium-dependent and occurs in a variety of cell types, including osteoblastic and fibroblastic cells. We also present evidence supporting a two-step mechanism of collagenase-3 binding and internalization involving both a specific collagenase-3 receptor and the low density lipoprotein receptor-related protein. Ligand blot analysis shows that (125)I-collagenase-3 binds specifically to two proteins ( approximately 170 kDa and approximately 600 kDa) present in UMR 106-01 cells. Western blotting identified the 600-kDa protein as the low density lipoprotein receptor-related protein. Our data suggest that the 170-kDa protein is a specific collagenase-3 receptor. Low density lipoprotein receptor-related protein-null mouse embryo fibroblasts bind but fail to internalize collagenase-3, whereas UMR 106-01 and wild-type mouse embryo fibroblasts bind and internalize collagenase-3. Internalization, but not binding, is inhibited by the 39-kDa receptor-associated protein. We conclude that the internalization of collagenase-3 requires the participation of the low density lipoprotein receptor-related protein and propose a model in which the cell surface interaction of this ligand requires a sequential contribution from two receptors, with the collagenase-3 receptor acting as a high affinity primary binding site and the low density lipoprotein receptor-related protein mediating internalization.

  12. Homeobox protein MSX-1 inhibits expression of bone morphogenetic protein 2, bone morphogenetic protein 4, and lymphoid enhancer-binding factor 1 via Wnt/β-catenin signaling to prevent differentiation of dental mesenchymal cells during the late bell stage.

    PubMed

    Feng, Xiao-Yu; Wu, Xiao-Shan; Wang, Jin-Song; Zhang, Chun-Mei; Wang, Song-Lin

    2018-02-01

    Homeobox protein MSX-1 (hereafter referred to as MSX-1) is essential for early tooth-germ development. Tooth-germ development is arrested at bud stage in Msx1 knockout mice, which prompted us to study the functions of MSX-1 beyond this stage. Here, we investigated the roles of MSX-1 during late bell stage. Mesenchymal cells of the mandibular first molar were isolated from mice at embryonic day (E)17.5 and cultured in vitro. We determined the expression levels of β-catenin, bone morphogenetic protein 2 (Bmp2), Bmp4, and lymphoid enhancer-binding factor 1 (Lef1) after knockdown or overexpression of Msx1. Our findings suggest that knockdown of Msx1 promoted expression of Bmp2, Bmp4, and Lef1, resulting in elevated differentiation of odontoblasts, which was rescued by blocking the expression of these genes. In contrast, overexpression of Msx1 decreased the expression of Bmp2, Bmp4, and Lef1, leading to a reduction in odontoblast differentiation. The regulation of Bmp2, Bmp4, and Lef1 by Msx1 was mediated by the Wnt/β-catenin signaling pathway. Additionally, knockdown of Msx1 impaired cell proliferation and slowed S-phase progression, while overexpression of Msx1 also impaired cell proliferation and prolonged G1-phase progression. We therefore conclude that MSX-1 maintains cell proliferation by regulating transition of cells from G1-phase to S-phase and prevents odontoblast differentiation by inhibiting expression of Bmp2, Bmp4, and Lef1 at the late bell stage via the Wnt/β-catenin signaling pathway. © 2017 Eur J Oral Sci.

  13. Endocytosis and membrane receptor internalization: implication of F-BAR protein Carom.

    PubMed

    Xu, Yanjie; Xia, Jixiang; Liu, Suxuan; Stein, Sam; Ramon, Cueto; Xi, Hang; Wang, Luqiao; Xiong, Xinyu; Zhang, Lixiao; He, Dingwen; Yang, William; Zhao, Xianxian; Cheng, Xiaoshu; Yang, Xiaofeng; Wang, Hong

    2017-03-01

    Endocytosis is a cellular process mostly responsible for membrane receptor internalization. Cell membrane receptors bind to their ligands and form a complex which can be internalized. We previously proposed that F-BAR protein initiates membrane curvature and mediates endocytosis via its binding partners. However, F-BAR protein partners involved in membrane receptor endocytosis and the regulatory mechanism remain unknown. In this study, we established database mining strategies to explore mechanisms underlying receptor-related endocytosis. We identified 34 endocytic membrane receptors and 10 regulating proteins in clathrin-dependent endocytosis (CDE), a major process of membrane receptor internalization. We found that F-BAR protein FCHSD2 (Carom) may facilitate endocytosis via 9 endocytic partners. Carom is highly expressed, along with highly expressed endocytic membrane receptors and partners, in endothelial cells and macrophages. We established 3 models of Carom-receptor complexes and their intracellular trafficking based on protein interaction and subcellular localization. We conclude that Carom may mediate receptor endocytosis and transport endocytic receptors to the cytoplasm for receptor signaling and lysosome/proteasome degradation, or to the nucleus for RNA processing, gene transcription and DNA repair.

  14. Mini G protein probes for active G protein-coupled receptors (GPCRs) in live cells.

    PubMed

    Wan, Qingwen; Okashah, Najeah; Inoue, Asuka; Nehmé, Rony; Carpenter, Byron; Tate, Christopher G; Lambert, Nevin A

    2018-05-11

    G protein-coupled receptors (GPCRs) are key signaling proteins that regulate nearly every aspect of cell function. Studies of GPCRs have benefited greatly from the development of molecular tools to monitor receptor activation and downstream signaling. Here, we show that mini G proteins are robust probes that can be used in a variety of assay formats to report GPCR activity in living cells. Mini G (mG) proteins are engineered GTPase domains of Gα subunits that were developed for structural studies of active-state GPCRs. Confocal imaging revealed that mG proteins fused to fluorescent proteins were located diffusely in the cytoplasm and translocated to sites of receptor activation at the cell surface and at intracellular organelles. Bioluminescence resonance energy transfer (BRET) assays with mG proteins fused to either a fluorescent protein or luciferase reported agonist, superagonist, and inverse agonist activities. Variants of mG proteins (mGs, mGsi, mGsq, and mG12) corresponding to the four families of Gα subunits displayed appropriate coupling to their cognate GPCRs, allowing quantitative profiling of subtype-specific coupling to individual receptors. BRET between luciferase-mG fusion proteins and fluorescent markers indicated the presence of active GPCRs at the plasma membrane, Golgi apparatus, and endosomes. Complementation assays with fragments of NanoLuc luciferase fused to GPCRs and mG proteins reported constitutive receptor activity and agonist-induced activation with up to 20-fold increases in luminescence. We conclude that mG proteins are versatile tools for studying GPCR activation and coupling specificity in cells and should be useful for discovering and characterizing G protein subtype-biased ligands. © 2018 Wan et al.

  15. Intervariability and intravariability of bone morphogenetic proteins in commercially available demineralized bone matrix products.

    PubMed

    Bae, Hyun W; Zhao, Li; Kanim, Linda E A; Wong, Pamela; Delamarter, Rick B; Dawson, Edgar G

    2006-05-20

    Enzyme-linked immunosorbent assay was used to detect bone morphogenetic proteins (BMPs) 2, 4, and 7 in 9 commercially available ("off the shelf") demineralized bone matrix (DBM) product formulations using 3 different manufacturer's production lots of each DBM formulation. To evaluate and compare the quantity of BMPs among several different DBM formulations (inter-product variability), as well as examine the variability of these proteins in different production lots within the same DBM formulation (intra-product variability). DBMs are commonly used to augment available bone graft in spinal fusion procedures. Surgeons are presented with an ever-increasing variety of commercially available human DBMs from which to choose. Yet, there is limited information on a specific DBM product's osteoinductive efficacy, potency, and constancy. There were protein extracts from each DBM sample separately dialyzed 4 times against distilled water at 4 degrees C for 48 hours. The amount of BMP-2, BMP-4, and BMP-7 was determined using enzyme-linked immunosorbent assay. RESULTS.: The concentrations of detected BMP-2 and BMP-7 were low for all DBM formulations, only nanograms of BMP were extracted from each gram of DBM (20.2-120.6 ng BMP-2/g DBM product; 54.2-226.8 ng BMP-7/g DBM). The variability of BMP concentrations among different lots of the same DBM formulation, intra-product variability, was higher than the variability of concentrations among different DBM formulations, inter-product variability (coefficient of variation range BMP-2 [16.34% to 76.01%], P < 0.01; BMP-7 [3.71% to 82.08%], P < 0.001). BMP-4 was undetectable. The relative quantities of BMPs in DBMs are low, in the order of 1 x 10(-9) g of BMP/g of DBM. There is higher variability in concentration of BMPs among 3 different lots of the same DBM formulation than among different DBM formulations. This variability questions DBM products' reliability and, possibly, efficacy in providing consistent osteoinduction.

  16. Oligomerization of G protein-coupled receptors: computational methods.

    PubMed

    Selent, J; Kaczor, A A

    2011-01-01

    Recent research has unveiled the complexity of mechanisms involved in G protein-coupled receptor (GPCR) functioning in which receptor dimerization/oligomerization may play an important role. Although the first high-resolution X-ray structure for a likely functional chemokine receptor dimer has been deposited in the Protein Data Bank, the interactions and mechanisms of dimer formation are not yet fully understood. In this respect, computational methods play a key role for predicting accurate GPCR complexes. This review outlines computational approaches focusing on sequence- and structure-based methodologies as well as discusses their advantages and limitations. Sequence-based approaches that search for possible protein-protein interfaces in GPCR complexes have been applied with success in several studies, but did not yield always consistent results. Structure-based methodologies are a potent complement to sequence-based approaches. For instance, protein-protein docking is a valuable method especially when guided by experimental constraints. Some disadvantages like limited receptor flexibility and non-consideration of the membrane environment have to be taken into account. Molecular dynamics simulation can overcome these drawbacks giving a detailed description of conformational changes in a native-like membrane. Successful prediction of GPCR complexes using computational approaches combined with experimental efforts may help to understand the role of dimeric/oligomeric GPCR complexes for fine-tuning receptor signaling. Moreover, since such GPCR complexes have attracted interest as potential drug target for diverse diseases, unveiling molecular determinants of dimerization/oligomerization can provide important implications for drug discovery.

  17. Follistatin-like 1 (Fstl1) is a bone morphogenetic protein (BMP) 4 signaling antagonist in controlling mouse lung development

    PubMed Central

    Geng, Yan; Dong, Yingying; Yu, Mingyan; Zhang, Long; Yan, Xiaohua; Sun, Jingxia; Qiao, Long; Geng, Huixia; Nakajima, Masahiro; Furuichi, Tatsuya; Ikegawa, Shiro; Gao, Xiang; Chen, Ye-Guang; Jiang, Dianhua; Ning, Wen

    2011-01-01

    Lung morphogenesis is a well orchestrated, tightly regulated process through several molecular pathways, including TGF-β/bone morphogenetic protein (BMP) signaling. Alteration of these signaling pathways leads to lung malformation. We investigated the role of Follistatin-like 1 (Fstl1), a secreted follistatin-module–containing glycoprotein, in lung development. Deletion of Fstl1 in mice led to postnatal lethality as a result of respiratory failure. Analysis of the mutant phenotype showed that Fstl1 is essential for tracheal cartilage formation and alveolar maturation. Deletion of the Fstl1 gene resulted in malformed tracheal rings manifested as discontinued rings and reduced ring number. Fstl1-deficient mice displayed septal hypercellularity and end-expiratory atelectasis, which were associated with impaired differentiation of distal alveolar epithelial cells and insufficient production of mature surfactant proteins. Mechanistically, Fstl1 interacted directly with BMP4, negatively regulated BMP4/Smad1/5/8 signaling, and inhibited BMP4-induced surfactant gene expression. Reducing BMP signaling activity by Noggin rescued pulmonary atelectasis of Fstl1-deficient mice. Therefore, we provide in vivo and in vitro evidence to demonstrate that Fstl1 modulates lung development and alveolar maturation, in part, through BMP4 signaling. PMID:21482757

  18. An emerging link between LIM domain proteins and nuclear receptors.

    PubMed

    Sala, Stefano; Ampe, Christophe

    2018-06-01

    Nuclear receptors are ligand-activated transcription factors that partake in several biological processes including development, reproduction and metabolism. Over the last decade, evidence has accumulated that group 2, 3 and 4 LIM domain proteins, primarily known for their roles in actin cytoskeleton organization, also partake in gene transcription regulation. They shuttle between the cytoplasm and the nucleus, amongst other as a consequence of triggering cells with ligands of nuclear receptors. LIM domain proteins act as important coregulators of nuclear receptor-mediated gene transcription, in which they can either function as coactivators or corepressors. In establishing interactions with nuclear receptors, the LIM domains are important, yet pleiotropy of LIM domain proteins and nuclear receptors frequently occurs. LIM domain protein-nuclear receptor complexes function in diverse physiological processes. Their association is, however, often linked to diseases including cancer.

  19. Receptor Activity-modifying Protein-directed G Protein Signaling Specificity for the Calcitonin Gene-related Peptide Family of Receptors.

    PubMed

    Weston, Cathryn; Winfield, Ian; Harris, Matthew; Hodgson, Rose; Shah, Archna; Dowell, Simon J; Mobarec, Juan Carlos; Woodlock, David A; Reynolds, Christopher A; Poyner, David R; Watkins, Harriet A; Ladds, Graham

    2016-10-14

    The calcitonin gene-related peptide (CGRP) family of G protein-coupled receptors (GPCRs) is formed through the association of the calcitonin receptor-like receptor (CLR) and one of three receptor activity-modifying proteins (RAMPs). Binding of one of the three peptide ligands, CGRP, adrenomedullin (AM), and intermedin/adrenomedullin 2 (AM2), is well known to result in a Gα s -mediated increase in cAMP. Here we used modified yeast strains that couple receptor activation to cell growth, via chimeric yeast/Gα subunits, and HEK-293 cells to characterize the effect of different RAMP and ligand combinations on this pathway. We not only demonstrate functional couplings to both Gα s and Gα q but also identify a Gα i component to CLR signaling in both yeast and HEK-293 cells, which is absent in HEK-293S cells. We show that the CGRP family of receptors displays both ligand- and RAMP-dependent signaling bias among the Gα s , Gα i , and Gα q/11 pathways. The results are discussed in the context of RAMP interactions probed through molecular modeling and molecular dynamics simulations of the RAMP-GPCR-G protein complexes. This study further highlights the importance of RAMPs to CLR pharmacology and to bias in general, as well as identifying the importance of choosing an appropriate model system for the study of GPCR pharmacology. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Mechanism of the G-protein mimetic nanobody binding to a muscarinic G-protein-coupled receptor.

    PubMed

    Miao, Yinglong; McCammon, J Andrew

    2018-03-20

    Protein-protein binding is key in cellular signaling processes. Molecular dynamics (MD) simulations of protein-protein binding, however, are challenging due to limited timescales. In particular, binding of the medically important G-protein-coupled receptors (GPCRs) with intracellular signaling proteins has not been simulated with MD to date. Here, we report a successful simulation of the binding of a G-protein mimetic nanobody to the M 2 muscarinic GPCR using the robust Gaussian accelerated MD (GaMD) method. Through long-timescale GaMD simulations over 4,500 ns, the nanobody was observed to bind the receptor intracellular G-protein-coupling site, with a minimum rmsd of 2.48 Å in the nanobody core domain compared with the X-ray structure. Binding of the nanobody allosterically closed the orthosteric ligand-binding pocket, being consistent with the recent experimental finding. In the absence of nanobody binding, the receptor orthosteric pocket sampled open and fully open conformations. The GaMD simulations revealed two low-energy intermediate states during nanobody binding to the M 2 receptor. The flexible receptor intracellular loops contribute remarkable electrostatic, polar, and hydrophobic residue interactions in recognition and binding of the nanobody. These simulations provided important insights into the mechanism of GPCR-nanobody binding and demonstrated the applicability of GaMD in modeling dynamic protein-protein interactions.

  1. Oestrogen receptor alpha in pulmonary hypertension.

    PubMed

    Wright, Audrey F; Ewart, Marie-Ann; Mair, Kirsty; Nilsen, Margaret; Dempsie, Yvonne; Loughlin, Lynn; Maclean, Margaret R

    2015-05-01

    Pulmonary arterial hypertension (PAH) occurs more frequently in women with mutations in bone morphogenetic protein receptor type 2 (BMPR2) and dysfunctional BMPR2 signalling underpinning heritable PAH. We have previously shown that serotonin can uncover a pulmonary hypertensive phenotype in BMPR2(+/-) mice and that oestrogen can increase serotinergic signalling in human pulmonary arterial smooth muscle cells (hPASMCs). Hence, here we wished to characterize the expression of oestrogen receptors (ERs) in male and female human pulmonary arteries and have examined the influence of oestrogen and serotonin on BMPR2 and ERα expression. By immunohistochemistry, we showed that ERα, ERβ, and G-protein-coupled receptors are expressed in human pulmonary arteries localizing mainly to the smooth muscle layer which also expresses the serotonin transporter (SERT). Protein expression of ERα protein was higher in female PAH patient hPASMCs compared with male and serotonin also increased the expression of ERα. 17β-estradiol induced proliferation of hPASMCs via ERα activation and this engaged mitogen-activated protein kinase and Akt signalling. Female mice over-expressing SERT (SERT(+) mice) develop PH and the ERα antagonist MPP attenuated the development of PH in normoxic and hypoxic female SERT(+) mice. The therapeutic effects of MPP were accompanied by increased expression of BMPR2 in mouse lung. ERα is highly expressed in female hPASMCs from PAH patients and mediates oestrogen-induced proliferation of hPASMCs via mitogen-activated protein kinase and Akt signalling. Serotonin can increase ERα expression in hPASMCs and antagonism of ERα reverses serotonin-dependent PH in the mouse and increases BMPR2 expression. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.

  2. Associations between variants of bone morphogenetic protein 7 gene and growth traits in chickens.

    PubMed

    Wang, Yan; Guo, Fuyou; Qu, Hao; Luo, Chenglong; Wang, Jie; Shu, Dingming

    2018-04-18

    1. Enhancing bone strength to solve leg disorders in poultry has become an important goal in broiler production. 2. Bone morphogenetic protein 7 (BMP7), a member of the BMP family, represents an attractive therapeutic target for bone regeneration in humans and plays critical roles in skeletal development. 3. The objective of this study was to investigate the relationship between BMP7 gene expression, single nucleotide polymorphisms (SNPs) and growth traits in chickens. Here, a SNP (c.1995T>C) in the chicken (Gallus gallus) BMP7 gene was identified, that was associated with growth and carcass traits. 4. Genotyping revealed that the T allele occurred more frequently in breeds with high growth rates, whereas the C allele was predominant in those with low growth rates. The expression level of BMP7 in the thigh bone of birds with the TT genotype was significantly higher than in those with the CC genotype at 21, 42 and 91 days of age. 5. These findings suggest that selecting the birds with the TT genotype of SNP c.1995T>C could improve bone growth, could reduce leg disorders in fast-growing birds. The SNP c.1995T>C may serve as a selective marker for improving bone growth and increasing the consistency of body weights in poultry breeding.

  3. Magnesium modification up-regulates the bioactivity of bone morphogenetic protein-2 upon calcium phosphate cement via enhanced BMP receptor recognition and Smad signaling pathway.

    PubMed

    Ding, Sai; Zhang, Jing; Tian, Yu; Huang, Baolin; Yuan, Yuan; Liu, Changsheng

    2016-09-01

    Efficient presentation of growth factors is one of the great challenges in tissue engineering. In living systems, bioactive factors exist in soluble as well as in matrix-bound forms, both of which play an integral role in regulating cell behaviors. Herein, effect of magnesium on osteogenic bioactivity of recombinant human bone morphogenetic protein-2 (rhBMP-2) was investigated systematically with a series of Mg modified calcium phosphate cements (xMCPCs, x means the content of magnesium phosphate cement wt%) as matrix model. The results indicated that the MCPC, especially 5MCPC, could promote the rhBMP-2-induced in vitro osteogenic differentiation via Smad signaling of C2C12 cells. Further studies demonstrated that all MCPC substrates exhibited similar rhBMP-2 release rate and preserved comparable conformation and biological activity of the released rhBMP-2. Also, the ionic extracts of MCPC made little difference to the bioactivity of rhBMP-2, either in soluble or in matrix-bound forms. However, with the quartz crystal microbalance (QCM), we observed a noticeable enhancement of rhBMP-2 mass-uptake on 5MCPC as well as a better recognition of the bound rhBMP-2 to BMPR IA and BMPR II. In vivo results demonstrated a better bone regeneration capacity of 5MCPC/rhBMP-2. From the above, our results demonstrated that it was the Mg anchored on the underlying substrates that tailored the way of rhBMP-2 bound on MCPC, and thus facilitated the recognition of BMPRs to stimulate osteogenic differentiation. The study will guide the development of Mg-doped bioactive bone implants for tissue regeneration. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Morphogenetic Pathway of Spore Wall Assembly in Saccharomyces cerevisiae

    PubMed Central

    Coluccio, Alison; Bogengruber, Edith; Conrad, Michael N.; Dresser, Michael E.; Briza, Peter; Neiman, Aaron M.

    2004-01-01

    The Saccharomyces cerevisiae spore is protected from environmental damage by a multilaminar extracellular matrix, the spore wall, which is assembled de novo during spore formation. A set of mutants defective in spore wall assembly were identified in a screen for mutations causing sensitivity of spores to ether vapor. The spore wall defects in 10 of these mutants have been characterized in a variety of cytological and biochemical assays. Many of the individual mutants are defective in the assembly of specific layers within the spore wall, leading to arrests at discrete stages of assembly. The localization of several of these gene products has been determined and distinguishes between proteins that likely are involved directly in spore wall assembly and probable regulatory proteins. The results demonstrate that spore wall construction involves a series of dependent steps and provide the outline of a morphogenetic pathway for assembly of a complex extracellular structure. PMID:15590821

  5. Bone Morphogenetic Protein-Induced Msx1 and Msx2 Inhibit Myocardin-Dependent Smooth Muscle Gene Transcription▿

    PubMed Central

    Hayashi, Ken'ichiro; Nakamura, Seiji; Nishida, Wataru; Sobue, Kenji

    2006-01-01

    During the onset and progression of atherosclerosis, the vascular smooth muscle cell (VSMC) phenotype changes from differentiated to dedifferentiated, and in some cases, this change is accompanied by osteogenic transition, resulting in vascular calcification. One characteristic of dedifferentiated VSMCs is the down-regulation of smooth muscle cell (SMC) marker gene expression. Bone morphogenetic proteins (BMPs), which are involved in the induction of osteogenic gene expression, are detected in calcified vasculature. In this study, we found that the BMP2-, BMP4-, and BMP6-induced expression of Msx transcription factors (Msx1 and Msx2) preceded the down-regulation of SMC marker expression in cultured differentiated VSMCs. Either Msx1 or Msx2 markedly reduced the myocardin-dependent promoter activities of SMC marker genes (SM22α and caldesmon). We further investigated interactions between Msx1 and myocardin/serum response factor (SRF)/CArG-box motif (cis element for SRF) using coimmunoprecipitation, gel-shift, and chromatin immunoprecipitation assays. Our results showed that Msx1 or Msx2 formed a ternary complex with SRF and myocardin and inhibited the binding of SRF or SRF/myocardin to the CArG-box motif, resulting in inhibition of their transcription. PMID:17030628

  6. Bone morphogenetic protein-induced MSX1 and MSX2 inhibit myocardin-dependent smooth muscle gene transcription.

    PubMed

    Hayashi, Ken'ichiro; Nakamura, Seiji; Nishida, Wataru; Sobue, Kenji

    2006-12-01

    During the onset and progression of atherosclerosis, the vascular smooth muscle cell (VSMC) phenotype changes from differentiated to dedifferentiated, and in some cases, this change is accompanied by osteogenic transition, resulting in vascular calcification. One characteristic of dedifferentiated VSMCs is the down-regulation of smooth muscle cell (SMC) marker gene expression. Bone morphogenetic proteins (BMPs), which are involved in the induction of osteogenic gene expression, are detected in calcified vasculature. In this study, we found that the BMP2-, BMP4-, and BMP6-induced expression of Msx transcription factors (Msx1 and Msx2) preceded the down-regulation of SMC marker expression in cultured differentiated VSMCs. Either Msx1 or Msx2 markedly reduced the myocardin-dependent promoter activities of SMC marker genes (SM22alpha and caldesmon). We further investigated interactions between Msx1 and myocardin/serum response factor (SRF)/CArG-box motif (cis element for SRF) using coimmunoprecipitation, gel-shift, and chromatin immunoprecipitation assays. Our results showed that Msx1 or Msx2 formed a ternary complex with SRF and myocardin and inhibited the binding of SRF or SRF/myocardin to the CArG-box motif, resulting in inhibition of their transcription.

  7. Chordin-Like 1 Suppresses Bone Morphogenetic Protein 4-Induced Breast Cancer Cell Migration and Invasion

    PubMed Central

    Cyr-Depauw, Chanèle; Northey, Jason J.; Tabariès, Sébastien; Annis, Matthew G.; Dong, Zhifeng; Cory, Sean; Hallett, Michael; Rennhack, Jonathan P.; Andrechek, Eran R.

    2016-01-01

    ShcA is an important mediator of ErbB2- and transforming growth factor β (TGF-β)-induced breast cancer cell migration, invasion, and metastasis. We show that in the context of reduced ShcA levels, the bone morphogenetic protein (BMP) antagonist chordin-like 1 (Chrdl1) is upregulated in numerous breast cancer cells following TGF-β stimulation. BMPs have emerged as important modulators of breast cancer aggressiveness, and we have investigated the ability of Chrdl1 to block BMP-induced increases in breast cancer cell migration and invasion. Breast cancer-derived conditioned medium containing elevated concentrations of endogenous Chrdl1, as well as medium containing recombinant Chrdl1, suppresses BMP4-induced signaling in multiple breast cancer cell lines. Live-cell migration assays reveal that BMP4 induces breast cancer migration, which is effectively blocked by Chrdl1. We demonstrate that BMP4 also stimulated breast cancer cell invasion and matrix degradation, in part, through enhanced metalloproteinase 2 (MMP2) and MMP9 activity that is antagonized by Chrdl1. Finally, high Chrdl1 expression was associated with better clinical outcomes in patients with breast cancer. Together, our data reveal that Chrdl1 acts as a negative regulator of malignant breast cancer phenotypes through inhibition of BMP signaling. PMID:26976638

  8. Bone morphogenetic protein and Notch signalling crosstalk in poor-prognosis, mesenchymal-subtype colorectal cancer.

    PubMed

    Irshad, Shazia; Bansal, Mukesh; Guarnieri, Paolo; Davis, Hayley; Al Haj Zen, Ayman; Baran, Brygida; Pinna, Claudia Maria Assunta; Rahman, Haseeb; Biswas, Sujata; Bardella, Chiara; Jeffery, Rosemary; Wang, Lai Mun; East, James Edward; Tomlinson, Ian; Lewis, Annabelle; Leedham, Simon John

    2017-06-01

    The functional role of bone morphogenetic protein (BMP) signalling in colorectal cancer (CRC) is poorly defined, with contradictory results in cancer cell line models reflecting the inherent difficulties of assessing a signalling pathway that is context-dependent and subject to genetic constraints. By assessing the transcriptional response of a diploid human colonic epithelial cell line to BMP ligand stimulation, we generated a prognostic BMP signalling signature, which was applied to multiple CRC datasets to investigate BMP heterogeneity across CRC molecular subtypes. We linked BMP and Notch signalling pathway activity and function in human colonic epithelial cells, and normal and neoplastic tissue. BMP induced Notch through a γ-secretase-independent interaction, regulated by the SMAD proteins. In homeostasis, BMP/Notch co-localization was restricted to cells at the top of the intestinal crypt, with more widespread interaction in some human CRC samples. BMP signalling was downregulated in the majority of CRCs, but was conserved specifically in mesenchymal-subtype tumours, where it interacts with Notch to induce an epithelial-mesenchymal transition (EMT) phenotype. In intestinal homeostasis, BMP-Notch pathway crosstalk is restricted to differentiating cells through stringent pathway segregation. Conserved BMP activity and loss of signalling stringency in mesenchymal-subtype tumours promotes a synergistic BMP-Notch interaction, and this correlates with poor patient prognosis. BMP signalling heterogeneity across CRC subtypes and cell lines can account for previous experimental contradictions. Crosstalk between the BMP and Notch pathways will render mesenchymal-subtype CRC insensitive to γ-secretase inhibition unless BMP activation is concomitantly addressed. © 2017 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. © 2017 The Authors. The Journal of Pathology published by John Wiley

  9. The Hedgehog Signal Induced Modulation of Bone Morphogenetic Protein Signaling: An Essential Signaling Relay for Urinary Tract Morphogenesis

    PubMed Central

    Nakagata, Naomi; Miyagawa, Shinichi; Suzuki, Kentaro; Kitazawa, Sohei; Yamada, Gen

    2012-01-01

    Background Congenital diseases of the urinary tract are frequently observed in infants. Such diseases present a number of developmental anomalies such as hydroureter and hydronephrosis. Although some genetically-modified mouse models of growth factor signaling genes reproduce urinary phenotypes, the pathogenic mechanisms remain obscure. Previous studies suggest that a portion of the cells in the external genitalia and bladder are derived from peri-cloacal mesenchymal cells that receive Hedgehog (Hh) signaling in the early developmental stages. We hypothesized that defects in such progenitor cells, which give rise to urinary tract tissues, may be a cause of such diseases. Methodology/Principal Findings To elucidate the pathogenic mechanisms of upper urinary tract malformations, we analyzed a series of Sonic hedgehog (Shh) deficient mice. Shh−/− displayed hydroureter and hydronephrosis phenotypes and reduced expression of several developmental markers. In addition, we suggested that Shh modulation at an early embryonic stage is responsible for such phenotypes by analyzing the Shh conditional mutants. Tissue contribution assays of Hh-responsive cells revealed that peri-cloacal mesenchymal cells, which received Hh signal secreted from cloacal epithelium, could contribute to the ureteral mesenchyme. Gain- and loss-of-functional mutants for Hh signaling revealed a correlation between Hh signaling and Bone morphogenetic protein (Bmp) signaling. Finally, a conditional ablation of Bmp receptor type IA (BmprIA) gene was examined in Hh-responsive cell lineages. This system thus made it possible to analyze the primary functions of the growth factor signaling relay. The defective Hh-to-Bmp signaling relay resulted in severe urinary tract phenotypes with a decrease in the number of Hh-responsive cells. Conclusions/Significance This study identified the essential embryonic stages for the pathogenesis of urinary tract phenotypes. These results suggested that Hh

  10. Open tibial fractures grade IIIC treated successfully with external fixation, negative-pressure wound therapy and recombinant human bone morphogenetic protein 7.

    PubMed

    Babiak, Ireneusz

    2014-10-01

    The aim of the therapy in open tibial fractures grade III was to cover the bone with soft tissue and achieve healed fracture without persistent infection. Open tibial fractures grade IIIC with massive soft tissue damage require combined orthopaedic, vascular and plastic-reconstructive procedures. Negative-pressure wound therapy (NPWT), used in two consecutive cases with open fracture grade IIIC of the tibia diaphysis, healed extensive soft tissue defect with exposure of the bone. NPWT eventually allowed for wound closure by split skin graft within 21-25 days. Ilizarov external fixator combined with application of recombinant human bone morphogenetic protein-7 at the site of delayed union enhanced definitive bone healing within 16-18 months. © 2012 The Authors. International Wound Journal © 2012 Medicalhelplines.com Inc and John Wiley & Sons Ltd.

  11. Direct bone morphogenetic protein 2 and Indian hedgehog gene transfer for articular cartilage repair using bone marrow coagulates.

    PubMed

    Sieker, J T; Kunz, M; Weißenberger, M; Gilbert, F; Frey, S; Rudert, M; Steinert, A F

    2015-03-01

    Bone morphogenetic protein 2 (BMP-2, encoded by BMP2) and Indian hedgehog protein (IHH, encoded by IHH) are well known regulators of chondrogenesis and chondrogenic hypertrophy. Despite being a potent chondrogenic factor BMP-2 was observed to induce chondrocyte hypertrophy in osteoarthritis (OA), growth plate cartilage and adult mesenchymal stem cells (MSCs). IHH might induce chondrogenic differentiation through different intracellular signalling pathways without inducing subsequent chondrocyte hypertrophy. The primary objective of this study is to test the efficacy of direct BMP2 and IHH gene delivery via bone marrow coagulates to influence histological repair cartilage quality in vivo. Vector-laden autologous bone marrow coagulates with 10(11) adenoviral vector particles encoding BMP2, IHH or the Green fluorescent protein (GFP) were delivered to 3.2 mm osteochondral defects in the trochlea of rabbit knees. After 13 weeks the histological repair cartilage quality was assessed using the ICRS II scoring system and the type II collagen positive area. IHH treatment resulted in superior histological repair cartilage quality than GFP controls in all of the assessed parameters (with P < 0.05 in five of 14 assessed parameters). Results of BMP2 treatment varied substantially, including severe intralesional bone formation in two of six joints after 13 weeks. IHH gene transfer is effective to improve repair cartilage quality in vivo, whereas BMP2 treatment, carried the risk intralesional bone formation. Therefore IHH protein can be considered as an attractive alternative candidate growth factor for further preclinical research and development towards improved treatments for articular cartilage defects. Copyright © 2014 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

  12. Nuclear Factor YY1 Inhibits Transforming Growth Factor β- and Bone Morphogenetic Protein-Induced Cell Differentiation

    PubMed Central

    Kurisaki, Keiko; Kurisaki, Akira; Valcourt, Ulrich; Terentiev, Alexei A.; Pardali, Katerina; ten Dijke, Peter; Heldin, Carl-Henrik; Ericsson, Johan; Moustakas, Aristidis

    2003-01-01

    Smad proteins transduce transforming growth factor β (TGF-β) and bone morphogenetic protein (BMP) signals that regulate cell growth and differentiation. We have identified YY1, a transcription factor that positively or negatively regulates transcription of many genes, as a novel Smad-interacting protein. YY1 represses the induction of immediate-early genes to TGF-β and BMP, such as the plasminogen activator inhibitor 1 gene (PAI-1) and the inhibitor of differentiation/inhibitor of DNA binding 1 gene (Id-1). YY1 inhibits binding of Smads to their cognate DNA elements in vitro and blocks Smad recruitment to the Smad-binding element-rich region of the PAI-1 promoter in vivo. YY1 interacts with the conserved N-terminal Mad homology 1 domain of Smad4 and to a lesser extent with Smad1, Smad2, and Smad3. The YY1 zinc finger domain mediates the association with Smads and is necessary for the repressive effect of YY1 on Smad transcriptional activity. Moreover, downregulation of endogenous YY1 by antisense and small interfering RNA strategies results in enhanced transcriptional responses to TGF-β or BMP. Ectopic expression of YY1 inhibits, while knockdown of endogenous YY1 enhances, TGF-β- and BMP-induced cell differentiation. In contrast, overexpression or knockdown of YY1 does not affect growth inhibition induced by TGF-β or BMP. Accordingly, YY1 does not interfere with the regulation of immediate-early genes involved in the TGF-β growth-inhibitory response, the cell cycle inhibitors p15 and p21, and the proto-oncogene c-myc. In conclusion, YY1 represses Smad transcriptional activities in a gene-specific manner and thus regulates cell differentiation induced by TGF-β superfamily pathways. PMID:12808092

  13. Structural organization of G-protein-coupled receptors

    NASA Astrophysics Data System (ADS)

    Lomize, Andrei L.; Pogozheva, Irina D.; Mosberg, Henry I.

    1999-07-01

    Atomic-resolution structures of the transmembrane 7-α-helical domains of 26 G-protein-coupled receptors (GPCRs) (including opsins, cationic amine, melatonin, purine, chemokine, opioid, and glycoprotein hormone receptors and two related proteins, retinochrome and Duffy erythrocyte antigen) were calculated by distance geometry using interhelical hydrogen bonds formed by various proteins from the family and collectively applied as distance constraints, as described previously [Pogozheva et al., Biophys. J., 70 (1997) 1963]. The main structural features of the calculated GPCR models are described and illustrated by examples. Some of the features reflect physical interactions that are responsible for the structural stability of the transmembrane α-bundle: the formation of extensive networks of interhelical H-bonds and sulfur-aromatic clusters that are spatially organized as 'polarity gradients' the close packing of side-chains throughout the transmembrane domain; and the formation of interhelical disulfide bonds in some receptors and a plausible Zn2+ binding center in retinochrome. Other features of the models are related to biological function and evolution of GPCRs: the formation of a common 'minicore' of 43 evolutionarily conserved residues; a multitude of correlated replacements throughout the transmembrane domain; an Na+-binding site in some receptors, and excellent complementarity of receptor binding pockets to many structurally dissimilar, conformationally constrained ligands, such as retinal, cyclic opioid peptides, and cationic amine ligands. The calculated models are in good agreement with numerous experimental data.

  14. G protein-coupled estrogen receptor 1/G protein-coupled receptor 30 localizes in the plasma membrane and traffics intracellularly on cytokeratin intermediate filaments.

    PubMed

    Sandén, Caroline; Broselid, Stefan; Cornmark, Louise; Andersson, Krister; Daszkiewicz-Nilsson, Joanna; Mårtensson, Ulrika E A; Olde, Björn; Leeb-Lundberg, L M Fredrik

    2011-03-01

    G protein-coupled receptor 30 [G protein-coupled estrogen receptor 1 (GPER1)], has been introduced as a membrane estrogen receptor and a candidate cancer biomarker and therapeutic target. However, several questions surround the subcellular localization and signaling of this receptor. In native cells, including mouse myoblast C(2)C(12) cells, Madin-Darby canine kidney epithelial cells, and human ductal breast epithelial tumor T47-D cells, G-1, a GPER1 agonist, and 17β-estradiol stimulated GPER1-dependent cAMP production, a defined plasma membrane (PM) event, and recruitment of β-arrestin2 to the PM. Staining of fixed and live cells showed that GPER1 was localized both in the PM and on intracellular structures. One such intracellular structure was identified as cytokeratin (CK) intermediate filaments, including those composed of CK7 and CK8, but apparently not endoplasmic reticulum, Golgi, or microtubules. Reciprocal coimmunoprecipitation of GPER1 and CKs confirmed an association of these proteins. Live staining also showed that the PM receptors constitutively internalize apparently to reach CK filaments. Receptor localization was supported using FLAG- and hemagglutinin-tagged GPER1. We conclude that GPER1-mediated stimulation of cAMP production and β-arrestin2 recruitment occur in the PM. Furthermore, the PM receptors constitutively internalize and localize intracellularly on CK. This is the first observation that a G protein-coupled receptor is capable of associating with intermediate filaments, which may be important for GPER1 regulation in epithelial cells and the relationship of this receptor to cancer.

  15. Structural studies of G protein-coupled receptors.

    PubMed

    Lu, Mengjie; Wu, Beili

    2016-11-01

    G protein-coupled receptors (GPCRs) comprise the largest membrane protein family. These receptors sense a variety of signaling molecules, activate multiple intracellular signal pathways, and act as the targets of over 40% of marketed drugs. Recent progress on GPCR structural studies provides invaluable insights into the structure-function relationship of the GPCR superfamily, deepening our understanding about the molecular mechanisms of GPCR signal transduction. Here, we review recent breakthroughs on GPCR structure determination and the structural features of GPCRs, and take the structures of chemokine receptor CCR5 and purinergic receptors P2Y 1 R and P2Y 12 R as examples to discuss the importance of GPCR structures on functional studies and drug discovery. In addition, we discuss the prospect of GPCR structure-based drug discovery. © 2016 IUBMB Life, 68(11):894-903, 2016. © 2016 International Union of Biochemistry and Molecular Biology.

  16. Bone Morphogenetic Proteins in Anterior Cervical Fusion: A Systematic Review and Meta-Analysis.

    PubMed

    Zadegan, Shayan Abdollah; Abedi, Aidin; Jazayeri, Seyed Behnam; Nasiri Bonaki, Hirbod; Jazayeri, Seyed Behzad; Vaccaro, Alexander R; Rahimi-Movaghar, Vafa

    2017-08-01

    Bone morphogenetic proteins (BMPs) have been commonly used as a graft substitute in spinal fusion. Although the U.S. Food and Drug Administration issued a warning on life-threatening complications of recombinant human BMPs (rhBMPs) in cervical spine fusion in 2008, their off-label use has been continued. This investigation aimed to review the evidence for the use of rhBMP-2 and rhBMP-7 in anterior cervical spine fusions. A comprehensive search was performed through Ovid (MEDLINE), PubMed, and Embase. The risk of bias assessment was according to the recommended criteria by the Cochrane Back and Neck group and MINORS (Methodological Index for Non-Randomized Studies). A wide array of radiographic and clinical outcomes including the adverse events were collated. Eighteen articles (1 randomized and 17 nonrandomized) were eligible for inclusion. The fusion rate was higher with use of rhBMP in most studies and our meta-analysis of the pooled data from 4782 patients confirmed this finding (odds ratio, 5.45; P < 0.00001). Altogether, the rhBMP and control groups were comparable in patient-reported outcomes. However, most studies tended to show a significantly higher incidence of overall complication rate, dysphagia/dysphonia, cervical swelling, readmission, wound complications, neurologic complications, and ossification. Application of rhBMPs in cervical spine fusion yields a significantly higher fusion rate with similar patient-reported outcomes, yet increased risk of life-threatening complications. Thus, we do not recommend the use of rhBMP in anterior cervical fusions. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Pharmacological characterization of receptor-activity-modifying proteins (RAMPs) and the human calcitonin receptor.

    PubMed

    Armour, S L; Foord, S; Kenakin, T; Chen, W J

    1999-12-01

    Receptor-activity-modifying proteins (RAMPs) are a family of single transmembrane domain proteins shown to be important for the transport and ligand specificity of the calcitonin gene-related peptide (CGRP) receptor. In this report, we describe the analysis of pharmacological properties of the human calcitonin receptor (hCTR) coexpressed with different RAMPs with the use of the Xenopus laevis melanophore expression system. We show that coexpression of RAMP3 with human calcitonin receptor changed the relative potency of hCTR to human calcitonin (hCAL) and rat amylin. RAMP1 and RAMP2, in contrast, had little effect on the change of hCTR potency to hCAL or rat amylin. When coexpressed with RAMP3, hCTR reversed the relative potency by a 3.5-fold loss in sensitivity to hCAL and a 19-fold increase in sensitivity to rat amylin. AC66, an inverse agonist, produced apparent simple competitive antagonism of hCAL and rat amylin, as indicated by linear Schild regressions. The potency of AC66 was changed in the blockade of rat amylin but not hCAL responses with RAMP3 coexpression. The mean pK(B) for AC66 to hCAL was 9.4 +/- 0.3 without RAMP3 and 9.45 +/- 0.07 with RAMP3. For the antagonism of AC66 to rat amylin, the pK(B) was 9.25 +/- 0.15 without RAMP3 and 8.2 +/- 0.35 with RAMP3. The finding suggests that RAMP3 might modify the active states of calcitonin receptor in such a way as to create a new receptor phenotype that is "amylin-like." Irrespective of the physiological association of the new receptor species, the finding that a coexpressed membrane protein can completely change agonist and antagonist affinities for a receptor raises implications for screening in recombinant receptor systems.

  18. Structure-based drug design for G protein-coupled receptors.

    PubMed

    Congreve, Miles; Dias, João M; Marshall, Fiona H

    2014-01-01

    Our understanding of the structural biology of G protein-coupled receptors has undergone a transformation over the past 5 years. New protein-ligand complexes are described almost monthly in high profile journals. Appreciation of how small molecules and natural ligands bind to their receptors has the potential to impact enormously how medicinal chemists approach this major class of receptor targets. An outline of the key topics in this field and some recent examples of structure- and fragment-based drug design are described. A table is presented with example views of each G protein-coupled receptor for which there is a published X-ray structure, including interactions with small molecule antagonists, partial and full agonists. The possible implications of these new data for drug design are discussed. © 2014 Elsevier B.V. All rights reserved.

  19. Spring-loaded model revisited: paramyxovirus fusion requires engagement of a receptor binding protein beyond initial triggering of the fusion protein.

    PubMed

    Porotto, Matteo; Devito, Ilaria; Palmer, Samantha G; Jurgens, Eric M; Yee, Jia L; Yokoyama, Christine C; Pessi, Antonello; Moscona, Anne

    2011-12-01

    During paramyxovirus entry into a host cell, receptor engagement by a specialized binding protein triggers conformational changes in the adjacent fusion protein (F), leading to fusion between the viral and cell membranes. According to the existing paradigm of paramyxovirus membrane fusion, the initial activation of F by the receptor binding protein sets off a spring-loaded mechanism whereby the F protein progresses independently through the subsequent steps in the fusion process, ending in membrane merger. For human parainfluenza virus type 3 (HPIV3), the receptor binding protein (hemagglutinin-neuraminidase [HN]) has three functions: receptor binding, receptor cleaving, and activating F. We report that continuous receptor engagement by HN activates F to advance through the series of structural rearrangements required for fusion. In contrast to the prevailing model, the role of HN-receptor engagement in the fusion process is required beyond an initiating step, i.e., it is still required even after the insertion of the fusion peptide into the target cell membrane, enabling F to mediate membrane merger. We also report that for Nipah virus, whose receptor binding protein has no receptor-cleaving activity, the continuous stimulation of the F protein by a receptor-engaged binding protein is key for fusion. We suggest a general model for paramyxovirus fusion activation in which receptor engagement plays an active role in F activation, and the continued engagement of the receptor binding protein is essential to F protein function until the onset of membrane merger. This model has broad implications for the mechanism of paramyxovirus fusion and for strategies to prevent viral entry.

  20. Trichoderma G protein-coupled receptors: functional characterisation of a cAMP receptor-like protein from Trichoderma atroviride.

    PubMed

    Brunner, Kurt; Omann, Markus; Pucher, Marion E; Delic, Marizela; Lehner, Sylvia M; Domnanich, Patrick; Kratochwill, Klaus; Druzhinina, Irina; Denk, Dagmar; Zeilinger, Susanne

    2008-12-01

    Galpha subunits act to regulate vegetative growth, conidiation, and the mycoparasitic response in Trichoderma atroviride. To extend our knowledge on G protein signalling, we analysed G protein-coupled receptors (GPCRs). As the genome sequence of T. atroviride is not publicly available yet, we carried out an in silico exploration of the genome database of the close relative T. reesei. Twenty genes encoding putative GPCRs distributed over eight classes and additional 35 proteins similar to the Magnaporthe grisea PTH11 receptor were identified. Subsequently, four T. atroviride GPCR-encoding genes were isolated and affiliated to the cAMP receptor-like family by phylogenetic and topological analyses. All four genes showed lowest expression on glycerol and highest mRNA levels upon carbon starvation. Transcription of gpr3 and gpr4 responded to exogenously added cAMP and the shift from liquid to solid media. gpr3 mRNA levels also responded to the presence of fungal hyphae or cellulose membranes. Further characterisation of mutants bearing a gpr1-silencing construct revealed that Gpr1 is essential for vegetative growth, conidiation and conidial germination. Four genes encoding the first GPCRs described in Trichoderma were isolated and their expression characterized. At least one of these GPCRs is important for several cellular processes, supporting the fundamental role of G protein signalling in this fungus.

  1. Regulation of bone morphogenetic protein signalling and cranial osteogenesis by Gpc1 and Gpc3.

    PubMed

    Dwivedi, Prem P; Grose, Randall H; Filmus, Jorge; Hii, Charles S T; Xian, Cory J; Anderson, Peter J; Powell, Barry C

    2013-08-01

    From birth, the vault of the skull grows at a prodigious rate, driven by the activity of osteoblastic cells at the fibrous joints (sutures) that separate the bony calvarial plates. One in 2500 children is born with a medical condition known as craniosynostosis because of premature bony fusion of the calvarial plates and a cessation of bone growth at the sutures. Bone morphogenetic proteins (BMPs) are potent growth factors that promote bone formation. Previously, we found that Glypican-1 (GPC1) and Glypican-3 (GPC3) are expressed in cranial sutures and are decreased during premature suture fusion in children. Although glypicans are known to regulate BMP signalling, a mechanistic link between GPC1, GPC3 and BMPs and osteogenesis has not yet been investigated. We now report that human primary suture mesenchymal cells coexpress GPC1 and GPC3 on the cell surface and release them into the media. We show that they inhibit BMP2, BMP4 and BMP7 activities, which both physically interact with BMP2 and that immunoblockade of endogenous GPC1 and GPC3 potentiates BMP2 activity. In contrast, increased levels of GPC1 and GPC3 as a result of overexpression or the addition of recombinant protein, inhibit BMP2 signalling and BMP2-mediated osteogenesis. We demonstrate that BMP signalling in suture mesenchymal cells is mediated by both SMAD-dependent and SMAD-independent pathways and that GPC1 and GPC3 inhibit both pathways. GPC3 inhibition of BMP2 activity is independent of attachment of the glypican on the cell surface and post-translational glycanation, and thus appears to be mediated by the core glypican protein. The discovery that GPC1 and GPC3 regulate BMP2-mediated osteogenesis, and that inhibition of endogenous GPC1 and GPC3 potentiates BMP2 responsiveness of human suture mesenchymal cells, indicates how downregulation of glypican expression could lead to the bony suture fusion that characterizes craniosynostosis. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. Mitogenic signaling of urokinase receptor-deficient kidney fibroblasts: actions of an alternative urokinase receptor and LDL receptor-related protein.

    PubMed

    Zhang, Guoqiang; Cai, Xiaohe; López-Guisa, Jesús M; Collins, Sarah J; Eddy, Allison A

    2004-08-01

    The urokinase receptor (uPAR) attenuates myofibroblast recruitment and fibrosis in the kidney. This study examined the role of uPAR and its co-receptor LDL receptor-related protein (LRP) in the regulation of kidney fibroblast proliferation and extracellular signal-regulated kinase (ERK) signaling. Compared with uPAR+/+ cells, uPAR-/- kidney fibroblasts were hyperproliferative. UPAR-/- fibroblast proliferation was 60% inhibited by an ERK kinase inhibitor. LRP protein was reduced and extracellular accumulation of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) proteins were greater in uPAR-/- cultures. Addition of functional uPA protein or LRP antisense RNA significantly increased ERK signaling and cell mitosis in both genotypes. Enhanced uPAR-/- fibroblast proliferation was reversed by a recombinant nonfunctional uPA peptide. The density of cell-bound fluor-uPA was similar between uPAR-/- and uPAR+/+ fibroblasts (78 +/- 6 versus 92 +/- 16 units). These data suggest that uPAR-deficient kidney fibroblasts express lower levels of its scavenger co-receptor LRP, resulting in greater extracellular accumulation of uPA and PAI-1. Enhanced proliferation of uPAR-/- fibroblasts seems to be mediated by uPA-dependent ERK signaling via an alternative urokinase receptor.

  3. (-)-Epigallocatechin gallate but not chlorogenic acid upregulates osteoprotegerin synthesis through regulation of bone morphogenetic protein-4 in osteoblasts.

    PubMed

    Fujita, Kazuhiko; Otsuka, Takanobu; Yamamoto, Naohiro; Kainuma, Shingo; Ohguchi, Reou; Kawabata, Tetsu; Sakai, Go; Kuroyanagi, Gen; Matsushima-Nishiwaki, Rie; Kozawa, Osamu; Tokuda, Haruhiko

    2017-07-01

    Chlorogenic acid (CGA) is a primary phenolic component of coffee and (-)-epigallocatechin gallate (EGCG) is a primary flavonoid component of green tea, both of which have been documented to possess beneficial health properties. A previous study by the present authors demonstrated that p38 mitogen-activated protein kinase (MAPK) may be associated with osteoprotegerin synthesis stimulated by bone morphogenetic protein-4 (BMP-4) in osteoblast-like MC3T3-E1 cells. In the present study, the effects of CGA and EGCG on BMP-4-stimulated osteoprotegerin synthesis in MC3T3-E1 cells were investigated. It was observed that CGA had no effect on osteoprotegerin release stimulated by BMP-4, whereas EGCG significantly enhanced BMP-4-stimulated osteoprotegerin release (P=0.003). Levels of osteoprotegerin mRNA expression induced by BMP-4 were also significantly increased by EGCG (P=0.03). By contrast, EGCG had no significant effect on phosphorylation of Smad1 or p38 MAPK induced by BMP-4. In addition, EGCG had little effect on BMP-induced phosphorylation of p70 S6 kinase; however rapamycin, as an inhibitor of p70 S6 kinase, significantly suppressed osteoprotegerin release (P=0.007). These data suggest that EGCG but not CGA may upregulate the synthesis of osteoprotegerin induced by BMP-4 in osteoblasts.

  4. G protein-coupled receptor mutations and human genetic disease.

    PubMed

    Thompson, Miles D; Hendy, Geoffrey N; Percy, Maire E; Bichet, Daniel G; Cole, David E C

    2014-01-01

    Genetic variations in G protein-coupled receptor genes (GPCRs) disrupt GPCR function in a wide variety of human genetic diseases. In vitro strategies and animal models have been used to identify the molecular pathologies underlying naturally occurring GPCR mutations. Inactive, overactive, or constitutively active receptors have been identified that result in pathology. These receptor variants may alter ligand binding, G protein coupling, receptor desensitization and receptor recycling. Receptor systems discussed include rhodopsin, thyrotropin, parathyroid hormone, melanocortin, follicle-stimulating hormone (FSH), luteinizing hormone, gonadotropin-releasing hormone (GNRHR), adrenocorticotropic hormone, vasopressin, endothelin-β, purinergic, and the G protein associated with asthma (GPRA or neuropeptide S receptor 1 (NPSR1)). The role of activating and inactivating calcium-sensing receptor (CaSR) mutations is discussed in detail with respect to familial hypocalciuric hypercalcemia (FHH) and autosomal dominant hypocalemia (ADH). The CASR mutations have been associated with epilepsy. Diseases caused by the genetic disruption of GPCR functions are discussed in the context of their potential to be selectively targeted by drugs that rescue altered receptors. Examples of drugs developed as a result of targeting GPCRs mutated in disease include: calcimimetics and calcilytics, therapeutics targeting melanocortin receptors in obesity, interventions that alter GNRHR loss from the cell surface in idiopathic hypogonadotropic hypogonadism and novel drugs that might rescue the P2RY12 receptor congenital bleeding phenotype. De-orphanization projects have identified novel disease-associated receptors, such as NPSR1 and GPR35. The identification of variants in these receptors provides genetic reagents useful in drug screens. Discussion of the variety of GPCRs that are disrupted in monogenic Mendelian disorders provides the basis for examining the significance of common

  5. Tissue engineering for lateral ridge augmentation with recombinant human bone morphogenetic protein 2 combination therapy: a case report.

    PubMed

    Mandelaris, George A; Spagnoli, Daniel B; Rosenfeld, Alan L; McKee, James; Lu, Mei

    2015-01-01

    This case report describes a tissue-engineered reconstruction with recombinant human bone morphogenetic protein 2/acellular collagen sponge (rhBMP-2/ ACS) + cancellous allograft and space maintenance via Medpor Contain mesh in the treatment of a patient requiring maxillary and mandibular horizontal ridge augmentation to enable implant placement. The patient underwent a previously unsuccessful corticocancellous bone graft at these sites. Multiple and contiguous sites in the maxilla and in the mandibular anterior, demonstrating advanced lateral ridge deficiencies, were managed using a tissue engineering approach as an alternative to autogenous bone harvesting. Four maxillary and three mandibular implants were placed 9 and 10 months, respectively, after tissue engineering reconstruction, and all were functioning successfully after 24 months of follow-up. Histomorphometric analysis of a bone core obtained at the time of the maxillary implant placement demonstrated a mean of 76.1% new vital bone formation, 22.2% marrow/cells, and 1.7% residual graft tissue. Tissue engineering for lateral ridge augmentation with combination therapy requires further research to determine predictability and limitations.

  6. Injectable hydrogels embedded with alginate microspheres for controlled delivery of bone morphogenetic protein-2.

    PubMed

    Zhu, Youjia; Wang, Jiulong; Wu, Jingjing; Zhang, Jun; Wan, Ying; Wu, Hua

    2016-03-23

    Some delivery carriers with injectable characteristics were built using the thermosensitive chitosan/dextran-polylactide/glycerophosphate hydrogel and selected alginate microspheres for the controllable release of bone morphogenetic protein-2 (BMP-2). BMP-2 was first loaded into the microspheres with an average size of around 20 μm and the resulting microspheres were then embedded into the gel in order to achieve well-controlled BMP-2 release. The microsphere-embedded gels show their incipient gelation temperature at around 32 °C and pH at about 7.1. Some gels had their elastic modulus close to 1400 Pa and the ratio of elastic modulus to viscous modulus at around 34, revealing that they behaved like mechanically strong gels. Optimized microsphere-embedded gels were found to be able to administer the BMP-2 release without significant initial burst release in an approximately linear manner over a period of time longer than four weeks. The release rate and the released amount of BMP-2 from these gels could be regulated individually or cooperatively by the initial BMP-2 load and the dextran-polylactide content in the gels. Measurements of the BMP-2 induced alkaline phosphatase activity in C2C12 cells confirmed that C2C12 cells responded to BMP-2 in a dose-dependent way and the released BMP-2 from the microsphere-embedded gels well retained their bioactivity. In vivo assessment of some gels revealed that the released BMP-2 maintained its osteogenesis functions.

  7. Frizzled Receptors in Development and Disease

    PubMed Central

    Wang, Yanshu; Chang, Hao; Rattner, Amir; Nathans, Jeremy

    2016-01-01

    Frizzled proteins are the principal receptors for the Wnt family of ligands. They mediate canonical Wnt signaling together with Lrp5 and Lrp6 coreceptors. In conjunction with Celsr, Vangl, and a small number of additional membrane and membrane-associated proteins, they also play a central role in tissue polarity/planar cell polarity (PCP) signaling. Targeted mutations in 9 of the 10 mammalian Frizzled genes have revealed their roles in an extraordinarily diverse set of developmental and homeostatic processes, including morphogenetic movements responsible for palate, ventricular septum, ocular furrow, and neural tube closure; survival of thalamic neurons; bone formation; central nervous system (CNS) angiogenesis and blood–brain barrier formation and maintenance; and a wide variety of processes that orient subcellular, cellular, and multicellular structures relative to the body axes. The last group likely reflects the mammalian equivalent of tissue polarity/PCP signaling, as defined in Drosophila, and it includes CNS axon guidance, hair follicle and tongue papilla orientation, and inner ear sensory hair bundle orientation. Frizzled receptors are ubiquitous among multicellular animals and, with other signaling molecules, they very likely evolved to permit the development of the complex tissue architectures that provide multicellular animals with their enormous selective advantage. PMID:26969975

  8. Constitutive dimerization of the G-protein coupled receptor, neurotensin receptor 1, reconstituted into phospholipid bilayers.

    PubMed

    Harding, Peter J; Attrill, Helen; Boehringer, Jonas; Ross, Simon; Wadhams, George H; Smith, Eleanor; Armitage, Judith P; Watts, Anthony

    2009-02-01

    Neurotensin receptor 1 (NTS1), a Family A G-protein coupled receptor (GPCR), was expressed in Escherichia coli as a fusion with the fluorescent proteins eCFP or eYFP. A fluorophore-tagged receptor was used to study the multimerization of NTS1 in detergent solution and in brain polar lipid bilayers, using fluorescence resonance energy transfer (FRET). A detergent-solubilized receptor was unable to form FRET-competent complexes at concentrations of up to 200 nM, suggesting that the receptor is monomeric in this environment. When reconstituted into a model membrane system at low receptor density, the observed FRET was independent of agonist binding, suggesting constitutive multimer formation. In competition studies, decreased FRET in the presence of untagged NTS1 excludes the possibility of fluorescent protein-induced interactions. A simulation of the experimental data indicates that NTS1 exists predominantly as a homodimer, rather than as higher-order multimers. These observations suggest that, in common with several other Family A GPCRs, NTS1 forms a constitutive dimer in lipid bilayers, stabilized through receptor-receptor interactions in the absence of other cellular signaling components. Therefore, this work demonstrates that well-characterized model membrane systems are useful tools for the study of GPCR multimerization, allowing fine control over system composition and complexity, provided that rigorous control experiments are performed.

  9. The association of metabotropic glutamate receptor type 5 with the neuronal Ca2+-binding protein 2 modulates receptor function.

    PubMed

    Canela, Laia; Fernández-Dueñas, Víctor; Albergaria, Catarina; Watanabe, Masahiko; Lluís, Carme; Mallol, Josefa; Canela, Enric I; Franco, Rafael; Luján, Rafael; Ciruela, Francisco

    2009-10-01

    Metabotropic glutamate (mGlu) receptors mediate in part the CNS effects of glutamate. These receptors interact with a large array of intracellular proteins in which the final role is to regulate receptor function. Here, using co-immunoprecipitation and pull-down experiments we showed a close and specific interaction between mGlu(5) receptor and NECAB2 in both transfected human embryonic kidney cells and rat hippocampus. Interestingly, in pull-down experiments increasing concentrations of calcium drastically reduced the ability of these two proteins to interact, suggesting that NECAB2 binds to mGlu(5) receptor in a calcium-regulated manner. Immunoelectron microscopy detection of NECAB2 and mGlu(5) receptor in the rat hippocampal formation indicated that both proteins are codistributed in the same subcellular compartment of pyramidal cells. In addition, the NECAB2/mGlu(5) receptor interaction regulated mGlu(5b)-mediated activation of both inositol phosphate accumulation and the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway. Overall, these findings indicate that NECAB2 by its physical interaction with mGlu(5b) receptor modulates receptor function.

  10. Heat shock proteins and toll-like receptors.

    PubMed

    Asea, Alexzander

    2008-01-01

    Researchers have only just begun to elucidate the relationship between heat shock proteins (HSP) and Toll-like receptors (TLR). HSP were originally described as an intracellular molecular chaperone of naïve, aberrantly folded, or mutated proteins and primarily implicated as a cytoprotective protein when cells are exposed to stressful stimuli. However, recent studies have ascribed novel functions to the Hsp70 protein depending on its localization: Surface-bound Hsp70 specifically activate natural killer (NK) cells, while Hsp70 released into the extracellular milieu specifically bind to Toll-like receptors (TLR) 2 and 4 on antigen-presenting cells (APC) and exerts immunoregulatory effects, including upregulation of adhesion molecules, co-stimulatory molecule expression, and cytokine and chemokine release-a process known as the chaperokine activity of Hsp70. This chapter discusses the most recent advances in the understanding of heat shock protein (HSP) and TLR interactions in general and highlights recent findings that demonstrate Hsp70 is a ligand for TLR and its biological significance.

  11. Bone morphogenetic protein Smads signaling in mesenchymal stem cells affected by osteoinductive calcium phosphate ceramics.

    PubMed

    Tang, Zhurong; Wang, Zhe; Qing, Fangzhu; Ni, Yilu; Fan, Yujiang; Tan, Yanfei; Zhang, Xingdong

    2015-03-01

    Porous calcium phosphate ceramics (CaP ceramics) could induce ectopic bone formation which was regulated by various signal molecules. In this work, bone marrow mesenchymal stem cells (MSCs) were cultured on the surface of osteoinductive hydroxyapatite (HA) and biphasic calcium phosphate (BCP) ceramics in comparison with control (culture plate) for up to 14 days to detect the signal molecules which might be affected by the CaP ceramics. Without adding osteogenic factors, MSCs cultured on HA and BCP both expressed higher Runx2, Osterix, collagen type I, osteopontin, bone sialoprotein, and osteocalcin at various stages compared with control, thus confirmed the osteoblastic differentiation of MSCs. Later study demonstrated the messenger RNA level of bone morphogenetic protein 2 (BMP2) and BMP4 were also significantly enhanced by HA and BCP. Furthermore, Smad1, 4, 5, and Dlx5, the main molecules in the BMP/Smads signaling pathway, were upregulated by HA and BCP. Moreover, the higher expression of Smads and BMP2, 4 in BCP over HA, corresponded to the better performance of BCP in stimulating in vitro osteoblastic differentiation of MSCs. This was in accordance with the better osteoinductivity of BCP over HA in vivo. Altogether, these results implied that the CaP ceramics may initiate the osteoblastic differentiation of MSCs by influencing the expression of molecules in BMP/Smads pathway. © 2014 Wiley Periodicals, Inc.

  12. Recombinant G protein-coupled receptor expression in Saccharomyces cerevisiae for protein characterization.

    PubMed

    Blocker, Kory M; Britton, Zachary T; Naranjo, Andrea N; McNeely, Patrick M; Young, Carissa L; Robinson, Anne S

    2015-01-01

    G protein-coupled receptors (GPCRs) are membrane proteins that mediate signaling across the cellular membrane and facilitate cellular responses to external stimuli. Due to the critical role that GPCRs play in signal transduction, therapeutics have been developed to influence GPCR function without an extensive understanding of the receptors themselves. Closing this knowledge gap is of paramount importance to improving therapeutic efficacy and specificity, where efforts to achieve this end have focused chiefly on improving our knowledge of the structure-function relationship. The purpose of this chapter is to review methods for the heterologous expression of GPCRs in Saccharomyces cerevisiae, including whole-cell assays that enable quantitation of expression, localization, and function in vivo. In addition, we describe methods for the micellular solubilization of the human adenosine A2a receptor and for reconstitution of the receptor in liposomes that have enabled its biophysical characterization. © 2015 Elsevier Inc. All rights reserved.

  13. Arrestin-dependent but G-protein coupled receptor kinase-independent uncoupling of D2-dopamine receptors.

    PubMed

    Celver, Jeremy; Sharma, Meenakshi; Thanawala, Vaidehi; Christopher Octeau, J; Kovoor, Abraham

    2013-10-01

    We reconstituted D2 like dopamine receptor (D2R) and the delta opioid receptor (DOR) coupling to G-protein gated inwardly rectifying potassium channels (K(ir)3) and directly compared the effects of co-expression of G-protein coupled receptor kinase (GRK) and arrestin on agonist-dependent desensitization of the receptor response. We found, as described previously, that co-expression of a GRK and an arrestin synergistically increased the rate of agonist-dependent desensitization of DOR. In contrast, only arrestin expression was required to produce desensitization of D2R responses. Furthermore, arrestin-dependent GRK-independent desensitization of D2R-K(ir)3 coupling could be transferred to DOR by substituting the third cytoplasmic loop of DOR with that of D2R. The arrestin-dependent GRK-independent desensitization of D2R desensitization was inhibited by staurosporine treatment, and blocked by alanine substitution of putative protein kinase C phosphorylation sites in the third cytoplasmic loop of D2R. Finally, the D2R construct in which putative protein kinase C phosphorylation sites were mutated did not undergo significant agonist-dependent desensitization even after GRK co-expression, suggesting that GRK phosphorylation of D2R does not play an important role in uncoupling of the receptor. © 2013 International Society for Neurochemistry.

  14. Joint morphogenetic cells in the adult mammalian synovium

    PubMed Central

    Roelofs, Anke J.; Zupan, Janja; Riemen, Anna H. K.; Kania, Karolina; Ansboro, Sharon; White, Nathan; Clark, Susan M.; De Bari, Cosimo

    2017-01-01

    The stem cells that safeguard synovial joints in adulthood are undefined. Studies on mesenchymal stromal/stem cells (MSCs) have mainly focused on bone marrow. Here we show that lineage tracing of Gdf5-expressing joint interzone cells identifies in adult mouse synovium an MSC population largely negative for the skeletal stem cell markers Nestin-GFP, Leptin receptor and Gremlin1. Following cartilage injury, Gdf5-lineage cells underpin synovial hyperplasia through proliferation, are recruited to a Nestin-GFPhigh perivascular population, and contribute to cartilage repair. The transcriptional co-factor Yap is upregulated after injury, and its conditional ablation in Gdf5-lineage cells prevents synovial lining hyperplasia and decreases contribution of Gdf5-lineage cells to cartilage repair. Cultured Gdf5-lineage cells exhibit progenitor activity for stable chondrocytes and are able to self-organize three-dimensionally to form a synovial lining-like layer. Finally, human synovial MSCs transduced with Bmp7 display morphogenetic properties by patterning a joint-like organ in vivo. Our findings further the understanding of the skeletal stem/progenitor cells in adult life. PMID:28508891

  15. The G protein Gi1 exhibits basal coupling but not preassembly with G protein-coupled receptors.

    PubMed

    Bondar, Alexey; Lazar, Josef

    2017-06-09

    The G i/o protein family transduces signals from a diverse group of G protein-coupled receptors (GPCRs). The observed specificity of G i/o -GPCR coupling and the high rate of G i/o signal transduction have been hypothesized to be enabled by the existence of stable associates between G i/o proteins and their cognate GPCRs in the inactive state (G i/o -GPCR preassembly). To test this hypothesis, we applied the recently developed technique of two-photon polarization microscopy (2PPM) to Gα i1 subunits labeled with fluorescent proteins and four GPCRs: the α 2A -adrenergic receptor, GABA B , cannabinoid receptor type 1 (CB 1 R), and dopamine receptor type 2. Our experiments with non-dissociating mutants of fluorescently labeled Gα i1 subunits (exhibiting impaired dissociation from activated GPCRs) showed that 2PPM is capable of detecting GPCR-G protein interactions. 2PPM experiments with non-mutated fluorescently labeled Gα i1 subunits and α 2A -adrenergic receptor, GABA B , or dopamine receptor type 2 receptors did not reveal any interaction between the G i1 protein and the non-stimulated GPCRs. In contrast, non-stimulated CB 1 R exhibited an interaction with the G i1 protein. Further experiments revealed that this interaction is caused solely by CB 1 R basal activity; no preassembly between CB 1 R and the G i1 protein could be observed. Our results demonstrate that four diverse GPCRs do not preassemble with non-active G i1 However, we also show that basal GPCR activity allows interactions between non-stimulated GPCRs and G i1 (basal coupling). These findings suggest that G i1 interacts only with active GPCRs and that the well known high speed of GPCR signal transduction does not require preassembly between G proteins and GPCRs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Osteogenic and chondrogenic master genes expression is dependent on the Kir2.1 potassium channel through the bone morphogenetic protein pathway.

    PubMed

    Pini, Jonathan; Giuliano, Serena; Matonti, Julia; Simkin, Dina; Rouleau, Matthieu; Bendahhou, Saïd

    2018-05-29

    Andersen's syndrome is a rare disorder affecting muscle, heart, and bone, that is associated with mutations leading to a loss of function of the inwardly rectifying K + channel Kir2.1. While the Kir2.1 function can be anticipated in excitable cells by controlling the electrical activity, its role in non-excitable cells remains to be investigated. Using Andersen's syndrome induced Pluripotent Stem cells, we investigated the cellular and molecular events during the osteoblastic and chondrogenic differentiation that are affected by the loss of the Ik1 current. We show that loss of Kir2.1 channel function impairs both osteoblastic and chondrogenic processes through the down regulation master gene expression. This down regulation is due to an impairment of the bone morphogenetic proteins signaling pathway through de-phosphorylation of the Smad proteins. Restoring Kir2.1 channel function in Andersen's syndrome cells rescued master genes expression, and restored normal osteoblasts and chondrocytes behavior. Our results show that Kir2.1-mediated activity controls endochondral and intramembranous ossification signaling pathways. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  17. Lifetime of muscarinic receptor-G-protein complexes determines coupling efficiency and G-protein subtype selectivity.

    PubMed

    Ilyaskina, Olga S; Lemoine, Horst; Bünemann, Moritz

    2018-05-08

    G-protein-coupled receptors (GPCRs) are essential for the detection of extracellular stimuli by cells and transfer the encoded information via the activation of functionally distinct subsets of heterotrimeric G proteins into intracellular signals. Despite enormous achievements toward understanding GPCR structures, major aspects of the GPCR-G-protein selectivity mechanism remain unresolved. As this can be attributed to the lack of suitable and broadly applicable assays, we set out to develop a quantitative FRET-based assay to study kinetics and affinities of G protein binding to activated GPCRs in membranes of permeabilized cells in the absence of nucleotides. We measured the association and dissociation kinetics of agonist-induced binding of G i/o , G q/11 , G s , and G 12/13 proteins to muscarinic M 1 , M 2 , and M 3 receptors in the absence of nucleotides between fluorescently labeled G proteins and receptors expressed in mammalian cells. Our results show a strong quantitative correlation between not the on-rates of G-protein-M 3 -R interactions but rather the affinities of G q and G o proteins to M 3 -Rs, their GPCR-G-protein lifetime and their coupling efficiencies determined in intact cells, suggesting that the G-protein subtype-specific affinity to the activated receptor in the absence of nucleotides is, in fact, a major determinant of the coupling efficiency. Our broadly applicable FRET-based assay represents a fast and reliable method to quantify the intrinsic affinity and relative coupling selectivity of GPCRs toward all G-protein subtypes.

  18. Functional capabilities of an N-formyl peptide receptor-G(alpha)(i)(2) fusion protein: assemblies with G proteins and arrestins.

    PubMed

    Shi, Mei; Bennett, Teresa A; Cimino, Daniel F; Maestas, Diane C; Foutz, Terry D; Gurevich, Vsevolod V; Sklar, Larry A; Prossnitz, Eric R

    2003-06-24

    G protein-coupled receptors (GPCRs) must constantly compete for interactions with G proteins, kinases, and arrestins. To evaluate the interactions of these proteins with GPCRs in greater detail, we generated a fusion protein between the N-formyl peptide receptor and the G(alpha)(i2) protein. The functional capabilities of this chimeric protein were determined both in vivo, in stably transfected U937 cells, and in vitro, using a novel reconstitution system of solubilized components. The chimeric protein exhibited a cellular ligand binding affinity indistinguishable from that of the wild-type receptor and existed as a complex, when solubilized, containing betagamma subunits, as demonstrated by sucrose density sedimentation. The chimeric protein mobilized intracellular calcium and desensitized normally in response to agonist. Furthermore, the chimeric receptor was internalized and recycled at rates similar to those of the wild-type FPR. Confocal fluorescence microscopy revealed that internalized chimeric receptors, as identified with fluorescent ligand, colocalized with arrestin, as well as G protein, unlike wild-type receptors. Soluble reconstitution experiments demonstrated that the chimeric receptor, even in the phosphorylated state, existed as a high ligand affinity G protein complex, in the absence of exogenous G protein. This interaction was only partially prevented through the addition of arrestins. Furthermore, our results demonstrate that the GTP-bound state of the G protein alpha subunit displays no detectable affinity for the receptor. Together, these results indicate that complex interactions exist between GPCRs, in their unphosphorylated and phosphorylated states, G proteins, and arrestins, which result in the highly regulated control of GPCR function.

  19. Salicylic Acid-Based Polymers for Guided Bone Regeneration Using Bone Morphogenetic Protein-2

    PubMed Central

    Subramanian, Sangeeta; Mitchell, Ashley; Yu, Weiling; Snyder, Sabrina; Uhrich, Kathryn

    2015-01-01

    Bone morphogenetic protein-2 (BMP-2) is used clinically to promote spinal fusion, treat complex tibia fractures, and to promote bone formation in craniomaxillofacial surgery. Excessive bone formation at sites where BMP-2 has been applied is an established complication and one that could be corrected by guided tissue regeneration methods. In this study, anti-inflammatory polymers containing salicylic acid [salicylic acid-based poly(anhydride-ester), SAPAE] were electrospun with polycaprolactone (PCL) to create thin flexible matrices for use as guided bone regeneration membranes. SAPAE polymers hydrolyze to release salicylic acid, which is a nonsteroidal anti-inflammatory drug. PCL was used to enhance the mechanical integrity of the matrices. Two different SAPAE-containing membranes were produced and compared: fast-degrading (FD-SAPAE) and slow-degrading (SD-SAPAE) membranes that release salicylic acid at a faster and slower rate, respectively. Rat femur defects were treated with BMP-2 and wrapped with FD-SAPAE, SD-SAPAE, or PCL membrane or were left unwrapped. The effects of different membranes on bone formation within and outside of the femur defects were measured by histomorphometry and microcomputed tomography. Bone formation within the defect was not affected by membrane wrapping at BMP-2 doses of 12 μg or more. In contrast, the FD-SAPAE membrane significantly reduced bone formation outside the defect compared with all other treatments. The rapid release of salicylic acid from the FD-SAPAE membrane suggests that localized salicylic acid treatment during the first few days of BMP-2 treatment can limit ectopic bone formation. The data support development of SAPAE polymer membranes for guided bone regeneration applications as well as barriers to excessive bone formation. PMID:25813520

  20. Altered receptor trafficking in Huntingtin Interacting Protein 1-transformed cells.

    PubMed

    Rao, Dinesh S; Bradley, Sarah V; Kumar, Priti D; Hyun, Teresa S; Saint-Dic, Djenann; Oravecz-Wilson, Katherine; Kleer, Celina G; Ross, Theodora S

    2003-05-01

    The clathrin-associated protein, Huntingtin Interacting Protein 1 (HIP1), is overexpressed in multiple human epithelial tumors. Here, we report that HIP1 is a novel oncoprotein that transforms cells. HIP1-transformed cells, in contrast to RasV12-transformed cells, have dysregulation of multiple receptors involved in clathrin trafficking. Examples include upregulation of the epidermal growth factor receptor (EGFR) and the transferrin receptor. Furthermore, accumulation of transferrin and EGF in the HIP1-transformed cells was increased, and breast tumors that had EGFR expressed also had HIP1 upregulated. Thus, HIP1 overexpression promotes tumor formation and is associated with a general alteration in receptor trafficking. HIP1 is the first endocytic protein to be directly implicated in tumor formation.

  1. Selective targeting of a TNFR decoy receptor pharmaceutical to the primate brain as a receptor-specific IgG fusion protein.

    PubMed

    Boado, Ruben J; Hui, Eric Ka-Wai; Lu, Jeff Zhiqiang; Zhou, Qing-Hui; Pardridge, William M

    2010-03-01

    Decoy receptors, such as the human tumor necrosis factor receptor (TNFR), are potential new therapies for brain disorders. However, decoy receptors are large molecule drugs that are not transported across the blood-brain barrier (BBB). To enable BBB transport of a TNFR decoy receptor, the human TNFR-II extracellular domain was re-engineered as a fusion protein with a chimeric monoclonal antibody (MAb) against the human insulin receptor (HIR). The HIRMAb acts as a molecular Trojan horse to ferry the TNFR therapeutic decoy receptor across the BBB. The HIRMAb-TNFR fusion protein was expressed in stably transfected CHO cells, and was analyzed with electrophoresis, Western blotting, size exclusion chromatography, and binding assays for the HIR and TNFalpha. The HIRMAb-TNFR fusion protein was radio-labeled by trititation, in parallel with the radio-iodination of recombinant TNFR:Fc fusion protein, and the proteins were co-injected in the adult Rhesus monkey. The TNFR:Fc fusion protein did not cross the primate BBB in vivo, but the uptake of the HIRMAb-TNFR fusion protein was high and 3% of the injected dose was taken up by the primate brain. The TNFR was selectively targeted to brain, relative to peripheral organs, following fusion to the HIRMAb. This study demonstrates that decoy receptors may be re-engineered as IgG fusion proteins with a BBB molecular Trojan horse that selectively targets the brain, and enables penetration of the BBB in vivo. IgG-decoy receptor fusion proteins represent a new class of human neurotherapeutics. Copyright 2010 Elsevier B.V. All rights reserved.

  2. Long-Segment Fusion for Adult Spinal Deformity Correction Using Low-Dose Recombinant Human Bone Morphogenetic Protein-2: A Retrospective Review of Fusion Rates.

    PubMed

    Schmitt, Paul J; Kelleher, John P; Ailon, Tamir; Heller, Joshua E; Kasliwal, Manish K; Shaffrey, Christopher I; Smith, Justin S

    2016-08-01

    Although use of very high-dose recombinant human bone morphogenetic protein-2 (rhBMP-2) has been reported to markedly improve fusion rates in adult spinal deformity (ASD) surgery, most centers use much lower doses due to cost constraints. How effective these lower doses are for fusion enhancement remains unclear. To assess fusion rates using relatively low-dose rhBMP-2 for ASD surgery. This was a retrospective review of consecutive ASD patients that underwent thoracic to sacral fusion. Patients that achieved 2-year follow-up were analyzed. Impact of patient and surgical factors on fusion rate was assessed, and fusion rates were compared with historical cohorts. Of 219 patients, 172 (78.5%) achieved 2-year follow-up and were analyzed. Using an average rhBMP-2 dose of 3.1 mg/level (average total dose = 35.9 mg/case), the 2-year fusion rate was 73.8%. Cancellous allograft, local autograft, and very limited iliac crest bone graft (<20 mL, obtained during iliac bolt placement) were also used. On multivariate analysis, female sex was associated with a higher fusion rate, whereas age, comorbidity score, deformity type, and 3-column osteotomy were not. There were no complications directly attributable to rhBMP-2. Fusion rates for ASD using low-dose rhBMP-2 were comparable to those reported for iliac crest bone graft but lower than for high-dose rhBMP-2. Importantly, there were substantial differences between patients in the present series and those in the historical comparison groups that could not be fully adjusted for based on available data. Prospective evaluation of rhBMP-2 dosing for ASD surgery is warranted to define the most appropriate dose that balances benefits, risks, and costs. ASD, adult spinal deformityICBG, iliac crest bone graftOR, odds ratiorhBMP-2, recombinant human bone morphogenetic protein-2RR, risk ratioTCO, 3-column osteotomy.

  3. Quality-of-Life Outcomes following Thoracolumbar and Lumbar Fusion with and without the Use of Recombinant Human Bone Morphogenetic Protein-2: Does Recombinant Human Bone Morphogenetic Protein-2 Make a Difference?

    PubMed Central

    Lubelski, Daniel; Alvin, Matthew D.; Torre-Healy, Andrew; Abdullah, Kalil G.; Nowacki, Amy S.; Whitmore, Robert G.; Steinmetz, Michael P.; Benzel, Edward C.; Mroz, Thomas E.

    2014-01-01

    Design Retrospective study. Objectives (1) To investigate the quality-of-life (QOL) outcomes in the population undergoing lumbar spine surgery with versus without recombinant human bone morphogenetic protein-2 (rhBMP-2); (2) to determine QOL outcomes for those patients who experience postoperative complications; and (3) to identify the effect of patient characteristics on postoperative QOL outcomes. Methods A retrospective review of QOL questionnaires, including the Patient Health Questionnaire-9, Patient Disability Questionnaire (PDQ), EuroQol-5D (EQ-5D), and quality of life-year (QALY), was performed for all patients who underwent thoracolumbar and lumbar fusion surgery with versus without rhBMP-2 between March 2008 and September 2010. Individual preoperative and postoperative QOL data were compared for each patient. Demographic factors and complications were reviewed. Results We identified 266 patients, including 60 with and 206 without rhBMP-2. Questionnaires were completed an average of 10.3 ± 5 months after surgery. For all measures, average scores improved postoperatively compared with preoperatively. No differences in postoperative QOL outcomes were identified between the rhBMP-2 and the control cohorts. Median annual household income was positively associated with EQ-5D and QALY. Compared with those without, patients with postoperative complications had fewer QOL improvements. Conclusions There was no difference in QOL outcomes in the rhBMP-2 compared with the control group. Socioeconomic status and postoperative complications affected QOL outcomes following surgery. The QOL questionnaires provide the clinician with information regarding the patients' self-perceived well-being and can be helpful in the selection of surgical candidates and for understanding the effectiveness of a given surgical procedure. PMID:25396105

  4. Association of dopamine D(3) receptors with actin-binding protein 280 (ABP-280).

    PubMed

    Li, Ming; Li, Chuanyu; Weingarten, Paul; Bunzow, James R; Grandy, David K; Zhou, Qun Yong

    2002-03-01

    Proteins that bind to G protein-coupled receptors have been identified as regulators of receptor localization and signaling. In our previous studies, a cytoskeletal protein, actin-binding protein 280 (ABP-280), was found to associate with the third cytoplasmic loop of dopamine D(2) receptors. In this study, we demonstrate that ABP-280 also interacts with dopamine D(3) receptors, but not with D(4) receptors. Similar to the dopamine D(2) receptor, the D(3)/ABP-280 association is of signaling importance. In human melanoma M2 cells lacking ABP-280, D(3) receptors were unable to inhibit forskolin-stimulated cyclic AMP (cAMP) production significantly. D(4) receptors, however, exhibited a similar degree of inhibition of forskolin-stimulated cAMP production in ABP-280-deficient M2 cells and ABP-280-replent M2 subclones (A7 cells). Further experiments revealed that the D(3)/ABP-280 interaction was critically dependent upon a 36 amino acid carboxyl domain of the D(3) receptor third loop, which is conserved in the D(2) receptor but not in the D(4) receptor. Our results demonstrate a subtype-specific regulation of dopamine D(2)-family receptor signaling by the cytoskeletal protein ABP-280.

  5. G-Protein Coupled Receptors: Surface Display and Biosensor Technology

    NASA Astrophysics Data System (ADS)

    McMurchie, Edward; Leifert, Wayne

    Signal transduction by G-protein coupled receptors (GPCRs) underpins a multitude of physiological processes. Ligand recognition by the receptor leads to the activation of a generic molecular switch involving heterotrimeric G-proteins and guanine nucleotides. With growing interest and commercial investment in GPCRs in areas such as drug targets, orphan receptors, high-throughput screening of drugs and biosensors, greater attention will focus on assay development to allow for miniaturization, ultrahigh-throughput and, eventually, microarray/biochip assay formats that will require nanotechnology-based approaches. Stable, robust, cell-free signaling assemblies comprising receptor and appropriate molecular switching components will form the basis of future GPCR/G-protein platforms, which should be able to be adapted to such applications as microarrays and biosensors. This chapter focuses on cell-free GPCR assay nanotechnologies and describes some molecular biological approaches for the construction of more sophisticated, surface-immobilized, homogeneous, functional GPCR sensors. The latter points should greatly extend the range of applications to which technologies based on GPCRs could be applied.

  6. Synergistic Effects of Vascular Endothelial Growth Factor on Bone Morphogenetic Proteins Induced Bone Formation In Vivo: Influencing Factors and Future Research Directions

    PubMed Central

    Li, Bo; Wang, Hai; Qiu, Guixing; Su, Xinlin

    2016-01-01

    Vascular endothelial growth factor (VEGF) and bone morphogenetic proteins (BMPs), as key mediators in angiogenesis and osteogenesis, are used in a combined delivery manner as a novel strategy in bone tissue engineering. VEGF has the potential to enhance BMPs induced bone formation. Both gene delivery and material-based delivery systems were incorporated in previous studies to investigate the synergistic effects of VEGF and BMPs. However, their results were controversial due to variation of methods incorporated in different studies. Factors influencing the synergistic effects of VEGF on BMPs induced bone formation were identified and analyzed in this review to reduce confusion on this issue. The potential mechanisms and directions of future studies were also proposed here. Further investigating mechanisms of the synergistic effects and optimizing these influencing factors will help to generate more effective bone regeneration. PMID:28070506

  7. Automated large-scale purification of a G protein-coupled receptor for neurotensin.

    PubMed

    White, Jim F; Trinh, Loc B; Shiloach, Joseph; Grisshammer, Reinhard

    2004-04-30

    Structure determination of integral membrane proteins requires milligram amounts of purified, functional protein on a regular basis. Here, we describe a protocol for the purification of a G protein-coupled neurotensin receptor fusion protein at the 3-mg or 10-mg level using immobilized metal affinity chromatography and a neurotensin column in a fully automated mode. Fermentation at a 200-l scale of Escherichia coli expressing functional receptors provides the material needed to feed into the purification routine. Constructs with tobacco etch virus protease recognition sites at either end of the receptor allow the isolation of neurotensin receptor devoid of its fusion partners. The presented expression and purification procedures are simple and robust, and provide the basis for crystallization experiments of receptors on a routine basis.

  8. (−)-Epigallocatechin gallate but not chlorogenic acid upregulates osteoprotegerin synthesis through regulation of bone morphogenetic protein-4 in osteoblasts

    PubMed Central

    Fujita, Kazuhiko; Otsuka, Takanobu; Yamamoto, Naohiro; Kainuma, Shingo; Ohguchi, Reou; Kawabata, Tetsu; Sakai, Go; Kuroyanagi, Gen; Matsushima-Nishiwaki, Rie; Kozawa, Osamu; Tokuda, Haruhiko

    2017-01-01

    Chlorogenic acid (CGA) is a primary phenolic component of coffee and (−)-epigallocatechin gallate (EGCG) is a primary flavonoid component of green tea, both of which have been documented to possess beneficial health properties. A previous study by the present authors demonstrated that p38 mitogen-activated protein kinase (MAPK) may be associated with osteoprotegerin synthesis stimulated by bone morphogenetic protein-4 (BMP-4) in osteoblast-like MC3T3-E1 cells. In the present study, the effects of CGA and EGCG on BMP-4-stimulated osteoprotegerin synthesis in MC3T3-E1 cells were investigated. It was observed that CGA had no effect on osteoprotegerin release stimulated by BMP-4, whereas EGCG significantly enhanced BMP-4-stimulated osteoprotegerin release (P=0.003). Levels of osteoprotegerin mRNA expression induced by BMP-4 were also significantly increased by EGCG (P=0.03). By contrast, EGCG had no significant effect on phosphorylation of Smad1 or p38 MAPK induced by BMP-4. In addition, EGCG had little effect on BMP-induced phosphorylation of p70 S6 kinase; however rapamycin, as an inhibitor of p70 S6 kinase, significantly suppressed osteoprotegerin release (P=0.007). These data suggest that EGCG but not CGA may upregulate the synthesis of osteoprotegerin induced by BMP-4 in osteoblasts. PMID:28672948

  9. Ligand screening system using fusion proteins of G protein-coupled receptors with G protein alpha subunits.

    PubMed

    Suga, Hinako; Haga, Tatsuya

    2007-01-01

    G protein-coupled receptors (GPCRs) constitute one of the largest families of genes in the human genome, and are the largest targets for drug development. Although a large number of GPCR genes have recently been identified, ligands have not yet been identified for many of them. Various assay systems have been employed to identify ligands for orphan GPCRs, but there is still no simple and general method to screen for ligands of such GPCRs, particularly of G(i)-coupled receptors. We have examined whether fusion proteins of GPCRs with G protein alpha subunit (Galpha) could be utilized for ligand screening and showed that the fusion proteins provide an effective method for the purpose. This article focuses on the followings: (1) characterization of GPCR genes and GPCRs, (2) identification of ligands for orphan GPCRs, (3) characterization of GPCR-Galpha fusion proteins, and (4) identification of ligands for orphan GPCRs using GPCR-Galpha fusion proteins.

  10. Segmental Bone Regeneration Using a Load Bearing Biodegradable Carrier of Bone Morphogenetic Protein-2

    PubMed Central

    Chu, Tien-Min G.; Warden, Stuart J.; Turner, Charles H.; Stewart, Rena L.

    2006-01-01

    Segmental defect regeneration has been a clinical challenge. Current tissue engineering approach using porous biodegradable scaffolds to delivery osteogenic cells and growth factors demonstrated success in facilitating bone regeneration in these cases. However, due to the lack of mechanical property, the porous scaffolds were evaluated in non-load bearing area or were stabilized with stress-shielding devices (bone plate or external fixation). In this paper, we tested a scaffold that does not require a bone plate because it has sufficient biomechanical strength. The tube-shaped scaffolds were manufactured from poly(propylene) fumarate/tricalcium phosphate (PPF/TCP) composites. Dicalcium phosphate dehydrate (DCPD) were used as bone morphogenetic protein -2 (BMP-2) carrier. Twenty two scaffolds were implanted in 5 mm segmental defects in rat femurs stabilized with k-wire for 6 and 15 weeks with and without 10 μg of rhBMP-2. Bridging of the segmental defect was evaluated first radiographically and was confirmed by histology and micro- computer tomography (μ-CT) imaging. The scaffolds in the BMP group maintained the bone length throughout the duration of the study and allow for bridging. The scaffolds in the control group failed to induce bridging and collapsed at 15 weeks. Peripheral computed tomography (pQCT) showed that BMP-2 does not increase the bone mineral density in the callus. Finally, the scaffold in BMP group was found to restore the mechanical property of the rat femur after 15 weeks. Our results demonstrated that the load-bearing BMP-2 scaffold can maintain bone length and allow successfully regeneration in segmental defects. PMID:16996588

  11. Bone morphogenetic proteins: a powerful osteoinductive compound with non-negligible side effects and limitations.

    PubMed

    Oryan, Ahmad; Alidadi, Soodeh; Moshiri, Ali; Bigham-Sadegh, Amin

    2014-01-01

    Healing and regeneration of large bone defects leading to non-unions is a great concern in orthopedic surgery. Since auto- and allografts have limitations, bone tissue engineering and regenerative medicine (TERM) has attempted to solve this issue. In TERM, healing promotive factors are necessary to regulate the several important events during healing. An ideal treatment strategy should provide osteoconduction, osteoinduction, osteogenesis, and osteointegration of the graft or biomaterials within the healing bone. Since many materials have osteoconductive properties, only a few biomaterials have osteoinductive properties which are important for osteogenesis and osteointegration. Bone morphogenetic proteins (BMPs) are potent inductors of the osteogenic and angiogenic activities during bone repair. The BMPs can regulate the production and activity of some growth factors which are necessary for the osteogenesis. Since the introduction of BMP, it has added a valuable tool to the surgeon's possibilities and is most commonly used in bone defects. Despite significant evidences suggesting their potential benefit on bone healing, there are some evidences showing their side effects such as ectopic bone formation, osteolysis and problems related to cost effectiveness. Bone tissue engineering may create a local environment, using the delivery systems, which enables BMPs to carry out their activities and to lower cost and complication rate associated with BMPs. This review represented the most important concepts and evidences regarding the role of BMPs on bone healing and regeneration from basic to clinical application. The major advantages and disadvantages of such biologic compounds together with the BMPs substitutes are also discussed. © 2014 International Union of Biochemistry and Molecular Biology.

  12. N-methyl pyrrolidone/bone morphogenetic protein-2 double delivery with in situ forming implants.

    PubMed

    Karfeld-Sulzer, Lindsay S; Ghayor, Chafik; Siegenthaler, Barbara; de Wild, Michael; Leroux, Jean-Christophe; Weber, Franz E

    2015-04-10

    Bone morphogenetic proteins (BMPs) are growth and differentiation factors involved during development in morphogenesis, organogenesis and later mainly in regeneration processes, in particular in bone where they are responsible for osteoinduction. For more than a decade, recombinant human (rh)BMP-2 has been used in the clinic for lumbar spinal fusion at non-physiological high dosages that appear to be causative for side effects, like male sterility. A possible strategy to reduce the effective amount of rhBMP-2 in the clinic is the co-delivery with an enhancer of BMPs' activity. In an earlier study, we showed that N-methylpyrrolidone (NMP) enhances BMP activity in vitro and in vivo. Here we report on the development of a slow and sustained double delivery of rhBMP-2 and NMP via an in situ forming implant based on poly(lactide-co-glycolide). The results showed that the release of NMP can be adjusted by varying the lactide/glycolide ratio and the polymer's molecular weight. The same applied to rhBMP-2, with release rates that could be sustained from two to three weeks. In the in vivo model of a critical size defect in the calvarial bone of rabbits, the implant containing 50mol% lactide performed better than the one having 75mol% lactide in terms of defect bridging and extent of bony regenerated area. In situ forming implants for the double delivery of the BMP enhancer NMP and rhBMP-2 appear to be promising delivery systems in bone regeneration. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. TAM receptors, Gas6, and protein S: roles in inflammation and hemostasis.

    PubMed

    van der Meer, Jonathan H M; van der Poll, Tom; van 't Veer, Cornelis

    2014-04-17

    TAM receptors (Tyro3, Axl, and Mer) belong to a family of receptor tyrosine kinases that have important effects on hemostasis and inflammation. Also, they affect cell proliferation, survival, adhesion, and migration. TAM receptors can be activated by the vitamin K-dependent proteins Gas6 and protein S. Protein S is more commonly known as an important cofactor for protein C as well as a direct inhibitor of multiple coagulation factors. To our knowledge, the functions of Gas6 are limited to TAM receptor activation. When activated, the TAM receptors have effects on primary hemostasis and coagulation and display an anti-inflammatory or a proinflammatory effect, depending on cell type. To comprehend the effects that the TAM receptors and their ligands have on hemostasis and inflammation, we compare studies that report the different phenotypes displayed by mice with deficiencies in the genes of this receptor family and its ligands (protein S(+/-), Gas6(-/-), TAM(-/-), and variations of these). In this manner, we aim to display which features are attributable to the different ligands. Because of the effects TAM receptors have on hemostasis, inflammation, and cancer growth, their modulation could make interesting therapeutic targets in thromboembolic disease, atherosclerosis, sepsis, autoimmune disease, and cancer.

  14. G Protein-Coupled Receptors in Anopheles gambiae

    NASA Astrophysics Data System (ADS)

    Hill, Catherine A.; Fox, A. Nicole; Pitts, R. Jason; Kent, Lauren B.; Tan, Perciliz L.; Chrystal, Mathew A.; Cravchik, Anibal; Collins, Frank H.; Robertson, Hugh M.; Zwiebel, Laurence J.

    2002-10-01

    We used bioinformatic approaches to identify a total of 276 G protein-coupled receptors (GPCRs) from the Anopheles gambiae genome. These include GPCRs that are likely to play roles in pathways affecting almost every aspect of the mosquito's life cycle. Seventy-nine candidate odorant receptors were characterized for tissue expression and, along with 76 putative gustatory receptors, for their molecular evolution relative to Drosophila melanogaster. Examples of lineage-specific gene expansions were observed as well as a single instance of unusually high sequence conservation.

  15. Arrestin–dependent but G–protein coupled receptor kinase–independent uncoupling of D2–dopamine receptors

    PubMed Central

    Celver, Jeremy; Sharma, Meenakshi; Thanawala, Vaidehi; Octeau, J. Christopher; Kovoor, Abraham

    2016-01-01

    We reconstituted D2 like dopamine receptor (D2R) and the delta opioid receptor (DOR) coupling to G-protein gated inwardly rectifying potassium channels (Kir3) and directly compared the effects of co-expression of G-protein coupled receptor kinase (GRK) and arrestin on agonist-dependent desensitization of the receptor response. We found, as described previously, that co-expression of a GRK and an arrestin synergistically increased the rate of agonist-dependent desensitization of DOR. In contrast, only arrestin expression was required to produce desensitization of D2R responses. Furthermore, arrestin-dependent GRK-independent desensitization of D2R-Kir3 coupling could be transferred to DOR by substituting the third cytoplasmic loop of DOR with that of D2R. The arrestin-dependent GRK-independent desensitization of D2R desensitization was inhibited by staurosporine treatment, and blocked by alanine substitution of putative protein kinase C phosphorylation sites in the third cytoplasmic loop of D2R. Finally, the D2R construct in which putative protein kinase C phosphorylation sites were mutated did not undergo significant agonist-dependent desensitization even after GRK co-expression, suggesting that GRK phosphorylation of D2R does not play an important role in uncoupling of the receptor. PMID:23815307

  16. A role for AT1 receptor-associated proteins in blood pressure regulation.

    PubMed

    Castrop, Hayo

    2015-04-01

    The renin angiotensin-system is one of the most important humoral regulators of blood pressure. The recently discovered angiotensin receptor-associated proteins serve as local modulators of the renin angiotensin-system. These proteins interact with the AT1 receptor in a tissue-specific manner and regulate the sensitivity of the target cell for angiotensin II. The predominant effect of the AT1 receptor-associated proteins on angiotensin II-induced signaling is the modulation of the surface expression of the AT1 receptor. This review provides an overview of our current knowledge with respect to the relevance of AT1 receptor-associated proteins for blood pressure regulation. Two aspects of blood pressure regulation will be discussed in detail: angiotensin II-dependent volume homoeostasis and vascular resistance. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Structural basis of G protein-coupled receptor-Gi protein interaction: formation of the cannabinoid CB2 receptor-Gi protein complex.

    PubMed

    Mnpotra, Jagjeet S; Qiao, Zhuanhong; Cai, Jian; Lynch, Diane L; Grossfield, Alan; Leioatts, Nicholas; Hurst, Dow P; Pitman, Michael C; Song, Zhao-Hui; Reggio, Patricia H

    2014-07-18

    In this study, we applied a comprehensive G protein-coupled receptor-Gαi protein chemical cross-linking strategy to map the cannabinoid receptor subtype 2 (CB2)-Gαi interface and then used molecular dynamics simulations to explore the dynamics of complex formation. Three cross-link sites were identified using LC-MS/MS and electrospray ionization-MS/MS as follows: 1) a sulfhydryl cross-link between C3.53(134) in TMH3 and the Gαi C-terminal i-3 residue Cys-351; 2) a lysine cross-link between K6.35(245) in TMH6 and the Gαi C-terminal i-5 residue, Lys-349; and 3) a lysine cross-link between K5.64(215) in TMH5 and the Gαi α4β6 loop residue, Lys-317. To investigate the dynamics and nature of the conformational changes involved in CB2·Gi complex formation, we carried out microsecond-time scale molecular dynamics simulations of the CB2 R*·Gαi1β1γ2 complex embedded in a 1-palmitoyl-2-oleoyl-phosphatidylcholine bilayer, using cross-linking information as validation. Our results show that although molecular dynamics simulations started with the G protein orientation in the β2-AR*·Gαsβ1γ2 complex crystal structure, the Gαi1β1γ2 protein reoriented itself within 300 ns. Two major changes occurred as follows. 1) The Gαi1 α5 helix tilt changed due to the outward movement of TMH5 in CB2 R*. 2) A 25° clockwise rotation of Gαi1β1γ2 underneath CB2 R* occurred, with rotation ceasing when Pro-139 (IC-2 loop) anchors in a hydrophobic pocket on Gαi1 (Val-34, Leu-194, Phe-196, Phe-336, Thr-340, Ile-343, and Ile-344). In this complex, all three experimentally identified cross-links can occur. These findings should be relevant for other class A G protein-coupled receptors that couple to Gi proteins. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. G-protein-coupled receptors signaling pathways in new antiplatelet drug development.

    PubMed

    Gurbel, Paul A; Kuliopulos, Athan; Tantry, Udaya S

    2015-03-01

    Platelet G-protein-coupled receptors influence platelet function by mediating the response to various agonists, including ADP, thromboxane A2, and thrombin. Blockade of the ADP receptor, P2Y12, in combination with cyclooxygenase-1 inhibition by aspirin has been among the most widely used pharmacological strategies to reduce cardiovascular event occurrence in high-risk patients. The latter dual pathway blockade strategy is one of the greatest advances in the field of cardiovascular medicine. In addition to P2Y12, the platelet thrombin receptor, protease activated receptor-1, has also been recently targeted for inhibition. Blockade of protease activated receptor-1 has been associated with reduced thrombotic event occurrence when added to a strategy using P2Y12 and cyclooxygenase-1 inhibition. At this time, the relative contributions of these G-protein-coupled receptor signaling pathways to in vivo thrombosis remain incompletely defined. The observation of treatment failure in ≈10% of high-risk patients treated with aspirin and potent P2Y12 inhibitors provides the rationale for targeting novel pathways mediating platelet function. Targeting intracellular signaling downstream from G-protein-coupled receptor receptors with phosphotidylionisitol 3-kinase and Gq inhibitors are among the novel strategies under investigation to prevent arterial ischemic event occurrence. Greater understanding of the mechanisms of G-protein-coupled receptor-mediated signaling may allow the tailoring of antiplatelet therapy. © 2015 American Heart Association, Inc.

  19. In vitro and in vivo evaluation of bone morphogenetic protein-2 (BMP-2) immobilized collagen-coated polyetheretherketone (PEEK)

    NASA Astrophysics Data System (ADS)

    Du, Ya-Wei; Zhang, Li-Nan; Ye, Xin; Nie, He-Min; Hou, Zeng-Tao; Zeng, Teng-Hui; Yan, Guo-Ping; Shang, Peng

    2015-03-01

    Polyetheretherketone (PEEK) is regarded as one of the most potential candidates of biomaterials in spinal implant applications. However, as a bioinert material, PEEK plays a limited role in osteoconduction and osseointegration. In this study, recombinant human bone morphogenetic protein-2 (rhBMP-2) was immobilized onto the surface of collagen-coated PEEK in order to prepare a multi-functional material. After adsorbed onto the PEEK surface by hydrophobic interaction, collagen was cross-linked with N-(3-dimethylaminopropyl)-N'-ethyl carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). EDC/NHS system also contributed to the immobilization of rhBMP-2. Water contact angle tests, XPS and SEM clearly demonstrated the surface changes. ELISA tests quantified the amount of rhBMP-2 immobilized and the release over a period of 30 d. In vitro evaluation proved that the osteogenesis differentiation rate was higher when cells were cultured on modified PEEK discs than on regular ones. In vivo tests were conducted and positive changes of major parameters were presented. This report demonstrates that the rhBMP-2 immobilized method for PEEK modification increase bioactivity in vitro and in vivo, suggesting its practicability in orthopedic and spinal clinical applications.

  20. Novel Wnt Regulator NEL-Like Molecule-1 Antagonizes Adipogenesis and Augments Osteogenesis Induced by Bone Morphogenetic Protein 2

    PubMed Central

    Shen, Jia; James, Aaron W.; Zhang, Xinli; Pang, Shen; Zara, Janette N.; Asatrian, Greg; Chiang, Michael; Lee, Min; Khadarian, Kevork; Nguyen, Alan; Lee, Kevin S.; Siu, Ronald K.; Tetradis, Sotirios; Ting, Kang; Soo, Chia

    2017-01-01

    The differentiation factor NEL-like molecule-1 (NELL-1) has been reported as osteoinductive in multiple in vivo preclinical models. Bone morphogenetic protein (BMP)-2 is used clinically for skeletal repair, but in vivo administration can induce abnormal, adipose-filled, poor-quality bone. We demonstrate that NELL-1 combined with BMP2 significantly optimizes osteogenesis in a rodent femoral segmental defect model by minimizing the formation of BMP2-induced adipose-filled cystlike bone. In vitro studies using the mouse bone marrow stromal cell line M2-10B4 and human primary bone marrow stromal cells have confirmed that NELL-1 enhances BMP2-induced osteogenesis and inhibits BMP2-induced adipogenesis. Importantly, the ability of NELL-1 to direct BMP2-treated cells toward osteogenesis and away from adipogenesis requires intact canonical Wnt signaling. Overall, these studies establish the feasibility of combining NELL-1 with BMP2 to improve clinical bone regeneration and provide mechanistic insight into canonical Wnt pathway activity during NELL-1 and BMP2 osteogenesis. The novel abilities of NELL-1 to stimulate Wnt signaling and to repress adipogenesis may highlight new treatment approaches for bone loss in osteoporosis. PMID:26772960

  1. Dephosphorylation of the linker regions of Smad1 and Smad2/3 by small C-terminal domain phosphatases has distinct outcomes for bone morphogenetic protein and transforming growth factor-beta pathways.

    PubMed

    Sapkota, Gopal; Knockaert, Marie; Alarcón, Claudio; Montalvo, Ermelinda; Brivanlou, Ali H; Massagué, Joan

    2006-12-29

    Smad proteins transduce bone morphogenetic protein (BMP) and transforming growth factor-beta (TGFbeta) signals upon phosphorylation of their C-terminal SXS motif by receptor kinases. The activity of Smad1 in the BMP pathway and Smad2/3 in the TGFbeta pathway is restricted by pathway cross-talk and feedback through protein kinases, including MAPK, CDK2/4, p38MAPK, JNK, and others. These kinases phosphorylate Smads 1-3 at the region that links the N-terminal DNA-binding domain and the C-terminal transcriptional domain. Phosphatases that dephosphorylate the linker region are therefore likely to play an integral part in the regulation of Smad activity. We reported previously that small C-terminal domain phosphatases 1, 2, and 3 (SCP1-3) dephosphorylate Smad1 C-terminal tail, thereby attenuating BMP signaling. Here we provide evidence that SCP1-3 also dephosphorylate the linker regions of Smad1 and Smad2/3 in vitro, in mammalian cells and in Xenopus embryos. Overexpression of SCP 1, 2, or 3 decreased linker phosphorylation of Smads 1, 2 and 3. Moreover, RNA interference-mediated knockdown of SCP1/2 increased the BMP-dependent phosphorylation of the Smad1 linker region as well as the C terminus. In contrast, SCP1/2 knockdown increased the TGFbeta-dependent linker phosphorylation of Smad2/3 but not the C-terminal phosphorylation. Consequently, SCP1/2 knockdown inhibited TGFbeta transcriptional responses, but it enhanced BMP transcriptional responses. Thus, by dephosphorylating Smad2/3 at the linker (inhibitory) but not the C-terminal (activating) site, the SCPs enhance TGFbeta signaling, and by dephosphorylating Smad1 at both sites, the SCPs reset Smad1 to the basal unphosphorylated state.

  2. Signaling through G protein coupled receptors.

    PubMed

    Tuteja, Narendra

    2009-10-01

    Heterotrimeric G proteins (Galpha, Gbeta/Ggamma subunits) constitute one of the most important components of cell signaling cascade. G Protein Coupled Receptors (GPCRs) perceive many extracellular signals and transduce them to heterotrimeric G proteins, which further transduce these signals intracellular to appropriate downstream effectors and thereby play an important role in various signaling pathways. GPCRs exist as a superfamily of integral membrane protein receptors that contain seven transmembrane alpha-helical regions, which bind to a wide range of ligands. Upon activation by a ligand, the GPCR undergoes a conformational change and then activate the G proteins by promoting the exchange of GDP/GTP associated with the Galpha subunit. This leads to the dissociation of Gbeta/Ggamma dimer from Galpha. Both these moieties then become free to act upon their downstream effectors and thereby initiate unique intracellular signaling responses. After the signal propagation, the GTP of Galpha-GTP is hydrolyzed to GDP and Galpha becomes inactive (Galpha-GDP), which leads to its re-association with the Gbeta/Ggamma dimer to form the inactive heterotrimeric complex. The GPCR can also transduce the signal through G protein independent pathway. GPCRs also regulate cell cycle progression. Till to date thousands of GPCRs are known from animal kingdom with little homology among them, but only single GPCR has been identified in plant system. The Arabidopsis GPCR was reported to be cell cycle regulated and also involved in ABA and in stress signaling. Here I have described a general mechanism of signal transduction through GPCR/G proteins, structure of GPCRs, family of GPCRs and plant GPCR and its role.

  3. Expression of G(alpha)(s) proteins and TSH receptor signalling in hyperfunctioning thyroid nodules with TSH receptor mutations.

    PubMed

    Holzapfel, Hans-Peter; Bergner, Beate; Wonerow, Peter; Paschke, Ralf

    2002-07-01

    Constitutively activating mutations of the thyrotrophin receptor (TSHR) are the main molecular cause of hyperfunctioning thyroid nodules (HTNs). The G protein coupling is an important and critical step in the TSHR signalling which mainly includes G(alpha)(s), G(alpha)(i) and G(alpha)(q)/11 proteins. We investigated the in vitro consequences of overexpressing G(alpha) proteins on signalling of the wild-type (WT) or mutated TSHR. Moreover, we investigated whether changes in G(alpha) protein expression are pathophysiologically relevant in HTNs or cold thyroid nodules (CTNs). Wild-type TSH receptor and mutated TSH receptors were coexpressed with G(alpha)(s), G(alpha)(i) or G(alpha)(q)/11, and cAMP and inositol phosphate (IP) production was measured after stimulation with TSH. The expression of G(alpha)(s), G(alpha)(i) and G(alpha)(q)/11 proteins was examined by Western blotting in 28 HTNs and 14 CTNs. Coexpression of G(alpha)(s) with the WT TSH receptor in COS 7 cells significantly increased the basal and TSH-stimulated cAMP accumulation while coexpression of the G(alpha)(q) or G(alpha)11 protein significantly increased the production of cAMP and inositol triphosphate (IP(3)). The coexpression of the TSH receptor mutants (I486F, DEL613-621), known to couple constitutively to G(alpha)(s) and G(alpha)(q) with G(alpha)(s) and G(alpha)(q)/11, significantly increased the basal and stimulated cAMP and IP(3) accumulation. Coexpression of the TSH receptor mutant V556F with G(alpha)(s) only increased the basal and stimulated cAMP production while its coexpression with G(alpha)(q)/11 increased the basal and stimulated IP(3) signalling. The expression of G(alpha)(s) protein subunits determined by Western blotting was significantly decreased in 14 HTNs with a constitutively activating TSH receptor mutation in comparison with the corresponding surrounding tissue, while in 14 HTNs without TSH receptor or G(alpha)(s) protein mutation and in 14 CTNs the expression of G

  4. How much do we know about the coupling of G-proteins to serotonin receptors?

    PubMed Central

    2014-01-01

    Serotonin receptors are G-protein-coupled receptors (GPCRs) involved in a variety of psychiatric disorders. G-proteins, heterotrimeric complexes that couple to multiple receptors, are activated when their receptor is bound by the appropriate ligand. Activation triggers a cascade of further signalling events that ultimately result in cell function changes. Each of the several known G-protein types can activate multiple pathways. Interestingly, since several G-proteins can couple to the same serotonin receptor type, receptor activation can result in induction of different pathways. To reach a better understanding of the role, interactions and expression of G-proteins a literature search was performed in order to list all the known heterotrimeric combinations and serotonin receptor complexes. Public databases were analysed to collect transcript and protein expression data relating to G-proteins in neural tissues. Only a very small number of heterotrimeric combinations and G-protein-receptor complexes out of the possible thousands suggested by expression data analysis have been examined experimentally. In addition this has mostly been obtained using insect, hamster, rat and, to a lesser extent, human cell lines. Besides highlighting which interactions have not been explored, our findings suggest additional possible interactions that should be examined based on our expression data analysis. PMID:25011628

  5. How much do we know about the coupling of G-proteins to serotonin receptors?

    PubMed

    Giulietti, Matteo; Vivenzio, Viviana; Piva, Francesco; Principato, Giovanni; Bellantuono, Cesario; Nardi, Bernardo

    2014-07-10

    Serotonin receptors are G-protein-coupled receptors (GPCRs) involved in a variety of psychiatric disorders. G-proteins, heterotrimeric complexes that couple to multiple receptors, are activated when their receptor is bound by the appropriate ligand. Activation triggers a cascade of further signalling events that ultimately result in cell function changes. Each of the several known G-protein types can activate multiple pathways. Interestingly, since several G-proteins can couple to the same serotonin receptor type, receptor activation can result in induction of different pathways. To reach a better understanding of the role, interactions and expression of G-proteins a literature search was performed in order to list all the known heterotrimeric combinations and serotonin receptor complexes. Public databases were analysed to collect transcript and protein expression data relating to G-proteins in neural tissues. Only a very small number of heterotrimeric combinations and G-protein-receptor complexes out of the possible thousands suggested by expression data analysis have been examined experimentally. In addition this has mostly been obtained using insect, hamster, rat and, to a lesser extent, human cell lines. Besides highlighting which interactions have not been explored, our findings suggest additional possible interactions that should be examined based on our expression data analysis.

  6. In vitro bone formation using muscle-derived cells: a new paradigm for bone tissue engineering using polymer-bone morphogenetic protein matrices.

    PubMed

    Lu, Helen H; Kofron, Michelle D; El-Amin, Saadiq F; Attawia, Mohammed A; Laurencin, Cato T

    2003-06-13

    Over 800,000 bone grafting procedures are performed in the United States annually, creating a demand for viable alternatives to autogenous bone, the grafting standard in osseous repair. The objective of this study was to examine the efficacy of a BMP-polymer matrix in inducing the expression of the osteoblastic phenotype and in vitro bone formation by muscle-derived cells. Specifically, we evaluated the ability of bone morphogenetic protein-7 (BMP-7), delivered from a poly(lactide-co-glycolide) (PLAGA) matrix, to induce the differentiation of cells derived from rabbit skeletal muscle into osteoblast-like cells and subsequently form mineralized tissue. Results confirmed that muscle-derived cells attached and proliferated on the PLAGA substrates. BMP-7 released from PLAGA induced the muscle-derived cells to increase bone marker expression and form mineralized cultures. These results demonstrate the efficacy of a BMP-polymer matrix in inducing the expression of the osteoblastic phenotype by muscle-derived cells and present a new paradigm for bone tissue engineering.

  7. Palatogenesis: morphogenetic and molecular mechanisms of secondary palate development

    PubMed Central

    Bush, Jeffrey O.; Jiang, Rulang

    2012-01-01

    Mammalian palatogenesis is a highly regulated morphogenetic process during which the embryonic primary and secondary palatal shelves develop as outgrowths from the medial nasal and maxillary prominences, respectively, remodel and fuse to form the intact roof of the oral cavity. The complexity of control of palatogenesis is reflected by the common occurrence of cleft palate in humans. Although the embryology of the palate has long been studied, the past decade has brought substantial new knowledge of the genetic control of secondary palate development. Here, we review major advances in the understanding of the morphogenetic and molecular mechanisms controlling palatal shelf growth, elevation, adhesion and fusion, and palatal bone formation. PMID:22186724

  8. Synaptic proteins and receptors defects in autism spectrum disorders

    PubMed Central

    Chen, Jianling; Yu, Shunying; Fu, Yingmei; Li, Xiaohong

    2014-01-01

    Recent studies have found that hundreds of genetic variants, including common and rare variants, rare and de novo mutations, and common polymorphisms contribute to the occurrence of autism spectrum disorders (ASDs). The mutations in a number of genes such as neurexin, neuroligin, postsynaptic density protein 95, SH3, and multiple ankyrin repeat domains 3 (SHANK3), synapsin, gephyrin, cadherin, and protocadherin, thousand-and-one-amino acid 2 kinase, and contactin, have been shown to play important roles in the development and function of synapses. In addition, synaptic receptors, such as gamma-aminobutyric acid receptors and glutamate receptors, have also been associated with ASDs. This review will primarily focus on the defects of synaptic proteins and receptors associated with ASDs and their roles in the pathogenesis of ASDs via synaptic pathways. PMID:25309321

  9. Promising efficacy of Escherichia coli recombinant human bone morphogenetic protein-2 in collagen sponge for ectopic and orthotopic bone formation and comparison with mammalian cell recombinant human bone morphogenetic protein-2.

    PubMed

    Kim, In Sook; Lee, Eui Nam; Cho, Tae Hyung; Song, Yun Mi; Hwang, Soon Jung; Oh, Ji Hye; Park, Eun Kyung; Koo, Tai Young; Seo, Young-Kwon

    2011-02-01

    Nonglycosylated recombinant human bone morphogenetic protein (rhBMP)-2 prepared in Escherichia coli (E. coli rhBMP-2) has recently been considered as an alternative to mammalian cell rhBMP-2. However, its clinical use is still limited owing to lack of evidence for osteogenic activity comparable with that of mammalian cell rhBMP-2 via microcomputed tomography-based analysis. Therefore, this study aimed to evaluate the ability of E. coli rhBMP-2 in absorbable collagen sponge to form ectopic and orthotopic bone and to compare it to that of mammalian rhBMP-2. In vitro investigation was performed to study osteoblast differentiation of human mesenchymal stromal cells. Both types of rhBMP-2 enhanced proliferation, alkaline phosphatase activity, and matrix mineralization of human mesenchymal stromal cells at similar levels. Similar tendencies were observed in microcomputed tomography analysis, which determined bone volume, fractional bone volume, trabecular thickness, trabecular separation, bone mineral density, and other characteristics. Histology from an in vivo osteoinductivity test and from a rat calvarial defect model demonstrated a dose-dependent increase in local bone formation. The E. coli rhBMP-2 group (5 μg) not only induced complete regeneration of an 8-mm critical-sized defect at 4 weeks, but also led to new bone with the same bone mineral density as normal bone at 8 weeks, with the same efficiency as that of mammalian cell rhBMP-2 (5 μg). These uniformly favorable results provide evidence that the osteogenic activity of E. coli rhBMP-2 is not inferior to that of mammalian cell rhBMP-2 despite its low solubility and lack of gylcosylation. These results suggest that the application of E. coli rhBMP-2 in absorbable collagen sponge may be a promising equivalent to mammalian cell rhBMP-2 in bone tissue engineering.

  10. Artificial Symmetry-Breaking for Morphogenetic Engineering Bacterial Colonies.

    PubMed

    Nuñez, Isaac N; Matute, Tamara F; Del Valle, Ilenne D; Kan, Anton; Choksi, Atri; Endy, Drew; Haseloff, Jim; Rudge, Timothy J; Federici, Fernan

    2017-02-17

    Morphogenetic engineering is an emerging field that explores the design and implementation of self-organized patterns, morphologies, and architectures in systems composed of multiple agents such as cells and swarm robots. Synthetic biology, on the other hand, aims to develop tools and formalisms that increase reproducibility, tractability, and efficiency in the engineering of biological systems. We seek to apply synthetic biology approaches to the engineering of morphologies in multicellular systems. Here, we describe the engineering of two mechanisms, symmetry-breaking and domain-specific cell regulation, as elementary functions for the prototyping of morphogenetic instructions in bacterial colonies. The former represents an artificial patterning mechanism based on plasmid segregation while the latter plays the role of artificial cell differentiation by spatial colocalization of ubiquitous and segregated components. This separation of patterning from actuation facilitates the design-build-test-improve engineering cycle. We created computational modules for CellModeller representing these basic functions and used it to guide the design process and explore the design space in silico. We applied these tools to encode spatially structured functions such as metabolic complementation, RNAPT7 gene expression, and CRISPRi/Cas9 regulation. Finally, as a proof of concept, we used CRISPRi/Cas technology to regulate cell growth by controlling methionine synthesis. These mechanisms start from single cells enabling the study of morphogenetic principles and the engineering of novel population scale structures from the bottom up.

  11. Guanine nucleotide regulatory protein co-purifies with the D/sub 2/-dopamine receptor

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Senogles, S.E.; Caron, M.G.

    1986-05-01

    The D/sub 2/-dopamine receptor from bovine anterior pituitary was purified approx.1000 fold by affinity chromatography on CMOS-Sepharose. Reconstitution of the affinity-purified receptor into phospholipid vesicles revealed the presence of high and low affinity agonist sites as detected by N-n-propylnorapomorphine (NPA) competition experiments with /sup 3/H-spiperone. High affinity agonist binding could be converted to the low affinity form by guanine nucleotides, indicating the presence of an endogenous guanine nucleotide binding protein (N protein) in the affinity-purified D/sub 2/ receptor preparations. Furthermore, this preparation contained an agonist-sensitive GTPase activity which was stimulated 2-3 fold over basal by 10 ..mu..M NPA. /sup 35/S-GTP..gamma..Smore » binding to these preparations revealed a stoichiometry of 0.4-0.7 mole N protein/mole receptor, suggesting the N protein may be specifically coupled with the purified D/sub 2/-dopamine receptor and not present as a contaminant. Pertussis toxin treatment of the affinity purified receptor preparations prevented high affinity agonist binding, as well as agonist stimulation of the GTPase activity, presumably by inactivating the associated N protein. Pertussis toxin lead to the ADP-ribosylation of a protein of 39-40K on SDS-PAGE. These findings indicate that an endogenous N protein, N/sub i/ or N/sub o/, co-purifies with the D/sub 2/-dopamine receptor which may reflect a precoupling of this receptor with an N protein within the membranes.« less

  12. G Protein-Coupled Receptor Signaling in Stem Cells and Cancer.

    PubMed

    Lynch, Jennifer R; Wang, Jenny Yingzi

    2016-05-11

    G protein-coupled receptors (GPCRs) are a large superfamily of cell-surface signaling proteins that bind extracellular ligands and transduce signals into cells via heterotrimeric G proteins. GPCRs are highly tractable drug targets. Aberrant expression of GPCRs and G proteins has been observed in various cancers and their importance in cancer stem cells has begun to be appreciated. We have recently reported essential roles for G protein-coupled receptor 84 (GPR84) and G protein subunit Gαq in the maintenance of cancer stem cells in acute myeloid leukemia. This review will discuss how GPCRs and G proteins regulate stem cells with a focus on cancer stem cells, as well as their implications for the development of novel targeted cancer therapies.

  13. G Protein-Coupled Receptor Signaling in Stem Cells and Cancer

    PubMed Central

    Lynch, Jennifer R.; Wang, Jenny Yingzi

    2016-01-01

    G protein-coupled receptors (GPCRs) are a large superfamily of cell-surface signaling proteins that bind extracellular ligands and transduce signals into cells via heterotrimeric G proteins. GPCRs are highly tractable drug targets. Aberrant expression of GPCRs and G proteins has been observed in various cancers and their importance in cancer stem cells has begun to be appreciated. We have recently reported essential roles for G protein-coupled receptor 84 (GPR84) and G protein subunit Gαq in the maintenance of cancer stem cells in acute myeloid leukemia. This review will discuss how GPCRs and G proteins regulate stem cells with a focus on cancer stem cells, as well as their implications for the development of novel targeted cancer therapies. PMID:27187360

  14. Spatially restricted G protein-coupled receptor activity via divergent endocytic compartments.

    PubMed

    Jean-Alphonse, Frederic; Bowersox, Shanna; Chen, Stanford; Beard, Gemma; Puthenveedu, Manojkumar A; Hanyaloglu, Aylin C

    2014-02-14

    Postendocytic sorting of G protein-coupled receptors (GPCRs) is driven by their interactions between highly diverse receptor sequence motifs with their interacting proteins, such as postsynaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1), zonula occludens-1 protein (zo-1) (PDZ) domain proteins. However, whether these diverse interactions provide an underlying functional specificity, in addition to driving sorting, is unknown. Here we identify GPCRs that recycle via distinct PDZ ligand/PDZ protein pairs that exploit their recycling machinery primarily for targeted endosomal localization and signaling specificity. The luteinizing hormone receptor (LHR) and β2-adrenergic receptor (B2AR), two GPCRs sorted to the regulated recycling pathway, underwent divergent trafficking to distinct endosomal compartments. Unlike B2AR, which traffics to early endosomes (EE), LHR internalizes to distinct pre-early endosomes (pre-EEs) for its recycling. Pre-EE localization required interactions of the LHR C-terminal tail with the PDZ protein GAIP-interacting protein C terminus, inhibiting its traffic to EEs. Rerouting the LHR to EEs, or EE-localized GPCRs to pre-EEs, spatially reprograms MAPK signaling. Furthermore, LHR-mediated activation of MAPK signaling requires internalization and is maintained upon loss of the EE compartment. We propose that combinatorial specificity between GPCR sorting sequences and interacting proteins dictates an unprecedented spatiotemporal control in GPCR signal activity.

  15. Competitive Binding Assay for the G-Protein-Coupled Receptor 30 (GPR30) or G-Protein-Coupled Estrogen Receptor (GPER).

    PubMed

    Thekkumkara, Thomas; Snyder, Russell; Karamyan, Vardan T

    2016-01-01

    The role of 2-methoxyestradiol is becoming a major area of investigation because of its therapeutic utility, though its mechanism is not fully explored. Recent studies have identified the G-protein-coupled receptor 30 (GPR30, GPER) as a high-affinity membrane receptor for 2-methoxyestradiol. However, studies aimed at establishing the binding affinities of steroid compounds for specific targets are difficult, as the tracers are highly lipophilic and often result in nonspecific binding in lipid-rich membrane preparations with low-level target receptor expression. 2-Methoxyestradiol binding studies are essential to elucidate the underlying effects of this novel estrogen metabolite and to validate its targets; therefore, this competitive receptor-binding assay protocol was developed in order to assess the membrane receptor binding and affinity of 2-methyoxyestradiol.

  16. Coordinated Proliferation and Differentiation of Human-Induced Pluripotent Stem Cell-Derived Cardiac Progenitor Cells Depend on Bone Morphogenetic Protein Signaling Regulation by GREMLIN 2

    PubMed Central

    Bylund, Jeffery B.; Trinh, Linh T.; Awgulewitsch, Cassandra P.; Paik, David T.; Jetter, Christopher; Jha, Rajneesh; Zhang, Jianhua; Nolan, Kristof; Xu, Chunhui; Thompson, Thomas B.; Kamp, Timothy J.

    2017-01-01

    Heart development depends on coordinated proliferation and differentiation of cardiac progenitor cells (CPCs), but how the two processes are synchronized is not well understood. Here, we show that the secreted Bone Morphogenetic Protein (BMP) antagonist GREMLIN 2 (GREM2) is induced in CPCs shortly after cardiac mesoderm specification during differentiation of human pluripotent stem cells. GREM2 expression follows cardiac lineage differentiation independently of the differentiation method used, or the origin of the pluripotent stem cells, suggesting that GREM2 is linked to cardiogenesis. Addition of GREM2 protein strongly increases cardiomyocyte output compared to established procardiogenic differentiation methods. Our data show that inhibition of canonical BMP signaling by GREM2 is necessary to promote proliferation of CPCs. However, canonical BMP signaling inhibition alone is not sufficient to induce cardiac differentiation, which depends on subsequent JNK pathway activation specifically by GREM2. These findings may have broader implications in the design of approaches to orchestrate growth and differentiation of pluripotent stem cell-derived lineages that depend on precise regulation of BMP signaling. PMID:28125926

  17. Coordinated Proliferation and Differentiation of Human-Induced Pluripotent Stem Cell-Derived Cardiac Progenitor Cells Depend on Bone Morphogenetic Protein Signaling Regulation by GREMLIN 2.

    PubMed

    Bylund, Jeffery B; Trinh, Linh T; Awgulewitsch, Cassandra P; Paik, David T; Jetter, Christopher; Jha, Rajneesh; Zhang, Jianhua; Nolan, Kristof; Xu, Chunhui; Thompson, Thomas B; Kamp, Timothy J; Hatzopoulos, Antonis K

    2017-05-01

    Heart development depends on coordinated proliferation and differentiation of cardiac progenitor cells (CPCs), but how the two processes are synchronized is not well understood. Here, we show that the secreted Bone Morphogenetic Protein (BMP) antagonist GREMLIN 2 (GREM2) is induced in CPCs shortly after cardiac mesoderm specification during differentiation of human pluripotent stem cells. GREM2 expression follows cardiac lineage differentiation independently of the differentiation method used, or the origin of the pluripotent stem cells, suggesting that GREM2 is linked to cardiogenesis. Addition of GREM2 protein strongly increases cardiomyocyte output compared to established procardiogenic differentiation methods. Our data show that inhibition of canonical BMP signaling by GREM2 is necessary to promote proliferation of CPCs. However, canonical BMP signaling inhibition alone is not sufficient to induce cardiac differentiation, which depends on subsequent JNK pathway activation specifically by GREM2. These findings may have broader implications in the design of approaches to orchestrate growth and differentiation of pluripotent stem cell-derived lineages that depend on precise regulation of BMP signaling.

  18. Rab15 Effector Protein: A Novel Protein for Receptor Recycling from the Endocytic Recycling CompartmentD⃞

    PubMed Central

    Strick, David J.; Elferink, Lisa A.

    2005-01-01

    Sorting endosomes and the endocytic recycling compartment are critical intracellular stores for the rapid recycling of internalized membrane receptors to the cell surface in multiple cell types. However, the molecular mechanisms distinguishing fast receptor recycling from sorting endosomes and slow receptor recycling from the endocytic recycling compartment remain poorly understood. We previously reported that Rab15 differentially regulates transferrin receptor trafficking through sorting endosomes and the endocytic recycling compartment, suggesting a role for distinct Rab15-effector interactions at these endocytic compartments. In this study, we identified the novel protein Rab15 effector protein (REP15) as a binding partner for Rab15-GTP. REP15 is compartment specific, colocalizing with Rab15 and Rab11 on the endocytic recycling compartment but not with Rab15, Rab4, or early endosome antigen 1 on sorting endosomes. REP15 interacts directly with Rab15-GTP but not with Rab5 or Rab11. Consistent with its localization, REP15 overexpression and small interfering RNA-mediated depletion inhibited transferrin receptor recycling from the endocytic recycling compartment, without affecting receptor entry into or recycling from sorting endosomes. Our data identify REP15 as a compartment-specific protein for receptor recycling from the endocytic recycling compartment, highlighting that the rapid and slow modes of transferrin receptor recycling are mechanistically distinct pathways. PMID:16195351

  19. Protease-Activated Receptors and other G-Protein-Coupled Receptors: the Melanoma Connection

    PubMed Central

    Rosero, Rebecca A.; Villares, Gabriel J.; Bar-Eli, Menashe

    2016-01-01

    The vast array of G-protein-coupled receptors (GPCRs) play crucial roles in both physiological and pathological processes, including vision, coagulation, inflammation, autophagy, and cell proliferation. GPCRs also affect processes that augment cell proliferation and metastases in many cancers including melanoma. Melanoma is the deadliest form of skin cancer, yet limited therapeutic modalities are available to patients with metastatic melanoma. Studies have found that both chemokine receptors and protease-activated receptors, both of which are GPCRs, are central to the metastatic melanoma phenotype and may serve as potential targets in novel therapies against melanoma and other cancers. PMID:27379162

  20. Protease-Activated Receptors and other G-Protein-Coupled Receptors: the Melanoma Connection.

    PubMed

    Rosero, Rebecca A; Villares, Gabriel J; Bar-Eli, Menashe

    2016-01-01

    The vast array of G-protein-coupled receptors (GPCRs) play crucial roles in both physiological and pathological processes, including vision, coagulation, inflammation, autophagy, and cell proliferation. GPCRs also affect processes that augment cell proliferation and metastases in many cancers including melanoma. Melanoma is the deadliest form of skin cancer, yet limited therapeutic modalities are available to patients with metastatic melanoma. Studies have found that both chemokine receptors and protease-activated receptors, both of which are GPCRs, are central to the metastatic melanoma phenotype and may serve as potential targets in novel therapies against melanoma and other cancers.

  1. Different Requirements for Proteolytic Processing of Bone Morphogenetic Protein 5/6/7/8 Ligands in Drosophila melanogaster*

    PubMed Central

    Fritsch, Cornelia; Sawala, Annick; Harris, Robin; Maartens, Aidan; Sutcliffe, Catherine; Ashe, Hilary L.; Ray, Robert P.

    2012-01-01

    Bone morphogenetic proteins (BMPs) are synthesized as proproteins that undergo proteolytic processing by furin/subtilisin proprotein convertases to release the active ligand. Here we study processing of BMP5/6/7/8 proteins, including the Drosophila orthologs Glass Bottom Boat (Gbb) and Screw (Scw) and human BMP7. Gbb and Scw have three functional furin/subtilisin proprotein convertase cleavage sites; two between the prodomain and ligand domain, which we call the Main and Shadow sites, and one within the prodomain, which we call the Pro site. In Gbb each site can be cleaved independently, although efficient cleavage at the Shadow site requires cleavage at the Main site, and remarkably, none of the sites is essential for Gbb function. Rather, Gbb must be processed at either the Pro or Main site to produce a functional ligand. Like Gbb, the Pro and Main sites in Scw can be cleaved independently, but cleavage at the Shadow site is dependent on cleavage at the Main site. However, both Pro and Main sites are essential for Scw function. Thus, Gbb and Scw have different processing requirements. The BMP7 ligand rescues gbb mutants in Drosophila, but full-length BMP7 cannot, showing that functional differences in the prodomain limit the BMP7 activity in flies. Furthermore, unlike Gbb, cleavage-resistant BMP7, although non-functional in rescue assays, activates the downstream signaling cascade and thus retains some functionality. Our data show that cleavage requirements evolve rapidly, supporting the notion that changes in post-translational processing are used to create functional diversity between BMPs within and between species. PMID:22199351

  2. Bone morphogenetic protein 2 and decorin expression in old fracture fragments and surrounding tissues.

    PubMed

    Han, X G; Wang, D K; Gao, F; Liu, R H; Bi, Z G

    2015-09-21

    Bone morphogenetic protein 2 (BMP-2) can promote fracture healing. Although the complex role BMP-2 in bone formation is increasingly understood, the role of endogenous BMP-2 in nonunion remains unclear. Decorin (DCN) can promote the formation of bone matrix and calcium deposition to control bone morphogenesis. In this study, tissue composition and expression of BMP-2 and DCN were detected in different parts of old fracture zones to explore inherent anti-fibrotic ability and osteogenesis. Twenty-three patients were selected, including eight cases of delayed union and 15 cases of nonunion. Average duration of delayed union or nonunion was 15 months. Fracture fragments and surrounding tissues, including bone grafts, marrow cavity contents, and sticking scars, were categorically sampled during surgery. Through observation and histological testing, component comparisons were made between fracture fragments and surrounding tissue. The expression levels of DCN and BMP-2 in different tissues were detected by immunohistochemical staining and real-time polymerase chain reaction. The expression of DCN and BMP- 2 in different parts of the nonunion area showed that, compared with bone graft and marrow cavity contents, sticking scars had the highest expression of BMP-2. Compared with the marrow cavity contents and sticking scars, bone grafts had the highest expression of DCN. The low antifibrotic and osteogenic activity of the nonunion area was associated with non-co-expression of BMP-2 and DCN. Therefore, the co-injection of osteogenic factor BMP and DCN into the nonunion area can improve the induction of bone formation and enhance the conversion of the old scar, thereby achieving better nonunion treatment.

  3. Potential role of centrioles in determining the morphogenetic status of animal somatic cells.

    PubMed

    Tkemaladze, J; Chichinadze, K

    2005-05-01

    Irreversible differentiation (change of morphogenetic status) and programmed death (apoptosis) are observed only in somatic cells. Cell division is the only way by which the morphogenetic status of the offspring cells may be modified. It is known that there is a fixed limit to the number of possible cell divisions, the so-called 'Hayflick limit'. Existing links between cell division, differentiation and apoptosis make it possible to conclude that all these processes could be controlled by a single self-reproducing structure. Potential candidates for this replicable structure in a somatic cell are chromosomes, mitochondria (both contain DNA), and centrioles. Centrioles (diplosome) are the most likely unit that can fully regulate the processes of irreversible differentiation, determination and modification of the morphogenetic status. It may contain differently encoded RNA molecules stacked in a definite order. During mitosis, these RNA molecules are released one by one into the cytoplasm. In the presence of reverse transcriptase and endonuclease, RNA can be embedded in nuclear DNA. This process presumably changes the status of repressed and potentially active genes and, subsequently, the morphogenetic status of a cell.

  4. [Experimental study on repair of the defect of the pars interarticularis in rat with bone morphogenetic protein and fibrin glue].

    PubMed

    Nakamura, T

    1992-07-01

    The possibility of repairing the defect of the pars interarticularis (pars defect) with Bone Morphogenetic Protein (BMP) and fibrin glue was studied. The pars defect established in the 5th lumbar vertebra of Wistar rat was treated with surgical implantation of a composite consisting of BMP, fibrin glue and autologous cancellous bone. At 3, 6, 9 and 12 weeks after implantation, the osteoinductive activity in the pars defect was observed histologically and compared with that of other composite implants such as BMP with fibrin glue, autologous cancellous bone alone and autologous cancellous bone with fibrin glue. Although perfect bone fusion was not obtained with any of the composites employed, a significant increase in bone formation was seen in a composite of BMP, fibrin glue and autologous cancellous bone (p less than 0.01) as compared with that seen in the others. Consequently, implantation of BMP and fibrin glue combined with some biomaterials which support osteo-induction of BMP and stabilize the pars defect might be successfully applied to repair the pars defect.

  5. Serial Femtosecond Crystallography of G Protein-Coupled Receptors

    PubMed Central

    Liu, Wei; Wacker, Daniel; Gati, Cornelius; Han, Gye Won; James, Daniel; Wang, Dingjie; Nelson, Garrett; Weierstall, Uwe; Katritch, Vsevolod; Barty, Anton; Zatsepin, Nadia A.; Li, Dianfan; Messerschmidt, Marc; Boutet, Sébastien; Williams, Garth J.; Koglin, Jason E.; Seibert, M. Marvin; Wang, Chong; Shah, Syed T.A.; Basu, Shibom; Fromme, Raimund; Kupitz, Christopher; Rendek, Kimberley N.; Grotjohann, Ingo; Fromme, Petra; Kirian, Richard A.; Beyerlein, Kenneth R.; White, Thomas A.; Chapman, Henry N.; Caffrey, Martin; Spence, John C.H.; Stevens, Raymond C.; Cherezov, Vadim

    2014-01-01

    X-ray crystallography of G protein-coupled receptors and other membrane proteins is hampered by difficulties associated with growing sufficiently large crystals that withstand radiation damage and yield high-resolution data at synchrotron sources. Here we used an x-ray free-electron laser (XFEL) with individual 50-fs duration x-ray pulses to minimize radiation damage and obtained a high-resolution room temperature structure of a human serotonin receptor using sub-10 µm microcrystals grown in a membrane mimetic matrix known as lipidic cubic phase. Compared to the structure solved by traditional microcrystallography from cryo-cooled crystals of about two orders of magnitude larger volume, the room temperature XFEL structure displays a distinct distribution of thermal motions and conformations of residues that likely more accurately represent the receptor structure and dynamics in a cellular environment. PMID:24357322

  6. Structural Basis of G Protein-coupled Receptor-Gi Protein Interaction

    PubMed Central

    Mnpotra, Jagjeet S.; Qiao, Zhuanhong; Cai, Jian; Lynch, Diane L.; Grossfield, Alan; Leioatts, Nicholas; Hurst, Dow P.; Pitman, Michael C.; Song, Zhao-Hui; Reggio, Patricia H.

    2014-01-01

    In this study, we applied a comprehensive G protein-coupled receptor-Gαi protein chemical cross-linking strategy to map the cannabinoid receptor subtype 2 (CB2)- Gαi interface and then used molecular dynamics simulations to explore the dynamics of complex formation. Three cross-link sites were identified using LC-MS/MS and electrospray ionization-MS/MS as follows: 1) a sulfhydryl cross-link between C3.53(134) in TMH3 and the Gαi C-terminal i-3 residue Cys-351; 2) a lysine cross-link between K6.35(245) in TMH6 and the Gαi C-terminal i-5 residue, Lys-349; and 3) a lysine cross-link between K5.64(215) in TMH5 and the Gαi α4β6 loop residue, Lys-317. To investigate the dynamics and nature of the conformational changes involved in CB2·Gi complex formation, we carried out microsecond-time scale molecular dynamics simulations of the CB2 R*·Gαi1β1γ2 complex embedded in a 1-palmitoyl-2-oleoyl-phosphatidylcholine bilayer, using cross-linking information as validation. Our results show that although molecular dynamics simulations started with the G protein orientation in the β2-AR*·Gαsβ1γ2 complex crystal structure, the Gαi1β1γ2 protein reoriented itself within 300 ns. Two major changes occurred as follows. 1) The Gαi1 α5 helix tilt changed due to the outward movement of TMH5 in CB2 R*. 2) A 25° clockwise rotation of Gαi1β1γ2 underneath CB2 R* occurred, with rotation ceasing when Pro-139 (IC-2 loop) anchors in a hydrophobic pocket on Gαi1 (Val-34, Leu-194, Phe-196, Phe-336, Thr-340, Ile-343, and Ile-344). In this complex, all three experimentally identified cross-links can occur. These findings should be relevant for other class A G protein-coupled receptors that couple to Gi proteins. PMID:24855641

  7. B61 is a ligand for the ECK receptor protein-tyrosine kinase.

    PubMed

    Bartley, T D; Hunt, R W; Welcher, A A; Boyle, W J; Parker, V P; Lindberg, R A; Lu, H S; Colombero, A M; Elliott, R L; Guthrie, B A

    1994-04-07

    A protein ligand for the ECK receptor protein-tyrosine kinase has been isolated by using the extracellular domain (ECK-X) of the receptor as an affinity reagent. Initially, concentrated cell culture supernatants were screened for receptor binding activity using immobilized ECK-X in a surface plasmon resonance detection system. Subsequently, supernatants from selected cell lines were fractionated directly by receptor affinity chromatography, resulting in the single-step purification of B61, a protein previously identified as the product of an early response gene induced by tumour necrosis factor-alpha. We report here that recombinant B61 induces autophosphorylation of ECK in intact cells, consistent with B61 being an authentic ligand for ECK. ECK is a member of a large orphan receptor protein-tyrosine kinase family headed by EPH, and we suggest that ligands for other members of this family will be related to B61, and can be isolated in the same way.

  8. G protein-coupled receptors: the inside story.

    PubMed

    Jalink, Kees; Moolenaar, Wouter H

    2010-01-01

    Recent findings necessitate revision of the traditional view of G protein-coupled receptor (GPCR) signaling and expand the diversity of mechanisms by which receptor signaling influences cell behavior in general. GPCRs elicit signals at the plasma membrane and are then rapidly removed from the cell surface by endocytosis. Internalization of GPCRs has long been thought to serve as a mechanism to terminate the production of second messengers such as cAMP. However, recent studies show that internalized GPCRs can continue to either stimulate or inhibit cAMP production in a sustained manner. They do so by remaining associated with their cognate G protein subunit and adenylyl cyclase at endosomal compartments. Once internalized, the GPCRs produce cellular responses distinct from those elicited at the cell surface.

  9. Recent Progress in Understanding Subtype Specific Regulation of NMDA Receptors by G Protein Coupled Receptors (GPCRs)

    PubMed Central

    Yang, Kai; Jackson, Michael F.; MacDonald, John F.

    2014-01-01

    G Protein Coupled Receptors (GPCRs) are the largest family of receptors whose ligands constitute nearly a third of prescription drugs in the market. They are widely involved in diverse physiological functions including learning and memory. NMDA receptors (NMDARs), which belong to the ionotropic glutamate receptor family, are likewise ubiquitously expressed in the central nervous system (CNS) and play a pivotal role in learning and memory. Despite its critical contribution to physiological and pathophysiological processes, few pharmacological interventions aimed directly at regulating NMDAR function have been developed to date. However, it is well established that NMDAR function is precisely regulated by cellular signalling cascades recruited downstream of G protein coupled receptor (GPCR) stimulation. Accordingly, the downstream regulation of NMDARs likely represents an important determinant of outcome following treatment with neuropsychiatric agents that target selected GPCRs. Importantly, the functional consequence of such regulation on NMDAR function varies, based not only on the identity of the GPCR, but also on the cell type in which relevant receptors are expressed. Indeed, the mechanisms responsible for regulating NMDARs by GPCRs involve numerous intracellular signalling molecules and regulatory proteins that vary from one cell type to another. In the present article, we highlight recent findings from studies that have uncovered novel mechanisms by which selected GPCRs regulate NMDAR function and consequently NMDAR-dependent plasticity. PMID:24562329

  10. G protein-coupled odorant receptors: From sequence to structure.

    PubMed

    de March, Claire A; Kim, Soo-Kyung; Antonczak, Serge; Goddard, William A; Golebiowski, Jérôme

    2015-09-01

    Odorant receptors (ORs) are the largest subfamily within class A G protein-coupled receptors (GPCRs). No experimental structural data of any OR is available to date and atomic-level insights are likely to be obtained by means of molecular modeling. In this article, we critically align sequences of ORs with those GPCRs for which a structure is available. Here, an alignment consistent with available site-directed mutagenesis data on various ORs is proposed. Using this alignment, the choice of the template is deemed rather minor for identifying residues that constitute the wall of the binding cavity or those involved in G protein recognition. © 2015 The Protein Society.

  11. Complete Reversible Refolding of a G-Protein Coupled Receptor on a Solid Support

    PubMed Central

    Di Bartolo, Natalie; Compton, Emma L. R.; Warne, Tony; Edwards, Patricia C.; Tate, Christopher G.; Schertler, Gebhard F. X.; Booth, Paula J.

    2016-01-01

    The factors defining the correct folding and stability of integral membrane proteins are poorly understood. Folding of only a few select membrane proteins has been scrutinised, leaving considerable deficiencies in knowledge for large protein families, such as G protein coupled receptors (GPCRs). Complete reversible folding, which is problematic for any membrane protein, has eluded this dominant receptor family. Moreover, attempts to recover receptors from denatured states are inefficient, yielding at best 40–70% functional protein. We present a method for the reversible unfolding of an archetypal family member, the β1-adrenergic receptor, and attain 100% recovery of the folded, functional state, in terms of ligand binding, compared to receptor which has not been subject to any unfolding and retains its original, folded structure. We exploit refolding on a solid support, which could avoid unwanted interactions and aggregation that occur in bulk solution. We determine the changes in structure and function upon unfolding and refolding. Additionally, we employ a method that is relatively new to membrane protein folding; pulse proteolysis. Complete refolding of β1-adrenergic receptor occurs in n-decyl-β-D-maltoside (DM) micelles from a urea-denatured state, as shown by regain of its original helical structure, ligand binding and protein fluorescence. The successful refolding strategy on a solid support offers a defined method for the controlled refolding and recovery of functional GPCRs and other membrane proteins that suffer from instability and irreversible denaturation once isolated from their native membranes. PMID:26982879

  12. Osteogenic potential of the human bone morphogenetic protein 2 gene activated nanobone putty.

    PubMed

    Tian, Xiao-bin; Sun, Li; Yang, Shu-hua; Zhang, Yu-kun; Hu, Ru-yin; Fu, De-hao

    2008-04-20

    Nanobone putty is an injectable and bioresorbable bone substitute. The neutral-pH putty resembles hard bone tissue, does not contain polymers or plasticizers, and is self-setting and nearly isothermic, properties which are helpful for the adhesion, proliferation, and function of bone cells. The aim of this study was to investigate the osteogenic potential of human bone morphogenetic protein 2 (hBMP2) gene activated nanobone putty in inducing ectopic bone formation, and the effects of the hBMP2 gene activated nanobone putty on repairing bone defects. Twenty four Kunming mice were randomly divided into two groups. The nanobone putty + hBMP2 plasmid was injected into the right thigh muscle pouches of the mice (experiment side). The nanobone putty + blank plasmid or nanobone putty was injected into the left thigh muscle pouches of the group 1 (control side 1) or group 2 (control side 2), respectively. The effects of ectopic bone formation were evaluated by radiography, histology, and molecular biology analysis at 2 and 4 weeks after operation. Bilateral 15 mm radial defects were made in forty-eight rabbits. These rabbits were randomly divided into three groups: Group A, nanobone putty + hBMP2 plasmid; Group B, putty + blank plasmid; Group C, nanobone putty only. Six rabbits with left radial defects served as blank controls. The effect of bone repairing was evaluated by radiography, histology, molecular biology, and biomechanical analysis at 4, 8, and 12 weeks after operation. The tissue from the experimental side of the mice expressed hBMP2. Obvious cartilage and island-distributed immature bone formation in implants of the experiment side were observed at 2 weeks after operation, and massive mature bone observed at 4 weeks. No bone formation was observed in the control side of the mice. The ALP activity in the experiment side of the mice was higher than that in the control side. The tissue of Group A rabbits expressed hBMP2 protein and higher ALP level. The new bone

  13. The incorporation of hydrophobic protein receptors and artificial lipid membranes.

    PubMed

    Reader, T A; Fiszer de Plazas, S; Salas, P J; de Robertis, E

    1976-01-01

    The mechanism of chemical synaptic transmission implies: 1) the existence of a specific protein receptor at the postsynaptic membrane, and 2) the interaction between the transmitter released and the receptor, thus producing a change in ionic permeability. Previous studies from our laboratory have shown that special hydrophobic proteins extracted from postsynpatic membranes of different tissues showed a high affinity binding for the different pharmacological agents. The present paper describes experiments in which different hydrophobic protein binding acetylcholine, noradrenaline, gamma-aminobutyric acid, and glutamate were incorporated into artificial lipid membranes, similar to those first described by Mueller et al. (19). The effect of the different pharmacological agents was tested under experimental conditions of voltage clamp and the d.c. current changes measured. The results were then compared for the different lipid-protein membranes employed during the steady state and during transient conductance changes. The specificity of the responses indicate that artificial lipid membranes containing these hydrophobic proteins from electroplax, myocardium, spleen capsule and shrimp muscle can be used as a model to study pharmacologic receptors.

  14. A Fluorescent Live Imaging Screening Assay Based on Translocation Criteria Identifies Novel Cytoplasmic Proteins Implicated in G Protein-coupled Receptor Signaling Pathways*

    PubMed Central

    Lecat, Sandra; Matthes, Hans W.D.; Pepperkok, Rainer; Simpson, Jeremy C.; Galzi, Jean-Luc

    2015-01-01

    Several cytoplasmic proteins that are involved in G protein-coupled receptor signaling cascades are known to translocate to the plasma membrane upon receptor activation, such as beta-arrestin2. Based on this example and in order to identify new cytoplasmic proteins implicated in the ON-and-OFF cycle of G protein-coupled receptor, a live-imaging screen of fluorescently labeled cytoplasmic proteins was performed using translocation criteria. The screening of 193 fluorescently tagged human proteins identified eight proteins that responded to activation of the tachykinin NK2 receptor by a change in their intracellular localization. Previously we have presented the functional characterization of one of these proteins, REDD1, that translocates to the plasma membrane. Here we report the results of the entire screening. The process of cell activation was recorded on videos at different time points and all the videos can be visualized on a dedicated website. The proteins BAIAP3 and BIN1, partially translocated to the plasma membrane upon activation of NK2 receptors. Proteins ARHGAP12 and PKM2 translocated toward membrane blebs. Three proteins that associate with the cytoskeleton were of particular interest : PLEKHH2 rearranged from individual dots located near the cell-substrate adhesion surface into lines of dots. The speriolin-like protein, SPATC1L, redistributed to cell-cell junctions. The Chloride intracellular Channel protein, CLIC2, translocated from actin-enriched plasma membrane bundles to cell-cell junctions upon activation of NK2 receptors. CLIC2, and one of its close paralogs, CLIC4, were further shown to respond with the same translocation pattern to muscarinic M3 and lysophosphatidic LPA receptors. This screen allowed us to identify potential actors in signaling pathways downstream of G protein-coupled receptors and could be scaled-up for high-content screening. PMID:25759509

  15. A Fluorescent Live Imaging Screening Assay Based on Translocation Criteria Identifies Novel Cytoplasmic Proteins Implicated in G Protein-coupled Receptor Signaling Pathways.

    PubMed

    Lecat, Sandra; Matthes, Hans W D; Pepperkok, Rainer; Simpson, Jeremy C; Galzi, Jean-Luc

    2015-05-01

    Several cytoplasmic proteins that are involved in G protein-coupled receptor signaling cascades are known to translocate to the plasma membrane upon receptor activation, such as beta-arrestin2. Based on this example and in order to identify new cytoplasmic proteins implicated in the ON-and-OFF cycle of G protein-coupled receptor, a live-imaging screen of fluorescently labeled cytoplasmic proteins was performed using translocation criteria. The screening of 193 fluorescently tagged human proteins identified eight proteins that responded to activation of the tachykinin NK2 receptor by a change in their intracellular localization. Previously we have presented the functional characterization of one of these proteins, REDD1, that translocates to the plasma membrane. Here we report the results of the entire screening. The process of cell activation was recorded on videos at different time points and all the videos can be visualized on a dedicated website. The proteins BAIAP3 and BIN1, partially translocated to the plasma membrane upon activation of NK2 receptors. Proteins ARHGAP12 and PKM2 translocated toward membrane blebs. Three proteins that associate with the cytoskeleton were of particular interest : PLEKHH2 rearranged from individual dots located near the cell-substrate adhesion surface into lines of dots. The speriolin-like protein, SPATC1L, redistributed to cell-cell junctions. The Chloride intracellular Channel protein, CLIC2, translocated from actin-enriched plasma membrane bundles to cell-cell junctions upon activation of NK2 receptors. CLIC2, and one of its close paralogs, CLIC4, were further shown to respond with the same translocation pattern to muscarinic M3 and lysophosphatidic LPA receptors. This screen allowed us to identify potential actors in signaling pathways downstream of G protein-coupled receptors and could be scaled-up for high-content screening. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Dual specificity of activin type II receptor ActRIIb in dorso-ventral patterning during zebrafish embryogenesis.

    PubMed

    Nagaso, H; Suzuki, A; Tada, M; Ueno, N

    1999-04-01

    Members of the transforming growth factor-beta (TGF-beta) superfamily are thought to regulate specification of a variety of tissue types in early embryogenesis. These effects are mediated through a cell surface receptor complex, consisting of two classes of ser/thr kinase receptor, type I and type II. In the present study, cDNA encoding zebrafish activin type II receptors, ActRIIa and ActRIIb was cloned and characterized. Overexpression of ActRIIb in zebrafish embryos caused dorsalization of embryos, as observed in activin-overexpressing embryos. However, in blastula stage embryos, ActRIIb induced formation of both dorsal and ventro-lateral mesoderm. It has been suggested that these inducing signals from ActRIIb are mediated through each specific type I receptor, TARAM-A and BMPRIA, depending on activin and bone morphogenetic protein (BMP), respectively. In addition, it was shown that a kinase-deleted form of ActRIIb (dnActRIIb) suppressed both activin- and BMP-like signaling pathways. These results suggest that ActRIIb at least has dual roles in both activin and BMP signaling pathways during zebrafish embryogenesis.

  17. Application of RGS box proteins to evaluate G-protein selectivity in receptor-promoted signaling.

    PubMed

    Hains, Melinda D; Siderovski, David P; Harden, T Kendall

    2004-01-01

    Regulator of G-protein signaling (RGS) domains bind directly to GTP-bound Galpha subunits and accelerate their intrinsic GTPase activity by up to several thousandfold. The selectivity of RGS proteins for individual Galpha subunits has been illustrated. Thus, the expression of RGS proteins can be used to inhibit signaling pathways activated by specific G protein-coupled receptors (GPCRs). This article describes the use of specific RGS domain constructs to discriminate among G(i/o), Gq-and G(12/13)-mediated activation of phospholipase C (PLC) isozymes in COS-7 cells. Overexpression of the N terminus of GRK2 (amino acids 45-178) or p115 RhoGEF (amino acids 1-240) elicited selective inhibition of Galphaq- or Galpha(12/13)-mediated signaling to PLC activation, respectively. In contrast, RGS2 overexpression was found to inhibit PLC activation by both G(i/o)- and Gq-coupled GPCRs. RGS4 exhibited dramatic receptor selectivity in its inhibitory actions; of the G(i/o)- and Gq-coupled GPCRs tested (LPA1, LPA2, P2Y1, S1P3), only the Gq-coupled lysophosphatidic acid-activated LPA2 receptor was found to be inhibited by RGS4 overexpression.

  18. Role of G protein-coupled receptor kinases in the homologous desensitization of the human and mouse melanocortin 1 receptors.

    PubMed

    Sánchez-Más, Jesús; Guillo, Lidia A; Zanna, Paola; Jiménez-Cervantes, Celia; García-Borrón, José C

    2005-04-01

    The melanocortin 1 receptor, a G protein-coupled receptor positively coupled to adenylyl cyclase, is a key regulator of epidermal melanocyte proliferation and differentiation and a determinant of human skin phototype and skin cancer risk. Despite its potential importance for regulation of pigmentation, no information is available on homologous desensitization of this receptor. We found that the human melanocortin 1 receptor (MC1R) and its mouse ortholog (Mc1r) undergo homologous desensitization in melanoma cells. Desensitization is not dependent on protein kinase A, protein kinase C, calcium mobilization, or MAPKs, but is agonist dose-dependent. Both melanoma cells and normal melanocytes express two members of the G protein-coupled receptor kinase (GRK) family, GRK2 and GRK6. Cotransfection of the receptor and GRK2 or GRK6 genes in heterologous cells demonstrated that GRK2 and GRK6 impair agonist-dependent signaling by MC1R or Mc1r. However, GRK6, but not GRK2, was able to inhibit MC1R agonist-independent constitutive signaling. Expression of a dominant negative GRK2 mutant in melanoma cells increased their cAMP response to agonists. Agonist-stimulated cAMP production decreased in melanoma cells enriched with GRK6 after stable transfection. Therefore, GRK2 and GRK6 seem to be key regulators of melanocortin 1 receptor signaling and may be important determinants of skin pigmentation.

  19. Activation of G-proteins by receptor-stimulated nucleoside diphosphate kinase in Dictyostelium.

    PubMed Central

    Bominaar, A A; Molijn, A C; Pestel, M; Veron, M; Van Haastert, P J

    1993-01-01

    Recently, interest in the enzyme nucleoside diphosphate kinase (EC2.7.4.6) has increased as a result of its possible involvement in cell proliferation and development. Since NDP kinase is one of the major sources of GTP in cells, it has been suggested that the effects of an altered NDP kinase activity on cellular processes might be the result of altered transmembrane signal transduction via guanine nucleotide-binding proteins (G-proteins). In the cellular slime mould Dictyostelium discoideum, extracellular cAMP induces an increase of phospholipase C activity via a surface cAMP receptor and G-proteins. In this paper it is demonstrated that part of the cellular NDP kinase is associated with the membrane and stimulated by cell surface cAMP receptors. The GTP produced by the action of NDP kinase is capable of activating G-proteins as monitored by altered G-protein-receptor interaction and the activation of the effector enzyme phospholipase C. Furthermore, specific monoclonal antibodies inhibit the effect of NDP kinase on G-protein activation. These results suggest that receptor-stimulated NDP kinase contributes to the mediation of hormone action by producing GTP for the activation of GTP-binding proteins. Images PMID:8389692

  20. [Roles of G protein-coupled estrogen receptor in the male reproductive system].

    PubMed

    Chen, Kai-hong; Zhang, Xian; Jiang, Xue-wu

    2016-02-01

    The G protein-coupled estrogen receptor (GPER), also known as G protein-coupled receptor 30 (GPR30), was identified in the recent years as a functional membrane receptor different from the classical nuclear estrogen receptors. This receptor is widely expressed in the cortex, cerebellum, hippocampus, heart, lung, liver, skeletal muscle, and the urogenital system. It is responsible for the mediation of nongenomic effects associated with estrogen and its derivatives, participating in the physiological activities of the body. The present study reviews the molecular structure, subcellular localization, signaling pathways, distribution, and function of GPER in the male reproductive system.

  1. 5-HT2A SEROTONIN RECEPTOR BIOLOGY: Interacting proteins, kinases and paradoxical regulation

    PubMed Central

    Roth, Bryan L

    2011-01-01

    5-hydroxytryptamine2A (5-HT2A) serotonin receptors are important pharmacological targets for a large number of central nervous system and peripheral serotonergic medications. In this review article I summarize work mainly from my lab regarding serotonin receptor anatomy, pharmacology, signaling and regulation. I highlight the role of serotonin receptor interacting proteins and the emerging paradigm of G-protein coupled receptor functional selectivity. PMID:21288474

  2. Protein-protein interactions in the RPS4/RRS1 immune receptor complex

    PubMed Central

    Sarris, Panagiotis F.

    2017-01-01

    Plant NLR (Nucleotide-binding domain and Leucine-rich Repeat) immune receptor proteins are encoded by Resistance (R) genes and confer specific resistance to pathogen races that carry the corresponding recognized effectors. Some NLR proteins function in pairs, forming receptor complexes for the perception of specific effectors. We show here that the Arabidopsis RPS4 and RRS1 NLR proteins are both required to make an authentic immune complex. Over-expression of RPS4 in tobacco or in Arabidopsis results in constitutive defense activation; this phenotype is suppressed in the presence of RRS1. RRS1 protein co-immunoprecipitates (co-IPs) with itself in the presence or absence of RPS4, but in contrast, RPS4 does not associate with itself in the absence of RRS1. In the presence of RRS1, RPS4 associates with defense signaling regulator EDS1 solely in the nucleus, in contrast to the extra-nuclear location found in the absence of RRS1. The AvrRps4 effector does not disrupt RPS4-EDS1 association in the presence of RRS1. In the absence of RRS1, AvrRps4 interacts with EDS1, forming nucleocytoplasmic aggregates, the formation of which is disturbed by the co-expression of PAD4 but not by SAG101. These data indicate that the study of an immune receptor protein complex in the absence of all components can result in misleading inferences, and reveals an NLR complex that dynamically interacts with the immune regulators EDS1/PAD4 or EDS1/SAG101, and with effectors, during the process by which effector recognition is converted to defense activation. PMID:28475615

  3. Muscarinic supersensitivity and impaired receptor desensitization in G protein-coupled receptor kinase 5-deficient mice.

    PubMed

    Gainetdinov, R R; Bohn, L M; Walker, J K; Laporte, S A; Macrae, A D; Caron, M G; Lefkowitz, R J; Premont, R T

    1999-12-01

    G protein-coupled receptor kinase 5 (GRK5) is a member of a family of enzymes that phosphorylate activated G protein-coupled receptors (GPCR). To address the physiological importance of GRK5-mediated regulation of GPCRs, mice bearing targeted deletion of the GRK5 gene (GRK5-KO) were generated. GRK5-KO mice exhibited mild spontaneous hypothermia as well as pronounced behavioral supersensitivity upon challenge with the nonselective muscarinic agonist oxotremorine. Classical cholinergic responses such as hypothermia, hypoactivity, tremor, and salivation were enhanced in GRK5-KO animals. The antinociceptive effect of oxotremorine was also potentiated and prolonged. Muscarinic receptors in brains from GRK5-KO mice resisted oxotremorine-induced desensitization, as assessed by oxotremorine-stimulated [5S]GTPgammaS binding. These data demonstrate that elimination of GRK5 results in cholinergic supersensitivity and impaired muscarinic receptor desensitization and suggest that a deficit of GPCR desensitization may be an underlying cause of behavioral supersensitivity.

  4. Structural prerequisites for G-protein activation by the neurotensin receptor

    PubMed Central

    Krumm, Brian E.; White, Jim F.; Shah, Priyanka; Grisshammer, Reinhard

    2015-01-01

    We previously determined the structure of neurotensin receptor NTSR1 in an active-like conformation with six thermostabilizing mutations bound to the peptide agonist neurotensin. This receptor was unable to activate G proteins, indicating that the mutations restricted NTSR1 to relate agonist binding to G-protein activation. Here we analyse the effect of three of those mutations (E166A3.49, L310A6.37, F358A7.42) and present two structures of NTSR1 able to catalyse nucleotide exchange at Gα. The presence of F3587.42 causes the conserved W3216.48 to adopt a side chain orientation parallel to the lipid bilayer sealing the collapsed Na+ ion pocket and linking the agonist with residues in the lower receptor part implicated in GPCR activation. In the intracellular receptor half, the bulkier L3106.37 side chain dictates the position of R1673.50 of the highly conserved D/ERY motif. These residues, together with the presence of E1663.49 provide determinants for G-protein activation by NTSR1. PMID:26205105

  5. A synthetic compound that potentiates bone morphogenetic protein-2-induced transdifferentiation of myoblasts into the osteoblastic phenotype

    PubMed Central

    Kato, Satoshi; Tomita, Katsuro; Titus, Louisa; Boden, Scott D.

    2011-01-01

    There is an urgent need to develop methods that lower costs of using recombinant human bone morphogenetic proteins (BMPs) to promote bone induction. In this study, we demonstrate the osteogenic effect of a low-molecular weight compound, SVAK-12, that potentiated the effects of BMP-2 in inducing transdifferentiation of C2C12 myoblasts into the osteoblastic phenotype. Here, we report a specific compound, SVAK-12, which was selected based on in silico screenings of small-molecule databases using the homology modeled interaction motif of Smurf1-WW2 domain. The enhancement of BMP-2 activity by SVAK-12 was characterized by evaluating a BMP-specific reporter activity and by monitoring the BMP-2-induced expression of mRNA for osteocalcin and alkaline phosphatase (ALP), which are widely accepted marker genes of osteoblast differentiation. Finally, we confirmed these results by also measuring the enhancement of BMP-2-induced activity of ALP. Smurf1 is an E3 ligase that targets osteogenic Smads for ubiquitin-mediated proteasomal degradation. Smurf1 is an interesting potential target to enhance bone formation based on the positive effects on bone of proteins that block Smurf1-binding to Smad targets or in Smurf1−/− knockout mice. Since Smads bind Smurf1 via its WW2 domain, we performed in silico screening to identify compounds that might interact with the Smurf1-WW2 domain. We recently reported the activity of a compound, SVAK-3. However, SVAK-3, while exhibiting BMP-potentiating activity, was not stable and thus warranted a new search for a more stable and efficacious compound among a selected group of candidates. In addition to being more stable, SVAK-12 exhibited a dose-dependent activity in inducing osteoblastic differentiation of myoblastic C2C12 cells even when multiple markers of the osteoblastic phenotype were parallelly monitored. PMID:21110071

  6. A synthetic compound that potentiates bone morphogenetic protein-2-induced transdifferentiation of myoblasts into the osteoblastic phenotype.

    PubMed

    Kato, Satoshi; Sangadala, Sreedhara; Tomita, Katsuro; Titus, Louisa; Boden, Scott D

    2011-03-01

    There is an urgent need to develop methods that lower costs of using recombinant human bone morphogenetic proteins (BMPs) to promote bone induction. In this study, we demonstrate the osteogenic effect of a low-molecular weight compound, SVAK-12, that potentiated the effects of BMP-2 in inducing transdifferentiation of C2C12 myoblasts into the osteoblastic phenotype. Here, we report a specific compound, SVAK-12, which was selected based on in silico screenings of small-molecule databases using the homology modeled interaction motif of Smurf1-WW2 domain. The enhancement of BMP-2 activity by SVAK-12 was characterized by evaluating a BMP-specific reporter activity and by monitoring the BMP-2-induced expression of mRNA for osteocalcin and alkaline phosphatase (ALP), which are widely accepted marker genes of osteoblast differentiation. Finally, we confirmed these results by also measuring the enhancement of BMP-2-induced activity of ALP. Smurf1 is an E3 ligase that targets osteogenic Smads for ubiquitin-mediated proteasomal degradation. Smurf1 is an interesting potential target to enhance bone formation based on the positive effects on bone of proteins that block Smurf1-binding to Smad targets or in Smurf1-/- knockout mice. Since Smads bind Smurf1 via its WW2 domain, we performed in silico screening to identify compounds that might interact with the Smurf1-WW2 domain. We recently reported the activity of a compound, SVAK-3. However, SVAK-3, while exhibiting BMP-potentiating activity, was not stable and thus warranted a new search for a more stable and efficacious compound among a selected group of candidates. In addition to being more stable, SVAK-12 exhibited a dose-dependent activity in inducing osteoblastic differentiation of myoblastic C2C12 cells even when multiple markers of the osteoblastic phenotype were parallelly monitored.

  7. Oocyte-somatic cell interactions in the human ovary-novel role of bone morphogenetic proteins and growth differentiation factors.

    PubMed

    Chang, Hsun-Ming; Qiao, Jie; Leung, Peter C K

    2016-12-01

    Initially identified for their capability to induce heterotopic bone formation, bone morphogenetic proteins (BMPs) are multifunctional growth factors that belong to the transforming growth factor β superfamily. Using cellular and molecular genetic approaches, recent studies have implicated intra-ovarian BMPs as potent regulators of ovarian follicular function. The bi-directional communication of oocytes and the surrounding somatic cells is mandatory for normal follicle development and oocyte maturation. This review summarizes the current knowledge on the physiological role and molecular determinants of these ovarian regulatory factors within the human germline-somatic regulatory loop. The regulation of ovarian function remains poorly characterized in humans because, while the fundamental process of follicular development and oocyte maturation is highly similar across species, most information on the regulation of ovarian function is obtained from studies using rodent models. Thus, this review focuses on the studies that used human biological materials to gain knowledge about human ovarian biology and disorders and to develop strategies for preventing, diagnosing and treating these abnormalities. Relevant English-language publications describing the roles of BMPs or growth differentiation factors (GDFs) in human ovarian biology and phenotypes were comprehensively searched using PubMed and the Google Scholar database. The publications included those published since the initial identification of BMPs in the mammalian ovary in 1999 through July 2016. Studies using human biological materials have revealed the expression of BMPs, GDFs and their putative receptors as well as their molecular signaling in the fundamental cells (oocyte, cumulus/granulosa cells (GCs) and theca/stroma cells) of the ovarian follicles throughout follicle development. With the availability of recombinant human BMPs/GDFs and the development of immortalized human cell lines, functional studies

  8. Bone morphogenetic protein-binding endothelial regulator of liver sinusoidal endothelial cells induces iron overload in a fatty liver mouse model.

    PubMed

    Hasebe, Takumu; Tanaka, Hiroki; Sawada, Koji; Nakajima, Shunsuke; Ohtake, Takaaki; Fujiya, Mikihiro; Kohgo, Yutaka

    2017-03-01

    Non-alcoholic fatty liver disease (NAFLD) is frequently accompanied by iron overload. However, because of the complex hepcidin-regulating molecules, the molecular mechanism underlying iron overload remains unknown. To identify the key molecule involved in NAFLD-associated iron dysregulation, we performed whole-RNA sequencing on the livers of obese mice. Male C57BL/6 mice were fed a regular or high-fat diet for 16 or 48 weeks. Internal iron was evaluated by plasma iron, ferritin or hepatic iron content. Whole-RNA sequencing was performed by transcriptome analysis using semiconductor high-throughput sequencer. Mouse liver tissues or isolated hepatocytes and sinusoidal endothelial cells were used to assess the expression of iron-regulating molecules. Mice fed a high-fat diet for 16 weeks showed excess iron accumulation. Longer exposure to a high-fat diet increased hepatic fibrosis and intrahepatic iron accumulation. A pathway analysis of the sequencing data showed that several inflammatory pathways, including bone morphogenetic protein (BMP)-SMAD signaling, were significantly affected. Sequencing analysis showed 2314 altered genes, including decreased mRNA expression of the hepcidin-coding gene Hamp. Hepcidin protein expression and SMAD phosphorylation, which induces Hamp, were found to be reduced. The expression of BMP-binding endothelial regulator (BMPER), which inhibits BMP-SMAD signaling by binding BMP extracellularly, was up-regulated in fatty livers. In addition, immunohistochemical and cell isolation analyses showed that BMPER was primarily expressed in the liver sinusoidal endothelial cells (LSECs) rather than hepatocytes. BMPER secretion by LSECs inhibits BMP-SMAD signaling in hepatocytes and further reduces hepcidin protein expression. These intrahepatic molecular interactions suggest a novel molecular basis of iron overload in NAFLD.

  9. G-protein-coupled receptors for neurotransmitter amino acids: C-terminal tails, crowded signalosomes.

    PubMed Central

    El Far, Oussama; Betz, Heinrich

    2002-01-01

    G-protein-coupled receptors (GPCRs) represent a superfamily of highly diverse integral membrane proteins that transduce external signals to different subcellular compartments, including nuclei, via trimeric G-proteins. By differential activation of diffusible G(alpha) and membrane-bound G(beta)gamma subunits, GPCRs might act on both cytoplasmic/intracellular and plasma-membrane-bound effector systems. The coupling efficiency and the plasma membrane localization of GPCRs are regulated by a variety of interacting proteins. In this review, we discuss recently disclosed protein interactions found with the cytoplasmic C-terminal tail regions of two types of presynaptic neurotransmitter receptors, the group III metabotropic glutamate receptors and the gamma-aminobutyric acid type-B receptors (GABA(B)Rs). Calmodulin binding to mGluR7 and other group III mGluRs may provide a Ca(2+)-dependent switch for unidirectional (G(alpha)) versus bidirectional (G(alpha) and G(beta)gamma) signalling to downstream effector proteins. In addition, clustering of mGluR7 by PICK1 (protein interacting with C-kinase 1), a polyspecific PDZ (PSD-95/Dlg1/ZO-1) domain containing synaptic organizer protein, sheds light on how higher-order receptor complexes with regulatory enzymes (or 'signalosomes') could be formed. The interaction of GABA(B)Rs with the adaptor protein 14-3-3 and the transcription factor ATF4 (activating transcription factor 4) suggests novel regulatory pathways for G-protein signalling, cytoskeletal reorganization and nuclear gene expression: processes that may all contribute to synaptic plasticity. PMID:12006104

  10. Bone Morphogenetic Protein-7 Enhances Degradation of Osteoinductive Bioceramic Implants in an Ectopic Model

    PubMed Central

    Klünter, Tim; Schulz, Peter; Deisinger, Ulrike; Diez, Claudius; Waiss, Waltraud; Kirschneck, Christian; Reichert, Torsten E.; Detsch, Rainer

    2017-01-01

    Background: The aim of the present study was to evaluate the degradation pattern of highly porous bioceramics as well as the bone formation in presence of bone morphogenetic protein 7 (BMP-7) in an ectopic site. Methods: Novel calcium phosphate ceramic cylinders sintered at 1,300°C with a total porosity of 92–94 vol%, 45 pores per inch, and sized 15 mm (Ø) × 5 mm were grafted on the musculus latissimus dorsi bilaterally in 10 Göttingen minipigs: group I (n = 5): hydroxyapatite (HA) versus biphasic calcium phosphate (BCP), a mixture of HA and tricalcium phosphate (TCP) in a ratio of 60/40 wt%; group II (n = 5): TCP versus BCP. A test side was supplied in situ with 250 μg BMP-7. Fluorochrome bone labeling and computed tomography were performed in vivo. Specimens were evaluated 14 weeks after surgery by environmental scanning electron microscopy, fluorescence microscopy, tartrate-resistant acid phosphatase, and pentachrome staining. Results: Bone formation was enhanced in the presence of BMP-7 in all ceramics (P = 0.001). Small spots of newly formed bone were observed in all implants in the absence of BMP-7. Degradation of HA and BCP was enhanced in the presence of BMP-7 (P = 0.001). In those ceramics, osteoclasts were observed. TCP ceramics were almost completely degraded independently of the effect of BMP-7 after 14 weeks (P = 0.76), osteoclasts were not observed. Conclusions: BMP-7 enhanced bone formation and degradation of HA and BCP ceramics via osteoclast resorption. TCP degraded via dissolution. All ceramics were osteoinductive. Novel degradable HA and BCP ceramics in the presence of BMP-7 are promising bone substitutes in the growing individual. PMID:28740783

  11. Spatial regulation of bone morphogenetic proteins (BMPs) in postnatal articular and growth plate cartilage

    PubMed Central

    Garrison, Presley; Yue, Shanna; Hanson, Jeffrey; Baron, Jeffrey; Lui, Julian C.

    2017-01-01

    Articular and growth plate cartilage both arise from condensations of mesenchymal cells, but ultimately develop important histological and functional differences. Each is composed of three layers—the superficial, mid and deep zones of articular cartilage and the resting, proliferative and hypertrophic zones of growth plate cartilage. The bone morphogenetic protein (BMP) system plays an important role in cartilage development. A gradient in expression of BMP-related genes has been observed across growth plate cartilage, likely playing a role in zonal differentiation. To investigate the presence of a similar expression gradient in articular cartilage, we used laser capture microdissection (LCM) to separate murine growth plate and articular cartilage from the proximal tibia into their six constituent zones, and used a solution hybridization assay with color-coded probes (nCounter) to quantify mRNAs for 30 different BMP-related genes in each zone. In situ hybridization and immunohistochemistry were then used to confirm spatial expression patterns. Expression gradients for Bmp2 and 6 were observed across growth plate cartilage with highest expression in hypertrophic zone. However, intracellular BMP signaling, assessed by phospho-Smad1/5/8 immunohistochemical staining, appeared to be higher in the proliferative zone and prehypertrophic area than in hypertrophic zone, possibly due to high expression of Smad7, an inhibitory Smad, in the hypertrophic zone. We also found BMP expression gradients across the articular cartilage with BMP agonists primarily expressed in the superficial zone and BMP functional antagonists primarily expressed in the deep zone. Phospho-Smad1/5/8 immunohistochemical staining showed a similar gradient. In combination with previous evidence that BMPs regulate chondrocyte proliferation and differentiation, the current findings suggest that BMP signaling gradients exist across both growth plate and articular cartilage and that these gradients may

  12. G protein-coupled receptor 30 in tumor development.

    PubMed

    Wang, Dengfeng; Hu, Lina; Zhang, Guonan; Zhang, Lin; Chen, Chen

    2010-08-01

    Estrogen plays several important physiological and pathological functions in not only reproductive system but many other systems as well. Its transcriptional activation has been traditionally described as being mediated by classic nuclear estrogen receptors (ERs). It is however established recently that a novel functional estrogen transmembrane receptor, G protein-coupled receptor 30 (GPR30), modulates both rapid non-genomic events and genomic transcriptional events of estrogen. It has been demonstrated that GPR30 promotes the progress of estrogen-related tumors through mitogen-activated protein kinase (MAPK) signaling pathways. Effects mediated by GPR30 are maintained when classic ERs are absent or blocked. In addition, GPR30 is involved in drug resistance, which is often occurring during cancer treatments. All these new findings strongly imply that GPR30 may be an important therapeutic target for estrogen-related tumors. Simultaneously blocking both GPR30 and classic ERs may be a better strategy for the treatment of estrogen-related tumors.

  13. PDZ Protein Regulation of G Protein-Coupled Receptor Trafficking and Signaling Pathways.

    PubMed

    Dunn, Henry A; Ferguson, Stephen S G

    2015-10-01

    G protein-coupled receptors (GPCRs) contribute to the regulation of every aspect of human physiology and are therapeutic targets for the treatment of numerous diseases. As a consequence, understanding the myriad of mechanisms controlling GPCR signaling and trafficking is essential for the development of new pharmacological strategies for the treatment of human pathologies. Of the many GPCR-interacting proteins, postsynaptic density protein of 95 kilodaltons, disc large, zona occludens-1 (PDZ) domain-containing proteins appear most abundant and have similarly been implicated in disease mechanisms. PDZ proteins play an important role in regulating receptor and channel protein localization within synapses and tight junctions and function to scaffold intracellular signaling protein complexes. In the current study, we review the known functional interactions between PDZ domain-containing proteins and GPCRs and provide insight into the potential mechanisms of action. These PDZ domain-containing proteins include the membrane-associated guanylate-like kinases [postsynaptic density protein of 95 kilodaltons; synapse-associated protein of 97 kilodaltons; postsynaptic density protein of 93 kilodaltons; synapse-associated protein of 102 kilodaltons; discs, large homolog 5; caspase activation and recruitment domain and membrane-associated guanylate-like kinase domain-containing protein 3; membrane protein, palmitoylated 3; calcium/calmodulin-dependent serine protein kinase; membrane-associated guanylate kinase protein (MAGI)-1, MAGI-2, and MAGI-3], Na(+)/H(+) exchanger regulatory factor proteins (NHERFs) (NHERF1, NHERF2, PDZ domain-containing kidney protein 1, and PDZ domain-containing kidney protein 2), Golgi-associated PDZ proteins (Gα-binding protein interacting protein, C-terminus and CFTR-associated ligand), PDZ domain-containing guanine nucleotide exchange factors (GEFs) 1 and 2, regulator of G protein signaling (RGS)-homology-RhoGEFs (PDZ domain-containing RhoGEF and

  14. A New Family of Nuclear Receptor Coregulators That Integrate Nuclear Receptor Signaling through CREB-Binding Protein

    PubMed Central

    Mahajan, Muktar A.; Samuels, Herbert H.

    2000-01-01

    We describe the cloning and characterization of a new family of nuclear receptor coregulators (NRCs) which modulate the function of nuclear hormone receptors in a ligand-dependent manner. NRCs are expressed as alternatively spliced isoforms which may exhibit different intrinsic activities and receptor specificities. The NRCs are organized into several modular structures and contain a single functional LXXLL motif which associates with members of the steroid hormone and thyroid hormone/retinoid receptor subfamilies with high affinity. Human NRC (hNRC) harbors a potent N-terminal activation domain (AD1), which is as active as the herpesvirus VP16 activation domain, and a second activation domain (AD2) which overlaps with the receptor-interacting LXXLL region. The C-terminal region of hNRC appears to function as an inhibitory domain which influences the overall transcriptional activity of the protein. Our results suggest that NRC binds to liganded receptors as a dimer and this association leads to a structural change in NRC resulting in activation. hNRC binds CREB-binding protein (CBP) with high affinity in vivo, suggesting that hNRC may be an important functional component of a CBP complex involved in mediating the transcriptional effects of nuclear hormone receptors. PMID:10866662

  15. Bone Morphogenetic Protein (BMP) signaling in animal reproductive system development and function.

    PubMed

    Lochab, Amaneet K; Extavour, Cassandra G

    2017-07-15

    In multicellular organisms, the specification, maintenance, and transmission of the germ cell lineage to subsequent generations are critical processes that ensure species survival. A number of studies suggest that the Bone Morphogenetic Protein (BMP) pathway plays multiple roles in this cell lineage. We wished to use a comparative framework to examine the role of BMP signaling in regulating these processes, to determine if patterns would emerge that might shed light on the evolution of molecular mechanisms that may play germ cell-specific or other reproductive roles across species. To this end, here we review evidence to date from the literature supporting a role for BMP signaling in reproductive processes across Metazoa. We focus on germ line-specific processes, and separately consider somatic reproductive processes. We find that from primordial germ cell (PGC) induction to maintenance of PGC identity and gametogenesis, BMP signaling regulates these processes throughout embryonic development and adult life in multiple deuterostome and protostome clades. In well-studied model organisms, functional genetic evidence suggests that BMP signaling is required in the germ line across all life stages, with the exception of PGC specification in species that do not use inductive signaling to induce germ cell formation. The current evidence is consistent with the hypothesis that BMP signaling is ancestral in bilaterian inductive PGC specification. While BMP4 appears to be the most broadly employed ligand for the reproductive processes considered herein, we also noted evidence for sex-specific usage of different BMP ligands. In gametogenesis, BMP6 and BMP15 seem to have roles restricted to oogenesis, while BMP8 is restricted to spermatogenesis. We hypothesize that a BMP-based mechanism may have been recruited early in metazoan evolution to specify the germ line, and was subsequently co-opted for use in other germ line-specific and somatic reproductive processes. We suggest

  16. Minireview: Role of Intracellular Scaffolding Proteins in the Regulation of Endocrine G Protein-Coupled Receptor Signaling

    PubMed Central

    Walther, Cornelia

    2015-01-01

    The majority of hormones stimulates and mediates their signal transduction via G protein-coupled receptors (GPCRs). The signal is transmitted into the cell due to the association of the GPCRs with heterotrimeric G proteins, which in turn activates an extensive array of signaling pathways to regulate cell physiology. However, GPCRs also function as scaffolds for the recruitment of a variety of cytoplasmic protein-interacting proteins that bind to both the intracellular face and protein interaction motifs encoded by GPCRs. The structural scaffolding of these proteins allows GPCRs to recruit large functional complexes that serve to modulate both G protein-dependent and -independent cellular signaling pathways and modulate GPCR intracellular trafficking. This review focuses on GPCR interacting PSD95-disc large-zona occludens domain containing scaffolds in the regulation of endocrine receptor signaling as well as their potential role as therapeutic targets for the treatment of endocrinopathies. PMID:25942107

  17. Expression, Purification, and Analysis of G-Protein-Coupled Receptor Kinases

    PubMed Central

    Sterne-Marr, Rachel; Baillargeon, Alison I.; Michalski, Kevin R.; Tesmer, John J.G.

    2015-01-01

    G-protein-coupled receptor (GPCR) kinases (GRKs) were first identified based on their ability to specifically phosphorylate activated GPCRs. Although many soluble substrates have since been identified, the chief physiological role of GRKs still remains the uncoupling of GPCRs from heterotrimeric G-proteins by promoting β-arrestin binding through the phosphorylation of the receptor. It is expected that GRKs recognize activated GPCRs through a docking site that not only recognizes the active conformation of the transmembrane domain of the receptor but also stabilizes a more catalytically competent state of the kinase domain. Many of the recent gains in understanding GRK-receptor interactions have been gleaned through biochemical and structural analysis of recombinantly expressed GRKs. Described herein are current techniques and procedures being used to express, purify, and assay GRKs in both in vitro and living cells. PMID:23351749

  18. Cardioprotective role of G-Protein Coupled Estrogen Receptor 1 (GPER1).

    PubMed

    Koganti, Sivaramakrishna

    2015-01-01

    G-Protein Coupled Estrogen Receptor 1 (GPER1), also known as G-Protein Coupled Receptor 30 (GPR30) and initially considered an orphan receptor, has become one of the most important pharmacological targets in cardiovascular research. Since the gene encoding this putative receptor was cloned nearly 20 years ago, researchers have addressed its role in various aspects of physiology, including cardioprotection. Although extensive research has been carried out to understand the role of GPER1 as a pharmacological target to treat cardiovascular diseases, there are few current reviews addressing the overall cardioprotective benefits of this receptor and the signaling intermediates involved. This review considers the origins of GPER1, its cell biology, its physiological and pharmacological roles as a therapeutic target in cardiovascular disease, and what future research on GPER1 might entail. More specifically, the review focuses on GPER1 regulation of Angiotensin Type I Receptor (AT1R) and the role of estrogen receptors, epidermal growth factor receptor (EGFR) and matrix metalloproteinases (MMPs) in bringing about the cardioprotective effects of GPER1. Areas where improved knowledge of GPER1 biology is still needed to better understand the receptor's cardioprotective effects are also discussed.

  19. Huntingtin interacting protein 1 modulates the transcriptional activity of nuclear hormone receptors.

    PubMed

    Mills, Ian G; Gaughan, Luke; Robson, Craig; Ross, Theodora; McCracken, Stuart; Kelly, John; Neal, David E

    2005-07-18

    Internalization of activated receptors regulates signaling, and endocytic adaptor proteins are well-characterized in clathrin-mediated uptake. One of these adaptor proteins, huntingtin interacting protein 1 (HIP1), induces cellular transformation and is overexpressed in some prostate cancers. We have discovered that HIP1 associates with the androgen receptor through a central coiled coil domain and is recruited to DNA response elements upon androgen stimulation. HIP1 is a novel androgen receptor regulator, significantly repressing transcription when knocked down using a silencing RNA approach and activating transcription when overexpressed. We have also identified a functional nuclear localization signal at the COOH terminus of HIP1, which contributes to the nuclear translocation of the protein. In conclusion, we have discovered that HIP1 is a nucleocytoplasmic protein capable of associating with membranes and DNA response elements and regulating transcription.

  20. Huntingtin interacting protein 1 modulates the transcriptional activity of nuclear hormone receptors

    PubMed Central

    Mills, Ian G.; Gaughan, Luke; Robson, Craig; Ross, Theodora; McCracken, Stuart; Kelly, John; Neal, David E.

    2005-01-01

    Internalization of activated receptors regulates signaling, and endocytic adaptor proteins are well-characterized in clathrin-mediated uptake. One of these adaptor proteins, huntingtin interacting protein 1 (HIP1), induces cellular transformation and is overexpressed in some prostate cancers. We have discovered that HIP1 associates with the androgen receptor through a central coiled coil domain and is recruited to DNA response elements upon androgen stimulation. HIP1 is a novel androgen receptor regulator, significantly repressing transcription when knocked down using a silencing RNA approach and activating transcription when overexpressed. We have also identified a functional nuclear localization signal at the COOH terminus of HIP1, which contributes to the nuclear translocation of the protein. In conclusion, we have discovered that HIP1 is a nucleocytoplasmic protein capable of associating with membranes and DNA response elements and regulating transcription. PMID:16027218

  1. Effects of simulated weightlessness on the kinase activity of MEK1 induced by bone morphogenetic protein-2 in rat osteosarcoma cells

    NASA Astrophysics Data System (ADS)

    Zhang, S.; Wang, B.; Cao, X. S.; Yang, Z.

    Objective The mRNA expression of alpha 1 chain of type I collagen COL-I alpha 1 in rat osteosarcoma ROS17 2 8 cells induced by bone morphogenetic protein-2 BMP-2 was reduced under simulated microgravity The protein kinase MEK1 of MAPK signal pathway plays an important role in the expression of COL-I alpha 1 mRNA The purpose of this study is to investigate the effects of simulated weightlessness on the activity of MEK1 induced by BMP-2 in ROS17 2 8 cells Methods ROS17 2 8 cells were cultured in 1G control and rotating clinostat simulated weightlessness for 24 h 48 h and 72 h BMP-2 500 ng ml was added into the medium 1 h before the culture ended There was a control group in which ROS17 2 8 cells were cultured in 1G condition without BMP-2 Then the total protein of cells was extracted and the expression of phosphated-ERK1 2 p-ERK1 2 protein was detected by means of Western Blotting to show the kinase activity of MEK1 Results There were no significant differences in the expression of total ERK1 2 among all groups The expression of p-ERK1 2 was unconspicuous in the control group without BMP-2 but increased significantly when BMP-2 was added P 0 01 The level of p-ERK1 2 in simulated weightlessness group was much more lower than that in 1G group in every time point P 0 01 The expression of p-ERK1 2 gradually decreased along with the time of weightlessness simulation P 0 01 Conclusions The kinase activity of MEK1 induced by BMP-2 in rat osteosarcoma cells was reduced under simulated weightlessness

  2. A protein that interacts with members of the nuclear hormone receptor family: identification and cDNA cloning.

    PubMed Central

    Zeiner, M; Gehring, U

    1995-01-01

    In search of proteins which interact with activated steroid hormone receptors, we screened a human liver lambda gt11 expression library with the glucocorticoid receptor. We identified and cloned a cDNA sequence of 1322 bp that encodes a protein of 274 aa. This protein consists predominantly of hydrophilic amino acids and contains a putative bipartite nuclear localization signal. The in vitro translated receptor-associating protein runs in SDS/polyacrylamide gels with an apparent molecular mass of 46 kDa. By use of the bacterially expressed fusion protein with glutathione S-transferase we have found that interaction is not limited to the glucocorticoid receptor but included other nuclear receptors--most notably, the estrogen and thyroid receptors. Binding also occurs with the glucocorticoid receptor complexed with the antiglucocorticoid RU 38486, with the estrogen receptor complexed with the antiestrogen 4-hydroxytamoxifen or ICI 164,384, and even with receptors not complexed with ligand. Association with steroid hormone receptors depends on prior receptor activation--i.e., release from heat shock proteins. The sequence identified here appears to be a general partner protein for nuclear hormone receptors, with the gene being expressed in a variety of mammalian tissues. Images Fig. 2 Fig. 3 Fig. 4 PMID:8524784

  3. Hydrogel Delivery of Mesenchymal Stem Cell–Expressing Bone Morphogenetic Protein-2 Enhances Bone Defect Repair

    PubMed Central

    Hsiao, Hui-Yi; Yang, Shu-Rui; Brey, Eric M.; Chu, I-Ming

    2016-01-01

    Background: The application of bone tissue engineering for repairing bone defects has gradually shown some satisfactory progress. One of the concerns raising scientific attention is the poor supply of growth factors. A number of growth factor delivery approaches have been developed for promoting bone formation. However, there is no systematic comparison of those approaches on efficiency of neobone formation. In this study, the approaches using periosteum, direct supply of growth factors, or gene transfection of growth factors were evaluated to determine the osteogenic capacity on the repair of bone defect. Methods: In total, 42 male 21-week-old Sprague-Dawley rats weighing 250 to 400 g were used as the bone defect model to evaluate the bone repair efficiency. Various tissue engineered constructs of poly(ethylene glycol)-poly(l-lactic acid) (PEG-PLLA) copolymer hydrogel with periosteum, with external supply of bone morphogenetic protein-2 (BMP2), or with BMP2-transfected bone marrow–derived mesenchymal stem cells (BMMSCs) were filled in a 7-mm bone defect region. Animals were euthanized at 3 months, and the hydrogel constructs were harvested. The evaluation with histological staining and radiography analysis were performed for the volume of new bone formation. Results: The PEG-PLLA scaffold with BMMSCs promotes bone regeneration with the addition of periosteum. The group with BMP2-transfected BMMSCs demonstrated the largest volume of new bone among all the testing groups. Conclusions: Altogether, the results of this study provide the evidence that the combination of PEG-PLLA hydrogels with BMMSCs and sustained delivery of BMP2 resulted in the maximal bone regeneration. PMID:27622106

  4. High doses of bone morphogenetic protein 2 induce structurally abnormal bone and inflammation in vivo.

    PubMed

    Zara, Janette N; Siu, Ronald K; Zhang, Xinli; Shen, Jia; Ngo, Richard; Lee, Min; Li, Weiming; Chiang, Michael; Chung, Jonguk; Kwak, Jinny; Wu, Benjamin M; Ting, Kang; Soo, Chia

    2011-05-01

    The major Food and Drug Association-approved osteoinductive factors in wide clinical use are bone morphogenetic proteins (BMPs). Although BMPs can promote robust bone formation, they also induce adverse clinical effects, including cyst-like bone formation and significant soft tissue swelling. In this study, we evaluated multiple BMP2 doses in a rat femoral segmental defect model and in a minimally traumatic rat femoral onlay model to determine its dose-dependent effects. Results of our femoral segmental defect model established a low BMP2 concentration range (5 and 10 μg/mL, total dose 0.375 and 0.75 μg in 75 μg total volume) unable to induce defect fusion, a mid-range BMP2 concentration range able to fuse the defect without adverse effects (30 μg/mL, total dose 2.25 μg in 75 μg total volume), and a high BMP2 concentration range (150, 300, and 600 μg/mL, total dose 11.25, 22.5, and 45 μg in 75 μg total volume) able to fuse the defect, but with formation of cyst-like bony shells filled with histologically confirmed adipose tissue. In addition, compared to control, 4 mg/mL BMP2 also induced significant tissue inflammatory infiltrates and exudates in the femoral onlay model that was accompanied by increased numbers of osteoclast-like cells at 3, 7, and 14 days. Overall, we consistently reproduced BMP2 side effects of cyst-like bone and soft tissue swelling using high BMP2 concentration approaching the typical human 1500 μg/mL.

  5. High Doses of Bone Morphogenetic Protein 2 Induce Structurally Abnormal Bone and Inflammation In Vivo

    PubMed Central

    Zara, Janette N.; Siu, Ronald K.; Zhang, Xinli; Shen, Jia; Ngo, Richard; Lee, Min; Li, Weiming; Chiang, Michael; Chung, Jonguk; Kwak, Jinny; Wu, Benjamin M.; Ting, Kang

    2011-01-01

    The major Food and Drug Association–approved osteoinductive factors in wide clinical use are bone morphogenetic proteins (BMPs). Although BMPs can promote robust bone formation, they also induce adverse clinical effects, including cyst-like bone formation and significant soft tissue swelling. In this study, we evaluated multiple BMP2 doses in a rat femoral segmental defect model and in a minimally traumatic rat femoral onlay model to determine its dose-dependent effects. Results of our femoral segmental defect model established a low BMP2 concentration range (5 and 10 μg/mL, total dose 0.375 and 0.75 μg in 75 μg total volume) unable to induce defect fusion, a mid-range BMP2 concentration range able to fuse the defect without adverse effects (30 μg/mL, total dose 2.25 μg in 75 μg total volume), and a high BMP2 concentration range (150, 300, and 600 μg/mL, total dose 11.25, 22.5, and 45 μg in 75 μg total volume) able to fuse the defect, but with formation of cyst-like bony shells filled with histologically confirmed adipose tissue. In addition, compared to control, 4 mg/mL BMP2 also induced significant tissue inflammatory infiltrates and exudates in the femoral onlay model that was accompanied by increased numbers of osteoclast-like cells at 3, 7, and 14 days. Overall, we consistently reproduced BMP2 side effects of cyst-like bone and soft tissue swelling using high BMP2 concentration approaching the typical human 1500 μg/mL. PMID:21247344

  6. G protein-coupled receptor (GPCR) signaling via heterotrimeric G proteins from endosomes.

    PubMed

    Tsvetanova, Nikoleta G; Irannejad, Roshanak; von Zastrow, Mark

    2015-03-13

    Some G protein-coupled receptors (GPCRs), in addition to activating heterotrimeric G proteins in the plasma membrane, appear to elicit a "second wave" of G protein activation after ligand-induced internalization. We briefly summarize evidence supporting this view and then discuss what is presently known about the functional significance of GPCR-G protein activation in endosomes. Endosomal activation can shape the cellular response temporally by prolonging its overall duration, and may shape the response spatially by moving the location of intracellular second messenger production relative to effectors. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Phenotypic regulation of the sphingosine 1-phosphate receptor miles apart by G protein-coupled receptor kinase 2.

    PubMed

    Burczyk, Martina; Burkhalter, Martin D; Blätte, Tamara; Matysik, Sabrina; Caron, Marc G; Barak, Lawrence S; Philipp, Melanie

    2015-01-27

    The evolutionarily conserved DRY motif at the end of the third helix of rhodopsin-like, class-A G protein-coupled receptors (GPCRs) is a major regulator of receptor stability, signaling activity, and β-arrestin-mediated internalization. Substitution of the DRY arginine with histidine in the human vasopressin receptor results in a loss-of-function phenotype associated with diabetes insipidus. The analogous R150H substitution of the DRY motif in zebrafish sphingosine-1 phosphate receptor 2 (S1p2) produces a mutation, miles apart m(93) (mil(m93)), that not only disrupts signaling but also impairs heart field migration. We hypothesized that constitutive S1p2 desensitization is the underlying cause of this strong zebrafish developmental defect. We observed in cell assays that the wild-type S1p2 receptor is at the cell surface whereas in distinct contrast the S1p2 R150H receptor is found in intracellular vesicles, blocking G protein but not arrestin signaling activity. Surface S1p2 R150H expression could be restored by inhibition of G protein-coupled receptor kinase 2 (GRK2). Moreover, we observed that β-arrestin 2 and GRK2 colocalize with S1p2 in developing zebrafish embryos and depletion of GRK2 in the S1p2 R150H miles apart zebrafish partially rescued cardia bifida. The ability of reduced GRK2 activity to reverse a developmental phenotype associated with constitutive desensitization supports efforts to genetically or pharmacologically target this kinase in diseases involving biased GPCR signaling.

  8. Phenotypic Regulation of the Sphingosine 1-Phosphate Receptor Miles Apart by G Protein-Coupled Receptor Kinase 2

    PubMed Central

    2016-01-01

    The evolutionarily conserved DRY motif at the end of the third helix of rhodopsin-like, class-A G protein-coupled receptors (GPCRs) is a major regulator of receptor stability, signaling activity, and β-arrestin-mediated internalization. Substitution of the DRY arginine with histidine in the human vasopressin receptor results in a loss-of-function phenotype associated with diabetes insipidus. The analogous R150H substitution of the DRY motif in zebrafish sphingosine-1 phosphate receptor 2 (S1p2) produces a mutation, miles apart m93 (milm93), that not only disrupts signaling but also impairs heart field migration. We hypothesized that constitutive S1p2 desensitization is the underlying cause of this strong zebrafish developmental defect. We observed in cell assays that the wild-type S1p2 receptor is at the cell surface whereas in distinct contrast the S1p2 R150H receptor is found in intracellular vesicles, blocking G protein but not arrestin signaling activity. Surface S1p2 R150H expression could be restored by inhibition of G protein-coupled receptor kinase 2 (GRK2). Moreover, we observed that β-arrestin 2 and GRK2 colocalize with S1p2 in developing zebrafish embryos and depletion of GRK2 in the S1p2 R150H miles apart zebrafish partially rescued cardia bifida. The ability of reduced GRK2 activity to reverse a developmental phenotype associated with constitutive desensitization supports efforts to genetically or pharmacologically target this kinase in diseases involving biased GPCR signaling. PMID:25555130

  9. Direct Modulation of Heterotrimeric G Protein-coupled Signaling by a Receptor Kinase Complex.

    PubMed

    Tunc-Ozdemir, Meral; Urano, Daisuke; Jaiswal, Dinesh Kumar; Clouse, Steven D; Jones, Alan M

    2016-07-01

    Plants and some protists have heterotrimeric G protein complexes that activate spontaneously without canonical G protein-coupled receptors (GPCRs). In Arabidopsis, the sole 7-transmembrane regulator of G protein signaling 1 (AtRGS1) modulates the G protein complex by keeping it in the resting state (GDP-bound). However, it remains unknown how a myriad of biological responses is achieved with a single G protein modulator. We propose that in complete contrast to G protein activation in animals, plant leucine-rich repeat receptor-like kinases (LRR RLKs), not GPCRs, provide this discrimination through phosphorylation of AtRGS1 in a ligand-dependent manner. G protein signaling is directly activated by the pathogen-associated molecular pattern flagellin peptide 22 through its LRR RLK, FLS2, and co-receptor BAK1. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Canola and hydrogenated soybean oils accelerate ectopic bone formation induced by implantation of bone morphogenetic protein in mice.

    PubMed

    Hashimoto, Yoko; Mori, Mayumi; Kobayashi, Shuichiro; Hanya, Akira; Watanabe, Shin-Ichi; Ohara, Naoki; Noguchi, Toshihide; Kawai, Tatsushi; Okuyama, Harumi

    2014-01-01

    Canola oil (Can) and hydrogenated soybean oil (H2-Soy) are commonly used edible oils. However, in contrast to soybean oil (Soy), they shorten the survival of stroke-prone spontaneously hypertensive (SHRSP) rats. It has been proposed that the adverse effects of these oils on the kidney and testis are caused at least in part by dihydro-vitamin K (VK) 1 in H2-Soy and unidentified component(s) in Can. Increased intake of dihydro-VK1 is associated with decreased tissue VK2 levels and bone mineral density in rats and humans, respectively. The aim of the present study was to determine the effects of these oils on bone morphogenetic protein (BMP)-induced ectopic bone formation, which is promoted by VK2 deficiency, in relation to the role of VK in the γ-carboxylation of osteocalcin and matrix Gla protein. A crude extract of BMPs was implanted into a gap in the fascia of the femoral muscle in 5-week-old mice maintained on a Soy, Can, or H2-Soy diet. Newly formed bone volume, assessed by three-dimensional X-ray micro-computed tomography and three-dimensional reconstruction imaging for bone, was 4-fold greater in the Can and H2-Soy groups than in the Soy group. The plasma carboxylated osteocalcin (Gla-OC) and total OC (Gla-OC plus undercarboxylated osteocalcin [Glu-OC]) levels were significantly lower in the Can group than in the Soy group ( p < 0.05). However, these levels did not significantly differ between the H2-Soy and Soy groups. The plasma Gla-OC/Glu-OC ratio in the Can and H2-Soy groups was significantly lower (in Can; p = 0.044) or was almost significantly lower (in H2-Soy; p = 0.053) than that in the Soy group. In conclusion, Can and H2-Soy accelerated BMP-induced bone formation in mice to a greater extent than Soy. Further research is required to evaluate whether the difference in accelerated ectopic bone formation is associated with altered levels of VK2 and VK-dependent protein(s) among the three dietary groups.

  11. The human mu opioid receptor: modulation of functional desensitization by calcium/calmodulin-dependent protein kinase and protein kinase C.

    PubMed

    Mestek, A; Hurley, J H; Bye, L S; Campbell, A D; Chen, Y; Tian, M; Liu, J; Schulman, H; Yu, L

    1995-03-01

    Opioids are some of the most efficacious analgesics used in humans. Prolonged administration of opioids, however, often causes the development of drug tolerance, thus limiting their effectiveness. To explore the molecular basis of those mechanisms that may contribute to opioid tolerance, we have isolated a cDNA for the human mu opioid receptor, the target of such opioid narcotics as morphine, codeine, methadone, and fentanyl. The receptor encoded by this cDNA is 400 amino acids long with 94% sequence similarity to the rat mu opioid receptor. Transient expression of this cDNA in COS-7 cells produced high-affinity binding sites to mu-selective agonists and antagonists. This receptor displays functional coupling to a recently cloned G-protein-activated K+ channel. When both proteins were expressed in Xenopus oocytes, functional desensitization developed upon repeated stimulation of the mu opioid receptor, as observed by a reduction in K+ current induced by the second mu receptor activation relative to that induced by the first. The extent of desensitization was potentiated by both the multifunctional calcium/calmodulin-dependent protein kinase and protein kinase C. These results demonstrate that kinase modulation is a molecular mechanism by which the desensitization of mu receptor signaling may be regulated at the cellular level, suggesting that this cellular mechanism may contribute to opioid tolerance in humans.

  12. Periplakin interferes with G protein activation by the melanin-concentrating hormone receptor-1 by binding to the proximal segment of the receptor C-terminal tail.

    PubMed

    Murdoch, Hannah; Feng, Gui-Jie; Bächner, Dietmar; Ormiston, Laura; White, Julia H; Richter, Dietmar; Milligan, Graeme

    2005-03-04

    In mice genetic ablation of expression of either melanin-concentrating hormone or the melanin-concentrating hormone-1 receptor results in alterations in energy metabolism and a lean phenotype. There is thus great interest in the function and regulation of this receptor. Using the yeast two-hybrid system we identified an interaction of the actin- and intermediate filament-binding protein periplakin with the intracellular C-terminal tail of the melanin-concentrating hormone-1 receptor. Direct association of these proteins was verified in pull-down and coimmunoprecipitation experiments. Truncations and internal deletions delineated the site of interaction to a group of 11 amino acids proximal to transmembrane helix VII, which was distinct from the binding site for the melanin-concentrating hormone-1 receptor-interacting zinc finger protein. Immunohistochemistry demonstrated coexpression of periplakin with melanin-concentrating hormone-1 receptor in specific cells of the piriform cortex, amygdala, and other structures of the adult mouse brain. Coexpression of the melanin-concentrating hormone-1 receptor with periplakin in human embryonic kidney 293 cells did not prevent agonist-mediated internalization of the receptor but did interfere with binding of (35)S-labeled guanosine 5'-3-O-(thio)triphosphate ([(35)S]GTPgammaS) to the G protein Galpha(o1) and the elevation of [Ca(2+)](i). Coexpression of the receptor with the interacting zinc finger protein did not modulate receptor internalization or G protein activation. The interaction of periplakin with receptors was selective. Coexpression of periplakin with the IP prostanoid receptor did not result in coimmunoprecipitation nor interfere with agonist-mediated binding of [(35)S]GTPgammaS to the G protein Galpha(s). Periplakin is the first protein described to modify the capacity of the melanin-concentrating hormone-1 receptor to initiate signal transduction.

  13. Estrogen receptor accessory proteins augment receptor-DNA interaction and DNA bending.

    PubMed

    Landel, C C; Potthoff, S J; Nardulli, A M; Kushner, P J; Greene, G L

    1997-01-01

    Increasing evidence suggests that accessory proteins play an important role in the ability of the estrogen receptor (ER) and other nuclear hormone receptors to modulate transcription when bound to cis-acting hormone response elements in target genes. We have previously shown that four proteins, hsp70, protein disulfide isomerase (PDI) and two unknown proteins (p48 and p45), copurify with ER that has been isolated by site-specific DNA chromatography (BERE) and influence the interaction of ER with DNA in vitro. To better define the nature of these effects, we used filter binding and electrophoretic mobility shift assays to study the ability of these proteins to alter the kinetics of ER-DNA interaction and to influence the ability of ER to bend DNA when bound to an estrogen response element (ERE). The results of both assays indicate that ERE-purified ER, with its four associated proteins (hsp70, PDI, p48, p45), has a greater ability to bind to the vitellogenin A2 ERE than ER purified by estradiol-Sepharose chromatography in the absence (ESeph) or presence (EATP) of ATP, in which p48, p45 (ESeph) and hsp70 (EATP) are removed. Surprisingly, the rates of association and dissociation of ER and ERE were essentially the same for all three mixtures, suggesting that one or more ER-associated proteins, especially p45 and p48, may be required for ER to attain maximum DNA binding activity. In addition, circular permutation and phasing analyses demonstrated that the same ER-associated proteins produced higher order ER-DNA complexes that significantly increased the magnitude of DNA distortion, but did not alter the direction of the ER-induced bend of ERE-containing DNA fragments, which was toward the major groove of the DNA helix. These results suggest that p45 and/or p48 and possibly hsp70, play an important role both in the specific DNA binding and bending activities of ER and thus contribute to the overall stimulation of transcription in target genes that contain cis

  14. Visualization of arrestin recruitment by a G Protein-Coupled Receptor

    PubMed Central

    Reis, Rosana I.; Huang, Li-Yin; Tripathi-Shukla, Prachi; Qian, Jiang; Li, Sheng; Blanc, Adi; Oleskie, Austin N.; Dosey, Anne M.; Su, Min; Liang, Cui-Rong; Gu, Ling-Ling; Shan, Jin-Ming; Chen, Xin; Hanna, Rachel; Choi, Minjung; Yao, Xiao Jie; Klink, Bjoern U.; Kahsai, Alem W.; Sidhu, Sachdev S.; Koide, Shohei; Penczek, Pawel A.; Kossiakoff, Anthony A.; Jr, Virgil L. Woods; Kobilka, Brian K.; Skiniotis, Georgios; Lefkowitz, Robert J.

    2014-01-01

    G Protein Coupled Receptors (GPCRs) are critically regulated by β-arrestins (βarrs), which not only desensitize G protein signaling but also initiate a G protein independent wave of signaling1-5. A recent surge of structural data on a number of GPCRs, including the β2 adrenergic receptor (β2AR)-G protein complex, has provided novel insights into the structural basis of receptor activation6-11. Lacking however has been complementary information on recruitment of βarrs to activated GPCRs primarily due to challenges in obtaining stable receptor-βarr complexes for structural studies. Here, we devised a strategy for forming and purifying a functional β2AR-βarr1 complex that allowed us to visualize its architecture by single particle negative stain electron microscopy (EM) and to characterize the interactions between β2AR and βarr1 using hydrogen-deuterium exchange mass spectrometry (HDXMS) and chemical cross-linking. EM 2D averages and 3D reconstructions reveal bimodal binding of βarr1 to the β2AR, involving two separate sets of interactions, one with the phosphorylated carboxy-terminus of the receptor and the other with its seven-transmembrane core. Areas of reduced HDX together with identification of cross-linked residues suggest engagement of the finger loop of βarr1 with the seven-transmembrane core of the receptor. In contrast, focal areas of increased HDX indicate regions of increased dynamics in both N and C domains of βarr1 when coupled to the β2AR. A molecular model of the β2AR-βarr signaling complex was made by docking activated βarr1 and β2AR crystal structures into the EM map densities with constraints provided by HDXMS and cross-linking, allowing us to obtain valuable insights into the overall architecture of a receptor-arrestin complex. The dynamic and structural information presented herein provides a framework for better understanding the basis of GPCR regulation by arrestins. PMID:25043026

  15. Structural prerequisites for G-protein activation by the neurotensin receptor

    DOE PAGES

    Krumm, Brian E.; White, Jim F.; Shah, Priyanka; ...

    2015-07-24

    We previously determined the structure of neurotensin receptor NTSR1 in an active-like conformation with six thermostabilizing mutations bound to the peptide agonist neurotensin. This receptor was unable to activate G proteins, indicating that the mutations restricted NTSR1 to relate agonist binding to G-protein activation. Here we analyse the effect of three of those mutations (E166A 3.49, L310A 6.37, F358A 7.42) and present two structures of NTSR1 able to catalyse nucleotide exchange at Gα. The presence of F358 7.42 causes the conserved W321 6.48 to adopt a side chain orientation parallel to the lipid bilayer sealing the collapsed Na+ ion pocketmore » and linking the agonist with residues in the lower receptor part implicated in GPCR activation. In the intracellular receptor half, the bulkier L310 6.37 side chain dictates the position of R167 3.50 of the highly conserved D/ERY motif. These residues, together with the presence of E166 3.49 provide determinants for G-protein activation by NTSR1.« less

  16. Structural prerequisites for G-protein activation by the neurotensin receptor

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Krumm, Brian E.; White, Jim F.; Shah, Priyanka

    We previously determined the structure of neurotensin receptor NTSR1 in an active-like conformation with six thermostabilizing mutations bound to the peptide agonist neurotensin. This receptor was unable to activate G proteins, indicating that the mutations restricted NTSR1 to relate agonist binding to G-protein activation. Here we analyse the effect of three of those mutations (E166A 3.49, L310A 6.37, F358A 7.42) and present two structures of NTSR1 able to catalyse nucleotide exchange at Gα. The presence of F358 7.42 causes the conserved W321 6.48 to adopt a side chain orientation parallel to the lipid bilayer sealing the collapsed Na+ ion pocketmore » and linking the agonist with residues in the lower receptor part implicated in GPCR activation. In the intracellular receptor half, the bulkier L310 6.37 side chain dictates the position of R167 3.50 of the highly conserved D/ERY motif. These residues, together with the presence of E166 3.49 provide determinants for G-protein activation by NTSR1.« less

  17. Type 1 angiotensin II receptor-associated protein ARAP1 binds and recycles the receptor to the plasma membrane.

    PubMed

    Guo, Deng-Fu; Chenier, Isabelle; Tardif, Valerie; Orlov, Sergei N; Inagami, Tadashi

    2003-10-31

    The carboxyl terminus of the type 1 angiotensin II receptor (AT(1)) plays an important role in receptor phosphorylation, desensitization, and internalization. The yeast two-hybrid system was employed to isolate proteins associated with the carboxyl terminal region of the AT(1A) receptor. In the present study, we report the isolation of a novel protein, ARAP1, which promotes recycling of AT(1A) to the plasma membrane in HEK-293 cells. ARAP1 cDNA encodes a 493-amino-acid protein and its mRNA is ubiquitously expressed in rat tissues. A complex of ARAP1 and AT(1A) was observed by immunoprecipitation and Western blotting in HEK-293 cells. In the presence of ARAP1, recycled AT(1A) showed a significant Ca(2+) release response to a second stimulation by Ang II 30 min after the first treatment. Immunocytochemical analysis revealed co-localization of recycled AT(1A) and ARAP1 in the plasma membrane 45 min after the initial exposure to Ang II. Taken together, these results indicate a role for ARAP1 in the recycling of the AT(1) receptor to the plasma membrane with presumable concomitant recovery of receptor signal functions.

  18. G-protein mediates voltage regulation of agonist binding to muscarinic receptors: effects on receptor-Na/sup +/ channel interaction

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Cohen-Armon, M.; Garty, H.; Sokolovsky, M.

    1988-01-12

    The authors previous experiments in membranes prepared from rat heart and brain led them to suggest that the binding of agonist to the muscarinic receptors and to the Na/sup +/ channels is a coupled event mediated by guanine nucleotide binding protein(s) (G-protein(s)). These in vitro findings prompted us to employ synaptoneurosomes from brain stem tissue to examine (i) the binding properties of (/sup 3/H) acetylcholine at resting potential and under depolarization conditions in the absence and presence of pertussis toxin; (ii) the binding of (/sup 3/H)batrachotoxin to Na/sup +/ channel(s) in the presence of the muscarinic agonists; and (iii) muscarinicallymore » induced /sup 22/Na/sup +/ uptake in the presence and absence of tetrodotoxin, which blocks Na/sup +/ channels. The findings indicate that agonist binding to muscarinic receptors is voltage dependent, that this process is mediated by G-protein(s), and that muscarinic agonists induce opening of Na/sup +/channels. The latter process persists even after pertussis toxin treatment, indicating that it is not likely to be mediated by pertussis toxin sensitive G-protein(s). The system with its three interacting components-receptor, G-protein, and Na/sup +/ channel-is such that at resting potential the muscarinic receptor induces opening of Na/sup +/ channels; this property may provide a possible physiological mechanism for the depolarization stimulus necessary for autoexcitation or repetitive firing in heart or brain tissues.« less

  19. Role of non-receptor protein kinases in spermatid transport during spermatogenesis*

    PubMed Central

    Wan, H. T.; Mruk, Dolores D.; Tang, Elizabeth I.; Xiao, Xiang; Cheng, Yan-ho; Wong, Elissa W.P.; Wong, Chris K. C.; Cheng, C. Yan

    2014-01-01

    Non-receptor protein tyrosine kinases are cytoplasmic kinases that activate proteins by phosphorylating target protein tyrosine residues, in turn affecting multiple functions in eukaryotic cells. Herein, we focus on the role of non-receptor protein tyrosine kinases, most notably, FAK, c-Yes and c-Src, in the transport of spermatids across the seminiferous epithelium during spermatogenesis. Since spermatids, which are formed from spermatocytes via meiosis, are immotile haploid cells, they must be transported by Sertoli cells across the seminiferous epithelium during the epithelial cycle of spermatogenesis. Without the timely transport of spermatids across the epithelium, the release of sperms at spermiation fails to occur, leading to infertility. Thus, the molecular event pertinent to spermatid transport is crucial to spermatogenesis. Herein, we provide a critical discussion based on recent findings in the field. We also provide a hypothetical model on spermatid transport, and the role of non-receptor protein tyrosine kinases in this event. We also highlight areas of research that deserve attention by investigators in the field. PMID:24727349

  20. Functional autoantibodies targeting G protein-coupled receptors in rheumatic diseases.

    PubMed

    Cabral-Marques, Otavio; Riemekasten, Gabriela

    2017-11-01

    G protein-coupled receptors (GPCRs) comprise the largest and most diverse family of integral membrane proteins that participate in different physiological processes such as the regulation of the nervous and immune systems. Besides the endogenous ligands of GPCRs, functional autoantibodies are also able to bind GPCRs to trigger or block intracellular signalling pathways, resulting in agonistic or antagonistic effects, respectively. In this Review, the effects of functional GPCR-targeting autoantibodies on the pathogenesis of autoimmune diseases, including rheumatic diseases, are discussed. Autoantibodies targeting β1 and β2 adrenergic receptors, which are expressed by cardiac and airway smooth muscle cells, respectively, have an important role in the development of asthma and cardiovascular diseases. In addition, high levels of autoantibodies against the muscarinic acetylcholine receptor M3 as well as those targeting endothelin receptor type A and type 1 angiotensin II receptor have several implications in the pathogenesis of rheumatic diseases such as Sjögren syndrome and systemic sclerosis. Expanding the knowledge of the pathophysiological roles of autoantibodies against GPCRs will shed light on the biology of these receptors and open avenues for new therapeutic approaches.

  1. Inhibitory Smads and bone morphogenetic protein (BMP) modulate anterior photoreceptor cell number during planarian eye regeneration.

    PubMed

    González-Sastre, Alejandro; Molina, Ma Dolores; Saló, Emili

    2012-01-01

    Planarians represent an excellent model to study the processes of body axis and organ re-specification during regeneration. Previous studies have revealed a conserved role for the bone morphogenetic protein (BMP) pathway and its intracellular mediators Smad1/5/8 and Smad4 in planarian dorsoventral (DV) axis re-establishment. In an attempt to gain further insight into the role of this signalling pathway in planarians, we have isolated and functionally characte-rized the inhibitory Smads (I-Smads) in Schmidtea mediterranea. Two I-Smad homologues have been identified: Smed-smad6/7-1 and Smed-smad6/7-2. Expression of smad6/7-1 was detected in the parenchyma, while smad6/7-2 was found to be ex-pressed in the central nervous system and the eyes. Neither single smad6/7-1 and smad6/7-2 nor double smad6/7-1,-2 silencing gave rise to any apparent disruption of the DV axis. However, both regenerating and intact smad6/7-2 (RNAi) planarians showed defects in eye morphogenesis and displayed small, rounded eyes that lacked the anterior subpopulation of photoreceptor cells. The number of pigment cells was also reduced in these animals at later stages of regeneration. In contrast, after low doses of Smed-bmp(RNAi), planarians regenerated larger eyes in which the anterior subpopulation of photoreceptor cells was expanded. Our results suggest that Smed-smad6/7-2 and Smed-bmp control the re-specification and maintenance of anterior photoreceptor cell number in S. mediterranea.

  2. Activity of bone morphogenetic protein-7 after treatment at various temperatures: freezing vs. pasteurization vs. allograft.

    PubMed

    Takata, Munetomo; Sugimoto, Naotoshi; Yamamoto, Norio; Shirai, Toshiharu; Hayashi, Katsuhiro; Nishida, Hideji; Tanzawa, Yoshikazu; Kimura, Hiroaki; Miwa, Shinji; Takeuchi, Akihiko; Tsuchiya, Hiroyuki

    2011-12-01

    Insufficient bone union is the occasional complication of biomechanical reconstruction after malignant bone tumor resection using temperature treated tumor bearing bone; freezing, pasteurization, and autoclaving. Since bone morphogenetic protein (BMP) plays an important role in bone formation, we assessed the amount and activity of BMP preserved after several temperature treatments, including -196 and -73°C for 20 min, 60 and 100°C for 30 min, 60°C for 10h following -80°C for 12h as an allograft model, and 4°C as the control. The material extracted from the human femoral bone was treated, and the amount of BMP-7 was analyzed using an enzyme-linked immunosorbent assay. Then, the activity of recombinant human BMP-7 after the treatment was assessed using a bioassay with NIH3T3 cells and immunoblotting analysis to measure the amount of phospho-Smad, one of the signaling substrates that reflect the intracellular reaction of BMPs. Both experiments revealed that BMP-7 was significantly better preserved in the hypothermia groups. The percentages of the amount of BMP-7 in which the control group was set at 100% were 114%, 108%, 70%, 49%, and 53% in the -196, -73, 60, 100°C, and the allograft-model group, respectively. The percentages of the amount of phospho-Smad were 89%, 87%, 24%, 4.9%, and 14% in the -196, -73, 60, 100°C, and the allograft-model group, respectively. These results suggested that freezing possibly preserves osteoinductive ability than hyperthermia treatment. Copyright © 2011 Elsevier Inc. All rights reserved.

  3. Signaling by Kit protein-tyrosine kinase--the stem cell factor receptor.

    PubMed

    Roskoski, Robert

    2005-11-11

    Signaling by stem cell factor and Kit, its receptor, plays important roles in gametogenesis, hematopoiesis, mast cell development and function, and melanogenesis. Moreover, human and mouse embryonic stem cells express Kit transcripts. Stem cell factor exists as both a soluble and a membrane-bound glycoprotein while Kit is a receptor protein-tyrosine kinase. The complete absence of stem cell factor or Kit is lethal. Deficiencies of either produce defects in red and white blood cell production, hypopigmentation, and sterility. Gain-of-function mutations of Kit are associated with several human neoplasms including acute myelogenous leukemia, gastrointestinal stromal tumors, and mastocytomas. Kit consists of an extracellular domain, a transmembrane segment, a juxtamembrane segment, and a protein kinase domain that contains an insert of about 80 amino acid residues. Binding of stem cell factor to Kit results in receptor dimerization and activation of protein kinase activity. The activated receptor becomes autophosphorylated at tyrosine residues that serve as docking sites for signal transduction molecules containing SH2 domains. The adaptor protein APS, Src family kinases, and Shp2 tyrosyl phosphatase bind to phosphotyrosine 568. Shp1 tyrosyl phosphatase and the adaptor protein Shc bind to phosphotyrosine 570. C-terminal Src kinase homologous kinase and the adaptor Shc bind to both phosphotyrosines 568 and 570. These residues occur in the juxtamembrane segment of Kit. Three residues in the kinase insert domain are phosphorylated and attract the adaptor protein Grb2 (Tyr703), phosphatidylinositol 3-kinase (Tyr721), and phospholipase Cgamma (Tyr730). Phosphotyrosine 900 in the distal kinase domain binds phosphatidylinositol 3-kinase which in turn binds the adaptor protein Crk. Phosphotyrosine 936, also in the distal kinase domain, binds the adaptor proteins APS, Grb2, and Grb7. Kit has the potential to participate in multiple signal transduction pathways as a result of

  4. Introduction to Sigma Proteins: Evolution of the Concept of Sigma Receptors.

    PubMed

    Kim, Felix J

    2017-01-01

    For over 40 years, scientists have endeavored to understand the so-called sigma receptors. During this time, the concept of sigma receptors has continuously and significantly evolved. With thousands of publications on the subject, these proteins have been implicated in various diseases, disorders, and physiological processes. Nevertheless, we are just beginning to understand what sigma proteins do and how they work. Two subtypes have been identified, Sigma1 and Sigma2. Whereas Sigma1 (also known as sigma-1 receptor, Sig1R, σ1 receptor, and several other names) was cloned over 20 years ago, Sigma2 (sigma-2 receptor, σ2 receptor) was cloned very recently and had remained a pharmacologically defined entity. In this volume, we will focus primarily on Sigma1. We will highlight several key subject areas in which Sigma1 has been well characterized as well as (re)emerging areas of interest. Despite the large number of publications regarding Sigma1, several fundamental questions remain unanswered or only partially answered. Most of what we know about Sigma1 comes from pharmacological studies; however, a clearly defined molecular mechanism of action remains elusive. One concept has become clear; Sigma1 is not a traditional receptor. Sigma1 is now considered a unique pharmacologically regulated integral membrane chaperone or scaffolding protein. A number of landmark discoveries over the past decade have begun to reshape the concept of sigma receptors. With the rapid emergence of new information, development of new tools, and changing conceptual frameworks, the field is poised for a period of accelerated progress.

  5. Lack of effect of bone morphogenetic protein 2 and 4 gene polymorphisms on bone density in postmenopausal Turkish women.

    PubMed

    Ozkan, Z S; Deveci, D; Onalan Etem, E; Yüce, H

    2010-11-30

    We investigated the effect of bone morphogenetic protein 2 and 4 (BMP-2 and -4) gene polymorphisms on bone density in postmenopausal Turkish women with osteoporosis. The frequency of single-nucleotide polymorphisms (SNPs) of BMP-2 and -4 genes was analyzed in 101 osteoporotic-postmenopausal women and 52 postmenopausal women with positive bone mineral density scores. We evaluated the frequency of the thymine→cytosine nucleotide variation at position 538 for BMP-4 and the transposition of adenine→thymine at codon 190 for BMP-2, with PCR. The proportions of genotypes observed for the BMP-2 SNP in the osteoporotic group were AA (47.5%), AT (39.6%), TT (12.9%), and in the non-osteoporotic group they were AA (48.1%), AT (40.4%), TT (11.5%). The corresponding frequencies for the BMP-4 SNP in the osteoporotic group were TT (30.7%), TC (45.5%), CC (23.8%), and in the non-osteoporotic group they were TT (26.9%), TC (40.4%), CC (32.7%). There were no significant differences in the frequencies of these genotypes between the patient and control groups. We conclude that genetic variations in BMP-2 and -4 do not substantially contribute to lumbar spine bone mineral density in postmenopausal Turkish women.

  6. Oxygen-glucose deprivation preconditioning protects neurons against oxygen-glucose deprivation/reperfusion induced injury via bone morphogenetic protein-7 mediated ERK, p38 and Smad signalling pathways.

    PubMed

    Guan, Junhong; Du, Shaonan; Lv, Tao; Qu, Shengtao; Fu, Qiang; Yuan, Ye

    2016-01-01

    Bone morphogenetic protein (BMP)-7 mediated neuroprotective effect of cerebral ischemic preconditioning (IPC) has been studied in an ischemic animal model, but the underlying cellular mechanisms have not been clearly clarified. In this study, primary cortical neurons and the SH-SY5Y cell line were used to investigate the role of BMP-7 and its downstream signals in the neuroprotective effects of oxygen-glucose deprivation preconditioning (OGDPC). Immunocytochemistry was used to detect the expression of neurofilament in neurons. MTT and lactate dehydrogenase activity assays were used to measure the cytotoxicity. Western blot was used to detect the protein expression of BMP-7 and downstream signals. BMP inhibitor, mitogen-activated protein kinase inhibitors, Smad inhibitor and siRNA of Smad 1 were used to investigate the role of corresponding signalling pathways in the OGDPC. Results showed that OGDPC-induced overexpression of BMP-7 in primary cortical neurons and SH-SY5Y cells. Both of endogenous and exogenous BMP-7 could replicate the neuroprotective effects seen in OGDPC pretreatment. In addition, extracellular regulated protein kinases, p38 and Smad signalling pathway were found to be involved in the neuroprotective effects mediated by OGDPC via BMP-7. This study primarily reveals the cellular mechanisms of the neuroprotection mediated by OGDPC, and provides evidence for better understanding of this intrinsic factor against ischemia. © 2015 Wiley Publishing Asia Pty Ltd.

  7. The Significance of G Protein-Coupled Receptor Crystallography for Drug Discovery

    PubMed Central

    Salon, John A.; Lodowski, David T.

    2011-01-01

    Crucial as molecular sensors for many vital physiological processes, seven-transmembrane domain G protein-coupled receptors (GPCRs) comprise the largest family of proteins targeted by drug discovery. Together with structures of the prototypical GPCR rhodopsin, solved structures of other liganded GPCRs promise to provide insights into the structural basis of the superfamily's biochemical functions and assist in the development of new therapeutic modalities and drugs. One of the greatest technical and theoretical challenges to elucidating and exploiting structure-function relationships in these systems is the emerging concept of GPCR conformational flexibility and its cause-effect relationship for receptor-receptor and receptor-effector interactions. Such conformational changes can be subtle and triggered by relatively small binding energy effects, leading to full or partial efficacy in the activation or inactivation of the receptor system at large. Pharmacological dogma generally dictates that these changes manifest themselves through kinetic modulation of the receptor's G protein partners. Atomic resolution information derived from increasingly available receptor structures provides an entrée to the understanding of these events and practically applying it to drug design. Supported by structure-activity relationship information arising from empirical screening, a unified structural model of GPCR activation/inactivation promises to both accelerate drug discovery in this field and improve our fundamental understanding of structure-based drug design in general. This review discusses fundamental problems that persist in drug design and GPCR structural determination. PMID:21969326

  8. Structure-Based Rational Design of a Toll-like Receptor 4 (TLR4) Decoy Receptor with High Binding Affinity for a Target Protein

    PubMed Central

    Lee, Sang-Chul; Hong, Seungpyo; Park, Keunwan; Jeon, Young Ho; Kim, Dongsup; Cheong, Hae-Kap; Kim, Hak-Sung

    2012-01-01

    Repeat proteins are increasingly attracting much attention as alternative scaffolds to immunoglobulin antibodies due to their unique structural features. Nonetheless, engineering interaction interface and understanding molecular basis for affinity maturation of repeat proteins still remain a challenge. Here, we present a structure-based rational design of a repeat protein with high binding affinity for a target protein. As a model repeat protein, a Toll-like receptor4 (TLR4) decoy receptor composed of leucine-rich repeat (LRR) modules was used, and its interaction interface was rationally engineered to increase the binding affinity for myeloid differentiation protein 2 (MD2). Based on the complex crystal structure of the decoy receptor with MD2, we first designed single amino acid substitutions in the decoy receptor, and obtained three variants showing a binding affinity (KD) one-order of magnitude higher than the wild-type decoy receptor. The interacting modes and contributions of individual residues were elucidated by analyzing the crystal structures of the single variants. To further increase the binding affinity, single positive mutations were combined, and two double mutants were shown to have about 3000- and 565-fold higher binding affinities than the wild-type decoy receptor. Molecular dynamics simulations and energetic analysis indicate that an additive effect by two mutations occurring at nearby modules was the major contributor to the remarkable increase in the binding affinities. PMID:22363519

  9. Hydrolysis and Sulfation Pattern Effects on Release of Bioactive Bone Morphogenetic Protein-2 from Heparin-Based Microparticles.

    PubMed

    Tellier, Liane E; Miller, Tobias; McDevitt, Todd C; Temenoff, Johnna S

    2015-10-28

    Glycosaminoglycans (GAGs) such as heparin are promising materials for growth factor delivery due to their ability to efficiently bind positively charged growth factors including bone morphogenetic protein-2 (BMP-2) through their negatively charged sulfate groups. Therefore, the goal of this study was to examine BMP-2 release from heparin-based microparticles (MPs) after first, incorporating a hydrolytically degradable crosslinker and varying heparin content within MPs to alter MP degradation and second, altering the sulfation pattern of heparin within MPs to vary BMP-2 binding and release. Using varied MP formulations, it was found that the time course of MP degradation for 1 wt% heparin MPs was ~4 days slower than 10 wt% heparin MPs, indicating that MP degradation was dependent on heparin content. After incubating 100 ng BMP-2 with 0.1 mg MPs, most MP formulations loaded BMP-2 with ~50% efficiency and significantly more BMP-2 release (60% of loaded BMP-2) was observed from more sulfated heparin MPs (MPs with ~100% and 80% of native sulfation). Similarly, BMP-2 bioactivity in more sulfated heparin MP groups was at least four-fold higher than soluble BMP-2 and less sulfated heparin MP groups, as determined by an established C2C12 cell alkaline phosphatase (ALP) assay. Ultimately, the two most sulfated 10 wt% heparin MP formulations were able to efficiently load and release BMP-2 while enhancing BMP-2 bioactivity, making them promising candidates for future growth factor delivery applications.

  10. Homology Modeling of Class A G Protein-Coupled Receptors

    PubMed Central

    Costanzi, Stefano

    2012-01-01

    G protein-coupled receptors (GPCRs) are a large superfamily of membrane bound signaling proteins that hold great pharmaceutical interest. Since experimentally elucidated structures are available only for a very limited number of receptors, homology modeling has become a widespread technique for the construction of GPCR models intended to study the structure-function relationships of the receptors and aid the discovery and development of ligands capable of modulating their activity. Through this chapter, various aspects involved in the constructions of homology models of the serpentine domain of the largest class of GPCRs, known as class A or rhodopsin family, are illustrated. In particular, the chapter provides suggestions, guidelines and critical thoughts on some of the most crucial aspect of GPCR modeling, including: collection of candidate templates and a structure-based alignment of their sequences; identification and alignment of the transmembrane helices of the query receptor to the corresponding domains of the candidate templates; selection of one or more templates receptor; election of homology or de novo modeling for the construction of specific extracellular and intracellular domains; construction of the three-dimensional models, with special consideration to extracellular regions, disulfide bridges, and interhelical cavity; validation of the models through controlled virtual screening experiments. PMID:22323225

  11. Structural–Functional Features of the Thyrotropin Receptor: A Class A G-Protein-Coupled Receptor at Work

    PubMed Central

    Kleinau, Gunnar; Worth, Catherine L.; Kreuchwig, Annika; Biebermann, Heike; Marcinkowski, Patrick; Scheerer, Patrick; Krause, Gerd

    2017-01-01

    The thyroid-stimulating hormone receptor (TSHR) is a member of the glycoprotein hormone receptors, a sub-group of class A G-protein-coupled receptors (GPCRs). TSHR and its endogenous ligand thyrotropin (TSH) are of essential importance for growth and function of the thyroid gland and proper function of the TSH/TSHR system is pivotal for production and release of thyroid hormones. This receptor is also important with respect to pathophysiology, such as autoimmune (including ophthalmopathy) or non-autoimmune thyroid dysfunctions and cancer development. Pharmacological interventions directly targeting the TSHR should provide benefits to disease treatment compared to currently available therapies of dysfunctions associated with the TSHR or the thyroid gland. Upon TSHR activation, the molecular events conveying conformational changes from the extra- to the intracellular side of the cell across the membrane comprise reception, conversion, and amplification of the signal. These steps are highly dependent on structural features of this receptor and its intermolecular interaction partners, e.g., TSH, antibodies, small molecules, G-proteins, or arrestin. For better understanding of signal transduction, pathogenic mechanisms such as autoantibody action and mutational modifications or for developing new pharmacological strategies, it is essential to combine available structural data with functional information to generate homology models of the entire receptor. Although so far these insights are fragmental, in the past few decades essential contributions have been made to investigate in-depth the involved determinants, such as by structure determination via X-ray crystallography. This review summarizes available knowledge (as of December 2016) concerning the TSHR protein structure, associated functional aspects, and based on these insights we suggest several receptor complex models. Moreover, distinct TSHR properties will be highlighted in comparison to other class A GPCRs to

  12. Seminal Plasma Proteins as Androgen Receptor Corregulators Promote Prostate Cancer Growth

    DTIC Science & Technology

    2016-12-01

    lines as well as the peptides described above, we will assess the efficacy of SgI peptides on tumor growth in a mouse xenograft model. Opportunities...Award Number: W81XWH-13-1-0412 TITLE: Seminal Plasma Proteins as Androgen Receptor Corregulators Promote Prostate Cancer Growth PRINCIPAL...SUBTITLE Seminal Plasma Proteins as Androgen Receptor Corregulators Promote Prostate Cancer Growth 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-13

  13. GIV/Girdin transmits signals from multiple receptors by triggering trimeric G protein activation.

    PubMed

    Garcia-Marcos, Mikel; Ghosh, Pradipta; Farquhar, Marilyn G

    2015-03-13

    Activation of trimeric G proteins has been traditionally viewed as the exclusive job of G protein-coupled receptors (GPCRs). This view has been challenged by the discovery of non-receptor activators of trimeric G proteins. Among them, GIV (a.k.a. Girdin) is the first for which a guanine nucleotide exchange factor (GEF) activity has been unequivocally associated with a well defined motif. Here we discuss how GIV assembles alternative signaling pathways by sensing cues from various classes of surface receptors and relaying them via G protein activation. We also describe the dysregulation of this mechanism in disease and how its targeting holds promise for novel therapeutics. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. G protein-coupled odorant receptors: From sequence to structure

    PubMed Central

    de March, Claire A; Kim, Soo-Kyung; Antonczak, Serge; Goddard, William A; Golebiowski, Jérôme

    2015-01-01

    Odorant receptors (ORs) are the largest subfamily within class A G protein-coupled receptors (GPCRs). No experimental structural data of any OR is available to date and atomic-level insights are likely to be obtained by means of molecular modeling. In this article, we critically align sequences of ORs with those GPCRs for which a structure is available. Here, an alignment consistent with available site-directed mutagenesis data on various ORs is proposed. Using this alignment, the choice of the template is deemed rather minor for identifying residues that constitute the wall of the binding cavity or those involved in G protein recognition. PMID:26044705

  15. Elevated extracellular calcium increases expression of bone morphogenetic protein-2 gene via a calcium channel and ERK pathway in human dental pulp cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Tada, Hiroyuki; Nemoto, Eiji, E-mail: e-nemoto@umin.ac.jp; Kanaya, Sousuke

    Dental pulp cells, which have been shown to share phenotypical features with osteoblasts, are capable of differentiating into odontoblast-like cells and generating a dentin-like mineral structure. Elevated extracellular Ca{sup 2+}Ca{sub o}{sup 2+} has been implicated in osteogenesis by stimulating the proliferation and differentiation of osteoblasts; however, the role of Ca{sub o}{sup 2+} signaling in odontogenesis remains unclear. We found that elevated Ca{sub o}{sup 2+} increases bone morphogenetic protein (BMP)-2 gene expression in human dental pulp cells. The increase was modulated not only at a transcriptional level but also at a post-transcriptional level, because treatment with Ca{sup 2+} increased the stabilitymore » of BMP-2 mRNA in the presence of actinomycin D, an inhibitor of transcription. A similar increase in BMP-2 mRNA level was observed in other human mesenchymal cells from oral tissue; periodontal ligament cells and gingival fibroblasts. However, the latter cells exhibited considerably lower expression of BMP-2 mRNA compared with dental pulp cells and periodontal ligament cells. The BMP-2 increase was markedly inhibited by pretreatment with an extracellular signal-regulated kinase (ERK) inhibitor, PD98059, and partially inhibited by the L-type Ca{sup 2+} channels inhibitor, nifedipine. However, pretreatment with nifedipine had no effect on ERK1/2 phosphorylation triggered by Ca{sup 2+}, suggesting that the Ca{sup 2+} influx from Ca{sup 2+} channels may operate independently of ERK signaling. Dental pulp cells do not express the transcript of Ca{sup 2+}-sensing receptors (CaSR) and only respond slightly to other cations such as Sr{sup 2+} and spermine, suggesting that dental pulp cells respond to Ca{sub o}{sup 2+} to increase BMP-2 mRNA expression in a manner different from CaSR and rather specific for Ca{sub o}{sup 2+} among cations.« less

  16. Lack of Association of Bone Morphogenetic Protein 2 Gene Haplotypes with Bone Mineral Density, Bone Loss, or Risk of Fractures in Men

    PubMed Central

    Varanasi, Satya S.; Tuck, Stephen P.; Mastana, Sarabjit S.; Dennison, Elaine; Cooper, Cyrus; Vila, Josephine; Francis, Roger M.; Datta, Harish K.

    2011-01-01

    Introduction. The association of bone morphogenetic protein 2 (BMP2) with BMD and risk of fracture was suggested by a recent linkage study, but subsequent studies have been contradictory. We report the results of a study of the relationship between BMP2 genotypes and BMD, annual change in BMD, and risk of fracture in male subjects. Materials and Methods. We tested three single-nucleotide polymorphisms (SNPs) across the BMP2 gene, including Ser37Ala SNP, in 342 Caucasian Englishmen, comprising 224 control and 118 osteoporotic subjects. Results. BMP2 SNP1 (Ser37Ala) genotypes were found to have similar low frequency in control subjects and men with osteoporosis. The major informative polymorphism, BMP2 SNP3 (Arg190Ser), showed no statistically significant association with weight, height, BMD, change in BMD at hip or lumbar spine, and risk of fracture. Conclusion. There were no genotypic or haplotypic effects of the BMP2 candidate gene on BMD, change in BMD, or fracture risk identified in this cohort. PMID:22013543

  17. Up-regulation of proproliferative genes and the ligand/receptor pair placental growth factor and vascular endothelial growth factor receptor 1 in hepatitis C cirrhosis.

    PubMed

    Huang, Xiao X; McCaughan, Geoffrey W; Shackel, Nicholas A; Gorrell, Mark D

    2007-09-01

    Cirrhosis can lead to hepatocellular carcinoma (HCC). Non-diseased liver and hepatitis C virus (HCV)-associated cirrhosis with or without HCC were compared. Proliferation pathway genes, immune response genes and oncogenes were analysed by a quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and immunostaining. Real-time RT-PCR showed up-regulation of genes in HCV cirrhosis including the proliferation-associated genes bone morphogenetic protein 3 (BMP3), placental growth factor 3 (PGF3), vascular endothelial growth factor receptor 1 (VEGFR1) and soluble VEGFR1, the oncogene FYN, and the immune response-associated genes toll-like receptor 9 (TLR9) and natural killer cell transcript 4 (NK4). Expressions of TLR2 and the oncogenes B-cell CLL/lymphoma 9 (BCL9) and PIM2 were decreased in HCV cirrhosis. In addition, PIM2 and TLR2 were increased in HCV cirrhosis with HCC compared with HCV cirrhosis. The ligand/receptor pair PGF and VEGFR1 was intensely expressed by the portal tract vascular endothelium. VEGFR1 was expressed in reactive biliary epithelial structures in fibrotic septum and in some stellate cells and macrophages. PGF and VEGFR1 may have an important role in the pathogenesis of the neovascular response in cirrhosis.

  18. GPR30: A G protein-coupled receptor for estrogen.

    PubMed

    Prossnitz, Eric R; Arterburn, Jeffrey B; Sklar, Larry A

    2007-02-01

    Estrogen is a critical steroid in human physiology exerting its effect both at the transcriptional level as well as at the level of rapid intracellular signaling through second messengers. Many of estrogen's transcriptional effects have long been known to be mediated through classical nuclear steroid receptors but recent studies also demonstrate the existence of a 7-transmembrane G protein-coupled receptor, GPR30 that responds to estrogen with rapid cellular signaling. There is currently controversy over the ability of classical estrogen receptors to recapitulate GPR30-mediated signaling mechanisms and vice versa. This article will summarize recent literature and address the relationship between GPR30 and conventional estrogen receptor signaling.

  19. Quartz crystal microbalance (QCM) with immobilized protein receptors: comparison of response to ligand binding for direct protein immobilization and protein attachment via disulfide linker.

    PubMed

    Baltus, Ruth E; Carmon, Kendra S; Luck, Linda A

    2007-03-27

    Results from an investigation of the frequency response resulting from ligand binding for a genetically engineered hormone-binding domain of the alpha-estrogen receptor immobilized to a piezoelectric quartz crystal are reported. Two different approaches were used to attach a genetically altered receptor to the gold electrode on the quartz surface: (1) the mutant receptor containing a single solvent-exposed cysteine was directly attached to the crystal via a sulfur to gold covalent bond, forming a self-assembled protein monolayer, and (2) the N-terminal histidine-tagged end was utilized to attach the receptor via a 3,3-dithiobis[N-(5-amino-5-carboxypentyl)propionamide-N',N'-diacetic acid] linker complexed with nickel. Previous studies have shown that these engineered constructs bind 17beta-estradiol and are fully functional. Exposure of the receptor directly attached to the piezoelectric crystal to the known ligand 17beta-estradiol resulted in a measurable frequency response, consistent with a change in conformation of the receptor with ligand binding. However, no response was observed when the receptor immobilized via the linker was exposed to the same ligand. The presence of the linker between the quartz surface and the protein receptor does not allow the crystal to sense the conformational change in the receptor that occurs with ligand binding. These results illustrate that the immobilization strategy used to bind the receptor to the sensor platform is key to eliciting an appropriate response from this biosensor. This study has important implications for the development of QCM-based sensors using protein receptors.

  20. Effects of LED phototherapy on bone defects grafted with MTA, bone morphogenetic proteins and guided bone regeneration: a Raman spectroscopic study.

    PubMed

    Pinheiro, Antonio L B; Soares, Luiz G P; Cangussú, Maria Cristina T; Santos, Nicole R S; Barbosa, Artur Felipe S; Silveira Júnior, Landulfo

    2012-09-01

    We studied peaks of calcium hydroxyapatite (CHA) and protein and lipid CH groups in defects grafted with mineral trioxide aggregate (MTA) treated or not with LED irradiation, bone morphogenetic proteins and guided bone regeneration. A total of 90 rats were divided into ten groups each of which was subdivided into three subgroups (evaluated at 15, 21 and 30 days after surgery). Defects were irradiated with LED light (wavelength 850 ± 10 nm) at 48-h intervals for 15 days. Raman readings were taken at the surface of the defects. There were no statistically significant differences in the CHA peaks among the nonirradiated defects at any of the experimental time-points. On the other hand, there were significant differences between the defects filled with blood clot and the irradiated defects at all time-points (p < 0.001, p = 0.02, p < 0.001). There were significant differences between the mean peak CHA in nonirradiated defects at all the experimental time-points (p < 0.01). The mean peak of the defects filled with blood clot was significantly different from that of the defects filled with MTA (p < 0.001). There were significant differences between the defects filled with blood clot and the irradiated defects (p < 0.001). The results of this study using Raman spectral analysis indicate that infrared LED light irradiation improves the deposition of CHA in healing bone grafted or not with MTA.

  1. Guanine nucleotide-binding protein regulation of melatonin receptors in lizard brain

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Rivkees, S.A.; Carlson, L.L.; Reppert, S.M.

    Melatonin receptors were identified and characterized in crude membrane preparations from lizard brain by using {sup 125}I-labeled melatonin ({sup 125}I-Mel), a potent melatonin agonist. {sup 125}I-Mel binding sites were saturable; Scatchard analysis revealed high-affinity and lower affinity binding sites, with apparent K{sub d} of 2.3 {plus minus} 1.0 {times} 10{sup {minus}11} M and 2.06 {plus minus} 0.43 {times} 10{sup {minus}10} M, respectively. Binding was reversible and inhibited by melatonin and closely related analogs but not by serotonin or norepinephrine. Treatment of crude membranes with the nonhydrolyzable GTP analog guanosine 5{prime}-({gamma}-thio)triphosphate (GTP({gamma}S)), significantly reduced the number of high-affinity receptors and increasedmore » the dissociation rate of {sup 125}I-Mel from its receptor. Furthermore, GTP({gamma}S) treatment of ligand-receptor complexes solubilized by Triton X-100 also led to a rapid dissociation of {sup 125}I-Mel from solubilized ligand-receptor complexes. Gel filtration chromatography of solubilized ligand-receptor complexes revealed two major peaks of radioactivity corresponding to M{sub r} > 400,000 and M{sub r} ca. 110,000. This elution profile was markedly altered by pretreatment with GTP({gamma}S) before solubilization; only the M{sub r} 110,000 peak was present in GTP({gamma}S)-pretreated membranes. The results strongly suggest that {sup 125}I-mel binding sites in lizard brain are melatonin receptors, with agonist-promoted guanine nucleotide-binding protein (G protein) coupling and that the apparent molecular size of receptors uncoupled from G proteins is about 110,000.« less

  2. Osseous regeneration in the rat calvarium using novel delivery systems for recombinant human bone morphogenetic protein-2 (rhBMP-2).

    PubMed

    Kenley, R; Marden, L; Turek, T; Jin, L; Ron, E; Hollinger, J O

    1994-10-01

    In the current investigation, we report osseous regeneration in critical-size rat calvarial defects using recombinant human bone morphogenetic protein-2 (rhBMP-2) and novel delivery systems based on biomaterials. The novel systems combine rhBMP-2 with dry powder microparticles of poly(D,L-lactide-co-glycolide) (PLGA). The mixture of rhBMP-2 with PLGA microparticles is added to an aqueous solution of biopolymer to yield a semisolid paste. The biopolymers tested include autologous blood clot, hydroxypropyl methylcellulose, and sodium alginate cross-linked with calcium ion. Insoluble collageneous bone matrix was also studied as a control. Test articles were made at 0-, 10-, and 30-micrograms doses of rhBMP-2 and imiplanted in 8-mm-diameter rat calvarial defects (which will not heal if left untreated). The animals were examined 21 days after implantation by radiography, radiomorphometry, histology, and histomorphometry. All tested materials containing rhBMP-2 restored radiopacity and normal contouring to the calvarial defects. Samples without added rhBMP-2 yielded only soft tissue within the defects. Histology showed restoration of inner and outer bone tables plus marrow constituents. The PLGA microparticles were significantly resorbed at the 21-day time point. Although small differences between delivery systems were evident at 0- and 10-micrograms rhBMP-2 doses, all test articles performed essentially equivalently at the 30-micrograms dose. Thus, novel delivery systems for rhBMP-2 offer the promise of combining the intrinsic bioactivity of the osteoinductive protein with pharmaceutically acceptable biomaterials.

  3. Characterization of the Caenorhabditis elegans G protein-coupled serotonin receptors.

    PubMed

    Carre-Pierrat, Maïté; Baillie, David; Johnsen, Robert; Hyde, Rhonda; Hart, Anne; Granger, Laure; Ségalat, Laurent

    2006-12-01

    Serotonin (5-HT) regulates a wide range of behaviors in Caenorhabditis elegans, including egg laying, male mating, locomotion and pharyngeal pumping. So far, four serotonin receptors have been described in the nematode C. elegans, three of which are G protein-coupled receptors (GPCR), (SER-1, SER-4 and SER-7), and one is an ion channel (MOD-1). By searching the C. elegans genome for additional 5-HT GPCR genes, we identified five further genes which encode putative 5-HT receptors, based on sequence similarities to 5-HT receptors from other species. Using loss-of-function mutants and RNAi, we performed a systematic study of the role of the eight GPCR genes in serotonin-modulated behaviors of C. elegans (F59C12.2, Y22D7AR.13, K02F2.6, C09B7.1, M03F4.3, F16D3.7, T02E9.3, C24A8.1). We also examined their expression patterns. Finally, we tested whether the most likely candidate receptors were able to modulate adenylate cyclase activity in transfected cells in a 5-HT-dependent manner. This paper is the first comprehensive study of G protein-coupled serotonin receptors of C. elegans. It provides a direct comparison of the expression patterns and functional roles for 5-HT receptors in C. elegans.

  4. Epidermal growth factor induces G protein-coupled receptor 30 expression in estrogen receptor-negative breast cancer cells.

    PubMed

    Albanito, Lidia; Sisci, Diego; Aquila, Saveria; Brunelli, Elvira; Vivacqua, Adele; Madeo, Antonio; Lappano, Rosamaria; Pandey, Deo Prakash; Picard, Didier; Mauro, Loredana; Andò, Sebastiano; Maggiolini, Marcello

    2008-08-01

    Different cellular receptors mediate the biological effects induced by estrogens. In addition to the classical nuclear estrogen receptors (ERs)-alpha and -beta, estrogen also signals through the seven-transmembrane G-protein-coupled receptor (GPR)-30. Using as a model system SkBr3 and BT20 breast cancer cells lacking the classical ER, the regulation of GPR30 expression by 17beta-estradiol, the selective GPR30 ligand G-1, IGF-I, and epidermal growth factor (EGF) was evaluated. Transient transfections with an expression plasmid encoding a short 5'-flanking sequence of the GPR30 gene revealed that an activator protein-1 site located within this region is required for the activating potential exhibited only by EGF. Accordingly, EGF up-regulated GPR30 protein levels, which accumulated predominantly in the intracellular compartment. The stimulatory role elicited by EGF on GPR30 expression was triggered through rapid ERK phosphorylation and c-fos induction, which was strongly recruited to the activator protein-1 site found in the short 5'-flanking sequence of the GPR30 gene. Of note, EGF activating the EGF receptor-MAPK transduction pathway stimulated a regulatory loop that subsequently engaged estrogen through GPR30 to boost the proliferation of SkBr3 and BT20 breast tumor cells. The up-regulation of GPR30 by ligand-activated EGF receptor-MAPK signaling provides new insight into the well-known estrogen and EGF cross talk, which, as largely reported, contributes to breast cancer progression. On the basis of our results, the action of EGF may include the up-regulation of GPR30 in facilitating a stimulatory role of estrogen, even in ER-negative breast tumor cells.

  5. G Protein-Coupled Receptor Rhodopsin: A Prospectus

    PubMed Central

    Filipek, Sławomir; Stenkamp, Ronald E.; Teller, David C.; Palczewski, Krzysztof

    2006-01-01

    Rhodopsin is a retinal photoreceptor protein of bipartite structure consisting of the transmembrane protein opsin and a light-sensitive chromophore 11-cis-retinal, linked to opsin via a protonated Schiff base. Studies on rhodopsin have unveiled many structural and functional features that are common to a large and pharmacologically important group of proteins from the G protein-coupled receptor (GPCR) superfamily, of which rhodopsin is the best-studied member. In this work, we focus on structural features of rhodopsin as revealed by many biochemical and structural investigations. In particular, the high-resolution structure of bovine rhodopsin provides a template for understanding how GPCRs work. We describe the sensitivity and complexity of rhodopsin that lead to its important role in vision. PMID:12471166

  6. Electron Tomography Imaging of Surface Glycoproteins on Human Parainfluenza Virus 3: Association of Receptor Binding and Fusion Proteins before Receptor Engagement

    PubMed Central

    Gui, Long; Jurgens, Eric M.; Ebner, Jamie L.

    2015-01-01

    ABSTRACT In order to deliver their genetic material to host cells during infection, enveloped viruses use specialized proteins on their surfaces that bind cellular receptors and induce fusion of the viral and host membranes. In paramyxoviruses, a diverse family of single-stranded RNA (ssRNA) viruses, including several important respiratory pathogens, such as parainfluenza viruses, the attachment and fusion machinery is composed of two separate proteins: a receptor binding protein (hemagglutinin-neuraminidase [HN]) and a fusion (F) protein that interact to effect membrane fusion. Here we used negative-stain and cryo-electron tomography to image the 3-dimensional ultrastructure of human parainfluenza virus 3 (HPIV3) virions in the absence of receptor engagement. We observed that HN exists in at least two organizations. The first were arrays of tetrameric HN that lacked closely associated F proteins: in these purely HN arrays, HN adopted a “heads-down” configuration. In addition, we observed regions of complex surface density that contained HN in an apparently extended “heads-up” form, colocalized with prefusion F trimers. This colocalization with prefusion F prior to receptor engagement supports a model for fusion in which HN in its heads-up state and F may interact prior to receptor engagement without activating F, and that interaction with HN in this configuration is not sufficient to activate F. Only upon receptor engagement by HN’s globular head does HN transmit its activating signal to F. PMID:25691596

  7. Bone Morphogenetic Protein 15 in the Pro-Mature Complex Form Enhances Bovine Oocyte Developmental Competence

    PubMed Central

    Sudiman, Jaqueline; Sutton-McDowall, Melanie L.; Ritter, Lesley J.; White, Melissa A.; Mottershead, David G.; Thompson, Jeremy G.; Gilchrist, Robert B.

    2014-01-01

    Developmental competence of in vitro matured (IVM) oocytes needs to be improved and this can potentially be achieved by adding recombinant bone morphogenetic protein 15 (BMP15) or growth differentiation factor (GDF9) to IVM. The aim of this study was to determine the effect of a purified pro-mature complex form of recombinant human BMP15 versus the commercially available bioactive forms of BMP15 and GDF9 (both isolated mature regions) during IVM on bovine embryo development and metabolic activity. Bovine cumulus oocyte complexes (COCs) were matured in vitro in control medium or treated with 100 ng/ml pro-mature BMP15, mature BMP15 or mature GDF9 +/− FSH. Metabolic measures of glucose uptake and lactate production from COCs and autofluorescence of NAD(P)H, FAD and GSH were measured in oocytes after IVM. Following in vitro fertilisation and embryo culture, day 8 blastocysts were stained for cell numbers. COCs matured in medium +/− FSH containing pro-mature BMP15 displayed significantly improved blastocyst development (57.7±3.9%, 43.5±4.2%) compared to controls (43.3±2.4%, 28.9±3.7%) and to mature GDF9+FSH (36.1±3.0%). The mature form of BMP15 produced intermediate levels of blastocyst development; not significantly different to control or pro-mature BMP15 levels. Pro-mature BMP15 increased intra-oocyte NAD(P)H, and reduced glutathione (GSH) levels were increased by both forms of BMP15 in the absence of FSH. Exogenous BMP15 in its pro-mature form during IVM provides a functional source of oocyte-secreted factors to improve bovine blastocyst development. This form of BMP15 may prove useful for improving cattle and human artificial reproductive technologies. PMID:25058588

  8. Bone morphogenetic protein 15 in the pro-mature complex form enhances bovine oocyte developmental competence.

    PubMed

    Sudiman, Jaqueline; Sutton-McDowall, Melanie L; Ritter, Lesley J; White, Melissa A; Mottershead, David G; Thompson, Jeremy G; Gilchrist, Robert B

    2014-01-01

    Developmental competence of in vitro matured (IVM) oocytes needs to be improved and this can potentially be achieved by adding recombinant bone morphogenetic protein 15 (BMP15) or growth differentiation factor (GDF9) to IVM. The aim of this study was to determine the effect of a purified pro-mature complex form of recombinant human BMP15 versus the commercially available bioactive forms of BMP15 and GDF9 (both isolated mature regions) during IVM on bovine embryo development and metabolic activity. Bovine cumulus oocyte complexes (COCs) were matured in vitro in control medium or treated with 100 ng/ml pro-mature BMP15, mature BMP15 or mature GDF9 +/- FSH. Metabolic measures of glucose uptake and lactate production from COCs and autofluorescence of NAD(P)H, FAD and GSH were measured in oocytes after IVM. Following in vitro fertilisation and embryo culture, day 8 blastocysts were stained for cell numbers. COCs matured in medium +/- FSH containing pro-mature BMP15 displayed significantly improved blastocyst development (57.7±3.9%, 43.5±4.2%) compared to controls (43.3±2.4%, 28.9±3.7%) and to mature GDF9+FSH (36.1±3.0%). The mature form of BMP15 produced intermediate levels of blastocyst development; not significantly different to control or pro-mature BMP15 levels. Pro-mature BMP15 increased intra-oocyte NAD(P)H, and reduced glutathione (GSH) levels were increased by both forms of BMP15 in the absence of FSH. Exogenous BMP15 in its pro-mature form during IVM provides a functional source of oocyte-secreted factors to improve bovine blastocyst development. This form of BMP15 may prove useful for improving cattle and human artificial reproductive technologies.

  9. Constitutive phospholipid scramblase activity of a G protein-coupled receptor

    NASA Astrophysics Data System (ADS)

    Goren, Michael A.; Morizumi, Takefumi; Menon, Indu; Joseph, Jeremiah S.; Dittman, Jeremy S.; Cherezov, Vadim; Stevens, Raymond C.; Ernst, Oliver P.; Menon, Anant K.

    2014-10-01

    Opsin, the rhodopsin apoprotein, was recently shown to be an ATP-independent flippase (or scramblase) that equilibrates phospholipids across photoreceptor disc membranes in mammalian retina, a process required for disc homoeostasis. Here we show that scrambling is a constitutive activity of rhodopsin, distinct from its light-sensing function. Upon reconstitution into vesicles, discrete conformational states of the protein (rhodopsin, a metarhodopsin II-mimic, and two forms of opsin) facilitated rapid (>10,000 phospholipids per protein per second) scrambling of phospholipid probes. Our results indicate that the large conformational changes involved in converting rhodopsin to metarhodopsin II are not required for scrambling, and that the lipid translocation pathway either lies near the protein surface or involves membrane packing defects in the vicinity of the protein. In addition, we demonstrate that β2-adrenergic and adenosine A2A receptors scramble lipids, suggesting that rhodopsin-like G protein-coupled receptors may play an unexpected moonlighting role in re-modelling cell membranes.

  10. Co-Expression of Regulator of G Protein Signaling 4 (RGS4) and the MU Opioid Receptor in Regions of Rat Brain: Evidence That RGS4 Attenuates MU Opioid Receptor Signaling

    DTIC Science & Technology

    2003-01-01

    coupled receptor signal transduction proposes that agonist-induced conformational changes in the receptor result in an enhanced release of GDP...Regulators of G protein Signalling (RGS) proteins influence G protein-coupled receptor signal transduction by enhancing the intrinsic GTPase activity...of G proteins. The RGS- enhanced GTPase activity of G proteins may be responsible for the desensitization of certain G protein-coupled receptors

  11. Modelling and simulation of biased agonism dynamics at a G protein-coupled receptor.

    PubMed

    Bridge, L J; Mead, J; Frattini, E; Winfield, I; Ladds, G

    2018-04-07

    Theoretical models of G protein-coupled receptor (GPCR) concentration-response relationships often assume an agonist producing a single functional response via a single active state of the receptor. These models have largely been analysed assuming steady-state conditions. There is now much experimental evidence to suggest that many GPCRs can exist in multiple receptor conformations and elicit numerous functional responses, with ligands having the potential to activate different signalling pathways to varying extents-a concept referred to as biased agonism, functional selectivity or pluri-dimensional efficacy. Moreover, recent experimental results indicate a clear possibility for time-dependent bias, whereby an agonist's bias with respect to different pathways may vary dynamically. Efforts towards understanding the implications of temporal bias by characterising and quantifying ligand effects on multiple pathways will clearly be aided by extending current equilibrium binding and biased activation models to include G protein activation dynamics. Here, we present a new model of time-dependent biased agonism, based on ordinary differential equations for multiple cubic ternary complex activation models with G protein cycle dynamics. This model allows simulation and analysis of multi-pathway activation bias dynamics at a single receptor for the first time, at the level of active G protein (α GTP ), towards the analysis of dynamic functional responses. The model is generally applicable to systems with N G G proteins and N* active receptor states. Numerical simulations for N G =N * =2 reveal new insights into the effects of system parameters (including cooperativities, and ligand and receptor concentrations) on bias dynamics, highlighting new phenomena including the dynamic inter-conversion of bias direction. Further, we fit this model to 'wet' experimental data for two competing G proteins (G i and G s ) that become activated upon stimulation of the adenosine A 1

  12. Modulation of Pain Transmission by G Protein-Coupled Receptors

    PubMed Central

    Pan, Hui-Lin; Wu, Zi-Zhen; Zhou, Hong-Yi; Chen, Shao-Rui; Zhang, Hong-Mei; Li, De-Pei

    2010-01-01

    The heterotrimeric G protein-coupled receptors (GPCRs) represent the largest and most diverse family of cell surface receptors and proteins. GPCRs are widely distributed in the peripheral and central nervous systems and are one of the most important therapeutic targets in pain medicine. GPCRs are present on the plasma membrane of neurons and their terminals along the nociceptive pathways and are closely associated with the modulation of pain transmission. GPCRs that can produce analgesia upon activation include opioid, cannabinoid, α2-adrenergic, muscarinic acetylcholine, γ-aminobutyric acidB (GABAB), group II and III metabotropic glutamate, and somatostatin receptors. Recent studies have led to a better understanding of the role of these GPCRs in the regulation of pain transmission. Here, we review the current knowledge about the cellular and molecular mechanisms that underlie the analgesic actions of GPCR agonists, with a focus on their effects on ion channels expressed on nociceptive sensory neurons and on synaptic transmission at the spinal cord level. PMID:17959251

  13. Spectral methods for study of the G-protein-coupled receptor rhodopsin. II. Magnetic resonance methods

    NASA Astrophysics Data System (ADS)

    Struts, A. V.; Barmasov, A. V.; Brown, M. F.

    2016-02-01

    This article continues our review of spectroscopic studies of G-protein-coupled receptors. Magnetic resonance methods including electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) provide specific structural and dynamical data for the protein in conjunction with optical methods (vibrational, electronic spectroscopy) as discussed in the accompanying article. An additional advantage is the opportunity to explore the receptor proteins in the natural membrane lipid environment. Solid-state 2H and 13C NMR methods yield information about both the local structure and dynamics of the cofactor bound to the protein and its light-induced changes. Complementary site-directed spin-labeling studies monitor the structural alterations over larger distances and correspondingly longer time scales. A multiscale reaction mechanism describes how local changes of the retinal cofactor unlock the receptor to initiate large-scale conformational changes of rhodopsin. Activation of the G-protein-coupled receptor involves an ensemble of conformational substates within the rhodopsin manifold that characterize the dynamically active receptor.

  14. An activation switch in the rhodopsin family of G protein-coupled receptors: the thyrotropin receptor.

    PubMed

    Urizar, Eneko; Claeysen, Sylvie; Deupí, Xavier; Govaerts, Cedric; Costagliola, Sabine; Vassart, Gilbert; Pardo, Leonardo

    2005-04-29

    We aimed at understanding molecular events involved in the activation of a member of the G protein-coupled receptor family, the thyrotropin receptor. We have focused on the transmembrane region and in particular on a network of polar interactions between highly conserved residues. Using molecular dynamics simulations and site-directed mutagenesis techniques we have identified residue Asn-7.49, of the NPxxY motif of TM 7, as a molecular switch in the mechanism of thyrotropin receptor (TSHr) activation. Asn-7.49 appears to adopt two different conformations in the inactive and active states. These two states are characterized by specific interactions between this Asn and polar residues in the transmembrane domain. The inactive gauche+ conformation is maintained by interactions with residues Thr-6.43 and Asp-6.44. Mutation of these residues into Ala increases the constitutive activity of the receptor by factors of approximately 14 and approximately 10 relative to wild type TSHr, respectively. Upon receptor activation Asn-7.49 adopts the trans conformation to interact with Asp-2.50 and a putatively charged residue that remains to be identified. In addition, the conserved Leu-2.46 of the (N/S)LxxxD motif also plays a significant role in restraining the receptor in the inactive state because the L2.46A mutation increases constitutive activity by a factor of approximately 13 relative to wild type TSHr. As residues Leu-2.46, Asp-2.50, and Asn-7.49 are strongly conserved, this molecular mechanism of TSHr activation can be extended to other members of the rhodopsin-like family of G protein-coupled receptors.

  15. Combination therapy of canine osteosarcoma with canine bone marrow stem cells, bone morphogenetic protein and carboplatin in an in vivo model.

    PubMed

    Rici, R E G; Will, S E A L; Luna, A C L; Melo, L F; Santos, A C; Rodrigues, R F; Leandro, R M; Maria, D A

    2018-05-20

    Osteosarcoma (OSA) is the most common malignant bone cancer in children and dogs. The therapeutic protocols adopted for dogs and humans are very similar, involving surgical options such as amputation. Besides surgical options, radiotherapy and chemotherapy also are adopted. However, hematologic, gastrointestinal and renal toxicity may occur because of chemotherapy treatments. Recent study clearly showed that mesenchymal stem cells (MSCs) combined with recombinant human bone morphogenetic protein (rhBMP-2) may be associated with decreases of the tumorigenic potential of canine OSA. The aim of this study was to analyse the efficacy of chemotherapy with carboplatin and rhBMP-2 with MSCs in a canine OSA in vivo model. Canine OSA cells were implanted in mice Balb-c/nude with MSCs, rhBMP-2 and carboplatin. Flow cytometry and PCR for markers involved in tumour suppression pathways were analysed. Results showed that the combination of MSCs and rhBMP-2 reduced tumour mass and infiltration of neoplastic cells in tissues more efficiently than carboplatin alone. Thus it was demonstrated that the use of rhBMP-2 and MSCs, in combination with conventional antineoplastic, may be an efficient treatment strategy. © 2018 John Wiley & Sons Ltd.

  16. Taste receptors and gustatory associated G proteins in channel catfish, Ictalurus punctatus.

    PubMed

    Gao, Sen; Liu, Shikai; Yao, Jun; Zhou, Tao; Li, Ning; Li, Qi; Dunham, Rex; Liu, Zhanjiang

    2017-03-01

    Taste sensation plays a pivotal role in nutrient identification and acquisition. This is particularly true for channel catfish (Ictalurus punctatus) that live in turbid waters with limited visibility. This biological process is mainly mediated by taste receptors expressed in taste buds that are distributed in several organs and tissues, including the barbels and skin. In the present study, we identified a complete repertoire of taste receptor and gustatory associated G protein genes in the channel catfish genome. A total of eight taste receptor genes were identified, including five type I and three type II taste receptor genes. Their genomic locations, phylogenetic relations, orthologies and expression were determined. Phylogenetic and collinear analyses provided understanding of the evolution dynamics of this gene family. Furthermore, the motif and dN/dS analyses indicated that selection pressures of different degrees were imposed on these receptors. Additionally, four genes of gustatory associated G proteins were also identified. It was indicated that expression patterns of catfish taste receptors and gustatory associated G proteins across organs mirror the distribution of taste buds across organs. Finally, the expression comparison between catfish and zebrafish organs provided evidence of potential roles of catfish skin and gill involved in taste sensation. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Transferrin receptor facilitates TGF-β and BMP signaling activation to control craniofacial morphogenesis

    PubMed Central

    Lei, R; Zhang, K; Liu, K; Shao, X; Ding, Z; Wang, F; Hong, Y; Zhu, M; Li, H; Li, H

    2016-01-01

    The Pierre Robin Sequence (PRS), consisting of cleft palate, glossoptosis and micrognathia, is a common human birth defect. However, how this abnormality occurs remains largely unknown. Here we report that neural crest cell (NCC)-specific knockout of transferrin receptor (Tfrc), a well known transferrin transporter protein, caused micrognathia, cleft palate, severe respiratory distress and inability to suckle in mice, which highly resemble human PRS. Histological and anatomical analysis revealed that the cleft palate is due to the failure of palatal shelves elevation that resulted from a retarded extension of Meckel's cartilage. Interestingly, Tfrc deletion dramatically suppressed both transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP) signaling in cranial NCCs-derived mandibular tissues, suggesting that Tfrc may act as a facilitator of these two signaling pathways during craniofacial morphogenesis. Together, our study uncovers an unknown function of Tfrc in craniofacial development and provides novel insight into the etiology of PRS. PMID:27362800

  18. Analysis of odorant receptor protein function in the yellow fever mosquito, aedes aegypti

    USDA-ARS?s Scientific Manuscript database

    Odorant receptors (ORs) in insects are ligand-gated ion channels comprised of two subunits: a variable receptor and an obligatory co-receptor (Orco). This protein receptor complex of unknown stoichiometry interacts with an odor molecule leading to changes in permeability of the sensory dendrite, th...

  19. Molecular evolution of a chordate specific family of G protein-coupled receptors

    PubMed Central

    2011-01-01

    Background Chordate evolution is a history of innovations that is marked by physical and behavioral specializations, which led to the development of a variety of forms from a single ancestral group. Among other important characteristics, vertebrates obtained a well developed brain, anterior sensory structures, a closed circulatory system and gills or lungs as blood oxygenation systems. The duplication of pre-existing genes had profound evolutionary implications for the developmental complexity in vertebrates, since mutations modifying the function of a duplicated protein can lead to novel functions, improving the evolutionary success. Results We analyzed here the evolution of the GPRC5 family of G protein-coupled receptors by comprehensive similarity searches and found that the receptors are only present in chordates and that the size of the receptor family expanded, likely due to genome duplication events in the early history of vertebrate evolution. We propose that a single GPRC5 receptor coding gene originated in a stem chordate ancestor and gave rise by duplication events to a gene family comprising three receptor types (GPRC5A-C) in vertebrates, and a fourth homologue present only in mammals (GPRC5D). Additional duplications of GPRC5B and GPRC5C sequences occurred in teleost fishes. The finding that the expression patterns of the receptors are evolutionarily conserved indicates an important biological function of these receptors. Moreover, we found that expression of GPRC5B is regulated by vitamin A in vivo, confirming previous findings that linked receptor expression to retinoic acid levels in tumor cell lines and strengthening the link between the receptor expression and the development of a complex nervous system in chordates, known to be dependent on retinoic acid signaling. Conclusions GPRC5 receptors, a class of G protein-coupled receptors with unique sequence characteristics, may represent a molecular novelty that helped non-chordates to become

  20. G protein-coupled receptors Flop1 and Flop2 inhibit Wnt/β-catenin signaling and are essential for head formation in Xenopus.

    PubMed

    Miyagi, Asuka; Negishi, Takefumi; Yamamoto, Takamasa S; Ueno, Naoto

    2015-11-01

    Patterning of the vertebrate anterior-posterior axis is regulated by the coordinated action of growth factors whose effects can be further modulated by upstream and downstream mediators and the cross-talk of different intracellular pathways. In particular, the inhibition of the Wnt/β-catenin signaling pathway by various factors is critically required for anterior specification. Here, we report that Flop1 and Flop2 (Flop1/2), G protein-coupled receptors related to Gpr4, contribute to the regulation of head formation by inhibiting Wnt/β-catenin signaling in Xenopus embryos. Using whole-mount in situ hybridization, we showed that flop1 and flop2 mRNAs were expressed in the neural ectoderm during early gastrulation. Both the overexpression and knockdown of Flop1/2 resulted in altered embryonic head phenotypes, while the overexpression of either Flop1/2 or the small GTPase RhoA in the absence of bone morphogenetic protein (BMP) signaling resulted in ectopic head induction. Examination of the Flops' function in Xenopus embryo animal cap cells showed that they inhibited Wnt/β-catenin signaling by promoting β-catenin degradation through both RhoA-dependent and -independent pathways in a cell-autonomous manner. These results suggest that Flop1 and Flop2 are essential regulators of Xenopus head formation that act as novel inhibitory components of the Wnt/β-catenin signaling pathway. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Serial femtosecond crystallography datasets from G protein-coupled receptors.

    PubMed

    White, Thomas A; Barty, Anton; Liu, Wei; Ishchenko, Andrii; Zhang, Haitao; Gati, Cornelius; Zatsepin, Nadia A; Basu, Shibom; Oberthür, Dominik; Metz, Markus; Beyerlein, Kenneth R; Yoon, Chun Hong; Yefanov, Oleksandr M; James, Daniel; Wang, Dingjie; Messerschmidt, Marc; Koglin, Jason E; Boutet, Sébastien; Weierstall, Uwe; Cherezov, Vadim

    2016-08-01

    We describe the deposition of four datasets consisting of X-ray diffraction images acquired using serial femtosecond crystallography experiments on microcrystals of human G protein-coupled receptors, grown and delivered in lipidic cubic phase, at the Linac Coherent Light Source. The receptors are: the human serotonin receptor 2B in complex with an agonist ergotamine, the human δ-opioid receptor in complex with a bi-functional peptide ligand DIPP-NH2, the human smoothened receptor in complex with an antagonist cyclopamine, and finally the human angiotensin II type 1 receptor in complex with the selective antagonist ZD7155. All four datasets have been deposited, with minimal processing, in an HDF5-based file format, which can be used directly for crystallographic processing with CrystFEL or other software. We have provided processing scripts and supporting files for recent versions of CrystFEL, which can be used to validate the data.

  2. Targeted Elimination of G Proteins and Arrestins Defines Their Specific Contributions to Both Intensity and Duration of G Protein-coupled Receptor Signaling.

    PubMed

    Alvarez-Curto, Elisa; Inoue, Asuka; Jenkins, Laura; Raihan, Sheikh Zahir; Prihandoko, Rudi; Tobin, Andrew B; Milligan, Graeme

    2016-12-30

    G protein-coupled receptors (GPCRs) can initiate intracellular signaling cascades by coupling to an array of heterotrimeric G proteins and arrestin adaptor proteins. Understanding the contribution of each of these coupling options to GPCR signaling has been hampered by a paucity of tools to selectively perturb receptor function. Here we employ CRISPR/Cas9 genome editing to eliminate selected G proteins (Gα q and Gα 11 ) or arrestin2 and arrestin3 from HEK293 cells together with the elimination of receptor phosphorylation sites to define the relative contribution of G proteins, arrestins, and receptor phosphorylation to the signaling outcomes of the free fatty acid receptor 4 (FFA4). A lack of FFA4-mediated elevation of intracellular Ca 2+ in Gα q /Gα 11 -null cells and agonist-mediated receptor internalization in arrestin2/3-null cells confirmed previously reported canonical signaling features of this receptor, thereby validating the genome-edited HEK293 cells. FFA4-mediated ERK1/2 activation was totally dependent on G q / 11 but intriguingly was substantially enhanced for FFA4 receptors lacking sites of regulated phosphorylation. This was not due to a simple lack of desensitization of G q / 11 signaling because the G q / 11 -dependent calcium response was desensitized by both receptor phosphorylation and arrestin-dependent mechanisms, whereas a substantially enhanced ERK1/2 response was only observed for receptors lacking phosphorylation sites and not in arrestin2/3-null cells. In conclusion, we validate CRISPR/Cas9 engineered HEK293 cells lacking G q / 11 or arrestin2/3 as systems for GPCR signaling research and employ these cells to reveal a previously unappreciated interplay of signaling pathways where receptor phosphorylation can impact on ERK1/2 signaling through a mechanism that is likely independent of arrestins. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Quaternary structure of a G-protein-coupled receptor heterotetramer in complex with Gi and Gs.

    PubMed

    Navarro, Gemma; Cordomí, Arnau; Zelman-Femiak, Monika; Brugarolas, Marc; Moreno, Estefania; Aguinaga, David; Perez-Benito, Laura; Cortés, Antoni; Casadó, Vicent; Mallol, Josefa; Canela, Enric I; Lluís, Carme; Pardo, Leonardo; García-Sáez, Ana J; McCormick, Peter J; Franco, Rafael

    2016-04-05

    G-protein-coupled receptors (GPCRs), in the form of monomers or homodimers that bind heterotrimeric G proteins, are fundamental in the transfer of extracellular stimuli to intracellular signaling pathways. Different GPCRs may also interact to form heteromers that are novel signaling units. Despite the exponential growth in the number of solved GPCR crystal structures, the structural properties of heteromers remain unknown. We used single-particle tracking experiments in cells expressing functional adenosine A1-A2A receptors fused to fluorescent proteins to show the loss of Brownian movement of the A1 receptor in the presence of the A2A receptor, and a preponderance of cell surface 2:2 receptor heteromers (dimer of dimers). Using computer modeling, aided by bioluminescence resonance energy transfer assays to monitor receptor homomerization and heteromerization and G-protein coupling, we predict the interacting interfaces and propose a quaternary structure of the GPCR tetramer in complex with two G proteins. The combination of results points to a molecular architecture formed by a rhombus-shaped heterotetramer, which is bound to two different interacting heterotrimeric G proteins (Gi and Gs). These novel results constitute an important advance in understanding the molecular intricacies involved in GPCR function.

  4. Regulation of prostate cancer by hormone-responsive G protein-coupled receptors.

    PubMed

    Wang, Wei; Chen, Zhao-Xia; Guo, Dong-Yu; Tao, Ya-Xiong

    2018-06-15

    Regulation of prostate cancer by androgen and androgen receptor (AR), and blockade of AR signaling by AR antagonists and steroidogenic enzyme inhibitors have been extensively studied. G protein-coupled receptors (GPCRs) are a family of membrane receptors that regulate almost all physiological processes. Nearly 40% of FDA-approved drugs in the market target GPCRs. A variety of GPCRs that mediate reproductive function have been demonstrated to be involved in the regulation of prostate cancer. These GPCRs include gonadotropin-releasing hormone receptor, luteinizing hormone receptor, follicle-stimulating hormone receptor, relaxin receptor, ghrelin receptor, and kisspeptin receptor. We highlight here GPCR regulation of prostate cancer by these GPCRs. Further therapeutic approaches targeting these GPCRs for the treatment of prostate cancer are summarized. Copyright © 2018. Published by Elsevier Inc.

  5. Fragile X Mental Retardation Protein Regulates the Levels of Scaffold Proteins and Glutamate Receptors in Postsynaptic Densities*

    PubMed Central

    Schütt, Janin; Falley, Katrin; Richter, Dietmar; Kreienkamp, Hans-Jürgen; Kindler, Stefan

    2009-01-01

    Functional absence of fragile X mental retardation protein (FMRP) causes the fragile X syndrome, a hereditary form of mental retardation characterized by a change in dendritic spine morphology. The RNA-binding protein FMRP has been implicated in regulating postsynaptic protein synthesis. Here we have analyzed whether the abundance of scaffold proteins and neurotransmitter receptor subunits in postsynaptic densities (PSDs) is altered in the neocortex and hippocampus of FMRP-deficient mice. Whereas the levels of several PSD components are unchanged, concentrations of Shank1 and SAPAP scaffold proteins and various glutamate receptor subunits are altered in both adult and juvenile knock-out mice. With the exception of slightly increased hippocampal SAPAP2 mRNA levels in adult animals, altered postsynaptic protein concentrations do not correlate with similar changes in total and synaptic levels of corresponding mRNAs. Thus, loss of FMRP in neurons appears to mainly affect the translation and not the abundance of particular brain transcripts. Semi-quantitative analysis of RNA levels in FMRP immunoprecipitates showed that in the mouse brain mRNAs encoding PSD components, such as Shank1, SAPAP1–3, PSD-95, and the glutamate receptor subunits NR1 and NR2B, are associated with FMRP. Luciferase reporter assays performed in primary cortical neurons from knock-out and wild-type mice indicate that FMRP silences translation of Shank1 mRNAs via their 3′-untranslated region. Activation of metabotropic glutamate receptors relieves translational suppression. As Shank1 controls dendritic spine morphology, our data suggest that dysregulation of Shank1 synthesis may significantly contribute to the abnormal spine development and function observed in brains of fragile X syndrome patients. PMID:19640847

  6. Low-intensity pulsed ultrasound enhances bone morphogenetic protein expression of human mandibular fracture haematoma-derived cells.

    PubMed

    Huang, W; Hasegawa, T; Imai, Y; Takeda, D; Akashi, M; Komori, T

    2015-07-01

    We previously demonstrated that human mandibular fracture haematoma-derived cells (MHCs) play an important role in mandibular fracture healing and that low-intensity pulsed ultrasound (LIPUS) accelerates this effect by stimulating various osteogenic cytokines. In the present study, we investigated how LIPUS affects the expression of bone morphogenetic proteins (BMPs), which are also known to have the ability to induce bone formation. MHCs were isolated from human mandibular fracture haematomas and the cells were divided into two groups: a LIPUS (+) group and a LIPUS (-) group, both of which were cultured in osteogenic medium. LIPUS was applied to the LIPUS (+) group 20 min a day for 4, 8, 14, and 20 days (1.5 MHz, 30 mW/cm(2)). Real-time PCR and immunofluorescence studies were carried out to determine the expression of BMP-2, 4, and 7. Compared to the LIPUS (-) group, gene expression levels were significantly increased in the LIPUS (+) group for BMP-2 on day 20 (67.38 ± 26.59 vs. 11.52 ± 3.42, P < 0.001), for BMP-4 on days 14 (45.12 ± 11.06 vs. 9.20 ± 2.88, P = 0.045) and 20 (40.96 ± 24.81 vs. 3.22 ± 1.53, P = 0.035), and for BMP-7 on day 8 (48.11 ± 35.36 vs. 7.03 ± 3.96, P = 0.034). These findings suggest that BMP-2, 4, and 7 may be mediated by LIPUS therapy during the bone repair process. Copyright © 2015 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

  7. Bone Morphogenetic Protein Regulation of Enteric Neuronal Phenotypic Diversity: Relationship to Timing of Cell Cycle Exit

    PubMed Central

    Chalazonitis, Alcmène; Pham, Tuan.D.; Li, Zhishan; Roman, Daniel; Guha, Udayan; Gomes, William; Kan, Lixin; Kessler, John A.; Gershon, Michael D.

    2008-01-01

    The effects of bone morphogenetic protein (BMP) signaling on enteric neuron development were examined in transgenic mice over expressing either the BMP inhibitor, noggin, or BMP4 under control of the neuron specific enolase (NSE) promoter. Noggin antagonism of BMP signaling increased total numbers of enteric neurons and those of subpopulations derived from precursors that exit the cell cycle early in neurogenesis (serotonin, calretinin, calbindin). In contrast, noggin overexpression decreased numbers of neurons derived from precursors that exit the cell cycle late (γ-aminobutyric acid, tyrosine hydroxylase [TH], dopamine transporter, calcitonin gene related peptide, TrkC). Numbers of TH- and TrkC-expressing neurons were increased by overexpression of BMP4. These observations are consistent with the idea that phenotypic expression in the enteric nervous system (ENS) is determined, in part, by the number of proliferative divisions neuronal precursors undergo before their terminal mitosis. BMP signaling may thus regulate enteric neuronal phenotypic diversity by promoting the exit of precursors from the cell cycle. BMP2 increased the numbers of TH- and TrkC-expressing neurons developing in vitro from immunoselected enteric crest-derived precursors; BMP signaling may thus also specify or promote the development of dopaminergic TrkC/NT-3-dependent neurons. The developmental defects in the ENS of noggin overexpressing mice caused a relatively mild disturbance of motility (irregular rapid transit and increased stool frequency, weight, and water content). Although the function of the gut thus displays a remarkable tolerance for ENS defects, subtle functional abnormalities in motility or secretion may arise when ENS defects short of aganglionosis occur during development. PMID:18537141

  8. The Efficacy of Cyclic Injection of Bone Morphogenetic Protein-2 in Large-Scale Calvarial Bone Defects.

    PubMed

    Choi, Jin Mi; Jeong, Woo Shik; Park, Eun Jung; Choi, Jong Woo

    2017-03-01

    Bone morphogenetic protein-2 (BMP-2) appears to be one of the most potent growth factors thus far studied. However, recent publications on the clinical application of BMP-2 revealed that its correct control is the paramount issue in clinical practice. For improving BMP-2 delivery, the cyclic administration might be an alternative. Accordingly, the authors cyclically injected BMP-2 in a cyclic injection model of large cranial defects to maintain the proper dosage during the bone healing process. A 10-mm diameter calvarial bone defect was produced using a round drill in 8-week-old Sprague-Dawley rats. Silk-hydroxyapatite scaffolds soaked in the appropriate concentration of BMP-2 were implanted into the defect. The animals were split into 4 single-injection groups and 3 multiple-injection groups; the latter groups received weekly subcutaneous injections of BMP-2 solution (1, 5, and 10 μg/mL) for 4 weeks, whereas the former groups received a single injection of BMP-2 at these concentrations. Each rat underwent computed tomography at 8 weeks. In terms of total volumes of the new bone, the 5 μg/mL multiple-injection BMP-2 group had significantly greater increases in bone volume than the single-injection groups. In terms of bone thickness, the multiple-injection groups had better outcomes than the single-injection groups. Thus, the cyclic injection protocol restored the original thickness without overgrowth. Cyclic injection of BMP-2 permits more accurate dosage control than single injection and improves thickness and dense bone regeneration. Therefore, it may represent a promising approach for future clinical trials. Further investigation using a greater number of animals is required.

  9. Hyaline cartilage regeneration by combined therapy of microfracture and long-term bone morphogenetic protein-2 delivery.

    PubMed

    Yang, Hee Seok; La, Wan-Geun; Bhang, Suk Ho; Kim, Hak-Jun; Im, Gun-Il; Lee, Haeshin; Park, Jung-Ho; Kim, Byung-Soo

    2011-07-01

    Microfracture of cartilage induces migration of bone-marrow-derived mesenchymal stem cells. However, this treatment often results in fibrocartilage regeneration. Growth factors such as bone morphogenetic protein (BMP)-2 induce the differentiation of bone-marrow-derived mesenchymal stem cells into chondrocytes, which can be used for hyaline cartilage regeneration. Here, we tested the hypothesis that long-term delivery of BMP-2 to cartilage defects subjected to microfracture results in regeneration of high-quality hyaline-like cartilage, as opposed to short-term delivery of BMP-2 or no BMP-2 delivery. Heparin-conjugated fibrin (HCF) and normal fibrin were used as carriers for the long- and short-term delivery of BMP-2, respectively. Rabbit articular cartilage defects were treated with microfracture combined with one of the following: no treatment, fibrin, short-term delivery of BMP-2, HCF, or long-term delivery of BMP-2. Eight weeks after treatment, histological analysis revealed that the long-term delivery of BMP-2 group (microfracture + HCF + BMP-2) showed the most staining with alcian blue. A biochemical assay, real-time polymerase chain reaction assay and Western blot analysis all revealed that the long-term delivery of BMP-2 group had the highest glucosaminoglycan content as well as the highest expression level of collagen type II. Taken together, the long-term delivery of BMP-2 to cartilage defects subjected to microfracture resulted in regeneration of hyaline-like cartilage, as opposed to short-term delivery or no BMP-2 delivery. Therefore, this method could be more convenient for hyaline cartilage regeneration than autologous chondrocyte implantation due to its less invasive nature and lack of cell implantation.

  10. Orphan nuclear receptor TLX regulates astrogenesis by modulating BMP signaling.

    PubMed

    Qin, Song; Niu, Wenze; Iqbal, Nida; Smith, Derek K; Zhang, Chun-Li

    2014-01-01

    Neural stem cells (NSCs) are self-renewing multipotent progenitors that generate both neurons and glia. The precise control of NSC behavior is fundamental to the architecture and function of the central nervous system. We previously demonstrated that the orphan nuclear receptor TLX is required for postnatal NSC activation and neurogenesis in the neurogenic niche. Here, we show that TLX modulates bone morphogenetic protein (BMP)-SMAD signaling to control the timing of postnatal astrogenesis. Genes involved in the BMP signaling pathway, such as Bmp4, Hes1, and Id3, are upregulated in postnatal brains lacking Tlx. Chromatin immunoprecipitation and electrophoretic mobility shift assays reveal that TLX can directly bind the enhancer region of Bmp4. In accordance with elevated BMP signaling, the downstream effectors SMAD1/5/8 are activated by phosphorylation in Tlx mutant mice. Consequently, Tlx mutant brains exhibit an early appearance and increased number of astrocytes with marker expression of glial fibrillary acidic protein (GFAP) and S100B. Taken together, these results suggest that TLX tightly controls postnatal astrogenesis through the modulation of BMP-SMAD signaling pathway activity.

  11. Design, Synthesis, and Evaluation of N- and C-Terminal Protein Bioconjugates as G Protein-Coupled Receptor Agonists.

    PubMed

    Healey, Robert D; Wojciechowski, Jonathan P; Monserrat-Martinez, Ana; Tan, Susan L; Marquis, Christopher P; Sierecki, Emma; Gambin, Yann; Finch, Angela M; Thordarson, Pall

    2018-02-21

    A G protein-coupled receptor (GPCR) agonist protein, thaumatin, was site-specifically conjugated at the N- or C-terminus with a fluorophore for visualization of GPCR:agonist interactions. The N-terminus was specifically conjugated using a synthetic 2-pyridinecarboxyaldehyde reagent. The interaction profiles observed for N- and C-terminal conjugates were varied; N-terminal conjugates interacted very weakly with the GPCR of interest, whereas C-terminal conjugates bound to the receptor. These chemical biology tools allow interactions of therapeutic proteins:GPCR to be monitored and visualized. The methodology used for site-specific bioconjugation represents an advance in application of 2-pyridinecarboxyaldehydes for N-terminal specific bioconjugations.

  12. Tissue organization by cadherin adhesion molecules: dynamic molecular and cellular mechanisms of morphogenetic regulation

    PubMed Central

    Niessen, Carien M.; Leckband, Deborah; Yap, Alpha S.

    2013-01-01

    This review addresses the cellular and molecular mechanisms of cadherin-based tissue morphogenesis. Tissue physiology is profoundly influenced by the distinctive organizations of cells in organs and tissues. In metazoa, adhesion receptors of the classical cadherin family play important roles in establishing and maintaining such tissue organization. Indeed, it is apparent that cadherins participate in a range of morphogenetic events that range from support of tissue integrity to dynamic cellular rearrangements. A comprehensive understanding of cadherin-based morphogenesis must then define the molecular and cellular mechanisms that support these distinct cadherin biologies. Here we focus on four key mechanistic elements: the molecular basis for adhesion through cadherin ectodomains; the regulation of cadherin expression at the cell surface; cooperation between cadherins and the actin cytoskeleton; and regulation by cell signaling. We discuss current progress and outline issues for further research in these fields. PMID:21527735

  13. Digital subtraction radiographic analysis of the combination of bioabsorbable membrane and bovine morphogenetic protein pool in human periodontal infrabony defects

    PubMed Central

    GUIMARÃES, Maria do Carmo Machado; PASSANEZI, Euloir; SANT’ANA, Adriana Campos Passanezi; GREGHI, Sebastião Luiz Aguiar; TABA JUNIOR, Mario

    2010-01-01

    Objectives This study assessed the bone density gain and its relationship with the periodontal clinical parameters in a case series of a regenerative therapy procedure. Material and Methods Using a split-mouth study design, 10 pairs of infrabony defects from 15 patients were treated with a pool of bovine bone morphogenetic proteins associated with collagen membrane (test sites) or collagen membrane only (control sites). The periodontal healing was clinically and radiographically monitored for six months. Standardized presurgical and 6-month postoperative radiographs were digitized for digital subtraction analysis, which showed relative bone density gain in both groups of 0.034 ± 0.423 and 0.105 ± 0.423 in the test and control group, respectively (p>0.05). Results As regards the area size of bone density change, the influence of the therapy was detected in 2.5 mm2 in the test group and 2 mm2 in the control group (p>0.05). Additionally, no correlation was observed between the favorable clinical results and the bone density gain measured by digital subtraction radiography (p>0.05). Conclusions The findings of this study suggest that the clinical benefit of the regenerative therapy observed did not come with significant bone density gains. Long-term evaluation may lead to a different conclusions. PMID:20835573

  14. Hepatitis B virus X protein modulates peroxisome proliferator-activated receptor gamma through protein-protein interaction.

    PubMed

    Choi, Youn-Hee; Kim, Ha-il; Seong, Je Kyung; Yu, Dae-Yeul; Cho, Hyeseong; Lee, Mi-Ock; Lee, Jae Myun; Ahn, Yong-ho; Kim, Se Jong; Park, Jeon Han

    2004-01-16

    Ligand activation of peroxisome proliferator-activated receptor gamma (PPARgamma) has been reported to induce growth inhibition and apoptosis in various cancers including hepatocellular carcinoma (HCC). However, the effect of hepatitis B virus X protein (HBx) on PPARgamma activation has not been characterized in hepatitis B virus (HBV)-associated HCC. Herein, we demonstrated that HBx counteracted growth inhibition caused by PPARgamma ligand in HBx-associated HCC cells. We found that HBx bound to DNA binding domain of PPARgamma and HBx/PPARgamma interaction blocked nuclear localization and binding to recognition site of PPARgamma. HBx significantly suppressed a PPARgamma-mediated transactivation. These results suggest that HBx modulates PPARgamma function through protein-protein interaction.

  15. Purification of family B G protein-coupled receptors using nanodiscs: Application to human glucagon-like peptide-1 receptor.

    PubMed

    Cai, Yingying; Liu, Yuting; Culhane, Kelly J; DeVree, Brian T; Yang, Yang; Sunahara, Roger K; Yan, Elsa C Y

    2017-01-01

    Family B G protein-coupled receptors (GPCRs) play vital roles in hormone-regulated homeostasis. They are drug targets for metabolic diseases, including type 2 diabetes and osteoporosis. Despite their importance, the signaling mechanisms for family B GPCRs at the molecular level remain largely unexplored due to the challenges in purification of functional receptors in sufficient amount for biophysical characterization. Here, we purified the family B GPCR human glucagon-like peptide-1 (GLP-1) receptor (GLP1R), whose agonists, e.g. exendin-4, are used for the treatment of type 2 diabetes mellitus. The receptor was expressed in HEK293S GnTl- cells using our recently developed protocol. The protocol incorporates the receptor into the native-like lipid environment of reconstituted high density lipoprotein (rHDL) particles, also known as nanodiscs, immediately after the membrane solubilization step followed by chromatographic purification, minimizing detergent contact with the target receptor to reduce denaturation and prolonging stabilization of receptor in lipid bilayers without extra steps of reconstitution. This method yielded purified GLP1R in nanodiscs that could bind to GLP-1 and exendin-4 and activate Gs protein. This nanodisc purification method can potentially be a general strategy to routinely obtain purified family B GPCRs in the 10s of microgram amounts useful for spectroscopic analysis of receptor functions and activation mechanisms.

  16. Purification of family B G protein-coupled receptors using nanodiscs: Application to human glucagon-like peptide-1 receptor

    PubMed Central

    Cai, Yingying; Liu, Yuting; Culhane, Kelly J.; DeVree, Brian T.; Yang, Yang; Sunahara, Roger K.; Yan, Elsa C. Y.

    2017-01-01

    Family B G protein-coupled receptors (GPCRs) play vital roles in hormone-regulated homeostasis. They are drug targets for metabolic diseases, including type 2 diabetes and osteoporosis. Despite their importance, the signaling mechanisms for family B GPCRs at the molecular level remain largely unexplored due to the challenges in purification of functional receptors in sufficient amount for biophysical characterization. Here, we purified the family B GPCR human glucagon-like peptide-1 (GLP-1) receptor (GLP1R), whose agonists, e.g. exendin-4, are used for the treatment of type 2 diabetes mellitus. The receptor was expressed in HEK293S GnTl- cells using our recently developed protocol. The protocol incorporates the receptor into the native-like lipid environment of reconstituted high density lipoprotein (rHDL) particles, also known as nanodiscs, immediately after the membrane solubilization step followed by chromatographic purification, minimizing detergent contact with the target receptor to reduce denaturation and prolonging stabilization of receptor in lipid bilayers without extra steps of reconstitution. This method yielded purified GLP1R in nanodiscs that could bind to GLP-1 and exendin-4 and activate Gs protein. This nanodisc purification method can potentially be a general strategy to routinely obtain purified family B GPCRs in the 10s of microgram amounts useful for spectroscopic analysis of receptor functions and activation mechanisms. PMID:28609478

  17. Structural analyses of von Willebrand factor C domains of collagen 2A and CCN3 reveal an alternative mode of binding to bone morphogenetic protein-2.

    PubMed

    Xu, Emma-Ruoqi; Blythe, Emily E; Fischer, Gerhard; Hyvönen, Marko

    2017-07-28

    Bone morphogenetic proteins (BMPs) are secreted growth factors that promote differentiation processes in embryogenesis and tissue development. Regulation of BMP signaling involves binding to a variety of extracellular proteins, among which are many von Willebrand factor C (vWC) domain-containing proteins. Although the crystal structure of the complex of crossveinless-2 (CV-2) vWC1 and BMP-2 previously revealed one mode of the vWC/BMP-binding mechanism, other vWC domains may bind to BMP differently. Here, using X-ray crystallography, we present for the first time structures of the vWC domains of two proteins thought to interact with BMP-2: collagen IIA and matricellular protein CCN3. We found that these two vWC domains share a similar N-terminal fold that differs greatly from that in CV-2 vWC, which comprises its BMP-2-binding site. We analyzed the ability of these vWC domains to directly bind to BMP-2 and detected an interaction only between the collagen IIa vWC and BMP-2. Guided by the collagen IIa vWC domain crystal structure and conservation of surface residues among orthologous domains, we mapped the BMP-binding epitope on the subdomain 1 of the vWC domain. This binding site is different from that previously observed in the complex between CV-2 vWC and BMP-2, revealing an alternative mode of interaction between vWC domains and BMPs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Periodontal tissue regeneration by combined applications of recombinant human osteogenic protein-1 and bone morphogenetic protein-2. A pilot study in Chacma baboons (Papio ursinus).

    PubMed

    Ripamonti, U; Crooks, J; Petit, J C; Rueger, D C

    2001-08-01

    Native and recombinant human bone morphogenetic/osteogenic proteins (BMPs/ OPs) singly initiate bone induction in vivo. The finding of synchronous but spatially different BMPs/OPs expression during periodontal tissue morphogenesis suggests novel therapeutic approaches using morphogen combinations based on recapitulation of embryonic development. Twelve furcation defects prepared in the first and second mandibular molars of three adult baboons (Papio ursinus) were used to assess whether qualitative histological aspects of periodontal tissue regeneration could be enhanced and tissue morphogenesis modified by combined or single applications of recombinant hOP-1 and hBMP-2. Doses of BMPs/OPs were 100 microg of each protein per 1 g of insoluble collagenous bone matrix as carrier. Approximately 200 mg of carrier matrix was used per furcation defect. Undecalcified sections cut for histological analysis 60 d after healing of hOP-1-treated specimens showed substantial cementogenesis with scattered remnants of the collagenous carrier. hBMP-2 applied alone induced greater amounts of mineralized bone and osteoid when compared to hOP-1 alone or to combined morphogen applications. Combined applications of hOP-1 and hBMP-2 did not enhance alveolar bone regeneration or new attachment formation over and above the single applications of the morphogens. The results of this study, which is the first to attempt to address the structure-activity relationship amongst BMP/OP family members, indicate that tissue morphogenesis induced by hOP-1 and hBMP-2 is qualitatively different when the morphogens are applied singly, with hOP-1 inducing substantial cementogenesis. hBMP-2 treated defects, on the other hand, showed limited cementum formation but a temporal enhancement of alveolar bone regeneration and remodelling. The demonstration of therapeutic mosaicism in periodontal regeneration will require extensive testing of ratios and doses of recombinant morphogen combinations for optimal tissue

  19. Vibrational resonance, allostery, and activation in rhodopsin-like G protein-coupled receptors

    PubMed Central

    Woods, Kristina N.; Pfeffer, Jürgen; Dutta, Arpana; Klein-Seetharaman, Judith

    2016-01-01

    G protein-coupled receptors are a large family of membrane proteins activated by a variety of structurally diverse ligands making them highly adaptable signaling molecules. Despite recent advances in the structural biology of this protein family, the mechanism by which ligands induce allosteric changes in protein structure and dynamics for its signaling function remains a mystery. Here, we propose the use of terahertz spectroscopy combined with molecular dynamics simulation and protein evolutionary network modeling to address the mechanism of activation by directly probing the concerted fluctuations of retinal ligand and transmembrane helices in rhodopsin. This approach allows us to examine the role of conformational heterogeneity in the selection and stabilization of specific signaling pathways in the photo-activation of the receptor. We demonstrate that ligand-induced shifts in the conformational equilibrium prompt vibrational resonances in the protein structure that link the dynamics of conserved interactions with fluctuations of the active-state ligand. The connection of vibrational modes creates an allosteric association of coupled fluctuations that forms a coherent signaling pathway from the receptor ligand-binding pocket to the G-protein activation region. Our evolutionary analysis of rhodopsin-like GPCRs suggest that specific allosteric sites play a pivotal role in activating structural fluctuations that allosterically modulate functional signals. PMID:27849063

  20. Serial femtosecond crystallography datasets from G protein-coupled receptors

    PubMed Central

    White, Thomas A.; Barty, Anton; Liu, Wei; Ishchenko, Andrii; Zhang, Haitao; Gati, Cornelius; Zatsepin, Nadia A.; Basu, Shibom; Oberthür, Dominik; Metz, Markus; Beyerlein, Kenneth R.; Yoon, Chun Hong; Yefanov, Oleksandr M.; James, Daniel; Wang, Dingjie; Messerschmidt, Marc; Koglin, Jason E.; Boutet, Sébastien; Weierstall, Uwe; Cherezov, Vadim

    2016-01-01

    We describe the deposition of four datasets consisting of X-ray diffraction images acquired using serial femtosecond crystallography experiments on microcrystals of human G protein-coupled receptors, grown and delivered in lipidic cubic phase, at the Linac Coherent Light Source. The receptors are: the human serotonin receptor 2B in complex with an agonist ergotamine, the human δ-opioid receptor in complex with a bi-functional peptide ligand DIPP-NH2, the human smoothened receptor in complex with an antagonist cyclopamine, and finally the human angiotensin II type 1 receptor in complex with the selective antagonist ZD7155. All four datasets have been deposited, with minimal processing, in an HDF5-based file format, which can be used directly for crystallographic processing with CrystFEL or other software. We have provided processing scripts and supporting files for recent versions of CrystFEL, which can be used to validate the data. PMID:27479354

  1. Protein receptor-independent plasma membrane remodeling by HAMLET: a tumoricidal protein-lipid complex.

    PubMed

    Nadeem, Aftab; Sanborn, Jeremy; Gettel, Douglas L; James, Ho C S; Rydström, Anna; Ngassam, Viviane N; Klausen, Thomas Kjær; Pedersen, Stine Falsig; Lam, Matti; Parikh, Atul N; Svanborg, Catharina

    2015-11-12

    A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This ''protein-centric" view is increasingly challenged by evidence for the involvement of specialized membrane domains in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. We identify a ''receptor independent" transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET accumulates within these de novo membrane conformations and define membrane blebs as cellular compartments for direct interactions of HAMLET with essential target proteins such as the Ras family of GTPases. Finally, we demonstrate lower sensitivity of healthy cell membranes to HAMLET challenge. These features suggest that HAMLET-induced curvature-dependent membrane conformations serve as surrogate receptors for initiating signal transduction cascades, ultimately leading to cell death.

  2. α1B-Adrenergic Receptors Differentially Associate with Rab Proteins during Homologous and Heterologous Desensitization

    PubMed Central

    Castillo-Badillo, Jean A.; Sánchez-Reyes, Omar B.; Alfonzo-Méndez, Marco A.; Romero-Ávila, M. Teresa; Reyes-Cruz, Guadalupe; García-Sáinz, J. Adolfo

    2015-01-01

    Internalization of G protein-coupled receptors can be triggered by agonists or by other stimuli. The process begins within seconds of cell activation and contributes to receptor desensitization. The Rab GTPase family controls endocytosis, vesicular trafficking, and endosomal fusion. Among their remarkable properties is the differential distribution of its members on the surface of various organelles. In the endocytic pathway, Rab 5 controls traffic from the plasma membrane to early endosomes, whereas Rab 4 and Rab 11 regulate rapid and slow recycling from early endosomes to the plasma membrane, respectively. Moreover, Rab 7 and Rab 9 regulate the traffic from late endosomes to lysosomes and recycling to the trans-Golgi. We explore the possibility that α1B-adrenergic receptor internalization induced by agonists (homologous) and by unrelated stimuli (heterologous) could involve different Rab proteins. This possibility was explored by Fluorescence Resonance Energy Transfer (FRET) using cells coexpressing α1B-adrenergic receptors tagged with the red fluorescent protein, DsRed, and different Rab proteins tagged with the green fluorescent protein. It was observed that when α1B-adrenergic receptors were stimulated with noradrenaline, the receptors interacted with proteins present in early endosomes, such as the early endosomes antigen 1, Rab 5, Rab 4, and Rab 11 but not with late endosome markers, such as Rab 9 and Rab 7. In contrast, sphingosine 1-phosphate stimulation induced rapid and transient α1B-adrenergic receptor interaction of relatively small magnitude with Rab 5 and a more pronounced and sustained one with Rab 9; interaction was also observed with Rab 7. Moreover, the GTPase activity of the Rab proteins appears to be required because no FRET was observed when dominant-negative Rab mutants were employed. These data indicate that α1B-adrenergic receptors are directed to different endocytic vesicles depending on the desensitization type (homologous vs

  3. RNA sequencing to determine the contribution of kinase receptor transactivation to G protein coupled receptor signalling in vascular smooth muscle cells.

    PubMed

    Kamato, Danielle; Bhaskarala, Venkata Vijayanand; Mantri, Nitin; Oh, Tae Gyu; Ling, Dora; Janke, Reearna; Zheng, Wenhua; Little, Peter J; Osman, Narin

    2017-01-01

    G protein coupled receptor (GPCR) signalling covers three major mechanisms. GPCR agonist engagement allows for the G proteins to bind to the receptor leading to a classical downstream signalling cascade. The second mechanism is via the utilization of the β-arrestin signalling molecule and thirdly via transactivation dependent signalling. GPCRs can transactivate protein tyrosine kinase receptors (PTKR) to activate respective downstream signalling intermediates. In the past decade GPCR transactivation dependent signalling was expanded to show transactivation of serine/threonine kinase receptors (S/TKR). Kinase receptor transactivation enormously broadens the GPCR signalling paradigm. This work utilizes next generation RNA-sequencing to study the contribution of transactivation dependent signalling to total protease activated receptor (PAR)-1 signalling. Transactivation, assessed as gene expression, accounted for 50 percent of the total genes regulated by thrombin acting through PAR-1 in human coronary artery smooth muscle cells. GPCR transactivation of PTKRs is approximately equally important as the transactivation of the S/TKR with 209 and 177 genes regulated respectively, via either signalling pathway. This work shows that genome wide studies can provide powerful insights into GPCR mediated signalling pathways.

  4. Internalization of G-protein-coupled receptors: Implication in receptor function, physiology and diseases.

    PubMed

    Calebiro, Davide; Godbole, Amod

    2018-04-01

    G protein-coupled receptors (GPCRs) are the largest family of membrane receptors and mediate the effects of numerous hormones and neurotransmitters. The nearly 1000 GPCRs encoded by the human genome regulate virtually all physiological functions and are implicated in the pathogenesis of prevalent human diseases such as thyroid disorders, hypertension or Parkinson's disease. As a result, 30-50% of all currently prescribed drugs are targeting these receptors. Once activated, GPCRs induce signals at the cell surface. This is often followed by internalization, a process that results in the transfer of receptors from the plasma membrane to membranes of the endosomal compartment. Internalization was initially thought to be mainly implicated in signal desensitization, a mechanism of adaptation to prolonged receptor stimulation. However, several unexpected functions have subsequently emerged. Most notably, accumulating evidence indicates that internalization can induce prolonged receptor signaling on intracellular membranes, which is apparently required for at least some biological effects of hormones like TSH, LH and adrenaline. These findings reveal an even stronger connection between receptor internalization and signaling than previously thought. Whereas new studies are just beginning to reveal an important physiological role for GPCR signaling after internalization and ways to exploit it for therapeutic purposes, future investigations will be required to explore its involvement in human disease. Copyright © 2018 Elsevier Ltd. All rights reserved.

  5. Cyst-Like Osteolytic Formations in Recombinant Human Bone Morphogenetic Protein-2 (rhBMP-2) Augmented Sheep Spinal Fusion.

    PubMed

    Pan, Hsin Chuan; Lee, Soonchul; Ting, Kang; Shen, Jia; Wang, Chenchao; Nguyen, Alan; Berthiaume, Emily A; Zara, Janette N; Turner, A Simon; Seim, Howard B; Kwak, Jin Hee; Zhang, Xinli; Soo, Chia

    2017-07-01

    Multiple case reports using recombinant human bone morphogenetic protein-2 (rhBMP-2) have reported complications. However, the local adverse effects of rhBMP-2 application are not well documented. In this report we show that, in addition to promoting lumbar spinal fusion through potent osteogenic effects, rhBMP-2 augmentation promotes local cyst-like osteolytic formations in sheep trabecular bones that have undergone anterior lumbar interbody fusion. Three months after operation, conventional computed tomography showed that the trabecular bones of the rhBMP-2 application groups could fuse, whereas no fusion was observed in the control group. Micro-computed tomography analysis revealed that the core implant area's bone volume fraction and bone mineral density increased proportionately with rhBMP-2 dose. Multiple cyst-like bone voids were observed in peri-implant areas when using rhBMP-2 applications, and these sites showed significant bone mineral density decreases in relation to the unaffected regions. Biomechanically, these areas decreased in strength by 32% in comparison with noncystic areas. Histologically, rhBMP-2-affected void sites had an increased amount of fatty marrow, thinner trabecular bones, and significantly more adiponectin- and cathepsin K-positive cells. Despite promoting successful fusion, rhBMP-2 use in clinical applications may result in local adverse structural alterations and compromised biomechanical changes to the bone. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  6. Activation of the novel estrogen receptor G protein-coupled receptor 30 (GPR30) at the plasma membrane.

    PubMed

    Filardo, E; Quinn, J; Pang, Y; Graeber, C; Shaw, S; Dong, J; Thomas, P

    2007-07-01

    G protein-coupled receptor 30 (GPR30), a seven-transmembrane receptor (7TMR), is associated with rapid estrogen-dependent, G protein signaling and specific estrogen binding. At present, the subcellular site of GPR30 action is unclear. Previous studies using antibodies and fluorochrome-labeled estradiol (E2) have failed to detect GPR30 on the cell surface, suggesting that GPR30 may function uniquely among 7TMRs as an intracellular receptor. Here, we show that detectable expression of GPR30 on the surface of transfected HEK-293 cells can be selected by fluorescence-activated cell sorting. Expression of GPR30 on the cell surface was confirmed by confocal microscopy using the lectin concanavalin A as a plasma membrane marker. Stimulation of GPR30-expressing HEK-293 cells with 17beta-E2 caused sequestration of GPR30 from the cell surface and resulted in its codistribution with clathrin and mobilization of intracellular calcium stores. Evidence that GPR30 signals from the cell surface was obtained from experiments demonstrating that the cell-impermeable E2-protein conjugates E2-BSA and E2-horseradish peroxidase promote GPR30-dependent elevation of intracellular cAMP concentrations. Subcellular fractionation studies further support the plasma membrane as a site of GPR30 action with specific [3H]17beta-E2 binding and G protein activation associated with plasma membrane but not microsomal, or other fractions, prepared from HEK-293 or SKBR3 breast cancer cells. These results suggest that GPR30, like other 7TMRs, functions as a plasma membrane receptor.

  7. Bone morphogenetic protein 4 induces differentiation of colorectal cancer stem cells and increases their response to chemotherapy in mice.

    PubMed

    Lombardo, Ylenia; Scopelliti, Alessandro; Cammareri, Patrizia; Todaro, Matilde; Iovino, Flora; Ricci-Vitiani, Lucia; Gulotta, Gaspare; Dieli, Francesco; de Maria, Ruggero; Stassi, Giorgio

    2011-01-01

    The limited clinical response observed in many patients with colorectal cancer may be related to the presence of chemoresistant colorectal cancer stem cells (CRC-SCs). Bone morphogenetic protein 4 (BMP4) promotes the differentiation of normal colonic stem cells. We investigated whether BMP4 might be used to induce differentiation of CRC-SCs and for therapeutic purposes. CRC-SCs were isolated from 25 tumor samples based on expression of CD133 or using a selection culture medium. BMP4 expression and activity on CRC-SCs were evaluated in vitro; progeny of the stem cells were evaluated by immunofluorescence, immunoblot, and flow cytometry analyses. The potential therapeutic effect of BMP4 was assessed in immunocompromised mice after injection of CRC-SCs that responded to chemotherapy (n = 4) or that did not (n = 2). CRC-SCs did not express BMP4 whereas differentiated cells did. Recombinant BMP4 promoted differentiation and apoptosis of CRC-SCs in 12 of 15 independent experiments; this effect did not depend on Small Mothers against decapentaplegic (Smad)4 expression level or microsatellite stability. BMP4 activated the canonical and noncanonical BMP signaling pathways, including phosphoInositide 3-kinase (PI3K) and PKB (protein kinase B)/AKT. Mutations in PI3K or loss of Phosphatase and Tensin homolog (PTEN) in Smad4-defective tumors made CRC-SCs unresponsive to BMP4. Administration of BMP4 to immunocompromised mice with tumors that arose from CRC-SCs increased the antitumor effects of 5-fluorouracil and oxaliplatin. BMP4 promotes terminal differentiation, apoptosis, and chemosensitization of CRC-SCs in tumors that do not have simultaneous mutations in Smad4 and constitutive activation of PI3K. BMP4 might be developed as a therapeutic agent against cancer stem cells in advanced colorectal tumors. Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.

  8. Interaction between G Protein-Coupled Receptor 143 and Tyrosinase: Implications for Understanding Ocular Albinism Type 1.

    PubMed

    De Filippo, Elisabetta; Schiedel, Anke C; Manga, Prashiela

    2017-02-01

    Developmental eye defects in X-linked ocular albinism type 1 are caused by G-protein coupled receptor 143 (GPR143) mutations. Mutations result in dysfunctional melanosome biogenesis and macromelanosome formation in pigment cells, including melanocytes and retinal pigment epithelium. GPR143, primarily expressed in pigment cells, localizes exclusively to endolysosomal and melanosomal membranes unlike most G protein-coupled receptors, which localize to the plasma membrane. There is some debate regarding GPR143 function and elucidating the role of this receptor may be instrumental for understanding neurogenesis during eye development and for devising therapies for ocular albinism type I. Many G protein-coupled receptors require association with other proteins to function. These G protein-coupled receptor-interacting proteins also facilitate fine-tuning of receptor activity and tissue specificity. We therefore investigated potential GPR143 interaction partners, with a focus on the melanogenic enzyme tyrosinase. GPR143 coimmunoprecipitated with tyrosinase, while confocal microscopy demonstrated colocalization of the proteins. Furthermore, tyrosinase localized to the plasma membrane when coexpressed with a GPR143 trafficking mutant. The physical interaction between the proteins was confirmed using fluorescence resonance energy transfer. This interaction may be required in order for GPR143 to function as a monitor of melanosome maturation. Identifying tyrosinase as a potential GPR143 binding protein opens new avenues for investigating the mechanisms that regulate pigmentation and neurogenesis. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  9. Alcohol action on a neuronal membrane receptor: evidence for a direct interaction with the receptor protein.

    PubMed Central

    Li, C; Peoples, R W; Weight, F F

    1994-01-01

    For almost a century, alcohols have been thought to produce their effects by actions on the membrane lipids of central nervous system neurons--the well known "lipid theory" of alcohol action. The rationale for this theory is the correlation of potency with oil/water or membrane/buffer partition coefficient. Although a number of recent studies have shown that alcohols can affect the function of certain neuronal neurotransmitter receptors, there is no evidence that the alcohols interact directly with these membrane proteins. In the present study, we report that inhibition of a neuronal neurotransmitter receptor, an ATP-gated ion channel, by a series of alcohols exhibits a distinct cutoff effect. For alcohols with a molecular volume of < or = 42.2 ml/mol, potency for inhibiting ATP-activated current was correlated with lipid solubility (order of potency: 1-propanol = trifluoroethanol > monochloroethanol > ethanol > methanol). However, despite increased lipid solubility, alcohols with a molecular volume of > or = 46.1 ml/mol (1-butanol, 1-pentanol, trichloroethanol, and dichloroethanol) were without effect on the ATP-activated current. The results suggest that alcohols inhibit the function of this neurotransmitter receptor by interacting with a small hydrophobic pocket on the receptor protein. PMID:8058780

  10. The prostaglandin EP1 receptor potentiates kainate receptor activation via a protein kinase C pathway and exacerbates status epilepticus

    PubMed Central

    Rojas, Asheebo; Gueorguieva, Paoula; Lelutiu, Nadia; Quan, Yi; Shaw, Renee; Dingledine, Raymond

    2014-01-01

    Prostaglandin E2 (PGE2) regulates membrane excitability, synaptic transmission, plasticity, and neuronal survival. The consequences of PGE2 release following seizures has been the subject of much study. Here we demonstrate that the prostaglandin E2 receptor 1 (EP1, or Ptger1) modulates native kainate receptors, a family of ionotropic glutamate receptors widely expressed throughout the central nervous system. Global ablation of the EP1 gene in mice (EP1-KO) had no effect on seizure threshold after kainate injection but reduced the likelihood to enter status epilepticus. EP1-KO mice that did experience typical status epilepticus had reduced hippocampal neurodegeneration and a blunted inflammatory response. Further studies with native prostanoid and kainate receptors in cultured cortical neurons, as well as with recombinant prostanoid and kainate receptors expressed in Xenopus oocytes, demonstrated that EP1 receptor activation potentiates heteromeric but not homomeric kainate receptors via a second messenger cascade involving phospholipase C, calcium and protein kinase C. Three critical GluK5 C-terminal serines underlie the potentiation of the GluK2/GluK5 receptor by EP1 activation. Taken together, these results indicate that EP1 receptor activation during seizures, through a protein kinase C pathway, increases the probability of kainic acid induced status epilepticus, and independently promotes hippocampal neurodegeneration and a broad inflammatory response. PMID:24952362

  11. [G protein-coupled receptors in the spot light].

    PubMed

    Benleulmi-Chaachoua, Abla; Wojciech, Stefanie; Jockers, Ralf

    2013-01-01

    G protein-coupled receptors (GPCRs), also known as seven transmembrane domain-spanning proteins (7TM), play an important role in tissue homeostasis and cellular and hormonal communication. GPCRs are targeted by a large panel of natural ligands such as photons, ions, metabolites, lipids and proteins but also by numerous drugs. Research efforts in the GPCR field have been rewarded in 2012 by the Nobel Price in Chemistry. The present article briefly summarizes our current knowledge on GPCRs and discusses future challenges in terms of fundamental aspects and therapeutic applications. © Société de Biologie, 2013.

  12. High-fat diet induces protein kinase A and G-protein receptor kinase phosphorylation of β2 -adrenergic receptor and impairs cardiac adrenergic reserve in animal hearts.

    PubMed

    Fu, Qin; Hu, Yuting; Wang, Qingtong; Liu, Yongming; Li, Ning; Xu, Bing; Kim, Sungjin; Chiamvimonvat, Nipavan; Xiang, Yang K

    2017-03-15

    Patients with diabetes show a blunted cardiac inotropic response to β-adrenergic stimulation despite normal cardiac contractile reserve. Acute insulin stimulation impairs β-adrenergically induced contractile function in isolated cardiomyocytes and Langendorff-perfused hearts. In this study, we aimed to examine the potential effects of hyperinsulinaemia associated with high-fat diet (HFD) feeding on the cardiac β 2 -adrenergic receptor signalling and the impacts on cardiac contractile function. We showed that 8 weeks of HFD feeding leads to reductions in cardiac functional reserve in response to β-adrenergic stimulation without significant alteration of cardiac structure and function, which is associated with significant changes in β 2 -adrenergic receptor phosphorylation at protein kinase A and G-protein receptor kinase sites in the myocardium. The results suggest that clinical intervention might be applied to subjects in early diabetes without cardiac symptoms to prevent further cardiac complications. Patients with diabetes display reduced exercise capability and impaired cardiac contractile reserve in response to adrenergic stimulation. We have recently uncovered an insulin receptor and adrenergic receptor signal network in the heart. The aim of this study was to understand the impacts of high-fat diet (HFD) on the insulin-adrenergic receptor signal network in hearts. After 8 weeks of HFD feeding, mice exhibited diabetes, with elevated insulin and glucose concentrations associated with body weight gain. Mice fed an HFD had normal cardiac structure and function. However, the HFD-fed mice displayed a significant elevation of phosphorylation of the β 2 -adrenergic receptor (β 2 AR) at both the protein kinase A site serine 261/262 and the G-protein-coupled receptor kinase site serine 355/356 and impaired adrenergic reserve when compared with mice fed on normal chow. Isolated myocytes from HFD-fed mice also displayed a reduced contractile response to

  13. Glucocorticoid acts on a putative G protein-coupled receptor to rapidly regulate the activity of NMDA receptors in hippocampal neurons.

    PubMed

    Zhang, Yanmin; Sheng, Hui; Qi, Jinshun; Ma, Bei; Sun, Jihu; Li, Shaofeng; Ni, Xin

    2012-04-01

    Glucocorticoids (GCs) have been demonstrated to act through both genomic and nongenomic mechanisms. The present study demonstrated that corticosterone rapidly suppressed the activity of N-methyl-D-aspartate (NMDA) receptors in cultured hippocampal neurons. The effect was maintained with corticosterone conjugated to bovine serum albumin and blocked by inhibition of G protein activity with intracellular GDP-β-S application. Corticosterone increased GTP-bound G(s) protein and cyclic AMP (cAMP) production, activated phospholipase Cβ(3) (PLC-β(3)), and induced inositol-1,4,5-triphosphate (IP(3)) production. Blocking PLC and the downstream cascades with PLC inhibitor, IP(3) receptor antagonist, Ca(2+) chelator, and protein kinase C (PKC) inhibitors prevented the actions of corticosterone. Blocking adenylate cyclase (AC) and protein kinase A (PKA) caused a decrease in NMDA-evoked currents. Application of corticosterone partly reversed the inhibition of NMDA currents caused by blockage of AC and PKA. Intracerebroventricular administration of corticosterone significantly suppressed long-term potentiation (LTP) in the CA1 region of the hippocampus within 30 min in vivo, implicating the possibly physiological significance of rapid effects of GC on NMDA receptors. Taken together, our results indicate that GCs act on a putative G protein-coupled receptor to activate multiple signaling pathways in hippocampal neurons, and the rapid suppression of NMDA activity by GCs is dependent on PLC and downstream signaling.

  14. Chaperoning G Protein-Coupled Receptors: From Cell Biology to Therapeutics

    PubMed Central

    Conn, P. Michael

    2014-01-01

    G protein-coupled receptors (GPCRs) are membrane proteins that traverse the plasma membrane seven times (hence, are also called 7TM receptors). The polytopic structure of GPCRs makes the folding of GPCRs difficult and complex. Indeed, many wild-type GPCRs are not folded optimally, and defects in folding are the most common cause of genetic diseases due to GPCR mutations. Both general and receptor-specific molecular chaperones aid the folding of GPCRs. Chemical chaperones have been shown to be able to correct the misfolding in mutant GPCRs, proving to be important tools for studying the structure-function relationship of GPCRs. However, their potential therapeutic value is very limited. Pharmacological chaperones (pharmacoperones) are potentially important novel therapeutics for treating genetic diseases caused by mutations in GPCR genes that resulted in misfolded mutant proteins. Pharmacoperones also increase cell surface expression of wild-type GPCRs; therefore, they could be used to treat diseases that do not harbor mutations in GPCRs. Recent studies have shown that indeed pharmacoperones work in both experimental animals and patients. High-throughput assays have been developed to identify new pharmacoperones that could be used as therapeutics for a number of endocrine and other genetic diseases. PMID:24661201

  15. A PAIR OF TRANSMEMBRANE RECEPTORS ESSENTIAL FOR THE RETENTION AND PIGMENTATION OF HAIR

    PubMed Central

    Han, Rong; Beppu, Hideyuki; Lee, Yun-Kyoung; Georgopoulos, Katia; Larue, Lionel; Li, En; Weiner, Lorin; Brissette, Janice L.

    2012-01-01

    Hair follicles are simple, accessible models for many developmental processes. Here, using mutant mice, we show that Bmpr2, a known receptor for bone morphogenetic proteins (Bmps), and Acvr2a, a known receptor for Bmps and activins, are individually redundant but together essential for multiple follicular traits. When Bmpr2/Acvr2a function is reduced in cutaneous epithelium, hair follicles undergo rapid cycles of hair generation and loss. Alopecia results from a failure to terminate hair development properly, as hair clubs never form, and follicular retraction is slowed. Hair regeneration is rapid due to premature activation of new hair-production programs. Hair shafts differentiate aberrantly due to impaired arrest of medullary-cell proliferation. When Bmpr2/Acvr2a function is reduced in melanocytes, gray hair develops, as melanosomes differentiate but fail to grow, resulting in organelle miniaturization. We conclude that Bmpr2 and Acvr2a normally play cell-type-specific, necessary roles in organelle biogenesis and the shutdown of developmental programs and cell division. PMID:22611050

  16. Pharmacological activation of CB1 receptor modulates long term potentiation by interfering with protein synthesis.

    PubMed

    Navakkode, Sheeja; Korte, Martin

    2014-04-01

    Cognitive impairment is one of the most important side effects associated with cannabis drug abuse, as well as the serious issue concerning the therapeutic use of cannabinoids. Cognitive impairments and neuropsychiatric symptoms are caused by early synaptic dysfunctions, such as loss of synaptic connections in different brain structures including the hippocampus, a region that is believed to play an important role in certain forms of learning and memory. We report here that metaplastic priming of synapses with a cannabinoid type 1 receptor (CB1 receptor) agonist, WIN55,212-2 (WIN55), significantly impaired long-term potentiation in the apical dendrites of CA1 pyramidal neurons. Interestingly, the CB1 receptor exerts its effect by altering the balance of protein synthesis machinery towards higher protein production. Therefore the activation of CB1 receptor, prior to strong tetanization, increased the propensity to produce new proteins. In addition, WIN55 priming resulted in the expression of late-LTP in a synaptic input that would have normally expressed early-LTP, thus confirming that WIN55 priming of LTP induces new synthesis of plasticity-related proteins. Furthermore, in addition to the effects on protein translation, WIN55 also induced synaptic deficits due to the ability of CB1 receptors to inhibit the release of acetylcholine, mediated by both muscarinic and nicotinic acetylcholine receptors. Taken together this supports the notion that the modulation of cholinergic activity by CB1 receptor activation is one mechanism that regulates the synthesis of plasticity-related proteins. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Molecular recognition of a model globular protein apomyoglobin by synthetic receptor cyclodextrin: effect of fluorescence modification of the protein and cavity size of the receptor in the interaction.

    PubMed

    Saha, Ranajay; Rakshit, Surajit; Pal, Samir Kumar

    2013-11-01

    Labelling of proteins with some extrinsic probe is unavoidable in molecular biology research. Particularly, spectroscopic studies in the optical region require fluorescence modification of native proteins by attaching polycyclic aromatic fluoroprobe with the proteins under investigation. Our present study aims to address the consequence of the attachment of a fluoroprobe at the protein surface in the molecular recognition of the protein by selectively small model receptor. A spectroscopic study involving apomyoglobin (Apo-Mb) and cyclodextrin (CyD) of various cavity sizes as model globular protein and synthetic receptors, respectively, using steady-state and picosecond-resolved techniques, is detailed here. A study involving Förster resonance energy transfer, between intrinsic amino acid tryptophan (donor) and N, N-dimethyl naphthalene moiety of the extrinsic dansyl probes at the surface of Apo-Mb, precisely monitor changes in donor acceptor distance as a consequence of interaction of the protein with CyD having different cavity sizes (β and γ variety). Molecular modelling studies on the interaction of tryptophan and dansyl probe with β-CyD is reported here and found to be consistent with the experimental observations. In order to investigate structural aspects of the interacting protein, we have used circular dichroism spectroscopy. Temperature-dependent circular dichroism studies explore the change in the secondary structure of Apo-Mb in association with CyD, before and after fluorescence modification of the protein. Overall, the study well exemplifies approaches to protein recognition by CyD as a synthetic receptor and offers a cautionary note on the use of hydrophobic fluorescent labels for proteins in biochemical studies involving recognition of molecules. Copyright © 2013 John Wiley & Sons, Ltd.

  18. The neuregulin receptor ErbB-4 interacts with PDZ-containing proteins at neuronal synapses

    PubMed Central

    Garcia, Rolando A. G.; Vasudevan, Kuzhalini; Buonanno, Andres

    2000-01-01

    Neuregulins regulate the expression of ligand- and voltage-gated channels in neurons and skeletal muscle by the activation of their cognate tyrosine kinase receptors, ErbB 1–4. The subcellular distribution and mechanisms that regulate the localization of ErbB receptors are unknown. We have found that ErbB receptors are present in brain subcellular fractions enriched for postsynaptic densities (PSD). The ErbB-4 receptor is unique among the ErbB proteins because its C-terminal tail (T-V-V) conforms to a sequence that binds to a protein motif known as the PDZ domain. Using the yeast two-hybrid system, we found that the C-terminal region of ErbB-4 interacts with the three related membrane-associated guanylate kinases (MAGUKs) PSD-95/SAP90, PSD-93/chapsyn-110, and SAP 102, which harbor three PDZ domains, as well as with β2-syntrophin, which has a single PDZ domain. As with N-methyl-d-aspartate (NMDA) receptors, ErbB4 interacts with the first two PDZ domains of PSD-95. Using coimmunoprecipitation assays, we confirmed the direct interactions between ErbB-4 and PSD-95 in transfected heterologous cells, as well as in vivo, where both proteins are coimmunoprecipitated from brain lysates. Moreover, evidence for colocalization of these proteins was also observed by immunofluorescence in cultured hippocampal neurons. ErbB-4 colocalizes with PSD-95 and NMDA receptors at a subset of excitatory synapses apposed to synaptophysin-positive presynaptic terminals. The capacity of ErbB receptors to interact with PDZ-domain proteins at cell junctions is conserved from invertebrates to mammals. As discussed, the interactions found between receptor tyrosine kinases and MAGUKs at neuronal synapses may have important implications for activity-dependent plasticity. PMID:10725395

  19. The structural basis of arrestin-mediated regulation of G-protein-coupled receptors

    PubMed Central

    Gurevich, Vsevolod V.; Gurevich, Eugenia V.

    2008-01-01

    The 4 mammalian arrestins serve as almost universal regulators of the largest known family of signaling proteins, G-protein-coupled receptors (GPCRs). Arrestins terminate receptor interactions with G proteins, redirect the signaling to a variety of alternative pathways, and orchestrate receptor internalization and subsequent intracellular trafficking. The elucidation of the structural basis and fine molecular mechanisms of the arrestin–receptor interaction paved the way to the targeted manipulation of this interaction from both sides to produce very stable or extremely transient complexes that helped to understand the regulation of many biologically important processes initiated by active GPCRs. The elucidation of the structural basis of arrestin interactions with numerous non-receptor-binding partners is long overdue. It will allow the construction of fully functional arrestins in which the ability to interact with individual partners is specifically disrupted or enhanced by targeted mutagenesis. These “custom-designed” arrestin mutants will be valuable tools in defining the role of various interactions in the intricate interplay of multiple signaling pathways in the living cell. The identification of arrestin-binding sites for various signaling molecules will also set the stage for designing molecular tools for therapeutic intervention that may prove useful in numerous disorders associated with congenital or acquired disregulation of GPCR signaling. PMID:16460808

  20. Endothelial nitric-oxide synthase (eNOS) is activated through G-protein-coupled receptor kinase-interacting protein 1 (GIT1) tyrosine phosphorylation and Src protein.

    PubMed

    Liu, Songling; Premont, Richard T; Rockey, Don C

    2014-06-27

    Nitric oxide (NO) is a critical regulator of vascular tone and plays an especially prominent role in liver by controlling portal blood flow and pressure within liver sinusoids. Synthesis of NO in sinusoidal endothelial cells by endothelial nitric-oxide synthase (eNOS) is regulated in response to activation of endothelial cells by vasoactive signals such as endothelins. The endothelin B (ETB) receptor is a G-protein-coupled receptor, but the mechanisms by which it regulates eNOS activity in sinusoidal endothelial cells are not well understood. In this study, we built on two previous strands of work, the first showing that G-protein βγ subunits mediated activation of phosphatidylinositol 3-kinase and Akt to regulate eNOS and the second showing that eNOS directly bound to the G-protein-coupled receptor kinase-interacting protein 1 (GIT1) scaffold protein, and this association stimulated NO production. Here we investigated the mechanisms by which the GIT1-eNOS complex is formed and regulated. GIT1 was phosphorylated on tyrosine by Src, and Y293F and Y554F mutations reduced GIT1 phosphorylation as well as the ability of GIT1 to bind to and activate eNOS. Akt phosphorylation activated eNOS (at Ser(1177)), and Akt also regulated the ability of Src to phosphorylate GIT1 as well as GIT1-eNOS association. These pathways were activated by endothelin-1 through the ETB receptor; inhibiting receptor-activated G-protein βγ subunits blocked activation of Akt, GIT1 tyrosine phosphorylation, and ET-1-stimulated GIT1-eNOS association but did not affect Src activation. These data suggest a model in which Src and Akt cooperate to regulate association of eNOS with the GIT1 scaffold to facilitate NO production. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Crucial role of neuron-enriched endosomal protein of 21 kDa in sorting between degradation and recycling of internalized G-protein-coupled receptors.

    PubMed

    Debaigt, Colin; Hirling, Harald; Steiner, Pascal; Vincent, Jean-Pierre; Mazella, Jean

    2004-08-20

    Recycling of endocytosed G-protein-coupled receptors involves a series of molecular events through early and recycling endosomes. The purpose of this work was to study the role of neuron-enriched endosomal protein of 21 kDa (NEEP21) in the recycling process of neurotensin receptors-1 and -2. Here we showed that suppression of NEEP21 expression does not modify the internalization rate of both receptors but strongly inhibited the recycling of the neurotensin receptor-2. In contrast, overexpression of NEEP21 changes the behavior of the neurotensin receptor-1 from a non-recycling to a recycling state. Recycling of the neurotensin receptor-2 involves both the phosphatidylinositol 3-kinase and the recycling endosome pathways, whereas recycling of the neurotensin receptor-1 induced by overexpression of NEEP21 only occurs by the phosphatidylinositol 3-kinase-dependent pathway. Taken together, these results confirm the essential role of NEEP21 in the recycling mechanism and show that this protein acts at the level of early endosomes to promote sorting of receptors toward a recycling pathway.

  2. Design and functional characterization of a novel, arrestin-biased designer G protein-coupled receptor.

    PubMed

    Nakajima, Ken-ichiro; Wess, Jürgen

    2012-10-01

    Mutational modification of distinct muscarinic receptor subtypes has yielded novel designer G protein-coupled receptors (GPCRs) that are unable to bind acetylcholine (ACh), the endogenous muscarinic receptor ligand, but can be efficiently activated by clozapine-N-oxide (CNO), an otherwise pharmacologically inert compound. These CNO-sensitive designer GPCRs [alternative name: designer receptors exclusively activated by designer drug (DREADDs)] have emerged as powerful new tools to dissect the in vivo roles of distinct G protein signaling pathways in specific cell types or tissues. As is the case with other GPCRs, CNO-activated DREADDs not only couple to heterotrimeric G proteins but can also recruit proteins of the arrestin family (arrestin-2 and -3). Accumulating evidence suggests that arrestins can act as scaffolding proteins to promote signaling through G protein-independent signaling pathways. To explore the physiological relevance of these arrestin-dependent signaling pathways, the availability of an arrestin-biased DREADD would be highly desirable. In this study, we describe the development of an M₃ muscarinic receptor-based DREADD [Rq(R165L)] that is no longer able to couple to G proteins but can recruit arrestins and promote extracellular signal-regulated kinase-1/2 phosphorylation in an arrestin- and CNO-dependent fashion. Moreover, CNO treatment of mouse insulinoma (MIN6) cells expressing the Rq(R165L) construct resulted in a robust, arrestin-dependent stimulation of insulin release, directly implicating arrestin signaling in the regulation of insulin secretion. This newly developed arrestin-biased DREADD represents an excellent novel tool to explore the physiological relevance of arrestin signaling pathways in distinct tissues and cell types.

  3. The Role of Rab Proteins in Neuronal Cells and in the Trafficking of Neurotrophin Receptors

    PubMed Central

    Bucci, Cecilia; Alifano, Pietro; Cogli, Laura

    2014-01-01

    Neurotrophins are a family of proteins that are important for neuronal development, neuronal survival and neuronal functions. Neurotrophins exert their role by binding to their receptors, the Trk family of receptor tyrosine kinases (TrkA, TrkB, and TrkC) and p75NTR, a member of the tumor necrosis factor (TNF) receptor superfamily. Binding of neurotrophins to receptors triggers a complex series of signal transduction events, which are able to induce neuronal differentiation but are also responsible for neuronal maintenance and neuronal functions. Rab proteins are small GTPases localized to the cytosolic surface of specific intracellular compartments and are involved in controlling vesicular transport. Rab proteins, acting as master regulators of the membrane trafficking network, play a central role in both trafficking and signaling pathways of neurotrophin receptors. Axonal transport represents the Achilles' heel of neurons, due to the long-range distance that molecules, organelles and, in particular, neurotrophin-receptor complexes have to cover. Indeed, alterations of axonal transport and, specifically, of axonal trafficking of neurotrophin receptors are responsible for several human neurodegenerative diseases, such as Huntington’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis and some forms of Charcot-Marie-Tooth disease. In this review, we will discuss the link between Rab proteins and neurotrophin receptor trafficking and their influence on downstream signaling pathways. PMID:25295627

  4. Quantification of Ligand Binding to G-Protein Coupled Receptors on Cell Membranes by Ellipsometry

    PubMed Central

    Kriechbaumer, Verena; Nabok, Alexei; Widdowson, Robert; Smith, David P.; Abell, Ben M.

    2012-01-01

    G-protein-coupled receptors (GPCRs) are prime drug targets and targeted by approximately 60% of current therapeutic drugs such as β-blockers, antipsychotics and analgesics. However, no biophysical methods are available to quantify their interactions with ligand binding in a native environment. Here, we use ellipsometry to quantify specific interactions of receptors within native cell membranes. As a model system, the GPCR-ligand CXCL12α and its receptor CXCR4 are used. Human-derived Ishikawa cells were deposited onto gold coated slides via Langmuir-Schaefer film deposition and interactions between the receptor CXCR4 on these cells and its ligand CXCL12α were detected via total internal reflection ellipsometry (TIRE). This interaction could be inhibited by application of the CXCR4-binding drug AMD3100. Advantages of this approach are that it allows measurement of interactions in a lipid environment without the need for labelling, protein purification or reconstitution of membrane proteins. This technique is potentially applicable to a wide variety of cell types and their membrane receptors, providing a novel method to determine ligand or drug interactions targeting GPCRs and other membrane proteins. PMID:23049983

  5. Induction of Cardiac Fibrosis by β-Blocker in G Protein-independent and G Protein-coupled Receptor Kinase 5/β-Arrestin2-dependent Signaling Pathways*

    PubMed Central

    Nakaya, Michio; Chikura, Satsuki; Watari, Kenji; Mizuno, Natsumi; Mochinaga, Koji; Mangmool, Supachoke; Koyanagi, Satoru; Ohdo, Shigehiro; Sato, Yoji; Ide, Tomomi; Nishida, Motohiro; Kurose, Hitoshi

    2012-01-01

    G-protein coupled receptors (GPCRs) have long been known as receptors that activate G protein-dependent cellular signaling pathways. In addition to the G protein-dependent pathways, recent reports have revealed that several ligands called “biased ligands” elicit G protein-independent and β-arrestin-dependent signaling through GPCRs (biased agonism). Several β-blockers are known as biased ligands. All β-blockers inhibit the binding of agonists to the β-adrenergic receptors. In addition to β-blocking action, some β-blockers are reported to induce cellular responses through G protein-independent and β-arrestin-dependent signaling pathways. However, the physiological significance induced by the β-arrestin-dependent pathway remains much to be clarified in vivo. Here, we demonstrate that metoprolol, a β1-adrenergic receptor-selective blocker, could induce cardiac fibrosis through a G protein-independent and β-arrestin2-dependent pathway. Metoprolol, a β-blocker, increased the expression of fibrotic genes responsible for cardiac fibrosis in cardiomyocytes. Furthermore, metoprolol induced the interaction between β1-adrenergic receptor and β-arrestin2, but not β-arrestin1. The interaction between β1-adrenergic receptor and β-arrestin2 by metoprolol was impaired in the G protein-coupled receptor kinase 5 (GRK5)-knockdown cells. Metoprolol-induced cardiac fibrosis led to cardiac dysfunction. However, the metoprolol-induced fibrosis and cardiac dysfunction were not evoked in β-arrestin2- or GRK5-knock-out mice. Thus, metoprolol is a biased ligand that selectively activates a G protein-independent and GRK5/β-arrestin2-dependent pathway, and induces cardiac fibrosis. This study demonstrates the physiological importance of biased agonism, and suggests that G protein-independent and β-arrestin-dependent signaling is a reason for the diversity of the effectiveness of β-blockers. PMID:22888001

  6. Reprogramming of G protein-coupled receptor recycling and signaling by a kinase switch

    PubMed Central

    Vistein, Rachel; Puthenveedu, Manojkumar A.

    2013-01-01

    The postendocytic recycling of signaling receptors is subject to multiple requirements. Why this is so, considering that many other proteins can recycle without apparent requirements, is a fundamental question. Here we show that cells can leverage these requirements to switch the recycling of the beta-2 adrenergic receptor (B2AR), a prototypic signaling receptor, between sequence-dependent and bulk recycling pathways, based on extracellular signals. This switch is determined by protein kinase A-mediated phosphorylation of B2AR on the cytoplasmic tail. The phosphorylation state of B2AR dictates its partitioning into spatially and functionally distinct endosomal microdomains mediating bulk and sequence-dependent recycling, and also regulates the rate of B2AR recycling and resensitization. Our results demonstrate that G protein-coupled receptor recycling is not always restricted to the sequence-dependent pathway, but may be reprogrammed as needed by physiological signals. Such flexible reprogramming might provide a versatile method for rapidly modulating cellular responses to extracellular signaling. PMID:24003153

  7. Effect of recombinant human bone morphogenetic protein-2 on bone regeneration and osseointegration of dental implants.

    PubMed

    Sykaras, N; Triplett, R G; Nunn, M E; Iacopino, A M; Opperman, L A

    2001-08-01

    Recombinant human bone morphogenetic protein-2 (rhBMP-2) induced bone regeneration and osseointegration was evaluated in bony defects created within the hollow chamber of endosseous dental implants in 14 foxhound dogs. Bilateral extractions of mandibular premolars were performed and surgical implantation of 104 hollow cylinder implants followed after 8 weeks of healing. Experimental implants had their hollow chamber filled with 20 microg of rhBMP-2 delivered with a bovine collagen carrier, whereas the control implants had their apical chamber left empty. Dogs were followed for 2, 4, 8 and 12 weeks. Histomorphometric evaluation and immunohistochemical analysis were performed. Minimal bone was regenerated at 2 weeks for both groups. At 4 weeks, bone fill averaged 23.48% for the rhBMP-2 and 5.98% for the control group (P<0.05). At 8 weeks, mean bone fill was 20.94% and 7.75% for the rhBMP-2 and the controls, respectively (P<0.05). At 12 weeks, mean bone fill was 31.39% and 24.31% for the rhBMP-2 and control implants, respectively (P>0.05). Bone-implant contact (BIC) increased for both groups over time and at 8 weeks the rhBMP-2 BIC value was 18.65% and for the control 7.22% (P<0.05). At 12 weeks, the BIC was 43.78% and 21.05% for the rhBMP-2 and the control group, respectively (P<0.05). Immunohistochemical staining for type II collagen was positive only for parts of the collagen carrier and formation of cartilaginous intermediate was not observed in any of the specimens. The results suggest that, in confined defects adjacent to dental implants, rhBMP-2 can induce bone regeneration in close apposition to the implant surface.

  8. Effects of Recombinant Human Bone Morphogenetic Protein-2 on Vertical Bone Augmentation in a Canine Model.

    PubMed

    Hsu, Yung-Ting; Al-Hezaimi, Khalid; Galindo-Moreno, Pablo; O'Valle, Francisco; Al-Rasheed, Abdulaziz; Wang, Hom-Lay

    2017-09-01

    Vertical bone augmentation (VBA) remains unpredictable and challenging for most clinicians. This study aims to compare hard tissue outcomes of VBA, with and without recombinant human bone morphogenetic protein (rhBMP)-2, under space-making titanium mesh in a canine model. Eleven male beagle dogs were used in the study. Experimental ridge defects were created to form atrophic ridges. VBA was performed via guided bone regeneration using titanium mesh and allografts. In experimental hemimandibles, rhBMP-2/absorbable collagen sponge was well mixed with allografts prior to procedures, whereas a control buffer was applied within controls. Dogs were euthanized after a 4-month healing period. Clinical and radiographic examinations were performed to assess ridge dimensional changes. In addition, specimens were used for microcomputed tomography (micro-CT) assessment and histologic analysis. Membrane exposure was found on five of 11 (45.5%) rhBMP-2-treated sites, whereas it was found on nine of 11 (81.8%) non-rhBMP-2-treated sites. Within 4 months of healing, rhBMP-2-treated sites showed better radiographic bone density, greater defect fill, and significantly more bone gain in ridge height (P <0.05) than controls. Experimental hemimandibles exhibited lower rates of membrane exposure and a noteworthy, ectopic bone formation above the mesh in 72% of sites. Results from micro-CT also suggested a trend of less vertical bone gain and bone mineral density in controls (P >0.05). Under light microscope, predominant lamellar patterns were found in the specimen obtained from rhBMP-2 sites. With inherent limitations of the canine model and the concern of such a demanding surgical technique, current findings suggest that the presence of rhBMP-2 in a composite graft allows an increase of vertical gain, with formation of ectopic bone over the titanium mesh in comparison with non-rhBMP-2 sites.

  9. Stepwise verification of bone regeneration using recombinant human bone morphogenetic protein-2 in rat fibula model

    PubMed Central

    2017-01-01

    Objectives The purpose of this study was to introduce our three experiments on bone morphogenetic protein (BMP) and its carriers performed using the critical sized segmental defect (CSD) model in rat fibula and to investigate development of animal models and carriers for more effective bone regeneration. Materials and Methods For the experiments, 14, 16, and 24 rats with CSDs on both fibulae were used in Experiments 1, 2, and 3, respectively. BMP-2 with absorbable collagen sponge (ACS) (Experiments 1 and 2), autoclaved autogenous bone (AAB) and fibrin glue (FG) (Experiment 3), and xenogenic bone (Experiment 2) were used in the experimental groups. Radiographic and histomorphological evaluations were performed during the follow-up period of each experiment. Results Significant new bone formation was commonly observed in all experimental groups using BMP-2 compared to control and xenograft (porcine bone) groups. Although there was some difference based on BMP carrier, regenerated bone volume was typically reduced by remodeling after initially forming excessive bone. Conclusion BMP-2 demonstrates excellent ability for bone regeneration because of its osteoinductivity, but efficacy can be significantly different depending on its delivery system. ACS and FG showed relatively good bone regeneration capacity, satisfying the essential conditions of localization and release-control when used as BMP carriers. AAB could not provide release-control as a BMP carrier, but its space-maintenance role was remarkable. Carriers and scaffolds that can provide sufficient support to the BMP/carrier complex are necessary for large bone defects, and AAB is thought to be able to act as an effective scaffold. The CSD model of rat fibula is simple and useful for initial estimate of bone regeneration by agents including BMPs. PMID:29333367

  10. Methyl farnesoate action, and morphogenetic signaling through the ligand binding pocket of the ortholog of the retinoid X receptor, in higher dipter

    USDA-ARS?s Scientific Manuscript database

    Most attention on metamorphic signaling by small terpenoids has focused action by juvenile hormone (JH) through bHLH-PAS proteins (e.g., MET and GCE), especially as that signaling axis intersects with ecdysteroid action through the receptor EcR. However, a long-standing series of endocrine and pharm...

  11. Spring-Loaded Model Revisited: Paramyxovirus Fusion Requires Engagement of a Receptor Binding Protein beyond Initial Triggering of the Fusion Protein▿

    PubMed Central

    Porotto, Matteo; DeVito, Ilaria; Palmer, Samantha G.; Jurgens, Eric M.; Yee, Jia L.; Yokoyama, Christine C.; Pessi, Antonello; Moscona, Anne

    2011-01-01

    During paramyxovirus entry into a host cell, receptor engagement by a specialized binding protein triggers conformational changes in the adjacent fusion protein (F), leading to fusion between the viral and cell membranes. According to the existing paradigm of paramyxovirus membrane fusion, the initial activation of F by the receptor binding protein sets off a spring-loaded mechanism whereby the F protein progresses independently through the subsequent steps in the fusion process, ending in membrane merger. For human parainfluenza virus type 3 (HPIV3), the receptor binding protein (hemagglutinin-neuraminidase [HN]) has three functions: receptor binding, receptor cleaving, and activating F. We report that continuous receptor engagement by HN activates F to advance through the series of structural rearrangements required for fusion. In contrast to the prevailing model, the role of HN-receptor engagement in the fusion process is required beyond an initiating step, i.e., it is still required even after the insertion of the fusion peptide into the target cell membrane, enabling F to mediate membrane merger. We also report that for Nipah virus, whose receptor binding protein has no receptor-cleaving activity, the continuous stimulation of the F protein by a receptor-engaged binding protein is key for fusion. We suggest a general model for paramyxovirus fusion activation in which receptor engagement plays an active role in F activation, and the continued engagement of the receptor binding protein is essential to F protein function until the onset of membrane merger. This model has broad implications for the mechanism of paramyxovirus fusion and for strategies to prevent viral entry. PMID:21976650

  12. Molecular Dynamics Simulations of G Protein-Coupled Receptors.

    PubMed

    Bruno, Agostino; Costantino, Gabriele

    2012-04-01

    G protein-coupled receptors (GPCRs) constitute the largest family of membrane-bound receptors with more than 800 members encoded by 351 genes in humans. It has been estimated that more than 50 % of clinically available drugs act on GPCRs, with an amount of 400, 50 and 25 druggable proteins for the class A, B and C, respectively. Furthermore, Class A GPCRs with approximately 25 % of marketed small drugs represent the most attractive pharmaceutical class. The recent availability of high-resolution 3-dimensional structures of some GPCRs supports the notion that GPCRs are dynamically versatile, and their functions can be modulated by several factors. In this scenario, molecular dynamics (MD) simulations techniques appear to be crucial when studying GPCR flexibility associated to functioning and ligand recognition. A general overview of biased and unbiased MD techniques is here presented with special emphasis on the recent results obtained in the GPCRs field. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Protein-tyrosine-phosphatase-mediated epidermal growth factor (EGF) receptor transinactivation and EGF receptor-independent stimulation of mitogen-activated protein kinase by bradykinin in A431 cells.

    PubMed Central

    Graness, A; Hanke, S; Boehmer, F D; Presek, P; Liebmann, C

    2000-01-01

    Transactivation of the epidermal growth factor (EGF) receptor (EGFR) has been proposed to represent an essential link between G-protein-coupled receptors and the mitogen-activated protein kinase (MAPK) pathway in various cell types. In the present work we report, in contrast, that in A431 cells bradykinin transinactivates the EGFR and stimulates MAPK activity independently of EGFR tyrosine phosphorylation. Both effects of bradykinin are mediated by a pertussis-toxin-insensitive G-protein. Three lines of evidence suggest the activation of a protein tyrosine phosphatase (PTP) by bradykinin: (i) treatment of A431 cells with bradykinin decreases both basal and EGF-induced EGFR tyrosine phosphorylation, (ii) this effect of bradykinin can be blocked by two different PTP inhibitors, and (iii) bradykinin significantly increased the PTP activity in total A431 cell lysates when measured in vitro. The transmembrane receptor PTP sigma was identified as a putative mediator of bradykinin-induced downregulation of EGFR autophosphorylation. Activation of MAPK in response to bradykinin was insensitive towards AG 1478, a specific inhibitor of EGFR tyrosine kinase, but was blocked by wortmannin or bisindolylmaleimide, inhibitors of phosphatidylinositol 3-kinase (PI3-K) and protein kinase C (PKC) respectively. These results also suggest that the bradykinin-induced activation of MAPK is independent of EGFR and indicate a pathway involving PI3-K and PKC. In addition, bradykinin evokes a rapid and transient increase in Src kinase activity. Although Src does not participate in bradykinin-induced stimulation of PTP activity, inhibition of Src by 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine leads to an increase in MAPK activation by bradykinin. Our results suggest that in A431 cells the G(q/11)-protein-coupled bradykinin B(2) receptor may stimulate PTP activity and thereby transinactivate the EGFR, and may simultaneously activate MAPK by an alternative signalling pathway

  14. Development of Protein Degradation Inducers of Androgen Receptor by Conjugation of Androgen Receptor Ligands and Inhibitor of Apoptosis Protein Ligands.

    PubMed

    Shibata, Norihito; Nagai, Katsunori; Morita, Yoko; Ujikawa, Osamu; Ohoka, Nobumichi; Hattori, Takayuki; Koyama, Ryokichi; Sano, Osamu; Imaeda, Yasuhiro; Nara, Hiroshi; Cho, Nobuo; Naito, Mikihiko

    2018-01-25

    Targeted protein degradation using small molecules is a novel strategy for drug development. We have developed hybrid molecules named specific and nongenetic inhibitor of apoptosis protein [IAP]-dependent protein erasers (SNIPERs) that recruit IAP ubiquitin ligases to degrade target proteins. Here, we show novel SNIPERs capable of inducing proteasomal degradation of the androgen receptor (AR). Through derivatization of the SNIPER(AR) molecule at the AR ligand and IAP ligand and linker, we developed 42a (SNIPER(AR)-51), which shows effective protein knockdown activity against AR. Consistent with the degradation of the AR protein, 42a inhibits AR-mediated gene expression and proliferation of androgen-dependent prostate cancer cells. In addition, 42a efficiently induces caspase activation and apoptosis in prostate cancer cells, which was not observed in the cells treated with AR antagonists. These results suggest that SNIPER(AR)s could be leads for an anticancer drug against prostate cancers that exhibit AR-dependent proliferation.

  15. Hematopoietic G-protein-coupled receptor kinase 2 deficiency decreases atherosclerotic lesion formation in LDL receptor-knockout mice

    PubMed Central

    Otten, Jeroen J. T.; de Jager, Saskia C. A.; Kavelaars, Annemieke; Seijkens, Tom; Bot, Ilze; Wijnands, Erwin; Beckers, Linda; Westra, Marijke M.; Bot, Martine; Busch, Matthias; Bermudez, Beatriz; van Berkel, Theo J. C.; Heijnen, Cobi J.; Biessen, Erik A. L.

    2013-01-01

    Leukocyte chemotaxis is deemed instrumental in initiation and progression of atherosclerosis. It is mediated by G-protein-coupled receptors (e.g., CCR2 and CCR5), the activity of which is controlled by G-protein-coupled receptor kinases (GRKs). In this study, we analyzed the effect of hematopoietic deficiency of a potent regulator kinase of chemotaxis (GRK2) on atherogenesis. LDL receptor-deficient (LDLr−/−) mice with heterozygous hematopoietic GRK2 deficiency, generated by bone marrow transplantation (n=15), displayed a dramatic attenuation of plaque development, with 79% reduction in necrotic core and increased macrophage content. Circulating monocytes decreased and granulocytes increased in GRK2+/− chimeras, which could be attributed to diminished granulocyte colony-forming units in bone marrow. Collectively, these data pointed to myeloid cells as major mediators of the impaired atherogenic response in GRK2+/− chimeras. LDLr−/− mice with macrophage/granulocyte-specific GRK2 deficiency (LysM-Cre GRK2flox/flox; n=8) failed to mimic the aforementioned phenotype, acquitting these cells as major responsible subsets for GRK2 deficiency-associated atheroprotection. To conclude, even partial hematopoietic GRK2 deficiency prevents atherosclerotic lesion progression beyond the fatty streak stage, identifying hematopoietic GRK2 as a potential target for intervention in atherosclerosis.—Otten, J. J. T., de Jager, S. C. A., Kavelaars, A., Seijkens, T., Bot, I., Wijnands, E., Beckers, L., Westra, M. M., Bot, M., Busch, M., Bermudez, B., van Berkel, T. J. C., Heijnen, C. J., Biessen, E. A. L. Hematopoietic G-protein-coupled receptor kinase 2 deficiency decreases atherosclerotic lesion formation in LDL receptor-knockout mice. PMID:23047899

  16. Protein receptor-independent plasma membrane remodeling by HAMLET: A tumoricidal protein-lipid complex

    DOE PAGES

    Nadeem, Aftab; Sanborn, Jeremy; Gettel, Douglas L.; ...

    2015-11-12

    A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This ‘’protein-centric” view is increasingly challenged by evidence for the involvement of specialized membrane domains in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. Wemore » identify a ‘’receptor independent” transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET accumulates within these de novo membrane conformations and define membrane blebs as cellular compartments for direct interactions of HAMLET with essential target proteins such as the Ras family of GTPases. In conclusion, we demonstrate lower sensitivity of healthy cell membranes to HAMLET challenge. These features suggest that HAMLET-induced curvature-dependent membrane conformations serve as surrogate receptors for initiating signal transduction cascades, ultimately leading to cell death.« less

  17. Protein receptor-independent plasma membrane remodeling by HAMLET: a tumoricidal protein-lipid complex

    PubMed Central

    Nadeem, Aftab; Sanborn, Jeremy; Gettel, Douglas L.; James, Ho C. S.; Rydström, Anna; Ngassam, Viviane N.; Klausen, Thomas Kjær; Pedersen, Stine Falsig; Lam, Matti; Parikh, Atul N.; Svanborg, Catharina

    2015-01-01

    A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This ‘’protein-centric” view is increasingly challenged by evidence for the involvement of specialized membrane domains in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. We identify a ‘’receptor independent” transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET accumulates within these de novo membrane conformations and define membrane blebs as cellular compartments for direct interactions of HAMLET with essential target proteins such as the Ras family of GTPases. Finally, we demonstrate lower sensitivity of healthy cell membranes to HAMLET challenge. These features suggest that HAMLET-induced curvature-dependent membrane conformations serve as surrogate receptors for initiating signal transduction cascades, ultimately leading to cell death. PMID:26561036

  18. Structure and Function of Serotonin G protein Coupled Receptors

    PubMed Central

    McCorvy, John D.; Roth, Bryan L.

    2015-01-01

    Serotonin receptors are prevalent throughout the nervous system and the periphery, and remain one of the most lucrative and promising drug discovery targets for disorders ranging from migraine headaches to neuropsychiatric disorders such as schizophrenia and depression. There are 14 distinct serotonin receptors, of which 13 are G protein coupled receptors (GPCRs), which are targets for approximately 40% of the approved medicines. Recent crystallographic and biochemical evidence has provided a converging understanding of the basic structure and functional mechanics of GPCR activation. Currently, two GPCR crystal structures exist for the serotonin family, the 5-HT1B and 5-HT2B receptor, with the antimigraine and valvulopathic drug ergotamine bound. The first serotonin crystal structures not only provide the first evidence of serotonin receptor topography but also provide mechanistic explanations into functional selectivity or biased agonism. This review will detail the findings of these crystal structures from a molecular and mutagenesis perspective for driving rational drug design for novel therapeutics incorporating biased signaling. PMID:25601315

  19. Calcitonin and Amylin Receptor Peptide Interaction Mechanisms: INSIGHTS INTO PEPTIDE-BINDING MODES AND ALLOSTERIC MODULATION OF THE CALCITONIN RECEPTOR BY RECEPTOR ACTIVITY-MODIFYING PROTEINS.

    PubMed

    Lee, Sang-Min; Hay, Debbie L; Pioszak, Augen A

    2016-04-15

    Receptor activity-modifying proteins (RAMP1-3) determine the selectivity of the class B G protein-coupled calcitonin receptor (CTR) and the CTR-like receptor (CLR) for calcitonin (CT), amylin (Amy), calcitonin gene-related peptide (CGRP), and adrenomedullin (AM) peptides. RAMP1/2 alter CLR selectivity for CGRP/AM in part by RAMP1 Trp-84 or RAMP2 Glu-101 contacting the distinct CGRP/AM C-terminal residues. It is unclear whether RAMPs use a similar mechanism to modulate CTR affinity for CT and Amy, analogs of which are therapeutics for bone disorders and diabetes, respectively. Here, we reproduced the peptide selectivity of intact CTR, AMY1 (CTR·RAMP1), and AMY2 (CTR·RAMP2) receptors using purified CTR extracellular domain (ECD) and tethered RAMP1- and RAMP2-CTR ECD fusion proteins and antagonist peptides. All three proteins bound salmon calcitonin (sCT). Tethering RAMPs to CTR enhanced binding of rAmy, CGRP, and the AMY antagonist AC413. Peptide alanine-scanning mutagenesis and modeling of receptor-bound sCT and AC413 supported a shared non-helical CGRP-like conformation for their TN(T/V)G motif prior to the C terminus. After this motif, the peptides diverged; the sCT C-terminal Pro was crucial for receptor binding, whereas the AC413/rAmy C-terminal Tyr had little or no influence on binding. Accordingly, mutant RAMP1 W84A- and RAMP2 E101A-CTR ECD retained AC413/rAmy binding. ECD binding and cell-based signaling assays with antagonist sCT/AC413/rAmy variants with C-terminal residue swaps indicated that the C-terminal sCT/rAmy residue identity affects affinity more than selectivity. rAmy(8-37) Y37P exhibited enhanced antagonism of AMY1 while retaining selectivity. These results reveal unexpected differences in how RAMPs determine CTR and CLR peptide selectivity and support the hypothesis that RAMPs allosterically modulate CTR peptide affinity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Polydopamine mediated assembly of hydroxyapatite nanoparticles and bone morphogenetic protein-2 on magnesium alloys for enhanced corrosion resistance and bone regeneration.

    PubMed

    Jiang, Yanan; Wang, Bi; Jia, Zhanrong; Lu, Xiong; Fang, Liming; Wang, Kefeng; Ren, Fuzeng

    2017-10-01

    Magnesium alloys have the great potential to be used as orthopedic implants due to their biodegradability and mechanical resemblance to human cortical bone. However, the rapid degradation in physiological environment with the evolution of hydrogen gas release hinders their clinical applications. In this study, we developed a novel functional and biocompatible coating strategy through polydopamine mediated assembly of hydroxyapatite nanoparticles and growth factor, bone morphogenetic protein-2 (BMP-2), onto the surface of AZ31 Mg alloys. Such functional coating has strong bonding with the substrate and can increase surface hydrophilicity of magnesium alloys. In vitro electrochemical corrosion and hydrogen evolution tests demonstrate that the coating can significantly enhance the corrosion resistance and therefore slow down the degradation of AZ31 Mg alloys. In vitro cell culture reveals that immobilization of HA nanoparticles and BMP-2 can obviously promote cell adhesion and proliferation. Furthermore, in vivo implantation tests indicate that with the synergistic effects of HA nanoparticles and BMP-2, the coating does not cause obvious inflammatory response and can significantly reduce the biodegradation rate of the magnesium alloys and induce the new bone formation adjacent to the implants. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2750-2761, 2017. © 2017 Wiley Periodicals, Inc.

  1. Contributions of protein kinases and β-arrestin to termination of protease-activated receptor 2 signaling.

    PubMed

    Jung, Seung-Ryoung; Seo, Jong Bae; Deng, Yi; Asbury, Charles L; Hille, Bertil; Koh, Duk-Su

    2016-03-01

    Activated Gq protein-coupled receptors (GqPCRs) can be desensitized by phosphorylation and β-arrestin binding. The kinetics and individual contributions of these two mechanisms to receptor desensitization have not been fully distinguished. Here, we describe the shut off of protease-activated receptor 2 (PAR2). PAR2 activates Gq and phospholipase C (PLC) to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol and inositol trisphosphate (IP3). We used fluorescent protein-tagged optical probes to monitor several consequences of PAR2 signaling, including PIP2 depletion and β-arrestin translocation in real time. During continuous activation of PAR2, PIP2 was depleted transiently and then restored within a few minutes, indicating fast receptor activation followed by desensitization. Knockdown of β-arrestin 1 and 2 using siRNA diminished the desensitization, slowing PIP2 restoration significantly and even adding a delayed secondary phase of further PIP2 depletion. These effects of β-arrestin knockdown on PIP2 recovery were prevented when serine/threonine phosphatases that dephosphorylate GPCRs were inhibited. Thus, PAR2 may continuously regain its activity via dephosphorylation when there is insufficient β-arrestin to trap phosphorylated receptors. Similarly, blockers of protein kinase C (PKC) and G protein-coupled receptor kinase potentiated the PIP2 depletion. In contrast, an activator of PKC inhibited receptor activation, presumably by augmenting phosphorylation of PAR2. Our interpretations were strengthened by modeling. Simulations supported the conclusions that phosphorylation of PAR2 by protein kinases initiates receptor desensitization and that recruited β-arrestin traps the phosphorylated state of the receptor, protecting it from phosphatases. Speculative thinking suggested a sequestration of phosphatidylinositol 4-phosphate 5 kinase (PIP5K) to the plasma membrane by β-arrestin to explain why knockdown of β-arrestin led to secondary

  2. Structure of metabotropic glutamate receptor C-terminal domains in contact with interacting proteins.

    PubMed

    Enz, Ralf

    2012-01-01

    Metabotropic glutamate receptors (mGluRs) regulate intracellular signal pathways that control several physiological tasks, including neuronal excitability, learning, and memory. This is achieved by the formation of synaptic signal complexes, in which mGluRs assemble with functionally related proteins such as enzymes, scaffolds, and cytoskeletal anchor proteins. Thus, mGluR associated proteins actively participate in the regulation of glutamatergic neurotransmission. Importantly, dysfunction of mGluRs and interacting proteins may lead to impaired signal transduction and finally result in neurological disorders, e.g., night blindness, addiction, epilepsy, schizophrenia, autism spectrum disorders and Parkinson's disease. In contrast to solved crystal structures of extracellular N-terminal domains of some mGluR types, only a few studies analyzed the conformation of intracellular receptor domains. Intracellular C-termini of most mGluR types are subject to alternative splicing and can be further modified by phosphorylation and SUMOylation. In this way, diverse interaction sites for intracellular proteins that bind to and regulate the glutamate receptors are generated. Indeed, most of the known mGluR binding partners interact with the receptors' C-terminal domains. Within the last years, different laboratories analyzed the structure of these domains and described the geometry of the contact surface between mGluR C-termini and interacting proteins. Here, I will review recent progress in the structure characterization of mGluR C-termini and provide an up-to-date summary of the geometry of these domains in contact with binding partners.

  3. MECHANISTIC PATHWAYS AND BIOLOGICAL ROLES FOR RECEPTOR-INDEPENDENT ACTIVATORS OF G-PROTEIN SIGNALING

    PubMed Central

    Blumer, Joe B.; Smrcka, Alan V.; Lanier, S.M.

    2007-01-01

    Signal processing via heterotrimeric G-proteins in response to cell surface receptors is a central and much investigated aspect of how cells integrate cellular stimuli to produce coordinated biological responses. The system is a target of numerous therapeutic agents, plays an important role in adaptive processes of organs, and aberrant processing of signals through these transducing systems is a component of various disease states. In addition to GPCR-mediated activation of G-protein signaling, nature has evolved creative ways to manipulate and utilize the Gαβγ heterotrimer or Gα and Gαβγ subunits independent of the cell surface receptor stimuli. In such situations, the G-protein subunits (Gα and Gαβγ) may actually be complexed with alternative binding partners independent of the typical heterotrimeric Gαβγ. Such regulatory accessory proteins include the family of RGS proteins that accelerate the GTPase activity of Gα and various entities that influence nucleotide binding properties and/or subunit interaction. The latter group of proteins includes receptor independent activators of G-protein signaling or AGS proteins that play surprising roles in signal processing. This review provides an overview of our current knowledge regarding AGS proteins. AGS proteins are indicative of a growing number of accessory proteins that influence signal propagation, facilitate cross talk between various types of signaling pathways and provide a platform for diverse functions of both the heterotrimeric Gαβγ and the individual Gα and Gαβγ subunits. PMID:17240454

  4. Dual Role of Jun N-Terminal Kinase Activity in Bone Morphogenetic Protein-Mediated Drosophila Ventral Head Development.

    PubMed

    Park, Sung Yeon; Stultz, Brian G; Hursh, Deborah A

    2015-12-01

    The Drosophila bone morphogenetic protein encoded by decapentaplegic (dpp) controls ventral head morphogenesis by expression in the head primordia, eye-antennal imaginal discs. These are epithelial sacs made of two layers: columnar disc proper cells and squamous cells of the peripodial epithelium. dpp expression related to head formation occurs in the peripodial epithelium; cis-regulatory mutations disrupting this expression display defects in sensory vibrissae, rostral membrane, gena, and maxillary palps. Here we document that disruption of this dpp expression causes apoptosis in peripodial cells and underlying disc proper cells. We further show that peripodial Dpp acts directly on the disc proper, indicating that Dpp must cross the disc lumen to act. We demonstrate that palp defects are mechanistically separable from the other mutant phenotypes; both are affected by the c-Jun N-terminal kinase pathway but in opposite ways. Slight reduction of both Jun N-terminal kinase and Dpp activity in peripodial cells causes stronger vibrissae, rostral membrane, and gena defects than Dpp alone; additionally, strong reduction of Jun N-terminal kinase activity alone causes identical defects. A more severe reduction of dpp results in similar vibrissae, rostral membrane, and gena defects, but also causes mutant maxillary palps. This latter defect is correlated with increased peripodial Jun N-terminal kinase activity and can be caused solely by ectopic activation of Jun N-terminal kinase. We conclude that formation of sensory vibrissae, rostral membrane, and gena tissue in head morphogenesis requires the action of Jun N-terminal kinase in peripodial cells, while excessive Jun N-terminal kinase signaling in these same cells inhibits the formation of maxillary palps. Copyright © 2015 by the Genetics Society of America.

  5. Complication Rates in Posterior Lumbar Interbody Fusion (PLIF) Surgery With Human Bone Morphogenetic Protein 2: Medicare Population.

    PubMed

    Alobaidaan, Raed; Cohen, Jeremiah R; Lord, Elizabeth L; Buser, Zorica; Yoon, S Tim; Youssef, Jim A; Park, Jong-Beom; Brodke, Darrel S; Wang, Jeffrey C; Meisel, Hans-Joerg

    2017-12-01

    Retrospective cohort study among Medicare beneficiaries who underwent posterior lumbar interbody fusion (PLIF) surgery. To identify the complication rates associated with the use of bone morphogenetic protein 2 (BMP2) in PLIF. Human BMP2 is commonly used in the "off-label" manner for various types of spine fusion procedures, including PLIF. However, recent studies have reported potential complications associated with the recombinant human BMP2 (rhBMP2) use in the posterior approach. Medicare records within the PearlDiver database were queried for patients undergoing PLIF procedure with and without rhBMP2 between 2005 and 2010. We evaluated complications within 1 year postoperatively. Chi-square was used to compare the complication rates between the 2 groups. A total of 8609 patients underwent PLIF procedure with or without rhBMP2. Individual complication rates in the rhBMP2 group ranged from 0.45% to 7.68% compared with 0.65% to 10.99 in the non-rhBMP2 group. Complication rates for cardiac, pulmonary, lumbosacral neuritis, infection, wound, and urinary tract (include acute kidney failure and post-operative complications) were significantly lower in the rhBMP2 group ( P < .05). There was no difference in the rates of central nervous system complications or radiculitis between the 2 groups. Our data showed that the patients who received rhBMP2 had lower complication rates compared to the non-rhBMP2 group. However, use of rhBMP2 was associated with a higher rate of pseudarthrosis. We did not observe any difference in radiculitis and central nervous system complications between the groups.

  6. Complication Rates in Posterior Lumbar Interbody Fusion (PLIF) Surgery With Human Bone Morphogenetic Protein 2: Medicare Population

    PubMed Central

    Alobaidaan, Raed; Cohen, Jeremiah R.; Lord, Elizabeth L.; Yoon, S. Tim; Youssef, Jim A.; Park, Jong-Beom; Brodke, Darrel S.; Wang, Jeffrey C.; Meisel, Hans-Joerg

    2017-01-01

    Study Design: Retrospective cohort study among Medicare beneficiaries who underwent posterior lumbar interbody fusion (PLIF) surgery. Objective: To identify the complication rates associated with the use of bone morphogenetic protein 2 (BMP2) in PLIF. Human BMP2 is commonly used in the “off-label” manner for various types of spine fusion procedures, including PLIF. However, recent studies have reported potential complications associated with the recombinant human BMP2 (rhBMP2) use in the posterior approach. Methods: Medicare records within the PearlDiver database were queried for patients undergoing PLIF procedure with and without rhBMP2 between 2005 and 2010. We evaluated complications within 1 year postoperatively. Chi-square was used to compare the complication rates between the 2 groups. Results: A total of 8609 patients underwent PLIF procedure with or without rhBMP2. Individual complication rates in the rhBMP2 group ranged from 0.45% to 7.68% compared with 0.65% to 10.99 in the non-rhBMP2 group. Complication rates for cardiac, pulmonary, lumbosacral neuritis, infection, wound, and urinary tract (include acute kidney failure and post-operative complications) were significantly lower in the rhBMP2 group (P < .05). There was no difference in the rates of central nervous system complications or radiculitis between the 2 groups. Conclusion: Our data showed that the patients who received rhBMP2 had lower complication rates compared to the non-rhBMP2 group. However, use of rhBMP2 was associated with a higher rate of pseudarthrosis. We did not observe any difference in radiculitis and central nervous system complications between the groups. PMID:29238641

  7. Estrogen receptor-independent catechol estrogen binding activity: protein binding studies in wild-type, Estrogen receptor-alpha KO, and aromatase KO mice tissues.

    PubMed

    Philips, Brian J; Ansell, Pete J; Newton, Leslie G; Harada, Nobuhiro; Honda, Shin-Ichiro; Ganjam, Venkataseshu K; Rottinghaus, George E; Welshons, Wade V; Lubahn, Dennis B

    2004-06-01

    Primary evidence for novel estrogen signaling pathways is based upon well-documented estrogenic responses not inhibited by estrogen receptor antagonists. In addition to 17beta-E2, the catechol estrogen 4-hydroxyestradiol (4OHE2) has been shown to elicit biological responses independent of classical estrogen receptors in estrogen receptor-alpha knockout (ERalphaKO) mice. Consequently, our research was designed to biochemically characterize the protein(s) that could be mediating the biological effects of catechol estrogens using enzymatically synthesized, radiolabeled 4-hydroxyestrone (4OHE1) and 4OHE2. Scatchard analyses identified a single class of high-affinity (K(d) approximately 1.6 nM), saturable cytosolic binding sites in several ERalphaKO estrogen-responsive tissues. Specific catechol estrogen binding was competitively inhibited by unlabeled catechol estrogens, but not by 17beta-E2 or the estrogen receptor antagonist ICI 182,780. Tissue distribution studies indicated significant binding differences both within and among various tissues in wild-type, ERalphaKO, and aromatase knockout female mice. Ligand metabolism experiments revealed extensive metabolism of labeled catechol estrogen, suggesting that catechol estrogen metabolites were responsible for the specific binding. Collectively, our data provide compelling evidence for the interaction of catechol estrogen metabolites with a novel binding protein that exhibits high affinity, specificity, and selective tissue distribution. The extensive biochemical characterization of this binding protein indicates that this protein may be a receptor, and thus may mediate ERalpha/beta-independent effects of catechol estrogens and their metabolites.

  8. Functional reconstitution of prostaglandin E receptor from bovine adrenal medulla with guanine nucleotide binding proteins

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Negishi, M.; Ito, S.; Yokohama, H.

    1988-05-15

    Prostaglandin E/sub 2/ (PEG/sub 2/) was found to bind specifically to a 100,000 x g pellet prepared from bovine adrenal medulla. The PGE receptor was associated with a GTP-binding protein (G-protein) and could be covalently cross-linked with this G-protein by dithiobis(succinimidyl propionate) in the 100,000 x g pellet. In order to characterize the G-protein associated with the PGE receptor and reconstitute these proteins in phospholipid vesicles, the authors purified the G-protein to apparent homogeneity from the 100,000 x g pellet. The G-protein served as a substrate of pertussis toxin but differed in its ..cap alpha.. subunit from two known pertussismore » toxin substrate G-proteins (G/sub i/ and G/sub 0/) purified from bovine brain. The molecular weight of the ..cap alpha.. subunit was 40,000, which is between those of G/sub i/ and G/sub 0/. The purified protein was also distinguished immunologically from G/sub i/ and G/sub 0/ and was referred to as G/sub am/. Reconstitution of the PGE receptor with pure C/sub am/, G/sub i/, or G/sub 0/ in phospholipid vesicles resulted in a remarkable restoration of (/sup 3/H)PGE/sub 2/ binding activity in a GTP-dependent manner. The efficiency of these three G-proteins in this capacity was roughly equal. When pertussis toxin- or N-ethylmaleimide-treated G-proteins, instead of the native ones, were reconstituted into vesicles, the restoration of binding activity was no longer observed. These results indicate that the PGE receptor can couple functionally with G/sub am/, G/sub i/, or G/sub 0/ in phospholipid vesicles and suggest that G/sub am/ may be involved in signal transduction of the PGE receptor in bovine adrenal medulla.« less

  9. Acetylation of pregnane X receptor protein determines selective function independent of ligand activation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Biswas, Arunima; Pasquel, Danielle; Tyagi, Rakesh Kumar

    2011-03-18

    Research highlights: {yields} Pregnane X receptor (PXR), a major regulatory protein, is modified by acetylation. {yields} PXR undergoes dynamic deacetylation upon ligand-mediated activation. {yields} SIRT1 partially mediates PXR deacetylation. {yields} PXR deacetylation per se induces lipogenesis mimicking ligand-mediated activation. -- Abstract: Pregnane X receptor (PXR), like other members of its class of nuclear receptors, undergoes post-translational modification [PTM] (e.g., phosphorylation). However, it is unknown if acetylation (a major and common form of protein PTM) is observed on PXR and, if it is, whether it is of functional consequence. PXR has recently emerged as an important regulatory protein with multiple ligand-dependentmore » functions. In the present work we show that PXR is indeed acetylated in vivo. SIRT1 (Sirtuin 1), a NAD-dependent class III histone deacetylase and a member of the sirtuin family of proteins, partially mediates deacetylation of PXR. Most importantly, the acetylation status of PXR regulates its selective function independent of ligand activation.« less

  10. G-Protein-Coupled Receptors in Adult Neurogenesis

    PubMed Central

    Doze, Van A.

    2012-01-01

    The importance of adult neurogenesis has only recently been accepted, resulting in a completely new field of investigation within stem cell biology. The regulation and functional significance of adult neurogenesis is currently an area of highly active research. G-protein-coupled receptors (GPCRs) have emerged as potential modulators of adult neurogenesis. GPCRs represent a class of proteins with significant clinical importance, because approximately 30% of all modern therapeutic treatments target these receptors. GPCRs bind to a large class of neurotransmitters and neuromodulators such as norepinephrine, dopamine, and serotonin. Besides their typical role in cellular communication, GPCRs are expressed on adult neural stem cells and their progenitors that relay specific signals to regulate the neurogenic process. This review summarizes the field of adult neurogenesis and its methods and specifies the roles of various GPCRs and their signal transduction pathways that are involved in the regulation of adult neural stem cells and their progenitors. Current evidence supporting adult neurogenesis as a model for self-repair in neuropathologic conditions, adult neural stem cell therapeutic strategies, and potential avenues for GPCR-based therapeutics are also discussed. PMID:22611178

  11. Design and Functional Characterization of a Novel, Arrestin-Biased Designer G Protein-Coupled Receptor

    PubMed Central

    Nakajima, Ken-ichiro

    2012-01-01

    Mutational modification of distinct muscarinic receptor subtypes has yielded novel designer G protein-coupled receptors (GPCRs) that are unable to bind acetylcholine (ACh), the endogenous muscarinic receptor ligand, but can be efficiently activated by clozapine-N-oxide (CNO), an otherwise pharmacologically inert compound. These CNO-sensitive designer GPCRs [alternative name: designer receptors exclusively activated by designer drug (DREADDs)] have emerged as powerful new tools to dissect the in vivo roles of distinct G protein signaling pathways in specific cell types or tissues. As is the case with other GPCRs, CNO-activated DREADDs not only couple to heterotrimeric G proteins but can also recruit proteins of the arrestin family (arrestin-2 and -3). Accumulating evidence suggests that arrestins can act as scaffolding proteins to promote signaling through G protein-independent signaling pathways. To explore the physiological relevance of these arrestin-dependent signaling pathways, the availability of an arrestin-biased DREADD would be highly desirable. In this study, we describe the development of an M3 muscarinic receptor-based DREADD [Rq(R165L)] that is no longer able to couple to G proteins but can recruit arrestins and promote extracellular signal-regulated kinase-1/2 phosphorylation in an arrestin- and CNO-dependent fashion. Moreover, CNO treatment of mouse insulinoma (MIN6) cells expressing the Rq(R165L) construct resulted in a robust, arrestin-dependent stimulation of insulin release, directly implicating arrestin signaling in the regulation of insulin secretion. This newly developed arrestin-biased DREADD represents an excellent novel tool to explore the physiological relevance of arrestin signaling pathways in distinct tissues and cell types. PMID:22821234

  12. Expression of bone morphogenetic proteins 4, 6 and 7 is downregulated in kidney allografts with interstitial fibrosis and tubular atrophy.

    PubMed

    Furic-Cunko, Vesna; Kes, Petar; Coric, Marijana; Hudolin, Tvrtko; Kastelan, Zeljko; Basic-Jukic, Nikolina

    2015-07-01

    Bone morphogenetic proteins (BMPs) are pleiotropic growth factors. This paper investigates the connection between the expression pattern of BMPs in kidney allograft tissue versus the cause of allograft dysfunction. The expression pattern of BMP2, BMP4, BMP6 and BMP7 in 50 kidney allografts obtained by transplant nephrectomy is investigated. Immunohistochemical staining is semiquantitatively evaluated for intensity to identify the expression pattern of BMPs in normal and allograft kidney tissues. The expression of BMP4 is unique between different tubular cell types in grafts without signs of fibrosis. This effect is not found in specimens with high grades of interstitial fibrosis and tubular atrophy (IFTA). In samples with IFTA grades II and III, the BMP7 expression is reduced in a significant fraction of specimens relative to those without signs of IFTA. The expression pattern of BMP6 indicates that its activation may be triggered by the act of transplantation and subsequent reperfusion injury. The expression of BMP2 is strong in all types of tubular epithelial cells and does not differ between the compared allografts and control kidney specimens. The intensity and expression pattern of BMP4, BMP6 and BMP7 in transplanted kidney tissue are found to be dependent upon the length of the transplanted period, the clinical indication for transplant nephrectomy and signs of IFTA in kidney tissue.

  13. Effect of nano-hydroxyapatite on bone morphogenetic protein-2-induced hard tissue formation and dentin resorption on a dentin surface

    NASA Astrophysics Data System (ADS)

    Tamagawa, Hiroki; Tenkumo, Taichi; Sugaya, Tsutomu; Kawanami, Masamitsu

    2012-12-01

    AimThe purpose of this study was to evaluate the effects of the addition of nano-hydroxyapatite to a collagen membrane-carrier of recombinant human bone morphogenetic protein-2 (rhBMP-2) on hard tissue formation and dentin resorption on dentin surfaces in vivo. Materials and methodsNano-hydroxyapatite collagen composite (nHAC) membranes or collagen (C) membranes were each immersed in either 100 or 400 μg/ml rhBMP-2 and placed on dentin chips that were implanted into rat thigh muscle. The implants were analyzed at 2 or 4 weeks after surgery by histological observation and histomorphometric analysis. ResultsThe percentage of the hard tissue formed by each nHAC group was significantly higher than that formed by any of the C groups, except for that formed by the group loaded with 400 μg/ml rhBMP-2 at 4 weeks after implantation. No significant differences were observed in the percentage of dentin resorption between the nHAC groups and C groups at any stage or at any rhBMP-2 concentration. ConclusionThese findings showed that addition of nano-hydroxyapatite to a collagen membrane accelerated the formation of hard tissue induced by a low dose of rhBMP-2 on dentin surfaces at an early stage after implantation into rat thigh muscle, without increasing dentin resorption.

  14. Bone Morphogenetic Protein-2 Promotes Human Mesenchymal Stem Cell Survival and Resultant Bone Formation When Entrapped in Photocrosslinked Alginate Hydrogels.

    PubMed

    Ho, Steve S; Vollmer, Nina L; Refaat, Motasem I; Jeon, Oju; Alsberg, Eben; Lee, Mark A; Leach, J Kent

    2016-10-01

    There is a substantial need to prolong cell persistence and enhance functionality in situ to enhance cell-based tissue repair. Bone morphogenetic protein-2 (BMP-2) is often used at high concentrations for osteogenic differentiation of mesenchymal stem cells (MSCs) but can induce apoptosis. Biomaterials facilitate the delivery of lower doses of BMP-2, reducing side effects and localizing materials at target sites. Photocrosslinked alginate hydrogels (PAHs) can deliver osteogenic materials to irregular-sized bone defects, providing improved control over material degradation compared to ionically cross-linked hydrogels. It is hypothesized that the delivery of MSCs and BMP-2 from a PAH increases cell persistence by reducing apoptosis, while promoting osteogenic differentiation and enhancing bone formation compared to MSCs in PAHs without BMP-2. BMP-2 significantly decreases apoptosis and enhances survival of photoencapsulated MSCs, while simultaneously promoting osteogenic differentiation in vitro. Bioluminescence imaging reveals increased MSC survival when implanted in BMP-2 PAHs. Bone defects treated with MSCs in BMP-2 PAHs demonstrate 100% union as early as 8 weeks and significantly higher bone volumes at 12 weeks, while defects with MSC-entrapped PAHs alone do not fully bridge. This study demonstrates that transplantation of MSCs with BMP-2 in PAHs achieves robust bone healing, providing a promising platform for bone repair. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. The morphogenetically active polymer, inorganic polyphosphate complexed with GdCl3, as an inducer of hydroxyapatite formation in vitro.

    PubMed

    Wang, Xiaohong; Huang, Jian; Wang, Kui; Neufurth, Meik; Schröder, Heinz C; Wang, Shunfeng; Müller, Werner E G

    2016-02-15

    Inorganic polyphosphate (polyP) is a physiological polymer composed of tens to hundreds of phosphate units linked together via phosphoanhydride bonds. Here we compared the biological activity of polyP (chain length of 40 phosphate units), complexed with Gd(3+) (polyP·Gd), with the one caused by polyP (as calcium salt) and by GdCl3 alone, regarding their potencies to induce hydroxyapatite (HA) formation in SaOS-2 cells in vitro. The three compounds, GdCl3, polyP and polyP·Gd were found to be non-toxic at concentrations up to at least 30μM. Selecting a low, 5μM, concentration it was found that polyP·Gd significantly induced HA formation, as determined by Alizarin Red S staining and by quantitative determinations using that dye. Under those conditions polyP·Gd and to a smaller extent also polyP or GdCl3 (5μM each) caused HA crystal formation arranged in a nest-like pattern. Exposure of cells to polyP·Gd resulted in a strong increase in alkaline phosphatase activity; this enzyme did not cause a distinct degradation of polyP but of polyP·Gd which was extensively hydrolyzed. The morphogenetic activity of gadolinium, in the form of polyP·Gd, is underscored by the finding that this polymer causes a strong upregulation of the genes encoding morphogenetic protein-2 (BMP2) as well as collagen type I. It is concluded that polyP·Gd is not an inert polymer but acts as a morphogenetically active polymer and induces HA formation in vitro. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Complications Related to the Recombinant Human Bone Morphogenetic Protein 2 Use in Posterior Cervical Fusion.

    PubMed

    Takahashi, Shinji; Buser, Zorica; Cohen, Jeremiah R; Roe, Allison; Myhre, Sue L; Meisel, Hans-Joerg; Brodke, Darrel S; Yoon, S Tim; Park, Jong-Beom; Wang, Jeffrey C; Youssef, Jim A

    2017-11-01

    A retrospective cohort study. To compare the complications between posterior cervical fusions with and without recombinant human bone morphogenetic protein 2 (rhBMP2). Use of rhBMP2 in anterior cervical spinal fusion procedures can lead to potential complications such as neck edema, resulting in airway complications or neurological compression. However, there are no data on the complications associated with the "off-label" use of rhBMP2 in upper and lower posterior cervical fusion approaches. Patients from the PearlDiver database who had a posterior cervical fusion between 2005 and 2011 were identified. We evaluated complications within 90 days after fusion and data was divided in 2 groups: (1) posterior cervical fusion including upper cervical spine O-C2 (upper group) and (2) posterior cervical fusion including lower cervical spine C3-C7 (lower group). Complications were divided into: any complication, neck-related complications, wound-related complications, and other complications. Of the 352 patients in the upper group, 73 patients (20.7%) received rhBMP2, and 279 patients (79.3%) did not. Likewise, in the lower group of 2372 patients, 378 patients (15.9%) had surgery with rhBMP2 and 1994 patients (84.1%) without. In the upper group, complications were observed in 7 patients (9.6%) with and 34 patients (12%) without rhBMP2. In the lower group, complications were observed in 42 patients (11%) with and 276 patients (14%) without rhBMP2. Furthermore, in the lower group the wound-related complications were significantly higher in the rhBMP2 group (23 patients, 6.1%) compared with the non-rhBMP2 group (75 patients, 3.8%). Our data showed that the use of rhBMP2 does not increase the risk of complications in upper cervical spine fusion procedures. However, in the lower cervical spine, rhBMP2 may elevate the risk of wound-related complications. Overall, there were no major complications associated with the use of rhBMP2 for posterior cervical fusion approaches. Level

  17. Synergistic roles of bone morphogenetic protein 15 and growth differentiation factor 9 in ovarian function.

    PubMed

    Yan, C; Wang, P; DeMayo, J; DeMayo, F J; Elvin, J A; Carino, C; Prasad, S V; Skinner, S S; Dunbar, B S; Dube, J L; Celeste, A J; Matzuk, M M

    2001-06-01

    Knockout mouse technology has been used over the last decade to define the essential roles of ovarian-expressed genes and uncover genetic interactions. In particular, we have used this technology to study the function of multiple members of the transforming growth factor-beta superfamily including inhibins, activins, and growth differentiation factor 9 (GDF-9 or Gdf9). Knockout mice lacking GDF-9 are infertile due to a block in folliculogenesis at the primary follicle stage. In addition, recombinant GDF-9 regulates multiple cumulus granulosa cell functions in the periovulatory period including hyaluronic acid synthesis and cumulus expansion. We have also cloned an oocyte-specific homolog of GDF-9 from mice and humans, which is termed bone morphogenetic protein 15 (BMP-15 or Bmp15). To define the function of BMP-15 in mice, we generated embryonic stem cells and knockout mice, which have a null mutation in this X-linked gene. Male chimeric and Bmp15 null mice are normal and fertile. In contrast to Bmp15 null males and Gdf9 knockout females, Bmp15 null females (Bmp15(-/-)) are subfertile and usually have minimal ovarian histopathological defects, but demonstrate decreased ovulation and fertilization rates. To further decipher possible direct or indirect genetic interactions between GDF-9 and BMP-15, we have generated double mutant mice lacking one or both alleles of these related homologs. Double homozygote females (Bmp15(-/-)Gdf9(-/-)) display oocyte loss and cysts and resemble Gdf9(-/-) mutants. In contrast, Bmp15(-/-)Gdf9(+/-) female mice have more severe fertility defects than Bmp15(-/-) females, which appear to be due to abnormalities in ovarian folliculogenesis, cumulus cell physiology, and fertilization. Thus, the dosage of intact Bmp15 and Gdf9 alleles directly influences the destiny of the oocyte during folliculogenesis and in the periovulatory period. These studies have important implications for human fertility control and the maintenance of fertility and

  18. Dose reduction of bone morphogenetic protein-2 for bone regeneration using a delivery system based on lyophilization with trehalose.

    PubMed

    Zhang, Xiaochen; Yu, Quan; Wang, Yan-An; Zhao, Jun

    2018-01-01

    To induce sufficient new bone formation, high doses of bone morphogenetic protein-2 (BMP-2) are applied in regenerative medicine that often induce serious side effects. Therefore, improved treatment strategies are required. Here, we investigate whether the delivery of BMP-2 lyophilized in the presence of trehalose reduced the dose of BMP-2 required for bone regeneration. A new growth factor delivery system was fabricated using BMP-2-loaded TiO 2 nanotubes by lyophilization with trehalose (TiO 2 -Lyo-Tre-BMP-2). We measured BMP-2 release characteristics, bioactivity, and stability, and determined the effects on the osteogenic differentiation of bone marrow stromal cells in vitro. Additionally, we evaluated the ability of this formulation to regenerate new bone around implants in rat femur defects by micro-computed tomography (micro-CT), sequential fluorescent labelling, and histological analysis. Compared with absorbed BMP-2-loaded TiO 2 nanotubes (TiO 2 -BMP-2), TiO 2 -Lyo-Tre-BMP-2 exhibited sustained release, consistent bioactivity, and higher stability of BMP-2, and resulted in greater osteogenic differentiation of BMSCs. Eight weeks post-operation, TiO 2 -Lyo-Tre-BMP-2 nanotubes, with various dosages of BMP-2, regenerated larger amounts of new bone than TiO 2 -BMP-2 nanotubes. Our findings indicate that delivery of BMP-2 lyophilized with trehalose may be a promising method to reduce the dose of BMP-2 and avoid the associated side effects.

  19. Biologically synthesized titanium oxide nanostructures combined with morphogenetic protein as wound healing agent in the femoral fracture after surgery.

    PubMed

    Zhang, Yushu; Zhang, Chuanlian; Liu, Kemiao; Zhu, Xia; Liu, Fang; Ge, Xiaofen

    2018-05-01

    The aim of the present study is to develop novel approach for the green synthesis of titanium oxide nanoparticles (TiO 2 NPs) using Eichhornia crassipes extract and calcined at different temperatures for evaluate the wound healing activity in the femoral fracture. The synthesized TiO 2 are formed different (plate and rod-like) nanostructures at various calcination temperatures. These samples were characterized by X-ray diffraction (XRD), Fourier transform-infrared spectroscopy (FTIR), Field emission scanning electron microscope (FE-SEM) and transmission electron microscope (TEM). Microscopic studies of TiO 2 NPs revealed that the synthesized TiO 2 NPs are formed well-defined rod-like structures at 400 °C with size ranged from 200 nm to 500 nm. The characterized plate and rod-like TiO 2 NPs are combined with human morphogenetic protein (HbMP) to improving its wound healing activity and osteoblast properties on femoral fractures. The biocompatibility was tested by using human bone marrow mesenchymal stem cells (BMSC) cells and antibacterial efficacy analyzed using human pathogenica bacteria Staphylococcus aureus and Escherichia coli through agar well diffusion assay. The green synthesized rod-like TiO 2 NPs combined with HbMP has been exhibited effective bone fusion behaviors with biomechanical properties and also improved antibacterial activity against pathogenic bacteria. From this study results, it is suggested that green synthesized TiO 2 NPs could be used effectively in biomedical application. Copyright © 2018. Published by Elsevier B.V.

  20. Exploring the stochastic dynamics of correlated movement of receptor proteins in plasma membranes in vivo

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Huang, Jung Y., E-mail: jyhuang@faculty.nctu.edu.tw; Lin, Chien Y.

    Ligand-induced receptor dimerization plays a crucial role in the signaling process of living cells. In this study, we developed a theoretical model and performed single-molecule tracking to explore the correlated diffusion processes of liganded epidermal growth factor receptors prior to dimer formation. We disclosed that both an attractive potential between liganded receptor proteins in proximity and correlated fluctuations in the local environments of the proteins play an important role to produce the observed correlated movement of the receptors. This result can serve as the foundation to shed light on the way in which receptor functions are regulated in plasma membranesmore » in vivo.« less

  1. Quantitative measurement of cell membrane receptor internalization by the nanoluciferase reporter: Using the G protein-coupled receptor RXFP3 as a model.

    PubMed

    Liu, Yu; Song, Ge; Shao, Xiao-Xia; Liu, Ya-Li; Guo, Zhan-Yun

    2015-02-01

    Nanoluciferase (NanoLuc) is a newly developed small luciferase reporter with the brightest bioluminescence to date. In the present work, we developed NanoLuc as a sensitive bioluminescent reporter to measure quantitatively the internalization of cell membrane receptors, based on the pH dependence of the reporter activity. The G protein-coupled receptor RXFP3, the cognate receptor of relaxin-3/INSL7, was used as a model receptor. We first generated stable HEK293T cells that inducibly coexpressed a C-terminally NanoLuc-tagged human RXFP3 and a C-terminally enhanced green fluorescent protein (EGFP)-tagged human RXFP3. The C-terminal EGFP-tag and NanoLuc-tag had no detrimental effects on the ligand-binding potency and intracellular trafficking of RXFP3. Based on the fluorescence of the tagged EGFP reporter, the ligand-induced RXFP3 internalization was visualized directly under a fluorescence microscope. Based on the bioluminescence of the tagged NanoLuc reporter, the ligand-induced RXFP3 internalization was measured quantitatively by a convenient bioluminescent assay. Coexpression of an EGFP-tagged inactive [E141R]RXFP3 had no detrimental effect on the ligand-binding potency and ligand-induced internalization of the NanoLuc-tagged wild-type RXFP3, suggesting that the mutant RXFP3 and wild-type RXFP3 worked independently. The present bioluminescent internalization assay could be extended to other G protein-coupled receptors and other cell membrane receptors to study ligand-receptor and receptor-receptor interactions. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Using Chou's general PseAAC to analyze the evolutionary relationship of receptor associated proteins (RAP) with various folding patterns of protein domains.

    PubMed

    Muthu Krishnan, S

    2018-05-14

    The receptor-associated protein (RAP) is an inhibitor of endocytic receptors that belong to the lipoprotein receptor gene family. In this study, a computational approach was tried to find the evolutionarily related fold of the RAP proteins. Through the structural and sequence-based analysis, found various protein folds that are very close to the RAP folds. Remote homolog datasets were used potentially to develop a different support vector machine (SVM) methods to recognize the homologous RAP fold. This study helps in understanding the relationship of RAP homologs folds based on the structure, function and evolutionary history. Copyright © 2018 Elsevier Ltd. All rights reserved.

  3. Identification of Phosphorylation Codes for Arrestin Recruitment by G Protein-Coupled Receptors.

    PubMed

    Zhou, X Edward; He, Yuanzheng; de Waal, Parker W; Gao, Xiang; Kang, Yanyong; Van Eps, Ned; Yin, Yanting; Pal, Kuntal; Goswami, Devrishi; White, Thomas A; Barty, Anton; Latorraca, Naomi R; Chapman, Henry N; Hubbell, Wayne L; Dror, Ron O; Stevens, Raymond C; Cherezov, Vadim; Gurevich, Vsevolod V; Griffin, Patrick R; Ernst, Oliver P; Melcher, Karsten; Xu, H Eric

    2017-07-27

    G protein-coupled receptors (GPCRs) mediate diverse signaling in part through interaction with arrestins, whose binding promotes receptor internalization and signaling through G protein-independent pathways. High-affinity arrestin binding requires receptor phosphorylation, often at the receptor's C-terminal tail. Here, we report an X-ray free electron laser (XFEL) crystal structure of the rhodopsin-arrestin complex, in which the phosphorylated C terminus of rhodopsin forms an extended intermolecular β sheet with the N-terminal β strands of arrestin. Phosphorylation was detected at rhodopsin C-terminal tail residues T336 and S338. These two phospho-residues, together with E341, form an extensive network of electrostatic interactions with three positively charged pockets in arrestin in a mode that resembles binding of the phosphorylated vasopressin-2 receptor tail to β-arrestin-1. Based on these observations, we derived and validated a set of phosphorylation codes that serve as a common mechanism for phosphorylation-dependent recruitment of arrestins by GPCRs. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Modeling G Protein-Coupled Receptors: a Concrete Possibility

    PubMed Central

    Costanzi, Stefano

    2010-01-01

    G protein-coupled receptors (GPCRs) are a large superfamily of membrane bound signaling proteins that are involved in the regulation of a wide range of physiological functions and constitute the most common target for therapeutic intervention. Due to the paucity of crystal structures, homology modeling has become a widespread technique for the construction of GPCR models, which have been applied to the study of their structure-function relationships and to the identification of lead ligands through virtual screening. Rhodopsin has been for years the only available template. However, recent breakthroughs in GPCR crystallography have led to the solution of the structures of a few additional receptors. In light of these newly elucidated crystal structures, we have been able to produce a substantial amount of data to demonstrate that accurate models of GPCRs in complex with their ligands can be constructed through homology modeling followed by fully flexible molecular docking. These results have been confirmed by our success in the first blind assessment of GPCR modeling and docking, organized in coordination with the solution of the X-ray structure of the adenosine A2A receptor. Taken together, these data indicate that: a) the transmembrane helical bundle can be modeled with considerable accuracy; b) predicting the binding mode of a ligand, although doable, is challenging; c) modeling of the extracellular and intracellular loops is still problematic. PMID:21253444

  5. Complications of anterior cervical discectomy and fusion using recombinant human bone morphogenetic protein-2

    PubMed Central

    Carp, Julia; Sethi, Anil; Bartol, Stephen; Craig, Joseph; Les, Clifford M.

    2007-01-01

    The use of bone morphogenetic protein-2 (rhBMP-2) in spinal fusion has increased dramatically since an FDA approval for its use in anterior lumbar fusion with the LT cage. There are several reports of its use in transforaminal lumbar interbody fusion, posterolateral fusion, and anterior cervical fusion. Reports on adverse effects of rhBMP-2 when used in spinal fusion are scarce in literature. An Institutional Review Board approved retrospective study was conducted in patients undergoing anterior spinal fusion and instrumentation following diskectomy at a single center. Forty-six consecutive patients were included. Twenty-two patients treated with rhBMP-2 and PEEK cages were compared to 24 in whom allograft spacers and demineralized bone matrix was used. Patients filled out Cervical Oswestry Scores, VAS for arm pain, neck pain, and had radiographs preoperatively as well at every follow up visit. Radiographic examination following surgery revealed end plate resorption in all patients in whom rhBMP-2 was used. This was followed by a period of new bone formation commencing at 6 weeks. In contrast, allograft patients showed a progressive blurring of end plate-allograft junction. Dysphagia was a common complication and it was significantly more frequent and more severe in patients in whom rhBMP-2 was used. Post operative swelling anterior to the vertebral body on lateral cervical spine X-ray was significantly larger in the rhBMP-2 group when measured from 1 to 6 weeks after which it was similar. These effects are possibly due to an early inflammatory response to rhBMP-2 and were observed to be dose related. With the parameters we used, there was no significant difference in the clinical outcome of patients in the two groups at 2 years. The cost of implants in patients treated with rhBMP-2 and PEEK spacers was more than three times the cost of allograft spacers and demineralized bone matrix in 1, 2, and 3-level cases. Despite providing consistently good fusion rates

  6. Pharmacoperone drugs: targeting misfolded proteins causing lysosomal storage-, ion channels-, and G protein-coupled receptors-associated conformational disorders.

    PubMed

    Hou, Zhi-Shuai; Ulloa-Aguirre, Alfredo; Tao, Ya-Xiong

    2018-06-01

    Conformational diseases are caused by structurally abnormal proteins that cannot fold properly and achieve their native conformation. Misfolded proteins frequently originate from genetic mutations that may lead to loss-of-function diseases involving a variety of structurally diverse proteins including enzymes, ion channels, and membrane receptors. Pharmacoperones are small molecules that cross the cell surface plasma membrane and reach their target proteins within the cell, serving as molecular scaffolds to stabilize the native conformation of misfolded or well-folded but destabilized proteins, to prevent their degradation and promote correct trafficking to their functional site of action. Because of their high specificity toward the target protein, pharmacoperones are currently the focus of intense investigation as therapy for several conformational diseases. Areas covered: This review summarizes data on the mechanisms leading to protein misfolding and the use of pharmacoperone drugs as an experimental approach to rescue function of distinct misfolded/misrouted proteins associated with a variety of diseases, such as lysosomal storage diseases, channelopathies, and G protein-coupled receptor misfolding diseases. Expert commentary: The fact that many misfolded proteins may retain function, offers a unique therapeutic opportunity to cure disease by directly correcting misrouting through administering pharmacoperone drugs thereby rescuing function of disease-causing, conformationally abnormal proteins.

  7. Large-scale production and study of a synthetic G protein-coupled receptor: Human olfactory receptor 17-4

    PubMed Central

    Cook, Brian L.; Steuerwald, Dirk; Kaiser, Liselotte; Graveland-Bikker, Johanna; Vanberghem, Melanie; Berke, Allison P.; Herlihy, Kara; Pick, Horst; Vogel, Horst; Zhang, Shuguang

    2009-01-01

    Although understanding of the olfactory system has progressed at the level of downstream receptor signaling and the wiring of olfactory neurons, the system remains poorly understood at the molecular level of the receptors and their interaction with and recognition of odorant ligands. The structure and functional mechanisms of these receptors still remain a tantalizing enigma, because numerous previous attempts at the large-scale production of functional olfactory receptors (ORs) have not been successful to date. To investigate the elusive biochemistry and molecular mechanisms of olfaction, we have developed a mammalian expression system for the large-scale production and purification of a functional OR protein in milligram quantities. Here, we report the study of human OR17-4 (hOR17-4) purified from a HEK293S tetracycline-inducible system. Scale-up of production yield was achieved through suspension culture in a bioreactor, which enabled the preparation of >10 mg of monomeric hOR17-4 receptor after immunoaffinity and size exclusion chromatography, with expression yields reaching 3 mg/L of culture medium. Several key post-translational modifications were identified using MS, and CD spectroscopy showed the receptor to be ≈50% α-helix, similar to other recently determined G protein-coupled receptor structures. Detergent-solubilized hOR17-4 specifically bound its known activating odorants lilial and floralozone in vitro, as measured by surface plasmon resonance. The hOR17-4 also recognized specific odorants in heterologous cells as determined by calcium ion mobilization. Our system is feasible for the production of large quantities of OR necessary for structural and functional analyses and research into OR biosensor devices. PMID:19581598

  8. Large-scale production and study of a synthetic G protein-coupled receptor: human olfactory receptor 17-4.

    PubMed

    Cook, Brian L; Steuerwald, Dirk; Kaiser, Liselotte; Graveland-Bikker, Johanna; Vanberghem, Melanie; Berke, Allison P; Herlihy, Kara; Pick, Horst; Vogel, Horst; Zhang, Shuguang

    2009-07-21

    Although understanding of the olfactory system has progressed at the level of downstream receptor signaling and the wiring of olfactory neurons, the system remains poorly understood at the molecular level of the receptors and their interaction with and recognition of odorant ligands. The structure and functional mechanisms of these receptors still remain a tantalizing enigma, because numerous previous attempts at the large-scale production of functional olfactory receptors (ORs) have not been successful to date. To investigate the elusive biochemistry and molecular mechanisms of olfaction, we have developed a mammalian expression system for the large-scale production and purification of a functional OR protein in milligram quantities. Here, we report the study of human OR17-4 (hOR17-4) purified from a HEK293S tetracycline-inducible system. Scale-up of production yield was achieved through suspension culture in a bioreactor, which enabled the preparation of >10 mg of monomeric hOR17-4 receptor after immunoaffinity and size exclusion chromatography, with expression yields reaching 3 mg/L of culture medium. Several key post-translational modifications were identified using MS, and CD spectroscopy showed the receptor to be approximately 50% alpha-helix, similar to other recently determined G protein-coupled receptor structures. Detergent-solubilized hOR17-4 specifically bound its known activating odorants lilial and floralozone in vitro, as measured by surface plasmon resonance. The hOR17-4 also recognized specific odorants in heterologous cells as determined by calcium ion mobilization. Our system is feasible for the production of large quantities of OR necessary for structural and functional analyses and research into OR biosensor devices.

  9. Receptor-like kinase SOBIR1/EVR interacts with receptor-like proteins in plant immunity against fungal infection.

    PubMed

    Liebrand, Thomas W H; van den Berg, Grardy C M; Zhang, Zhao; Smit, Patrick; Cordewener, Jan H G; America, Antoine H P; America, Antione H P; Sklenar, Jan; Jones, Alexandra M E; Tameling, Wladimir I L; Robatzek, Silke; Thomma, Bart P H J; Joosten, Matthieu H A J

    2013-06-11

    The plant immune system is activated by microbial patterns that are detected as nonself molecules. Such patterns are recognized by immune receptors that are cytoplasmic or localized at the plasma membrane. Cell surface receptors are represented by receptor-like kinases (RLKs) that frequently contain extracellular leucine-rich repeats and an intracellular kinase domain for activation of downstream signaling, as well as receptor-like proteins (RLPs) that lack this signaling domain. It is therefore hypothesized that RLKs are required for RLPs to activate downstream signaling. The RLPs Cf-4 and Ve1 of tomato (Solanum lycopersicum) mediate resistance to the fungal pathogens Cladosporium fulvum and Verticillium dahliae, respectively. Despite their importance, the mechanism by which these immune receptors mediate downstream signaling upon recognition of their matching ligand, Avr4 and Ave1, remained enigmatic. Here we show that the tomato ortholog of the Arabidopsis thaliana RLK Suppressor Of BIR1-1/Evershed (SOBIR1/EVR) and its close homolog S. lycopersicum (Sl)SOBIR1-like interact in planta with both Cf-4 and Ve1 and are required for the Cf-4- and Ve1-mediated hypersensitive response and immunity. Tomato SOBIR1/EVR interacts with most of the tested RLPs, but not with the RLKs FLS2, SERK1, SERK3a, BAK1, and CLV1. SOBIR1/EVR is required for stability of the Cf-4 and Ve1 receptors, supporting our observation that these RLPs are present in a complex with SOBIR1/EVR in planta. We show that SOBIR1/EVR is essential for RLP-mediated immunity and propose that the protein functions as a regulatory RLK of this type of cell-surface receptors.

  10. 0610009K11Rik, a testis-specific and germ cell nuclear receptor-interacting protein

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhang Heng; Denhard, Leslie A.; Zhou Huaxin

    Using an in silico approach, a putative nuclear receptor-interacting protein 0610009K11Rik was identified in mouse testis. We named this gene testis-specific nuclear receptor-interacting protein-1 (Tnrip-1). Tnrip-1 was predominantly expressed in the testis of adult mouse tissues. Expression of Tnrip-1 in the testis was regulated during postnatal development, with robust expression in 14-day-old or older testes. In situ hybridization analyses showed that Tnrip-1 is highly expressed in pachytene spermatocytes and spermatids. Consistent with its mRNA expression, Tnrip-1 protein was detected in adult mouse testes. Immunohistochemical studies showed that Tnrip-1 is a nuclear protein and mainly expressed in pachytene spermatocytes and roundmore » spermatids. Moreover, co-immunoprecipitation analyses showed that endogenous Tnrip-1 protein can interact with germ cell nuclear receptor (GCNF) in adult mouse testes. Our results suggest that Tnrip-1 is a testis-specific and GCNF-interacting protein which may be involved in the modulation of GCNF-mediated gene transcription in spermatogenic cells within the testis.« less

  11. LDL receptor-related protein 1 regulates the abundance of diverse cell-signaling proteins in the plasma membrane proteome.

    PubMed

    Gaultier, Alban; Simon, Gabriel; Niessen, Sherry; Dix, Melissa; Takimoto, Shinako; Cravatt, Benjamin F; Gonias, Steven L

    2010-12-03

    LDL receptor-related protein 1 (LRP1) is an endocytic receptor, reported to regulate the abundance of other receptors in the plasma membrane, including uPAR and tissue factor. The goal of this study was to identify novel plasma membrane proteins, involved in cell-signaling, that are regulated by LRP1. Membrane protein ectodomains were prepared from RAW 264.7 cells in which LRP1 was silenced and control cells using protease K. Peptides were identified by LC-MS/MS. By analysis of spectral counts, 31 transmembrane and secreted proteins were regulated in abundance at least 2-fold when LRP1 was silenced. Validation studies confirmed that semaphorin4D (Sema4D), plexin domain-containing protein-1 (Plxdc1), and neuropilin-1 were more abundant in the membranes of LRP1 gene-silenced cells. Regulation of Plxdc1 by LRP1 was confirmed in CHO cells, as a second model system. Plxdc1 coimmunoprecipitated with LRP1 from extracts of RAW 264.7 cells and mouse liver. Although Sema4D did not coimmunoprecipitate with LRP1, the cell-surface level of Sema4D was increased by RAP, which binds to LRP1 and inhibits binding of other ligands. These studies identify Plxdc1, Sema4D, and neuropilin-1 as novel LRP1-regulated cell-signaling proteins. Overall, LRP1 emerges as a generalized regulator of the plasma membrane proteome.

  12. The protein arginine methyltransferase PRMT5 promotes D2-like dopamine receptor signaling

    PubMed Central

    Likhite, Neah; Jackson, Christopher A.; Liang, Mao-Shih; Krzyzanowski, Michelle C.; Lei, Pedro; Wood, Jordan F.; Birkaya, Barbara; Michaels, Kerry L.; Andreadis, Stelios T.; Clark, Stewart D.; Yu, Michael C.; Ferkey, Denise M.

    2017-01-01

    Protein arginine methylation regulates diverse functions of eukaryotic cells, including gene expression, the DNA damage response, and circadian rhythms. We showed that arginine residues within the third intracellular loop of the human D2 dopamine receptor, which are conserved in the DOP-3 receptor in the nematode Caenorhabditis elegans, were methylated by protein arginine methyl-transferase 5 (PRMT5). By mutating these arginine residues, we further showed that their methylation enhanced the D2 receptor–mediated inhibition of cyclic adenosine monophosphate (cAMP) signaling in cultured human embryonic kidney (HEK) 293T cells. Analysis of prmt-5–deficient worms indicated that methylation promoted the dopamine-mediated modulation of chemosensory and locomotory behaviors in C. elegans through the DOP-3 receptor. In addition to delineating a previously uncharacterized means of regulating GPCR (heterotrimeric guanine nucleotide–binding protein–coupled receptor) signaling, these findings may lead to the development of a new class of pharmacological therapies that modulate GPCR signaling by changing the methylation status of these key proteins. PMID:26554819

  13. Hydrophobic cluster analysis of G protein-coupled receptors: a powerful tool to derive structural and functional information from 2D-representation of protein sequences.

    PubMed

    Lentes, K U; Mathieu, E; Bischoff, R; Rasmussen, U B; Pavirani, A

    1993-01-01

    Current methods for comparative analyses of protein sequences are 1D-alignments of amino acid sequences based on the maximization of amino acid identity (homology) and the prediction of secondary structure elements. This method has a major drawback once the amino acid identity drops below 20-25%, since maximization of a homology score does not take into account any structural information. A new technique called Hydrophobic Cluster Analysis (HCA) has been developed by Lemesle-Varloot et al. (Biochimie 72, 555-574), 1990). This consists of comparing several sequences simultaneously and combining homology detection with secondary structure analysis. HCA is primarily based on the detection and comparison of structural segments constituting the hydrophobic core of globular protein domains, with or without transmembrane domains. We have applied HCA to the analysis of different families of G-protein coupled receptors, such as catecholamine receptors as well as peptide hormone receptors. Utilizing HCA the thrombin receptor, a new and as yet unique member of the family of G-protein coupled receptors, can be clearly classified as being closely related to the family of neuropeptide receptors rather than to the catecholamine receptors for which the shape of the hydrophobic clusters and the length of their third cytoplasmic loop are very different. Furthermore, the potential of HCA to predict relationships between new putative and already characterized members of this family of receptors will be presented.

  14. Cellular and molecular biology of orphan G protein-coupled receptors.

    PubMed

    Oh, Da Young; Kim, Kyungjin; Kwon, Hyuk Bang; Seong, Jae Young

    2006-01-01

    The superfamily of G protein-coupled receptors (GPCRs) is the largest and most diverse group of membrane-spanning proteins. It plays a variety of roles in pathophysiological processes by transmitting extracellular signals to cells via heterotrimeric G proteins. Completion of the human genome project revealed the presence of approximately 168 genes encoding established nonsensory GPCRs, as well as 207 genes predicted to encode novel GPCRs for which the natural ligands remained to be identified, the so-called orphan GPCRs. Eighty-six of these orphans have now been paired to novel or previously known molecules, and 121 remain to be deorphaned. A better understanding of the GPCR structures and classification; knowledge of the receptor activation mechanism, either dependent on or independent of an agonist; increased understanding of the control of GPCR-mediated signal transduction; and development of appropriate ligand screening systems may improve the probability of discovering novel ligands for the remaining orphan GPCRs.

  15. The Host Cell Receptors for Measles Virus and Their Interaction with the Viral Hemagglutinin (H) Protein

    PubMed Central

    Lin, Liang-Tzung; Richardson, Christopher D.

    2016-01-01

    The hemagglutinin (H) protein of measles virus (MeV) interacts with a cellular receptor which constitutes the initial stage of infection. Binding of H to this host cell receptor subsequently triggers the F protein to activate fusion between virus and host plasma membranes. The search for MeV receptors began with vaccine/laboratory virus strains and evolved to more relevant receptors used by wild-type MeV. Vaccine or laboratory strains of measles virus have been adapted to grow in common cell lines such as Vero and HeLa cells, and were found to use membrane cofactor protein (CD46) as a receptor. CD46 is a regulator that normally prevents cells from complement-mediated self-destruction, and is found on the surface of all human cells, with the exception of erythrocytes. Mutations in the H protein, which occur during adaptation and allow the virus to use CD46 as a receptor, have been identified. Wild-type isolates of measles virus cannot use the CD46 receptor. However, both vaccine/laboratory and wild-type strains can use an immune cell receptor called signaling lymphocyte activation molecule family member 1 (SLAMF1; also called CD150) and a recently discovered epithelial receptor known as Nectin-4. SLAMF1 is found on activated B, T, dendritic, and monocyte cells, and is the initial target for infections by measles virus. Nectin-4 is an adherens junction protein found at the basal surfaces of many polarized epithelial cells, including those of the airways. It is also over-expressed on the apical and basal surfaces of many adenocarcinomas, and is a cancer marker for metastasis and tumor survival. Nectin-4 is a secondary exit receptor which allows measles virus to replicate and amplify in the airways, where the virus is expelled from the body in aerosol droplets. The amino acid residues of H protein that are involved in binding to each of the receptors have been identified through X-ray crystallography and site-specific mutagenesis. Recombinant measles “blind” to

  16. The Host Cell Receptors for Measles Virus and Their Interaction with the Viral Hemagglutinin (H) Protein.

    PubMed

    Lin, Liang-Tzung; Richardson, Christopher D

    2016-09-20

    The hemagglutinin (H) protein of measles virus (MeV) interacts with a cellular receptor which constitutes the initial stage of infection. Binding of H to this host cell receptor subsequently triggers the F protein to activate fusion between virus and host plasma membranes. The search for MeV receptors began with vaccine/laboratory virus strains and evolved to more relevant receptors used by wild-type MeV. Vaccine or laboratory strains of measles virus have been adapted to grow in common cell lines such as Vero and HeLa cells, and were found to use membrane cofactor protein (CD46) as a receptor. CD46 is a regulator that normally prevents cells from complement-mediated self-destruction, and is found on the surface of all human cells, with the exception of erythrocytes. Mutations in the H protein, which occur during adaptation and allow the virus to use CD46 as a receptor, have been identified. Wild-type isolates of measles virus cannot use the CD46 receptor. However, both vaccine/laboratory and wild-type strains can use an immune cell receptor called signaling lymphocyte activation molecule family member 1 (SLAMF1; also called CD150) and a recently discovered epithelial receptor known as Nectin-4. SLAMF1 is found on activated B, T, dendritic, and monocyte cells, and is the initial target for infections by measles virus. Nectin-4 is an adherens junction protein found at the basal surfaces of many polarized epithelial cells, including those of the airways. It is also over-expressed on the apical and basal surfaces of many adenocarcinomas, and is a cancer marker for metastasis and tumor survival. Nectin-4 is a secondary exit receptor which allows measles virus to replicate and amplify in the airways, where the virus is expelled from the body in aerosol droplets. The amino acid residues of H protein that are involved in binding to each of the receptors have been identified through X-ray crystallography and site-specific mutagenesis. Recombinant measles "blind" to each of

  17. Isoform composition and stoichiometry of the approx. 90-kDa heat shock protein associated with glucocorticoid receptors

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mendel, D.B.; Orti, E.

    1988-05-15

    The authors observed that the approx. 90-kDa non-steroid-binding component of nonactivated glucocorticoid receptors purified from WEHI-7 mouse thymoma cells (which has been identified as the approx. 90-kDa heat shock protein) consistently migrates as a doublet during polyacrylamide gel electrophoresis under denaturing and reducing conditions. It has recently been reported that murine Meth A cells contain a tumor-specific transplantation antigen (TSTA) which is related or identical to the approx. 90-kDa heat shock protein. The observation that TSTA and the approx. 90-kDa heat shock protein isolated from these cells exists as two isoforms of similar molecular mass and charge has suggested thatmore » the doublet observed is also due to the existence of two isoforms. They have therefore conducted this study to determine whether TSTA and the approx. 90-kDa component of glucocorticoid receptors are indeed related, to establish whether the receptor preferentially binds one isoform of the approx. 90-kDa heat shock protein, and to investigate the stoichiometry of the nonactivated receptor complex. They used the BuGr1 and AC88 monoclonal antibodies to purify, respectively, receptor-associated and free approx. 90-kDa heat shock protein from WEHI-7 cells grown for 48 h with (/sup 35/S)methionine to metabolically label proteins to steady state. The long-term metabolic labeling approach has also enabled them to directly determine that the purified non-activated glucocorticoid receptor contains a single steroid-binding protein and two approx. 90-kDa non-steroid-binding subunits. The consistency with which a approx. 1:2 stoichiometric ratio of steroid binding to approx. 90-kDa protein is observed supports the view that the approx. 90-kDa heat shock protein is a true component of nonactivated glucocorticoid-receptor complexes.« less

  18. Ku proteins function as corepressors to regulate farnesoid X receptor-mediated gene expression

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ohno, Masae; Kunimoto, Masaaki; Nishizuka, Makoto

    2009-12-18

    The farnesoid X receptor (FXR; NR1H4) is a member of the nuclear receptor superfamily and regulates the expression of genes involved in enterohepatic circulation and the metabolism of bile acids. Based on functional analyses, nuclear receptors are divided into regions A-F. To explore the cofactors interacting with FXR, we performed a pull-down assay using GST-fused to the N-terminal A/B region and the C region, which are required for the ligand-independent transactivation and DNA-binding, respectively, of FXR, and nuclear extracts from HeLa cells. We identified DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Ku80, and Ku70 as FXR associated factors. These proteins aremore » known to have an important role in DNA repair, recombination, and transcription. DNA-PKcs mainly interacted with the A/B region of FXR, whereas the Ku proteins interacted with the C region and with the D region (hinge region). Chromatin immunoprecipitation assays revealed that the Ku proteins associated with FXR on the bile salt export pump (BSEP) promoter. Furthermore, we demonstrated that ectopic expression of the Ku proteins decreased the promoter activity and expression of BSEP gene mediated by FXR. These results suggest that the Ku proteins function as corepressors for FXR.« less

  19. Imaging of persistent cAMP signaling by internalized G protein-coupled receptors.

    PubMed

    Calebiro, Davide; Nikolaev, Viacheslav O; Lohse, Martin J

    2010-07-01

    G protein-coupled receptors (GPCRs) are the largest family of plasma membrane receptors. They mediate the effects of several endogenous cues and serve as important pharmacological targets. Although many biochemical events involved in GPCR signaling have been characterized in great detail, little is known about their spatiotemporal dynamics in living cells. The recent advent of optical methods based on fluorescent resonance energy transfer allows, for the first time, to directly monitor GPCR signaling in living cells. Utilizing these methods, it has been recently possible to show that the receptors for two protein/peptide hormones, the TSH and the parathyroid hormone, continue signaling to cAMP after their internalization into endosomes. This type of intracellular signaling is persistent and apparently triggers specific cellular outcomes. Here, we review these recent data and explain the optical methods used for such studies. Based on these findings, we propose a revision of the current model of the GPCR-cAMP signaling pathway to accommodate receptor signaling at endosomes.

  20. The mapping of yeast's G-protein coupled receptor with an atomic force microscope

    NASA Astrophysics Data System (ADS)

    Takenaka, Musashi; Miyachi, Yusuke; Ishii, Jun; Ogino, Chiaki; Kondo, Akihiko

    2015-03-01

    An atomic force microscope (AFM) can measure the adhesion force between a sample and a cantilever while simultaneously applying a rupture force during the imaging of a sample. An AFM should be useful in targeting specific proteins on a cell surface. The present study proposes the use of an AFM to measure the adhesion force between targeting receptors and their ligands, and to map the targeting receptors. In this study, Ste2p, one of the G protein-coupled receptors (GPCRs), was chosen as the target receptor. The specific force between Ste2p on a yeast cell surface and a cantilever modified with its ligand, α-factor, was measured and found to be approximately 250 pN. In addition, through continuous measuring of the cell surface, a mapping of the receptors on the cell surface could be performed, which indicated the differences in the Ste2p expression levels. Therefore, the proposed AFM system is accurate for cell diagnosis.

  1. Stargazin regulates AMPA receptor trafficking through adaptor protein complexes during long-term depression

    NASA Astrophysics Data System (ADS)

    Matsuda, Shinji; Kakegawa, Wataru; Budisantoso, Timotheus; Nomura, Toshihiro; Kohda, Kazuhisa; Yuzaki, Michisuke

    2013-11-01

    Long-term depression (LTD) underlies learning and memory in various brain regions. Although postsynaptic AMPA receptor trafficking mediates LTD, its underlying molecular mechanisms remain largely unclear. Here we show that stargazin, a transmembrane AMPA receptor regulatory protein, forms a ternary complex with adaptor proteins AP-2 and AP-3A in hippocampal neurons, depending on its phosphorylation state. Inhibiting the stargazin-AP-2 interaction disrupts NMDA-induced AMPA receptor endocytosis, and inhibiting that of stargazin-AP-3A abrogates the late endosomal/lysosomal trafficking of AMPA receptors, thereby upregulating receptor recycling to the cell surface. Similarly, stargazin’s interaction with AP-2 or AP-3A is necessary for low-frequency stimulus-evoked LTD in CA1 hippocampal neurons. Thus, stargazin has a crucial role in NMDA-dependent LTD by regulating two trafficking pathways of AMPA receptors—transport from the cell surface to early endosomes and from early endosomes to late endosomes/lysosomes—through its sequential binding to AP-2 and AP-3A.

  2. Development and optimization of a cell-based assay for the selection of synthetic compounds that potentiate bone morphogenetic protein-2 activity‡

    PubMed Central

    Okada, Motohiro; Sangadala, Sreedhara; Liu, Yunshan; Yoshida, Munehito; Reddy, Boojala Vijay B.; Titus, Louisa; Boden, Scott D.

    2010-01-01

    The requirement of large amounts of the recombinant human bone morphogenetic protein-2 (BMP-2) produces a huge translational barrier for its routine clinical use due to high cost. This leads to an urgent need to develop alternative methods to lower costs and/or increase efficacies for using BMP-2. In this study, we describe the development and optimization of a cell-based assay that is sensitive, reproducible, and reliable in identifying reagents that potentiate the effects of BMP-2 in inducing transdifferentiation of C2C12 myoblasts into the osteoblastic phenotype. The assay is based on a BMP-responsive Smad1-driven luciferase reporter gene. LIM mineralization protein-1 (LMP-1) is a novel intracellular LIM domain protein that has been shown by our group to enhance cellular responsiveness to BMP-2. Our previous report elucidated that the binding of LMP-1 with the WW2 domain in Smad ubiquitin regulatory factor-1 (Smurf1) rescues the osteogenic Smads from degradation. Here, using the optimized cell-based assay, we first evaluated the activity of the recombinantly prepared proteins, LMP-1, and its mutant (LMP-1ΔSmurf1) that lacks the Smurf1-WW2 domain-binding motif. Both the wild type and the mutant proteins were engineered to contain an 11-amino acid HIV-TAT protein derived membrane transduction domain to aid the cellular delivery of recombinant proteins. The cell-based reporter assay confirmed that LMP-1 potentiates the BMP-induced stimulation of C2C12 cells towards the osteoblastic phenotype. The potentiating effect of LMP-1 was significantly reduced when a specific-motif known to interact with Smurf1 was mutated. We validated the results obtained in the reporter assay by also monitoring the expression of mRNA for osteocalcin and alkaline phosphatase (ALP) which is widely accepted osteoblast differentiation marker genes. Finally, we provide further confirmation of our results by measuring the activity of alkaline phosphatase in support of the accuracy and

  3. Development and optimization of a cell-based assay for the selection of synthetic compounds that potentiate bone morphogenetic protein-2 activity.

    PubMed

    Okada, Motohiro; Sangadala, Sreedhara; Liu, Yunshan; Yoshida, Munehito; Reddy, Boojala Vijay B; Titus, Louisa; Boden, Scott D

    2009-12-01

    The requirement of large amounts of the recombinant human bone morphogenetic protein-2 (BMP-2) produces a huge translational barrier for its routine clinical use due to high cost. This leads to an urgent need to develop alternative methods to lower costs and/or increase efficacies for using BMP-2. In this study, we describe the development and optimization of a cell-based assay that is sensitive, reproducible, and reliable in identifying reagents that potentiate the effects of BMP-2 in inducing transdifferentiation of C2C12 myoblasts into the osteoblastic phenotype. The assay is based on a BMP-responsive Smad1-driven luciferase reporter gene. LIM mineralization protein-1 (LMP-1) is a novel intracellular LIM domain protein that has been shown by our group to enhance cellular responsiveness to BMP-2. Our previous report elucidated that the binding of LMP-1 with the WW2 domain in Smad ubiquitin regulatory factor-1 (Smurf1) rescues the osteogenic Smads from degradation. Here, using the optimized cell-based assay, we first evaluated the activity of the recombinantly prepared proteins, LMP-1, and its mutant (LMP-1DeltaSmurf1) that lacks the Smurf1-WW2 domain-binding motif. Both the wild type and the mutant proteins were engineered to contain an 11-amino acid HIV-TAT protein derived membrane transduction domain to aid the cellular delivery of recombinant proteins. The cell-based reporter assay confirmed that LMP-1 potentiates the BMP-induced stimulation of C2C12 cells towards the osteoblastic phenotype. The potentiating effect of LMP-1 was significantly reduced when a specific-motif known to interact with Smurf1 was mutated. We validated the results obtained in the reporter assay by also monitoring the expression of mRNA for osteocalcin and alkaline phosphatase (ALP) which is widely accepted osteoblast differentiation marker genes. Finally, we provide further confirmation of our results by measuring the activity of alkaline phosphatase in support of the accuracy and

  4. Cellular progesterone receptor phosphorylation in response to ligands activating protein kinases

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Rao, K.V.; Peralta, W.D.; Greene, G.L.

    1987-08-14

    Progesterone receptors were immunoprecipitated with monoclonal antibodies KD68 from lysates of human breast carcinoma T47D cells labelled to steady state specific activity with /sup 32/Pi. The 120 kDa /sup 32/P-labelled progesterone receptor band was resolved by polyacrylamide gel electrophoresis and identified by autoradiography. Phosphoamino acid analysis revealed serine phosphorylation, but no threonine or tyrosine phosphorylation. Treatment of the /sup 32/Pi-labelled cells with EGF, TPA or dibutyryl cAMP had no significant quantitative effect on progesterone receptor phosphorylation, though the EGF receptor and the cAMP-dependent protein kinases have been reported to catalyze phosphorylation of purified avian progesterone receptor preparations in cell freemore » systems. Progesterone receptor phosphorylation on serine residues was increased by 2-fold in cells treated with 10 nM progesterone; EGF had no effect on progesterone-mediated progesterone receptor phosphorylation.« less

  5. Heterotrimeric G Protein-coupled Receptor Signaling in Yeast Mating Pheromone Response.

    PubMed

    Alvaro, Christopher G; Thorner, Jeremy

    2016-04-08

    The DNAs encoding the receptors that respond to the peptide mating pheromones of the budding yeastSaccharomyces cerevisiaewere isolated in 1985, and were the very first genes for agonist-binding heterotrimeric G protein-coupled receptors (GPCRs) to be cloned in any organism. Now, over 30 years later, this yeast and its receptors continue to provide a pathfinding experimental paradigm for investigating GPCR-initiated signaling and its regulation, as described in this retrospective overview. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Selective targeting of G-protein-coupled receptor subtypes with venom peptides.

    PubMed

    Näreoja, K; Näsman, J

    2012-02-01

    The G-protein-coupled receptor (GPCR) family is one of the largest gene superfamilies with approx. 370 members responding to endogenous ligands in humans and a roughly equal amount of receptors sensitive to external stimuli from the surrounding. A number of receptors from this superfamily are well recognized targets for medical treatment of various disease conditions, whereas for many others the potential medical benefit of interference is still obscure. A general problem associated with GPCR research and therapeutics is the insufficient specificity of available ligands to differentiate between closely homologous receptor subtypes. In this context, venom peptides could make a significant contribution to the development of more specific drugs. Venoms from certain animals specialized in biochemical hunting contain a mixture of molecules that are directed towards a variety of membrane proteins. Peptide toxins isolated from these mixtures usually exhibit high specificity for their targets. Muscarinic toxins found from mamba snakes attracted much attention during the 1990s. These are 65-66 amino acid long peptides with a structural three-finger folding similar to the α-neurotoxins and they target the muscarinic acetylcholine receptors in a subtype-selective manner. Recently, several members of the three-finger toxins from mamba snakes as well as conotoxins from marine cone snails have been shown to selectively interact with subtypes of adrenergic receptors. In this review, we will discuss the GPCR-directed peptide toxins found from different venoms and how some of these can be useful in exploring specific roles of receptor subtypes. © 2011 The Authors. Acta Physiologica © 2011 Scandinavian Physiological Society.

  7. Characterization of B61, the ligand for the Eck receptor protein-tyrosine kinase.

    PubMed

    Shao, H; Pandey, A; O'Shea, K S; Seldin, M; Dixit, V M

    1995-03-10

    B61 was originally described as a novel secreted tumor necrosis factor-alpha-inducible gene product in endothelial cells (Holzman, L. B., Marks, R. M., and Dixit, V. M. (1990) Mol. Cell. Biol. 10, 5830-5838). It was recently discovered that soluble recombinant B61 could serve as a ligand for the Eck receptor protein-tyrosine kinase, a member of the Eph/Eck subfamily of receptor protein-tyrosine kinases (Bartley, T.D., Hunt, R. W., Welcher, A. A., Boyle, W. J., Parker, V. P., Lindberg, R. A., Lu, H. S., Colombero, A. M., Elliott, R. L., Guthrie, R. A., Holst, P. L., Skrine, J. D., Toso, R. J., Zhang, M., Fernandez, E., Trail, G., Yarnum, B., Yarden, Y., Hunter, T., and Fox, G. M. (1994) Nature 368, 558-560). We now show that B61 can also exist as a cell surface glycosylphosphatidyl-inositol-linked protein that is capable of activating the Eck receptor protein-tyrosine kinase, the first such report of a receptor protein-tyrosine kinase ligand that is glycosylphosphatidylinositol-linked. In addition, the expression patterns of B61 and Eck during mouse ontogeny were determined by in situ hybridization. Both were found to be highly expressed in the developing lung and gut, while Eck was preferentially expressed in the thymus. Finally, the gene for B61 was localized to a specific position on mouse chromosome 3 by interspecific back-cross analysis.

  8. Protein partners in the life history of activated fibroblast growth factor receptors.

    PubMed

    Vecchione, Anna; Cooper, Helen J; Trim, Kimberley J; Akbarzadeh, Shiva; Heath, John K; Wheldon, Lee M

    2007-12-01

    Fibroblast growth factor receptors (FGFRs) are a family of four transmembrane (TM) receptor tyrosine kinases (RTKs) which bind to a large family of fibroblast growth factor (FGF) ligands with varying affinity and specificity. FGFR signaling regulates many physiological and pathological processes in development and tissue homeostasis. Understanding FGFR signaling processes requires the identification of partner proteins which regulate receptor function and biological outputs. In this study, we employ an epitope-tagged, covalently dimerized, and constitutively activated form of FGFR1 to identify potential protein partners by MS. By this approach, we sample candidate FGFR effectors throughout the life history of the receptor. Functional classification of the partners identified revealed specific subclasses involved in protein biosynthesis and folding; structural and regulatory components of the cytoskeleton; known signaling effectors and small GTPases implicated in endocytosis and vesicular trafficking. The kinase dependency of the interaction was determined for a subset of previously unrecognized partners by coimmunoprecipitation, Western blotting, and immunocytochemistry. From this group, the small GTPase Rab5 was selected for functional interrogation. We show that short hairpin (sh) RNA-mediated depletion of Rab5 attenuates the activation of the extracellular-regulated kinase (ERK) 1/2 pathway by FGFR signaling. The strategic approach adopted in this study has revealed bona fide novel effectors of the FGFR signaling pathway.

  9. Orphan nuclear receptor TLX regulates astrogenesis by modulating BMP signaling

    PubMed Central

    Qin, Song; Niu, Wenze; Iqbal, Nida; Smith, Derek K.; Zhang, Chun-Li

    2014-01-01

    Neural stem cells (NSCs) are self-renewing multipotent progenitors that generate both neurons and glia. The precise control of NSC behavior is fundamental to the architecture and function of the central nervous system. We previously demonstrated that the orphan nuclear receptor TLX is required for postnatal NSC activation and neurogenesis in the neurogenic niche. Here, we show that TLX modulates bone morphogenetic protein (BMP)-SMAD signaling to control the timing of postnatal astrogenesis. Genes involved in the BMP signaling pathway, such as Bmp4, Hes1, and Id3, are upregulated in postnatal brains lacking Tlx. Chromatin immunoprecipitation and electrophoretic mobility shift assays reveal that TLX can directly bind the enhancer region of Bmp4. In accordance with elevated BMP signaling, the downstream effectors SMAD1/5/8 are activated by phosphorylation in Tlx mutant mice. Consequently, Tlx mutant brains exhibit an early appearance and increased number of astrocytes with marker expression of glial fibrillary acidic protein (GFAP) and S100B. Taken together, these results suggest that TLX tightly controls postnatal astrogenesis through the modulation of BMP-SMAD signaling pathway activity. PMID:24782704

  10. Functional assay for T4 lysozyme-engineered G protein-coupled receptors with an ion channel reporter.

    PubMed

    Niescierowicz, Katarzyna; Caro, Lydia; Cherezov, Vadim; Vivaudou, Michel; Moreau, Christophe J

    2014-01-07

    Structural studies of G protein-coupled receptors (GPCRs) extensively use the insertion of globular soluble protein domains to facilitate their crystallization. However, when inserted in the third intracellular loop (i3 loop), the soluble protein domain disrupts their coupling to G proteins and impedes the GPCRs functional characterization by standard G protein-based assays. Therefore, activity tests of crystallization-optimized GPCRs are essentially limited to their ligand binding properties using radioligand binding assays. Functional characterization of additional thermostabilizing mutations requires the insertion of similar mutations in the wild-type receptor to allow G protein-activation tests. We demonstrate that ion channel-coupled receptor technology is a complementary approach for a comprehensive functional characterization of crystallization-optimized GPCRs and potentially of any engineered GPCR. Ligand-induced conformational changes of the GPCRs are translated into electrical signal and detected by simple current recordings, even though binding of G proteins is sterically blocked by the added soluble protein domain. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Effects of estrogen receptor modulators on cytoskeletal proteins in the central nervous system.

    PubMed

    Segura-Uribe, Julia J; Pinto-Almazán, Rodolfo; Coyoy-Salgado, Angélica; Fuentes-Venado, Claudia E; Guerra-Araiza, Christian

    2017-08-01

    Estrogen receptor modulators are compounds of interest because of their estrogenic agonistic/antagonistic effects and tissue specificity. These compounds have many clinical applications, particularly for breast cancer treatment and osteoporosis in postmenopausal women, as well as for the treatment of climacteric symptoms. Similar to estrogens, neuroprotective effects of estrogen receptor modulators have been described in different models. However, the mechanisms of action of these compounds in the central nervous system have not been fully described. We conducted a systematic search to investigate the effects of estrogen receptor modulators in the central nervous system, focusing on the modulation of cytoskeletal proteins. We found that raloxifene, tamoxifen, and tibolone modulate some cytoskeletal proteins such as tau, microtuble-associated protein 1 (MAP1), MAP2, neurofilament 38 (NF38) by different mechanisms of action and at different levels: neuronal microfilaments, intermediate filaments, and microtubule-associated proteins. Finally, we emphasize the importance of the study of these compounds in the treatment of neurodegenerative diseases since they present the benefits of estrogens without their side effects.

  12. Effects of estrogen receptor modulators on cytoskeletal proteins in the central nervous system

    PubMed Central

    Segura-Uribe, Julia J.; Pinto-Almazán, Rodolfo; Coyoy-Salgado, Angélica; Fuentes-Venado, Claudia E.; Guerra-Araiza, Christian

    2017-01-01

    Estrogen receptor modulators are compounds of interest because of their estrogenic agonistic/antagonistic effects and tissue specificity. These compounds have many clinical applications, particularly for breast cancer treatment and osteoporosis in postmenopausal women, as well as for the treatment of climacteric symptoms. Similar to estrogens, neuroprotective effects of estrogen receptor modulators have been described in different models. However, the mechanisms of action of these compounds in the central nervous system have not been fully described. We conducted a systematic search to investigate the effects of estrogen receptor modulators in the central nervous system, focusing on the modulation of cytoskeletal proteins. We found that raloxifene, tamoxifen, and tibolone modulate some cytoskeletal proteins such as tau, microtuble-associated protein 1 (MAP1), MAP2, neurofilament 38 (NF38) by different mechanisms of action and at different levels: neuronal microfilaments, intermediate filaments, and microtubule-associated proteins. Finally, we emphasize the importance of the study of these compounds in the treatment of neurodegenerative diseases since they present the benefits of estrogens without their side effects. PMID:28966632

  13. Novel Mechanisms in the Regulation of G Protein-coupled Receptor Trafficking to the Plasma Membrane*

    PubMed Central

    Tholanikunnel, Baby G.; Joseph, Kusumam; Kandasamy, Karthikeyan; Baldys, Aleksander; Raymond, John R.; Luttrell, Louis M.; McDermott, Paul J.; Fernandes, Daniel J.

    2010-01-01

    β2-Adrenergic receptors (β2-AR) are low abundance, integral membrane proteins that mediate the effects of catecholamines at the cell surface. Whereas the processes governing desensitization of activated β2-ARs and their subsequent removal from the cell surface have been characterized in considerable detail, little is known about the mechanisms controlling trafficking of neo-synthesized receptors to the cell surface. Since the discovery of the signal peptide, the targeting of the integral membrane proteins to plasma membrane has been thought to be determined by structural features of the amino acid sequence alone. Here we report that localization of translationally silenced β2-AR mRNA to the peripheral cytoplasmic regions is critical for receptor localization to the plasma membrane. β2-AR mRNA is recognized by the nucleocytoplasmic shuttling RNA-binding protein HuR, which silences translational initiation while chaperoning the mRNA-protein complex to the cell periphery. When HuR expression is down-regulated, β2-AR mRNA translation is initiated prematurely in perinuclear polyribosomes, leading to overproduction of receptors but defective trafficking to the plasma membrane. Our results underscore the importance of the spatiotemporal relationship between β2-AR mRNA localization, translation, and trafficking to the plasma membrane, and establish a novel mechanism whereby G protein-coupled receptor (GPCR) responsiveness is regulated by RNA-based signals. PMID:20739277

  14. The Concise Guide to Pharmacology 2013/14: G Protein-Coupled Receptors

    PubMed Central

    Alexander, Stephen PH; Benson, Helen E; Faccenda, Elena; Pawson, Adam J; Sharman, Joanna L; Spedding, Michael; Peters, John A; Harmar, Anthony J

    2013-01-01

    The Concise Guide to PHARMACOLOGY 2013/14 provides concise overviews of the key properties of over 2000 human drug targets with their pharmacology, plus links to an open access knowledgebase of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. The full contents can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.12444/full. G protein-coupled receptors are one of the seven major pharmacological targets into which the Guide is divided, with the others being G protein-coupled receptors, ligand-gated ion channels, ion channels, catalytic receptors, nuclear hormone receptors, transporters and enzymes. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. A new landscape format has easy to use tables comparing related targets. It is a condensed version of material contemporary to late 2013, which is presented in greater detail and constantly updated on the website www.guidetopharmacology.org, superseding data presented in previous Guides to Receptors and Channels. It is produced in conjunction with NC-IUPHAR and provides the official IUPHAR classification and nomenclature for human drug targets, where appropriate. It consolidates information previously curated and displayed separately in IUPHAR-DB and the Guide to Receptors and Channels, providing a permanent, citable, point-in-time record that will survive database updates. PMID:24517644

  15. Mapping allosteric connections from the receptor to the nucleotide-binding pocket of heterotrimeric G proteins

    PubMed Central

    Oldham, William M.; Van Eps, Ned; Preininger, Anita M.; Hubbell, Wayne L.; Hamm, Heidi E.

    2007-01-01

    Heterotrimeric G proteins function as molecular relays that mediate signal transduction from heptahelical receptors in the cell membrane to intracellular effector proteins. Crystallographic studies have demonstrated that guanine nucleotide exchange on the Gα subunit causes specific conformational changes in three key “switch” regions of the protein, which regulate binding to Gβγ subunits, receptors, and effector proteins. In the present study, nitroxide side chains were introduced at sites within the switch I region of Gαi to explore the structure and dynamics of this region throughout the G protein cycle. EPR spectra obtained for each of the Gα(GDP), Gα(GDP)βγ heterotrimer and Gα(GTPγS) conformations are consistent with the local environment observed in the corresponding crystal structures. Binding of the heterotrimer to activated rhodopsin to form the nucleotide-free (empty) complex, for which there is no crystal structure, causes prominent changes relative to the heterotrimer in the structure of switch I and contiguous sequences. The data identify a putative pathway of allosteric changes triggered by receptor binding and, together with previously published data, suggest elements of a mechanism for receptor-catalyzed nucleotide exchange. PMID:17463080

  16. G protein-coupled estrogen receptor inhibits the P2Y receptor-mediated Ca(2+) signaling pathway in human airway epithelia.

    PubMed

    Hao, Yuan; Chow, Alison W; Yip, Wallace C; Li, Chi H; Wan, Tai F; Tong, Benjamin C; Cheung, King H; Chan, Wood Y; Chen, Yangchao; Cheng, Christopher H; Ko, Wing H

    2016-08-01

    P2Y receptor activation causes the release of inflammatory cytokines in the bronchial epithelium, whereas G protein-coupled estrogen receptor (GPER), a novel estrogen (E2) receptor, may play an anti-inflammatory role in this process. We investigated the cellular mechanisms underlying the inhibitory effect of GPER activation on the P2Y receptor-mediated Ca(2+) signaling pathway and cytokine production in airway epithelia. Expression of GPER in primary human bronchial epithelial (HBE) or 16HBE14o- cells was confirmed on both the mRNA and protein levels. Stimulation of HBE or 16HBE14o- cells with E2 or G1, a specific agonist of GPER, attenuated the nucleotide-evoked increases in [Ca(2+)]i, whereas this effect was reversed by G15, a GPER-specific antagonist. G1 inhibited the secretion of two proinflammatory cytokines, interleukin (IL)-6 and IL-8, in cells stimulated by adenosine 5'-(γ-thio)triphosphate (ATPγS). G1 stimulated a real-time increase in cAMP levels in 16HBE14o- cells, which could be inhibited by adenylyl cyclase inhibitors. The inhibitory effects of E2 or G1 on P2Y receptor-induced increases in Ca(2+) were reversed by treating the cells with a protein kinase A (PKA) inhibitor. These results demonstrated that the inhibitory effects of G1 or E2 on P2Y receptor-mediated Ca(2+) mobilization and cytokine secretion were due to GPER-mediated activation of a cAMP-dependent PKA pathway. This study has reported, for the first time, the expression and function of GPER as an anti-inflammatory component in human bronchial epithelia, which may mediate through its opposing effects on the pro-inflammatory pathway activated by the P2Y receptors in inflamed airway epithelia.

  17. G protein-coupled receptor 30 (GPR30) forms a plasma membrane complex with membrane-associated guanylate kinases (MAGUKs) and protein kinase A-anchoring protein 5 (AKAP5) that constitutively inhibits cAMP production.

    PubMed

    Broselid, Stefan; Berg, Kelly A; Chavera, Teresa A; Kahn, Robin; Clarke, William P; Olde, Björn; Leeb-Lundberg, L M Fredrik

    2014-08-08

    GPR30, or G protein-coupled estrogen receptor, is a G protein-coupled receptor reported to bind 17β-estradiol (E2), couple to the G proteins Gs and Gi/o, and mediate non-genomic estrogenic responses. However, controversies exist regarding the receptor pharmacological profile, effector coupling, and subcellular localization. We addressed the role of the type I PDZ motif at the receptor C terminus in receptor trafficking and coupling to cAMP production in HEK293 cells and CHO cells ectopically expressing the receptor and in Madin-Darby canine kidney cells expressing the native receptor. GPR30 was localized both intracellularly and in the plasma membrane and subject to limited basal endocytosis. E2 and G-1, reported GPR30 agonists, neither stimulated nor inhibited cAMP production through GPR30, nor did they influence receptor localization. Instead, GPR30 constitutively inhibited cAMP production stimulated by a heterologous agonist independently of Gi/o. Moreover, siRNA knockdown of native GPR30 increased cAMP production. Deletion of the receptor PDZ motif interfered with inhibition of cAMP production and increased basal receptor endocytosis. GPR30 interacted with membrane-associated guanylate kinases, including SAP97 and PSD-95, and protein kinase A-anchoring protein (AKAP) 5 in the plasma membrane in a PDZ-dependent manner. Knockdown of AKAP5 or St-Ht31 treatment, to disrupt AKAP interaction with the PKA RIIβ regulatory subunit, decreased inhibition of cAMP production, and St-Ht31 increased basal receptor endocytosis. Therefore, GPR30 forms a plasma membrane complex with a membrane-associated guanylate kinase and AKAP5, which constitutively attenuates cAMP production in response to heterologous agonists independently of Gi/o and retains receptors in the plasma membrane. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Sch proteins are localized on endoplasmic reticulum membranes and are redistributed after tyrosine kinase receptor activation.

    PubMed Central

    Lotti, L V; Lanfrancone, L; Migliaccio, E; Zompetta, C; Pelicci, G; Salcini, A E; Falini, B; Pelicci, P G; Torrisi, M R

    1996-01-01

    The intracellular localization of Shc proteins was analyzed by immunofluorescence and immunoelectron microscopy in normal cells and cells expressing the epidermal growth factor receptor or the EGFR/erbB2 chimera. In unstimulated cells, the immunolabeling was localized in the central perinuclear area of the cell and mostly associated with the cytosolic side of rough endoplasmic reticulum membranes. Upon epidermal growth factor treatment and receptor tyrosine kinase activation, the immunolabeling became peripheral and was found to be associated with the cytosolic surface of the plasma membrane and endocytic structures, such as coated pits and endosomes, and with the peripheral cytosol. Receptor activation in cells expressing phosphorylation-defective mutants of Shc and erbB-2 kinase showed that receptor autophosphorylation, but not Shc phosphorylation, is required for redistribution of Shc proteins. The rough endoplasmic reticulum localization of Shc proteins in unstimulated cells and their massive recruitment to the plasma membrane, endocytic structures, and peripheral cytosol following receptor tyrosine kinase activation could account for multiple putative functions of the adaptor protein. PMID:8628261

  19. Case Study: Organotypic human in vitro models of embryonic morphogenetic fusion

    EPA Science Inventory

    Morphogenetic fusion of tissues is a common event in embryonic development and disruption of fusion is associated with birth defects of the eye, heart, neural tube, phallus, palate, and other organ systems. Embryonic tissue fusion requires precise regulation of cell-cell and cell...

  20. The orphan receptor ALK7 and the Activin receptor ALK4 mediate signaling by Nodal proteins during vertebrate development.

    PubMed

    Reissmann, E; Jörnvall, H; Blokzijl, A; Andersson, O; Chang, C; Minchiotti, G; Persico, M G; Ibáñez, C F; Brivanlou, A H

    2001-08-01

    Nodal proteins have crucial roles in mesendoderm formation and left-right patterning during vertebrate development. The molecular mechanisms of signal transduction by Nodal and related ligands, however, are not fully understood. In this paper, we present biochemical and functional evidence that the orphan type I serine/threonine kinase receptor ALK7 acts as a receptor for mouse Nodal and Xenopus Nodal-related 1 (Xnr1). Receptor reconstitution experiments indicate that ALK7 collaborates with ActRIIB to confer responsiveness to Xnr1 and Nodal. Both receptors can independently bind Xnr1. In addition, Cripto, an extracellular protein genetically implicated in Nodal signaling, can independently interact with both Xnr1 and ALK7, and its expression greatly enhances the ability of ALK7 and ActRIIB to respond to Nodal ligands. The Activin receptor ALK4 is also able to mediate Nodal signaling but only in the presence of Cripto, with which it can also interact directly. A constitutively activated form of ALK7 mimics the mesendoderm-inducing activity of Xnr1 in Xenopus embryos, whereas a dominant-negative ALK7 specifically blocks the activities of Nodal and Xnr1 but has little effect on other related ligands. In contrast, a dominant-negative ALK4 blocks all mesoderm-inducing ligands tested, including Nodal, Xnr1, Xnr2, Xnr4, and Activin. In agreement with a role in Nodal signaling, ALK7 mRNA is localized to the ectodermal and organizer regions of Xenopus gastrula embryos and is expressed during early stages of mouse embryonic development. Therefore, our results indicate that both ALK4 and ALK7 can mediate signal transduction by Nodal proteins, although ALK7 appears to be a receptor more specifically dedicated to Nodal signaling.

  1. The orphan receptor ALK7 and the Activin receptor ALK4 mediate signaling by Nodal proteins during vertebrate development

    PubMed Central

    Reissmann, Eva; Jörnvall, Henrik; Blokzijl, Andries; Andersson, Olov; Chang, Chenbei; Minchiotti, Gabriella; Persico, M. Graziella; Ibáñez, Carlos F.; Brivanlou, Ali H.

    2001-01-01

    Nodal proteins have crucial roles in mesendoderm formation and left–right patterning during vertebrate development. The molecular mechanisms of signal transduction by Nodal and related ligands, however, are not fully understood. In this paper, we present biochemical and functional evidence that the orphan type I serine/threonine kinase receptor ALK7 acts as a receptor for mouse Nodal and Xenopus Nodal-related 1 (Xnr1). Receptor reconstitution experiments indicate that ALK7 collaborates with ActRIIB to confer responsiveness to Xnr1 and Nodal. Both receptors can independently bind Xnr1. In addition, Cripto, an extracellular protein genetically implicated in Nodal signaling, can independently interact with both Xnr1 and ALK7, and its expression greatly enhances the ability of ALK7 and ActRIIB to respond to Nodal ligands. The Activin receptor ALK4 is also able to mediate Nodal signaling but only in the presence of Cripto, with which it can also interact directly. A constitutively activated form of ALK7 mimics the mesendoderm-inducing activity of Xnr1 in Xenopus embryos, whereas a dominant-negative ALK7 specifically blocks the activities of Nodal and Xnr1 but has little effect on other related ligands. In contrast, a dominant-negative ALK4 blocks all mesoderm-inducing ligands tested, including Nodal, Xnr1, Xnr2, Xnr4, and Activin. In agreement with a role in Nodal signaling, ALK7 mRNA is localized to the ectodermal and organizer regions of Xenopus gastrula embryos and is expressed during early stages of mouse embryonic development. Therefore, our results indicate that both ALK4 and ALK7 can mediate signal transduction by Nodal proteins, although ALK7 appears to be a receptor more specifically dedicated to Nodal signaling. PMID:11485994

  2. Signaling, physiological functions and clinical relevance of the G protein-coupled estrogen receptor GPER.

    PubMed

    Prossnitz, Eric R; Barton, Matthias

    2009-09-01

    GPR30, now named GPER1 (G protein-coupled estrogen receptor1) or GPER here, was first identified as an orphan 7-transmembrane G protein-coupled receptor by multiple laboratories using either homology cloning or differential expression and subsequently shown to be required for estrogen-mediated signaling in certain cancer cells. The actions of estrogen are extensive in the body and are thought to be mediated predominantly by classical nuclear estrogen receptors that act as transcription factors/regulators. Nevertheless, certain aspects of estrogen function remain incompatible with the generally accepted mechanisms of classical estrogen receptor action. Many recent studies have revealed that GPER contributes to some of the actions of estrogen, including rapid signaling events and rapid transcriptional activation. With the introduction of GPER-selective ligands and GPER knockout mice, the functions of GPER are becoming more clearly defined. In many cases, there appears to be a complex interplay between the two receptor systems, suggesting that estrogen-mediated physiological responses may be mediated by either receptor or a combination of both receptor types, with important medical implications.

  3. Genome wide identification, phylogeny, and expression of bone morphogenetic protein genes in tetraploidized common carp (Cyprinus carpio).

    PubMed

    Chen, Lin; Dong, Chuanju; Kong, Shengnan; Zhang, Jiangfan; Li, Xuejun; Xu, Peng

    2017-09-05

    Bone morphogenetic proteins (Bmps) are a group of signaling molecules known to play important roles during formation and maintenance of various organs, not only bone, but also muscle, blood and so on. Common carp (Cyprinus carpio) is one of the most intensively studied fish due to its economic and environmental importance. Besides, common carp has encountered an additional round of whole genome duplication (WGD) compared with many closely related diploid teleost, which make it one of the most important models for genome evolutionary studies in teleost. Comprehensive genome resources of common carp have been developed recently, which facilitate the thorough characterization of bmp gene family in the tetraploidized common carp genome. We identified a total of 44 bmps from the common carp genome, which are twice as many as that of zebrafish. Phylogenetic analysis revealed that most of bmps are highly conserved. Comparative analysis was performed across six typical vertebrate genomes. It appeared that all the bmp genes in common carp were duplicated. Obviously, the expansion of the bmp gene family in common carp was due to the latest additional round of whole genome duplication and made it more abundant than other diploid teleosts. Expression signatures were assessed in major tissues, including gill, intestine, liver, spleen, skin, heart, gonad, muscle, kidney, head kidney, brain and blood, which demonstrated the comprehensive expression profiles of bmp genes in the tetraploidized genome. Significant gene expression divergences were observed which revealed substantial functional divergences of those duplicated bmp genes post the latest WGD event. The conserved synteny blocks of bmp5s revealed the genome rearrangement of common carp post the 4R WGD. The whole set of bmp gene family in common carp provides insight into gene fate of tetraploidized common carp genome post recent WGD. Copyright © 2017. Published by Elsevier B.V.

  4. Tricalcium phosphate and glutaraldehyde crosslinked gelatin incorporating bone morphogenetic protein--a viable scaffold for bone tissue engineering.

    PubMed

    Yang, Shu-Hua; Hsu, Chung-King; Wang, Kuo-Cheng; Hou, Sheng-Mou; Lin, Feng-Huei

    2005-07-01

    Bone defects caused by various etiologies must be filled with suitable substances to promote bone repair. Autogenous iliac crest graft is most frequently used, but is often associated with morbidities. Several bone graft substitutes have been developed to provide osteoconductive matrices as well as to enhance osteoinductivity. A tricalcium phosphate and glutaraldehyde crosslinked gelatin (GTG) scaffold, incorporated with bone morphogenetic proteins (BMPs), was developed to provide an alternative mean of bone tissue engineering. This study investigated differences between GTG and BMP-4 immobilized GTG (GTG-BMP) scaffolds on neonatal rat calvaria osteoblast activities. The GTG scaffold possessed an average pore size of 200 microm and a porosity of 75%. HE staining revealed uniform cell distribution throughout the scaffold 24 h post cell seeding. Alkaline phosphatase (ALP) activity of the GTG samples increased initially and then stabilized at 3 weeks postseeding. ALP activity of the GTG-BMP samples was similar to that of the GTG samples in the second and third weeks, but it continued increasing and became significantly greater than that of the GTG samples by the fourth week. Gla-type osteocalcin (Gla-OC) activity of the GTG-BMP samples was initially lower, but also became significantly greater than that of the GTG samples by the fourth week. An HE stain revealed greater numbers of attached cells and a richer matrix deposits in the GTG-BMP samples. A von Kossa stain showed larger mineralizing nodules, in greater numbers, after 4 weeks of in vitro cultivation. These findings suggest that the GTG scaffold provides an excellent porous structure, conductive to greater cell attachment and osteoblast differentiation, and that utility can be significantly enhanced by the inclusion of BMPs. A GTG-BMP scaffold holds promise as a superior bioactive material for bone tissue engineering. Copyright 2005 Wiley Periodicals, Inc.

  5. Modulation of neurosteroid potentiation by protein kinases at synaptic- and extrasynaptic-type GABAA receptors

    PubMed Central

    Adams, Joanna M.; Thomas, Philip; Smart, Trevor G.

    2015-01-01

    GABAA receptors are important for inhibition in the CNS where neurosteroids and protein kinases are potent endogenous modulators. Acting individually, these can either enhance or depress receptor function, dependent upon the type of neurosteroid or kinase and the receptor subunit combination. However, in vivo, these modulators probably act in concert to fine-tune GABAA receptor activity and thus inhibition, although how this is achieved remains unclear. Therefore, we investigated the relationship between these modulators at synaptic-type α1β3γ2L and extrasynaptic-type α4β3δ GABAA receptors using electrophysiology. For α1β3γ2L, potentiation of GABA responses by tetrahydro-deoxycorticosterone was reduced after inhibiting protein kinase C, and enhanced following its activation, suggesting this kinase regulates neurosteroid modulation. In comparison, neurosteroid potentiation was reduced at α1β3S408A,S409Aγ2L receptors, and unaltered by PKC inhibitors or activators, indicating that phosphorylation of β3 subunits is important for regulating neurosteroid activity. To determine whether extrasynaptic-type GABAA receptors were similarly modulated, α4β3δ and α4β3S408A,S409Aδ receptors were investigated. Neurosteroid potentiation was reduced at both receptors by the kinase inhibitor staurosporine. By contrast, neurosteroid-mediated potentiation at α4S443Aβ3S408A,S409Aδ receptors was unaffected by protein kinase inhibition, strongly suggesting that phosphorylation of α4 and β3 subunits is required for regulating neurosteroid activity at extrasynaptic receptors. Western blot analyses revealed that neurosteroids increased phosphorylation of β3S408,S409 implying that a reciprocal pathway exists for neurosteroids to modulate phosphorylation of GABAA receptors. Overall, these findings provide important insight into the regulation of GABAA receptors in vivo, and into the mechanisms by which GABAergic inhibitory transmission may be simultaneously tuned by

  6. A dopamine D2 receptor mutant capable of G protein-mediated signaling but deficient in arrestin binding.

    PubMed

    Lan, Hongxiang; Liu, Yong; Bell, Michal I; Gurevich, Vsevolod V; Neve, Kim A

    2009-01-01

    Arrestins mediate G protein-coupled receptor desensitization, internalization, and signaling. Dopamine D(2) and D(3) receptors have similar structures but distinct characteristics of interaction with arrestins. The goals of this study were to compare arrestin-binding determinants in D(2) and D(3) receptors other than phosphorylation sites and to create a D(2) receptor that is deficient in arrestin binding. We first assessed the ability of purified arrestins to bind to glutathione transferase (GST) fusion proteins containing the receptor third intracellular loops (IC3). Arrestin3 bound to IC3 of both D(2) and D(3) receptors, with the affinity and localization of the binding site indistinguishable between the receptor subtypes. Mutagenesis of the GST-IC3 fusion proteins identified an important determinant of the binding of arrestin3 in the N-terminal region of IC3. Alanine mutations of this determinant (IYIV212-215) in the full-length D(2) receptor generated a signaling-biased receptor with intact ligand binding and G-protein coupling and activation, but deficient in receptor-mediated arrestin3 translocation to the membrane, agonist-induced receptor internalization, and agonist-induced desensitization in human embryonic kidney 293 cells. This mutation also decreased arrestin-dependent activation of extracellular signal-regulated kinases. The finding that nonphosphorylated D(2)-IC3 and D(3)-IC3 have similar affinity for arrestin is consistent with previous suggestions that the differential effects of D(2) and D(3) receptor activation on membrane translocation of arrestin and receptor internalization are due, at least in part, to differential phosphorylation of the receptors. In addition, these results imply that the sequence IYIV212-215 at the N terminus of IC3 of the D(2) receptor is a key element of the arrestin binding site.

  7. Opioid and GABAB receptors differentially couple to an adenylyl cyclase/protein kinase A downstream effector after chronic morphine treatment

    PubMed Central

    Bagley, Elena E.

    2014-01-01

    Opioids are intensely addictive, and cessation of their chronic use is associated with a highly aversive withdrawal syndrome. A cellular hallmark of withdrawal is an opioid sensitive protein kinase A-dependent increase in GABA transporter-1 (GAT-1) currents in periaqueductal gray (PAG) neurons. Elevated GAT-1 activity directly increases GABAergic neuronal excitability and synaptic GABA release, which will enhance GABAergic inhibition of PAG output neurons. This reduced activity of PAG output neurons to several brain regions, including the hypothalamus and medulla, contributes to many of the PAG-mediated signs of opioid withdrawal. The GABAB receptor agonist baclofen reduces some of the PAG mediated signs of opioid withdrawal. Like the opioid receptors the GABAB receptor is a Gi/Go coupled G-protein coupled receptor. This suggests it could be modulating GAT-1 activity in PAG neurons through its inhibition of the adenylyl cyclase/protein kinase A pathway. Opioid modulation of the GAT-1 activity can be detected by changes in the reversal potential of opioid membrane currents. We found that when opioids are reducing the GAT-1 cation conductance and increasing the GIRK conductance the opioid agonist reversal potential is much more negative than Ek. Using this approach for GABAB receptors we show that the GABAB receptor agonist, baclofen, does not couple to inhibition of GAT-1 currents during opioid withdrawal. It is possible this differential signaling of the two Gi/Go coupled G-protein coupled receptors is due to the strong compartmentalization of the GABAB receptor that does not favor signaling to the adenylyl cyclase/protein kinase A/GAT-1 pathway. This highlights the importance of studying the effects of G-protein coupled receptors in native tissue with endogenous G-protein coupled receptors and the full complement of relevant proteins and signaling molecules. This study suggests that baclofen reduces opioid withdrawal symptoms through a non-GAT-1 effector. PMID

  8. Opioid and GABAB receptors differentially couple to an adenylyl cyclase/protein kinase A downstream effector after chronic morphine treatment.

    PubMed

    Bagley, Elena E

    2014-01-01

    Opioids are intensely addictive, and cessation of their chronic use is associated with a highly aversive withdrawal syndrome. A cellular hallmark of withdrawal is an opioid sensitive protein kinase A-dependent increase in GABA transporter-1 (GAT-1) currents in periaqueductal gray (PAG) neurons. Elevated GAT-1 activity directly increases GABAergic neuronal excitability and synaptic GABA release, which will enhance GABAergic inhibition of PAG output neurons. This reduced activity of PAG output neurons to several brain regions, including the hypothalamus and medulla, contributes to many of the PAG-mediated signs of opioid withdrawal. The GABAB receptor agonist baclofen reduces some of the PAG mediated signs of opioid withdrawal. Like the opioid receptors the GABAB receptor is a Gi/Go coupled G-protein coupled receptor. This suggests it could be modulating GAT-1 activity in PAG neurons through its inhibition of the adenylyl cyclase/protein kinase A pathway. Opioid modulation of the GAT-1 activity can be detected by changes in the reversal potential of opioid membrane currents. We found that when opioids are reducing the GAT-1 cation conductance and increasing the GIRK conductance the opioid agonist reversal potential is much more negative than E k . Using this approach for GABAB receptors we show that the GABAB receptor agonist, baclofen, does not couple to inhibition of GAT-1 currents during opioid withdrawal. It is possible this differential signaling of the two Gi/Go coupled G-protein coupled receptors is due to the strong compartmentalization of the GABAB receptor that does not favor signaling to the adenylyl cyclase/protein kinase A/GAT-1 pathway. This highlights the importance of studying the effects of G-protein coupled receptors in native tissue with endogenous G-protein coupled receptors and the full complement of relevant proteins and signaling molecules. This study suggests that baclofen reduces opioid withdrawal symptoms through a non-GAT-1 effector.

  9. Coupling of G Proteins to Reconstituted Monomers and Tetramers of the M2 Muscarinic Receptor*

    PubMed Central

    Redka, Dar'ya S.; Morizumi, Takefumi; Elmslie, Gwendolynne; Paranthaman, Pranavan; Shivnaraine, Rabindra V.; Ellis, John; Ernst, Oliver P.; Wells, James W.

    2014-01-01

    G protein-coupled receptors can be reconstituted as monomers in nanodiscs and as tetramers in liposomes. When reconstituted with G proteins, both forms enable an allosteric interaction between agonists and guanylyl nucleotides. Both forms, therefore, are candidates for the complex that controls signaling at the level of the receptor. To identify the biologically relevant form, reconstituted monomers and tetramers of the purified M2 muscarinic receptor were compared with muscarinic receptors in sarcolemmal membranes for the effect of guanosine 5′-[β,γ-imido]triphosphate (GMP-PNP) on the inhibition of N-[3H]methylscopolamine by the agonist oxotremorine-M. With monomers, a stepwise increase in the concentration of GMP-PNP effected a lateral, rightward shift in the semilogarithmic binding profile (i.e. a progressive decrease in the apparent affinity of oxotremorine-M). With tetramers and receptors in sarcolemmal membranes, GMP-PNP effected a vertical, upward shift (i.e. an apparent redistribution of sites from a state of high affinity to one of low affinity with no change in affinity per se). The data were analyzed in terms of a mechanistic scheme based on a ligand-regulated equilibrium between uncoupled and G protein-coupled receptors (the “ternary complex model”). The model predicts a rightward shift in the presence of GMP-PNP and could not account for the effects at tetramers in vesicles or receptors in sarcolemmal membranes. Monomers present a special case of the model in which agonists and guanylyl nucleotides interact within a complex that is both constitutive and stable. The results favor oligomers of the M2 receptor over monomers as the biologically relevant state for coupling to G proteins. PMID:25023280

  10. Function of the cytoplasmic tail of human calcitonin receptor-like receptor in complex with receptor activity-modifying protein 2

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kuwasako, Kenji, E-mail: kuwasako@fc.miyazaki-u.ac.jp; Kitamura, Kazuo; Nagata, Sayaka

    2010-02-12

    Receptor activity-modifying protein 2 (RAMP2) enables calcitonin receptor-like receptor (CRLR) to form an adrenomedullin (AM)-specific receptor. Here we investigated the function of the cytoplasmic C-terminal tail (C-tail) of human (h)CRLR by co-transfecting its C-terminal mutants into HEK-293 cells stably expressing hRAMP2. Deleting the C-tail from CRLR disrupted AM-evoked cAMP production or receptor internalization, but did not affect [{sup 125}I]AM binding. We found that CRLR residues 428-439 are required for AM-evoked cAMP production, though deleting this region had little effect on receptor internalization. Moreover, pretreatment with pertussis toxin (100 ng/mL) led to significant increases in AM-induced cAMP production via wild-type CRLR/RAMP2more » complexes. This effect was canceled by deleting CRLR residues 454-457, suggesting Gi couples to this region. Flow cytometric analysis revealed that CRLR truncation mutants lacking residues in the Ser/Thr-rich region extending from Ser{sup 449} to Ser{sup 467} were unable to undergo AM-induced receptor internalization and, in contrast to the effect on wild-type CRLR, overexpression of GPCR kinases-2, -3 and -4 failed to promote internalization of CRLR mutants lacking residues 449-467. Thus, the hCRLR C-tail is crucial for AM-evoked cAMP production and internalization of the CRLR/RAMP2, while the receptor internalization is dependent on the aforementioned GPCR kinases, but not Gs coupling.« less

  11. The C Terminus of the Saccharomyces cerevisiae α-Factor Receptor Contributes to the Formation of Preactivation Complexes with Its Cognate G Protein

    PubMed Central

    Dosil, Mercedes; Schandel, Kimberly A.; Gupta, Ekta; Jenness, Duane D.; Konopka, James B.

    2000-01-01

    Binding of the α-factor pheromone to its G-protein-coupled receptor (encoded by STE2) activates the mating pathway in MATa yeast cells. To investigate whether specific interactions between the receptor and the G protein occur prior to ligand binding, we analyzed dominant-negative mutant receptors that compete with wild-type receptors for G proteins, and we analyzed the ability of receptors to suppress the constitutive signaling activity of mutant Gα subunits in an α-factor-independent manner. Although the amino acid substitution L236H in the third intracellular loop of the receptor impairs G-protein activation, this substitution had no influence on the ability of the dominant-negative receptors to sequester G proteins or on the ability of receptors to suppress the GPA1-A345T mutant Gα subunit. In contrast, removal of the cytoplasmic C-terminal domain of the receptor eliminated both of these activities even though the C-terminal domain is unnecessary for G-protein activation. Moreover, the α-factor-independent signaling activity of ste2-P258L mutant receptors was inhibited by the coexpression of wild-type receptors but not by coexpression of truncated receptors lacking the C-terminal domain. Deletion analysis suggested that the distal half of the C-terminal domain is critical for sequestration of G proteins. The C-terminal domain was also found to influence the affinity of the receptor for α-factor in cells lacking G proteins. These results suggest that the C-terminal cytoplasmic domain of the α-factor receptor, in addition to its role in receptor downregulation, promotes the formation of receptor–G-protein preactivation complexes. PMID:10866688

  12. Metabotropic glutamate receptor 5 couples cellular prion protein to intracellular signalling in Alzheimer’s disease

    PubMed Central

    Haas, Laura T.; Salazar, Santiago V.; Kostylev, Mikhail A.; Um, Ji Won; Kaufman, Adam C.

    2016-01-01

    Alzheimer’s disease-related phenotypes in mice can be rescued by blockade of either cellular prion protein or metabotropic glutamate receptor 5. We sought genetic and biochemical evidence that these proteins function cooperatively as an obligate complex in the brain. We show that cellular prion protein associates via transmembrane metabotropic glutamate receptor 5 with the intracellular protein mediators Homer1b/c, calcium/calmodulin-dependent protein kinase II, and the Alzheimer’s disease risk gene product protein tyrosine kinase 2 beta. Coupling of cellular prion protein to these intracellular proteins is modified by soluble amyloid-β oligomers, by mouse brain Alzheimer’s disease transgenes or by human Alzheimer’s disease pathology. Amyloid-β oligomer-triggered phosphorylation of intracellular protein mediators and impairment of synaptic plasticity in vitro requires Prnp–Grm5 genetic interaction, being absent in transheterozygous loss-of-function, but present in either single heterozygote. Importantly, genetic coupling between Prnp and Grm5 is also responsible for signalling, for survival and for synapse loss in Alzheimer’s disease transgenic model mice. Thus, the interaction between metabotropic glutamate receptor 5 and cellular prion protein has a central role in Alzheimer’s disease pathogenesis, and the complex is a potential target for disease-modifying intervention. PMID:26667279

  13. G Protein and β-Arrestin Signaling Bias at the Ghrelin Receptor*

    PubMed Central

    Evron, Tama; Peterson, Sean M.; Urs, Nikhil M.; Bai, Yushi; Rochelle, Lauren K.; Caron, Marc G.; Barak, Larry S.

    2014-01-01

    The G protein-coupled ghrelin receptor GHSR1a is a potential pharmacological target for treating obesity and addiction because of the critical role ghrelin plays in energy homeostasis and dopamine-dependent reward. GHSR1a enhances growth hormone release, appetite, and dopamine signaling through Gq/11, Gi/o, and G12/13 as well as β-arrestin-based scaffolds. However, the contribution of individual G protein and β-arrestin pathways to the diverse physiological responses mediated by ghrelin remains unknown. To characterize whether a signaling bias occurs for GHSR1a, we investigated ghrelin signaling in a number of cell-based assays, including Ca2+ mobilization, serum response factor response element, stress fiber formation, ERK1/2 phosphorylation, and β-arrestin translocation, utilizing intracellular second loop and C-tail mutants of GHSR1a. We observed that GHSR1a and β-arrestin rapidly form metastable plasma membrane complexes following exposure to an agonist, but replacement of the GHSR1a C-tail by the tail of the vasopressin 2 receptor greatly stabilizes them, producing complexes observable on the plasma membrane and also in endocytic vesicles. Mutations of the contiguous conserved amino acids Pro-148 and Leu-149 in the GHSR1a intracellular second loop generate receptors with a strong bias to G protein and β-arrestin, respectively, supporting a role for conformation-dependent signaling bias in the wild-type receptor. Our results demonstrate more balance in GHSR1a-mediated ERK signaling from G proteins and β-arrestin but uncover an important role for β-arrestin in RhoA activation and stress fiber formation. These findings suggest an avenue for modulating drug abuse-associated changes in synaptic plasticity via GHSR1a and indicate the development of GHSR1a-biased ligands as a promising strategy for selectively targeting downstream signaling events. PMID:25261469

  14. Using constitutive activity to define appropriate high-throughput screening assays for orphan g protein-coupled receptors.

    PubMed

    Ngo, Tony; Coleman, James L J; Smith, Nicola J

    2015-01-01

    Orphan G protein-coupled receptors represent an underexploited resource for drug discovery but pose a considerable challenge for assay development because their cognate G protein signaling pathways are often unknown. In this methodological chapter, we describe the use of constitutive activity, that is, the inherent ability of receptors to couple to their cognate G proteins in the absence of ligand, to inform the development of high-throughput screening assays for a particular orphan receptor. We specifically focus on a two-step process, whereby constitutive G protein coupling is first determined using yeast Gpa1/human G protein chimeras linked to growth and β-galactosidase generation. Coupling selectivity is then confirmed in mammalian cells expressing endogenous G proteins and driving accumulation of transcription factor-fused luciferase reporters specific to each of the classes of G protein. Based on these findings, high-throughput screening campaigns can be performed on the already miniaturized mammalian reporter system.

  15. Human Coronavirus HKU1 Spike Protein Uses O-Acetylated Sialic Acid as an Attachment Receptor Determinant and Employs Hemagglutinin-Esterase Protein as a Receptor-Destroying Enzyme.

    PubMed

    Huang, Xingchuan; Dong, Wenjuan; Milewska, Aleksandra; Golda, Anna; Qi, Yonghe; Zhu, Quan K; Marasco, Wayne A; Baric, Ralph S; Sims, Amy C; Pyrc, Krzysztof; Li, Wenhui; Sui, Jianhua

    2015-07-01

    Human coronavirus (hCoV) HKU1 is one of six hCoVs identified to date and the only one with an unidentified cellular receptor. hCoV-HKU1 encodes a hemagglutinin-esterase (HE) protein that is unique to the group a betacoronaviruses (group 2a). The function of HKU1-HE remains largely undetermined. In this study, we examined binding of the S1 domain of hCoV-HKU1 spike to a panel of cells and found that the S1 could specifically bind on the cell surface of a human rhabdomyosarcoma cell line, RD. Pretreatment of RD cells with neuraminidase (NA) and trypsin greatly reduced the binding, suggesting that the binding was mediated by sialic acids on glycoproteins. However, unlike other group 2a CoVs, e.g., hCoV-OC43, for which 9-O-acetylated sialic acid (9-O-Ac-Sia) serves as a receptor determinant, HKU1-S1 bound with neither 9-O-Ac-Sia-containing glycoprotein(s) nor rat and mouse erythrocytes. Nonetheless, the HKU1-HE was similar to OC43-HE, also possessed sialate-O-acetylesterase activity, and acted as a receptor-destroying enzyme (RDE) capable of eliminating the binding of HKU1-S1 to RD cells, whereas the O-acetylesterase-inactive HKU1-HE mutant lost this capacity. Using primary human ciliated airway epithelial (HAE) cell cultures, the only in vitro replication model for hCoV-HKU1 infection, we confirmed that pretreatment of HAE cells with HE but not the enzymatically inactive mutant blocked hCoV-HKU1 infection. These results demonstrate that hCoV-HKU1 exploits O-Ac-Sia as a cellular attachment receptor determinant to initiate the infection of host cells and that its HE protein possesses the corresponding sialate-O-acetylesterase RDE activity. Human coronaviruses (hCoV) are important human respiratory pathogens. Among the six hCoVs identified to date, only hCoV-HKU1 has no defined cellular receptor. It is also unclear whether hemagglutinin-esterase (HE) protein plays a role in viral entry. In this study, we found that, similarly to other members of the group 2a CoVs, sialic

  16. Addition of bone morphogenetic protein type 2 to ascorbate and β-glycerophosphate supplementation did not enhance osteogenic differentiation of human adipose-derived stem cells.

    PubMed

    Cruz, Ariadne Cristiane Cabral; Silva, Mariana Lúcia; Caon, Thiago; Simões, Cláudia Maria Oliveira

    2012-01-01

    Bone morphogenetic protein type 2 (BMP-2) is a potent local factor, which promotes bone formation and has been used as an osteogenic supplement for mesenchymal stem cells. This study evaluated the effect of a recombinant BMP-2 as well as the endogenous BMP-4 and BMP-7 in the osteogenic differentiation of adipose-derived stem cells (ASCs) in medium supplemented with ascorbate and β-glycerophosphate. Human ASCs were treated with osteogenic medium in the presence (ASCs+OM+BMP-2) or absence (ASCs+OM) of BMP-2. The alkaline phosphatase (ALP) activity was determined and the extracellular matrix mineralization was evaluated by Von Kossa staining and calcium quantification. The expressions of BMP-4, BMP-7, Smad1, Smad4, and phosphorylated Smad1/5/8 were analyzed by western blotting. Relative mRNA expressions of Smad1, BMP receptor type II (BMPR-II), osteonectin, and osteocalcin were evaluated by qPCR. ASCs+OM demonstrated the highest expression of BMP-4 and BMP-7 at days 21 and 7, respectively, the highest levels of BMPR-II mRNA expression at day 28, and the highest levels of Smad1 mRNA at days 14 and 28. ASCs+OM+BMP-2 demonstrated the highest levels of Smad1 mRNA expression at days 1, 7, and 21, the highest expression of Smad1 at day 7, the highest expression of Smad4 at day 14, the highest ALP activity at days 14 and 21, and expression of phosphorylated Smad1/5/8 at day 7. ASCs+OM and ASCs+OM+BMP2 showed similar ALP activity at days 7 and 28, similar osteonectin and osteocalcin mRNA expression at all time periods, and similar calcium depositions at all time periods. We concluded that human ASCs expressed endogenous BMP-4 and BMP-7. Moreover, the supplementation of ASCs with BMP-2 did not increase the level of osteogenic markers in the initial (ALP activity), intermediate (osteonectin and osteocalcin), or final (calcium deposition) phases, suggesting that the exogenous addition of BMP-2 did not improve the in vitro osteogenesis process of human ASCs.

  17. Addition of bone morphogenetic protein type 2 to ascorbate and β-glycerophosphate supplementation did not enhance osteogenic differentiation of human adipose-derived stem cells

    PubMed Central

    CRUZ, Ariadne Cristiane Cabral; SILVA, Mariana Lúcia; CAON, Thiago; SIMÕES, Cláudia Maria Oliveira

    2012-01-01

    Bone morphogenetic protein type 2 (BMP-2) is a potent local factor, which promotes bone formation and has been used as an osteogenic supplement for mesenchymal stem cells. Objectives This study evaluated the effect of a recombinant BMP-2 as well as the endogenous BMP-4 and BMP-7 in the osteogenic differentiation of adipose-derived stem cells (ASCs) in medium supplemented with ascorbate and β-glycerophosphate. Material and Methods Human ASCs were treated with osteogenic medium in the presence (ASCs+OM+BMP-2) or absence (ASCs+OM) of BMP-2. The alkaline phosphatase (ALP) activity was determined and the extracellular matrix mineralization was evaluated by Von Kossa staining and calcium quantification. The expressions of BMP-4, BMP-7, Smad1, Smad4, and phosphorylated Smad1/5/8 were analyzed by western blotting. Relative mRNA expressions of Smad1, BMP receptor type II (BMPR-II), osteonectin, and osteocalcin were evaluated by qPCR. Results: ASCs+OM demonstrated the highest expression of BMP-4 and BMP-7 at days 21 and 7, respectively, the highest levels of BMPR-II mRNA expression at day 28, and the highest levels of Smad1 mRNA at days 14 and 28. ASCs+OM+BMP-2 demonstrated the highest levels of Smad1 mRNA expression at days 1, 7, and 21, the highest expression of Smad1 at day 7, the highest expression of Smad4 at day 14, the highest ALP activity at days 14 and 21, and expression of phosphorylated Smad1/5/8 at day 7. ASCs+OM and ASCs+OM+BMP2 showed similar ALP activity at days 7 and 28, similar osteonectin and osteocalcin mRNA expression at all time periods, and similar calcium depositions at all time periods. Conclusions We concluded that human ASCs expressed endogenous BMP-4 and BMP-7. Moreover, the supplementation of ASCs with BMP-2 did not increase the level of osteogenic markers in the initial (ALP activity), intermediate (osteonectin and osteocalcin), or final (calcium deposition) phases, suggesting that the exogenous addition of BMP-2 did not improve the in vitro

  18. Synergistic effects of dimethyloxallyl glycine and recombinant human bone morphogenetic protein-2 on repair of critical-sized bone defects in rats

    PubMed Central

    Qi, Xin; Liu, Yang; Ding, Zhen-yu; Cao, Jia-qing; Huang, Jing-huan; Zhang, Jie-yuan; Jia, Wei-tao; Wang, Jing; Liu, Chang-sheng; Li, Xiao-lin

    2017-01-01

    In bone remodeling, osteogenesis is closely coupled to angiogenesis. Bone tissue engineering using multifunctional bioactive materials is a promising technique which has the ability to simultaneously stimulate osteogenesis and angiogenesis for repair of bone defects. We developed mesoporous bioactive glass (MBG)-doped poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) composite scaffolds as delivery vehicle. Two bioactive molecules, dimethyloxalylglycine (DMOG), a small-molecule angiogenic drug, and recombinant human bone morphogenetic protein-2 (rhBMP-2), an osteoinductive growth factor, were co-incorporated into the scaffold. The synergistic effects of DMOG and rhBMP-2 released in the composite scaffolds on osteogenic and angiogenic differentiation of hBMSCs were investigated using real-time quantitative polymerase chain reaction and western blotting. Moreover, in vivo studies were conducted to observe bone regeneration and vascular formation of critical-sized bone defects in rats using micro-computed tomography, histological analyses, Microfil® perfusion, fluorescence labeling, and immunohistochemical analysis. The results showed that DMOG and rhBMP-2 released in the MBG-PHBHHx scaffolds did exert synergistic effects on the osteogenic and angiogenic differentiation of hBMSCs. Moreover, DMOG and rhBMP-2 produced significant increases in newly-formed bone and neovascularization of calvarial bone defects in rats. It is concluded that the co-delivery strategy of both rhBMP-2 and DMOG can significantly improve the critical-sized bone regeneration. PMID:28230059

  19. Synergistic effects of dimethyloxallyl glycine and recombinant human bone morphogenetic protein-2 on repair of critical-sized bone defects in rats

    NASA Astrophysics Data System (ADS)

    Qi, Xin; Liu, Yang; Ding, Zhen-Yu; Cao, Jia-Qing; Huang, Jing-Huan; Zhang, Jie-Yuan; Jia, Wei-Tao; Wang, Jing; Liu, Chang-Sheng; Li, Xiao-Lin

    2017-02-01

    In bone remodeling, osteogenesis is closely coupled to angiogenesis. Bone tissue engineering using multifunctional bioactive materials is a promising technique which has the ability to simultaneously stimulate osteogenesis and angiogenesis for repair of bone defects. We developed mesoporous bioactive glass (MBG)-doped poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) composite scaffolds as delivery vehicle. Two bioactive molecules, dimethyloxalylglycine (DMOG), a small-molecule angiogenic drug, and recombinant human bone morphogenetic protein-2 (rhBMP-2), an osteoinductive growth factor, were co-incorporated into the scaffold. The synergistic effects of DMOG and rhBMP-2 released in the composite scaffolds on osteogenic and angiogenic differentiation of hBMSCs were investigated using real-time quantitative polymerase chain reaction and western blotting. Moreover, in vivo studies were conducted to observe bone regeneration and vascular formation of critical-sized bone defects in rats using micro-computed tomography, histological analyses, Microfil® perfusion, fluorescence labeling, and immunohistochemical analysis. The results showed that DMOG and rhBMP-2 released in the MBG-PHBHHx scaffolds did exert synergistic effects on the osteogenic and angiogenic differentiation of hBMSCs. Moreover, DMOG and rhBMP-2 produced significant increases in newly-formed bone and neovascularization of calvarial bone defects in rats. It is concluded that the co-delivery strategy of both rhBMP-2 and DMOG can significantly improve the critical-sized bone regeneration.

  20. Inactivation of bone morphogenetic protein 2 may predict clinical outcome and poor overall survival for renal cell carcinoma through epigenetic pathways

    PubMed Central

    Mitsui, Yozo; Hirata, Hiroshi; Arichi, Naoko; Hiraki, Miho; Yasumoto, Hiroaki; Chang, Inik; Fukuhara, Shinichiro; Yamamura, Soichiro; Shahryari, Varahram; Deng, Guoren; Saini, Sharanjot; Majid, Shahana; Dahiya, Rajvir; Tanaka, Yuichiro; Shiina, Hiroaki

    2015-01-01

    We investigated whether impaired regulation of bone morphogenetic protein-2 (BMP-2) via epigenetic pathways is associated with renal cell carcinoma (RCC) pathogenesis. Expression and CpG methylation of the BMP-2 gene were analyzed using RCC cell lines, and 96 matched RCC and normal renal tissues. We also performed functional analysis using BMP-2 restored RCC cells. A significant association of BMP-2 mRNA expression was also found with advanced tumor stage and lymph node involvement, while lower BMP-2 mRNA expression was significantly associated with poor overall survival after radical nephrectomy. In RCC cells, BMP-2 restoration significantly inhibited cell proliferation, migration, invasion, and colony formation. In addition, BMP-2 overexpression induced p21WAF1/CIP1 and p27KIP1 expression, and cellular apoptosis in RCC cells. BMP-2 mRNA expression was significantly enhanced in RCC cells by 5-aza-2′-deoxycitidine treatment. The prevalence of BMP-2 promoter methylation was significantly greater and BMP-2 mRNA expression was significantly lower in RCC samples as compared to normal kidney samples. Furthermore, a significant correlation was found between BMP-2 promoter methylation and mRNA transcription in tumors. Aberrant BMP-2 methylation and the resultant loss of BMP-2 expression may be a useful molecular marker for designing improved diagnostic and therapeutic strategies for RCC. PMID:25797254

  1. Molecular recognition of parathyroid hormone by its G protein-coupled receptor

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Pioszak, Augen A.; Xu, H. Eric

    Parathyroid hormone (PTH) is central to calcium homeostasis and bone maintenance in vertebrates, and as such it has been used for treating osteoporosis. It acts primarily by binding to its receptor, PTH1R, a member of the class B G protein-coupled receptor (GPCR) family that also includes receptors for glucagon, calcitonin, and other therapeutically important peptide hormones. Despite considerable interest and much research, determining the structure of the receptor-hormone complex has been hindered by difficulties in purifying the receptor and obtaining diffraction-quality crystals. Here, we present a method for expression and purification of the extracellular domain (ECD) of human PTH1R engineeredmore » as a maltose-binding protein (MBP) fusion that readily crystallizes. The 1.95-{angstrom} structure of PTH bound to the MBP-PTH1R-ECD fusion reveals that PTH docks as an amphipathic helix into a central hydrophobic groove formed by a three-layer {alpha}-{beta}-{beta}{alpha} fold of the PTH1R ECD, resembling a hot dog in a bun. Conservation in the ECD scaffold and the helical structure of peptide hormones emphasizes this hot dog model as a general mechanism of hormone recognition common to class B GPCRs. Our findings reveal critical insights into PTH actions and provide a rational template for drug design that targets this hormone signaling pathway.« less

  2. Highly aligned porous Ti scaffold coated with bone morphogenetic protein-loaded silica/chitosan hybrid for enhanced bone regeneration.

    PubMed

    Jung, Hyun-Do; Yook, Se-Won; Han, Cheol-Min; Jang, Tae-Sik; Kim, Hyoun-Ee; Koh, Young-Hag; Estrin, Yuri

    2014-07-01

    Porous Ti has been widely investigated for orthopedic and dental applications on account of their ability to promote implant fixation via bone ingrowth into pores. In this study, highly aligned porous Ti scaffolds coated with a bone morphogenetic protein (BMP)-loaded silica/chitosan hybrid were produced, and their bone regeneration ability was evaluated by in vivo animal experiments. Reverse freeze casting allowed for the creation of highly aligned pores, resulting in a high compressive strength of 254 ± 21 MPa of the scaffolds at a porosity level of ∼51 vol %. In addition, a BMP-loaded silica/chitosan hybrid coating layer with a thickness of ∼1 μm was uniformly deposited on the porous Ti scaffold, which enabled the sustained release of the BMP over a prolonged period of time up to 26 days. The cumulative amount of the BMP released was ∼4 μg, which was much higher than that released from the specimen without a hybrid coating layer. In addition, the bone regeneration ability of the porous Ti scaffold with a BMP-loaded silica/chitosan coating layer was examined by in vivo animal testing using a rabbit calvarial defect model and compared with those of the as-produced porous Ti scaffold and porous Ti scaffold with a silica/chitosan coating layer. After 4 weeks of healing, the specimen coated with a BMP-loaded silica/chitosan hybrid showed a much higher bone regeneration volume (∼36%) than the as-produced specimen (∼15%) (p < 0.005) and even the specimen coated with a silica/chitosan hybrid (∼25%) (p < 0.05). © 2013 Wiley Periodicals, Inc.

  3. The G-protein coupled estrogen receptor, GPER: The inside and inside-out story.

    PubMed

    Gaudet, H M; Cheng, S B; Christensen, E M; Filardo, E J

    2015-12-15

    GPER possesses structural and functional characteristics shared by members of the G-protein-coupled receptor (GPCR) superfamily, the largest class of plasma membrane receptors. This newly appreciated estrogen receptor is localized predominately within intracellular membranes in most, but not all, cell types and its surface expression is modulated by steroid hormones and during tissue injury. An intracellular staining pattern is not unique among GPCRs, which employ a diverse array of molecular mechanisms that restrict cell surface expression and effectively regulating receptor binding and activation. The finding that GPER displays an intracellular predisposition has created some confusion as the estrogen-inducible transcription factors, ERα and ERβ, also reside intracellularly, and has led to complex suggestions of receptor interaction. GPER undergoes constitutive retrograde trafficking from the plasma membrane to the endoplasmic reticulum and recent studies indicate its interaction with PDZ binding proteins that sort transmembrane receptors to synaptosomes and endosomes. Genetic targeting and selective ligand approaches as well as cell models that express GPER in the absence of ERs clearly supports GPER as a bonafide "stand alone" receptor. Here, the molecular details that regulate GPER action, its cell biological activities and its implicated roles in physiological and pathological processes are reviewed. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  4. Expression of nuclear receptor interacting proteins TIF-1, SUG-1, receptor interacting protein 140, and corepressor SMRT in tamoxifen-resistant breast cancer.

    PubMed

    Chan, C M; Lykkesfeldt, A E; Parker, M G; Dowsett, M

    1999-11-01

    Regulation of gene transcription as a consequence of steroid receptor-DNA interaction is mediated via nuclear receptor interacting proteins (RIPs), including coactivator or corepressor proteins, which interact with both the receptor and components of the basic transcriptional unit and vary between cell types. The aim of this study was to test the hypothesis that resistance of some breast carcinomas to tamoxifen was associated with inappropriate expression of some of these RIPs. Using Northern analysis, we observed no significant difference between the amount of either TIF-1 or SUG-1 mRNA expressed in parental MCF-7 and MCF-7 tamoxifen-resistant cell lines. However, the expression of RIP140 mRNA was lower in the resistant cell line and in the presence of estradiol, the level of RIP140 mRNA was higher in the resistant cells but not in the parental cells. In a cohort of 19 tamoxifen-resistant breast tumor samples, there was no significant difference in the level of the RIP140 and TIF-1 and corepressor SMRT mRNA compared with tamoxifen-treated tumors (n = 6) or untreated tumors (n = 21). However, SUG-1 mRNA was lower in resistant breast tumors. These data provide no support for increased expression of these RIPs or decreased expression of corepressor SMRT for being a mechanism for resistance of breast tumors to tamoxifen.

  5. Biotin-transfer from a trifunctional crosslinker for identification of cell surface receptors of soluble protein ligands

    PubMed Central

    Tremblay, Tammy-Lynn; Hill, Jennifer J.

    2017-01-01

    Here we describe a novel crosslinker and its application as a biotin-transfer reagent to identify cell surface receptors of soluble protein ligands on live cells. This crosslinker contains three functional groups: an aldehyde-reactive aminooxy group, a sulfhydryl, and a biotin (ASB). It is readily synthesized via a 3-step addition reaction using standard solid-phase peptide synthesis methods and commercially available intermediates, allowing access to laboratories without specialized synthetic chemistry capabilities. For the biotin-transfer method, ASB is linked to a protein ligand through the sulfhydryl group in a two-step process that allows the introduction of a disulfide bond between the ligand and the crosslinker. Incubation of the labelled ligand with oxidized live cells leads to the formation of crosslinks with aldehyde-containing glycans on the cell surface receptor. Subsequent reduction of the disulfide bond results in biotin transfer from the ligand to the cell surface receptor. Protein biotinylation that is mediated by ligand binding to its receptor is differentiated from background biotinylation events by comparison with a similarly labelled control protein using comparative proteomic mass spectrometry to quantify streptavidin-bound proteins. Using this method, we successfully identified the cell surface receptors of a peptide hormone, a monoclonal antibody, and a single-domain antibody-Fc fusion construct. PMID:28422167

  6. Proliferation of NS0 cells in protein-free medium: the role of cell-derived proteins, known growth factors and cellular receptors.

    PubMed

    Spens, Erika; Häggström, Lena

    2009-05-20

    NS0 cells proliferate without external supply of growth factors in protein-free media. We hypothesize that the cells produce their own factors to support proliferation. Understanding the mechanisms behind this autocrine regulation of proliferation may open for the novel approaches to improve animal cell processes. The following proteins were identified in NS0 conditioned medium (CM): cyclophilin A, cyclophilin B (CypB), cystatin C, D-dopachrome tautomerase, IL-25, isopentenyl-diphosphate delta-isomerase, macrophage migration inhibitory factor (MIF), beta(2)-microglobulin, Niemann pick type C2, secretory leukocyte protease inhibitor, thioredoxin-1, TNF-alpha, tumour protein translationally controlled 1 and ubiquitin. Further, cDNA microarray analysis indicated that the genes for IL-11, TNF receptor 6, TGF-beta receptor 1 and the IFN-gamma receptor were transcribed. CypB, IFN-alpha/beta/gamma, IL-11, IL-25, MIF, TGF-beta and TNF-alpha as well as the known growth factors EGF, IGF-I/II, IL-6, leukaemia inhibitory factor and oncostatin M (OSM) were excluded as involved in autocrine regulation of NS0 cell proliferation. The receptors for TGF-beta, IGF and OSM are however present in NS0 cell membranes since TGF-beta(1) caused cell death, and IGF-I/II and OSM improved cell growth. Even though no ligand was found, the receptor subunit gp130, active in signal transduction of the IL-6 like proteins, was shown to be essential for NS0 cells as demonstrated by siRNA gene silencing.

  7. REEPs Are Membrane Shaping Adapter Proteins That Modulate Specific G Protein-Coupled Receptor Trafficking by Affecting ER Cargo Capacity

    PubMed Central

    Ho, Vincent K.; Angelotti, Timothy

    2013-01-01

    Receptor expression enhancing proteins (REEPs) were identified by their ability to enhance cell surface expression of a subset of G protein-coupled receptors (GPCRs), specifically GPCRs that have proven difficult to express in heterologous cell systems. Further analysis revealed that they belong to the Yip (Ypt-interacting protein) family and that some REEP subtypes affect ER structure. Yip family comparisons have established other potential roles for REEPs, including regulation of ER-Golgi transport and processing/neuronal localization of cargo proteins. However, these other potential REEP functions and the mechanism by which they selectively enhance GPCR cell surface expression have not been clarified. By utilizing several REEP family members (REEP1, REEP2, and REEP6) and model GPCRs (α2A and α2C adrenergic receptors), we examined REEP regulation of GPCR plasma membrane expression, intracellular processing, and trafficking. Using a combination of immunolocalization and biochemical methods, we demonstrated that this REEP subset is localized primarily to ER, but not plasma membranes. Single cell analysis demonstrated that these REEPs do not specifically enhance surface expression of all GPCRs, but affect ER cargo capacity of specific GPCRs and thus their surface expression. REEP co-expression with α2 adrenergic receptors (ARs) revealed that this REEP subset interacts with and alter glycosidic processing of α2C, but not α2A ARs, demonstrating selective interaction with cargo proteins. Specifically, these REEPs enhanced expression of and interacted with minimally/non-glycosylated forms of α2C ARs. Most importantly, expression of a mutant REEP1 allele (hereditary spastic paraplegia SPG31) lacking the carboxyl terminus led to loss of this interaction. Thus specific REEP isoforms have additional intracellular functions besides altering ER structure, such as enhancing ER cargo capacity, regulating ER-Golgi processing, and interacting with select cargo proteins

  8. Reduced expression of G protein-coupled receptor kinases in schizophrenia but not in schizoaffective disorder

    PubMed Central

    Bychkov, ER; Ahmed, MR; Gurevich, VV; Benovic, JL; Gurevich, EV

    2011-01-01

    Alterations of multiple G protein-mediated signaling pathways are detected in schizophrenia. G protein-coupled receptor kinases (GRKs) and arrestins terminate signaling by G protein-coupled receptors exerting powerful influence on receptor functions. Modifications of arrestin and/or GRKs expression may contribute to schizophrenia pathology. Cortical expression of arrestins and GRKs was measured postmortem in control and subjects with schizophrenia or schizoaffective disorder. Additionally, arrestin/GRK expression was determined in elderly patients with schizophrenia and age-matched control. Patients with schizophrenia, but not schizoaffective disorder, displayed reduced concentration of arrestin and GRK mRNAs and GRK3 protein. Arrestins and GRK significantly decreased with age. In elderly patients, GRK6 was reduced, with other GRKs and arrestins unchanged. Reduced cortical concentration of GRKs in schizophrenia (resembling that in aging) may result in altered G protein-dependent signaling, thus contributing to prefrontal deficits in schizophrenia. The data suggest distinct molecular mechanisms underlying schizophrenia and schizoaffective disorder. PMID:21784156

  9. Sodium modulates opioid receptors through a membrane component different from G-proteins. Demonstration by target size analysis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ott, S.; Costa, T.; Herz, A.

    1988-07-25

    The target size for opioid receptor binding was studied after manipulations known to affect the interactions between receptor and GTP-binding regulatory proteins (G-proteins). Addition of GTP or its analogs to the binding reaction, exposure of intact cells to pertussis toxin prior to irradiation, or treatment of irradiated membranes with N-ethylmaleimide did not change the target size (approximately equal to 100 kDa) for opioid receptors in NG 108-15 cells and rat brain. These data suggest that the 100-kDa species does not include an active subunit of a G-protein or alternatively that GTP does not promote the dissociation of the receptor-G-protein complex.more » The presence of Na+ (100 mM) in the radioligand binding assay induced a biphasic decay curve for agonist binding and a flattening of the monoexponential decay curve for a partial agonist. In both cases the effect was explained by an irradiation-induced loss of the low affinity state of the opioid receptor produced by the addition of Na+. This suggests that an allosteric inhibitor that mediates the effect of sodium on the receptor is destroyed at low doses of irradiation, leaving receptors which are no longer regulated by sodium. The effect of Na+ on target size was slightly increased by the simultaneous addition of GTP but was not altered by pertussis toxin treatment. Thus, the sodium unit is distinct from G-proteins and may represent a new component of the opioid receptor complex. Assuming a simple bimolecular model of one Na+ unit/receptor, the size of this inhibitor can be measured as 168 kDa.« less

  10. Expression of the Grb2-related protein of the lymphoid system in B cell subsets enhances B cell antigen receptor signaling through mitogen-activated protein kinase pathways.

    PubMed

    Yankee, Thomas M; Solow, Sasha A; Draves, Kevin D; Clark, Edward A

    2003-01-01

    Adapter proteins play a critical role in regulating signals triggered by Ag receptor cross-linking. These small molecules link receptor proximal events with downstream signaling pathways. In this study, we explore the expression and function of the Grb2-related protein of the lymphoid system (GrpL)/Grb2-related adaptor downstream of Shc adapter protein in human B cells. GrpL is expressed in naive B cells and is down-regulated following B cell Ag receptor ligation. By contrast, germinal center and memory B cells express little or no GrpL. Using human B cell lines, we detected constitutive interactions between GrpL and B cell linker protein, Src homology (SH)2 domain-containing leukocyte protein of 76 kDa, hemopoietic progenitor kinase 1, and c-Cbl. The N-terminal SH3 domain of GrpL binds c-Cbl while the C-terminal SH3 domain binds B cell linker protein and SH2 domain-containing leukocyte protein of 76 kDa. Exogenous expression of GrpL in a GrpL-negative B cell line leads to enhanced Ag receptor-induced extracellular signal-related kinase and p38 mitogen-activated protein kinase phosphorylation. Thus, GrpL expression in human B cell subsets appears to regulate Ag receptor-mediated signaling events.

  11. Few residues within an extensive binding interface drive receptor interaction and determine the specificity of arrestin proteins.

    PubMed

    Vishnivetskiy, Sergey A; Gimenez, Luis E; Francis, Derek J; Hanson, Susan M; Hubbell, Wayne L; Klug, Candice S; Gurevich, Vsevolod V

    2011-07-08

    Arrestins bind active phosphorylated forms of G protein-coupled receptors, terminating G protein activation, orchestrating receptor trafficking, and redirecting signaling to alternative pathways. Visual arrestin-1 preferentially binds rhodopsin, whereas the two non-visual arrestins interact with hundreds of G protein-coupled receptor subtypes. Here we show that an extensive surface on the concave side of both arrestin-2 domains is involved in receptor binding. We also identified a small number of residues on the receptor binding surface of the N- and C-domains that largely determine the receptor specificity of arrestins. We show that alanine substitution of these residues blocks the binding of arrestin-1 to rhodopsin in vitro and of arrestin-2 and -3 to β2-adrenergic, M2 muscarinic cholinergic, and D2 dopamine receptors in intact cells, suggesting that these elements critically contribute to the energy of the interaction. Thus, in contrast to arrestin-1, where direct phosphate binding is crucial, the interaction of non-visual arrestins with their cognate receptors depends to a lesser extent on phosphate binding and more on the binding to non-phosphorylated receptor elements.

  12. Few Residues within an Extensive Binding Interface Drive Receptor Interaction and Determine the Specificity of Arrestin Proteins*

    PubMed Central

    Vishnivetskiy, Sergey A.; Gimenez, Luis E.; Francis, Derek J.; Hanson, Susan M.; Hubbell, Wayne L.; Klug, Candice S.; Gurevich, Vsevolod V.

    2011-01-01

    Arrestins bind active phosphorylated forms of G protein-coupled receptors, terminating G protein activation, orchestrating receptor trafficking, and redirecting signaling to alternative pathways. Visual arrestin-1 preferentially binds rhodopsin, whereas the two non-visual arrestins interact with hundreds of G protein-coupled receptor subtypes. Here we show that an extensive surface on the concave side of both arrestin-2 domains is involved in receptor binding. We also identified a small number of residues on the receptor binding surface of the N- and C-domains that largely determine the receptor specificity of arrestins. We show that alanine substitution of these residues blocks the binding of arrestin-1 to rhodopsin in vitro and of arrestin-2 and -3 to β2-adrenergic, M2 muscarinic cholinergic, and D2 dopamine receptors in intact cells, suggesting that these elements critically contribute to the energy of the interaction. Thus, in contrast to arrestin-1, where direct phosphate binding is crucial, the interaction of non-visual arrestins with their cognate receptors depends to a lesser extent on phosphate binding and more on the binding to non-phosphorylated receptor elements. PMID:21471193

  13. Palmitoylation and membrane cholesterol stabilize μ-opioid receptor homodimerization and G protein coupling

    PubMed Central

    2012-01-01

    Background A cholesterol-palmitoyl interaction has been reported to occur in the dimeric interface of the β2-adrenergic receptor crystal structure. We sought to investigate whether a similar phenomenon could be observed with μ-opioid receptor (OPRM1), and if so, to assess the role of cholesterol in this class of G protein-coupled receptor (GPCR) signaling. Results C3.55(170) was determined to be the palmitoylation site of OPRM1. Mutation of this Cys to Ala did not affect the binding of agonists, but attenuated receptor signaling and decreased cholesterol associated with the receptor signaling complex. In addition, both attenuation of receptor palmitoylation (by mutation of C3.55[170] to Ala) and inhibition of cholesterol synthesis (by treating the cells with simvastatin, a HMG-CoA reductase inhibitor) impaired receptor signaling, possibly by decreasing receptor homodimerization and Gαi2 coupling; this was demonstrated by co-immunoprecipitation, immunofluorescence colocalization and fluorescence resonance energy transfer (FRET) analyses. A computational model of the OPRM1 homodimer structure indicated that a specific cholesterol-palmitoyl interaction can facilitate OPRM1 homodimerization at the TMH4-TMH4 interface. Conclusions We demonstrate that C3.55(170) is the palmitoylation site of OPRM1 and identify a cholesterol-palmitoyl interaction in the OPRM1 complex. Our findings suggest that this interaction contributes to OPRM1 signaling by facilitating receptor homodimerization and G protein coupling. This conclusion is supported by computational modeling of the OPRM1 homodimer. PMID:22429589

  14. Cloning the promoter for transforming growth factor-beta type III receptor. Basal and conditional expression in fetal rat osteoblasts

    NASA Technical Reports Server (NTRS)

    Ji, C.; Chen, Y.; McCarthy, T. L.; Centrella, M.

    1999-01-01

    Transforming growth factor-beta binds to three high affinity cell surface molecules that directly or indirectly regulate its biological effects. The type III receptor (TRIII) is a proteoglycan that lacks significant intracellular signaling or enzymatic motifs but may facilitate transforming growth factor-beta binding to other receptors, stabilize multimeric receptor complexes, or segregate growth factor from activating receptors. Because various agents or events that regulate osteoblast function rapidly modulate TRIII expression, we cloned the 5' region of the rat TRIII gene to assess possible control elements. DNA fragments from this region directed high reporter gene expression in osteoblasts. Sequencing showed no consensus TATA or CCAAT boxes, whereas several nuclear factors binding sequences within the 3' region of the promoter co-mapped with multiple transcription initiation sites, DNase I footprints, gel mobility shift analysis, or loss of activity by deletion or mutation. An upstream enhancer was evident 5' proximal to nucleotide -979, and a silencer region occurred between nucleotides -2014 and -2194. Glucocorticoid sensitivity mapped between nucleotides -687 and -253, whereas bone morphogenetic protein 2 sensitivity co-mapped within the silencer region. Thus, the TRIII promoter contains cooperative basal elements and dispersed growth factor- and hormone-sensitive regulatory regions that can control TRIII expression by osteoblasts.

  15. The therapeutic potential of G-protein coupled receptors in Huntington's disease.

    PubMed

    Dowie, Megan J; Scotter, Emma L; Molinari, Emanuela; Glass, Michelle

    2010-11-01

    Huntington's disease is a late-onset autosomal dominant inherited neurodegenerative disease characterised by increased symptom severity over time and ultimately premature death. An expanded CAG repeat sequence in the huntingtin gene leads to a polyglutamine expansion in the expressed protein, resulting in complex dysfunctions including cellular excitotoxicity and transcriptional dysregulation. Symptoms include cognitive deficits, psychiatric changes and a movement disorder often referred to as Huntington's chorea, which involves characteristic involuntary dance-like writhing movements. Neuropathologically Huntington's disease is characterised by neuronal dysfunction and death in the striatum and cortex with an overall decrease in cerebral volume (Ho et al., 2001). Neuronal dysfunction begins prior to symptom presentation, and cells of particular vulnerability include the striatal medium spiny neurons. Huntington's is a devastating disease for patients and their families and there is currently no cure, or even an effective therapy for disease symptoms. G-protein coupled receptors are the most abundant receptor type in the central nervous system and are linked to complex downstream pathways, manipulation of which may have therapeutic application in many neurological diseases. This review will highlight the potential of G-protein coupled receptor drug targets as emerging therapies for Huntington's disease. Copyright © 2010 Elsevier Inc. All rights reserved.

  16. Crystal structures of botulinum neurotoxin DC in complex with its protein receptors synaptotagmin I and II.

    PubMed

    Berntsson, Ronnie Per-Arne; Peng, Lisheng; Svensson, Linda Marie; Dong, Min; Stenmark, Pål

    2013-09-03

    Botulinum neurotoxins (BoNTs) can cause paralysis at exceptionally low concentrations and include seven serotypes (BoNT/A-G). The chimeric BoNT/DC toxin has a receptor binding domain similar to the same region in BoNT/C. However, BoNT/DC does not share protein receptor with BoNT/C. Instead, it shares synaptotagmin (Syt) I and II as receptors with BoNT/B, despite their low sequence similarity. Here, we present the crystal structures of the binding domain of BoNT/DC in complex with the recognition domains of its protein receptors, Syt-I and Syt-II. The structures reveal that BoNT/DC possesses a Syt binding site, distinct from the established Syt-II binding site in BoNT/B. Structure-based mutagenesis further shows that hydrophobic interactions play a key role in Syt binding. The structures suggest that the BoNT/DC ganglioside binding sites are independent of the protein receptor binding site. Our results reveal the remarkable versatility in the receptor recognition of the BoNTs. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. Decreased expression of thyroid receptor-associated protein 220 in temporal lobe tissue of patients with refractory epilepsy

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Li Jinmei; Wang Xuefeng; Xi Zhiqin

    2006-10-06

    Purpose: TRAP220 (thyroid hormone receptor-associated protein) functions as a coactivator for nuclear receptors and stimulates transcription by recruiting the TRAP mediator complex to hormone responsive promoter regions. Thus, TRAP220 enhances the function of thyroid/steroid hormone receptors such as thyroid hormone and oestrogen receptors. This study investigated the expression of TRAP220 mRNA and protein level in epileptic brains comparing with human control. Methods: We examined the expression of TRAP220 mRNA and protein levels in temporal lobes from patients with chronic pharmacoresistant epilepsy who have undergone surgery. Results: Expression of TRAP220 mRNA and protein was shown to be decreased significantly in themore » temporal cortex of the patients with epilepsy. Conclusions: Our work showed that a decrease in TRAP220 mRNA and protein levels may be involved in the pathophysiology of epilepsy and may be associated with impairment of the brain caused by frequent seizures.« less

  18. An expressed sequence tag (EST) data mining strategy succeeding in the discovery of new G-protein coupled receptors.

    PubMed

    Wittenberger, T; Schaller, H C; Hellebrand, S

    2001-03-30

    We have developed a comprehensive expressed sequence tag database search method and used it for the identification of new members of the G-protein coupled receptor superfamily. Our approach proved to be especially useful for the detection of expressed sequence tag sequences that do not encode conserved parts of a protein, making it an ideal tool for the identification of members of divergent protein families or of protein parts without conserved domain structures in the expressed sequence tag database. At least 14 of the expressed sequence tags found with this strategy are promising candidates for new putative G-protein coupled receptors. Here, we describe the sequence and expression analysis of five new members of this receptor superfamily, namely GPR84, GPR86, GPR87, GPR90 and GPR91. We also studied the genomic structure and chromosomal localization of the respective genes applying in silico methods. A cluster of six closely related G-protein coupled receptors was found on the human chromosome 3q24-3q25. It consists of four orphan receptors (GPR86, GPR87, GPR91, and H963), the purinergic receptor P2Y1, and the uridine 5'-diphosphoglucose receptor KIAA0001. It seems likely that these receptors evolved from a common ancestor and therefore might have related ligands. In conclusion, we describe a data mining procedure that proved to be useful for the identification and first characterization of new genes and is well applicable for other gene families. Copyright 2001 Academic Press.

  19. A Retrospective Analysis of Complications Associated With Bone Morphogenetic Protein 2 in Anterior Lumbar Interbody Fusion.

    PubMed

    Hindoyan, Kevork; Tilan, Justin; Buser, Zorica; Cohen, Jeremiah R; Brodke, Darrel S; Youssef, Jim A; Park, Jong-Beom; Yoon, S Tim; Meisel, Hans-Joerg; Wang, Jeffrey C

    2017-04-01

    Retrospective review. The aim of our study was to quantify the frequency of complications associated with recombinant human bone morphogenetic protein 2 (rhBMP-2) use in anterior lumbar interbody fusion (ALIF). The orthopedic subset of the Medicare database (PearlDiver) was queried for this retrospective cohort study using International Statistical Classification of Diseases 9 (ICD-9) and Current Procedure Terminology (CPT) codes for ALIF procedures with and without rhBMP-2 between 2005 and 2010. Frequencies of complications and reoperations were then identified within 1 year from the index procedure. Complications included reoperations, pulmonary embolus, deep vein thrombosis, myocardial infarction, nerve-related complications, incision and drainage procedures, wound, sepsis, pneumonia, urinary tract infections, respiratory, heterotopic ossification, retrograde ejaculation, radiculopathy, and other medical complications. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to assess the statistical significance. We identified a total of 41 865 patients who had an ALIF procedure. A total of 14 384 patients received rhBMP-2 while 27 481 did not. Overall, 6016 (41.8%) complications within 1 year from surgery were noted within the group who received rhBMP-2 and 12 950 (47.1%) complications within 1 year from surgery were recorded in those who did not receive rhBMP-2 (OR = 0.81, CI = 0.77-0.84). Overall, exposure to rhBMP-2 was associated with significantly decreased odds of complications with exception to reoperation rates (0.9% rhBMP-2 vs 1.0% no rhBMP-2; OR = 0.88, CI = 0.71-1.09) and radiculopathy (4.4% rhBMP-2 vs 4.3% no rhBMP-2; OR = 1.02, CI = 0.93-1.13). The use of rhBMP-2 in patients undergoing ALIF procedure was associated with a significantly decreased rate of complications. Further studies are needed to elucidate a true incidence of complication.

  20. A Retrospective Analysis of Complications Associated With Bone Morphogenetic Protein 2 in Anterior Lumbar Interbody Fusion

    PubMed Central

    Hindoyan, Kevork; Tilan, Justin; Cohen, Jeremiah R.; Brodke, Darrel S.; Youssef, Jim A.; Park, Jong-Beom; Yoon, S. Tim; Meisel, Hans-Joerg; Wang, Jeffrey C.

    2017-01-01

    Study Design: Retrospective review. Objective: The aim of our study was to quantify the frequency of complications associated with recombinant human bone morphogenetic protein 2 (rhBMP-2) use in anterior lumbar interbody fusion (ALIF). Methods: The orthopedic subset of the Medicare database (PearlDiver) was queried for this retrospective cohort study using International Statistical Classification of Diseases 9 (ICD-9) and Current Procedure Terminology (CPT) codes for ALIF procedures with and without rhBMP-2 between 2005 and 2010. Frequencies of complications and reoperations were then identified within 1 year from the index procedure. Complications included reoperations, pulmonary embolus, deep vein thrombosis, myocardial infarction, nerve-related complications, incision and drainage procedures, wound, sepsis, pneumonia, urinary tract infections, respiratory, heterotopic ossification, retrograde ejaculation, radiculopathy, and other medical complications. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to assess the statistical significance. Results: We identified a total of 41 865 patients who had an ALIF procedure. A total of 14 384 patients received rhBMP-2 while 27 481 did not. Overall, 6016 (41.8%) complications within 1 year from surgery were noted within the group who received rhBMP-2 and 12 950 (47.1%) complications within 1 year from surgery were recorded in those who did not receive rhBMP-2 (OR = 0.81, CI = 0.77-0.84). Overall, exposure to rhBMP-2 was associated with significantly decreased odds of complications with exception to reoperation rates (0.9% rhBMP-2 vs 1.0% no rhBMP-2; OR = 0.88, CI = 0.71-1.09) and radiculopathy (4.4% rhBMP-2 vs 4.3% no rhBMP-2; OR = 1.02, CI = 0.93-1.13). Conclusions: The use of rhBMP-2 in patients undergoing ALIF procedure was associated with a significantly decreased rate of complications. Further studies are needed to elucidate a true incidence of complication. PMID:28507884