The Epidermal Growth Factor Receptor (EGFR) Promotes Uptake of Influenza A Viruses (IAV) into Host Cells

PubMed Central

Eierhoff, Thorsten; Hrincius, Eike R.; Rescher, Ursula; Ludwig, Stephan; Ehrhardt, Christina

2010-01-01

Influenza A viruses (IAV) bind to sialic-acids at cellular surfaces and enter cells by using endocytotic routes. There is evidence that this process does not occur constitutively but requires induction of specific cellular signals, including activation of PI3K that promotes virus internalization. This implies engagement of cellular signaling receptors during viral entry. Here, we present first indications for an interplay of IAV with receptor tyrosine kinases (RTKs). As representative RTK family-members the epidermal growth factor receptor (EGFR) and the c-Met receptor were studied. Modulation of expression or activity of both RTKs resulted in altered uptake of IAV, showing that these receptors transmit entry relevant signals upon virus binding. More detailed studies on EGFR function revealed that virus binding lead to clustering of lipid-rafts, suggesting that multivalent binding of IAV to cells induces a signaling platform leading to activation of EGFR and other RTKs that in turn facilitates IAV uptake. PMID:20844577

Aqueous and Ethanolic Valeriana officinalis Extracts Change the Binding of Ligands to Glutamate Receptors

PubMed Central

Del Valle-Mojica, Lisa M.; Cordero-Hernández, José M.; González-Medina, Giselle; Ramos-Vélez, Igmeris; Berríos-Cartagena, Nairimer; Torres-Hernández, Bianca A.; Ortíz, José G.

2011-01-01

The effects of two valerian extracts (aqueous and hydroalcoholic) were investigated through [3H]Glutamate ([3H]Glu) and [3H]Fluorowillardine ([3H]FW) receptor binding assays using rat synaptic membranes in presence of different receptor ligands. In addition, the extract stability was monitored spectrophotometrically. Both extracts demonstrated interaction with ionotropic glutamate receptors (iGluRs). However, the extracts displayed considerable differences in receptor selectivity. The hydroalcoholic extract selectively interacted with quisqualic acid (QA), group I metabotropic glutamate receptor (mGluR) ligand, while the aqueous extract did not alter the binding of QA. The stability of the extracts was examined during several weeks. Freshly prepared extract inhibited 38–60% of [3H]FW binding (AMPA). After 10 days, the aqueous extract inhibited 85% of [3H]FW binding while the hydroalcoholic extract markedly potentiated (200%) [3H]FW binding to AMPA receptors. Thus, our results showed that factors such as extraction solvent and extract stability determine the selectivity for glutamate receptor (GluR) interactions. PMID:21151614

Aqueous and Ethanolic Valeriana officinalis Extracts Change the Binding of Ligands to Glutamate Receptors.

PubMed

Del Valle-Mojica, Lisa M; Cordero-Hernández, José M; González-Medina, Giselle; Ramos-Vélez, Igmeris; Berríos-Cartagena, Nairimer; Torres-Hernández, Bianca A; Ortíz, José G

2011-01-01

The effects of two valerian extracts (aqueous and hydroalcoholic) were investigated through [(3)H]Glutamate ([(3)H]Glu) and [(3)H]Fluorowillardine ([(3)H]FW) receptor binding assays using rat synaptic membranes in presence of different receptor ligands. In addition, the extract stability was monitored spectrophotometrically. Both extracts demonstrated interaction with ionotropic glutamate receptors (iGluRs). However, the extracts displayed considerable differences in receptor selectivity. The hydroalcoholic extract selectively interacted with quisqualic acid (QA), group I metabotropic glutamate receptor (mGluR) ligand, while the aqueous extract did not alter the binding of QA. The stability of the extracts was examined during several weeks. Freshly prepared extract inhibited 38-60% of [(3)H]FW binding (AMPA). After 10 days, the aqueous extract inhibited 85% of [(3)H]FW binding while the hydroalcoholic extract markedly potentiated (200%) [(3)H]FW binding to AMPA receptors. Thus, our results showed that factors such as extraction solvent and extract stability determine the selectivity for glutamate receptor (GluR) interactions.

Effects of heparin-binding epidermal growth factor-like growth factor on cell repopulation and signal transduction in periodontal ligament cells after scratch wounding in vitro.

PubMed

Lee, J S; Kim, J M; Hong, E K; Kim, S-O; Yoo, Y-J; Cha, J-H

2009-02-01

A growing amount of attention has been placed on periodontal regeneration and wound healing for periodontal therapy. This study was conducted in an effort to determine the effects of heparin-binding epidermal growth factor-like growth factor on cell repopulation and signal transduction in periodontal ligament cells after scratch wounding in vitro. Human periodontal ligament cells were acquired from explant tissue of human healthy periodontal ligament. After the wounding of periodontal ligament cells, the change in expression of heparin-binding epidermal growth factor-like growth factor and epidermal growth factor receptors 1-4 mRNA was assessed. The effects of heparin-binding epidermal growth factor-like growth factor on periodontal ligament cell proliferation and repopulation were assessed in vitro via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and by photographing the injuries, respectively. Extracellular signal-regulated kinase (Erk)1/2, p38 and Akt phosphorylation was characterized via western blotting. Scratch wounding resulted in a significant up-regulation of heparin-binding epidermal growth factor-like growth factor mRNA expression, whereas wounding had no effect on the expression levels of epidermal growth factor receptors 1-4. Interestingly, no expression of epidermal growth factor receptors 2 and 4 was detectable prior to or after wounding. Heparin-binding epidermal growth factor-like growth factor treatment promoted the proliferation and repopulation of periodontal ligament cells. The scratch wounding also stimulated the phosphorylation of Erk1/2 and p38, but not of Akt, in periodontal ligament cells, and heparin-binding epidermal growth factor-like growth factor treatment applied after wounding amplified and extended the activations of Erk1/2 and p38, but not of Akt. Furthermore, Erk1/2 inhibition blocked the process of cell repopulation induced by heparin-binding epidermal growth factor-like growth factor, whereas the inhibition of p38 delayed the process. These results indicate that heparin-binding epidermal growth factor-like growth factor may constitute a critical factor in the wound healing of human periodontal ligament cells by a mechanism that requires the activation of Erk1/2 via specific interaction with epidermal growth factor receptor 1.

Structural basis for ligand and innate immunity factor uptake by the trypanosome haptoglobin-haemoglobin receptor.

PubMed

Lane-Serff, Harriet; MacGregor, Paula; Lowe, Edward D; Carrington, Mark; Higgins, Matthew K

2014-12-12

The haptoglobin-haemoglobin receptor (HpHbR) of African trypanosomes allows acquisition of haem and provides an uptake route for trypanolytic factor-1, a mediator of innate immunity against trypanosome infection. In this study, we report the structure of Trypanosoma brucei HpHbR in complex with human haptoglobin-haemoglobin (HpHb), revealing an elongated ligand-binding site that extends along its membrane distal half. This contacts haptoglobin and the β-subunit of haemoglobin, showing how the receptor selectively binds HpHb over individual components. Lateral mobility of the glycosylphosphatidylinositol-anchored HpHbR, and a ∼50° kink in the receptor, allows two receptors to simultaneously bind one HpHb dimer. Indeed, trypanosomes take up dimeric HpHb at significantly lower concentrations than monomeric HpHb, due to increased ligand avidity that comes from bivalent binding. The structure therefore reveals the molecular basis for ligand and innate immunity factor uptake by trypanosomes and identifies adaptations that allow efficient ligand uptake in the context of the complex trypanosome cell surface.

The C-terminal SH2 domain of p85 accounts for the high affinity and specificity of the binding of phosphatidylinositol 3-kinase to phosphorylated platelet-derived growth factor beta receptor.

PubMed Central

Klippel, A; Escobedo, J A; Fantl, W J; Williams, L T

1992-01-01

Upon stimulation by its ligand, the platelet-derived growth factor (PDGF) receptor associates with the 85-kDa subunit of phosphatidylinositol (PI) 3-kinase. The 85-kDa protein (p85) contains two Src homology 2 (SH2) domains and one SH3 domain. To define the part of p85 that interacts with the PDGF receptor, a series of truncated p85 mutants was analyzed for association with immobilized PDGF receptor in vitro. We found that a fragment of p85 that contains a single Src homology domain, the C-terminal SH2 domain (SH2-C), was sufficient for directing the high-affinity interaction with the receptor. Half-maximal binding of SH2-C to the receptor was observed at an SH2-C concentration of 0.06 nM. SH2-C, like full-length p85, was able to distinguish between wild-type PDGF receptor and a mutant receptor lacking the PI 3-kinase binding site. An excess of SH2-C blocked binding of full-length p85 and PI 3-kinase to the receptor but did not interfere with the binding of two other SH2-containing proteins, phospholipase C-gamma and GTPase-activating protein. These results demonstrate that a region of p85 containing a single SH2 domain accounts both for the high affinity and specificity of binding of PI 3-kinase to the PDGF receptor. Images PMID:1312663

Neomycin is a platelet-derived growth factor (PDGF) antagonist that allows discrimination of PDGF alpha- and beta-receptor signals in cells expressing both receptor types.

PubMed

Vassbotn, F S; Ostman, A; Siegbahn, A; Holmsen, H; Heldin, C H

1992-08-05

The aminoglycoside neomycin has recently been found to affect certain platelet-derived growth factor (PDGF) responses in C3H/10T1/2 C18 fibroblasts. Using porcine aortic endothelial cells transfected with PDGF alpha- or beta-receptors, we explored the possibility that neomycin interferes with the interaction between the different PDGF isoforms and their receptors. We found that neomycin (5 mM) inhibited the binding of 125I-PDGF-BB to the alpha-receptor with only partial effect on the binding of 125I-PDGF-AA; in contrast, the binding of 125I-PDGF-BB to the beta-receptor was not affected by the aminoglycoside. Scatchard analyses showed that neomycin (5 mM) decreased the number of binding sites for PDGF-BB on alpha-receptor-expressing cells by 87%. Together with cross-competition studies with 125I-labeled PDGF homodimers, the effect of neomycin indicates that PDGF-AA and PDGF-BB bind to both common and unique structures on the PDGF alpha-receptor. Neomycin specifically inhibited the autophosphorylation of the alpha-receptor by PDGF-BB, with less effect on the phosphorylation induced by PDGF-AA and no effect on the phosphorylation of the beta-receptor by PDGF-BB. Thus, neomycin is a PDGF isoform- and receptor-specific antagonist that provides a possibility to compare the signal transduction pathways of alpha- and beta-receptors in cells expressing both receptor types. This approach was used to show that activation of PDGF beta-receptors by PDGF-BB mediated a chemotactic response in human fibroblasts, whereas activation of alpha-receptors by the same ligand inhibited chemotaxis.

Transcriptional switches in the control of macronutrient metabolism.

PubMed

Wise, Alan

2008-06-01

This review shows how some transcription factors respond to alterations in macronutrients. Carbohydrates induce enzymes for their metabolism and fatty acid synthesis. Fatty acids reduce carbohydrate processing, induce enzymes for their metabolism, and increase both gluconeogenesis and storage of fat. Fat stores help control carbohydrate uptake by other cells. The following main transcription factors are discussed: carbohydrate response element-binding protein; sterol regulatory element-binding protein-1c, cyclic AMP response element-binding protein, peroxisome proliferator-activated receptor-alpha, and peroxisome proliferator-activated receptor-gamma.

NMR study of the transforming growth factor-alpha (TGF-alpha)-epidermal growth factor receptor complex. Visualization of human TGF-alpha binding determinants through nuclear Overhauser enhancement analysis.

PubMed

McInnes, C; Hoyt, D W; Harkins, R N; Pagila, R N; Debanne, M T; O'Connor-McCourt, M; Sykes, B D

1996-12-13

The study of human transforming growth factor-alpha (TGF-alpha) in complex with the epidermal growth factor (EGF) receptor extracellular domain has been undertaken in order to generate information on the interactions of these molecules. Analysis of 1H NMR transferred nuclear Overhauser enhancement data for titration of the ligand with the receptor has yielded specific data on the residues of the growth factor involved in contact with the larger protein. Significant increases and decreases in nuclear Overhauser enhancement cross-peak intensity occur upon complexation, and interpretation of these changes indicates that residues of the A- and C-loops of TGF-alpha form the major binding interface, while the B-loop provides a structural scaffold for this site. These results corroborate the conclusions from NMR relaxation studies (Hoyt, D. W., Harkins, R. N., Debanne, M. T., O'Connor-McCourt, M., and Sykes, B. D. (1994) Biochemistry 33, 15283-15292), which suggest that the C-terminal residues of the polypeptide are immobilized upon receptor binding, while the N terminus of the molecule retains considerable flexibility, and are consistent with structure-function studies of the TGF-alpha/EGF system indicating a multidomain binding model. These results give a visualization, for the first time, of native TGF-alpha in complex with the EGF receptor and generate a picture of the ligand-binding site based upon the intact molecule. This will undoubtedly be of utility in the structure-based design of TGF-alpha/EGF agonists and/or antagonists.

An Antagonistic Vascular Endothelial Growth Factor (VEGF) Variant Inhibits VEGF-Stimulated Receptor Autophosphorylation and Proliferation of Human Endothelial Cells

NASA Astrophysics Data System (ADS)

Siemeister, Gerhard; Schirner, Michael; Reusch, Petra; Barleon, Bernhard; Marme, Dieter; Martiny-Baron, Georg

1998-04-01

Vascular endothelial growth factor (VEGF) is a potent mitogen with a unique specificity for endothelial cells and a key mediator of aberrant endothelial cell proliferation and vascular permeability in a variety of human pathological situations, such as tumor angiogenesis, diabetic retinopathy, rheumatoid arthritis, or psoriasis. VEGF is a symmetric homodimeric molecule with two receptor binding interfaces lying on each pole of the molecule. Herein we report on the construction and recombinant expression of an asymmetric heterodimeric VEGF variant with an intact receptor binding interface at one pole and a mutant receptor binding interface at the second pole of the dimer. This VEGF variant binds to VEGF receptors but fails to induce receptor activation. In competition experiments, the heterodimeric VEGF variant antagonizes VEGF-stimulated receptor autophosphorylation and proliferation of endothelial cells. A 15-fold excess of the heterodimer was sufficient to inhibit VEGF-stimulated endothelial cell proliferation by 50%, and a 100-fold excess resulted in an almost complete inhibition. By using a rational approach that is based on the structure of VEGF, we have shown the feasibility to construct a VEGF variant that acts as an VEGF antagonist.

DEPENDENCE OF PPAR LIGAND-INDUCED MAPK SIGNALING ON EPIDERMAL GROWTH FACTOR RECEPTOR TRANSACTIVATION HEPARIN-BINDING EGF CLEAVAGE MEDIATES ZINC-INDUCED EGF RECEPTOR PHOSPHORYLATION

EPA Science Inventory

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that function as ligand-activated transcription factors regulating lipid metabolism and homeostasis. In addition to their ability to regulate PPAR-mediated gene transcription, PPARalpha and gamma li...

Regulation of insulin-like growth factor I receptors on vascular smooth muscle cells by growth factors and phorbol esters.

PubMed

Ververis, J J; Ku, L; Delafontaine, P

1993-06-01

Insulin-like growth factor I (IGF I) is an important mitogen for vascular smooth muscle cells. To characterize regulation of vascular IGF I receptors, we performed radioligand displacement experiments using rat aortic smooth muscle cells (RASMs). Serum deprivation for 48 hours caused a 40% decrease in IGF I receptor number. Exposure of quiescent RASMs to platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), or angiotensin II (Ang II) caused a 1.5-2.0-fold increase in IGF I receptors per cell. After FGF exposure, there was a marked increase in the mitogenic response to IGF I. IGF I downregulated its receptors in the presence of platelet-poor plasma. Stimulation of protein kinase C (PKC) by exposure of quiescent RASMs to phorbol 12-myristate 13-acetate caused a biphasic response in IGF I binding; there was a 42% decrease in receptor number at 45 minutes and a 238% increase at 24 hours. To determine the role of PKC in growth factor-induced regulation of IGF I receptors, we downregulated PKC by exposing RASMs to phorbol 12,13-dibutyrate (PDBu) for 48 hours. PDGF- and FGF- but not Ang II-mediated upregulation of IGF I receptors was completely inhibited in PDBu-treated cells. Thus, acute PKC activation by phorbol esters inhibits IGF I binding, whereas chronic PKC activation increases IGF I binding. PDGF and FGF but not Ang II regulate vascular IGF I receptors through a PKC-dependent pathway. These data provide new insights into the regulation of vascular smooth muscle cell IGF I receptors in vitro and are of potential importance in characterizing vascular proliferative responses in vivo.

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 interaction with several enzymes and adaptor proteins.

CCN2/CTGF binds to fibroblast growth factor receptor 2 and modulates its signaling.

PubMed

Aoyama, Eriko; Kubota, Satoshi; Takigawa, Masaharu

2012-12-14

CCN2 plays a critical role in the development of mesenchymal tissues such as cartilage and bone, and the binding of CCN2 to various cytokines and receptors regulates their signaling.By screening a protein array, we found that CCN2 could bind to fibroblast growth factor receptors (FGFRs) 2 and 3, with a higher affinity toward FGFR2.We ascertained that FGFR2 bound to CCN2 and that the binding of FGFR2 to FGF2 and FGF4 was enhanced by CCN2.CCN2 and FGF2 had a collaborative effect on the phosphorylation of ERK and the differentiation of osteoblastic cells.The present results indicate the biological significance of the binding of CCN2 to FGFR2 in bone metabolism. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Receptor binding proteins of Listeria monocytogenes bacteriophages A118 and P35 recognize serovar-specific teichoic acids

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

Bielmann, Regula; Habann, Matthias; Eugster, Marcel R.

Adsorption of a bacteriophage to the host requires recognition of a cell wall-associated receptor by a receptor binding protein (RBP). This recognition is specific, and high affinity binding is essential for efficient virus attachment. The molecular details of phage adsorption to the Gram-positive cell are poorly understood. We present the first description of receptor binding proteins and a tail tip structure for the siphovirus group infecting Listeria monocytogenes. The host-range determining factors in two phages, A118 and P35 specific for L. monocytogenes serovar 1/2 have been determined. Two proteins were identified as RBPs in phage A118. Rhamnose residues in wallmore » teichoic acids represent the binding ligands for both proteins. In phage P35, protein gp16 could be identified as RBP and the role of both rhamnose and N-acetylglucosamine in phage adsorption was confirmed. Immunogold-labeling and transmission electron microscopy allowed the creation of a topological model of the A118 phage tail. - Highlights: • We present the first description of receptor binding proteins and a tail tip structure for the Siphovirus group infecting Listeria monocytogenes. • The host-range determining factors in two phages, A118 and P35 specific for L. monocytogenes serovar 1/2 have been determined. • Rhamnose residues in wall teichoic acids represent the binding ligands for both receptor binding proteins in phage A118. • Rhamnose and N-acetylglucosamine are required for adsorption of phage P35. • We preset a topological model of the A118 phage tail.« less

Takifugu rubripes cation independent mannose 6-phosphate receptor: Cloning, expression and functional characterization of the IGF-II binding domain.

PubMed

A, Ajith Kumar; Nadimpalli, Siva Kumar

2018-07-01

Mannose 6-phosphate/IGF-II receptor mediated lysosomal clearance of insulin-like growth factor-II is significantly associated with the evolution of placental mammals. The protein is also referred to as the IGF-II receptor. Earlier studies suggested relatively low binding affinity between the receptor and ligand in prototherian and metatherian mammals. In the present study, we cloned the IGF-II binding domain of the early vertebrate fugu fish and expressed it in bacteria. A 72000Da truncated receptor containing the IGF-II binding domain was obtained. Analysis of this protein (covering domains 11-13 of the CIMPR) for its affinity to fish and human IGF-II by ligand blot assays and ELISA showed that the expressed receptor can specifically bind to both fish and human IGF-II. Additionally, a peptide-specific antibody raised against the region of the IGF-II binding domain also was able to recognize the IGF-II binding regions of mammalian and non-mammalian cation independent MPR protein. These interactions were further characterized by Surface Plasma resonance support that the receptor binds to fish IGF-II, with a dissociation constant of 548nM. Preliminary analysis suggests that the binding mechanism as well as the affinity of the fish and human receptor for IGF-II may have varied according to different evolutionary pressures. Copyright © 2018. Published by Elsevier B.V.

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.

N-3 polyunsaturated fatty acid regulation of hepatic gene transcription

PubMed Central

Jump, Donald B.

2009-01-01

Purpose of review The liver plays a central role in whole body lipid metabolism and adapts rapidly to changes in dietary fat composition. This adaption involves changes in the expression of genes involved in glycolysis, de-novo lipogenesis, fatty acid elongation, desaturation and oxidation. This review brings together metabolic and molecular studies that help explain n-3 (omega-3) polyunsaturated fatty acid regulation of hepatic gene transcription. Recent findings Dietary n-3 polyunsaturated fatty acid regulates hepatic gene expression by targeting three major transcriptional regulatory networks: peroxisome proliferator-activated receptor α, sterol regulatory element binding protein-1 and the carbohydrate regulatory element binding protein/Max-like factor X heterodimer. 22 : 6,n-3, the most prominent n-3 polyunsaturated fatty acid in tissues, is a weak activator of peroxisome proliferator-activated receptor α. Hepatic metabolism of 22 : 6,n-3, however, generates 20 : 5,n-3, a strong peroxisome proliferator-activated receptor α activator. In contrast to peroxisome proliferator-activated receptor α, 22 : 6,n-3 is the most potent fatty acid regulator of hepatic sterol regulatory element binding protein-1. 22 : 6,n-3 suppresses sterol regulatory element binding protein-1 gene expression while enhancing degradation of nuclear sterol regulatory element binding protein-1 through 26S proteasome and Erk1/2-dependent mechanisms. Both n-3 and n-6 polyunsaturated fatty acid suppress carbohydrate regulatory element binding protein and Max-like factor X nuclear abundance and interfere with glucose-regulated hepatic metabolism. Summary These studies have revealed unique mechanisms by which specific polyunsaturated fatty acids control peroxisome proliferator activated receptor α, sterol regulatory element binding protein-1 and carbohydrate regulatory element binding protein/Max-like factor X function. As such, specific metabolic and signal transduction pathways contribute significantly to the fatty acid regulation of these transcription factors and their corresponding regulatory networks. PMID:18460914

Transformation of glucocorticoid receptors bound to the antagonist RU 486: Effects of alkaline phosphatase

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

Gruol, D.J.; Wolfe, K.A.

1990-08-28

RU 486 is a synthetic steroid that binds avidly to glucocorticoid receptors without promoting their transformation into activated transcription factors. A significant part of this behavior has been shown to be due to a failure of the RU 486 bound receptor to be efficiently released from a larger (sedimenting at 8-9 S) multimeric complex containing the 90-kDa heat shock protein. The studies have found that in vitro at 15{degree}C the RU 486-receptor was slowly released from the 8-9S complex and converted into a DNA binding protein by a process that could be blocked by sodium fluoride. Moreover, this transition wasmore » significantly accelerated by treatment with alkaline phosphatase. High-resolution anion-exchange chromatography showed that the profile of receptor subspecies released from the 8-9S complex was different for the RU 486 bound receptor when compared to the receptor occupied by the agonist triamcinolone acetonide. Production of the earliest eluting receptor form (peak A) was inhibited with RU 486. Treatment of the Ru 486-receptor with alkaline phosphatase increased the formation of the peak A subspecies as well as the capacity of receptor to bind DNA-cellulose. Taken together, the results indicate that phosphorylation of the receptor or a tightly bound factor contributes to defining the capacity with which individual steroids can promote dissociation of the 8-9S complex and conversion of the glucocorticoid receptor into a DNA-binding protein.« less

Specific receptors for epidermal growth factor in rat intestinal microvillus membranes

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

Thompson, J.F.

Epidermal growth factor (EGF) is present in high concentrations in milk, salivary, and pancreaticobiliary secretions. EGF, delivered to the intestinal lumen by these fluids, appears to influence intestinal proliferation. Because EGF exerts its mitogenic effect through binding to specific membrane-bound receptors, binding studies of {sup 125}I-labeled EGF to purified microvillus membrane (MVM) preparations fetal, newborn, and adult rat small intestine were performed. Using the membrane filter technique, binding of {sup 125}I-EGF to adult MVM was specific, saturable, and reversible. Adult and fetal MVM binding was rapid and reached a plateau after 30 min at both 20 and 37{degree}C. No bindingmore » was detected at 4{degree}C. Specific binding increased linearly from 0 to 75 {mu}g MVM protein. Scatchard analysis revealed a single class of receptors in fetal and adult MVM with an association constant of 1.0 {+-} 0.35 {times} 10{sup 9} and 2.3 {+-} 1.6 {times} 10{sup 9} M{sup {minus}1}, respectively. Binding capacity was 435.0 {+-} 89 and 97.7 {+-} 41.3 fmol {sup 125}I-EGF bound/mg MVM protein for fetal and adult MVM, respectively. Newborn MVM binding was negligible. After binding, cross-linking utilizing disuccinimidyl suberate, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography revealed a 170-kDa receptor. These data demonstrate specific receptors for EGF on MVM of rat small intestine and, thus, suggest a mechanism for the intraluminal regulation of enterocyte proliferation by EGF.« less

The overexpressed human 46-kDa mannose 6-phosphate receptor mediates endocytosis and sorting of. beta. -glucuronidase

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

Watanabe, H.; Grubb, J.H.; Sly, W.S.

1990-10-01

The authors studied the function of the human small (46-kDa) mannose 6-phosphate receptor (SMPR) in transfected mouse L cells that do not express the larger insulin-like growth factor II/mannose 6-phosphate receptor. Cells overexpressing human SMPR were studied for enzyme binding to cell surface receptors, for binding to intracellular receptors in permeabilized cells, and for receptor-mediated endocytosis of recombinant human {beta}-glucuronidase. Specific binding to human SMPR in permeabilized cells showed a pH optimum between pH 6.0 and pH 6.5. Binding was significant in the present of EDTA but was enhanced by added divalent cations. Up to 2.3{percent} of the total functionalmore » receptor could be detected on the cell surface by enzyme binding. They present experiments showing that at very high levels of overexpression, and at pH 6.5, human SMPR mediated the endocytosis of {beta}-glucuronidase. At pH 7.5, the rate of endocytosis was only 14{percent} the rate seen at pH 6.5. Cells overexpressing human SMPR also showed reduced secretion of newly synthesized {beta}-glucuronidase when compared to cells transfected with vector only, suggesting that overexpressed human SMPR can participate in sorting of newly synthesized {beta}-glucuronidase and partially correct the sorting defect in mouse L cells that do not express the insulin-like growth factor II/mannose 6-phosphate receptor.« less

Effect of receptor binding specificity on the immunogenicity and protective efficacy of influenza virus A H1 vaccines

PubMed Central

Sun, Xiangjie; Cao, Weiping; Pappas, Claudia; Liu, Feng; Katz, Jacqueline M.; Tumpey, Terrence M.

2018-01-01

The biological basis for the poor immunogenicity of unadjuvanted avian influenza A virus vaccines in mammals is not well understood. Here, we mutated the hemagglutinin (HA) of two H1N1 virus vaccines to determine whether virus receptor binding specificity contributes to the low immunogenicity of avian influenza virus vaccines. Mutations were introduced into the HA of an avian influenza virus, A/Duck/New York/15024–21/96 (Dk/96) which switched the binding preference from α2,3- to α2,6-linked sialic acid (SA). A switch in receptor specificity of the human A/South Carolina/1/18 (SC/18) virus generated a mutant virus with α2,3 SA (avian) binding preference. Inactivated vaccines were generated and administered to mice and ferrets intramuscularly. We found that the vaccines with human receptor binding preference induced slightly higher antibody titers and cell-mediated immune responses compared to their isogenic viruses with avian receptor binding specificity. Upon challenge with DK/96 or SC18 virus, differences in lung virus titers between the vaccine groups with different receptor-binding specificities were minimal. Overall, our data suggest that receptor binding specificity contributes only marginally to the immunogenicity of avian influenza vaccines and that other factors may also be involved. PMID:25078114

The mannose 6-phosphate-binding sites of M6P/IGF2R determine its capacity to suppress matrix invasion by squamous cell carcinoma cells

PubMed Central

Probst, Olivia C.; Karayel, Evren; Schida, Nicole; Nimmerfall, Elisabeth; Hehenberger, Elisabeth; Puxbaum, Verena; Mach, Lukas

2013-01-01

The M6P (mannose 6-phosphate)/IGF2R (insulin-like growth factor II receptor) interacts with a variety of factors that impinge on tumour invasion and metastasis. It has been shown that expression of wild-type M6P/IGF2R reduces the tumorigenic and invasive properties of receptor-deficient SCC-VII squamous cell carcinoma cells. We have now used mutant forms of M6P/IGF2R to assess the relevance of the different ligand-binding sites of the receptor for its biological activities in this cellular system. The results of the present study demonstrate that M6P/IGF2R does not require a functional binding site for insulin-like growth factor II for inhibition of anchorage-independent growth and matrix invasion by SCC-VII cells. In contrast, the simultaneous mutation of both M6P-binding sites is sufficient to impair all cellular functions of the receptor tested. These findings highlight that the interaction between M6P/IGF2R and M6P-modified ligands is not only important for intracellular accumulation of lysosomal enzymes and formation of dense lysosomes, but is also crucial for the ability of the receptor to suppress SCC-VII growth and invasion. The present study also shows that some of the biological activities of M6P/IGF2R in SCC-VII cells strongly depend on a functional M6P-binding site within domain 3, thus providing further evidence for the non-redundant cellular functions of the individual carbohydrate-binding domains of the receptor. PMID:23347038

Factor VIII Interacts with the Endocytic Receptor Low-density Lipoprotein Receptor-related Protein 1 via an Extended Surface Comprising "Hot-Spot" Lysine Residues.

PubMed

van den Biggelaar, Maartje; Madsen, Jesper J; Faber, Johan H; Zuurveld, Marleen G; van der Zwaan, Carmen; Olsen, Ole H; Stennicke, Henning R; Mertens, Koen; Meijer, Alexander B

2015-07-03

Lysine residues are implicated in driving the ligand binding to the LDL receptor family. However, it has remained unclear how specificity is regulated. Using coagulation factor VIII as a model ligand, we now study the contribution of individual lysine residues in the interaction with the largest member of the LDL receptor family, low-density lipoprotein receptor-related protein (LRP1). Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and SPR interaction analysis on a library of lysine replacement variants as two independent approaches, we demonstrate that the interaction between factor VIII (FVIII) and LRP1 occurs over an extended surface containing multiple lysine residues. None of the individual lysine residues account completely for LRP1 binding, suggesting an additive binding model. Together with structural docking studies, our data suggest that FVIII interacts with LRP1 via an extended surface of multiple lysine residues that starts at the bottom of the C1 domain and winds around the FVIII molecule. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Synthetic heparin-binding factor analogs

DOEpatents

Pena, Louis A [Poquott, NY; Zamora, Paul O [Gaithersburg, MD; Lin, Xinhua [Plainview, NY; Glass, John D [Shoreham, NY

2010-04-20

The invention provides synthetic heparin-binding growth factor analogs having at least one peptide chain, and preferably two peptide chains branched from a dipeptide branch moiety composed of two trifunctional amino acid residues, which peptide chain or chains bind a heparin-binding growth factor receptor and are covalently bound to a non-signaling peptide that includes a heparin-binding domain, preferably by a linker, which may be a hydrophobic linker. The synthetic heparin-binding growth factor analogs are useful as pharmaceutical agents, soluble biologics or as surface coatings for medical devices.

The pure estrogen receptor antagonist ICI 182,780 promotes a novel interaction of estrogen receptor-alpha with the 3',5'-cyclic adenosine monophosphate response element-binding protein-binding protein/p300 coactivators.

PubMed

Jaber, Basem M; Gao, Tong; Huang, Luping; Karmakar, Sudipan; Smith, Carolyn L

2006-11-01

Estrogen receptor-alpha (ERalpha) is a member of the nuclear receptor superfamily of ligand-activated transcription factors. Abundant evidence demonstrates that ERalpha agonists promote, whereas antagonists inhibit, receptor binding to coactivators. In this report we demonstrate that binding of the ICI 182,780 (ICI) pure antiestrogen to ERalpha promotes its interaction with the cAMP response element-binding protein-binding protein (CBP)/p300 but not the p160 family of coactivators, demonstrating the specificity of this interaction. Amino acid mutations within the coactivator binding surface of the ERalpha ligand-binding domain revealed that CBP binds to this region of the ICI-liganded receptor. The carboxy-terminal cysteine-histidine rich domain 3 of CBP, rather than its amino-terminal nuclear interacting domain, shown previously to mediate agonist-dependent interactions of CBP with nuclear receptors, is required for binding to ICI-liganded ERalpha. Chromatin immunoprecipitation assays revealed that ICI but not the partial agonist/antagonist 4-hydroxytamoxifen is able to recruit CBP to the pS2 promoter, and this distinguishes ICI from this class of antiestrogens. Chromatin immunoprecipitation assays for pS2 and cytochrome P450 1B1 promoter regions revealed that ICI-dependent recruitment of CBP, but not receptor, to ERalpha targets is gene specific. ICI treatment did not recruit the steroid receptor coactivator 1 to the pS2 promoter, and it failed to induce the expression of this gene. Taken together, these data indicate that recruitment of the CBP coactivator/cointegrator without steroid receptor coactivator 1 to ERalpha is insufficient to promote transcription of ERalpha target genes.

Diminished hepatic growth hormone receptor binding in sex-linked dwarf broiler and leghorn chickens.

PubMed

Leung, F C; Styles, W J; Rosenblum, C I; Lilburn, M S; Marsh, J A

1987-02-01

Hepatic growth hormone (GH) receptor binding was compared in normal and sex-linked dwarfs (SLD) from both Hubbard and Cornell strain chickens. At 6, 8, and 20 weeks of age, hepatic GH receptor binding in the Hubbard SLD chickens was significantly lower than that of normal fast-growing birds. At 20 weeks of age, only 2 of 22 SLD chickens in the Hubbard broiler strain showed positive binding at a high enough level to allow for Scatchard analysis. The affinity constants and binding capacities of these two SLD chickens were numerically (but not significantly) lower than those of the normal fast-growing birds. We further examined hepatic GH receptor binding in two closely related White Leghorn strains of chickens that have been maintained as closed breeding populations for many years. We observed no detectable hepatic GH binding in the Cornell SLD chickens (N = 20), as compared to the normal-growing control strain (K strain). In both SLD strains, pretreatment with 4 M MgCl2 did not enhance GH binding, suggesting that there was no endogenous GH binding to the receptor. Based on these data, we suggest that the lack, or greatly reduced number, of GH receptors may be a major contributing factor to the dwarfism observed in these strains.

Structure of nerve growth factor complexed with the shared neurotrophin receptor p75.

PubMed

He, Xiao-Lin; Garcia, K Christopher

2004-05-07

Neurotrophins are secreted growth factors critical for the development and maintenance of the vertebrate nervous system. Neurotrophins activate two types of cell surface receptors, the Trk receptor tyrosine kinases and the shared p75 neurotrophin receptor. We have determined the 2.4 A crystal structure of the prototypic neurotrophin, nerve growth factor (NGF), complexed with the extracellular domain of p75. Surprisingly, the complex is composed of an NGF homodimer asymmetrically bound to a single p75. p75 binds along the homodimeric interface of NGF, which disables NGF's symmetry-related second p75 binding site through an allosteric conformational change. Thus, neurotrophin signaling through p75 may occur by disassembly of p75 dimers and assembly of asymmetric 2:1 neurotrophin/p75 complexes, which could potentially engage a Trk receptor to form a trimolecular signaling complex.

The Fn14 cytoplasmic tail binds tumour-necrosis-factor-receptor-associated factors 1, 2, 3 and 5 and mediates nuclear factor-kappaB activation.

PubMed Central

Brown, Sharron A N; Richards, Christine M; Hanscom, Heather N; Feng, Sheau-Line Y; Winkles, Jeffrey A

2003-01-01

Fn14 is a growth-factor-inducible immediate-early-response gene encoding a 102-amino-acid type I transmembrane protein. The human Fn14 protein was recently identified as a cell-surface receptor for the tumour necrosis factor (TNF) superfamily member named TWEAK (TNF-like weak inducer of apoptosis). In the present paper, we report that the human TWEAK extracellular domain can also bind the murine Fn14 protein. Furthermore, site-specific mutagenesis and directed yeast two-hybrid interaction assays revealed that the TNFR-associated factor (TRAF) 1, 2, 3 and 5 adaptor molecules bind the murine Fn14 cytoplasmic tail at an overlapping, but non-identical, amino acid sequence motif. We also found that TWEAK treatment of quiescent NIH 3T3 cells stimulates inhibitory kappaBalpha phosphorylation and transcriptional activation of a nuclear factor-kappaB (NF-kappaB) enhancer/luciferase reporter construct. Fn14 overexpression in transiently transfected NIH 3T3 cells also promotes NF-kappaB activation, and this cellular response requires an intact TRAF binding site. These results indicate that Fn14 is a functional TWEAK receptor that can associate with four distinct TRAF family members and stimulate the NF-kappaB transcription factor signalling pathway. PMID:12529173

Conformational stability of the epidermal growth factor (EGF) receptor as influenced by glycosylation, dimerization and EGF hormone binding.

PubMed

Taylor, Eric S; Pol-Fachin, Laercio; Lins, Roberto D; Lower, Steven K

2017-04-01

The epidermal growth factor receptor (EGFR) is an important transmembrane glycoprotein kinase involved the initiation or perpetuation of signal transduction cascades within cells. These processes occur after EGFR binds to a ligand [epidermal growth factor (EGF)], thus inducing its dimerization and tyrosine autophosphorylation. Previous publications have highlighted the importance of glycosylation and dimerization for promoting proper function of the receptor and conformation in membranes; however, the effects of these associations on the protein conformational stability have not yet been described. Molecular dynamics simulations were performed to characterize the conformational preferences of the monomeric and dimeric forms of the EGFR extracellular domain upon binding to EGF in the presence and absence of N-glycan moieties. Structural stability analyses revealed that EGF provides the most conformational stability to EGFR, followed by glycosylation and dimerization, respectively. The findings also support that EGF-EGFR binding takes place through a large-scale induced-fitting mechanism. Proteins 2017; 85:561-570. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Transcription control and neuronal differentiation by agents that activate the LXR nuclear receptor family.

PubMed

Schmidt, A; Vogel, R; Holloway, M K; Rutledge, S J; Friedman, O; Yang, Z; Rodan, G A; Friedman, E

1999-09-10

LXR and PPAR receptors belong to the nuclear receptor superfamily of transcriptional activating factors. Using ligand-dependent transcription assays, we found that 5-tetradecyloxy-2-furancarboxylic acid (TOFA) transactivates chimeric receptors composed of the glucocorticoid receptor DNA binding domain and the ligand binding regions of PPARalpha, PPARbeta (NUC-1) and LXRbeta (NER) receptors. In the same assays, ligands for PPARs (oleic acid, WY-14643 and L-631,033) and LXRs (hydroxycholesterols) maintain their respective receptor selectivity. TOFA and hydroxycholesterols also stimulate transcription from a minimal fibrinogen promoter that is under the control of AP-1 or NF-kappaB transcription factor binding sites. In addition to their effects on transcription, these LXRbeta activators induce neuronal differentiation in rat pheochromocytoma cells. TOFA and the natural LXR agonist, 22 (R)-hydroxycholesterol, stimulate neurite outgrowth in 55 and 28% of cells, respectively. No neurite outgrowth was induced by the related 22(S)-hydroxycholesterol, which does not activate the LXR family. These results suggest that the hydroxycholesterol signaling pathway has a complex effect on transcription that mediates the activity of TOFA and hydroxycholesterol on neuronal differentiation in pheochromocytoma cells.

Azadirachtin interacts with retinoic acid receptors and inhibits retinoic acid-mediated biological responses.

PubMed

Thoh, Maikho; Babajan, Banaganapalli; Raghavendra, Pongali B; Sureshkumar, Chitta; Manna, Sunil K

2011-02-11

Considering the role of retinoids in regulation of more than 500 genes involved in cell cycle and growth arrest, a detailed understanding of the mechanism and its regulation is useful for therapy. The extract of the medicinal plant Neem (Azadirachta indica) is used against several ailments especially for anti-inflammatory, anti-itching, spermicidal, anticancer, and insecticidal activities. In this report we prove the detailed mechanism on the regulation of retinoic acid-mediated cell signaling by azadirachtin, active components of neem extract. Azadirachtin repressed all trans-retinoic acid (ATRA)-mediated nuclear transcription factor κB (NF-κB) activation, not the DNA binding but the NF-κB-dependent gene expression. It did not inhibit IκBα degradation, IκBα kinase activity, or p65 phosphorylation and its nuclear translocation but inhibited NF-κB-dependent reporter gene expression. Azadirachtin inhibited TRAF6-mediated, but not TRAF2-mediated NF-κB activation. It inhibited ATRA-induced Sp1 and CREB (cAMP-response element-binding protein) DNA binding. Azadirachtin inhibited ATRA binding with retinoid receptors, which is supported by biochemical and in silico evidences. Azadirachtin showed strong interaction with retinoid receptors. It suppressed ATRA-mediated removal of retinoid receptors, bound with DNA by inhibiting ATRA binding to its receptors. Overall, our data suggest that azadirachtin interacts with retinoic acid receptors and suppresses ATRA binding, inhibits falling off the receptors, and activates transcription factors like CREB, Sp1, NF-κB, etc. Thus, azadirachtin exerts anti-inflammatory and anti-metastatic responses by a novel pathway that would be beneficial for further anti-inflammatory and anti-cancer therapies.

The first three domains of the insulin receptor differ structurally from the insulin-like growth factor 1 receptor in the regions governing ligand specificity

PubMed Central

Lou, Meizhen; Garrett, Thomas P. J.; McKern, Neil M.; Hoyne, Peter A.; Epa, V. Chandana; Bentley, John D.; Lovrecz, George O.; Cosgrove, Leah J.; Frenkel, Maurice J.; Ward, Colin W.

2006-01-01

The insulin receptor (IR) and the type-1 insulin-like growth factor receptor (IGF1R) are homologous multidomain proteins that bind insulin and IGF with differing specificity. Here we report the crystal structure of the first three domains (L1–CR–L2) of human IR at 2.3 Å resolution and compare it with the previously determined structure of the corresponding fragment of IGF1R. The most important differences seen between the two receptors are in the two regions governing ligand specificity. The first is at the corner of the ligand-binding surface of the L1 domain, where the side chain of F39 in IR forms part of the ligand binding surface involving the second (central) β-sheet. This is very different to the location of its counterpart in IGF1R, S35, which is not involved in ligand binding. The second major difference is in the sixth module of the CR domain, where IR contains a larger loop that protrudes further into the ligand-binding pocket. This module, which governs IGF1-binding specificity, shows negligible sequence identity, significantly more α-helix, an additional disulfide bond, and opposite electrostatic potential compared to that of the IGF1R. PMID:16894147

IL-3 specifically inhibits GM-CSF binding to the higher affinity receptor

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

Taketazu, F.; Chiba, S.; Shibuya, K.

1991-02-01

The inhibition of binding between human granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptor by human interleukin-3 (IL-3) was observed in myelogenous leukemia cell line KG-1 which bore the receptors both for GM-CSF and IL-3. In contrast, this phenomenon was not observed in histiocytic lymphoma cell line U-937 or in gastric carcinoma cell line KATO III, both of which have apparent GM-CSF receptor but an undetectable IL-3 receptor. In KG-1 cells, the cross-inhibition was preferentially observed when the binding of GM-CSF was performed under the high-affinity binding condition; i.e., a low concentration of 125I-GM-CSF was incubated. Scatchard analysis of 125I-GM-CSF bindingmore » to KG-1 cells in the absence and in the presence of unlabeled IL-3 demonstrated that IL-3 inhibited GM-CSF binding to the higher-affinity component of GM-CSF receptor on KG-1 cells. Moreover, a chemical cross-linking study has revealed that the cross-inhibition of the GM-CSF binding observed in KG-1 cells is specific for the beta-chain, Mr 135,000 binding protein which has been identified as a component forming the high-affinity GM-CSF receptor existing specifically on hemopoietic cells.« less

Pirenzepine binding to membrane-bound, solubilized and purified muscarinic receptor subtypes

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

Baumgold, J.

1986-05-01

Muscarinic receptors were purified to near-homogeneity from bovine cortex, an area rich in the putative M1 subtype, and from bovine pons/medulla, an area rich in the putative M2 subtype. In both cases, the receptors were solubilized in digitonin and purified over an affinity column. Both the cortical and pons/medulla preparations yielded receptor proteins of 70,000 daltons. Pirenzepine binding was deduced from its competition with /sup 3/H-N-methyl scopolamine. The binding of pirenzepine to membrane-bound receptors from cortex was best described by a two site model, with approximately half the sites having a Ki of 6.4 x 10/sup -9/ M and themore » remaining sites having a Ki of 3.5 x 10/sup -7/ M. Membrane-bound receptors from pons/medulla bound pirenzepine according to a one-site model with a Ki of 1.1 x 10/sup -7/ M. After solubilization the two-site binding of cortical receptors became a one-site binding, Ki = 1.1 x 10/sup -7/M. This value was still five-fold lower than that of soluble receptors from pons/medulla. After purification however the affinity of pirenzepine for the pons/medulla receptor increased so that the two putative subtypes bound pirenzepine with approximately the same affinity. These findings suggest that the different pirenzepine binding characteristics used to define muscarinic receptor subtypes are not inherent in the receptor protein itself but may be due to coupling factors associated with the receptor.« less

Digested wheat gluten inhibits binding between leptin and its receptor.

PubMed

Jönsson, Tommy; Memon, Ashfaque A; Sundquist, Kristina; Sundquist, Jan; Olsson, Stefan; Nalla, Amarnadh; Bauer, Mikael; Linse, Sara

2015-01-20

Leptin resistance is considered a primary risk factor for obesity. It has been hypothesized that dietary cereal grain protein could cause leptin resistance by preventing leptin from binding to its receptor. Non-degraded dietary wheat protein has been found in human serum at a mean level of 41 ng/mL. Here, we report our findings from testing whether enzymatically digested gluten from wheat prevents leptin from binding to the leptin receptor in vitro. Gluten from wheat was digested with pepsin and trypsin under physiological conditions. Pepsin and trypsin activity was removed from the gluten digest with a 10 kDa spin-filter or by heat treatment at 100°C for 30 min. Binding to the leptin receptor of leptin mixed with gluten digest at a series of concentrations was measured using surface plasmon resonance technology. Binding of the gluten digest to the leptin receptor was not detected. Spin-filtered gluten digest inhibited binding of leptin to the leptin receptor, with 50% inhibition at a gluten digest concentration of ~10 ng/mL. Heat-treated gluten digest did not inhibit leptin binding. Digested wheat gluten inhibits binding of leptin to the leptin receptor, with half-maximal inhibition at 10 ng/mL. The inhibition is significant at clinically relevant concentrations and could therefore serve as a novel pathway to investigate to understand the molecular basis of leptin resistance, obesity and associated disorders.

The Evolving Field of Human Papillomavirus Receptor Research: a Review of Binding and Entry

PubMed Central

Raff, Adam B.; Woodham, Andrew W.; Raff, Laura M.; Skeate, Joseph G.; Yan, Lisa; Da Silva, Diane M.; Schelhaas, Mario

2013-01-01

Human papillomaviruses (HPVs) infect epithelia and can lead to the development of lesions, some of which have malignant potential. HPV type 16 (HPV16) is the most oncogenic genotype and causes various types of cancer, including cervical, anal, and head and neck cancers. However, despite significant research, our understanding of the mechanism by which HPV16 binds to and enters host cells remains fragmented. Over several decades, many HPV receptors and entry pathways have been described. This review puts those studies into context and offers a model of HPV16 binding and entry as a framework for future research. Our model suggests that HPV16 binds to heparin sulfate proteoglycans (HSPGs) on either the epithelial cell surface or basement membrane through interactions with the L1 major capsid protein. Growth factor receptors may also become activated through HSPG/growth factor/HPV16 complexes that initiate signaling cascades during early virion-host cell interactions. After binding to HSPGs, the virion undergoes conformational changes, leading to isomerization by cyclophilin B and proprotein convertase-mediated L2 minor capsid protein cleavage that increases L2 N terminus exposure. Along with binding to HSPGs, HPV16 binds to α6 integrins, which initiate further intracellular signaling events. Following these primary binding events, HPV16 binds to a newly identified L2-specific receptor, the annexin A2 heterotetramer. Subsequently, clathrin-, caveolin-, lipid raft-, flotillin-, cholesterol-, and dynamin-independent endocytosis of HPV16 occurs. PMID:23536685

Dual chain synthetic heparin-binding growth factor analogs

DOEpatents

Zamora, Paul O [Gaithersburg, MD; Pena, Louis A [Poquott, NY; Lin, Xinhua [Plainview, NY

2012-04-24

The invention provides synthetic heparin-binding growth factor analogs having two peptide chains each branched from a branch moiety, such as trifunctional amino acid residues, the branch moieties separated by a first linker of from 3 to about 20 backbone atoms, which peptide chains bind a heparin-binding growth factor receptor and are covalently bound to a non-signaling peptide that includes a heparin-binding domain, preferably by a second linker, which may be a hydrophobic second linker. The synthetic heparin-binding growth factor analogs are useful as pharmaceutical agents, soluble biologics or as surface coatings for medical devices.

Dual chain synthetic heparin-binding growth factor analogs

DOEpatents

Zamora, Paul O [Gaithersburg, MD; Pena, Louis A [Poquott, NY; Lin, Xinhua [Plainview, NY

2009-10-06

The invention provides synthetic heparin-binding growth factor analogs having two peptide chains each branched from a branch moiety, such as trifunctional amino acid residues, the branch moieties separated by a first linker of from 3 to about 20 backbone atoms, which peptide chains bind a heparin-binding growth factor receptor and are covalently bound to a non-signaling peptide that includes a heparin-binding domain, preferably by a second linker, which may be a hydrophobic second linker. The synthetic heparin-binding growth factor analogs are useful as pharmaceutical agents, soluble biologics or as surface coatings for medical devices.

A Pro-Nerve Growth Factor (proNGF) and NGF Binding Protein, α2-Macroglobulin, Differentially Regulates p75 and TrkA Receptors and Is Relevant to Neurodegeneration Ex Vivo and In Vivo

PubMed Central

Barcelona, Pablo F.

2015-01-01

Nerve growth factor (NGF) is generated from a precursor, proNGF, that is proteolytically processed. NGF preferentially binds a trophic tyrosine kinase receptor, TrkA, while proNGF binds a neurotrophin receptor (NTR), p75NTR, that can have neurotoxic activity. Previously, we along with others showed that the soluble protein α2-macroglobulin (α2M) is neurotoxic. Toxicity is due in part to α2M binding to NGF and inhibiting trophic activity, presumably by preventing NGF binding to TrkA. However, the mechanisms remained unclear. Here, we show ex vivo and in vivo three mechanisms for α2M neurotoxicity. First, unexpectedly the α2M-NGF complexes do bind TrkA receptors but do not induce TrkA dimerization or activation, resulting in deficient trophic support. Second, α2M makes stable complexes with proNGF, conveying resistance to proteolysis that results in more proNGF and less NGF. Third, α2M-proNGF complexes bind p75NTR and are more potent agonists than free proNGF, inducing tumor necrosis factor alpha (TNF-α) production. Hence, α2M regulates proNGF/p75NTR positively and mature NGF/TrkA negatively, causing neuronal death ex vivo. These three mechanisms are operative in vivo, and α2M causes neurodegeneration in a p75NTR- and proNGF-dependent manner. α2M could be exploited as a therapeutic target, or as a modifier of neurotrophin signals. PMID:26217017

A Plant Immune Receptor Detects Pathogen Effectors that Target WRKY Transcription Factors.

PubMed

Sarris, Panagiotis F; Duxbury, Zane; Huh, Sung Un; Ma, Yan; Segonzac, Cécile; Sklenar, Jan; Derbyshire, Paul; Cevik, Volkan; Rallapalli, Ghanasyam; Saucet, Simon B; Wirthmueller, Lennart; Menke, Frank L H; Sohn, Kee Hoon; Jones, Jonathan D G

2015-05-21

Defense against pathogens in multicellular eukaryotes depends on intracellular immune receptors, yet surveillance by these receptors is poorly understood. Several plant nucleotide-binding, leucine-rich repeat (NB-LRR) immune receptors carry fusions with other protein domains. The Arabidopsis RRS1-R NB-LRR protein carries a C-terminal WRKY DNA binding domain and forms a receptor complex with RPS4, another NB-LRR protein. This complex detects the bacterial effectors AvrRps4 or PopP2 and then activates defense. Both bacterial proteins interact with the RRS1 WRKY domain, and PopP2 acetylates lysines to block DNA binding. PopP2 and AvrRps4 interact with other WRKY domain-containing proteins, suggesting these effectors interfere with WRKY transcription factor-dependent defense, and RPS4/RRS1 has integrated a "decoy" domain that enables detection of effectors that target WRKY proteins. We propose that NB-LRR receptor pairs, one member of which carries an additional protein domain, enable perception of pathogen effectors whose function is to target that domain. Copyright © 2015 Elsevier Inc. All rights reserved.

Metformin is a novel suppressor for transforming growth factor (TGF)-β1

NASA Astrophysics Data System (ADS)

Xiao, Han; Zhang, Jianshu; Xu, Zhonghe; Feng, Yenan; Zhang, Mingliang; Liu, Jianli; Chen, Ruifei; Shen, Jing; Wu, Jimin; Lu, Zhizhen; Fang, Xiaohong; Li, Jingyuan; Zhang, Youyi

2016-06-01

Metformin is a widely used first-line antidiabetic drug that has been shown to protect against a variety of specific diseases in addition to diabetes, including cardiovascular disorders, polycystic ovary syndrome, and cancer. However, the precise mechanisms underlying the diverse therapeutic effects of metformin remain elusive. Here, we report that transforming growth factor-β1 (TGF-β1), which is involved in the pathogenesis of numerous diseases, is a novel target of metformin. Using a surface plasmon resonance-based assay, we identified the direct binding of metformin to TGF-β1 and found that metformin inhibits [125I]-TGF-β1 binding to its receptor. Furthermore, based on molecular docking and molecular dynamics simulations, metformin was predicted to interact with TGF-β1 at its receptor-binding domain. Single-molecule force spectroscopy revealed that metformin reduces the binding probability but not the binding force of TGF-β1 to its type II receptor. Consequently, metformin suppresses type II TGF-β1 receptor dimerization upon exposure to TGF-β1, which is essential for downstream signal transduction. Thus, our results indicate that metformin is a novel TGF-β suppressor with therapeutic potential for numerous diseases in which TGF-β1 hyperfunction is indicated.

Single-molecule photobleaching reveals increased MET receptor dimerization upon ligand binding in intact cells

PubMed Central

2013-01-01

Background The human receptor tyrosine kinase MET and its ligand hepatocyte growth factor/scatter factor are essential during embryonic development and play an important role during cancer metastasis and tissue regeneration. In addition, it was found that MET is also relevant for infectious diseases and is the target of different bacteria, amongst them Listeria monocytogenes that induces bacterial uptake through the surface protein internalin B. Binding of ligand to the MET receptor is proposed to lead to receptor dimerization. However, it is also discussed whether preformed MET dimers exist on the cell membrane. Results To address these issues we used single-molecule fluorescence microscopy techniques. Our photobleaching experiments show that MET exists in dimers on the membrane of cells in the absence of ligand and that the proportion of MET dimers increases significantly upon ligand binding. Conclusions Our results indicate that partially preformed MET dimers may play a role in ligand binding or MET signaling. The addition of the bacterial ligand internalin B leads to an increase of MET dimers which is in agreement with the model of ligand-induced dimerization of receptor tyrosine kinases. PMID:23731667

Identification of heparin-binding EGF-like growth factor as a target in intercellular regulation of epidermal basal cell growth by suprabasal retinoic acid receptors.

PubMed Central

Xiao, J H; Feng, X; Di, W; Peng, Z H; Li, L A; Chambon, P; Voorhees, J J

1999-01-01

The role of retinoic acid receptors (RARs) in intercellular regulation of cell growth was assessed by targeting a dominant-negative RARalpha mutant (dnRARalpha) to differentiated suprabasal cells of mouse epidermis. dnRARalpha lacks transcriptional activation but not DNA-binding and receptor dimerization functions. Analysis of transgenic mice revealed that dnRARalpha dose-dependently impaired induction of basal cell proliferation and epidermal hyperplasia by all-trans RA (tRA). dnRARalpha formed heterodimers with endogenous retinoid X receptor-alpha (RXRalpha) over RA response elements in competition with remaining endogenous RARgamma-RXRalpha heterodimers, and dose-dependently impaired retinoid-dependent gene transcription. To identify genes regulated by retinoid receptors and involved in cell growth control, we analyzed the retinoid effects on expression of the epidermal growth factor (EGF) receptor, EGF, transforming growth factor-alpha, heparin-binding EGF-like growth factor (HB-EGF) and amphiregulin genes. In normal epidermis, tRA rapidly and selectively induced expression of HB-EGF but not the others. This induction occurred exclusively in suprabasal cells. In transgenic epidermis, dnRARalpha dose-dependently inhibited tRA induction of suprabasal HB-EGF and subsequent basal cell hyperproliferation. Together, our observations suggest that retinoid receptor heterodimers located in differentiated suprabasal cells mediate retinoid induction of HB-EGF, which in turn stimulates basal cell growth via intercellular signaling. These events may underlie retinoid action in epidermal regeneration during wound healing. PMID:10075925

Rational Design of Potent Antagonists to the Human Growth Hormone Receptor

NASA Astrophysics Data System (ADS)

Fuh, Germaine; Cunningham, Brian C.; Fukunaga, Rikiro; Nagata, Shigekazu; Goeddel, David V.; Wells, James A.

1992-06-01

A hybrid receptor was constructed that contained the extracellular binding domain of the human growth hormone (hGH) receptor linked to the transmembrane and intracellular domains of the murine granulocyte colony-stimulating factor receptor. Addition of hGH to a myeloid leukemia cell line (FDC-P1) that expressed the hybrid receptor caused proliferation of these cells. The mechanism for signal transduction of the hybrid receptor required dimerization because monoclonal antibodies to the hGH receptor were agonists whereas their monovalent fragments were not. Receptor dimerization occurs sequentially-a receptor binds to site 1 on hGH, and then a second receptor molecule binds to site 2 on hGH. On the basis of this sequential mechanism, which may occur in many other cytokine receptors, inactive hGH analogs were designed that were potent antagonists to hGH-induced cell proliferation. Such antagonists could be useful for treating clinical conditions of hGH excess, such as acromegaly.

Azadirachtin Interacts with Retinoic Acid Receptors and Inhibits Retinoic Acid-mediated Biological Responses*

PubMed Central

Thoh, Maikho; Babajan, Banaganapalli; Raghavendra, Pongali B.; Sureshkumar, Chitta; Manna, Sunil K.

2011-01-01

Considering the role of retinoids in regulation of more than 500 genes involved in cell cycle and growth arrest, a detailed understanding of the mechanism and its regulation is useful for therapy. The extract of the medicinal plant Neem (Azadirachta indica) is used against several ailments especially for anti-inflammatory, anti-itching, spermicidal, anticancer, and insecticidal activities. In this report we prove the detailed mechanism on the regulation of retinoic acid-mediated cell signaling by azadirachtin, active components of neem extract. Azadirachtin repressed all trans-retinoic acid (ATRA)-mediated nuclear transcription factor κB (NF-κB) activation, not the DNA binding but the NF-κB-dependent gene expression. It did not inhibit IκBα degradation, IκBα kinase activity, or p65 phosphorylation and its nuclear translocation but inhibited NF-κB-dependent reporter gene expression. Azadirachtin inhibited TRAF6-mediated, but not TRAF2-mediated NF-κB activation. It inhibited ATRA-induced Sp1 and CREB (cAMP-response element-binding protein) DNA binding. Azadirachtin inhibited ATRA binding with retinoid receptors, which is supported by biochemical and in silico evidences. Azadirachtin showed strong interaction with retinoid receptors. It suppressed ATRA-mediated removal of retinoid receptors, bound with DNA by inhibiting ATRA binding to its receptors. Overall, our data suggest that azadirachtin interacts with retinoic acid receptors and suppresses ATRA binding, inhibits falling off the receptors, and activates transcription factors like CREB, Sp1, NF-κB, etc. Thus, azadirachtin exerts anti-inflammatory and anti-metastatic responses by a novel pathway that would be beneficial for further anti-inflammatory and anti-cancer therapies. PMID:21127062

Azadirachtin interacts with the tumor necrosis factor (TNF) binding domain of its receptors and inhibits TNF-induced biological responses.

PubMed

Thoh, Maikho; Kumar, Pankaj; Nagarajaram, Hampathalu A; Manna, Sunil K

2010-02-19

The role of azadirachtin, an active component of a medicinal plant Neem (Azadirachta indica), on TNF-induced cell signaling in human cell lines was investigated. Azadirachtin blocks TNF-induced activation of nuclear factor kappaB (NF-kappaB) and also expression of NF-kappaB-dependent genes such as adhesion molecules and cyclooxygenase 2. Azadirachtin inhibits the inhibitory subunit of NF-kappaB (IkappaB alpha) phosphorylation and thereby its degradation and RelA (p65) nuclear translocation. It blocks IkappaB alpha kinase (IKK) activity ex vivo, but not in vitro. Surprisingly, azadirachtin blocks NF-kappaB DNA binding activity in transfected cells with TNF receptor-associated factor (TRAF)2, TNF receptor-associated death domain (TRADD), IKK, or p65, but not with TNFR, suggesting its effect is at the TNFR level. Azadirachtin blocks binding of TNF, but not IL-1, IL-4, IL-8, or TNF-related apoptosis-inducing ligand (TRAIL) with its respective receptors. Anti-TNFR antibody or TNF protects azadirachtin-mediated down-regulation of TNFRs. Further, in silico data suggest that azadirachtin strongly binds in the TNF binding site of TNFR. Overall, our data suggest that azadirachtin modulates cell surface TNFRs thereby decreasing TNF-induced biological responses. Thus, azadirachtin exerts an anti-inflammatory response by a novel pathway, which may be beneficial for anti-inflammatory therapy.

Azadirachtin Interacts with the Tumor Necrosis Factor (TNF) Binding Domain of Its Receptors and Inhibits TNF-induced Biological Responses*

PubMed Central

Thoh, Maikho; Kumar, Pankaj; Nagarajaram, Hampathalu A.; Manna, Sunil K.

2010-01-01

The role of azadirachtin, an active component of a medicinal plant Neem (Azadirachta indica), on TNF-induced cell signaling in human cell lines was investigated. Azadirachtin blocks TNF-induced activation of nuclear factor κB (NF-κB) and also expression of NF-κB-dependent genes such as adhesion molecules and cyclooxygenase 2. Azadirachtin inhibits the inhibitory subunit of NF-κB (IκBα) phosphorylation and thereby its degradation and RelA (p65) nuclear translocation. It blocks IκBα kinase (IKK) activity ex vivo, but not in vitro. Surprisingly, azadirachtin blocks NF-κB DNA binding activity in transfected cells with TNF receptor-associated factor (TRAF)2, TNF receptor-associated death domain (TRADD), IKK, or p65, but not with TNFR, suggesting its effect is at the TNFR level. Azadirachtin blocks binding of TNF, but not IL-1, IL-4, IL-8, or TNF-related apoptosis-inducing ligand (TRAIL) with its respective receptors. Anti-TNFR antibody or TNF protects azadirachtin-mediated down-regulation of TNFRs. Further, in silico data suggest that azadirachtin strongly binds in the TNF binding site of TNFR. Overall, our data suggest that azadirachtin modulates cell surface TNFRs thereby decreasing TNF-induced biological responses. Thus, azadirachtin exerts an anti-inflammatory response by a novel pathway, which may be beneficial for anti-inflammatory therapy. PMID:20018848

Docking simulations suggest that all-trans retinoic acid could bind to retinoid X receptors.

PubMed

Tsuji, Motonori; Shudo, Koichi; Kagechika, Hiroyuki

2015-10-01

Retinoid X receptors (RXRs) are ligand-controlled transcription factors which heterodimerize with other nuclear receptors to regulate gene transcriptions associated with crucial biological events. 9-cis retinoic acid (9cRA), which transactivates RXRs, is believed to be an endogenous RXR ligand. All-trans retinoic acid (ATRA) is a natural ligand for retinoic acid receptors (RARs), which heterodimerize with RXRs. Although the concentration of 9cRA in tissues is very low, ATRA is relatively abundant and some reports show that ATRA activates RXRs. We computationally studied the possibility of ATRA binding to RXRs using two different docking methods with our developed programs to assess the binding affinities of naturally occurring retinoids. The simulations showed good correlations to the reported binding affinities of these molecules for RXRs and RARs.

EGF receptor ligands: recent advances.

PubMed

Singh, Bhuminder; Carpenter, Graham; Coffey, Robert J

2016-01-01

Seven ligands bind to and activate the mammalian epidermal growth factor (EGF) receptor (EGFR/ERBB1/HER1): EGF, transforming growth factor-alpha (TGFA), heparin-binding EGF-like growth factor (HBEGF), betacellulin (BTC), amphiregulin (AREG), epiregulin (EREG), and epigen (EPGN). Of these, EGF, TGFA, HBEGF, and BTC are thought to be high-affinity ligands, whereas AREG, EREG, and EPGN constitute low-affinity ligands. This focused review is meant to highlight recent studies related to actions of the individual EGFR ligands, the interesting biology that has been uncovered, and relevant advances related to ligand interactions with the EGFR.

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

Characterization of the Igf-II Binding Site of the IGF-II/MAN-6-P Receptor Extracellular Domain.

NASA Astrophysics Data System (ADS)

Garmroudi, Farideh

1995-01-01

In mammals, insulin-like growth factor II (IGF -II) and glycoproteins bearing the mannose 6-phosphate (Man -6-P) recognition marker bind with high affinity to the same receptor. The functional consequences of IGF-II binding to the receptor at the cell surface are not clear. In these studies, we sought to broaden our understanding of the functional regions of the receptor regarding its IGF -II binding site. The IGF-II binding/cross-linking domain of the IGF-II/Man-6-P receptor was mapped by sequencing receptor fragments covalently attached to IGF-II. Purified rat placental or bovine liver receptors were affinity-labeled, with ^{125}I-IGF-II and digested with endoproteinase Glu-C. Analysis of digests by gel electrophoresis revealed a major radiolabeled band of 18 kDa, which was purified by gel filtration chromatography followed by reverse-phase HPLC and electroblotting. Sequence analysis revealed that, the peptide S(H)VNSXPMF, located within extracellular repeat 10 and beginning with serine 1488 of the bovine receptor, was the best candidate for the IGF-II cross-linked peptide. These data indicated that residues within repeats 10-11 were important for IGF -II binding. To define the location of the IGF-II binding site further, a nested set of six human receptor cDNA constructs was designed to produce epitope-tagged fusion proteins encompassing the region between repeats 8 and 11 of the human IGF-II/Man-6-P receptor extracellular domain. These truncated receptors were transiently expressed in COS-7 cells, immunoprecipitated and analyzed for their abilities to bind and cross-link to IGF-II. All of the constructs were capable of binding/cross-linking to IGF-II, except for the 9.0-11 construct. Displacement curve analysis indicated that the truncated receptors were approximately equivalent in IGF-II binding affinity, but were of 5- to 10-fold lower affinity than full-length receptors. Sequencing of the 9.0-11 construct indicated the presence of a point mutation substituting threonine for isoleucine at position 1621, which is located in the N-terminal half of repeat 11, and was found to abrogate IGF-II binding. Collectively, our work indicates that repeat 11 of the IGF-II/Man-6-P receptor's extracellular domain encompasses the elements both for binding and cross-linking to IGF-II.

Tomography of epidermal growth factor receptor binding to fluorescent Affibody in vivo studied with magnetic resonance guided fluorescence recovery in varying orthotopic glioma sizes

NASA Astrophysics Data System (ADS)

Holt, Robert W.; Demers, Jennifer-Lynn H.; Sexton, Kristian J.; Gunn, Jason R.; Davis, Scott C.; Samkoe, Kimberley S.; Pogue, Brian W.

2015-02-01

The ability to image targeted tracer binding to epidermal growth factor receptor (EGFR) was studied in vivo in orthotopically grown glioma tumors of different sizes. The binding potential was quantified using a dual-tracer approach, which employs a fluorescently labeled peptide targeted to EGFR and a reference tracer with similar pharmacokinetic properties but no specific binding, to estimate the relative bound fraction from kinetic compartment modeling. The recovered values of binding potential did not vary significantly as a function of tumor size (1 to 33 mm3), suggesting that binding potential may be consistent in the U251 tumors regardless of size or stage after implantation. However, the fluorescence yield of the targeted fluorescent tracers in the tumor was affected significantly by tumor size, suggesting that dual-tracer imaging helps account for variations in absolute uptake, which plague single-tracer imaging techniques. Ex vivo analysis showed relatively high spatial heterogeneity in each tumor that cannot be resolved by tomographic techniques. Nonetheless, the dual-tracer tomographic technique is a powerful tool for longitudinal bulk estimation of receptor binding.

Smad2-dependent glycosaminoglycan elongation in aortic valve interstitial cells enhances binding of LDL to proteoglycans.

PubMed

Osman, Narin; Grande-Allen, K Jane; Ballinger, Mandy L; Getachew, Robel; Marasco, Silvana; O'Brien, Kevin D; Little, Peter J

2013-01-01

Calcific aortic valve disease is a progressive condition that shares some common pathogenic features with atherosclerosis. Transforming growth factor-β1 is a recognized mediator of atherosclerosis and is expressed in aortic valve lesions. Transforming growth factorβ1 stimulates glycosaminoglycan elongation of proteoglycans that is associated with increased lipid binding. We investigated the presence of transforming growth factor-β1 and downstream signaling intermediates in diseased human aortic valves and the effects of activated transforming growth factor-β1 receptor signaling on aortic valve interstitial cell proteoglycan synthesis and lipid binding as a possible mechanism for the initiation of the early lesion of calcific aortic valve disease. Diseased human aortic valve leaflets demonstrated strong immunohistochemical staining for transforming growth factor-β1 and phosphorylated Smad2/3. In primary porcine aortic valve interstitial cells, Western blots showed that transforming growth factor-β1 stimulated phosphorylation in both the carboxy and linker regions of Smad2/3, which was inhibited by the transforming growth factor-β1 receptor inhibitor SB431542. Gel electrophoresis and size exclusion chromatography demonstrated that SB431542 decreased transforming growth factor-β1-mediated [(35)S]-sulfate incorporation into proteoglycans in a dose-dependent manner. Further, in proteoglycans derived from transforming growth factor-β1-treated valve interstitial cells, gel mobility shift assays demonstrated that inhibition of transforming growth factor-β1 receptor signaling resulted in decreased lipid binding. Classic transforming growth factor-β1 signaling is present in human aortic valves in vivo and contributes to the modification of proteoglycans expressed by valve interstitial cells in vitro. These findings suggest that transforming growth factor-β1 may promote increased low-density lipoprotein binding in the early phases of calcific aortic valve disease. Copyright © 2013 Elsevier Inc. All rights reserved.

Coarse-Grained Molecular Simulation of Epidermal Growth Factor Receptor Protein Tyrosine Kinase Multi-Site Self-Phosphorylation

PubMed Central

Koland, John G.

2014-01-01

Upon the ligand-dependent dimerization of the epidermal growth factor receptor (EGFR), the intrinsic protein tyrosine kinase (PTK) activity of one receptor monomer is activated, and the dimeric receptor undergoes self-phosphorylation at any of eight candidate phosphorylation sites (P-sites) in either of the two C-terminal (CT) domains. While the structures of the extracellular ligand binding and intracellular PTK domains are known, that of the ∼225-amino acid CT domain is not, presumably because it is disordered. Receptor phosphorylation on CT domain P-sites is critical in signaling because of the binding of specific signaling effector molecules to individual phosphorylated P-sites. To investigate how the combination of conventional substrate recognition and the unique topological factors involved in the CT domain self-phosphorylation reaction lead to selectivity in P-site phosphorylation, we performed coarse-grained molecular simulations of the P-site/catalytic site binding reactions that precede EGFR self-phosphorylation events. Our results indicate that self-phosphorylation of the dimeric EGFR, although generally believed to occur in trans, may well occur with a similar efficiency in cis, with the P-sites of both receptor monomers being phosphorylated to a similar extent. An exception was the case of the most kinase-proximal P-site-992, the catalytic site binding of which occurred exclusively in cis via an intramolecular reaction. We discovered that the in cis interaction of P-site-992 with the catalytic site was facilitated by a cleft between the N-terminal and C-terminal lobes of the PTK domain that allows the short CT domain sequence tethering P-site-992 to the PTK core to reach the catalytic site. Our work provides several new mechanistic insights into the EGFR self-phosphorylation reaction, and demonstrates the potential of coarse-grained molecular simulation approaches for investigating the complexities of self-phosphorylation in molecules such as EGFR (HER/ErbB) family receptors and growth factor receptor PTKs in general. PMID:24453959

Absence of C-type natriuretic peptide receptors in hamster glomeruli.

PubMed

Luk, J K; Wong, E F; Wong, N L

1994-01-01

The distribution of atrial natriuretic peptide receptor B (ANPR-B) varies between tissues and species. The aim of this study is to determine whether ANPR-B is present in the hamster glomeruli. In vitro C-type natriuretic peptide (CNP)- and atrial natriuretic factor (ANF)-stimulated cGMP accumulation studies were performed in hamster glomeruli. Elevated cGMP accumulations were observed upon ANF addition. No cGMP response was seen with CNP. Competitive receptor-binding experiments were performed with 125I-CNP and 125I-ANF against their respective cold peptides in hamster glomeruli. Although no CNP binding was detected, positive ANF binding was found and two types of ANF receptor were demonstrated. The affinity (Kdl) and maximum binding capacity (Bmaxl) of the high-affinity ANF receptor were 0.014 +/- 0.001 nM and 60.4 +/- 10.2 fmol/mg protein, respectively. Those of the low-affinity receptor (Kd2 and Bmax2) were 45.7 +/- 6.2 nM and 28.3 +/- 6.3 pmol/mg protein, respectively. Similarly, saturation binding experiments also failed to show any CNP receptor binding in hamster glomeruli. This finding suggests that ANPR-B is not present in hamster glomeruli and CNP is not a direct physiological regulator of hamster renal function.

Role of G protein-coupled estrogen receptor-1, matrix metalloproteinases 2 and 9, and heparin binding epidermal growth factor-like growth factor in estradiol-17β-stimulated bovine satellite cell proliferation.

PubMed

Kamanga-Sollo, E; Thornton, K J; White, M E; Dayton, W R

2014-10-01

In feedlot steers, estradiol-17β (E2) and combined E2 and trenbolone acetate (a testosterone analog) implants enhance rate and efficiency of muscle growth; and, consequently, these compounds are widely used as growth promoters. Although the positive effects of E2 on rate and efficiency of bovine muscle growth are well established, the mechanisms involved in these effects are not well understood. Combined E2 and trenbolone acetate implants result in significantly increased muscle satellite cell number in feedlot steers. Additionally, E2 treatment stimulates proliferation of cultured bovine satellite cells (BSC). Studies in nonmuscle cells have shown that binding of E2 to G protein-coupled estrogen receptor (GPER)-1 results in activation of matrix metalloproteinases 2 and 9 (MMP2/9) resulting in proteolytic release of heparin binding epidermal growth factor-like growth factor (hbEGF) from the cell surface. Released hbEGF binds to and activates the epidermal growth factor receptor resulting in increased proliferation. To assess if GPER-1, MMP2/9, and/or hbEGF are involved in the mechanism of E2-stimulated BSC proliferation, we have examined the effects of G36 (a specific inhibitor of GPER-1), CRM197 (a specific inhibitor of hbEGF), and MMP-2/MMP-9 Inhibitor II (an inhibitor of MMP2/9 activity) on E2-stimulated BSC proliferation. Inhibition of GPER-1, MMP2/9, or hbEGF suppresses E2-stimulated BSC proliferation (P < 0.001) suggesting that all these are required in order for E2 to stimulate BSC proliferation. These results strongly suggest that E2 may stimulate BSC proliferation by binding to GPER-1 resulting in MMP2/9-catalyzed release of cell membrane-bound hbEGF and subsequent activation of epidermal growth factor receptor by binding of released hbEGF. Copyright © 2014 Elsevier Inc. All rights reserved.

Gene Transfer and Molecular Cloning of the Human NGF Receptor

NASA Astrophysics Data System (ADS)

Chao, Moses V.; Bothwell, Mark A.; Ross, Alonzo H.; Koprowski, Hilary; Lanahan, Anthony A.; Buck, C. Randall; Sehgal, Amita

1986-04-01

Nerve growth factor (NGF) and its receptor are important in the development of cells derived from the neural crest. Mouse L cell transformants have been generated that stably express the human NGF receptor gene transfer with total human DNA. Affinity cross-linking, metabolic labeling and immunoprecipitation, and equilibrium binding with 125I-labeled NGF revealed that this NGF receptor had the same size and binding characteristics as the receptor from human melanoma cells and rat PC12 cells. The sequences encoding the NGF receptor were molecularly cloned using the human Alu repetitive sequence as a probe. A cosmid clone that contained the human NGF receptor gene allowed efficient transfection and expression of the receptor.

Ligand-independent activation of the oestrogen receptor by mutation of a conserved tyrosine.

PubMed Central

White, R; Sjöberg, M; Kalkhoven, E; Parker, M G

1997-01-01

The oestrogen receptor is a member of the nuclear receptor family of transcription factors which, on binding the steroid hormone 17beta-oestradiol, interacts with co-activator proteins and stimulates gene expression. Replacement of a single tyrosine in the hormone-binding domain generated activated forms of the receptor which stimulated transcription in the absence of hormone. This increased activation is related to a decrease in hydrophobicity and a reduction in size of the side chain of the amino acid with which the tyrosine is replaced. Ligand-independent, in common with ligand-dependent transcriptional activation, requires an amphipathic alpha-helix at the C-terminus of the ligand-binding domain which is essential for the interaction of the receptor with a number of potential co-activator proteins. In contrast to the wild-type protein, constitutively active receptors were able to bind both the receptor-interacting protein RIP-140 and the steroid receptor co-activator SRC-1 in a ligand-independent manner, although in the case of SRC-1 this was only evident when the receptors were prebound to DNA. We propose, therefore, that this tyrosine is required to maintain the receptor in a transcriptionally inactive state in the absence of hormone. Modification of this residue may generate a conformational change in the ligand-binding domain of the receptor to form an interacting surface which allows the recruitment of co-activators independent of hormone binding. This suggests that this tyrosine may be a target for a different signalling pathway which forms an alternative mechanism of activating oestrogen receptor-mediated transcription. PMID:9135157

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

Freemark, M.; Comer, M.; Mularoni, T.

We have recently identified and purified from fetal liver a distinct receptor that mediates the effects of placental lactogen (PL) on amino acid transport, glycogen synthesis, and somatomedin production in fetal tissues. At present, the factors that regulate the number and affinity of PL receptors in the fetus are unknown. Since maternal nutrition plays a critical role in fetal metabolism and growth, we have examined the role of nutrition in the regulation of the PL receptor in fetal lambs. Pregnant ewes at 123-126 days gestation were fed ad libitum (FED), fasted for 3 days (FASTED), or fasted for 3 daysmore » and then refed for an additional 3 days (REFED). The ewes were then killed, and the binding of (125I)ovine (o) PL to hepatic microsomes from the fetal lambs was examined. Maternal fasting caused a 60-75% reduction in the specific binding of oPL to fetal liver; the effect of fasting was reversed in part by refeeding. The decrease in oPL binding resulted from an 80% reduction in the number of fetal oPL-binding sites (Scatchard analysis); there were no changes in the affinity of the oPL receptor (Kd, 0.6 nM), the subunit structure of the receptor, or the degree of occupancy of the receptor in vivo by endogenous fetal hormones. The specific bindings of GH (0.6%), PRL (0.3%), and insulin (35%) to fetal liver were not affected by maternal fasting, indicating that caloric restriction exerted a specific effect on oPL binding in the fetus. The number of fetal oPL-binding sites was positively correlated with the fetal liver glycogen content (r = 0.69; P less than 0.01) and the fetal plasma concentrations of glucose (r = 0.68; P less than 0.01) and insulin-like growth factor-I (r = 0.74; P less than 0.001), suggesting a role for the PL receptor in the regulation of fetal carbohydrate metabolism and growth.« less

Antitumor activity of a novel anti-vascular endothelial growth factor receptor-1 monoclonal antibody that does not interfere with ligand binding

PubMed Central

Tentori, Lucio; Scimeca, Manuel; Dorio, Annalisa S.; Atzori, Maria Grazia; Failla, Cristina M.; Morea, Veronica; Bonanno, Elena; D'Atri, Stefania; Lacal, Pedro M.

2016-01-01

Vascular endothelial growth factor receptor-1 (VEGFR-1) is a tyrosine kinase transmembrane receptor that has also a soluble isoform containing most of the extracellular ligand binding domain (sVEGFR-1). VEGF-A binds to both VEGFR-2 and VEGFR-1, whereas placenta growth factor (PlGF) interacts exclusively with VEGFR-1. In this study we generated an anti-VEGFR-1 mAb (D16F7) by immunizing BALB/C mice with a peptide that we had previously reported to inhibit angiogenesis and endothelial cell migration induced by PlGF. D16F7 did not affect binding of VEGF-A or PlGF to VEGFR-1, thus allowing sVEGFR-1 to act as decoy receptor for these growth factors, but it hampered receptor homodimerization and activation. D16F7 inhibited both the chemotactic response of human endothelial, myelomonocytic and melanoma cells to VEGFR-1 ligands and vasculogenic mimicry by tumor cells. Moreover, D16F7 exerted in vivo antiangiogenic effects in a matrigel plug assay. Importantly, D16F7 inhibited tumor growth and was well tolerated by B6D2F1 mice injected with syngeneic B16F10 melanoma cells. The antitumor effect was associated with melanoma cell apoptosis, vascular abnormalities and decrease of both monocyte/macrophage infiltration and myeloid progenitor mobilization. For all the above, D16F7 may be exploited in the therapy of metastatic melanoma and other tumors or pathological conditions involving VEGFR-1 activation. PMID:27655684

Phospholipase C-gamma 1 binding to intracellular receptors for activated protein kinase C.

PubMed

Disatnik, M H; Hernandez-Sotomayor, S M; Jones, G; Carpenter, G; Mochly-Rosen, D

1994-01-18

Phospholipase C-gamma 1 (PLC-gamma 1; EC 3.1.4.11) hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol and inositol 1,4,5-trisphosphate and is activated in response to growth factor stimulation and tyrosine phosphorylation. Concomitantly, the enzyme translocates from the cytosol to the particulate cell fraction. A similar process of activation-induced translocation from the cytosol to the cell particulate fraction has also been described for protein kinase C (PKC). We have previously shown that activated PKC binds to specific receptor proteins, receptors for activated C kinase, or RACKs, of approximately 30 kDa. Here, we show that PLC-gamma 1 bound to these RACKs and inhibited subsequent PKC binding to RACKs. However, unlike PKC, the binding of PLC-gamma 1 to RACKs did not require phospholipids and calcium. After epidermal growth factor treatment of intact A-431 cells, the binding of PLC-gamma 1 to RACKs increased as compared with PLC-gamma 1 from control cells. This increase in PLC-gamma 1 binding to RACKs was due to the phosphorylation of PLC-gamma 1. Additional data indicated that PLC-gamma 1 binds to RACKs in solution; epidermal growth factor receptor-dependent PLC-gamma 1 phosphorylation and activation decreased in the presence of RACKs. It is possible that, in vivo, PLC-gamma 1 associates with RACKs or with other PLC-gamma 1-specific anchoring proteins in the particulate cell fraction. Since a PKC C2 homologous region is present in PLC-gamma 1, the C2 region may mediate the activation-induced translocation of the enzyme to the cell particulate fraction and the anchoring protein-PLC-gamma 1 complex may be the active translocated form of PLC-gamma 1.

Expression of human peroxisome proliferator-activated receptors ligand binding domain-maltose binding protein fusion protein in Escherichia coli: a convenient and reliable method for preparing receptor for screening ligands.

PubMed

Li, Changqing; Tian, Mi; Yuan, Ye; Zhou, Qinxin

2008-12-01

Human peroxisome proliferator-activated receptors (hPPARs) are ligand-activated transcription factors and are the target for the treatment of many diseases. Screening of their ligands is mainly based on assays of ligand binding to the ligand binding domain (LBD) of hPPARs.However, such assays are difficult because of the preparation of hPPARs LBD. In order to yield functional hPPARs LBD for screening ligands, hPPARs LBD was fused with maltose-binding protein(MBP) using the pMAL-p2x expression system through the gene engineering technique. The radioligand binding assay showed that MBP did not affect ligand binding with hPPARs LBD in the fusion proteins, which means that MBP-hPPARs LBD can be used instead of hPPARs LBD in ligand screening work. The results show that the new strategy using MBP as a fusion tag for preparing hPPARs LBD for screening ligands is a convenient and reliable method. It may be used to easily obtain the other nuclear receptors.

Mutant protein of recombinant human granulocyte colony-stimulating factor for receptor binding assay.

PubMed

Watanabe, M; Fukamachi, H; Uzumaki, H; Kabaya, K; Tsumura, H; Ishikawa, M; Matsuki, S; Kusaka, M

1991-05-15

A new mutant protein of recombinant human granulocyte colony-stimulating factor (rhG-CSF) was produced for the studies on receptors for human G-CSF. The mutant protein [(Tyr1, Tyr3]rhG-CSF), the biological activity of which was almost equal to that of rhG-CSF, was prepared by the replacement of threonine-1 and leucine-3 of rhG-CSF with tyrosine. The radioiodinated preparation of the mutant protein showed high specific radioactivity and retained full biological activity for at least 3 weeks. The binding capacity of the radioiodinated ligand was compared with that of [35S]rhG-CSF. Both radiolabeled ligands showed specific binding to murine bone marrow cells. Unlabeled rhG-CSF and human G-CSF purified from the culture supernatant of the human bladder carcinoma cell line 5637 equally competed for the binding of labeled rhG-CSFs in a dose-dependent manner, demonstrating that the sugar moiety of human G-CSF made no contribution to the binding of human G-CSF to target cells. In contrast, all other colony-stimulating factors and lymphokines examined did not affect the binding. Scatchard analysis of the specific binding of both labeled ligands revealed a single class of binding site with an apparent dissociation constant (Kd) of 20-30 pM and 100-200 maximal binding sites per cell. These data indicate that the radioiodinated preparation of the mutant protein binds the same specific receptor with the same affinity as [35S]rhG-CSF. The labeled mutant protein also showed specific binding to human circulating neutrophils.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid Activation of Bone Morphogenic Protein 9 by Receptor-mediated Displacement of Pro-domains*

PubMed Central

Kienast, Yvonne; Jucknischke, Ute; Scheiblich, Stefan; Thier, Martina; de Wouters, Mariana; Haas, Alexander; Lehmann, Christian; Brand, Verena; Bernicke, Dirk; Honold, Konrad; Lorenz, Stefan

2016-01-01

By non-covalent association after proteolytic cleavage, the pro-domains modulate the activities of the mature growth factor domains across the transforming growth factor-β family. In the case of bone morphogenic protein 9 (BMP9), however, the pro-domains do not inhibit the bioactivity of the growth factor, and the BMP9·pro-domain complexes have equivalent biological activities as the BMP9 mature ligand dimers. By using real-time surface plasmon resonance, we could demonstrate that either binding of pro-domain-complexed BMP9 to type I receptor activin receptor-like kinase 1 (ALK1), type II receptors, co-receptor endoglin, or to mature BMP9 domain targeting antibodies leads to immediate and complete displacement of the pro-domains from the complex. Vice versa, pro-domain binding by an anti-pro-domain antibody results in release of the mature BMP9 growth factor. Based on these findings, we adjusted ELISA assays to measure the protein levels of different BMP9 variants. Although mature BMP9 and inactive precursor BMP9 protein were directly detectable by ELISA, BMP9·pro-domain complex could only be measured indirectly as dissociated fragments due to displacement of mature growth factor and pro-domains after antibody binding. Our studies provide a model in which BMP9 can be readily activated upon getting into contact with its receptors. This increases the understanding of the underlying biology of BMP9 activation and also provides guidance for ELISA development for the detection of circulating BMP9 variants. PMID:26677222

The opposing roles of laminin-binding integrins in cancer.

PubMed

Ramovs, Veronika; Te Molder, Lisa; Sonnenberg, Arnoud

2017-01-01

Integrins play an important role in cell adhesion by linking the cytoskeleton of cells to components in the extracellular matrix. In this capacity, integrins cooperate with different cell surface receptors, including growth factor receptors and G-protein coupled receptors, to regulate intracellular signaling pathways that control cell polarization, spreading, migration, survival, and gene expression. A distinct subfamily of molecules in the integrin family of adhesion receptors is formed by receptors that mediate cell adhesion to laminins, major components of the basement membrane that lie under clusters of cells or surround them, separating them from other cells and/or adjacent connective tissue. During the past decades, many studies have provided evidence for a role of laminin-binding integrins in tumorigenesis, and both tumor-promoting and suppressive activities have been identified. In this review we discuss the dual role of the laminin-binding integrins α3β1 and α6β4 in tumor development and progression, and examine the factors and mechanisms involved in these opposing effects. Copyright © 2016 Elsevier B.V. All rights reserved.

A Major Binding Protein for Leukemia Inhibitory Factor in Normal Mouse Serum: Identification as a Soluble Form of the Cellular Receptor

NASA Astrophysics Data System (ADS)

Layton, Meredith J.; Cross, Bronwyn A.; Metcalf, Donald; Ward, Larry D.; Simpson, Richard J.; Nicola, Nicos A.

1992-09-01

A protein that specifically binds leukemia inhibitory factor (LIF) has been isolated from normal mouse serum by using four successive fractionation steps: chromatography on a LIF affinity matrix, anion-exchange chromatography, size-exclusion chromatography, and preparative native gel electrophoresis. The purified LIF-binding protein (LBP) is a glycoprotein with an apparent molecular mass of 90 kDa that specifically binds 125I-labeled murine LIF with an affinity comparable to that of the low-affinity cellular LIF receptor (K_d = 600 pM). N-terminal sequencing has identified this protein as a soluble truncated form of the α chain of the cellular LIF receptor. LBP is present in normal mouse serum at high levels (1 μg/ml) and these levels are elevated in pregnant mice and reduced in neonatal mice. Since normal serum concentrations of LBP can block the biological actions of LIF in culture, LBP may serve as an inhibitor of the systemic effects of locally produced LIF.

Effects of the Insulin-like Growth Factor Pathway on the Regulation of Mammary Gland Development.

PubMed

Ha, Woo Tae; Jeong, Ha Yeon; Lee, Seung Yoon; Song, Hyuk

2016-09-01

The insulin-like growth factor (IGF) pathway is a key signal transduction pathway involved in cell proliferation, migration, and apoptosis. In dairy cows, IGF family proteins and binding receptors, including their intracellular binding partners, regulate mammary gland development. IGFs and IGF receptor interactions in mammary glands influence the early stages of mammogenesis, i.e., mammary ductal genesis until puberty. The IGF pathway includes three major components, IGFs (such as IGF-I, IGF-II, and insulin), their specific receptors, and their high-affinity binding partners (IGF binding proteins [IGFBPs]; i.e., IGFBP1-6), including specific proteases for each IGFBP. Additionally, IGFs and IGFBP interactions are critical for the bioactivities of various intracellular mechanisms, including cell proliferation, migration, and apoptosis. Notably, the interactions between IGFs and IGFBPs in the IGF pathway have been difficult to characterize during specific stages of bovine mammary gland development. In this review, we aim to describe the role of the interaction between IGFs and IGFBPs in overall mammary gland development in dairy cows.

Comparison of Nerve Growth Factor Receptor Binding Models Using Heterodimeric Muteins

PubMed Central

Mehta, Hrishikesh M.; Woo, Sang B.; Neet, Kenneth E.

2013-01-01

Nerve growth factor (NGF) is a homodimer that binds to two distinct receptor types, TrkA and p75, to support survival and differentiation of neurons. The high-affinity binding on the cell surface is believed to involve a heteroreceptor complex, but its exact nature is unclear. We developed a heterodimer (heteromutein) of two NGF muteins that can bind p75 and TrkA on opposite sides of the heterodimer, but not two TrkA receptors. Previously described muteins are Δ9/13 that is TrkA negative and 7-84-103 that is signal selective through TrkA. The heteromutein (Htm1) was used to study the heteroreceptor complex formation and function, in the putative absence of NGF-induced TrkA dimerization. Cellular binding assays indicated that Htm1 does not bind TrkA as efficiently as wild-type (wt) NGF but has better affinity than either homodimeric mutein. Htm1, 7-84-103, and Δ9/13 were each able to compete for cold-temperature, cold-chase stable binding on PC12 cells, indicating that binding to p75 was required for a portion of this high-affinity binding. Survival, neurite outgrowth, and MAPK signaling in PC12 cells also showed a reduced response for Htm1, compared with wtNGF, but was better than the parent muteins in the order wtNGF > Htm1 > 7-84-103 >> Δ9/13. Htm1 and 7-84-103 demonstrated similar levels of survival on cells expressing only TrkA. In the longstanding debate on the NGF receptor binding mechanism, our data support the ligand passing of NGF from p75 to TrkA involving a transient heteroreceptor complex of p75-NGF-TrkA. PMID:22903500

H-2RIIBP, a member of the nuclear hormone receptor superfamily that binds to both the regulatory element of major histocompatibility class I genes and the estrogen response element.

PubMed

Hamada, K; Gleason, S L; Levi, B Z; Hirschfeld, S; Appella, E; Ozato, K

1989-11-01

Transcription of major histocompatibility complex (MHC) class I genes is regulated by the conserved MHC class I regulatory element (CRE). The CRE has two factor-binding sites, region I and region II, both of which elicit enhancer function. By screening a mouse lambda gt 11 library with the CRE as a probe, we isolated a cDNA clone that encodes a protein capable of binding to region II of the CRE. This protein, H-2RIIBP (H-2 region II binding protein), bound to the native region II sequence, but not to other MHC cis-acting sequences or to mutant region II sequences, similar to the naturally occurring region II factor in mouse cells. The deduced amino acid sequence of H-2RIIBP revealed two putative zinc fingers homologous to the DNA-binding domain of steroid/thyroid hormone receptors. Although sequence similarity in other regions was minimal, H-2RIIBP has apparent modular domains characteristic of the nuclear hormone receptors. Further analyses showed that both H-2RIIBP and the natural region II factor bind to the estrogen response element (ERE) of the vitellogenin A2 gene. The ERE is composed of a palindrome, and half of this palindrome resembles the region II binding site of the MHC CRE. These results indicate that H-2RIIBP (i) is a member of the superfamily of nuclear hormone receptors and (ii) may regulate not only MHC class I genes but also genes containing the ERE and related sequences. Sequences homologous to the H-2RIIBP gene are widely conserved in the animal kingdom. H-2RIIBP mRNA is expressed in many mouse tissues, in agreement with the distribution of the natural region II factor.

Ligand binding and dynamics of the monomeric epidermal growth factor receptor ectodomain

PubMed Central

Loeffler, Hannes H; Winn, Martyn D

2013-01-01

The ectodomain of the human epidermal growth factor receptor (hEGFR) controls input to several cell signalling networks via binding with extracellular growth factors. To gain insight into the dynamics and ligand binding of the ectodomain, the hEGFR monomer was subjected to molecular dynamics simulation. The monomer was found to be substantially more flexible than the ectodomain dimer studied previously. Simulations where the endogeneous ligand EGF binds to either Subdomain I or Subdomain III, or where hEGFR is unbound, show significant differences in dynamics. The molecular mechanics Poisson–Boltzmann surface area method has been used to derive relative free energies of ligand binding, and we find that the ligand is capable of binding either subdomain with a slight preference for III. Alanine-scanning calculations for the effect of selected ligand mutants on binding reproduce the trends of affinity measurements. Taken together, these results emphasize the possible role of the ectodomain monomer in the initial step of ligand binding, and add details to the static picture obtained from crystal structures. Proteins 2013; 81:1931–1943. © 2013 The Authors. Proteins published by Wiley Periodicals, Inc. PMID:23760854

Identification of the cellular receptor for anthrax toxin

NASA Astrophysics Data System (ADS)

Bradley, Kenneth A.; Mogridge, Jeremy; Mourez, Michael; Collier, R. John; Young, John A. T.

2001-11-01

The tripartite toxin secreted by Bacillus anthracis, the causative agent of anthrax, helps the bacterium evade the immune system and can kill the host during a systemic infection. Two components of the toxin enzymatically modify substrates within the cytosol of mammalian cells: oedema factor (OF) is an adenylate cyclase that impairs host defences through a variety of mechanisms including inhibiting phagocytosis; lethal factor (LF) is a zinc-dependent protease that cleaves mitogen-activated protein kinase kinase and causes lysis of macrophages. Protective antigen (PA), the third component, binds to a cellular receptor and mediates delivery of the enzymatic components to the cytosol. Here we describe the cloning of the human PA receptor using a genetic complementation approach. The receptor, termed ATR (anthrax toxin receptor), is a type I membrane protein with an extracellular von Willebrand factor A domain that binds directly to PA. In addition, a soluble version of this domain can protect cells from the action of the toxin.

EGF receptor ligands: recent advances

PubMed Central

Singh, Bhuminder; Carpenter, Graham; Coffey, Robert J.

2016-01-01

Seven ligands bind to and activate the mammalian epidermal growth factor (EGF) receptor (EGFR/ERBB1/HER1): EGF, transforming growth factor-alpha (TGFA), heparin-binding EGF-like growth factor (HBEGF), betacellulin (BTC), amphiregulin (AREG), epiregulin (EREG), and epigen (EPGN). Of these, EGF, TGFA, HBEGF, and BTC are thought to be high-affinity ligands, whereas AREG, EREG, and EPGN constitute low-affinity ligands. This focused review is meant to highlight recent studies related to actions of the individual EGFR ligands, the interesting biology that has been uncovered, and relevant advances related to ligand interactions with the EGFR. PMID:27635238

Structural Basis of Chemokine Sequestration by CrmD, a Poxvirus-Encoded Tumor Necrosis Factor Receptor

PubMed Central

Wang, Dongli; Chen, Dongwei; He, Guangjun; Huang, Li; Wang, Hanzhong; Wang, Xinquan

2011-01-01

Pathogens have evolved sophisticated mechanisms to evade detection and destruction by the host immune system. Large DNA viruses encode homologues of chemokines and their receptors, as well as chemokine-binding proteins (CKBPs) to modulate the chemokine network in host response. The SECRET domain (smallpox virus-encoded chemokine receptor) represents a new family of viral CKBPs that binds a subset of chemokines from different classes to inhibit their activities, either independently or fused with viral tumor necrosis factor receptors (vTNFRs). Here we present the crystal structures of the SECRET domain of vTNFR CrmD encoded by ectromelia virus and its complex with chemokine CX3CL1. The SECRET domain adopts a β-sandwich fold and utilizes its β-sheet I surface to interact with CX3CL1, representing a new chemokine-binding manner of viral CKBPs. Structure-based mutagenesis and biochemical analysis identified important basic residues in the 40s loop of CX3CL1 for the interaction. Mutation of corresponding acidic residues in the SECRET domain also affected the binding for other chemokines, indicating that the SECRET domain binds different chemokines in a similar manner. We further showed that heparin inhibited the binding of CX3CL1 by the SECRET domain and the SECRET domain inhibited RAW264.7 cell migration induced by CX3CL1. These results together shed light on the structural basis for the SECRET domain to inhibit chemokine activities by interfering with both chemokine-GAG and chemokine-receptor interactions. PMID:21829356

Cellular level models as tools for cytokine design.

PubMed

Radhakrishnan, Mala L; Tidor, Bruce

2010-01-01

Cytokines and growth factors are critical regulators that connect intracellular and extracellular environments through binding to specific cell-surface receptors. They regulate a wide variety of immunological, growth, and inflammatory response processes. The overall signal initiated by a population of cytokine molecules over long time periods is controlled by the subtle interplay of binding, signaling, and trafficking kinetics. Building on the work of others, we abstract a simple kinetic model that captures relevant features from cytokine systems as well as related growth factor systems. We explore a large range of potential biochemical behaviors, through systematic examination of the model's parameter space. Different rates for the same reaction topology lead to a dramatic range of biochemical network properties and outcomes. Evolution might productively explore varied and different portions of parameter space to create beneficial behaviors, and effective human therapeutic intervention might be achieved through altering network kinetic properties. Quantitative analysis of the results reveals the basis for tensions among a number of different network characteristics. For example, strong binding of cytokine to receptor can increase short-term receptor activation and signal initiation but decrease long-term signaling due to internalization and degradation. Further analysis reveals the role of specific biochemical processes in modulating such tensions. For instance, the kinetics of cytokine binding and receptor activation modulate whether ligand-receptor dissociation can generally occur before signal initiation or receptor internalization. Beyond analysis, the same models and model behaviors provide an important basis for the design of more potent cytokine therapeutics by providing insight into how binding kinetics affect ligand potency. (c) 2010 American Institute of Chemical Engineers

Bio-nanocapsule-based scaffold improves the sensitivity and ligand-binding capacity of mammalian receptors on the sensor chip.

PubMed

Iijima, Masumi; Yoshimoto, Nobuo; Niimi, Tomoaki; Maturana, Andrés D; Kuroda, Shun'ichi

2016-06-01

Mammalian receptors are recognized as target molecules for drug discovery, and chemical libraries have been screened for both potential antagonists and agonists mainly by ligand-binding assays using immobilized receptors. A bio-nanocapsule (BNC) of approximately 30 nm that displays a tandem form of the protein A-derived immunoglobulin G (IgG) Fc-binding Z domains (denoted as ZZ-BNC) has been developed for both clustering and oriented immobilization of IgGs on the solid phase of immunosensors. In this study, human IgG1 Fc-fused vascular endothelial growth factor (VEGF) receptor was immobilized through ZZ-BNC on the sensor chip of quartz crystal microbalance (ZZ-BNC-coating). When compared with direct adsorption and protein A-coating, the sensor chip showed higher sensitivity (∽46- and ∽165-fold, respectively) and larger ligand-binding capacity (∽4- and ∽18-fold, respectively). Furthermore, the number of VEGF molecules bound to its receptor increased from 0.20 (direct adsorption) to 2.06 by ZZ-BNC-coating, strongly suggesting that ZZ-BNC reduced the steric hindrance near ligand recognition sites through oriented immobilization. Similarly, the sensitivity and ligand-binding capacity of leptin and prolactin receptors were both enhanced at a level comparable to that observed for the VEGF receptor. Thus, the combination of ZZ-BNC and Fc-fused receptors could significantly improve the function of ligand-binding assays. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification of COUP-TFII Orphan Nuclear Receptor as a Retinoic Acid-Activated Receptor

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

Kruse, Schoen W; Suino-Powell, Kelly; Zhou, X Edward

2010-01-12

The chicken ovalbumin upstream promoter-transcription factors (COUP-TFI and II) make up the most conserved subfamily of nuclear receptors that play key roles in angiogenesis, neuronal development, organogenesis, cell fate determination, and metabolic homeostasis. Although the biological functions of COUP-TFs have been studied extensively, little is known of their structural features or aspects of ligand regulation. Here we report the ligand-free 1.48 {angstrom} crystal structure of the human COUP-TFII ligand-binding domain. The structure reveals an autorepressed conformation of the receptor, where helix {alpha}10 is bent into the ligand-binding pocket and the activation function-2 helix is folded into the cofactor binding site,more » thus preventing the recruitment of coactivators. In contrast, in multiple cell lines, COUP-TFII exhibits constitutive transcriptional activity, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, and ligand binding, substantially reduce the COUP-TFII transcriptional activity. Importantly, retinoid acids are able to promote COUP-TFII to recruit coactivators and activate a COUP-TF reporter construct. Although the concentration needed is higher than the physiological levels of retinoic acids, these findings demonstrate that COUP-TFII is a ligand-regulated nuclear receptor, in which ligands activate the receptor by releasing it from the autorepressed conformation.« less

Phosphorylation of a conserved serine in the deoxyribonucleic acid binding domain of nuclear receptors alters intracellular localization.

PubMed

Sun, Kai; Montana, Vedrana; Chellappa, Karthikeyani; Brelivet, Yann; Moras, Dino; Maeda, Yutaka; Parpura, Vladimir; Paschal, Bryce M; Sladek, Frances M

2007-06-01

Nuclear receptors (NRs) are a superfamily of transcription factors whose genomic functions are known to be activated by lipophilic ligands, but little is known about how to deactivate them or how to turn on their nongenomic functions. One obvious mechanism is to alter the nuclear localization of the receptors. Here, we show that protein kinase C (PKC) phosphorylates a highly conserved serine (Ser) between the two zinc fingers of the DNA binding domain of orphan receptor hepatocyte nuclear factor 4alpha (HNF4alpha). This Ser (S78) is adjacent to several positively charged residues (Arg or Lys), which we show here are involved in nuclear localization of HNF4alpha and are conserved in nearly all other NRs, along with the Ser/threonine (Thr). A phosphomimetic mutant of HNF4alpha (S78D) reduced DNA binding, transactivation ability, and protein stability. It also impaired nuclear localization, an effect that was greatly enhanced in the MODY1 mutant Q268X. Treatment of the hepatocellular carcinoma cell line HepG2 with PKC activator phorbol 12-myristate 13-acetate also resulted in increased cytoplasmic localization of HNF4alpha as well as decreased endogenous HNF4alpha protein levels in a proteasome-dependent fashion. We also show that PKC phosphorylates the DNA binding domain of other NRs (retinoic acid receptor alpha, retinoid X receptor alpha, and thyroid hormone receptor beta) and that phosphomimetic mutants of the same Ser/Thr result in cytoplasmic localization of retinoid X receptor alpha and peroxisome proliferator-activated receptor alpha. Thus, phosphorylation of this conserved Ser between the two zinc fingers may be a common mechanism for regulating the function of NRs.

Principles of antibody-mediated TNF receptor activation

PubMed Central

Wajant, H

2015-01-01

From the beginning of research on receptors of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF), agonistic antibodies have been used to stimulate TNFRSF receptors in vitro and in vivo. Indeed, CD95, one of the first cloned TNFRSF receptors, was solely identified as the target of cell death-inducing antibodies. Early on, it became evident from in vitro studies that valency and Fcγ receptor (FcγR) binding of antibodies targeting TNFRSF receptors can be of crucial relevance for agonistic activity. TNFRSF receptor-specific antibodies of the IgM subclass and secondary cross-linked or aggregation prone dimeric antibodies typically display superior agonistic activity compared with dimeric antibodies. Likewise, anchoring of antibodies to cell surface-expressed FcγRs potentiate their ability to trigger TNFRSF receptor signaling. However, only recently has the relevance of oligomerization and FcγR binding for the in vivo activity of antibody-induced TNFRSF receptor activation been straightforwardly demonstrated in vivo. This review discusses the crucial role of oligomerization and/or FcγR binding for antibody-mediated TNFRSF receptor stimulation in light of current models of TNFRSF receptor activation and especially the overwhelming relevance of these issues for the rational development of therapeutic TNFRSF receptor-targeting antibodies. PMID:26292758

Photochemically enhanced binding of small molecules to the tumor necrosis factor receptor-1 inhibits the binding of TNF-[alpha

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

Carter, Percy H.; Scherle, Peggy A.; Muckelbauer, Jodi K.

2010-03-05

The binding of tumor necrosis factor alpha (TNF-{alpha}) to the type-1 TNF receptor (TNFRc1) plays an important role in inflammation. Despite the clinical success of biologics (antibodies, soluble receptors) for treating TNF-based autoimmune conditions, no potent small molecule antagonists have been developed. Our screening of chemical libraries revealed that N-alkyl 5-arylidene-2-thioxo-1,3-thiazolidin-4-ones were antagonists of this protein-protein interaction. After chemical optimization, we discovered IW927, which potently disrupted the binding of TNF-{alpha} to TNFRc1 (IC{sub 50} = 50 nM) and also blocked TNF-stimulated phosphorylation of I{kappa}-B in Ramos cells (IC{sub 50} = 600 nM). This compound did not bind detectably to themore » related cytokine receptors TNFRc2 or CD40, and did not display any cytotoxicity at concentrations as high as 100 {micro}M. Detailed evaluation of this and related molecules revealed that compounds in this class are 'photochemically enhanced' inhibitors, in that they bind reversibly to the TNFRc1 with weak affinity (ca. 40-100 mM) and then covalently modify the receptor via a photochemical reaction. We obtained a crystal structure of IV703 (a close analog of IW927) bound to the TNFRc1. This structure clearly revealed that one of the aromatic rings of the inhibitor was covalently linked to the receptor through the main-chain nitrogen of Ala-62, a residue that has already been implicated in the binding of TNF-{alpha} to the TNFRc1. When combined with the fact that our inhibitors are reversible binders in light-excluded conditions, the results of the crystallography provide the basis for the rational design of nonphotoreactive inhibitors of the TNF-{alpha}-TNFRc1 interaction.« less

A monoclonal antibody against PDGF B-chain inhibits PDGF-induced DNA synthesis in C3H fibroblasts and prevents binding of PDGF to its receptor.

PubMed

Vassbotn, F S; Langeland, N; Hagen, I; Holmsen, H

1990-09-01

A monoclonal antibody (MAb 6D11) against platelet-derived growth factor (PDGF) was studied. We found that the MAb 6D11 in concentrations equimolar to PDGF blocked the [3H]thymidine incorporation in C3H/10T1/2 C18 fibroblasts stimulated by PDGF B-B and PDGF A-B. This inhibition was overcome by high doses of PDGF. The [3H]thymidine incorporation stimulated by other growth factors (aFGF, bFGF and bombesin) was not inhibited by the antibody. The MAb 6D11 blocked receptor binding of PDGF B-B, but not PDGF A-A. These findings suggest that the MAb 6D11 abolishes PDGF-induced DNA synthesis by blocking PDGF receptor binding. In this communication we demonstrate an isoform-specific monoclonal antibody against PDGF.

The Phosphorylation of Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) by Engineered Surfaces with Electrostatically or Covalently Immobilized VEGF

PubMed Central

Anderson, Sean M.; Chen, Tom T.; Iruela-Arispe, M. Luisa; Segura, Tatiana

2010-01-01

Growth factors are a class of signaling proteins that direct cell fate through interaction with cell surface receptors. Although a myriad of possible cell fates stem from a growth factor binding to its receptor, the signaling cascades that result in one fate over another are still being elucidated. One possible mechanism by which nature modulates growth factor signaling is through the method of presentation of the growth factor – soluble or immobilized (matrix bound). Here we present the methodology to study signaling of soluble versus immobilized VEGF through VEGFR-2. We have designed a strategy to covalently immobilize VEGF using its heparin-binding domain to orient the molecule (bind) and a secondary functional group to mediate covalent binding (lock). This bind-and-lock approach aims to allow VEGF to assume a bioactive orientation before covalent immobilization. Surface plasmon resonance (SPR) demonstrated heparin and VEGF binding with surface densities of 60 ng/cm2 and 100 pg/cm2, respectively. ELISA experiments confirmed VEGF surface density and showed that electrostatically bound VEGF releases in cell medium and heparin solutions while covalently bound VEGF remains immobilized. Electrostatically bound VEGF and covalently bound VEGF phosphorylate VEGFR-2 in both VEGFR-2 transfected cells and VEGFR-2 endogenously producing cells. HUVECs plated on VEGF functionalized surfaces showed different morphologies between surface-bound VEGF and soluble VEGF. The surfaces synthesized in these studies allow for the study of VEGF/VEGFR-2 signaling induced by covalently bound, electrostatically bound, and soluble VEGF and may provide further insight into the design of materials for the generation of a mature and stable vasculature. PMID:19540581

Insulin Mimetic Peptide Disrupts the Primary Binding Site of the Insulin Receptor*

PubMed Central

Lawrence, Callum F.; Margetts, Mai B.; Menting, John G.; Smith, Nicholas A.; Smith, Brian J.; Ward, Colin W.; Lawrence, Michael C.

2016-01-01

Sets of synthetic peptides that interact with the insulin receptor ectodomain have been discovered by phage display and reported in the literature. These peptides were grouped into three classes termed Site 1, Site 2, and Site 3 based on their mutual competition of binding to the receptor. Further refinement has yielded, in particular, a 36-residue Site 2-Site 1 fusion peptide, S519, that binds the insulin receptor with subnanomolar affinity and exhibits agonist activity in both lipogenesis and glucose uptake assays. Here, we report three-dimensional crystallographic detail of the interaction of the C-terminal, 16-residue Site 1 component (S519C16) of S519 with the first leucine-rich repeat domain (L1) of the insulin receptor. Our structure shows that S519C16 binds to the same site on the L1 surface as that occupied by a critical component of the primary binding site, namely the helical C-terminal segment of the insulin receptor α-chain (termed αCT). In particular, the two phenylalanine residues within the FYXWF motif of S519C16 are seen to engage the insulin receptor L1 domain surface in a fashion almost identical to the respective αCT residues Phe701 and Phe705. The structure provides a platform for the further development of peptidic and/or small molecule agents directed toward the insulin receptor and/or the type 1 insulin-like growth factor receptor. PMID:27281820

Analysis of molecular determinants of affinity and relative efficacy of a series of R- and S-2-(dipropylamino)tetralins at the 5-HT1A serotonin receptor

PubMed Central

Alder, J Tracy; Hacksell, Uli; Strange, Philip G

2003-01-01

Factors influencing agonist affinity and relative efficacy have been studied for the 5-HT1A serotonin receptor using membranes of CHO cells expressing the human form of the receptor and a series of R-and S-2-(dipropylamino)tetralins (nonhydroxylated and monohydroxylated (5-OH, 6-OH, 7-OH, 8-OH) species). Ligand binding studies were used to determine dissociation constants for agonist binding to the 5-HT1A receptor: Ki values for agonists were determined in competition versus the binding of the agonist [3H]-8-OH DPAT. Competition data were all fitted best by a one-binding site model.Ki values for agonists were also determined in competition versus the binding of the antagonist [3H]-NAD-199. Competition data were all fitted best by a two-binding site model, and agonist affinities for the higher (Kh) and lower affinity (Kl) sites were determined. The ability of the agonists to activate the 5-HT1A receptor was determined using stimulation of [35S]-GTPγS binding. Maximal effects of agonists (Emax) and their potencies (EC50) were determined from concentration/response curves for stimulation of [35S]-GTPγS binding. Kl/Kh determined from ligand binding assays correlated with the relative efficacy (relative Emax) of agonists determined in [35S]-GTPγS binding assays. There was also a correlation between Kl/Kh and Kl/EC50 for agonists determined from ligand binding and [35S]-GTPγS binding assays. Simulations of agonist binding and effect data were performed using the Ternary Complex Model in order to assess the use of Kl/Kh for predicting the relative efficacy of agonists. PMID:12684269

Targeting the Neural Microenvironment in Prostate Cancer

DTIC Science & Technology

2016-10-01

neurotrophic factor (GDNF), which is expressed by peripheral nerves. GDNF binds to RET, a receptor tyrosine kinase, in conjunction with its co-receptor...kinase, in conjunction with its co- receptor GFRα1 and activates cellular signaling. Studies in pancreatic cancer strongly implicate RET signaling as

Affinity States of Striatal Dopamine D2 Receptors in Antipsychotic-Free Patients with Schizophrenia

PubMed Central

Kubota, Manabu; Nagashima, Tomohisa; Takano, Harumasa; Kodaka, Fumitoshi; Fujiwara, Hironobu; Takahata, Keisuke; Moriguchi, Sho; Higuchi, Makoto; Okubo, Yoshiro; Takahashi, Hidehiko; Ito, Hiroshi

2017-01-01

Abstract Background Dopamine D2 receptors are reported to have high-affinity (D2High) and low-affinity (D2Low) states. Although an increased proportion of D2High has been demonstrated in animal models of schizophrenia, few clinical studies have investigated this alteration of D2High in schizophrenia in vivo. Methods Eleven patients with schizophrenia, including 10 antipsychotic-naive and 1 antipsychotic-free individuals, and 17 healthy controls were investigated. Psychopathology was assessed by Positive and Negative Syndrome Scale, and a 5-factor model was used. Two radioligands, [11C]raclopride and [11C]MNPA, were employed to quantify total dopamine D2 receptor and D2High, respectively, in the striatum by measuring their binding potentials. Binding potential values of [11C]raclopride and [11C]MNPA and the binding potential ratio of [11C]MNPA to [11C]raclopride in the striatal subregions were statistically compared between the 2 diagnostic groups using multivariate analysis of covariance controlling for age, gender, and smoking. Correlations between binding potential and Positive and Negative Syndrome Scale scores were also examined. Results Multivariate analysis of covariance demonstrated a significant effect of diagnosis (schizophrenia and control) on the binding potential ratio (P=.018), although the effects of diagnosis on binding potential values obtained with either [11C]raclopride or [11C]MNPA were nonsignificant. Posthoc test showed that the binding potential ratio was significantly higher in the putamen of patients (P=.017). The Positive and Negative Syndrome Scale “depressed” factor in patients was positively correlated with binding potential values of both ligands in the caudate. Conclusions The present study indicates the possibilities of: (1) a higher proportion of D2High in the putamen despite unaltered amounts of total dopamine D2 receptors; and (2) associations between depressive symptoms and amounts of caudate dopamine D2 receptors in patients with schizophrenia. PMID:29016872

Regulation of platelet activating factor receptor coupled phosphoinositide-specific phospholipase C activity

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

Morrison, W.J.

1988-01-01

The major objectives of this study were two-fold. The first was to establish whether binding of platelet activating factor (PAF) to its receptor was integral to the stimulation of polyphosphoinositide-specific phospholipase C (PLC) in rabbit platelets. The second was to determine regulatory features of this receptor-coupled mechanism. ({sup 3}H)PAF binding demonstrated two binding sites, a high affinity site with a inhibitory constant (Ki) of 2.65 nM and a low affinity site with a Ki of 0.80 {mu}M. PAF receptor coupled activation of phosphoinositide-specific PLC was studied in platelets which were made refractory, by short term pretreatments, to either PAF ormore » thrombin. Saponin-permeabilized rabbit platelets continue to regulate the mechanism(s) coupling PAF receptors to PLC stimulation. However, TRP{gamma}S and GDP{beta}S, which affect guanine nucleotide regulatory protein functions, were unable to modulate the PLC activity to any appreciable extent as compared to PAF. The possible involvement of protein kinase C (PKC) activation in regulating PAF-stimulated PLC activity was studied in rabbit platelets pretreated with staurosporine followed by pretreatments with PAF or phorbol 12-myristate 13-acetate (PMA).« less

Differential CLE peptide perception by plant receptors implicated from structural and functional analyses of TDIF-TDR interactions

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

Li, Zhijie; Chakraborty, Sayan; Xu, Guozhou

Tracheary Element Differentiation Inhibitory Factor (TDIF) belongs to the family of post-translationally modified CLE (CLAVATA3/embryo surrounding region (ESR)-related) peptide hormones that control root growth and define the delicate balance between stem cell proliferation and differentiation in SAM (shoot apical meristem) or RAM (root apical meristem). In Arabidopsis, Tracheary Element Differentiation Inhibitory Factor Receptor (TDR) and its ligand TDIF signaling pathway is involved in the regulation of procambial cell proliferation and inhibiting its differentiation into xylem cells. Here we present the crystal structures of the extracellular domains (ECD) of TDR alone and in complex with its ligand TDIF resolved at 2.65more » Åand 2.75 Å respectively. These structures provide insights about the ligand perception and specific interactions between the CLE peptides and their cognate receptors. Our in vitro biochemical studies indicate that the interactions between the ligands and the receptors at the C-terminal anchoring site provide conserved binding. While the binding interactions occurring at the N-terminal anchoring site dictate differential binding specificities between different ligands and receptors. Our studies will open different unknown avenues of TDR-TDIF signaling pathways that will enhance our knowledge in this field highlighting the receptor ligand interaction, receptor activation, signaling network, modes of action and will serve as a structure function relationship model between the ligand and the receptor for various similar leucine-rich repeat receptor-like kinases (LRR-RLKs).« less

Structure-function relationship in the binding of snake neurotoxins to the torpedo membrane receptor.

PubMed

Chicheportiche, R; Vincent, J P; Kopeyan, C; Schweitz, H; Lazdunski, M

1975-05-20

The Cys30-Cus34 bridge present in all long neutotoxins (71-74 amino acids, 5 disulfide bridges), but not in short toxins (60-63 amino acids, 4 disulfide bridges), is exposed at the surface since it can be reduced rapidly and selectively by sodium borohydride. Reduction and alkylation of the Cys30-Cys34 bridge of Naja haje neurotoxin III hardly alter the conformational properties of this model long toxin. Although alkylation by iodoacetic acid of th -SH groups liberated by reduction abolishes the toxicity, alkylation by iodoacetamide or ethylenimine does not affect the curarizing efficacy of the toxin. The Cys30-Cys34 bridge is not very important for the toxic activity of long neurotoxins. Reduction of the Cys30-Cys34 bridge followed by alkylation with radioactive iodoacetamide gave a labeled and active toxin which is a convenient derivative for binding experiments to the toxin receptor in membranes of the Torpedo electric organ. The binding capacity of these membrane is 1200 pmol of toxin/mg of membrane protein. The dissociation constant of the modified toxin-receptor complex at pH 7.4, 20 degrees is 10 minus 8m. Reduction with carbroxamidomethylation of the Cys30-Cys34 bridge decreases the affinity of the native Naja haje toxin only by a factor of 15. Carboxymethylation after reduction prevents binding to the membrane receptor. The binding properties of the derivative obtained by reduction and aminoethylation of Cys30-Cys34 are very similar to those of native neurotoxin III; the affinity is decreased only by a factor of 5. Binding properties to Toredo membrane of long neurotoxins (Naja haje neurotoxin III) and short neurotoxins (Naje haje toxin I and Naja mossambica toxin I) have been compared. Dissociation constants of receptor-long neurotoxin and receptor-short neurotoxin complexes are very similar (5.7 minus 8.2 times 10(-10) M at pH 7.4, 20degrees. However, the kinetics of complex formation and complex dissociation are quite different. Short neurotoxins associate 6-7 times faster with the toxin receptor and dissociate about 5-9 times faster that long neurotoxins. Acetylation and dansylation of Lys53 and Lys 27 decrease the affinity of long and short toxins for their receptor by a factor of about 200 at pH 7.4, 20 degrees, mainly because of the slower rate of association with the receptor.

Cargo binding promotes KDEL receptor clustering at the mammalian cell surface

PubMed Central

Becker, Björn; Shaebani, M. Reza; Rammo, Domenik; Bubel, Tobias; Santen, Ludger; Schmitt, Manfred J.

2016-01-01

Transmembrane receptor clustering is a ubiquitous phenomenon in pro- and eukaryotic cells to physically sense receptor/ligand interactions and subsequently translate an exogenous signal into a cellular response. Despite that receptor cluster formation has been described for a wide variety of receptors, ranging from chemotactic receptors in bacteria to growth factor and neurotransmitter receptors in mammalian cells, a mechanistic understanding of the underlying molecular processes is still puzzling. In an attempt to fill this gap we followed a combined experimental and theoretical approach by dissecting and modulating cargo binding, internalization and cellular response mediated by KDEL receptors (KDELRs) at the mammalian cell surface after interaction with a model cargo/ligand. Using a fluorescent variant of ricin toxin A chain as KDELR-ligand (eGFP-RTAH/KDEL), we demonstrate that cargo binding induces dose-dependent receptor cluster formation at and subsequent internalization from the membrane which is associated and counteracted by anterograde and microtubule-assisted receptor transport to preferred docking sites at the plasma membrane. By means of analytical arguments and extensive numerical simulations we show that cargo-synchronized receptor transport from and to the membrane is causative for KDELR/cargo cluster formation at the mammalian cell surface. PMID:27353000

Cargo binding promotes KDEL receptor clustering at the mammalian cell surface

NASA Astrophysics Data System (ADS)

Becker, Björn; Shaebani, M. Reza; Rammo, Domenik; Bubel, Tobias; Santen, Ludger; Schmitt, Manfred J.

2016-06-01

Transmembrane receptor clustering is a ubiquitous phenomenon in pro- and eukaryotic cells to physically sense receptor/ligand interactions and subsequently translate an exogenous signal into a cellular response. Despite that receptor cluster formation has been described for a wide variety of receptors, ranging from chemotactic receptors in bacteria to growth factor and neurotransmitter receptors in mammalian cells, a mechanistic understanding of the underlying molecular processes is still puzzling. In an attempt to fill this gap we followed a combined experimental and theoretical approach by dissecting and modulating cargo binding, internalization and cellular response mediated by KDEL receptors (KDELRs) at the mammalian cell surface after interaction with a model cargo/ligand. Using a fluorescent variant of ricin toxin A chain as KDELR-ligand (eGFP-RTAH/KDEL), we demonstrate that cargo binding induces dose-dependent receptor cluster formation at and subsequent internalization from the membrane which is associated and counteracted by anterograde and microtubule-assisted receptor transport to preferred docking sites at the plasma membrane. By means of analytical arguments and extensive numerical simulations we show that cargo-synchronized receptor transport from and to the membrane is causative for KDELR/cargo cluster formation at the mammalian cell surface.

The kangaroo cation-independent mannose 6-phosphate receptor binds insulin-like growth factor II with low affinity.

PubMed

Yandell, C A; Dunbar, A J; Wheldrake, J F; Upton, Z

1999-09-17

The mammalian cation-independent mannose 6-phosphate receptor (CI-MPR) binds mannose 6-phosphate-bearing glycoproteins and insulin-like growth factor (IGF)-II. However, the CI-MPR from the opossum has been reported to bind bovine IGF-II with low affinity (Dahms, N. M., Brzycki-Wessell, M. A., Ramanujam, K. S., and Seetharam, B. (1993) Endocrinology 133, 440-446). This may reflect the use of a heterologous ligand, or it may represent the intrinsic binding affinity of this receptor. To examine the binding of IGF-II to a marsupial CI-MPR in a homologous system, we have previously purified kangaroo IGF-II (Yandell, C. A., Francis, G. L., Wheldrake, J. F., and Upton, Z. (1998) J. Endocrinol. 156, 195-204), and we now report the purification and characterization of the CI-MPR from kangaroo liver. The interaction of the kangaroo CI-MPR with IGF-II has been examined by ligand blotting, radioreceptor assay, and real-time biomolecular interaction analysis. Using both a heterologous and homologous approach, we have demonstrated that the kangaroo CI-MPR has a lower binding affinity for IGF-II than its eutherian (placental mammal) counterparts. Furthermore, real-time biomolecular interaction analysis revealed that the kangaroo CI-MPR has a higher affinity for kangaroo IGF-II than for human IGF-II. The cDNA sequence of the kangaroo CI-MPR indicates that there is considerable divergence in the area corresponding to the IGF-II binding site of the eutherian receptor. Thus, the acquisition of a high-affinity binding site for regulating IGF-II appears to be a recent event specific to the eutherian lineage.

Characterization of insulin-like growth factor I receptor on human erythrocytes.

PubMed

Hizuka, N; Takano, K; Tanaka, I; Honda, N; Tsushima, T; Shizume, K

1985-12-01

[125I]Insulin-like growth factor I (IGF-I) specifically bound to erythrocytes; the binding was saturable, and time and temperature dependent. Steady state binding was reached at 16 h at 4 C, and specific binding averaged 14.3 +/- 0.7% (+/- SEM) at a concentration of 3.6 X 10(9) cells/ml in seven normal subjects. [125I]IGF-I binding to the cells was displaced by unlabeled IGF-I in a dose-dependent manner. Scatchard analysis indicated a linear plot, and Ka and number of binding sites/cell were 1.43 +/- 0.07 X 10(9) M-1 and 20.7 +/- 2.2, respectively. Compared to IGF-I, the relative potencies of multiplication-stimulating activity and insulin for displacing [125I]IGF-I binding were 20% and 1%, respectively. [125I]IGF-I binding to erythrocytes from patients with acromegaly was lower than binding to cells from pituitary dwarfs. An inverse correlation between plasma IGF-I level and the number of IGF-I-binding sites per cell was found (r = -0.75; P less than 0.005). These results demonstrate that [125I]IGF-I binding to erythrocytes can be used for clinical measurement of the IGF-I receptor.

Probing receptor-ligand interactions by sedimentation equilibrium

NASA Astrophysics Data System (ADS)

Philo, John S.

1997-05-01

While sedimentation equilibrium is most commonly used to characterize the molecular weight and state of association of single proteins, this technique is also a very powerful tool for probing the interactions between two or more different proteins, and can characterize both the binding stoichiometry and the equilibrium constants. To resolve the complex binding interactions that can occur in such systems, it is crucial to globally fit data from many experiments to a common binding model, including samples made with different mixing ratios and a wide range of total concentration. It is often also essential to constrain the parameters during fitting so that the fits correctly reproduce the molar ratio of proteins used in making each sample. We have applied this methodology to probe mechanisms of receptor activation for a number of hematopoietic receptors and their cognate ligands, using receptor extracellular domains expressed as soluble proteins. Such data can potentially help in the design of improved or new protein therapeutics, as well as in efforts to create small- molecular mimetics of protein hormones through structure- based drug design. Sedimentation equilibrium has shown that stem cell factor, erythropoietin, and granulocyte-colony stimulating factor can each dimerize their respective receptors in solution, but the mechanism of ligand-induced receptor dimerization for these three systems are strikingly different.

Ligand-independent Dimer Formation of Epidermal Growth Factor Receptor (EGFR) Is a Step Separable from Ligand-induced EGFR Signaling

PubMed Central

Yu, Xiaochun; Sharma, Kailash D.; Takahashi, Tsuyoshi; Iwamoto, Ryo; Mekada, Eisuke

2002-01-01

Dimerization and phosphorylation of the epidermal growth factor (EGF) receptor (EGFR) are the initial and essential events of EGF-induced signal transduction. However, the mechanism by which EGFR ligands induce dimerization and phosphorylation is not fully understood. Here, we demonstrate that EGFRs can form dimers on the cell surface independent of ligand binding. However, a chimeric receptor, comprising the extracellular and transmembrane domains of EGFR and the cytoplasmic domain of the erythropoietin receptor (EpoR), did not form a dimer in the absence of ligands, suggesting that the cytoplasmic domain of EGFR is important for predimer formation. Analysis of deletion mutants of EGFR showed that the region between 835Ala and 918Asp of the EGFR cytoplasmic domain is required for EGFR predimer formation. In contrast to wild-type EGFR ligands, a mutant form of heparin-binding EGF-like growth factor (HB2) did not induce dimerization of the EGFR-EpoR chimeric receptor and therefore failed to activate the chimeric receptor. However, when the dimerization was induced by a monoclonal antibody to EGFR, HB2 could activate the chimeric receptor. These results indicate that EGFR can form a ligand-independent inactive dimer and that receptor dimerization and activation are mechanistically distinct and separable events. PMID:12134089

Is receptor oligomerization causally linked to activation of the EGF receptor kinase?

NASA Technical Reports Server (NTRS)

Rintoul, D. A.; Spooner, B. S. (Principal Investigator)

1992-01-01

Transduction of a signal from an extracellular peptide hormone to produce an intracellular response is often mediated by a cell surface receptor, which is usually a glycoprotein. The secondary intracellular signal(s) generated after hormone binding to the receptor have been intensively studied. The nature of the primary signal generated by ligand binding to the receptor is understood less well in most cases. The particular case of the epidermal growth factor (EGF) receptor is analyzed, and evidence for or against two dissimilar models of primary signal transduction is reviewed. Evidence for the most widely accepted current model is found to be unconvincing. Evidence for the other model is substantial but indirect; a direct test of this model remains to be done.

Increased abundance of insulin/insulin-like growth factor-I hybrid receptors in skeletal muscle of obese subjects is correlated with in vivo insulin sensitivity.

PubMed

Federici, M; Porzio, O; Lauro, D; Borboni, P; Giovannone, B; Zucaro, L; Hribal, M L; Sesti, G

1998-08-01

We reported that in noninsulin-dependent diabetes melitus (NIDDM) patients expression of insulin/insulin-like growth factor I (IGF-I) hybrid receptors is increased in insulin target tissues. Whether this is a defect associated with NIDDM or represents a generalized abnormality associated with insulin resistant states is still unsettled. To address this, we applied a microwell-based immunoassay to measure abundance of insulin receptors, type 1 IGF receptors, and hybrid receptors in muscle of eight normal and eight obese subjects. Maximal insulin binding to insulin receptors was lower in obese than in control subjects (B/T = 1.8 +/- 0.20 and 2.6 +/- 0.30; P < 0.03, respectively) and was negatively correlated with insulinemia (r = -0.60; P < 0.01). Maximal IGF-I binding to type 1 IGF receptors was higher in obese than in controls (B/T = 1.9 +/- 0.20 and 0.86 +/- 0.10; P < 0.0001, respectively) and was negatively correlated with plasma IGF-I levels (r = -0.69; P < 0.003). Hybrid receptor abundance was higher in obese than in normal subjects (B/T = 1.21 +/- 0.14 and 0.44 +/- 0.06; P < 0.0003, respectively) and was negatively correlated with insulin binding (r = -0.60; P < 0.01) and positively correlated with IGF-I binding (r = 0.92; P < 0.0001). Increased abundance of hybrids was correlated with insulinemia (r = 0.70; P < 0.002) and body mass index (r = 0.71; P < 0.0019), whereas it was negatively correlated with in vivo insulin sensitivity measured by ITT (r = -0.67; P < 0.016). These results indicate that downregulation of insulin receptors or upregulation of type 1 IGF receptors because of changes in plasma insulin and IGF-I levels may result in modifications in hybrid receptor abundance.

The Growth Factor Progranulin Binds to TNF Receptors and Is Therapeutic Against Inflammatory Arthritis in Mice

PubMed Central

Tang, Wei; Lu, Yi; Tian, Qing-Yun; Zhang, Yan; Guo, Feng-Jin; Liu, Guang-Yi; Syed, Nabeel Muzaffar; Lai, Yongjie; Lin, Edward Alan; Kong, Li; Su, Jeffrey; Yin, Fangfang; Ding, Ai-Hao; Zanin-Zhorov, Alexandra; Dustin, Michael L.; Tao, Jian; Craft, Joseph; Yin, Zhinan; Feng, Jian Q.; Abramson, Steven B.; Yu, Xiu-Ping; Liu, Chuan-ju

2011-01-01

The growth factor progranulin (PGRN) has been implicated in embryonic development, tissue repair, tumorigenesis, and inflammation, but its receptors remain unidentified. We report that PGRN bound directly to tumor necrosis factor receptors (TNFR), and disturbed the TNFα/TNFR interaction. PGRN-deficient mice were susceptible to collagen-induced arthritis, and administration of PGRN reversed inflammatory arthritis. Atsttrin, an engineered protein composed of three PGRN fragments, exhibited selective TNFR binding. PGRN and Atsttrin prevented inflammation in multiple arthritis mouse models and inhibited TNFα-activated intracellular signaling. Collectively, these findings demonstrate that PGRN is a ligand of TNFR, an antagonist of TNFα signaling and plays a critical role in the pathogenesis of inflammatory arthritis in mice. They also suggest new potential therapeutic interventions for various TNFα-mediated pathologies and conditions, including rheumatoid arthritis. PMID:21393509

[Advanced glycation end products: A risk factor for human health].

PubMed

Wautier, M-P; Tessier, F J; Wautier, J-L

2014-11-01

Advanced glycation end products (AGE) result from a chemical reaction between the carbonyl group of reducing sugar and the nucleophilic NH2 of a free amino acid or a protein; lysine and arginine being the main reactive amino acids on proteins. Following this first step, a molecular rearrangement occurs, rearrangement of Amadori resulting to the formation of Maillard products. Glycation can cause the clouding of the lens by inducing reactions crosslinking proteins. Specialized receptors (RAGE, Galectin 3…) bind AGE. The binding to the receptor causes the formation of free radicals, which have a deleterious effect because they are powerful oxidizing agents, but also play the role of intracellular messenger, altering the cell functions. This is especially true at the level of endothelial cells: the attachment of AGE to RAGE receptor causes an increase in vascular permeability. AGE binding to endothelium RAGE and to monocytes-macrophages, led to the production of cytokines, growth factors, to the expression of adhesion molecules, and the production of procoagulant activity. Diabetic retinopathy is related to excessive secretion of vascular growth factor (vascular endothelial growth factor [VEGF]). AGE-RAGE receptor binding causes the synthesis and secretion of VEGF. Increased permeability, facilitation of leukocyte migration, the production of reactive oxygen species, cytokines and VEGF suggest that the AGE could be an element of a cascade of reactions responsible for the diabetic angiopathy and vascular damages observed during aging and chronic renal failure. Balanced diet or some drugs can limit the deleterious effect of AGE. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Molecular Modeling, de novo Design and Synthesis of a Novel, Extracellular Binding Fibroblast Growth Factor Receptor 2 Inhibitor Alofanib (RPT835).

PubMed

Tsimafeyeu, Ilya; Daeyaert, Frits; Joos, Jean-Baptiste; Aken, Koen V; Ludes-Meyers, John; Byakhov, Mikhail; Tjulandin, Sergei

2016-01-01

Fibroblast growth factor (FGF) receptors (FGFRs) play a key role in tumor growth and angiogenesis. The present report describes our search for an extracellularly binding FGFR inhibitor using a combined molecular modeling and de novo design strategy. Based upon crystal structures of the receptor with its native ligand and knowledge of inhibiting peptides, we have developed a computational protocol that predicts the putative binding of a molecule to the extracellular domains of the receptor. This protocol, or scoring function, was used in combination with the de novo synthesis program 'SYNOPSIS' to generate high scoring and synthetically accessible compounds. Eight compounds belonging to 3 separate chemical classes were synthesized. One of these compounds, alofanib (RPT835), was found to be an effective inhibitor of the FGF/FGFR2 pathway. The preclinical in vitro data support an allosteric inhibition mechanism of RPT835. RPT835 potently inhibited growth of KATO III gastric cancer cells expressing FGFR2, with GI50 value of 10 nmol/L. These results provide strong rationale for the evaluation of compound in advanced cancers.

Macrophage Migration Inhibitory Factor-CXCR4 Receptor Interactions

PubMed Central

Rajasekaran, Deepa; Gröning, Sabine; Schmitz, Corinna; Zierow, Swen; Drucker, Natalie; Bakou, Maria; Kohl, Kristian; Mertens, André; Lue, Hongqi; Weber, Christian; Xiao, Annie; Luker, Gary; Kapurniotu, Aphrodite; Lolis, Elias; Bernhagen, Jürgen

2016-01-01

An emerging number of non-chemokine mediators are found to bind to classical chemokine receptors and to elicit critical biological responses. Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine that exhibits chemokine-like activities through non-cognate interactions with the chemokine receptors CXCR2 and CXCR4, in addition to activating the type II receptor CD74. Activation of the MIF-CXCR2 and -CXCR4 axes promotes leukocyte recruitment, mediating the exacerbating role of MIF in atherosclerosis and contributing to the wealth of other MIF biological activities. Although the structural basis of the MIF-CXCR2 interaction has been well studied and was found to engage a pseudo-ELR and an N-like loop motif, nothing is known about the regions of CXCR4 and MIF that are involved in binding to each other. Using a genetic strain of Saccharomyces cerevisiae that expresses a functional CXCR4 receptor, site-specific mutagenesis, hybrid CXCR3/CXCR4 receptors, pharmacological reagents, peptide array analysis, chemotaxis, fluorescence spectroscopy, and circular dichroism, we provide novel molecular information about the structural elements that govern the interaction between MIF and CXCR4. The data identify similarities with classical chemokine-receptor interactions but also provide evidence for a partial allosteric agonist compared with CXCL12 that is possible due to the two binding sites of CXCR4. PMID:27226569

Cloning retinoid and peroxisome proliferator-activated nuclear receptors of the Pacific oyster and in silico binding to environmental chemicals

PubMed Central

Vogeler, Susanne; Galloway, Tamara S.; Isupov, Michail

2017-01-01

Disruption of nuclear receptors, a transcription factor superfamily regulating gene expression in animals, is one proposed mechanism through which pollution causes effects in aquatic invertebrates. Environmental pollutants have the ability to interfere with the receptor’s functions through direct binding and inducing incorrect signals. Limited knowledge of invertebrate endocrinology and molecular regulatory mechanisms, however, impede the understanding of endocrine disruptive effects in many aquatic invertebrate species. Here, we isolated three nuclear receptors of the Pacific oyster, Crassostrea gigas: two isoforms of the retinoid X receptor, CgRXR-1 and CgRXR-2, a retinoic acid receptor ortholog CgRAR, and a peroxisome proliferator-activated receptor ortholog CgPPAR. Computer modelling of the receptors based on 3D crystal structures of human proteins was used to predict each receptor’s ability to bind to different ligands in silico. CgRXR showed high potential to bind and be activated by 9-cis retinoic acid and the organotin tributyltin (TBT). Computer modelling of CgRAR revealed six residues in the ligand binding domain, which prevent the successful interaction with natural and synthetic retinoid ligands. This supports an existing theory of loss of retinoid binding in molluscan RARs. Modelling of CgPPAR was less reliable due to high discrepancies in sequence to its human ortholog. Yet, there are suggestions of binding to TBT, but not to rosiglitazone. The effect of potential receptor ligands on early oyster development was assessed after 24h of chemical exposure. TBT oxide (0.2μg/l), all-trans retinoic acid (ATRA) (0.06 mg/L) and perfluorooctanoic acid (20 mg/L) showed high effects on development (>74% abnormal developed D-shelled larvae), while rosiglitazone (40 mg/L) showed no effect. The results are discussed in relation to a putative direct (TBT) disruption effect on nuclear receptors. The inability of direct binding of ATRA to CgRAR suggests either a disruptive effect through a pathway excluding nuclear receptors or an indirect interaction. Our findings provide valuable information on potential mechanisms of molluscan nuclear receptors and the effects of environmental pollution on aquatic invertebrates. PMID:28426724

Quantitative pharmacological analysis of antagonist binding kinetics at CRF1 receptors in vitro and in vivo

PubMed Central

Ramsey, Simeon J; Attkins, Neil J; Fish, Rebecca; van der Graaf, Piet H

2011-01-01

BACKGROUND AND PURPOSE A series of novel non-peptide corticotropin releasing factor type-1 receptor (CRF1) antagonists were found to display varying degrees of insurmountable and non-competitive behaviour in functional in vitro assays. We describe how we attempted to relate this behaviour to ligand receptor-binding kinetics in a quantitative manner and how this resulted in the development and implementation of an efficient pharmacological screening method based on principles described by Motulsky and Mahan. EXPERIMENTAL APPROACH A non-equilibrium binding kinetic assay was developed to determine the receptor binding kinetics of non-peptide CRF1 antagonists. Nonlinear, mixed-effects modelling was used to obtain estimates of the compounds association and dissociation rates. We present an integrated pharmacokinetic–pharmacodynamic (PKPD) approach, whereby the time course of in vivo CRF1 receptor binding of novel compounds can be predicted on the basis of in vitro assays. KEY RESULTS The non-competitive antagonist behaviour appeared to be correlated to the CRF1 receptor off-rate kinetics. The integrated PKPD model suggested that, at least in a qualitative manner, the in vitro assay can be used to triage and select compounds for further in vivo investigations. CONCLUSIONS AND IMPLICATIONS This study provides evidence for a link between ligand offset kinetics and insurmountable/non-competitive antagonism at the CRF1 receptor. The exact molecular pharmacological nature of this association remains to be determined. In addition, we have developed a quantitative framework to study and integrate in vitro and in vivo receptor binding kinetic behaviour of CRF1 receptor antagonists in an efficient manner in a drug discovery setting. PMID:21449919

Nuclear actions of insulin-like growth factor binding protein-3.

PubMed

Baxter, Robert C

2015-09-10

In addition to its actions outside the cell, cellular uptake and nuclear import of insulin-like growth factor binding protein-3 (IGFBP-3) has been recognized for almost two decades, but knowledge of its nuclear actions has been slow to emerge. IGFBP-3 has a functional nuclear localization signal and interacts with the nuclear transport protein importin-β. Within the nucleus IGFBP-3 appears to have a role in transcriptional regulation. It can bind to the nuclear receptor, retinoid X receptor-α and several of its dimerization partners, including retinoic acid receptor, vitamin D receptor (VDR), and peroxisome proliferator-activated receptor-γ (PPARγ). These interactions modulate the functions of these receptors, for example inhibiting VDR-dependent transcription in osteoblasts and PPARγ-dependent transcription in adipocytes. Nuclear IGFBP-3 can be detected by immunohistochemistry in cancer and other tissues, and its presence in the nucleus has been shown in many cell culture studies to be necessary for its pro-apoptotic effect, which may also involve interaction with the nuclear receptor Nur77, and export from the nucleus. IGFBP-3 is p53-inducible and in response to DNA damage, forms a complex with the epidermal growth factor receptor (EGFR), translocating to the nucleus to interact with DNA-dependent protein kinase. Inhibition of EGFR kinase activity or downregulation of IGFBP-3 can inhibit DNA double strand-break repair by nonhomologous end joining. IGFBP-3 thus has the ability to influence many cell functions through its interactions with intranuclear pathways, but the importance of these interactions in vivo, and their potential to be targeted for therapeutic benefit, require further investigation. Copyright © 2015 Elsevier B.V. All rights reserved.

Tumour necrosis factors modulate the affinity state of the leukotriene B4 receptor on human neutrophils.

PubMed Central

Brom, J; Knöller, J; Köller, M; König, W

1988-01-01

Pre-incubation of human polymorphonuclear granulocytes with recombinant human tumour necrosis factors (TNF) revealed a time- and dose-dependent reduction of the expression of leukotriene B4-receptor sites. Analysis of the binding data by Scatchard plots showed a shift from a heterologous receptor population (indicating high- and low-affinity subsets) to a homologous population. From the results it is considered that TNF can influence host defence through the modulation of leukotriene B4 receptor affinity. PMID:2851543

Molecular Determinants of Epidermal Growth Factor Binding: A Molecular Dynamics Study

PubMed Central

Sanders, Jeffrey M.; Wampole, Matthew E.; Thakur, Mathew L.; Wickstrom, Eric

2013-01-01

The epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinase family that plays a role in multiple cellular processes. Activation of EGFR requires binding of a ligand on the extracellular domain to promote conformational changes leading to dimerization and transphosphorylation of intracellular kinase domains. Seven ligands are known to bind EGFR with affinities ranging from sub-nanomolar to near micromolar dissociation constants. In the case of EGFR, distinct conformational states assumed upon binding a ligand is thought to be a determining factor in activation of a downstream signaling network. Previous biochemical studies suggest the existence of both low affinity and high affinity EGFR ligands. While these studies have identified functional effects of ligand binding, high-resolution structural data are lacking. To gain a better understanding of the molecular basis of EGFR binding affinities, we docked each EGFR ligand to the putative active state extracellular domain dimer and 25.0 ns molecular dynamics simulations were performed. MM-PBSA/GBSA are efficient computational approaches to approximate free energies of protein-protein interactions and decompose the free energy at the amino acid level. We applied these methods to the last 6.0 ns of each ligand-receptor simulation. MM-PBSA calculations were able to successfully rank all seven of the EGFR ligands based on the two affinity classes: EGF>HB-EGF>TGF-α>BTC>EPR>EPG>AR. Results from energy decomposition identified several interactions that are common among binding ligands. These findings reveal that while several residues are conserved among the EGFR ligand family, no single set of residues determines the affinity class. Instead we found heterogeneous sets of interactions that were driven primarily by electrostatic and Van der Waals forces. These results not only illustrate the complexity of EGFR dynamics but also pave the way for structure-based design of therapeutics targeting EGF ligands or the receptor itself. PMID:23382875

GPR30: a seven-transmembrane-spanning estrogen receptor that triggers EGF release.

PubMed

Filardo, Edward J; Thomas, Peter

2005-10-01

Heterotrimeric G proteins and seven-transmembrane-spanning (7TM) receptors are implicated in rapid estrogen signaling. The orphan 7TM receptor GPR30 is linked to estrogen-mediated activation of adenylyl cyclase, release of epidermal growth factor (EGF)-related ligands, and specific estrogen binding. GPR30 acts independently of estrogen receptors, ERalpha and ERbeta, and probably functions as a heptahelical ER. 7TM receptors elicit signals that stimulate second messengers, and convey intracellular signals via EGF receptors. Identification of GPR30 as a Gs-coupled 7TM receptor that triggers release of heparin-binding EGF establishes its role in cell signaling cascades initiated by estrogens, and explains their capacity to activate second messengers and promote EGF-like effects. Thus, estrogen can signal by the same mechanism as various other hormones, through a specific 7TM receptor.

Receptor Signaling Directs Global Recruitment of Pre-existing Transcription Factors to Inducible Elements.

PubMed

Cockerill, Peter N

2016-12-01

Gene expression programs are largely regulated by the tissue-specific expression of lineage-defining transcription factors or by the inducible expression of transcription factors in response to specific stimuli. Here I will review our own work over the last 20 years to show how specific activation signals also lead to the wide-spread re-distribution of pre-existing constitutive transcription factors to sites undergoing chromatin reorganization. I will summarize studies showing that activation of kinase signaling pathways creates open chromatin regions that recruit pre-existing factors which were previously unable to bind to closed chromatin. As models I will draw upon genes activated or primed by receptor signaling in memory T cells, and genes activated by cytokine receptor mutations in acute myeloid leukemia. I also summarize a hit-and-run model of stable epigenetic reprograming in memory T cells, mediated by transient Activator Protein 1 (AP-1) binding, which enables the accelerated activation of inducible enhancers.

Tomato pistil factor STIG1 promotes in vivo pollen tube growth by binding to phosphatidylinositol 3-phosphate and the extracellular domain of the pollen receptor kinase LePRK2

USDA-ARS?s Scientific Manuscript database

The speed of pollen tube growth is a major determinant of reproductive success in flowering plants. Tomato (Solanum lycopersicum) STIGMA-SPECIFIC PROTEIN1 (STIG1), a small Cys-rich protein from the pistil, was previously identified as a binding partner of the pollen receptor kinase LePRK2 and shown ...

Modeling hypertrophic IP3 transients in the cardiac myocyte.

PubMed

Cooling, Michael; Hunter, Peter; Crampin, Edmund J

2007-11-15

Cardiac hypertrophy is a known risk factor for heart disease, and at the cellular level is caused by a complex interaction of signal transduction pathways. The IP3-calcineurin pathway plays an important role in stimulating the transcription factor NFAT which binds to DNA cooperatively with other hypertrophic transcription factors. Using available kinetic data, we construct a mathematical model of the IP3 signal production system after stimulation by a hypertrophic alpha-adrenergic agonist (endothelin-1) in the mouse atrial cardiac myocyte. We use a global sensitivity analysis to identify key controlling parameters with respect to the resultant IP3 transient, including the phosphorylation of cell-membrane receptors, the ligand strength and binding kinetics to precoupled (with G(alpha)GDP) receptor, and the kinetics associated with precoupling the receptors. We show that the kinetics associated with the receptor system contribute to the behavior of the system to a great extent, with precoupled receptors driving the response to extracellular ligand. Finally, by reparameterizing for a second hypertrophic alpha-adrenergic agonist, angiotensin-II, we show that differences in key receptor kinetic and membrane density parameters are sufficient to explain different observed IP3 transients in essentially the same pathway.

A chemokine-binding domain in the tumor necrosis factor receptor from variola (smallpox) virus

PubMed Central

Alejo, Alí; Ruiz-Argüello, M. Begoña; Ho, Yin; Smith, Vincent P.; Saraiva, Margarida; Alcami, Antonio

2006-01-01

Variola virus (VaV) is the causative agent of smallpox, one of the most devastating diseases encountered by man, that was eradicated in 1980. The deliberate release of VaV would have catastrophic consequences on global public health. However, the mechanisms that contribute to smallpox pathogenesis are poorly understood at the molecular level. The ability of viruses to evade the host defense mechanisms is an important determinant of viral pathogenesis. Here we show that the tumor necrosis factor receptor (TNFR) homologue CrmB encoded by VaV functions not only as a soluble decoy TNFR but also as a highly specific binding protein for several chemokines that mediate recruitment of immune cells to mucosal surfaces and the skin, sites of virus entry and viral replication at late stages of smallpox. CrmB binds chemokines through its C-terminal domain, which is unrelated to TNFRs, was named smallpox virus-encoded chemokine receptor (SECRET) domain and uncovers a family of poxvirus chemokine inhibitors. An active SECRET domain was found in another viral TNFR (CrmD) and three secreted proteins encoded by orthopoxviruses. These findings identify a previously undescribed chemokine-binding and inhibitory domain unrelated to host chemokine receptors and a mechanism of immune modulation in VaV that may influence smallpox pathogenesis. PMID:16581912

A chemokine-binding domain in the tumor necrosis factor receptor from variola (smallpox) virus.

PubMed

Alejo, Alí; Ruiz-Argüello, M Begoña; Ho, Yin; Smith, Vincent P; Saraiva, Margarida; Alcami, Antonio

2006-04-11

Variola virus (VaV) is the causative agent of smallpox, one of the most devastating diseases encountered by man, that was eradicated in 1980. The deliberate release of VaV would have catastrophic consequences on global public health. However, the mechanisms that contribute to smallpox pathogenesis are poorly understood at the molecular level. The ability of viruses to evade the host defense mechanisms is an important determinant of viral pathogenesis. Here we show that the tumor necrosis factor receptor (TNFR) homologue CrmB encoded by VaV functions not only as a soluble decoy TNFR but also as a highly specific binding protein for several chemokines that mediate recruitment of immune cells to mucosal surfaces and the skin, sites of virus entry and viral replication at late stages of smallpox. CrmB binds chemokines through its C-terminal domain, which is unrelated to TNFRs, was named smallpox virus-encoded chemokine receptor (SECRET) domain and uncovers a family of poxvirus chemokine inhibitors. An active SECRET domain was found in another viral TNFR (CrmD) and three secreted proteins encoded by orthopoxviruses. These findings identify a previously undescribed chemokine-binding and inhibitory domain unrelated to host chemokine receptors and a mechanism of immune modulation in VaV that may influence smallpox pathogenesis.

Dynamic Regulation of FoxA1 by Steroid Receptors | Center for Cancer Research

Cancer.gov

The estrogen receptor (ER) is a key regulator in breast cancer initiation and progression. A widely discussed model proposes that forkhead box protein A1 (FoxA1) acts as a pioneer factor in cancer by binding and penetrating closed chromatin to allow access by transcription factors (TFs), including ER.

Variable Dependence of Signaling Output on Agonist Occupancy of Ste2p, a G Protein-coupled Receptor in Yeast.

PubMed

Sridharan, Rajashri; Connelly, Sara M; Naider, Fred; Dumont, Mark E

2016-11-11

We report here on the relationship between ligand binding and signaling responses in the yeast pheromone response pathway, a well characterized G protein-coupled receptor system. Responses to agonist (α-factor) by cells expressing widely varying numbers of receptors depend primarily on fractional occupancy, not the absolute number of agonist-bound receptors. Furthermore, the concentration of competitive antagonist required to inhibit α-factor-dependent signaling is more than 10-fold higher than predicted based on the known ligand affinities. Thus, responses to a particular number of agonist-bound receptors can vary greatly, depending on whether there are unoccupied or antagonist-bound receptors present on the same cell surface. This behavior does not appear to be due to pre-coupling of receptors to G protein or to the Sst2p regulator of G protein signaling. The results are consistent with a signaling response that is determined by the integration of positive signals from agonist-occupied receptors and inhibitory signals from unoccupied receptors, where the inhibitory signals can be diminished by antagonist binding. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Binding of Signal Recognition Particle Gives Ribosome/Nascent Chain Complexes a Competitive Advantage in Endoplasmic Reticulum Membrane Interaction

PubMed Central

Neuhof, Andrea; Rolls, Melissa M.; Jungnickel, Berit; Kalies, Kai-Uwe; Rapoport, Tom A.

1998-01-01

Most secretory and membrane proteins are sorted by signal sequences to the endoplasmic reticulum (ER) membrane early during their synthesis. Targeting of the ribosome-nascent chain complex (RNC) involves the binding of the signal sequence to the signal recognition particle (SRP), followed by an interaction of ribosome-bound SRP with the SRP receptor. However, ribosomes can also independently bind to the ER translocation channel formed by the Sec61p complex. To explain the specificity of membrane targeting, it has therefore been proposed that nascent polypeptide-associated complex functions as a cytosolic inhibitor of signal sequence- and SRP-independent ribosome binding to the ER membrane. We report here that SRP-independent binding of RNCs to the ER membrane can occur in the presence of all cytosolic factors, including nascent polypeptide-associated complex. Nontranslating ribosomes competitively inhibit SRP-independent membrane binding of RNCs but have no effect when SRP is bound to the RNCs. The protective effect of SRP against ribosome competition depends on a functional signal sequence in the nascent chain and is also observed with reconstituted proteoliposomes containing only the Sec61p complex and the SRP receptor. We conclude that cytosolic factors do not prevent the membrane binding of ribosomes. Instead, specific ribosome targeting to the Sec61p complex is provided by the binding of SRP to RNCs, followed by an interaction with the SRP receptor, which gives RNC–SRP complexes a selective advantage in membrane targeting over nontranslating ribosomes. PMID:9436994

Proteomic Identification of Dengue Virus Binding Proteins in Aedes aegypti Mosquitoes and Aedes albopictus Cells

PubMed Central

Muñoz, Maria de Lourdes; Limón-Camacho, Gustavo; Tovar, Rosalinda; Diaz-Badillo, Alvaro; Mendoza-Hernández, Guillermo; Black, William C.

2013-01-01

The main vector of dengue in America is the mosquito Aedes aegypti, which is infected by dengue virus (DENV) through receptors of midgut epithelial cells. The envelope protein (E) of dengue virus binds to receptors present on the host cells through its domain III that has been primarily recognized to bind cell receptors. In order to identify potential receptors, proteins from mosquito midgut tissue and C6/36 cells were purified by affinity using columns with the recombinant E protein domain III (rE-DIII) or DENV particles bound covalently to Sepharose 4B to compare and evaluate their performance to bind proteins including putative receptors from female mosquitoes of Ae. aegypti. To determine their identity mass spectrometric analysis of purified proteins separated by polyacrylamide gel electrophoresis was performed. Our results indicate that both viral particles and rE-DIII bound proteins with the same apparent molecular weights of 57 and 67 kDa. In addition, viral particles bound high molecular weight proteins. Purified proteins identified were enolase, beta-adrenergic receptor kinase (beta-ARK), translation elongation factor EF-1 alpha/Tu, and cadherin. PMID:24324976

Specificity of binding of clathrin adaptors to signals on the mannose-6-phosphate/insulin-like growth factor II receptor.

PubMed Central

Glickman, J N; Conibear, E; Pearse, B M

1989-01-01

Adaptors mediate the interaction of clathrin with select groups of receptors. Two distinct types of adaptors, the HA-II adaptors (found in plasma membrane coated pits) and the HA-I adaptors (localized to Golgi coated pits) bind to the cytoplasmic portion of the 270 kd mannose 6-phosphate (M6P) receptor-a receptor which is concentrated in coated pits on both the plasma membrane and in the trans-Golgi network. Neither type of adaptor appears to compete with the other for binding, suggesting that each type recognizes a distinct site on the M6P receptor tail. Mutation of the two tyrosines in the tail essentially eliminates the interaction with the HA-II plasma membrane adaptor, which recognizes a 'tyrosine' signal on other endocytosed receptors (for example, the LDL receptor and the poly Ig receptor). In contrast, the wild type and the mutant M6P receptor tail (lacking tyrosines) are equally effective at binding HA-I adaptors. This suggests that there is an HA-I recognition signal in another region of the M6P receptor tail, C-terminal to the tyrosine residues, which remains intact in the mutant. This signal is presumably responsible for the concentration of the M6P receptor, with bound lysosomal enzymes, into coated pits which bud from the trans-Golgi network, thus mediating efficient transfer of these enzymes to lysosomes. Images PMID:2545438

RNA-induced silencing complex (RISC) Proteins PACT, TRBP, and Dicer are SRA binding nuclear receptor coregulators

PubMed Central

Redfern, Andrew D.; Colley, Shane M.; Beveridge, Dianne J.; Ikeda, Naoya; Epis, Michael R.; Li, Xia; Foulds, Charles E.; Stuart, Lisa M.; Barker, Andrew; Russell, Victoria J.; Ramsay, Kerry; Kobelke, Simon J.; Li, Xiaotao; Hatchell, Esme C.; Payne, Christine; Giles, Keith M.; Messineo, Adriana; Gatignol, Anne; Lanz, Rainer B.; O’Malley, Bert W.; Leedman, Peter J.

2013-01-01

The cytoplasmic RNA-induced silencing complex (RISC) contains dsRNA binding proteins, including protein kinase RNA activator (PACT), transactivation response RNA binding protein (TRBP), and Dicer, that process pre-microRNAs into mature microRNAs (miRNAs) that target specific mRNA species for regulation. There is increasing evidence for important functional interactions between the miRNA and nuclear receptor (NR) signaling networks, with recent data showing that estrogen, acting through the estrogen receptor, can modulate initial aspects of nuclear miRNA processing. Here, we show that the cytoplasmic RISC proteins PACT, TRBP, and Dicer are steroid receptor RNA activator (SRA) binding NR coregulators that target steroid-responsive promoters and regulate NR activity and downstream gene expression. Furthermore, each of the RISC proteins, together with Argonaute 2, associates with SRA and specific pre-microRNAs in both the nucleus and cytoplasm, providing evidence for links between NR-mediated transcription and some of the factors involved in miRNA processing. PMID:23550157

RNA-induced silencing complex (RISC) Proteins PACT, TRBP, and Dicer are SRA binding nuclear receptor coregulators.

PubMed

Redfern, Andrew D; Colley, Shane M; Beveridge, Dianne J; Ikeda, Naoya; Epis, Michael R; Li, Xia; Foulds, Charles E; Stuart, Lisa M; Barker, Andrew; Russell, Victoria J; Ramsay, Kerry; Kobelke, Simon J; Li, Xiaotao; Hatchell, Esme C; Payne, Christine; Giles, Keith M; Messineo, Adriana; Gatignol, Anne; Lanz, Rainer B; O'Malley, Bert W; Leedman, Peter J

2013-04-16

The cytoplasmic RNA-induced silencing complex (RISC) contains dsRNA binding proteins, including protein kinase RNA activator (PACT), transactivation response RNA binding protein (TRBP), and Dicer, that process pre-microRNAs into mature microRNAs (miRNAs) that target specific mRNA species for regulation. There is increasing evidence for important functional interactions between the miRNA and nuclear receptor (NR) signaling networks, with recent data showing that estrogen, acting through the estrogen receptor, can modulate initial aspects of nuclear miRNA processing. Here, we show that the cytoplasmic RISC proteins PACT, TRBP, and Dicer are steroid receptor RNA activator (SRA) binding NR coregulators that target steroid-responsive promoters and regulate NR activity and downstream gene expression. Furthermore, each of the RISC proteins, together with Argonaute 2, associates with SRA and specific pre-microRNAs in both the nucleus and cytoplasm, providing evidence for links between NR-mediated transcription and some of the factors involved in miRNA processing.

[Targeting of membrane receptor tyrosine kinases: is there resistance in the HER?].

PubMed

Monnier, Lucile; Milano, Gérard; Penault-Llorca, Frédérique; Merlin, Jean-Louis

2004-09-01

Human Epidermal growth factor Receptors (HER) play an important role in cellular proliferation, and differentiation. Their overexpression in tumor tissues is often associated with a poor prognosis. Consequently, HER receptors are interesting therapeutic targets for cancer treatment. Two strategies are proposed. First, monoclonal antibodies can be used to inhibit the binding of one ligand to its receptor. The second approach is based upon the designing of tyrosine kinase inhibitors capable to bind into the phosphorylation site of the receptor. Consequently, both approaches block the signal transduction downstream. Resistance to anti receptor tyrosine kinase therapy can lead to enhanced morbidity associated with high therapeutic cost. Different mechanisms can be implicated. Non specific mechanisms include alterations of the signal transduction pathways (PI3K/AKT), recruitment of alternative receptor tyrosine kinase pathways (IGFR, VEGFR) and proteasome degradation inhibition. Other mechanisms are specific to HER and rely on inhibition of the binding of monoclonal antibodies (sialomucin-MUC4), heterodimerisation of HER, truncated soluble receptors intervention and mutated variants, as demonstrated very recently with EGF receptors, or genetic polymorphism. This paper reviews these different resistance mechanisms that have been identified in preclinical and clinical situations.

CD86 and beta2-adrenergic receptor signaling pathways, respectively, increase Oct-2 and OCA-B Expression and binding to the 3'-IgH enhancer in B cells.

PubMed

Podojil, Joseph R; Kin, Nicholas W; Sanders, Virginia M

2004-05-28

Stimulation of CD86 (formerly known as B7-2) and/or the beta2-adrenergic receptor on a CD40 ligand/interleukin-4-activated B cell increased the rate of mature IgG1 transcription. To identify the mechanism responsible for this effect, we determined whether CD86 and/or beta2-adrenergic receptor stimulation regulated transcription factor expression and binding to the 3'-IgH enhancer in vitro and in vivo. We showed that CD86 stimulation increased the nuclear localization of NF-kappaB1 (p50) and phosphorylated RelA (p65) and increased Oct-2 expression and binding to the 3'-IgH enhancer, in a protein kinase C-dependent manner. These effects were lost when CD86-deficient or NF-kappaB1-deficient B cells were used. CD86 stimulation also increased the level of IkappaB-alpha phosphorylation but in a protein kinase C-independent manner. Beta2-adrenergic receptor stimulation increased CREB phosphorylation, OCA-B expression, and OCA-B binding to the 3'-IgH enhancer in a protein kinase A-dependent manner, an effect lost when beta2-adrenergic receptor-deficient B cells were used. Also, the beta2-adrenergic receptor-induced increase in the level of mature IgG1 transcript was lost when OCA-B-deficient B cells were used. These data are the first to show that CD86 stimulation up-regulates the expression of the transcription factor Oct-2 in a protein kinase C- and NF-kappaB1-dependent manner, and that beta2-adrenergic receptor stimulation up-regulates the expression of the coactivator OCA-B in a protein kinase A-dependent manner to cooperate with Oct-2 binding to the 3'-IgH enhancer.

Platelet activating factor receptor binding plays a critical role in jet fuel-induced immune suppression.

PubMed

Ramos, Gerardo; Kazimi, Nasser; Nghiem, Dat X; Walterscheid, Jeffrey P; Ullrich, Stephen E

2004-03-15

Applying military jet fuel (JP-8) or commercial jet fuel (Jet-A) to the skin of mice suppresses the immune response in a dose-dependent manner. The release of biological response modifiers, particularly prostaglandin E2 (PGE2), is a critical step in activating immune suppression. Previous studies have shown that injecting selective cyclooxygenase-2 inhibitors into jet fuel-treated mice blocks immune suppression. Because the inflammatory phospholipid mediator, platelet-activating factor (PAF), up-regulates cyclooxygenase-2 production and PGE2 synthesis by keratinocytes, we tested the hypothesis that PAF-receptor binding plays a role in jet fuel-induced immune suppression. Treating keratinocyte cultures with PAF and/or jet fuel (JP-8 and Jet-A) stimulates PGE2 secretion. Jet fuel-induced PGE2 production was suppressed by treating the keratinocytes with specific PAF-receptor antagonists. Injecting mice with PAF, or treating the skin of the mice with JP-8, or Jet-A, induced immune suppression. Jet fuel-induced immune suppression was blocked when the jet fuel-treated mice were injected with PAF-receptor antagonists before treatment. Jet fuel treatment has been reported to activate oxidative stress and treating the mice with anti-oxidants (Vitamins C, or E or beta-hydroxy toluene), before jet fuel application, interfered with immune suppression. These findings confirm previous studies showing that PAF-receptor binding can modulate immune function. Furthermore, they suggest that PAF-receptor binding may be an early event in the induction of immune suppression by immunotoxic environmental agents that target the skin.

Europium-labeled epidermal growth factor and neurotensin: novel probes for receptor-binding studies.

PubMed

Mazor, Ohad; Hillairet de Boisferon, Marc; Lombet, Alain; Gruaz-Guyon, Anne; Gayer, Batya; Skrzydelsky, Delphine; Kohen, Fortune; Forgez, Patricia; Scherz, Avigdor; Rostene, William; Salomon, Yoram

2002-02-01

We investigated the possibility of labeling two biologically active peptides, epidermal growth factor (EGF) and neurotensin (NT), with europium (Eu)-diethylenetriaminepentaacetic acid. More specifically, we tested them as probes in studying receptor binding using time-resolved fluorescence of Eu3+. The relatively simple synthesis yields ligands with acceptable binding characteristics similar to isotopically labeled derivatives. The binding affinity (Kd) of labeled Eu-EGF to human A431 epidermal carcinoid cells was 3.6 +/- 1.2 nM, similar to the reported Kd values of EGF, whereas the Kd of Eu-NT to human HT29 colon cancer cells (7.4 +/- 0.5 nM) or to Chinese hamster ovary (CHO) cells transfected with the high-affinity NT receptor (CHO-NT1) were about 10-fold higher than the Kd values of NT. The bioactivity of the Eu-labeled EGF as determined by stimulation of cultured murine D1 hematopoietic cell proliferation was nearly the same as that obtained with native EGF. The maximal stimulation of Ca2+ influx with NT and Eu-NT in CHO-NT1 cells was similar, but the respective K0.5 values were 20 pM and 1 nM, corresponding to differences in the binding affinities previously described. The results of these studies indicate that Eu labeling of peptide hormones and growth factor molecules ranging from 10(3) to 10(5) Da can be conveniently accomplished. Importantly, the Eu-labeled products are stable for approximately 2 years and are completely safe for laboratory use compared to the biohazardous radioligands. Thus, Eu-labeled peptides present an attractive alternative for commonly used radiolabeled ligands in biological studies in general and in receptor assays in particular.

Plasticity in interactions of fibroblast growth factor 1 (FGF1) N terminus with FGF receptors underlies promiscuity of FGF1.

PubMed

Beenken, Andrew; Eliseenkova, Anna V; Ibrahimi, Omar A; Olsen, Shaun K; Mohammadi, Moosa

2012-01-27

Tissue-specific alternative splicing in the second half of Ig-like domain 3 (D3) of fibroblast growth factor receptors 1-3 (FGFR1 to -3) generates epithelial FGFR1b-FGFR3b and mesenchymal FGFR1c-FGFR3c splice isoforms. This splicing event establishes a selectivity filter to restrict the ligand binding specificity of FGFRb and FGFRc isoforms to mesenchymally and epithelially derived fibroblast growth factors (FGFs), respectively. FGF1 is termed the "universal FGFR ligand" because it overrides this specificity barrier. To elucidate the molecular basis for FGF1 cross-reactivity with the "b" and "c" splice isoforms of FGFRs, we determined the first crystal structure of FGF1 in complex with an FGFRb isoform, FGFR2b, at 2.1 Å resolution. Comparison of the FGF1-FGFR2b structure with the three previously published FGF1-FGFRc structures reveals that plasticity in the interactions of the N-terminal region of FGF1 with FGFR D3 is the main determinant of FGF1 cross-reactivity with both isoforms of FGFRs. In support of our structural data, we demonstrate that substitution of three N-terminal residues (Gly-19, His-25, and Phe-26) of FGF2 (a ligand that does not bind FGFR2b) for the corresponding residues of FGF1 (Phe-16, Asn-22, and Tyr-23) enables the FGF2 triple mutant to bind and activate FGFR2b. These findings taken together with our previous structural data on receptor binding specificity of FGF2, FGF8, and FGF10 conclusively show that sequence divergence at the N termini of FGFs is the primary regulator of the receptor binding specificity and promiscuity of FGFs.

Characterization of the swine adipocyte A1 adenosine receptor using an optimized assay system.

PubMed

Dong, Q; Schuchman, J; Carey, G B

1994-07-01

The radioligand binding assay of A1 adenosine receptors in adipocyte crude plasma membrane from Yucatan miniature swine was optimized by evaluating 17 factors involved in the assay. Significant effects of CHAPS, adenosine deaminase, EDTA, pre-rinsing glass fiber filters and pH were found for the binding measurements. Using the optimized procedure, [3H]8-cyclopentyl-1,3-dipropylxanthine, ([3H]-DPCPX) binding to A1 adenosine receptors in swine subcutaneous adipocyte crude plasma membrane was measured; Bmax and Kd values were 479 +/- 77 fmol/mg protein and 0.87 +/- 0.10 nM, respectively. Values for mesenteric adipose tissue from sedentary swine and subcutaneous adipose tissue from exercise-trained swine were also measured.

Structural Basis for Activation of the Receptor Tyrosine Kinase KIT by Stem Cell Factor

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

Yuzawa,S.; Opatowsky, Y.; Zhang, Z.

2007-01-01

Stem Cell Factor (SCF) initiates its multiple cellular responses by binding to the ectodomain of KIT, resulting in tyrosine kinase activation. We describe the crystal structure of the entire ectodomain of KIT before and after SCF stimulation. The structures show that KIT dimerization is driven by SCF binding whose sole role is to bring two KIT molecules together. Receptor dimerization is followed by conformational changes that enable lateral interactions between membrane proximal Ig-like domains D4 and D5 of two KIT molecules. Experiments with cultured cells show that KIT activation is compromised by point mutations in amino acids critical for D4-D4more » interaction. Moreover, a variety of oncogenic mutations are mapped to the D5-D5 interface. Since key hallmarks of KIT structures, ligand-induced receptor dimerization, and the critical residues in the D4-D4 interface, are conserved in other receptors, the mechanism of KIT stimulation unveiled in this report may apply for other receptor activation.« less

6-Gingerol reduces Pseudomonas aeruginosa biofilm formation and virulence via quorum sensing inhibition.

PubMed

Kim, Han-Shin; Lee, Sang-Hoon; Byun, Youngjoo; Park, Hee-Deung

2015-03-02

Pseudomonas aeruginosa is a well-known pathogenic bacterium that forms biofilms and produces virulence factors via quorum sensing (QS). Interfering with normal QS interactions between signal molecules and their cognate receptors is a developing strategy for attenuating its virulence. Here we tested the hypothesis that 6-gingerol, a pungent oil of fresh ginger, reduces biofilm formation and virulence by antagonistically binding to P. aeruginosa QS receptors. In silico studies demonstrated molecular binding occurs between 6-gingerol and the QS receptor LasR through hydrogen bonding and hydrophobic interactions. Experimentally 6-gingerol reduced biofilm formation, several virulence factors (e.g., exoprotease, rhamnolipid, and pyocyanin), and mice mortality. Further transcriptome analyses demonstrated that 6-gingerol successfully repressed QS-induced genes, specifically those related to the production of virulence factors. These results strongly support our hypothesis and offer insight into the molecular mechanism that caused QS gene repression.

6-Gingerol reduces Pseudomonas aeruginosa biofilm formation and virulence via quorum sensing inhibition

PubMed Central

Kim, Han-Shin; Lee, Sang-Hoon; Byun, Youngjoo; Park, Hee-Deung

2015-01-01

Pseudomonas aeruginosa is a well-known pathogenic bacterium that forms biofilms and produces virulence factors via quorum sensing (QS). Interfering with normal QS interactions between signal molecules and their cognate receptors is a developing strategy for attenuating its virulence. Here we tested the hypothesis that 6-gingerol, a pungent oil of fresh ginger, reduces biofilm formation and virulence by antagonistically binding to P. aeruginosa QS receptors. In silico studies demonstrated molecular binding occurs between 6-gingerol and the QS receptor LasR through hydrogen bonding and hydrophobic interactions. Experimentally 6-gingerol reduced biofilm formation, several virulence factors (e.g., exoprotease, rhamnolipid, and pyocyanin), and mice mortality. Further transcriptome analyses demonstrated that 6-gingerol successfully repressed QS-induced genes, specifically those related to the production of virulence factors. These results strongly support our hypothesis and offer insight into the molecular mechanism that caused QS gene repression. PMID:25728862

Generation of tumour-necrosis-factor-alpha-specific affibody molecules capable of blocking receptor binding in vitro.

PubMed

Jonsson, Andreas; Wållberg, Helena; Herne, Nina; Ståhl, Stefan; Frejd, Fredrik Y

2009-08-17

Affibody molecules specific for human TNF-alpha (tumour necrosis factor-alpha) were selected by phage-display technology from a library based on the 58-residue Protein A-derived Z domain. TNF-alpha is a proinflammatory cytokine involved in several inflammatory diseases and, to this day, four TNF-alpha-blocking protein pharmaceuticals have been approved for clinical use. The phage selection generated 18 unique cysteine-free affibody sequences of which 12 were chosen, after sequence cluster analysis, for characterization as proteins. Biosensor binding studies of the 12 Escherichia coli-produced and IMAC (immobilized-metal-ion affinity chromatography)-purified affibody molecules revealed three variants that demonstrated the strongest binding to human TNF-alpha. These three affibody molecules were subjected to kinetic binding analysis and also tested for their binding to mouse, rat and pig TNF-alpha. For ZTNF-alpha:185, subnanomolar affinity (KD=0.1-0.5 nM) for human TNF-alpha was demonstrated, as well as significant binding to TNF-alpha from the other species. Furthermore, the binding site was found to overlap with the binding site for the TNF-alpha receptor, since this interaction could be efficiently blocked by the ZTNF-alpha:185 affibody. When investigating six dimeric affibody constructs with different linker lengths, and one trimeric construct, it was found that the inhibition of the TNF-alpha binding to its receptor could be further improved by using dimers with extended linkers and/or a trimeric affibody construct. The potential implication of the results for the future design of affibody-based reagents for the diagnosis of inflammation is discussed.

Rpn1 provides adjacent receptor sites for substrate binding and deubiquitination by the proteasome

PubMed Central

Shi, Yuan; Chen, Xiang; Elsasser, Suzanne; Stocks, Bradley B.; Tian, Geng; Lee, Byung-Hoon; Shi, Yanhong; Zhang, Naixia; de Poot, Stefanie A. H.; Tuebing, Fabian; Sun, Shuangwu; Vannoy, Jacob; Tarasov, Sergey G.; Engen, John R.; Finley, Daniel; Walters, Kylie J.

2016-01-01

Structured Abstract INTRODUCTION The ubiquitin-proteasome system comprises hundreds of distinct pathways of degradation, which converge at the step of ubiquitin recognition by the proteasome. Five proteasomal ubiquitin receptors have been identified, two that are intrinsic to the proteasome (Rpn10 and Rpn13) and three reversibly associated proteasomal ubiquitin receptors (Rad23, Dsk2, and Ddi1). RATIONALE We found that the five known proteasomal ubiquitin receptors of yeast are collectively nonessential for ubiquitin recognition by the proteasome. We therefore screened for additional ubiquitin receptors in the proteasome and identified subunit Rpn1 as a candidate. We used nuclear magnetic resonance (NMR) spectroscopy to characterize the structure of the binding site within Rpn1, which we term the T1 site. Mutational analysis of this site showed its functional importance within the context of intact proteasomes. T1 binds both ubiquitin and ubiquitin-like (UBL) proteins, in particular the substrate-delivering shuttle factor Rad23. A second site within the Rpn1 toroid, T2, recognizes the UBL domain of deubiquitinating enzyme Ubp6, as determined by hydrogen-deuterium exchange mass spectrometry analysis and validated by amino acid substitution and functional assays. The Rpn1 toroid thus serves a critical scaffolding role within the proteasome, helping to assemble multiple proteasome cofactors as well as substrates. RESULTS Our results indicate that proteasome subunit Rpn1 can recognize both ubiquitin and UBL domains of substrate shuttling factors that themselves bind ubiquitin and function as reversibly-associated proteasomal ubiquitin receptors. Recognition is mediated by the T1 site within the Rpn1 toroid, which supports proteasome function in vivo. We found that the capacity of T1 to recognize both ubiquitin and UBL proteins was shared with Rpn10 and Rpn13. The surprising multiplicity of ubiquitin-recognition domains within the proteasome may promote enhanced, multipoint binding of ubiquitin chains. The structures of the T1 site in its free state and complexed with monoubiquitin or K48-linked diubiquitin were solved, revealing that three neighboring outer helices from the T1 toroid engage two ubiquitins. This binding mode leads to a preference for certain ubiquitin chain types, especially K6- and K48-linked chains, in a distinct configuration that can position substrates close to the entry port of the proteasome. The fate of proteasome-docked ubiquitin conjugates is determined by a competition between deubiquitination and substrate degradation. We find that proximal to the T1 site within the Rpn1 toroid is a second UBL-binding site, T2, that does not assist in ubiquitin chain recognition, but rather in chain disassembly, by binding to the UBL domain of deubiquitinating enzyme Ubp6. Importantly, the UBL interactors at T1 and T2 are distinct, assigning substrate localization to T1 and substrate deubiquitination to T2. CONCLUSION A ligand-binding hotspot was identified in the Rpn1 toroid, consisting of two adjacent receptor sites, T1 and T2. The Rpn1 toroid represents a novel class of binding domains for ubiquitin and UBL proteins. This study thus defines a novel two-site recognition domain intrinsic to the proteasome that uses homologous ubiquitin/UBL-class ligands to assemble substrates, substrate shuttling factors, and a deubiquitinating enzyme in close proximity. A ligand-binding hotspot in the proteasome for assembling substrates and cofactors Schematic (top) and model structure (bottom, left) mapping the UBL-binding Rpn1 T1 (indigo) and T2 (orange) sites. (Bottom, right) Enlarged region of the proteasome to illustrate the Rpn1 T1 and T2 sites bound to a ubiquitin chain (yellow) and deubiquitinating enzyme Ubp6 (green), respectively. PDB 4CR2 and 2B9R were used for this figure. Hundreds of pathways for degradation converge at ubiquitin recognition by proteasome. Here we found that the five known proteasomal ubiquitin receptors are collectively nonessential for ubiquitin recognition, and identified a sixth receptor, Rpn1. A site (T1) in the Rpn1 toroid recognized ubiquitin and ubiquitin-like (UBL) domains of substrate shuttling factors. T1 structures with monoubiquitin or K48 diubiquitin show three neighboring outer helices engaging two ubiquitins. T1 contributes a distinct substrate-binding pathway with preference for K48-linked chains. Proximal to T1 within the Rpn1 toroid is a second UBL-binding site (T2) that assists in ubiquitin chain disassembly, by binding the UBL of deubiquitinating enzyme Ubp6. Thus a two-site recognition domain intrinsic to the proteasome uses homologous ubiquitin/UBL-class ligands to assemble substrates, shuttling factors, and a deubiquitinating enzyme. PMID:26912900

Molecular Recognition of Corticotropin releasing Factor by Its G protein-coupled Receptor CRFR1

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

Pioszak, Augen A.; Parker, Naomi R.; Suino-Powell, Kelly

2009-01-15

The bimolecular interaction between corticotropin-releasing factor (CRF), a neuropeptide, and its type 1 receptor (CRFR1), a class B G-protein-coupled receptor (GPCR), is crucial for activation of the hypothalamic-pituitary-adrenal axis in response to stress, and has been a target of intense drug design for the treatment of anxiety, depression, and related disorders. As a class B GPCR, CRFR1 contains an N-terminal extracellular domain (ECD) that provides the primary ligand binding determinants. Here we present three crystal structures of the human CRFR1 ECD, one in a ligand-free form and two in distinct CRF-bound states. The CRFR1 ECD adopts the alpha-beta-betaalpha fold observedmore » for other class B GPCR ECDs, but the N-terminal alpha-helix is significantly shorter and does not contact CRF. CRF adopts a continuous alpha-helix that docks in a hydrophobic surface of the ECD that is distinct from the peptide-binding site of other class B GPCRs, thereby providing a basis for the specificity of ligand recognition between CRFR1 and other class B GPCRs. The binding of CRF is accompanied by clamp-like conformational changes of two loops of the receptor that anchor the CRF C terminus, including the C-terminal amide group. These structural studies provide a molecular framework for understanding peptide binding and specificity by the CRF receptors as well as a template for designing potent and selective CRFR1 antagonists for therapeutic applications.« less

SH3 domain-mediated binding of the Drk protein to Dos is an important step in signaling of Drosophila receptor tyrosine kinases.

PubMed

Feller, Stephan M; Wecklein, Heike; Lewitzky, Marc; Kibler, Eike; Raabe, Thomas

2002-08-01

Activation of the Sevenless (Sev) receptor tyrosine kinase (RTK) in the developing Drosophila eye is required for the specification of the R7 photoreceptor cell fate. Daughter of Sevenless (Dos), a putative multi-site adaptor protein, is a substrate of the Sev kinase and is known to associate with the tyrosine phosphatase Corkscrew (Csw). Binding of Csw to Dos depends on the Csw Src homology 2 (SH2) domains and is an essential step for signaling by the Sev RTK. Dos, however, lacks a recognizable phosphotyrosine interaction domain and it was previously unclear how it is recruited to the Sev receptor. Here it is shown that the SH2/SH3 domain adaptor protein Drk can provide this link. Drk binds with its SH2 domain to the autophosphorylated Sev receptor while the C-terminal SH3 domain is able to associate with Dos. The Drk SH3 domain binding motifs on Dos were mapped to two sites which do not conform the known Drk SH3 domain binding motif (PxxPxR) but instead have the consensus PxxxRxxKP. Mutational analysis in vitro and in vivo provided evidence that both Drk binding sites fulfil an important function in the context of Sev and Drosophila epidermal growth factor receptor mediated signaling processes.

Cholinergic Receptor Binding in Alzheimer Disease and Healthy Aging: Assessment In Vivo with Positron Emission Tomography Imaging.

PubMed

Sultzer, David L; Melrose, Rebecca J; Riskin-Jones, Hannah; Narvaez, Theresa A; Veliz, Joseph; Ando, Timothy K; Juarez, Kevin O; Harwood, Dylan G; Brody, Arthur L; Mandelkern, Mark A

2017-04-01

To compare regional nicotinic cholinergic receptor binding in older adults with Alzheimer disease (AD) and healthy older adults in vivo and to assess relationships between receptor binding and clinical symptoms. Using cross-sectional positron emission tomography (PET) neuroimaging and structured clinical assessment, outpatients with mild to moderate AD (N = 24) and healthy older adults without cognitive complaints (C group; N = 22) were studied. PET imaging of α4β2* nicotinic cholinergic receptor binding using 2-[ 18 F]fluoro-3-(2(S)azetidinylmethoxy)pyridine (2FA) and clinical measures of global cognition, attention/processing speed, verbal memory, visuospatial memory, and neuropsychiatric symptoms were used. 2FA binding was lower in the AD group compared with the C group in the medial thalamus, medial temporal cortex, anterior cingulate, insula/opercula, inferior caudate, and brainstem (p < 0.05, corrected cluster), but binding was not associated with cognition. The C group had significant inverse correlations between 2FA binding in the thalamus (left: r s  = -0.55, p = 0.008; right: r s  = -0.50, p = 0.02; N = 22) and hippocampus (left: r s  = -0.65, p = 0.001; right: r s  = -0.55, p = 0.009; N = 22) and the Trails A score. The AD group had inverse correlation between 2FA binding in anterior cingulate (left: r s  = -0.50, p = 0.01; right: r s  = -0.50, p = 0.01; N = 24) and Neurobehavioral Rating Scale agitation/disinhibition factor score. Cholinergic receptor binding is reduced in specific brain regions in mild to moderate AD and is related to neuropsychiatric symptoms. Among healthy older adults, lower receptor binding may be associated with slower processing speed. Cholinergic receptor binding in vivo may reveal links to other key brain changes associated with aging and AD and may provide a potential molecular treatment target. Published by Elsevier Inc.

Intracellular signaling of the Ufo/Axl receptor tyrosine kinase is mediated mainly by a multi-substrate docking-site.

PubMed

Braunger, J; Schleithoff, L; Schulz, A S; Kessler, H; Lammers, R; Ullrich, A; Bartram, C R; Janssen, J W

1997-06-05

Ufo/Axl belongs to a new family of receptor tyrosine kinases with an extracellular structure similar to that of neural cell adhesion molecules. In order to elucidate intracellular signaling, the cytoplasmic moiety of Ufo/Axl was used to screen an expression library according to the CORT (cloning of receptor targets) method. Three putative Ufo substrates were identified: phospholipase Cgamma1 (PLCgamma), as well as p85alpha and p85beta subunits of phosphatidylinositol 3'-kinase (PI3-kinase). Subsequently, chimeric EGFR/Ufo receptors consisting of the extracellular domains of the epidermal growth factor receptor (EGFR) and the transmembrane and intracellular moiety of Ufo were engineered. Using different far-Western blot analyses and coimmunoprecipitation assays, receptor binding of PLCgamma and p85 proteins as well as GRB2, c-src and lck was examined in vitro and in vivo. Competitive inhibition of substrate binding and mutagenesis experiments with EGFR/Ufo constructs revealed C-terminal tyrosine 821 (EILpYVNMDEG) as a docking site for multiple effectors, namely PLCgamma, p85 proteins, GRB2, c-src and lck. Tyrosine 779 (DGLpYALMSRC) demonstrated an additional, but lower binding affinity for the p85 proteins in vitro. In addition, binding of PLCgamma occurred through tyrosine 866 (AGRpYVLCPST). Moreover, our in vivo data indicate that further direct or indirect binding sites for PLCgamma, GRB2, c-src and lck on the human Ufo receptor may exist.

The SH2 and SH3 domains of mammalian Grb2 couple the EGF receptor to the Ras activator mSos1.

PubMed

Rozakis-Adcock, M; Fernley, R; Wade, J; Pawson, T; Bowtell, D

1993-05-06

Many tyrosine kinases, including the receptors for hormones such as epidermal growth factor (EGF), nerve growth factor and insulin, transmit intracellular signals through Ras proteins. Ligand binding to such receptors stimulates Ras guanine-nucleotide-exchange activity and increases the level of GTP-bound Ras, suggesting that these tyrosine kinases may activate a guanine-nucleotide releasing protein (GNRP). In Caenorhabditis elegans and Drosophila, genetic studies have shown that Ras activation by tyrosine kinases requires the protein Sem-5/drk, which contains a single Src-homology (SH) 2 domain and two flanking SH3 domains. Sem-5 is homologous to the mammalian protein Grb2, which binds the autophosphorylated EGF receptor and other phosphotyrosine-containing proteins such as Shc through its SH2 domain. Here we show that in rodent fibroblasts, the SH3 domains of Grb2 are bound to the proline-rich carboxy-terminal tail of mSos1, a protein homologous to Drosophila Sos. Sos is required for Ras signalling and contains a central domain related to known Ras-GNRPs. EGF stimulation induces binding of the Grb2-mSos1 complex to the autophosphorylated EGF receptor, and mSos1 phosphorylation. Grb2 therefore appears to link tyrosine kinases to a Ras-GNRP in mammalian cells.

Residues remote from the binding pocket control the antagonist selectivity towards the corticotropin-releasing factor receptor-1

NASA Astrophysics Data System (ADS)

Sun, Xianqiang; Cheng, Jianxin; Wang, Xu; Tang, Yun; Ågren, Hans; Tu, Yaoquan

2015-01-01

The corticotropin releasing factors receptor-1 and receptor-2 (CRF1R and CRF2R) are therapeutic targets for treating neurological diseases. Antagonists targeting CRF1R have been developed for the potential treatment of anxiety disorders and alcohol addiction. It has been found that antagonists targeting CRF1R always show high selectivity, although CRF1R and CRF2R share a very high rate of sequence identity. This has inspired us to study the origin of the selectivity of the antagonists. We have therefore built a homology model for CRF2R and carried out unbiased molecular dynamics and well-tempered metadynamics simulations for systems with the antagonist CP-376395 in CRF1R or CRF2R to address this issue. We found that the side chain of Tyr6.63 forms a hydrogen bond with the residue remote from the binding pocket, which allows Tyr6.63 to adopt different conformations in the two receptors and results in the presence or absence of a bottleneck controlling the antagonist binding to or dissociation from the receptors. The rotameric switch of the side chain of Tyr3566.63 allows the breaking down of the bottleneck and is a perquisite for the dissociation of CP-376395 from CRF1R.

Residues remote from the binding pocket control the antagonist selectivity towards the corticotropin-releasing factor receptor-1.

PubMed

Sun, Xianqiang; Cheng, Jianxin; Wang, Xu; Tang, Yun; Ågren, Hans; Tu, Yaoquan

2015-01-28

The corticotropin releasing factors receptor-1 and receptor-2 (CRF1R and CRF2R) are therapeutic targets for treating neurological diseases. Antagonists targeting CRF1R have been developed for the potential treatment of anxiety disorders and alcohol addiction. It has been found that antagonists targeting CRF1R always show high selectivity, although CRF1R and CRF2R share a very high rate of sequence identity. This has inspired us to study the origin of the selectivity of the antagonists. We have therefore built a homology model for CRF2R and carried out unbiased molecular dynamics and well-tempered metadynamics simulations for systems with the antagonist CP-376395 in CRF1R or CRF2R to address this issue. We found that the side chain of Tyr(6.63) forms a hydrogen bond with the residue remote from the binding pocket, which allows Tyr(6.63) to adopt different conformations in the two receptors and results in the presence or absence of a bottleneck controlling the antagonist binding to or dissociation from the receptors. The rotameric switch of the side chain of Tyr356(6.63) allows the breaking down of the bottleneck and is a perquisite for the dissociation of CP-376395 from CRF1R.

Preliminary crystallographic analysis of mouse Elf3 C-terminal DNA-binding domain in complex with type II TGF-[beta] receptor promoter DNA

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

Agarkar, Vinod B.; Babayeva, Nigar D.; Rizzino, Angie

2010-10-08

Ets proteins are transcription factors that activate or repress the expression of genes that are involved in various biological processes, including cellular proliferation, differentiation, development, transformation and apoptosis. Like other Ets-family members, Elf3 functions as a sequence-specific DNA-binding transcriptional factor. A mouse Elf3 C-terminal fragment (amino-acid residues 269-371) containing the DNA-binding domain has been crystallized in complex with mouse type II TGF-{beta} receptor promoter (TR-II) DNA. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 42.66, b = 52, c = 99.78 {angstrom}, and diffracted to a resolution of 2.2 {angstrom}.

Latent myostatin has significant activity and this activity is controlled more efficiently by WFIKKN1 than by WFIKKN2

PubMed Central

Szláma, György; Trexler, Mária; Patthy, László

2013-01-01

Myostatin, a negative regulator of skeletal muscle growth, is produced from myostatin precursor by multiple steps of proteolytic processing. After cleavage by a furin-type protease, the propeptide and growth factor domains remain associated, forming a noncovalent complex, the latent myostatin complex. Mature myostatin is liberated from latent myostatin by bone morphogenetic protein 1/tolloid proteases. Here, we show that, in reporter assays, latent myostatin preparations have significant myostatin activity, as the noncovalent complex dissociates at an appreciable rate, and both mature and semilatent myostatin (a complex in which the dimeric growth factor domain interacts with only one molecule of myostatin propeptide) bind to myostatin receptor. The interaction of myostatin receptor with semilatent myostatin is efficiently blocked by WAP, Kazal, immunoglobulin, Kunitz and NTR domain-containing protein 1 or growth and differentiation factor-associated serum protein 2 (WFIKKN1), a large extracellular multidomain protein that binds both mature myostatin and myostatin propeptide [Kondás et al. (2008) J Biol Chem 283, 23677–23684]. Interestingly, the paralogous protein WAP, Kazal, immunoglobulin, Kunitz and NTR domain-containing protein 2 or growth and differentiation factor-associated serum protein 1 (WFIKKN2) was less efficient than WFIKKN1 as an antagonist of the interactions of myostatin receptor with semilatent myostatin. Our studies have shown that this difference is attributable to the fact that only WFIKKN1 has affinity for the propeptide domain, and this interaction increases its potency in suppressing the receptor-binding activity of semilatent myostatin. As the interaction of WFIKKN1 with various forms of myostatin permits tighter control of myostatin activity until myostatin is liberated from latent myostatin by bone morphogenetic protein 1/tolloid proteases, WFIKKN1 may have greater potential as an antimyostatic agent than WFIKKN2. Structured digital abstract Furin cleaves Promyostatin by protease assay (View interaction) myostatin binds to PRO by surface plasmon resonance (View interaction) BMP-1 cleaves Promyostatin by protease assay (View interaction) ACR IIB physically interacts with Latent Myostatin by surface plasmon resonance (View interaction) Promyostatin and Promyostatin bind by comigration in gel electrophoresis (View interaction) WFIKKN1 binds to Latent Myostatin by pull down (View interaction) ACR IIB binds to Mature Myostatin by surface plasmon resonance (View Interaction: 1, 2, 3) WFIKKN1 binds to Myostatin Prodomain by surface plasmon resonance (View Interaction: 1, 2, 3) PMID:23829672

Binding Mode and Structure-Activity Relationships of ITE as an Aryl Hydrocarbon Receptor (AhR) Agonist.

PubMed

Dolciami, Daniela; Gargaro, Marco; Cerra, Bruno; Scalisi, Giulia; Bagnoli, Luana; Servillo, Giuseppe; Fazia, Maria Agnese Della; Puccetti, Paolo; Quintana, Francisco J; Fallarino, Francesca; Macchiarulo, Antonio

2018-02-06

Discovered as a modulator of the toxic response to environmental pollutants, aryl hydrocarbon receptor (AhR) has recently gained attention for its involvement in various physiological and pathological pathways. AhR is a ligand-dependent transcription factor activated by a large array of chemical compounds, which include metabolites of l-tryptophan (l-Trp) catabolism as endogenous ligands of the receptor. Among these, 2-(1'H-indole-3'-carbonyl)thiazole-4-carboxylic acid methyl ester (ITE) has attracted interest in the scientific community, being endowed with nontoxic, immunomodulatory, and anticancer AhR-mediated functions. So far, no information about the binding mode and interactions of ITE with AhR is available. In this study, we used docking and molecular dynamics to propose a putative binding mode of ITE into the ligand binding pocket of AhR. Mutagenesis studies were then instrumental in validating the proposed binding mode, identifying His 285 and Tyr 316 as important key residues for ligand-dependent receptor activation. Finally, a set of ITE analogues was synthesized and tested to further probe molecular interactions of ITE to AhR and characterize the relevance of specific functional groups in the chemical structure for receptor activity. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Photoaffinity labelling of the ATP-binding site of the epidermal growth factor-dependent protein kinase.

PubMed

Kudlow, J E; Leung, Y

1984-06-15

Epidermal growth factor (EGF), after binding to its receptor, activates a tyrosine-specific protein kinase which phosphorylates several substrates, including the EGF receptor itself. The effects of a photoaffinity analogue of ATP, 3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)adenosine 5'-triphosphate (arylazido-beta-alanyl-ATP) on the EGF-dependent protein kinase in A431 human tumour cell plasma membrane vesicles was investigated. This analogue was capable of inactivating the EGF-receptor kinase in a photodependent manner. Partial inactivation occurred at an analogue concentration of 1 microM and complete inactivation occurred at 10 microM when a 2 min light exposure was used. Arylazido-beta-alanine at 100 microM and ATP at 100 microM were incapable of inactivating the enzyme with 2 min of light exposure. The photodependent inactivation of the enzyme by the analogue could be partially blocked by 20 mM-ATP and more effectively blocked by either 20 mM-adenosine 5'-[beta gamma-imido]triphosphate or 20 mM-guanosine 5'-[beta gamma-imido]triphosphate, indicating nucleotide-binding site specificity. Arylazido-beta-alanyl-[alpha-32P]ATP was capable of labelling membrane proteins in a photodependent manner. Numerous proteins were labelled, the most prominent of which ran with an apparent Mr of 53000 on polyacrylamide-gel electrophoresis. A band of minor intensity was seen of Mr corresponding to the EGF receptor (170000). Immunoprecipitation of affinity-labelled and solubilized membranes with an anti-(EGF receptor) monoclonal antibody demonstrated that the Mr 170000 receptor protein was photoaffinity labelled by the analogue. The Mr 53000 peptide was not specifically bound by the anti-receptor antibody. The affinity labelling of the receptor was not enhanced by EGF, suggesting that EGF stimulation of the kinase activity does not result from changes in the affinity of the kinase for ATP. These studies demonstrate that arylazido-beta-alanyl-ATP interacts with the ATP-binding site of the EGF-receptor kinase with apparent high affinity and that this analogue is an effective photoaffinity label for the kinase. Furthermore, these studies demonstrate that the EGF receptor, identified by using monoclonal antibodies, contains an ATP-binding site, providing further confirmation that the EGF receptor and EGF-dependent protein kinase are domains of the Mr 170000 protein.

Recent Progress in the Design and Discovery of RXR Modulators Targeting Alternate Binding Sites of the Receptor.

PubMed

Su, Ying; Zeng, Zhiping; Chen, Ziwen; Xu, Dan; Zhang, Weidong; Zhang, Xiao-Kun

2017-01-01

Retinoid X receptors (RXRs) occupy a central position within the nuclear receptor superfamily. They not only function as important transcriptional factors but also exhibit diverse nongenomic biological activities. The pleiotropic actions of RXRs under both physiological and pathophysiological conditions confer RXRs important drug targets for the treatment of cancer, and metabolic and neurodegenerative diseases. RXR modulators have been studied for the purpose of developing both drug molecules and chemical tools for biological investigation of RXR. Development of RXR modulators has focused on small molecules targeting the canonical ligand-binding pocket. However, accumulating results have demonstrated that there are other binding mechanisms by which small molecules interact with RXR to act as RXR modulators. This review discusses the recent development in the design and discovery of RXR modulators with a focus on those targeting novel binding sites on RXR.

Common and divergent structural features of a series of corticotropin releasing factor-related peptides.

PubMed

Grace, Christy Rani R; Perrin, Marilyn H; Cantle, Jeffrey P; Vale, Wylie W; Rivier, Jean E; Riek, Roland

2007-12-26

Members of the corticoliberin family include the corticotropin releasing factors (CRFs), sauvagine, the urotensins, and urocortin 1 (Ucn1), which bind to both the CRF receptors CRF-R1 and CRF-R2, and the urocortins 2 (Ucn2) and 3 (Ucn3), which are selective agonists of CRF-R2. Structure activity relationship studies led to several potent and long-acting analogues with selective binding to either one of the receptors. NMR structures of six ligands of this family (the antagonists astressin B and astressin2-B, the agonists stressin1, and the natural ligands human Ucn1, Ucn2, and Ucn3) were determined in DMSO. These six peptides show differences in binding affinities, receptor-selectivity, and NMR structure. Overall, their backbones are alpha-helical, with a small kink or a turn around residues 25-27, resulting in a helix-loop-helix motif. The C-terminal helices are of amphipathic nature, whereas the N-terminal helices vary in their amphipathicity. The C-terminal helices thereby assume a conformation very similar to that of astressin bound to the ECD1 of CRF-R2 recently reported by our group.1 On the basis of an analysis of the observed 3D structures and relative potencies of [Ala]-substituted analogues, it is proposed that both helices could play a crucial role in receptor binding and selectivity. In conclusion, the C-terminal helices may interact along their hydrophobic faces with the ECD1, whereas the entire N-terminal helical surface may be involved in receptor activation. On the basis of the common and divergent features observed in the 3D structures of these ligands, multiple binding models are proposed that may explain their plurality of actions.

Loop III region of platelet-derived growth factor (PDGF) B-chain mediates binding to PDGF receptors and heparin.

PubMed Central

Schilling, D; Reid IV, J D; Hujer, A; Morgan, D; Demoll, E; Bummer, P; Fenstermaker, R A; Kaetzel, D M

1998-01-01

Site-directed mutagenesis of the platelet-derived growth factor (PDGF) B-chain was conducted to determine the importance of cationic amino acid residues (Arg160-Lys161-Lys162; RKK) located within the loop III region in mediating the biological and cell-association properties of the molecule. Binding to both PDGF alpha-and beta-receptors was inhibited by the conversion of all three cationic residues into anionic glutamates (RKK-->EEE), whereas an RKK-->SSS mutant also exhibited a modest loss in affinity for beta-receptors. Replacements with serine at either Arg160 (RKK-->SKK) or at all three positions (RKK-->SSS) had little effect on binding to alpha-receptors. Replacements with either glutamic or serine residues at any of the three positions also resulted in significant inhibition of heparin-binding activity. Furthermore, the RKK-->EEE mutant exhibited decreased association with the cell surface and accumulated in the culture medium as 29-32 kDa forms. Stable transfection of U87 astrocytoma cells with RKK-->EEE mutants of either the A-chain or the B-chain inhibited malignant growth in athymic nude mice. Despite altered receptor-binding activities, each of the loop III mutants retained full mitogenic activity when applied to cultured Swiss 3T3 cells. CD spectrophotometric analysis of the RKK-->EEE mutant revealed a secondary structure indistinguishable from the wild type, with a high degree of beta-sheet structure and random coil content (50% and 43% respectively). These findings indicate an important role of the Arg160-Lys161-Lys162 sequence in mediating the biological and cell-associative activities of the PDGF-BB homodimer, and reveal that the mitogenic activity of PDGF-BB is insufficient to mediate its full oncogenic properties. PMID:9677323

Enhanced In Vivo Tumor Detection by Active Tumor Cell Targeting Using Multiple Tumor Receptor-Binding Peptides Presented on Genetically Engineered Human Ferritin Nanoparticles.

PubMed

Kwon, Koo Chul; Ko, Ho Kyung; Lee, Jiyun; Lee, Eun Jung; Kim, Kwangmeyung; Lee, Jeewon

2016-08-01

Human ferritin heavy-chain nanoparticle (hFTH) is genetically engineered to present tumor receptor-binding peptides (affibody and/or RGD-derived cyclic peptides, named 4CRGD here) on its surface. The affibody and 4CRGD specifically and strongly binds to human epidermal growth factor receptor I (EGFR) and human integrin αvβ3, respectively, which are overexpressed on various tumor cells. Through in vitro culture of EGFR-overexpressing adenocarcinoma (MDA-MB-468) and integrin-overexpressing glioblastoma cells (U87MG), it is clarified that specific interactions between receptors on tumor cells and receptor-binding peptides on engineered hFTH is critical in active tumor cell targeting. After labeling with the near-infrared fluorescence dye (Cy5.5) and intravenouse injection into MDA-MB-468 or U87MG tumor-bearing mice, the recombinant hFTHs presenting either peptide or both of affibody and 4CRGD are successfully delivered to and retained in the tumor for a prolonged period of time. In particular, the recombinant hFTH presenting both affibody and 4CRGD notably enhances in vivo detection of U87MG tumors that express heterogeneous receptors, integrin and EGFR, compared to the other recombinant hFTHs presenting either affibody or 4CRGD only. Like affibody and 4CRGD used in this study, other multiple tumor receptor-binding peptides can be also genetically introduced to the hFTH surface for actively targeting of in vivo tumors with heterogenous receptors. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Inter-species chimeras of leukaemia inhibitory factor define a major human receptor-binding determinant.

PubMed Central

Owczarek, C M; Layton, M J; Metcalf, D; Lock, P; Willson, T A; Gough, N M; Nicola, N A

1993-01-01

Human leukaemia inhibitory factor (hLIF) binds to both human and mouse LIF receptors (LIF-R), while mouse LIF (mLIF) binds only to mouse LIF-R. Moreover, hLIF binds with higher affinity to the mLIF-R than does mLIF. In order to define the regions of the hLIF molecule responsible for species-specific interaction with the hLIF-R and for the unusual high-affinity binding to the mLIF-R, a series of 15 mouse/human LIF hybrids has been generated. Perhaps surprisingly, both of these properties mapped to the same region of the hLIF molecule. The predominant contribution was from residues in the loop linking the third and fourth helices, with lesser contributions from residues in the third helix and the loop connecting the second and third helices in the predicted three-dimensional structure. Since all chimeras retained full biological activity and receptor-binding activity on mouse cells, and there was little variation in the specific biological activity of the purified proteins, it can be concluded that the overall secondary and tertiary structures of each chimera were intact. This observation also implied that the primary binding sites on mLIF and hLIF for the mLIF-R were unaltered by inter-species domain swapping. Consequently, the site on the hLIF molecule that confers species-specific binding to the hLIF-R and higher affinity binding to the mLIF-R, must constitute an additional interaction site to that used by both mLIF and hLIF to bind to the mLIF-R. These studies define a maximum of 15 amino acid differences between hLIF and mLIF that are responsible for the different properties of these proteins. Images PMID:8253075

Dissection of androgen receptor-promoter interactions: Steroid receptors partition their interaction energetics in parallel with their phylogenetic divergence

PubMed Central

De Angelis, Rolando W; Yang, Qin; Miura, Michael T; Bain, David L

2013-01-01

Steroid receptors comprise a homologous family of ligand-activated transcription factors. The members include androgen receptor (AR), estrogen receptor (ER), glucocorticoid receptor (GR), mineralocorticoid receptor (MR) and progesterone receptor (PR). Phylogenetic studies demonstrate that AR, GR, MR and PR are most closely related, falling into subgroup 3C. ER is more distantly related, falling into subgroup 3A. To determine the quantitative basis by which receptors generate their unique transcriptional responses, we are systematically dissecting the promoter-binding energetics of all receptors under a single “standard state” condition. Here we examine the self-assembly and promoter-binding energetics of full-length AR and a mutant associated with prostate cancer, T877A. We first demonstrate that both proteins exist only as monomers, showing no evidence of dimerization. Although this result contradicts the traditional understanding that steroid receptors dimerize in the absence of DNA, it is fully consistent with our previous work demonstrating that GR and two PR isoforms either do not dimerize or dimerize only weakly. Moreover, both AR proteins exhibit substantial cooperativity between binding sites, again as seen for GR and PR. In sharp contrast, the more distantly related ER-α dimerizes so strongly that energetics can only be measured indirectly, yet cooperativity is negligible. Thus homologous receptors partition their promoter-binding energetics quite differently. Moreover, since receptors most closely related by phylogeny partition their energetics similarly, such partitioning appears to be evolutionarily conserved. We speculate that such differences in energetics, coupled with different promoter architectures, serve as the basis for generating receptor-specific promoter occupancy and thus function. PMID:23917122

Binding Mode Analysis of Zerumbone to Key Signal Proteins in the Tumor Necrosis Factor Pathway

PubMed Central

Fatima, Ayesha; Abdul, Ahmad Bustamam Hj.; Abdullah, Rasedee; Karjiban, Roghayeh Abedi; Lee, Vannajan Sanghiran

2015-01-01

Breast cancer is the second most common cancer among women worldwide. Several signaling pathways have been implicated as causative and progression agents. The tumor necrosis factor (TNF) α protein plays a dual role in promoting and inhibiting cancer depending largely on the pathway initiated by the binding of the protein to its receptor. Zerumbone, an active constituent of Zingiber zerumbet, Smith, is known to act on the tumor necrosis factor pathway upregulating tumour necrosis factor related apoptosis inducing ligand (TRAIL) death receptors and inducing apoptosis in cancer cells. Zerumbone is a sesquiterpene that is able to penetrate into the hydrophobic pockets of proteins to exert its inhibiting activity with several proteins. We found a good binding with the tumor necrosis factor, kinase κB (IKKβ) and the Nuclear factor κB (NF-κB) component proteins along the TNF pathway. Our results suggest that zerumbone can exert its apoptotic activities by inhibiting the cytoplasmic proteins. It inhibits the IKKβ kinase that activates the NF-κB and also binds to the NF-κB complex in the TNF pathway. Blocking both proteins can lead to inhibition of cell proliferating proteins to be downregulated and possibly ultimate induction of apoptosis. PMID:25629232

Insulin, Central Dopamine D2 Receptors, and Monetary Reward Discounting in Obesity

PubMed Central

Eisenstein, Sarah A.; Gredysa, Danuta M.; Antenor–Dorsey, Jo Ann; Green, Leonard; Arbeláez, Ana Maria; Koller, Jonathan M.; Black, Kevin J.; Perlmutter, Joel S.; Moerlein, Stephen M.; Hershey, Tamara

2015-01-01

Animal research finds that insulin regulates dopamine signaling and reward behavior, but similar research in humans is lacking. We investigated whether individual differences in body mass index, percent body fat, pancreatic β-cell function, and dopamine D2 receptor binding were related to reward discounting in obese and non-obese adult men and women. Obese (n = 27; body mass index>30) and non-obese (n = 20; body mass index<30) adults were assessed for percent body fat with dual-energy X-ray absorptiometry and for β-cell function using disposition index. Choice of larger, but delayed or less certain, monetary rewards relative to immediate, certain smaller monetary rewards was measured using delayed and probabilistic reward discounting tasks. Positron emission tomography using a non-displaceable D2-specific radioligand, [11C](N-methyl)benperidol quantified striatal D2 receptor binding. Groups differed in body mass index, percent body fat, and disposition index, but not in striatal D2 receptor specific binding or reward discounting. Higher percent body fat in non-obese women related to preference for a smaller, certain reward over a larger, less likely one (greater probabilistic discounting). Lower β-cell function in the total sample and lower insulin sensitivity in obese related to stronger preference for an immediate and smaller monetary reward over delayed receipt of a larger one (greater delay discounting). In obese adults, higher striatal D2 receptor binding related to greater delay discounting. Interestingly, striatal D2 receptor binding was not significantly related to body mass index, percent body fat, or β-cell function in either group. Our findings indicate that individual differences in percent body fat, β-cell function, and striatal D2 receptor binding may each contribute to altered reward discounting behavior in non-obese and obese individuals. These results raise interesting questions about whether and how striatal D2 receptor binding and metabolic factors, including β-cell function, interact to affect reward discounting in humans. PMID:26192187

Insulin, Central Dopamine D2 Receptors, and Monetary Reward Discounting in Obesity.

PubMed

Eisenstein, Sarah A; Gredysa, Danuta M; Antenor-Dorsey, Jo Ann; Green, Leonard; Arbeláez, Ana Maria; Koller, Jonathan M; Black, Kevin J; Perlmutter, Joel S; Moerlein, Stephen M; Hershey, Tamara

2015-01-01

Animal research finds that insulin regulates dopamine signaling and reward behavior, but similar research in humans is lacking. We investigated whether individual differences in body mass index, percent body fat, pancreatic β-cell function, and dopamine D2 receptor binding were related to reward discounting in obese and non-obese adult men and women. Obese (n = 27; body mass index>30) and non-obese (n = 20; body mass index<30) adults were assessed for percent body fat with dual-energy X-ray absorptiometry and for β-cell function using disposition index. Choice of larger, but delayed or less certain, monetary rewards relative to immediate, certain smaller monetary rewards was measured using delayed and probabilistic reward discounting tasks. Positron emission tomography using a non-displaceable D2-specific radioligand, [11C](N-methyl)benperidol quantified striatal D2 receptor binding. Groups differed in body mass index, percent body fat, and disposition index, but not in striatal D2 receptor specific binding or reward discounting. Higher percent body fat in non-obese women related to preference for a smaller, certain reward over a larger, less likely one (greater probabilistic discounting). Lower β-cell function in the total sample and lower insulin sensitivity in obese related to stronger preference for an immediate and smaller monetary reward over delayed receipt of a larger one (greater delay discounting). In obese adults, higher striatal D2 receptor binding related to greater delay discounting. Interestingly, striatal D2 receptor binding was not significantly related to body mass index, percent body fat, or β-cell function in either group. Our findings indicate that individual differences in percent body fat, β-cell function, and striatal D2 receptor binding may each contribute to altered reward discounting behavior in non-obese and obese individuals. These results raise interesting questions about whether and how striatal D2 receptor binding and metabolic factors, including β-cell function, interact to affect reward discounting in humans.

The Influence of Adnectin Binding on the Extracellular Domain of Epidermal Growth Factor Receptor

NASA Astrophysics Data System (ADS)

Iacob, Roxana E.; Chen, Guodong; Ahn, Joomi; Houel, Stephane; Wei, Hui; Mo, Jingjie; Tao, Li; Cohen, Daniel; Xie, Dianlin; Lin, Zheng; Morin, Paul E.; Doyle, Michael L.; Tymiak, Adrienne A.; Engen, John R.

2014-12-01

The precise and unambiguous elucidation and characterization of interactions between a high affinity recognition entity and its cognate protein provides important insights for the design and development of drugs with optimized properties and efficacy. In oncology, one important target protein has been shown to be the epidermal growth factor receptor (EGFR) through the development of therapeutic anticancer antibodies that are selective inhibitors of EGFR activity. More recently, smaller protein derived from the 10th type III domain of human fibronectin termed an adnectin has also been shown to inhibit EGFR in clinical studies. The mechanism of EGFR inhibition by either an adnectin or an antibody results from specific binding of the high affinity protein to the extracellular portion of EGFR (exEGFR) in a manner that prevents phosphorylation of the intracellular kinase domain of the receptor and thereby blocks intracellular signaling. Here, the structural changes induced upon binding were studied by probing the solution conformations of full length exEGFR alone and bound to a cognate adnectin through hydrogen/deuterium exchange mass spectrometry (HDX MS). The effects of binding in solution were identified and compared with the structure of a bound complex determined by X-ray crystallography.

SHP-1 Binds and Negatively Modulates the c-Kit Receptor by Interaction with Tyrosine 569 in the c-Kit Juxtamembrane Domain

PubMed Central

Kozlowski, Maya; Larose, Louise; Lee, Fai; Le, Duc Mingh; Rottapel, Robert; Siminovitch, Katherine A.

1998-01-01

The SH2 domain-containing SHP-1 tyrosine phosphatase has been shown to negatively regulate a broad spectrum of growth factor- and cytokine-driven mitogenic signaling pathways. Included among these is the cascade of intracellular events evoked by stem cell factor binding to c-Kit, a tyrosine kinase receptor which associates with and is dephosphorylated by SHP-1. Using a series of glutathione S-transferase (GST) fusion proteins containing either tyrosine-phosphorylated segments of the c-Kit cytosolic region or the SH2 domains of SHP-1, we have shown that SHP-1 interacts with c-Kit by binding selectively to the phosphorylated c-Kit juxtamembrane region and that the association of c-Kit with the larger of the two SHP-1 isoforms may be mediated through either the N-terminal or C-terminal SHP-1 SH2 domain. The results of binding assays with mutagenized GST-Kit juxtamembrane fusion proteins and competitive inhibition assays with phosphopeptides encompassing each c-Kit juxtamembrane region identified the tyrosine residue at position 569 as the major site for binding of SHP-1 to c-Kit and suggested that tyrosine 567 contributes to, but is not required for, this interaction. By analysis of Ba/F3 cells retrovirally transduced to express c-Kit receptors, phenylalanine substitution of c-Kit tyrosine residue 569 was shown to be associated with disruption of c-Kit–SHP-1 binding and induction of hyperproliferative responses to stem cell factor. Although phenylalanine substitution of c-Kit tyrosine residue 567 in the Ba/F3–c-Kit cells did not alter SHP-1 binding to c-Kit, the capacity of a second c-Kit-binding tyrosine phosphatase, SHP-2, to associate with c-Kit was markedly reduced, and the cells again showed hyperproliferative responses to stem cell factor. These data therefore identify SHP-1 binding to tyrosine 569 on c-Kit as an interaction pivotal to SHP-1 inhibitory effects on c-Kit signaling, but they indicate as well that cytosolic protein tyrosine phosphatases other than SHP-1 may also negatively regulate the coupling of c-Kit engagement to proliferation. PMID:9528781

Structural Determinants for Naturally Evolving H5N1 Hemagglutinin to Switch its Receptor Specificity

PubMed Central

Tharakaraman, Kannan; Raman, Rahul; Viswanathan, Karthik; Stebbins, Nathan W.; Jayaraman, Akila; Krishnan, Arvind; Sasisekharan, V.; Sasisekharan, Ram

2013-01-01

SUMMARY Of the factors governing human-to-human transmission of the highly pathogenic avian-adapted H5N1 virus, the most critical is the acquisition of mutations on the viral hemagglutinin (HA) to “quantitatively switch” its binding from avian to human glycan receptors. Herein, we describe a structural framework that outlines a necessary set of H5 HA receptor binding site (RBS) features required for the H5 HA to quantitatively switch its preference to human receptors. We show here that the same RBS HA mutations that lead to aerosol transmission of A/Vietnam/1203/04 and A/Indonesia/5/05 viruses, when introduced in currently circulating H5N1, do not lead to quantitative switch in receptor preference. We demonstrate that HAs from circulating clades require as few as a single base-pair mutation to quantitatively switch their binding to human receptors. The mutations identified by this study can be used to monitor the emergence of strains having human-to-human transmission potential. PMID:23746829

Regulation of atrial natriuretic peptide clearance receptors in mesangial cells by growth factors.

PubMed

Paul, R V; Wackym, P S; Budisavljevic, M; Everett, E; Norris, J S

1993-08-25

Rat mesangial cells can express both 130-kDa guanylyl cyclase-coupled and 66-kDa non-coupled atrial natriuretic peptide (ANP) receptors (ANPR-A and ANPR-C, respectively). Exposure of mesangial cells, grown in 20% fetal calf serum, to 0.1% serum for 24 h increased total ANP receptor density more than 2-fold (Bmax = 87 versus 37 fmol/mg of cell protein) without changing binding affinity (Kd = 94 versus 88 pM). Radioligand binding and cross-linking studies demonstrated that up-regulation of ANP binding after serum deprivation was entirely due to an increase in ANPR-C, with little or no change in ANPR-A. Inhibition of protein synthesis with cycloheximide blocked up-regulation after serum deprivation. Steady-state ANPR-C mRNA level was increased 15-fold by serum deprivation, as judged by Northern blotting. There was no change in ANPR-A mRNA. Platelet-derived growth factor and phorbol myristate acetate, when added to low serum medium, blocked or reversed the effect of serum deprivation on ANPR-C. We conclude that synthesis and expression of ANPR-C but not ANPR-A is suppressed by serum, platelet-derived growth factor, and phorbol myristate acetate. Suppression of ANPR-C in vivo could contribute to mesangial cell proliferative responses to growth factors.

Non-conventional Frizzled ligands and Wnt receptors.

PubMed

Hendrickx, Marijke; Leyns, Luc

2008-05-01

The Wnt family of secreted signaling factors plays numerous roles in embryonic development and in stem cell biology. In the adult, Wnt signaling is involved in tissue homeostasis and mutations that lead to the overexpression of Wnt can be linked to cancer. Wnt signaling is transduced intracellularly by the Frizzled (Fzd) family of receptors. In the canonical pathway, accumulation of beta-catenin and the subsequent formation of a complex with T cell factors (TCF) or lymphoid enhancing factors (Lef) lead to target gene activation. The identification of Ryk as an alternative Wnt receptor and the discovery of the novel Fzd ligands Norrie disease protein (NDP) and R-Spondin, changed the traditional view of Wnts binding to Fzd receptors. Mouse R-Spondin cooperates with Wnt signaling and Low density lipoprotein (LDL) receptor related protein (LRP) to activate beta-catenin dependent gene expression and is involved in processes such as limb and placental development in the mouse. NDP is the product of the Norrie disease gene and controls vascular development in the retina, inner ear and in the female reproductive system during pregnancy. In this review a functional overview of the interactions of the different Wnt and non-Wnt ligands with the Fzd receptors is given as well as a survey of Wnts binding to Ryk and we discuss the biological significance of these interactions.

Fibroblast growth factor receptors, developmental corruption and malignant disease.

PubMed

Kelleher, Fergal C; O'Sullivan, Hazel; Smyth, Elizabeth; McDermott, Ray; Viterbo, Antonella

2013-10-01

Fibroblast growth factors (FGF) are a family of ligands that bind to four different types of cell surface receptor entitled, FGFR1, FGFR2, FGFR3 and FGFR4. These receptors differ in their ligand binding affinity and tissue distribution. The prototypical receptor structure is that of an extracellular region comprising three immunoglobulin (Ig)-like domains, a hydrophobic transmembrane segment and a split intracellular tyrosine kinase domain. Alternative gene splicing affecting the extracellular third Ig loop also creates different receptor isoforms entitled FGFRIIIb and FGFRIIIc. Somatic fibroblast growth factor receptor (FGFR) mutations are implicated in different types of cancer and germline FGFR mutations occur in developmental syndromes particularly those in which craniosynostosis is a feature. The mutations found in both conditions are often identical. Many somatic FGFR mutations in cancer are gain-of-function mutations of established preclinical oncogenic potential. Gene amplification can also occur with 19-22% of squamous cell lung cancers for example having amplification of FGFR1. Ontologic comparators can be informative such as aberrant spermatogenesis being implicated in both spermatocytic seminomas and Apert syndrome. The former arises from somatic FGFR3 mutations and Apert syndrome arises from germline FGFR2 mutations. Finally, therapeutics directed at inhibiting the FGF/FGFR interaction are a promising subject for clinical trials.

Unveiling the molecular mechanism of brassinosteroids: Insights from structure-based molecular modeling studies.

PubMed

Lei, Beilei; Liu, Jiyuan; Yao, Xiaojun

2015-12-01

Brassinosteroid (BR) phytohormones play indispensable roles in plant growth and development. Brassinolide (BL) and 24-epibrassinolide (24-epiBL) are the most active ones among the BRs reported thus far. Unfortunately, the extremely low natural content and intricate synthesis process limit their popularization in agricultural production. Earlier reports to discover alternative compounds have resulted in molecules with nearly same scaffold structure and without diversity in chemical space. In the present study, receptors structure based BRs regulation mechanism was analyzed. First, we examined the detailed binding interactions and their dynamic stability between BL and its receptor BRI1 and co-receptor BAK1. Then, the binding modes and binding free energies for 24-epiBL and a series of representative BRs binding with BRI1 and BRI1-BAK1 were carried out by molecular docking, energy minimization and MM-PBSA free energy calculation. The obtained binding structures and energetic results provided vital insights into the structural factors affecting the activity from both receptors and BRs aspects. Subsequently, the obtained knowledge will serve as valuable guidance to build pharmacophore models for rational screening of new scaffold alternative BRs. Copyright © 2015 Elsevier Inc. All rights reserved.

Selective localization of oxytocin receptors and vasopressin 1a receptors in the human brainstem

PubMed Central

Freeman, Sara M.; Smith, Aaron L.; Goodman, Mark M.; Bales, Karen L.

2017-01-01

Intranasal oxytocin affects a suite of human social behaviors, including trust, eye contact, and emotion recognition. However, it is unclear where oxytocin receptors (OXTR) and the structurally related vasopressin 1a receptors (AVPR1a) are expressed in the human brain. We have previously described a reliable, pharmacologically informed receptor autoradiography protocol for visualizing these receptors in postmortem primate brain tissue. We used this technique in human brainstem tissue to identify the neural targets of oxytocin and vasopressin. To determine binding selectivity of the OXTR radioligand and AVPR1a radioligand, sections were incubated in four conditions: radioligand alone, radioligand with the selective AVPR1a competitor SR49059, and radioligand with a low or high concentration of the selective OXTR competitor ALS-II-69. We found selective OXTR binding in the spinal trigeminal nucleus, a conserved region of OXTR expression in all primate species investigated to date. We found selective AVPR1a binding in the nucleus prepositus, an area implicated in eye gaze stabilization. The tissue's postmortem interval was not correlated with either the specific or nonspecific binding of either radioligand, indicating that it will not likely be a factor in similar postmortem studies. This study provides critical data for future studies of OXTR and AVPR1a in human brain tissue. PMID:26911439

Design and Synthesis of Benzimidazoles As Novel Corticotropin-Releasing Factor 1 Receptor Antagonists.

PubMed

Mochizuki, Michiyo; Kori, Masakuni; Kobayashi, Katsumi; Yano, Takahiko; Sako, Yuu; Tanaka, Maiko; Kanzaki, Naoyuki; Gyorkos, Albert C; Corrette, Christopher P; Cho, Suk Young; Pratt, Scott A; Aso, Kazuyoshi

2016-03-24

Benzazole derivatives with a flexible aryl group bonded through a one-atom linker as a new scaffold for a corticotropin-releasing factor 1 (CRF1) receptor antagonist were designed, synthesized, and evaluated. We expected that structural diversity could be expanded beyond that of reported CRF1 receptor antagonists. In a structure-activity relationship study, 4-chloro-N(2)-(4-chloro-2-methoxy-6-methylphenyl)-1-methyl-N(7),N(7)-dipropyl-1H-benzimidazole-2,7-diamine 29g had the most potent binding activity against a human CRF1 receptor and the antagonistic activity (IC50 = 9.5 and 88 nM, respectively) without concerns regarding cytotoxicity at 30 μM. Potent CRF1 receptor-binding activity in brain in an ex vivo test and suppression of stress-induced activation of the hypothalamus-pituitary-adrenocortical (HPA) axis were also observed at 138 μmol/kg of compound 29g after oral administration in mice. Thus, the newly designed benzimidazole 29g showed in vivo CRF1 receptor antagonistic activity and good brain penetration, indicating that it is a promising lead for CRF1 receptor antagonist drug discovery research.

Tumor microenvironment: Modulation by decorin and related molecules harboring leucine-rich tandem motifs.

PubMed

Goldoni, Silvia; Iozzo, Renato V

2008-12-01

Decorin, the prototype member of the small leucine-rich proteoglycans, resides in the tumor microenvironment and affects the biology of various types of cancer by downregulating the activity of several receptors involved in cell growth and survival. Decorin binds to and modulates the signaling of the epidermal growth factor receptor and other members of the ErbB family of receptor tyrosine kinases. It exerts its antitumor activity by a dual mechanism: via inhibition of these key receptors through their physical downregulation coupled with attenuation of their signaling, and by binding to and sequestering TGFbeta. Decorin also modulates the insulin-like growth factor receptor and the low-density lipoprotein receptor-related protein 1, which indirectly affects the TGFbeta receptor pathway. When expressed in tumor xenograft-bearing mice or injected systemically, decorin inhibits both primary tumor growth and metastatic spreading. In this review, we summarize the latest reports on decorin and related molecules that are relevant to cancer and bring forward the idea of decorin as an anticancer therapeutic and possible prognostic marker for patients affected by various types of tumors. We also discuss the role of lumican and LRIG1, a novel cell growth inhibitor homologous to decorin. (c) 2008 Wiley-Liss, Inc.

Role of CRF Receptor Signaling in Stress Vulnerability, Anxiety, and Depression

PubMed Central

Hauger, Richard L.; Risbrough, Victoria; Oakley, Robert H.; Olivares-Reyes, J. Alberto; Dautzenberg, Frank M.

2011-01-01

Markers of hyperactive central corticotropin releasing factor (CRF) systems and CRF-related single nucleotide polymorphisms (SNPs) have been identified in patients with anxiety and depressive disorders. Designing more effective antagonists may now be guided by data showing that small molecules bind to transmembrane domains. Specifically, CRF1 receptor antagonists have been developed as novel anxiolytic and antidepressant treatments. Because CRF1 receptors become rapidly desensitized by G protein-coupled receptor kinase (GRK) and β-arrestin mechanisms in the presence of high agonist concentrations, neuronal hypersecretion of synaptic CRF alone may be insufficient to account for excessive central CRF neurotransmission in stress-induced affective pathophysiology. In addition to desensitizing receptor function, GRK phosphorylation and β-arrestin binding can shift a G protein-coupled receptor (GPCR) to signal selectively via the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK-MAPK) or Akt pathways independent of G proteins. Also, Epac-dependent CRF1 receptor signaling via the ERK-MAPK pathway has been found to potentiate brain-derived neurotrophic factor (BDNF)-stimulated TrkB signaling. Thus, genetic or acquired abnormalities in GRK and β-arrestin function may be involved in the pathophysiology of stress-induced anxiety and depression. PMID:19906236

Effects of vitamin D(3)-binding protein-derived macrophage activating factor (GcMAF) on angiogenesis.

PubMed

Kanda, Shigeru; Mochizuki, Yasushi; Miyata, Yasuyoshi; Kanetake, Hiroshi; Yamamoto, Nobuto

2002-09-04

The vitamin D(3)-binding protein (Gc protein)-derived macrophage activating factor (GcMAF) activates tumoricidal macrophages against a variety of cancers indiscriminately. We investigated whether GcMAF also acts as an antiangiogenic factor on endothelial cells. The effects of GcMAF on angiogenic growth factor-induced cell proliferation, chemotaxis, and tube formation were examined in vitro by using cultured endothelial cells (murine IBE cells, porcine PAE cells, and human umbilical vein endothelial cells [HUVECs]) and in vivo by using a mouse cornea micropocket assay. Blocking monoclonal antibodies to CD36, a receptor for the antiangiogenic factor thrombospondin-1, which is also a possible receptor for GcMAF, were used to investigate the mechanism of GcMAF action. GcMAF inhibited the endothelial cell proliferation, chemotaxis, and tube formation that were all stimulated by fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor-A, or angiopoietin 2. FGF-2-induced neovascularization in murine cornea was also inhibited by GcMAF. Monoclonal antibodies against murine and human CD36 receptor blocked the antiangiogenic action of GcMAF on the angiogenic factor stimulation of endothelial cell chemotaxis. In addition to its ability to activate tumoricidal macrophages, GcMAF has direct antiangiogenic effects on endothelial cells independent of tissue origin. The antiangiogenic effects of GcMAF may be mediated through the CD36 receptor.

Interaction of PF4 (CXCL4) with the vasculature: a role in atherosclerosis and angiogenesis.

PubMed

Aidoudi, Sallouha; Bikfalvi, Andreas

2010-11-01

Platelet factor-4 (PF4), a platelet-derived chemokine, has two important functions in the vasculature. It has a pro-atherogenic role while also having anti-angiogenic effects. The activity of platelet factor-4 (PF4), unlike other chemokines that bind to specific receptors, depends on its unusually high affinity for proteoglycans and other negatively charged molecules. High affinity for heparan sulfates was thought to be central to all of PF4's biological functions. However, other mechanisms have been described such as direct growth factor binding, activation of the CXCR3B chemokine receptor isoform that is present in some vascular cells or binding to lipoprotein-related protein-1 (LRP1). Furthermore, PF4 also binds to integrins with affinities similar to matrix molecules. These interactions may explain the effects of PF4 in healthy and pathological tissues. However, the mechanisms involved in PF4's activity are complex and may depend on a given tissue or localisation. Overall, while much is already known about PF4, its specific role in atherosclerosis and angiogenesis remains still to be clarified.

Adaptation of avian influenza A (H6N1) virus from avian to human receptor-binding preference

PubMed Central

Wang, Fei; Qi, Jianxun; Bi, Yuhai; Zhang, Wei; Wang, Min; Zhang, Baorong; Wang, Ming; Liu, Jinhua; Yan, Jinghua; Shi, Yi; Gao, George F

2015-01-01

The receptor-binding specificity of influenza A viruses is a major determinant for the host tropism of the virus, which enables interspecies transmission. In 2013, the first human case of infection with avian influenza A (H6N1) virus was reported in Taiwan. To gather evidence concerning the epidemic potential of H6 subtype viruses, we performed comprehensive analysis of receptor-binding properties of Taiwan-isolated H6 HAs from 1972 to 2013. We propose that the receptor-binding properties of Taiwan-isolated H6 HAs have undergone three major stages: initially avian receptor-binding preference, secondarily obtaining human receptor-binding capacity, and recently human receptor-binding preference, which has been confirmed by receptor-binding assessment of three representative virus isolates. Mutagenesis work revealed that E190V and G228S substitutions are important to acquire the human receptor-binding capacity, and the P186L substitution could reduce the binding to avian receptor. Further structural analysis revealed how the P186L substitution in the receptor-binding site of HA determines the receptor-binding preference change. We conclude that the human-infecting H6N1 evolved into a human receptor preference. PMID:25940072

A Cell-Permeable Phospholipase C[gamma]1-Binding Peptide Transduces Neurons and Impairs Long-Term Spatial Memory

ERIC Educational Resources Information Center

Blum, Sonja; Dash, Pramod K.

2004-01-01

Growth factor-mediated signaling has emerged as an essential component of memory formation. In this study, we used a phospholipase C gamma 1 (PLC[gamma]1) binding, cell-penetrating peptide to sequester PLC[gamma]1 away from its target, the phosphotyrosine residues within the activated growth factor receptor. Peptides appear to transduce neurons…

Ligand binding was acquired during evolution of nuclear receptors

PubMed Central

Escriva, Hector; Safi, Rachid; Hänni, Catherine; Langlois, Marie-Claire; Saumitou-Laprade, Pierre; Stehelin, Dominique; Capron, André; Pierce, Raymond; Laudet, Vincent

1997-01-01

The nuclear receptor (NR) superfamily comprises, in addition to ligand-activated transcription factors, members for which no ligand has been identified to date. We demonstrate that orphan receptors are randomly distributed in the evolutionary tree and that there is no relationship between the position of a given liganded receptor in the tree and the chemical nature of its ligand. NRs are specific to metazoans, as revealed by a screen of NR-related sequences in early- and non-metazoan organisms. The analysis of the NR gene duplication pattern during the evolution of metazoans shows that the present NR diversity arose from two waves of gene duplications. Strikingly, our results suggest that the ancestral NR was an orphan receptor that acquired ligand-binding ability during subsequent evolution. PMID:9192646

Growth hormone-like factor produced by the tapeworm, Spirometra mansonoides, displaces human growth hormone (hGH) from its receptors on cultured human lymphocytes

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

Watts, D.J.; Phares, C.K.

1986-03-01

An analogue of hGH isolated from plerocercoids of the tapeworm Spirometra mansonoides displaces (/sup 125/I)hGH from its receptors in rabbit, rat, and hamster liver membranes. Biologically, plerocercoid growth factor (PGF) is more similar to hGH than to other mammalian GH's but has not been shown to bond human cells. Receptors specific for hGH have been described on cultured human lymphocytes (IM-9). In this study, the authors compared the binding of PGF and hGH in IM-9 cells and in rabbit hepatic membranes. IM-9 lymphocytes (12 x 10/sup 6/ cells/tube) were incubated with (/sup 125/I)hGH and increasing concentrations of hGH (ng/ml) ormore » PGF (serial dilutions) for 90 min at 30/sup 0/ C. Specific binding (B/sub 0/ - NSB) was determined for each dose of hGH or PGF and the binding curves were analyzed by logit-log regression. The results show that PGF displaced (/sup 125/I)hGH from human cells in a dose dependent manner (r = 0.98). Based on the IM-9 assay, 1 ml of the PGF had an activity equivalent to 625 ng of the hGH standard (ngE). However, the binding activity of the PGF in the rabbit liver RRA was 1653 ngE/ml, indicating that the binding potency of PGF in IM-9 cells was only 38% of that in the rabbit liver. These results clearly demonstrate that PGF binds hGH receptors in cells of human origin, suggesting that PGF will be effective in humans.« less

Agonistic Human Monoclonal Antibodies against Death Receptor 4 (DR4) | NCI Technology Transfer Center | TTC

Cancer.gov

The National Cancer Institute is seeking parties interested in licensing human monoclonal antibodies (mAbs) that bind to death receptor 4 ("DR4"). The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and its functional receptors, DR4 and DR5, have been recognized as promising targets for cancer treatment.

Transcriptional activation of human mu-opioid receptor gene by insulin-like growth factor-I in neuronal cells is modulated by the transcription factor REST.

PubMed

Bedini, Andrea; Baiula, Monica; Spampinato, Santi

2008-06-01

The human mu-opioid receptor gene (OPRM1) promoter contains a DNA sequence binding the repressor element 1 silencing transcription factor (REST) that is implicated in transcriptional repression. We investigated whether insulin-like growth factor I (IGF-I), which affects various aspects of neuronal induction and maturation, regulates OPRM1 transcription in neuronal cells in the context of the potential influence of REST. A series of OPRM1-luciferase promoter/reporter constructs were transfected into two neuronal cell models, neuroblastoma-derived SH-SY5Y cells and PC12 cells. In the former, endogenous levels of human mu-opioid receptor (hMOPr) mRNA were evaluated by real-time PCR. IGF-I up-regulated OPRM1 transcription in: PC12 cells lacking REST, in SH-SY5Y cells transfected with constructs deficient in the REST DNA binding element, or when REST was down-regulated in retinoic acid-differentiated cells. IGF-I activates the signal transducer and activator of transcription-3 signaling pathway and this transcription factor, binding to the signal transducer and activator of transcription-1/3 DNA element located in the promoter, increases OPRM1 transcription. We propose that a reduction in REST is a critical switch enabling IGF-I to up-regulate hMOPr. These findings help clarify how hMOPr expression is regulated in neuronal cells.

Crystal structure of the tyrosine kinase domain of the hepatocyte growth factor receptor c-Met and its complex with the microbial alkaloid K-252a.

PubMed

Schiering, Nikolaus; Knapp, Stefan; Marconi, Marina; Flocco, Maria M; Cui, Jean; Perego, Rita; Rusconi, Luisa; Cristiani, Cinzia

2003-10-28

The protooncogene c-met codes for the hepatocyte growth factor receptor tyrosine kinase. Binding of its ligand, hepatocyte growth factor/scatter factor, stimulates receptor autophosphorylation, which leads to pleiotropic downstream signaling events in epithelial cells, including cell growth, motility, and invasion. These events are mediated by interaction of cytoplasmic effectors, generally through Src homology 2 (SH2) domains, with two phosphotyrosine-containing sequence motifs in the unique C-terminal tail of c-Met (supersite). There is a strong link between aberrant c-Met activity and oncogenesis, which makes this kinase an important cancer drug target. The furanosylated indolocarbazole K-252a belongs to a family of microbial alkaloids that also includes staurosporine. It was recently shown to be a potent inhibitor of c-Met. Here we report the crystal structures of an unphosphorylated c-Met kinase domain harboring a human cancer mutation and its complex with K-252a at 1.8-A resolution. The structure follows the well established architecture of protein kinases. It adopts a unique, inhibitory conformation of the activation loop, a catalytically noncompetent orientation of helix alphaC, and reveals the complete C-terminal docking site. The first SH2-binding motif (1349YVHV) adopts an extended conformation, whereas the second motif (1356YVNV), a binding site for Grb2-SH2, folds as a type II Beta-turn. The intermediate portion of the supersite (1353NATY) assumes a type I Beta-turn conformation as in an Shc-phosphotyrosine binding domain peptide complex. K-252a is bound in the adenosine pocket with an analogous binding mode to those observed in previously reported structures of protein kinases in complex with staurosporine.

Binding of the Ras activator son of sevenless to insulin receptor substrate-1 signaling complexes.

PubMed

Baltensperger, K; Kozma, L M; Cherniack, A D; Klarlund, J K; Chawla, A; Banerjee, U; Czech, M P

1993-06-25

Signal transmission by insulin involves tyrosine phosphorylation of a major insulin receptor substrate (IRS-1) and exchange of Ras-bound guanosine diphosphate for guanosine triphosphate. Proteins containing Src homology 2 and 3 (SH2 and SH3) domains, such as the p85 regulatory subunit of phosphatidylinositol-3 kinase and growth factor receptor-bound protein 2 (GRB2), bind tyrosine phosphate sites on IRS-1 through their SH2 regions. Such complexes in COS cells were found to contain the heterologously expressed putative guanine nucleotide exchange factor encoded by the Drosophila son of sevenless gene (dSos). Thus, GRB2, p85, or other proteins with SH2-SH3 adapter sequences may link Sos proteins to IRS-1 signaling complexes as part of the mechanism by which insulin activates Ras.

EGFR oligomerization organizes kinase-active dimers into competent signalling platforms

PubMed Central

Needham, Sarah R.; Roberts, Selene K.; Arkhipov, Anton; Mysore, Venkatesh P.; Tynan, Christopher J.; Zanetti-Domingues, Laura C.; Kim, Eric T.; Losasso, Valeria; Korovesis, Dimitrios; Hirsch, Michael; Rolfe, Daniel J.; Clarke, David T.; Winn, Martyn D.; Lajevardipour, Alireza; Clayton, Andrew H. A.; Pike, Linda J.; Perani, Michela; Parker, Peter J.; Shan, Yibing; Shaw, David E.; Martin-Fernandez, Marisa L.

2016-01-01

Epidermal growth factor receptor (EGFR) signalling is activated by ligand-induced receptor dimerization. Notably, ligand binding also induces EGFR oligomerization, but the structures and functions of the oligomers are poorly understood. Here, we use fluorophore localization imaging with photobleaching to probe the structure of EGFR oligomers. We find that at physiological epidermal growth factor (EGF) concentrations, EGFR assembles into oligomers, as indicated by pairwise distances of receptor-bound fluorophore-conjugated EGF ligands. The pairwise ligand distances correspond well with the predictions of our structural model of the oligomers constructed from molecular dynamics simulations. The model suggests that oligomerization is mediated extracellularly by unoccupied ligand-binding sites and that oligomerization organizes kinase-active dimers in ways optimal for auto-phosphorylation in trans between neighbouring dimers. We argue that ligand-induced oligomerization is essential to the regulation of EGFR signalling. PMID:27796308

Characterization of the primary interaction between the mating pheromone, alpha-factor, and its receptor in Saccharomyces cerevisiae

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

Raths, S.K.

1987-01-01

Alpha-factor is a peptide of thirteen amino acids which is required for mating between the haploid mating types, a and ..cap alpha.., in Saccharomyces cerevisiae. An analogue of alpha-factor, DHP/sup 8/ DHP/sup 11/ Nle/sup 12/ tridecapeptide, was catalytically reduced in the presence of /sup 3/H gas for production of a radiolabeled pheromone suitable for use in binding studies. Incorporation of tritium resulted in /sup 3/H-alpha-factor with high specific activity, purity, biological activity and long shelf-life. Binding studies revealed that alpha-factor interacts with its receptor via a simple, reversible process which obeys the law of mass action. Association and dissociation kineticsmore » indicate values of 2.92 x 10/sup 6/ M/sup /minus/1/ min/sup -1/ for k/sub 1/ and between 4 and 7 x 10/sup /minus/2/ min/sup /minus/1/ for k/sub /minus/1/. Saturation binding studies reveal an equilibrium dissociation constant equal to 2.32 x 10/sup /minus/8/ M which approximate the kinetically-derived K/sub D/ of 2.12 x 10/sup /minus/8/ M. Scatchard and Hill analyses as well as dissociation behavior in the presence of excess unlabeled ligand indicate alpha-factor interacts with a homogeneous population of binding sites which do not interact and exhibit one affinity for the alpha-factor pheromone.« less

Inhibitory Effects of Polyacetylene Compounds from Panax ginseng on Neurotrophin Receptor-Mediated Hair Growth.

PubMed

Suzuki, Aoi; Matsuura, Daisuke; Kanatani, Hirotoshi; Yano, Shingo; Tsunakawa, Mitsuo; Matsuyama, Shigeru; Shigemori, Hideyuki

2017-01-01

Neurotrophins play an important role in the control of the hair growth cycle. Therefore, neurotrophin receptor antagonists have therapeutic potential for the treatment of hair growth disorders. In this study, we investigated the inhibitory effect of Panax ginseng, a medicinal plant commonly used to treat alopecia, on the binding of neurotrophins to their receptors. In addition, we isolated and characterized the bioactive compounds of P. ginseng extracts. P. ginseng hexane extracts strongly inhibited brain-derived neurotrophic factor (BDNF)-TrkB and β-nerve growth factor (β-NGF)-p75 neurotrophin receptor (p75NTR) binding. Furthermore, we identified the following 6 polyacetylene compounds as the bioactive components in P. ginseng hexane extract: panaxynol (1), panaxydol (2), panaxydol chlorohydrin (3), 1,8-heptadecadiene-4,6-diyne-3,10-diol (4), panaxytriol (5), and dihydropanaxacol (6). In particular, compounds 4, 5, and 6 significantly inhibited BDNF-TrkB binding in a dose-dependent manner. To identify the structural component mediating the inhibitory effect, we investigated the effects of the hydroxyl moiety in these compounds. We found that the inhibitory effect of panaxytriol (5) was strong, whereas the inhibitory effect of Ac-panaxytriol (7) was relatively weak. Our findings suggest that P. ginseng-derived polyacetylenes with a hydroxyl moiety might provide therapeutic benefits to patients with hair growth disorders such as alopecia by inhibiting the binding of neurotrophins to their receptors. Although saponins have been proposed to be the primary mediators of the effects of P. ginseng on hair growth, this study revealed that polyacetylene compounds exert similar effects.

Gab1 Is Required for Cell Cycle Transition, Cell Proliferation, and Transformation Induced by an Oncogenic Met Receptor

PubMed Central

Mood, Kathleen; Saucier, Caroline; Bong, Yong-Sik; Lee, Hyun-Shik; Park, Morag

2006-01-01

We have shown previously that either Grb2- or Shc-mediated signaling from the oncogenic Met receptor Tpr-Met is sufficient to trigger cell cycle progression in Xenopus oocytes. However, direct binding of these adaptors to Tpr-Met is dispensable, implying that another Met binding partner mediates these responses. In this study, we show that overexpression of Grb2-associated binder 1 (Gab1) promotes cell cycle progression when Tpr-Met is expressed at suboptimal levels. This response requires that Gab1 possess an intact Met-binding motif, the pleckstrin homology domain, and the binding sites for phosphatidylinositol 3-kinase and tyrosine phosphatase SHP-2, but not the Grb2 and CrkII/phospholipase Cγ binding sites. Importantly, we establish that Gab1-mediated signals are critical for cell cycle transition promoted by the oncogenic Met and fibroblast growth factor receptors, but not by progesterone, the natural inducer of cell cycle transition in Xenopus oocytes. Moreover, Gab1 is essential for Tpr-Met–mediated morphological transformation and proliferation of fibroblasts. This study provides the first evidence that Gab1 is a key binding partner of the Met receptor for induction of cell cycle progression, proliferation, and oncogenic morphological transformation. This study identifies Gab1 and its associated signaling partners as potential therapeutic targets to impair proliferation or transformation of cancer cells in human malignancies harboring a deregulated Met receptor. PMID:16775003

Expression of receptors for atrial natriuretic peptide on the murine bone marrow-derived stromal cells.

PubMed

Agui, T; Yamada, T; Legros, G; Nakajima, T; Clark, M; Peschel, C; Matsumoto, K

1992-05-01

Atrial natriuretic peptide (ANP) receptors were identified on both murine bone marrow-derived stromal cell lines A-3 and ALC and primary cultured cells using [125I]ANP binding assays and Northern blot analyses. The binding of [125I] ANP to the stromal cells was rapid, saturable, and of high affinity. The dissociation constants between ANP and its receptors on these cells showed no difference among cell types, while maximal binding capacity values were different among cell types. Competitive inhibition of [125I]ANP binding with C-atrial natriuretic factor, specific for ANP clearance receptor (ANPR-C), revealed that most of [125I]ANP-binding sites corresponded to ANPR-C. Northern blotting data corroborated that bone marrow-derived stromal cells expressed ANPR-C. However, in ALC cells, ANP biological receptors (either ANPR-A or ANPR-B), the mol wt of which is approximately 130K, were detected, and cGMP was accumulated after stimulation with ANP. On the other hand, in another stromal cell clone, A-3 cells, the expression of biological receptor was not detected in the affinity cross-linking and competitive inhibition experiments using [125I]ANP. However, A-3 cells accumulated cGMP by responding to ANPR-B-specific ligand, C-type natriuretic peptide. These results suggest that ALC cells equally express ANPR-A and ANPR-B, while A-3 cells express ANPR-B dominantly. Although the physiological roles of these receptors in the bone marrow is still not resolved, ANP is expected to play a role in the regulation of stromal cell functions in bone marrow.

Tyr721 regulates specific binding of the CSF-1 receptor kinase insert to PI 3'-kinase SH2 domains: a model for SH2-mediated receptor-target interactions.

PubMed Central

Reedijk, M; Liu, X; van der Geer, P; Letwin, K; Waterfield, M D; Hunter, T; Pawson, T

1992-01-01

Efficient binding of active phosphatidylinositol (PI) 3'-kinase to the autophosphorylated macrophage colony stimulating factor receptor (CSF-1R) requires the noncatalytic kinase insert (KI) region of the receptor. To test whether this region could function independently to bind PI 3'-kinase, the isolated CSF-1R KI was expressed in Escherichia coli, and was inducibly phosphorylated on tyrosine. The tyrosine phosphorylated form of the CSF-1R KI bound PI 3'-kinase in vitro, whereas the unphosphorylated form had no binding activity. The p85 alpha subunit of PI 3'-kinase contains two Src homology (SH)2 domains, which are implicated in the interactions of signalling proteins with activated receptors. Bacterially expressed p85 alpha SH2 domains complexed in vitro with the tyrosine phosphorylated CSF-1R KI. Binding of the CSF-1R KI to PI 3'-kinase activity, and to the p85 alpha SH2 domains, required phosphorylation of Tyr721 within the KI domain, but was independent of phosphorylation at Tyr697 and Tyr706. Tyr721 was also critical for the association of activated CSF-1R with PI 3'-kinase in mammalian cells. Complex formation between the CSF-1R and PI 3'-kinase can therefore be reconstructed in vitro in a specific interaction involving the phosphorylated receptor KI and the SH2 domains of p85 alpha. Images PMID:1314163

Solution structure of the chick TGFbeta type II receptor ligand-binding domain.

PubMed

Marlow, Michael S; Brown, Christopher B; Barnett, Joey V; Krezel, Andrzej M

2003-02-28

The transforming growth factor beta (TGFbeta) signaling pathway influences cell proliferation, immune responses, and extracellular matrix reorganization throughout the vertebrate life cycle. The signaling cascade is initiated by ligand-binding to its cognate type II receptor. Here, we present the structure of the chick type II TGFbeta receptor determined by solution NMR methods. Distance and angular constraints were derived from 15N and 13C edited NMR experiments. Torsion angle dynamics was used throughout the structure calculations and refinement. The 20 final structures were energy minimized using the generalized Born solvent model. For these 20 structures, the average backbone root-mean-square distance from the average structure is below 0.6A. The overall fold of this 109-residue domain is conserved within the superfamily of these receptors. Chick receptors fully recognize and respond to human TGFbeta ligands despite only 60% identity at the sequence level. Comparison with the human TGFbeta receptor determined by X-ray crystallography reveals different conformations in several regions. Sequence divergence and crystal packing interactions under low pH conditions are likely causes. This solution structure identifies regions were structural changes, however subtle, may occur upon ligand-binding. We also identified two very well conserved molecular surfaces. One was found to bind ligand in the crystallized human TGFbeta3:TGFbeta type II receptor complex. The other, newly identified area can be the interaction site with type I and/or type III receptors of the TGFbeta signaling complex.

Identification of a human erythrocyte receptor for colonization factor antigen I pili expressed by H10407 enterotoxigenic Escherichia coli.

PubMed Central

Pieroni, P; Worobec, E A; Paranchych, W; Armstrong, G D

1988-01-01

We have identified a receptor for colonization factor antigen I (CFA/I) pili in human erythrocyte membranes. Erythrocyte binding assays, using whole organisms, suggested that the CFA/I receptor was a glycoprotein containing important sialic acid moieties. Subsequently, human erythrocyte membranes were extracted with lithium diiodosalicylate to obtain a soluble glycoprotein fraction from which to isolate receptors. The extracted material caused agglutination of the CFA/I+ but not the CFA/I- organisms at a protein concentration of 0.5 mg/ml. The CFA/I receptor was identified in iodinated extract by an affinity isolation procedure, using whole bacterial cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of the washed, extract-coated H10407 CFA/I+ organisms revealed a band with an apparent molecular weight of 26,000 which was present in the original extract but was not observed on extract-coated H10407 CFA/I- bacteria. The addition of purified CFA/I pili reduced binding of the 26,000-molecular-weight receptor to CFA/I+ bacteria. The CFA/I-specific receptor species also bound to wheat germ agglutinin-agarose. This observation supported the suggestion that the CFA/I receptor identified in this report is a sialoglycoprotein. Images PMID:2895745

The effects of abnormalities of glucose homeostasis on the expression and binding of muscarinic receptors in cerebral cortex of rats.

PubMed

Sherin, Antony; Peeyush, Kumar T; Naijil, George; Nandhu, Mohan Sobhana; Jayanarayanan, Sadanandan; Jes, Paul; Paulose, Cheramadathikudiyil Skaria

2011-01-25

Glucose homeostasis in humans is an important factor for the functioning of nervous system. Both hypo and hyperglycemia contributes to neuronal functional deficit. In the present study, effect of insulin induced hypoglycemia and streptozotocin induced diabetes on muscarinic receptor binding, cholinergic enzymes; AChE, ChAT expression and GLUT3 in the cerebral cortex of experimental rats were analysed. Total muscarinic, muscarinic M(1) receptor showed a significant decrease and muscarinic M(3) receptor subtype showed a significant increased binding in the cerebral cortex of hypoglycemic rats compared to diabetic and control. Real-Time PCR analysis of muscarinic M(1), M(3) receptor subtypes confirmed the receptor binding studies. Immunohistochemistry of muscarinic M(1), M(3) receptors using specific antibodies were also carried out. AChE and GLUT3 expression up regulated and ChAT expression down regulated in hypoglycemic rats compared to diabetic and control rats. Our results showed that hypo/hyperglycemia caused impaired glucose transport in neuronal cells as shown by altered expression of GLUT3. Increased AChE and decreased ChAT expression is suggested to alter cortical acetylcholine metabolism in experimental rats along with altered muscarinic receptor binding in hypo/hyperglycemic rats, impair cholinergic transmission, which subsequently lead to cholinergic dysfunction thereby causing learning and memory deficits. We observed a prominent cholinergic functional disturbance in hypoglycemic condition than in hyperglycemia. Hypoglycemia exacerbated the neurochemical changes in cerebral cortex induced by hyperglycemia. These findings have implications for both therapy and identification of causes contributing to neuronal dysfunction in diabetes. Copyright © 2010 Elsevier B.V. All rights reserved.

High-mobility group (HMG) protein HMG-1 and TATA-binding protein-associated factor TAF(II)30 affect estrogen receptor-mediated transcriptional activation.

PubMed

Verrier, C S; Roodi, N; Yee, C J; Bailey, L R; Jensen, R A; Bustin, M; Parl, F F

1997-07-01

The estrogen receptor (ER) belongs to a family of ligand-inducible nuclear receptors that exert their effects by binding to cis-acting DNA elements in the regulatory region of target genes. The detailed mechanisms by which ER interacts with the estrogen response element (ERE) and affects transcription still remain to be elucidated. To study the ER-ERE interaction and transcription initiation, we employed purified recombinant ER expressed in both the baculovirus-Sf9 and his-tagged bacterial systems. The effect of high-mobility group (HMG) protein HMG-1 and purified recombinant TATA-binding protein-associated factor TAF(II)30 on ER-ERE binding and transcription initiation were assessed by electrophoretic mobility shift assay and in vitro transcription from an ERE-containing template (pERE2LovTATA), respectively. We find that purified, recombinant ER fails to bind to ERE in spite of high ligand-binding activity and electrophoretic and immunological properties identical to ER in MCF-7 breast cancer cells. HMG-1 interacts with ER and promotes ER-ERE binding in a concentration- and time-dependent manner. The effectiveness of HMG-1 to stimulate ER-ERE binding in the electrophoretic mobility shift assay depends on the sequence flanking the ERE consensus as well as the position of the latter in the oligonucleotide. We find that TAF(II)30 has no effect on ER-ERE binding either alone or in combination with ER and HMG-1. Although HMG-1 promotes ER-ERE binding, it fails to stimulate transcription initiation either in the presence or absence of hormone. In contrast, TAF(II)30, while not affecting ER-ERE binding, stimulates transcription initiation 20-fold in the presence of HMG-1. These results indicate that HMG-1 and TAF(II)30 act in sequence, the former acting to promote ER-ERE binding followed by the latter to stimulate transcription initiation.

Blue News Update: BODIPY-GTP Binds to the Blue-Light Receptor YtvA While GTP Does Not

PubMed Central

Schmieder, Peter

2012-01-01

Light is an important environmental factor for almost all organisms. It is mainly used as an energy source but it is also a key factor for the regulation of multiple cellular functions. Light as the extracellular stimulus is thereby converted into an intracellular signal by photoreceptors that act as signal transducers. The blue-light receptor YtvA, a bacterial counterpart of plant phototropins, is involved in the stress response of Bacillus subtilis. The mechanism behind its activation, however, remains unknown. It was suggested based on fluorescence spectroscopic studies that YtvA function involves GTP binding and that this interaction is altered by absorption of light. We have investigated this interaction by several biophysical methods and show here using fluorescence spectroscopy, ITC titrations, and three NMR spectroscopic assays that while YtvA interacts with BODIPY-GTP as a fluorescent GTP analogue originally used for the detection of GTP binding, it does not bind GTP. PMID:22247770

AOP description: Androgen receptor agonism leading to reproductive dysfunction (in fish)

EPA Science Inventory

This adverse outcome pathway details the linkage between binding and activation of androgen receptor as a nuclear transcription factor in females and the adverse effect of reduced cumulative fecundity in repeat-spawning fish species. Cumulative fecundity is the most apical endpoi...

Residues remote from the binding pocket control the antagonist selectivity towards the corticotropin-releasing factor receptor-1

PubMed Central

Sun, Xianqiang; Cheng, Jianxin; Wang, Xu; Tang, Yun; Ågren, Hans; Tu, Yaoquan

2015-01-01

The corticotropin releasing factors receptor-1 and receptor-2 (CRF1R and CRF2R) are therapeutic targets for treating neurological diseases. Antagonists targeting CRF1R have been developed for the potential treatment of anxiety disorders and alcohol addiction. It has been found that antagonists targeting CRF1R always show high selectivity, although CRF1R and CRF2R share a very high rate of sequence identity. This has inspired us to study the origin of the selectivity of the antagonists. We have therefore built a homology model for CRF2R and carried out unbiased molecular dynamics and well-tempered metadynamics simulations for systems with the antagonist CP-376395 in CRF1R or CRF2R to address this issue. We found that the side chain of Tyr6.63 forms a hydrogen bond with the residue remote from the binding pocket, which allows Tyr6.63 to adopt different conformations in the two receptors and results in the presence or absence of a bottleneck controlling the antagonist binding to or dissociation from the receptors. The rotameric switch of the side chain of Tyr3566.63 allows the breaking down of the bottleneck and is a perquisite for the dissociation of CP-376395 from CRF1R. PMID:25628267

Computer-aided structure-affinity relationships in a set of piperazine and 3,8-diazabicyclo[3.2.1]octane derivatives binding to the μ-opioid receptor

NASA Astrophysics Data System (ADS)

Barlocco, Daniela; Cignarella, Giorgio; Greco, Giovanni; Novellino, Ettore

1993-10-01

Molecular modeling studies were carried out on a set of piperazine and 3,8-diazabicyclo[3.2.1]octane derivatives with the aim to highlight the main factors modulating their affinity for the μ-opioid receptor. Structure-affinity relationships were developed with the aid of molecular mechanics and semiempirical quantum-mechanics methods. According to our proposed pharmacodynamic model, the binding to the μ-receptor is promoted by the following physico-chemical features: the presence of hydrocarbon fragments on the nitrogen ring frame capable of interacting with one of two hypothesized hydrophobic receptor pockets; a `correct' orientation of an N-propionyl side chain so as to avoid a sterically hindered region of the receptor; the possibility of accepting a hydrogen bond from a receptor site complementary to the morphine phenol oxygen.

Platelet-derived growth factor receptor mediates activation of ras through different signaling pathways in different cell types.

PubMed Central

Satoh, T; Fantl, W J; Escobedo, J A; Williams, L T; Kaziro, Y

1993-01-01

A series of pieces of evidence have shown that Ras protein acts as a transducer of the platelet-derived growth factor (PDGF) receptor-mediated signaling pathway: (i) formation of Ras.GTP is detected immediately on PDGF stimulation, and (ii) a dominant inhibitory mutant Ras, as well as a neutralizing anti-Ras antibody, can interfere with PDGF-induced responses. On the other hand, several signal transducing molecules including phosphatidylinositol 3-kinase (PI3-K), GTPase-activating protein (GAP), and phospholipase C gamma (PLC gamma) bind directly to the PDGF receptor and become tyrosine phosphorylated. Recently, it was shown that specific phosphorylated tyrosines of the PDGF receptor are responsible for interaction between the receptor and each signaling molecule. However, the roles of these signaling molecules have not been elucidated, and it remains unclear which molecules are implicated in the Ras pathway. In this study, we measured Ras activation in cell lines expressing mutant PDGF receptors that are deficient in coupling with specific molecules. In fibroblast CHO cells, a mutant receptor (Y708F/Y719F [PI3-K-binding sites]) was unable to stimulate Ras, whereas another mutant (Y739F [the GAP-binding site]) could do so, suggesting an indispensable role of PI3-K or a protein that binds to the same sites as PI3-K for PDGF-stimulated Ras activation. By contrast, both of the above mutants were capable of stimulating Ras protein in a pro-B-cell line, BaF3. Furthermore, a mutant receptor (Y977F/Y989F [PLC gamma-binding sites]) could fully activate Ras, and the direct activation of protein kinase C and calcium mobilization had almost no effect on the GDP/GTP state of Ras in this cell line. These results suggest that, in the pro-B-cell transfectants, each of the above pathways (PI3-K, GAP, and PLC gamma) can be eliminated without a loss of Ras activation. It remains unclear whether another unknown essential pathway which regulates Ras protein exists within BaF3 cells. Therefore, it is likely that several different PDGF receptor-mediated signaling pathways function upstream of Ras, and the extent of the contribution of each pathway for the regulation of Ras may differ among different cell types. Images PMID:8388543

IL-17s adopt a cystine knot fold: structure and activity of a novel cytokine, IL-17F, and implications for receptor binding

PubMed Central

Hymowitz, Sarah G.; Filvaroff, Ellen H.; Yin, JianPing; Lee, James; Cai, Liping; Risser, Philip; Maruoka, Miko; Mao, Weiguang; Foster, Jessica; Kelley, Robert F.; Pan, Guohua; Gurney, Austin L.; de Vos, Abraham M.; Starovasnik, Melissa A.

2001-01-01

The proinflammatory cytokine interleukin 17 (IL-17) is the founding member of a family of secreted proteins that elicit potent cellular responses. We report a novel human IL-17 homolog, IL-17F, and show that it is expressed by activated T cells, can stimulate production of other cytokines such as IL-6, IL-8 and granulocyte colony-stimulating factor, and can regulate cartilage matrix turnover. Unexpectedly, the crystal structure of IL-17F reveals that IL-17 family members adopt a monomer fold typical of cystine knot growth factors, despite lacking the disulfide responsible for defining the canonical ‘knot’ structure. IL-17F dimerizes in a parallel manner like neurotrophins, and features an unusually large cavity on its surface. Remarkably, this cavity is located in precisely the same position where nerve growth factor binds its high affinity receptor, TrkA, suggesting further parallels between IL-17s and neurotrophins with respect to receptor recognition. PMID:11574464

Surveying GPCR solubilisation conditions using surface plasmon resonance.

PubMed

Navratilova, Iva Hopkins; Aristotelous, Tonia; Bird, Louise E; Hopkins, Andrew L

2018-06-15

Biophysical screening techniques, such as surface plasmon resonance, enable detailed kinetic analysis of ligands binding to solubilised G-protein coupled receptors. The activity of a receptor solubilised out of the membrane is crucially dependent on the environment in which it is suspended. Finding the right conditions is challenging due to the number of variables to investigate in order to determine the optimum solubilisation buffer for any given receptor. In this study we used surface plasmon resonance technology to screen a variety of solubilisation conditions including buffers and detergents for two model receptors: CXCR4 and CCR5. We tested 950 different combinations of solubilisation conditions for both receptors. The activity of both receptors was monitored by using conformation dependent monoclonal antibodies and the binding of small molecule ligands. Despite both receptors belonging to the chemokine receptor family they show some differences in their preference for solubilisation conditions that provide the highest level of binding for both the conformation dependent antibodies and small molecules. The study described here is focused not only on finding the best solubilisation conditions for each receptor, but also on factors that determine the sensitivity of the assay for each receptor. We also suggest how these data about different buffers and detergents can be used as a guide for selecting solubilisation conditions for other membrane proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

Steroid hormone and epidermal growth factor receptors in meningiomas.

PubMed

Horsfall, D J; Goldsmith, K G; Ricciardelli, C; Skinner, J M; Tilley, W D; Marshall, V R

1989-11-01

A prospective study of steroid hormone and epidermal growth factor receptor expression in 57 meningiomas is presented. Scatchard analysis of radioligand binding identified 20% of meningiomas as expressing classical oestrogen receptors (ER) at levels below that normally accepted for positivity, the remainder being negative. ER could not be visualized in any meningioma using immunocytochemistry. Alternatively, 74% of meningiomas demonstrated the presence of progesterone receptors (PR) by Scatchard analysis, the specificity of which could not be attributed to glucocorticoid or androgen receptors. Confirmation of classical PR presence was determined by immunocytochemical staining. The presence of epidermal growth factor receptor (EGFR) was demonstrated in 100% of meningiomas using immunocytochemical staining. These data are reviewed in the context of previously reported results and are discussed in relation to the potential for medical therapy as an adjunct to surgery.

Insight on Mutation-Induced Resistance from Molecular Dynamics Simulations of the Native and Mutated CSF-1R and KIT.

PubMed

Da Silva Figueiredo Celestino Gomes, Priscila; Chauvot De Beauchêne, Isaure; Panel, Nicolas; Lopez, Sophie; De Sepulveda, Paulo; Geraldo Pascutti, Pedro; Solary, Eric; Tchertanov, Luba

2016-01-01

The receptors tyrosine kinases (RTKs) for the colony stimulating factor-1, CSF-1R, and for the stem cell factor, SCFR or KIT, are important mediators of signal transduction. The abnormal function of these receptors, promoted by gain-of-function mutations, leads to their constitutive activation, associated with cancer or other proliferative diseases. A secondary effect of the mutations is the alteration of receptors' sensitivity to tyrosine kinase inhibitors, compromising effectiveness of these molecules in clinical treatment. In particular, the mutation V560G in KIT increases its sensitivity to Imatinib, while the D816V in KIT, and D802V in CSF-1R, triggers resistance to the drug. We analyzed the Imatinib binding affinity to the native and mutated KIT (mutations V560G, S628N and D816V) and CSF-1R (mutation D802V) by using molecular dynamics simulations and energy calculations of Imatinib•target complexes. Further, we evaluated the sensitivity of the studied KIT receptors to Imatinib by measuring the inhibition of KIT phosphorylation. Our study showed that (i) the binding free energy of Imatinib to the targets is highly correlated with their experimentally measured sensitivity; (ii) the electrostatic interactions are a decisive factor affecting the binding energy; (iii) the most deleterious impact to the Imatinib sensitivity is promoted by D802V (CSF-1R) and D816V (KIT) mutations; (iv) the role of the juxtamembrane region, JMR, in the imatinib binding is accessory. These findings contribute to a better description of the mutation-induced effects alternating the targets sensitivity to Imatinib.

Functional properties of an isolated. cap alpha beta. heterodimeric human placenta insulin-like growth factor 1 receptor complex

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

Feltz, S.M.; Swanson, M.L.; Wemmie, J.A.

1988-05-03

Treatment of human placenta membranes at pH 8.5 in the presence of 2.0 mM dithiothreitol (DTT) for 5 min, followed by the simultaneous removal of the DTT and pH adjustment of pH 7.6, resulted in the formation of a functional ..cap alpha beta.. heterodimeric insulin-like growth factor 1 (IGF-1) receptor complex from the native ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric disulfide-linked state. The membrane-bound ..cap alpha beta.. heterodimeric complex displayed similar curvilinear /sup 125/I-IGF-1 equilibrium binding compared to the ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric complex. /sup 125/I-IGF-1 binding to both the isolated ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric and ..cap alpha beta..more » heterodimeric complexes demonstrated a marked straightening of the Scatchard plots, compared to the placenta membrane-bound IGF-1 receptors, with a 2-fold increase in the high-affinity binding component. IGF-1 stimulation of IGF-1 receptor autophosphorylation indicated that the ligand-dependent activation of ..cap alpha beta.. heterodimeric protein kinase activity occurred concomitant with the reassociation into a covalent ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric state. These data demonstrate that (i) a combination of alkaline pH and DTT treatment of human placenta membranes results in the formation of an ..cap alpha beta.. heterodimeric IGF-1 receptor complex, (ii) unlike the insulin receptor, high-affinity homogeneous IGF-1 binding occurs in both the ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric and ..cap alpha beta.. heterodimeric complexes, and (iii) IGF-1-dependent autophosphorylation of the ..cap alpha beta.. heterodimeric IGF-1 receptor complex correlates wit an IGF-1 dependent covalent reassociation into an ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric disulfide-linked state.« less

HEPARIN-BINDING EGF CLEAVAGE MEDIATES ZINC-INDUCED EGF RECEPTOR PHOSPHORYLATION

EPA Science Inventory

We have previously shown that exposure to zinc ions can activate epidermal growth factor (EGF) receptor (EGFR) signaling in murine fibroblasts and A431 cells through a mechanism involving Src kinase. While studying the effects of zinc ions in normal human bronchial epithelial cel...

The transcriptional co-factor RIP140 regulates mammary gland development by promoting the generation of key mitogenic signals.

PubMed

Nautiyal, Jaya; Steel, Jennifer H; Mane, Meritxell Rosell; Oduwole, Olayiwola; Poliandri, Ariel; Alexi, Xanthippi; Wood, Nicholas; Poutanen, Matti; Zwart, Wilbert; Stingl, John; Parker, Malcolm G

2013-03-01

Nuclear receptor interacting protein (Nrip1), also known as RIP140, is a co-regulator for nuclear receptors that plays an essential role in ovulation by regulating the expression of the epidermal growth factor-like family of growth factors. Although several studies indicate a role for RIP140 in breast cancer, its role in the development of the mammary gland is unclear. By using RIP140-null and RIP140 transgenic mice, we demonstrate that RIP140 is an essential factor for normal mammary gland development and that it functions by mediating oestrogen signalling. RIP140-null mice exhibit minimal ductal elongation with no side-branching, whereas RIP140-overexpressing mice show increased cell proliferation and ductal branching with age. Tissue recombination experiments demonstrate that RIP140 expression is required in both the mammary epithelial and stromal compartments for ductal elongation during puberty and that loss of RIP140 leads to a catastrophic loss of the mammary epithelium, whereas RIP140 overexpression augments the mammary basal cell population and shifts the progenitor/differentiated cell balance within the luminal cell compartment towards the progenitors. For the first time, we present a genome-wide global view of oestrogen receptor-α (ERα) binding events in the developing mammary gland, which unravels 881 ERα binding sites. Unbiased evaluation of several ERα binding sites for RIP140 co-occupancy reveals selectivity and demonstrates that RIP140 acts as a co-regulator with ERα to regulate directly the expression of amphiregulin (Areg), the progesterone receptor (Pgr) and signal transducer and activator of transcription 5a (Stat5a), factors that influence key mitogenic pathways that regulate normal mammary gland development.

Identification of the functional interleukin-22 (IL-22) receptor complex: the IL-10R2 chain (IL-10Rbeta ) is a common chain of both the IL-10 and IL-22 (IL-10-related T cell-derived inducible factor, IL-TIF) receptor complexes.

PubMed

Kotenko, S V; Izotova, L S; Mirochnitchenko, O V; Esterova, E; Dickensheets, H; Donnelly, R P; Pestka, S

2001-01-26

Interleukin-10 (IL-10)-related T cell-derived inducible factor (IL-TIF; provisionally designated IL-22) is a cytokine with limited homology to IL-10. We report here the identification of a functional IL-TIF receptor complex that consists of two receptor chains, the orphan CRF2-9 and IL-10R2, the second chain of the IL-10 receptor complex. Expression of the CRF2-9 chain in monkey COS cells renders them sensitive to IL-TIF. However, in hamster cells both chains, CRF2-9 and IL-10R2, must be expressed to assemble the functional IL-TIF receptor complex. The CRF2-9 chain (or the IL-TIF-R1 chain) is responsible for Stat recruitment. Substitution of the CRF2-9 intracellular domain with the IFN-gammaR1 intracellular domain changes the pattern of IL-TIF-induced Stat activation. The CRF2-9 gene is expressed in normal liver and kidney, suggesting a possible role for IL-TIF in regulating gene expression in these tissues. Each chain, CRF2-9 and IL-10R2, is capable of binding IL-TIF independently and can be cross-linked to the radiolabeled IL-TIF. However, binding of IL-TIF to the receptor complex is greater than binding to either receptor chain alone. Sharing of the common IL-10R2 chain between the IL-10 and IL-TIF receptor complexes is the first such case for receptor complexes with chains belonging to the class II cytokine receptor family, establishing a novel paradigm for IL-10-related ligands similar to the shared use of the gamma common chain (gamma(c)) by several cytokines, including IL-2, IL-4, IL-7, IL-9, and IL-15.

Mutagenesis of Paramyxovirus Hemagglutinin-Neuraminidase Membrane-Proximal Stalk Region Influences Stability, Receptor Binding, and Neuraminidase Activity.

PubMed

Adu-Gyamfi, Emmanuel; Kim, Lori S; Jardetzky, Theodore S; Lamb, Robert A

2016-09-01

Paramyxoviridae consist of a large family of enveloped, negative-sense, nonsegmented single-stranded RNA viruses that account for a significant number of human and animal diseases. The fusion process for nearly all paramyxoviruses involves the mixing of the host cell plasma membrane and the virus envelope in a pH-independent fashion. Fusion is orchestrated via the concerted action of two surface glycoproteins: an attachment protein called hemagglutinin-neuraminidase (HN [also called H or G depending on virus type and substrate]), which acts as a receptor binding protein, and a fusion (F) protein, which undergoes a major irreversible refolding process to merge the two membranes. Recent biochemical evidence suggests that receptor binding by HN is dispensable for cell-cell fusion. However, factors that influence the stability and/or conformation of the HN 4-helix bundle (4HB) stalk have not been studied. Here, we used oxidative cross-linking as well as functional assays to investigate the role of the structurally unresolved membrane-proximal stalk region (MPSR) (residues 37 to 58) of HN in the context of headless and full-length HN membrane fusion promotion. Our data suggest that the receptor binding head serves to stabilize the stalk to regulate fusion. Moreover, we found that the MPSR of HN modulates receptor binding and neuraminidase activity without a corresponding regulation of F triggering. Paramyxoviruses require two viral membrane glycoproteins, the attachment protein variously called HN, H, or G and the fusion protein (F), to couple host receptor recognition to virus-cell fusion. The HN protein has a globular head that is attached to a membrane-anchored flexible stalk of ∼80 residues and has three activities: receptor binding, neuraminidase, and fusion activation. In this report, we have identified the functional significance of the membrane-proximal stalk region (MPSR) (HN, residues 37 to 56) of the paramyxovirus parainfluenza virus (PIV5), a region of the HN stalk that has not had its structure determined by X-ray crystallography. Our data suggest that the MPSR influences receptor binding and neuraminidase activity via an indirect mechanism. Moreover, the receptor binding head group stabilizes the 4HB stalk as part of the general mechanism to fine-tune F-activation. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Phosphorylated Nuclear Receptor CAR Forms a Homodimer To Repress Its Constitutive Activity for Ligand Activation

PubMed Central

Shizu, Ryota; Osabe, Makoto; Perera, Lalith; Moore, Rick; Sueyoshi, Tatsuya

2017-01-01

ABSTRACT The nuclear receptor CAR (NR1I3) regulates hepatic drug and energy metabolism as well as cell fate. Its activation can be a critical factor in drug-induced toxicity and the development of diseases, including diabetes and tumors. CAR inactivates its constitutive activity by phosphorylation at threonine 38. Utilizing receptor for protein kinase 1 (RACK1) as the regulatory subunit, protein phosphatase 2A (PP2A) dephosphorylates threonine 38 to activate CAR. Here we demonstrate that CAR undergoes homodimer-monomer conversion to regulate this dephosphorylation. By coexpression of two differently tagged CAR proteins in Huh-7 cells, mouse primary hepatocytes, and mouse livers, coimmunoprecipitation and two-dimensional gel electrophoresis revealed that CAR can form a homodimer in a configuration in which the PP2A/RACK1 binding site is buried within its dimer interface. Epidermal growth factor (EGF) was found to stimulate CAR homodimerization, thus constraining CAR in its inactive form. The agonistic ligand CITCO binds directly to the CAR homodimer and dissociates phosphorylated CAR into its monomers, exposing the PP2A/RACK1 binding site for dephosphorylation. Phenobarbital, which is not a CAR ligand, binds the EGF receptor, reversing the EGF signal to monomerize CAR for its indirect activation. Thus, the homodimer-monomer conversion is the underlying molecular mechanism that regulates CAR activation, by placing phosphorylated threonine 38 as the common target for both direct and indirect activation of CAR. PMID:28265001

Artemin Crystal Structure Reveals Insights into Heparan Sulfate Binding

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

Silvian,L.; Jin, P.; Carmillo, P.

2006-01-01

Artemin (ART) promotes the growth of developing peripheral neurons by signaling through a multicomponent receptor complex comprised of a transmembrane tyrosine kinase receptor (cRET) and a specific glycosylphosphatidylinositol-linked co-receptor (GFR{alpha}3). Glial cell line-derived neurotrophic factor (GDNF) signals through a similar ternary complex but requires heparan sulfate proteoglycans (HSPGs) for full activity. HSPG has not been demonstrated as a requirement for ART signaling. We crystallized ART in the presence of sulfate and solved its structure by isomorphous replacement. The structure reveals ordered sulfate anions bound to arginine residues in the pre-helix and amino-terminal regions that were organized in a triad arrangementmore » characteristic of heparan sulfate. Three residues in the pre-helix were singly or triply substituted with glutamic acid, and the resulting proteins were shown to have reduced heparin-binding affinity that is partly reflected in their ability to activate cRET. This study suggests that ART binds HSPGs and identifies residues that may be involved in HSPG binding.« less

The effect of thyroid hormone and a long-acting somatostatin analogue on TtT-97 murine thyrotropic tumors.

PubMed

Woodmansee, W W; Gordon, D F; Dowding, J M; Stolz, B; Lloyd, R V; James, R A; Wood, W M; Ridgway, E C

2000-07-01

Thyroid hormone inhibits thyrotropin (TSH) production and thyrotrope growth. Somatostatin has been implicated as a synergistic factor in the inhibition of thyrotrope function. We have previously shown that pharmacological doses of thyroid hormone (levothyroxine [LT4]) inhibit growth of murine TtT-97 thyrotropic tumors in association with upregulation of somatostatin receptor type 5 (sst5) mRNA and somatostatin receptor binding. In the current study, we examined the effect of physiological thyroid hormone replacement alone or in combination with the long-acting somatostatin analogue, Sandostatin LAR, on thyrotropic tumor growth, thyrotropin growth factor-beta (TSH-beta), and sst5 mRNA expression, as well as somatostatin receptor binding sites. Physiological LT4 replacement therapy resulted in tumor shrinkage in association with increased sst5 mRNA levels, reduced TSH-beta mRNA levels and enhanced somatostatin receptor binding. Sandostatin LAR alone had no effect on any parameter measured. However, Sandostatin LAR combined with LT4 synergistically inhibited TSH-beta mRNA production and reduced final tumor weights to a greater degree. In this paradigm, Sandostatin LAR required a euthyroid status to alter thyrotrope parameters. These data suggest an important interaction between the somatostatinergic system and thyroid hormone in the regulation of thyrotrope cell structure and function.

Tyrosine dephosphorylation enhances the therapeutic target activity of epidermal growth factor receptor (EGFR) by disrupting its interaction with estrogen receptor (ER).

PubMed

Ma, Shao; Yin, Ning; Qi, Xiaomei; Pfister, Sandra L; Zhang, Mei-Jie; Ma, Rong; Chen, Guan

2015-05-30

Protein-protein interactions can increase or decrease its therapeutic target activity and the determining factors involved, however, are largely unknown. Here, we report that tyrosine-dephosphorylation of epidermal growth factor receptor (EGFR) increases its therapeutic target activity by disrupting its interaction with estrogen receptor (ER). Protein tyrosine phosphatase H1 (PTPH1) dephosphorylates the tyrosine kinase EGFR, disrupts its interaction with the nuclear receptor ER, and increases breast cancer sensitivity to small molecule tyrosine kinase inhibitors (TKIs). These effects require PTPH1 catalytic activity and its interaction with EGFR, suggesting that the phosphatase may increase the sensitivity by dephosphorylating EGFR leading to its dissociation with ER. Consistent with this notion, a nuclear-localization defective ER has a higher EGFR-binding activity and confers the resistance to TKI-induced growth inhibition. Additional analysis show that PTPH1 stabilizes EGFR, stimulates the membranous EGFR accumulation, and enhances the growth-inhibitory activity of a combination therapy of TKIs with an anti-estrogen. Since EGFR and ER both are substrates for PTPH1 in vitro and in intact cells, these results indicate that an inhibitory EGFR-ER protein complex can be switched off through a competitive enzyme-substrate binding. Our results would have important implications for the treatment of breast cancer with targeted therapeutics.

Measles virus fusion machinery activated by sialic acid binding globular domain.

PubMed

Talekar, Aparna; Moscona, Anne; Porotto, Matteo

2013-12-01

Paramyxoviruses, including the human pathogen measles virus (MV) and the avian Newcastle disease virus (NDV), enter host cells through fusion of the viral envelope with the target cell membrane. This fusion is driven by the concerted action of two viral envelope glycoproteins: the receptor binding protein and the fusion protein (F). The MV receptor binding protein (hemagglutinin [H]) attaches to proteinaceous receptors on host cells, while the receptor binding protein of NDV (hemagglutinin-neuraminidase [HN]) interacts with sialic acid-containing receptors. The receptor-bound HN/H triggers F to undergo conformational changes that render it competent to mediate fusion of the viral and cellular membranes. The mechanism of fusion activation has been proposed to be different for sialic acid-binding viruses and proteinaceous receptor-binding viruses. We report that a chimeric protein containing the NDV HN receptor binding region and the MV H stalk domain can activate MV F to fuse, suggesting that the signal to the stalk of a protein-binding receptor binding molecule can be transmitted from a sialic acid binding domain. By engineering the NDV HN globular domain to interact with a proteinaceous receptor, the fusion activation signal was preserved. Our findings are consistent with a unified mechanism of fusion activation, at least for the Paramyxovirinae subfamily, in which the receptor binding domains of the receptor binding proteins are interchangeable and the stalk determines the specificity of F activation.

Yttrium-90 and indium-111 labelling, receptor binding and biodistribution of [DOTA0,d-Phe1,Tyr3]octreotide, a promising somatostatin analogue for radionuclide therapy.

PubMed

de Jong, M; Bakker, W H; Krenning, E P; Breeman, W A; van der Pluijm, M E; Bernard, B F; Visser, T J; Jermann, E; Béhé, M; Powell, P; Mäcke, H R

1997-04-01

In vitro octreotide receptor binding of [111In-DOTA0,d-Phe1, Tyr3]octreotide (111In-DOTATOC) and the in vivo metabolism of 90Y- or 111In-labelled DOTATOC were investigated in rats in comparison with [111In-DTPA0]octreotide [111In-DTPAOC). 111In-DOTATOC was found to have an affinity similar to octreotide itself for the octreotide receptor in rat cerebral cortex microsomes. Twenty-four hours after injection of 90Y- or 111In-labelled DOTATOC, uptake of radioactivity in the octreotide receptor-expressing tissues pancreas, pituitary, adrenals and tumour was a factor of 2-6 that after injection of 111In-DTPAOC. Uptake of labelled DOTATOC in pituitary, pancreas, adrenals and tumour was almost completely blocked by pretreatment with 0.5 mg unlabelled octreotide, indicating specific binding to the octreotide receptors. These findings strongly indicate that 90Y-DOTATOC is a promising radiopharmaceutical for radiotherapy and that 111In-DOTATOC is of potential value for diagnosis of patients with octreotide receptor-positive lesions, such as most neuroendocrine tumours.

Regulatory Features for Odorant Receptor Genes in the Mouse Genome.

PubMed

Degl'Innocenti, Andrea; D'Errico, Anna

2017-01-01

The odorant receptor genes, seven transmembrane receptor genes constituting the vastest mammalian gene multifamily, are expressed monogenically and monoallelicaly in each sensory neuron in the olfactory epithelium. This characteristic, often referred to as the one neuron-one receptor rule, is driven by mostly uncharacterized molecular dynamics, generally named odorant receptor gene choice . Much attention has been paid by the scientific community to the identification of sequences regulating the expression of odorant receptor genes within their loci , where related genes are usually arranged in genomic clusters. A number of studies identified transcription factor binding sites on odorant receptor promoter sequences. Similar binding sites were also found on a number of enhancers that regulate in cis their transcription, but have been proposed to form interchromosomal networks. Odorant receptor gene choice seems to occur via the local removal of strongly repressive epigenetic markings, put in place during the maturation of the sensory neuron on each odorant receptor locus . Here we review the fast-changing state of art for the study of regulatory features for odorant receptor genes.

Molecular mechanisms of immunosuppression by cyclosporins.

PubMed

Zenke, G; Baumann, G; Wenger, R; Hiestand, P; Quesniaux, V; Andersen, E; Schreier, M H

1993-06-23

Despite the successful clinical application of the immunosuppressive drug cyclosporin A (CsA, Sandimmun), its precise mechanism of action in the process of T cell activation remains elusive. CsA binds to the high-affinity cytosolic receptor cyclophilin whose peptidyl-prolyl cis-trans isomerase activity is inhibited upon binding. The linkage of this effect with the inhibition of the T cell receptor-mediated signal transduction pathway, which leads to a suppression of lymphokine gene transcription, is still unclear. We analyzed the relationship between cyclophilin-binding and immunosuppressive activity (e.g., effect on IL-2 transcription) of cyclosporin derivatives in vitro. The results show that binding to cyclophilin is required, but not sufficient for immunosuppression. Cyclosporin analogues which completely lack immunosuppressive activity but fully retained their cyclophilin-binding capacity antagonize the immunosuppressive activity of CsA. These derivatives inhibit the isomerase activity of cyclophilin, which clearly demonstrates that inhibition of the cyclophilin isomerase activity does not lead to immunosuppression. In analogy to the other immunosuppressants of microbial origin, FK-506 and rapamycin, a specific structure of the "effector" domain of CsA, which is unrelated to the cyclophilin-binding domain, determines the biological activity. In the nucleus, CsA interferes with the DNA-binding of inducible transcription factors to their respective DNA motifs within lymphokine promoters by affecting intracellular translocation of transcription factor subunits.

Single-molecule analysis of steroid receptor and cofactor action in living cells

PubMed Central

Paakinaho, Ville; Presman, Diego M.; Ball, David A.; Johnson, Thomas A.; Schiltz, R. Louis; Levitt, Peter; Mazza, Davide; Morisaki, Tatsuya; Karpova, Tatiana S.; Hager, Gordon L.

2017-01-01

Population-based assays have been employed extensively to investigate the interactions of transcription factors (TFs) with chromatin and are often interpreted in terms of static and sequential binding. However, fluorescence microscopy techniques reveal a more dynamic binding behaviour of TFs in live cells. Here we analyse the strengths and limitations of in vivo single-molecule tracking and performed a comprehensive analysis on the intranuclear dwell times of four steroid receptors and a number of known cofactors. While the absolute residence times estimates can depend on imaging acquisition parameters due to sampling bias, our results indicate that only a small proportion of factors are specifically bound to chromatin at any given time. Interestingly, the glucocorticoid receptor and its cofactors affect each other’s dwell times in an asymmetric manner. Overall, our data indicate transient rather than stable TF-cofactors chromatin interactions at response elements at the single-molecule level. PMID:28635963

Allosteric Pathways in the PPARγ-RXRα nuclear receptor complex

NASA Astrophysics Data System (ADS)

Ricci, Clarisse G.; Silveira, Rodrigo L.; Rivalta, Ivan; Batista, Victor S.; Skaf, Munir S.

2016-01-01

Understanding the nature of allostery in DNA-nuclear receptor (NR) complexes is of fundamental importance for drug development since NRs regulate the transcription of a myriad of genes in humans and other metazoans. Here, we investigate allostery in the peroxisome proliferator-activated/retinoid X receptor heterodimer. This important NR complex is a target for antidiabetic drugs since it binds to DNA and functions as a transcription factor essential for insulin sensitization and lipid metabolism. We find evidence of interdependent motions of Ω-loops and PPARγ-DNA binding domain with contacts susceptible to conformational changes and mutations, critical for regulating transcriptional functions in response to sequence-dependent DNA dynamics. Statistical network analysis of the correlated motions, observed in molecular dynamics simulations, shows preferential allosteric pathways with convergence centers comprised of polar amino acid residues. These findings are particularly relevant for the design of allosteric modulators of ligand-dependent transcription factors.

Productive interaction between transmembrane mutants of the bovine papillomavirus E5 protein and the platelet-derived growth factor beta receptor.

PubMed

Lai, Char-Chang; Edwards, Anne P B; DiMaio, Daniel

2005-02-01

The bovine papillomavirus E5 protein is a 44-amino-acid transmembrane protein that transforms cells by binding to the transmembrane region of the cellular platelet-derived growth factor (PDGF) beta receptor, resulting in sustained receptor signaling. However, there are published reports that certain mutants with amino acid substitutions in the membrane-spanning segment of the E5 protein transform cells without activating the PDGF beta receptor. We re-examined several of these transmembrane mutants, and here we present five lines of evidence that these mutants do in fact activate the PDGF beta receptor, resulting in cellular signaling and transformation.

Activation of the protein tyrosine phosphatase SHP2 via the interleukin-6 signal transducing receptor protein gp130 requires tyrosine kinase Jak1 and limits acute-phase protein expression.

PubMed

Schaper, F; Gendo, C; Eck, M; Schmitz, J; Grimm, C; Anhuf, D; Kerr, I M; Heinrich, P C

1998-11-01

Stimulation of the interleukin-6 (IL-6) signalling pathway occurs via the IL-6 receptor-glycoprotein 130 (IL-6R-gp130) receptor complex and results in the regulation of acute-phase protein genes in liver cells. Ligand binding to the receptor complex leads to tyrosine phosphorylation and activation of Janus kinases (Jak), phosphorylation of the signal transducing subunit gp130, followed by recruitment and phosphorylation of the signal transducer and activator of transcription factors STAT3 and STAT1 and the src homology domain (SH2)-containing protein tyrosine phosphatase (SHP2). The tyrosine phosphorylated STAT factors dissociate from the receptor, dimerize and translocate to the nucleus where they bind to enhancer sequences of IL-6 target genes. Phosphorylated SHP2 is able to bind growth factor receptor bound protein (grb2) and thus might link the Jak/STAT pathway to the ras/raf/mitogen-activated protein kinase pathway. Here we present data on the dose-dependence, kinetics and kinase requirements for SHP2 phosphorylation after the activation of the signal transducer, gp130, of the IL-6-type family receptor complex. When human fibrosarcoma cell lines deficient in Jak1, Jak2 or tyrosine kinase 2 (Tyk2) were stimulated with IL-6-soluble IL-6R complexes it was found that only in Jak1-, but not in Jak 2- or Tyk2-deficient cells, SHP2 activation was greatly impaired. It is concluded that Jak1 is required for the tyrosine phosphorylation of SHP2. This phosphorylation depends on Tyr-759 in the cytoplasmatic domain of gp130, since a Tyr-759-->Phe exchange abrogates SHP2 activation and in turn leads to elevated and prolonged STAT3 and STAT1 activation as well as enhanced acute-phase protein gene induction. Therefore, SHP2 plays an important role in acute-phase gene regulation.

Krüppel-like factors are effectors of nuclear receptor signaling

PubMed Central

Knoedler, Joseph R.; Denver, Robert J.

2015-01-01

Binding of steroid and thyroid hormones to their cognate nuclear receptors (NRs) impacts virtually every aspect of postembryonic development, physiology and behavior, and inappropriate signaling by NRs may contribute to disease. While NRs regulate genes by direct binding to hormone response elements in the genome, their actions may depend on the activity of other transcription factors (TFs) that may or may not bind DNA. The Krüppel-like family of transcription factors (KLF) is an evolutionarily conserved class of DNA-binding proteins that influence many aspects of development and physiology. Several members of this family have been shown to play diverse roles in NR signaling. For example, KLFs 1) act as accessory transcription factors for NR actions, 2) regulate expression of NR genes, and 3) as gene products of primary NR response genes function as key players in NR-dependent transcriptional networks. In mouse models, deletion of different KLFs leads to aberrant transcriptional and physiological responses to hormones, underscoring the importance of these proteins in the regulation of hormonal signaling. Understanding the functional relationships between NRs and KLFs will yield important insights into mechanisms of NR signaling. In this review we present a conceptual framework for understanding how KLFs participate in NR signaling, and we provide examples of how these proteins function to effect hormone action. PMID:24642391

Opiate antagonist prevents μ- and δ-opiate receptor dimerization to facilitate ability of agonist to control ethanol-altered natural killer cell functions and mammary tumor growth.

PubMed

Sarkar, Dipak K; Sengupta, Amitabha; Zhang, Changqing; Boyadjieva, Nadka; Murugan, Sengottuvelan

2012-05-11

In the natural killer (NK) cells, δ-opiate receptor (DOR) and μ-opioid receptor (MOR) interact in a feedback manner to regulate cytolytic function with an unknown mechanism. Using RNK16 cells, a rat NK cell line, we show that MOR and DOR monomer and dimer proteins existed in these cells and that chronic treatment with a receptor antagonist reduced protein levels of the targeted receptor but increased levels of opposing receptor monomer and homodimer. The opposing receptor-enhancing effects of MOR and DOR antagonists were abolished following receptor gene knockdown by siRNA. Ethanol treatment increased MOR and DOR heterodimers while it decreased the cellular levels of MOR and DOR monomers and homodimers. The opioid receptor homodimerization was associated with an increased receptor binding, and heterodimerization was associated with a decreased receptor binding and the production of cytotoxic factors. Similarly, in vivo, opioid receptor dimerization, ligand binding of receptors, and cell function in immune cells were promoted by chronic treatment with an opiate antagonist but suppressed by chronic ethanol feeding. Additionally, a combined treatment of an MOR antagonist and a DOR agonist was able to reverse the immune suppressive effect of ethanol and reduce the growth and progression of mammary tumors in rats. These data identify a role of receptor dimerization in the mechanism of DOR and MOR feedback interaction in NK cells, and they further elucidate the potential for the use of a combined opioid antagonist and agonist therapy for the treatment of immune incompetence and cancer and alcohol-related diseases.

Homocysteine directly interacts and activates the angiotensin II type I receptor to aggravate vascular injury.

PubMed

Li, Tuoyi; Yu, Bing; Liu, Zhixin; Li, Jingyuan; Ma, Mingliang; Wang, Yingbao; Zhu, Mingjiang; Yin, Huiyong; Wang, Xiaofeng; Fu, Yi; Yu, Fang; Wang, Xian; Fang, Xiaohong; Sun, Jinpeng; Kong, Wei

2018-01-02

Hyperhomocysteinemia (HHcy) is a risk factor for various cardiovascular diseases. However, the mechanism underlying HHcy-aggravated vascular injury remains unclear. Here we show that the aggravation of abdominal aortic aneurysm by HHcy is abolished in mice with genetic deletion of the angiotensin II type 1 (AT1) receptor and in mice treated with an AT1 blocker. We find that homocysteine directly activates AT1 receptor signalling. Homocysteine displaces angiotensin II and limits its binding to AT1 receptor. Bioluminescence resonance energy transfer analysis reveals distinct conformational changes of AT1 receptor upon binding to angiotensin II and homocysteine. Molecular dynamics and site-directed mutagenesis experiments suggest that homocysteine regulates the conformation of the AT1 receptor both orthosterically and allosterically by forming a salt bridge and a disulfide bond with its Arg 167 and Cys 289 residues, respectively. Together, these findings suggest that strategies aimed at blocking the AT1 receptor may mitigate HHcy-associated aneurysmal vascular injuries.

Analysis of Ligand Binding ErbB Receptor Tyrosine Kinases

DTIC Science & Technology

2000-07-01

epidermal growth factor receptor. Protein Sci 9, 310- 324 . 10 UNPUBLISHED DATA Principal Investigator (last, first, middle); Ferguson, Kathryn, M...and hetero-oligomerization. Purified Muaei- anto ane titse in tdi0ML protein (15 p.l) was loaded at a concentration of I mg/ml, and the gel Multi

Steroids and the scientist.

PubMed

Gustafsson, Jan-Ake

2005-06-01

Our interest in nuclear receptors (NRs) originated from early studies on hepatic steroid metabolism. We discovered a new hypothalamo-pituitary-liver axis, imprinted neonatally by androgens and operating through sexually differentiated GH secretory patterns. Male and female patterns have opposite effects on sexually differentiated hepatic genes, explaining sexually dimorphic liver patterns. To further understand steroid action, we purified the glucocorticoid receptor (GR) leading to our discovery of the NR three-domain structure, with separable DNA binding domain and ligand binding domains and a third domain now known to have transcriptional regulatory properties. Knowledge of this domain structure has been immensely important for deciphering NR actions. Using this first purified NR, we collaborated with Keith Yamamoto and first demonstrated specific NR binding to DNA. This also was the first demonstration of a mammalian transcription factor, a breakthrough that led to discovery of NR response elements. In further collaboration with Yamamoto, we cloned the first NR cDNA sequences, leading to cloning of the superfamily of NR genes. With Yamamoto and Kaptein, we determined the first three-dimensional NR structure, that of DNA binding domain. Later work on orphan receptors resulted in the first discovery of: 1) endogenous ligands for an orphan receptor (fatty acids as activators of peroxisomal proliferator-activated receptor alpha); 2) liver X receptor beta (OR-1) and its role in central nervous system cholesterol homeostasis; and 3) estrogen receptor beta, leading to a paradigm shift in understanding of estrogen signaling, of importance in endocrinology, immunology, and oncology and to development of estrogen receptor beta agonists for treatment of autoimmune diseases, prostate disease, depression, and ovulatory dysfunction.

Different Epidermal Growth Factor (EGF) Receptor Ligands Show Distinct Kinetics and Biased or Partial Agonism for Homodimer and Heterodimer Formation*

PubMed Central

Macdonald-Obermann, Jennifer L.; Pike, Linda J.

2014-01-01

The EGF receptor has seven different cognate ligands. Previous work has shown that these different ligands are capable of inducing different biological effects, even in the same cell. To begin to understand the molecular basis for this variation, we used luciferase fragment complementation to measure ligand-induced dimer formation and radioligand binding to study the effect of the ligands on subunit-subunit interactions in EGF receptor (EGFR) homodimers and EGFR/ErbB2 heterodimers. In luciferase fragment complementation imaging studies, amphiregulin (AREG) functioned as a partial agonist, inducing only about half as much total dimerization as the other three ligands. However, unlike the other ligands, AREG showed biphasic kinetics for dimer formation, suggesting that its path for EGF receptor activation involves binding to both monomers and preformed dimers. EGF, TGFα, and betacellulin (BTC) appear to mainly stimulate receptor activation through binding to and dimerization of receptor monomers. In radioligand binding assays, EGF and TGFα exhibited increased affinity for EGFR/ErbB2 heterodimers compared with EGFR homodimers. By contrast, BTC and AREG showed a similar affinity for both dimers. Thus, EGF and TGFα are biased agonists, whereas BTC and AREG are balanced agonists with respect to selectivity of dimer formation. These data suggest that the differences in biological response to different EGF receptor ligands may result from partial agonism for dimer formation, differences in the kinetic pathway utilized to generate activated receptor dimers, and biases in the formation of heterodimers versus homodimers. PMID:25086039

Analysis of Activated Platelet-Derived Growth Factor β Receptor and Ras-MAP Kinase Pathway in Equine Sarcoid Fibroblasts

PubMed Central

Altamura, Gennaro; Corteggio, Annunziata; Nasir, Lubna; Yuan, Zheng Qiang; Roperto, Franco; Borzacchiello, Giuseppe

2013-01-01

Equine sarcoids are skin tumours of fibroblastic origin affecting equids worldwide. Bovine papillomavirus type-1 (BPV-1) and, less commonly, type-2 are recognized as etiological factors of sarcoids. The transforming activity of BPV is related to the functions of its major oncoprotein E5 which binds to the platelet-derived growth factor β receptor (PDGFβR) causing its phosphorylation and activation. In this study, we demonstrate, by coimmunoprecipitation and immunoblotting, that in equine sarcoid derived cell lines PDGFβR is phosphorylated and binds downstream molecules related to Ras-mitogen-activated protein kinase-ERK pathway thus resulting in Ras activation. Imatinib mesylate is a tyrosine kinase receptors inhibitor which selectively inhibits the activation of PDGFβR in the treatment of several human and animal cancers. Here we show that imatinib inhibits receptor phosphorylation, and cell viability assays demonstrate that this drug decreases sarcoid fibroblasts viability in a dose-dependent manner. This study contributes to a better understanding of the molecular mechanisms involved in the pathology of sarcoids and paves the way to a new therapeutic approach for the treatment of this common equine skin neoplasm. PMID:23936786

Ethylene and pollination decrease transcript abundance of an ethylene receptor gene in Dendrobium petals.

PubMed

Thongkum, Monthathip; Burns, Parichart; Bhunchoth, Anjana; Warin, Nuchnard; Chatchawankanphanich, Orawan; van Doorn, Wouter G

2015-03-15

We studied the expression of a gene encoding an ethylene receptor, called Ethylene Response Sensor 1 (Den-ERS1), in the petals of Dendrobium orchid flowers. Transcripts accumulated during the young floral bud stage and declined by the time the flowers had been open for several days. Pollination or exposure to exogenous ethylene resulted in earlier flower senescence, an increase in ethylene production and a lower Den-ERS1 transcript abundance. Treatment with 1-methylcyclopropene (1-MCP), an inhibitor of the ethylene receptor, decreased ethylene production and resulted in high transcript abundance. The literature indicates two kinds of ethylene receptor genes with regard to the effects of ethylene. One group shows ethylene-induced down-regulated transcription, while the other has ethylene-induced up-regulation. The present gene is an example of the first group. The 5' flanking region showed binding sites for Myb and myb-like, homeodomain, MADS domain, NAC, TCP, bHLH and EIN3-like transcription factors. The binding site for the EIN3-like factor might explain the ethylene effect on transcription. A few other transcription factors (RAV1 and NAC) seem also related to ethylene effects. Copyright © 2015 Elsevier GmbH. All rights reserved.

NMR spectroscopy of the ligand binding core of ionotropic glutamate receptor 2 bound to 5-substituted willardiine partial agonists

PubMed Central

Fenwick, Michael K.; Oswald, Robert E.

2008-01-01

Glutamate receptors mediate neuronal intercommunication in the central nervous system by coupling extracellular neurotransmitter-receptor interactions to ion channel conductivity. To gain insight into structural and dynamical factors that underlie this coupling, solution NMR experiments were performed on the bi-lobed ligand-binding core of glutamate receptor 2 in complexes with a set of willardiine partial agonists. These agonists are valuable for studying structure-function relationships because their 5-position substituent size is correlated with ligand efficacy and extent of receptor desensitization whereas the substituent electronegativity is correlated with ligand potency. NMR results show that the protein backbone amide chemical shift deviations correlate mainly with efficacy and extent of desensitization. Pronounced deviations occur at specific residues in the ligand-binding site and in the two helical segments that join the lobes by a disulfide bond. Experiments detecting conformational exchange show that micro- to millisecond timescale motions also occur near the disulfide bond and vary largely with efficacy and extent of desensitization. These results thus identify regions displaying structural and dynamical dissimilarity arising from differences in ligand-protein interactions and lobe closure which may play a critical role in receptor response. Furthermore, measures of line broadening and conformational exchange for a portion of the ligand-binding site correlate with ligand EC50 data. These results do not have any correlate in the currently available crystal structures and thus provide a novel view of ligand-binding events that may be associated with agonist potency differences. PMID:18387631

Molecular Docking and Drug Discovery in β-Adrenergic Receptors.

PubMed

Vilar, Santiago; Sobarzo-Sanchez, Eduardo; Santana, Lourdes; Uriarte, Eugenio

2017-01-01

Evolution in computer engineering, availability of increasing amounts of data and the development of new and fast docking algorithms and software have led to improved molecular simulations with crucial applications in virtual high-throughput screening and drug discovery. Moreover, analysis of protein-ligand recognition through molecular docking has become a valuable tool in drug design. In this review, we focus on the applicability of molecular docking on a particular class of G protein-coupled receptors: the β-adrenergic receptors, which are relevant targets in clinic for the treatment of asthma and cardiovascular diseases. We describe the binding site in β-adrenergic receptors to understand key factors in ligand recognition along with the proteins activation process. Moreover, we focus on the discovery of new lead compounds that bind the receptors, on the evaluation of virtual screening using the active/ inactive binding site states, and on the structural optimization of known families of binders to improve β-adrenergic affinity. We also discussed strengths and challenges related to the applicability of molecular docking in β-adrenergic receptors. Molecular docking is a valuable technique in computational chemistry to deeply analyze ligand recognition and has led to important breakthroughs in drug discovery and design in the field of β-adrenergic receptors. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Expression of transforming growth factor alpha and epidermal growth factor receptor messenger RNA in neoplastic and nonneoplastic human kidney tissue.

PubMed

Mydlo, J H; Michaeli, J; Cordon-Cardo, C; Goldenberg, A S; Heston, W D; Fair, W R

1989-06-15

Using Northern blot analysis, we have demonstrated that mRNA for transforming growth factor alpha (TGF-alpha) was expressed in five malignant kidney tissue specimens but was not detected in their autologous nonneoplastic homologues. In addition, the expression of epidermal growth factor (EGF) receptor mRNA in these malignant tissues was 2- to 3-fold greater than in nontransformed tissues. In two cases examined using immunohistochemistry, we were able to correlate the increased expression of the mRNA with an increase in protein expression. Since TGF-alpha is known to bind to the EGF receptor, the finding of an increased expression of both TGF-alpha and EGF receptor mRNA in kidney tumor tissue suggests that interaction between TGF-alpha and the EGF receptor may play a role in promoting transformation and/or proliferation of kidney neoplasms, perhaps by an autocrine mechanism.

Analysis of Ethylene Receptors: Ethylene-Binding Assays.

PubMed

Binder, Brad M; Schaller, G Eric

2017-01-01

Plant ethylene receptors bind ethylene with high affinity. Most of the characterization of ethylene binding to the receptors has been carried out using a radioligand-binding assay on functional receptors expressed in yeast. In this chapter, we describe methods for expressing ethylene receptors in yeast and conducting ethylene-binding assays on intact yeast and yeast membranes. The ethylene-binding assays can be modified to analyze ethylene binding to intact plants and other organisms as well as membranes isolated from any biological source.

Impaired growth in Rabson-Mendenhall syndrome: lack of effect of growth hormone and insulin-like growth factor-I.

PubMed

Longo, N; Singh, R; Griffin, L D; Langley, S D; Parks, J S; Elsas, L J

1994-09-01

Mutations in the insulin receptor gene cause the severe insulin-resistant syndromes leprechaunism and Rabson-Mendenhall syndrome. There is no accepted therapy for these inherited conditions. Here we report the results of recombinant human GH (rhGH) and recombinant human insulin-like growth factor-I (rhIGF-I) treatment of a male patient, Atl-2, with Rabson-Mendenhall syndrome. The patient was small for gestational age, had premature dentition, absence of sc fat, acanthosis nigricans, fasting hypoglycemia and postprandial hyperglycemia, and extremely high concentrations of circulating insulin (up to 8500 microU/mL). Fibroblasts and lymphoblasts established from this patient had reduced insulin binding, which was 20-30% of the control value. Binding of epidermal growth factor, IGF-I, and GH to the patient's fibroblasts was normal. The growth of fibroblasts cultured from patient Atl-2 in vitro was intermediate between that of fibroblasts from patients with leprechaunism and control values. The patient's growth curve in vivo was far below the fifth percentile despite adequate nutrition. To stimulate growth, therapy with rhGH was initiated, the rationale being to stimulate hepatic IGF-I production and IGF-I receptor signaling, and bypass the inherited block in insulin receptor signaling. Therapy with rhGH (up to 0.5 mg/kg.week) did not improve growth and failed to increase the levels of circulating IGF-I and IGF-binding protein-3 over a 14-month period. As rhGH could not stimulate growth, rhIGF-I (up to 100 micrograms/kg.day) was given by daily sc injection. No increase in growth velocity was observed over a 14-month period. These results indicate that both GH and IGF-I fail to correct growth in a patient with severe inherited insulin resistance. The lack of efficacy of IGF-I treatment may be related to multiple factors, such as the poor metabolic state of the patient, the deficiency of serum carrier protein for IGF-I, an increased clearance of the growth factor, IGF-I resistance in target cells at a receptor or postreceptor level, or an inhibitory action of the mutant insulin receptors on IGF-I receptor signaling.

Enterovirus D68 receptor requirements unveiled by haploid genetics

PubMed Central

Baggen, Jim; Thibaut, Hendrik Jan; Staring, Jacqueline; Jae, Lucas T.; Liu, Yue; Guo, Hongbo; Slager, Jasper J.; de Bruin, Jost W.; van Vliet, Arno L. W.; Blomen, Vincent A.; Overduin, Pieter; Sheng, Ju; de Haan, Cornelis A. M.; de Vries, Erik; Meijer, Adam; Rossmann, Michael G.; Brummelkamp, Thijn R.; van Kuppeveld, Frank J. M.

2016-01-01

Enterovirus D68 (EV-D68) is an emerging pathogen that can cause severe respiratory disease and is associated with cases of paralysis, especially among children. Heretofore, information on host factor requirements for EV-D68 infection is scarce. Haploid genetic screening is a powerful tool to reveal factors involved in the entry of pathogens. We performed a genome-wide haploid screen with the EV-D68 prototype Fermon strain to obtain a comprehensive overview of cellular factors supporting EV-D68 infection. We identified and confirmed several genes involved in sialic acid (Sia) biosynthesis, transport, and conjugation to be essential for infection. Moreover, by using knockout cell lines and gene reconstitution, we showed that both α2,6- and α2,3-linked Sia can be used as functional cellular EV-D68 receptors. Importantly, the screen did not reveal a specific protein receptor, suggesting that EV-D68 can use multiple redundant sialylated receptors. Upon testing recent clinical strains, we identified strains that showed a similar Sia dependency, whereas others could infect cells lacking surface Sia, indicating they can use an alternative, nonsialylated receptor. Nevertheless, these Sia-independent strains were still able to bind Sia on human erythrocytes, raising the possibility that these viruses can use multiple receptors. Sequence comparison of Sia-dependent and Sia-independent EV-D68 strains showed that many changes occurred near the canyon that might allow alternative receptor binding. Collectively, our findings provide insights into the identity of the EV-D68 receptor and suggest the possible existence of Sia-independent viruses, which are essential for understanding tropism and disease. PMID:26787879

Localization of a gene for a glutamate binding subunit of a NMDA receptor (GRINA) to 8q24

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

Lewis, T.B.; DuPont, B.R.; Leach, R.

1996-02-15

This article reports on the localization of a gene for a glutamate binding subunit of an N-methyl-D-aspartate (NMDA) receptor, called GRINA, to human chromosome 8q24 using fluorescence in situ hybridization and radiation hybridization mapping. This gene mapped outside the critical region for benign familial neonatal convulsions (BFNC), a rare form of epilepsy; however, GRINA could be the causative genetic factor inducing idiopathic generalized epilepsy. Further studies need to be conducted. 15 refs., 2 figs.

Chronic hypoxia up-regulates expression of adenosine A1 receptors in DDT1-MF2 cells.

PubMed

Hammond, Lucy C; Bonnet, Claire; Kemp, Paul J; Yates, Michael S; Bowmer, Christopher J

2004-02-01

As the first step to understand how chronic hypoxia might regulate smooth muscle function in health and disease, we have employed an established immortalised cell model of smooth muscle, DDT1-MF2 cells, to address the hypothesis that adenosine A1 receptor density is modulated by O2 availability. Maximal specific binding (Bmax) of the selective adenosine A1 receptor antagonist, [3H]-DPCPX, to cell membranes increased 3.5-fold from 0.48 +/- 0.02 pmol/mg to 1.7 +/- 0.5 pmol/mg protein after 16 hr of hypoxia and this effect was not accompanied by any statistically significant changes in either binding affinity (0.84 +/- 0.2 nM vs. 1.2 +/- 0.3 nM) or Hill coefficient (1.1 +/- 0.1 vs. 0.99 +/- 0.03). Hypoxia-evoked increases in membrane receptor density were paralleled in intact DDT1-MF2 cells. In addition, the increase in [3H]-DPCPX binding to intact cells was inhibited by co-incubation during hypoxia with the translational inhibitor cycloheximide, the transcriptional blocker actinomycin D and the NFkappaB inhibitor sulphasalazine. Together, these data show that adenosine A1 receptor density is modulated, at least in part, by O2-dependent activation of the transcription factor NFkappaB and adds to the list of processes dynamically regulated by ambient oxygen availability. Since hypoxia is an initiating factor in acute renal failure, similar changes in transcription may account for up-regulation of adenosine A1 receptors noted previously in the renal vasculature of rats with acute renal failure.

NR4A nuclear receptors are orphans but not lonesome.

PubMed

Kurakula, Kondababu; Koenis, Duco S; van Tiel, Claudia M; de Vries, Carlie J M

2014-11-01

The NR4A subfamily of nuclear receptors consists of three mammalian members: Nur77, Nurr1, and NOR-1. The NR4A receptors are involved in essential physiological processes such as adaptive and innate immune cell differentiation, metabolism and brain function. They act as transcription factors that directly modulate gene expression, but can also form trans-repressive complexes with other transcription factors. In contrast to steroid hormone nuclear receptors such as the estrogen receptor or the glucocorticoid receptor, no ligands have been described for the NR4A receptors. This lack of known ligands might be explained by the structure of the ligand-binding domain of NR4A receptors, which shows an active conformation and a ligand-binding pocket that is filled with bulky amino acid side-chains. Other mechanisms, such as transcriptional control, post-translational modifications and protein-protein interactions therefore seem to be more important in regulating the activity of the NR4A receptors. For Nur77, over 80 interacting proteins (the interactome) have been identified so far, and roughly half of these interactions has been studied in more detail. Although the NR4As show some overlap in interacting proteins, less information is available on the interactome of Nurr1 and NOR-1. Therefore, the present review will describe the current knowledge on the interactomes of all three NR4A nuclear receptors with emphasis on Nur77. Copyright © 2014 Elsevier B.V. All rights reserved.

Chemical Composition of Pinus roxburghii Bark Volatile Oil and Validation of Its Anti-Inflammatory Activity Using Molecular Modelling and Bleomycin-Induced Inflammation in Albino Mice.

PubMed

Labib, Rola M; Youssef, Fadia S; Ashour, Mohamed L; Abdel-Daim, Mohamed M; Ross, Samir A

2017-08-29

The chemical composition of Pinus roxburghii bark essential oil (PRO) was qualitatively and quantitatively determined using GC/FID and GC/MS. The anti-inflammatory activity was assessed in vitro by evaluating the binding percentages on the cannabinoids and opioids receptors. Bleomycin (BLM)-induced pulmonary inflammation in albino mice was adopted to assess PRO anti-inflammatory efficacy in vivo. In silico molecular modelling of its major components was performed on human glucocorticoids receptor (GR). Seventy-five components were identified in which longifolene (33.13%) and palmitic acid (9.34%) constituted the predominant components. No binding was observed on cannabinoid receptor type 1 (CB1), whereas mild binding was observed on cannabinoid receptor type 2 (CB2), delta , kappa , and mu receptors accounting for 2.9%, 6.9%, 10.9% and 22% binding. A significant in vivo activity was evidenced by reduction of the elevated malondialdehyde (MDA), nitric oxide (NO), myeloperoxidase (MPO), interleukin-6 (IL-6), and tumor necrosis factor- α (TNF- α ) levels by 55.56%, 55.66%, 64.64%, 58.85% and 77.78% with concomitant elevation of superoxide dismutase (SOD) and catalase (CAT) activities comparable to BLM-treated group at 100 mg/kg body weight. In silico studies showed that palmitic acid exerted the fittest binding. PRO could serve as a potent anti-inflammatory natural candidate that should be supported by further clinical trials.

Lipopolysaccharides in liver injury: molecular mechanisms of Kupffer cell activation.

PubMed

Su, Grace L

2002-08-01

Endogenous gut-derived bacterial lipopolysaccharides have been implicated as important cofactors in the pathogenesis of liver injury. However, the molecular mechanisms by which lipopolysaccharides exert their effect are not entirely clear. Recent studies have pointed to proinflammatory cytokines such as tumor necrosis factor-alpha as mediators of hepatocyte injury. Within the liver, Kupffer cells are major sources of proinflammatory cytokines that are produced in response to lipopolysaccharides. This review will focus on three important molecular components of the pathway by which lipopolysaccharides activate Kupffer cells: CD14, Toll-like receptor 4, and lipopolysaccharide binding protein. Within the liver, lipopolysaccharides bind to lipopolysaccharide binding protein, which then facilitates its transfer to membrane CD14 on the surface of Kupffer cells. Signaling of lipopolysaccharide through CD14 is mediated by the downstream receptor Toll-like receptor 4 and results in activation of Kupffer cells. The role played by these molecules in liver injury will be examined.

Differential Receptor Binding and Regulatory Mechanisms for the Lymphangiogenic Growth Factors Vascular Endothelial Growth Factor (VEGF)-C and -D*

PubMed Central

Davydova, Natalia; Harris, Nicole C.; Roufail, Sally; Paquet-Fifield, Sophie; Ishaq, Musarat; Streltsov, Victor A.; Williams, Steven P.; Karnezis, Tara; Stacker, Steven A.; Achen, Marc G.

2016-01-01

VEGF-C and VEGF-D are secreted glycoproteins that induce angiogenesis and lymphangiogenesis in cancer, thereby promoting tumor growth and spread. They exhibit structural homology and activate VEGFR-2 and VEGFR-3, receptors on endothelial cells that signal for growth of blood vessels and lymphatics. VEGF-C and VEGF-D were thought to exhibit similar bioactivities, yet recent studies indicated distinct signaling mechanisms (e.g. tumor-derived VEGF-C promoted expression of the prostaglandin biosynthetic enzyme COX-2 in lymphatics, a response thought to facilitate metastasis via the lymphatic vasculature, whereas VEGF-D did not). Here we explore the basis of the distinct bioactivities of VEGF-D using a neutralizing antibody, peptide mapping, and mutagenesis to demonstrate that the N-terminal α-helix of mature VEGF-D (Phe93–Arg108) is critical for binding VEGFR-2 and VEGFR-3. Importantly, the N-terminal part of this α-helix, from Phe93 to Thr98, is required for binding VEGFR-3 but not VEGFR-2. Surprisingly, the corresponding part of the α-helix in mature VEGF-C did not influence binding to either VEGFR-2 or VEGFR-3, indicating distinct determinants of receptor binding by these growth factors. A variant of mature VEGF-D harboring a mutation in the N-terminal α-helix, D103A, exhibited enhanced potency for activating VEGFR-3, was able to promote increased COX-2 mRNA levels in lymphatic endothelial cells, and had enhanced capacity to induce lymphatic sprouting in vivo. This mutant may be useful for developing protein-based therapeutics to drive lymphangiogenesis in clinical settings, such as lymphedema. Our studies shed light on the VEGF-D structure/function relationship and provide a basis for understanding functional differences compared with VEGF-C. PMID:27852824

Inhibition of Herpes Simplex Virus gD and Lymphotoxin-α Binding to HveA by Peptide Antagonists

PubMed Central

Sarrias, Maria Rosa; Whitbeck, J. Charles; Rooney, Isabelle; Spruce, Lynn; Kay, Brian K.; Montgomery, Rebecca I.; Spear, Patricia G.; Ware, Carl F.; Eisenberg, Roselyn J.; Cohen, Gary H.; Lambris, John D.

1999-01-01

The herpesvirus entry mediator A (HveA) is a recently characterized member of the tumor necrosis factor receptor family that mediates the entry of most herpes simplex virus type 1 (HSV-1) strains into mammalian cells. Studies on the interaction of HSV-1 with HveA have shown that of all the viral proteins involved in uptake, only gD has been shown to bind directly to HveA, and this binding mediates viral entry into cells. In addition to gD binding to HveA, the latter has been shown to interact with proteins of tumor necrosis factor receptor-associated factor family, lymphotoxin-α (LT-α), and a membrane-associated protein referred to as LIGHT. To study the relationship between HveA, its natural ligands, and the viral proteins involved in HSV entry into cells, we have screened two phage-displayed combinatorial peptide libraries for peptide ligands of a recombinant form of HveA. Affinity selection experiments yielded two peptide ligands, BP-1 and BP-2, which could block the interaction between gD and HveA. Of the two peptides, only BP-2 inhibited HSV entry into CHO cells transfected with an HveA-expressing plasmid. When we analyzed these peptides for the ability to interfere with HveA binding to its natural ligand LT-α, we found that BP-1 inhibited the interaction of cellular LT-α with HveA. Thus, we have dissected the sites of interaction between the cell receptor, its natural ligand LT-α and gD, the virus-specific protein involved in HSV entry into cells. PMID:10364318

Non-canonical dynamic mechanisms of interaction between the p66Shc protein and Met receptor

PubMed Central

Landry, Mélissa; Pomerleau, Véronique; Saucier, Caroline

2016-01-01

Met receptor tyrosine kinase (RTK) is known to bind to the three distinct protein isoforms encoded by the ShcA (Shc) gene. Structure–function studies have unveiled critical roles for p52Shc-dependent signalling pathways in Met-regulated biological functions. The molecular basis of the interaction between the Met and p52Shc proteins is well-defined, but not for the longest protein isoform, p66Shc. In the present study, co-immunoprecipitation assays were performed in human embryonic kidney 293 (HEK293) cells, transiently co-transfected with Met and p66Shc mutants, in order to define the molecular determinants involved in mediating Met–p66Shc interaction. Our results show that p66Shc interacts constitutively with the receptor Met, and the Grb2 (growth factor receptor-bound protein-2) and Gab1 (Grb2-associated binder-1) adaptor proteins. Although its phosphotyrosine-binding domain (PTB) and Src homology 2 (SH2) domains co-ordinate p66Shc binding to non-activated Met receptor, these phosphotyrosine-binding modules, and its collagen homology domain 2 (CH2) region, exert negative constraints. In contrast, p66Shc interaction with the activated Met depends mainly on the integrity of its PTB domain, and to a lesser extent of its SH2 domain. Even though not required for the recruitment of p66Shc, tyrosine phosphorylation of p66Shc by activated Met enhances these interactions by mechanisms not reliant on the integrity of the Met multisubstrate-binding site. In turn, this increases phosphotyrosine-dependent p66Shc–Grb2–Gab1 complex formation away from the receptor, while blocking Grb2 and Gab1 recruitment to activated Met. In conclusion, we identify, for the first time, a novel non-canonical dynamic mode of interaction between Met and the p66 protein isoform of Shc and its effects on rewiring binding effector complexes according to the activation state of the receptor. PMID:27048591

Structural Analysis of Botulinum Neurotoxin Type G Receptor Binding

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

Schmitt, John; Karalewitz, Andrew; Benefield, Desire A.

2010-10-19

Botulinum neurotoxin (BoNT) binds peripheral neurons at the neuromuscular junction through a dual-receptor mechanism that includes interactions with ganglioside and protein receptors. The receptor identities vary depending on BoNT serotype (A-G). BoNT/B and BoNT/G bind the luminal domains of synaptotagmin I and II, homologous synaptic vesicle proteins. We observe conditions under which BoNT/B binds both Syt isoforms, but BoNT/G binds only SytI. Both serotypes bind ganglioside G{sub T1b}. The BoNT/G receptor-binding domain crystal structure provides a context for examining these binding interactions and a platform for understanding the physiological relevance of different Syt receptor isoforms in vivo.

The heterodimeric sweet taste receptor has multiple potential ligand binding sites.

PubMed

Cui, Meng; Jiang, Peihua; Maillet, Emeline; Max, Marianna; Margolskee, Robert F; Osman, Roman

2006-01-01

The sweet taste receptor is a heterodimer of two G protein coupled receptors, T1R2 and T1R3. This discovery has increased our understanding at the molecular level of the mechanisms underlying sweet taste. Previous experimental studies using sweet receptor chimeras and mutants show that there are at least three potential binding sites in this heterodimeric receptor. Receptor activity toward the artificial sweeteners aspartame and neotame depends on residues in the amino terminal domain of human T1R2. In contrast, receptor activity toward the sweetener cyclamate and the sweet taste inhibitor lactisole depends on residues within the transmembrane domain of human T1R3. Furthermore, receptor activity toward the sweet protein brazzein depends on the cysteine rich domain of human T1R3. Although crystal structures are not available for the sweet taste receptor, useful homology models can be developed based on appropriate templates. The amino terminal domain, cysteine rich domain and transmembrane helix domain of T1R2 and T1R3 have been modeled based on the crystal structures of metabotropic glutamate receptor type 1, tumor necrosis factor receptor, and bovine rhodopsin, respectively. We have used homology models of the sweet taste receptors, molecular docking of sweet ligands to the receptors, and site-directed mutagenesis of the receptors to identify potential ligand binding sites of the sweet taste receptor. These studies have led to a better understanding of the structure and function of this heterodimeric receptor, and can act as a guide for rational structure-based design of novel non-caloric sweeteners, which can be used in the fighting against obesity and diabetes.

Membrane and Integrative Nuclear Fibroblastic Growth Factor Receptor (FGFR) Regulation of FGF-23*

PubMed Central

Han, Xiaobin; Xiao, Zhousheng; Quarles, L. Darryl

2015-01-01

Fibroblastic growth factor receptor 1 (FGFR1) signaling pathways are implicated in the regulation of FGF-23 gene transcription, but the molecular pathways remain poorly defined. We used low molecular weight (LMW, 18 kDa) FGF-2 and high molecular weight (HMW) FGF-2 isoforms, which, respectively, activate cell surface FGF receptors and intranuclear FGFR1, to determine the roles of membrane FGFRs and integrative nuclear FGFR1 signaling (INFS) in the regulation of FGF-23 gene transcription in osteoblasts. We found that LMW-FGF-2 induced NFAT and Ets1 binding to conserved cis-elements in the proximal FGF-23 promoter and stimulated FGF-23 promoter activity through PLCγ/calcineurin/NFAT and MAPK pathways in SaOS-2 and MC3T3-E1 osteoblasts. In contrast, HMW-FGF-2 stimulated FGF-23 promoter activity in osteoblasts through a cAMP-dependent binding of FGFR1 and cAMP-response element-binding protein (CREB) to a conserved cAMP response element (CRE) contiguous with the NFAT binding site in the FGF-23 promoter. Mutagenesis of the NFAT and CRE binding sites, respectively, inhibited the effects of LMW-FGF-2 and HMW-FGF-23 to stimulate FGF-23 promoter activity. FGF-2 activation of both membrane FGFRs and INFS-dependent FGFR1 pathways may provide a means to integrate systemic and local regulation of FGF-23 transcription under diverse physiological and pathological conditions. PMID:25752607

Gab-family adapter molecules in signal transduction of cytokine and growth factor receptors, and T and B cell antigen receptors.

PubMed

Hibi, M; Hirano, T

2000-04-01

Gab1 and Gab2 (Grb2 associated binder 1 and 2) are scaffolding adapter molecules that display sequence similarity with Drosophila DOS (daughter of sevenless), which is a potential substrate for the protein tyrosine phosphatase, Corkscrew, Both Gab1 and Gab2, like DOS, have a pleckstrin homology domain and potential binding sites for SH2 and SH3 domains. Gab1 and Gab2 are phosphorylated on tyrosine upon the stimulation of various cytokines, growth factors, and antigen receptors, and interact with signaling molecules, such as Grb2, SHP-2, and PI-3 kinase. Overexpression of Gab1 or Gab2 mimics or enhances growth factor or cytokine-mediated biological processes and activates ERK MAP kinase. These data imply that Gab1 and Gab2 act downstream of a broad range of cytokine and growth factor receptors, as well as T and B antigen receptors, and link these receptors to ERK MAP kinase and biological actions.

Anti-Tumor Activity of a Novel HS-Mimetic-Vascular Endothelial Growth Factor Binding Small Molecule

PubMed Central

Sugahara, Kazuyuki; Thimmaiah, Kuntebommanahalli N.; Bid, Hemant K.; Houghton, Peter J.; Rangappa, Kanchugarakoppal S.

2012-01-01

The angiogenic process is controlled by variety of factors of which the vascular endothelial growth factor (VEGF) pathway plays a major role. A series of heparan sulfate mimetic small molecules targeting VEGF/VEGFR pathway has been synthesized. Among them, compound 8 (2-butyl-5-chloro-3-(4-nitro-benzyl)-3H-imidazole-4-carbaldehyde) was identified as a significant binding molecule for the heparin-binding domain of VEGF, determined by high-throughput-surface plasmon resonance assay. The data predicted strong binding of compound 8 with VEGF which may prevent the binding of VEGF to its receptor. We compared the structure of compound 8 with heparan sulfate (HS), which have in common the functional ionic groups such as sulfate, nitro and carbaldehyde that can be located in similar positions of the disaccharide structure of HS. Molecular docking studies predicted that compound 8 binds at the heparin binding domain of VEGF through strong hydrogen bonding with Lys-30 and Gln-20 amino acid residues, and consistent with the prediction, compound 8 inhibited binding of VEGF to immobilized heparin. In vitro studies showed that compound 8 inhibits the VEGF-induced proliferation migration and tube formation of mouse vascular endothelial cells, and finally the invasion of a murine osteosarcoma cell line (LM8G7) which secrets high levels of VEGF. In vivo, these effects produce significant decrease of tumor burden in an experimental model of liver metastasis. Collectively, these data indicate that compound 8 may prevent tumor growth through a direct effect on tumor cell proliferation and by inhibition of endothelial cell migration and angiogenesis mediated by VEGF. In conclusion, compound 8 may normalize the tumor vasculature and microenvironment in tumors probably by inhibiting the binding of VEGF to its receptor. PMID:22916091

Bradykinin-induced growth inhibition of normal rat kidney (NRK) cells is paralleled by a decrease in epidermal-growth-factor receptor expression.

PubMed Central

Van Zoelen, E J; Peters, P H; Afink, G B; Van Genesen, S; De Roos, D G; Van Rotterdam, W; Theuvenet, A P

1994-01-01

Normal rat kidney fibroblasts, grown to density arrest in the presence of epidermal growth factor (EGF), can be induced to undergo phenotypic transformation by treatment with transforming growth factor beta or retinoic acid. Here we show that bradykinin blocks this growth-stimulus-induced loss of density-dependent growth arrest by a specific receptor-mediated mechanism. The effects of bradykinin are specific, and are not mimicked by other phosphoinositide-mobilizing agents such as prostaglandin F2 alpha. Northern-blot analysis and receptor-binding studies demonstrate that bradykinin also inhibits the retinoic acid-induced increase in EGF receptor levels in these cells. These studies provide additional evidence that EGF receptor levels modulate EGF-induced expression of the transformed phenotype in these cells. Images Figure 5 PMID:8135739

Detecting Tie2, an endothelial growth factor receptor, by using immunohistochemistry in mouse lungs.

PubMed

Guha, Prajna P; David, Sascha A; Ghosh, Chandra C

2014-01-01

Immunohistochemical (IHC) staining is an invaluable, sensitive, and effective method to detect the presence and localization of proteins in the cellular compartment in tissues. The basic concept of IHC is detecting the antigen in tissues by means of specific antibody binding, which is then demonstrated with a colored histochemical reaction that can be observed under a light microscope. The most challenging aspect of IHC techniques is optimizing the precise experimental conditions that are required to get a specific and a strong signal. The critical steps of IHC are specimen acquisition, fixation, permeabilization, detection system, and selection of the antigen specific antibody and its optimization. Here, we elaborate the technique using the endothelial growth factor binding receptor Tie2 in mouse lungs.

Insulin-like growth factor (IGF) binding protein from human decidua inhibits the binding and biological action of IGF-I in cultured choriocarcinoma cells

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

Ritvos, O.; Ranta, T.; Jalkanen, J.

1988-05-01

The placenta expresses genes for insulin-like growth factors (IGFs) and possesses IGF-receptors, suggesting that placental growth is regulated by IGFs in an autocrine manner. We have previously shown that human decidua, but not placenta, synthesizes and secretes a 34 K IGF-binding protein (34 K IGF-BP) called placental protein 12. We now used human choriocarcinoma JEG-3 cell monolayer cultures and recombinant (Thr59)IGF-I as a model to study whether the decidual 34 K IGF-BP is able to modulate the receptor binding and biological activity of IGFs in trophoblasts. JEG-3 cells, which possess type I IGF receptors, were unable to produce IGF-BPs. Purifiedmore » 34 K IGF-BP specifically bound (125I)iodo-(Thr59)IGF-I. Multiplication-stimulating activity had 2.5% the potency of (Thr59)IGF-I, and insulin had no effect on the binding of (125I) iodo-(Thr59)IGF-I. 34 K IGF-BP inhibited the binding of (125I) iodo-(Thr59)IGF-I to JEG-3 monolayers in a concentration-dependent manner by forming with the tracer a soluble complex that could not bind to the cell surface as demonstrated by competitive binding and cross-linking experiments. After incubating the cell monolayers with (125I)iodo-(Thr59)IGF-I in the presence of purified binding protein, followed by cross-linking, no affinity labeled bands were seen on autoradiography. In contrast, an intensely labeled band at 40 K was detected when the incubation medium was analyzed, suggesting that (Thr59)IGF-I and 34 K IGF-BP formed a complex in a 1:1 molar ratio. Also, 34 K IGF-BP inhibited both basal and IGF-I-stimulated uptake of alpha-(3H)aminoisobutyric acid in JEG-3 cells. RNA analysis revealed that IGF-II is expressed in JEG-3 cells.« less

The DNA replication licensing factor miniature chromosome maintenance 7 is essential for RNA splicing of epidermal growth factor receptor, c-Met, and platelet-derived growth factor receptor.

PubMed

Chen, Zhang-Hui; Yu, Yan P; Michalopoulos, George; Nelson, Joel; Luo, Jian-Hua

2015-01-16

Miniature chromosome maintenance 7 (MCM7) is an essential component of DNA replication licensing complex. Recent studies indicate that MCM7 is amplified and overexpressed in a variety of human malignancies. In this report, we show that MCM7 binds SF3B3. The binding motif is located in the N terminus (amino acids 221-248) of MCM7. Knockdown of MCM7 or SF3B3 significantly increased unspliced RNA of epidermal growth factor receptor, platelet-derived growth factor receptor, and c-Met. A dramatic drop of reporter gene expression of the oxytocin exon 1-intron-exon 2-EGFP construct was also identified in SF3B3 and MCM7 knockdown PC3 and DU145 cells. The MCM7 or SF3B3 depleted cell extract failed to splice reporter RNA in in vitro RNA splicing analyses. Knockdown of SF3B3 and MCM7 leads to an increase of cell death of both PC3 and DU145 cells. Such cell death induction is partially rescued by expressing spliced c-Met. To our knowledge, this is the first report suggesting that MCM7 is a critical RNA splicing factor, thus giving significant new insight into the oncogenic activity of this protein. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Overcoming Endocrine Resistance by Targeting ER/FoxA1/IL 8 Axis

DTIC Science & Technology

2016-10-01

INTRODUCTION Approximately 75% of breast cancers express the hormone estrogen receptor α (ER). As a critical determinant in estrogen response and oncogenic...factor of estrogen receptor α (ER)–chromatin binding and function, yet its aberration in endocrine-resistant (Endo-R) breast cancer is unknown. Here, we...positive tumors. FOXA1 | estrogen receptor | breast cancer | transcriptional reprogramming | endocrine resistance About 75% of breast cancers express

The multifaceted subunit interfaces of ionotropic glutamate receptors.

PubMed

Green, Tim; Nayeem, Naushaba

2015-01-01

The past fifteen years has seen a revolution in our understanding of ionotropic glutamate receptor (iGluR) structure, starting with the first view of the ligand binding domain (LBD) published in 1998, and in many ways culminating in the publication of the full-length structure of GluA2 in 2009. These reports have revealed not only the central role played by subunit interfaces in iGluR function, but also myriad binding sites within interfaces for endogenous and exogenous factors. Changes in the conformation of inter-subunit interfaces are central to transmission of ligand gating into pore opening (itself a rearrangement of interfaces), and subsequent closure through desensitization. With the exception of the agonist binding site, which is located entirely within individual subunits, almost all modulatory factors affecting iGluRs appear to bind to sites in subunit interfaces. This review seeks to summarize what we currently understand about the diverse roles interfaces play in iGluR function, and to highlight questions for future research. © 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.

HCV NS3 protease enhances liver fibrosis via binding to and activating TGF-β type I receptor

NASA Astrophysics Data System (ADS)

Sakata, Kotaro; Hara, Mitsuko; Terada, Takaho; Watanabe, Noriyuki; Takaya, Daisuke; Yaguchi, So-Ichi; Matsumoto, Takehisa; Matsuura, Tomokazu; Shirouzu, Mikako; Yokoyama, Shigeyuki; Yamaguchi, Tokio; Miyazawa, Keiji; Aizaki, Hideki; Suzuki, Tetsuro; Wakita, Takaji; Imoto, Masaya; Kojima, Soichi

2013-11-01

Viruses sometimes mimic host proteins and hijack the host cell machinery. Hepatitis C virus (HCV) causes liver fibrosis, a process largely mediated by the overexpression of transforming growth factor (TGF)-β and collagen, although the precise underlying mechanism is unknown. Here, we report that HCV non-structural protein 3 (NS3) protease affects the antigenicity and bioactivity of TGF-β2 in (CAGA)9-Luc CCL64 cells and in human hepatic cell lines via binding to TGF-β type I receptor (TβRI). Tumor necrosis factor (TNF)-α facilitates this mechanism by increasing the colocalization of TβRI with NS3 protease on the surface of HCV-infected cells. An anti-NS3 antibody against computationally predicted binding sites for TβRI blocked the TGF-β mimetic activities of NS3 in vitro and attenuated liver fibrosis in HCV-infected chimeric mice. These data suggest that HCV NS3 protease mimics TGF-β2 and functions, at least in part, via directly binding to and activating TβRI, thereby enhancing liver fibrosis.

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.

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

Diminished survival of human cytotrophoblast cells exposed to hypoxia/reoxygenation injury and associated reduction of heparin-binding epidermal growth factor-like growth factor.

PubMed

Leach, Richard E; Kilburn, Brian A; Petkova, Anelia; Romero, Roberto; Armant, D Randall

2008-04-01

The antiapoptotic action of heparin-binding epidermal growth factor (HBEGF)-like growth factor and its regulation by O(2) constitutes a key factor for trophoblast survival. The hypothesis that cytotrophoblast survival is compromised by exposure to hypoxia-reoxygenation (H/R) injury, which may contribute to preeclampsia and some missed abortions, prompted us to investigate HBEGF regulation and its role as a survival factor during H/R in cytotrophoblast cells. A transformed human first-trimester cytotrophoblast cell line HTR-8/SVneo was exposed to H/R (2% O(2) followed by 20% O(2)) and assessed for HBEGF expression and cell death. Cellular HBEGF declined significantly within 30 minutes of reoxygenation after culture at 2% O(2). H/R significantly reduced proliferation and increased cell death when compared with trophoblast cells cultured continuously at 2% or 20% O(2). Restoration of cell survival also was achieved by adding recombinant HBEGF during reoxygenation. HBEGF inhibited apoptosis through its binding to either human epidermal receptor (HER)-1 or HER4, its cognate receptors. These results provide evidence that cytotrophoblast exposure to H/R induces apoptosis and decreased cell proliferation. HBEGF accumulation is diminished under these conditions, whereas restoration of HBEGF signaling improves trophoblast survival.

Signalling properties and pharmacological analysis of two sulfakinin receptors from the red flour beetle, Tribolium castaneum

USDA-ARS?s Scientific Manuscript database

Sulfakinin is an insect neuropeptide that constitutes an important component of the complex network of hormonal and neural factors that regulate feeding and digestion. The key modulating functions of sulfakinin are mediated by binding and signaling via G-protein coupled receptors. Although a subst...

Neither bST nor Growth Hormone Releasing Factor Alter Expression of Thyroid Hormone Receptors in Liver and Mammary Tissues

USDA-ARS?s Scientific Manuscript database

Physiological effects of thyroid hormones are mediated primarily by binding of triiodothyronine, to specific nuclear receptors. It has been hypothesized that organ-specific changes in production of triiodothyronine from its prohormone, thyroxine, target the action of thyroid hormones to the mammary...

Dimerization-induced corepressor binding and relaxed DNA-binding specificity are critical for PML/RARA-induced immortalization

PubMed Central

Zhou, Jun; Pérès, Laurent; Honoré, Nicole; Nasr, Rihab; Zhu, Jun; de Thé, Hugues

2006-01-01

The pathogenesis of acute promyelocytic leukemia involves the transcriptional repression of master genes of myeloid differentiation by the promyelocytic leukemia–retinoic acid receptor α (PML/RARA) oncogene. PML-enforced RARA homodimerization allows the tighter binding of corepressors, silencing RARA target genes. In addition, homodimerization dramatically extends the spectrum of DNA-binding sites of the fusion protein compared with those of normal RARA. Yet, any contribution of these two properties of PML/RARA to differentiation arrest and immortalization of primary mouse hematopoietic progenitors was unknown. We demonstrate that dimerization-induced silencing mediator of retinoid and thyroid receptors (SMRT)-enhanced binding and relaxed DNA-binding site specificity are both required for efficient immortalization. Thus, enforced RARA dimerization is critical not only for triggering transcriptional repression but also for extending the repertoire of target genes. Our studies exemplify how dimerization-induced gain of functions converts an unessential transcription factor into a dominant oncogenic protein. PMID:16757557

Characterization of binding affinity of CJ-023,423 for human prostanoid EP4 receptor.

PubMed

Murase, Akio; Nakao, Kazunari; Takada, Junji

2008-01-01

In order to characterize the receptor binding pharmacology of CJ-023,423, a potent and selective EP4 antagonist, we performed a radioligand receptor binding assay under various assay conditions. An acidic (pH 6) and hypotonic buffer is a conventional, well-known buffer for prostaglandin E2 receptor binding assays. CJ-023,423 showed moderate binding affinity for human EP4 receptor under conventional buffer conditions. However, its binding affinity was greatly increased under neutral (pH 7.4) and isotonic buffer conditions. In this report, the binding mechanism between CJ-023,423 and human EP4 receptor is discussed based on the binding affinities determined under various assay conditions. Copyright 2008 S. Karger AG, Basel.

Placental hormones, nutrition, and fetal development.

PubMed

Mulay, S; Browne, C A; Varma, D R; Solomon, S

1980-02-01

Fetal growth retardation due to maternal malnutrition is widespread especially in the Third World. Little is known about the mechanisms that regulate the growth of the fetus and placenta during protein malnutrition. It is known that the placental size and levels of circulating placental hormones such as human chorionic gonadotrophins (hCG), human placental lactogen (hPL), and estrogens are affected by the nutritional status of the mother. There is suggestive evidence that during malnutrition, hPL may increase lipolysis and exert a glucose sparing effect in the mother, thereby promoting glucose availability to the fetus. We have studied the influence of dietary protein deficiency on the binding of dexamethasone to the specific cytosol receptors in adult and fetal tissues. A low protein diet in adult male rats is associated with a decrease in dexamethasone binding to liver cytosol receptors. On the other hand, protein deprivation in pregnant female rats leads to an increase in dexamethasone binding to liver cytosol receptors of both the mother and fetus. However, the influences of maternal protein deprivation on dexamethasone receptors in the fetal liver and lungs are not similar. At 21 days gestation the binding of dexamethasone to fetal lung receptors of protein-deficient mothers is lower than that in the controls. These differences at a critical time in the fetal lung development indicate that a fall in receptors for dexamethasone may lead to impaired phospholipid synthesis in fetuses of protein-deficient mothers and point to the importance of nutritional factors in the biochemistry of fetal development.

In Vivo Fluorescence Lifetime Imaging Monitors Binding of Specific Probes to Cancer Biomarkers

PubMed Central

Ardeshirpour, Yasaman; Chernomordik, Victor; Zielinski, Rafal; Capala, Jacek; Griffiths, Gary; Vasalatiy, Olga; Smirnov, Aleksandr V.; Knutson, Jay R.; Lyakhov, Ilya; Achilefu, Samuel; Gandjbakhche, Amir; Hassan, Moinuddin

2012-01-01

One of the most important factors in choosing a treatment strategy for cancer is characterization of biomarkers in cancer cells. Particularly, recent advances in Monoclonal Antibodies (MAB) as primary-specific drugs targeting tumor receptors show that their efficacy depends strongly on characterization of tumor biomarkers. Assessment of their status in individual patients would facilitate selection of an optimal treatment strategy, and the continuous monitoring of those biomarkers and their binding process to the therapy would provide a means for early evaluation of the efficacy of therapeutic intervention. In this study we have demonstrated for the first time in live animals that the fluorescence lifetime can be used to detect the binding of targeted optical probes to the extracellular receptors on tumor cells in vivo. The rationale was that fluorescence lifetime of a specific probe is sensitive to local environment and/or affinity to other molecules. We attached Near-InfraRed (NIR) fluorescent probes to Human Epidermal Growth Factor 2 (HER2/neu)-specific Affibody molecules and used our time-resolved optical system to compare the fluorescence lifetime of the optical probes that were bound and unbound to tumor cells in live mice. Our results show that the fluorescence lifetime changes in our model system delineate HER2 receptor bound from the unbound probe in vivo. Thus, this method is useful as a specific marker of the receptor binding process, which can open a new paradigm in the “image and treat” concept, especially for early evaluation of the efficacy of the therapy. PMID:22384092

BXR, an embryonic orphan nuclear receptor activated by a novel class of endogenous benzoate metabolites

PubMed Central

Blumberg, Bruce; Kang, Heonjoong; Bolado, Jack; Chen, Hongwu; Craig, A. Grey; Moreno, Tanya A.; Umesono, Kazuhiko; Perlmann, Thomas; De Robertis, Eddy M.; Evans, Ronald M.

1998-01-01

Nuclear receptors are ligand-modulated transcription factors that respond to steroids, retinoids, and thyroid hormones to control development and body physiology. Orphan nuclear receptors, which lack identified ligands, provide a unique, and largely untapped, resource to discover new principles of physiologic homeostasis. We describe the isolation and characterization of the vertebrate orphan receptor, BXR, which heterodimerizes with RXR and binds high-affinity DNA sites composed of a variant thyroid hormone response element. A bioactivity-guided screen of embryonic extracts revealed that BXR is activatable by low-molecular-weight molecules with spectral patterns distinct from known nuclear receptor ligands. Mass spectrometry and 1H NMR analysis identified alkyl esters of amino and hydroxy benzoic acids as potent, stereoselective activators. In vitro cofactor association studies, along with competable binding of radiolabeled compounds, establish these molecules as bona fide ligands. Benzoates comprise a new molecular class of nuclear receptor ligand and their activity suggests that BXR may control a previously unsuspected vertebrate signaling pathway. PMID:9573044

In Silico Molecular Interaction of Bisphenol Analogues with Human Nuclear Receptors Reveals their Stronger Affinity vs. Classical Bisphenol A.

PubMed

Sharma, Shikha; Ahmad, Shahzad; Faraz Khan, Mohemmed; Parvez, Suhel; Raisuddin, Sheikh

2018-06-21

Bisphenol A (BPA) is known for endocrine disrupting activity. In order to replace BPA a number of bisphenol analogues have been designed. However, their activity profile is poorly described and little information exists about their endocrine disrupting potential and interactions with nuclear receptors. An understanding of such interaction may unravel mechanism of their molecular action and provide valuable inputs for risk assessment. BPA binds and activates peroxisome proliferator-activated receptors (PPARs) and retinoid X receptors (RXRs) which act as transcription factors and regulate genes involved in glucose, lipid, and cholesterol metabolism and adipogenesis. We studied binding efficiency of 18 bisphenol analogues and BPA with human PPARs and RXRs. Using Maestro Schrodinger 9.4, docking scores of bisphenols were compared with the known endogenous and exogenous ligands of hPPARs and hRXRs. BPA showed good binding efficiency. Several analogues also showed higher binding efficiency than BPA. BPPH which has high tendency to be absorbed in tissues showed the strongest binding with hPPARα, hPPARβ, hPPARγ and hRXRα whereas two of the most toxic bisphenols, BPM and BPAF showed strongest binding with hRXRβ and hRXRγ. Some of the bisphenol analogues showed a stronger binding affinity with PPAR and RXR compared to BPA implying that BPA substitutes may not be fully safe and chemico-biological interactions indicate their toxic potential. These results may also serve to plan further studies for determining safety profile of bisphenol analogues and be helpful in risk characterization.

Insulin-like growth factor II messenger RNA-binding protein-3 is an independent prognostic factor in uterine leiomyosarcoma.

PubMed

Yasutake, Nobuko; Ohishi, Yoshihiro; Taguchi, Kenichi; Hiraki, Yuka; Oya, Masafumi; Oshiro, Yumi; Mine, Mari; Iwasaki, Takeshi; Yamamoto, Hidetaka; Kohashi, Kenichi; Sonoda, Kenzo; Kato, Kiyoko; Oda, Yoshinao

2018-04-01

The aim of this study was to identify the prognostic factors of uterine leiomyosarcoma (ULMS). We reviewed 60 cases of surgically resected ULMSs and investigated conventional clinicopathological factors, together with the expression of insulin-like growth factor II messenger RNA-binding protein-3 (IMP3), hormone receptors and cell cycle regulatory markers by immunohistochemistry. Mediator complex subunit 12 (MED12) mutation analysis was also performed. Univariate analyses revealed that advanced stage (P < 0.0001), older age (P = 0.0244) and IMP3 expression (P = 0.0011) were significant predictors of a poor outcome. Multivariate analysis revealed advanced stage (P < 0.0001) and IMP3 (P = 0.0373) as independent predictors of a poor prognosis. Expressions of cell cycle markers and hormone receptors, and MED12 mutations (12% in ULMSs) were not identified as prognostic markers in this study. IMP3 expression in ULMS could be a marker of a poor prognosis. © 2017 John Wiley & Sons Ltd.

Angiotensin II AT1 receptor blocker candesartan prevents the fast up-regulation of cerebrocortical benzodiazepine-1 receptors induced by acute inflammatory and restraint stress

PubMed Central

Sánchez-Lemus, Enrique; Honda, Masaru; Saavedra, Juan M.

2012-01-01

Centrally acting Angiotensin II AT1 receptor blockers (ARBs) protect from stress-induced disorders and decrease anxiety in a model of inflammatory stress, the systemic injection of bacterial endotoxin lipopolysaccharide (LPS). In order to better understand the anxiolytic effect of ARBs, we treated rats with LPS (50 µg/kg) with or without three days of pretreatment with the ARB candesartan (1 mg/kg/day), and studied cortical benzodiazepine (BZ) and corticotrophin-releasing factor (CRF) receptors. We compared the cortical BZ and CRF receptors expression pattern induced by LPS with that produced in restraint stress. Inflammation stress produced a generalized increase in cortical BZ1 receptors and reduced mRNA expression of the GABAA receptor γ2 subunit in cingulate cortex; changes were prevented by candesartan pretreatment. Moreover, restraint stress produced similar increases in cortical BZ1 receptor binding, and candesartan prevented these changes. Treatment with candesartan alone increased cortical BZ1 binding, and decreased γ2 subunit mRNA expression in the cingulate cortex. Conversely, we did not find changes in CRF1 receptor expression in any of the cortical areas studied, either after inflammation or restraint stress. Cortical CRF2 receptor binding was undetectable, but CRF2 mRNA expression was decreased by inflammation stress, a change prevented by candesartan. We conclude that stress promotes rapid and widespread changes in cortical BZ1 receptor expression; and that the stress-induced BZ1 receptor expression is under the control of AT1 receptor activity. The results suggest that the anti-anxiety effect of ARBs may be associated with their capacity to regulate stress-induced alterations in cortical BZ1 receptors. PMID:22503782

Biocompatible medical implant materials with binding sites for a biodegradable drug-delivery system

PubMed Central

Al-Dubai, Haifa; Pittner, Gisela; Pittner, Fritz; Gabor, Franz

2011-01-01

Feasibility studies have been carried out for development of a biocompatible coating of medical implant materials allowing the binding of biodegradable drug-delivery systems in a way that their reloading might be possible. These novel coatings, able to bind biodegradable nanoparticles, may serve in the long run as drug carriers to mediate local pharmacological activity. After biodegradation of the nanoparticles, the binding sites could be reloaded with fresh drug-delivering particles. As a suitable receptor system for the nanoparticles, antibodies are anchored. The design of the receptor is of great importance as any bio- or chemorecognitive interaction with other components circulating in the blood has to be avoided. Furthermore, the binding between receptor and the particles has to be strong enough to keep them tightly bound during their lifetime, but on the other hand allow reloading after final degradation of the particles. The nanoparticles suggested as a drug-delivery system for medical implants can be loaded with different pharmaceuticals such as antibiotics, growth factors, or immunosuppressives. This concept may enable the changing of medication, even after implantation of the medical device, if afforded by patients’ needs. PMID:24198488

Insight into Buffalo (Bubalus bubalis) RIG1 and MDA5 Receptors: A Comparative Study on dsRNA Recognition and In-Vitro Antiviral Response

PubMed Central

Singh, Manvender; Brahma, Biswajit; Maharana, Jitendra; Patra, Mahesh Chandra; Kumar, Sushil; Mishra, Purusottam; Saini, Megha; De, Bidhan Chandra; Mahanty, Sourav; Datta, Tirtha Kumar; De, Sachinandan

2014-01-01

RIG1 and MDA5 have emerged as important intracellular innate pattern recognition receptors that recognize viral RNA and mediate cellular signals controlling Type I interferon (IFN-I) response. Buffalo RIG1 and MDA5 genes were investigated to understand the mechanism of receptor induced antiviral response. Sequence analysis revealed that RIG1 and MDA5 maintain a domain arrangement that is common in mammals. Critical binding site residues of the receptors are evolutionary conserved among mammals. Molecular dynamics simulations suggested that RIG1 and MDA5 follow a similar, if not identical, dsRNA binding pattern that has been previously reported in human. Moreover, binding free energy calculation revealed that MDA5 had a greater affinity towards dsRNA compared to RIG1. Constitutive expressions of RLR genes were ubiquitous in different tissues without being specific to immune organs. Poly I:C stimulation induced elevated expressions of IFN-β and IFN-stimulated genes (ISGs) through interferon regulatory factors (IRFs) mediated pathway in buffalo foetal fibroblast cells. The present study provides crucial insights into the structure and function of RIG1 and MDA5 receptors in buffalo. PMID:24587036

The Macrophage Galactose-Type Lectin Can Function as an Attachment and Entry Receptor for Influenza Virus

PubMed Central

Ng, Wy Ching; Liong, Stella; Tate, Michelle D.; Irimura, Tatsuro; Denda-Nagai, Kaori; Brooks, Andrew G.; Londrigan, Sarah L.

2014-01-01

Specific protein receptors that mediate internalization and entry of influenza A virus (IAV) have not been identified for any cell type. Sialic acid (SIA), the primary attachment factor for IAV hemagglutinin, is expressed by numerous cell surface glycoproteins and glycolipids, confounding efforts to identify specific receptors involved in virus infection. Lec1 Chinese hamster ovary (CHO) epithelial cells express cell surface SIA and bind IAV yet are largely resistant to infection. Here, we demonstrate that expression of the murine macrophage galactose-type lectin 1 (MGL1) by Lec1 cells enhanced Ca2+-dependent IAV binding and restored permissivity to infection. Lec1 cells expressing MGL1 were infected in the presence or absence of cell surface SIA, indicating that MGL1 can act as a primary receptor or as a coreceptor with SIA. Lec1 cells expressing endocytosis-deficient MGL1 mediated Ca2+-dependent IAV binding but were less sensitive to IAV infection, indicating that direct internalization via MGL1 can result in cellular infection. Together, these studies identify MGL1 as a cell surface glycoprotein that can act as an authentic receptor for both attachment and infectious entry of IAV. PMID:24257596

Transforming Growth Factor β Suppresses Peroxisome Proliferator–Activated Receptor γ Expression via Both SMAD Binding and Novel TGF-β Inhibitory Elements

PubMed Central

Lakshmi, Sowmya P.; Reddy, Aravind T.; Reddy, Raju C.

2017-01-01

Transforming growth factor β (TGF-β) contributes to wound healing and, when dysregulated, to pathological fibrosis. TGF-β and the anti-fibrotic nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) repress each other’s expression, and such PPARγ downregulation is prominent in fibrosis and mediated, via previously unknown SMAD-signaling mechanisms. Here we show that TGF-β induces association of SMAD3 with both SMAD4, needed for translocation of the complex into the nucleus, and the essential context-sensitive corepressors E2F4 and p107. The complex mediates TGF-β-induced repression by binding to regulatory elements in the target promoter. In the PPARG promoter, we found that the SMAD3-SMAD4 complex binds both to a previously unknown consensus TGF-β inhibitory element (TIE) and also to canonical SMAD-binding elements (SBEs). Furthermore, the TIE and SBEs independently mediated partial repression of PPARG transcription, the first demonstration of a TIE and SBEs functioning within the same promoter. Also, TGF-β-treated fibroblasts contained SMAD complexes that activated a SMAD target gene in addition to those repressing PPARG transcription, the first finding of such dual activity within the same cell. These findings describe in detail novel mechanisms by which TGF-β represses PPARG transcription, thereby facilitating its own pro-fibrotic activity. PMID:28100650

Hypoxia-inducible factor 1 mediates hypoxia-induced cardiomyocyte lipid accumulation by reducing the DNA binding activity of peroxisome proliferator-activated receptor {alpha}/retinoid X receptor

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

Belanger, Adam J.; Luo Zhengyu; Vincent, Karen A.

2007-12-21

In response to cellular hypoxia, cardiomyocytes adapt to consume less oxygen by shifting ATP production from mitochondrial fatty acid {beta}-oxidation to glycolysis. The transcriptional activation of glucose transporters and glycolytic enzymes by hypoxia is mediated by hypoxia-inducible factor 1 (HIF-1). In this study, we examined whether HIF-1 was involved in the suppression of mitochondrial fatty acid {beta}-oxidation in hypoxic cardiomyocytes. We showed that either hypoxia or adenovirus-mediated expression of a constitutively stable hybrid form (HIF-1{alpha}/VP16) suppressed mitochondrial fatty acid metabolism, as indicated by an accumulation of intracellular neutral lipid. Both treatments also reduced the mRNA levels of muscle carnitine palmitoyltransferasemore » I which catalyzes the rate-limiting step in the mitochondrial import of fatty acids for {beta}-oxidation. Furthermore, adenovirus-mediated expression of HIF-1{alpha}/VP16 in cardiomyocytes under normoxic conditions also mimicked the reduction in the DNA binding activity of peroxisome proliferator-activated receptor {alpha} (PPAR{alpha})/retinoid X receptor (RXR), in the presence or absence of a PPAR{alpha} ligand. These results suggest that HIF-1 may be involved in hypoxia-induced suppression of fatty acid metabolism in cardiomyocytes by reducing the DNA binding activity of PPAR{alpha}/RXR.« less

The retinol esterifying enzyme LRAT supports cell signaling by retinol-binding protein and its receptor STRA6.

PubMed

Marwarha, Gurdeep; Berry, Daniel C; Croniger, Colleen M; Noy, Noa

2014-01-01

Vitamin A, retinol, circulates in blood bound to retinol-binding protein (RBP). At some tissues, holo-RBP is recognized by a plasma membrane receptor termed STRA6, which serves a dual role: it mediates transport of retinol from RBP into cells, and it functions as a cytokine receptor that, on binding holo-RBP, activates JAK2/STAT5 signaling. As STAT target genes include SOCS3, an inhibitor of insulin receptor, holo-RBP suppresses insulin responses in STRA6-expressing cells. We have shown previously that the two functions of STRA6 are interdependent. These observations suggest factors that regulate STRA6-mediated retinol transport may also control STRA6-mediated cell signaling. One such factor is retinol metabolism, which enables cellular uptake of retinol by maintaining an inward-directed concentration gradient. We show here that lecithin:retinol acyl transferase (LRAT), which catalyzes esterification of retinol to its storage species retinyl esters, is necessary for activation of the STRA6/JAK2/STAT5 cascade by holo-RBP. In accordance, LRAT-null mice are protected from holo-RBP-induced suppression of insulin responses. Hence, STRA6 signaling, which requires STRA6-mediated retinol transport, is supported by LRAT-catalyzed retinol metabolism. The observations demonstrate that STRA6 regulates key cellular processes by coupling circulating holo-RBP levels and intracellular retinol metabolism to cell signaling.

In vitro pharmacological characterization of CJ-042794, a novel, potent, and selective prostaglandin EP(4) receptor antagonist.

PubMed

Murase, Akio; Taniguchi, Yasuhito; Tonai-Kachi, Hiroko; Nakao, Kazunari; Takada, Junji

2008-01-16

Activation of the prostaglandin E(2) (PGE(2)) EP(4) receptor, a G-protein-coupled receptor (GPCR), results in increases in intracellular cyclic AMP (cAMP) levels via stimulation of adenylate cyclase. Here we describe the in vitro pharmacological characterization of a novel EP(4) receptor antagonist, CJ-042794 (4-{(1S)-1-[({5-chloro-2-[(4-fluorophenyl)oxy]phenyl}carbonyl)amino]ethyl}benzoic acid). CJ-042794 inhibited [(3)H]-PGE(2) binding to the human EP(4) receptor with a mean pK(i) of 8.5, a binding affinity that was at least 200-fold more selective for the human EP(4) receptor than other human EP receptor subtypes (EP(1), EP(2), and EP(3)). CJ-042794 did not exhibit any remarkable binding to 65 additional proteins, including GPCRs, enzymes, and ion channels, suggesting that CJ-042794 is highly selective for the EP(4) receptor. CJ-042794 competitively inhibited PGE(2)-evoked elevations of intracellular cAMP levels in HEK293 cells overexpressing human EP(4) receptor with a mean pA(2) value of 8.6. PGE(2) inhibited the lipopolysaccharide (LPS)-induced production of tumor necrosis factor alpha (TNFalpha) in human whole blood (HWB); CJ-042794 reversed the inhibitory effects of PGE(2) on LPS-induced TNFalpha production in a concentration-dependent manner. These results suggest that CJ-042794, a novel, potent, and selective EP(4) receptor antagonist, has excellent pharmacological properties that make it a useful tool for exploring the physiological role of EP(4) receptors.

FcγRII-binding Centyrins mediate agonism and antibody-dependent cellular phagocytosis when fused to an anti-OX40 antibody

PubMed Central

Zhang, Di; Whitaker, Brian; Derebe, Mehabaw G.; Chiu, Mark L.

2018-01-01

ABSTRACT Immunostimulatory antibodies against the tumor necrosis factor receptors (TNFR) are emerging as promising cancer immunotherapies. The agonism activity of such antibodies depends on crosslinking to Fc gamma RIIB receptor (FcγRIIB) to enable the antibody multimerization that drives TNFR activation. Previously, Fc engineering was used to enhance the binding of such antibodies to Fcγ receptors. Here, we report the identification of Centyrins as alternative scaffold proteins with binding affinities to homologous FcγRIIB and FcγRIIA, but not to other types of Fcγ receptors. One Centyrin, S29, was engineered at distinct positions of an anti-OX40 SF2 antibody to generate bispecific and tetravalent molecules named as mAbtyrins. Regardless of the position of S29 on the SF2 antibody, SF2-S29 mAbtyrins could bind FcγRIIB and FcγRIIA specifically while maintaining binding to OX40 receptors. In a NFκB reporter assay, attachment of S29 Centyrin molecules at the C-termini, but not the N-termini, resulted in SF2 antibodies with increased agonism owing to FcγRIIB crosslinking. The mAbtyrins also showed agonism in T-cell activation assays with immobilized FcγRIIB and FcγRIIA, but this activity was confined to mAbtyrins with S29 specifically at the C-termini of antibody heavy chains. Furthermore, regardless of the position of the molecule, S29 Centyrin could equip an otherwise Fc-silent antibody with antibody-dependent cellular phagocytosis activity without affecting the antibody's intrinsic antibody-dependent cell-meditated cytotoxicity and complement-dependent cytotoxicity. In summary, the appropriate adoption FcγRII-binding Centyrins as functional modules represents a novel strategy to engineer therapeutic antibodies with improved functionalities. PMID:29359992

Inhibition of estrogen-responsive gene activation by the retinoid X receptor beta: evidence for multiple inhibitory pathways.

PubMed

Segars, J H; Marks, M S; Hirschfeld, S; Driggers, P H; Martinez, E; Grippo, J F; Brown, M; Wahli, W; Ozato, K

1993-04-01

The retinoid X receptor beta (RXR beta; H-2RIIBP) forms heterodimers with various nuclear hormone receptors and binds multiple hormone response elements, including the estrogen response element (ERE). In this report, we show that endogenous RXR beta contributes to ERE binding activity in nuclear extracts of the human breast cancer cell line MCF-7. To define a possible regulatory role of RXR beta regarding estrogen-responsive transcription in breast cancer cells, RXR beta and a reporter gene driven by the vitellogenin A2 ERE were transfected into estrogen-treated MCF-7 cells. RXR beta inhibited ERE-driven reporter activity in a dose-dependent and element-specific fashion. This inhibition occurred in the absence of the RXR ligand 9-cis retinoic acid. The RXR beta-induced inhibition was specific for estrogen receptor (ER)-mediated ERE activation because inhibition was observed in ER-negative MDA-MB-231 cells only following transfection of the estrogen-activated ER. No inhibition of the basal reporter activity was observed. The inhibition was not caused by simple competition of RXR beta with the ER for ERE binding, since deletion mutants retaining DNA binding activity but lacking the N-terminal or C-terminal domain failed to inhibit reporter activity. In addition, cross-linking studies indicated the presence of an auxiliary nuclear factor present in MCF-7 cells that contributed to RXR beta binding of the ERE. Studies using known heterodimerization partners of RXR beta confirmed that RXR beta/triiodothyronine receptor alpha heterodimers avidly bind the ERE but revealed the existence of another triiodothyronine-independent pathway of ERE inhibition. These results indicate that estrogen-responsive genes may be negatively regulated by RXR beta through two distinct pathways.

Residues W320 and Y328 within the binding site of the μ-opioid receptor influence opiate ligand bias.

PubMed

Hothersall, J Daniel; Torella, Rubben; Humphreys, Sian; Hooley, Monique; Brown, Alastair; McMurray, Gordon; Nickolls, Sarah A

2017-05-15

The development of G protein-biased agonists for the μ-opioid receptor (MOR) offers a clear drug discovery rationale for improved analgesia and reduced side-effects of opiate pharmacotherapy. However, our understanding of the molecular mechanisms governing ligand bias is limited, which hinders our ability to rationally design biased compounds. We have investigated the role of MOR binding site residues W320 and Y328 in controlling bias, by receptor mutagenesis. The pharmacology of a panel of ligands in a cAMP and a β-arrestin2 assay were compared between the wildtype and mutated receptors, with bias factors calculated by operational analysis using ΔΔlog(τ/K A ) values. [ 3 H]diprenorphine competition binding was used to estimate affinity changes. Introducing the mutations W320A and Y328F caused changes in pathway bias, with different patterns of change between ligands. For example, DAMGO increased relative β-arrestin2 activity at the W320A mutant, whilst its β-arrestin2 response was completely lost at Y328F. In contrast, endomorphin-1 gained activity with Y328F but lost activity at W320A, in both pathways. For endomorphin-2 there was a directional shift from cAMP bias at the wildtype towards more β-arrestin2 bias at W320A. We also observe clear uncoupling between mutation-driven changes in function and binding affinity. These findings suggest that the mutations influenced the balance of pathway activation in a ligand-specific manner, thus identifying residues in the MOR binding pocket that govern ligand bias. This increases our understanding of how ligand/receptor binding interactions can be translated into agonist-specific pathway activation. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Analysis of Nuclear Factor-κB (NF-κB) Essential Modulator (NEMO) Binding to Linear and Lysine-linked Ubiquitin Chains and Its Role in the Activation of NF-κB*

PubMed Central

Kensche, Tobias; Tokunaga, Fuminori; Ikeda, Fumiyo; Goto, Eiji; Iwai, Kazuhiro; Dikic, Ivan

2012-01-01

Nuclear factor-κB (NF-κB) essential modulator (NEMO), a component of the inhibitor of κB kinase (IKK) complex, controls NF-κB signaling by binding to ubiquitin chains. Structural studies of NEMO provided a rationale for the specific binding between the UBAN (ubiquitin binding in ABIN and NEMO) domain of NEMO and linear (Met-1-linked) di-ubiquitin chains. Full-length NEMO can also interact with Lys-11-, Lys-48-, and Lys-63-linked ubiquitin chains of varying length in cells. Here, we show that purified full-length NEMO binds preferentially to linear ubiquitin chains in competition with lysine-linked ubiquitin chains of defined length, including long Lys-63-linked deca-ubiquitins. Linear di-ubiquitins were sufficient to activate both the IKK complex in vitro and to trigger maximal NF-κB activation in cells. In TNFα-stimulated cells, NEMO chimeras engineered to bind exclusively to Lys-63-linked ubiquitin chains mediated partial NF-κB activation compared with cells expressing NEMO that binds to linear ubiquitin chains. We propose that NEMO functions as a high affinity receptor for linear ubiquitin chains and a low affinity receptor for long lysine-linked ubiquitin chains. This phenomenon could explain quantitatively distinct NF-κB activation patterns in response to numerous cell stimuli. PMID:22605335

[Molecular mechanism of Bupleuri Radix and Scutellariae Radix drug pair for depression based on integrative pharmacology platform of traditional Chinese medicine].

PubMed

Wang, Jian-Ting; Wang, Shang; Liu, Song-Lin; Wang, Yan-Chun; Li, Jia-Geng; Chen, Yu

2018-04-01

Xiaochaihu decoction is a classic prescription of traditional Chinese medicine. Modern research has proved its anti-depression effect. However, its pharmacological mechanism for anti-depression effect is difficult to be unveiled because of the complexity of compound Chinese medicines. Bupleuri Radix and Scutellariae Radix is the core drug pair of Xiaochaihu decoction. In this research, Bupleuri Radix and Scutellariae Radix were analyzed by the integrative pharmacology platform to study its molecular mechanism for anti-depression. One hundred and sixteen active ingredients were predicted, 62 for Bupleuri Radix, mainly including saikosaponins, acids, alcohols, and 54 for Scutellariae Radix, mainly including flavonoids and glycosides. Its anti-depression effect was relevant to 118 core targets, including 22 known disease targets, such as serotonin receptor(HTR2C), activating transcription factor(ATF1, ATF2), δ opioid receptor(OPRD1), μ opioid receptor (OPRM1), κ opioid receptor(OPRK1), inositol monophosphatase(IMPA1), Toll-like receptor 4 (TLR4), histamine H1 receptor(HRH1), neurotrophic factor tyrosine kinase receptor1 (NTRK1), Glycogen synthetase kinase 3β(GSK3β), etc. The antidepressant effect involved positive regulation of transcription from RNA polymerase Ⅱ promoter, transcription factor binding, cytosol, transcriptional regulation of DNA template, enzyme binding, endocrine system, nervous system, neurotrophin signaling pathway, cell growth and death, signal transduction, thyroid hormone signaling pathway and other related biological processes and metabolic pathways. This study provides a scientific evidence for further study of the anti-depression mechanism of this drug pair. Copyright© by the Chinese Pharmaceutical Association.

Crystal Structure of the Pseudomonas aeruginosa Virulence Factor Regulator

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

Cordes, Timothy J.; Worzalla, Gregory A.; Ginster, Aaron M.

2012-09-07

Virulence factor regulator (Vfr) enhances Pseudomonas aeruginosa pathogenicity through its role as a global transcriptional regulator. The crystal structure of Vfr shows that it is a winged-helix DNA-binding protein like its homologue cyclic AMP receptor protein (CRP). In addition to an expected primary cyclic AMP-binding site, a second ligand-binding site is nestled between the N-terminal domain and the C-terminal helix-turn-helix domain. Unlike CRP, Vfr is a symmetric dimer in the absence of DNA. Removal of seven disordered N-terminal residues of Vfr prvents the growth of P. aeruginosa.

Phosphorylation and Intramolecular Stabilization of the Ligand Binding Domain in the Nuclear Receptor Steroidogenic Factor 1

PubMed Central

Desclozeaux, Marion; Krylova, Irina N.; Horn, Florence; Fletterick, Robert J.; Ingraham, Holly A.

2002-01-01

Steroidogenic factor 1 (SF-1) is an orphan nuclear receptor with no known ligand. We showed previously that phosphorylation at serine 203 located N′-terminal to the ligand binding domain (LBD) enhanced cofactor recruitment, analogous to the ligand-mediated recruitment in ligand-dependent receptors. In this study, results of biochemical analyses and an LBD helix assembly assay suggest that the SF-1 LBD adopts an active conformation, with helices 1 and 12 packed against the predicted alpha-helical bundle, in the apparent absence of ligand. Fine mapping of the previously defined proximal activation function in SF-1 showed that the activation function mapped fully to helix 1 of the LBD. Limited proteolyses demonstrate that phosphorylation of S203 in the hinge region mimics the stabilizing effects of ligand on the LBD. Moreover, similar effects were observed in an SF-1/thyroid hormone LBD chimera receptor, illustrating that the S203 phosphorylation effects are transferable to a heterologous ligand-dependent receptor. Our collective data suggest that the hinge together with helix 1 is an individualized specific motif, which is tightly associated with its cognate LBD. For SF-1, we find that this intramolecular association and hence receptor activity are further enhanced by mitogen-activated protein kinase phosphorylation, thus mimicking many of the ligand-induced changes observed for ligand-dependent receptors. PMID:12242296

Structural Biology and Evolution of the TGF-β Family

PubMed Central

Hinck, Andrew P.; Mueller, Thomas D.; Springer, Timothy A.

2017-01-01

We review the evolution and structure of members of the transforming growth factor β (TGF-β) family, antagonistic or agonistic modulators, and receptors that regulate TGF-β signaling in extracellular environments. The growth factor (GF) domain common to all family members and many of their antagonists evolved from a common cystine knot growth factor (CKGF) domain. The CKGF superfamily comprises six distinct families in primitive metazoans, including the TGF-β and Dan families. Compared with Wnt/Frizzled and Notch/Delta families that also specify body axes, cell fate, tissues, and other families that contain CKGF domains that evolved in parallel, the TGF-β family was the most fruitful in evolution. Complexes between the prodomains and GFs of the TGF-β family suggest a new paradigm for regulating GF release by conversion from closed- to open-arm procomplex conformations. Ternary complexes of the final step in extracellular signaling show how TGF-β GF dimers bind type I and type II receptors on the cell surface, and enable understanding of much of the specificity and promiscuity in extracellular signaling. However, structures suggest that when GFs bind repulsive guidance molecule (RGM) family coreceptors, type I receptors do not bind until reaching an intracellular, membrane-enveloped compartment, blurring the line between extra- and intracellular signaling. Modulator protein structures show how structurally diverse antagonists including follistatins, noggin, and members of the chordin family bind GFs to regulate signaling; complexes with the Dan family remain elusive. Much work is needed to understand how these molecular components assemble to form signaling hubs in extracellular environments in vivo. PMID:27638177

Growth hormone receptor deficiency in Ecuador: clinical and biochemical phenotype in two populations.

PubMed

Guevara-Aguirre, J; Rosenbloom, A L; Fielder, P J; Diamond, F B; Rosenfeld, R G

1993-02-01

We have identified 56 patients with GH receptor deficiency (Laron syndrome) from two provinces in southern Ecuador, one group of 26 (Loja province) with a 4:1 female predominance and 30 patients from neighboring El Oro province with a normal sex ratio. There were no significant differences between the Loja and El Oro populations in stature (-5.3 to -11.5 standard deviation score), other auxologic measures, or in biochemical measures. GH binding protein, the circulating extracellular domain of the GH receptor, was measured by ligand immunofunction assay and found to be comparably low in children and adults. Levels of insulin-like growth factor (IGF)-I and -II and the GH-dependent IGF binding protein-3 (measured by RIA) were significantly greater, and GH and IGF binding protein-2 levels significantly lower in adults than children. Levels of IGF-I (adults) and IGF binding protein-3 (children and adults) correlated inversely with statural deviation from normal (P < 0.01). School performance was at an exceptionally high level, 41 out of 47 who had attended school being in the top 3 in classes of 15-50 persons.

(/sup 3/H)Ethylketocyclazocine binding to mouse brain membranes: evidence for a kappa opioid receptor type

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

Garzon, J.; Sanchez-Blazquez, P.; Lee, N.M.

1984-10-01

The binding of the putative kappa agonist ethylketocyclazocine (EKC) to synaptosomal membranes of mouse brain was studied. This benzomorphan was able to bind to different opioid receptors. A portion of this binding was not inhibited by the agonist naloxone, even at high concentrations (10 microM). This population of receptors, to which opioate alkaloids and opiod peptides display very low affinity, is probably the sigma receptor. Another class of binding sites was identified by the simultaneous addition of the selective agonists Sandoz FK-33824 and D-Ala2-D-Leu5-enkephalin, which blocked the access of EKC to mu and delta opioid receptors, respectively, leaving a portionmore » of naloxone-displaceable benzomorphan binding still detectable. Analysis of this remaining binding revealed a small population of receptors of high affinity, the kappa receptor. Therefore, EKC binds to the mu, delta, kappa and sigma receptors in the mouse brain, with similar affinities for the mu and kappa (0.22 and 0.15 nM). These results confirm the existence of a kappa opioid receptor type in the mouse brain.« less

NOD2 Down-Regulates Colonic Inflammation by IRF4-Mediated Inhibition of K63-Linked Polyubiquitination of RICK and TRAF6

PubMed Central

Watanabe, Tomohiro; Asano, Naoki; Meng, Guangxun; Yamashita, Kouhei; Arai, Yasuyuki; Sakurai, Toshiharu; Kudo, Masatoshi; Fuss, Ivan J; Kitani, Atsushi; Shimosegawa, Tooru; Chiba, Tsutomu; Strober, Warren

2014-01-01

It is well established that polymorphisms of the nucleotide-binding oligomerization domain 2 (NOD2) gene, a major risk factor in Crohn's disease (CD), lead to loss of NOD2 function. However, a molecular explanation of how such loss of function leads to increased susceptibility to CD has remained unclear. In a previous study exploring this question we reported that activation of NOD2 in human dendritic cells by its ligand, muramyl dipeptide (MDP) negatively regulates Toll-like receptor (TLR)-mediated inflammatory responses. Here we show that NOD2 activation results in increased interferon regulatory factor 4 (IRF4) expression and binding to TNF receptor associated factor 6 (TRAF6) and receptor interacting serine-threonine kinase (RICK). We then show that such binding leads to IRF4-mediated inhibition of Lys63-linked polyubiquitination of TRAF6 and RICK and thus to down-regulation of NF-κB activation. Finally, we demonstrate that protection of mice from the development of experimental colitis by MDP or IRF4 administration is accompanied by similar IRF4-mediated effects on polyubiquitination of TRAF6 and RICK in colonic lamina propria mononuclear cells. These findings thus define a mechanism of NOD2-mediated regulation of innate immune responses to intestinal microflora that could explain the relation of NOD2 polymorphisms and resultant NOD2 dysfunction to CD. PMID:24670424

Negative Cooperativity in the EGF Receptor

PubMed Central

Pike, Linda J.

2012-01-01

Scatchard analyses of the binding of EGF to its receptor yield concave up Scatchard plots, indicative of some type of heterogenity in ligand binding affinity. This was typically interpreted as being due to the presence of two independent binding site–one of high affinity representing ≤10% of the receptor population and one of low affinity making up the bulk of the receptors. However, the concept of two independent binding sites is difficult to reconcile with the X-ray structures of the dimerized EGF receptor that show symmetric binding of the two ligands. A new approach to the analysis of 125I-EGF binding data combined with the structure of the singly-occupied Drosophila EGF receptor have now shown that this heterogeneity is due to the presence of negative cooperativity in the EGF receptor. Concerns that negative cooperativity precludes ligand-induced dimerization of the EGF receptor confuse the concepts of linkage cooperativity. Linkage refers to the effect of ligand on the assembly of dimers while cooperativity refers to the effect of ligand binding to one subunit on ligand binding to the other subunit within a preassembled dimer. Binding of EGF to its receptor is positively linked with dimer assembly but shows negative cooperativity within the dimer. PMID:22260659

Participation of Water in the Binding of Estrogen Receptor with Estrogen Responsive Element in vitro.

PubMed

Zhu, Guo-Zhang; Tang, Guo-Qing; Ruan, Kang-Cheng; Gong, Yue-Ting; Zhang, Yong-Lian

1998-01-01

Many reports have showed that bound water was involved in the interaction between/among the macromolecules. However, it has not been reported whether bound water is also involved in the binding of trans-factors and cis-elements in the regulation of the eukaryotic gene trans-cription or not. Preliminary studies have been made on the effect of bound water on the binding of estrogen receptor with estrogen responsive element in vitro. In the gel retardation assay using the cytosol extract of rat uterus as the supplier of estrogen receptor and 32 bp oligonucleotide containing a concensus vitellogenin A(2) ERE as the probe, various cosolvents, such as glycerol, sucrose, N-dimethylformamide and dimethylsulfoxide, were added respectively to the reaction mixture in varying concentrations to regulate the osmotic pressure. The results indicated that the binding of ER-ERE was enhanced with the increase in the final concentration of these individual cosolvents. On the other hand, when the reaction was carried out under an increasing hydrostatic pressure, the ER-ERE binding was decreased sharply. After decompression the binding of ER-ERE was gradually restored to the normal level with the lapse of time. These results suggested that bound water was directly involved in the binding of ER-ERE and may play an important role in the regulation of the eukaryotic gene transcription.

Structural Characterization of the Interaction of the Fibroblast Growth Factor Receptor with a Small Molecule Allosteric Inhibitor.

PubMed

Kappert, Franziska; Sreeramulu, Sridhar; Jonker, Hendrik R A; Richter, Christian; Rogov, Vladimir V; Proschak, Ewgenij; Hargittay, Bruno; Saxena, Krishna; Schwalbe, Harald

2018-06-04

The interaction of fibroblast growth factors (FGFs) with their fibroblast growth factor receptors (FGFRs) are important in the signaling network of cell growth and development. SSR128129E (SSR), a ligand of small molecular weight with potential anti-cancer properties, acts allosterically on the extracellular domains of FGFRs. Up to now, the structural basis of SSR binding to the D3 domain of FGFR remained elusive. This work reports the structural characterization of the interaction of SSR with one specific receptor, FGFR3, by NMR spectroscopy. This information provides a basis for rational drug design for allosteric FGFR inhibitors. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Insulin-like growth factor binding protein-3 (IGFBP-3): Novel ligands mediate unexpected functions.

PubMed

Baxter, Robert C

2013-08-01

In addition to its important role in the regulation of somatic growth by acting as the major circulating transport protein for the insulin-like growth factors (IGFs), IGF binding protein-3 (IGFBP-3) has a variety of intracellular ligands that point to its function within major signaling pathways. The discovery of its interaction with the retinoid X receptor has led to the elucidation of roles in regulating the function of several nuclear hormone receptors including retinoic acid receptor-α, Nur77 and vitamin D receptor. Its interaction with the nuclear hormone receptor peroxisome proliferator-activated receptor-γ is believed to be involved in regulating adipocyte differentiation, which is also modulated by IGFBP-3 through an interaction with TGFβ/Smad signaling. IGFBP-3 can induce apoptosis alone or in conjunction with other agents, and in different systems can activate caspases -8 and -9. At least two unrelated proteins (LRP1 and TMEM219) have been designated as receptors for IGFBP-3, the latter with a demonstrated role in inducing caspase-8-dependent apoptosis. In contrast, IGFBP-3 also has demonstrated roles in survival-related functions, including the repair of DNA double-strand breaks through interaction with the epidermal growth factor receptor and DNA-dependent protein kinase, and the induction of autophagy through interaction with GRP78. The ability of IGFBP-3 to modulate the balance between pro-apoptotic and pro-survival sphingolipids by regulating sphingosine kinase 1 and sphingomyelinases may be integral to its role at the crossroads between cell death and survival in response to a variety of stimuli. The pleiotropic nature of IGFBP-3 activity supports the idea that IGFBP-3 itself, or pathways with which it interacts, should be investigated as targets of therapy for a variety of diseases.

G Protein-regulated inducer of neurite outgrowth (GRIN) modulates Sprouty protein repression of mitogen-activated protein kinase (MAPK) activation by growth factor stimulation.

PubMed

Hwangpo, Tracy Anh; Jordan, J Dedrick; Premsrirut, Prem K; Jayamaran, Gomathi; Licht, Jonathan D; Iyengar, Ravi; Neves, Susana R

2012-04-20

Gα(o/i) interacts directly with GRIN (G protein-regulated inducer of neurite outgrowth). Using the yeast two-hybrid system, we identified Sprouty2 as an interacting partner of GRIN. Gα(o) and Sprouty2 bind to overlapping regions of GRIN, thus competing for GRIN binding. Imaging experiments demonstrated that Gα(o) expression promoted GRIN translocation to the plasma membrane, whereas Sprouty2 expression failed to do so. Given the role of Sprouty2 in the regulation of growth factor-mediated MAPK activation, we examined the contribution of the GRIN-Sprouty2 interaction to CB1 cannabinoid receptor regulation of FGF receptor signaling. In Neuro-2A cells, a system that expresses all of the components endogenously, modulation of GRIN levels led to regulation of MAPK activation. Overexpression of GRIN potentiated FGF activation of MAPK and decreased tyrosine phosphorylation of Sprouty2. Pretreatment with G(o/i)-coupled CB1 receptor agonist attenuated subsequent FGF activation of MAPK. Decreased expression of GRIN both diminished FGF activation of MAPK and blocked CB1R attenuation of MAPK activation. These observations indicate that Gα(o) interacts with GRIN and outcompetes GRIN from bound Sprouty. Free Sprouty then in turn inhibits growth factor signaling. Thus, here we present a novel mechanism of how G(o/i)-coupled receptors can inhibit growth factor signaling to MAPK.

Dimensionality of Motion and Binding Valency Govern Receptor-Ligand Kinetics As Revealed by Agent-Based Modeling.

PubMed

Lehnert, Teresa; Figge, Marc Thilo

2017-01-01

Mathematical modeling and computer simulations have become an integral part of modern biological research. The strength of theoretical approaches is in the simplification of complex biological systems. We here consider the general problem of receptor-ligand binding in the context of antibody-antigen binding. On the one hand, we establish a quantitative mapping between macroscopic binding rates of a deterministic differential equation model and their microscopic equivalents as obtained from simulating the spatiotemporal binding kinetics by stochastic agent-based models. On the other hand, we investigate the impact of various properties of B cell-derived receptors-such as their dimensionality of motion, morphology, and binding valency-on the receptor-ligand binding kinetics. To this end, we implemented an algorithm that simulates antigen binding by B cell-derived receptors with a Y-shaped morphology that can move in different dimensionalities, i.e., either as membrane-anchored receptors or as soluble receptors. The mapping of the macroscopic and microscopic binding rates allowed us to quantitatively compare different agent-based model variants for the different types of B cell-derived receptors. Our results indicate that the dimensionality of motion governs the binding kinetics and that this predominant impact is quantitatively compensated by the bivalency of these receptors.

TRAF molecules in cell signaling and in human diseases

PubMed Central

2013-01-01

The tumor necrosis factor receptor (TNF-R)-associated factor (TRAF) family of intracellular proteins were originally identified as signaling adaptors that bind directly to the cytoplasmic regions of receptors of the TNF-R superfamily. The past decade has witnessed rapid expansion of receptor families identified to employ TRAFs for signaling. These include Toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), T cell receptor, IL-1 receptor family, IL-17 receptors, IFN receptors and TGFβ receptors. In addition to their role as adaptor proteins, most TRAFs also act as E3 ubiquitin ligases to activate downstream signaling events. TRAF-dependent signaling pathways typically lead to the activation of nuclear factor-κBs (NF-κBs), mitogen-activated protein kinases (MAPKs), or interferon-regulatory factors (IRFs). Compelling evidence obtained from germ-line and cell-specific TRAF-deficient mice demonstrates that each TRAF plays indispensable and non-redundant physiological roles, regulating innate and adaptive immunity, embryonic development, tissue homeostasis, stress response, and bone metabolism. Notably, mounting evidence implicates TRAFs in the pathogenesis of human diseases such as cancers and autoimmune diseases, which has sparked new appreciation and interest in TRAF research. This review presents an overview of the current knowledge of TRAFs, with an emphasis on recent findings concerning TRAF molecules in signaling and in human diseases. PMID:23758787

An engineered transforming growth factor β (TGF-β) monomer that functions as a dominant negative to block TGF-β signaling

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

Kim, Sun Kyung; Barron, Lindsey; Hinck, Cynthia S.

The transforming growth factor β isoforms, TGF-β1, -β2, and -β3, are small secreted homodimeric signaling proteins with essential roles in regulating the adaptive immune system and maintaining the extracellular matrix. However, dysregulation of the TGF-β pathway is responsible for promoting the progression of several human diseases, including cancer and fibrosis. Despite the known importance of TGF-βs in promoting disease progression, no inhibitors have been approved for use in humans. Herein, we describe an engineered TGF-β monomer, lacking the heel helix, a structural motif essential for binding the TGF-β type I receptor (TβRI) but dispensable for binding the other receptor requiredmore » for TGF-β signaling, the TGF-β type II receptor (TβRII), as an alternative therapeutic modality for blocking TGF-β signaling in humans. As shown through binding studies and crystallography, the engineered monomer retained the same overall structure of native TGF-β monomers and bound TβRII in an identical manner. Cell-based luciferase assays showed that the engineered monomer functioned as a dominant negative to inhibit TGF-β signaling with a Ki of 20–70 nM. Investigation of the mechanism showed that the high affinity of the engineered monomer for TβRII, coupled with its reduced ability to non-covalently dimerize and its inability to bind and recruit TβRI, enabled it to bind endogenous TβRII but prevented it from binding and recruiting TβRI to form a signaling complex. Such engineered monomers provide a new avenue to probe and manipulate TGF-β signaling and may inform similar modifications of other TGF-β family members.« less

Identification and grafting of a unique peptide-binding site in the Fab framework of monoclonal antibodies

DOE PAGES

Donaldson, Joshua M.; Zer, Cindy; Avery, Kendra N.; ...

2013-10-07

Capitalizing on their extraordinary specificity, monoclonal antibodies (mAbs) have become one of the most reengineered classes of biological molecules. A major goal in many of these engineering efforts is to add new functionality to the parental mAb, including the addition of cytotoxins and imaging agents for medical applications. Herein, we present a unique peptide-binding site within the central cavity of the fragment antigen binding framework region of the chimeric, anti-epidermal growth factor receptor mAb cetuximab. We demonstrate through diffraction methods, biophysical studies, and sequence analysis that this peptide, a meditope, has moderate affinity for the Fab, is specific to cetuximabmore » (i.e., does not bind to human IgGs), and has no significant effect on antigen binding. We further demonstrate by diffraction studies and biophysical methods that the meditope binding site can be grafted onto the anti-human epidermal growth factor receptor 2 mAb trastuzumab, and that the antigen binding affinity of the grafted trastuzumab is indistinguishable from the parental mAb. Lastly, we demonstrate a bivalent meditope variant binds specifically and stably to antigen-bearing cells only in the presence of the meditope-enabled mAbs. Collectively, this finding and the subsequent characterization and engineering efforts indicate that this unique interface could serve as a noncovalent “linker” for any meditope-enabled mAb with applications in multiple mAb-based technologies including diagnostics, imaging, and therapeutic delivery.« less

Bacterial production and structure-functional validation of a recombinant antigen-binding fragment (Fab) of an anti-cancer therapeutic antibody targeting epidermal growth factor receptor.

PubMed

Kim, Ji-Hun; Sim, Dae-Won; Park, Dongsun; Jung, Tai-Geun; Lee, Seonghwan; Oh, Taeheun; Ha, Jong-Ryul; Seok, Seung-Hyeon; Seo, Min-Duk; Kang, Ho Chul; Kim, Young Pil; Won, Hyung-Sik

2016-12-01

Fragment engineering of monoclonal antibodies (mAbs) has emerged as an excellent paradigm to develop highly efficient therapeutic and/or diagnostic agents. Engineered mAb fragments can be economically produced in bacterial systems using recombinant DNA technologies. In this work, we established recombinant production in Escherichia coli for monovalent antigen-binding fragment (Fab) adopted from a clinically used anticancer mAB drug cetuximab targeting epidermal growth factor receptor (EGFR). Recombinant DNA constructs were designed to express both polypeptide chains comprising Fab in a single vector and to secrete them to bacterial periplasmic space for efficient folding. Particularly, a C-terminal engineering to confer an interchain disulfide bond appeared to be able to enhance its heterodimeric integrity and EGFR-binding activity. Conformational relevance of the purified final product was validated by mass spectrometry and crystal structure at 1.9 Å resolution. Finally, our recombinant cetuximab-Fab was found to have strong binding affinity to EGFR overexpressed in human squamous carcinoma model (A431) cells. Its binding ability was comparable to that of cetuximab. Its EGFR-binding affinity was estimated at approximately 0.7 nM of Kd in vitro, which was quite stronger than the binding affinity of natural ligand EGF. Hence, the results validate that our construction could serve as an efficient platform to produce a recombinant cetuximab-Fab with a retained antigen-binding functionality.

Binding of N-methylscopolamine to the extracellular domain of muscarinic acetylcholine receptors

NASA Astrophysics Data System (ADS)

Jakubík, Jan; Randáková, Alena; Zimčík, Pavel; El-Fakahany, Esam E.; Doležal, Vladimír

2017-01-01

Interaction of orthosteric ligands with extracellular domain was described at several aminergic G protein-coupled receptors, including muscarinic acetylcholine receptors. The orthosteric antagonists quinuclidinyl benzilate (QNB) and N-methylscopolamine (NMS) bind to the binding pocket of the muscarinic acetylcholine receptor formed by transmembrane α-helices. We show that high concentrations of either QNB or NMS slow down dissociation of their radiolabeled species from all five subtypes of muscarinic acetylcholine receptors, suggesting allosteric binding. The affinity of NMS at the allosteric site is in the micromolar range for all receptor subtypes. Using molecular modelling of the M2 receptor we found that E172 and E175 in the second extracellular loop and N419 in the third extracellular loop are involved in allosteric binding of NMS. Mutation of these amino acids to alanine decreased affinity of NMS for the allosteric binding site confirming results of molecular modelling. The allosteric binding site of NMS overlaps with the binding site of some allosteric, ectopic and bitopic ligands. Understanding of interactions of NMS at the allosteric binding site is essential for correct analysis of binding and action of these ligands.

Membrane type 1-matrix metalloproteinase cleaves off the NH2-terminal portion of heparin-binding epidermal growth factor and converts it into a heparin-independent growth factor.

PubMed

Koshikawa, Naohiko; Mizushima, Hiroto; Minegishi, Tomoko; Iwamoto, Ryo; Mekada, Eisuke; Seiki, Motoharu

2010-07-15

Epidermal growth factor (EGF) receptors (ErbB) and EGF family members represent promising targets for cancer therapy. Heparin-binding EGF (HB-EGF) is a member of the EGF family and is an important target for therapy in some types of human cancers. Processing of HB-EGF by proprotein convertases, and successively, by ADAM family proteases, generates a soluble growth factor that requires heparin as a cofactor. Although heparin potentiates HB-EGF activity in vitro, it is not clear how the heparin-binding activity of HB-EGF is regulated. Here, we show that membrane type 1-matrix metalloproteinase (MT1-MMP; MMP14), a potent invasion-promoting protease, markedly enhances HB-EGF-dependent tumor formation in mice. MT1-MMP additionally cleaves HB-EGF and removes the NH(2)-terminal 20 amino acids that are important for binding heparin. Consequently, the processing of HB-EGF by MT1-MMP converts HB-EGF into a heparin-independent growth factor with enhanced mitogenic activity, and thereby, expression of both proteins costimulates tumor cell growth in vitro and in vivo. The ErbB family of receptors expressed in human gastric carcinoma cells play a role in mediating enhanced HB-EGF activity by MT1-MMP during invasive cell growth in collagen. Thus, we shed light on a new mechanism whereby HB-EGF activity is regulated that should be considered when designing HB-EGF-targeted cancer therapy. (c)2010 AACR.

Human blood-brain barrier insulin-like growth factor receptor

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

Duffy, K.R.; Pardridge, W.M.; Rosenfeld, R.G.

1988-02-01

Insulin-like growth factor (IGF)-1 and IGF-2, may be important regulatory molecules in the CNS. Possible origins of IGFs in brain include either de novo synthesis or transport of circulating IGFs from blood into brain via receptor mediated transcytosis mechanisms at the brain capillary endothelial wall, ie, the blood-brain barrier (BBB). In the present studies, isolated human brain capillaries are used as an in vitro model system of the human BBB and the characteristics of IGF-1 or IGF-2 binding to this preparation were assessed. The total binding of IGF-2 at 37 degrees C exceeded 130% per mg protein and was threefoldmore » greater than the total binding for IGF-1. However, at 37 degrees C nonsaturable binding equaled total binding, suggesting that endocytosis is rate limiting at physiologic temperatures. Binding studies performed at 4 degrees C slowed endocytosis to a greater extent than membrane binding, and specific binding of either IGF-1 or IGF-2 was detectable. Scatchard plots for either peptide were linear and the molar dissociation constant of IGF-1 and IGF-2 binding was 2.1 +/- 0.4 and 1.1 +/- 0.1 nmol/L, respectively. Superphysiologic concentrations of porcine insulin inhibited the binding of both IGF-1 (ED50 = 2 micrograms/mL) and IGF-2 (ED50 = 0.5 microgram/mL). Affinity cross linking of /sup 125/I-IGF-1, /sup 125/I-IGF-2, and /sup 125/I-insulin to isolated human brain capillaries was performed using disuccinimidylsuberate (DSS). These studies revealed a 141 kd binding site for both IGF-1 and IGF-2, and a 133 kd binding site for insulin.« less

Binding of Free and Immune Complex-Associated Hepatitis C Virus to Erythrocytes Is Mediated by the Complement System.

PubMed

Salam, Kazi Abdus; Wang, Richard Y; Grandinetti, Teresa; De Giorgi, Valeria; Alter, Harvey J; Allison, Robert D

2018-05-09

Erythrocytes bind circulating immune complexes (IC) and facilitate IC clearance from the circulation. Chronic hepatitis C virus (HCV) infection is associated with IC-related disorders. In this study we investigated the kinetics and mechanism of HCV and HCV-IC binding to and dissociation from erythrocytes. Cell culture-produced HCV was mixed with erythrocytes from healthy blood donors and erythrocyte-associated virus particles were quantified. Purified complement proteins, complement-depleted serum, and complement receptor antibodies were used to investigate complement-mediated HCV-erythrocyte binding. Purified HCV-specific immunoglobulin G from a chronic HCV-infected patient was used to study complement-mediated HCV-IC-erythrocyte binding. Binding of HCV to erythrocytes increased 200 to 1,000 fold after adding complement active human serum in the absence of antibody. Opsonization of free HCV occurred within 10 minutes and peak binding to erythrocytes was observed at 20-30 minutes. Complement protein C1 was required for binding, while C2, C3 and C4 significantly enhanced binding. Complement receptor 1 (CR1, CD35) antibodies blocked the binding of HCV to erythrocytes isolated from chronically infected HCV patients and healthy blood donors. HCV-ICs significantly enhanced complement-mediated binding to erythrocytes compared to unbound HCV. Dissociation of complement-opsonized HCV from erythrocytes depended on the presence of Factor I. HCV released by Factor I bound preferentially to CD19+ B cells compared to other leukocytes. These results demonstrate that complement mediates the binding of free and IC-associated HCV to CR1 on erythrocytes, and provide a mechanistic rationale for investigating the differential phenotypic expression of HCV-IC-related disease. This article is protected by copyright. All rights reserved. © 2018 by the American Association for the Study of Liver Diseases.

Nuclear factor ETF specifically stimulates transcription from promoters without a TATA box.

PubMed

Kageyama, R; Merlino, G T; Pastan, I

1989-09-15

Transcription factor ETF stimulates the expression of the epidermal growth factor receptor (EGFR) gene which does not have a TATA box in the promoter region. Here, we show that ETF recognizes various GC-rich sequences including stretches of deoxycytidine or deoxyguanosine residues and GC boxes with similar affinities. ETF also binds to TATA boxes but with a lower affinity. ETF stimulated in vitro transcription from several promoters without TATA boxes but had little or no effect on TATA box-containing promoters even though they had strong ETF-binding sites. These inactive ETF-binding sites became functional when placed upstream of the EGFR promoter whose own ETF-binding sites were removed. Furthermore, when a TATA box was introduced into the EGFR promoter, the responsiveness to ETF was abolished. These results indicate that ETF is a specific transcription factor for promoters which do not contain TATA elements.

Identification and Biological Evaluation of Coactivator Binding Inhibitors for the Estrogen Receptor

ERIC Educational Resources Information Center

Gunther, Jillian Rebecca

2009-01-01

The physiologic effects of estrogen action through the estrogen receptor (ER) are widespread, as this hormone exerts actions in both reproductive (e.g., uterus) and non-reproductive (e.g., bone, brain) tissues in both men and women. As such, the regulation of the activity of this ligand-activated transcription factor is highly relevant to the…

In vivo binding properties of SH2 domains from GTPase-activating protein and phosphatidylinositol 3-kinase.

PubMed Central

Cooper, J A; Kashishian, A

1993-01-01

We have used a transient expression system and mutant platelet-derived growth factor (PDGF) receptors to study the binding specificities of the Src homology 2 (SH2) regions of the Ras GTPase-activator protein (GAP) and the p85 alpha subunit of phosphatidylinositol 3-kinase (PI3 kinase). A number of fusion proteins, each tagged with an epitope allowing recognition by a monoclonal antibody, were expressed at levels comparable to those of endogenous GAP. Fusion proteins containing the central SH2-SH3-SH2 region of GAP or the C-terminal region of p85 alpha, which includes two SH2 domains, bound to PDGF receptors in response to PDGF stimulation. Both fusion proteins showed the same requirements for tyrosine phosphorylation sites in the PDGF receptor as the full-length proteins from which they were derived, i.e., binding of the GAP fusion protein was reduced by mutation of Tyr-771, and binding of the p85 fusion protein was reduced by mutation of Tyr-740, Tyr-751, or both residues. Fusion proteins containing single SH2 domains from either GAP or p85 alpha did not bind detectably to PDGF receptors in this system, suggesting that two SH2 domains in a single polypeptide cooperate to raise the affinity of binding. The sequence specificities of individual SH2 domains were deduced from the binding properties of fusion proteins containing one SH2 domain from GAP and another from p85. The results suggest that the C-terminal GAP SH2 domain specifies binding to Tyr-771, the C-terminal p85 alpha SH2 domain binds to either Tyr-740 or Tyr-751, and each protein's N-terminal SH2 domain binds to unidentified phosphorylation sites.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:8382774

Phenobarbital indirectly activates the constitutive active androstane receptor (CAR) by inhibition of epidermal growth factor receptor signaling

PubMed Central

Mutoh, Shingo; Sobhany, Mack; Moore, Rick; Perera, Lalith; Pedersen, Lee; Sueyoshi, Tatsuya; Negishi, Masahiko

2017-01-01

Phenobarbital is a central nervous system depressant that also indirectly activates nuclear receptor constitutive active androstane receptor (CAR), which promotes drug and energy metabolism, as well as cell growth (and death), in the liver. We found that phenobarbital activated CAR by inhibiting epidermal growth factor receptor (EGFR) signaling. Phenobarbital bound to EGFR and potently inhibited the binding of EGF, which prevented the activation of EGFR. This abrogation of EGFR signaling induced the dephosphorylation of receptor for activated C kinase 1 (RACK1) at Tyr52, which then promoted the dephosphorylation of CAR at Thr38 by the catalytic core subunit of protein phosphatase 2A. The findings demonstrated that the phenobarbital-induced mechanism of CAR dephosphorylation and activation is mediated through its direct interaction with and inhibition of EGFR. PMID:23652203

Phenobarbital indirectly activates the constitutive active androstane receptor (CAR) by inhibition of epidermal growth factor receptor signaling.

PubMed

Mutoh, Shingo; Sobhany, Mack; Moore, Rick; Perera, Lalith; Pedersen, Lee; Sueyoshi, Tatsuya; Negishi, Masahiko

2013-05-07

Phenobarbital is a central nervous system depressant that also indirectly activates nuclear receptor constitutive active androstane receptor (CAR), which promotes drug and energy metabolism, as well as cell growth (and death), in the liver. We found that phenobarbital activated CAR by inhibiting epidermal growth factor receptor (EGFR) signaling. Phenobarbital bound to EGFR and potently inhibited the binding of EGF, which prevented the activation of EGFR. This abrogation of EGFR signaling induced the dephosphorylation of receptor for activated C kinase 1 (RACK1) at Tyr(52), which then promoted the dephosphorylation of CAR at Thr(38) by the catalytic core subunit of protein phosphatase 2A. The findings demonstrated that the phenobarbital-induced mechanism of CAR dephosphorylation and activation is mediated through its direct interaction with and inhibition of EGFR.

Direct labelling of the human P2X7 receptor and identification of positive and negative cooperativity of binding.

PubMed

Michel, A D; Chambers, L J; Clay, W C; Condreay, J P; Walter, D S; Chessell, I P

2007-05-01

The P2X(7) receptor exhibits complex pharmacological properties. In this study, binding of a [(3)H]-labelled P2X(7) receptor antagonist to human P2X(7) receptors has been examined to further understand ligand interactions with this receptor. The P2X(7) receptor antagonist, N-[2-({2-[(2-hydroxyethyl)amino]ethyl}amino)-5-quinolinyl]-2-tricyclo[3.3.1.1(3,7)]dec-1-ylacetamide (compound-17), was radiolabelled with tritium and binding studies were performed using membranes prepared from U-2 OS or HEK293 cells expressing human recombinant P2X(7) receptors. Binding of [(3)H]-compound-17 was higher in membranes prepared from cells expressing P2X(7) receptors than from control cells and was inhibited by ATP suggesting labelled sites represented human P2X(7) receptors. Binding was reversible, saturable and modulated by P2X(7) receptor ligands (Brilliant Blue G, KN62, ATP, decavanadate). Furthermore, ATP potency was reduced in the presence of divalent cations or NaCl. Radioligand binding exhibited both positive and negative cooperativity. Positive cooperativity was evident from bell shaped Scatchard plots, reduction in radioligand dissociation rate by unlabelled compound-17 and enhancement of radioligand binding by KN62 and unlabelled compound-17. ATP and decavanadate inhibited binding in a negative cooperative manner as they enhanced radioligand dissociation. These data demonstrate that human P2X(7) receptors can be directly labelled and provide novel insights into receptor function. The positive cooperativity observed suggests that binding of compound-17 to one subunit in the P2X(7) receptor complex enhances subsequent binding to other P2X(7) subunits in the same complex. The negative cooperative effects of ATP suggest that ATP and compound-17 bind at separate, interacting, sites on the P2X(7) receptor.

Direct labelling of the human P2X7 receptor and identification of positive and negative cooperativity of binding

PubMed Central

Michel, A D; Chambers, L J; Clay, W C; Condreay, J P; Walter, D S; Chessell, I P

2007-01-01

Background and Purpose: The P2X7 receptor exhibits complex pharmacological properties. In this study, binding of a [3H]-labelled P2X7 receptor antagonist to human P2X7 receptors has been examined to further understand ligand interactions with this receptor. Experimental Approach: The P2X7 receptor antagonist, N-[2-({2-[(2-hydroxyethyl)amino]ethyl}amino)-5-quinolinyl]-2-tricyclo[3.3.1.13,7]dec-1-ylacetamide (compound-17), was radiolabelled with tritium and binding studies were performed using membranes prepared from U-2 OS or HEK293 cells expressing human recombinant P2X7 receptors. Key Results: Binding of [3H]-compound-17 was higher in membranes prepared from cells expressing P2X7 receptors than from control cells and was inhibited by ATP suggesting labelled sites represented human P2X7 receptors. Binding was reversible, saturable and modulated by P2X7 receptor ligands (Brilliant Blue G, KN62, ATP, decavanadate). Furthermore, ATP potency was reduced in the presence of divalent cations or NaCl. Radioligand binding exhibited both positive and negative cooperativity. Positive cooperativity was evident from bell shaped Scatchard plots, reduction in radioligand dissociation rate by unlabelled compound-17 and enhancement of radioligand binding by KN62 and unlabelled compound-17. ATP and decavanadate inhibited binding in a negative cooperative manner as they enhanced radioligand dissociation. Conclusions: These data demonstrate that human P2X7 receptors can be directly labelled and provide novel insights into receptor function. The positive cooperativity observed suggests that binding of compound-17 to one subunit in the P2X7 receptor complex enhances subsequent binding to other P2X7 subunits in the same complex. The negative cooperative effects of ATP suggest that ATP and compound-17 bind at separate, interacting, sites on the P2X7 receptor. PMID:17339830

Role of transcription factor Sp1 and RNA binding protein HuR in the down-regulation of Dr+ Escherichia coli receptor protein Decay Accelerating Factor (DAF or CD55) by Nitric oxide

PubMed Central

Banadakoppa, Manu; Liebenthal, Daniel; Nowak, David E; Urvil, Petri; Yallampalli, Uma; Wilson, Gerald M; Kishor, Aparna; Yallampalli, Chandra

2012-01-01

We previously reported that nitric oxide (NO) reduces the rate of bacteremia and maternal mortality in pregnant rats with uterine infection by Escherichia coli expressing the Dr Fimbria (Dr+). The epithelial invasion of Dr+ E. coli is dependent on the expression level of its cellular receptor decay accelerating factor (DAF). NO reduces the rate of bacteremia by down-regulating the expression of DAF. In this study, we elucidated the role of transcription factor Sp1 and RNA binding protein HuR in the down-regulation of human DAF by NO. We generated a series of deletion mutant constructs of DAF gene 5′-untranslated region and mapped NO-response region upstream to the core promoter region of the DAF gene. One of the several Sp1 binding sites in the DAF 5′-untranslated region was located within the NO-response region. The binding of Sp1 to this site was inhibited by NO. Furthermore, NO also promoted the degradation of DAF mRNA. The 3′-untranslated region of DAF harbors an AU-rich element and this element destabilized the mRNA transcript. The NO promoted the rapid degradation of DAF mRNA by inhibiting the binding of mRNA stabilizing protein HuR to this AU-rich region. The inhibition of binding of HuR to AU-rich region was due to the S-nitrosylation of one or more cysteine residues by NO. Thus, these data reveal the molecular mediators of transcriptional and post-transcriptional regulation of DAF by NO with implications in pathophysiology related to DAF. PMID:23176121

Role of transcription factor Sp1 and RNA binding protein HuR in the downregulation of Dr+ Escherichia coli receptor protein decay accelerating factor (DAF or CD55) by nitric oxide.

PubMed

Banadakoppa, Manu; Liebenthal, Daniel; Nowak, David E; Urvil, Petri; Yallampalli, Uma; Wilson, Gerald M; Kishor, Aparna; Yallampalli, Chandra

2013-02-01

We previously reported that nitric oxide (NO) reduces the rate of bacteremia and maternal mortality in pregnant rats with uterine infection by Escherichia coli expressing the Dr Fimbria (Dr(+) ). The epithelial invasion of Dr(+) E. coli is dependent on the expression level of its cellular receptor decay accelerating factor (DAF). NO reduces the rate of bacteremia by downregulating the expression of DAF. In this study, we elucidated the role of transcription factor Sp1 and RNA binding protein HuR in the downregulation of human DAF by NO. We generated a series of deletion mutant constructs of DAF gene 5'-untranslated region and mapped the NO-response region upstream to the core promoter region of the DAF gene. One of the several Sp1 binding sites in the DAF 5'-untranslated region was located within the NO-response region. The binding of Sp1 to this site was inhibited by NO. Furthermore, NO also promoted the degradation of DAF mRNA. The 3'-untranslated region of DAF harbors an AU-rich element and this element destabilized the mRNA transcript. NO promoted the rapid degradation of DAF mRNA by inhibiting the binding of mRNA stabilizing protein HuR to this AU-rich region. The inhibition of binding of HuR to the AU-rich region was due to the S-nitrosylation of one or more cysteine residues by NO. Thus, these data reveal the molecular mediators of transcriptional and post-transcriptional regulation of DAF by NO with implications in pathophysiology related to DAF. © 2012 The Authors Journal compilation © 2012 FEBS.

Epidermal Growth Factor Receptor mediated cellular and subcellular targeted delivery of Iron oxide core-Titanium dioxide shell nanoparticles

NASA Astrophysics Data System (ADS)

Yuan, Ye

TiO2 nanomaterials can carry a multitude of therapeutic and diagnostic agents and the semiconductor properties of TiO2 allow for the production of cytotoxic reactive oxygen species following photoactivation. However, the delivery of these nanomaterials to specific cancer cells and specific subcellular organelles within these cells can have a substantial impact on the efficacy and safety of TiO2 nanoparticle therapeutics. Targeting cell surface receptors that are overexpressed by cancer cells is one strategy to improve the specificity of nanoparticle delivery. Therefore we decided to target the Epidermal Growth Factor Receptor (EGFR) because ligand- binding induces rapid receptor endocytosis and ligand-bound EGFR can translocate to the nucleus of cancer cells. To create NPs that can bind EGFR, we identified a peptide derived from the B-loop of Epidermal Growth Factor (EGF) that has been shown to bind and activate EGFR and conjugated it to the surface of Fe3O4 core-TiO2 shell NPs to produce B-loop NCs. We then devised a pulldown assay to show that B-loop NCs, but not bare NPs or NCs carrying a scrambled B-loop peptide, can bind and extract EGFR from HeLa cell protein extracts. Interestingly, B-loop NCs can also pulldown importin-beta, a protein that can transport EGFR to the nucleus. Furthermore, we used flow cytometry and fluorescently labeled NPs to show that B-loop peptides can significantly improve the internalization of NPs by EGFR-expressing HeLa cells. We determined that B-loop NCs can bind EGFR on the membrane of HeLa cells and that these NCs can be transported to the nucleus, by using a combination of confocal microscopy and X-ray Fluorescence Microscopy (XFM) to indirectly and directly track the subcellular distribution of NCs. Finally, we demonstrate how the Bionanoprobe, a novel high-resolution XFM apparatus that can scan whole-mounted, frozen-hydrated cells at multiple angles can be used to verify the subcellular distribution of B-loop NCs.

A structural perspective on nuclear receptors as targets of environmental compounds

PubMed Central

Delfosse, Vanessa; Maire, Albane le; Balaguer, Patrick; Bourguet, William

2015-01-01

Nuclear receptors (NRs) are members of a large superfamily of evolutionarily related transcription factors that control a plethora of biological processes. NRs orchestrate complex events such as development, organ homeostasis, metabolism, immune function, and reproduction. Approximately one-half of the 48 human NRs have been shown to act as ligand-regulated transcription factors and respond directly to a large variety of endogenous hormones and metabolites that are generally hydrophobic and small in size (eg, retinoic acid or estradiol). The second half of the NR family comprises the so-called orphan receptors, for which regulatory ligands are still unknown or may not exist despite the presence of a C-terminal ligand-binding domain, which is the hallmark of all NRs. Several chemicals released into the environment (eg, bisphenols, phthalates, parabens, etc) share some physicochemical properties with natural ligands, allowing them to bind to NRs and activate or inhibit their action. Collectively referred to as endocrine disruptors or endocrine-disrupting chemicals (EDCs), these environmental pollutants are highly suspected to cause a wide range of developmental, reproductive, neurological, or metabolic defects in humans and wildlife. Crystallographic studies are revealing unanticipated mechanisms by which chemically diverse EDCs interact with the ligand-binding domain of NRs. These studies thereby provide a rational basis for designing novel chemicals with lower impacts on human and animal health. In this review, we provide a structural and mechanistic view of endocrine disrupting action using estrogen receptors α and β, (ERα/β), peroxisome proliferator activated receptor γ (PPARγ), and their respective environmental ligands as representative examples. PMID:25500867

Activation‐Induced Killer Cell Immunoglobulin‐like Receptor 3DL2 Binding to HLA–B27 Licenses Pathogenic T Cell Differentiation in Spondyloarthritis

PubMed Central

Ridley, Anna; Hatano, Hiroko; Wong‐Baeza, Isabel; Shaw, Jacqueline; Matthews, Katherine K.; Al‐Mossawi, Hussein; Ladell, Kristin; Price, David A.; Bowness, Paul

2016-01-01

Objective In the spondyloarthritides (SpA), increased numbers of CD4+ T cells express killer cell immunoglobulin‐like receptor 3DL2 (KIR‐3DL2). The aim of this study was to determine the factors that induce KIR‐3DL2 expression, and to characterize the relationship between HLA–B27 and the phenotype and function of KIR‐3DL2–expressing CD4+ T cells in SpA. Methods In total, 34 B27+ patients with SpA, 28 age‐ and sex‐matched healthy controls (20 B27− and 8 B27+), and 9 patients with rheumatoid arthritis were studied. KIR-3DL2 expression and other phenotypic characteristics of peripheral blood and synovial fluid CD4+ T cells were studied by flow cytometry, quantitative polymerase chain reaction, and Western blotting. T cell receptor clonality was determined by template‐switch anchored reverse transcription–polymerase chain reaction and sequencing analysis. Cytokines were measured by enzyme‐linked immunosorbent assay. Results Cellular activation induced KIR‐3DL2 expression on both naive and effector CD4+ T cells. KIR‐3DL2 binding to B27+ cells promoted expression of KIR‐3DL2, the Th17‐specific transcription factor retinoic acid receptor–related orphan nuclear receptor γt, and the antiapoptotic factor B cell lymphoma 2. KIR‐3DL2+CD4+ T cells in patients with ankylosing spondylitis were oligoclonal and enriched for markers of T cell activation and for the gut homing receptor CCR9. In the presence of B27+ antigen‐presenting cells, KIR‐3DL2+CD4+ T cells produced less interleukin‐2 (IL‐2) but more IL‐17. This effect was blocked by HC10, an antibody that inhibits the binding of KIR‐3DL2 to B27 heavy chains. Conclusion KIR‐3DL2 binding to HLA–B27 licenses Th17 cell differentiation in SpA. These findings raise the therapeutic potential of targeting HLA–B27–KIR‐3DL2 interactions for the treatment of B27+ patients with SpA. PMID:26841353

Structural basis for the inhibition of insulin-like growth factors by insulin-like growth factor-binding proteins

PubMed Central

Sitar, Tomasz; Popowicz, Grzegorz M.; Siwanowicz, Igor; Huber, Robert; Holak, Tad A.

2006-01-01

Insulin-like growth factor-binding proteins (IGFBPs) control bioavailability, activity, and distribution of insulin-like growth factor (IGF)1 and -2 through high-affinity IGFBP/IGF complexes. IGF-binding sites are found on N- and C-terminal fragments of IGFBPs, the two conserved domains of IGFBPs. The relative contributions of these domains to IGFBP/IGF complexation has been difficult to analyze, in part, because of the lack of appropriate three-dimensional structures. To analyze the effects of N- and C-terminal domain interactions, we determined several x-ray structures: first, of a ternary complex of N- and C-terminal domain fragments of IGFBP4 and IGF1 and second, of a “hybrid” ternary complex using the C-terminal domain fragment of IGFBP1 instead of IGFBP4. We also solved the binary complex of the N-terminal domains of IGFBP4 and IGF1, again to analyze C- and N-terminal domain interactions by comparison with the ternary complexes. The structures reveal the mechanisms of IGF signaling regulation via IGFBP binding. This finding supports research into the design of IGFBP variants as therapeutic IGF inhibitors for diseases of IGF disregulation. In IGFBP4, residues 1–38 form a rigid disulphide bond ladder-like structure, and the first five N-terminal residues bind to IGF and partially mask IGF residues responsible for the type 1 IGF receptor binding. A high-affinity IGF1-binding site is located in a globular structure between residues 39 and 82. Although the C-terminal domains do not form stable binary complexes with either IGF1 or the N-terminal domain of IGFBP4, in the ternary complex, the C-terminal domain contacts both and contributes to blocking of the IGF1 receptor-binding region of IGF1. PMID:16924115

Desensitization of the nicotinic acetylcholine receptor by diisopropylfluorophosphate.

PubMed

Eldefrawi, M E; Schweizer, G; Bakry, N M; Valdes, J J

1988-01-01

The interaction of diisopropylfluorophosphate (DFP) with the nicotinic acetylcholine (ACh) receptor of Torpedo electric organ was studied, using [3H]-phencyclidine ([3H]-PCP) as a reporter probe. Phencyclidine binds with different kinetics to resting, activated, and desensitized receptor conformations. Although DFP did not inhibit binding of [3H]-ACh or 125I-alpha-bungarotoxin (BGT) to the receptor recognition sites and potentiated in a time-dependent manner [3H]-PCP binding to the receptor's high-affinity allosteric site, it inhibited the ACh- or carbamylcholine-stimulated [3H]-PCP binding. This suggested that DFP bound to a third kind of site on the receptor and affected receptor conformation. Preincubation of the membranes with DFP increased the receptor's affinity for carbamylcholine by eightfold and raised the pseudo-first-order rate of [3H]-PCP binding to that of an agonist-desensitized receptor. Accordingly, it is suggested that DFP induces receptor desensitization by binding to a site that is distinct from the recognition or high-affinity noncompetitive sites.

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.

Detection of angiotensin II binding to single adrenal zona glomerulosa cells by confocal Raman microspectroscopy

NASA Astrophysics Data System (ADS)

McCoy, Michael J.; Habermann, Timothy J.; Hanke, Craig J.; Adar, Fran; Campbell, William B.; Nithipatikom, Kasem

1999-04-01

We developed a confocal Raman microspectroscopic technique to study ligand-receptor bindings in single cells using Raman-labeled ligands and surface-enhanced Raman scattering (SERS). The adrenal zona glomerulosa (ZG) cells were used as a model in this study. ZG cells have a high density of angiotensin II (AII) receptors on the cellular membrane. There are two identified subtypes of AII receptors,namely AT1 and AT2 receptors. AII is a peptidic hormone, which upon binding to its receptors, stimulates the release of aldosterone from ZG cells. The cellular localization of these receptors subtypes was detected in single ZG cells by using immunocomplexation of receptors with specific antibodies and confocal Raman microspectroscopy. In the binding study, we used biotin-labeled AII to bind to its receptors in ZG cells. Then, avidin and Raman-labeled AII. The binding was measure directly on the single ZG cells. The results showed that the binding was displaced with unlabeled AII and specific AII antagonists. This is a rapid and sensitive technique for detection of cellular ligand bindings as well as antagonists screening in drug discovery.

A peptide representing the carboxyl-terminal tail of the met receptor inhibits kinase activity and invasive growth.

PubMed

Bardelli, A; Longati, P; Williams, T A; Benvenuti, S; Comoglio, P M

1999-10-08

Interaction of the hepatocyte growth factor (HGF) with its receptor, the Met tyrosine kinase, results in invasive growth, a genetic program essential to embryonic development and implicated in tumor metastasis. Met-mediated invasive growth requires autophosphorylation of the receptor on tyrosines located in the kinase activation loop (Tyr(1234)-Tyr(1235)) and in the carboxyl-terminal tail (Tyr(1349)-Tyr(1356)). We report that peptides derived from the Met receptor tail, but not from the activation loop, bind the receptor and inhibit the kinase activity in vitro. Cell delivery of the tail receptor peptide impairs HGF-dependent Met phosphorylation and downstream signaling. In normal and transformed epithelial cells, the tail receptor peptide inhibits HGF-mediated invasive growth, as measured by cell migration, invasiveness, and branched morphogenesis. The Met tail peptide inhibits the closely related Ron receptor but does not significantly affect the epidermal growth factor, platelet-derived growth factor, or vascular endothelial growth factor receptor activities. These experiments show that carboxyl-terminal sequences impair the catalytic properties of the Met receptor, thus suggesting that in the resting state the nonphosphorylated tail acts as an intramolecular modulator. Furthermore, they provide a strategy to selectively target the MET proto-oncogene by using small, cell-permeable, peptide derivatives.

Angiogenesis Inhibitors

MedlinePlus

... Leukemia Liver Cancer Lung Cancer Lymphoma Pancreatic Cancer Prostate Cancer Skin Cancer Thyroid Cancer Uterine Cancer All Cancer ... as vascular endothelial growth factor (VEGF) , bind to receptors on the surface of normal endothelial cells. When ...

Ligand affinity of the 67-kD elastin/laminin binding protein is modulated by the protein's lectin domain: visualization of elastin/laminin-receptor complexes with gold-tagged ligands

PubMed Central

1991-01-01

Video-enhanced microscopy was used to examine the interaction of elastin- or laminin-coated gold particles with elastin binding proteins on the surface of live cells. By visualizing the binding events in real time, it was possible to determine the specificity and avidity of ligand binding as well as to analyze the motion of the receptor-ligand complex in the plane of the plasma membrane. Although it was difficult to interpret the rates of binding and release rigorously because of the possibility for multiple interactions between particles and the cell surface, relative changes in binding have revealed important aspects of the regulation of affinity of ligand-receptor interaction in situ. Both elastin and laminin were found to compete for binding to the cell surface and lactose dramatically decreased the affinity of the receptor(s) for both elastin and laminin. These findings were supported by in vitro studies of the detergent-solubilized receptor. Further, immobilization of the ligand-receptor complexes through binding to the cytoskeleton dramatically decreased the ability of bound particles to leave the receptor. The changes in the kinetics of ligand-coated gold binding to living cells suggest that both laminin and elastin binding is inhibited by lactose and that attachment of receptor to the cytoskeleton increases its affinity for the ligand. PMID:1848864

Effects of serotonin-2A receptor binding and gender on personality traits and suicidal behavior in borderline personality disorder.

PubMed

Soloff, Paul H; Chiappetta, Laurel; Mason, Neale Scott; Becker, Carl; Price, Julie C

2014-06-30

Impulsivity and aggressiveness are personality traits associated with a vulnerability to suicidal behavior. Behavioral expression of these traits differs by gender and has been related to central serotonergic function. We assessed the relationships between serotonin-2A receptor function, gender, and personality traits in borderline personality disorder (BPD), a disorder characterized by impulsive-aggression and recurrent suicidal behavior. Participants, who included 33 BPD patients and 27 healthy controls (HC), were assessed for Axis I and II disorders with the Structured Clinical Interview for DSM-IV and the International Personality Disorders Examination, and with the Diagnostic Interview for Borderline Patients-Revised for BPD. Depressed mood, impulsivity, aggression, and temperament were assessed with standardized measures. Positron emission tomography with [(18)F]altanserin as ligand and arterial blood sampling was used to determine the binding potentials (BPND) of serotonin-2A receptors in 11 regions of interest. Data were analyzed using Logan graphical analysis, controlling for age and non-specific binding. Among BPD subjects, aggression, Cluster B co-morbidity, antisocial PD, and childhood abuse were each related to altanserin binding. BPND values predicted impulsivity and aggression in BPD females (but not BPD males), and in HC males (but not HC females.) Altanserin binding was greater in BPD females than males in every contrast, but it did not discriminate suicide attempters from non-attempters. Region-specific differences in serotonin-2A receptor binding related to diagnosis and gender predicted clinical expression of aggression and impulsivity. Vulnerability to suicidal behavior in BPD may be related to serotonin-2A binding through expression of personality risk factors. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Structural determinants of ubiquitin-CXC chemokine receptor 4 interaction.

PubMed

Saini, Vikas; Marchese, Adriano; Tang, Wei-Jen; Majetschak, Matthias

2011-12-23

Ubiquitin, a post-translational protein modifier inside the cell, functions as a CXC chemokine receptor (CXCR) 4 agonist outside the cell. However, the structural determinants of the interaction between extracellular ubiquitin and CXCR4 remain unknown. Utilizing C-terminal truncated ubiquitin and ubiquitin mutants, in which surface residues that are known to interact with ubiquitin binding domains in interacting proteins are mutated (Phe-4, Leu-8, Ile-44, Asp-58, Val-70), we provide evidence that the ubiquitin-CXCR4 interaction follows a two-site binding mechanism in which the hydrophobic surfaces surrounding Phe-4 and Val-70 are important for receptor binding, whereas the flexible C terminus facilitates receptor activation. Based on these findings and the available crystal structures, we then modeled the ubiquitin-CXCR4 interface with the RosettaDock software followed by small manual adjustments, which were guided by charge complementarity and anticipation of a conformational switch of CXCR4 upon activation. This model suggests three residues of CXCR4 (Phe-29, Phe-189, Lys-271) as potential interaction sites. Binding studies with HEK293 cells overexpressing wild type and CXCR4 after site-directed mutagenesis confirm that these residues are important for ubiquitin binding but that they do not contribute to the binding of stromal cell-derived factor 1α. Our findings suggest that the structural determinants of the CXCR4 agonist activity of ubiquitin mimic the typical structure-function relationship of chemokines. Furthermore, we provide evidence for separate and specific ligand binding sites on CXCR4. As exogenous ubiquitin has been shown to possess therapeutic potential, our findings are expected to facilitate the structure-based design of new compounds with ubiquitin-mimetic actions on CXCR4.

The Role of the EGF Receptor First Intron in Its Regulation in Breast Canceer.

DTIC Science & Technology

1997-08-01

of EGFR, to malignantly transform mammary glands in transgenic mice (46). Future work could determine if these putative oncogene factor binding sites...general transcription factors, and the use or overuse of one promoter could monopolize the abundance of these transcription factors within a cell. The ideal

CSL311, a novel, potent, therapeutic monoclonal antibody for the treatment of diseases mediated by the common β chain of the IL-3, GM-CSF and IL-5 receptors

PubMed Central

Panousis, Con; Dhagat, Urmi; Edwards, Kirsten M.; Rayzman, Veronika; Hardy, Matthew P.; Braley, Hal; Gauvreau, Gail M.; Hercus, Timothy R.; Smith, Steven; Sehmi, Roma; McMillan, Laura; Dottore, Mara; McClure, Barbara J.; Fabri, Louis J.; Vairo, Gino; Lopez, Angel F; Parker, Michael W.; Nash, Andrew D.; Wilson, Nicholas J.; Wilson, Michael J.; Owczarek, Catherine M.

2016-01-01

ABSTRACT The β common-signaling cytokines interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-5 stimulate pro-inflammatory activities of haematopoietic cells via a receptor complex incorporating cytokine-specific α and shared β common (βc, CD131) receptor. Evidence from animal models and recent clinical trials demonstrate that these cytokines are critical mediators of the pathogenesis of inflammatory airway disease such as asthma. However, no therapeutic agents, other than steroids, that specifically and effectively target inflammation mediated by all 3 of these cytokines exist. We employed phage display technology to identify and optimize a novel, human monoclonal antibody (CSL311) that binds to a unique epitope that is specific to the cytokine-binding site of the human βc receptor. The binding epitope of CSL311 on the βc receptor was defined by X-ray crystallography and site-directed mutagenesis. CSL311 has picomolar binding affinity for the human βc receptor, and at therapeutic concentrations is a highly potent antagonist of the combined activities of IL-3, GM-CSF and IL-5 on primary eosinophil survival in vitro. Importantly, CSL311 inhibited the survival of inflammatory cells present in induced sputum from human allergic asthmatic subjects undergoing allergen bronchoprovocation. Due to its high potency and ability to simultaneously suppress the activity of all 3 β common cytokines, CSL311 may provide a new strategy for the treatment of chronic inflammatory diseases where the human βc receptor is central to pathogenesis. The coordinates for the βc/CSL311 Fab complex structure have been deposited with the RCSB Protein Data Bank (PDB 5DWU). PMID:26651396

Changes in the hemagglutinin of H5N1 viruses during human infection – Influence on receptor binding☆

PubMed Central

Crusat, Martin; Liu, Junfeng; Palma, Angelina S.; Childs, Robert A.; Liu, Yan; Wharton, Stephen A.; Lin, Yi Pu; Coombs, Peter J.; Martin, Stephen R.; Matrosovich, Mikhail; Chen, Zi; Stevens, David J.; Hien, Vo Minh; Thanh, Tran Tan; Nhu, Le Nguyen Truc; Nguyet, Lam Anh; Ha, Do Quang; van Doorn, H.Rogier; Hien, Tran Tinh; Conradt, Harald S.; Kiso, Makoto; Gamblin, Steve J.; Chai, Wengang; Skehel, John J.; Hay, Alan J.; Farrar, Jeremy; de Jong, Menno D.; Feizi, Ten

2013-01-01

As avian influenza A(H5N1) viruses continue to circulate in Asia and Africa, global concerns of an imminent pandemic persist. Recent experimental studies suggest that efficient transmission between humans of current H5N1 viruses only requires a few genetic changes. An essential step is alteration of the virus hemagglutinin from preferential binding to avian receptors for the recognition of human receptors present in the upper airway. We have identified receptor-binding changes which emerged during H5N1 infection of humans, due to single amino acid substitutions, Ala134Val and Ile151Phe, in the hemagglutinin. Detailed biological, receptor-binding, and structural analyses revealed reduced binding of the mutated viruses to avian-like receptors, but without commensurate increased binding to the human-like receptors investigated, possibly reflecting a receptor-binding phenotype intermediate in adaptation to more human-like characteristics. These observations emphasize that evolution in nature of avian H5N1 viruses to efficient binding of human receptors is a complex multistep process. PMID:24050651

Binding specificity of Bacillus thuringiensis Cry1Aa for purified, native Bombyx mori aminopeptidase N and cadherin-like receptors

PubMed Central

Jenkins, Jeremy L; Dean, Donald H

2001-01-01

Background To better understand the molecular interactions of Bt toxins with non-target insects, we have examined the real-time binding specificity and affinity of Cry1 toxins to native silkworm (Bombyx mori) midgut receptors. Previous studies on B. mori receptors utilized brush border membrane vesicles or purifed receptors in blot-type assays. Results The Bombyx mori (silkworm) aminopeptidase N (APN) and cadherin-like receptors for Bacillus thuringiensis insecticidal Cry1Aa toxin were purified and their real-time binding affinities for Cry toxins were examined by surface plasmon resonance. Cry1Ab and Cry1Ac toxins did not bind to the immobilized native receptors, correlating with their low toxicities. Cry1Aa displayed moderate affinity for B. mori APN (75 nM), and unusually tight binding to the cadherin-like receptor (2.6 nM), which results from slow dissociation rates. The binding of a hybrid toxin (Aa/Aa/Ac) was identical to Cry1Aa. Conclusions These results indicate domain II of Cry1Aa is essential for binding to native B. mori receptors and for toxicity. Moreover, the high-affinity binding of Cry1Aa to native cadherin-like receptor emphasizes the importance of this receptor class for Bt toxin research. PMID:11722800

Molecular characterization of the receptor binding structure-activity relationships of influenza B virus hemagglutinin.

PubMed

Carbone, V; Kim, H; Huang, J X; Baker, M A; Ong, C; Cooper, M A; Li, J; Rockman, S; Velkov, T

2013-01-01

Selectivity of α2,6-linked human-like receptors by B hemagglutinin (HA) is yet to be fully understood. This study integrates binding data with structure-recognition models to examine the impact of regional-specific sequence variations within the receptor-binding pocket on selectivity and structure activity relationships (SAR). The receptor-binding selectivity of influenza B HAs corresponding to either B/Victoria/2/1987 or the B/Yamagata/16/88 lineages was examined using surface plasmon resonance, solid-phase ELISA and gel-capture assays. Our SAR data showed that the presence of asialyl sugar units is the main determinant of receptor preference of α2,6 versus α2,3 receptor binding. Changes to the type of sialyl-glycan linkage present on receptors exhibit only a minor effect upon binding affinity. Homology-based structural models revealed that structural properties within the HA pocket, such as a glyco-conjugate at Asn194 on the 190-helix, sterically interfere with binding to avian receptor analogs by blocking the exit path of the asialyl sugars. Similarly, naturally occurring substitutions in the C-terminal region of the 190-helix and near the N-terminal end of the 140-loop narrows the horizontal borders of the binding pocket, which restricts access of the avian receptor analog LSTa. This study helps bridge the gap between ligand structure and receptor recognition for influenza B HA; and provides a consensus SAR model for the binding of human and avian receptor analogs to influenza B HA.

Virtual and biomolecular screening converge on a selective agonist for GPR30.

PubMed

Bologa, Cristian G; Revankar, Chetana M; Young, Susan M; Edwards, Bruce S; Arterburn, Jeffrey B; Kiselyov, Alexander S; Parker, Matthew A; Tkachenko, Sergey E; Savchuck, Nikolay P; Sklar, Larry A; Oprea, Tudor I; Prossnitz, Eric R

2006-04-01

Estrogen is a hormone critical in the development, normal physiology and pathophysiology of numerous human tissues. The effects of estrogen have traditionally been solely ascribed to estrogen receptor alpha (ERalpha) and more recently ERbeta, members of the soluble, nuclear ligand-activated family of transcription factors. We have recently shown that the seven-transmembrane G protein-coupled receptor GPR30 binds estrogen with high affinity and resides in the endoplasmic reticulum, where it activates multiple intracellular signaling pathways. To differentiate between the functions of ERalpha or ERbeta and GPR30, we used a combination of virtual and biomolecular screening to isolate compounds that selectively bind to GPR30. Here we describe the identification of the first GPR30-specific agonist, G-1 (1), capable of activating GPR30 in a complex environment of classical and new estrogen receptors. The development of compounds specific to estrogen receptor family members provides the opportunity to increase our understanding of these receptors and their contribution to estrogen biology.

Structure of the Full-length VEGFR-1 Extracellular Domain in Complex with VEGF-A.

PubMed

Markovic-Mueller, Sandra; Stuttfeld, Edward; Asthana, Mayanka; Weinert, Tobias; Bliven, Spencer; Goldie, Kenneth N; Kisko, Kaisa; Capitani, Guido; Ballmer-Hofer, Kurt

2017-02-07

Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel development upon activation of three receptor tyrosine kinases: VEGFR-1, -2, and -3. Partial structures of VEGFR/VEGF complexes based on single-particle electron microscopy, small-angle X-ray scattering, and X-ray crystallography revealed the location of VEGF binding and domain arrangement of individual receptor subdomains. Here, we describe the structure of the full-length VEGFR-1 extracellular domain in complex with VEGF-A at 4 Å resolution. We combined X-ray crystallography, single-particle electron microscopy, and molecular modeling for structure determination and validation. The structure reveals the molecular details of ligand-induced receptor dimerization, in particular of homotypic receptor interactions in immunoglobulin homology domains 4, 5, and 7. Functional analyses of ligand binding and receptor activation confirm the relevance of these homotypic contacts and identify them as potential therapeutic sites to allosterically inhibit VEGFR-1 activity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Reassessment of the Unique Mode of Binding between Angiotensin II Type 1 Receptor and Their Blockers

PubMed Central

Matsuo, Yoshino; Saku, Keijiro; Karnik, Sadashiva S.

2013-01-01

While the molecular structures of angiotensin II (Ang II) type 1 (AT1) receptor blockers (ARBs) are very similar, they are also slightly different. Although each ARB has been shown to exhibit a unique mode of binding to AT1 receptor, different positions of the AT1 receptor have been analyzed and computational modeling has been performed using different crystal structures for the receptor as a template and different kinds of software. Therefore, we systematically analyzed the critical positions of the AT1 receptor, Tyr113, Tyr184, Lys199, His256 and Gln257 using a mutagenesis study, and subsequently performed computational modeling of the binding of ARBs to AT1 receptor using CXCR4 receptor as a new template and a single version of software. The interactions between Tyr113 in the AT1 receptor and the hydroxyl group of olmesartan, between Lys199 and carboxyl or tetrazole groups, and between His256 or Gln257 and the tetrazole group were studied. The common structure, a tetrazole group, of most ARBs similarly bind to Lys199, His256 and Gln257 of AT1 receptor. Lys199 in the AT1 receptor binds to the carboxyl group of EXP3174, candesartan and azilsartan, whereas oxygen in the amidecarbonyl group of valsartan may bind to Lys199. The benzimidazole portion of telmisartan may bind to a lipophilic pocket that includes Tyr113. On the other hand, the n-butyl group of irbesartan may bind to Tyr113. In conclusion, we confirmed that the slightly different structures of ARBs may be critical for binding to AT1 receptor and for the formation of unique modes of binding. PMID:24260317

Activation-Induced Killer Cell Immunoglobulin-like Receptor 3DL2 Binding to HLA-B27 Licenses Pathogenic T Cell Differentiation in Spondyloarthritis.

PubMed

Ridley, Anna; Hatano, Hiroko; Wong-Baeza, Isabel; Shaw, Jacqueline; Matthews, Katherine K; Al-Mossawi, Hussein; Ladell, Kristin; Price, David A; Bowness, Paul; Kollnberger, Simon

2016-04-01

In the spondyloarthritides (SpA), increased numbers of CD4+ T cells express killer cell immunoglobulin-like receptor 3DL2 (KIR-3DL2). The aim of this study was to determine the factors that induce KIR-3DL2 expression, and to characterize the relationship between HLA-B27 and the phenotype and function of KIR-3DL2-expressing CD4+ T cells in SpA. In total, 34 B27+ patients with SpA, 28 age- and sex-matched healthy controls (20 B27- and 8 B27+), and 9 patients with rheumatoid arthritis were studied. KIR-3DL2 expression and other phenotypic characteristics of peripheral blood and synovial fluid CD4+ T cells were studied by flow cytometry, quantitative polymerase chain reaction, and Western blotting. T cell receptor clonality was determined by template-switch anchored reverse transcription-polymerase chain reaction and sequencing analysis. Cytokines were measured by enzyme-linked immunosorbent assay. Cellular activation induced KIR-3DL2 expression on both naive and effector CD4+ T cells. KIR-3DL2 binding to B27+ cells promoted expression of KIR-3DL2, the Th17-specific transcription factor retinoic acid receptor-related orphan nuclear receptor γt, and the antiapoptotic factor B cell lymphoma 2. KIR-3DL2+CD4+ T cells in patients with ankylosing spondylitis were oligoclonal and enriched for markers of T cell activation and for the gut homing receptor CCR9. In the presence of B27+ antigen-presenting cells, KIR-3DL2+CD4+ T cells produced less interleukin-2 (IL-2) but more IL-17. This effect was blocked by HC10, an antibody that inhibits the binding of KIR-3DL2 to B27 heavy chains. KIR-3DL2 binding to HLA-B27 licenses Th17 cell differentiation in SpA. These findings raise the therapeutic potential of targeting HLA-B27-KIR-3DL2 interactions for the treatment of B27+ patients with SpA. © 2016 The Authors. Arthritis & Rheumatology published by Wiley Periodicals, Inc. on behalf of the American College of Rheumatology.

Accelerated molecular dynamics simulations of ligand binding to a muscarinic G-protein-coupled receptor.

PubMed

Kappel, Kalli; Miao, Yinglong; McCammon, J Andrew

2015-11-01

Elucidating the detailed process of ligand binding to a receptor is pharmaceutically important for identifying druggable binding sites. With the ability to provide atomistic detail, computational methods are well poised to study these processes. Here, accelerated molecular dynamics (aMD) is proposed to simulate processes of ligand binding to a G-protein-coupled receptor (GPCR), in this case the M3 muscarinic receptor, which is a target for treating many human diseases, including cancer, diabetes and obesity. Long-timescale aMD simulations were performed to observe the binding of three chemically diverse ligand molecules: antagonist tiotropium (TTP), partial agonist arecoline (ARc) and full agonist acetylcholine (ACh). In comparison with earlier microsecond-timescale conventional MD simulations, aMD greatly accelerated the binding of ACh to the receptor orthosteric ligand-binding site and the binding of TTP to an extracellular vestibule. Further aMD simulations also captured binding of ARc to the receptor orthosteric site. Additionally, all three ligands were observed to bind in the extracellular vestibule during their binding pathways, suggesting that it is a metastable binding site. This study demonstrates the applicability of aMD to protein-ligand binding, especially the drug recognition of GPCRs.

Selective labelling of diazepam-insensitive GABAA receptors in vivo using [3H]Ro 15-4513.

PubMed

Pym, Luanda J; Cook, Susan M; Rosahl, Thomas; McKernan, Ruth M; Atack, John R

2005-11-01

Classical benzodiazepines (BZs), such as diazepam, bind to GABAA receptors containing alpha1, alpha2, alpha3 or alpha5 subunits that are therefore described as diazepam-sensitive (DS) receptors. However, the corresponding binding site of GABAA receptors containing either an alpha4 or alpha6 subunit do not bind the classical BZs and are therefore diazepam-insensitive (DIS) receptors; a difference attributable to a single amino acid (histidine in alpha1, alpha2, alpha3 and alpha5 subunits and arginine in alpha4 and alpha6). Unlike classical BZs, the imidazobenzodiazepines Ro 15-4513 and bretazenil bind to both DS and DIS populations of GABAA receptors. In the present study, an in vivo assay was developed using lorazepam to fully occupy DS receptors such that [3H]Ro 15-4513 was then only able to bind to DIS receptors. When dosed i.v., [3H]Ro 15-4513 rapidly entered and was cleared from the brain, with approximately 70% of brain radioactivity being membrane-bound. Essentially all membrane binding to DS+DIS receptors could be displaced by unlabelled Ro 15-4513 or bretazenil, with respective ID50 values of 0.35 and 1.2 mg kg(-1). A dose of 30 mg kg(-1) lorazepam was used to block all DS receptors in a [3H]Ro 15-1788 in vivo binding assay. When predosed in a [3H]Ro 15-4513 binding assay, lorazepam blocked [3H]Ro 15-4513 binding to DS receptors, with the remaining binding to DIS receptors accounting for 5 and 23% of the total (DS plus DIS) receptors in the forebrain and cerebellum, respectively. The in vivo binding of [3H]Ro 15-4513 to DIS receptors in the presence of lorazepam was confirmed using alpha1H101R knock-in mice, in which alpha1-containing GABAA receptors are rendered diazepam insensitive by mutation of the histidine that confers diazepam sensitivity to arginine. In these mice, and in the presence of lorazepam, there was an increase of in vivo [3H]Ro 15-4513 binding in the forebrain and cerebellum from 4 and 15% to 36 and 59% of the total (i.e. DS plus DIS) [3H]Ro 15-4513 binding observed in the absence of lorazepam.

Monitoring the kinetics of the pH driven transition of the anthrax toxin prepore to the pore by biolayer interferometry and surface plasmon resonance

PubMed Central

Naik, Subhashchandra; Brock, Susan; Akkaladevi, Narahari; Tally, Jon; Mcginn-Straub, Wesley; Zhang, Na; Gao, Phillip; Gogol, E. P.; Pentelute, B. L.; Collier, R. John; Fisher, Mark T.

2013-01-01

Domain 2 of the anthrax protective antigen (PA) prepore heptamer unfolds and refolds during endosome acidification to generate an extended 100 Å beta barrel pore that inserts into the endosomal membrane. The PA pore facilitates the pH dependent unfolding and translocation of bound toxin enzymic components, lethal factor (LF) and/or edema factor (EF), from the endosome into the cytoplasm. We constructed immobilized complexes of the prepore with the PA-binding domain of LF (LFN) to monitor the real-time prepore to pore kinetic transition using surface plasmon resonance (SPR) and bio-layer interferometry (BLI). The kinetics of this transition increased as the solution pH was decreased from pH 7.5 to pH 5.0, mirroring acidification of the endosome. Once transitioned, the LFN-PA pore complex was removed from the BLI biosensor tip and deposited onto EM grids, where the PA pore formation was confirmed by negative stain electron microscopy. When the soluble receptor domain (ANTRX2/CMG2) binds the immobilized PA prepore, the transition to the pore state was observed only after the pH was lowered to early or late endosomal pH conditions (5.5 to 5.0 respectively). Once the pore formed, the soluble receptor readily dissociated from the PA pore. Separate binding experiments with immobilized PA pores and soluble receptor indicate that the receptor has a weakened propensity to bind to the transitioned pore. This immobilized anthrax toxin platform can be used to identify or validate potential antimicrobial lead compounds capable of regulating and/or inhibiting anthrax toxin complex formation or pore transitions. PMID:23964683

Monitoring the kinetics of the pH-driven transition of the anthrax toxin prepore to the pore by biolayer interferometry and surface plasmon resonance.

PubMed

Naik, Subhashchandra; Brock, Susan; Akkaladevi, Narahari; Tally, Jon; McGinn-Straub, Wesley; Zhang, Na; Gao, Phillip; Gogol, E P; Pentelute, B L; Collier, R John; Fisher, Mark T

2013-09-17

Domain 2 of the anthrax protective antigen (PA) prepore heptamer unfolds and refolds during endosome acidification to generate an extended 100 Å β barrel pore that inserts into the endosomal membrane. The PA pore facilitates the pH-dependent unfolding and translocation of bound toxin enzymic components, lethal factor (LF) and/or edema factor, from the endosome to the cytoplasm. We constructed immobilized complexes of the prepore with the PA-binding domain of LF (LFN) to monitor the real-time prepore to pore kinetic transition using surface plasmon resonance and biolayer interferometry (BLI). The kinetics of this transition increased as the solution pH was decreased from 7.5 to 5.0, mirroring acidification of the endosome. Once it had undergone the transition, the LFN-PA pore complex was removed from the BLI biosensor tip and deposited onto electron microscopy grids, where PA pore formation was confirmed by negative stain electron microscopy. When the soluble receptor domain (ANTRX2/CMG2) binds the immobilized PA prepore, the transition to the pore state was observed only after the pH was lowered to early (pH 5.5) or late (pH 5.0) endosomal pH conditions. Once the pore formed, the soluble receptor readily dissociated from the PA pore. Separate binding experiments with immobilized PA pores and the soluble receptor indicate that the receptor has a weakened propensity to bind to the transitioned pore. This immobilized anthrax toxin platform can be used to identify or validate potential antimicrobial lead compounds capable of regulating and/or inhibiting anthrax toxin complex formation or pore transitions.

Insulin-Like Growth Factor (IGF) Binding Protein-2, Independently of IGF-1, Induces GLUT-4 Translocation and Glucose Uptake in 3T3-L1 Adipocytes

PubMed Central

Assefa, Biruhalem; Mahmoud, Ayman M.; Pfeiffer, Andreas F. H.; Birkenfeld, Andreas L.; Spranger, Joachim

2017-01-01

Insulin-like growth factor binding protein-2 (IGFBP-2) is the predominant IGF binding protein produced during adipogenesis and is known to increase the insulin-stimulated glucose uptake (GU) in myotubes. We investigated the IGFBP-2-induced changes in basal and insulin-stimulated GU in adipocytes and the underlying mechanisms. We further determined the role of insulin and IGF-1 receptors in mediating the IGFBP-2 and the impact of IGFBP-2 on the IGF-1-induced GU. Fully differentiated 3T3-L1 adipocytes were treated with IGFBP-2 in the presence and absence of insulin and IGF-1. Insulin, IGF-1, and IGFBP-2 induced a dose-dependent increase in GU. IGFBP-2 increased the insulin-induced GU after long-term incubation. The IGFBP-2-induced impact on GU was neither affected by insulin or IGF-1 receptor blockage nor by insulin receptor knockdown. IGFBP-2 significantly increased the phosphorylation of PI3K, Akt, AMPK, TBC1D1, and PKCζ/λ and induced GLUT-4 translocation. Moreover, inhibition of PI3K and AMPK significantly reduced IGFBP-2-stimulated GU. In conclusion, IGFBP-2 stimulates GU in 3T3-L1 adipocytes through activation of PI3K/Akt, AMPK/TBC1D1, and PI3K/PKCζ/λ/GLUT-4 signaling. The stimulatory effect of IGFBP-2 on GU is independent of its binding to IGF-1 and is possibly not mediated through the insulin or IGF-1 receptor. This study highlights the potential role of IGFBP-2 in glucose metabolism. PMID:29422987

Insulin-Like Growth Factor (IGF) Binding Protein-2, Independently of IGF-1, Induces GLUT-4 Translocation and Glucose Uptake in 3T3-L1 Adipocytes.

PubMed

Assefa, Biruhalem; Mahmoud, Ayman M; Pfeiffer, Andreas F H; Birkenfeld, Andreas L; Spranger, Joachim; Arafat, Ayman M

2017-01-01

Insulin-like growth factor binding protein-2 (IGFBP-2) is the predominant IGF binding protein produced during adipogenesis and is known to increase the insulin-stimulated glucose uptake (GU) in myotubes. We investigated the IGFBP-2-induced changes in basal and insulin-stimulated GU in adipocytes and the underlying mechanisms. We further determined the role of insulin and IGF-1 receptors in mediating the IGFBP-2 and the impact of IGFBP-2 on the IGF-1-induced GU. Fully differentiated 3T3-L1 adipocytes were treated with IGFBP-2 in the presence and absence of insulin and IGF-1. Insulin, IGF-1, and IGFBP-2 induced a dose-dependent increase in GU. IGFBP-2 increased the insulin-induced GU after long-term incubation. The IGFBP-2-induced impact on GU was neither affected by insulin or IGF-1 receptor blockage nor by insulin receptor knockdown. IGFBP-2 significantly increased the phosphorylation of PI3K, Akt, AMPK, TBC1D1, and PKC ζ / λ and induced GLUT-4 translocation. Moreover, inhibition of PI3K and AMPK significantly reduced IGFBP-2-stimulated GU. In conclusion, IGFBP-2 stimulates GU in 3T3-L1 adipocytes through activation of PI3K/Akt, AMPK/TBC1D1, and PI3K/PKC ζ / λ /GLUT-4 signaling. The stimulatory effect of IGFBP-2 on GU is independent of its binding to IGF-1 and is possibly not mediated through the insulin or IGF-1 receptor. This study highlights the potential role of IGFBP-2 in glucose metabolism.

Impact of morphine on the expression of insulin receptor and protein levels of insulin/IGFs in rat neural stem cells.

PubMed

Salarinasab, Sadegh; Nourazarian, AliReza; Nikanfar, Masoud; Abdyazdani, Nima; Kazemi, Masoumeh; Feizy, Navid; Rahbarghazi, Reza

2017-11-01

Alzheimer's disease is correlated with neuronal degeneration and loss of neuronal precursors in different parts of the brain. It has been found disturbance in the homeostasis neural stem cells (NSCs) can cause neurodegeneration. Morphine, an analgesic agent, can disrupt the dynamic and normal state of NSCs. However, more investigations are required to clearly address underlying mechanisms. The current experiment aimed to investigate the effects of morphine on the cell distribution of insulin factor and receptor and insulin-like growth factors (IGF1, IGF2) in NSCs. NSCs were isolated from rats and stemness feature confirmed by antibodies against nestin and Sox2. The cells were exposed to 100μM morphine, 50μM naloxone and combination of these two drugs for 72h. The neural cell growth, changes in levels of insulin and insulin-like growth factors secreted by NSCs as well as the insulin-receptor-gene expression were assessed by flow cytometry, ELlSA, and real-time PCR, respectively. Cell cycle assay revealed the exposure of cells to morphine for 72h increased cell apoptosis and decreased neural stem cell growth. The biosynthesis of insulin, insulin-like growth factors, and insulin receptor were reduced (p<0.05) after NSCs exposure to morphine at the concentration of 100μM for 24, 48 and 72h. Naloxone is a competitive antagonist which binds MOR where morphine (and endogenous opioids) bind, and reversed the detrimental effects of morphine. It can be concluded that morphine initiated irregularity in NSCs kinetics and activity by reducing the secretion of insulin and insulin-like growth factors and down-regulation of insulin receptor. Copyright © 2017 Elsevier B.V. All rights reserved.

Quantitative analysis reveals how EGFR activation and downregulation are coupled in normal but not in cancer cells

PubMed Central

Capuani, Fabrizio; Conte, Alexia; Argenzio, Elisabetta; Marchetti, Luca; Priami, Corrado; Polo, Simona; Di Fiore, Pier Paolo; Sigismund, Sara; Ciliberto, Andrea

2015-01-01

Ubiquitination of the epidermal growth factor receptor (EGFR) that occurs when Cbl and Grb2 bind to three phosphotyrosine residues (pY1045, pY1068 and pY1086) on the receptor displays a sharp threshold effect as a function of EGF concentration. Here we use a simple modelling approach together with experiments to show that the establishment of the threshold requires both the multiplicity of binding sites and cooperative binding of Cbl and Grb2 to the EGFR. While the threshold is remarkably robust, a more sophisticated model predicted that it could be modulated as a function of EGFR levels on the cell surface. We confirmed experimentally that the system has evolved to perform optimally at physiological levels of EGFR. As a consequence, this system displays an intrinsic weakness that causes—at the supraphysiological levels of receptor and/or ligand associated with cancer—uncoupling of the mechanisms leading to signalling through phosphorylation and attenuation through ubiquitination. PMID:26264748

Activation of epidermal growth factor receptor mediates receptor axon sorting and extension in the developing olfactory system of the moth Manduca sexta.

PubMed

Gibson, Nicholas J; Tolbert, Leslie P

2006-04-10

During development of the adult olfactory system of the moth Manduca sexta, olfactory receptor neurons extend axons from the olfactory epithelium in the antenna into the brain. As they arrive at the brain, interactions with centrally derived glial cells cause axons to sort and fasciculate with other axons destined to innervate the same glomeruli. Here we report studies indicating that activation of the epidermal growth factor receptor (EGFR) is involved in axon ingrowth and targeting. Blocking the EGFR kinase domain pharmacologically leads to stalling of many axons in the sorting zone and nerve layer as well as abnormal axonal fasciculation in the sorting zone. We also find that neuroglian, an IgCAM known to activate the EGFR through homophilic interactions in other systems, is transiently present on olfactory receptor neuron axons and on glia during the critical stages of the sorting process. The neuroglian is resistant to extraction with Triton X-100 in the sorting zone and nerve layer, possibly indicating its stabilization by homophilic binding in these regions. Our results suggest a mechanism whereby neuroglian molecules on axons and possibly sorting zone glia bind homophilically, leading to activation of EGFRs, with subsequent effects on axon sorting, pathfinding, and extension, and glomerulus development. Copyright 2006 Wiley-Liss, Inc.

Atrial natriuretic factor receptor heterogeneity in rat tissues

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

Andresen, J.W.; Kuno, T.; Kamisaki, Y.

1986-03-01

Rat /sup 125/I-atrial natriuretic factor (ANF, 8-33) was used to identify ANF receptors in membrane preparations from rat adrenal gland and lung. When solubilized with Lubrol-PX, the receptors retained a binding profile and properties that correspond to the high affinity and specificity found in crude membranes. Single peaks of binding activity were observed in gel permeation HPLC and density gradient centrifugation analysis of the solubilized preparations. However, when membranes and solubilized preparations were labeled with /sup 125/I-ANF, treated with crosslinking reagent (disuccinimidyl suberate), and analyzed by SDS gel electrophoresis several specifically labeled bands (120,000, 70,000, and 60,000 daltons) were identifiedmore » by autoradiography. The relative distribution of the specifically labeled proteins varied significantly between rat adrenal gland and lung. In adrenal glands the 120K dalton band was the most prominent specifically labeled protein, while the 60K and 70K dalton proteins were labeled to a lesser degree. In lung membranes the lower molecular weight proteins were more prominent. These results suggest the presence of multiple ANF receptor subtypes, the distribution of which varies among tissues. Chromatographic separation and further characterization of these receptors are currently in progress, and preliminary purification studies support this hypothesis.« less

Characterization of dFOXO binding sites upstream of the Insulin Receptor P2 promoter across the Drosophila phylogeny

PubMed Central

Orengo, Dorcas J.; Aguadé, Montserrat

2017-01-01

The insulin/TOR signal transduction pathway plays a critical role in determining such important traits as body and organ size, metabolic homeostasis and life span. Although this pathway is highly conserved across the animal kingdom, the affected traits can exhibit important differences even between closely related species. Evolutionary studies of regulatory regions require the reliable identification of transcription factor binding sites. Here we have focused on the Insulin Receptor (InR) expression from its P2 promoter in the Drosophila genus, which in D. melanogaster is up-regulated by hypophosphorylated Drosophila FOXO (dFOXO). We have finely characterized this transcription factor binding sites in vitro along the 1.3 kb region upstream of the InR P2 promoter in five Drosophila species. Moreover, we have tested the effect of mutations in the characterized dFOXO sites of D. melanogaster in transgenic flies. The number of experimentally established binding sites varies across the 1.3 kb region of any particular species, and their distribution also differs among species. In D. melanogaster, InR expression from P2 is differentially affected by dFOXO binding sites at the proximal and distal halves of the species 1.3 kb fragment. The observed uneven distribution of binding sites across this fragment might underlie their differential contribution to regulate InR transcription. PMID:29200426

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.

Role of G protein-coupled receptors (GPCR), matrix metalloproteinases 2 and 9 (MMP2 and MMP9), heparin-binding epidermal growth factor-like growth factor (hbEGF), epidermal growth factor receptor (EGFR), erbB2, and insulin-like growth factor 1 receptor (IGF-1R) in trenbolone acetate-stimulated bovine satellite cell proliferation.

PubMed

Thornton, K J; Kamange-Sollo, E; White, M E; Dayton, W R

2015-09-01

Implanting cattle with steroids significantly enhances feed efficiency, rate of gain, and muscle growth. However, the mechanisms responsible for these improvements in muscle growth have not been fully elucidated. Trenbolone acetate (TBA), a testosterone analog, has been shown to increase proliferation rate in bovine satellite cell (BSC) cultures. The classical genomic actions of testosterone have been well characterized; however, our results indicate that TBA may also initiate a quicker, nongenomic response that involves activation of G protein-coupled receptors (GPCR) resulting in activation of matrix metalloproteinases 2 and 9 (MMP2 and MMP9) that release membrane-bound heparin-binding epidermal growth factor-like growth factor (hbEGF), which then binds to and activates the epidermal growth factor receptor (EGFR) and/or erbB2. Furthermore, the EGFR has been shown to regulate expression of the IGF-1 receptor (IGF-1R), which is well known for its role in modulating muscle growth. To determine whether this nongenomic pathway is potentially involved in TBA-stimulated BSC proliferation, we analyzed the effects of treating BSC with guanosine 5'-O-2-thiodiphosphate (GDPβS), an inhibitor of all GPCR; a MMP2 and MMP9 inhibitor (MMPI); CRM19, a specific inhibitor of hbEGF; AG1478, a specific EGFR tyrosine kinase inhibitor; AG879, a specific erbB2 kinase inhibitor; and AG1024, an IGF-1R tyrosine kinase inhibitor on TBA-stimulated proliferation rate (H-thymidine incorporation). Assays were replicated at least 9 times for each inhibitor experiment using BSC cultures obtained from at least 3 different animals. Bovine satellite cell cultures were obtained from yearling steers that had no previous exposure to androgenic or estrogenic compounds. As expected, BSC cultures treated with 10 n TBA showed ( < 0.05) increased proliferation rate when compared with control cultures. Additionally, treatment with 5 ng hbEGF/mL stimulated proliferation in BSC cultures ( < 0.05). Treatment with GDPβS, MMPI, CRM197, AG1024, AG1478, and/or AG879 all suppressed ( < 0.05) TBA-induced increases in proliferation. These data indicate that TBA likely initiates a nongenomic response involving GPCR, MMP2 and MMP9, hbEGF, EGFR, erbB2, and IGF-1R, which may play a role in TBA-mediated increases in BSC proliferation.

Ligand-dependent nucleo-cytoplasmic shuttling of peroxisome proliferator-activated receptors, PPARα and PPARγ.

PubMed

Umemoto, Tomoe; Fujiki, Yukio

2012-07-01

Peroxisome proliferator-activated receptors (PPARs) play important roles in diverse biological processes including metabolisms of sugars and lipids and differentiation of cells such as adipocytes. PPARs are transcription factors belonging to the ligand-dependent hormone receptor group. To function as transcription factors, PPARs translocate into nucleus where they associate with transcription apparatus. However, mechanisms underlying nuclear transport of PPARs remain enigmatic. We show here that PPARα and PPARγ dynamically shuttle between nucleus and cytoplasm, although they constitutively and predominantly appear in nucleus. With a series of truncation mutants, we identify that PPAR nuclear transport is mediated by at least two nuclear localization signals (NLSs) in DNA-binding domain (DBD)-hinge and activation function 1 (AF1) regions and their respective receptors including importinα/β, importin 7, and an unidentified receptor. PPARs also harbor two nuclear export signals in DBD and ligand-binding domain regions that are recognized by distinct export receptors, calreticulin and CRM1. Moreover, we show that nuclear-cytoplasmic shuttling of PPARs is regulated by respective PPAR ligands and Ca2+ concentration. Taken together, we suggest that the multiple pathways for the nuclear-cytoplasmic transport of PPARs regulate the biological functions of PPARs in response to external signals. © 2012 The Authors Journal compilation © 2012 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

Signaling properties and pharmacological analysis of two sulfakinin receptors from the red flour beetle, Tribolium castaneum.

PubMed

Zels, Sven; Verlinden, Heleen; Dillen, Senne; Vleugels, Rut; Nachman, Ronald J; Vanden Broeck, Jozef

2014-01-01

Sulfakinin is an insect neuropeptide that constitutes an important component of the complex network of hormonal and neural factors that regulate feeding and digestion. The key modulating functions of sulfakinin are mediated by binding and signaling via G-protein coupled receptors. Although a substantial amount of functional data have already been reported on sulfakinins in different insect species, only little information is known regarding the properties of their respective receptors. In this study, we report on the molecular cloning, functional expression and characterization of two sulfakinin receptors in the red flour beetle, Tribolium castaneum. Both receptor open reading frames show extensive sequence similarity with annotated sulfakinin receptors from other insects. Comparison of the sulfakinin receptor sequences with homologous vertebrate cholecystokinin receptors reveals crucial conserved regions for ligand binding and receptor activation. Quantitative reverse transcriptase PCR shows that transcripts of both receptors are primarily expressed in the central nervous system of the beetle. Pharmacological characterization using 29 different peptide ligands clarified the essential requirements for efficient activation of these sulfakinin receptors. Analysis of the signaling pathway in multiple cell lines disclosed that the sulfakinin receptors of T. castaneum can stimulate both the Ca²⁺ and cyclic AMP second messenger pathways. This in depth characterization of two insect sulfakinin receptors may provide useful leads for the further development of receptor ligands with a potential applicability in pest control and crop protection.

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.

Lactose-containing starburst dendrimers: influence of dendrimer generation and binding-site orientation of receptors (plant/animal lectins and immunoglobulins) on binding properties.

PubMed

André, S; Ortega, P J; Perez, M A; Roy, R; Gabius, H J

1999-11-01

Starburst glycodendrimers offer the potential to serve as high-affinity ligands for clinically relevant sugar receptors. In order to define areas of application, their binding behavior towards sugar receptors with differential binding-site orientation but identical monosaccharide specificity must be evaluated. Using poly(amidoamine) starburst dendrimers of five generations, which contain the p-isothiocyanato derivative of p-aminophenyl-beta-D-lactoside as ligand group, four different types of galactoside-binding proteins were chosen for this purpose, i.e., the (AB)(2)-toxic agglutinin from mistletoe, a human immunoglobulin G fraction, the homodimeric galectin-1 with its two binding sites at opposite ends of the jelly-roll-motif-harboring protein and monomeric galectin-3. Direct solid-phase assays with surface-immobilized glycodendrimers resulted in obvious affinity enhancements by progressive core branching for the plant agglutinin and less pronounced for the antibody and galectin-1. High density of binding of galectin-3 with modest affinity increases only from the level of the 32-mer onwards points to favorable protein-protein interactions of the monomeric lectin and a spherical display of the end groups without a major share of backfolding. When the inhibitory potency of these probes was evaluated as competitor of receptor binding to an immobilized neoglycoprotein or to asialofetuin, a marked selectivity was detected. The 32- and 64-mers were second to none as inhibitors for the plant agglutinin against both ligand-exposing matrices and for galectin-1 on the matrix with a heterogeneous array of interglycoside distances even on the per-sugar basis. In contrast, a neoglycoprotein with the same end group was superior in the case of the antibody and, less pronounced, monomeric galectin-3. Intimate details of topological binding-site presentation and the ligand display on different generations of core assembly are major operative factors which determine the potential of dendrimers for applications as lectin-targeting device, as attested by these observations.

High vascular delivery of EGF, but low receptor binding rate is observed in AsPC-1 tumors as compared to normal pancreas.

PubMed

Samkoe, Kimberley S; Sexton, Kristian; Tichauer, Kenneth M; Hextrum, Shannon K; Pardesi, Omar; Davis, Scott C; O'Hara, Julia A; Hoopes, P Jack; Hasan, Tayyaba; Pogue, Brian W

2012-08-01

Cellular receptor targeted imaging agents present the potential to target extracellular molecular expression in cancerous lesions; however, the image contrast in vivo does not reflect the magnitude of overexpression expected from in vitro data. Here, the in vivo delivery and binding kinetics of epidermal growth factor receptor (EGFR) was determined for normal pancreas and AsPC-1 orthotopic pancreatic tumors known to overexpress EGFR. EGFR in orthotopic xenograft AsPC-1 tumors was targeted with epidermal growth factor (EGF) conjugated with IRDye800CW. The transfer rate constants (k(e), K₁₂, k₂₁, k₂₃, and k₃₂) associated with a three-compartment model describing the vascular delivery, leakage rate and binding of targeted agents were determined experimentally. The plasma excretion rate, k (e), was determined from extracted blood plasma samples. K₁₂, k₂₁, and k₃₂ were determined from ex vivo tissue washing studies at time points ≥ 24 h. The measured in vivo uptake of IRDye800CW-EGF and a non-targeted tracer dye, IRDye700DX-carboxylate, injected simultaneously was used to determined k₂₃. The vascular exchange of IRDye800CW-EGF in the orthotopic tumor (K₁₂ and k₂₁) was higher than in the AsPC-1 tumor as compared to normal pancreas, suggesting that more targeted agent can be taken up in tumor tissue. However, the cellular associated (binding) rate constant (k₂₃) was slightly lower for AsPC-1 pancreatic tumor (4.1 × 10(-4) s(-1)) than the normal pancreas (5.5 × 10(-4) s(-1)), implying that less binding is occurring. Higher vascular delivery but low cellular association in the AsPC-1 tumor compared to the normal pancreas may be indicative of low receptor density due to low cellular content. This attribute of the AsPC-1 tumor may indicate one contributing cause of the difficulty in treating pancreatic tumors with cellular targeted agents.

Estrogen regulation of chicken riboflavin carrier protein gene is mediated by ERE half sites without direct binding of estrogen receptor.

PubMed

Bahadur, Urvashi; Ganjam, Goutham K; Vasudevan, Nandini; Kondaiah, Paturu

2005-02-28

Estrogen is an important steroid hormone that mediates most of its effects on regulation of gene expression by binding to intracellular receptors. The consensus estrogen response element (ERE) is a 13bp palindromic inverted repeat with a three nucleotide spacer. However, several reports suggest that many estrogen target genes are regulated by diverse elements, such as imperfect EREs and ERE half sites (ERE 1/2), which are either the proximal or the distal half of the palindrome. To gain more insight into ERE half site-mediated gene regulation, we used a region from the estrogen-regulated chicken riboflavin carrier protein (RCP) gene promoter that contains ERE half sites. Using moxestrol, an analogue of estrogen and transient transfection of deletion and mutation containing RCP promoter/reporter constructs in chicken hepatoma (LMH2A) cells, we identified an estrogen response unit (ERU) composed of two consensus ERE 1/2 sites and one non-consensus ERE 1/2 site. Mutation of any of these sites within this ERU abolishes moxestrol response. Further, the ERU is able to confer moxestrol responsiveness to a heterologous promoter. Interestingly, RCP promoter is regulated by moxestrol in estrogen responsive human MCF-7 cells, but not in other cell lines such as NIH3T3 and HepG2 despite estrogen receptor-alpha (ER-alpha) co transfection. Electrophoretic mobility shift assays (EMSAs) with promoter regions encompassing the half sites and nuclear extracts from LMH2A cells show the presence of a moxestrol-induced complex that is abolished by a polyclonal anti-ERalpha antibody. Surprisingly, estrogen receptor cannot bind to these promoter elements in isolation. Thus, there appears to be a definite requirement for some other factor(s) in addition to estrogen receptor, for the generation of a suitable response of this promoter to estrogen. Our studies therefore suggest a novel mechanism of gene regulation by estrogen, involving ERE half sites without direct binding of ER to the cognate elements.

Elucidating the Activation Mechanism of the Insulin-Family Proteins with Molecular Dynamics Simulations.

PubMed

Papaioannou, Anastasios; Kuyucak, Serdar; Kuncic, Zdenka

2016-01-01

The insulin-family proteins bind to their own receptors, but insulin-like growth factor II (IGF-II) can also bind to the A isoform of the insulin receptor (IR-A), activating unique and alternative signaling pathways from those of insulin. Although extensive studies of insulin have revealed that its activation is associated with the opening of the B chain-C terminal (BC-CT), the activation mechanism of the insulin-like growth factors (IGFs) still remains unknown. Here, we present the first comprehensive study of the insulin-family proteins comparing their activation process and mechanism using molecular dynamics simulations to reveal new insights into their specificity to the insulin receptor. We have found that all the proteins appear to exhibit similar stochastic dynamics in their conformational change to an active state. For the IGFs, our simulations show that activation involves two opening locations: the opening of the BC-CT section away from the core, similar to insulin; and the additional opening of the BC-CT section away from the C domain. Furthermore, we have found that these two openings occur simultaneously in IGF-I, but not in IGF-II, where they can occur independently. This suggests that the BC-CT section and the C domain behave as a unified domain in IGF-I, but as two independent domains in IGF-II during the activation process, implying that the IGFs undergo different activation mechanisms for receptor binding. The probabilities of the active and inactive states of the proteins suggest that IGF-II is hyperactive compared to IGF-I. The hinge residue and the hydrophobic interactions in the core are found to play a critical role in the stability and activity of IGFs. Overall, our simulations have elucidated the crucial differences and similarities in the activation mechanisms of the insulin-family proteins, providing new insights into the molecular mechanisms responsible for the observed differences between IGF-I and IGF-II in receptor binding.

Opposite actions of transforming growth factor-beta 1 on the gene expression of atrial natriuretic peptide biological and clearance receptors in a murine thymic stromal cell line.

PubMed

Agui, T; Xin, X; Cai, Y; Shim, G; Muramatsu, Y; Yamada, T; Fujiwara, H; Matsumoto, K

1995-09-01

The regulation of the gene expression of the atrial natriuretic peptide receptor (ANPR) subtypes, ANPR-A, ANPR-B, and ANPR-C, was investigated in a murine thymic stromal cell line, MRL 104.8a. When MRL 104.8a cells were cultured with transforming growth factor (TGF)-beta1, [125I]ANP binding sites increased with increasing dose of TGF-beta1. These binding sites were identified as ANPR-C by a displacement experiment with ANPR-C-specific ligand, C-ANF, and by the affinity cross-linking of the [125I]ANP binding sites with a chemical cross-linker to determine the molecular weight of the ANPR. This augmentation of the ANPR-C expression was elucidated to occur at the transcriptional level by Northern blot experiment, comparison of the relative amounts of mRNA by reverse transcription (RT)-PCR, and in vitro nuclear transcription assay. Conversely, the expression of the ANP biological receptors, ANPR-A and ANPR-B, was shown to be down-regulated by TGF-beta1. These data suggest that TGF-beta1 regulates the gene expression of ANPRs in the thymic stromal cells and that ANP and TGF-beta1 might affect the thymic stromal cell functions.

Loss of EGF binding and cation transport response during differentiation of mouse neuroblastoma cells.

PubMed

Mummery, C L; van der Saag, P T; de Laat, S W

1983-01-01

Mouse neuroblastoma cells (clone N1E-115) differentiate in culture upon withdrawal of serum growth factors and acquire the characteristics of neurons. We have shown tht exponentially growing N1E-115 cells possess functional epidermal growth factor (EGF) receptors but that the capacity for binding EGF and for stimulation of DNA synthesis is lost as the cells differentiate. Furthermore, in exponentially growing cells, EGF induces a rapid increase in amiloride-sensitive Na+ influx, followed by stimulation of the (Na+-K+)ATPase, indicating that activation of the Na+/H+ exchange mechanism in N1E-115 cells [1] may be induced by EGF. The ionic response is also lost during differentiation, but we have shown that the stimulation of both Na+ and K+ influx is directly proportional to the number of occupied receptors in all cells whether exponentially growing or differentiating, thus only indirectly dependent on the external EGF concentration. The linearity of the relationships indicates that there is no rate-limiting step between EGF binding and the ionic response. Our data would suggest that as neuroblastoma cells differentiate and acquire neuronal properties, their ability to respond to mitogens, both biologically and in the activation of cation transport processes, progressively decreases owing to the loss of the appropriate receptors.

Receptor-Mediated Uptake and Intracellular Sorting of Multivalent Lipid Nanoparticles Against the Epidermal Growth Factor Receptor (EGFR) and the Human EGFR 2 (HER2)

NASA Astrophysics Data System (ADS)

Tran, David Tu

In the area of receptor-targeted lipid nanoparticles for drug delivery, efficiency has been mainly focused on cell-specificity, endocytosis, and subsequently effects on bioactivity such as cell growth inhibition. Aspects of targeted liposomal uptake and intracellular sorting are not well defined. This dissertation assessed a series of ligands as targeted functional groups against HER2 and EGFR for liposomal drug delivery. Receptor-mediated uptake, both mono-targeted and dual-targeted to multiple receptors of different ligand valence, and the intracellular sorting of lipid nanoparticles were investigated to improve the delivery of drugs to cancer cells. Lipid nanoparticles were functionalized through a new sequential micelle transfer---conjugation method, while the micelle transfer method was extended to growth factors. Through a combination of both techniques, anti-HER2 and anti-EGFR dual-targeted immunoliposomes with different combinations of ligand valence were developed for comparative studies. With the array of lipid nanoparticles, the uptake and cytotoxicity of lipid nanoparticles in relationship to ligand valence, both mono-targeting and dual-targeting, were evaluated on a small panel of breast cancer cell lines that express HER2 and EGFR of varying levels. Comparable uptake ratios of ligand to expressed receptor and apparent cooperativity were observed. For cell lines that express both receptors, additive dose-uptake effects were also observed with dual-targeted immunoliposomes, which translated to marginal improvements in cell growth inhibition with doxorubicin delivery. Colocalization analysis revealed that ligand-conjugated lipid nanoparticles settle to endosomal compartments similar to their attached ligands. Pathway transregulation and pathway saturation were also observed to affect trafficking. In the end, liposomes routed to the recycling endosomes were never observed to traffic beyond the endosomes nor to be exocytose like recycled ligands. Based on the experimental data, models were developed to help interpret and predict the binding and trafficking of lipid nanoparticles. The crosslink multivalent binding model of lipid nanoparticles to monovalent receptors was able to predict ligand valence for optimum binding, cell association concentrations, offer explanations to the antagonistic effects observed from high ligand valence, and predict the binding limitations of both ligand valence and ligand affinity. Hopefully, the models will serve as valuable tools for future optimizations in targeted liposomal drug delivery.

Salmonella enterica serotype Typhimurium Std fimbriae bind terminal alpha(1,2)fucose residues in the cecal mucosa.

PubMed

Chessa, Daniela; Winter, Maria G; Jakomin, Marcello; Bäumler, Andreas J

2009-02-01

The std operon encodes a fimbrial adhesin of Salmonella enterica serotype Typhimurium that is required for attachment to intestinal epithelial cells and for cecal colonization in the mouse. To study the mechanism by which this virulence factor contributes to colonization we characterized its binding specificity. Std-mediated binding to human colonic epithelial (Caco-2) cells could be abrogated by removing N-linked glycans. Adherence of Std fimbriated S. Typhimurium to Caco-2 cells could be blocked by co-incubation with H type 2 oligosaccharide (Fucalpha1-2Galbeta1-4GlcNAc) or by pretreatment of cells with alpha1-2 fucosidase. In contrast, pretreatment of Caco-2 cells with neuraminidase or co-incubation with the type 2 disaccharide precursor (Galbeta1-4GlcNAc) did not reduce adherence of Std fimbriated S. Typhimurium. Binding of purified Std fimbriae to Fucalpha1-2Galbeta1-4GlcNAc in a solid phase binding assay was competitively inhibited by Ulex europaeus agglutinin-I (UEA-I), a lectin specific for Fucalpha1-2 moieties. Purified Std fimbriae and UEA both bound to a receptor localized in the mucus layer of the murine cecum. These data suggest that the std operon encodes an adhesin that binds an alpha1-2 fucosylated receptor(s) present in the cecal mucosa.

Binding of GDNF and neurturin to human GDNF family receptor alpha 1 and 2. Influence of cRET and cooperative interactions.

PubMed

Cik, M; Masure, S; Lesage, A S; Van Der Linden, I; Van Gompel, P; Pangalos, M N; Gordon, R D; Leysen, J E

2000-09-08

The members of the glial cell line-derived neurotrophic factor (GDNF) family signal via binding to the glycosyl phosphatidylinositol-anchored membrane proteins, the GDNF family receptors alpha (GFRalpha), and activation of cRET. We performed a detailed analysis of the binding of GDNF and neurturin to their receptors and investigated the influence of cRET on the binding affinities. We show that the rate of dissociation of (125)I-GDNF from GFRalpha1 is increased in the presence of 50 nm GDNF, an effect that can be explained by the occurrence of negative cooperativity. Scatchard plots of the ligand concentration binding isotherms reveal a pronounced downward curvature at low (125)I-GDNF concentrations suggesting the presence of positive cooperativity. This effect is observed in the range of GDNF concentrations responsible for biological activity (1-20 pm) and may have an important role in cRET-independent signaling. A high affinity site with a K(D) of 11 pm for (125)I-GDNF is detected only when GFRalpha1 is co-expressed with cRET at a DNA ratio of 1:3. These results suggest an interaction of GFRalpha1 and cRET in the absence of GDNF and demonstrate that the high affinity binding can be measured only when cRET is present.

Salmonella enterica serotype Typhimurium Std fimbriae bind terminal α (1,2)fucose residues in the cecal mucosa

PubMed Central

Chessa, Daniela; Winter, Maria G.; Jakomin, Marcello; Bäumler, Andreas J.

2013-01-01

SUMMARY The std operon encodes a fimbrial adhesin of Salmonella enterica serotype Typhimurium that is required for attachment to intestinal epithelial cells and for cecal colonization in the mouse. To study the mechanism by which this virulence factor contributes to colonization we characterized its binding specificity. Std-mediated binding to human colonic epithelial (Caco-2) cells could be abrogated by removing N-linked glycans. Adherence of Std fimbriated S. Typhimurium to Caco-2 cells could be blocked by co-incubation with H type 2 oligosaccharide (Fucα1-2Galβ1-4GlcNAc) or by pretreatment of cells with α1-2 fucosidase. In contrast, pretreatment of Caco-2 cells with neuraminidase or co-incubation with the type 2 disaccharide precursor (Galβ1-4GlcNAc) did not reduce adherence of Std fimbriated S. Typhimurium. Binding of purified Std fimbriae to Fucα1-2Galβ1-4GlcNAc in a solid phase binding assay was competitively inhibited by Ulex europaeus agglutinin-I (UEA-I), a lectin specific for Fucα1-2 moieties. Purified Std fimbriae and UEA both bound to a receptor localized in the mucus layer of the murine cecum. These data suggest that the std operon encodes an adhesin that binds an α1-2 fucosylated receptor(s) present in the cecal mucosa. PMID:19183274

Identification of amino acids in the transmembrane and juxtamembrane domains of the platelet-derived growth factor receptor required for productive interaction with the bovine papillomavirus E5 protein.

PubMed

Petti, L M; Reddy, V; Smith, S O; DiMaio, D

1997-10-01

The bovine papillomavirus E5 protein forms a stable complex with the cellular platelet-derived growth factor (PDGF) beta receptor, resulting in receptor activation and cell transformation. Amino acids in both the putative transmembrane domain and extracytoplasmic carboxyl-terminal domain of the E5 protein appear important for PDGF receptor binding and activation. Previous analysis indicated that the transmembrane domain of the receptor was also required for complex formation and receptor activation. Here we analyzed receptor chimeras and point mutants to identify specific amino acids in the PDGF beta receptor required for productive interaction with the E5 protein. These receptor mutants were analyzed in murine Ba/F3 cells, which do not express endogenous receptor. Our results confirmed the importance of the transmembrane domain of the receptor for complex formation, receptor tyrosine phosphorylation, and mitogenic signaling in response to the E5 protein and established that the threonine residue in this domain is required for these activities. In addition, a positive charge in the extracellular juxtamembrane domain of the receptor was required for E5 interaction and signaling, whereas replacement of the wild-type lysine with either a neutral or acidic amino acid inhibited E5-induced receptor activation and transformation. All of the receptor mutants defective for activation by the E5 protein responded to acute treatment with PDGF and to stable expression of v-Sis, a form of PDGF. The required juxtamembrane lysine and transmembrane threonine are predicted to align precisely on the same face of an alpha helix packed in a left-handed coiled-coil geometry. These results establish that the E5 protein and v-Sis recognize distinct binding sites on the PDGF beta receptor and further clarify the nature of the interaction between the viral transforming protein and its cellular target.

The role of small molecule platelet-derived growth factor receptor (PDGFR) inhibitors in the treatment of neoplastic disorders.

PubMed

Roskoski, Robert

2018-03-01

Platelet-derived growth factor (PDGF) was discovered as a serum-derived component necessary for the growth of smooth muscle cells, fibroblasts, and glial cells. The PDGF family is a product of four gene products and consists of five dimeric isoforms: PDGF-AA, PDGF-BB, PDGF-CC, PDGF-DD, and the PDGF-AB heterodimer. This growth factor family plays an essential role in embryonic development and in wound healing in the adult. These growth factors mediate their effects by binding to and activating their receptor protein-tyrosine kinases, which are encoded by two genes: PDGFRA and PDGFRB. The functional receptors consist of the PDGFRα/α and PDGFRβ/β homodimers and the PDGFRα/β heterodimer. Although PDGF signaling is most closely associated with mesenchymal cells, PDGFs and PDGF receptors are widely expressed in the mammalian central nervous system. The PDGF receptors contain an extracellular domain that is made up of five immunoglobulin-like domains (Ig-d1/2/3/4/5), a transmembrane segment, a juxtamembrane segment, a protein-tyrosine kinase domain that contains an insert of about 100 amino acid residues, and a carboxyterminal tail. Although uncommon, activating mutations in the genes for PDGF or PDGF receptors have been documented in various neoplasms including dermatofibrosarcoma protuberans (DFSP) and gastrointestinal stromal tumors (GIST). In most neoplastic diseases, PDGF expression and action appear to involve the tumor stroma. Moreover, this family is pro-angiogenic. More than ten PDGFRα/β multikinase antagonists have been approved by the FDA for the treatment of several neoplastic disorders and interstitial pulmonary fibrosis (www.brimr.org/PKI/PKIs.htm). Type I protein kinase inhibitors interact with the active enzyme form with DFG-D of the proximal activation segment directed inward toward the active site (DFG-D in ). In contrast, type II inhibitors bind to their target with the DFG-D pointing away from the active site (DFG-D out ). We used the Schrödinger induced-fit docking protocol to model the interaction of several antagonists with PDGFRα including imatinib, sorafenib, and sunitinib. The results indicate that these antagonists are able to bind to the DFG-D out conformation of the receptor and are thus classified as type II inhibitors. Owing to the multiplicity of less active protein kinase conformations when compared with the canonical more active conformation, it was hypothesized that type II drugs would be less promiscuous than type I drugs which bind to the typical active conformation. Although type II inhibitors may be more selective, most - if not all - inhibit more than one target protein kinase and the differences are a matter of degree only. Copyright © 2018 Elsevier Ltd. All rights reserved.

Differential manipulation of arrestin-3 binding to basal and agonist-activated G protein-coupled receptors.

PubMed

Prokop, Susanne; Perry, Nicole A; Vishnivetskiy, Sergey A; Toth, Andras D; Inoue, Asuka; Milligan, Graeme; Iverson, Tina M; Hunyady, Laszlo; Gurevich, Vsevolod V

2017-08-01

Non-visual arrestins interact with hundreds of different G protein-coupled receptors (GPCRs). Here we show that by introducing mutations into elements that directly bind receptors, the specificity of arrestin-3 can be altered. Several mutations in the two parts of the central "crest" of the arrestin molecule, middle-loop and C-loop, enhanced or reduced arrestin-3 interactions with several GPCRs in receptor subtype and functional state-specific manner. For example, the Lys139Ile substitution in the middle-loop dramatically enhanced the binding to inactive M 2 muscarinic receptor, so that agonist activation of the M 2 did not further increase arrestin-3 binding. Thus, the Lys139Ile mutation made arrestin-3 essentially an activation-independent binding partner of M 2 , whereas its interactions with other receptors, including the β 2 -adrenergic receptor and the D 1 and D 2 dopamine receptors, retained normal activation dependence. In contrast, the Ala248Val mutation enhanced agonist-induced arrestin-3 binding to the β 2 -adrenergic and D 2 dopamine receptors, while reducing its interaction with the D 1 dopamine receptor. These mutations represent the first example of altering arrestin specificity via enhancement of the arrestin-receptor interactions rather than selective reduction of the binding to certain subtypes. Copyright © 2017. Published by Elsevier Inc.

Phosphatidylserine recognition and induction of apoptotic cell clearance by Drosophila engulfment receptor Draper.

PubMed

Tung, Tran Thanh; Nagaosa, Kaz; Fujita, Yu; Kita, Asana; Mori, Hiroki; Okada, Ryo; Nonaka, Saori; Nakanishi, Yoshinobu

2013-05-01

The membrane phospholipid phosphatidylserine is exposed on the cell surface during apoptosis and acts as an eat-me signal in the phagocytosis of apoptotic cells in mammals and nematodes. However, whether this is also true in insects was unclear. When milk fat globule-epidermal growth factor 8, a phosphatidylserine-binding protein of mammals, was ectopically expressed in Drosophila, the level of phagocytosis was reduced, whereas this was not the case for the same protein lacking a domain responsible for the binding to phosphatidylserine. We found that the extracellular region of Draper, an engulfment receptor of Drosophila, binds to phosphatidylserine in an enzyme-linked immunosorbent assay-like solid-phase assay and in an assay for surface plasmon resonance. A portion of Draper containing domains EMI and NIM located close to the N-terminus was required for binding to phosphatidylserine, and a Draper protein lacking this region was not active in Drosophila. Finally, the level of tyrosine-phosphorylated Draper, indicative of the activation of Draper, in a hemocyte-derived cell line was increased after treatment with phosphatidylserine-containing liposome. These results indicated that phosphatidylserine serves as an eat-me signal in the phagocytic removal of apoptotic cells in Drosophila and that Draper is a phosphatidylserine-binding receptor for phagocytosis.

HIP1 and HIP1r stabilize receptor tyrosine kinases and bind 3-phosphoinositides via epsin N-terminal homology domains.

PubMed

Hyun, Teresa S; Rao, Dinesh S; Saint-Dic, Djenann; Michael, L Evan; Kumar, Priti D; Bradley, Sarah V; Mizukami, Ikuko F; Oravecz-Wilson, Katherine I; Ross, Theodora S

2004-04-02

Huntingtin-interacting protein 1-related (HIP1r) is the only known mammalian relative of huntingtin-interacting protein 1 (HIP1), a protein that transforms fibroblasts via undefined mechanisms. Here we demonstrate that both HIP1r and HIP1 bind inositol lipids via their epsin N-terminal homology (ENTH) domains. In contrast to other ENTH domain-containing proteins, lipid binding is preferential to the 3-phosphate-containing inositol lipids, phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,5-bisphosphate. Furthermore, the HIP1r ENTH domain, like that of HIP1, is necessary for lipid binding, and expression of an ENTH domain-deletion mutant, HIP1r/deltaE, induces apoptosis. Consistent with the ability of HIP1r and HIP1 to affect cell survival, full-length HIP1 and HIP1r stabilize pools of growth factor receptors by prolonging their half-life following ligand-induced endocytosis. Although HIP1r and HIP1 display only a partially overlapping pattern of protein interactions, these data suggest that both proteins share a functional homology by binding 3-phosphorylated inositol lipids and stabilizing receptor tyrosine kinases in a fashion that may contribute to their ability to alter cell growth and survival.

Insight on Mutation-Induced Resistance from Molecular Dynamics Simulations of the Native and Mutated CSF-1R and KIT

PubMed Central

Da Silva Figueiredo Celestino Gomes, Priscila; Chauvot De Beauchêne, Isaure; Panel, Nicolas; Lopez, Sophie; De Sepulveda, Paulo; Geraldo Pascutti, Pedro; Solary, Eric; Tchertanov, Luba

2016-01-01

The receptors tyrosine kinases (RTKs) for the colony stimulating factor-1, CSF-1R, and for the stem cell factor, SCFR or KIT, are important mediators of signal transduction. The abnormal function of these receptors, promoted by gain-of-function mutations, leads to their constitutive activation, associated with cancer or other proliferative diseases. A secondary effect of the mutations is the alteration of receptors’ sensitivity to tyrosine kinase inhibitors, compromising effectiveness of these molecules in clinical treatment. In particular, the mutation V560G in KIT increases its sensitivity to Imatinib, while the D816V in KIT, and D802V in CSF-1R, triggers resistance to the drug. We analyzed the Imatinib binding affinity to the native and mutated KIT (mutations V560G, S628N and D816V) and CSF-1R (mutation D802V) by using molecular dynamics simulations and energy calculations of Imatinib•target complexes. Further, we evaluated the sensitivity of the studied KIT receptors to Imatinib by measuring the inhibition of KIT phosphorylation. Our study showed that (i) the binding free energy of Imatinib to the targets is highly correlated with their experimentally measured sensitivity; (ii) the electrostatic interactions are a decisive factor affecting the binding energy; (iii) the most deleterious impact to the Imatinib sensitivity is promoted by D802V (CSF-1R) and D816V (KIT) mutations; (iv) the role of the juxtamembrane region, JMR, in the imatinib binding is accessory. These findings contribute to a better description of the mutation-induced effects alternating the targets sensitivity to Imatinib. PMID:27467080

SRY-box-containing Gene 2 Regulation of Nuclear Receptor Tailless (Tlx) Transcription in Adult Neural Stem Cells*

PubMed Central

Shimozaki, Koji; Zhang, Chun-Li; Suh, Hoonkyo; Denli, Ahmet M.; Evans, Ronald M.; Gage, Fred H.

2012-01-01

Adult neurogenesis is maintained by self-renewable neural stem cells (NSCs). Their activity is regulated by multiple signaling pathways and key transcription factors. However, it has been unclear whether these factors interplay with each other at the molecular level. Here we show that SRY-box-containing gene 2 (Sox2) and nuclear receptor tailless (TLX) form a molecular network in adult NSCs. We observed that both Sox2 and TLX proteins bind to the upstream region of Tlx gene. Sox2 positively regulates Tlx expression, whereas the binding of TLX to its own promoter suppresses its transcriptional activity in luciferase reporter assays. Such TLX-mediated suppression can be antagonized by overexpressing wild-type Sox2 but not a mutant lacking the transcriptional activation domain. Furthermore, through regions involved in DNA-binding activity, Sox2 and TLX physically interact to form a complex on DNAs that contain a consensus binding site for TLX. Finally, depletion of Sox2 revealed the potential negative feedback loop of TLX expression that is antagonized by Sox2 in adult NSCs. These data suggest that Sox2 plays an important role in Tlx transcription in cultured adult NSCs. PMID:22194602

SRY-box-containing gene 2 regulation of nuclear receptor tailless (Tlx) transcription in adult neural stem cells.

PubMed

Shimozaki, Koji; Zhang, Chun-Li; Suh, Hoonkyo; Denli, Ahmet M; Evans, Ronald M; Gage, Fred H

2012-02-17

Adult neurogenesis is maintained by self-renewable neural stem cells (NSCs). Their activity is regulated by multiple signaling pathways and key transcription factors. However, it has been unclear whether these factors interplay with each other at the molecular level. Here we show that SRY-box-containing gene 2 (Sox2) and nuclear receptor tailless (TLX) form a molecular network in adult NSCs. We observed that both Sox2 and TLX proteins bind to the upstream region of Tlx gene. Sox2 positively regulates Tlx expression, whereas the binding of TLX to its own promoter suppresses its transcriptional activity in luciferase reporter assays. Such TLX-mediated suppression can be antagonized by overexpressing wild-type Sox2 but not a mutant lacking the transcriptional activation domain. Furthermore, through regions involved in DNA-binding activity, Sox2 and TLX physically interact to form a complex on DNAs that contain a consensus binding site for TLX. Finally, depletion of Sox2 revealed the potential negative feedback loop of TLX expression that is antagonized by Sox2 in adult NSCs. These data suggest that Sox2 plays an important role in Tlx transcription in cultured adult NSCs.

A mammalian model for Laron syndrome produced by targeted disruption of the mouse growth hormone receptor/binding protein gene (the Laron mouse)

PubMed Central

Zhou, Yihua; Xu, Bixiong C.; Maheshwari, Hiralal G.; He, Li; Reed, Michael; Lozykowski, Maria; Okada, Shigeru; Cataldo, Lori; Coschigamo, Karen; Wagner, Thomas E.; Baumann, Gerhard; Kopchick, John J.

1997-01-01

Laron syndrome [growth hormone (GH) insensitivity syndrome] is a hereditary dwarfism resulting from defects in the GH receptor (GHR) gene. GHR deficiency has not been reported in mammals other than humans. Many aspects of GHR dysfunction remain unknown because of ethical and practical limitations in studying humans. To create a mammalian model for this disease, we generated mice bearing a disrupted GHR/binding protein (GHR/BP) gene through a homologous gene targeting approach. Homozygous GHR/BP knockout mice showed severe postnatal growth retardation, proportionate dwarfism, absence of the GHR and GH binding protein, greatly decreased serum insulin-like growth factor I and elevated serum GH concentrations. These characteristics represent the phenotype typical of individuals with Laron syndrome. Animals heterozygous for the GHR/BP defect show only minimal growth impairment but have an intermediate biochemical phenotype, with decreased GHR and GH binding protein expression and slightly diminished insulin-like growth factor I levels. These findings indicate that the GHR/BP-deficient mouse (Laron mouse) is a suitable model for human Laron syndrome that will prove useful for the elucidation of many aspects of GHR/BP function that cannot be obtained in humans. PMID:9371826

A mammalian model for Laron syndrome produced by targeted disruption of the mouse growth hormone receptor/binding protein gene (the Laron mouse).

PubMed

Zhou, Y; Xu, B C; Maheshwari, H G; He, L; Reed, M; Lozykowski, M; Okada, S; Cataldo, L; Coschigamo, K; Wagner, T E; Baumann, G; Kopchick, J J

1997-11-25

Laron syndrome [growth hormone (GH) insensitivity syndrome] is a hereditary dwarfism resulting from defects in the GH receptor (GHR) gene. GHR deficiency has not been reported in mammals other than humans. Many aspects of GHR dysfunction remain unknown because of ethical and practical limitations in studying humans. To create a mammalian model for this disease, we generated mice bearing a disrupted GHR/binding protein (GHR/BP) gene through a homologous gene targeting approach. Homozygous GHR/BP knockout mice showed severe postnatal growth retardation, proportionate dwarfism, absence of the GHR and GH binding protein, greatly decreased serum insulin-like growth factor I and elevated serum GH concentrations. These characteristics represent the phenotype typical of individuals with Laron syndrome. Animals heterozygous for the GHR/BP defect show only minimal growth impairment but have an intermediate biochemical phenotype, with decreased GHR and GH binding protein expression and slightly diminished insulin-like growth factor I levels. These findings indicate that the GHR/BP-deficient mouse (Laron mouse) is a suitable model for human Laron syndrome that will prove useful for the elucidation of many aspects of GHR/BP function that cannot be obtained in humans.

Enhanced Human-Type Receptor Binding by Ferret-Transmissible H5N1 with a K193T Mutation.

PubMed

Peng, Wenjie; Bouwman, Kim M; McBride, Ryan; Grant, Oliver C; Woods, Robert J; Verheije, Monique H; Paulson, James C; de Vries, Robert P

2018-05-15

All human influenza pandemics have originated from avian influenza viruses. Although multiple changes are needed for an avian virus to be able to transmit between humans, binding to human-type receptors is essential. Several research groups have reported mutations in H5N1 viruses that exhibit specificity for human-type receptors and promote respiratory droplet transmission between ferrets. Upon detailed analysis, we have found that these mutants exhibit significant differences in fine receptor specificity compared to human H1N1 and H3N2 and retain avian-type receptor binding. We have recently shown that human influenza viruses preferentially bind to α2-6-sialylated branched N-linked glycans, where the sialic acids on each branch can bind to receptor sites on two protomers of the same hemagglutinin (HA) trimer. In this binding mode, the glycan projects over the 190 helix at the top of the receptor-binding pocket, which in H5N1 would create a stearic clash with lysine at position 193. Thus, we hypothesized that a K193T mutation would improve binding to branched N-linked receptors. Indeed, the addition of the K193T mutation to the H5 HA of a respiratory-droplet-transmissible virus dramatically improves both binding to human trachea epithelial cells and specificity for extended α2-6-sialylated N-linked glycans recognized by human influenza viruses. IMPORTANCE Infections by avian H5N1 viruses are associated with a high mortality rate in several species, including humans. Fortunately, H5N1 viruses do not transmit between humans because they do not bind to human-type receptors. In 2012, three seminal papers have shown how these viruses can be engineered to transmit between ferrets, the human model for influenza virus infection. Receptor binding, among others, was changed, and the viruses now bind to human-type receptors. Receptor specificity was still markedly different compared to that of human influenza viruses. Here we report an additional mutation in ferret-transmissible H5N1 that increases human-type receptor binding. K193T seems to be a common receptor specificity determinant, as it increases human-type receptor binding in multiple subtypes. The K193T mutation can now be used as a marker during surveillance of emerging viruses to assess potential pandemic risk. Copyright © 2018 American Society for Microbiology.

Hyperekplexia mutation R271L of alpha1 glycine receptors potentiates allosteric interactions of nortropeines, propofol and glycine with [3H]strychnine binding.

PubMed

Maksay, Gábor; Bíró, Tímea; Laube, Bodo; Nemes, Péter

2008-01-01

Human alpha1 and hyperekplexia mutant alpha1(R271L) glycine receptors (GlyRs) were transiently expressed in human embryonic kidney 293 cells for [3H]strychnine binding. Binding parameters were determined using a ternary allosteric model. The hyperekplexia mutation increased the positive cooperativity of 0.3 mM propofol and glycine binding by about six times: the cooperativity factor beta was 0.26 for alpha1 GlyRs and 0.04 for alpha1(R271L) GlyRs. Thus, propofol restored the potency of glycine impaired by the mutation. Five nortropeines, i.e. substituted benzoates of nortropine and a new compound, nortropisetron were prepared and also examined on [3H]strychnine binding. They showed nanomolar displacing potencies amplified by the hyperekplexia mutation. The affinity of nor-O-zatosetron (2.6 nM) is one of the highest reported for GlyRs. This binding test offers an in vitro method to evaluate agents against neurological disorders associated with inherited mutations of GlyRs.

Transforming growth factor β suppresses peroxisome proliferator-activated receptor γ expression via both SMAD binding and novel TGF-β inhibitory elements.

PubMed

Lakshmi, Sowmya P; Reddy, Aravind T; Reddy, Raju C

2017-04-24

Transforming growth factor β (TGF-β) contributes to wound healing and, when dysregulated, to pathological fibrosis. TGF-β and the anti-fibrotic nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) repress each other's expression, and such PPARγ down-regulation is prominent in fibrosis and mediated, via previously unknown SMAD-signaling mechanisms. Here, we show that TGF-β induces the association of SMAD3 with both SMAD4, needed for translocation of the complex into the nucleus, and the essential context-sensitive co-repressors E2F4 and p107. The complex mediates TGF-β-induced repression by binding to regulatory elements in the target promoter. In the PPARG promoter, we found that the SMAD3-SMAD4 complex binds both to a previously unknown consensus TGF-β inhibitory element (TIE) and also to canonical SMAD-binding elements (SBEs). Furthermore, the TIE and SBEs independently mediated the partial repression of PPARG transcription, the first demonstration of a TIE and SBEs functioning within the same promoter. Also, TGF-β-treated fibroblasts contained SMAD complexes that activated a SMAD target gene in addition to those repressing PPARG transcription, the first finding of such dual activity within the same cell. These findings describe in detail novel mechanisms by which TGF-β represses PPARG transcription, thereby facilitating its own pro-fibrotic activity. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

Medicinal mushroom Lingzhi or Reishi, Ganoderma lucidum (W.Curt.:Fr.) P. Karst., beta-glucan induces Toll-like receptors and fails to induce inflammatory cytokines in NF-kappaB inhibitor-treated macrophages.

PubMed

Batbayar, Sainkhuu; Kim, Mi Jeong; Kim, Ha Won

2011-01-01

Beta-Glucan of medicinal Lingzhi or Reishi mushroom, Ganoderma lucidum (BGG), possesses immunostimulatory and anti-tumor activities. Innate immune cells are activated by the binding of beta-glucan to the dectin-1 receptor. The present study investigated the immunostimulating activities of BGG, including binding to dectin-1, secretion of cytokines and reactive oxygen species, and induction of Toll-like receptors (TLRs) in RAW264.7 mouse macrophages. Reverse transcription-polymerase chain reaction and flow cytometry were used for the cytokine and TLR analyses. A mouse inflammation antibody array was used for protein-level cytokine analysis. BGG bound to dectin-1 and induced RAW264.7 cell secretion of several cytokines, including granulocyte colony-stimulating factor, interleukin (IL)-6, regulated upon activation normal T cell expressed and secreted (RANTES), tissue inhibitor of metalloproteinase-1, and tumor necrosis factor-alpha. The secretion of these cytokines was further increased by the addition of lipopolysaccharide (LPS). BGG also induced both nitric oxide and inducible nitric oxide synthase (iNOS). Treatment with an inhibitor of nuclear factor-kappa B (NF-kappaB) reduced the induction of IL-1, IL-6, and iNOS in a concentration-dependent manner. Expressions of TLR2, TLR4, and TLR6 were increased by BGG treatment, and addition of LPS induced further induction of TLR4 and TLR6. Our result indicates that BGG induces macrophage secretion of inflammatory cytokines, which can be potentiated by the presence of LPS, likely by binding to dectin-1 and TLR-2/6 receptors, which activate NF-kappaB and prompt the secretion of cytokines.

Combination of two insulin-like growth factor-I receptor inhibitory antibodies targeting distinct epitopes leads to an enhanced antitumor response.

PubMed

Dong, Jianying; Demarest, Stephen J; Sereno, Arlene; Tamraz, Susan; Langley, Emma; Doern, Adam; Snipas, Tracey; Perron, Keli; Joseph, Ingrid; Glaser, Scott M; Ho, Steffan N; Reff, Mitchell E; Hariharan, Kandasamy

2010-09-01

The insulin-like growth factor-I receptor (IGF-IR) is a cell surface receptor tyrosine kinase that mediates cell survival signaling and supports tumor progression in multiple tumor types. We identified a spectrum of inhibitory IGF-IR antibodies with diverse binding epitopes and ligand-blocking properties. By binding distinct inhibitory epitopes, two of these antibodies, BIIB4 and BIIB5, block both IGF-I and IGF-II binding to IGF-IR using competitive and allosteric mechanisms, respectively. Here, we explored the inhibitory effects of combining BIIB4 and BIIB5. In biochemical assays, the combination of BIIB4 and BIIB5 improved both the potency and extent of IGF-I and IGF-II blockade compared with either antibody alone. In tumor cells, the combination of BIIB4 and BIIB5 accelerated IGF-IR downregulation and more efficiently inhibited IGF-IR activation as well as downstream signaling, particularly AKT phosphorylation. In several carcinoma cell lines, the antibody combination more effectively inhibited ligand-driven cell growth than either BIIB4 or BIIB5 alone. Notably, the enhanced tumor growth-inhibitory activity of the BIIB4 and BIIB5 combination was much more pronounced at high ligand concentrations, where the individual antibodies exhibited substantially reduced activity. Compared with single antibodies, the BIIB4 and BIIB5 combination also significantly further enhanced the antitumor activity of the epidermal growth factor receptor inhibitor erlotinib and the mTOR inhibitor rapamycin. Moreover, in osteosarcoma and hepatocellular carcinoma xenograft models, the BIIB4 and BIIB5 combination significantly reduced tumor growth to a greater degree than each single antibody. Taken together, our results suggest that targeting multiple distinct inhibitory epitopes on IGF-IR may be a more effective strategy of affecting the IGF-IR pathway in cancer.

A diketopiperazine factor from Rheinheimera aquimaris QSI02 exhibits anti-quorum sensing activity.

PubMed

Sun, Shiwei; Dai, Xiaoyun; Sun, Jiao; Bu, Xiangguo; Weng, Caihong; Li, Hui; Zhu, Hu

2016-12-21

An ethyl acetate (EtOAc) extract isolated from the marine bacterium, Rheinheimera aquimaris QSI02, was found to exhibit anti-quorum sensing (anti-QS) activity. A subsequent bioassay-guided isolation protocol led to the detection of an active diketopiperazine factor, cyclo(Trp-Ser). Biosensor assay data showed that the minimum inhibitory concentration (MIC) of cyclo(Trp-Ser) ranged from 3.2 mg/ml to 6.4 mg/m for several microorganisms, including Escherichia coli, Chromobacterium violaceum CV026, Pseudomonas aeruginosa PA01, Staphylococcus aureus, and Candida albicans. Additionally, sub-MICs of cyclo(Trp-Ser) decreased the QS-regulated violacein production in C. violaceum CV026 by 67%. Furthermore, cyclo(Trp-Ser) can decrease QS-regulated pyocyanin production, elastase activity and biofilm formation in P. aeruginosa PA01 by 65%, 40% and 59.9%, respectively. Molecular docking results revealed that cyclo(Trp-Ser) binds to CviR receptor more rigidly than C 6 HSL with lower docking energy -8.68 kcal/mol, while with higher binding energy of -8.40 kcal/mol than 3-oxo-C 12 HSL in LasR receptor. Molecular dynamics simulation suggested that cyclo(Trp-Ser) is more easy to bind to CviR receptor than natural signaling molecule, but opposite in LasR receptor. These results suggest that cyclo(Trp-Ser) can be used as a potential inhibitor to control QS systems of C. violaceum and P. aeruginosa and provide increased the understanding of molecular mechanism that influences QS-regulated behaviors.

Contribution of vascular endothelial growth factor receptor-2 sialylation to the process of angiogenesis.

PubMed

Chiodelli, P; Rezzola, S; Urbinati, C; Federici Signori, F; Monti, E; Ronca, R; Presta, M; Rusnati, M

2017-11-23

Vascular endothelial growth factor receptor-2 (VEGFR2) is the main pro-angiogenic receptor expressed by endothelial cells (ECs). Using surface plasmon resonance, immunoprecipitation, enzymatic digestion, immunofluorescence and cross-linking experiments with specific sugar-binding lectins, we demonstrated that VEGFR2 bears both α,1-fucose and α(2,6)-linked sialic acid (NeuAc). However, only the latter is required for VEGF binding to VEGFR2 and consequent VEGF-dependent VEGFR2 activation and motogenic response in ECs. Notably, downregulation of β-galactoside α(2,6)-sialyltransferase expression by short hairpin RNA transduction inhibits VEGFR2 α(2,6) sialylation that is paralleled by an increase of β-galactoside α(2,3)-sialyltransferase expression. This results in an ex-novo α(2,3)-NeuAc sialylation of the receptor that functionally replaces the lacking α(2,6)-NeuAc, thus allowing VEGF/VEGFR2 interaction. In keeping with the role of VEGFR2 sialylation in angiogenesis, the α(2,6)-NeuAc-binding lectin Sambucus nigra (SNA) prevents VEGF-dependent VEGFR2 autophosphorylation and EC motility, proliferation and motogenesis. In addition, SNA exerts a VEGF-antagonist activity in tridimensional angiogenesis models in vitro and in the chick-embryo chorioallantoic membrane neovascularization assay and mouse matrigel plug assay in vivo. In conclusion, VEGFR2-associated NeuAc plays an important role in modulating VEGF/VEGFR2 interaction, EC pro-angiogenic activation and neovessel formation. VEGFR2 sialylation may represent a target for the treatment of angiogenesis-dependent diseases.

GPER Mediates Non-Genomic Effects of Estrogen.

PubMed

Pupo, Marco; Maggiolini, Marcello; Musti, Anna Maria

2016-01-01

Estrogens are important modulators of a broad spectrum of physiological functions in humans. However, despite their beneficial actions, a number of lines of evidence correlate the sustained exposure to exogenous estrogen with increased risk of the onset of various cancers. Mainly these steroid hormones induce their effects by binding and activating estrogen receptors (ERα and ERβ). These receptors belong to the family of ligand-regulated transcription factors, and upon activation they regulate the expression of different target genes by binding directly to specific DNA sequences. On the other hand, in recent years it has become clear that the G protein-coupled estrogen receptor 30 (GPR30/GPER) is able to mediate non-genomic action of estrogens in different cell contexts. In particular, GPER has been shown to specifically bind estrogens, and in turn to functionally cross-react with diverse cell signaling systems such as the epidermal growth factor receptor (EGFR) pathway, the Notch signaling pathway and the mitogen-activated protein kinases (MAPK) pathway. In this chapter we will present some of the different experimental techniques currently used to demonstrate the functional role of GPER in mediating non-genomic actions of estrogens, such as the dual luciferase assay, assessment of the involvement of GPER in the stimulation of cell migration in breast cancer cell lines and in cancer-associated fibroblasts, and chromatin immunoprecipitation assay. Overall, the experimental procedures described herein represent key instruments for assessing the biological role of GPER in mediating non-genomic signals of estrogen.

Role of the thrombin receptor in restenosis and atherosclerosis.

PubMed

Baykal, D; Schmedtje, J F; Runge, M S

1995-02-23

Thrombus generation is central to thrombosis at vascular lesion sites, including post-PCTA acute reocclusion and chronic restenosis. Thrombin stimulates platelet activation, monocyte and neutrophil chemotaxis, and endothelial production of prothrombotic factors. The varied physiologic effects of thrombin are due to the widespread presence of thrombin receptors in many cell types. The receptor is uniquely activated: thrombin binds to the receptor at the thrombin anion-binding exosite, the receptor ligand ("tethered ligand") apparently being a sequence of 6 amino acids (SFLLRN). Thus, peptides corresponding to the sequence of the tethered ligand can stimulate almost all functions of native thrombin itself. Several intracellular signaling pathways have been identified as important in the restenosis process: the G protein-related pathway, cyclic adenosine monophosphate (cAMP) mediator pathway, and tyrosine kinase activation pathway. In situ hybridization has demonstrated an increase in thrombin receptor mRNA throughout the period of neointimal and vascular lesion development. The mechanism of this increase is unknown, but may be mediated by multiple inflammatory modulators. Several strategies have been tested in animal models for inhibiting thrombin: (1) Hirudin not only prevents thrombin from cleaving fibrinogen, but also prevents thrombin receptor activation. (2) Thrombin receptor antagonist peptides block platelet aggregation effects of thrombin. (3) Mono- and polyclonal antibodies inhibit thrombin receptor activation. (4) Antisense oligonucleotides block thrombin receptor expression.

TIM-1 Mediates Dystroglycan-Independent Entry of Lassa Virus.

PubMed

Brouillette, Rachel B; Phillips, Elisabeth K; Patel, Radhika; Mahauad-Fernandez, Wadie; Moller-Tank, Sven; Rogers, Kai J; Dillard, Jacob A; Cooney, Ashley L; Martinez-Sobrido, Luis; Okeoma, Chioma; Maury, Wendy

2018-06-06

Lassa virus (LASV) is an Old World arenavirus responsible for hundreds of thousands of infections in West Africa every year. LASV entry into a variety of cell types is mediated by interactions with glycosyltransferase LARGE-modified O-linked glycans present on the ubiquitous receptor, α-dystroglycan (αDG). Yet, cells lacking αDG are permissive to LASV infection, suggesting that alternative receptors exist. Previous studies demonstrate that phosphatidylserine (PtdSer)-binding receptors, Axl and Tyro3 along with C-type lectin receptors, mediate αDG-independent entry. Here, we demonstrate that another PtdSer receptor, TIM-1, mediates LASV glycoprotein (GP) pseudotyped virions entry into αDG knocked out HEK 293T and wild-type (WT) Vero which express αDG lacking appropriate glycosylation. To investigate the mechanism by which TIM-1 mediates enhancement of entry, we demonstrate that mutagenesis of the TIM-1 IgV domain PtdSer-binding pocket abrogated transduction. Further, the human TIM-1 IgV domain binding monoclonal antibody, ARD5, blocked transduction of pseudovirions bearing LASV GP in a dose-dependent manner. Finally, as we showed previously for other viruses that use TIM-1 for entry, a chimeric TIM-1 protein that substitutes the proline rich region (PRR) from murine leukemia virus envelope (Env) for the mucin-like domain served as a competent receptor. These studies provide evidence that, in the absence of a functional αDG, TIM-1 mediates entry of LASV pseudoviral particles through interactions of virions with the IgV PtdSer binding pocket of TIM-1. Importance PtdSer receptors, such as TIM-1, are emerging as critical entry factors for many enveloped viruses. Most recently, Hepatitis C virus and Zika virus have been added to a growing list. PtdSer receptors engage with enveloped viruses through binding of PtdSer embedded in the viral envelope, defining them as GP-independent receptors. This GP-independent entry mechanism should effectively mediate entry of all enveloped viruses, yet LASV GP pseudotyped viruses were previously found to be unresponsive to PtdSer receptor enhancement in HEK 293T cells. Here we demonstrate that LASV pseudovirions can utilize the PtdSer receptor TIM-1, but only in the absence of appropriately glycosylated α-dystroglycan (αDG), the high affinity cell surface receptor for LASV. Our studies shed light on LASV receptor utilization and explain why earlier studies performed in α-DG-expressing cells did not find that LASV pseudovirions utilize PtdSer receptors for virus uptake. Copyright © 2018 American Society for Microbiology.

Tyrosine Phosphorylation of the Guanine Nucleotide Exchange Factor GIV Promotes Activation of PI3K During Cell Migration

PubMed Central

Lin, Changsheng; Ear, Jason; Pavlova, Yelena; Mittal, Yash; Kufareva, Irina; Ghassemian, Majid; Abagyan, Ruben; Garcia-Marcos, Mikel; Ghosh, Pradipta

2014-01-01

GIV (Gα-interacting vesicle-associated protein; also known as Girdin), enhances Akt activation downstream of multiple growth factor– and G-protein–coupled receptors to trigger cell migration and cancer invasion. Here we demonstrate that GIV is a tyrosine phosphoprotein that directly binds to and activates phosphoinositide 3-kinase (PI3K). Upon ligand stimulation of various receptors, GIV was phosphorylated at Tyr1764 and Tyr1798 by both receptor and non-receptor tyrosine kinases. These phosphorylation events enabled direct binding of GIV to the N- and C-terminal SH2 domains of p85α, a regulatory subunit of PI3K, stabilized receptor association with PI3K, and enhanced PI3K activity at the plasma membrane to trigger cell migration. Tyrosine phosphorylation of GIV and its association with p85α increased during metastatic progression of a breast carcinoma. These results suggest a mechanism by which multiple receptors activate PI3K through tyrosine phosphorylation of GIV, thereby making the GIVPI3K interaction a potential therapeutic target within the PI3K-Akt pathway. PMID:21954290

A method for acetylcholinesterase staining of brain sections previously processed for receptor autoradiography.

PubMed

Lim, M M; Hammock, E A D; Young, L J

2004-02-01

Receptor autoradiography using selective radiolabeled ligands allows visualization of brain receptor distribution and density on film. The resolution of specific brain regions on the film often can be difficult to discern owing to the general spread of the radioactive label and the lack of neuroanatomical landmarks on film. Receptor binding is a chemically harsh protocol that can render the tissue virtually unstainable by Nissl and other conventional stains used to delineate neuroanatomical boundaries of brain regions. We describe a method for acetylcholinesterase (AChE) staining of slides previously processed for receptor binding. AChE staining is a useful tool for delineating major brain nuclei and tracts. AChE staining on sections that have been processed for receptor autoradiography provides a direct comparison of brain regions for more precise neuroanatomical description. We report a detailed thiocholine protocol that is a modification of the Koelle-Friedenwald method to amplify the AChE signal in brain sections previously processed for autoradiography. We also describe several temporal and experimental factors that can affect the density and clarity of the AChE signal when using this protocol.

Differences in the binding mechanism of RU486 and progesterone to the progesterone receptor

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

Skafar, D.F.

1991-11-12

The binding mechanism of the antagonist RU486 to the progesterone receptor was compared with that of the agonists progesterone and R5020. Both progesterone and RU486 bound to the receptor with a Hill coefficient of 1.2, indicating the binding of each ligand is positive cooperative. However, when each ligand was used to compete with ({sup 3}H)progesterone for binding to the receptor at receptor concentrations near 8 nM, at which the receptor is likely a dimer, the competition curve for RU486 was significantly steeper than the curves for progesterone and R5020. This indicated that a difference in the binding mechanism of RU486more » and progesterone can be detected when both ligands are present. In contrast, at receptor concentrations near 1 nM, at which the receptor is likely a monomer, the competition curves for all three ligands were indistinguishable. These results indicate that RU486 and agonists have different binding mechanisms for the receptor and further suggest that this difference may be related to site-site interactions within the receptor.« less

Engineering high-potency R-spondin adult stem cell growth factors.

PubMed

Warner, Margaret L; Bell, Tufica; Pioszak, Augen A

2015-01-01

Secreted R-spondin proteins (RSPOs1-4) function as adult stem cell growth factors by potentiating Wnt signaling. Simultaneous binding of distinct regions of the RSPO Fu1-Fu2 domain module to the extracellular domains (ECDs) of the LGR4 G protein-coupled receptor and the ZNRF3 transmembrane E3 ubiquitin ligase regulates Wnt receptor availability. Here, we examine the molecular basis for the differing signaling strengths of RSPOs1-4 using purified RSPO Fu1-Fu2, LGR4 ECD, and ZNRF3 ECD proteins in Wnt signaling and receptor binding assays, and we engineer novel high-potency RSPOs. RSPO2/3/4 had similar signaling potencies that were stronger than that of RSPO1, whereas RSPO1/2/3 had similar efficacies that were greater than that of RSPO4. The RSPOs bound LGR4 with affinity rank order RSPO4 > RSPO2/3 > RSPO1 and ZNRF3 with affinity rank order RSPO2/3 > > RSPO1 > RSPO4. An RSPO2-4 chimera combining RSPO2 ZNRF3 binding with RSPO4 LGR4 binding was a "Superspondin" that exhibited enhanced ternary complex formation and 10-fold stronger signaling potency than RSPO2 and efficacy equivalent to RSPO2. An RSPO4-1 chimera combining RSPO4 ZNRF3 binding with RSPO1 LGR4 binding was a "Poorspondin" that exhibited signaling potency similar to RSPO1 and efficacy equivalent to RSPO4. Conferring increased ZNRF3 binding upon RSPO4 with amino acid substitutions L56F, I58L, and I63M enhanced its signaling potency and efficacy. Our results reveal the molecular basis for RSPOs1-4 activity differences and suggest that signaling potency is determined by ternary complex formation ability, whereas efficacy depends on ZNRF3 recruitment. High-potency RSPOs may be of value for regenerative medicine and/or therapeutic applications. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Time course of the estradiol-dependent induction of oxytocin receptor binding in the ventromedial hypothalamic nucleus of the rat

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

Johnson, A.E.; Ball, G.F.; Coirini, H.

1989-09-01

Oxytocin (OT) transmission is involved in the steroid-dependent display of sexual receptivity in rats. One of the biochemical processes stimulated by the ovarian steroid 17 beta-estradiol (E2) that is relevant to reproduction is the induction of OT receptor binding in the ventromedial hypothalamic nucleus (VMN). The purpose of these experiments was to determine if E2-induced changes in OT receptor binding in the VMN occur within a time frame relevant to cyclic changes in ovarian steroid secretion. OT receptor binding was measured in the VMN of ovariectomized rats implanted for 0-96 h with E2-containing Silastic capsules. The rate of decay ofmore » OT receptor binding was measured in another group of animals 6-48 h after capsule removal. Receptors were labeled with the specific OT receptor antagonist ({sup 125}I)d(CH2)5(Tyr(Me)2,Thr4,Tyr-NH2(9))OVT, and binding was measured with quantitative autoradiographic methods. In addition, plasma E2 levels and uterine weights were assessed in animals from each treatment condition. Significant increases in E2-dependent OT receptor binding and uterine weight occurred within 24 h of steroid treatment. After E2 withdrawal, OT receptor binding and uterine weight decreased significantly within 24 h. These results are consistent with the hypothesis that steroid modulation of OT receptor binding is necessary for the induction of sexual receptivity.« less

A PPARgamma mutant serves as a dominant negative inhibitor of PPAR signaling and is localized in the nucleus.

PubMed

Berger, J; Patel, H V; Woods, J; Hayes, N S; Parent, S A; Clemas, J; Leibowitz, M D; Elbrecht, A; Rachubinski, R A; Capone, J P; Moller, D E

2000-04-25

The peroxisomal proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily that act as ligand-activated transcription factors. PPARgamma plays a critical role in regulating adipocyte differentiation and lipid metabolism. Recently, thiazolidinedione (TZD) and select non-TZD antidiabetic agents have been identified as PPARgamma agonists. To further characterize this receptor subclass, a mutant hPPARgamma lacking five carboxyl-terminal amino acids was produced (hPPARgamma2Delta500). In COS-1 cells transfected with PPAR-responsive reporter constructs, the mutant receptor could not be activated by a potent PPARgamma agonist. When cotransfected with hPPARgamma2 or hPPARalpha, hPPARgamma2Delta500 abrogated wild-type receptor activity in a dose-responsive manner. hPPARgamma2Delta500 was also impaired with respect to binding of a high-affinity radioligand. In addition, its conformation was unaffected by normally saturating concentrations of PPARgamma agonist as determined by protease protection experiments. Electrophoretic mobility shift assays demonstrated that hPPARgamma2Delta500 and hPPARgamma2 both formed heterodimeric complexes with human retinoidxreceptor alpha (hRXRalpha) and could bind a peroxisome proliferator-responsive element (PPRE) with similar affinity. Therefore, hPPARgamma2Delta500 appears to repress PPAR activity by competing with wild type receptor to dimerize with RXR and bind the PPRE. In addition, the mutant receptor may titrate out factors required for PPAR-regulated transcriptional activation. Both hPPARgamma2 and hPPARgamma2Delta500 localized to the nucleus of transiently transfected COS-1 cells as determined by immunofluorescence using a PPARgamma-specific antibody. Thus, nuclear localization of PPARgamma occurs independently of its activation state. The dominant negative mutant, hPPARgamma2Delta500, may prove useful in further studies to characterize PPAR functions both in vitro and in vivo

Neurotensin receptor binding levels in basal ganglia are not altered in Huntington's chorea or schizophrenia

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

Palacios, J.M.; Chinaglia, G.; Rigo, M.

1991-02-01

Autoradiographic techniques were used to examine the distribution and levels of neurotensin receptor binding sites in the basal ganglia and related regions of the human brain. Monoiodo ({sup 125}I-Tyr3)neurotensin was used as a ligand. High amounts of neurotensin receptor binding sites were found in the substantia nigra pars compacta. Lower but significant quantities of neurotensin receptor binding sites characterized the caudate, putamen, and nucleus accumbens, while very low quantities were seen in both medial and lateral segments of the globus pallidus. In Huntington's chorea, the levels of neurotensin receptor binding sites were found to be comparable to those of controlmore » cases. Only slight but not statistically significant decreases in amounts of receptor binding sites were detected in the dorsal part of the head and in the body of caudate nucleus. No alterations in the levels of neurotensin receptor binding sites were observed in the substantia nigra pars compacta and reticulata. These results suggest that a large proportion of neurotensin receptor binding sites in the basal ganglia are located on intrinsic neurons and on extrinsic afferent fibers that do not degenerate in Huntington's disease.« less

Adrenergic Agonists Bind to Adrenergic-Receptor-Like Regions of the Mu Opioid Receptor, Enhancing Morphine and Methionine-Enkephalin Binding: A New Approach to "Biased Opioids"?

PubMed

Root-Bernstein, Robert; Turke, Miah; Subhramanyam, Udaya K Tiruttani; Churchill, Beth; Labahn, Joerg

2018-01-17

Extensive evidence demonstrates functional interactions between the adrenergic and opioid systems in a diversity of tissues and organs. While some effects are due to receptor and second messenger cross-talk, recent research has revealed an extracellular, allosteric opioid binding site on adrenergic receptors that enhances adrenergic activity and its duration. The present research addresses whether opioid receptors may have an equivalent extracellular, allosteric adrenergic binding site that has similar enhancing effects on opioid binding. Comparison of adrenergic and opioid receptor sequences revealed that these receptors share very significant regions of similarity, particularly in some of the extracellular and transmembrane regions associated with adrenergic binding in the adrenergic receptors. Five of these shared regions from the mu opioid receptor (muOPR) were synthesized as peptides and tested for binding to adrenergic, opioid and control compounds using ultraviolet spectroscopy. Adrenergic compounds bound to several of these muOPR peptides with low micromolar affinity while acetylcholine, histamine and various adrenergic antagonists did not. Similar studies were then conducted with purified, intact muOPR with similar results. Combinations of epinephrine with methionine enkephalin or morphine increased the binding of both by about half a log unit. These results suggest that muOPR may be allosterically enhanced by adrenergic agonists.

Old dance with a new partner: EGF receptor as the phenobarbital receptor mediating Cyp2B expression.

PubMed

Meyer, Sharon A; Jirtle, Randy L

2013-05-07

The decades-long quest for the phenobarbital (PhB) receptor that mediates activation of Cyp2B would appear fulfilled with the discovery by Mutoh et al., who found that PhB binds with pharmacological affinity to the epidermal growth factor receptor (EGFR). This finding provides a molecular basis for the suppression of hepatocyte EGFR signaling observed with PhB treatment, as previously noted in the context of tumor promotion. Although the PhB-mediated induction of Cyp2B expression through the association of a canonical nuclear receptor with the 5'-enhancer PBREM of Cyp2B is well known, direct binding of PhB to constitutive active androstane receptor (CAR, also known as NR1I3) typical of other xenobiotic-activated nuclear receptors has eluded detection. One EGF-activated pathway affected by the PhB-EGFR interaction is the loss of tyrosine phosphorylation of the scaffold protein RACK1. Dephosphorylated RACK1 provides the mechanistic link between the binding of PhB to EGFR and its effects on CAR by facilitating the interaction of serine/threonine phosphatase PP2A with inactive phosphorylated CAR. The dephosphorylation of CAR enables its translocation to the nucleus and activation of Cyp2B expression. Because EGFR and transducers RACK1, PP2A, and other partners are highly networked in numerous cellular pathways, this newly discovered partnership will surely reveal new fundamental roles for PhB beyond the regulation of drug metabolism.

Nuclear binding of progesterone in hen oviduct. Binding to multiple sites in vitro.

PubMed Central

Pikler, G M; Webster, R A; Spelsberg, T C

1976-01-01

Steroid hormones, including progesterone, are known to bind with high affinity (Kd approximately 1x10(-10)M) to receptor proteins once they enter target cells. This complex (the progesterone-receptor) then undergoes a temperature-and/or salt-dependent activation which allows it to migrate to the cell nucleus and to bind to the deoxyribonucleoproteins. The present studies demonstrate that binding the hormone-receptor complex in vitro to isolated nuclei from the oviducts of laying hens required the same conditions as do other studies of bbinding in vitro reported previously, e.g. the hormone must be complexed to intact and activated receptor. The assay of the nuclear binding by using multiple concentrations of progesterone receptor reveals the presence of more than one class of binding site in the oviduct nuclei. The affinity of each of these classes of binding sites range from Kd approximately 1x10(-9)-1x10(-8)M. Assays using free steroid (not complexed with receptor) show no binding to these sites. The binding to each of the classes of sites, displays a differential stability to increasing ionic concentrations, suggesting primarily an ionic-type interaction for all classes. Only the highest-affinity class of binding site is capable of binding progesterone receptor under physioligical-saline conditions. This class represent 6000-10000 sites per cell nucleus and resembles the sites detected in vivo (Spelsberg, 1976, Biochem. J. 156, 391-398) which cause maximal transcriptional response when saturated with the progesterone receptor. The multiple binding sites for the progesterone receptor either are not present or are found in limited numbers in the nuclei of non-target organs. Differences in extent of binding to the nuclear material between a target tissue (oviduct) and other tissues (spleen or erythrocyte) are markedly dependent on the ionic conditions, and are probably due to binding to different classes of sites in the nuclei. PMID:182147

Design of an Insulin Analog with Enhanced Receptor Binding Selectivity

PubMed Central

Zhao, Ming; Wan, Zhu-li; Whittaker, Linda; Xu, Bin; Phillips, Nelson B.; Katsoyannis, Panayotis G.; Ismail-Beigi, Faramarz; Whittaker, Jonathan; Weiss, Michael A.

2009-01-01

Insulin binds with high affinity to the insulin receptor (IR) and with low affinity to the type 1 insulin-like growth factor (IGF) receptor (IGFR). Such cross-binding, which reflects homologies within the insulin-IGF signaling system, is of clinical interest in relation to the association between hyperinsulinemia and colorectal cancer. Here, we employ nonstandard mutagenesis to design an insulin analog with enhanced affinity for the IR but reduced affinity for the IGFR. Unnatural amino acids were introduced by chemical synthesis at the N- and C-capping positions of a recognition α-helix (residues A1 and A8). These sites adjoin the hormone-receptor interface as indicated by photocross-linking studies. Specificity is enhanced more than 3-fold on the following: (i) substitution of GlyA1 by d-Ala or d-Leu, and (ii) substitution of ThrA8 by diaminobutyric acid (Dab). The crystal structure of [d-AlaA1,DabA8]insulin, as determined within a T6 zinc hexamer to a resolution of 1.35 Å, is essentially identical to that of human insulin. The nonstandard side chains project into solvent at the edge of a conserved receptor-binding surface shared by insulin and IGF-I. Our results demonstrate that modifications at this edge discriminate between IR and IGFR. Because hyperinsulinemia is typically characterized by a 3-fold increase in integrated postprandial insulin concentrations, we envisage that such insulin analogs may facilitate studies of the initiation and progression of cancer in animal models. Future development of clinical analogs lacking significant IGFR cross-binding may enhance the safety of insulin replacement therapy in patients with type 2 diabetes mellitus at increased risk of colorectal cancer. PMID:19773552

The Roles of Hemagglutinin Phe-95 in Receptor Binding and Pathogenicity of Influenza B Virus

PubMed Central

Ni, Fengyun; Mbawuike, Innocent Nnadi; Kondrashkina, Elena; Wang, Qinghua

2014-01-01

Diverged ~4,000 years ago, influenza B virus has several important differences from influenza A virus, including lower receptor-binding affinity and highly restricted host range. Based on our prior structural studies, we hypothesized that a single-residue difference in the receptor-binding site of hemagglutinin (HA), Phe-95 in influenza B virus versus Tyr-98 in influenza A/H1~H15, is possibly a key determinant for the low receptor-binding affinity. Here we demonstrate that the mutation Phe95→Tyr in influenza B virus HA restores all three hydrogen bonds made by Tyr-98 in influenza A/H3 HA and has the potential to enhance receptor binding. However, the full realization of this potential is influenced by the local environment into which the mutation is introduced. The binding and replication of the recombinant viruses correlate well with the receptor-binding capabilities of HA. These results are discussed in relation to the roles of Phe-95 in receptor binding and pathogenicity of influenza B virus. PMID:24503069

The bovine papillomavirus E5 protein requires a juxtamembrane negative charge for activation of the platelet-derived growth factor beta receptor and transformation of C127 cells.

PubMed

Klein, O; Kegler-Ebo, D; Su, J; Smith, S; DiMaio, D

1999-04-01

The bovine papillomavirus E5 gene encodes a 44-amino-acid, homodimeric transmembrane protein that is the smallest known transforming protein. The E5 protein transforms cultured fibroblasts by forming a stable complex with the endogenous platelet-derived growth factor (PDGF) beta receptor through transmembrane and juxtamembrane interactions, leading to sustained receptor activation. Aspartic acid 33 in the extracellular juxtamembrane region of the E5 protein is important for cell transformation and interaction with the PDGF beta receptor. A. N. Meyer et al. (Proc. Natl. Acad. Sci USA 91:4634-4638, 1994) speculated that this residue interacted with lysine 499 on the receptor. We constructed E5 mutants containing all possible substitutions at position 33, as well as several double mutants containing substitutions at aspartic acid 33 and at glutamic acid 36, and we examined the ability of these mutants to transform C127 mouse fibroblasts and to bind to and induce activation of the PDGF beta receptor. There was an excellent correlation between the transformation activities of the various mutants and their ability to bind to and activate the PDGF beta receptor. Analysis of the mutants demonstrated that a juxtamembrane negative charge on the E5 protein was required for cell transformation and for productive interaction with the PDGF beta receptor and indicated that aspartic acid 33 was more important for these activities than was glutamic acid 36. These results are consistent with the existence of an essential juxtamembrane salt bridge between lysine 499 on the PDGF beta receptor and an acidic residue in the C terminus of the E5 protein and lend support to our proposed model for the complex between the E5 dimer and the PDGF beta receptor.

Improved efficacy of soluble human receptor activator of nuclear factor kappa B (RANK) fusion protein by site-directed mutagenesis.

PubMed

Son, Young Jun; Han, Jihye; Lee, Jae Yeon; Kim, HaHyung; Chun, Taehoon

2015-06-01

Soluble human receptor activator of nuclear factor kappa B fusion immunoglobulin (hRANK-Ig) has been considered as one of the therapeutic agents to treat osteoporosis or diseases associated with bone destruction by blocking the interaction between RANK and the receptor activator of nuclear factor kappa B ligand (RANKL). However, no scientific record showing critical amino acid residues within the structural interface between the human RANKL and RANK complex is yet available. In this study, we produced several mutants of hRANK-Ig by replacement of amino acid residue(s) and tested whether the mutants had increased binding affinity to human RANKL. Based on the results from flow cytometry and surface plasmon resonance analyses, the replacement of E(125) with D(125), or E(125) and C(127) with D(125) and F(127) within loop 3 of cysteine-rich domain 3 of hRANK-Ig increases binding affinity to human RANKL over the wild-type hRANK-Ig. This result may provide the first example of improvement in the efficacy of hRANK-Ig by protein engineering and may give additional information to understand a more defined structural interface between hRANK and RANKL.

Muscarinic and alpha 1-adrenergic receptor binding characteristics of saw palmetto extract in rat lower urinary tract.

PubMed

Suzuki, Mayumi; Oki, Tomomi; Sugiyama, Tomomi; Umegaki, Keizo; Uchida, Shinya; Yamada, Shizuo

2007-06-01

To elucidate the in vitro and ex vivo effects of saw palmetto extract (SPE) on autonomic receptors in the rat lower urinary tract. The in vitro binding affinities for alpha 1-adrenergic, muscarinic, and purinergic receptors in the rat prostate and bladder were measured by radioligand binding assays. Rats received vehicle or SPE (0.6 to 60 mg/kg/day) orally for 4 weeks, and alpha 1-adrenergic and muscarinic receptor binding in tissues of these rats were measured. Saw palmetto extract inhibited specific binding of [3H]prazosin and [N-methyl-3H]scopolamine methyl chloride (NMS) but not alpha, beta-methylene adenosine triphosphate [2,8-(3)H]tetrasodium salt in the rat prostate and bladder. The binding activity of SPE for muscarinic receptors was four times greater than that for alpha 1-adrenergic receptors. Scatchard analysis revealed that SPE significantly reduced the maximal number of binding sites (Bmax) for each radioligand in the prostate and bladder under in vitro condition. Repeated oral administration of SPE to rats brought about significant alteration in Bmax for prostatic [3H]prazosin binding and for bladder [3H]NMS binding. Such alteration by SPE was selective to the receptors in the lower urinary tract. Saw palmetto extract exerts significant binding activity on autonomic receptors in the lower urinary tract under in vitro and in vivo conditions.

Heterogeneity of binding of muscarinic receptor antagonists in rat brain homogenates

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

Lee, J.H.; el-Fakahany, E.E.

1985-06-01

The binding properties of (-)-(/sup 3/H)quinuclidinyl benzilate and (/sup 3/H) N-methylscopolamine to muscarinic acetylcholine receptors have been investigated in rat brain homogenates. The binding of both antagonists demonstrated high affinity and saturability. Analysis of the binding data resulted in linear Scatchard plots. However, (-)-(/sup 3/H)quinuclidinyl benzilate showed a significantly higher maximal binding capacity than that of (/sup 3/H)N-methylscopolamine. Displacement of both ligands with several muscarinic receptor antagonists resulted in competition curves in accordance with the law of mass-action for quinuclidinyl benzilate, atropine and scopolamine. A similar profile was found for the quaternary ammonium analogs of atropine and scopolamine when (/supmore » 3/H)N-methylscopolamine was used to label the receptors. However, when these hydrophilic antagonists were used to displace (-)-(/sup 3/H) quinuclidinyl benzilate binding, they showed interaction with high- and low-affinity binding sites. On the other hand, the nonclassical muscarinic receptor antagonist, pirenzepine, was able to displace both ligands from two binding sites. The present data are discussed in terms of the relationship of this anomalous heterogenity of binding of these hydrophilic muscarinic receptor antagonists and the proposed M1 and M2 receptor subtypes.« less

Role of Insulin-Like Growth Factor-1 Signaling Pathway in Cisplatin-Resistant Lung Cancer Cells

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

Sun Yunguang; Zheng Siyuan; Torossian, Artour

2012-03-01

Purpose: The development of drug-resistant phenotypes has been a major obstacle to cisplatin use in non-small-cell lung cancer. We aimed to identify some of the molecular mechanisms that underlie cisplatin resistance using microarray expression analysis. Methods and Materials: H460 cells were treated with cisplatin. The differences between cisplatin-resistant lung cancer cells and parental H460 cells were studied using Western blot, MTS, and clonogenic assays, in vivo tumor implantation, and microarray analysis. The cisplatin-R cells were treated with human recombinant insulin-like growth factor (IGF) binding protein-3 and siRNA targeting IGF-1 receptor. Results: Cisplatin-R cells illustrated greater expression of the markers CD133more » and aldehyde dehydrogenase, more rapid in vivo tumor growth, more resistance to cisplatin- and etoposide-induced apoptosis, and greater survival after treatment with cisplatin or radiation than the parental H460 cells. Also, cisplatin-R demonstrated decreased expression of insulin-like growth factor binding protein-3 and increased activation of IGF-1 receptor signaling compared with parental H460 cells in the presence of IGF-1. Human recombinant IGF binding protein-3 reversed cisplatin resistance in cisplatin-R cells and targeting of IGF-1 receptor using siRNA resulted in sensitization of cisplatin-R-cells to cisplatin and radiation. Conclusions: The IGF-1 signaling pathway contributes to cisplatin-R to cisplatin and radiation. Thus, this pathway represents a potential target for improved lung cancer response to treatment.« less

Gamma-Aminobutyric acid and benzodiazepine receptors in the kindling model of epilepsy: a quantitative radiohistochemical study

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

Shin, C.; Pedersen, H.B.; McNamara, J.O.

1985-10-01

Quantitative radiohistochemistry was utilized to study alterations of gamma-aminobutyric acid (GABA) and benzodiazepine receptors in the kindling model of epilepsy. The radioligands used for GABA and benzodiazepine receptors were (TH) muscimol and (TH)flunitrazepam, respectively. GABA receptor binding was increased by 22% in fascia dentata of the hippocampal formation but not in neocortex or substantia nigra of kindled rats. Within fascia dentata, GABA receptor binding was increased to an equivalent extent in stratum granulosum and throughout stratum moleculare; no increase was found in dentate hilus or stratum lacunosummoleculare or stratum radiatum of CA1. The increased binding was present at 24 hrmore » but not at 28 days after the last kindled seizure. The direction, anatomic distribution, and time course of the increased GABA receptor binding were paralleled by increased benzodiazepine receptor binding. The anatomic distribution of the increased GABA receptor binding is consistent with a localization to somata and dendritic trees of dentate granule cells. The authors suggest that increased GABA and benzodiazepine receptor binding may contribute to enhanced inhibition of dentate granule cells demonstrated electrophysiologically in kindled animals.« less

A natural variant of the cysteine protease virulence factor of group A Streptococcus with an arginine-glycine-aspartic acid (RGD) motif preferentially binds human integrins alphavbeta3 and alphaIIbbeta3.

PubMed

Stockbauer, K E; Magoun, L; Liu, M; Burns, E H; Gubba, S; Renish, S; Pan, X; Bodary, S C; Baker, E; Coburn, J; Leong, J M; Musser, J M

1999-01-05

The human pathogenic bacterium group A Streptococcus produces an extracellular cysteine protease [streptococcal pyrogenic exotoxin B (SpeB)] that is a critical virulence factor for invasive disease episodes. Sequence analysis of the speB gene from 200 group A Streptococcus isolates collected worldwide identified three main mature SpeB (mSpeB) variants. One of these variants (mSpeB2) contains an Arg-Gly-Asp (RGD) sequence, a tripeptide motif that is commonly recognized by integrin receptors. mSpeB2 is made by all isolates of the unusually virulent serotype M1 and several other geographically widespread clones that frequently cause invasive infections. Only the mSpeB2 variant bound to transfected cells expressing integrin alphavbeta3 (also known as the vitronectin receptor) or alphaIIbbeta3 (platelet glycoprotein IIb-IIIa), and binding was blocked by a mAb that recognizes the streptococcal protease RGD motif region. In addition, mSpeB2 bound purified platelet integrin alphaIIbbeta3. Defined beta3 mutants that are altered for fibrinogen binding were defective for SpeB binding. Synthetic peptides with the mSpeB2 RGD motif, but not the RSD sequence present in other mSpeB variants, blocked binding of mSpeB2 to transfected cells expressing alphavbeta3 and caused detachment of cultured human umbilical vein endothelial cells. The results (i) identify a Gram-positive virulence factor that directly binds integrins, (ii) identify naturally occurring variants of a documented Gram-positive virulence factor with biomedically relevant differences in their interactions with host cells, and (iii) add to the theme that subtle natural variation in microbial virulence factor structure alters the character of host-pathogen interactions.

GNL3L Inhibits Estrogen Receptor-Related Protein Activities by Competing for Coactivator Binding

PubMed Central

Yasumoto, Hiroaki; Meng, Lingjun; Lin, Tao; Zhu, Qubo; Tsai, Robert Y.L.

2010-01-01

Summary Guanine-nucleotide binding protein 3-like (GNL3L) is the closest homologue of a stem cell-enriched factor nucleostemin in vertebrates. They share the same yeast orthologue, Grn1p, but only GNL3L can rescue the growth-deficient phenotype in Grn1p-null yeasts. To determine the unique function of GNL3L, we identified estrogen receptor-related protein-γ (ERRγ) as a GNL3L-specific binding protein. GNL3L and ERRγ are coexpressed in the eye, kidney and muscle, and co-reside in the nucleoplasm. The interaction between GNL3L and ERRγ requires the intermediate domain of GNL3L and the AF2-domain of ERRγ. Gain- and loss-of-function experiments show that GNL3L can inhibit the transcriptional activities of ERR genes in a cell-based reporter system, which does not require the nucleolar localization of GNL3L. We further demonstrate that GNL3L is able to reduce the steroid receptor coactivator (SRC) binding and the SRC-mediated transcriptional coactivation of ERRγ. This work reveals a novel mechanism that negatively regulates the transcriptional function of ERRγ by GNL3L through coactivator competition. PMID:17623774

Profile of neratinib and its potential in the treatment of breast cancer

PubMed Central

Feldinger, Katharina; Kong, Anthony

2015-01-01

The HER (ErbB) receptor tyrosine kinase receptors are implicated in many cancers and several anti-HER treatments are now approved. In recent years, a new group of compounds that bind irreversibly to the adenosine triphosphate binding pocket of HER receptors have been developed. One of these compounds, neratinib, has passed preclinical phases and is currently undergoing various clinical trials. This manuscript reviews the preclinical as well as clinical data on neratinib. As a pan-HER inhibitor, this irreversible tyrosine kinase inhibitor binds and inhibits the tyrosine kinase activity of epidermal growth factor receptors, EGFR (or HER1), HER2 and HER4, which leads to reduced phosphorylation and activation of downstream signaling pathways. Neratinib has been shown to be effective against HER2-overexpressing or mutant tumors in vitro and in vivo. Neratinib is currently being investigated in various clinical trials in breast cancers and other solid tumors, including those with HER2 mutation. Earlier studies have already shown promising clinical activity for neratinib. However, more translational research is required to investigate biomarkers that could help to predict response and resistance for selection of appropriate patients for treatment with neratinib, either as monotherapy or in combination with other drug(s). PMID:26089701

Profile of neratinib and its potential in the treatment of breast cancer.

PubMed

Feldinger, Katharina; Kong, Anthony

2015-01-01

The HER (ErbB) receptor tyrosine kinase receptors are implicated in many cancers and several anti-HER treatments are now approved. In recent years, a new group of compounds that bind irreversibly to the adenosine triphosphate binding pocket of HER receptors have been developed. One of these compounds, neratinib, has passed preclinical phases and is currently undergoing various clinical trials. This manuscript reviews the preclinical as well as clinical data on neratinib. As a pan-HER inhibitor, this irreversible tyrosine kinase inhibitor binds and inhibits the tyrosine kinase activity of epidermal growth factor receptors, EGFR (or HER1), HER2 and HER4, which leads to reduced phosphorylation and activation of downstream signaling pathways. Neratinib has been shown to be effective against HER2-overexpressing or mutant tumors in vitro and in vivo. Neratinib is currently being investigated in various clinical trials in breast cancers and other solid tumors, including those with HER2 mutation. Earlier studies have already shown promising clinical activity for neratinib. However, more translational research is required to investigate biomarkers that could help to predict response and resistance for selection of appropriate patients for treatment with neratinib, either as monotherapy or in combination with other drug(s).

The tertiary structures of porcine AhR and ARNT proteins and molecular interactions within the TCDD/AhR/ARNT complex.

PubMed

Orlowska, Karina; Molcan, Tomasz; Swigonska, Sylwia; Sadowska, Agnieszka; Jablonska, Monika; Nynca, Anna; Jastrzebski, Jan P; Ciereszko, Renata E

2016-06-01

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that can be activated by structurally diverse synthetic and natural chemicals, including toxic environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In the present study, homology models of the porcine AhR-ligand binding domain (LBD) and the porcine aryl hydrocarbon receptor nuclear translocator-ligand binding domain (ARNT-LBD) were created on the basis of structures of closely related respective proteins i.e., human Hif-2α and ARNT. Molecular docking of TCDD to the porcine AhR-LBD model revealed high binding affinity (-8.8kcal/mol) between TCDD and the receptor. Moreover, formation of the TCDD/AhR-LBD complex was confirmed experimentally with the use of electrophoretic mobility shift assay (EMSA). It was found that TCDD (10nM, 2h of incubation) not only bound to the AhR in the porcine granulosa cells but also activated the receptor. The current study provides a framework for examining the key events involved in the ligand-dependent activation of the AhR. Copyright © 2016 Elsevier Inc. All rights reserved.

RP-1551s, a family of azaphilones produced by Penicillium sp., inhibit the binding of PDGF to the extracellular domain of its receptor.

PubMed

Toki, S; Tanaka, T; Uosaki, Y; Yoshida, M; Suzuki, Y; Kita, K; Mihara, A; Ando, K; Lokker, N A; Giese, N A; Matsuda, Y

1999-03-01

Nine azaphilones designated RP-1551-1, -2, -3, -4, -5, -6, -7, -M1, and -M2 were isolated from the culture broth of Penicillium sp. SPC-21609 as inhibitors of PDGF binding to its receptor. RP-1551s inhibit the binding of PDGF AA to the extracellular domain of PDGF alpha-receptor with IC50 values ranging from 0.1 to 2 microM without affecting PDGF BB binding to the extracellular domain of PDGF beta-receptor. PDGF binding was not restored after the PDGF alpha-receptor extracellular domain was washed in an attempt to remove the RP-1551-1 bound to the receptor. This result suggests that RP-1551-1 may irreversibly interact with the PDGF alpha-receptor. Since many azaphilone compounds possess high reactivity with an amino group, RP-1551-1 may prevent PDGF AA binding by reacting with amino groups on the alpha-receptor extracellular domain.

Structure of unliganded HSV gD reveals a mechanism for receptor-mediated activation of virus entry

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

Krummenacher, Claude; Supekar, Vinit M.; Whitbeck, J. Charles

2010-07-19

Herpes simplex virus (HSV) entry into cells requires binding of the envelope glycoprotein D (gD) to one of several cell surface receptors. The 50 C-terminal residues of the gD ectodomain are essential for virus entry, but not for receptor binding. We have determined the structure of an unliganded gD molecule that includes these C-terminal residues. The structure reveals that the C-terminus is anchored near the N-terminal region and masks receptor-binding sites. Locking the C-terminus in the position observed in the crystals by an intramolecular disulfide bond abolished receptor binding and virus entry, demonstrating that this region of gD moves uponmore » receptor binding. Similarly, a point mutant that would destabilize the C-terminus structure was nonfunctional for entry, despite increased affinity for receptors. We propose that a controlled displacement of the gD C-terminus upon receptor binding is an essential feature of HSV entry, ensuring the timely activation of membrane fusion.« less

One amino acid in mouse activated factor VII defines its endothelial protein C receptor (EPCR) binding and modulates its EPCR-dependent hemostatic activity in vivo.

PubMed

Pavani, G; Zintner, S M; Ivanciu, L; Small, J C; Stafford, K A; Szeto, J H; Margaritis, P

2017-03-01

Essentials The lack of factor (F) VIIa-endothelial protein C receptor (EPCR) binding in mice is unresolved. A single substitution of Leu4 to Phe in mouse FVIIa (mFVIIa) enables its interaction with EPCR. mFVIIa with a Phe4 shows EPCR binding-dependent enhanced hemostatic function in vivo vs. mFVIIa. Defining the FVIIa-EPCR interaction in mice allows for further investigating its biology in vivo. Background Human activated factor VII (hFVIIa), which is used in hemophilia treatment, binds to the endothelial protein C (PC) receptor (EPCR) with unclear hemostatic consequences. Interestingly, mice lack the activated FVII (FVIIa)-EPCR interaction. Therefore, to investigate the hemostatic consequences of this interaction in hemophilia, we previously engineered a mouse FVIIa (mFVIIa) molecule that bound mouse EPCR (mEPCR) by using three substitutions from mouse PC (mPC), i.e. Leu4→Phe, Leu8→Met, and Trp9→Arg. The resulting molecule, mFVIIa-FMR, modeled the EPCR-binding properties of hFVIIa and showed enhanced hemostatic capacity in hemophilic mice versus mFVIIa. These data implied a role of EPCR in the action of hFVIIa in hemophilia treatment. However, the substitutions in mFVIIa-FMR only broadly defined the sequence determinants for its mEPCR interaction and enhanced function in vivo. Objectives To determine the individual contributions of mPC Phe4, Met8 and Arg9 to the in vitro/in vivo properties of mFVIIa-FMR. Methods The mEPCR-binding properties of single amino acid variants of mFVIIa or mPC at position 4, 8 or 9 were investigated. Results and conclusions Phe4 in mFVIIa or mPC was solely critical for interaction with mEPCR. In hemophilic mice, administration of mFVIIa harboring a Phe4 resulted in a 1.9-2.5-fold increased hemostatic capacity versus mFVIIa that was EPCR binding-dependent. This recapitulated previous observations made with triple-mutant mFVIIa-FMR. As Leu8 is crucial for hFVIIa-EPCR binding, we describe the sequence divergence of this interaction in mice, now allowing its further characterization in vivo. We also illustrate that modulation of the EPCR-FVIIa interaction may lead to improved FVIIa therapeutics. © 2016 International Society on Thrombosis and Haemostasis.

The N54-αs Mutant Has Decreased Affinity for βγ and Suggests a Mechanism for Coupling Heterotrimeric G Protein Nucleotide Exchange with Subunit Dissociation.

PubMed

Cleator, John H; Wells, Christopher A; Dingus, Jane; Kurtz, David T; Hildebrandt, John D

2018-05-01

Ser54 of G s α binds guanine nucleotide and Mg 2+ as part of a conserved sequence motif in GTP binding proteins. Mutating the homologous residue in small and heterotrimeric G proteins generates dominant-negative proteins, but by protein-specific mechanisms. For α i/o , this results from persistent binding of α to βγ , whereas for small GTP binding proteins and α s this results from persistent binding to guanine nucleotide exchange factor or receptor. This work examined the role of βγ interactions in mediating the properties of the Ser54-like mutants of G α subunits. Unexpectedly, WT- α s or N54- α s coexpressed with α 1B -adrenergic receptor in human embryonic kidney 293 cells decreased receptor stimulation of IP3 production by a cAMP-independent mechanism, but WT- α s was more effective than the mutant. One explanation for this result would be that α s , like Ser47 α i/o , blocks receptor activation by sequestering βγ ; implying that N54- α S has reduced affinity for βγ since it was less effective at blocking IP3 production. This possibility was more directly supported by the observation that WT- α s was more effective than the mutant in inhibiting βγ activation of phospholipase C β 2. Further, in vitro synthesized N54- α s bound biotinylated- βγ with lower apparent affinity than did WT- α s The Cys54 mutation also decreased βγ binding but less effectively than N54- α s Substitution of the conserved Ser in α o with Cys or Asn increased βγ binding, with the Cys mutant being more effective. This suggests that Ser54 of α s is involved in coupling changes in nucleotide binding with altered subunit interactions, and has important implications for how receptors activate G proteins. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

Both host and parasite MIF molecules bind to chicken macrophages via CD74 surface receptor

USDA-ARS?s Scientific Manuscript database

Macrophage migration inhibitory factor (MIF) is recognized as a soluble factor that inhibits the random migration of macrophages and plays a pivotal immunoregulatory function in innate and adaptive immunity. Our group has identified both chicken and Eimeria MIFs, and characterized their function in...

Human sex hormone-binding globulin binding affinities of 125 structurally diverse chemicals and comparison with their binding to androgen receptor, estrogen receptor, and α-fetoprotein.

PubMed

Hong, Huixiao; Branham, William S; Ng, Hui Wen; Moland, Carrie L; Dial, Stacey L; Fang, Hong; Perkins, Roger; Sheehan, Daniel; Tong, Weida

2015-02-01

One endocrine disruption mechanism is through binding to nuclear receptors such as the androgen receptor (AR) and estrogen receptor (ER) in target cells. The concentration of a chemical in serum is important for its entry into the target cells to bind the receptors, which is regulated by the serum proteins. Human sex hormone-binding globulin (SHBG) is the major transport protein in serum that can bind androgens and estrogens and thus change a chemical's availability to enter the target cells. Sequestration of an androgen or estrogen in the serum can alter the chemical elicited AR- and ER-mediated responses. To better understand the chemical-induced endocrine activity, we developed a competitive binding assay using human pregnancy plasma and measured the binding to the human SHBG for 125 structurally diverse chemicals, most of which were known to bind AR and ER. Eighty seven chemicals were able to bind the human SHBG in the assay, whereas 38 chemicals were nonbinders. Binding data for human SHBG are compared with that for rat α-fetoprotein, ER and AR. Knowing the binding profiles between serum and nuclear receptors will improve assessment of a chemical's potential for endocrine disruption. The SHBG binding data reported here represent the largest data set of structurally diverse chemicals tested for human SHBG binding. Utilization of the SHBG binding data with AR and ER binding data could enable better evaluation of endocrine disrupting potential of chemicals through AR- and ER-mediated responses since sequestration in serum could be considered. Published by Oxford University Press on behalf of the Society of Toxicology 2014. This work is written by US Government employees and is in the public domain in the US.

Modular scanning FCS quantifies receptor-ligand interactions in living multicellular organisms.

PubMed

Ries, Jonas; Yu, Shuizi Rachel; Burkhardt, Markus; Brand, Michael; Schwille, Petra

2009-09-01

Analysis of receptor-ligand interactions in vivo is key to biology but poses a considerable challenge to quantitative microscopy. Here we combine static-volume, two-focus and dual-color scanning fluorescence correlation spectroscopy to solve this task at cellular resolution in complex biological environments. We quantified the mobility of fibroblast growth factor receptors Fgfr1 and Fgfr4 in cell membranes of living zebrafish embryos and determined their in vivo binding affinities to their ligand Fgf8.

The alpha-fetoprotein third domain receptor binding fragment: in search of scavenger and associated receptor targets.

PubMed

Mizejewski, G J

2015-01-01

Recent studies have demonstrated that the carboxyterminal third domain of alpha-fetoprotein (AFP-CD) binds with various ligands and receptors. Reports within the last decade have established that AFP-CD contains a large fragment of amino acids that interact with several different receptor types. Using computer software specifically designed to identify protein-to-protein interaction at amino acid sequence docking sites, the computer searches identified several types of scavenger-associated receptors and their amino acid sequence locations on the AFP-CD polypeptide chain. The scavenger receptors (SRs) identified were CD36, CD163, Stabilin, SSC5D, SRB1 and SREC; the SR-associated receptors included the mannose, low-density lipoprotein receptors, the asialoglycoprotein receptor, and the receptor for advanced glycation endproducts (RAGE). Interestingly, some SR interaction sites were localized on the AFP-derived Growth Inhibitory Peptide (GIP) segment at amino acids #480-500. Following the detection studies, a structural subdomain analysis of both the receptor and the AFP-CD revealed the presence of epidermal growth factor (EGF) repeats, extracellular matrix-like protein regions, amino acid-rich motifs and dimerization subdomains. For the first time, it was reported that EGF-like sequence repeats were identified on each of the three domains of AFP. Thereafter, the localization of receptors on specific cell types were reviewed and their functions were discussed.

Thrombin-dependent Incorporation of von Willebrand Factor into a Fibrin Network*

PubMed Central

Miszta, Adam; Pelkmans, Leonie; Lindhout, Theo; Krishnamoorthy, Ganeshram; de Groot, Philip G.; Hemker, Coenraad H.; Heemskerk, Johan W. M.; Kelchtermans, Hilde; de Laat, Bas

2014-01-01

Attachment of platelets from the circulation onto a growing thrombus is a process involving multiple platelet receptors, endothelial matrix components, and coagulation factors. It has been indicated previously that during a transglutaminase reaction activated factor XIII (FXIIIa) covalently cross-links von Willebrand factor (VWF) to polymerizing fibrin. Bound VWF further recruits and activates platelets via interactions with the platelet receptor complex glycoprotein Ib (GPIb). In the present study we found proof for binding of VWF to a fibrin monomer layer during the process of fibrinogen-to-fibrin conversion in the presence of thrombin, arvin, or a snake venom from Crotalus atrox. Using a domain deletion mutant we demonstrated the involvement of the C domains of VWF in this binding. Substantial binding of VWF to fibrin monomers persisted in the presence of the FXIIIa inhibitor K9-DON, illustrating that cross-linking via factor XIII is not essential for this phenomenon and suggesting the identification of a second mechanism through which VWF multimers incorporate into a fibrin network. Under high shear conditions, platelets were shown to adhere to fibrin only if VWF had been incorporated. In conclusion, our experiments show that the C domains of VWF and the E domain of fibrin monomers are involved in the incorporation of VWF during the polymerization of fibrin and that this incorporation fosters binding and activation of platelets. Fibrin thus is not an inert end product but partakes in further thrombus growth. Our findings help to elucidate the mechanism of thrombus growth and platelet adhesion under conditions of arterial shear rate. PMID:25381443

Nectin-like interactions between poliovirus and its receptor trigger conformational changes associated with cell entry.

PubMed

Strauss, Mike; Filman, David J; Belnap, David M; Cheng, Naiqian; Noel, Roane T; Hogle, James M

2015-04-01

Poliovirus infection is initiated by attachment to a receptor on the cell surface called Pvr or CD155. At physiological temperatures, the receptor catalyzes an irreversible expansion of the virus to form an expanded form of the capsid called the 135S particle. This expansion results in the externalization of the myristoylated capsid protein VP4 and the N-terminal extension of the capsid protein VP1, both of which become inserted into the cell membrane. Structures of the expanded forms of poliovirus and of several related viruses have recently been reported. However, until now, it has been unclear how receptor binding triggers viral expansion at physiological temperature. Here, we report poliovirus in complex with an enzymatically partially deglycosylated form of the 3-domain ectodomain of Pvr at a 4-Å resolution, as determined by cryo-electron microscopy. The interaction of the receptor with the virus in this structure is reminiscent of the interactions of Pvr with its natural ligands. At a low temperature, the receptor induces very few changes in the structure of the virus, with the largest changes occurring within the footprint of the receptor, and in a loop of the internal protein VP4. Changes in the vicinity of the receptor include the displacement of a natural lipid ligand (called "pocket factor"), demonstrating that the loss of this ligand, alone, is not sufficient to induce particle expansion. Finally, analogies with naturally occurring ligand binding in the nectin family suggest which specific structural rearrangements in the virus-receptor complex could help to trigger the irreversible expansion of the capsid. The cell-surface receptor (Pvr) catalyzes a large structural change in the virus that exposes membrane-binding protein chains. We fitted known atomic models of the virus and Pvr into three-dimensional experimental maps of the receptor-virus complex. The molecular interactions we see between poliovirus and its receptor are reminiscent of the nectin family, by involving the burying of otherwise-exposed hydrophobic groups. Importantly, poliovirus expansion is regulated by the binding of a lipid molecule within the viral capsid. We show that receptor binding either causes this molecule to be expelled or requires it, but that its loss is not sufficient to trigger irreversible expansion. Based on our model, we propose testable hypotheses to explain how the viral shell becomes destabilized, leading to RNA uncoating. These findings give us a better understanding of how poliovirus has evolved to exploit a natural process of its host to penetrate the membrane barrier. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The amphiphilic peptide adenoregulin enhances agonist binding to A1-adenosine receptors and [35S]GTP gamma S to brain membranes.

PubMed

Moni, R W; Romero, F S; Daly, J W

1995-08-01

1. Adenoregulin is an amphilic peptide isolated from skin mucus of the tree frog, Phyllomedusa bicolor. Synthetic adenoregulin enhanced the binding of agonists to several G-protein-coupled receptors in rat brain membranes. 2. The maximal enhancement of agonist binding, and in parentheses, the concentration of adenoregulin affording maximal enhancement were as follows: 60% (20 microM) for A1-adenosine receptors, 30% (100 microM) for A2a-adenosine receptors, 20% (2 microM) for alpha 2-adrenergic receptors, and 30% (10 microM) for 5HT1A receptors. High affinity agonist binding for A1-, alpha 2-, and 5HT1A-receptors was virtually abolished by GTP gamma S in the presence of adenoregulin, but was only partially abolished in its absence. Magnesium ions increased the binding of agonists to receptors and reduced the enhancement elicited by adenoregulin. 3. The effect of adenoregulin on binding of N6-cyclohexyladenosine ([3H]CHA) to A1-receptors was relatively slow and was irreversible. Adenoregulin increased the Bmax value for [3H]CHA binding sites, and the proportion of high affinity states, and slowed the rate of [3H]CHA dissociation. Binding of the A1-selective antagonist, [3H]DPCPX, was maximally enhanced by only 13% at 2 microM adenoregulin. Basal and A1-adenosine receptor-stimulated binding of [35S]GTP gamma S were maximally enhanced 45% and 23%, respectively, by 50 microM adenoregulin. In CHAPS-solubilized membranes from rat cortex, the binding of both [3H]CHA and [3H]DPCPX were enhanced by adenoregulin. Binding of [3H]CHA to membranes from DDT1 MF-2 cells was maximally enhanced 17% at 20 microM adenoregulin. In intact DDT1 MF-2 cells, 20 microM adenoregulin did not potentiate the inhibition of cyclic AMP accumulation mediated via the adenosine A1 receptor. 4. It is proposed that adenoregulin enhances agonist binding through a mechanism involving enhancement of guanyl nucleotide exchange at G-proteins, resulting in a conversion of receptors into a high affinity state complexed with guanyl nucleotide-free G-protein.

Precipitation of the thyrotropin receptor and identification of thyroid autoantigens using Graves' disease immunoglobulins.

PubMed Central

Heyma, P; Harrison, L C

1984-01-01

The thyrotropin (TSH) receptor is a putative target for autoantibodies in Graves' hyperthyroidism and therefore, should be capable of being identified, isolated, and structurally characterized by immunological means. To this end, four sera from patients with hyperthyroidism, three of which inhibited the binding of 125I-TSH to Triton-solubilized human thyroid membranes, were used to isolate TSH receptors by immunoprecipitation. To account for an effect of TSH binding or receptor occupancy on the ability of Graves' immunoglobulins to precipitate TSH receptors, two approaches were taken: (a) specific 125I-TSH binding activity was measured after solubilized thyroid membranes had been incubated with Graves' sera followed by precipitation with Staphylococcus protein A ("receptor depletion"); (b) TSH binding sites were labeled with 125I-TSH and the complexes were precipitated using Graves' sera and Staphylococcus protein A ("receptor precipitation"). The three sera which inhibited 125I-TSH binding depleted 125I-TSH binding activity between 30-80%. Preformed complexes between Staphylococcus protein A and immunoglobulins in these sera were also able to deplete 125I-TSH binding activity. However, after receptor depletion, the one serum that did not inhibit 125I-TSH binding was associated with a significant increase in 125I-TSH binding. All four sera specifically precipitated 80-100% of receptors identified by prelabeling with 125I-TSH. The dilutions of sera that precipitated 50% of 125I-TSH-receptor complexes ranged from 1:150-1:20. Complexes were partially precipitated by high concentrations of control sera (1:20), but the relative potency of control sera was at least fourfold less than Graves' sera. Immunoprecipitates of 125I-labeled thyroid membranes were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography to reveal Graves'-specific bands of reduced molecular weights of 100-110,000, 80-90,000, and 70-75,000. These bands were similar to those obtained from 125I-labeled thyroid membranes purified by TSH affinity chromatography. Thus, Graves' immunoglobulins: (a) precipitate unoccupied and occupied TSH receptors, (b) in one case, neither inhibit binding nor immunodeplete the unoccupied receptor but immunoprecipitate 125I-TSH-receptor complexes, suggesting that binding of TSH may initiate an interaction between the binding site and a separate immunoreactive molecule, and (c) identify the molecular structure of Graves' autoantigens, putatively, the TSH receptor. Images PMID:6088581

Predictive model of thrombospondin-1 and vascular endothelial growth factor in breast tumor tissue.

PubMed

Rohrs, Jennifer A; Sulistio, Christopher D; Finley, Stacey D

2016-01-01

Angiogenesis, the formation of new blood capillaries from pre-existing vessels, is a hallmark of cancer. Thus far, strategies for reducing tumor angiogenesis have focused on inhibiting pro-angiogenic factors, while less is known about the therapeutic effects of mimicking the actions of angiogenesis inhibitors. Thrombospondin-1 (TSP1) is an important endogenous inhibitor of angiogenesis that has been investigated as an anti-angiogenic agent. TSP1 impedes the growth of new blood vessels in many ways, including crosstalk with pro-angiogenic factors. Due to the complexity of TSP1 signaling, a predictive systems biology model would provide quantitative understanding of the angiogenic balance in tumor tissue. Therefore, we have developed a molecular-detailed, mechanistic model of TSP1 and vascular endothelial growth factor (VEGF), a promoter of angiogenesis, in breast tumor tissue. The model predicts the distribution of the angiogenic factors in tumor tissue, revealing that TSP1 is primarily in an inactive, cleaved form due to the action of proteases, rather than bound to its cellular receptors or to VEGF. The model also predicts the effects of enhancing TSP1's interactions with its receptors and with VEGF. To provide additional predictions that can guide the development of new anti-angiogenic drugs, we simulate administration of exogenous TSP1 mimetics that bind specific targets. The model predicts that the CD47-binding TSP1 mimetic dramatically decreases the ratio of receptor-bound VEGF to receptor-bound TSP1, in favor of anti-angiogenesis. Thus, we have established a model that provides a quantitative framework to study the response to TSP1 mimetics.

Signal transduction by beta1 integrin receptors in human chondrocytes in vitro: collaboration with the insulin-like growth factor-I receptor.

PubMed

Shakibaei, M; John, T; De Souza, P; Rahmanzadeh, R; Merker, H J

1999-09-15

We have examined the mechanism by which collagen-binding integrins co-operate with insulin-like growth factor-I (IGF-I) receptors (IGF-IR) to regulate chondrocyte phenotype and differentiation. Adhesion of chondrocytes to anti-beta1 integrin antibodies or collagen type II leads to phosphorylation of cytoskeletal and signalling proteins localized at focal adhesions, including alpha-actinin, vinculin, paxillin and focal adhesion kinase (FAK). These stimulate docking proteins such as Shc (Src-homology collagen). Moreover, exposure of collagen type II-cultured chondrocytes to IGF-I leads to co-immunoprecipitation of Shc protein with the IGF-IR and with beta1, alpha1 and alpha5 integrins, but not with alpha3 integrin. Shc then associates with growth factor receptor-bound protein 2 (Grb2), an adaptor protein and extracellular signal-regulated kinase. The expression of the docking protein Shc occurs only when chondrocytes are bound to collagen type II or integrin antibodies and increases when IGF-I is added, suggesting a collaboration between integrins and growth factors in a common/shared biochemical signalling pathway. Furthermore, these results indicate that focal adhesion assembly may facilitate signalling via Shc, a potential common target for signal integration between integrin and growth-factor signalling regulatory pathways. Thus, the collagen-binding integrins and IGF-IR co-operate to regulate focal adhesion components and these signalling pathways have common targets (Shc-Grb2 complex) in subcellular compartments, thereby linking to the Ras-mitogen-activated protein kinase signalling pathway. These events may play a role during chondrocyte differentiation.

Role of the chromatin landscape and sequence in determining cell type-specific genomic glucocorticoid receptor binding and gene regulation

PubMed Central

Huska, Matthew R.; Jurk, Marcel; Schöpflin, Robert; Starick, Stephan R.; Schwahn, Kevin; Cooper, Samantha B.; Yamamoto, Keith R.; Thomas-Chollier, Morgane; Vingron, Martin

2017-01-01

Abstract The genomic loci bound by the glucocorticoid receptor (GR), a hormone-activated transcription factor, show little overlap between cell types. To study the role of chromatin and sequence in specifying where GR binds, we used Bayesian modeling within the universe of accessible chromatin. Taken together, our results uncovered that although GR preferentially binds accessible chromatin, its binding is biased against accessible chromatin located at promoter regions. This bias can only be explained partially by the presence of fewer GR recognition sequences, arguing for the existence of additional mechanisms that interfere with GR binding at promoters. Therefore, we tested the role of H3K9ac, the chromatin feature with the strongest negative association with GR binding, but found that this correlation does not reflect a causative link. Finally, we find a higher percentage of promoter–proximal GR binding for genes regulated by GR across cell types than for cell type-specific target genes. Given that GR almost exclusively binds accessible chromatin, we propose that cell type-specific regulation by GR preferentially occurs via distal enhancers, whose chromatin accessibility is typically cell type-specific, whereas ubiquitous target gene regulation is more likely to result from binding to promoter regions, which are often accessible regardless of cell type examined. PMID:27903902

HIV-1 Tat binds to SH3 domains: cellular and viral outcome of Tat/Grb2 interaction

PubMed Central

Rom, Slava; Pacifici, Marco; Passiatore, Giovanni; Aprea, Susanna; Waligorska, Agnieszka; Valle, Luis Del; Peruzzi, Francesca

2011-01-01

The Src-homology 3 (SH3) domain is one of the most frequent protein recognition modules (PRMs), being represented in signal transduction pathways and in several pathologies such as cancer and AIDS. Grb2 (growth factor receptor-bound protein 2) is an adaptor protein that contains two SH3 domains and is involved in receptor tyrosine kinase (RTK) signal transduction pathways. The HIV-1 transactivator factor Tat is required for viral replication and it has been shown to bind directly or indirectly to several host proteins, deregulating their functions. In this study, we show interaction between the cellular factor Grb2 and the HIV-1 trans-activating protein Tat. The binding is mediated by the proline-rich sequence of Tat and the SH3 domain of Grb2. As the adaptor protein Grb2 participates in a wide variety of signaling pathways, we characterized at least one of the possible downstream effects of the Tat/Grb2 interaction on the well-known IGF-1R/Raf/MAPK cascade. We show that the binding of Tat to Grb2 impairs activation of the Raf/MAPK pathway, while potentiating the PKA/Raf inhibitory pathway. The Tat/Grb2 interaction affects also viral function by inhibiting the Tat-mediated transactivation of HIV-1 LTR and viral replication in infected primary microglia. PMID:21745501

Structure-based receptor MIMICS targeted against bacterial superantigen toxins

DOEpatents

Gupta, Goutam [Santa Fe, NM; Hong-Geller, Elizabeth [Los Alamos, NM; Shiflett, Patrick R [Los Alamos, NM; Lehnert, Nancy M [Albuquerque, NM

2009-08-18

The invention provides therapeutic compositions useful in the treatment of bacterial superantigen mediated conditions, such as Toxic Shock Syndrome. The compositions comprise genetically engineered bifunctional polypeptides containing a specific T-cell receptor binding domain and a specific MHC class II receptor binding domain, each targeting non-overlapping epitopes on a superantigen molecule against which they are designed. The anti-superantigen "receptor mimetics" or "chimeras" are rationally designed to recreate the modality of superantigen binding directly to both the TCR and the MHC-II receptor, and are capable of acting as decoys for superantigen binding, effectively out-competing the host T-cell and MHC-II receptors, the natural host receptors.

Actin-Sorting Nexin 27 (SNX27)-Retromer Complex Mediates Rapid Parathyroid Hormone Receptor Recycling*

PubMed Central

McGarvey, Jennifer C.; Xiao, Kunhong; Bowman, Shanna L.; Mamonova, Tatyana; Zhang, Qiangmin; Bisello, Alessandro; Sneddon, W. Bruce; Ardura, Juan A.; Jean-Alphonse, Frederic; Vilardaga, Jean-Pierre; Puthenveedu, Manojkumar A.; Friedman, Peter A.

2016-01-01

The G protein-coupled parathyroid hormone receptor (PTHR) regulates mineral-ion homeostasis and bone remodeling. Upon parathyroid hormone (PTH) stimulation, the PTHR internalizes into early endosomes and subsequently traffics to the retromer complex, a sorting platform on early endosomes that promotes recycling of surface receptors. The C terminus of the PTHR contains a type I PDZ ligand that binds PDZ domain-containing proteins. Mass spectrometry identified sorting nexin 27 (SNX27) in isolated endosomes as a PTHR binding partner. PTH treatment enriched endosomal PTHR. SNX27 contains a PDZ domain and serves as a cargo selector for the retromer complex. VPS26, VPS29, and VPS35 retromer subunits were isolated with PTHR in endosomes from cells stimulated with PTH. Molecular dynamics and protein binding studies establish that PTHR and SNX27 interactions depend on the PDZ recognition motif in PTHR and the PDZ domain of SNX27. Depletion of either SNX27 or VPS35 or actin depolymerization decreased the rate of PTHR recycling following agonist stimulation. Mutating the PDZ ligand of PTHR abolished the interaction with SNX27 but did not affect the overall rate of recycling, suggesting that PTHR may directly engage the retromer complex. Coimmunoprecipitation and overlay experiments show that both intact and mutated PTHR bind retromer through the VPS26 protomer and sequentially assemble a ternary complex with PTHR and SNX27. SNX27-independent recycling may involve N-ethylmaleimide-sensitive factor, which binds both PDZ intact and mutant PTHRs. We conclude that PTHR recycles rapidly through at least two pathways, one involving the ASRT complex of actin, SNX27, and retromer and another possibly involving N-ethylmaleimide-sensitive factor. PMID:27008860

Klotho converts canonical FGF receptor into a specific receptor for FGF23.

PubMed

Urakawa, Itaru; Yamazaki, Yuji; Shimada, Takashi; Iijima, Kousuke; Hasegawa, Hisashi; Okawa, Katsuya; Fujita, Toshiro; Fukumoto, Seiji; Yamashita, Takeyoshi

2006-12-07

FGF23 is a unique member of the fibroblast growth factor (FGF) family because it acts as a hormone that derives from bone and regulates kidney functions, whereas most other family members are thought to regulate various cell functions at a local level. The renotropic activity of circulating FGF23 indicates the possible presence of an FGF23-specific receptor in the kidney. Here we show that a previously undescribed receptor conversion by Klotho, a senescence-related molecule, generates the FGF23 receptor. Using a renal homogenate, we found that Klotho binds to FGF23. Forced expression of Klotho enabled the high-affinity binding of FGF23 to the cell surface and restored the ability of a renal cell line to respond to FGF23 treatment. Moreover, FGF23 incompetence was induced by injecting wild-type mice with an anti-Klotho monoclonal antibody. Thus, Klotho is essential for endogenous FGF23 function. Because Klotho alone seemed to be incapable of intracellular signalling, we searched for other components of the FGF23 receptor and found FGFR1(IIIc), which was directly converted by Klotho into the FGF23 receptor. Thus, the concerted action of Klotho and FGFR1(IIIc) reconstitutes the FGF23 receptor. These findings provide insights into the diversity and specificity of interactions between FGF and FGF receptors.

Hormone Binding to Recombinant Estrogen Receptors from Human, Alligator, Quail, Salamander, and Fathead Minnow

EPA Science Inventory

In this work, a 96-well plate estrogen receptor binding assay was developed to facilitate the direct comparison of chemical binding to full-length recombinant estrogen receptors across vertebrate classes. Receptors were generated in a baculovirus expression system. This approach ...

Analysis of the hormone-binding domain of steroid receptors using chimeras generated by homologous recombination

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

Martinez, Elisabeth D.; Pattabiraman, Nagarajan; Department of Oncology, Georgetown University School of Medicine, Washington, DC 20057

2005-08-15

The glucocorticoid receptor and the mineralocorticoid receptor are members of the steroid receptor family that exhibit ligand cross-reactivity. Specificity of steroid receptor action is investigated in the present work by the construction and characterization of chimeras between the glucocorticoid receptor and the mineralocorticoid receptor. We used an innovative approach to make novel steroid receptor proteins in vivo that in general, contrary to our expectations, show increased ligand specificity compared to the parental receptors. We describe a receptor that is specific for the potent synthetic glucocorticoid triamcinolone acetonide and does not bind aldosterone. A further set of chimeras has an increasedmore » ability to discriminate between ligands, responding potently to mineralocorticoids and only very weakly to synthetic glucocorticoids. A chimera with the fusion site in the hinge highlights the importance of the region between the DNA-binding and the hormone-binding domains since, unlike both the glucocorticoid and mineralocorticoid receptors, it only responds to mineralocorticoids. One chimera has reduced specificity in that it acts as a general corticoid receptor, responding to glucocorticoids and mineralocorticoids with similar potency and efficacy. Our data suggest that regions of the glucocorticoid and mineralocorticoid receptor hormone-binding domains are functionally non-reciprocal. We present transcriptional, hormone-binding, and structure-modeling evidence that suggests that receptor-specific interactions within and across domains mediate aspects of specificity in transcriptional responses to steroids.« less

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

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.

Identification of a novel A20-binding inhibitor of nuclear factor-kappa B activation termed ABIN-2.

PubMed

Van Huffel, S; Delaei, F; Heyninck, K; De Valck, D; Beyaert, R

2001-08-10

The nuclear factor kappaB (NF-kappaB) plays a central role in the regulation of genes implicated in immune responses, inflammatory processes, and apoptotic cell death. The zinc finger protein A20 is a cellular inhibitor of NF-kappaB activation by various stimuli and plays a critical role in terminating NF-kappaB responses. The underlying mechanism for NF-kappaB inhibition by A20 is still unknown. A20 has been shown to interact with several proteins including tumor necrosis factor (TNF) receptor-associated factors 2 and 6, as well as the inhibitory protein of kappaB kinase (IKK) gamma protein. Here we report the cloning and characterization of ABIN-2, a previously unknown protein that binds to the COOH-terminal zinc finger domain of A20. NF-kappaB activation induced by TNF and interleukin-1 is inhibited by overexpression of ABIN-2. The latter also inhibits NF-kappaB activation induced by overexpression of receptor-interacting protein or TNF receptor-associated factor 2. In contrast, NF-kappaB activation by overexpression of IKKbeta or direct activators of the IKK complex, such as Tax, cannot be inhibited by ABIN-2. These results indicate that ABIN-2 interferes with NF-kappaB activation upstream of the IKK complex and that it might contribute to the NF-kappaB-inhibitory function of A20.

Human adenovirus serotypes 3 and 5 bind to two different cellular receptors via the fiber head domain.

PubMed Central

Stevenson, S C; Rollence, M; White, B; Weaver, L; McClelland, A

1995-01-01

The adenovirus fiber protein is responsible for attachment of the virion to cell surface receptors. The identity of the cellular receptor which mediates binding is unknown, although there is evidence suggesting that two distinct adenovirus receptors interact with the group C (adenovirus type 5 [Ad5]) and the group B (Ad3) adenoviruses. In order to define the determinants of adenovirus receptor specificity, we have carried out a series of competition binding experiments using recombinant native fiber polypeptides from Ad5 and Ad3 and chimeric fiber proteins in which the head domains of Ad5 and Ad3 were exchanged. Specific binding of fiber to HeLa cell receptors was assessed with radiolabeled protein synthesized in vitro, and by competition analysis with baculovirus-expressed fiber protein. Fiber produced in vitro was found as both monomer and trimer, but only the assembled trimers had receptor binding activity. Competition data support the conclusion that Ad5 and Ad3 interact with different cellular receptors. The Ad5 receptor distribution on several cell lines was assessed with a fiber binding flow cytometric assay. HeLa cells were found to express high levels of receptor, while CHO and human diploid fibroblasts did not. A chimeric fiber containing the Ad5 fiber head domain blocked the binding of Ad5 fiber but not Ad3 fiber. Similarly, a chimeric fiber containing the Ad3 fiber head blocked the binding of labeled Ad3 fiber but not Ad5 fiber. In addition, the isolated Ad3 fiber head domain competed effectively with labeled Ad3 fiber for binding to HeLa cell receptors. These results demonstrate that the determinants of receptor binding are located in the head domain of the fiber and that the isolated head domain is capable of trimerization and binding to cellular receptors. Our results also show that it is possible to change the receptor specificity of the fiber protein by manipulation of sequences contained in the head domain. Modification or replacement of the fiber head domain with novel ligands may permit adenovirus vectors with new receptor specificities which could be useful for targeted gene delivery in vivo to be engineered. PMID:7707507

Use of polyclonal and monoclonal antibodies to study hCG-receptor interactions

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

Milius, R.P.

1985-01-01

Although the glycoprotein hormones lutropin (LH), follitropin (FSH), and thyrotropin (TSH) bind to different receptors, each contains an identical alpha subunit. Specificity is somehow endowed by theta subunits which are distinct for each hormone. Human choriogonadotropin (hCG) is a natural LH analog that contains a beta subunit nearly identical to that of LH. The roles of these subunits in the recognition and high affinity binding of hCG to receptor was examined. Polyclonal and monoclonal antibodies specific for the individual subunits of hCG were used to probe the hormone-receptor interaction. Conformation-specific and sequence-specific antibodies were examined for their abilities to bindmore » Triton X-100-solubilized /sup 125/I-hCG-receptor complex and to inhibit hormone binding to crude rat ovarian membranes containing receptor. Even though the immunoreactive sites are not located on the receptor binding surface of the beta subunit, most, but not all, of these polyclonal and monoclonal antibodies were able to inhibit /sup 125/I-hCG binding to receptor. Although the inhibition of binding may be due to steric interference due to the size of the antibody molecules, a two-step model for hCG binding to receptor is presented that also explains these results. In this model, the beta subunit initially binds with the receptor with a highly specific but low affinity interaction. This activates a site for the high affinity binding of the alpha subunit and stabilization of the complex. This is an attractive model as it may be applied to other glycoprotein hormones sharing an alpha subunit.« less

SCM, the M Protein of Streptococcus canis Binds Immunoglobulin G

PubMed Central

Bergmann, Simone; Eichhorn, Inga; Kohler, Thomas P.; Hammerschmidt, Sven; Goldmann, Oliver; Rohde, Manfred; Fulde, Marcus

2017-01-01

The M protein of Streptococcus canis (SCM) is a virulence factor and serves as a surface-associated receptor with a particular affinity for mini-plasminogen, a cleavage product of the broad-spectrum serine protease plasmin. Here, we report that SCM has an additional high-affinity immunoglobulin G (IgG) binding activity. The ability of a particular S. canis isolate to bind to IgG significantly correlates with a scm-positive phenotype, suggesting a dominant role of SCM as an IgG receptor. Subsequent heterologous expression of SCM in non-IgG binding S. gordonii and Western Blot analysis with purified recombinant SCM proteins confirmed its IgG receptor function. As expected for a zoonotic agent, the SCM-IgG interaction is species-unspecific, with a particular affinity of SCM for IgGs derived from human, cats, dogs, horses, mice, and rabbits, but not from cows and goats. Similar to other streptococcal IgG-binding proteins, the interaction between SCM and IgG occurs via the conserved Fc domain and is, therefore, non-opsonic. Interestingly, the interaction between SCM and IgG-Fc on the bacterial surface specifically prevents opsonization by C1q, which might constitute another anti-phagocytic mechanism of SCM. Extensive binding analyses with a variety of different truncated SCM fragments defined a region of 52 amino acids located in the central part of the mature SCM protein which is important for IgG binding. This binding region is highly conserved among SCM proteins derived from different S. canis isolates but differs significantly from IgG-Fc receptors of S. pyogenes and S. dysgalactiae sub. equisimilis, respectively. In summary, we present an additional role of SCM in the pathogen-host interaction of S. canis. The detailed analysis of the SCM-IgG interaction should contribute to a better understanding of the complex roles of M proteins in streptococcal pathogenesis. PMID:28401063

3- and 4-O-sulfoconjugated and methylated dopamine: highly reduced binding affinity to dopamine D2 receptors in rat striatal membranes.

PubMed

Werle, E; Lenz, T; Strobel, G; Weicker, H

1988-07-01

The binding properties of 3- and 4-O-sulfo-conjugated dopamine (DA-3-O-S, DA-4-O-S) as well as 3-O-methylated dopamine (MT) to rat striatal dopamine D2 receptors were investigated. 3H-spiperone was used as a radioligand in the binding studies. In saturation binding experiments (+)butaclamol, which has been reported to bind to dopaminergic D2 and serotoninergic 5HT2 receptors, was used in conjunction with ketanserin and sulpiride, which preferentially label 5HT2 and D2 receptors, respectively, in order to discriminate between 3H-spiperone binding to D2 and to 5HT2 receptors. Under our particular membrane preparation and assay conditions, 3H-spiperone binds to D2 and 5HT2 receptors with a maximal binding capacity (Bmax) of 340 fmol/mg protein in proportions of about 75%:25% with similar dissociation constants KD (35 pmol/l; 43 pmol/l). This result was verified by the biphasic competition curve of ketanserin, which revealed about 20% high (KD = 24 nmol/l) and 80% low (KD = 420 nmol/l) affinity binding sites corresponding to 5HT2 and D2 receptors, respectively. Therefore, all further competition experiments at a tracer concentration of 50 pmol/l were performed in the presence of 0.1 mumol/l ketanserin to mask the 5HT2 receptors. DA competition curves were best fitted assuming two binding sites, with high (KH = 0.12 mumol/l) and low (KL = 18 mumol/l) affinity, present in a ratio of 3:1. The high affinity binding sites were interconvertible by 100 mumol/l guanyl-5-yl imidodiphosphate [Gpp(NH)p], resulting in a homogenous affinity state of DA receptors (KD = 2.8 mumol/l).2+ off

The two-state dimer receptor model: a general model for receptor dimers.

PubMed

Franco, Rafael; Casadó, Vicent; Mallol, Josefa; Ferrada, Carla; Ferré, Sergi; Fuxe, Kjell; Cortés, Antoni; Ciruela, Francisco; Lluis, Carmen; Canela, Enric I

2006-06-01

Nonlinear Scatchard plots are often found for agonist binding to G-protein-coupled receptors. Because there is clear evidence of receptor dimerization, these nonlinear Scatchard plots can reflect cooperativity on agonist binding to the two binding sites in the dimer. According to this, the "two-state dimer receptor model" has been recently derived. In this article, the performance of the model has been analyzed in fitting data of agonist binding to A(1) adenosine receptors, which are an example of receptor displaying concave downward Scatchard plots. Analysis of agonist/antagonist competition data for dopamine D(1) receptors using the two-state dimer receptor model has also been performed. Although fitting to the two-state dimer receptor model was similar to the fitting to the "two-independent-site receptor model", the former is simpler, and a discrimination test selects the two-state dimer receptor model as the best. This model was also very robust in fitting data of estrogen binding to the estrogen receptor, for which Scatchard plots are concave upward. On the one hand, the model would predict the already demonstrated existence of estrogen receptor dimers. On the other hand, the model would predict that concave upward Scatchard plots reflect positive cooperativity, which can be neither predicted nor explained by assuming the existence of two different affinity states. In summary, the two-state dimer receptor model is good for fitting data of binding to dimeric receptors displaying either linear, concave upward, or concave downward Scatchard plots.

Rapid molecular evolution across amniotes of the IIS/TOR network

PubMed Central

McGaugh, Suzanne E.; Bronikowski, Anne M.; Kuo, Chih-Horng; Reding, Dawn M.; Addis, Elizabeth A.; Flagel, Lex E.; Janzen, Fredric J.

2015-01-01

The insulin/insulin-like signaling and target of rapamycin (IIS/TOR) network regulates lifespan and reproduction, as well as metabolic diseases, cancer, and aging. Despite its vital role in health, comparative analyses of IIS/TOR have been limited to invertebrates and mammals. We conducted an extensive evolutionary analysis of the IIS/TOR network across 66 amniotes with 18 newly generated transcriptomes from nonavian reptiles and additional available genomes/transcriptomes. We uncovered rapid and extensive molecular evolution between reptiles (including birds) and mammals: (i) the IIS/TOR network, including the critical nodes insulin receptor substrate (IRS) and phosphatidylinositol 3-kinase (PI3K), exhibit divergent evolutionary rates between reptiles and mammals; (ii) compared with a proxy for the rest of the genome, genes of the IIS/TOR extracellular network exhibit exceptionally fast evolutionary rates; and (iii) signatures of positive selection and coevolution of the extracellular network suggest reptile- and mammal-specific interactions between members of the network. In reptiles, positively selected sites cluster on the binding surfaces of insulin-like growth factor 1 (IGF1), IGF1 receptor (IGF1R), and insulin receptor (INSR); whereas in mammals, positively selected sites clustered on the IGF2 binding surface, suggesting that these hormone-receptor binding affinities are targets of positive selection. Further, contrary to reports that IGF2R binds IGF2 only in marsupial and placental mammals, we found positively selected sites clustered on the hormone binding surface of reptile IGF2R that suggest that IGF2R binds to IGF hormones in diverse taxa and may have evolved in reptiles. These data suggest that key IIS/TOR paralogs have sub- or neofunctionalized between mammals and reptiles and that this network may underlie fundamental life history and physiological differences between these amniote sister clades. PMID:25991861

Single amino acid substitutions at 2 of 14 positions in an ultra-conserved region of the androgen receptor yield an androgen-binding domain that is reversibly thermolabile

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

Vasiliou, M.; Lumbroso, R.; Alvarado, C.

1994-09-01

The stereochemistry of the androgen receptor (AR) that is responsible for androgen-specific binding and for its contribution to the transregulatory attributes of an androgen-receptor complex are unknown. Our objective is to define structure-function relations of the human AR by correlating germline missense mutations at its X-linked locus with its resultant misbehavior. Subjects with Arg773Cys have complete androgen insensitivity. We and several other laboratories have reported that their genital skin fibroblasts (GSF) have negligible androgen-binding activity at 37{degrees}. We have found that Phe763Leu also causes CAI, but with approximately 10 fmol/mg protein androgen-binding activity at 37{degrees} (R-deficient). Within COS-1 cells transfectedmore » with each mutant AR cDNA, Phe763Leu and Arg773Cys androgen-binding activities are reversibly thermolabile, by a factor of 2, at 37{degrees} versus 22{degrees}, only in the presence of androgen; in the absence of androgen they are thermostable at 37{degrees}. We have discovered that (for a reason yet unknown) the GSF from a third family with Arg773Cys (and no other coding sequence mutation) have 20-40 mol/mg protein of androgen-binding activity at 37{degrees} when measured with 3-6 nFM androgen. This activity reversibly doubles at 22{degrees}. The reversible thermolability of an AR with Arg773Cys (and probably with Phe763Leu) is demonstrable within GSF. Ligand-dependence of this thermolability implies that ligand induces these mutant AR to undergo a deviant conformational change in, or near, a 14-aa region that shares 90% identity/similarity with its closest receptor relatives.« less

Rapid molecular evolution across amniotes of the IIS/TOR network.

PubMed

McGaugh, Suzanne E; Bronikowski, Anne M; Kuo, Chih-Horng; Reding, Dawn M; Addis, Elizabeth A; Flagel, Lex E; Janzen, Fredric J; Schwartz, Tonia S

2015-06-02

The insulin/insulin-like signaling and target of rapamycin (IIS/TOR) network regulates lifespan and reproduction, as well as metabolic diseases, cancer, and aging. Despite its vital role in health, comparative analyses of IIS/TOR have been limited to invertebrates and mammals. We conducted an extensive evolutionary analysis of the IIS/TOR network across 66 amniotes with 18 newly generated transcriptomes from nonavian reptiles and additional available genomes/transcriptomes. We uncovered rapid and extensive molecular evolution between reptiles (including birds) and mammals: (i) the IIS/TOR network, including the critical nodes insulin receptor substrate (IRS) and phosphatidylinositol 3-kinase (PI3K), exhibit divergent evolutionary rates between reptiles and mammals; (ii) compared with a proxy for the rest of the genome, genes of the IIS/TOR extracellular network exhibit exceptionally fast evolutionary rates; and (iii) signatures of positive selection and coevolution of the extracellular network suggest reptile- and mammal-specific interactions between members of the network. In reptiles, positively selected sites cluster on the binding surfaces of insulin-like growth factor 1 (IGF1), IGF1 receptor (IGF1R), and insulin receptor (INSR); whereas in mammals, positively selected sites clustered on the IGF2 binding surface, suggesting that these hormone-receptor binding affinities are targets of positive selection. Further, contrary to reports that IGF2R binds IGF2 only in marsupial and placental mammals, we found positively selected sites clustered on the hormone binding surface of reptile IGF2R that suggest that IGF2R binds to IGF hormones in diverse taxa and may have evolved in reptiles. These data suggest that key IIS/TOR paralogs have sub- or neofunctionalized between mammals and reptiles and that this network may underlie fundamental life history and physiological differences between these amniote sister clades.

Gene regulation by NMDA receptor activation in the SDN-POA neurons of male rats during sexual development.

PubMed

Hsu, Hseng-Kuang; Shao, Pei-Lin; Tsai, Ke-Li; Shih, Huei-Chuan; Lee, Tzu-Ying; Hsu, Chin

2005-04-01

The present study was designed to identify possible signaling pathways, which may play a role in prevention of neuronal apoptosis in the sexually dimorphic nucleus of the preoptic area (SDN-POA) after physiological activation of the N-methyl-D-aspartate (NMDA) receptor. Gene response to the blockage of the NMDA receptor by an antagonist (dizocilpine hydrogen maleate; MK-801) was screened after suppression subtractive hybridization (SSH). The results showed that differential screening after SSH detected the presence of some neurotrophic genes (RNA binding motif protein 3 (RBM3), alpha-tubulin) as well as apoptosis-related genes (Bcl-2, cytochrome oxidase subunit II, cytochrome oxidase subunit III) in the SDN-POA of male rats, which were down-regulated by blocking the NMDA receptor. The RT-PCR products of the aforementioned genes in MK-801-treated males were significantly less than that in untreated males. In particular, the expression of Bcl-2 mRNA, including Bcl-2 protein, in male rats were significantly suppressed by MK-801 treatment. Moreover, the binding activity of nuclear factor kappaB (NFkappaB) was significantly higher in male rats than in females, but significantly diminished by blocking the NMDA receptor with MK-801 in male rats. No significant difference in cAMP response element-binding protein (CREB) binding activity was observed among untreated male, MK-801-treated male, untreated female and MK-801-treated female groups. These results suggest that genes regulated by NMDA receptor activation might participate in neuronal growth and/or anti-apoptosis, and support an important signaling pathway of NFkappaB activation and its target gene, Bcl-2, in preventing neuronal apoptosis in the SDN-POA of male rats during sexual development.

Role of decoy molecules in neuronal ischemic preconditioning

PubMed Central

Panneerselvam, Mathivadhani; Patel, Piyush M.; Roth, David M.; Kidd, Michael W.; Chin-Lee, Blake; Head, Brian P.; Niesman, Ingrid R.; Inoue, Satoki; Patel, Hemal H.; Davis, Daniel P.

2011-01-01

Decoy receptors bind with TNF related apoptosis inducing ligands (TRAIL) but do not contain the cytoplasmic domains necessary to transduce apoptotic signals. We hypothesized that decoy receptors may confer neuronal protection against lethal ischemia after ischemic preconditioning (IPC). Mixed cortical neurons were exposed to IPC one day prior to TRAIL treatment or lethal ischemia. IPC increased decoy receptor but reduced death receptor expression compared to lethal ischemia. IPC-induced increase in decoy receptor expression was reduced by prior treatment with CAPE, a nuclear factor-kappa B inhibitor (NFκB). Expression of decoy molecules, dependent on NFκB, may mediate neuronal survival induced by IPC. PMID:21315738

Prediction of consensus binding mode geometries for related chemical series of positive allosteric modulators of adenosine and muscarinic acetylcholine receptors.

PubMed

Sakkal, Leon A; Rajkowski, Kyle Z; Armen, Roger S

2017-06-05

Following insights from recent crystal structures of the muscarinic acetylcholine receptor, binding modes of Positive Allosteric Modulators (PAMs) were predicted under the assumption that PAMs should bind to the extracellular surface of the active state. A series of well-characterized PAMs for adenosine (A 1 R, A 2A R, A 3 R) and muscarinic acetylcholine (M 1 R, M 5 R) receptors were modeled using both rigid and flexible receptor CHARMM-based molecular docking. Studies of adenosine receptors investigated the molecular basis of the probe-dependence of PAM activity by modeling in complex with specific agonist radioligands. Consensus binding modes map common pharmacophore features of several chemical series to specific binding interactions. These models provide a rationalization of how PAM binding slows agonist radioligand dissociation kinetics. M 1 R PAMs were predicted to bind in the analogous M 2 R PAM LY2119620 binding site. The M 5 R NAM (ML-375) was predicted to bind in the PAM (ML-380) binding site with a unique induced-fit receptor conformation. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Pharmacological activities of Vitex agnus-castus extracts in vitro.

PubMed

Meier, B; Berger, D; Hoberg, E; Sticher, O; Schaffner, W

2000-10-01

The pharmacological effects of ethanolic Vitex agnus-castus fruit-extracts (especially Ze 440) and various extract fractions of different polarities were evaluated both by radioligand binding studies and by superfusion experiments. A relative potent binding inhibition was observed for dopamine D2 and opioid (micro and kappa subtype) receptors with IC50 values of the native extract between 20 and 70 mg/mL. Binding, neither to the histamine H1, benzodiazepine and OFQ receptor, nor to the binding-site of the serotonin (5-HT) transporter, was significantly inhibited. The lipophilic fractions contained the diterpenes rotun-difuran and 6beta,7beta-diacetoxy-13-hydroxy-labda-8,14-dien . They exhibited inhibitory actions on dopamine D2 receptor binding. While binding inhibition to mu and kappa opioid receptors was most pronounced in lipophilic fractions, binding to delta opioid receptors was inhibited mainly by a aqueous fraction. Standardised Ze 440 extracts of different batches were of constant pharmacological quality according to their potential to inhibit the binding to D2 receptors. In superfusion experiments, the aqueous fraction of a methanolic extract inhibited the release of acetylcholine in a concentration-dependent manner. In addition, the potent D2 receptor antagonist spiperone antagonised the effect of the extract suggesting a dopaminergic action mediated by D2 receptor activation. Our results indicate a dopaminergic effect of Vitex agnus-castus extracts and suggest additional pharmacological actions via opioid receptors.

Adrenergic Agonists Bind to Adrenergic-Receptor-Like Regions of the Mu Opioid Receptor, Enhancing Morphine and Methionine-Enkephalin Binding: A New Approach to “Biased Opioids”?

PubMed Central

Turke, Miah; Subhramanyam, Udaya K. Tiruttani; Churchill, Beth; Labahn, Joerg

2018-01-01

Extensive evidence demonstrates functional interactions between the adrenergic and opioid systems in a diversity of tissues and organs. While some effects are due to receptor and second messenger cross-talk, recent research has revealed an extracellular, allosteric opioid binding site on adrenergic receptors that enhances adrenergic activity and its duration. The present research addresses whether opioid receptors may have an equivalent extracellular, allosteric adrenergic binding site that has similar enhancing effects on opioid binding. Comparison of adrenergic and opioid receptor sequences revealed that these receptors share very significant regions of similarity, particularly in some of the extracellular and transmembrane regions associated with adrenergic binding in the adrenergic receptors. Five of these shared regions from the mu opioid receptor (muOPR) were synthesized as peptides and tested for binding to adrenergic, opioid and control compounds using ultraviolet spectroscopy. Adrenergic compounds bound to several of these muOPR peptides with low micromolar affinity while acetylcholine, histamine and various adrenergic antagonists did not. Similar studies were then conducted with purified, intact muOPR with similar results. Combinations of epinephrine with methionine enkephalin or morphine increased the binding of both by about half a log unit. These results suggest that muOPR may be allosterically enhanced by adrenergic agonists. PMID:29342106

von Willebrand Factor Test

MedlinePlus

... Cancer Therapy Glucose Tests Gonorrhea Testing Gram Stain Growth Hormone Haptoglobin hCG Pregnancy hCG Tumor Marker HDL Cholesterol ... Semen Analysis Serotonin Serum Free Light Chains Sex Hormone Binding Globulin ... Transferrin Receptor Stool Culture Stool Elastase Strep ...

A20-binding inhibitor of NF-κB (ABIN1) controls Toll-like receptor-mediated CCAAT/enhancer-binding protein β activation and protects from inflammatory disease.

PubMed

Zhou, Jingran; Wu, Ruiqiong; High, Anthony A; Slaughter, Clive A; Finkelstein, David; Rehg, Jerold E; Redecke, Vanessa; Häcker, Hans

2011-11-01

Toll-like receptors (TLRs) are expressed on innate immune cells and trigger inflammation upon detection of pathogens and host tissue injury. TLR-mediated proinflammatory-signaling pathways are counteracted by partially characterized anti-inflammatory mechanisms that prevent exaggerated inflammation and host tissue damage as manifested in inflammatory diseases. We biochemically identified a component of TLR-signaling pathways, A20-binding inhibitor of NF-κB (ABIN1), which recently has been linked by genome-wide association studies to the inflammatory diseases systemic lupus erythematosus and psoriasis. We generated ABIN1-deficient mice to study the function of ABIN1 in vivo and during TLR activation. Here we show that ABIN1-deficient mice develop a progressive, lupus-like inflammatory disease characterized by expansion of myeloid cells, leukocyte infiltrations in different parenchymatous organs, activated T and B lymphocytes, elevated serum Ig levels, and the appearance of autoreactive antibodies. Kidneys develop glomerulonephritis and proteinuria, reflecting tissue injury. Surprisingly, ABIN1-deficient macrophages exhibit normal regulation of major proinflammatory signaling pathways and mediators but show selective deregulation of the transcription factor CCAAT/enhancer binding protein β (C/EBPβ) and its target genes, such as colony-stimulating factor 3 (Csf3), nitric oxide synthase, inducible (Nos2), and S100 calcium-binding protein A8 (S100a8). Their gene products, which are intimately linked to innate immune cell expansion (granulocyte colony-stimulating factor), cytotoxicity (inducible nitric oxide synthase), and host factor-derived inflammation (S100A8), may explain, at least in part, the inflammatory phenotype observed. Together, our data reveal ABIN1 as an essential anti-inflammatory component of TLR-signaling pathways that controls C/EBPβ activity.

Target gene analysis by microarrays and chromatin immunoprecipitation identifies HEY proteins as highly redundant bHLH repressors.

PubMed

Heisig, Julia; Weber, David; Englberger, Eva; Winkler, Anja; Kneitz, Susanne; Sung, Wing-Kin; Wolf, Elmar; Eilers, Martin; Wei, Chia-Lin; Gessler, Manfred

2012-01-01

HEY bHLH transcription factors have been shown to regulate multiple key steps in cardiovascular development. They can be induced by activated NOTCH receptors, but other upstream stimuli mediated by TGFß and BMP receptors may elicit a similar response. While the basic and helix-loop-helix domains exhibit strong similarity, large parts of the proteins are still unique and may serve divergent functions. The striking overlap of cardiac defects in HEY2 and combined HEY1/HEYL knockout mice suggested that all three HEY genes fulfill overlapping function in target cells. We therefore sought to identify target genes for HEY proteins by microarray expression and ChIPseq analyses in HEK293 cells, cardiomyocytes, and murine hearts. HEY proteins were found to modulate expression of their target gene to a rather limited extent, but with striking functional interchangeability between HEY factors. Chromatin immunoprecipitation revealed a much greater number of potential binding sites that again largely overlap between HEY factors. Binding sites are clustered in the proximal promoter region especially of transcriptional regulators or developmental control genes. Multiple lines of evidence suggest that HEY proteins primarily act as direct transcriptional repressors, while gene activation seems to be due to secondary or indirect effects. Mutagenesis of putative DNA binding residues supports the notion of direct DNA binding. While class B E-box sequences (CACGYG) clearly represent preferred target sequences, there must be additional and more loosely defined modes of DNA binding since many of the target promoters that are efficiently bound by HEY proteins do not contain an E-box motif. These data clearly establish the three HEY bHLH factors as highly redundant transcriptional repressors in vitro and in vivo, which explains the combinatorial action observed in different tissues with overlapping expression.

Target Gene Analysis by Microarrays and Chromatin Immunoprecipitation Identifies HEY Proteins as Highly Redundant bHLH Repressors

PubMed Central

Englberger, Eva; Winkler, Anja; Kneitz, Susanne; Sung, Wing-Kin; Wolf, Elmar; Eilers, Martin; Wei, Chia-Lin; Gessler, Manfred

2012-01-01

HEY bHLH transcription factors have been shown to regulate multiple key steps in cardiovascular development. They can be induced by activated NOTCH receptors, but other upstream stimuli mediated by TGFß and BMP receptors may elicit a similar response. While the basic and helix-loop-helix domains exhibit strong similarity, large parts of the proteins are still unique and may serve divergent functions. The striking overlap of cardiac defects in HEY2 and combined HEY1/HEYL knockout mice suggested that all three HEY genes fulfill overlapping function in target cells. We therefore sought to identify target genes for HEY proteins by microarray expression and ChIPseq analyses in HEK293 cells, cardiomyocytes, and murine hearts. HEY proteins were found to modulate expression of their target gene to a rather limited extent, but with striking functional interchangeability between HEY factors. Chromatin immunoprecipitation revealed a much greater number of potential binding sites that again largely overlap between HEY factors. Binding sites are clustered in the proximal promoter region especially of transcriptional regulators or developmental control genes. Multiple lines of evidence suggest that HEY proteins primarily act as direct transcriptional repressors, while gene activation seems to be due to secondary or indirect effects. Mutagenesis of putative DNA binding residues supports the notion of direct DNA binding. While class B E-box sequences (CACGYG) clearly represent preferred target sequences, there must be additional and more loosely defined modes of DNA binding since many of the target promoters that are efficiently bound by HEY proteins do not contain an E-box motif. These data clearly establish the three HEY bHLH factors as highly redundant transcriptional repressors in vitro and in vivo, which explains the combinatorial action observed in different tissues with overlapping expression. PMID:22615585

The cholesterol-dependent cytolysins pneumolysin and streptolysin O require binding to red blood cell glycans for hemolytic activity

PubMed Central

Shewell, Lucy K.; Harvey, Richard M.; Higgins, Melanie A.; Day, Christopher J.; Hartley-Tassell, Lauren E.; Chen, Austen Y.; Gillen, Christine M.; James, David B. A.; Alonzo, Francis; Torres, Victor J.; Walker, Mark J.; Paton, Adrienne W.; Paton, James C.; Jennings, Michael P.

2014-01-01

The cholesterol-dependent cytolysin (CDC) pneumolysin (Ply) is a key virulence factor of Streptococcus pneumoniae. Membrane cholesterol is required for the cytolytic activity of this toxin, but it is not clear whether cholesterol is the only cellular receptor. Analysis of Ply binding to a glycan microarray revealed that Ply has lectin activity and binds glycans, including the Lewis histo-blood group antigens. Surface plasmon resonance analysis showed that Ply has the highest affinity for the sialyl LewisX (sLeX) structure, with a Kd of 1.88 × 10−5 M. Ply hemolytic activity against human RBCs showed dose-dependent inhibition by sLeX. Flow cytometric analysis and Western blots showed that blocking binding of Ply to the sLeX glycolipid on RBCs prevents deposition of the toxin in the membrane. The lectin domain responsible for sLeX binding is in domain 4 of Ply, which contains candidate carbohydrate-binding sites. Mutagenesis of these predicted carbohydrate-binding residues of Ply resulted in a decrease in hemolytic activity and a reduced affinity for sLeX. This study reveals that this archetypal CDC requires interaction with the sLeX glycolipid cellular receptor as an essential step before membrane insertion. A similar analysis conducted on streptolysin O from Streptococcus pyogenes revealed that this CDC also has glycan-binding properties and that hemolytic activity against RBCs can be blocked with the glycan lacto-N-neotetraose by inhibiting binding to the cell surface. Together, these data support the emerging paradigm shift that pore-forming toxins, including CDCs, have cellular receptors other than cholesterol that define target cell tropism. PMID:25422425

The cholesterol-dependent cytolysins pneumolysin and streptolysin O require binding to red blood cell glycans for hemolytic activity.

PubMed

Shewell, Lucy K; Harvey, Richard M; Higgins, Melanie A; Day, Christopher J; Hartley-Tassell, Lauren E; Chen, Austen Y; Gillen, Christine M; James, David B A; Alonzo, Francis; Torres, Victor J; Walker, Mark J; Paton, Adrienne W; Paton, James C; Jennings, Michael P

2014-12-09

The cholesterol-dependent cytolysin (CDC) pneumolysin (Ply) is a key virulence factor of Streptococcus pneumoniae. Membrane cholesterol is required for the cytolytic activity of this toxin, but it is not clear whether cholesterol is the only cellular receptor. Analysis of Ply binding to a glycan microarray revealed that Ply has lectin activity and binds glycans, including the Lewis histo-blood group antigens. Surface plasmon resonance analysis showed that Ply has the highest affinity for the sialyl LewisX (sLeX) structure, with a K(d) of 1.88 × 10(-5) M. Ply hemolytic activity against human RBCs showed dose-dependent inhibition by sLeX. Flow cytometric analysis and Western blots showed that blocking binding of Ply to the sLeX glycolipid on RBCs prevents deposition of the toxin in the membrane. The lectin domain responsible for sLeX binding is in domain 4 of Ply, which contains candidate carbohydrate-binding sites. Mutagenesis of these predicted carbohydrate-binding residues of Ply resulted in a decrease in hemolytic activity and a reduced affinity for sLeX. This study reveals that this archetypal CDC requires interaction with the sLeX glycolipid cellular receptor as an essential step before membrane insertion. A similar analysis conducted on streptolysin O from Streptococcus pyogenes revealed that this CDC also has glycan-binding properties and that hemolytic activity against RBCs can be blocked with the glycan lacto-N-neotetraose by inhibiting binding to the cell surface. Together, these data support the emerging paradigm shift that pore-forming toxins, including CDCs, have cellular receptors other than cholesterol that define target cell tropism.

Understanding Cytokine and Growth Factor Receptor Activation Mechanisms

PubMed Central

Atanasova, Mariya; Whitty, Adrian

2012-01-01

Our understanding of the detailed mechanism of action of cytokine and growth factor receptors – and particularly our quantitative understanding of the link between structure, mechanism and function – lags significantly behind our knowledge of comparable functional protein classes such as enzymes, G protein-coupled receptors, and ion channels. In particular, it remains controversial whether such receptors are activated by a mechanism of ligand-induced oligomerization, versus a mechanism in which the ligand binds to a pre-associated receptor dimer or oligomer that becomes activated through subsequent conformational rearrangement. A major limitation to progress has been the relative paucity of methods for performing quantitative mechanistic experiments on unmodified receptors expressed at endogenous levels on live cells. In this article we review the current state of knowledge on the activation mechanisms of cytokine and growth factor receptors, critically evaluate the evidence for and against the different proposed mechanisms, and highlight other key questions that remain unanswered. New approaches and techniques have led to rapid recent progress in this area, and the field is poised for major advances in the coming years, which promises to revolutionize our understanding of this large and biologically and medically important class of receptors. PMID:23046381

A glioma-derived analog to platelet-derived growth factor: demonstration of receptor competing activity and immunological crossreactivity.

PubMed Central

Nistér, M; Heldin, C H; Wasteson, A; Westermark, B

1984-01-01

A human clonal glioma cell line, U-343 MGa Cl 2, cultured under serum-free conditions, was found to release a factor that competed with 125I-labeled platelet-derived growth factor (125I-PDGF) for binding to human foreskin fibroblasts. The concentration of competing activity in conditioned medium was equal to 20-30 ng of PDGF per ml. The PDGF receptor competing activity had an elution position on Sephadex G-200 close to that of tracer PDGF. The same fractions in the chromatogram also contained growth-promoting activity and material active in a PDGF radioimmunoassay. Incubation of partially purified, 125I-labeled glioma factor with fibroblasts, or rabbit anti-PDGF serum, led to the selective binding of a component with an estimated Mr of 31,000, as shown by NaDodSO4/gel electrophoresis under nonreducing conditions. After reduction this component migrated as a Mr 18,000 protein. Thus, the behavior in NaDodSO4/gel electrophoresis was similar to that of PDGF. Furthermore, incubation of partially purified glioma factor with immobilized PDGF antibodies markedly decreased the amount of PDGF receptor competing activity remaining in the supernatant. These results suggest that the factor produced by glioma cells has structural, immunological, and functional resemblance to PDGF. We previously reported that a human osteosarcoma cell line produces a PDGF-like molecule with growth-promoting activity. Taken together with the recent finding that PDGF is homologous to the transforming gene product of simian sarcoma virus, our present data give additional support for the idea that an autocrine activation of the PDGF receptor may be operational in the growth of human tumors of mesenchymal or glial origin. Images PMID:6322178

Analysis of ligand-receptor cross-linked fragments by mass spectrometry

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

Son, C.D.; Sargsyan, H.; Hurst, Gregory

G-protein coupled receptors (GPCRs) are a class of integral membrane receptor proteins that are characterized by a signature seven-transmembrane (7-TM) configuration. The a-factor receptor (Ste2p) from Saccharomyces cerevisiae is a GPCR that, upon binding of a peptide ligand, transduces a signal to initiate a cascade of events leading to the mating of haploid yeast cells. This study summarizes the application of affinity purification and of matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) experiments using biotinylated photoactivatable a-factor analogs. Affinity purification and enrichment of biotinylated peptides by monomeric avidin beads resulted in mass spectrometric detection of specific signals corresponding to crosslinked fragments ofmore » Ste2p. Data obtained from cyanogen bromide (CNBr) fragments of receptor cross-linked to an a-factor analog with the photoaffinity group p-benzoyl-L-phenylalanine on position 1 were in agreement with the previous results reported by our laboratory suggesting the cross-linking between position 1 of a-factor and a region of Ste2p covering residues 251 294.« less

Muscarinic Receptor Binding in Rat Bladder Urothelium and Detrusor Muscle by Intravesical Solifenacin.

PubMed

Ito, Yoshihiko; Kashiwabara, Michishi; Yoshida, Akira; Hikiyama, Eriko; Onoue, Satomi; Yamada, Shizuo

2016-01-01

Solifenacin is an antimuscarinic agent used to treat symptoms of overactive bladder. Pharmacologically significant amounts of solifenacin were excreted in the urine of humans taking a clinical dose of this drug. The aim of this study is to measure muscarinic receptor binding in the bladder urothelium and detrusor muscles of rats following the intravesical instillation of solifenacin. Muscarinic receptors were measured by radioreceptor assay using [N-methyl-(3)H]scopolamine methyl chloride ([(3)H]NMS), a selective radioligand of muscarinic receptors. Solifenacin showed concentration-dependent inhibition of specific [(3)H]NMS binding in the bladder urothelium and detrusor muscle of rats, with no significant difference in Ki values or Hill coefficients between these tissues. Following the intravesical instillation of solifenacin, there was significant muscarinic receptor binding (increase in Kd for specific [(3)H]NMS binding) in the bladder urothelium and detrusor muscle of rats. Similar bladder muscarinic receptor binding was observed by the intravesical instillation of oxybutynin, but not with trospium. In conclusion, the present study has demonstrated that solifenacin binds muscarinic receptors not only in the detrusor muscle but also in the bladder urothelium with high affinity. These bladder muscarinic receptors may be significantly affected by solifenacin excreted in the urine.

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.

Transcriptional Network Analysis in Muscle Reveals AP-1 as a Partner of PGC-1α in the Regulation of the Hypoxic Gene Program

PubMed Central

Baresic, Mario; Salatino, Silvia; Kupr, Barbara

2014-01-01

Skeletal muscle tissue shows an extraordinary cellular plasticity, but the underlying molecular mechanisms are still poorly understood. Here, we use a combination of experimental and computational approaches to unravel the complex transcriptional network of muscle cell plasticity centered on the peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a regulatory nexus in endurance training adaptation. By integrating data on genome-wide binding of PGC-1α and gene expression upon PGC-1α overexpression with comprehensive computational prediction of transcription factor binding sites (TFBSs), we uncover a hitherto-underestimated number of transcription factor partners involved in mediating PGC-1α action. In particular, principal component analysis of TFBSs at PGC-1α binding regions predicts that, besides the well-known role of the estrogen-related receptor α (ERRα), the activator protein 1 complex (AP-1) plays a major role in regulating the PGC-1α-controlled gene program of the hypoxia response. Our findings thus reveal the complex transcriptional network of muscle cell plasticity controlled by PGC-1α. PMID:24912679

Luminal expression of cubilin is impaired in Imerslund-Gräsbeck syndrome with compound AMN mutations in intron 3 and exon 7

PubMed Central

Namour, Fares; Dobrovoljski, Gabriele; Chery, Celine; Audonnet, Sandra; Feillet, François; Sperl, Wolfgang; Gueant, Jean-Louis

2011-01-01

Juvenile megaloblastic anaemia 1 (OMIM # 261100) is a rare autosomic disorder characterized by selective cobalamin mal-absorption and inconstant proteinuria produced by mutations in either CUBN or AMN genes. Amnionless, the gene product of AMN, is a transmembrane protein that binds tightly to the N-terminal end of cubilin, the gene product of CUBN. Cubilin binds to intrinsic factor-cobalamin complex and is expressed in the distal intestine and the proximal renal tubule. We report a compound AMN heterozygosity with c.742C>T, p.Gln248X and c.208-2A>G mutations in 2 siblings that led to premature termination codon in exon 7 and exon 6, respectively. It produced a dramatic decrease in receptor activity in urine, despite absence of CUBN mutation and normal affinity of the receptor for intrinsic factor binding. Heterozygous carriers for c.742T and c.208-2G had no pathological signs. These results indicate that amnionless is essential for the correct luminal expression of cubilin in humans. PMID:21750092

Ondansetron and granisetron binding orientation in the 5-HT(3) receptor determined by unnatural amino acid mutagenesis.

PubMed

Duffy, Noah H; Lester, Henry A; Dougherty, Dennis A

2012-10-19

The serotonin type 3 receptor (5-HT(3)R) is a ligand-gated ion channel found in the central and peripheral nervous systems. The 5-HT(3)R is a therapeutic target, and the clinically available drugs ondansetron and granisetron inhibit receptor activity. Their inhibitory action is through competitive binding to the native ligand binding site, although the binding orientation of the drugs at the receptor has been a matter of debate. Here we heterologously express mouse 5-HT(3)A receptors in Xenopus oocytes and use unnatural amino acid mutagenesis to establish a cation-π interaction for both ondansetron and granisetron to tryptophan 183 in the ligand binding pocket. This cation-π interaction establishes a binding orientation for both ondansetron and granisetron within the binding pocket.

Ondansetron and Granisetron Binding Orientation in the 5-HT3 Receptor Determined by Unnatural Amino Acid Mutagenesis

PubMed Central

Duffy, Noah H.; Lester, Henry A.; Dougherty, Dennis A.

2012-01-01

The serotonin type 3 receptor (5-HT3R) is a ligand-gated ion channel that mediates fast synaptic transmission in the central and peripheral nervous systems. The 5-HT3R is a therapeutic target, and the clinically available drugs ondansetron and granisetron inhibit receptor activity. Their inhibitory action is through competitive binding to the native ligand binding site, although the binding orientation of the drugs at the receptor has been a matter of debate. Here we heterologously express mouse 5-HT3A receptors in Xenopus oocytes and use unnatural amino acid mutagenesis to establish a cation-π interaction for both ondansetron and granisetron to tryptophan 183 in the ligand binding pocket. This cation-π interaction establishes a binding orientation for both ondansetron and granisetron within the binding pocket. PMID:22873819

Vitamin D receptor signaling and its therapeutic implications: Genome-wide and structural view.

PubMed

Carlberg, Carsten; Molnár, Ferdinand

2015-05-01

Vitamin D3 is one of the few natural compounds that has, via its metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) and the transcription factor vitamin D receptor (VDR), a direct effect on gene regulation. For efficiently applying the therapeutic and disease-preventing potential of 1,25(OH)2D3 and its synthetic analogs, the key steps in vitamin D signaling need to be understood. These are the different types of molecular interactions with the VDR, such as (i) the complex formation of VDR with genomic DNA, (ii) the interaction of VDR with its partner transcription factors, (iii) the binding of 1,25(OH)2D3 or its synthetic analogs within the ligand-binding pocket of the VDR, and (iv) the resulting conformational change on the surface of the VDR leading to a change of the protein-protein interaction profile of the receptor with other proteins. This review will present the latest genome-wide insight into vitamin D signaling, and will discuss its therapeutic implications.

How glucocorticoid receptors modulate the activity of other transcription factors: a scope beyond tethering.

PubMed

Ratman, Dariusz; Vanden Berghe, Wim; Dejager, Lien; Libert, Claude; Tavernier, Jan; Beck, Ilse M; De Bosscher, Karolien

2013-11-05

The activity of the glucocorticoid receptor (GR), a nuclear receptor transcription factor belonging to subclass 3C of the steroid/thyroid hormone receptor superfamily, is typically triggered by glucocorticoid hormones. Apart from driving gene transcription via binding onto glucocorticoid response elements in regulatory regions of particular target genes, GR can also inhibit gene expression via transrepression, a mechanism largely based on protein:protein interactions. Hereby GR can influence the activity of other transcription factors, without contacting DNA itself. GR is known to inhibit the activity of a growing list of immune-regulating transcription factors. Hence, GCs still rule the clinic for treatments of inflammatory disorders, notwithstanding concomitant deleterious side effects. Although patience is a virtue when it comes to deciphering the many mechanisms GR uses to influence various signaling pathways, the current review is testimony of the fact that groundbreaking mechanistic work has been accumulating over the past years and steadily continues to grow. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Fibroblast growth factor receptor inhibitors.

PubMed

Kumar, Suneel B V S; Narasu, Lakshmi; Gundla, Rambabu; Dayam, Raveendra; J A R P, Sarma

2013-01-01

Fibroblast growth factor receptors (FGFRs) play an important role in embryonic development, angiogenesis, wound healing, cell proliferation and differentiation. The fibroblast growth factor receptor (FGFR) isoforms have been under intense scrutiny for effective anticancer drug candidates. The fibroblast growth factor (FGF) and its receptor (FGFR) provide another pathway that seems critical to monitoring angiogenesis. Recent findings suggest that FGFR mediates signaling, regulates the PKM2 activity, and plays a crucial role in cancer metabolism. The current review also covers the recent findings on the role of FGFR1 in cancer metabolism. This paper reviews the progress, mechanism, and binding modes of recently known kinase inhibitors such as PD173074, SU series and other inhibitors still under clinical development. Some of the structural classes that will be highlighted in this review include Pyrido[2,3-d]pyrimidines, Indolin- 2-one, Pyrrolo[2,1-f][1,2,4]triazine, Pyrido[2,3-d]pyrimidin-7(8H)-one, and 1,6- Naphthyridin-2(1H)-ones.

Identification of a nonsense mutation in the granulocyte-colony-stimulating factor receptor in severe congenital neutropenia

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

Dong, F.; Loewenberg, B.; Hoefsloot, L.H.

Severe congenital neutropenia (Kostmann syndrome) is characterized by profound absolute neutropenia and a maturation arrest of marrow progenitor cells at the promyelocyte-myelocyte stage. Marrow cells from such patients frequently display a reduced responsiveness to granulocyte-colony-stimulating factor (G-CSF). G-CSF binds to and activates a specific receptor which transduces signals critical for the proliferation and maturation of granulocytic progenitor cells. Here the authors report the identification of a somatic point mutation in one allele of the G-CSF receptor gene in a patient with severe congenital neutropenia. The mutation results in a cytoplasmic truncation of the receptor. When expressed in murine myeloid cells,more » the mutant receptor transduced a strong growth signal but, in contrast to the wild-type G-CSF receptor, was defective in maturation induction. This mutant receptor chain may act in a dominant negative manner to block granulocytic maturation. 40 refs., figs., 2 tabs.« less

Elimination of a ligand gating site generates a supersensitive olfactory receptor.

PubMed

Sharma, Kanika; Ahuja, Gaurav; Hussain, Ashiq; Balfanz, Sabine; Baumann, Arnd; Korsching, Sigrun I

2016-06-21

Olfaction poses one of the most complex ligand-receptor matching problems in biology due to the unparalleled multitude of odor molecules facing a large number of cognate olfactory receptors. We have recently deorphanized an olfactory receptor, TAAR13c, as a specific receptor for the death-associated odor cadaverine. Here we have modeled the cadaverine/TAAR13c interaction, exchanged predicted binding residues by site-directed mutagenesis, and measured the activity of the mutant receptors. Unexpectedly we observed a binding site for cadaverine at the external surface of the receptor, in addition to an internal binding site, whose mutation resulted in complete loss of activity. In stark contrast, elimination of the external binding site generated supersensitive receptors. Modeling suggests this site to act as a gate, limiting access of the ligand to the internal binding site and thereby downregulating the affinity of the native receptor. This constitutes a novel mechanism to fine-tune physiological sensitivity to socially relevant odors.

Elimination of a ligand gating site generates a supersensitive olfactory receptor

PubMed Central

Sharma, Kanika; Ahuja, Gaurav; Hussain, Ashiq; Balfanz, Sabine; Baumann, Arnd; Korsching, Sigrun I.

2016-01-01

Olfaction poses one of the most complex ligand-receptor matching problems in biology due to the unparalleled multitude of odor molecules facing a large number of cognate olfactory receptors. We have recently deorphanized an olfactory receptor, TAAR13c, as a specific receptor for the death-associated odor cadaverine. Here we have modeled the cadaverine/TAAR13c interaction, exchanged predicted binding residues by site-directed mutagenesis, and measured the activity of the mutant receptors. Unexpectedly we observed a binding site for cadaverine at the external surface of the receptor, in addition to an internal binding site, whose mutation resulted in complete loss of activity. In stark contrast, elimination of the external binding site generated supersensitive receptors. Modeling suggests this site to act as a gate, limiting access of the ligand to the internal binding site and thereby downregulating the affinity of the native receptor. This constitutes a novel mechanism to fine-tune physiological sensitivity to socially relevant odors. PMID:27323929

Structure of colicin I receptor bound to the R-domain of colicin Ia: implications for protein import

PubMed Central

Buchanan, Susan K; Lukacik, Petra; Grizot, Sylvestre; Ghirlando, Rodolfo; Ali, Maruf M U; Barnard, Travis J; Jakes, Karen S; Kienker, Paul K; Esser, Lothar

2007-01-01

Colicin Ia is a 69 kDa protein that kills susceptible Escherichia coli cells by binding to a specific receptor in the outer membrane, colicin I receptor (70 kDa), and subsequently translocating its channel forming domain across the periplasmic space, where it inserts into the inner membrane and forms a voltage-dependent ion channel. We determined crystal structures of colicin I receptor alone and in complex with the receptor binding domain of colicin Ia. The receptor undergoes large and unusual conformational changes upon colicin binding, opening at the cell surface and positioning the receptor binding domain of colicin Ia directly above it. We modelled the interaction with full-length colicin Ia to show that the channel forming domain is initially positioned 150 Å above the cell surface. Functional data using full-length colicin Ia show that colicin I receptor is necessary for cell surface binding, and suggest that the receptor participates in translocation of colicin Ia across the outer membrane. PMID:17464289

Preclinical pharmacology of bilastine, a new selective histamine H1 receptor antagonist: receptor selectivity and in vitro antihistaminic activity.

PubMed

Corcóstegui, Reyes; Labeaga, Luis; Innerárity, Ana; Berisa, Agustin; Orjales, Aurelio

2005-01-01

This study aimed to establish the receptor selectivity and antihistaminic activity of bilastine, a new selective antihistamine receptor antagonist. In vitro experiments were conducted using a receptor binding screening panel and guinea-pig and rat tissues. Antihistaminic activity was determined using H1 receptor binding studies and in vitro H1 antagonism studies conducted in guinea-pig tissues and human cell lines. Receptor selectivity was established using a receptor binding screening panel and a receptor antagonism screening conducted in guinea-pig, rat and rabbit tissues. Inhibition of inflammatory mediators was determined through the Schultz-Dale reaction in sensitised guinea-pig ileum. Bilastine binds to histamine H1-receptors as indicated by its displacement of [3H]-pyrilamine from H1-receptors expressed in guinea-pig cerebellum and human embryonic kidney (HEK) cell lines. The studies conducted on guinea-pig smooth muscle demonstrated the capability of bilastine to antagonise H1-receptors. Bilastine is selective for histamine H1-receptors as shown in receptor-binding screening conducted to determine the binding capacity of bilastine to 30 different receptors. The specificity of its H1-receptor antagonistic activity was also demonstrated in a series of in vitro experiments conducted on guinea-pig and rat tissues. The results of these studies confirmed the lack of significant antagonism against serotonin, bradykinin, leukotriene D4, calcium, muscarinic M3-receptors, alpha1-adrenoceptors, beta2-adrenoceptors, and H2- and H3-receptors. The results of the in vitro Schultz-Dale reaction demonstrated that bilastine also has anti-inflammatory activity. These preclinical studies provide evidence that bilastine has H1- antihistamine activity, with high specificity for H1-receptors, and poor or no affinity for other receptors. Bilastine has also been shown to have anti-inflammatory properties.

Combined sodium ion sensitivity in agonist binding and internalization of vasopressin V1b receptors.

PubMed

Koshimizu, Taka-Aki; Kashiwazaki, Aki; Taniguchi, Junichi

2016-05-03

Reducing Na(+) in the extracellular environment may lead to two beneficial effects for increasing agonist binding to cell surface G-protein coupled receptors (GPCRs): reduction of Na(+)-mediated binding block and reduce of receptor internalization. However, such combined effects have not been explored. We used Chinese Hamster Ovary cells expressing vasopressin V1b receptors as a model to explore Na(+) sensitivity in agonist binding and receptor internalization. Under basal conditions, a large fraction of V1b receptors is located intracellularly, and a small fraction is in the plasma membrane. Decreases in external Na(+) increased cell surface [(3)H]AVP binding and decreased receptor internalization. Substitution of Na(+) by Cs(+) or NH4(+) inhibited agonist binding. To suppress receptor internalization, the concentration of NaCl, but not of CsCl, had to be less than 50 mM, due to the high sensitivity of the internalization machinery to Na(+) over Cs(+). Iso-osmotic supplementation of glucose or NH4Cl maintained internalization of the V1b receptor, even in a low-NaCl environment. Moreover, iodide ions, which acted as a counter anion, inhibited V1b agonist binding. In summary, we found external ionic conditions that could increase the presence of high-affinity state receptors at the cell surface with minimum internalization during agonist stimulations.

Combined sodium ion sensitivity in agonist binding and internalization of vasopressin V1b receptors

PubMed Central

Koshimizu, Taka-aki; Kashiwazaki, Aki; Taniguchi, Junichi

2016-01-01

Reducing Na+ in the extracellular environment may lead to two beneficial effects for increasing agonist binding to cell surface G-protein coupled receptors (GPCRs): reduction of Na+-mediated binding block and reduce of receptor internalization. However, such combined effects have not been explored. We used Chinese Hamster Ovary cells expressing vasopressin V1b receptors as a model to explore Na+ sensitivity in agonist binding and receptor internalization. Under basal conditions, a large fraction of V1b receptors is located intracellularly, and a small fraction is in the plasma membrane. Decreases in external Na+ increased cell surface [3H]AVP binding and decreased receptor internalization. Substitution of Na+ by Cs+ or NH4+ inhibited agonist binding. To suppress receptor internalization, the concentration of NaCl, but not of CsCl, had to be less than 50 mM, due to the high sensitivity of the internalization machinery to Na+ over Cs+. Iso-osmotic supplementation of glucose or NH4Cl maintained internalization of the V1b receptor, even in a low-NaCl environment. Moreover, iodide ions, which acted as a counter anion, inhibited V1b agonist binding. In summary, we found external ionic conditions that could increase the presence of high-affinity state receptors at the cell surface with minimum internalization during agonist stimulations. PMID:27138239

Alternative dimerization interfaces in the glucocorticoid receptor-α ligand binding domain.

PubMed

Bianchetti, Laurent; Wassmer, Bianca; Defosset, Audrey; Smertina, Anna; Tiberti, Marion L; Stote, Roland H; Dejaegere, Annick

2018-04-30

Nuclear hormone receptors (NRs) constitute a large family of multi-domain ligand-activated transcription factors. Dimerization is essential for their regulation, and both DNA binding domain (DBD) and ligand binding domain (LBD) are implicated in dimerization. Intriguingly, the glucocorticoid receptor-α (GRα) presents a DBD dimeric architecture similar to that of the homologous estrogen receptor-α (ERα), but an atypical dimeric architecture for the LBD. The physiological relevance of the proposed GRα LBD dimer is a subject of debate. We analyzed all GRα LBD homodimers observed in crystals using an energetic analysis based on the PISA and on the MM/PBSA methods and a sequence conservation analysis, using the ERα LBD dimer as a reference point. Several dimeric assemblies were observed for GRα LBD. The assembly generally taken to be physiologically relevant showed weak binding free energy and no significant residue conservation at the contact interface, while an alternative homodimer mediated by both helix 9 and C-terminal residues showed significant binding free energy and residue conservation. However, none of the GRα LBD assemblies found in crystals are as stable or conserved as the canonical ERα LBD dimer. GRα C-terminal sequence (F-domain) forms a steric obstacle to the canonical dimer assembly in all available structures. Our analysis calls for a re-examination of the currently accepted GRα homodimer structure and experimental investigations of the alternative architectures. This work questions the validity of the currently accepted architecture. This has implications for interpreting physiological data and for therapeutic design pertaining to glucocorticoid research. Copyright © 2018. Published by Elsevier B.V.

Positive allosteric modulators as an approach to nicotinic acetylcholine receptor- targeted therapeutics: advantages and limitations

PubMed Central

Williams, Dustin K.; Wang, Jingyi; Papke, Roger L.

2011-01-01

Neuronal nicotinic acetylcholine receptors (nAChR), recognized targets for drug development in cognitive and neuro-degenerative disorders, are allosteric proteins with dynamic interconversions between multiple functional states. Activation of the nAChR ion channel is primarily controlled by the binding of ligands (agonists, partial agonists, competitive antagonists) at conventional agonist binding sites, but is also regulated in either negative or positive ways by the binding of ligands to other modulatory sites. In this review, we discuss models for the activation and desensitization of nAChR, and the discovery of multiple types of ligands that influence those processes in both heteromeric nAChR, such as the high affinity nicotine receptors of the brain, and homomeric α7-type receptors. In recent years, α7 nAChRs have been identified as a potential target for therapeutic indications leading to the development of α7-selective agonists and partial agonists. However, unique properties of α7 nAChR, including low probability of channel opening and rapid desensitization, may limit the therapeutic usefulness of ligands binding exclusively to conventional agonist binding sites. New enthusiasm for the therapeutic targeting of α7 has come from the identification of α7-selective positive allosteric modulators (PAMs) that work effectively on the intrinsic factors that limit α7 ion channel activation. While these new drugs appear promising for therapeutic development, we also consider potential caveats and possible limitations for their use, including PAM-insensitive forms of desensitization and cytotoxicity issues. PMID:21575610

Positive allosteric modulators as an approach to nicotinic acetylcholine receptor-targeted therapeutics: advantages and limitations.

PubMed

Williams, Dustin K; Wang, Jingyi; Papke, Roger L

2011-10-15

Neuronal nicotinic acetylcholine receptors (nAChR), recognized targets for drug development in cognitive and neuro-degenerative disorders, are allosteric proteins with dynamic interconversions between multiple functional states. Activation of the nAChR ion channel is primarily controlled by the binding of ligands (agonists, partial agonists, competitive antagonists) at conventional agonist binding sites, but is also regulated in either negative or positive ways by the binding of ligands to other modulatory sites. In this review, we discuss models for the activation and desensitization of nAChR, and the discovery of multiple types of ligands that influence those processes in both heteromeric nAChR, such as the high-affinity nicotine receptors of the brain, and homomeric α7-type receptors. In recent years, α7 nAChRs have been identified as a potential target for therapeutic indications leading to the development of α7-selective agonists and partial agonists. However, unique properties of α7 nAChR, including low probability of channel opening and rapid desensitization, may limit the therapeutic usefulness of ligands binding exclusively to conventional agonist binding sites. New enthusiasm for the therapeutic targeting of α7 has come from the identification of α7-selective positive allosteric modulators (PAMs) that work effectively on the intrinsic factors that limit α7 ion channel activation. While these new drugs appear promising for therapeutic development, we also consider potential caveats and possible limitations for their use, including PAM-insensitive forms of desensitization and cytotoxicity issues. Copyright © 2011 Elsevier Inc. All rights reserved.

Heterogeneity of D2 dopamine receptors in different brain regions.

PubMed Central

Leonard, M N; Macey, C A; Strange, P G

1987-01-01

The binding of [3H]spiperone has been examined in membranes derived from different regions of bovine brain. In caudate nucleus, nucleus accumbens, olfactory tubercle and putamen binding is to D2 dopamine and 5HT2 serotonin receptors, whereas in cingulate cortex only serotonin 5HT2 receptor binding can be detected. D2 dopamine receptors were examined in detail in caudate nucleus, olfactory tubercle and putamen using [3H]spiperone binding in the presence of 0.3 microM-mianserin (to block 5HT2 serotonin receptors). No evidence for heterogeneity among D2 dopamine receptors either between brain regions or within a brain region was found from the displacements of [3H]spiperone binding by a range of antagonists, including dibenzazepines and substituted benzamides. Regulation of agonist binding by guanine nucleotides did, however, differ between regions. In caudate nucleus a population of agonist binding sites appeared resistant to guanine nucleotide regulation, whereas this was not the case in olfactory tubercle and putamen. PMID:2963621

Functional map of arrestin binding to phosphorylated opsin, with and without agonist.

PubMed

Peterhans, Christian; Lally, Ciara C M; Ostermaier, Martin K; Sommer, Martha E; Standfuss, Jörg

2016-06-28

Arrestins desensitize G protein-coupled receptors (GPCRs) and act as mediators of signalling. Here we investigated the interactions of arrestin-1 with two functionally distinct forms of the dim-light photoreceptor rhodopsin. Using unbiased scanning mutagenesis we probed the individual contribution of each arrestin residue to the interaction with the phosphorylated apo-receptor (Ops-P) and the agonist-bound form (Meta II-P). Disruption of the polar core or displacement of the C-tail strengthened binding to both receptor forms. In contrast, mutations of phosphate-binding residues (phosphosensors) suggest the phosphorylated receptor C-terminus binds arrestin differently for Meta II-P and Ops-P. Likewise, mutations within the inter-domain interface, variations in the receptor-binding loops and the C-edge of arrestin reveal different binding modes. In summary, our results indicate that arrestin-1 binding to Meta II-P and Ops-P is similarly dependent on arrestin activation, although the complexes formed with these two receptor forms are structurally distinct.

Characterization of (/sup 3/H)pirenzepine binding to muscarinic cholinergic receptors solubilized from rat brain

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

Luthin, G.R.; Wolfe, B.B.

Membranes prepared from rat cerebral cortex were solubilized in buffer containing 1% digitonin. Material present in the supernatant after centrifugation at 147,000 X g was shown to contain binding sites for both (/sup 3/H)quinuclidinyl benzilate ((/sup 3/H)QNB) and (/sup 3/H)pirenzepine ((/sup 3/H)PZ). Recovery of binding sites was approximately 25% of the initial membrane-bound (/sup 3/H)QNB binding sites. The Kd values for (/sup 3/H)QNB and (/sup 3/H)PZ binding to solubilized receptors were 0.3 nM and 0.1 microM, respectively. As has been observed previously in membrane preparations, (/sup 3/H)PZ appeared to label fewer solubilized binding sites than did (/sup 3/H)QNB. Maximum bindingmore » values for (/sup 3/H)PZ and (/sup 3/H)QNB binding to solubilized receptors were approximately 400 and 950 fmol/mg of protein, respectively. Competition curves for PZ inhibiting the binding of (/sup 3/H)QNB, however, had Hill slopes of 1, with a Ki value of 0.24 microM. The k1 and k-1 for (/sup 3/H)PZ binding were 3.5 X 10(6) M-1 min-1 and 0.13 min-1, respectively. The muscarinic receptor antagonists atropine, scopolamine and PZ inhibited the binding of (/sup 3/H)QNB and (/sup 3/H)PZ to solubilized receptors with Hill slopes of 1, as did the muscarinic receptor agonist oxotremorine. The muscarinic receptor agonist carbachol competed for (/sup 3/H)QNB and (/sup 3/H)PZ binding with a Hill slope of less than 1 in cerebral cortex, but not in cerebellum. GTP did not alter the interactions of carbachol or oxotremorine with the solubilized receptor. Together, these data suggest that muscarinic receptor sites solubilized from rat brain retain their abilities to interact selectively with muscarinic receptor agonists and antagonists.« less

The Receptor-Binding Site of the Measles Virus Hemagglutinin Protein Itself Constitutes a Conserved Neutralizing Epitope

PubMed Central

Ohno, Shinji; Sakai, Kouji; Ito, Yuri; Fukuhara, Hideo; Komase, Katsuhiro; Brindley, Melinda A.; Rota, Paul A.; Plemper, Richard K.; Maenaka, Katsumi; Takeda, Makoto

2013-01-01

Here, we provide direct evidence that the receptor-binding site of measles virus (MV) hemagglutinin protein itself forms an effective conserved neutralizing epitope (CNE). Several receptor-interacting residues constitute the CNE. Thus, viral escape from neutralization has to be associated with loss of receptor-binding activity. Since interactions with both the signaling lymphocyte activation molecule (SLAM) and nectin4 are critical for MV pathogenesis, its escape, which results from loss of receptor-binding activity, should not occur in nature. PMID:23283964

In brain, Axl recruits Grb2 and the p85 regulatory subunit of Pl3 kinase; in vitro mutagenesis defines th requisite binding sites for downstream Akt activation

PubMed Central

Weinger, Jason G.; Gohari, Pouyan; Yan, Ying; Backer, Jonathan M.; Varnum, Brian; Shafit-Zagardo, Bridget

2010-01-01

Axl is a receptor tyrosine kinase implicated in cell survival following growth factor withdrawal and other stressors. The binding of Axl's ligand, growth arrest-specific protein 6 (Gas6), results in Axl autophosphorylation, recruitment of signaling molecules, and activation of downstream survival pathways. Pull-down assays and immunoprecipitations using wildtype and mutant Axl transfected cells determined that Axl directly binds growth factor receptor-bound protein 2 (Grb2) at pYVN and the p85 subunit of phosphatidylinositol-3 kinase (PI3 kinase) at two pYXXM sites (pY779 and pY821). Also, p85 can indirectly bind to Axl via an interaction between p85's second proline-rich region and the N-terminal SH3 domain of Grb2. Further, Grb2 and p85 can compete for binding at the pY821VNM site. Gas6-stimulation of Axl-transfected COS7 cells recruited activated PI3 kinase and phosphorylated Akt. An interaction between Axl, p85 and Grb2 was confirmed in brain homogenates, enriched populations of O4+ oligodendrocytes, and O4– flow-through prepared from day 10 mouse brain, indicating that cells with active Gas6/Axl signal through Grb2 and the PI3 kinase/Akt pathways. PMID:18346204

Central peptidergic mechanisms controlling reproductive hormone secretion: novel methodology reveals a role for the natriuretic peptides.

PubMed

Samson, W K; Alexander, B D; Skala, K D; Huang, F L; Fulton, R J

1992-05-01

A variety of neural factors can influence reproductive hormone secretion by neuromodulatory actions within the hypothalamus or neuroendocrine actions within the anterior pituitary gland. Passive immunoneutralization and antagonist administration protocols have suggested physiological roles for a number of these factors; however, both experimental approaches have severe technical limitations. We have developed novel methodology utilizing cytotoxin cell targeting with neuropeptides linked to the toxic A chain of the plant cytotoxin ricin. With this methodology we can target and destroy in vivo or in vitro cells bearing receptors for that peptide. Ricin A chain conjugated to atrial natriuretic peptide (ANP), a neuropeptide known to pharmacologically inhibit luteinizing hormone-releasing hormone (LHRH) release, was injected into the cerebroventricular system of intact, cycling rats and ovariectomized rats. Cytotoxin conjugate treatment significantly lengthened the estrous cycle. In ovariectomized rats the luteinizing hormone surge induced by steroid priming was completely inhibited. LHRH content of the median eminences of these rats was not significantly altered. These data suggest that ANP binding to clearance receptors in the hypothalamus displaces the C-type natriuretic peptide (CNP) from the shared clearance receptor, making more CNP available to inhibit LHRH release. In the absence of cells bearing the clearance receptor all available CNP binds to the ANPR-B receptor and exerts its effect via an inhibitory interneuron, since LHRH fibers are spared by this treatment.

Integrating data on the Arabidopsis NPR1/NPR3/NPR4 salicylic acid receptors; a differentiating argument.

PubMed

Kuai, Xiahezi; MacLeod, Brandon J; Després, Charles

2015-01-01

Salicylic acid (SA) is a mandatory plant metabolite in the deployment of systemic acquired resistance (SAR), a broad-spectrum systemic immune response induced by local inoculation with avirulent pathogens. The NPR1 transcription co-activator is the central node positively regulating SAR. SA was the last of the major hormones to be without a known receptor. Recently, NPR1 was shown to be the direct link between SA and gene activation. This discovery seems to be controversial. NPR1 being an SA-receptor is reminiscent of the mammalian steroid receptors, which are transcription factors whose binding to DNA is dependent on the interaction with a ligand. Unlike steroid receptors, NPR1 does not bind directly to DNA, but is recruited to promoters by the TGA family of transcription factors to form an enhanceosome. In Arabidopsis, NPR1 is part of a multigene family in which two other members, NPR3 and NPR4, have also been shown to interact with SA. NPR3/NPR4 are negative regulators of immunity and act as substrate adaptors for the recruitment of NPR1 to an E3-ubiquitin ligase, leading to its subsequent degradation by the proteasome. In this perspective, we will stress-test in a friendly way the current NPR1/NPR3/NPR4 model.

Substance P receptor binding sites are expressed by glia in vivo after neuronal injury

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

Mantyh, P.W.; Johnson, D.J.; Boehmer, C.G.

1989-07-01

In vitro studies have demonstrated that glia can express functional receptors for a variety of neurotransmitters. To determine whether similar neurotransmitter receptors are also expressed by glia in vivo, the authors examined the glial scar in the transected optic nerve of the albino rabbit by quantitative receptor autoradiography. Receptor binding sites for radiolabeled calcitonin gene-related peptide, cholecystokinin, galanin, glutamate, somatostatin, substance P, and vasoactive intestinal peptide were examined. Specific receptor binding sites for each of these neurotransmitters were identified in the rabbit forebrain but were not detected in the normal optic nerve or tract. In the transected optic nerve andmore » tract, only receptor binding sites for substance P were expressed at detectable levels. The density of substance P receptor binding sites observed in this glial scar is among the highest observed in the rabbit forebrain. Ligand displacement and saturation experiments indicate that the substance P receptor binding site expressed by the glial scar has pharmacological characteristics similar to those of substance P receptors in the rabbit striatum, rat brain, and rat and canine gut. The present study demonstrates that glial cells in vivo express high concentrations of substance P receptor binding sites after transection of retinal ganglion cell axons. Because substance P has been shown to regulate inflammatory and immune responses in peripheral tissues, substance P may also, by analogy, be involved in regulating the glial response to injury in the central nervous system.« less

MIBE acts as antagonist ligand of both estrogen receptor α and GPER in breast cancer cells

PubMed Central

2012-01-01

Introduction The multiple biological responses to estrogens are mainly mediated by the classical estrogen receptors ERα and ERβ, which act as ligand-activated transcription factors. ERα exerts a main role in the development of breast cancer; therefore, the ER antagonist tamoxifen has been widely used although its effectiveness is limited by de novo and acquired resistance. Recently, GPR30/GPER, a member of the seven-transmembrane G protein-coupled receptor family, has been implicated in mediating the effects of estrogens in various normal and cancer cells. In particular, GPER triggered gene expression and proliferative responses induced by estrogens and even ER antagonists in hormone-sensitive tumor cells. Likewise, additional ER ligands showed the ability to bind to GPER eliciting promiscuous and, in some cases, opposite actions through the two receptors. We synthesized a novel compound (ethyl 3-[5-(2-ethoxycarbonyl-1-methylvinyloxy)-1-methyl-1H-indol-3-yl]but-2-enoate), referred to as MIBE, and investigated its properties elicited through ERα and GPER in breast cancer cells. Methods Molecular modeling, binding experiments and functional assays were performed in order to evaluate the biological action exerted by MIBE through ERα and GPER in MCF7 and SkBr3 breast cancer cells. Results MIBE displayed the ability to act as an antagonist ligand for ERα and GPER as it elicited inhibitory effects on gene transcription and growth effects by binding to both receptors in breast cancer cells. Moreover, GPER was required for epidermal growth factor receptor (EGFR) and ERK activation by EGF as ascertained by using MIBE and performing gene silencing experiments. Conclusions Our findings provide novel insights on the functional cross-talk between GPER and EGFR signaling. Furthermore, the exclusive antagonistic activity exerted by MIBE on ERα and GPER could represent an innovative pharmacological approach targeting breast carcinomas which express one or both receptors at the beginning and/or during tumor progression. Hence, the simultaneous inhibition of both ERα and GPER may guarantee major therapeutic benefits in respect to the use of a selective estrogen receptor antagonist. PMID:22251451

MIBE acts as antagonist ligand of both estrogen receptor α and GPER in breast cancer cells.

PubMed

Lappano, Rosamaria; Santolla, Maria Francesca; Pupo, Marco; Sinicropi, Maria Stefania; Caruso, Anna; Rosano, Camillo; Maggiolini, Marcello

2012-01-17

The multiple biological responses to estrogens are mainly mediated by the classical estrogen receptors ERα and ERβ, which act as ligand-activated transcription factors. ERα exerts a main role in the development of breast cancer; therefore, the ER antagonist tamoxifen has been widely used although its effectiveness is limited by de novo and acquired resistance. Recently, GPR30/GPER, a member of the seven-transmembrane G protein-coupled receptor family, has been implicated in mediating the effects of estrogens in various normal and cancer cells. In particular, GPER triggered gene expression and proliferative responses induced by estrogens and even ER antagonists in hormone-sensitive tumor cells. Likewise, additional ER ligands showed the ability to bind to GPER eliciting promiscuous and, in some cases, opposite actions through the two receptors. We synthesized a novel compound (ethyl 3-[5-(2-ethoxycarbonyl-1-methylvinyloxy)-1-methyl-1H-indol-3-yl]but-2-enoate), referred to as MIBE, and investigated its properties elicited through ERα and GPER in breast cancer cells. Molecular modeling, binding experiments and functional assays were performed in order to evaluate the biological action exerted by MIBE through ERα and GPER in MCF7 and SkBr3 breast cancer cells. MIBE displayed the ability to act as an antagonist ligand for ERα and GPER as it elicited inhibitory effects on gene transcription and growth effects by binding to both receptors in breast cancer cells. Moreover, GPER was required for epidermal growth factor receptor (EGFR) and ERK activation by EGF as ascertained by using MIBE and performing gene silencing experiments. Our findings provide novel insights on the functional cross-talk between GPER and EGFR signaling. Furthermore, the exclusive antagonistic activity exerted by MIBE on ERα and GPER could represent an innovative pharmacological approach targeting breast carcinomas which express one or both receptors at the beginning and/or during tumor progression. Hence, the simultaneous inhibition of both ERα and GPER may guarantee major therapeutic benefits in respect to the use of a selective estrogen receptor antagonist.

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.

An intracellular loop 2 amino acid residue determines differential binding of arrestin to the dopamine D2 and D3 receptors.

PubMed

Lan, Hongxiang; Teeter, Martha M; Gurevich, Vsevolod V; Neve, Kim A

2009-01-01

Dopamine D(2) and D(3) receptors are similar subtypes with distinct interactions with arrestins; the D(3) receptor mediates less agonist-induced translocation of arrestins than the D(2) receptor. The goals of this study were to compare nonphosphorylated arrestin-binding determinants in the second intracellular domain (IC2) of the D(2) and D(3) receptors to identify residues that contribute to the differential binding of arrestin to the subtypes. Arrestin 3 bound to glutathione transferase (GST) fusion proteins of the D(2) receptor IC2 more avidly than to the D(3) receptor IC2. Mutagenesis of the fusion proteins identified a residue at the C terminus of IC2, Lys149, that was important for the preferential binding of arrestin 3 to D(2)-IC2; arrestin binding to D(2)-IC2-K149C was greatly decreased compared with wild-type D(2)-IC2, whereas binding to the reciprocal mutant D(3)-IC2-C147K was enhanced compared with wild-type D(3)-IC2. Mutating this lysine in the full-length D(2) receptor to cysteine decreased the ability of the D(2) receptor to mediate agonist-induced arrestin 3 translocation to the membrane and decreased agonist-induced receptor internalization in human embryonic kidney 293 cells. The reciprocal mutation in the D(3) receptor increased receptor-mediated translocation of arrestin 3 without affecting agonist-induced receptor internalization. G protein-coupled receptor crystal structures suggest that Lys149, at the junction of IC2 and the fourth membrane-spanning helix, has intramolecular interactions that contribute to maintaining an inactive receptor state. It is suggested that the preferential agonist-induced binding of arrestin3 to the D(2) receptor over the D(3) receptor is due in part to Lys149, which could be exposed as a result of receptor activation.

Benzodiazepine and kainate receptor binding sites in the RCS rat retina.

PubMed

Stasi, Kalliopi; Naskar, Rita; Thanos, Solon; Kouvelas, Elias D; Mitsacos, Ada

2003-02-01

The effect of age and photoreceptor degeneration on the kainate subtype of glutamate receptors and on the benzodiazepine-sensitive gamma-aminobutyric acid-A receptors (GABA(A)) in normal and RCS (Royal College of Surgeons) rats were investigated. [(3)H]Kainate and [(3)H]flunitrazepam were used as radioligands for kainate and GABA(A)/benzodiazepine()receptors, respectively, using the quantitative receptor autoradiography technique. In both normal and RCS rat retina we observed that [(3)Eta]flunitrazepam and [(3)Eta]kainate binding levels were several times higher in inner plexiform layer (IPL) than in outer plexiform layer (OPL) at all four ages studied (P17, P35, P60 and P180). Age-related changes in receptor binding were observed in normal rat retina: [(3)Eta]flunitrazepam binding showed a significant decrease of 25% between P17 and P60 in IPL,and [(3)Eta]kainate binding showed significant decreases between P17 and P35 in both synaptic layers (71% in IPL and 63% in OPL). Degeneration-related changes in benzodiazepine and kainate receptor binding were observed in RCS rat retina. In IPL, [(3)Eta]flunitrazepam and [(3)Eta]kainate binding levels were higher than in normal retina at P35 (by 24% and 86%, respectively). In OPL, [(3)Eta]flunitrazepam binding was higher in RCS than in normal retina on P35 (74%) and also on P60 (62%). The results indicate that postnatal changes occur in kainate and benzodiazepine receptor binding sites in OPL and IPL of the rat retina up to 6 months of age. The data also suggest that the receptor binding changes observed in the RCS retina could be a consequence of the primary photoreceptor degeneration.

Dexamethasone effects on creatine kinase activity and insulin-like growth factor receptors in cultured muscle cells

NASA Technical Reports Server (NTRS)

Whitson, Peggy A.; Stuart, Charles A.; Huls, M. H.; Sams, Clarence F.; Cintron, Nitza M.

1989-01-01

The effect of dexamethasone on the activity of creatine kinase (CK) and the insulin-like growth factor I (IGF-I) binding were investigated using skeletal- and cardiac-muscle-derived cultured cell lines (mouse, C2C12; rat, L6 and H9c2). It was found that, in skeletal muscle cells, dexamethasone treatment during differentiation of skeletal-muscle cells caused dose-dependent increases in CK activity and increases in the degree of myotube formation, whereas cardiac cells (H9c2) exhibited very low CK activity during culture or dexamethasone treatment. Results for IGF-I binding were similar in all three cell lines. The IGF-I binding to dexamethasone-treated cells (50 nM for 24 hr on the day prior to confluence) resulted in an increased number of available binding sites, with no effect on the binding affinities.

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.

Live Cell Imaging Confocal Microscopy Analysis of HBV Myr-PreS1 Peptide Binding and Uptake in NTCP-GFP Expressing HepG2 Cells.

PubMed

König, Alexander; Glebe, Dieter

2017-01-01

To obtain basic knowledge about specific molecular mechanisms involved in the entry of pathogens into cells is the basis for establishing pharmacologic substances blocking initial viral binding, infection, and subsequent viral spread. Lack of information about key cellular factors involved in the initial steps of HBV infection has hampered the characterization of HBV binding and entry for decades. However, recently, the liver-specific sodium-dependent taurocholate cotransporting polypeptide (NTCP) has been discovered as a functional receptor for HBV and HDV, thus opening the field for new concepts of basic binding and entry of HBV and HDV. Here, we describe practical issues of a basic in vitro assay system to examine kinetics and mechanisms of receptor-dependent HBV binding, uptake, and intracellular trafficking by live-cell imaging confocal microscopy. The assay system is comprised of HepG2 cells expressing a NTCP-GFP fusion-protein and chemically synthesized, fluorophore-labeled part of HBV surface protein, spanning the first N-terminal 48 amino acids of preS1 of the large hepatitis B virus surface protein.

Estrogen Receptor Alpha Binding to ERE is Required for Full Tlr7- and Tlr9-Induced Inflammation

PubMed Central

Cunningham, Melissa A; Wirth, Jena R; Naga, Osama; Eudaly, Jackie; Gilkeson, Gary S

2014-01-01

We previously found that a maximum innate inflammatory response induced by stimulation of Toll-like receptors (TLRs) 3, 7 and 9 requires ERα, but does not require estrogen in multiple cell types from both control and lupus-prone mice. Given the estrogen-independence, we hypothesized that ERα mediates TLR signaling by tethering to, and enhancing, the activity of downstream transcription factors such as NFκB, rather than acting classically by binding EREs on target genes. To investigate the mechanism of ERα impact on TLR signaling, we utilized mice with a knock-in ERα mutant that is unable to bind ERE. After stimulation with TLR ligands, both ex vivo spleen cells and bone marrow-derived dendritic cells (BM-DCs) isolated from mutant ERα (“KIKO”) mice produced significantly less IL-6 compared with cells from wild-type (WT) littermates. These results suggest that ERα modulation of TLR signaling does indeed require ERE binding for its effect on the innate immune response. PMID:25061615

Quantitative in vivo receptor binding. I. Theory and application to the muscarinic cholinergic receptor

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

Frey, K.A.; Ehrenkaufer, R.L.; Beaucage, S.

1985-02-01

A novel approach to in vivo receptor binding experiments is presented which allows direct quantitation of binding site densities. The method is based on an equilibrium model of tracer uptake and is designed to produce a static distribution proportional to receptor density and to minimize possible confounding influences of regional blood flow, blood-brain barrier permeability, and nonspecific binding. This technique was applied to the measurement of regional muscarinic cholinergic receptor densities in rat brain using (/sup 3/H)scopolamine. Specific in vivo binding of scopolamine demonstrated saturability, a pharmacologic profile, and regional densities which are consistent with interaction of the tracer withmore » the muscarinic receptor. Estimates of receptor density obtained with the in vivo method and in vitro measurements in homogenates were highly correlated. Furthermore, reduction in striatal muscarinic receptors following ibotenic acid lesions resulted in a significant decrease in tracer uptake in vivo, indicating that the correlation between scopolamine distribution and receptor density may be used to demonstrate pathologic conditions. We propose that the general method presented here is directly applicable to investigation of high affinity binding sites for a variety of radioligands.« less

Opioid agonists binding and responses in SH-SY5Y cells

NASA Technical Reports Server (NTRS)

Costa, E. M.; Hoffmann, B. B.; Loew, G. H.

1992-01-01

SH-SY5Y (human neuroblastoma) cultured cells, known to have mu-opioid receptors, have been used to assess and compare the ability of eight representative mu-selective compounds from diverse opioid families to recognize and activate these receptors. A wide range of receptor affinities spanning a factor of 10,000 was found between the highest affinity fentanyl analogs (Ki = 0.1nM) and the lowest affinity analog, meperidine (Ki = 1 microM). A similar range was found for inhibition of PGE1-stimulated cAMP accumulation with a rank order of activities that closely paralleled binding affinities. Maximum inhibition of cAMP accumulation by each compound was about 80%. Maximum stimulation of GTPase activity (approximately 50%) was also similar for all compounds except the lowest affinity meperidine. Both effects were naloxone reversible. These results provide further evidence that mu-receptors are coupled to inhibition of adenylate cyclase and that the SH-SY5Y cell line is a good system for assessment of mu-agonists functional responses.

Robo4 maintains vessel integrity and inhibits angiogenesis by interacting with UNC5B.

PubMed

Koch, Alexander W; Mathivet, Thomas; Larrivée, Bruno; Tong, Raymond K; Kowalski, Joe; Pibouin-Fragner, Laurence; Bouvrée, Karine; Stawicki, Scott; Nicholes, Katrina; Rathore, Nisha; Scales, Suzie J; Luis, Elizabeth; del Toro, Raquel; Freitas, Catarina; Bréant, Christiane; Michaud, Annie; Corvol, Pierre; Thomas, Jean-Léon; Wu, Yan; Peale, Franklin; Watts, Ryan J; Tessier-Lavigne, Marc; Bagri, Anil; Eichmann, Anne

2011-01-18

Robo4 is an endothelial cell-specific member of the Roundabout axon guidance receptor family. To identify Robo4 binding partners, we performed a protein-protein interaction screen with the Robo4 extracellular domain. We find that Robo4 specifically binds to UNC5B, a vascular Netrin receptor, revealing unexpected interactions between two endothelial guidance receptors. We show that Robo4 maintains vessel integrity by activating UNC5B, which inhibits signaling downstream of vascular endothelial growth factor (VEGF). Function-blocking monoclonal antibodies against Robo4 and UNC5B increase angiogenesis and disrupt vessel integrity. Soluble Robo4 protein inhibits VEGF-induced vessel permeability and rescues barrier defects in Robo4(-/-) mice, but not in mice treated with anti-UNC5B. Thus, Robo4-UNC5B signaling maintains vascular integrity by counteracting VEGF signaling in endothelial cells, identifying a novel function of guidance receptor interactions in the vasculature. Copyright © 2011 Elsevier Inc. All rights reserved.

Structural basis of GM-CSF and IL-2 sequestration by the viral decoy receptor GIF

PubMed Central

Felix, Jan; Kandiah, Eaazhisai; De Munck, Steven; Bloch, Yehudi; van Zundert, Gydo C.P.; Pauwels, Kris; Dansercoer, Ann; Novanska, Katka; Read, Randy J.; Bonvin, Alexandre M.J.J.; Vergauwen, Bjorn; Verstraete, Kenneth; Gutsche, Irina; Savvides, Savvas N.

2016-01-01

Subversion of the host immune system by viruses is often mediated by molecular decoys that sequester host proteins pivotal to mounting effective immune responses. The widespread mammalian pathogen parapox Orf virus deploys GIF, a member of the poxvirus immune evasion superfamily, to antagonize GM-CSF (granulocyte macrophage colony-stimulating factor) and IL-2 (interleukin-2), two pleiotropic cytokines of the mammalian immune system. However, structural and mechanistic insights into the unprecedented functional duality of GIF have remained elusive. Here we reveal that GIF employs a dimeric binding platform that sequesters two copies of its target cytokines with high affinity and slow dissociation kinetics to yield distinct complexes featuring mutually exclusive interaction footprints. We illustrate how GIF serves as a competitive decoy receptor by leveraging binding hotspots underlying the cognate receptor interactions of GM-CSF and IL-2, without sharing any structural similarity with the cytokine receptors. Our findings contribute to the tracing of novel molecular mimicry mechanisms employed by pathogenic viruses. PMID:27819269

Role of molecular chaperones and TPR-domain proteins in the cytoplasmic transport of steroid receptors and their passage through the nuclear pore.

PubMed

Galigniana, Mario D; Echeverría, Pablo C; Erlejman, Alejandra G; Piwien-Pilipuk, Graciela

2010-01-01

In the absence of hormone, corticosteroid receptors such as GR (glucocorticoid receptor) and (mineralocorticoid receptor) are primarily located in the cytoplasm. Upon steroid-binding, they rapidly accumulate in the nucleus. Regardless of their primary location, these receptors and many other nuclear factors undergo a constant and dynamic nucleocytoplasmic shuttling. All members of the steroid receptor family are known to form large oligomeric structures with the heat-shock proteins of 90-kDa (hsp90) and 70-kDa (hsp70), the small acidic protein p23, and a tetratricopeptide repeat (TPR) -domain protein such as FK506-binding proteins (FKBPs), cyclophilins (CyPs) or the serine/threonine protein phosphatase 5 (PP5). It has always been stated that the dissociation of the chaperone heterocomplex (a process normally referred to as receptor "transformation") is the first step that permits the nuclear import of steroid receptors. However the experimental evidence is consistent with a model where the chaperone machinery is required for the retrotransport of the receptor through the cytoplasm and also facilitates the passage through the nuclear pore. Recent evidence indicates that the hsp90-based chaperone system also interacts with structures of the nuclear pore such as importin β and the integral nuclear pore glycoprotein Nup62 facilitating the passage of the untransformed receptor through the nuclear pore.

DNA residence time is a regulatory factor of transcription repression

PubMed Central

Clauß, Karen; Popp, Achim P.; Schulze, Lena; Hettich, Johannes; Reisser, Matthias; Escoter Torres, Laura; Uhlenhaut, N. Henriette

2017-01-01

Abstract Transcription comprises a highly regulated sequence of intrinsically stochastic processes, resulting in bursts of transcription intermitted by quiescence. In transcription activation or repression, a transcription factor binds dynamically to DNA, with a residence time unique to each factor. Whether the DNA residence time is important in the transcription process is unclear. Here, we designed a series of transcription repressors differing in their DNA residence time by utilizing the modular DNA binding domain of transcription activator-like effectors (TALEs) and varying the number of nucleotide-recognizing repeat domains. We characterized the DNA residence times of our repressors in living cells using single molecule tracking. The residence times depended non-linearly on the number of repeat domains and differed by more than a factor of six. The factors provoked a residence time-dependent decrease in transcript level of the glucocorticoid receptor-activated gene SGK1. Down regulation of transcription was due to a lower burst frequency in the presence of long binding repressors and is in accordance with a model of competitive inhibition of endogenous activator binding. Our single molecule experiments reveal transcription factor DNA residence time as a regulatory factor controlling transcription repression and establish TALE-DNA binding domains as tools for the temporal dissection of transcription regulation. PMID:28977492

Binding of /sup 125/I alpha-bungarotoxin to the thymus of mice

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

Ohshima, F.; Kondo, K.; Tsubaki, T.

1978-01-01

Alpha-bungarotoxin is known to bind with nicotinic acetylcholine receptors of skeletal muscle. Binding of iodine 125-labeled alpha bungarotoxin to the murine thymus, muscle, and liver was estimated. The toxin was bound to the muscle. The thymus was also capable of binding a considerable amount of the toxin, and the binding was obviously blocked by tubocurarine chloride. Binding to the liver, an organ containing no nicotinic acetylcholine receptor, was very slight. These results may indicate the presence of nicotinic acetylcholine receptors in the thymus, which could have implications in the pathogenesis of myasthenia gravis. Degenerating myoid cells and their receptors maymore » represent autoantigens that induce an immunological cross-reaction with the receptors of skeletal muscles, giving rise to myasthenia gravis.« less

[Effect of calcium and magnesium ions on the interaction of corticosterone with the cytosol receptor(s) in the rat brain].

PubMed

Ueda, M

1981-01-01

The effects of calcium and magnesium ions on the corticosterone binding to rat brain cytosol receptor protein(s) were investigated. The increasing amounts of CaCl2 or MgCl2 up to 5.0 mM were added, the specific [3H] corticosterone binding increased 1.3-fold and 1.5 respectively. The addition of MnCl2 and KCl did not affect this binding. The binding of corticosterone with rat brain cytosol receptor(s) were decreased by increasing amounts of EDTA and complete inhibition was observed at concentration equal to and greater than 2.5 mM. Inhibition of this binding by EDTA was less than by EGTA. Either theophylline or dibutyryl cyclic AMP had no effect on this binding.

p56Lck and p59Fyn Regulate CD28 Binding to Phosphatidylinositol 3-Kinase, Growth Factor Receptor-Bound Protein GRB-2, and T Cell-Specific Protein-Tyrosine Kinase ITK: Implications for T-Cell Costimulation

NASA Astrophysics Data System (ADS)

Raab, Monika; Cai, Yun-Cai; Bunnell, Stephen C.; Heyeck, Stephanie D.; Berg, Leslie J.; Rudd, Christopher E.

1995-09-01

T-cell activation requires cooperative signals generated by the T-cell antigen receptor ξ-chain complex (TCRξ-CD3) and the costimulatory antigen CD28. CD28 interacts with three intracellular proteins-phosphatidylinositol 3-kinase (PI 3-kinase), T cell-specific protein-tyrosine kinase ITK (formerly TSK or EMT), and the complex between growth factor receptor-bound protein 2 and son of sevenless guanine nucleotide exchange protein (GRB-2-SOS). PI 3-kinase and GRB-2 bind to the CD28 phosphotyrosine-based Tyr-Met-Asn-Met motif by means of intrinsic Src-homology 2 (SH2) domains. The requirement for tyrosine phosphorylation of the Tyr-Met-Asn-Met motif for SH2 domain binding implicates an intervening protein-tyrosine kinase in the recruitment of PI 3-kinase and GRB-2 by CD28. Candidate kinases include p56Lck, p59Fyn, ξ-chain-associated 70-kDa protein (ZAP-70), and ITK. In this study, we demonstrate in coexpression studies that p56Lck and p59Fyn phosphorylate CD28 primarily at Tyr-191 of the Tyr-Met-Asn-Met motif, inducing a 3- to 8-fold increase in p85 (subunit of PI 3-kinase) and GRB-2 SH2 binding to CD28. Phosphatase digestion of CD28 eliminated binding. In contrast to Src kinases, ZAP-70 and ITK failed to induce these events. Further, ITK binding to CD28 was dependent on the presence of p56Lck and is thus likely to act downstream of p56Lck/p59Fyn in a signaling cascade. p56Lck is therefore likely to be a central switch in T-cell activation, with the dual function of regulating CD28-mediated costimulation as well as TCR-CD3-CD4 signaling.

Cannabinoid inhibition of adenylate cyclase-mediated signal transduction and interleukin 2 (IL-2) expression in the murine T-cell line, EL4.IL-2.

PubMed

Condie, R; Herring, A; Koh, W S; Lee, M; Kaminski, N E

1996-05-31

Cannabinoid receptors negatively regulate adenylate cyclase through a pertussis toxin-sensitive GTP-binding protein. In the present studies, signaling via the adenylate cyclase/cAMP pathway was investigated in the murine thymoma-derived T-cell line, EL4.IL-2. Northern analysis of EL4.IL-2 cells identified the presence of 4-kilobase CB2 but not CB1 receptor-subtype mRNA transcripts. Southern analysis of genomic DNA digests for the CB2 receptor demonstrated identical banding patterns for EL4.IL-2 cells and mouse-derived DNA, both of which were dissimilar to DNA isolated from rat. Treatment of EL4.IL-2 cells with either cannabinol or Delta9-THC disrupted the adenylate cyclase signaling cascade by inhibiting forskolin-stimulated cAMP accumulation which consequently led to a decrease in protein kinase A activity and the binding of transcription factors to a CRE consensus sequence. Likewise, an inhibition of phorbol 12-myristate 13-acetate (PMA)/ionomycin-induced interleukin 2 (IL-2) protein secretion, which correlated to decreased IL-2 gene transcription, was induced by both cannabinol and Delta9-THC. Further, cannabinoid treatment also decreased PMA/ionomycin-induced nuclear factor binding to the AP-1 proximal site of the IL-2 promoter. Conversely, forskolin enhanced PMA/ionomycin-induced AP-1 binding. These findings suggest that inhibition of signal transduction via the adenylate cyclase/cAMP pathway induces T-cell dysfunction which leads to a diminution in IL-2 gene transcription.

A diketopiperazine factor from Rheinheimera aquimaris QSI02 exhibits anti-quorum sensing activity

PubMed Central

Sun, Shiwei; Dai, Xiaoyun; Sun, Jiao; Bu, Xiangguo; Weng, Caihong; Li, Hui; Zhu, Hu

2016-01-01

An ethyl acetate (EtOAc) extract isolated from the marine bacterium, Rheinheimera aquimaris QSI02, was found to exhibit anti-quorum sensing (anti-QS) activity. A subsequent bioassay-guided isolation protocol led to the detection of an active diketopiperazine factor, cyclo(Trp-Ser). Biosensor assay data showed that the minimum inhibitory concentration (MIC) of cyclo(Trp-Ser) ranged from 3.2 mg/ml to 6.4 mg/m for several microorganisms, including Escherichia coli, Chromobacterium violaceum CV026, Pseudomonas aeruginosa PA01, Staphylococcus aureus, and Candida albicans. Additionally, sub-MICs of cyclo(Trp-Ser) decreased the QS-regulated violacein production in C. violaceum CV026 by 67%. Furthermore, cyclo(Trp-Ser) can decrease QS-regulated pyocyanin production, elastase activity and biofilm formation in P. aeruginosa PA01 by 65%, 40% and 59.9%, respectively. Molecular docking results revealed that cyclo(Trp-Ser) binds to CviR receptor more rigidly than C6HSL with lower docking energy −8.68 kcal/mol, while with higher binding energy of −8.40 kcal/mol than 3-oxo-C12HSL in LasR receptor. Molecular dynamics simulation suggested that cyclo(Trp-Ser) is more easy to bind to CviR receptor than natural signaling molecule, but opposite in LasR receptor. These results suggest that cyclo(Trp-Ser) can be used as a potential inhibitor to control QS systems of C. violaceum and P. aeruginosa and provide increased the understanding of molecular mechanism that influences QS-regulated behaviors. PMID:28000767

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.

Characterizing carbohydrate-protein interactions by NMR

PubMed Central

Bewley, Carole A.; Shahzad-ul-Hussan, Syed

2013-01-01

Interactions between proteins and soluble carbohydrates and/or surface displayed glycans are central to countless recognition, attachment and signaling events in biology. The physical chemical features associated with these binding events vary considerably, depending on the biological system of interest. For example, carbohydrate-protein interactions can be stoichiometric or multivalent, the protein receptors can be monomeric or oligomeric, and the specificity of recognition can be highly stringent or rather promiscuous. Equilibrium dissociation constants for carbohydrate binding are known to vary from micromolar to millimolar, with weak interactions being far more prevalent; and individual carbohydrate binding sites can be truly symmetrical or merely homologous, and hence, the affinities of individual sites within a single protein can vary, as can the order of binding. Several factors, including the weak affinities with which glycans bind their protein receptors, the dynamic nature of the glycans themselves, and the non-equivalent interactions among oligomeric carbohydrate receptors, have made NMR an especially powerful tool for studying and defining carbohydrate-protein interactions. Here we describe those NMR approaches that have proven to be the most robust in characterizing these systems, and explain what type of information can (or cannot) be obtained from each. Our goal is to provide to the reader the information necessary for selecting the correct experiment or sets of experiments to characterize their carbohydrate-protein interaction of interest. PMID:23784792