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Sample records for laminin receptor activation

  1. Presence of Laminin Receptors in Staphylococcus aureus

    NASA Astrophysics Data System (ADS)

    Lopes, J. D.; Dos Reis, M.; Brentani, R. R.

    1985-07-01

    A characteristic feature of infection by Staphylococcus aureus is bloodstream invasion and widespread metastatic abscess formation. The ability to extravasate, which entails crossing the vascular basement membrane, appears to be critical for the organism's pathogenicity. Extravasation by normal and neoplastic mammalian cells has been correlated with the presence of specific cell surface receptors for the basement membrane glycoprotein laminin. Similar laminin receptors were found in Staphylococcus aureus but not in Staphylococcus epidermidis, a noninvasive pathogen. There were about 100 binding sites per cell, with an apparent binding affinity of 2.9 nanomolar. The molecular weight of the receptor was 50,000 and pI was 4.2. Eukaryotic laminin receptors were visualized by means of the binding of S. aureus in the presence of laminin. Prokaryotic and eukaryotic invasive cells might utilize similar, if not identical, mechanisms for invasion.

  2. Laminin receptor on platelets is the integrin VLA-6

    NASA Astrophysics Data System (ADS)

    Sonnenberg, Arnoud; Modderman, Piet W.; Hogervorst, Frans

    1988-12-01

    Adhesion of platelets to the subendothelial matrix of an injured vessel wall is an essential step in triggering the formation of a haemostatic plug. Fibronectin, collagen and laminin are three major components of the subendothelial matrix which support platelet adhesion1. Receptors for fibronectin and collagen have been identified on platelets and are included in the integrin family2-11. Here we report that adhesion of platelets to laminin is inhibited by a rat monoclonal antibody against the integrin family member, VLA-6. This antibody does not affect platelet adhesion to fibrinogen, fibronectin or to type I and III collagen. Binding to laminin does not require platelet activation and is not inhibited by fibronectin and laminin cell-attachment peptides. Platelet adhesion to laminin is supported by Mn2+, Co2+and Mg2+, but not by Ca2+, Zn2+ and Cu2+. This cation preference is distinct from that characteristic for other platelet-adhesive glycoproteins8,12,13.

  3. The laminin-binding activity of the alpha 7 integrin receptor is defined by developmentally regulated splicing in the extracellular domain.

    PubMed Central

    Ziober, B L; Chen, Y; Kramer, R H

    1997-01-01

    The expression pattern of the laminin-binding alpha 7 beta 1 integrin is developmentally regulated in skeletal, cardiac, and smooth muscle. The X1/X2 alternative splicing in the extracellular domain of alpha 7 is found in the variable region between conserved alpha-chain homology repeat domains III and IV, a site implicated in ligand binding. To assess differences in X1/X2 isoform activity, we generated MCF-7 cell lines transfected with alpha 7-X1/X2 cDNAs. Transfectants expressing the alpha 7-X2 variant adhered rapidly to laminin 1, whereas those expressing alpha 7-X1 failed to attach. That alpha 7-X1 exists in an inactive state was established in assays using an activating beta 1 antibody that induced X1-dependent cell adhesion and spreading. Furthermore, the activation of alpha 7-X1 was cell type specific, and when expressed in HT1080 cells, the integrin was converted into a fully functional receptor capable of promoting adhesion. Thus, the expression of the alpha 7-X1/X2 integrin is a novel mechanism that regulates receptor affinity states in a cell-specific context and may modulate integrin-dependent events during muscle development and repair. Images PMID:9307969

  4. Knock-Down of the 37kDa/67kDa Laminin Receptor LRP/LR Impedes Telomerase Activity

    PubMed Central

    Otgaar, Tyrone C.; Bernert, Martin; Ferreira, Eloise; Reusch, Uwe; Knackmuss, Stefan; Little, Melvyn; Weiss, Stefan F. T.; Letsolo, Boitelo T.

    2015-01-01

    Cancer has become a major problem worldwide due to its increasing incidence and mortality rates. Both the 37kDa/67kDa laminin receptor (LRP/LR) and telomerase are overexpressed in cancer cells. LRP/LR enhances the invasiveness of cancer cells thereby promoting metastasis, supporting angiogenesis and hampering apoptosis. An essential component of telomerase, hTERT is overexpressed in 85–90% of most cancers. hTERT expression and increased telomerase activity are associated with tumor progression. As LRP/LR and hTERT both play a role in cancer progression, we investigated a possible correlation between LRP/LR and telomerase. LRP/LR and hTERT co-localized in the perinuclear compartment of tumorigenic breast cancer (MDA_MB231) cells and non-tumorigenic human embryonic kidney (HEK293) cells. FLAG® Co-immunoprecipitation assays confirmed an interaction between LRP/LR and hTERT. In addition, flow cytometry revealed that both cell lines displayed high cell surface and intracellular LRP/LR and hTERT levels. Knock-down of LRP/LR by RNAi technology significantly reduced telomerase activity. These results suggest for the first time a novel function of LRP/LR in contributing to telomerase activity. siRNAs targeting LRP/LR may act as a potential alternative therapeutic tool for cancer treatment by (i) blocking metastasis (ii) promoting angiogenesis (iii) inducing apoptosis and (iv) impeding telomerase activity. PMID:26545108

  5. The Human Integrin VLA-2 is a Collagen Receptor on Some Cells and a Collagen/Laminin Receptor on Others

    NASA Astrophysics Data System (ADS)

    Elices, Mariano J.; Hemler, Martin E.

    1989-12-01

    The integrin heterodimer VLA-2, previously known as a collagen receptor, is now shown also to be a laminin receptor. Adhesion of the human melanoma cell line LOX to laminin was inhibited by anti-VLA ? 2 antibodies. Because VLA-2-mediated LOX cell attachment to laminin was not inhibited by digestion with collagenase, collagen contamination of laminin was not a factor. In addition, VLA-2 from LOX cells bound to immobilized laminin, and binding was disrupted by EDTA but not by Arg-Gly-Asp (RGD) peptides. VLA-3 also bound to laminin-Sepharose, although less avidly than VLA-2. Thus, at least four separate members of the integrin ? 1 subfamily serve as laminin receptors--i.e., VLA-2 and VLA-3 (this study) together with VLA-1 and VLA-6 (other reports). Whereas LOX and other cell lines used VLA-2 as both a laminin and collagen receptor, fibroblast VLA-2 mediated collagen but not laminin binding. Likewise, VLA-2 from platelets did not interact with laminin. Despite this functional discordancy, VLA-2 from laminin-binding and nonbinding sources was indistinguishable by all immunochemical and biochemical criteria examined. Thus, functional differences in VLA-2 may be due to cell type-specific modulation.

  6. The Laminin Receptor Is a Cellular Attachment Receptor for Classical Swine Fever Virus

    PubMed Central

    Chen, Jianing; He, Wen-Rui; Shen, Liang; Dong, Hong; Yu, Jiahui; Wang, Xiao; Yu, Shaoxiong; Li, Yongfeng; Li, Su; Luo, Yuzi; Sun, Yuan

    2015-01-01

    ABSTRACT Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious, economically important viral disease in many countries. The Erns and E2 envelope glycoproteins are responsible for the binding to and entry into the host cell by CSFV. To date, only one cellular receptor, heparan sulfate (HS), has been identified as being involved in CSFV attachment. HS is also present on the surface of various cells that are nonpermissive to CSFV. Hence, there must be another receptor(s) that has been unidentified to date. In this study, we used a set of small interfering RNAs (siRNAs) against a number of porcine cell membrane protein genes to screen cellular proteins involved in CSFV infection. This approach resulted in the identification of several proteins, and of these, the laminin receptor (LamR) has been demonstrated to be a cellular receptor for several viruses. Confocal analysis showed that LamR is colocalized with CSFV virions on the membrane, and a coimmunoprecipitation assay indicated that LamR interacts with the CSFV Erns protein. In inhibition assays, anti-LamR antibodies, soluble laminin, or LamR protein significantly inhibited CSFV infection in a dose-dependent manner. Transduction of PK-15 cells with a recombinant lentivirus expressing LamR yielded higher viral titers. Moreover, an attachment assay demonstrated that LamR functions during virus attachment. We also demonstrate that LamR acts as an alternative attachment receptor, especially in SK6 cells. These results indicate that LamR is a cellular attachment receptor for CSFV. IMPORTANCE Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), an economically important viral disease affecting the pig industry in many countries. To date, only heparan sulfate (HS) has been identified to be an attachment receptor for CSFV. Here, using RNA interference screening with small interfering RNAs (siRNAs) against a number of porcine membrane protein genes, we identified the laminin receptor (LamR) to be another attachment receptor. We demonstrate the involvement of LamR together with HS in virus attachment, and we elucidate the relationship between LamR and HS. LamR also serves as an attachment receptor for many viral pathogens, including dengue virus, a fatal human flavivirus. The study will help to enhance our understanding of the life cycle of flaviviruses and the development of antiviral strategies for flaviviruses. PMID:25694590

  7. The cell substrate attachment (CSAT) antigen has properties of a receptor for laminin and fibronectin

    PubMed Central

    1985-01-01

    The cell substrate attachment (CSAT) antigen is an integral membrane glycoprotein complex that participates in the adhesion of cells to extracellular molecules. The CSAT monoclonal antibody, directed against this complex, inhibited adhesion of cardiac and tendon fibroblasts and skeletal myoblasts to both laminin and fibronectin, thus implicating the CSAT antigen in adhesion to these extracellular molecules. Equilibrium gel filtration was used to explore the hypothesis that the CSAT antigen functions as a cell surface receptor for both laminin and fibronectin. In this technique, designed for rapidly exchanging equilibria, the gel filtration column is pre-equilibrated with extracellular ligand to ensure receptor occupancy during its journey through the column. Both laminin and fibronectin formed complexes with the CSAT antigen. The association with laminin was inhibited by the CSAT monoclonal antibody; the associations with both fibronectin and laminin were inhibited by synthetic peptides containing the fibronectin cell-binding sequence. Estimates of the dissociation constants by equilibrium gel filtration agree well with those available from other measurements. This suggests that these associations are biologically significant. SDS PAGE showed that all three glycoproteins comprising the CSAT antigen were present in the antigen-ligand complexes. Gel filtration and velocity sedimentation were used to show that the three bands comprise and oligomeric complex, which provides an explanation for their functional association. The inhibition of adhesion by the CSAT monoclonal antibody and the association of the purified antigen with extracellular ligands are interpreted as strongly implicating the CSAT antigen as a receptor for both fibronectin and laminin and perhaps for other extracellular molecules as well. PMID:2933413

  8. Laminin isoforms differentially regulate adhesion, spreading, proliferation, and ERK activation of h1 integrin-null cells

    E-print Network

    Campbell, Kevin P.

    of h1 integrin-null cells Yamato Kikkawaa , Hao Yua , Elke Generscha , Noriko Sanzenb , Kiyotoshi The presence of many laminin receptors of the h1 integrin family on most cells makes it difficult to define the biological functions of other major laminin receptors such as integrin a6h4 and dystroglycan. We therefore

  9. 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

  10. Laminin-derived peptide AG73 regulates migration, invasion, and protease activity of human oral squamous cell carcinoma cells through syndecan-1 and beta1 integrin.

    PubMed

    Siqueira, Adriane S; Gama-de-Souza, Letícia N; Arnaud, Maria Vanda C; Pinheiro, Joăo J V; Jaeger, Ruy G

    2010-01-01

    Squamous cell carcinoma is a prevalent head and neck tumor with high mortality. We studied the role played by laminin alpha1 chain peptide AG73 on migration, invasion, and protease activity of cells (OSCC) from human oral squamous cell carcinoma. Immunohistochemistry and immunofluorescence analyzed expression of laminin alpha1 chain and MMP9 in oral squamous cells carcinoma in vivo and in vitro. Migratory activity of AG73-treated OSCC cells was investigated by monolayer wound assays and in chemotaxis chambers. AG73-induced invasion was assessed in Boyden chambers. Invasion depends on MMPs. Conditioned media from cells grown on AG73 was subjected to zymography. We searched for AG73 receptors related to these activities in OSCC cells. Immunofluorescence analyzed AG73-induced colocalization of syndecan-1 and beta1 integrin. Cells had these receptors silenced by siRNA, followed by treatment with AG73 and analysis of migration, invasion, and protease activity. Oral squamous cell carcinoma expresses laminin alpha1 chain and MMP9. OSCC cells treated with AG73 showed increased migration, invasion, and protease activity. AG73 induced colocalization of syndecan-1 and beta1 integrin. Knockdown of these receptors decreased AG73-dependent migration, invasion, and protease activity. Syndecan-1 and beta1 integrin signaling downstream of AG73 regulate migration, invasion, and MMP production by OSCC cells. PMID:20237901

  11. Molecular cloning of the rat integrin alpha 1-subunit: a receptor for laminin and collagen

    PubMed Central

    1990-01-01

    Integrin heterodimers mediate a variety of adhesive interactions, including neuronal attachment to and process outgrowth on laminin. We report here the cloning and primary sequence of an M-200 kD integrin alpha subunit that associates with the integrin beta 1 subunit to form a receptor for both laminin and collagen. Similarities in ligand- binding specificity, relative molecular mass and NH2-terminal sequence make this a strong candidate for the rat homologue of the alpha subunit of the human integrin VLA-1. The full-length rat alpha 1 cDNAs encode a protein containing a purative signal sequence and a mature polypeptide of 1,152 amino acids, with extracellular, transmembrane and cytoplasmic domains. Several structural features are conserved with other integrin alpha chains, including (a) a sequence motif repeated seven times in the NH2-terminal half; (b) potential Ca2+/Mg2+ binding sites in repeats 5, 6, and 7, and (c) alignment of at least 14 of 23 cysteine residues. This rat alpha 1 sequence also contains a 206-amino acid I domain, inserted between repeats 2 and 3, that is homologous to I domains found in the same position in the alpha subunits of several integrins (VLA-2, Mac-1, LFA-1, p150). The rat alpha 1 and human VLA-2 apha subunits share greater than 50% sequence identity in the seven repeats and I domain, suggesting that these sequence identities may underlie some of their similar ligand-binding specificities. However, the rat integrin alpha 1 subunit has several unique features, including a 38-residue insert between two Ca2+/Mg2+ binding domains, and a divergent 15- residue cytoplasmic sequence, that may potentially account for unique functions of this integrin. PMID:2380249

  12. Calcium channels link the muscle-derived synapse organizer laminin ?2 to Bassoon and CAST/Erc2 to organize presynaptic active zones.

    PubMed

    Chen, Jie; Billings, Sara E; Nishimune, Hiroshi

    2011-01-12

    Synapse formation requires the organization of presynaptic active zones, the synaptic vesicle release sites, in precise apposition to postsynaptic neurotransmitter receptor clusters; however, the molecular mechanisms responsible for these processes remain unclear. Here, we show that P/Q-type and N-type voltage-dependent calcium channels (VDCCs) play essential roles as scaffolding proteins in the organization of presynaptic active zones. The neuromuscular junction of double knock-out mice for P/Q- and N-type VDCCs displayed a normal size but had significantly reduced numbers of active zones and docked vesicles and featured an attenuation of the active-zone proteins Bassoon, Piccolo, and CAST/Erc2. Consistent with this phenotype, direct interactions of the VDCC ?1b or ?4 subunits and the active zone-specific proteins Bassoon or CAST/Erc2 were confirmed by immunoprecipitation. A decrease in the number of active zones caused by a loss of presynaptic VDCCs resembled the pathological conditions observed in the autoimmune neuromuscular disorder Lambert-Eaton myasthenic syndrome. At the synaptic cleft of double knock-out mice, we also observed a decrease of the synaptic organizer laminin ?2 protein, an extracellular ligand of P/Q- and N-type VDCCs. However, the transcription level of laminin ?2 did not decrease in double knock-out mice, suggesting that the synaptic accumulation of laminin ?2 protein required its interaction with presynaptic VDCCs. These results suggest that presynaptic VDCCs link the target-derived synapse organizer laminin ?2 to active-zone proteins and function as scaffolding proteins to anchor active-zone proteins to the presynaptic membrane. PMID:21228161

  13. Deciphering the complex three-way interaction between the non-integrin laminin receptor, galectin-3 and Neisseria meningitidis

    PubMed Central

    Alqahtani, Fulwah; Mahdavi, Jafar; Wheldon, Lee M.; Vassey, Matthew; Pirinccioglu, Necmettin; Royer, Pierre-Joseph; Qarani, Suzan M.; Morroll, Shaun; Stoof, Jeroen; Holliday, Nicholas D.; Teo, Siew Y.; Oldfield, Neil J.; Wooldridge, Karl G.; Ala'Aldeen, Dlawer A. A.

    2014-01-01

    The non-integrin laminin receptor (LAMR1/RPSA) and galectin-3 (Gal-3) are multi-functional host molecules with roles in diverse pathological processes, particularly of infectious or oncogenic origins. Using bimolecular fluorescence complementation and confocal imaging, we demonstrate that the two proteins homo- and heterodimerize, and that each isotype forms a distinct cell surface population. We present evidence that the 37 kDa form of LAMR1 (37LRP) is the precursor of the previously described 67 kDa laminin receptor (67LR), whereas the heterodimer represents an entity that is distinct from this molecule. Site-directed mutagenesis confirmed that the single cysteine (C173) of Gal-3 or lysine (K166) of LAMR1 are critical for heterodimerization. Recombinant Gal-3, expressed in normally Gal-3-deficient N2a cells, dimerized with endogenous LAMR1 and led to a significantly increased number of internalized bacteria (Neisseria meningitidis), confirming the role of Gal-3 in bacterial invasion. Contact-dependent cross-linking determined that, in common with LAMR1, Gal-3 binds the meningococcal secretin PilQ, in addition to the major pilin PilE. This study adds significant new mechanistic insights into the bacterial–host cell interaction by clarifying the nature, role and bacterial ligands of LAMR1 and Gal-3 isotypes during colonization. PMID:25274119

  14. A Keratinocyte Hypermotility/Growth-Arrest Response Involving Laminin 5 and p16INK4A Activated in Wound Healing and Senescence

    PubMed Central

    Natarajan, Easwar; Omobono, John D.; Guo, Zongyou; Hopkinson, Susan; Lazar, Alexander J.F.; Brenn, Thomas; Jones, Jonathan C.; Rheinwald, James G.

    2006-01-01

    Keratinocytes become migratory to heal wounds, during early neoplastic invasion, and when undergoing telomere-unrelated senescence in culture. All three settings are associated with expression of the cell cycle inhibitor p16INK4A (p16) and of the basement membrane protein laminin 5 (LN5). We have investigated cause-and-effect relationships among laminin 5, p16, hypermotility, and growth arrest. Plating primary human keratinocytes on the ?2 precursor form of laminin 5 (LN5?) immediately induced directional hypermotility at ?125 ?m/hour, followed by p16 expression and growth arrest. Cells deficient in p16 and either p14ARF or p53 became hypermotile in response to LN5? but did not arrest growth. Plating on LN5? triggered smad nuclear translocation, and all LN5? effects were blocked by a transforming growth factor (TGF) ? receptor I (TGF?RI) kinase inhibitor. In contrast, plating cells on collagen I triggered a TGF?RI kinase-independent hypermotility unaccompanied by smad translocation or growth arrest. Plating on control surfaces with TGF? induced hypermotility after a 1-day lag time and growth arrest by a p16-independent mechanism. Keratinocytes serially cultured with TGF?RI kinase inhibitor exhibited an extended lifespan, and immortalization was facilitated following transduction to express the catalytic subunit of telomerase (TERT). These results reveal fundamental features of a keratinocyte hypermotility/growth-arrest response that is activated in wound healing, tumor suppression, and during serial cul-ture. PMID:16723698

  15. Laminin therapy for the promotion of muscle regeneration.

    PubMed

    Riederer, Ingo; Bonomo, Adriana Cesar; Mouly, Vincent; Savino, Wilson

    2015-11-14

    Muscle regeneration is essentially due to activation of satellite cells, which can be isolated and amplified ex vivo, thus representing good candidates for cell therapy. Accumulating data show that the local microenvironment plays a major role during muscle regeneration. In the satellite cell niche, a major extracellular matrix protein is laminin. Human myoblasts transplanted into immunodeficient mice are preferentially located in laminin-enriched areas. Additionally, laminin-111 enhances myoblast proliferation in vitro and increases expression of the ?7?1 integrin-type laminin receptor. Intramuscular injection of laminin-111 ameliorates muscular pathology in mdx mice, protecting muscle fibers from damage. Moreover, transplantation of human myoblasts with laminin-111 into Rag/mdx immunodeficient recipients improved efficacy of myoblast transplantation, increasing the number of human dystrophin-positive myofibres. Taken together, these data strongly indicate that exogenous laminin can ameliorate the regeneration process in different models of muscular dystrophies and can be instrumental for improving cell therapy aiming at repairing the degeneration/regeneration process in skeletal muscle. PMID:26459029

  16. Presynaptic calcium channels and ?3-integrins are complexed with synaptic cleft laminins, cytoskeletal elements and active zone components.

    PubMed

    Carlson, Steven S; Valdez, Gregorio; Sanes, Joshua R

    2010-11-01

    At chemical synapses, synaptic cleft components interact with elements of the nerve terminal membrane to promote differentiation and regulate function. Laminins containing the ?2 subunit are key cleft components, and they act in part by binding the pore-forming subunit of a pre-synaptic voltage-gated calcium channel (Ca(v)?) (Nishimune et al. 2004). In this study, we identify Ca(v)?-associated intracellular proteins that may couple channel-anchoring to assembly or stabilization of neurotransmitter release sites called active zones. Using Ca(v)?-antibodies, we isolated a protein complex from Torpedo electric organ synapses, which resemble neuromuscular junctions but are easier to isolate in bulk. We identified 10 components of the complex: six cytoskeletal proteins (?2/?2 spectrins, plectin 1, AHNAK/desmoyokin, dystrophin, and myosin 1), two active zone components (bassoon and piccolo), synaptic laminin, and a calcium channel ? subunit. Immunocytochemistry confirmed these proteins in electric organ synapses, and PCR analysis revealed their expression by developing mammalian motor neurons. Finally, we show that synaptic laminins also interact with pre-synaptic integrins containing the ?3 subunit. Together with our previous finding that a distinct synaptic laminin interacts with SV2 on nerve terminals (Son et al. 2000), our results identify three paths by which synaptic cleft laminins can send developmentally important signals to nerve terminals. PMID:20731762

  17. Adhesomes: specific granules containing receptors for laminin, C3bi/fibrinogen, fibronectin, and vitronectin in human polymorphonuclear leukocytes and monocytes

    PubMed Central

    1989-01-01

    We have localized several major extracellular matrix protein receptors in the specific granules of human polymorphonuclear (PMN) and monocytic leukocytes using double label immunoelectron microscopy (IEM) with ultrathin frozen sections and colloidal-gold conjugates. Rabbit antibodies to 67-kD human laminin receptor (LNR) were located on the inner surface of the specific granule membrane and within its internal matrix. LNR antigens co-distributed with lactoferrin, a marker of specific granules, but did not co-localize with elastase in azurophilic granules of PMNs. Further, CD11b/CD18 (leukocyte receptor for C3bi, fibrinogen, endothelial cells, and endotoxin), mammalian fibronectin receptor (FNR), and vitronectin receptor (VNR) antigens were also co- localized with LNR in PMN specific granules. A similar type of granule was found in monocytes which stained for LNR, FNR, VNR, CD18, and lysozyme. Activation of PMNs with either PMA, f-met-leu-phe (fMLP), tumor necrosis factor (TNF), or monocytic leukocytes with lipopolysaccharide (LPS), induced fusion of specific granules with the cell membrane and expression of both LNR and CD18 antigens on the outer cell surface. Further, stimulation led to augmented PMN adhesion on LN substrata, and six- to eightfold increases in specific binding of soluble LN that was inhibited by LNR antibody. These results indicate that four types of extracellular matrix receptors are located in leukocyte specific granules, and suggest that up-regulation of these receptors during inflammation may mediate leukocyte adhesion and extravasation. We have thus termed leukocyte specific granules adhesomes. PMID:2480353

  18. Green tea polyphenol epigallocatechin-3-gallate inhibits TLR4 signaling through the 67-kDa laminin receptor on lipopolysaccharide-stimulated dendritic cells

    SciTech Connect

    Byun, Eui-Baek; Choi, Han-Gyu; Sung, Nak-Yun; Byun, Eui-Hong

    2012-10-05

    Highlights: Black-Right-Pointing-Pointer Expressions of CD80, CD86, and MHC class I/II were inhibited by EGCG via 67LR. Black-Right-Pointing-Pointer EGCG-treated DCs inhibited LPS-induced pro-inflammatory cytokines via 67LR. Black-Right-Pointing-Pointer EGCG-treated DCs inhibited MAPKs activation and NF-{kappa}B p65 translocation via 67LR. Black-Right-Pointing-Pointer EGCG elevated the expression of the Tollip protein through 67LR in DCs. -- Abstract: Epigallocatechin-3-gallate (EGCG), a major active polyphenol of green tea, has been shown to down-regulate inflammatory responses in dendritic cells (DCs); however, the underlying mechanism has not been understood. Recently, we identified the 67-kDa laminin receptor (67LR) as a cell-surface EGCG receptor. In this study, we showed the molecular basis for the down-regulation of toll-like receptor 4 (TLR4) signal transduction by EGCG in DCs. The expressions of CD80, CD86, and MHC class I and II, which are molecules essential for antigen presentation by DCs, were inhibited by EGCG via 67LR. In addition, EGCG-treated DCs inhibited lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines (tumor necrosis factor [TNF]-{alpha}, interleukin [IL]-1{beta}, and IL-6) and activation of mitogen-activated protein kinases (MAPKs), e.g., extracellular signal-regulated kinase 1/2 (ERK1/2), p38, c-Jun N-terminal kinase (JNK), and nuclear factor {kappa}B (NF-{kappa}B) p65 translocation through 67LR. Interestingly, we also found that EGCG markedly elevated the expression of the Tollip protein, a negative regulator of TLR signaling, through 67LR. These novel findings provide new insight into the understanding of negative regulatory mechanisms of the TLR4 signaling pathway and consequent inflammatory responses that are implicated in the development and progression of many chronic diseases.

  19. Laminin receptor specific therapeutic gold nanoparticles ((AuNP)-Au-198-EGCg) show efficacy in treating prostate cancer

    SciTech Connect

    Shukla, Ravi; Chanda, Nripen; Zambre, Ajit; Katti, Kavita; Upendran, Anandhi; Kulkarni, Rajesh R.; Nune, Satish K.; Casteel, Stan W.; Smith, C. J.; Boote, Evan; Robertson, J. D.; Kan, Para; Engelbrecht, Hendrik; Watkinson, Lisa D.; Carmack, Terry L.; Lever, John R.; Cutler, Cathy; Caldwell, Charles; Kannan, Raghuraman; Katti, Kattesh V.

    2012-07-31

    Systemic delivery of therapeutic agents to solid tumors is hindered by vascular and interstitial barriers. We hypothesized that prostate tumor specific epigallocatechingallate( EGCg) functionalized radioactive gold nanoparticles, when delivered intratumorally (IT), will circumvent transport barriers, resulting in targeted delivery of therapeutic payloads. The results described herein provide unequivocal validation of our hypothesis. We report the development of inherently therapeutic gold nanoparticles derived from Au-198 isotope; the range of 198Au ?-particle ( ~ 11 mm in tissue or ~1100 cell diameters) is sufficiently long to provide cross-fire effects of radiation dose delivered to cells within the prostate gland and short enough to minimize radiation dose to critical tissues near the periphery of the capsule. The formulation of biocompatible 198AuNPs utilizes the redox chemistry of prostate tumor specific phytochemical EGCg as it converts gold salt into gold nanoparticles and also selectively binds with excellent affinity to Laminin67R receptors which are over expressed in prostate tumor cells. Pharmacokinetic studies in PC-3 xenograft SCID mice showed ~72% retention of 198AuNP-EGCg in tumors 24 h after intratumoral administration. Therapeutic studies showed 80% reduction of tumor volumes after 28 days demonstrating significant inhibition of tumor growth compared to controls. This innovative “green nanotechnological“approach serves as a basis for designing target specific antineoplastic agents. This novel intratumorally injectable 198AuNP-EGCg nanotherapeutic agent may provide significant advances in oncology for use as an effective treatment for prostate and other solid tumors.

  20. Photodynamic treatment of epithelial tissue derived from patients with endometrial cancer: a contribution to the role of laminin and epidermal growth factor receptor in photodynamic therapy

    NASA Astrophysics Data System (ADS)

    Ziolkowski, Piotr P.; Symonowicz, Krzysztof; Osiecka, Beata J.; Rabczynski, Jerzy; Gerber, Jerzy

    1999-07-01

    Photodynamic therapy (PDT) was used to treat endometrial G1 cancer tissue derived from patients who had undergone a total hysterectomy and bilateral salpingo-oophorectomy. After surgical treatment the cancerous tissue was kept in a medium containing Dulbecco solution, fetal calf serum, and antibiotics. The tissue was then exposed to hematoporphyrin derivative (0.1 mg/l) and 24 h later exposed to light (total light dose--18 J/sq cm). Necrosis depth was evaluated 24 h later using a light microscope. In order to assess the possible role of the basal membrane component laminin, as well as epidermal growth factor receptor susceptibility to PDT, immunohistochemical studies were carried out. Additionally, nucleolar organizer regions evaluation was performed. Our experiment confirmed that PDT results in the necrosis in the treated endometrial cancer, while not affecting the laminin in the cancerous tissue. In contrast, PDT strongly affects the epidermal growth factor receptor and nucleolar organizer regions in cancer cells. We suggest that laminin may contribute to the prevention of cancer dissemination in the cases where PDT has to be repeated, and that after PDT the cells become less susceptible to a mitogen, like, e.g., epidermal growth factor.

  1. Aberrant Development of Thymocytes in Mice Lacking Laminin-2

    PubMed Central

    Magner, William J.; Chang, Andrew C.; Owens, Jennie; Hong, M-J. P.; Brooks, Andrew

    2000-01-01

    In previous in vitro studies, we proposed a role for the extracellular matrix component, laminin- 2, and its integrin receptor, VLA-6, in thymocyte development. The characterization of two dystrophic mouse strains with different defects in laminin-2 allowed us to examine this proposal in vivo. Mice deficient in laminin-2, dy/dy, show a significant reduction in thymus size and number of thymocytes compared to normal littermates. These mice also exhibited apparent alterations of thymic architecture. Examination of the CD4/CD8 populations in dy/dy thymi showed large relative increases in the DN (CD4-CD8-) and SP (CD4+CD8-, CD4-CD8+) populations and a significant decrease in the DP (CD4+CD8+) population. Further examination of the DN population for CD44 and CD25 expression showed a remarkable decrease in the more mature pre-T cell populations. Analysis of apoptosis in situ, and by flow cytometry, in dy/dy thymi revealed a significant increase in apoptotic DN thymocytes in the capsule and subcapsular regions. Interestingly, thymocyte development appeared to proceed normally in dystrophic mice expressing a mutant form of laminin-2, dy2J, as well as, in fetal and neonatal dy/dy mice. We propose that laminin-2 plays an active role in thymocyte development by delivering cell survival and differentiation signals at specific stages of development in young adult mice. PMID:11097211

  2. Laminin receptor specific therapeutic gold nanoparticles (198AuNP-EGCg) show efficacy in treating prostate cancer.

    PubMed

    Shukla, Ravi; Chanda, Nripen; Zambre, Ajit; Upendran, Anandhi; Katti, Kavita; Kulkarni, Rajesh R; Nune, Satish Kumar; Casteel, Stan W; Smith, Charles Jeffrey; Vimal, Jatin; Boote, Evan; Robertson, J David; Kan, Para; Engelbrecht, Hendrik; Watkinson, Lisa D; Carmack, Terry L; Lever, John R; Cutler, Cathy S; Caldwell, Charles; Kannan, Raghuraman; Katti, Kattesh V

    2012-07-31

    Systemic delivery of therapeutic agents to solid tumors is hindered by vascular and interstitial barriers. We hypothesized that prostate tumor specific epigallocatechin-gallate (EGCg) functionalized radioactive gold nanoparticles, when delivered intratumorally (IT), would circumvent transport barriers, resulting in targeted delivery of therapeutic payloads. The results described herein support our hypothesis. We report the development of inherently therapeutic gold nanoparticles derived from the Au-198 isotope; the range of the (198)Au ?-particle (approximately 11 mm in tissue or approximately 1100 cell diameters) is sufficiently long to provide cross-fire effects of a radiation dose delivered to cells within the prostate gland and short enough to minimize the radiation dose to critical tissues near the periphery of the capsule. The formulation of biocompatible (198)AuNPs utilizes the redox chemistry of prostate tumor specific phytochemical EGCg as it converts gold salt into gold nanoparticles and also selectively binds with excellent affinity to Laminin67R receptors, which are over expressed in prostate tumor cells. Pharmacokinetic studies in PC-3 xenograft SCID mice showed approximately 72% retention of (198)AuNP-EGCg in tumors 24 h after intratumoral administration. Therapeutic studies showed 80% reduction of tumor volumes after 28 d demonstrating significant inhibition of tumor growth compared to controls. This innovative nanotechnological approach serves as a basis for designing biocompatible target specific antineoplastic agents. This novel intratumorally injectable (198)AuNP-EGCg nanotherapeutic agent may provide significant advances in oncology for use as an effective treatment for prostate and other solid tumors. PMID:22802668

  3. Title: Laminin-5 activates extracellular matrix production and osteogenic gene focusing in human mesenchymal stem cells

    E-print Network

    Vandenberg, Scott

    mesenchymal stem cells Authors: Robert F. Klees1 , Roman M. Salasznyk1 , Scott Vandenberg3 , Kristin P@rpi.edu George E. Plopper: ploppg@rpi.edu Key words: Laminin-5, extracellular matrix, mesenchymal stem cells by human mesenchymal stem cells (hMSC), stimulates osteogenic gene expression in these cells in the absence

  4. The 37/67-Kilodalton Laminin Receptor Is a Receptor for Adeno-Associated Virus Serotypes 8, 2, 3, and 9

    PubMed Central

    Akache, Bassel; Grimm, Dirk; Pandey, Kusum; Yant, Stephen R.; Xu, Hui; Kay, Mark A.

    2006-01-01

    Adeno-associated virus serotype 8 (AAV8) is currently emerging as a powerful gene transfer vector, owing to its capability to efficiently transduce many different tissues in vivo. While this is believed to be in part due to its ability to uncoat more readily than other AAV serotypes such as AAV2, understanding all the processes behind AAV8 transduction is important for its application and optimal use in human gene therapy. Here, we provide the first report of a cellular receptor for AAV8, the 37/67-kDa laminin receptor (LamR). We document binding of LamR to AAV8 capsid proteins and intact virions in vitro and demonstrate its contribution to AAV8 transduction of cultured cells and mouse liver in vivo. We also show that LamR plays a role in transduction by three other closely related serotypes (AAV2, -3, and -9). Sequence and deletion analysis allowed us to map LamR binding to two protein subdomains predicted to be exposed on the AAV capsid exterior. Use of LamR, which is constitutively expressed in many clinically relevant tissues and is overexpressed in numerous cancers, provides a molecular explanation for AAV8's broad tissue tropism. Along with its robust transduction efficiency, our findings support the continued development of AAV8-based vectors for clinical applications in humans, especially for tumor gene therapy. PMID:16973587

  5. Extraribosomal Functions Associated with the C Terminus of the 37/67 kDa Laminin Receptor are Required for Maintaining Cell Viability

    SciTech Connect

    J Scheiman; K Jamieson; J Ziello; J Tseng; D Meruelo

    2011-12-31

    The 37/67 kDa laminin receptor (LAMR) is a multifunctional protein, acting as an extracellular receptor, localizing to the nucleus, and playing roles in rRNA processing and ribosome assembly. LAMR is important for cell viability; however, it is unclear which of its functions are essential. We developed a silent mutant LAMR construct, resistant to siRNA, to rescue the phenotypic effects of knocking down endogenous LAMR, which include inhibition of protein synthesis, cell cycle arrest, and apoptosis. In addition, we generated a C-terminal-truncated silent mutant LAMR construct structurally homologous to the Archaeoglobus fulgidus S2 ribosomal protein and missing the C-terminal 75 residues of LAMR, which displays more sequence divergence. We found that HT1080 cells stably expressing either silent mutant LAMR construct still undergo arrest in the G{sub 1} phase of the cell cycle when treated with siRNA. However, the expression of full-length silent mutant LAMR rescues cell viability, whereas the expression of the C-terminal-truncated LAMR does not. Interestingly, we also found that both silent mutant constructs restore protein translation and localize to the nucleus. Our findings indicate that the ability of LAMR to regulate viability is associated with its C-terminal 75 residues. Furthermore, this function is distinct from its role in cell proliferation, independent of its ribosomal functions, and may be regulated by a nonnuclear localization.

  6. Laminin ?4-Null Mutant Mice Develop Chronic Kidney Disease with Persistent Overexpression of Platelet-Derived Growth Factor

    PubMed Central

    Abrass, Christine K.; Hansen, Kim M.; Patton, Bruce L.

    2010-01-01

    Each extracellular matrix compartment in the kidney has a unique composition, with regional specificity in the expression of various laminin isoforms. Although null mutations in the majority of laminin chains lead to specific developmental abnormalities in the kidney, Lama4?/? mice have progressive glomerular and tubulointerstitial fibrosis. These mice have a significant increase in expression of platelet-derived growth factor (PDGF)-BB, PDGF-DD, and PDGF receptor ? in association with immature glomerular and peritubular capillaries. In addition, mesangial cell exposure to ?4-containing laminins, but not other isoforms, results in down-regulation of PDGF receptor mRNA and protein, suggesting a direct effect of LN411/LN421 on vessel maturation. Given the known role of overexpression of PDGF-BB and PDGF-DD on glomerular and tubulointerstitial fibrosis, these data suggest that failure of laminin ?4-mediated down-regulation of PDGF activity contributes to the progressive renal lesions in this animal model. Given the recent demonstration that individuals with laminin ?4 mutations develop cardiomyopathy, these findings may be relevant to kidney disease in humans. PMID:20035058

  7. Our understanding of the structure and function of dystroglycan, a cell surface laminin/agrin receptor, has

    E-print Network

    Campbell, Kevin P.

    with Duchenne muscular dystrophy and also in dystrophin deficient mdx mice [1­3]. In skeletal muscle in the pathogenesis of several muscular dystrophies and serves as a receptor for a human pathogen as well as being Fukuyama-type congenital muscular dystrophy Gal galactose GlcNAc N-acetyl glucosamine Man mannose NMJ

  8. Simplified Purification Procedure of Laminin-332 and Laminin-511 from Human Cell Lines

    PubMed Central

    Sroka, Isis C.; Chen, Man Ling; Cress, Anne E.

    2008-01-01

    Laminins are glycoproteins expressed in the basement membrane of multiple epithelial tissues. Previously described purification procedures for the human laminin variants laminin-5 (LN-332) and laminin-10 (LN-511) use tissue as starting material and have multiple steps. We demonstrate a two-step laminin immunoaffinity purification method to produce consistent quantities of intact and biologically active LN-332 and LN-511 from human keratinocyte (HaCaT) and human lung carcinoma (A549) cell lines, respectively. The purification of LN-332 and LN-551 was demonstrated by PAGE analysis, silver staining and Western blot analysis. The purification procedure includes instruction on removing a cell adhesion contaminant known as galectin-3 binding protein from purified LN-511. The biological activity of purified laminin was tested in a standard cell adhesion assay and compared to commercially available LN-111. This rapid and reproducible purification method will contribute to understanding the role of LN-332 and LN-511 in cell behavior, signaling and gene expression. PMID:18713621

  9. Laminin Mediates Tissue-specific Gene Expression in Mammary Epithelia

    SciTech Connect

    Streuli, Charles H; Schmidhauser, Christian; Bailey, Nina; Yurchenco, Peter; Skubitz, Amy P. N.; Roskelley, Calvin; Bissell, Mina J

    1995-04-01

    Tissue-specific gene expression in mammary epithelium is dependent on the extracellular matrix as well as hormones. There is good evidence that the basement membrane provides signals for regulating beta-casein expression, and that integrins are involved in this process. Here, we demonstrate that in the presence of lactogenic hormones, laminin can direct expression of the beta-casein gene. Mouse mammary epithelial cells plated on gels of native laminin or laminin-entactin undergo functional differentiation. On tissue culture plastic, mammary cells respond to soluble basement membrane or purified laminin, but not other extracellular matrix components, by synthesizing beta-casein. In mammary cells transfected with chloramphenicol acetyl transferase reporter constructs, laminin activates transcription from the beta-casein promoter through a specific enhancer element. The inductive effect of laminin on casein expression was specifically blocked by the E3 fragment of the carboxy terminal region of the alpha 1 chain of laminin, by antisera raised against the E3 fragment, and by a peptide corresponding to a sequence within this region. Our results demonstrate that laminin can direct tissue-specific gene expression in epithelial cells through its globular domain.

  10. Interaction of Human Laminin Receptor with Sup35, the [PSI+] Prion-Forming Protein from S. cerevisiae: A Yeast Model for Studies of LamR Interactions with Amyloidogenic Proteins

    PubMed Central

    Pampeno, Christine; Derkatch, Irina L.; Meruelo, Daniel

    2014-01-01

    The laminin receptor (LamR) is a cell surface receptor for extracellular matrix laminin, whereas the same protein within the cell interacts with ribosomes, nuclear proteins and cytoskeletal fibers. LamR has been shown to be a receptor for several bacteria and viruses. Furthermore, LamR interacts with both cellular and infectious forms of the prion protein, PrPC and PrPSc. Indeed, LamR is a receptor for PrPC. Whether LamR interacts with PrPSc exclusively in a capacity of the PrP receptor, or LamR specifically recognizes prion determinants of PrPSc, is unclear. In order to explore whether LamR has a propensity to interact with prions and amyloids, we examined LamR interaction with the yeast prion-forming protein, Sup35. Sup35 is a translation termination factor with no homology or functional relationship to PrP. Plasmids expressing LamR or LamR fused with the green fluorescent protein (GFP) were transformed into yeast strain variants differing by the presence or absence of the prion conformation of Sup35, respectively [PSI+] and [psi?]. Analyses by immunoprecipitation, centrifugal fractionation and fluorescent microscopy reveal interaction between LamR and Sup35 in [PSI+] strains. The presence of [PSI+] promotes LamR co-precipitation with Sup35 as well as LamR aggregation. In [PSI+] cells, LamR tagged with GFP or mCherry forms bright fluorescent aggregates that co-localize with visible [PSI+] foci. The yeast prion model will facilitate studying the interaction of LamR with amyloidogenic prions in a safe and easily manipulated system that may lead to a better understanding and treatment of amyloid diseases. PMID:24416454

  11. Differential Activation of Peroxisome Proliferator-activated Receptors by Eicosanoids*

    E-print Network

    Brown, Myles

    Differential Activation of Peroxisome Proliferator-activated Receptors by Eicosanoids* (Received, Pennsylvania 19104 Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors occurring compounds activate each of the PPAR subtypes. To address this issue, a screening assay

  12. Neuronal migration on laminin in vitro.

    PubMed

    Liang, S; Crutcher, K A

    1992-03-20

    Chick sympathetic (E-9) or telencephalic (E-7) neurons were cultured at low density on poly-DL-ornithine (PORN), poly-L-lysine (POLS), laminin or laminin-covered PORN or POLS and monitored with time-lapse videomicroscopy. Neurons migrated on laminin, or laminin-covered PORN or POLS, but not on PORN or POLS alone. Neuronal migration did not involve interactions with other cells indicating that neurons are capable of independent migration when exposed to a laminin substrate. PMID:1600626

  13. Astrocytic laminin regulates pericyte differentiation and maintains blood brain barrier integrity

    NASA Astrophysics Data System (ADS)

    Yao, Yao; Chen, Zu-Lin; Norris, Erin H.; Strickland, Sidney

    2014-03-01

    Blood brain barrier (BBB) breakdown is not only a consequence of but also contributes to many neurological disorders, including stroke and Alzheimer’s disease. How the basement membrane (BM) contributes to the normal functioning of the BBB remains elusive. Here we use conditional knockout mice and an acute adenovirus-mediated knockdown model to show that lack of astrocytic laminin, a brain-specific BM component, induces BBB breakdown. Using functional blocking antibody and RNAi, we further demonstrate that astrocytic laminin, by binding to integrin ?2 receptor, prevents pericyte differentiation from the BBB-stabilizing resting stage to the BBB-disrupting contractile stage, and thus maintains the integrity of BBB. Additionally, loss of astrocytic laminin decreases aquaporin-4 (AQP4) and tight junction protein expression. Altogether, we report a critical role for astrocytic laminin in BBB regulation and pericyte differentiation. These results indicate that astrocytic laminin maintains the integrity of BBB through, at least in part, regulation of pericyte differentiation.

  14. Mechanisms regulating chemokine receptor activity

    PubMed Central

    Bennett, Laura D; Fox, James M; Signoret, Nathalie

    2011-01-01

    Co-ordinated movement and controlled positioning of leucocytes is key to the development, maintenance and proper functioning of the immune system. Chemokines and their receptors play an essential role in these events by mediating directed cell migration, often referred to as chemotaxis. The chemotactic property of these molecules is also thought to contribute to an array of pathologies where inappropriate recruitment of specific chemokine receptor-expressing leucocytes is observed, including cancer and inflammatory diseases. As a result, chemokine receptors have become major targets for therapeutic intervention, and during the past 15 years much research has been devoted to understanding the regulation of their biological activity. From these studies, processes which govern the availability of functional chemokine receptors at the cell surface have emerged as playing a central role. In this review, we summarize and discuss current knowledge on the molecular mechanisms contributing to the regulation of chemokine receptor surface expression, from gene transcription and protein degradation to post-translational modifications, multimerization, intracellular transport and cross-talk. PMID:21977995

  15. Responses of cultured neural retinal cells to substratum-bound laminin and other extracellular matrix molecules.

    PubMed

    Adler, R; Jerdan, J; Hewitt, A T

    1985-11-01

    The responses of cultured chick embryo retinal neurons to several extracellular matrix molecules are described. Retinal cell suspensions in serum-free medium containing the "N1" supplement (J. E. Bottenstein, S. D. Skaper, S. Varon, and J. Sato, 1980, Exp. Cell Res. 125, 183-190) were seeded on tissue culture plastic surfaces pretreated with polyornithine (PORN) and with one of the factors to be tested. Substantial cell survival could be observed after 72 hr in vitro on PORN pretreated with serum or laminin, whereas most cells appeared to be degenerating on untreated PORN, PORN-fibronectin, and PORN-chondronectin. Cell attachment, although quantitatively similar for all these substrata, was temperature-dependent on serum and laminin but not on fibronectin or untreated PORN. In a short-term bioassay, neurite development was abundant on laminin, scarce on serum and fibronectin, and absent on PORN. No positive correlation between cell spreading and neurite production could be seen: cell spreading was more extensive on PORN and fibronectin than on laminin or serum, while on laminin-treated dishes, spreading was similar for neurite-bearing and non-neurite-bearing cells. Laminin effects on retinal neurons were clearly substratum dependent. When bound to tissue culture plastic, laminin showed a dose-dependent inhibitory effect on cell attachment and did not stimulate neurite development. PORN-bound laminin, on the other hand, did not affect cell attachment but caused marked stimulation of neurite development, suggesting that laminin conformation and/or the spatial distribution of active sites play an important role in the neurite-promoting function of this extracellular matrix molecule. Investigation of the embryonic retina with ELISA and immunocytochemical methods showed that laminin is present in this organ during development. Therefore, in vivo and in vitro observations are consistent with the possibility that laminin might influence neuronal development in the retina. PMID:3902534

  16. Keratinocyte-derived Laminin-332 Protein Promotes Melanin Synthesis via Regulation of Tyrosine Uptake*

    PubMed Central

    Chung, Heesung; Jung, Hyejung; Lee, Jung-hyun; Oh, Hye Yun; Kim, Ok Bin; Han, Inn-Oc; Oh, Eok-Soo

    2014-01-01

    Melanocytes, which produce the pigment melanin, are known to be closely regulated by neighboring keratinocytes. However, how keratinocytes regulate melanin production is unclear. Here we report that melanin production in melanoma cells (B16F10 and MNT-1) was increased markedly on a keratinocyte-derived extracellular matrix compared with a melanoma cell-derived extracellular matrix. siRNA-mediated reduction of keratinocyte-derived laminin-332 expression decreased melanin synthesis in melanoma cells, and laminin-332, but not fibronectin, enhanced melanin content and ?-melanocyte-stimulating hormone-regulated melanin production in melanoma cells. Similar effects were observed in human melanocytes. Interestingly, however, laminin-332 did not affect the expression or activity of tyrosinase. Instead, laminin-332 promoted the uptake of extracellular tyrosine and, subsequently, increased intracellular levels of tyrosine in both melanocytes and melanoma cells. Taken together, these data strongly suggest that keratinocyte-derived laminin-332 contributes to melanin production by regulating tyrosine uptake. PMID:24951591

  17. Characterization of adhesive interactions between mast cells and laminin isoforms: evidence of a principal role for alpha 6 integrin.

    PubMed Central

    Vliagoftis, H; Metcalfe, D D

    1997-01-01

    Mast cells are known to adhere to laminin, although there is limited information on the characteristics of this event. To further examine this adhesive interaction, we thus determined the adherence of murine mast cell lines and primary bone marrow cultured mast cells (BMCMC) to murine laminin (mLN), human placental laminin-1 (hLN), merosin (laminin-2) and various laminin fragments, concentrating on activating stimuli, the involvement of integrins, and the effect on mast cell activation. Murine mast cells were found to adhere to both mLN and hLN and to merosin, not only following exposure to phorbol 12-myristate 13-acetate (PMA), but also after Fc epsilon RI aggregation or addition of stem cell factor (SCF). Adhesion to laminin was partially inhibited by soluble E8 and PA22-2, both fragments of laminin that promote mast-cell adhesion when bound on surfaces. Mast-cell lines and BMCMC consistently expressed high levels of alpha 6 integrin. Antibody to alpha 6 blocked spontaneous and inhibited activated mast-cell adhesion to hLN, and inhibited mast-cell adhesion to mLN and its fragment E8. Mast-cell adhesion to both laminin isoforms increased Fc epsilon RI-mediated mast-cell secretion. These observations demonstrate that mast-cell attachment to laminin is promoted by physiological stimuli, is mediated principally by alpha 6 integrin, and results in enhanced cell activation. Images Figure 4 PMID:9497498

  18. Mechanism for the activation of glutamate receptors

    Cancer.gov

    Scientists at the NIH have used a technique called cryo-electron microscopy to determine a molecular mechanism for the activation and desensitization of ionotropic glutamate receptors, a prominent class of neurotransmitter receptors in the brain and spina

  19. Laminin peptide YIGSR induces collagen synthesis in Hs27 human dermal fibroblasts

    SciTech Connect

    Yoon, Jong Hyuk; Kim, Jaeyoon; Lee, Hyeongjoo; Kim, So Young; Department of Convergence Medicine and Pharmaceutical Biosciences, Graduate School, Chung-Ang University, Seoul 156-756 ; Jang, Hwan-Hee; Ryu, Sung Ho; Kim, Beom Joon; Department of Convergence Medicine and Pharmaceutical Biosciences, Graduate School, Chung-Ang University, Seoul 156-756 ; Lee, Taehoon G.

    2012-11-23

    Highlights: Black-Right-Pointing-Pointer We identify a function of the YIGSR peptide to enhance collagen synthesis in Hs27. Black-Right-Pointing-Pointer YIGSR peptide enhanced collagen type 1 synthesis both of gene and protein levels. Black-Right-Pointing-Pointer There were no changes in cell proliferation and MMP-1 level in YIGSR treatment. Black-Right-Pointing-Pointer The YIGSR effect on collagen synthesis mediated activation of FAK, pyk2 and ERK. Black-Right-Pointing-Pointer The YIGSR-induced FAK and ERK activation was modulated by FAK and MEK inhibitors. -- Abstract: The dermal ECM is synthesized from fibroblasts and is primarily compromised of fibrillar collagen and elastic fibers, which support the mechanical strength and resiliency of skin, respectively. Laminin, a major glycoprotein located in the basement membrane, promotes cell adhesion, cell growth, differentiation, and migration. The laminin tyrosine-isoleucine-glycine-serine-arginine (YIGSR) peptide, corresponding to the 929-933 sequence of the {beta}1 chain, is known to be a functional motif with effects on the inhibition of tumor metastasis, the regulation of sensory axonal response and the inhibition of angiogenesis through high affinity to the 67 kDa laminin receptor. In this study, we identified a novel function of the YIGSR peptide to enhance collagen synthesis in human dermal fibroblasts. To elucidate this novel function regarding collagen synthesis, we treated human dermal fibroblasts with YIGSR peptide in both a time- and dose-dependent manner. According to subsequent experiments, we found that the YIGSR peptide strongly enhanced collagen type 1 synthesis without changing cell proliferation or cellular MMP-1 level. This YIGSR peptide-mediated collagen type 1 synthesis was modulated by FAK inhibitor and MEK inhibitor. This study clearly reveals that YIGSR peptide plays a novel function on the collagen type 1 synthesis of dermal fibroblasts and also suggests that YIGSR is a strong candidate peptide for the treatment of skin aging and wrinkles.

  20. Expression of s-laminin and laminin in the developing rat central nervous system.

    PubMed

    Hunter, D D; Llinas, R; Ard, M; Merlie, J P; Sanes, J R

    1992-09-01

    The extracellular matrix component, s-laminin, is a homologue of the B1 subunit of laminin. S-laminin is concentrated in the synaptic cleft at the neuromuscular junction and contains a site that is adhesive for motor neurons, suggesting that it may influence neuromuscular development. To ascertain whether s-laminin may also play roles in the genesis of the central nervous system, we have examined its expression in the brain and spinal cord of embryonic and postnatal rats. S-laminin was not detectable in synapse-rich areas of adults. However, s-laminin was present in discrete subsets of three laminin-containing structures: (1) In the developing cerebral cortex, laminin and s-laminin were expressed in the subplate, a transient layer through which neuroblasts migrate and cortical afferents grow. Both laminin and s-laminin disappeared as embryogenesis proceeded; however, laminin was more widely distributed and present longer than s-laminin. (2) In the developing spinal cord, laminin was present throughout the pia. In contrast, s-laminin was concentrated in the pia that overlies the floor plate, a region in which extracellular cues have been postulated to guide growing axons. (3) In central capillaries, s-laminin appeared perinatally, an interval during which the blood-brain barrier matures. In contrast, laminin was present in capillary walls of both embryos and adults. To extend our immunohistochemical results, we used biochemical methods to characterize s-laminin in brain. We found that authentic s-laminin mRNA is present in the embryonic brain, but that brain-derived s-laminin differs (perhaps by a posttranslational modification) from that derived from nonneural tissues. We also used tissue culture methods to show that glia are capable of synthesizing "brain-like" s-laminin, and of assembling it into an extracellular matrix. Thus, glia may be one cellular source of s-laminin in brain. Together, these results demonstrate that s-laminin is present in the developing central nervous system, and raise the possibility that this molecule may influence developmental processes. PMID:1401258

  1. Laminin-based Nanomaterials for Peripheral Nerve Tissue Engineering

    NASA Astrophysics Data System (ADS)

    Neal, Rebekah Anne

    Peripheral nerve transection occurs commonly in traumatic injury, causing motor and sensory deficits distal to the site of injury. One option for surgical repair is the nerve conduit. Conduits currently on the market are hollow tubes into which the nerve ends are sutured. Although these conduits fill the gap, they often fail due to the slow rate of regeneration over long gaps. To facilitate increased speed of regeneration and greater potential for functional recovery, the ideal conduit should provide biochemically relevant signals and physical guidance cues, thus playing an active role in peripheral nerve regeneration. In this dissertation, I fabricated laminin-1 and laminin-polycaprolactone (PCL) blend nanofibers that mimic the geometry and functionality of the peripheral nerve basement membrane. These fibers resist hydration in aqueous media and require no harsh chemical crosslinkers. Adhesion and differentiation of both neuron-like and neuroprogenitor cells is improved on laminin nanofibrous meshes over two-dimensional laminin substrates. Blend meshes with varying laminin content were characterized for composition, tensile properties, degradation rates, and bioactivity in terms of cell attachment and axonal elongation. I have established that 10% (wt) laminin content is sufficient to retain the significant neurite-promoting effects of laminin critical in peripheral nerve repair. In addition, I utilized modified collector plate design to manipulate electric field gradients during electrospinning for the fabrication of aligned nanofibers. These aligned substrates provide enhanced directional guidance cues to the regenerating axons. Finally, I replicated the clinical problem of peripheral nerve transection using a rat tibial nerve defect model for conduit implantation. When the lumens of conduits were filled with nanofiber meshes of varying laminin content and alignment, I observed significant recovery of sensory and motor function over six weeks. This recovery was supported by nerve conduction studies and electromyography which described impulse transmission, muscle stimulation, and foot twitch through the region of regeneration. These studies provide a firm foundation for the use of natural-synthetic blend electrospun nanofibers to enhance existing hollow nerve guidance conduits. The similarity in surgical technique and obvious benefit to the patient should lead to rapid translation into clinical application.

  2. Laminins and retinal vascular development

    PubMed Central

    Edwards, Malia M.; Lefebvre, Olivier

    2013-01-01

    The mechanisms controlling vascular development, both normal and pathological, are not yet fully understood. Many diseases, including cancer and diabetic retinopathy, involve abnormal blood vessel formation. Therefore, increasing knowledge of these mechanisms may help develop novel therapeutic targets. The identification of novel proteins or cells involved in this process would be particularly useful. The retina is an ideal model for studying vascular development because it is easy to access, particularly in rodents where this process occurs post-natally. Recent studies have suggested potential roles for laminin chains in vascular development of the retina. This review will provide an overview of these studies, demonstrating the importance of further research into the involvement of laminins in retinal blood vessel formation. PMID:23154403

  3. Identification of different laminin binding proteins in basolateral cell membranes of human colorectal carcinomas and normal colonic mucosa.

    PubMed Central

    Stallmach, A; Riese, H H; Schuppan, D; Bornhöft, G; Stein, H; Riecken, E O

    1991-01-01

    The adhesive properties of tumour cells to laminin, the major glycoprotein of basement membranes, play a crucial part in the complex process of tumour invasion and metastasis. We therefore investigated the expression of laminin binding proteins in isolated basolateral cell membranes of human colorectal carcinomas and the adjacent normal colonic mucosa. Cell membrane binding assays and immunoblotting experiments showed appreciable quantitative and qualitative differences in the expression of these proteins in neoplastic and normal tissue. Epithelial basolateral cell membranes of colorectal carcinomas bound five to eight times more radioactive labelled laminin than basolateral cell membranes of the adjacent normal colonic epithelium. The expression of laminin binding proteins with Mr 66,000-69,000 daltons corresponding to the so called 'Mr 67,000 dalton laminin receptor' was three to four times higher in colorectal carcinomas than in normal colonic epithelium. In addition, laminin binding proteins with higher molecular weights, which may be related to the family of integrins, were also increased in colorectal carcinomas. In particular, laminin binding proteins with Mr 180,000 daltons were exclusively expressed on neoplastic epithelial cells of human colorectal carcinomas. Our data suggest that certain classes of laminin binding proteins may be selectively expressed on colonic tumour cells, leading to an increased capacity for migration, invasion, and metastasis. Images Figure 1 PMID:2013422

  4. Cellular Interaction of Integrin ?3?1 with Laminin 5 Promotes Gap Junctional Communication

    PubMed Central

    Lampe, Paul D.; Nguyen, Beth P.; Gil, Susana; Usui, Marcia; Olerud, John; Takada, Yoshikazu; Carter, William G.

    1998-01-01

    Wounding of skin activates epidermal cell migration over exposed dermal collagen and fibronectin and over laminin 5 secreted into the provisional basement membrane. Gap junctional intercellular communication (GJIC) has been proposed to integrate the individual motile cells into a synchronized colony. We found that outgrowths of human keratinocytes in wounds or epibole cultures display parallel changes in the expression of laminin 5, integrin ?3?1, E-cadherin, and the gap junctional protein connexin 43. Adhesion of keratinocytes on laminin 5, collagen, and fibronectin was found to differentially regulate GJIC. When keratinocytes were adhered on laminin 5, both structural (assembly of connexin 43 in gap junctions) and functional (dye transfer) assays showed a two- to threefold increase compared with collagen and five- to eightfold over fibronectin. Based on studies with immobilized integrin antibody and integrin-transfected Chinese hamster ovary cells, the interaction of integrin ?3?1 with laminin 5 was sufficient to promote GJIC. Mapping of intermediate steps in the pathway linking ?3?1–laminin 5 interactions to GJIC indicated that protein trafficking and Rho signaling were both required. We suggest that adhesion of epithelial cells to laminin 5 in the basement membrane via ?3?1 promotes GJIC that integrates individual cells into synchronized epiboles. PMID:9852164

  5. A Review on the Potential Role of Basement Membrane Laminin in the Pathogenesis of Psoriasis.

    PubMed

    McFadden, J P; Kimber, I

    2016-01-01

    We have previously reviewed alterations to basement membrane laminin in psoriasis and how disruption of this layer could lead to at least some of the pathological changes observed. We here postulate that basement membrane laminin is the key antigen in driving psoriasis, inducing a T cell-mediated autoimmune response. For laminin to be considered as the key autoantigen in psoriasis, it would be reasonable to expect the following to be demonstrable: (1) that autoantigens are present in psoriatic inflammation; (2) that basement membrane laminin is perturbed in involved and uninvolved skin, and that some of the pathological changes associated with psoriasis could be predicted as a sequel to this; (3) that disruption of the basement membrane is among the earliest events in the evolution of psoriatic lesions; (4) that as streptococcal pharyngitis is the most clearly defined event to trigger or exacerbate psoriasis, then a T cell-mediated autoimmune response to laminin should be anticipated as a potential sequelae to streptococcal pharyngitis; (5) that T cells in psoriasis can be shown to react to peptides with homology to laminin; (6) that HLACw6, as the most closely related gene associated with psoriasis and which is involved in antigen expression, should be preferentially expressed within lesional psoriasis towards the basement membrane, together with other proximal associated immune activity; and (7) that there is some association between antilaminin pemphigoid, a humorally mediated autoimmune disease to skin basement membrane laminin, and psoriasis. We here review the data relevant to each of these requirements. PMID:26346598

  6. Laminins: Roles and Utility in Wound Repair

    PubMed Central

    Iorio, Valentina; Troughton, Lee D.; Hamill, Kevin J.

    2015-01-01

    Significance: Laminins are complex extracellular macromolecules that are major players in the control of a variety of core cell processes, including regulating rates of cell proliferation, differentiation, adhesion, and migration. Laminins, and related extracellular matrix components, have essential roles in tissue homeostasis; however, during wound healing, the same proteins are critical players in re-epithelialization and angiogenesis. Understanding how these proteins influence cell behavior in these different conditions holds great potential in identifying new strategies to enhance normal wound closure or to treat chronic/nonhealing wounds. Recent Advances: Laminin-derived bioactive peptides and, more recently, laminin-peptide conjugated scaffolds, have been designed to improve tissue regeneration after injuries. These peptides have been shown to be effective in decreasing inflammation and granulation tissue, and in promoting re-epithelialization, angiogenesis, and cell migration. Critical Issues: Although there is now a wealth of knowledge concerning laminin form and function, there are still areas of some controversy. These include the relative contribution of two laminin-based adhesive devices (focal contacts and hemidesmosomes) to the re-epithelialization process, the impact and implications of laminin proteolytic processing, and the importance of laminin polymer formation on cell behavior. In addition, the roles in wound healing of the laminin-related proteins, netrins, and LaNts are still to be fully defined. Future Directions: The future of laminin-based therapeutics potentially lies in the bioengineering of specific substrates to support laminin deposition for ex vivo expansion of autologous cells for graft formation and transplantation. Significant recent advances suggest that this goal is within sight. PMID:25945287

  7. Laminin and Fibronectin in Cell Adhesion: Enhanced Adhesion of Cells from Regenerating Liver to Laminin

    NASA Astrophysics Data System (ADS)

    Carlsson, Roland; Engvall, Eva; Freeman, Aaron; Ruoslahti, Erkki

    1981-04-01

    Laminin, a basement membrane glycoprotein isolated from cultures of mouse endodermal cells and rat yolk sac carcinoma cells, promoted the attachment of liver cells obtained from regenerating mouse liver. Cells from normal mouse liver attached readily to dishes coated with fibronectin but attached poorly to surfaces coated with laminin. Both proteins efficiently promoted the attachment of cells from livers undergoing regeneration. After regeneration, the attachment to laminin returned to the low levels found in animals not subjected to partial hepatectomy but attachment to fibronectin remained high. Immunofluorescent staining of sections of normal liver with antilaminin revealed the presence of laminin in or adjacent to the walls of the bile ducts and blood vessels. After induction of regeneration by partial hepatectomy, increased amounts of laminin appeared in the sinusoidal areas. After carbon tetrachloride poisoning, staining for laminin was especially pronounced in the necrotic and postnecrotic areas around the central veins. This additional expression of laminin was transient. It reached a maximum around 5-6 days after the injury and then gradually disappeared. These findings show that laminin is an adhesive protein. The increase of laminin in regenerating liver and the adhesiveness of cells from such livers to laminin suggest a role for laminin in the maintenance of a proper tissue organization during liver regeneration.

  8. Rethinking Molecular Mimicry in Rheumatic Heart Disease and Autoimmune Myocarditis: Laminin, Collagen IV, CAR, and B1AR as Initial Targets of Disease

    PubMed Central

    Root-Bernstein, Robert

    2014-01-01

    Rationale: Molecular mimicry theory (MMT) suggests that epitope mimicry between pathogens and human proteins can activate autoimmune disease. Group A streptococci (GAS) mimics human cardiac myosin in rheumatic heart disease (RHD) and coxsackie viruses (CX) mimic actin in autoimmune myocarditis (AM). But myosin and actin are immunologically inaccessible and unlikely initial targets. Extracellular cardiac proteins that mimic GAS and CX would be more likely. Objectives: To determine whether extracellular cardiac proteins such as coxsackie and adenovirus receptor (CAR), beta 1 adrenergic receptor (B1AR), CD55/DAF, laminin, and collagen IV mimic GAS, CX, and/or cardiac myosin or actin. Methods: BLAST 2.0 and LALIGN searches of the UniProt protein database were employed to identify potential molecular mimics. Quantitative enzyme-linked immunosorbent assay was used to measure antibody cross-reactivity. Measurements: Similarities were considered to be significant if a sequence contained at least 5 identical amino acids in 10. Antibodies were considered to be cross-reactive if the binding constant had a Kd less than 10-9 M. Main results: Group A streptococci mimics laminin, CAR, and myosin. CX mimics actin and collagen IV and B1AR. The similarity search results are mirrored by antibody cross-reactivities. Additionally, antibodies against laminin recognize antibodies against collagen IV; antibodies against actin recognize antibodies against myosin, and antibodies against GAS recognize antibodies against CX. Thus, there is both mimicry of extracellular proteins and antigenic complementarity between GAS-CX in RHD/AM. Conclusion: Rheumatic heart disease/AM may be due to combined infections of GAS with CX localized at cardiomyocytes that may produce a synergistic, hyperinflammatory response that cross-reacts with laminin, collagen IV, CAR, and/or B1AR. Epitope drift shifts the immune response to myosin and actin after cardiomyocytes become damaged. PMID:25191648

  9. Isolation of a laminin-binding protein from the protozoan parasite Leishmania donovani that may mediate cell adhesion.

    PubMed Central

    Ghosh, A; Bandyopadhyay, K; Kole, L; Das, P K

    1999-01-01

    Extracellular matrix (ECM)-binding proteins on the surface of Leishmania are thought to play a crucial role in the onset of leishmaniasis, as these parasites invade mononuclear phagocytes in various organs after migrating through the ECM. In a previous report, we presented several lines of evidence suggesting that Leishmania has a specific receptor for laminin, a major ECM protein, with a Kd in the nanomolar range. Here we describe the identification, purification and biochemical characterization of the Leishmania laminin receptor. When the outer membrane proteins of L. donovani were blotted on to nitrocellulose paper and probed with laminin, a prominent laminin-binding protein of 67 kDa was identified. The purified protein was isolated by a three-step process involving DEAE-cellulose, Con A (concanavalin A)-Sepharose and laminin-Sepharose affinity chromatography and was used to raise a monospecific antibody. The same protein was obtained when parasite membrane extracts were adsorbed to antibody affinity matrix and eluted with glycine. The affinity-purified protein bound to laminin in a detergent-solubilized form as well as after integration into artificial bilayers, and was subsequently characterized as an integral membrane protein. Metaperiodate oxidation and metabolic inhibition of glycosylation studies indicate the binding protein to be glycoprotein in nature and that N-linked oligosaccharides play a part in the interaction of laminin with the binding protein. Surface-labelled parasites attached to microtitre wells coated with laminin and the 67 kDa protein blocked the adhesion to laminin substrate. We propose that the 67 kDa protein is an adhesin involved in the attachment of Leishmania to host tissues. PMID:9895301

  10. Interdomain movements in metabotropic glutamate receptor activation

    PubMed Central

    Huang, Siluo; Cao, Jianhua; Jiang, Ming; Labesse, Gilles; Liu, Jianfeng; Pin, Jean-Philippe; Rondard, Philippe

    2011-01-01

    Many cell surface receptors are multimeric proteins, composed of several structural domains, some involved in ligand recognition, whereas others are responsible for signal transduction. In most cases, the mechanism of how ligand interaction in the extracellular domains leads to the activation of effector domains remains largely unknown. Here we examined how the extracellular ligand binding to the venus flytrap (VFT) domains of the dimeric metabotropic glutamate receptors activate the seven transmembrane (7TM) domains responsible for G protein activation. These two domains are interconnected by a cysteine-rich domain (CRD). We show that any of the four disulfide bridges of the CRD are required for the allosteric coupling between the VFT and the 7TM domains. More importantly, we show that a specific association of the two CRDs corresponds to the active state of the receptor. Indeed, a specific crosslinking of the CRDs with intersubunit disulfide bridges leads to fully constitutively active receptors, no longer activated by agonists nor by allosteric modulators. These data demonstrate that intersubunit movement at the level of the CRDs represents a key step in metabotropic glutamate receptor activation. PMID:21896740

  11. Using Nuclear Receptor Activity to Stratify Hepatocarcinogens

    PubMed Central

    Shah, Imran; Houck, Keith; Judson, Richard S.; Kavlock, Robert J.; Martin, Matthew T.; Reif, David M.; Wambaugh, John; Dix, David J.

    2011-01-01

    Background Nuclear receptors (NR) are a superfamily of ligand-activated transcription factors that control a range of cellular processes. Persistent stimulation of some NR is a non-genotoxic mechanism of rodent liver cancer with unclear relevance to humans. Here we report on a systematic analysis of new in vitro human NR activity data on 309 environmental chemicals in relationship to their liver cancer-related chronic outcomes in rodents. Results The effects of 309 environmental chemicals on human constitutive androstane receptors (CAR/NR1I3), pregnane X receptor (PXR/NR1I2), aryl hydrocarbon receptor (AhR), peroxisome proliferator-activated receptors (PPAR/NR1C), liver X receptors (LXR/NR1H), retinoic X receptors (RXR/NR2B) and steroid receptors (SR/NR3) were determined using in vitro data. Hepatic histopathology, observed in rodents after two years of chronic treatment for 171 of the 309 chemicals, was summarized by a cancer lesion progression grade. Chemicals that caused proliferative liver lesions in both rat and mouse were generally more active for the human receptors, relative to the compounds that only affected one rodent species, and these changes were significant for PPAR (p0.001), PXR (p0.01) and CAR (p0.05). Though most chemicals exhibited receptor promiscuity, multivariate analysis clustered them into relatively few NR activity combinations. The human NR activity pattern of chemicals weakly associated with the severity of rodent liver cancer lesion progression (p0.05). Conclusions The rodent carcinogens had higher in vitro potency for human NR relative to non-carcinogens. Structurally diverse chemicals with similar NR promiscuity patterns weakly associated with the severity of rodent liver cancer progression. While these results do not prove the role of NR activation in human liver cancer, they do have implications for nuclear receptor chemical biology and provide insights into putative toxicity pathways. More importantly, these findings suggest the utility of in vitro assays for stratifying environmental contaminants based on a combination of human bioactivity and rodent toxicity. PMID:21339822

  12. Deletion of integrin ?7 subunit does not aggravate the phenotype of laminin ?2 chain-deficient mice

    PubMed Central

    Gawlik, Kinga I.; Durbeej, Madeleine

    2015-01-01

    Laminin-211 is a major constituent of the skeletal muscle basement membrane, exerting its biological functions by binding to cell surface receptors integrin ?7?1 and dystroglycan (the latter is part of the dystrophin-glycoprotein complex). The importance of these molecules for normal muscle function is underscored by the fact that their respective deficiency leads to different forms of muscular dystrophy with different severity in humans and animal models. We recently demonstrated that laminin ?2 chain and members of the dystrophin-glycoprotein complex have overlapping but non-redundant roles despite being part of the same adhesion complex. To analyse whether laminin-211 and integrin ?7 subunit have non-redundant functions we generated mice deficient in laminin ?2 chain and integrin ?7 subunit (dy3K/itga7). We show that lack of both molecules did not exacerbate the severe phenotype of laminin ?2-chain deficient animals. They displayed the same weight, survival and dystrophic pattern of muscle biopsy, with similar degree of inflammation and fibrosis. These data suggest that laminin-211 and integrin ?7?1 have intersecting roles in skeletal muscle. PMID:26355035

  13. Nuclear estrogen receptors co-activation mechanisms.

    PubMed

    Skrzypczak, M; Kapka-Skrzypczak, L; Cyranka, M; Treeck, O; Wrobel, A; Matosiuk, D

    2013-01-01

    Estrogens play very important role in opening the transcription event, which is a final step of activation of the first order mediators as receptors or channels in the cell wall by information coming from the outside of the cell. For the long time the exact step by step mechanism of cellular transfer of information to the cell nuclei was not known. Currently many new informations are available. Very important seems the step of phosphorylation and therefore desensitization of the target proteins. All peptide kinases, especially serine and threonine, like protein kinases A and C, RAS and MAP kinases, cycline kinases are potential or confirmed biological targets. Except them elements of the transcription complexes like p160.SRC-1, histon acetyltransferase and histon deacetylase, CBP/p300, TRAP/DRIP, NSD1, PPAR?/PGC-1, NCOR1, SMRT, REA were also found useful. Finally estrogens are able to activate other receptors, namely aryl hydrocarbon receptors (AhR) and estrogen receptor related proteins (ERR). It is well known that many types of cancer are related to the direct or indirect excessive activation of nuclear estrogen receptors, therefore their inhibition could be crucial in many estrogen-related cancers. Understanding the interactions in such complexes would help in developing new and better multi-target cures and finding new ligands with better pharmacological and pharmacokinetic properties. PMID:23651307

  14. Alignment and composition of laminin–polycaprolactone nanofiber blends enhance peripheral nerve regeneration

    PubMed Central

    Neal, Rebekah A.; Tholpady, Sunil S.; Foley, Patricia L.; Swami, Nathan; Ogle, Roy C.; Botchwey, Edward A.

    2012-01-01

    Peripheral nerve transection occurs commonly in traumatic injury, causing deficits distal to the injury site. Conduits for repair currently on the market are hollow tubes; however, they often fail due to slow regeneration over long gaps. To facilitate increased regeneration speed and functional recovery, the ideal conduit should provide biochemically relevant signals and physical guidance cues, thus playing an active role in regeneration. To that end, laminin and laminin–polycaprolactone (PCL) blend nanofibers were fabricated to mimic peripheral nerve basement membrane. In vitro assays established 10% (wt) laminin content is sufficient to retain neurite-promoting effects of laminin. In addition, modified collector plate design to introduce an insulating gap enabled the fabrication of aligned nanofibers. The effects of laminin content and fiber orientation were evaluated in rat tibial nerve defect model. The lumens of conduits were filled with nanofiber meshes of varying laminin content and alignment to assess changes in motor and sensory recovery. Retrograde nerve conduction speed at 6 weeks was significantly faster in animals receiving aligned nanofiber conduits than in those receiving random nanofiber conduits. Animals receiving nanofiber-filled conduits showed some conduction in both anterograde and retrograde directions, whereas in animals receiving hollow conduits, no impulse conduction was detected. Aligned PCL nanofibers significantly improved motor function; aligned laminin blend nanofibers yielded the best sensory function recovery. In both cases, nanofiber-filled conduits resulted in better functional recovery than hollow conduits. These studies provide a firm foundation for the use of natural–synthetic blend electrospun nanofibers to enhance existing hollow nerve guidance conduits. PMID:22106069

  15. Activation of insulin receptors by lagerstroemin.

    PubMed

    Hattori, Katsuji; Sukenobu, Naoe; Sasaki, Tomo; Takasuga, Shunsuke; Hayashi, Takeo; Kasai, Ryoji; Yamasaki, Kazuo; Hazeki, Osamu

    2003-09-01

    Lagerstroemin, an ellagitannin isolated from the leaves of Lagerstroemia speciosa (L.) Pers. (Lythraceae), was examined for its biological activities. In rat adipocytes, the compound increased the rate of glucose uptake and decreased the isoproterenol-induced glycerol release. In Chinese hamster ovary cells expressing human insulin receptors, it increased the Erk activity. These insulin-like actions were accompanied by the increased tyrosine-phosphorylation of the beta-subunit of the insulin receptors. Tryptic digestion of the extracellular sites of the insulin receptors markedly increased the effective concentrations of insulin without changing those of lagerstroemin. Thus lagerstroemin was considered to cause its insulin-like actions by a mechanism different from that employed by insulin. PMID:14501154

  16. Dystroglycan Matrix Receptor Function in Cardiac Myocytes Is Important for Limiting Activity-Induced

    E-print Network

    Campbell, Kevin P.

    in dystrophin and each of the four sarcoglycans can cause Duchenne/Becker and limb girdle muscular dystrophies of dystroglycan and loss of its laminin-binding activity in genetic forms of human muscular dystrophy. Human of dystroglycan in cardiac muscle and smooth muscle in the development of cardiomyopathy in muscular dystrophies

  17. Regeneration of Aplysia Bag Cell Neurons is Synergistically Enhanced by Substrate-Bound Hemolymph Proteins and Laminin

    NASA Astrophysics Data System (ADS)

    Hyland, Callen; Dufrense, Eric R.; Forscher, Paul

    2014-04-01

    We have investigated Aplysia hemolymph as a source of endogenous factors to promote regeneration of bag cell neurons. We describe a novel synergistic effect between substrate-bound hemolymph proteins and laminin. This combination increased outgrowth and branching relative to either laminin or hemolymph alone. Notably, the addition of hemolymph to laminin substrates accelerated growth cone migration rate over ten-fold. Our results indicate that the active factor is either a high molecular weight protein or protein complex and is not the respiratory protein hemocyanin. Substrate-bound factor(s) from central nervous system-conditioned media also had a synergistic effect with laminin, suggesting a possible cooperation between humoral proteins and nervous system extracellular matrix. Further molecular characterization of active factors and their cellular targets is warranted on account of the magnitude of the effects reported here and their potential relevance for nervous system repair.

  18. Using Nuclear Receptor Activity to Stratify Hepatocarcinogens

    EPA Science Inventory

    Nuclear receptors (NR) are a superfamily of ligand-activated transcription factors that control a range of cellular processes. Persistent stimulation of some NR is a non-genotoxic mechanism of rodent liver cancer with unclear relevance to humans. Here we report on a systematic an...

  19. Receptor Dissociation and B-Cell Activation.

    PubMed

    Yang, Jianying; Reth, Michael

    2016-01-01

    The B-cell antigen receptor (BCR) is one of the most abundant receptors on the surface of B cells with roughly 100,000-200,000 copies per cell. Signaling through the BCR is crucial for the activation and differentiation of B cells. Unlike other receptors, the BCR can be activated by a large set of structurally different ligands, but the molecular mechanism of BCR activation is still a matter of controversy. Although dominant for a long time, the cross-link model (CLM) of BCR activation is not supported by recent studies of the nanoscale organization of the BCR on the surface of resting B cells. In contrast to the prediction of CLM, the numerous BCR complexes on these cells are not randomly distributed monomers but rather form oligomers which reside within membrane confinements. This finding is more in line with the dissociation activation model (DAM) , wherein B-cell activation is accompanied by an opening of the auto-inhibited BCR oligomers instead of a cross-linking of the BCR monomers . In this review, we discuss in detail the new findings and their implications for BCR signaling. PMID:26428245

  20. Proteinase-activated receptors (PARs) – focus on receptor-receptor-interactions and their physiological and pathophysiological impact

    PubMed Central

    2013-01-01

    Proteinase-activated receptors (PARs) are a subfamily of G protein-coupled receptors (GPCRs) with four members, PAR1, PAR2, PAR3 and PAR4, playing critical functions in hemostasis, thrombosis, embryonic development, wound healing, inflammation and cancer progression. PARs are characterized by a unique activation mechanism involving receptor cleavage by different proteinases at specific sites within the extracellular amino-terminus and the exposure of amino-terminal “tethered ligand“ domains that bind to and activate the cleaved receptors. After activation, the PAR family members are able to stimulate complex intracellular signalling networks via classical G protein-mediated pathways and beta-arrestin signalling. In addition, different receptor crosstalk mechanisms critically contribute to a high diversity of PAR signal transduction and receptor-trafficking processes that result in multiple physiological effects. In this review, we summarize current information about PAR-initiated physical and functional receptor interactions and their physiological and pathological roles. We focus especially on PAR homo- and heterodimerization, transactivation of receptor tyrosine kinases (RTKs) and receptor serine/threonine kinases (RSTKs), communication with other GPCRs, toll-like receptors and NOD-like receptors, ion channel receptors, and on PAR association with cargo receptors. In addition, we discuss the suitability of these receptor interaction mechanisms as targets for modulating PAR signalling in disease. PMID:24215724

  1. Activation of a nicotinic acetylcholine receptor.

    PubMed

    Sine, S M; Steinbach, J H

    1984-01-01

    We studied activation of the nicotinic acetylcholine (ACh) receptor on cells of a mouse clonal muscle cell line (BC3H1). We analyzed single-channel currents through outside-out patches elicited with various concentrations of acetylcholine (ACh), carbamylcholine (Carb) and suberyldicholine (Sub). Our goal is to determine a likely reaction scheme for receptor activation by agonist and to determine values of rate constants for transitions in that scheme. Over a wide range of agonist concentrations the open-time duration histograms are not described by single exponential functions, but are well-described by the sum of two exponentials, a brief-duration and a long-duration component. At high concentration, channel openings occur in groups and these groups contain an excess number of brief openings. We conclude that there are two open states of the ACh receptor with different mean open times and that a single receptor may open to either open state. The concentration dependence of the numbers of brief and long openings indicates that brief openings do not result from the opening of channels of receptors which have only one agonist molecule bound to them. Closed-time duration histograms exhibit a major brief component at low concentrations. We have used the method proposed by Colquhoun and Sakmann (1981) to analyze these brief closings and to extract estimates for the rates of channel opening (beta) and agonist dissociation (k-2). We find that this estimate of beta does not predict our closed-time histograms at high agonist concentration (ACh: 30-300 microM; Carb: 300-1,000 microM). We conclude that brief closings at low agonist concentrations do not result solely from transitions between the doubly-liganded open and the doubly-liganded closed states. Instead, we postulate the existence of a second closed-channel state coupled to the open state. PMID:6324901

  2. Monoclonal Antibodies to the Human Insulin Receptor that Activate Glucose Transport but not Insulin Receptor Kinase Activity

    NASA Astrophysics Data System (ADS)

    Forsayeth, John R.; Caro, Jose F.; Sinha, Madhur K.; Maddux, Betty A.; Goldfine, Ira D.

    1987-05-01

    Three mouse monoclonal antibodies were produced that reacted with the ? subunit of the human insulin receptor. All three both immunoprecipitated 125I-labeled insulin receptors from IM-9 lymphocytes and competitively inhibited 125I-labeled insulin binding to its receptor. Unlike insulin, the antibodies failed to stimulate receptor autophosphorylation in both intact IM-9 lymphocytes and purified human placental insulin receptors. Moreover, unlike insulin, the antibodies failed to stimulate receptor-mediated phosphorylation of exogenous substrates. However, like insulin, two of the three antibodies stimulated glucose transport in isolated human adipocytes. One antibody, on a molar basis, was as potent as insulin. These studies indicate, therefore, that monoclonal antibodies to the insulin receptor can mimic a major function of insulin without activating receptor kinase activity. They also raise the possibility that certain actions of insulin such as stimulation of glucose transport may not require the activation of receptor kinase activity.

  3. Peroxynitrous acid induces structural and functional modifications to basement membranes and its key component, laminin.

    PubMed

    Degendorfer, Georg; Chuang, Christine Y; Hammer, Astrid; Malle, Ernst; Davies, Michael J

    2015-12-01

    Basement membranes (BM) are specialized extracellular matrices underlying endothelial cells in the artery wall. Laminin, the most abundant BM glycoprotein, is a structural and biologically active component. Peroxynitrous acid (ONOOH), a potent oxidizing and nitrating agent, is formed in vivo at sites of inflammation from superoxide and nitric oxide radicals. Considerable data supports ONOOH formation in human atherosclerotic lesions, and an involvement of this oxidant in atherosclerosis development and lesion rupture. These effects may be mediated, at least in part, via extracellular matrix damage. In this study we demonstrate co-localization of 3-nitrotyrosine (a product of tyrosine damage by ONOOH) and laminin in human atherosclerotic lesions. ONOOH-induced damage to BM was characterized for isolated murine BM, and purified murine laminin-111. Exposure of laminin-111 to ONOOH resulted in dose-dependent loss of protein tyrosine and tryptophan residues, and formation of 3-nitrotyrosine, 6-nitrotryptophan and the cross-linked material di-tyrosine, as detected by amino acid analysis and Western blotting. These changes were accompanied by protein aggregation and fragmentation as detected by SDS-PAGE. Endothelial cell adhesion to isolated laminin-111 exposed to 10?M or higher levels of ONOOH was significantly decreased (~25%) compared to untreated controls. These data indicate that laminin is oxidized by equimolar or greater concentrations of ONOOH, with this resulting in structural and functional changes. These modifications, and resulting compromised cell-matrix interactions, may contribute to endothelial cell dysfunction, a weakening of the structure of atherosclerotic lesions, and an increased propensity to rupture. PMID:26453917

  4. Activation of Peroxisome Proliferator-Activated Receptor (PPAR ) by Rosiglitazone Suppresses

    E-print Network

    Abraham, Nader G.

    Activation of Peroxisome Proliferator-Activated Receptor (PPAR ) by Rosiglitazone Suppresses) that are ligands for peroxisome proliferator-activated receptor (PPAR ). Stimulation of PPAR suppresses bone formation and enhances marrow adipogenesis. We hypothe- sized that activation of PPAR down

  5. POMT1 mutation results in defective glycosylation and loss of laminin-binding

    E-print Network

    Campbell, Kevin P.

    reduced glycosylation and loss of laminin- binding activity of -DG in skeletal muscle. Methods. Patient was added 21 days after birth. Mild microphthalmia and corneal clouding also were observed. Serum creatine markedly delayed milestones. He could not control his head, roll over, or sit. He showed lack of facial

  6. The optical activity of bleached retinal receptors

    PubMed Central

    Villermet, Gabrielle M.; Weale, R. A.

    1969-01-01

    1. A study was made of the birefringence of retinal receptors obtained from the retinae of frogs, cats, rabbits and goldfish. 2. Normal rods and cones show similar optical activity. 3. The effects of fixatives, lipid solvents, de- and rehydration, and temperature were examined. 4. Rods fixed with formaldehyde preserve normal optical activity for a long time while glutaraldehyde changes it swiftly in a manner similar to that resulting from the application of heat. 5. Alcohol acts like a denaturing agent rather than as a solvent for rod lipids. 6. The action of glutaraldehyde on goldfish rods is instantaneous but cones preserve their normal activity for up to 1 hr. 7. A number of striking parallels between the presence of the R2 component of the early receptor potential (as reported in the literature) and optical activity are listed; it is suggested that normal optical activity is a prerequisite for the recording, if not the elicitation, of R2. Images(a)(b)(a)(b)Plate 2 PMID:5780551

  7. Therapeutic implications of constitutive activity of receptors: the example of the histamine H3 receptor.

    PubMed

    Schwartz, J C; Morisset, S; Rouleau, A; Ligneau, X; Gbahou, F; Tardivel-Lacombe, J; Stark, H; Schunack, W; Ganellin, C R; Arrang, J M

    2003-01-01

    Some G-protein-coupled receptors display constitutive activity, that is spontaneous activity in the absence of agonist: a proportion of the receptor population adopts a conformation that can bind and activate G proteins. Whereas this was mainly shown to occur with recombinant or pathologically mutated receptors, the physiological relevance of the process has remained debated. We have adressed this question in the case of the histamine H3 receptor, a presynaptic inhibitory receptor regulating histamine release in brain. Having identified a neutral antagonist and inverse agonists with variable intrinsic activity, we show that the native H3 receptor in brain displays high constitutive activity in vitro and, in vivo, controls the release of endogenous histamine. This implies that inverse agonists with high intrinsic activity should be preferred for therapeutic application as "cognitive enhancers" in several psychiatric disorders. PMID:12830926

  8. Activation of estrogen receptor increases and estrogen receptor decreases apolipoprotein E

    E-print Network

    Brinton, Roberta Diaz

    Activation of estrogen receptor increases and estrogen receptor decreases apolipoprotein E that, in carriers of apolipoprotein E4 (ApoE4), estrogen therapy increased the risk of late:1949­1954]. Estrogen mechanisms of action are mediated by two estrogen receptors (ERs), ER and ER . In this study, we

  9. Aryl-Hydrocarbon Receptor Activation Regulates Constitutive Androstane Receptor Levels in Murine

    E-print Network

    Perdew, Gary

    Aryl-Hydrocarbon Receptor Activation Regulates Constitutive Androstane Receptor Levels in Murine and Human Liver Rushang D. Patel, Brett D. Hollingshead, Curtis J. Omiecinski, and Gary H. Perdew The aryl-hydrocarbon 2007;46:209-218.) T he aryl-hydrocarbon receptor (AhR) is a ligand- dependent basic helix

  10. Angiogenic Laminin-Derived Peptides Stimulate Wound Healing

    PubMed Central

    Malinda, Katherine M.; Wysocki, Annette B.; Koblinski, Jennifer E.; Kleinman, Hynda K.; Ponce, M. Lourdes

    2008-01-01

    Acceleration of the wound healing process by using angiogenic peptides has been demonstrated previously. Here we used select laminin-111 peptides, A13 and C16, from the laminin ?1 and ?1 chain, respectively, to test whether they are able to stimulate wound healing in a rat full thickness wound model. The 12-mer peptides C16 and A13 are highly angiogenic and bind to integrins ?v?3 and ?5?1. We show that A13 increases wound reepithelialization as much as 17% over controls by day 4 and C16 increases coverage by 11%. Contraction of the treated wounds was increased as much as 11% for A13 and 8% for C16 at day 4. No differences were observed at day 7 with either peptide. The peptides also stimulated fibroblast migration in Boyden chamber assays. A13 increased cell migration as much as 2.4-fold on uncoated filters and as much as 16-fold on collagen type IV-coated filters over negative controls. Similarly, C16 also stimulated migration 1.8-fold on uncoated filters and as much as 12-fold on collagen-coated filters. A13 and C16 significantly decreased expression of the pro and active forms of matrix metalloproteinase 2 in foreskin fibroblasts indicating their role in collagen accumulation. We conclude that small bioactive angiogenic peptides can promote dermal wound healing and may offer a new class of stable and chemically manipulable therapeutics for wound healing. PMID:18603014

  11. Laminin-Mediated Interactions in Thymocyte Migration and Development

    PubMed Central

    Savino, Wilson; Mendes-da-Cruz, Daniella Aręas; Golbert, Daiane Cristina Ferreira; Riederer, Ingo; Cotta-de-Almeida, Vinicius

    2015-01-01

    Intrathymic T-cell differentiation is a key process for the development and maintenance of cell-mediated immunity, and occurs concomitantly to highly regulated migratory events. We have proposed a multivectorial model for describing intrathymic thymocyte migration. One of the individual vectors comprises interactions mediated by laminins (LMs), a heterotrimeric protein family of the extracellular matrix. Several LMs are expressed in the thymus, being produced by microenvironmental cells, particularly thymic epithelial cells (TECs). Also, thymocytes and epithelial cells express integrin-type LM receptors. Functionally, it has been reported that the dy/dy mutant mouse (lacking the LM isoform 211) exhibits defective thymocyte differentiation. Several data show haptotactic effects of LMs upon thymocytes, as well as their adhesion on TECs; both effects being prevented by anti-LM or anti-LM receptor antibodies. Interestingly, LM synergizes with chemokines to enhance thymocyte migration, whereas classe-3 semaphorins and B ephrins, which exhibit chemorepulsive effects in the thymus, downregulate LM-mediated migratory responses of thymocytes. More recently, we showed that knocking down the ITGA6 gene (which encodes the ?6 integrin chain of LM receptors) in human TECs modulates a large number of cell migration-related genes and results in changes of adhesion pattern of thymocytes onto the thymic epithelium. Overall, LM-mediated interactions can be placed at the cross-road of the multivectorial process of thymocyte migration, with a direct influence per se, as well as by modulating other molecular interactions associated with the intrathymic-trafficking events. PMID:26635793

  12. How IGF-1 activates its receptor

    PubMed Central

    Kavran, Jennifer M; McCabe, Jacqueline M; Byrne, Patrick O; Connacher, Mary Katherine; Wang, Zhihong; Ramek, Alexander; Sarabipour, Sarvenaz; Shan, Yibing; Shaw, David E; Hristova, Kalina; Cole, Philip A; Leahy, Daniel J

    2014-01-01

    The type I insulin-like growth factor receptor (IGF1R) is involved in growth and survival of normal and neoplastic cells. A ligand-dependent conformational change is thought to regulate IGF1R activity, but the nature of this change is unclear. We point out an underappreciated dimer in the crystal structure of the related Insulin Receptor (IR) with Insulin bound that allows direct comparison with unliganded IR and suggests a mechanism by which ligand regulates IR/IGF1R activity. We test this mechanism in a series of biochemical and biophysical assays and find the IGF1R ectodomain maintains an autoinhibited state in which the TMs are held apart. Ligand binding releases this constraint, allowing TM association and unleashing an intrinsic propensity of the intracellular regions to autophosphorylate. Enzymatic studies of full-length and kinase-containing fragments show phosphorylated IGF1R is fully active independent of ligand and the extracellular-TM regions. The key step triggered by ligand binding is thus autophosphorylation. DOI: http://dx.doi.org/10.7554/eLife.03772.001 PMID:25255214

  13. Adhesion of membranes via actively switched receptors

    E-print Network

    Bartosz Rozycki; Reinhard Lipowsky; Thomas R. Weikl

    2005-12-20

    We consider a theoretical model for membranes with adhesive receptors, or stickers, that are actively switched between two conformational states. In their 'on'-state, the stickers bind to ligands in an apposing membrane, whereas they do not interact with the ligands in their 'off'-state. We show that the adhesiveness of the membranes depends sensitively on the rates of the conformational switching process. This dependence is reflected in a resonance at intermediate switching rates, which can lead to large membrane separations and unbinding. Our results may provide insights into novel mechanisms for the controlled adhesion of biological or biomimetic membranes.

  14. 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 Fc? receptor (Fc?R) 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 Fc?Rs potentiate their ability to trigger TNFRSF receptor signaling. However, only recently has the relevance of oligomerization and Fc?R 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 Fc?R 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

  15. Identification of Gene Markers for Activation of the Nuclear Receptor Pregnane X Receptor

    EPA Science Inventory

    Many environmentally-relevant chemicals and drugs activate the nuclear receptor pregnane X receptor (PXR). Activation of PXR in the mouse liver can lead to increases in liver weight in part through increased hepatocyte replication similar to chemicals that activate other nuclear ...

  16. Model for growth hormone receptor activation based on subunit rotation within a receptor dimer

    SciTech Connect

    Brown, Richard J.; Adams, Julian J.; Pelekanos, Rebecca A.; Wan, Yu; McKinstry, William J.; Palethorpe, Kathryn; Seeber, Ruth M.; Monks, Thea A.; Eidne, Karin A.; Parker, Michael W.; Waters, Michael J.

    2010-07-13

    Growth hormone is believed to activate the growth hormone receptor (GHR) by dimerizing two identical receptor subunits, leading to activation of JAK2 kinase associated with the cytoplasmic domain. However, we have reported previously that dimerization alone is insufficient to activate full-length GHR. By comparing the crystal structure of the liganded and unliganded human GHR extracellular domain, we show here that there is no substantial change in its conformation on ligand binding. However, the receptor can be activated by rotation without ligand by inserting a defined number of alanine residues within the transmembrane domain. Fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET) and coimmunoprecipitation studies suggest that receptor subunits undergo specific transmembrane interactions independent of hormone binding. We propose an activation mechanism involving a relative rotation of subunits within a dimeric receptor as a result of asymmetric placement of the receptor-binding sites on the ligand.

  17. Receptor-activating autoantibodies and disease: preeclampsia and beyond

    PubMed Central

    Xia, Yang; Kellems, Rodney E

    2012-01-01

    The research reviewed in this article provides examples of autoantibody-mediated receptor activation that likely contributes to disease. The classic example is Graves’ hyperthyroidism, in which autoantibodies activate the thyroid-stimulating hormone receptor resulting in overproduction of thyroid hormones. Other compelling examples come from the cardiovascular literature and include agonistic autoantibodies targeting the cardiac ?1-adrenergic receptor, which are associated with dilated cardiomyopathy. Autoantibodies capable of activating ?1-adrenergic receptors are associated with refractory hypertension and cardiomyopathy. A prominent example is preeclampsia, a hypertensive disease of pregnancy, characterized by the presence of autoantibodies that activate the major angiotensin receptor, AT1. AT1 receptor-activating autoantibodies are also observed in kidney transplant recipients suffering from severe vascular rejection and malignant hypertension. AT1 receptor-activating autoantibodies and antibodies that activate the endothelin-1 receptor, ETA, are prevalent in individuals diagnosed with systemic sclerosis. Thus, the presence of agonistic autoantibodies directed to G protein-coupled receptors has been observed in numerous cardiovascular disease states. Rapidly emerging evidence indicates that receptor-activating autoantibodies contribute to disease, and that efforts to detect and remove these pathogenic autoantibodies or block their actions will provide promising therapeutic possibilities. PMID:21895478

  18. Receptor-activating autoantibodies and disease: preeclampsia and beyond.

    PubMed

    Xia, Yang; Kellems, Rodney E

    2011-09-01

    The research reviewed in this article provides examples of autoantibody-mediated receptor activation that likely contributes to disease. The classic example is Graves' hyperthyroidism, in which autoantibodies activate the thyroid-stimulating hormone receptor resulting in overproduction of thyroid hormones. Other compelling examples come from the cardiovascular literature and include agonistic autoantibodies targeting the cardiac ?(1)-adrenergic receptor, which are associated with dilated cardiomyopathy. Autoantibodies capable of activating ?(1)-adrenergic receptors are associated with refractory hypertension and cardiomyopathy. A prominent example is preeclampsia, a hypertensive disease of pregnancy, characterized by the presence of autoantibodies that activate the major angiotensin receptor, AT(1). AT(1) receptor-activating autoantibodies are also observed in kidney transplant recipients suffering from severe vascular rejection and malignant hypertension. AT(1) receptor-activating autoantibodies and antibodies that activate the endothelin-1 receptor, ET(A), are prevalent in individuals diagnosed with systemic sclerosis. Thus, the presence of agonistic autoantibodies directed to G protein-coupled receptors has been observed in numerous cardiovascular disease states. Rapidly emerging evidence indicates that receptor-activating autoantibodies contribute to disease, and that efforts to detect and remove these pathogenic autoantibodies or block their actions will provide promising therapeutic possibilities. PMID:21895478

  19. A New First Step in Activation of Steroid Receptors

    E-print Network

    Abraham, Nader G.

    A New First Step in Activation of Steroid Receptors HORMONE-INDUCED SWITCHING OF FKBP51 AND FKBP52 known event in steroid receptor signaling and provide the first evi- dence of differential roles for FKBP51 and FKBP52 im- munophilins in the control of steroid receptor subcellu- lar localization

  20. Activation of neurotensin receptor type 1 attenuates locomotor activity.

    PubMed

    Vadnie, Chelsea A; Hinton, David J; Choi, Sun; Choi, YuBin; Ruby, Christina L; Oliveros, Alfredo; Prieto, Miguel L; Park, Jun Hyun; Choi, Doo-Sup

    2014-10-01

    Intracerebroventricular administration of neurotensin (NT) suppresses locomotor activity. However, the brain regions that mediate the locomotor depressant effect of NT and receptor subtype-specific mechanisms involved are unclear. Using a brain-penetrating, selective NT receptor type 1 (NTS1) agonist PD149163, we investigated the effect of systemic and brain region-specific NTS1 activation on locomotor activity. Systemic administration of PD149163 attenuated the locomotor activity of C57BL/6J mice both in a novel environment and in their homecage. However, mice developed tolerance to the hypolocomotor effect of PD149163 (0.1 mg/kg, i.p.). Since NTS1 is known to modulate dopaminergic signaling, we examined whether PD149163 blocks dopamine receptor-mediated hyperactivity. Pretreatment with PD149163 (0.1 or 0.05 mg/kg, i.p.) inhibited D2R agonist bromocriptine (8 mg/kg, i.p.)-mediated hyperactivity. D1R agonist SKF-81297 (8 mg/kg, i.p.)-induced hyperlocomotion was only inhibited by 0.1 mg/kg of PD149163. Since the nucleus accumbens (NAc) and medial prefrontal cortex (mPFC) have been implicated in the behavioral effects of NT, we examined whether microinjection of PD149163 into these regions reduces locomotion. Microinjection of PD149163 (2 pmol) into the NAc, but not the mPFC suppressed locomotor activity. In summary, our results indicate that systemic and intra-NAc activation of NTS1 is sufficient to reduce locomotion and NTS1 activation inhibits D2R-mediated hyperactivity. Our study will be helpful to identify pharmacological factors and a possible therapeutic window for NTS1-targeted therapies for movement disorders. PMID:24929110

  1. Modulation of Glucagon Receptor Pharmacology by Receptor Activity-modifying Protein-2 (RAMP2)*

    PubMed Central

    Weston, Cathryn; Lu, Jing; Li, Naichang; Barkan, Kerry; Richards, Gareth O.; Roberts, David J.; Skerry, Timothy M.; Poyner, David; Pardamwar, Meenakshi; Reynolds, Christopher A.; Dowell, Simon J.; Willars, Gary B.; Ladds, Graham

    2015-01-01

    The glucagon and glucagon-like peptide-1 (GLP-1) receptors play important, opposing roles in regulating blood glucose levels. Consequently, these receptors have been identified as targets for novel diabetes treatments. However, drugs acting at the GLP-1 receptor, although having clinical efficacy, have been associated with severe adverse side-effects, and targeting of the glucagon receptor has yet to be successful. Here we use a combination of yeast reporter assays and mammalian systems to provide a more complete understanding of glucagon receptor signaling, considering the effect of multiple ligands, association with the receptor-interacting protein receptor activity-modifying protein-2 (RAMP2), and the role of individual G protein ?-subunits. We demonstrate that RAMP2 alters both ligand selectivity and G protein preference of the glucagon receptor. Importantly, we also uncover novel cross-reactivity of therapeutically used GLP-1 receptor ligands at the glucagon receptor that is abolished by RAMP2 interaction. This study reveals the glucagon receptor as a previously unidentified target for GLP-1 receptor agonists and highlights a role for RAMP2 in regulating its pharmacology. Such previously unrecognized functions of RAMPs highlight the need to consider all receptor-interacting proteins in future drug development. PMID:26198634

  2. Defining the metabolic effect of peroxisome proliferator-activated receptor ? activation

    E-print Network

    Roberts, Lee D

    2010-06-08

    Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that function as ligand activated transcription factors. There are three identified isotypes: PPAR alpha, PPAR gamma and PPAR delta, together controlling the expression...

  3. Identification of a Treponema pallidum laminin-binding protein.

    PubMed

    Cameron, Caroline E

    2003-05-01

    Host extracellular matrix (ECM) components represent ideal microbial adhesion targets that many pathogens use for colonization of tissues and initiation of infection. This study investigated the interaction of the spirochete Treponema pallidum with the ECM component laminin. To identify candidate laminin-binding adhesins, the T. pallidum genome was analyzed to predict open reading frames that encode putative outer membrane proteins, as these proteins interact directly with host ECM components. Subsequent recombinant expression of these proteins and analysis of their laminin-binding potential identified one protein, Tp0751, that demonstrated specific attachment to laminin. Tp0751 attached to laminin in a dose-dependent, saturable manner but did not attach to the ECM component collagen type I or IV or to the negative control proteins fetuin or bovine serum albumin. Sodium metaperiodate treatment of laminin reduced the Tp0751-laminin interaction in a concentration-dependent manner, suggesting that oligosaccharides play a role in this interaction. In addition, Tp0751-specific antibodies were detected in serum samples collected from both experimental and natural syphilis infections, indicating that Tp0751 is expressed in vivo during the course of infection. Collectively, these experiments identified Tp0751 as a laminin-binding protein that is expressed during infection and may be involved in attachment of T. pallidum to host tissues. PMID:12704124

  4. GLYCOSYLATION INFLUENCES THE LIGAND BINDING ACTIVITIES OF MANNOSE RECEPTOR

    E-print Network

    8 GLYCOSYLATION INFLUENCES THE LIGAND BINDING ACTIVITIES OF MANNOSE RECEPTOR Yunpeng Roc Su1QU, Oxford, UK Murine mannose receptor (MR) contains seven N-linked and three O-linked oligosac modified with Man 5 GlcNAc 2 sugars (Man-5 MR) completely lost its mannose-internalisation activity, which

  5. Glycosylation Influences the Lectin Activities of the Macrophage Mannose Receptor*

    E-print Network

    Glycosylation Influences the Lectin Activities of the Macrophage Mannose Receptor* Received, University of Oxford, South Parks Road, Oxford OX1 3RE, United Kingdom The mannose receptor (MR) is a heavily mannose internaliza- tion activity, but could internalize sulfated glycans. Accordingly, purified MR

  6. Sustained activation of STAT5 is essential for chromatin remodeling and maintenance of mammary-specific function

    SciTech Connect

    Xu, Ren; Nelson, Celeste M.; Muschler, John L.; Veiseh, Mandana; Vonderhaar, Barbara K.; Bissell, Mina J.

    2009-06-03

    Epithelial cells, once dissociated and placed in two-dimensional (2D) cultures, rapidly lose tissue-specific functions. We showed previously that in addition to prolactin, signaling by laminin-111 was necessary to restore functional differentiation of mammary epithelia. Here, we elucidate two additional aspects of laminin-111 action. We show that in 2D cultures, the prolactin receptor is basolaterally localized and physically segregated from its apically placed ligand. Detachment of the cells exposes the receptor to ligation by prolactin leading to signal transducers and activators of transcription protein 5 (STAT5) activation, but only transiently and not sufficiently for induction of milk protein expression. We show that laminin-111 reorganizes mammary cells into polarized acini, allowing both the exposure of the prolactin receptor and sustained activation of STAT5. The use of constitutively active STAT5 constructs showed that the latter is necessary and sufficient for chromatin reorganization and {beta}-casein transcription. These results underscore the crucial role of continuous laminin signaling and polarized tissue architecture in maintenance of transcription factor activation, chromatin organization, and tissue-specific gene expression.

  7. Identification of Modulators of the Nuclear Receptor Peroxisome Proliferator-Activated Receptor ? (PPAR?) in a Mouse Liver Gene Expression Compendium

    EPA Science Inventory

    The nuclear receptor family member peroxisome proliferator-activated receptor ? (PPAR?) is activated by therapeutic hypolipidemic drugs and environmentally-relevant chemicals to regulate genes involved in lipid transport and catabolism. Chronic activation of PPAR? in rodents inc...

  8. The effect of laminin-1-doped nanoroughened implant surfaces: gene expression and morphological evaluation.

    PubMed

    Schwartz-Filho, Humberto Osvaldo; Bougas, Kostas; Coelho, Paulo G; Xue, Ying; Hayashi, Mariko; Faeda, Rafael Silveira; Marcantonio, Rosemary Adriana Chiérici; Ono, Daisuke; Kobayashi, Fumio; Mustafa, Kamal; Wennerberg, Ann; Jimbo, Ryo

    2012-01-01

    Aim. This study aimed to observe the morphological and molecular effect of laminin-1 doping to nanostructured implant surfaces in a rabbit model. Materials and Methods. Nanostructured implants were coated with laminin-1 (test; dilution, 100??g/mL) and inserted into the rabbit tibiae. Noncoated implants were used as controls. After 2 weeks of healing, the implants were removed and subjected to morphological analysis using scanning electron microscopy (SEM) and gene expression analysis using the real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Results. SEM revealed bony tissue attachment for both control and test implants. Real-time RT-PCR analysis showed that the expression of osteoblast markers RUNX-2, osteocalcin, alkaline phosphatase, and collagen I was higher (1.62-fold, 1.53-fold, 1.97-fold, and 1.04-fold, resp.) for the implants modified by laminin-1 relative to the control. All osteoclast markers investigated in the study presented higher expression on the test implants than controls as follows: tartrate-resistant acid phosphatase (1.67-fold), calcitonin receptor (1.35-fold), and ATPase (1.25-fold). The test implants demonstrated higher expression of inflammatory markers interleukin-10 (1.53-fold) and tumour necrosis factor-? (1.61-fold) relative to controls. Conclusion. The protein-doped surface showed higher gene expression of typical genes involved in the osseointegration cascade than the control surface. PMID:23304151

  9. Peroxisome proliferator-activated receptors-alpha modulate dopamine cell activity through nicotinic receptors

    PubMed Central

    Melis, Miriam; Carta, Stefano; Fattore, Liana; Tolu, Stefania; Yasar, Sevil; Goldberg, Steven R.; Fratta, Walter; Maskos, Uwe; Pistis, Marco

    2010-01-01

    Background Modulation of midbrain dopamine neurons by nicotinic acetylcholine receptors (nAChRs) plays an important role in behavior, cognition, motivation and reward. Specifically, nAChRs containing ?2 subunits (?2-nAChRs) switch dopamine cells from a resting to an excited state. However, how ?2-nAChRs can be modulated and thereby dopamine firing activity be affected is still elusive. Because changes in dopamine cell activity are reflected in the dynamics of micro-circuits generating altered responses to stimuli/inputs, factors regulating their state are fundamental. Among these, endogenous ligands to the nuclear receptor-transcription factor peroxisome proliferator-activated receptors type-alpha (PPAR?) have been recently found to suppress nicotine-induced responses of dopamine neurons. Methods We used both in vitro and in vivo electrophysiological techniques together with behavioral analysis to investigate on the effects of modulation of PPAR? in Sprague Dawley rat and C57BLJ/6 mouse dopamine neurons, and their interactions with ?2-nAChRs. To this aim, we took advantage of a selective re-expression of ?2-nAChR exclusively in dopamine cells by stereotaxically injecting a lentiviral vector in the mouse ventral tegmental area. Results We found that activation of PPAR? decreases in vitro both dopamine cell activity and ventral tegmental area net output through negative modulation of ?2-nAChRs. Additionally, PPAR? activation in vivo reduces both the number of spontaneously active dopamine neurons and nicotine-induced increased locomotion. Conclusions Our combined findings suggest PPAR? ligands as important negative modulators of ?2-nAChRs on dopamine neurons. Thus, PPAR? ligands might prove beneficial in treating those disorders where dopamine dysfunction plays a prominent role, such as schizophrenia and nicotine addiction. PMID:20570248

  10. Neuropeptide Y receptor mediates activation of ERK1/2 via transactivation of the IGF receptor.

    PubMed

    Lecat, Sandra; Belemnaba, Lazare; Galzi, Jean-Luc; Bucher, Bernard

    2015-07-01

    Neuropeptide Y binds to G-protein coupled receptors whose action results in inhibition of adenylyl cyclase activity. Using HEK293 cells stably expressing the native neuropeptide Y Y1 receptors, we found that the NPY agonist elicits a transient phosphorylation of the extracellular signal-regulated kinases (ERK1/2). We first show that ERK1/2 activation following Y1 receptor stimulation is dependent on heterotrimeric Gi/o since it is completely inhibited by pre-treatment with pertussis toxin. In addition, ERK1/2 activation is internalization-independent since mutant Y1 receptors unable to recruit ?-arrestins, can still activate ERK signaling to the same extent as wild-type receptors. We next show that this activation of the MAPK pathway is inhibited by the MEK inhibitor U0126, is not dependent on calcium signaling at the Y1 receptor (no effect upon inhibition of phospholipase C, protein kinase C or protein kinase D) but instead dependent on G?/? and associated signaling pathways that activate PI3-kinase. Although inhibition of the epidermal-growth factor receptor tyrosine kinase did not influence NPY-induced ERK1/2 activation, we show that the inhibition of insulin growth factor receptor IGFR by AG1024 completely blocks activation of ERK1/2 by the Y1 receptor. This G?/?-PI3K-AG1024-sensitive pathway does not involve activation of IGFR through the release of a soluble ligand by metalloproteinases since it is not affected by the metalloproteinase inhibitor marimastat. Finally, we found that a similar pathway, sensitive to wortmannin-AG1024 but insensitive to marimastat, is implicated in activation of ERK signaling in HEK293 cells by endogenously expressed GPCRs coupled to Gq-protein (muscarinic M3 receptors) or coupled to Gs-protein (endothelin ETB receptors). Our analysis is the first to show that ?-arrestin recruitment to the NPY Y1 receptor is not necessary for MAPK activation by this receptor but that transactivation of the IGFR receptor is required. PMID:25817573

  11. The Orphan Nuclear Receptor TR4 Is a Vitamin A-activated Nuclear Receptor

    SciTech Connect

    Zhou, X. Edward; Suino-Powell, Kelly M.; Xu, Yong; Chan, Cee-Wah; Tanabe, Osamu; Kruse, Schoen W.; Reynolds, Ross; Engel, James Douglas; Xu, H. Eric

    2015-11-30

    Testicular receptors 2 and 4 (TR2/4) constitute a subgroup of orphan nuclear receptors that play important roles in spermatogenesis, lipid and lipoprotein regulation, and the development of the central nervous system. Currently, little is known about the structural features and the ligand regulation of these receptors. Here we report the crystal structure of the ligand-free TR4 ligand binding domain, which reveals an autorepressed conformation. The ligand binding pocket of TR4 is filled by the C-terminal half of helix 10, and the cofactor binding site is occupied by the AF-2 helix, thus preventing ligand-independent activation of the receptor. However, TR4 exhibits constitutive transcriptional activity on multiple promoters, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, or ligand binding substantially reduce the transcriptional activity of this receptor. Importantly, both retinol and retinoic acid are able to promote TR4 to recruit coactivators and to activate a TR4-regulated reporter. These findings demonstrate that TR4 is a ligand-regulated nuclear receptor and suggest that retinoids might have a much wider regulatory role via activation of orphan receptors such as TR4.

  12. Plant cysteine proteases that evoke itch activate protease-activated receptors

    PubMed Central

    Reddy, V.B.; Lerner, E.A.

    2013-01-01

    Background Bromelain, ficin and papain are cysteine proteases from plants that produce itch upon injection into skin. Their mechanism of action has not been considered previously. Objectives To determine the mechanism by which these proteases function. Methods The ability of these proteases to activate protease-activated receptors was determined by ratiometric calcium imaging. Results We show here that bromelain, ficin and papain activate protease-activated receptors 2 and 4. Conclusions Bromelain, ficin and papain function as signalling molecules and activate protease-activated receptors. Activation of these receptors is the likely mechanism by which these proteases evoke itch. PMID:20491769

  13. Interdicting Gq Activation in Airway Disease by Receptor-Dependent and Receptor-Independent Mechanisms.

    PubMed

    Carr, Richard; Koziol-White, Cynthia; Zhang, Jie; Lam, Hong; An, Steven S; Tall, Gregory G; Panettieri, Reynold A; Benovic, Jeffrey L

    2016-01-01

    G?q?? heterotrimer (Gq), an important mediator in the pathology of airway disease, plays a central role in bronchoconstriction and airway remodeling, including airway smooth muscle growth and inflammation. Current therapeutic strategies to treat airway disease include the use of muscarinic and leukotriene receptor antagonists; however, these pharmaceuticals demonstrate a limited clinical efficacy as multiple Gq-coupled receptor subtypes contribute to these pathologies. Thus, broadly inhibiting the activation of Gq may be an advantageous therapeutic approach. Here, we investigated the effects of broadly inhibiting Gq activation in vitro and ex vivo using receptor-dependent and receptor-independent strategies. P4pal-10 is a protease activated receptor 4-derived pepducin that exhibits efficacy toward multiple Gq-coupled receptors. Mechanistic studies demonstrated that P4pal-10 selectively inhibits all G protein coupling to several Gq-coupled receptors, including protease activated receptor 1, muscarinic acetylcholine M3, and histamine H1 receptors, while demonstrating no direct effect on Gq. We also evaluated the ability of FR900359, also known as UBO-QIC, to directly inhibit Gq activation. FR900359 inhibited spontaneous G?q nucleotide exchange, while having little effect on G?s??, G?i??, or G?12/13?? heterotrimer activity. Both P4pal-10 and FR900359 inhibited Gq-mediated intracellular signaling and primary human airway smooth muscle growth, whereas only FR900359 effectively interdicted agonist-promoted airway contraction in human precision cut lung slices. These studies serve as a proof of concept that the broad-based inhibition of Gq activation may be a useful therapeutic approach to treat multiple common pathologies of airway disease. PMID:26464325

  14. The ? Opioid Receptor Agonist SNC80 Selectively Activates Heteromeric ?–? Opioid Receptors

    PubMed Central

    2012-01-01

    Coexpressed and colocalized ?- and ?-opioid receptors have been established to exist as heteromers in cultured cells and in vivo. However the biological significance of opioid receptor heteromer activation is less clear. To explore this significance, the efficacy of selective activation of opioid receptors by SNC80 was assessed in vitro in cells singly and coexpressing opioid receptors using a chimeric G-protein-mediated calcium fluorescence assay, SNC80 produced a substantially more robust response in cells expressing ?–? heteromers than in all other cell lines. Intrathecal SNC80 administration in ?- and ?-opioid receptor knockout mice produced diminished antinociceptive activity compared with wild type. The combined in vivo and in vitro results suggest that SNC80 selectively activates ?–? heteromers to produce maximal antinociception. These data contrast with the current view that SNC80 selectively activates ?-opioid receptor homomers to produce antinociception. Thus, the data suggest that heteromeric ?–? receptors should be considered as a target when SNC80 is employed as a pharmacological tool in vivo. PMID:22860219

  15. Characterization of peroxisome proliferator-activiated receptor alpha (PPARalpha)-independent effects of PPARalpha activators in the rodent liver: Di(2-ethylehexyl) phthalate activates the constitutive activated receptor

    EPA Science Inventory

    Peroxisome proliferator chemicals (PPC) are thought to mediate their effects in rodents on hepatocyte growth and liver cancer through the nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha). Recent studies indicate that the plasticizer di-2-ethylhexyl ph...

  16. Constitutively active ALK2 receptor mutants require type II receptor cooperation.

    PubMed

    Bagarova, Jana; Vonner, Ashley J; Armstrong, Kelli A; Börgermann, Jan; Lai, Carol S C; Deng, Donna Y; Beppu, Hideyuki; Alfano, Ivan; Filippakopoulos, Panagis; Morrell, Nicholas W; Bullock, Alex N; Knaus, Petra; Mishina, Yuji; Yu, Paul B

    2013-06-01

    Constitutively activating mutations in receptor kinases recruit downstream effector pathways independently of upstream signaling, with consequences ranging from developmental syndromes to cancer. Classic fibrodysplasia ossificans progressiva (FOP) is a congenital syndrome resulting from highly conserved activating mutations of the glycine-serine-rich (GS) regulatory domain of ACVR1, encoding bone morphogenetic protein (BMP) type I receptor ALK2, which lead to inappropriate signaling and heterotopic ossification of soft tissues. It is unclear if constitutively active mutant ALK2 receptors (caALK2) can function independently of signaling complexes with type II receptors and ligands. We found that ablation of BmpRII and ActRIIa abrogated BMP ligand-mediated and caALK2-mediated signaling and transcription in cells and disrupted caALK2-induced heterotopic ossification in mice. Signaling via GS domain ALK2 mutants could be restored by the expression of either BMP type II receptor. The contribution of BMP type II receptors was independent of their ligand-binding or kinase function but was dependent upon an intact cytoplasmic domain. These data demonstrate that GS domain ALK2 mutants act independently of upstream signaling but may require a nonenzymatic scaffolding function provided by type II receptors to form functional, apparently ligand-independent signaling complexes. These findings define the minimal requirements for signaling of GS domain ALK2 mutants, with implications for the therapeutic targeting of their activity in disease. PMID:23572558

  17. Activation of the orphan receptor tyrosine kinase ALK by zinc.

    PubMed

    Bennasroune, Aline; Mazot, Pierre; Boutterin, Marie-Claude; Vigny, Marc

    2010-08-01

    Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase essentially and transiently expressed during development of the central and peripheral nervous system. The nature of the cognate ligand of this receptor in Vertebrates is still a matter of debate. During synaptic transmission the release of ionic zinc found in vesicles of certain glutamatergic and gabaergic terminals may act as a neuromodulator by binding to pre- or post-synaptic receptors. Recently, zinc has been shown to activate the receptor tyrosine kinase, TrkB, independently of neurotrophins. This activation occurs via increasing the Src family kinase activity. In the present study, we investigated whether the ALK activity could be modulated by extracellular zinc. We first showed that zinc alone rapidly activates ALK. This activation is dependent of ALK tyrosine kinase activity and dimerization of the receptor but is independent of Src family kinase activity. In contrast, addition of sodium pyrithione, a zinc ionophore, led to a further activation of ALK. This stronger activation is dependent of Src family kinase but independent of ALK activity and dimerization. In conclusion, zinc could constitute an endogenous ligand of ALK in vertebrates. PMID:20621063

  18. 5-HT7 receptor activation promotes an increase in TrkB receptor expression and phosphorylation

    PubMed Central

    Samarajeewa, Anshula; Goldemann, Lolita; Vasefi, Maryam S.; Ahmed, Nawaz; Gondora, Nyasha; Khanderia, Chandni; Mielke, John G.; Beazely, Michael A.

    2014-01-01

    The serotonin (5-HT) type 7 receptor is expressed throughout the CNS including the cortex and hippocampus. We have previously demonstrated that the application of 5-HT7 receptor agonists to primary hippocampal neurons and SH-SY5Y cells increases platelet-derived growth factor (PDGF) receptor expression and promotes neuroprotection against N-methyl-D-aspartate-(NMDA)-induced toxicity. The tropomyosin-related kinase B (TrkB) receptor is one of the receptors for brain-derived neurotrophic factor (BDNF) and is associated with neurodevelopmental and neuroprotective effects. Application of LP 12 to primary cerebral cortical cultures, SH-SY5Y cells, as well as the retinal ganglion cell line, RGC-5, increased both the expression of full length TrkB as well as its basal phosphorylation state at tyrosine 816. The increase in TrkB expression and phosphorylation was observed as early as 30 min after 5-HT7 receptor activation. In addition to full-length TrkB, kinase domain-deficient forms may be expressed and act as dominant-negative proteins toward the full length receptor. We have identified distinct patterns of TrkB isoform expression across our cell lines and cortical cultures. Although TrkB receptor expression is regulated by cyclic AMP and G?s-coupled GPCRs in several systems, we demonstrate that, depending on the model system, pathways downstream of both G?s and G?12 are involved in the regulation of TrkB expression by 5-HT7 receptors. Given the number of psychiatric and degenerative diseases associated with TrkB/BDNF deficiency and the current interest in developing 5-HT7 receptor ligands as pharmaceuticals, identifying signaling relationships between these two receptors will aid in our understanding of the potential therapeutic effects of 5-HT7 receptor ligands. PMID:25426041

  19. Identification of a bioactive core sequence from human laminin and its applicability to tissue engineering.

    PubMed

    Yeo, In-Sung; Min, Seung-Ki; Kang, Hyun Ki; Kwon, Taek-Ka; Jung, Sung Youn; Min, Byung-Moo

    2015-12-01

    Finding bioactive short peptides derived from proteins is a critical step to the advancement of tissue engineering and regenerative medicine, because the former maintains the functions of the latter without immunogenicity in biological systems. Here, we discovered a bioactive core nonapeptide sequence, PPFEGCIWN (residues 2678-2686; Ln2-LG3-P2-DN3), from the human laminin ?2 chain, and investigated the role of this peptide in binding to transmembrane proteins to promote intracellular events leading to cell functions. This minimum bioactive sequence had neither secondary nor tertiary structures in a computational structure prediction. Nonetheless, Ln2-LG3-P2-DN3 bound to various cell types as actively as laminin in cell adhesion assays. The in vivo healing tests using rats revealed that Ln2-LG3-P2-DN3 promoted bone formation without any recognizable antigenic activity. Ln2-LG3-P2-DN3-treated titanium (Ti) discs and Ti implant surfaces caused the enhancement of bone cell functions in vitro and induced faster osseointegration in vivo, respectively. These findings established a minimum bioactive sequence within human laminin, and its potential application value for regenerative medicine, especially for bone tissue engineering. PMID:26406450

  20. Laminin Mediates Tissue-specific Gene Expression in Mammary Epithelia

    E-print Network

    Streuli, Charles H

    2011-01-01

    tissue culture plastic, synthesized no ?-caseins (Fig. 1 c).plastic, mammary cells respond to soluble basement membrane or purified laminin, but not other extracellular matrix components, by synthesizing ?-casein.

  1. Hedgehog Signaling and Laminin Play Unique and Synergistic Roles in Muscle Development

    PubMed Central

    Peterson, Matthew T.; Henry, Clarissa A.

    2010-01-01

    Hedgehog (Hh) signaling and laminin-111, a basement membrane protein, are required for early muscle development. Hh signaling specifies different populations of muscle fibers and laminin-111 is critical for early muscle morphogenesis. However, additional requirements for Hh signaling and laminin during later phases of muscle development are not known. Furthermore, interactions between Hh signaling and laminin in this context are unknown. We used laminin gamma1 mutant zebrafish and cyclopamine to block Hh signal transduction separately and in combination to investigate their functions and interactions. We found that both Hh signaling and laminin are required for normal myosin chain expression. In addition, Hh signaling and laminin act synergistically during fast-twitch fiber elongation: fast muscle cells do not elongate in embryos deficient for both Hh signaling and laminin. Finally, we present evidence which suggests that Hh signaling is indirectly required via slow fiber specification for recovery of fast fiber elongation in laminin gamma1 mutant embryos. PMID:20063418

  2. 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

  3. Transient expression of laminin {alpha}1 chain in regenerating murine liver: Restricted localization of laminin chains and nidogen-1

    SciTech Connect

    Kikkawa, Yamato . E-mail: yamato@sapmed.ac.jp; Mochizuki, Yoichi; Miner, Jeffrey H.; Mitaka, Toshihiro

    2005-04-15

    Most interstitia between epithelial and endothelial cells contain basal laminae (BLs), as defined by electron microscopy. However, in liver, the sinusoidal interstitium (called space of Disse) between hepatocytes and sinusoidal endothelial cells (SECs) lacks BLs. Because laminins are major components of BLs throughout the body, whether laminins exist in sinusoids has been a controversial issue. Despite recent advances, the distribution and expression of laminin chains have not been well defined in mammalian liver. Here, using a panel of antibodies, we examined laminins in normal and regenerating mouse livers. Of {alpha} chains, {alpha}5 was widely observed in all BLs except for sinusoids, while the other {alpha} chains were variously expressed in Glisson's sheath and central veins. Laminin {gamma}1 was also distributed to all BLs except for sinusoids. Although the {beta}2 chain was observed in all BLs and sinusoids, the expression of {beta}1 chain was restricted to Glisson's sheath. Detailed analysis of regenerating liver revealed that {alpha}1 and {gamma}1 chains appeared in sinusoids and were produced by stellate cells. The staining of {alpha}1 and {gamma}1 chains reached its maximum intensity at 6 days after two-thirds partial hepatectomy (PHx). Moreover, in vitro studies showed that {alpha}1-containing laminin promoted spreading of sinusoidal endothelial cells (SECs) isolated from normal liver, but not other hepatic cells. In addition, SECs isolated from regenerating liver elongated pseudopodia on {alpha}1-containing laminin more so than did cells from normal liver. The transient expression of laminin {alpha}1 may promote formation of sinusoids after PHx.

  4. Analyzing the activation of the melanocortin-2 receptor of tetrapods.

    PubMed

    Dores, Robert M; Liang, Liang

    2014-07-01

    Following the biochemical characterization of the pituitary hormone, adrenocorticotropin (ACTH), in the 1950's, a number of structure/function studies were done which identifies two amino acid motifs in ACTH, the HFRW motif and KKRR motif, as critical for the activation of the "ACTH" receptor on adrenal cortex cells. In the 1990's the "ACTH" receptor was identified as a member of the melanocortin receptor gene family, and given the name melanocortin-2 receptor (MC2R). Since that time a number of studies on both tetrapod and teleost MC2R orthologs have established that these orthologs can only be activated by ACTH, but not by any of the MSH-sized melanocortin ligands, and these orthologs require interaction with the melanocortin-2 receptor accessory protein (MRAP) for functional expression. This review summarizes recent structure/function studies on human ACTH, and points out the importance of the GKPVG motif in ACTH for the activation of the receptor. In this regard, a multiple-step model for the activation of tetrapod and teleost MC2R orthologs is presented, and the evolution of gnathostome MC2R ligand selectivity and the requirement for MRAP interaction is discussed in light of a recent study on a cartilaginous fish MC2R ortholog. This review contains excerpts from the Gorbman/Bern Lecture presented at the Second Meeting of the North American Society for Comparative Endocrinology (NASCE). PMID:24713445

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

    SciTech Connect

    Kruse, Schoen W.; Suino-Powell, Kelly; Zhou, X. Edward; Kretschman, Jennifer E.; Reynolds, Ross; Vonrhein, Clemens; Xu, Yong; Wang, Liliang; Tsai, Sophia Y.; Tsai, Ming-Jer; Xu, H. Eric

    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, 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.

  6. NMDA receptors are expressed in oligodendrocytes and activated in ischaemia.

    PubMed

    Káradóttir, Ragnhildur; Cavelier, Pauline; Bergersen, Linda H; Attwell, David

    2005-12-22

    Glutamate-mediated damage to oligodendrocytes contributes to mental or physical impairment in periventricular leukomalacia (pre- or perinatal white matter injury leading to cerebral palsy), spinal cord injury, multiple sclerosis and stroke. Unlike neurons, white matter oligodendrocytes reportedly lack NMDA (N-methyl-d-aspartate) receptors. It is believed that glutamate damages oligodendrocytes, especially their precursor cells, by acting on calcium-permeable AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)/kainate receptors alone or by reversing cystine-glutamate exchange and depriving cells of antioxidant protection. Here we show that precursor, immature and mature oligodendrocytes in the white matter of the cerebellum and corpus callosum exhibit NMDA-evoked currents, mediated by receptors that are blocked only weakly by Mg2+ and that may contain NR1, NR2C and NR3 NMDA receptor subunits. NMDA receptors are present in the myelinating processes of oligodendrocytes, where the small intracellular space could lead to a large rise in intracellular ion concentration in response to NMDA receptor activation. Simulating ischaemia led to development of an inward current in oligodendrocytes, which was partly mediated by NMDA receptors. These results point to NMDA receptors of unusual subunit composition as a potential therapeutic target for preventing white matter damage in a variety of diseases. PMID:16372011

  7. Recombinant disintegrin (r-Cam-dis) from Crotalus adamanteus inhibits adhesion of human pancreatic cancer cell lines to laminin-1 and vitronectin.

    PubMed

    Suntravat, Montamas; Barret, Henriquez S; Jurica, Cameron A; Lucena, Sara E; Perez, John C; Sánchez, Elda E

    2015-01-01

    Pancreatic cancer is a malignant cancer common worldwide having poor prognosis, even when diagnosed at its early stage. Cell adhesion plays a critical role in cancer invasion and metastasis. Integrins are major mediators of cell adhesion and play an important role in invasion and metastatic growth of human pancreatic cancer cells. Snake disintegrins are the most potent ligands of several integrins and have potential therapeutic applications for cancers. We have previously cloned and expressed a new recombinant RGD-disintegrin from Crotalus adamanteus (r-Cam-dis). This recently published r-Cam-dis has an extra nine amino acids derived from the vector (SPGARGSEF) at the N-terminus end and has strong anti-platelet activity. However, this r-Cam-dis contains the contamination of the cleavage of the N-terminal end of the pET-43.1a cloning vector. In this study, we have cloned r-Cam-dis in a different cloning vector (pGEX-4T-1) showing five different amino acids (GSPEF) at the N-terminal part. This new r-Cam-dis was expressed and tested for inhibition of platelet aggregation, specific binding activity with seven different integrins, and inhibition of adhesion of three different pancreatic cancer cell lines on laminin-1 and vitronectin. The r-Cam-dis showed potent binding to ?v?3 integrin, but was moderate to weak with ?v?5, ?v?6, ?2?1, and ?6?1. Interestingly, the inhibition of r-Cam-dis on pancreatic cancer cell lines adhesion to laminin-1 was more effective than that to vitronectin. Based on our binding results to integrin receptors and previous adhesion studies using function-blocking monoclonal antibodies, it is suggested that r-Cam-dis could be inhibiting adhesion of pancreatic cancer cell lines through integrins ?2?1, ?6?1, ?v?5, and ?v?6. PMID:26045944

  8. Recombinant disintegrin (r-Cam-dis) from Crotalus adamanteus inhibits adhesion of human pancreatic cancer cell lines to laminin-1 and vitronectin

    PubMed Central

    Suntravat, Montamas; Barret, Henriquez S; Jurica, Cameron A; Lucena, Sara E; Perez, John C; Sánchez, Elda E

    2015-01-01

    Pancreatic cancer is a malignant cancer common worldwide having poor prognosis, even when diagnosed at its early stage. Cell adhesion plays a critical role in cancer invasion and metastasis. Integrins are major mediators of cell adhesion and play an important role in invasion and metastatic growth of human pancreatic cancer cells. Snake disintegrins are the most potent ligands of several integrins and have potential therapeutic applications for cancers. We have previously cloned and expressed a new recombinant RGD-disintegrin from Crotalus adamanteus (r-Cam-dis). This recently published r-Cam-dis has an extra nine amino acids derived from the vector (SPGARGSEF) at the N-terminus end and has strong anti-platelet activity. However, this r-Cam-dis contains the contamination of the cleavage of the N-terminal end of the pET-43.1a cloning vector. In this study, we have cloned r-Cam-dis in a different cloning vector (pGEX-4T-1) showing five different amino acids (GSPEF) at the N-terminal part. This new r-Cam-dis was expressed and tested for inhibition of platelet aggregation, specific binding activity with seven different integrins, and inhibition of adhesion of three different pancreatic cancer cell lines on laminin-1 and vitronectin. The r-Cam-dis showed potent binding to ?v?3 integrin, but was moderate to weak with ?v?5, ?v?6, ?2?1, and ?6?1. Interestingly, the inhibition of r-Cam-dis on pancreatic cancer cell lines adhesion to laminin-1 was more effective than that to vitronectin. Based on our binding results to integrin receptors and previous adhesion studies using function-blocking monoclonal antibodies, it is suggested that r-Cam-dis could be inhibiting adhesion of pancreatic cancer cell lines through integrins ?2?1, ?6?1, ?v?5, and ?v?6. PMID:26045944

  9. Receptors involved in cell activation by antiphospholipid antibodies.

    PubMed

    Brandt, Karim J; Kruithof, Egbert K O; de Moerloose, Philippe

    2013-10-01

    The antiphospholipid syndrome (APS) is an autoimmune disease associated with arterial or venous thrombosis and/or recurrent fetal loss and is caused by pathogenic antiphospholipid antibodies (aPLA). The plasma protein ?2-glycoprotein 1 (?2GP1) has been identified as a major target of aPLA associated with APS. Cell activation by aPLA appears to be a major pathogenic cause in the pathogenesis of APS. Receptors, co-receptors and accessory molecules are known to assist the pathogenic effects of aPLA. Members of the TLR family and the platelet receptor apolipoprotein E receptor 2' (apoER2'), a receptor belonging to the low-density lipoprotein receptor (LDL-R) family, as well as GPIb?, were identified as putative candidates for aPLA recognition. CD14, a co-receptor for TLR2 and TLR4, and annexin A2, a ubiquitous Ca2+ -binding protein that is essential for actin-dependent vesicle transport, could serve as important accessory molecules in mediating the pathogenic effects of aPLA. Finally, complement activation has been reported in association with the pathogenicity of APS. The relative contribution of these different mechanisms in the pathogenesis of APS is controversial. Here, we review the various in vivo and in vitro models that have been used to investigate the pathogenic mechanisms of aPLA in APS. PMID:24054056

  10. Flavonoids as dietary regulators of nuclear receptor activity

    PubMed Central

    Avior, Yishai; Bomze, David; Ramon, Ory

    2013-01-01

    Metabolic diseases such as obesity, type II diabetes, and dyslipidemia are a rising cause of mortality worldwide. The progression of many metabolic diseases is fundamentally regulated on the transcriptional level by a family of ligand-activated transcription factors, called nuclear receptors, which detect and respond to metabolic changes. Their role in maintaining metabolic homeostasis makes nuclear receptors an important pharmaceutical and dietary target. This review will present the growing evidence that flavonoids, natural secondary plant metabolites, are important regulators of nuclear receptor activity. Structural similarities between flavonoids and cholesterol derivatives combined with the promiscuous nature of most nuclear receptors provide a wealth of possibilities for pharmaceutical and dietary modulation of metabolism. While the challenges of bringing flavonoid-derived therapeutics to the market are significant, we consider this rapidly growing field to be an essential aspect of the functional food initiative and an important mine for pharmaceutical compounds. PMID:23598551

  11. Serotonin augments gut pacemaker activity via 5-HT3 receptors.

    PubMed

    Liu, Hong-Nian; Ohya, Susumu; Nishizawa, Yuji; Sawamura, Kenta; Iino, Satoshi; Syed, Mohsin Md; Goto, Kazunori; Imaizumi, Yuji; Nakayama, Shinsuke

    2011-01-01

    Serotonin (5-hydroxytryptamine: 5-HT) affects numerous functions in the gut, such as secretion, muscle contraction, and enteric nervous activity, and therefore to clarify details of 5-HT's actions leads to good therapeutic strategies for gut functional disorders. The role of interstitial cells of Cajal (ICC), as pacemaker cells, has been recognised relatively recently. We thus investigated 5-HT actions on ICC pacemaker activity. Muscle preparations with myenteric plexus were isolated from the murine ileum. Spatio-temporal measurements of intracellular Ca(2+) and electric activities in ICC were performed by employing fluorescent Ca(2+) imaging and microelectrode array (MEA) systems, respectively. Dihydropyridine (DHP) Ca(2+) antagonists and tetrodotoxin (TTX) were applied to suppress smooth muscle and nerve activities, respectively. 5-HT significantly enhanced spontaneous Ca(2+) oscillations that are considered to underlie electric pacemaker activity in ICC. LY-278584, a 5-HT(3) receptor antagonist suppressed spontaneous Ca(2+) activity in ICC, while 2-methylserotonin (2-Me-5-HT), a 5-HT(3) receptor agonist, restored it. GR113808, a selective antagonist for 5-HT(4), and O-methyl-5-HT (O-Me-5-HT), a non-selective 5-HT receptor agonist lacking affinity for 5-HT(3) receptors, had little effect on ICC Ca(2+) activity. In MEA measurements of ICC electric activity, 5-HT and 2-Me-5-HT caused excitatory effects. RT-PCR and immunostaining confirmed expression of 5-HT(3) receptors in ICC. The results indicate that 5-HT augments ICC pacemaker activity via 5-HT(3) receptors. ICC appear to be a promising target for treatment of functional motility disorders of the gut, for example, irritable bowel syndrome. PMID:21949791

  12. Motogenic and morphogenic activity of epithelial receptor tyrosine kinases

    PubMed Central

    1996-01-01

    Receptor tyrosine kinases play essential roles in morphogenesis and differentiation of epithelia. Here we examined various tyrosine kinase receptors, which are preferentially expressed in epithelia (c-met, c- ros, c-neu, and the keratin growth factor [KGF] receptor), for their capacity to induce cell motility and branching morphogenesis of epithelial cells. We exchanged the ligand-binding domain of these receptors by the ectodomain of trkA and could thus control signaling by the new ligand, NGF. We demonstrate here that the tyrosine kinases of c- met, c-ros, c-neu, the KGF receptor, and trkA, but not the insulin receptor, induced scattering and increased motility of kidney epithelial cells in tissue culture. Mutational analysis suggests that SHC binding is essential for scattering and increased cell motility induced by trkA. The induction of motility in epithelial cells is thus an important feature of various receptor tyrosine kinases, which in vivo play a role in embryogenesis and metastasis. In contrast, only the c-met receptor promoted branching morphogenesis of kidney epithelial cells in three-dimensional matrices, which resemble the formation of tubular epithelia in development. Interestingly, the ability of c-met to induce morphogenesis could be transferred to trkA, when in a novel receptor hybrid COOH-terminal sequences of c-met (including Y14 to Y16) were fused to the trkA kinase domain. These data demonstrate that tubulogenesis of epithelia is a restricted activity of tyrosine kinases, as yet only demonstrated for the c-met receptor. We predict the existence of specific substrates that mediate this morphogenesis signal. PMID:8655582

  13. Ghrelin receptor conformational dynamics regulate the transition from a preassembled to an active receptor:Gq complex

    PubMed Central

    Damian, Marjorie; Mary, Sophie; Maingot, Mathieu; M'Kadmi, Céline; Gagne, Didier; Leyris, Jean-Philippe; Denoyelle, Séverine; Gaibelet, Gérald; Gavara, Laurent; Garcia de Souza Costa, Mauricio; Perahia, David; Trinquet, Eric; Mouillac, Bernard; Galandrin, Ségolčne; Galčs, Céline; Fehrentz, Jean-Alain; Floquet, Nicolas; Martinez, Jean; Marie, Jacky; Bančres, Jean-Louis

    2015-01-01

    How G protein-coupled receptor conformational dynamics control G protein coupling to trigger signaling is a key but still open question. We addressed this question with a model system composed of the purified ghrelin receptor assembled into lipid discs. Combining receptor labeling through genetic incorporation of unnatural amino acids, lanthanide resonance energy transfer, and normal mode analyses, we directly demonstrate the occurrence of two distinct receptor:Gq assemblies with different geometries whose relative populations parallel the activation state of the receptor. The first of these assemblies is a preassembled complex with the receptor in its basal conformation. This complex is specific of Gq and is not observed with Gi. The second one is an active assembly in which the receptor in its active conformation triggers G protein activation. The active complex is present even in the absence of agonist, in a direct relationship with the high constitutive activity of the ghrelin receptor. These data provide direct evidence of a mechanism for ghrelin receptor-mediated Gq signaling in which transition of the receptor from an inactive to an active conformation is accompanied by a rearrangement of a preassembled receptor:G protein complex, ultimately leading to G protein activation and signaling. PMID:25605885

  14. Enhancing neural stem cell response to SDF-1? gradients through hyaluronic acid-laminin hydrogels.

    PubMed

    Addington, C P; Heffernan, J M; Millar-Haskell, C S; Tucker, E W; Sirianni, R W; Stabenfeldt, S E

    2015-12-01

    Traumatic brain injury (TBI) initiates an expansive biochemical insult that is largely responsible for the long-term dysfunction associated with TBI; however, current clinical treatments fall short of addressing these underlying sequelae. Pre-clinical investigations have used stem cell transplantation with moderate success, but are plagued by staggeringly low survival and engraftment rates (2-4%). As such, providing cell transplants with the means to better dynamically respond to injury-related signals within the transplant microenvironment may afford improved transplantation survival and engraftment rates. The chemokine stromal cell-derived factor-1? (SDF-1?) is a potent chemotactic signal that is readily present after TBI. In this study, we sought to develop a transplantation vehicle to ultimately enhance the responsiveness of neural transplants to injury-induced SDF-1?. Specifically, we hypothesize that a hyaluronic acid (HA) and laminin (Lm) hydrogel would promote 1. upregulated expression of the SDF-1? receptor CXCR4 in neural progenitor/stem cells (NPSCs) and 2. enhanced NPSC migration in response to SDF-1? gradients. We demonstrated successful development of a HA-Lm hydrogel and utilized standard protein and cellular assays to probe NPSC CXCR4 expression and NPSC chemotactic migration. The findings demonstrated that NPSCs significantly increased CXCR4 expression after 48 h of culture on the HA-Lm gel in a manner critically dependent on both HA and laminin. Moreover, the HA-Lm hydrogel significantly increased NPSC chemotactic migration in response to SDF-1? at 48 h, an effect that was critically dependent on HA, laminin and the SDF-1? gradient. Therefore, this hydrogel serves to 1. prime NPSCs for the injury microenvironment and 2. provide the appropriate infrastructure to support migration into the surrounding tissue, equipping cells with the tools to more effectively respond to the injury microenvironment. PMID:26340314

  15. Laminin-332 (Laminin-5) is the major motility ligand for B cell chronic lymphocytic leukemia.

    PubMed

    Spessotto, Paola; Zucchetto, Antonella; Degan, Massimo; Wasserman, Bruna; Danussi, Carla; Bomben, Riccardo; Perris, Roberto; Canzonieri, Vincenzo; Radillo, Oriano; Colombatti, Alfonso; Gattei, Valter

    2007-07-01

    Cell adhesion and motility are central aspects in the pathophysiology of B cell chronic lymphocytic leukemia (B-CLL), but the role of specific extracellular matrix proteins is still to be completely unveiled. Purified peripheral blood neoplastic cells of B-CLL patients migrated poorly on laminins-111,-411,-511, but showed pronounced motility on laminin (LM)-332 in a high percentage of cases. B-CLL cell motility on LM-332 was mediated by the alpha3beta1 integrin and was preferentially observed in cells carrying a mutated IgV(H) gene profile. Within normal lymph nodes, LM-332 was circumscribed around blood vessels and to areas corresponding to marginal zones, where it was deposited in a pattern reminiscent of reticular fibers. Conversely, in B-CLL involved lymph nodes, a positive LM-332 reticular mesh was diffusely evident, throughout the disrupted nodal architecture. In the present study we identified LM-332 as a crucial motility-promoting factor for B-CLL lymphocytes and as a potential constituent favoring the dissemination of B-CLL lymphocytes through vascular basement membranes and possibly lymph node compartments. PMID:17482449

  16. Regulation of Proteome Maintenance Gene Expression by Activators of Peroxisome Proliferator-Activated Receptor a (PPARa)

    EPA Science Inventory

    The nuclear receptor peroxisome proliferator-activated receptor alpha (PPARa) is activated by a large number of xenobiotic and hypolipidemic compounds called peroxisome proliferator chemicals (PPC). One agonist of PPARa (WY-14,643) regulates responses in the mouse liver to chemic...

  17. Activation of CR3-mediated phagocytosis by MSP requires the RON receptor, tyrosine kinase activity,

    E-print Network

    Correll, Pamela H.

    Activation of CR3-mediated phagocytosis by MSP requires the RON receptor, tyrosine kinase activity: Macrophage-stimulating protein (MSP) promotes the phagocytosis of C3bi-coated erythro- cytes by resident-induced com- plement-mediated phagocytosis requires the RON receptor tyrosine kinase and the M 2 integrin

  18. Toll-like receptors: Activation, signalling and transcriptional modulation.

    PubMed

    De Nardo, Dominic

    2015-08-01

    Families of innate immune receptors serve as the bodies primary defence system by recognising and rapidly responding to infection by microorganisms or to endogenous danger signals and initiating inflammatory processes. Whilst Toll-like receptors (TLRs) were the first family to be discovered, important and exciting discoveries continue to emerge into the molecular mechanisms that control their activation and regulation. Herein, I will provide an overview of TLR activation and their downstream signalling cascades, and discuss some of the recent findings concerning the assembly of a TLR oligomeric signalling platform, known as the Myddosome. Further, a brief examination of the importance of crosstalk between multiple TLRs or between TLRs and other innate immune receptors for appropriate and coordinated immune responses will be presented. Finally, I will discuss the importance of mechanisms that regulate TLRs with a focus on the role of activating transcription factor 3 (ATF3) in modulating transcriptional responses downstream of TLRs. PMID:25846205

  19. Interfering with mineralocorticoid receptor activation: the past, present, and future

    PubMed Central

    2014-01-01

    Aldosterone is a potent mineralocorticoid produced by the adrenal gland. Aldosterone binds to and activates the mineralocorticoid receptor (MR) in a plethora of tissues, but the cardiovascular actions of aldosterone are of primary interest clinically. Although MR antagonists were developed as antihypertensive agents, they are now considered to be important therapeutic options for patients with heart failure. Specifically, blocking only the MR has proven to be a difficult task because of its similarity to other steroid receptors, including the androgen and progesterone receptors. This lack of specificity caused the use of the first-generation mineralocorticoid receptor antagonists to be fraught with difficulty because of the side effects produced by drug administration. However, in recent years, several advances have been made that could potentially increase the clinical use of agents that inhibit the actions of aldosterone. These will be discussed here along with some examples of the beneficial effects of these new therapeutic agents. PMID:25165560

  20. Role of peroxisome proliferator-activated receptor gamma and retinoid X receptor heterodimer in hepatogastroenterological diseases.

    PubMed

    Dubuquoy, Laurent; Dharancy, Sébastien; Nutten, Sophie; Pettersson, Sven; Auwerx, Johan; Desreumaux, Pierre

    2002-11-01

    The peroxisome proliferator-activated receptor gamma (PPARgamma) and its partner the retinoid X receptor (RXR) are two nuclear receptors that are expressed mainly in adipose tissue and which have a role in lipid metabolism and insulin sensitisation. New sites of PPARgamma/RXR expression have been described, especially in the intestinal tract, pancreas, and liver. Concomitantly, new functions have been attributed to this heterodimer in regulation of inflammation, by its inhibition of nuclear factor (NF)-kappaB and via stress-kinase pathways. These new sites and functions of PPARgamma/RXR have led to novel ideas about pathophysiology of different inflammatory digestive diseases and to development of innovative treatment strategies with PPARgamma activators. PMID:12424006

  1. Modulation of Opioid Receptor Ligand Affinity and Efficacy Using Active and Inactive State Receptor Models

    PubMed Central

    Anand, Jessica P.; Purington, Lauren C.; Pogozheva, Irina D.; Traynor, John R.; Mosberg, Henry I.

    2012-01-01

    Mu opioid receptor (MOR) agonists are widely used for the treatment of pain; however chronic use results in the development of tolerance and dependence. It has been demonstrated that co-administration of a MOR agonist with a delta opioid receptor (DOR) antagonist maintains the analgesia associated with MOR agonists, but with reduced negative side effects. Using our newly refined opioid receptor models for structure-based ligand design, we have synthesized several pentapeptides with tailored affinity and efficacy profiles. In particular, we have obtained pentapeptides 8, Tyr-c(S-S)[DCys-1Nal-Nle-Cys]NH2, and 12, Tyr-c(S-S)[DCys-1Nal-Nle-Cys]OH, which demonstrates high affinity and full agonist behavior at MOR, high affinity but very low efficacy for DOR, and minimal affinity for the kappa opioid receptor (KOR). Functional properties of these peptides as MOR agonists/DOR antagonists lacking undesired KOR activity make them promising candidates for future in vivo studies of MOR/DOR interactions. Subtle structural variation of 12, by substituting D-Cys5 for L-Cys5, generated analog 13 which maintains low nanomolar MOR and DOR affinity, but which displays no efficacy at either receptor. These results demonstrate the power and utility of accurate receptor models for structure-based ligand design, as well as the profound sensitivity of ligand function on its structure. PMID:22882801

  2. TGF-?1 activates two distinct type I receptors in neurons

    PubMed Central

    König, Hans-Georg; Kögel, Donat; Rami, Abdelhaq; Prehn, Jochen H.M.

    2005-01-01

    Transforming growth factor-?s (TGF-?s) are pleiotropic cytokines involved in development and maintenance of the nervous system. In several neural lesion paradigms, TGF-?1 exerts potent neuroprotective effects. Neurons treated with TGF-?1 activated the canonical TGF-? receptor I/activin-like kinase receptor 5 (ALK5) pathway. The transcription factor nuclear factor-?B (NF-?B) plays a fundamental role in neuroprotection. Treatment with TGF-?1 enhanced NF-?B activity in gelshift and reporter gene analyses. However, ectopic expression of a constitutively active ALK5 failed to mimic these effects. ALK1 has been described as an alternative TGF-? receptor in endothelial cells. Interestingly, we detected significant basal expression of ALK1 and its injury-induced up-regulation in neurons. Treatment with TGF-?1 also induced a pronounced increase in downstream Smad1 phosphorylation. Overexpression of a constitutively active ALK1 mimicked the effect of TGF-?1 on NF-?B activation and neuroprotection. Our data suggest that TGF-?1 simultaneously activates two distinct receptor pathways in neurons and that the ALK1 pathway mediates TGF-?1–induced NF-?B survival signaling. PMID:15781474

  3. Protease-activated receptor 1-dependent neuronal damage involves NMDA receptor function

    PubMed Central

    Hamill, Cecily E.; Mannaioni, Guido; Lyuboslavsky, Polina; Sastre, Aristide A.; Traynelis, Stephen F.

    2009-01-01

    Protease-activated receptor 1 (PAR1) is a G-protein coupled receptor that is expressed throughout the central nervous system. PAR1 activation by brain-derived as well as blood-derived proteases has been shown to have variable and complex effects in a variety of animal models of neuronal injury and inflammation. In this study, we have evaluated the effects of PAR1 on lesion volume in wild-type or PAR1?/? C57Bl/6 mice subjected to transient occlusion of the middle cerebral artery or injected with NMDA in the striatum. We found that removal of PAR1 reduced infarct volume following transient focal ischemia to 57% of control. Removal of PAR1 or application of a PAR1 antagonist also reduced the neuronal injury associated with intrastriatal injection of NMDA to 60% of control. To explore whether NMDA receptor potentiation by PAR1 activation contributes to the harmful effects of PAR1, we investigated the effect of NMDA receptor antagonists on the neuroprotective phenotype of PAR1?/? mice. We found that MK801 reduced penumbral but not core neuronal injury in mice subjected to transient middle cerebral artery occlusion or intrastriatal NMDA injection. Lesion volumes in both models were not significantly different between PAR1?/? mice treated with and without MK801. Use of the NMDA receptor antagonist and dissociative anesthetic ketamine also renders NMDA-induced lesion volumes identical in PAR1?/? mice and wild-type mice. These data suggest that the ability of PAR1 activation to potentiate NMDA receptor function may underlie its harmful actions during injury. PMID:19416668

  4. Mechanisms of Activation of Receptor Tyrosine Kinases: Monomers or Dimers

    PubMed Central

    Maruyama, Ichiro N.

    2014-01-01

    Receptor tyrosine kinases (RTKs) play essential roles in cellular processes, including metabolism, cell-cycle control, survival, proliferation, motility and differentiation. RTKs are all synthesized as single-pass transmembrane proteins and bind polypeptide ligands, mainly growth factors. It has long been thought that all RTKs, except for the insulin receptor (IR) family, are activated by ligand-induced dimerization of the receptors. An increasing number of diverse studies, however, indicate that RTKs, previously thought to exist as monomers, are present as pre-formed, yet inactive, dimers prior to ligand binding. The non-covalently associated dimeric structures are reminiscent of those of the IR family, which has a disulfide-linked dimeric structure. Furthermore, recent progress in structural studies has provided insight into the underpinnings of conformational changes during the activation of RTKs. In this review, I discuss two mutually exclusive models for the mechanisms of activation of the epidermal growth factor receptor, the neurotrophin receptor and IR families, based on these new insights. PMID:24758840

  5. Liver X Receptor and Peroxisome Proliferator-Activated Receptor Agonist from Cornus alternifolia

    PubMed Central

    He, Yang-Qing; Ma, Guo-Yi; Peng, Jiang-nan; Ma, Zhan-Ying; Hamann, Mark T.

    2012-01-01

    Background Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear receptors superfamily and are transcription factors activated by specific ligands. Liver X receptors (LXR) belong to the nuclear hormone receptors and have been shown to play an important role in cholesterol homeostasis. From the previous screening of several medicinal plants for potential partial PPAR? agonists, the extracts of Cornus alternifolia were found to exhibit promising bioactivity. In this paper, we report the isolation and structural elucidation of four new compounds and their potential as ligands for PPAR. Methods The new compounds were extracted from the leaves of Cornus alternifolia and fractionated by high-performance liquid chromatography. Their structures were elucidated on the basis of spectroscopic evidence and analysis of their hydrolysis products. Results Three new iridoid glycosides including an iridolactone, alternosides A-C (1–3), a new megastigmane glycoside, cornalternoside (4) and 10 known compounds, were obtained from the leaves of Cornus alternifolia. Kaempferol-3-O-?-glucopyranoside (5) exhibited potent agonistic activities for PPAR?, PPAR? and LXR with EC50 values of 0.62, 3.0 and 1.8 ? M, respectively. Conclusions We isolated four new and ten known compounds from Cornus alternifolia, and one known compound showed agonistic activities for PPAR?, PPAR? and LXR. General significance Compound 1 is the first example of a naturally occurring iridoid glycoside containing a ?-glucopyranoside moiety at C-6. PMID:22353334

  6. Ecdysone receptor expression and activity in adult Drosophila melanogaster.

    PubMed

    Schwedes, Christoph; Tulsiani, Siddharth; Carney, Ginger E

    2011-07-01

    Disrupting components of the ecdysone/EcR/USP signaling pathway in insects leads to morphological defects and developmental arrest. In adult Drosophila melanogaster decreased EcR function affects fertility, lifespan, behavior, learning, and memory; however we lack a clear understanding of how EcR/USP expression and activity impacts these phenotypes. To shed light on this issue, we characterized the wild-type expression patterns and activity of EcR/USP in individual tissues during early adult life. EcR and usp were expressed in numerous adult tissues, but receptor activity varied depending on tissue type and adult age. Receptor activity did not detectably change in response to mating status, environmental stress, ecdysone treatment or gender but is reduced when a constitutively inactive ecdysone receptor is present. Since only a subset of adult tissues expressing EcR and usp contain active receptors, it appears that an important adult function of EcR/USP in some tissues may be repression of genes containing EcRE's. PMID:21507325

  7. Structural basis for selective activation of ABA receptors

    SciTech Connect

    Peterson, Francis C.; Burgie, E. Sethe; Park, Sang-Youl; Jensen, Davin R.; Weiner, Joshua J.; Bingman, Craig A.; Chang, Chia-En A.; Cutler, Sean R.; Phillips, Jr., George N.; Volkman, Brian F.

    2010-11-01

    Changing environmental conditions and lessening fresh water supplies have sparked intense interest in understanding and manipulating abscisic acid (ABA) signaling, which controls adaptive responses to drought and other abiotic stressors. We recently discovered a selective ABA agonist, pyrabactin, and used it to discover its primary target PYR1, the founding member of the PYR/PYL family of soluble ABA receptors. To understand pyrabactin's selectivity, we have taken a combined structural, chemical and genetic approach. We show that subtle differences between receptor binding pockets control ligand orientation between productive and nonproductive modes. Nonproductive binding occurs without gate closure and prevents receptor activation. Observations in solution show that these orientations are in rapid equilibrium that can be shifted by mutations to control maximal agonist activity. Our results provide a robust framework for the design of new agonists and reveal a new mechanism for agonist selectivity.

  8. Activation of glycine receptors modulates spontaneous epileptiform activity in the immature rat hippocampus.

    PubMed

    Chen, Rongqing; Okabe, Akihito; Sun, Haiyan; Sharopov, Salim; Hanganu-Opatz, Ileana L; Kolbaev, Sergei N; Fukuda, Atsuo; Luhmann, Heiko J; Kilb, Werner

    2014-05-15

    While the expression of glycine receptors in the immature hippocampus has been shown, no information about the role of glycine receptors in controlling the excitability in the immature CNS is available. Therefore, we examined the effect of glycinergic agonists and antagonists in the CA3 region of an intact corticohippocampal preparation of the immature (postnatal days 4-7) rat using field potential recordings. Bath application of 100 ?M taurine or 10 ?M glycine enhanced the occurrence of recurrent epileptiform activity induced by 20 ?M 4-aminopyridine in low Mg(2+) solution. This proconvulsive effect was prevented by 3 ?M strychnine or after incubation with the loop diuretic bumetanide (10 ?M), suggesting that it required glycine receptors and an active NKCC1-dependent Cl(-) accumulation. Application of higher doses of taurine (? 1 mM) or glycine (100 ?M) attenuated recurrent epileptiform discharges. The anticonvulsive effect of taurine was also observed in the presence of the GABAA receptor antagonist gabazine and was attenuated by strychnine, suggesting that it was partially mediated by glycine receptors. Bath application of the glycinergic antagonist strychnine (0.3 ?M) induced epileptiform discharges. We conclude from these results that in the immature hippocampus, activation of glycine receptors can mediate both pro- and anticonvulsive effects, but that a persistent activation of glycine receptors is required to suppress epileptiform activity. In summary, our study elucidated the important role of glycine receptors in the control of neuronal excitability in the immature hippocampus. PMID:24665103

  9. Nuclear Receptor Activity and Liver Cancer Lesion Progression

    EPA Science Inventory

    Nuclear receptors (NRs) are ligand-activated transcription factors that control diverse cellular processes. Chronic stimulation of some NRs is a non-genotoxic mechanism of rodent liver cancer with unclear relevance to humans. We explored this question using human CAR, PXR, PPAR?,...

  10. INTRODUCTION Peroxisome proliferator-activated receptor gamma (PPAR)

    E-print Network

    Lee, Keun Woo

    71 INTRODUCTION Peroxisome proliferator-activated receptor gamma (PPAR) belongs to the nuclear commonly generated as hormones or in the intermediary met- abolic pathways (Evans et al., 2004). PPAR al., 2004; Tontonoz and Spiegelman, 2008). Recently, there have been a few reports of PPAR- mediated

  11. Soluble Extracts from Helicobacter pylori Induce Dome Formation in Polarized Intestinal Epithelial Monolayers in a Laminin-Dependent Manner

    PubMed Central

    Terrés, A. M.; Windle, H. J.; Ardini, E.; Kelleher, D. P.

    2003-01-01

    Helicobacter pylori colonizes the stomach at the interface between the mucus layer and the apical pole of gastric epithelial cells. A number of secreted and shed products from the bacteria, such as proteins and lipopolysaccharide, are likely to have a role in the pathogenesis at the epithelial level. To determine the physiological response of transporting polarized epithelia to released soluble factors from the bacterium, we used the T84 cell line. Monolayers of T84 cells were exposed to soluble extracts from H. pylori. The extracts induced rapid “dome” formation as well as an immediate decrease in transepithelial electrical resistance. Domes are fluid-filled blister-like structures unique to polarized epithelia. Their formation has been linked to sodium-transporting events as well as to diminished adherence of the cells to the substrate. H. pylori-induced dome formation in T84 monolayers was exacerbated by amiloride and inhibited by ouabain. Furthermore, it was associated with changes in the expression of the laminin binding ?6?4 integrin and the 67-kDa laminin receptor. Domes formed primarily on laminin-coated filters, rather than on fibronectin or collagen matrices, and their formation was inhibited by preincubating the bacterial extract with soluble laminin. This effect was specific to H. pylori and independent of the urease, vacA, cagA, and Lewis phenotype of the strains. These data indicate that released elements from H. pylori can alter the physiological balance and integrity of the epithelium in the absence of an underlying immune response. PMID:12819097

  12. The aryl hydrocarbon receptor and glucocorticoid receptor interact to activate human metallothionein 2A

    SciTech Connect

    Sato, Shoko; Shirakawa, Hitoshi; Tomita, Shuhei; Tohkin, Masahiro; Gonzalez, Frank J.; Komai, Michio

    2013-11-15

    Although the aryl hydrocarbon receptor (AHR) and glucocorticoid receptor (GR) play essential roles in mammalian development, stress responses, and other physiological events, crosstalk between these receptors has been the subject of much debate. Metallothioneins are classic glucocorticoid-inducible genes that were reported to increase upon treatment with AHR agonists in rodent tissues and cultured human cells. In this study, the mechanism of human metallothionein 2A (MT2A) gene transcription activation by AHR was investigated. Cotreatment with 3-methylcholanthrene and dexamethasone, agonists of AHR and GR respectively, synergistically increased MT2A mRNA levels in HepG2 cells. MT2A induction was suppressed by RNA interference against AHR or GR. Coimmunoprecipitation experiments revealed a physical interaction between AHR and GR proteins. Moreover, chromatin immunoprecipitation assays indicated that AHR was recruited to the glucocorticoid response element in the MT2A promoter. Thus, we provide a novel mechanism whereby AHR modulates expression of human MT2A via the glucocorticoid response element and protein–protein interactions with GR. - Highlights: • Aryl hydrocarbon receptor forms a complex with glucocorticoid receptor in cells. • Human metallothionein gene is regulated by the AHR and GR interaction. • AHR–GR complex binds to glucocorticoid response element in metallothionein gene. • We demonstrated a novel transcriptional mechanism via AHR and GR interaction.

  13. Luciferase Reporter Assays to Assess Liver X Receptor Transcriptional Activity.

    PubMed

    Gage, Matthew C; Pourcet, Benoit; Pineda-Torra, Inés

    2016-01-01

    Luciferase reporter assays are sensitive and accurate tests that enable the analysis of regulatory sequences, the magnitude of transcriptional activity by transcription factors, and the discovery of gene regulatory elements and small-molecule modulators with high levels of precision. This is made possible through detection of bioluminescence produced by luciferase-coding reporters in a wide range of cellular environments. These assays are routinely used to analyze the activity of transcription factors, including the lipid-activated liver X receptor (LXR), in response to different stimuli as well as for the identification of their ligands. In this chapter we describe in detail the assays performed to investigate LXR activity in a macrophage-like cell line (RAW 267.4). These can be easily adapted to other nuclear receptors and transcription factors. PMID:26552676

  14. Propagation of conformational changes during ?-opioid receptor activation.

    PubMed

    Sounier, Rémy; Mas, Camille; Steyaert, Jan; Laeremans, Toon; Manglik, Aashish; Huang, Weijiao; Kobilka, Brian K; Déméné, Héléne; Granier, Sébastien

    2015-08-20

    µ-Opioid receptors (µORs) are G-protein-coupled receptors that are activated by a structurally diverse spectrum of natural and synthetic agonists including endogenous endorphin peptides, morphine and methadone. The recent structures of the ?OR in inactive and agonist-induced active states (Huang et al., ref. 2) provide snapshots of the receptor at the beginning and end of a signalling event, but little is known about the dynamic sequence of events that span these two states. Here we use solution-state NMR to examine the process of ?OR activation using a purified receptor (mouse sequence) preparation in an amphiphile membrane-like environment. We obtain spectra of the ?OR in the absence of ligand, and in the presence of the high-affinity agonist BU72 alone, or with BU72 and a G protein mimetic nanobody. Our results show that conformational changes in transmembrane segments 5 and 6 (TM5 and TM6), which are required for the full engagement of a G protein, are almost completely dependent on the presence of both the agonist and the G protein mimetic nanobody, revealing a weak allosteric coupling between the agonist-binding pocket and the G-protein-coupling interface (TM5 and TM6), similar to that observed for the ?2-adrenergic receptor. Unexpectedly, in the presence of agonist alone, we find larger spectral changes involving intracellular loop 1 and helix 8 compared to changes in TM5 and TM6. These results suggest that one or both of these domains may play a role in the initial interaction with the G protein, and that TM5 and TM6 are only engaged later in the process of complex formation. The initial interactions between the G protein and intracellular loop 1 and/or helix 8 may be involved in G-protein coupling specificity, as has been suggested for other family A G-protein-coupled receptors. PMID:26245377

  15. Human mast cells adhere to and migrate on epithelial and vascular basement membrane laminins LM-332 and LM-511 via alpha3beta1 integrin.

    PubMed

    Sime, Wondossen; Lunderius-Andersson, Carolina; Enoksson, Mattias; Rousselle, Patricia; Tryggvason, Karl; Nilsson, Gunnar; Harvima, Ilkka; Patarroyo, Manuel

    2009-10-01

    Mast cells (MCs) are multifunctional effectors of the immune system that are distributed in many tissues, often in close association with the basement membrane of blood vessels, epithelium and nerves. Laminins (LMs), a family of large alphabetagamma heterotrimeric proteins, are major components of basement membrane that strongly promote cell adhesion and migration. In this study, we investigated the role of LM isoforms and their integrin receptors in human MC biology in vitro. In functional assays, alpha3-(LM-332) and alpha5-(LM-511) LMs, but not alpha1-(LM-111), alpha2-(LM-211), or alpha4-(LM-411) LMs, readily promoted adhesion and migration of cultured MCs. These activities were strongly enhanced by various stimuli. alpha3-LM was also able to costimulate IL-8 production. Among LM-binding integrins, MCs expressed alpha(3)beta(1), but not alpha(6)beta(1), alpha(7)beta(1), or alpha(6)beta(4), integrins. Blocking Abs to alpha(3)beta(1) integrin caused inhibition of both cell adhesion and migration on alpha3- and alpha5-LMs. Immunohistochemical studies on skin showed that MCs colocalized with epithelial and vascular basement membranes that expressed alpha3- and alpha5-LMs and that MCs expressed alpha(3) integrin but not alpha(6) integrin(s). These results demonstrate a role for alpha3- and alpha5-LMs and their alpha(3)beta(1) integrin receptor in MC biology. This may explain the intimate structural and functional interactions that MCs have with specific basement membranes. PMID:19752234

  16. Structural insights into µ-opioid receptor activation.

    PubMed

    Huang, Weijiao; Manglik, Aashish; Venkatakrishnan, A J; Laeremans, Toon; Feinberg, Evan N; Sanborn, Adrian L; Kato, Hideaki E; Livingston, Kathryn E; Thorsen, Thor S; Kling, Ralf C; Granier, Sébastien; Gmeiner, Peter; Husbands, Stephen M; Traynor, John R; Weis, William I; Steyaert, Jan; Dror, Ron O; Kobilka, Brian K

    2015-08-20

    Activation of the ?-opioid receptor (?OR) is responsible for the efficacy of the most effective analgesics. To shed light on the structural basis for ?OR activation, here we report a 2.1 Ĺ X-ray crystal structure of the murine ?OR bound to the morphinan agonist BU72 and a G protein mimetic camelid antibody fragment. The BU72-stabilized changes in the ?OR binding pocket are subtle and differ from those observed for agonist-bound structures of the ?2-adrenergic receptor (?2AR) and the M2 muscarinic receptor. Comparison with active ?2AR reveals a common rearrangement in the packing of three conserved amino acids in the core of the ?OR, and molecular dynamics simulations illustrate how the ligand-binding pocket is conformationally linked to this conserved triad. Additionally, an extensive polar network between the ligand-binding pocket and the cytoplasmic domains appears to play a similar role in signal propagation for all three G-protein-coupled receptors. PMID:26245379

  17. Impaired T cell receptor activation in Interleukin-1 Receptor-associated Kinase-4-deficient patients

    PubMed Central

    McDonald, Douglas R.; Goldman, Frederick; Gomez-Duarte, Oscar D.; Issekutz, Andrew C.; Kumararatne, Dinakantha S.; Doffinger, Rainer; Geha, Raif S.

    2010-01-01

    Background Interleukin-1 receptor associated kinase-4 (IRAK-4) is an effector of the Toll-like receptor and interleukin-1 receptor pathways, which plays a critical role in innate immune responses. The role of IRAK-4 in adaptive immune functions in humans is incompletely understood. Objective To evaluate T cell function in Interleukin-1 receptor associated kinase-4 deficient patients. Methods We compared upregulation of CD25 and CD69 on T cells, and production of interleukin-2, interleukin-6, and interferon gamma following stimulation of peripheral blood mononuclear cells from four IRAK-4 deficient patients and normal controls with anti-CD3 and anti-CD28. Results Upregulation of CD25 and CD69 on T cells and production of interleukin-6 and interferon-gamma, but not interleukin-2, was significantly reduced in IRAK-4 deficient patients. Conclusions IRAK-4 deficient patients have defects in T cell activation. Clinical Implications Defects in T cell activation may contribute to the susceptibility of IRAK-4 deficient patients to infections. PMID:20621347

  18. The Laminin 511/521 Binding Site on the Lutheran Blood Group Glycoprotein is Located at theFlexible Junction of Ig Domains 2 and 3

    SciTech Connect

    Mankelow, Tosti J.; Burton, Nicholas; Stedansdottir, Fanney O.; Spring, Frances A.; Parsons, Stephen F.; Pesersen, Jan S.; Oliveira, Cristiano L.P.; Lammie, Donna; Wess, Timothy; Mohandas, Narla; Chasis, Joel A.; Brady, R. Leo; Anstee, David J.

    2007-07-01

    The Lutheran blood group glycoprotein, first discovered on erythrocytes, is widely expressed in human tissues. It is a ligand for the {alpha}5 subunit of Laminin 511/521, an extracellular matrix protein. This interaction may contribute to vasocclusive events that are an important cause of morbidity in sickle cell disease. Using X-ray crystallography, small angle X-ray scattering and site directed mutagenesis we show that the extracellular region of Lutheran forms an extended structure with a distinctive bend between the second and third immunoglobulin-like domains. The linker between domains 2 and 3 appears to be flexible and is a critical determinant in maintaining an overall conformation for Lutheran that is capable of binding to Laminin. Mutagenesis studies indicate that Asp312 of Lutheran and the surrounding cluster of negatively charged residues in this linker region form the Laminin binding site. Unusually, receptor binding is therefore not a function of the domains expected to be furthermost from the plasma membrane. These studies imply that structural flexibility of Lutheran may be essential for its interaction with Laminin and present a novel opportunity for the development of therapeutics for sickle cell disease.

  19. A highly efficient and sensitive screening method for trans-activation activity of estrogen receptors

    E-print Network

    Zhao, Huimin

    A highly efficient and sensitive screening method for trans-activation activity of estrogen describe a highly efficient and sensitive yeast-based screening method for isolating human estrogen that estrogen receptor is a ligand-activated transcription factor, and links the transactivation activity

  20. Activation of Peroxisome Proliferator-Activated Receptor by Substituted Urea-Derived Soluble Epoxide Hydrolase Inhibitors

    E-print Network

    Hammock, Bruce D.

    effectively inhibiting sEH, increased peroxisome proliferator- activated receptor (PPAR) activity. In PPAR-adamantanyl-N -dodecanoic acid urea (AUDA) produced 6- and 3-fold increases, respec- tively, in PPAR activation. Neither CUDA nor AUDA activated PPAR or PPAR directly, indicating selectivity for PPAR . CUDA did not alter

  1. Molecular identification of receptor for pituitary adenylate cyclase activating polypeptide.

    PubMed

    Ohtaki, T; Watanabe, T; Ishibashi, Y; Kitada, C; Tsuda, M; Gottschall, P E; Arimura, A; Fujino, M

    1990-09-14

    Pituitary adenylate cyclase activating polypeptide (PACAP) is a novel hypothalamic peptide with 38 (PACAP38) or 27 (PACAP27) amino acid residues, structurally related to vasoactive intestinal peptide (VIP). Bovine brain membrane has a PACAP specific receptor interacting with both PACAP27 and PACAP38. Affinity-labeling of the receptor with [125I]PACAP27 identified a dominant band of Mr = 60 k. The labeling density of the 60 k band decreased in the presence of unlabeled PACAP27 or PACAP38, whereas the 60 k band remained in the presence of unlabeled VIP. Binding of [125I]PACAP27 to the membrane decreased in the presence of GTP and the labeling density of the 60 k band decreased concomitantly. The results indicate that bovine brain has a specific PACAP receptor, whose apparent molecular weight is 57 k (substracting the molecular weight of [125I]PACAP27 from 60 k). PMID:2169736

  2. Human peroxisome proliferator-activated receptor mRNA and protein expression during development

    EPA Science Inventory

    The peroxisome proliferator-activated receptors (PPAR) are nuclear hormone receptors that regulate lipid and glucose homeostasis and are important in reproduction and development. PPARs are targets ofpharmaceuticals and are also activated by environmental contaminants, including ...

  3. Protease-activated-receptor-2 affects protease-activated-receptor-1-driven breast cancer.

    PubMed

    Jaber, Mohammad; Maoz, Miriam; Kancharla, Arun; Agranovich, Daniel; Peretz, Tamar; Grisaru-Granovsky, Sorina; Uziely, Beatrice; Bar-Shavit, Rachel

    2014-07-01

    Mammalian protease-activated-receptor-1 and -2 (PAR1 and PAR2) are activated by proteases found in the flexible microenvironment of a tumor and play a central role in breast cancer. We propose in the present study that PAR1 and PAR2 act together as a functional unit during malignant and physiological invasion processes. This notion is supported by assessing pro-tumor functions in the presence of short hairpin; shRNA knocked-down hPar2 or by the use of a truncated PAR2 devoid of the entire cytoplasmic tail. Silencing of hPar2 by shRNA-attenuated thrombin induced PAR1 signaling as recapitulated by inhibiting the assembly of Etk/Bmx or Akt onto PAR1-C-tail, by thrombin-instigated colony formation and invasion. Strikingly, shRNA-hPar2 also inhibited the TFLLRN selective PAR1 pro-tumor functions. In addition, while evaluating the physiological invasion process of placenta extravillous trophoblast (EVT) organ culture, we observed inhibition of both thrombin or the selective PAR1 ligand; TFLLRNPNDK induced EVT invasion by shRNA-hPar2 but not by scrambled shRNA-hPar2. In parallel, when a truncated PAR2 was utilized in a xenograft mouse model, it inhibited PAR1-PAR2-driven tumor growth in vivo. Similarly, it also attenuated the interaction of Etk/Bmx with the PAR1-C-tail in vitro and decreased markedly selective PAR1-induced Matrigel invasion. Confocal images demonstrated co-localization of PAR1 and PAR2 in HEK293T cells over-expressing YFP-hPar2 and HA-hPar1. Co-immuno-precipitation analyses revealed PAR1-PAR2 complex formation but no PAR1-CXCR4 complex was formed. Taken together, our observations show that PAR1 and PAR2 act as a functional unit in tumor development and placenta-uterus interactions. This conclusion may have significant consequences on future breast cancer therapeutic modalities and improved late pregnancy outcome. PMID:24177339

  4. CINPA1 is an inhibitor of constitutive androstane receptor that does not activate pregnane X receptor.

    PubMed

    Cherian, Milu T; Lin, Wenwei; Wu, Jing; Chen, Taosheng

    2015-05-01

    Constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are xenobiotic sensors that enhance the detoxification and elimination of xenobiotics and endobiotics by modulating the expression of genes encoding drug-metabolizing enzymes and transporters. Elevated levels of drug-metabolizing enzymes and efflux transporters, resulting from CAR activation in various cancers, promote the elimination of chemotherapeutic agents, leading to reduced therapeutic effectiveness and acquired drug resistance. CAR inhibitors, in combination with existing chemotherapeutics, could therefore be used to attenuate multidrug resistance in cancers. Interestingly, all previously reported CAR inverse-agonists are also activators of PXR, rendering them mechanistically counterproductive in tissues where both these xenobiotic receptors are present and active. We used a directed high-throughput screening approach, followed by subsequent mechanistic studies, to identify novel, potent, and specific small-molecule CAR inhibitors that do not activate PXR. We describe here one such inhibitor, CINPA1 (CAR inhibitor not PXR activator 1), capable of reducing CAR-mediated transcription with an IC50 of ?70 nM. CINPA1 1) is a specific xenobiotic receptor inhibitor and has no cytotoxic effects up to 30 µM; 2) inhibits CAR-mediated gene expression in primary human hepatocytes, where CAR is endogenously expressed; 3) does not alter the protein levels or subcellular localization of CAR; 4) increases corepressor and reduces coactivator interaction with the CAR ligand-binding domain in mammalian two-hybrid assays; and 5) disrupts CAR binding to the promoter regions of target genes in chromatin immunoprecipitation assays. CINPA1 could be used as a novel molecular tool for understanding CAR function. PMID:25762023

  5. Activation of NMDA receptors thickens the postsynaptic density via proteolysis.

    PubMed

    Fukunaga, Yuko; Nakajima, Eri; Hatano, Erika; Itoh, Sayaka; Kashino, Yasuhiro; Miyazawa, Atsuo

    2015-12-01

    The postsynaptic density (PSD) is a protein complex that is critical for synaptic transmission. Ultrastructural changes in the PSD are therefore likely to modify synaptic functions. In this study, we investigated the ultrastructural changes in the PSD in the hippocampal CA1 stratum radiatum following neuronal excitation. Oxygen-glucose deprivation-induced PSD thickening in hippocampal slice cultures was blocked by the N-methyl-d-aspartate (NMDA) receptor antagonist MK801. To gain more insight into the mechanisms underlying NMDA receptor-mediated PSD thickening, we assessed the area, length, and thickness of the PSD after NMDA treatment. The PSDs thickened with just 2min of NMDA receptor stimulation, and this treatment was considered sublethal. When N-acetyl-leucyl-leucyl-norleucinal, an inhibitor of calpain, cathepsins, and the proteasome, was applied, NMDA-induced PSD thickening was abolished. Furthermore, the calcium-induced calcium release inhibitor, ryanodine, reduced NMDA receptor-mediated PSD thickening. These results suggest that NMDA receptor activation induces PSD thickening by proteolysis through intracellular calcium increase, including that induced by calcium. PMID:26188126

  6. Laminin and biomimetic extracellular elasticity enhance functional differentiation in mammary

    E-print Network

    Nelson, Celeste M.

    EMBO open Laminin and biomimetic extracellular elasticity enhance functional differentiation-casein expression using atomic force microscopy to measure the elasticity of the cells and their underlying' extracellular matrix, as is the case in normal tissues in vivo, and biomi- metic intracellular elasticity

  7. The Orphan Nuclear Receptor SHP Utilizes Conserved LXXLL-Related Motifs for Interactions with Ligand-Activated Estrogen Receptors

    PubMed Central

    Johansson, Lotta; Bĺvner, Ann; Thomsen, Jane S.; Färnegĺrdh, MatHias; Gustafsson, Jan-?ke; Treuter, Eckardt

    2000-01-01

    SHP (short heterodimer partner) is an unusual orphan nuclear receptor consisting only of a ligand-binding domain, and it exhibits unique features of interaction with conventional nuclear receptors. While the mechanistic basis of these interactions has remained enigmatic, SHP has been suggested to inhibit nuclear receptor activation by at least three alternatives; inhibition of DNA binding via dimerization, direct antagonism of coactivator function via competition, and possibly transrepression via recruitment of putative corepressors. We now show that SHP binds directly to estrogen receptors via LXXLL-related motifs. Similar motifs, referred to as NR (nuclear receptor) boxes, are usually critical for the binding of coactivators to the ligand-regulated activation domain AF-2 within nuclear receptors. In concordance with the NR box dependency, SHP requires the intact AF-2 domain of agonist-bound estrogen receptors for interaction. Mutations within the ligand-binding domain helix 12, or binding of antagonistic ligands, which are known to result in an incomplete AF-2 surface, abolish interactions with SHP. Supporting the idea that SHP directly antagonizes receptor activation via AF-2 binding, we demonstrate that SHP variants, carrying either interaction-defective NR box mutations or a deletion of the repressor domain, have lost the capacity to inhibit agonist-dependent transcriptional estrogen receptor activation. Furthermore, our studies indicate that SHP may function as a cofactor via the formation of ternary complexes with dimeric receptors on DNA. These novel insights provide a mechanistic explanation for the inhibitory role of SHP in nuclear receptor signaling, and they may explain how SHP functions as a negative coregulator or corepressor for ligand-activated receptors, a novel and unique function for an orphan nuclear receptor. PMID:10648597

  8. Conserved phosphorylation sites in the activation loop of the Arabidopsis phytosulfokine receptor PSKR1 differentially affect kinase and receptor activity

    PubMed Central

    Hartmann, Jens; Linke, Dennis; Bönniger, Christine; Tholey, Andreas; Sauter, Margret

    2015-01-01

    PSK (phytosulfokine) is a plant peptide hormone perceived by a leucine-rich repeat receptor kinase. Phosphosite mapping of epitope-tagged PSKR1 (phytosulfokine receptor 1) from Arabidopsis thaliana plants identified Ser696 and Ser698 in the JM (juxtamembrane) region and probably Ser886 and/or Ser893 in the AL (activation loop) as in planta phosphorylation sites. In vitro-expressed kinase was autophosphorylated at Ser717 in the JM, and at Ser733, Thr752, Ser783, Ser864, Ser911, Ser958 and Thr998 in the kinase domain. The LC–ESI–MS/MS spectra provided support that up to three sites (Thr890, Ser893 and Thr894) in the AL were likely to be phosphorylated in vitro. These sites are evolutionarily highly conserved in PSK receptors, indicative of a conserved function. Site-directed mutagenesis of the four conserved residues in the activation segment, Thr890, Ser893, Thr894 and Thr899, differentially altered kinase activity in vitro and growth-promoting activity in planta. The T899A and the quadruple-mutated TSTT-A (T890A/S893A/T894A/T899A) mutants were both kinase-inactive, but PSKR1(T899A) retained growth-promoting activity. The T890A and S893A/T894A substitutions diminished kinase activity and growth promotion. We hypothesize that phosphorylation within the AL activates kinase activity and receptor function in a gradual and distinctive manner that may be a means to modulate the PSK response. PMID:26472115

  9. Conserved phosphorylation sites in the activation loop of the Arabidopsis phytosulfokine receptor PSKR1 differentially affect kinase and receptor activity.

    PubMed

    Hartmann, Jens; Linke, Dennis; Bönniger, Christine; Tholey, Andreas; Sauter, Margret

    2015-12-15

    PSK (phytosulfokine) is a plant peptide hormone perceived by a leucine-rich repeat receptor kinase. Phosphosite mapping of epitope-tagged PSKR1 (phytosulfokine receptor 1) from Arabidopsis thaliana plants identified Ser(696) and Ser(698) in the JM (juxtamembrane) region and probably Ser(886) and/or Ser(893) in the AL (activation loop) as in planta phosphorylation sites. In vitro-expressed kinase was autophosphorylated at Ser(717) in the JM, and at Ser(733), Thr(752), Ser(783), Ser(864), Ser(911), Ser(958) and Thr(998) in the kinase domain. The LC-ESI-MS/MS spectra provided support that up to three sites (Thr(890), Ser(893) and Thr(894)) in the AL were likely to be phosphorylated in vitro. These sites are evolutionarily highly conserved in PSK receptors, indicative of a conserved function. Site-directed mutagenesis of the four conserved residues in the activation segment, Thr(890), Ser(893), Thr(894) and Thr(899), differentially altered kinase activity in vitro and growth-promoting activity in planta. The T899A and the quadruple-mutated TSTT-A (T890A/S893A/T894A/T899A) mutants were both kinase-inactive, but PSKR1(T899A) retained growth-promoting activity. The T890A and S893A/T894A substitutions diminished kinase activity and growth promotion. We hypothesize that phosphorylation within the AL activates kinase activity and receptor function in a gradual and distinctive manner that may be a means to modulate the PSK response. PMID:26472115

  10. Sigma-1 receptor activation inhibits osmotic swelling of rat retinal glial (Müller) cells by transactivation of glutamatergic and purinergic receptors.

    PubMed

    Vogler, Stefanie; Winters, Helge; Pannicke, Thomas; Wiedemann, Peter; Reichenbach, Andreas; Bringmann, Andreas

    2016-01-01

    Water accumulation in retinal glial (Müller) and neuronal cells resulting in cellular swelling contributes to the development of retinal edema and neurodegeneration. Sigma (?) receptor activation is known to have neuroprotective effects in the retina. Here, we show that the nonselective ? receptor agonist ditolylguanidine, and the selective ?1 receptor agonist PRE-084, inhibit the osmotic swelling of Müller cell somata induced by superfusion of rat retinal slices with a hypoosmotic solution containing barium ions. In contrast, PRE-084 did not inhibit the osmotic swelling of bipolar cell somata. The effects of ? receptor agonists on the Müller cell swelling were abrogated in the presence of blockers of metabotropic glutamate and purinergic P2Y1 receptors, respectively, suggesting that ? receptor activation triggers activation of a glutamatergic-purinergic signaling cascade which is known to prevent the osmotic Müller cell swelling. The swelling-inhibitory effect of 17?-estradiol was prevented by the ?1 receptor antagonist BD1047, suggesting that the effect is mediated by ?1 receptor activation. The data may suggest that the neuroprotective effect of ? receptor activation in the retina is in part mediated by prevention of the cytotoxic swelling of retinal glial cells. PMID:26499958

  11. Platelet Specific Promoters Are Insufficient to Express Protease Activated Receptor 1 (PAR1) Transgene in

    E-print Network

    Platelet Specific Promoters Are Insufficient to Express Protease Activated Receptor 1 (PAR1 study of protease activated receptors (PARs) in platelets is complicated due to species specific to Express Protease Activated Receptor 1 (PAR1) Transgene in Mouse Platelets. PLoS ONE 9(5): e97724. doi:10

  12. Pyrimidinergic Receptor Activation Controls Toxoplasma gondii Infection in Macrophages.

    PubMed

    Moreira-Souza, Aline Cristina Abreu; Marinho, Ygor; Correa, Gladys; Santoro, Giani França; Coutinho, Claudia Mara Lara Melo; Vommaro, Rossiane Claudia; Coutinho-Silva, Robson

    2015-01-01

    Infection by the protozoan parasite Toxoplasma gondii is highly prevalent worldwide and may have serious clinical manifestations in immunocompromised patients. T. gondii is an obligate intracellular parasite that infects almost any cell type in mammalian hosts, including immune cells. The immune cells express purinergic P2 receptors in their membrane--subdivided into P2Y and P2X subfamilies--whose activation is important for infection control. Here, we examined the effect of treatment with UTP and UDP in mouse peritoneal macrophages infected with T. gondii tachyzoites. Treatment with these nucleotides reduced parasitic load by 90%, but did not increase the levels of the inflammatory mediators NO and ROS, nor did it modulate host cell death by apoptosis or necrosis. On the other hand, UTP and UDP treatments induced early egress of tachyzoites from infected macrophages, in a Ca2+-dependent manner, as shown by scanning electron microscopy analysis, and videomicroscopy. In subsequent infections, prematurely egressed parasites had reduced infectivity, and could neither replicate nor inhibit the fusion of lysosomes to the parasitophorous vacuole. The use of selective agonists and antagonists of the receptor subtypes P2Y2 and P2Y4 and P2Y6 showed that premature parasite egress may be mediated by the activation of these receptor subtypes. Our results suggest that the activity of P2Y host cell receptors controls T. gondii infection in macrophages, highlighting the importance of pyrimidinergic signaling for innate immune system response against infection. Finally the P2Y receptors should be considered as new target for the development of drugs against T. gondii infection. PMID:26192447

  13. Pyrimidinergic Receptor Activation Controls Toxoplasma gondii Infection in Macrophages

    PubMed Central

    Moreira-Souza, Aline Cristina Abreu; Marinho, Ygor; Correa, Gladys; Santoro, Giani França; Coutinho, Claudia Mara Lara Melo; Vommaro, Rossiane Claudia; Coutinho-Silva, Robson

    2015-01-01

    Infection by the protozoan parasite Toxoplasma gondii is highly prevalent worldwide and may have serious clinical manifestations in immunocompromised patients. T. gondii is an obligate intracellular parasite that infects almost any cell type in mammalian hosts, including immune cells. The immune cells express purinergic P2 receptors in their membrane – subdivided into P2Y and P2X subfamilies - whose activation is important for infection control. Here, we examined the effect of treatment with UTP and UDP in mouse peritoneal macrophages infected with T. gondii tachyzoites. Treatment with these nucleotides reduced parasitic load by 90%, but did not increase the levels of the inflammatory mediators NO and ROS, nor did it modulate host cell death by apoptosis or necrosis. On the other hand, UTP and UDP treatments induced early egress of tachyzoites from infected macrophages, in a Ca2+-dependent manner, as shown by scanning electron microscopy analysis, and videomicroscopy. In subsequent infections, prematurely egressed parasites had reduced infectivity, and could neither replicate nor inhibit the fusion of lysosomes to the parasitophorous vacuole. The use of selective agonists and antagonists of the receptor subtypes P2Y2 and P2Y4 and P2Y6 showed that premature parasite egress may be mediated by the activation of these receptor subtypes. Our results suggest that the activity of P2Y host cell receptors controls T. gondii infection in macrophages, highlighting the importance of pyrimidinergic signaling for innate immune system response against infection. Finally the P2Y receptors should be considered as new target for the development of drugs against T. gondii infection. PMID:26192447

  14. Differential effects on bone of estrogen receptor ? and androgen receptor activation in orchidectomized adult male mice

    PubMed Central

    Movérare, Sofia; Venken, Katrien; Eriksson, Anna-Lena; Andersson, Niklas; Skrtic, Stanko; Wergedal, Jon; Mohan, Subburaman; Salmon, Phil; Bouillon, Roger; Gustafsson, Jan-Ĺke; Vanderschueren, Dirk; Ohlsson, Claes

    2003-01-01

    Androgens may regulate the male skeleton either directly by stimulation of the androgen receptor (AR) or indirectly by aromatization of androgens into estrogens and, thereafter, by stimulation of the estrogen receptors (ERs). To directly compare the effect of ER activation on bone in vivo with the effect of AR activation, 9-month-old orchidectomized wild-type and ER-inactivated mice were treated with the nonaromatizable androgen 5?-dihydrotestosterone, 17?-estradiol, or vehicle. Both ER? and AR but not ER? activation preserved the amount of trabecular bone. ER? activation resulted both in a preserved thickness and number of trabeculae. In contrast, AR activation exclusively preserved the number of trabeculae, whereas the thickness of the trabeculae was unaffected. Furthermore, the effects of 17?-estradiol could not be mediated by the AR, and the effects of 5?-dihydrotestosterone were increased rather than decreased in ER-inactivated mice. ER?, but not AR or ER?, activation resulted in preserved thickness, volumetric density, and mechanical strength of the cortical bone. ER? activation increased serum levels of insulin-like growth factor I, which were positively correlated with all the cortical and trabecular bone parameters that were specifically preserved by ER? activation but not by AR activation, suggesting that insulin-like growth factor I might mediate these effects of ER? activation. Thus, the in vivo bone-sparing effect of ER? activation is distinct from the bone-sparing effect of AR activation in adult male mice. Because these two pathways are clearly distinct from each other, one may speculate that a combined treatment of selective ER modulators and selective AR modulators might be beneficial in the treatment of osteoporosis. PMID:14573701

  15. The thrombopoietin receptor P106L mutation functionally separates receptor signaling activity from thrombopoietin homeostasis.

    PubMed

    Stockklausner, Clemens; Klotter, Anne-Christine; Dickemann, Nicole; Kuhlee, Isabelle N; Duffert, Christin M; Kerber, Carolin; Gehring, Niels H; Kulozik, Andreas E

    2015-02-12

    The interaction between thrombopoietin (THPO) and its receptor c-Mpl regulates downstream cytokine signaling and platelet homeostasis. Hereditary mutations of c-Mpl can either result in loss-of-function and thrombocytopenia or in gain-of-function and thrombocythemia (HT), and are important models to analyze the mechanism of c-Mpl activity. We have analyzed the effect of the c-Mpl P106L gain-of-function and the nearby loss-of-function R102P and F104S mutations, which cause HT or thrombocytopenia, respectively, on posttranslational processing, intracellular trafficking, cell surface expression, and cell proliferation. In contrast to R102P and F104S, the P106L mutant confers cytokine-independent growth and stimulates downstream signaling after THPO treatment in Ba/F3 cells. Despite their opposite function, R102P and P106L, both lead to abnormal subcellular receptor distribution, lack of membrane localization, impaired glycosylation, and elevated THPO serum levels in effected patients. These findings indicate that the activation of downstream signaling by c-Mpl P106L does not require correct processing, trafficking, and cell surface expression of c-Mpl, whereas the negative feedback loop controlling THPO serum levels requires cell surface expression of the receptor. Thus, we propose that the P106L mutation functionally separates the activity of c-Mpl in downstream signaling from that in maintaining platelet homeostasis. PMID:25538044

  16. The peroxisome proliferator-activated receptor ?, an integrator of transcriptional repression and nuclear receptor signaling

    PubMed Central

    Shi, Yanhong; Hon, Michelle; Evans, Ronald M.

    2002-01-01

    The three PPAR (peroxisome proliferator-activated receptor) isoforms are critical regulators of lipid homeostasis by controlling the balance between the burning and storage of long chain fatty acids. Whereas PPAR? and PPAR? have been studied extensively, the function of PPAR? remains the most elusive. Intriguingly, in cotransfection experiments, PPAR? is a potent inhibitor of ligand-induced transcriptional activity of PPAR? and PPAR?. This inhibition is achieved, in part, by binding of PPAR? to a peroxisome proliferator response element and the association of nonliganded PPAR? with corepressors SMRT (silencing mediator for retinoid and thyroid hormone receptors), SHARP (SMRT and histone deacetylase-associated repressor protein), and class I histone deacetylases. Stable expression of PPAR? inhibits the expression of endogenous PPAR? target gene expression in 3T3-PPAR? cells, whereas a PPAR? mutant that does not interact with the corepressor SMRT loses its ability to repress the induction of PPAR? target gene. Similarly, stable expression of PPAR? in 3T3-PPAR? cells leads to inhibition of PPAR? target gene expression and PPAR?-mediated adipogenesis. Given the widespread expression of PPAR? and the restricted pattern for PPAR? and PPAR?, these results suggest a role for PPAR? as a gateway receptor whose relative levels of expression can be used to modulate PPAR? and PPAR? activity. PMID:11867749

  17. Protease activated receptor-1 mediated dual kinase receptor transactivation stimulates the expression of glycosaminoglycan synthesizing genes.

    PubMed

    Kamato, Danielle; Thach, Lyna; Getachew, Robel; Burch, Micah; Hollenberg, Morley D; Zheng, Wenhua; Little, Peter J; Osman, Narin

    2016-01-01

    G protein-coupled receptors (GPCR) are one of the most important targets for therapeutics due to their abundance and diversity. The G protein-coupled receptor for thrombin can transactivate protein tyrosine kinase receptors (PTKR) and we have recently established that it can also transactivate serine/threonine kinase receptors (S/TKR). A comprehensive knowledge of the signalling pathways that GPCR transactivation elicits is necessary to fully understand the implications of both GPCR activation and the impact of target drugs. Here, we demonstrate that thrombin elicits dual transactivation-dependent signalling pathways to stimulate mRNA expression of glycosaminoglycan synthesizing enzymes chondroitin 4-O-sulfotransferase 1 and chondroitin sulfate synthase 1. The PTKR mediated response involves matrix metalloproteinases and the phosphorylation of the MAP kinase Erk. The S/TKR mediated response differs markedly and involves the phosphorylation of Smad2 carboxy terminal serine residues and does not involve matrix metalloproteinases. This work shows that all of the thrombin mediated signalling to glycosaminoglycan synthesizing enzyme gene expression occurs via transactivation-dependent pathways and does not involve transactivation-independent signalling. These findings highlight the complexity of thrombin-mediated transactivation signalling and the broader implications of GPCR targeted therapeutics. PMID:26548632

  18. Modulation of Retinoic Acid Receptor-related Orphan Receptor ? and ? Activity by 7-Oxygenated Sterol Ligands*

    PubMed Central

    Wang, Yongjun; Kumar, Naresh; Solt, Laura A.; Richardson, Timothy I.; Helvering, Leah M.; Crumbley, Christine; Garcia-Ordonez, Ruben D.; Stayrook, Keith R.; Zhang, Xi; Novick, Scott; Chalmers, Michael J.; Griffin, Patrick R.; Burris, Thomas P.

    2010-01-01

    The retinoic acid receptor-related orphan receptors ? and ? (ROR? (NR1F1) and ROR? (NR1F3)) are orphan nuclear receptors and perform critical roles in regulation of development, metabolism, and immune function. Cholesterol and cholesterol sulfate have been suggested to be ROR? ligands, but the physiological significance is unclear. To date, no endogenous ROR? ligands have been described. Here, we demonstrate that 7-oxygenated sterols function as high affinity ligands for both ROR? and ROR? by directly binding to their ligand-binding domains (Ki ?20 nm), modulating coactivator binding, and suppressing the transcriptional activity of the receptors. One of the 7-oxygenated sterols, 7?-hydroxycholesterol (7?-OHC), serves as a key intermediate in bile acid metabolism, and we show that 7?-OHC modulates the expression of ROR target genes, including Glc-6-Pase and phosphoenolpyruvate carboxykinase, in an ROR-dependent manner. Furthermore, glucose output from hepatocytes is suppressed by 7?-OHC functioning as an ROR?/? ligand. Thus, ROR? and ROR? are ligand-regulated members of the NR superfamily and may serve as sensors for 7-oxygenated sterols. PMID:19965867

  19. Role of Peroxisome Proliferator-Activated Receptor ? in Ocular Diseases

    PubMed Central

    Zhang, Su; Gu, Hongwei; Hu, Nan

    2015-01-01

    Peroxisome proliferator-activated receptor ? (PPAR ?), a member of the nuclear receptor superfamily, is a ligand-activated transcription factor that plays an important role in the control of a variety of physiological processes. The last decade has witnessed an increasing interest for the role played by the agonists of PPAR ? in antiangiogenesis, antifibrosis, anti-inflammation effects and in controlling oxidative stress response in various organs. As the pathologic mechanisms of major blinding diseases, such as age-related macular degeneration (AMD), diabetic retinopathy (DR), keratitis, and optic neuropathy, often involve neoangiogenesis and inflammation- and oxidative stress-mediated cell death, evidences are accumulating on the potential benefits of PPAR ? to improve or prevent these vision threatening eye diseases. In this paper we describe what is known about the role of PPAR ? in the ocular pathophysiological processes and PPAR ? agonists as novel adjuvants in the treatment of eye diseases. PMID:26146566

  20. Artificial synthetic receptors as regulators of protein activity.

    PubMed

    Dutt, Som; Wilch, Constanze; Schrader, Thomas

    2011-05-21

    This article discusses most recent work and progress in the direction of a rational design of small molecule receptors that efficiently interfere with the biological function of a particular receptor or enzyme-some of which are therapeutically relevant. More specifically, the following topics are highlighted here: the inhibition of voltage-dependent potassium channels of the K(v)1.x family by designed porphyrin and calix[4]arene ligands, the structural and functional recovery of the tetramerization domain of mutated P53 protein by tailored calix[4]arene ligands and the control over LDH activity by supramolecular signaling. Finally a new way to modulate NAD(+)-dependent enzymatic activities by molecular clips and tweezers is presented. PMID:21394349

  1. Selenoprotein W controls epidermal growth factor receptor surface expression, activation and degradation via receptor ubiquitination.

    PubMed

    Alkan, Zeynep; Duong, Frank L; Hawkes, Wayne C

    2015-05-01

    Epidermal growth factor (EGF) receptor (EGFR) is the founding member of the ErbB family of growth factor receptors that modulate a complex network of intracellular signaling pathways controlling growth, proliferation, differentiation, and motility. Selenoprotein W (SEPW1) is a highly conserved, diet-regulated 9kDa thioredoxin-like protein required for normal cell cycle progression. We report here that SEPW1 is required for EGF-induced EGFR activation and that it functions by suppressing EGFR ubiquitination and receptor degradation. SEPW1 depletion inhibited EGF-dependent cell cycle entry in breast and prostate epithelial cells. In prostate cells, SEPW1 depletion decreased EGFR auto-phosphorylation, while SEPW1 overexpression increased EGFR auto-phosphorylation. SEPW1 depletion increased the rate of EGFR degradation, which decreased total and surface EGFR and suppressed EGF-dependent EGFR endocytosis, EGFR dimer formation, and activation of EGF-dependent pathways. EGFR ubiquitination was increased in SEPW1-depleted cells--in agreement with the increased rate of EGFR degradation, and suggests that SEPW1 suppresses EGFR ubiquitination. Ubiquitination-directed lysozomal degradation controls post-translational EGFR expression and is dysregulated in many cancers. Thus, suppression of EGFR ubiquitination by SEPW1 may be related to the putative increase in cancer risk associated with high selenium intakes. Knowledge of the mechanisms underlying SEPW1's regulation of EGFR ubiquitination may reveal new opportunities for nutritional cancer prevention or cancer drug development. PMID:25721765

  2. Ultrastructural and biochemical analysis of fibrinogen receptors on activated thrombocytes

    SciTech Connect

    O'Toole, E.T.

    1989-01-01

    The present studies have been concerned with the role of fibrinogen and its receptor, GP IIb/IIIa, during the activation and early aggregation of pigeon thrombocytes. Thrombocytes were surface labeled with {sup 125}I then separated on SDS-PAGE. Analysis by gel autoradiography revealed major bands at MW 145 kd and 98 kd, which corresponded to human GPIIb and GPIIIa. Immunologic similarity of the pigeon and human receptor components was established by dot blot analysis using polyclonal antibodies directed against human GPIIb and GPIIIa. Pigeon fibrinogen, isolated by plasma precipitation with PEG-1000 and purified over Sepharose 4B, was used to study receptor-ligand interaction. Separation of pigeon fibrinogen on SDS-PAGE resulted in three peptides having apparent MW of 62kd, 55kd, and 47kd which are comparable to human fibrinogen. Further similarity of human and pigeon fibrinogen was verified by immonodiffusion against an antibody specific for the human protein. The role of fibrinogen and its receptor in thrombocyte function was established by turbidimetric aggregation using thrombin as an agonist under conditions requiring Ca++ and fibrinogen.

  3. A recombinant tail-less integrin beta 4 subunit disrupts hemidesmosomes, but does not suppress alpha 6 beta 4-mediated cell adhesion to laminins

    PubMed Central

    1995-01-01

    To examine the function of the alpha 6 beta 4 integrin we have determined its ligand-binding ability and overexpressed two potentially dominant negative mutant beta 4 subunits, lacking either the cytoplasmic or extracellular domain, in bladder epithelial 804G cells. The results of cell adhesion and radioligand-binding assays showed that alpha 6 beta 4 is a receptor for several laminin isoforms, including laminin 1, 2, 4, and 5. Overexpression of the tail-less or head-less mutant beta 4 subunit did not suppress alpha 6 beta 4-mediated adhesion to laminins, as both types of transfectants adhered to these ligands in the presence of blocking anti-beta 1 antibodies as well as the controls. However, immunofluorescence experiments indicated that the endogenous alpha 6 beta 4 integrin and other hemidesmosomal markers were not concentrated in hemidesmosomes in cells overexpressing tail- less beta 4, while the distribution of these molecules was not altered in cells overexpressing the head-less subunit. Electron microscopic studies confirmed that cells overexpressing tail-less beta 4 had a drastically reduced number of hemidesmosomes, while cells expressing the head-less subunit had a normal number of these structures. Thus, expression of a tail-less, but not a head-less mutant beta 4 subunit leads to a dominant negative effect on hemidesmosome assembly without suppressing initial adhesion to laminins. We conclude that the alpha 6 beta 4 integrin binds to several laminins and plays an essential role in the assembly and/or stability of hemidesmosomes, that alpha 6 beta 4- mediated adhesion and hemidesmosome assembly have distinct requirements, and that it is possible to use a dominant negative approach to selectively interfere with a specific function of an integrin. PMID:7721947

  4. Liver X Receptors Regulate the Transcriptional Activity of the Glucocorticoid Receptor: Implications for the Carbohydrate Metabolism

    PubMed Central

    Nader, Nancy; Ng, Sinnie Sin Man; Wang, Yonghong; Abel, Brent S.; Chrousos, George P.; Kino, Tomoshige

    2012-01-01

    GLUCOCORTICOIDS are steroid hormones that strongly influence intermediary carbohydrate metabolism by increasing the transcription rate of glucose-6-phosphatase (G6Pase), a key enzyme of gluconeogenesis, and suppress the immune system through the glucocorticoid receptor (GR). The liver X receptors (LXRs), on the other hand, bind to cholesterol metabolites, heterodimerize with the retinoid X receptor (RXR), and regulate the cholesterol turnover, the hepatic glucose metabolism by decreasing the expression of G6Pase, and repress a set of inflammatory genes in immune cells. Since the actions of these receptors overlap with each other, we evaluated the crosstalk between the GR- and LXR-mediated signaling systems. Transient transfection-based reporter assays and gene silencing methods using siRNAs for LXRs showed that overexpression/ligand (GW3965) activation of LXRs/RXRs repressed GR-stimulated transactivation of certain glucocorticoid response element (GRE)-driven promoters in a gene-specific fashion. Activation of LXRs by GW3965 attenuated dexamethasone-stimulated elevation of circulating glucose in rats. It also suppressed dexamethasone-induced mRNA expression of hepatic glucose-6-phosphatase (G6Pase) in rats, mice and human hepatoma HepG2 cells, whereas endogenous, unliganded LXRs were required for dexamethasone-induced mRNA expression of phosphoenolpyruvate carboxylase. In microarray transcriptomic analysis of rat liver, GW3965 differentially regulated glucocorticoid-induced transcriptional activity of about 15% of endogenous glucocorticoid-responsive genes. To examine the mechanism through which activated LXRs attenuated GR transcriptional activity, we examined LXR?/RXR? binding to GREs. Endogenous LXR?/RXR? bound GREs and inhibited GR binding to these DNA sequences both in in vitro and in vivo chromatin immunoprecipitation assays, while their recombinant proteins did so on classic or G6Pase GREs in gel mobility shift assays. We propose that administration of LXR agonists may be beneficial in glucocorticoid treatment- or stress-associated dysmetabolic states by directly and gene-specifically attenuating the transcriptional activity of the GR on glucose and/or lipid metabolism. PMID:22457708

  5. Murine atopic dermatitis responds to peroxisome proliferator-activated receptor ?, ?/?(but not ?), and liver-X-receptor activators

    PubMed Central

    Hatano, Yutaka; Man, Mao-Qiang; Uchida, Yoshikazu; Crumrine, Debra; Mauro, Theodora M.; Feingold, Kenneth R.; Elias, Peter M.; Holleran, Walter M.

    2010-01-01

    Background Atopic dermatitis (AD) is a chronic inflammatory dermatosis now increasingly linked to mutations that alter the structure and function of the stratum corneum (SC). Activators of peroxisome proliferators-activated receptor (PPAR)?, ?/?, ? and liver-X-receptor (LXR) regulate epidermal protein and lipid production, leading to superior barrier function. Additionally, some of these activators exhibit potent anti-hyperplastic and anti-inflammatory activity in irritant contact dermatitis and acute allergic contact dermatitis mouse models. Objective We evaluated the efficacy of PPAR/LXR activation in a hapten (oxazolone [Ox])-induced atopic dermatitis-like model (Ox-AD) in hairless mice. Methods Ox-AD was established with ten Ox challenges (every-other day) on the flank. After the establishment of Ox-AD, twice daily topical application with individual PPAR/LXR activators was then performed for 4 days, with continued Ox challenges every other day. The efficacy of topical PPAR/LXR activators to reduce parameters of Ox-AD was assessed physiologically, morphologically and immunologically. Results Certain topical activators of PPAR?, PPAR?/?, and LXR, but not activators of PPAR?, reversed the clinical dermatosis, significantly improved barrier function, and increased SC hydration in Ox-AD mice. In addition, the same activators, but again not PPAR?, largely reversed the immunologic abnormalities in Ox-AD mice, including the elevated TH2 markers, such as tissue eosinophil/mast cell density, serum TARC levels, density of CRTH2-positive lymphocytes (but not serum IgE levels), and reduced IL-1? and TNF? activation, despite on-going hapten challenges. Conclusion These results suggest that topical applications of certain activators/ligands of PPAR?, ?/?, and LXR could be useful for the treatment of AD in humans. PMID:19818482

  6. In vitro modulation of estrogen receptor activity by norfluoxetine

    PubMed Central

    LUPU, DIANA; POP, ANCA; CHERFAN, JULIEN; KISS, BÉLA; LOGHIN, FELICIA

    2015-01-01

    Background and aims Selective serotonin reuptake inhibitors (SSRIs) are antidepressants increasingly prescribed for pregnancy and postpartum depression. However, these compounds can cross the placenta and also pass into breast milk, thus reaching the fetus and infant during critical developmental stages, potentially causing adverse effects. Fluoxetine, a widely used SSRI, has been shown to affect (neuro)endocrine signaling in various organisms, including humans. This compound can also interact with estrogen receptors in vitro and cause an estrogen-dependent uterotrophic response in rodents. Consequently, the aim of the present study was to assess if the active metabolite of fluoxetine, namely norfluoxetine (NFLX), shares the same capacity for estrogen receptor interaction. Methods The in vitro (anti)estrogenic activity of norfluoxetine was assessed using a firefly luciferase reporter construct in the T47D-Kbluc breast cancer cell line. These cells express nuclear estrogen receptors (ERs) that can activate the transcription of the luciferase reporter gene upon binding of ER agonists. Light emission was monitored in case of cells exposed to norfluoxetine or mixtures of norfluoxetine-estradiol. Cell viability was assessed using a resazurin-based assay. Results During individual testing, NFLX was able to induce a significant increase in luciferase activity compared to control, but only at the highest concentration tested (10 ?M). In binary mixtures with estradiol (30 pM constant concentration) a significant increase in luminescence was observed at low submicromolar norfluoxetine concentrations compared to estradiol alone. Conclusion Norfluoxetine can induce estrogenic effects in vitro and can potentiate the activity of estradiol. However, further studies are needed to clarify if these observed estrogenic effects may have detrimental consequences for human exposure. PMID:26609274

  7. Acute activation of the calcium-sensing receptor inhibits plasma renin activity in vivo

    PubMed Central

    Atchison, Douglas K.; Ortiz-Capisano, M. Cecilia

    2010-01-01

    In vitro, the renin-secreting juxtaglomerular cells express the calcium-sensing receptor, and its activation with the calcimimetic cinacalcet inhibits renin release. To test whether the activation of calcium-sensing receptor similarly inhibits plasma renin activity (PRA) in vivo, we hypothesized that the calcium-sensing receptor is expressed in juxtaglomerular cells in vivo, and acutely administered cinacalcet would inhibit renin activity in anesthetized rats. Since cinacalcet inhibits parathyroid hormone, which may stimulate renin activity, we sought to determine whether cinacalcet inhibits renin activity by decreasing parathyroid hormone. Lastly, we hypothesized that chronically administered cinacalcet would inhibit basal and stimulated renin in conscious rats. Calcium-sensing receptors and renin were localized in the same juxtaglomerular cells using immunofluorescence in rat cortical slices fixed in vivo. Cinacalcet was administered acutely via intravenous bolus in anesthetized rats and chronically in conscious rats by oral gavage. Acute administration of cinacalcet decreased basal renin activity from 13.6 ± 2.4 to 6.1 ± 1.1 ng ANG I·ml?1·h?1 (P < 0.001). Likewise, cinacalcet decreased furosemide-stimulated renin from 30.6 ± 2.3 to 21.3 ± 2.3 ng ANG I·ml?1·h?1 (P < 0.001). In parathyroidectomized rats, cinacalcet decreased renin activity from 9.3 ± 1.3 to 5.2 ± 0.5 ng ANG I·ml?1·h?1 (P < 0.05) similar to sham-operated controls (13.5 ± 2.2 to 6.6 ± 0.8 ng ANG I·ml?1·h?1, P < 0.05). Chronic administration of cinacalcet over 7 days had no significant effect on PRA under basal or stimulated conditions. In conclusion, calcium-sensing receptors are expressed in juxtaglomerular cells in vivo, and acute activation of these receptors with cinacalcet inhibits PRA in anesthetized rats, independent of parathyroid hormone. PMID:20660105

  8. Depersonalization disorder may be related to glutamate receptor activation imbalance.

    PubMed

    Pikwer, Andreas

    2011-10-01

    Low-dose ketamine administration mimics, both clinically and on gross neuroimaging, depersonalization disorder. The perceptual effects of ketamine may be due to secondary stimulation of glutamate release and lamotrigine, possibly by inhibited glutamate release, may reduce some of ketamine's so-called dissociative effects. However, lamotrigine does not seem to be useful in the treatment of depersonalization disorder. Glutamate release in prefrontal cortex is increased by subanaesthetic doses of ketamine, resulting in increased inhibition, possibly via intercalated GABAerg cells, of projections from amygdala, affecting structures critically involved in depersonalization. I speculate that, in depersonalization disorder, the increased glutamate activity in prefrontal cortex is due to intrinsic imbalance, resulting in long-term potentiation, at the postsynaptic glutamate receptors on the GABAerg interneurons while the same receptor abnormality at the synapses on the intercalated GABAerg cells of the amygdala result in long-term depression in the case of either normal or high glutamate release. PMID:21742442

  9. Feedback control of T-cell receptor activation.

    PubMed Central

    Chan, Cliburn; Stark, Jaroslav; George, Andrew J. T.

    2004-01-01

    The specificity and sensitivity of T-cell recognition is vital to the immune response. Ligand engagement with the T-cell receptor (TCR) results in the activation of a complex sequence of signalling events, both on the cell membrane and intracellularly. Feedback is an integral part of these signalling pathways, yet is often ignored in standard accounts of T-cell signalling. Here we show, using a mathematical model, that these feedback loops can explain the ability of the TCR to discriminate between ligands with high specificity and sensitivity, as well as provide a mechanism for sustained signalling. The model also explains the recent counter-intuitive observation that endogenous 'null' ligands can significantly enhance T-cell signalling. Finally, the model may provide an archetype for receptor switching based on kinase-phosphatase switches, and thus be of interest to the wider signalling community. PMID:15255048

  10. Role of peroxisome proliferator-activated receptors in mechanisms of rejection in heart transplantation

    E-print Network

    Binello, Emanuela

    2004-01-01

    Peroxisome proliferator-activated receptors (PPARs) belong to a nuclear receptor superfamily; two major isoforms, PPAR? and PPAR[gamma], are primarily involved in lipid and glucose homeostasis. However, evidence also ...

  11. Emergence of constitutively active estrogen receptor-? mutations in pretreated advanced estrogen receptor positive breast cancer

    PubMed Central

    Meric-Bernstam, Funda; Gonzalez-Angulo, Ana Maria; Ferrer-Lozano, Jaime; Perez-Fidalgo, Jose A.; Cristofanilli, Massimo; Gómez, Henry; Arteaga, Carlos L.; Giltnane, Jennifer; Balko, Justin M.; Cronin, Maureen T; Jarosz, Mirna; Sun, James; Hawryluk, Matthew; Lipson, Doron; Otto, Geoff; Ross, Jeffrey S; Dvir, Addie; Soussan-Gutman, Lior; Wolf, Ido; Rubinek, Tamar; Gilmore, Lauren; Schnitt, Stuart; Come, Steven E.; Pusztai, Lajos; Stephens, Philip; Brown, Myles; Miller, Vincent A.

    2014-01-01

    Purpose We undertook this study to determine the prevalence of estrogen receptor (ER) ? (ESR1) mutations throughout the natural history of hormone dependent breast cancer and to delineate the functional roles of the most commonly detected alterations. Experimental Design We studied a total of 249 tumor specimens from 208 patients. The specimens include 134 ER positive (ER+/HER2–) and, as controls, 115 ER negative (ER?) tumors. The ER+ samples consist of 58 primary breast cancers and 76 metastatic samples. All tumors were sequenced to high unique coverage using next generation sequencing targeting the coding sequence of the estrogen receptor and an additional 182 cancer-related genes. Results Recurring somatic mutations in codons 537 and 538 within the ligand-binding domain of ER were detected in ER+ metastatic disease. Overall, the frequency of these mutations was 12% (9/76, 95% CI 6%-21%) in metastatic tumors and in a subgroup of patients who received an average of 7 lines of treatment the frequency was 20% (5/25, 95% CI 7%-41%). These mutations were not detected in primary or treatment naďve ER+ cancer or in any stage of ER? disease. Functional studies in cell line models demonstrate that these mutations render estrogen receptor constitutive activity and confer partial resistance to currently available endocrine treatments. Conclusions In this study we show evidence for the temporal selection of functional ESR1 mutations as potential drivers of endocrine resistance during the progression of ER positive breast cancer. PMID:24398047

  12. Phorbol esters cause sequential activation and deactivation of complement receptors on polymorphonuclear leukocytes

    SciTech Connect

    Wright, S.D.; Meyer, B.C.

    1986-03-01

    Phorbol myristate acetate (PMA) exerts a biphasic effect on receptors for C3b and C3bi of human polymorphonuclear leukocytes (PMN). The addition of PMA for 10 min enhances the capacity of these receptors to promote binding and phagocytosis of C3b- and C3bi-coated erythrocytes. Upon additional incubation for 60 min, the capacity of these receptors to bind and ingest ligand-coated erythrocytes decreases the levels below those of resting cells. Although PMA does cause increased expression of cell surface C3b and C3bi receptors, the sequential rise and fall of receptor activity cannot be accounted for the alterations in the number of surface receptors. It appears, rather, that PMA causes qualitative changes in these receptors, first an increase in receptor activity (activation) and then a decrease in receptor activity. To explore the role of phosphorylation in the sequential activation and deactivation of phagocytosis-promoting receptors, the authors loaded PMN with thiophosphate (thioP). This compound is incorporated into cellular nucleotides and proteins, and the resultant (thio)phosphorylated proteins are resistant to phosphatases. thioP-loaded cells show enhanced receptor activity, suggesting that activation of receptors is mediated by a phosphorylation event. Cells loaded with thioP and treated with PMA for 70 min do not deactivate C3 or Fc reactors, suggesting that the deactivation is the result of a dephosphorylation event.

  13. Dual activities of odorants on olfactory and nuclear hormone receptors.

    PubMed

    Pick, Horst; Etter, Sylvain; Baud, Olivia; Schmauder, Ralf; Bordoli, Lorenza; Schwede, Torsten; Vogel, Horst

    2009-10-30

    We have screened an odorant compound library and discovered molecules acting as chemical signals that specifically activate both G-protein-coupled olfactory receptors (ORs) on the cell surface of olfactory sensory neurons and the human nuclear estrogen receptor alpha (ER) involved in transcriptional regulation of cellular differentiation and proliferation in a wide variety of tissues. Hence, these apparent dual active odorants induce distinct signal transduction pathways at different subcellular localizations, which affect both neuronal signaling, resulting in odor perception, and the ER-dependent transcriptional control of specific genes. We demonstrate these effects using fluorescence-based in vitro and cellular assays. Among these odorants, we have identified synthetic sandalwood compounds, an important class of molecules used in the fragrance industry. For one estrogenic odorant we have also identified the cognate OR. This prompted us to compare basic molecular recognition principles of odorants on the two structurally and apparent functionally non-related receptors using computational modeling in combination with functional assays. Faced with the increasing evidence that ORs may perform chemosensory functions in a number of tissues outside of the nasal olfactory epithelium, the unraveling of these molecular ligand-receptor interaction principles is of critical importance. In addition the evidence that certain olfactory sensory neurons naturally co-express ORs and ERs may provide a direct functional link between the olfactory and hormonal systems in humans. Our results are therefore useful for defining the structural and functional characteristics of ER-specific odorants and the role of odorant molecules in cellular processes other than olfaction. PMID:19723634

  14. Trans-activation between 7TM domains: implication in heterodimeric GABAB receptor activation

    PubMed Central

    Monnier, Carine; Tu, Haijun; Bourrier, Emmanuel; Vol, Claire; Lamarque, Laurent; Trinquet, Eric; Pin, Jean-Philippe; Rondard, Philippe

    2011-01-01

    Seven-transmembrane domain (7TM) receptors have important functions in cell–cell communication and can assemble into dimers or oligomers. Such complexes may allow specific functional cross-talk through trans-activation of interacting 7TMs, but this hypothesis requires further validation. Herein, we used the GABAB receptor, which is composed of two distinct subunits, GABAB1, which binds the agonist, and GABAB2, which activates G proteins, as a model system. By using a novel orthogonal-labelling approach compatible with time-resolved FRET and based on ACP- and SNAP-tag technologies to verify the heterodimerization of wild-type and mutated GABAB subunits, we demonstrate the existence of a direct allosteric coupling between the 7TMs of GABAB heterodimers. Indeed, a GABAB receptor, in which the GABAB2 extracellular domain was deleted, was still capable of activating G proteins. Furthermore, synthetic ligands for the GABAB2 7TM could increase agonist affinity at the GABAB1 subunit in this mutated receptor. In addition to bringing new information on GABAB receptor activation, these data clearly demonstrate the existence of direct trans-activation between the 7TM of two interacting proteins. PMID:21063387

  15. NMDA RECEPTOR ACTIVATION STRENGTHENS WEAK ELECTRICAL COUPLING IN MAMMALIAN BRAIN

    PubMed Central

    Turecek, Josef; Yuen, Genevieve S.; Han, Victor Z.; Zeng, Xiao-Hui; Bayer, K. Ulrich; Welsh, John P.

    2014-01-01

    SUMMARY Electrical synapses are formed by gap junctions and permit electrical coupling that shapes the synchrony of neuronal ensembles. Here, we provide the first direct demonstration of receptormediated strengthening of electrical coupling in mammalian brain. Electrical coupling in the inferior olive of rats was strengthened by activation of NMDA-type glutamate-receptors (NMDARs), which were found at synaptic loci and at extrasynaptic loci 20–100 nm proximal to gap junctions. Electrical coupling was strengthened by pharmacological and synaptic activation of NMDARs, while co-stimulation of ionotropic non-NMDAR glutamate-receptors transiently antagonized the effect of NMDAR activation. NMDAR-dependent strengthening (i) occurred despite increased input conductance, (ii) induced Ca2+-influx microdomains near dendritic spines, (iii) required activation of the Ca2+/calmodulin-dependent protein-kinase II, (iv) was restricted to neurons that were weakly coupled, and thus, (v) strengthened coupling mainly between non-adjacent neurons. This provided a mechanism to expand the synchronization of rhythmic membrane potential oscillations by chemical neurotransmitter input. PMID:24656255

  16. DHEA metabolites activate estrogen receptors alpha and beta

    PubMed Central

    Michael Miller, Kristy K.; Al-Rayyan, Numan; Ivanova, Margarita M.; Mattingly, Kathleen A.; Ripp, Sharon L.; Klinge, Carolyn M.; Prough, Russell A.

    2012-01-01

    Dehydroepiandrosterone (DHEA) levels were reported to associate with increased breast cancer risk in postmenopausal women, but some carcinogen-induced rat mammary tumor studies question this claim. The purpose of this study was to determine how DHEA and its metabolites affect estrogen receptors ? or ? (ER? or ER?) -regulated gene transcription and cell proliferation. In transiently transfected HEK-293 cells, androstenediol, DHEA, and DHEA-S activated ER?. In ER? transfected HepG2 cells, androstenedione, DHEA, androstenediol, and 7-oxo DHEA stimulated reporter activity. ER antagonists ICI 182,780 (fulvestrant) and 4-hydroxytamoxifen, general P450 inhibitor miconazole, and aromatase inhibitor exemestane inhibited activation by DHEA or metabolites in transfected cells. ER?-selective antagonist R,R-THC (R,R-cis-diethyl tetrahydrochrysene) inhibited DHEA and DHEA metabolite transcriptional activity in ER?-transfected cells. Expression of endogenous estrogen-regulated genes: pS2, progesterone receptor, cathepsin D1, and nuclear respiratory factor-1 was increased by DHEA and its metabolites in an ER-subtype, gene, and cell-specific manner. DHEA metabolites, but not DHEA, competed with 17?-estradiol for ER? and ER? binding and stimulated MCF-7 cell proliferation, demonstrating that DHEA metabolites interact directly with ER? and ER? in vitro, modulating estrogen target genes in vivo. PMID:23123738

  17. An Improved Ivermectin-activated Chloride Channel Receptor for Inhibiting Electrical Activity in Defined Neuronal Populations*

    PubMed Central

    Lynagh, Timothy; Lynch, Joseph W.

    2010-01-01

    The ability to silence the electrical activity of defined neuronal populations in vivo is dramatically advancing our understanding of brain function. This technology may eventually be useful clinically for treating a variety of neuropathological disorders caused by excessive neuronal activity. Several neuronal silencing methods have been developed, with the bacterial light-activated halorhodopsin and the invertebrate allatostatin-activated G protein-coupled receptor proving the most successful to date. However, both techniques may be difficult to implement clinically due to their requirement for surgically implanted stimulus delivery methods and their use of nonhuman receptors. A third silencing method, an invertebrate glutamate-gated chloride channel receptor (GluClR) activated by ivermectin, solves the stimulus delivery problem as ivermectin is a safe, well tolerated drug that reaches the brain following systemic administration. However, the limitations of this method include poor functional expression, possibly due to the requirement to coexpress two different subunits in individual neurons, and the nonhuman origin of GluClR. Here, we describe the development of a modified human ?1 glycine receptor as an improved ivermectin-gated silencing receptor. The crucial development was the identification of a mutation, A288G, which increased ivermectin sensitivity almost 100-fold, rendering it similar to that of GluClR. Glycine sensitivity was eliminated via the F207A mutation. Its large unitary conductance, homomeric expression, and human origin may render the F207A/A288G ?1 glycine receptor an improved silencing receptor for neuroscientific and clinical purposes. As all known highly ivermectin-sensitive GluClRs contain an endogenous glycine residue at the corresponding location, this residue appears essential for exquisite ivermectin sensitivity. PMID:20308070

  18. Mode of action framework analysis for receptor-mediated toxicity: the Peroxisome Proliferator-Activated Receptor alpha (PPAR?) as a case study

    EPA Science Inventory

    Therapeutic hypolipidemic agents and industrial chemicals that cause peroxisome proliferation and induce liver tumors in rodents activate the nuclear receptor peroxisome proliferator-activated receptor alpha (PPAR?). Research has elucidated the cellular and molecular events by w...

  19. Attenuation of Colon Inflammation through Activators of the Retinoid X Receptor (Rxr)/Peroxisome Proliferator–Activated Receptor ? (Ppar?) Heterodimer

    PubMed Central

    Desreumaux, Pierre; Dubuquoy, Laurent; Nutten, Sophie; Peuchmaur, Michel; Englaro, Walter; Schoonjans, Kristina; Derijard, Benoit; Desvergne, Beatrice; Wahli, Walter; Chambon, Pierre; Leibowitz, Mark D.; Colombel, Jean-Frédéric; Auwerx, Johan

    2001-01-01

    The peroxisome proliferator–activated receptor ? (PPAR?) is highly expressed in the colon mucosa and its activation has been reported to protect against colitis. We studied the involvement of PPAR? and its heterodimeric partner, the retinoid X receptor (RXR) in intestinal inflammatory responses. PPAR?1/? and RXR?1/? mice both displayed a significantly enhanced susceptibility to 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis compared with their wild-type littermates. A role for the RXR/PPAR? heterodimer in the protection against colon inflammation was explored by the use of selective RXR and PPAR? agonists. TNBS-induced colitis was significantly reduced by the administration of both PPAR? and RXR agonists. This beneficial effect was reflected by increased survival rates, an improvement of macroscopic and histologic scores, a decrease in tumor necrosis factor ? and interleukin 1? mRNA levels, a diminished myeloperoxidase concentration, and reduction of nuclear factor ?B DNA binding activity, c-Jun NH2-terminal kinase, and p38 activities in the colon. When coadministered, a significant synergistic effect of PPAR? and RXR ligands was observed. In combination, these data demonstrate that activation of the RXR/PPAR? heterodimer protects against colon inflammation and suggest that combination therapy with both RXR and PPAR? ligands might hold promise in the clinic due to their synergistic effects. PMID:11283155

  20. Changes in Laminin Expression Pattern during Early Differentiation of Human Embryonic Stem Cells

    PubMed Central

    Pook, Martin; Teino, Indrek; Kallas, Ade; Maimets, Toivo; Ingerpuu, Sulev; Jaks, Viljar

    2015-01-01

    Laminin isoforms laminin-511 and -521 are expressed by human embryonic stem cells (hESC) and can be used as a growth matrix to culture these cells under pluripotent conditions. However, the expression of these laminins during the induction of hESC differentiation has not been studied in detail. Furthermore, the data regarding the expression pattern of laminin chains in differentiating hESC is scarce. In the current study we aimed to fill this gap and investigated the potential changes in laminin expression during early hESC differentiation induced by retinoic acid (RA). We found that laminin-511 but not -521 accumulates in the committed cells during early steps of hESC differentiation. We also performed a comprehensive analysis of the laminin chain repertoire and found that pluripotent hESC express a more diverse range of laminin chains than shown previously. In particular, we provide the evidence that in addition to ?1, ?5, ?1, ?2 and ?1 chains, hESC express ?2, ?3, ?3, ?2 and ?3 chain proteins and mRNA. Additionally, we found that a variant of laminin ?3 chain—145 kDa—accumulated in RA-treated hESC showing that these cells produce prevalently specifically modified version of ?3 chain in early phase of differentiation. PMID:26378917

  1. Aryl hydrocarbon receptor-independent activation of estrogen receptor-dependent transcription by 3-methycholanthrene

    SciTech Connect

    Shipley, Jonathan M.; Waxman, David J. . E-mail: djw@bu.edu

    2006-06-01

    Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that stimulates transcription directed by xenobiotic response elements upstream of target genes. Recently, AhR ligands were reported to induce formation of an AhR-estrogen receptor (ER) complex, which can bind to estrogen response elements (EREs) and stimulate transcription of ER target genes. Presently, we investigate the effect of the AhR ligands 3-methylcholanthrene (3MC), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3,3',4,4',5-pentachlorobiphenyl (BZ126) on ERE-regulated luciferase reporter activity and endogenous ER target gene expression. In MCF-7 human breast cancer cells, 3MC induced transcription of ER reporter genes containing native promoter sequences of the ER-responsive genes complement 3 and pS2 and heterologous promoters regulated by isolated EREs. Dose-response studies revealed that the concentration of 3MC required to half-maximally activate transcription (EC{sub 5}) was >100-fold higher for an ER reporter (27-57 {mu}M) than for an AhR reporter (86-250 nM) in both MCF-7 cells and in human endometrial cancer Ishikawa cells. 3MC also stimulated expression of the endogenous ER target genes amphiregulin, cathepsin D and progesterone receptor, albeit to a much lower extent than was achieved following stimulation with 17{beta}-estradiol. In Ishikawa cells, 3MC, but not BZ126 or TCDD, stimulated ER{alpha}-dependent reporter activity but did not induce expression of endogenous ER target genes. Finally, studies carried out in the AhR-positive rat hepatoma cell line 5L and the AhR-deficient variant BP8 demonstrated that ER reporter activity could be induced by 3MC in a manner that was independent of AhR and thus distinct from the AhR-ER 'hijacking' mechanism described recently. 3MC may thus elicit estrogenic activity by multiple mechanisms.

  2. Dopamine Receptor Activation Increases HIV Entry into Primary Human Macrophages

    PubMed Central

    Gaskill, Peter J.; Yano, Hideaki H.; Kalpana, Ganjam V.; Javitch, Jonathan A.; Berman, Joan W.

    2014-01-01

    Macrophages are the primary cell type infected with HIV in the central nervous system, and infection of these cells is a major component in the development of neuropathogenesis and HIV-associated neurocognitive disorders. Within the brains of drug abusers, macrophages are exposed to increased levels of dopamine, a neurotransmitter that mediates the addictive and reinforcing effects of drugs of abuse such as cocaine and methamphetamine. In this study we examined the effects of dopamine on HIV entry into primary human macrophages. Exposure to dopamine during infection increased the entry of R5 tropic HIV into macrophages, irrespective of the concentration of the viral inoculum. The entry pathway affected was CCR5 dependent, as antagonizing CCR5 with the small molecule inhibitor TAK779 completely blocked entry. The effect was dose-dependent and had a steep threshold, only occurring above 108 M dopamine. The dopamine-mediated increase in entry required dopamine receptor activation, as it was abrogated by the pan-dopamine receptor antagonist flupenthixol, and could be mediated through both subtypes of dopamine receptors. These findings indicate that the effects of dopamine on macrophages may have a significant impact on HIV pathogenesis. They also suggest that drug-induced increases in CNS dopamine may be a common mechanism by which drugs of abuse with distinct modes of action exacerbate neuroinflammation and contribute to HIV-associated neurocognitive disorders in infected drug abusers. PMID:25268786

  3. Peroxisome proliferator-activated receptor {alpha}-independent peroxisome proliferation

    SciTech Connect

    Zhang Xiuguo; Tanaka, Naoki . E-mail: naopi@hsp.md.shinshu-u.ac.jp; Nakajima, Takero; Kamijo, Yuji; Gonzalez, Frank J.; Aoyama, Toshifumi

    2006-08-11

    Hepatic peroxisome proliferation, increases in the numerical and volume density of peroxisomes, is believed to be closely related to peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}) activation; however, it remains unknown whether peroxisome proliferation depends absolutely on this activation. To verify occurrence of PPAR{alpha}-independent peroxisome proliferation, fenofibrate treatment was used, which was expected to significantly enhance PPAR{alpha} dependence in the assay system. Surprisingly, a novel type of PPAR{alpha}-independent peroxisome proliferation and enlargement was uncovered in PPAR{alpha}-null mice. The increased expression of dynamin-like protein 1, but not peroxisome biogenesis factor 11{alpha}, might be associated with the PPAR{alpha}-independent peroxisome proliferation at least in part.

  4. Oxidatively fragmented phosphatidylcholines activate human neutrophils through the receptor for platelet-activating factor.

    PubMed

    Smiley, P L; Stremler, K E; Prescott, S M; Zimmerman, G A; McIntyre, T M

    1991-06-15

    Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) activates neutrophils (polymorphonuclear leukocytes, PMN) through a receptor that specifically recognizes short sn-2 residues. We oxidized synthetic [2-arachidonoyl]phosphatidylcholine to fragment and shorten the sn-2 residue, and then examined the phospholipid products for the ability to stimulate PMN. 1-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine was fragmented by ozonolysis to 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine. This phospholipid activated human neutrophils at submicromolar concentrations, and is effects were inhibited by specific PAF receptor antagonists WEB2086, L659,989, and CV3988. 1-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine next was fragmented by an uncontrolled free radical-catalyzed reaction: it was treated with soybean lipoxygenase to form its sn-2 15-hydroperoxy derivative (which did not activate neutrophils) and then allowed to oxidize under air. The secondary oxidation resulted in the formation of numerous fragmented phospholipids (Stremler, K. E., Stafforini, D. M., Prescott, S. M., and McIntyre, T. M. (1991) J. Biol. Chem. 266, 11095-11103), some of which activated PMN. Hydrolysis of sn-2 residues with phospholipase A2 destroyed biologic activity, as did hydrolysis with PAF acetylhydrolase. PAF acetylhydrolase is specific for short or intermediate length sn-2 residues and does not hydrolyze the starting material (Stremler, K. E., Stafforini, D. M., Prescott, S. M., and McIntyre, T. M. (1991) J. Biol. Chem. 266, 11095-11103). Neutrophil activation was completely blocked by L659,989, a specific PAF receptor antagonist. We conclude that diacylphosphatidylcholines containing an sn-2 polyunsaturated fatty acyl residue can be oxidatively fragmented to species with sn-2 residues short enough to activate the PAF receptor of neutrophils. This suggests a new mechanism for the appearance of biologically active phospholipids, and shows that PAF receptor antagonists block the action of both PAF and these PAF-like lipids. PMID:1645725

  5. Shear stress activation of nuclear receptor PXR in endothelial detoxification.

    PubMed

    Wang, Xiaohong; Fang, Xi; Zhou, Jing; Chen, Zhen; Zhao, Beilei; Xiao, Lei; Liu, Ao; Li, Yi-Shuan J; Shyy, John Y-J; Guan, Youfei; Chien, Shu; Wang, Nanping

    2013-08-01

    Endothelial cells (ECs) are constantly exposed to xenobiotics and endobiotics or their metabolites, which perturb EC function, as well as to shear stress, which plays a crucial role in vascular homeostasis. Pregnane X receptor (PXR) is a nuclear receptor and a key regulator of the detoxification of xeno- and endobiotics. Here we show that laminar shear stress (LSS), the atheroprotective flow, activates PXR in ECs, whereas oscillatory shear stress, the atheroprone flow, suppresses PXR. LSS activation of PXR in cultured ECs led to the increased expression of a PXR target gene, multidrug resistance 1 (MDR1). An in vivo study using rats showed that the expression of MDR1 was significantly higher in the endothelium from the descending thoracic aorta, where flow is mostly laminar, than from the inner curvature of aortic arch, where flow is disturbed. Functionally, LSS-activated PXR protects ECs from apoptosis triggered by doxorubicin via the induction of MDR1 and other detoxification genes. PXR also suppressed the expression of proinflammatory adhesion molecules and monocyte adhesion in response to TNF-? and lipopolysaccharide. Overexpression of a constitutively active PXR in rat carotid arteries potently attenuated proinflammatory responses. In addition, cDNA microarray revealed a large number of the PXR-activated endothelial genes whose products are responsible for major steps of detoxification, including phase I and II metabolizing enzymes and transporters. These detoxification genes in ECs are induced by LSS in ECs in a PXR-dependent manner. In conclusion, our results indicate that PXR represents a flow-activated detoxification system to protect ECs against damage by xeno- and endobiotics. PMID:23878263

  6. The glucagon-like peptide 1 receptor agonist enhances intrinsic peroxisome proliferator-activated receptor ? activity in endothelial cells

    SciTech Connect

    Onuma, Hirohisa; Inukai, Kouichi Kitahara, Atsuko; Moriya, Rie; Nishida, Susumu; Tanaka, Toshiaki; Katsuta, Hidenori; Takahashi, Kazuto; Sumitani, Yoshikazu; Hosaka, Toshio; Ishida, Hitoshi

    2014-08-22

    Highlights: • PPAR? activation was involved in the GLP-1-mediated anti-inflammatory action. • Exendin-4 enhanced endogenous PPAR? transcriptional activity in HUVECs. • H89, a PKA inhibitor, abolished GLP-1-induced PPAR? enhancement. • The anti-inflammatory effects of GLP-1 may be explained by PPAR? activation. - Abstract: Recent studies have suggested glucagon-like peptide-1 (GLP-1) signaling to exert anti-inflammatory effects on endothelial cells, although the precise underlying mechanism remains to be elucidated. In the present study, we investigated whether PPAR? activation is involved in the GLP-1-mediated anti-inflammatory action on endothelial cells. When we treated HUVEC cells with 0.2 ng/ml exendin-4, a GLP-1 receptor agonist, endogenous PPAR? transcriptional activity was significantly elevated, by approximately 20%, as compared with control cells. The maximum PPAR? activity enhancing effect of exendin-4 was observed 12 h after the initiation of incubation with exendin-4. As H89, a PKA inhibitor, abolished GLP-1-induced PPAR? enhancement, the signaling downstream from GLP-1 cross-talk must have been involved in PPAR? activation. In conclusion, our results suggest that GLP-1 has the potential to induce PPAR? activity, partially explaining the anti-inflammatory effects of GLP-1 on endothelial cells. Cross-talk between GLP-1 signaling and PPAR? activation would have major impacts on treatments for patients at high risk for cardiovascular disease.

  7. An antiglycolipid antibody inhibits Madin-Darby canine kidney cell adhesion to laminin and interferes with basolateral polarization and tight junction formation

    PubMed Central

    1996-01-01

    Epithelial cells polarize not only in response to cell-cell contacts, but also to contacts with a substratum composed of extracellular matrix molecules. To probe the role of specific matrix constituents in epithelial cell polarization, we investigated the effects of an adhesion-blocking mAb, 12B12, on initial polarization of MDCK cells. The 12B12 antibody, raised against whole MDCK cells, blocks adhesion to laminin by 65% but has no effect on adhesion of cells to collagen type I. Taking advantage of this antibody's function-blocking activity, as well as the fact that MDCK cells secrete laminin, the role of endogenous laminin in polarization was examined by plating cells on collagen-coated substrata in the presence of the antibody. Under these conditions, cell spreading was reduced 1.5h after plating, and cells were flatter and had fewer microvilli after 24 h. Even though lateral cell membranes were closely apposed, transepithelial resistance in the presence of the antibody was significantly reduced relative to controls. When the polarization of specific apical and basolateral markers was examined both biochemically and immunocytochemically in the presence of the antibody, we observed that the apical marker polarized at normal rates while basolateral markers did not. Surprisingly, the 12B12 antibody was not directed against any known cell adhesion protein but reacted specifically with Forssman antigen, a glycosphingolipid. These results suggest that glycolipids may play a significant role in cell adhesion via laminin and in epithelial cell polarization. PMID:8636242

  8. Separate endocytic pathways of kinase-defective and -active EGF receptor mutants expressed in same cells

    SciTech Connect

    Honegger, A.M.; Schmidt, A.; Ullrich, A.; Schlessinger, J. )

    1990-05-01

    Ligand binding to the membrane receptor for EGF induces its clustering and internalization. Both receptor and ligand are then degraded by lysosomal enzymes. A kinase defective point mutant (K721A) of EGF receptor undergoes internalization similarly to the wild-type receptor. However, while internalized EGF molecules bound to either the wild-type or mutant receptors are degraded, the K721A mutant receptor molecules recycle to the cell surface for reutilization. To investigate the mechanism of receptor trafficking, we have established transfected NIH-3T3 cells coexpressing the kinase-negative mutant (K721A) together with a mutant EGF receptor (CD63) with active kinase. CD63 was chosen because it behaves like wild-type EGF receptor with respect to biological responsiveness and cellular routing but afforded immunological distinction between kinase active and inactive mutants. Although expressed in the same cells, the two receptor mutants followed their separate endocytic itineraries. Like wild-type receptor, the CD63 mutant was downregulated and degraded in response to EFG while the kinase-negative mutant K721A returned to the cell surface for reutilization. Intracellular trafficking of EGF receptor must be determined by a sorting mechanism that specifically recognizes EGF receptor molecules according to their intrinsic kinase activity.

  9. High constitutive activity of native H3 receptors regulates histamine neurons in brain.

    PubMed

    Morisset, S; Rouleau, A; Ligneau, X; Gbahou, F; Tardivel-Lacombe, J; Stark, H; Schunack, W; Ganellin, C R; Schwartz, J C; Arrang, J M

    2000-12-14

    Some G-protein-coupled receptors display 'constitutive activity', that is, spontaneous activity in the absence of agonist. This means that a proportion of the receptor population spontaneously undergoes an allosteric transition, leading to a conformation that can bind G proteins. The process has been shown to occur with recombinant receptors expressed at high density, and/or mutated, but also non-mutated recombinant receptors expressed at physiological concentrations. Transgenic mice that express a constitutively active mutant of the beta2-adrenergic receptor display cardiac anomalies; and spontaneous receptor mutations leading to constitutive activity are at the origin of some human diseases. Nevertheless, this process has not previously been found to occur in animals expressing normal levels of receptor. Here we show that two isoforms of the recombinant rat H3 receptor display high constitutive activity. Using drugs that abrogate this activity ('inverse agonists') and a drug that opposes both agonists and inverse agonists ('neutral antagonist'), we show that constitutive activity of native H3 receptors is present in rodent brain and that it controls histaminergic neuron activity in vivo. Inverse agonists may therefore find therapeutic applications, even in the case of diseases involving non-mutated receptors expressed at normal levels. PMID:11130725

  10. Muscarinic receptor activation elicits sustained, recurring depolarizations in reticulospinal neurons.

    PubMed

    Smetana, R W; Alford, S; Dubuc, R

    2007-05-01

    In lampreys, brain stem reticulospinal (RS) neurons constitute the main descending input to the spinal cord and activate the spinal locomotor central pattern generators. Cholinergic nicotinic inputs activate RS neurons, and consequently, induce locomotion. Cholinergic muscarinic agonists also induce locomotion when applied to the brain stem of birds. This study examined whether bath applications of muscarinic agonists could activate RS neurons and initiate motor output in lampreys. Bath applications of 25 microM muscarine elicited sustained, recurring depolarizations (mean duration of 5.0 +/- 0.5 s recurring with a mean period of 55.5 +/- 10.3 s) in intracellularly recorded rhombencephalic RS neurons. Calcium imaging experiments revealed that muscarine induced oscillations in calcium levels that occurred synchronously within the RS neuron population. Bath application of TTX abolished the muscarine effect, suggesting the sustained depolarizations in RS neurons are driven by other neurons. A series of lesion experiments suggested the caudal half of the rhombencephalon was necessary. Microinjections of muscarine (75 microM) or the muscarinic receptor (mAchR) antagonist atropine (1 mM) lateral to the rostral pole of the posterior rhombencephalic reticular nucleus induced or prevented, respectively, the muscarinic RS neuron response. Cells immunoreactive for muscarinic receptors were found in this region and could mediate this response. Bath application of glutamatergic antagonists (6-cyano-7-nitroquinoxaline-2,3-dione/D-2-amino-5-phosphonovaleric acid) abolished the muscarine effect, suggesting that glutamatergic transmission is needed for the effect. Ventral root recordings showed spinal motor output coincides with RS neuron sustained depolarizations. We propose that unilateral mAchR activation on specific cells in the caudal rhombencephalon activates a circuit that generates synchronous sustained, recurring depolarizations in bilateral populations of RS neurons. PMID:17344371

  11. EP2 receptor signaling pathways regulate classical activation of microglia.

    PubMed

    Quan, Yi; Jiang, Jianxiong; Dingledine, Ray

    2013-03-29

    Activation of EP2 receptors by prostaglandin E2 (PGE2) promotes brain inflammation in neurodegenerative diseases, but the pathways responsible are unclear. EP2 receptors couple to G?s and increase cAMP, which associates with protein kinase A (PKA) and cAMP-regulated guanine nucleotide exchange factors (Epacs). Here, we studied EP2 function and its signaling pathways in rat microglia in their resting state or undergoing classical activation in vitro following treatment with low concentrations of lipopolysaccharide and interferon-?. Real time PCR showed that PGE2 had no effect on expression of CXCL10, TGF-?1, and IL-11 and exacerbated the rapid up-regulation of mRNAs encoding cyclooxygenase-2, inducible NOS, IL-6, and IL-1? but blunted the production of mRNAs encoding TNF-?, IL-10, CCL3, and CCL4. These effects were mimicked fully by the EP2 agonist butaprost but only weakly by the EP1/EP3 agonist 17-phenyl trinor PGE2 or the EP4 agonist CAY10598 and not at all by the EP3/EP1 agonist sulprostone and confirmed by protein measurements of cyclooxygenase-2, IL-6, IL-10, and TNF-?. In resting microglia, butaprost induced cAMP formation and altered the mRNA expression of inflammatory mediators, but protein expression was unchanged. The PKA inhibitor H89 had little or no effect on inflammatory mediators modulated by EP2, whereas the Epac activator 8-(4-chlorophenylthio)-2'-O-methyladenosine 3',5'-cyclic monophosphate acetoxymethyl ester mimicked all butaprost effects. These results indicate that EP2 activation plays a complex immune regulatory role during classical activation of microglia and that Epac pathways are prominent in this role. PMID:23404506

  12. Canonical Wnt signalling regulates epithelial patterning by modulating levels of laminins in zebrafish appendages

    PubMed Central

    Nagendran, Monica; Arora, Prateek; Gori, Payal; Mulay, Aditya; Ray, Shinjini; Jacob, Tressa; Sonawane, Mahendra

    2015-01-01

    The patterning and morphogenesis of body appendages – such as limbs and fins – is orchestrated by the activities of several developmental pathways. Wnt signalling is essential for the induction of limbs. However, it is unclear whether a canonical Wnt signalling gradient exists and regulates the patterning of epithelium in vertebrate appendages. Using an evolutionarily old appendage – the median fin in zebrafish – as a model, we show that the fin epithelium exhibits graded changes in cellular morphology along the proximo-distal axis. This epithelial pattern is strictly correlated with the gradient of canonical Wnt signalling activity. By combining genetic analyses with cellular imaging, we show that canonical Wnt signalling regulates epithelial cell morphology by modulating the levels of laminins, which are extracellular matrix components. We have unravelled a hitherto unknown mechanism involved in epithelial patterning, which is also conserved in the pectoral fins – evolutionarily recent appendages that are homologous to tetrapod limbs. PMID:25519245

  13. Saturated Fatty Acid Activates but Polyunsaturated Fatty Acid Inhibits Toll-like Receptor 2 Dimerized with

    E-print Network

    Lee, Won-Ha

    Saturated Fatty Acid Activates but Polyunsaturated Fatty Acid Inhibits Toll-like Receptor 2 Dimerized with Toll-like Receptor 6 or 1* Received for publication, November 30, 2003, and in revised form, Massachusetts 01605 Toll-like receptor 4 (TLR4) and TLR2 agonists from bacterial origin require acylated

  14. Nuclear receptor peroxisome proliferator activated receptor (PPAR) ?/? in skin wound healing and cancer.

    PubMed

    Montagner, Alexandra; Wahli, Walter; Tan, Nguan Soon

    2015-04-01

    We review the functions of peroxisome proliferator activated receptor (PPAR) ?/? in skin wound healing and cancer. In particular, we highlight the roles of PPAR?/? in inhibiting keratinocyte apoptosis at wound edges via activation of the PI3K/PKB?/Akt1 pathway and its role during re-epithelialization in regulating keratinocyte adhesion and migration. In fibroblasts, PPAR?/? controls IL-1 signalling and thereby contributes to the homeostatic control of keratinocyte proliferation. We discuss its therapeutic potential for treating diabetic wounds and inflammatory skin diseases such as psoriasis and acne vulgaris. PPAR?/? is classified as a tumour growth modifier; it is activated by chronic low-grade inflammation, which promotes the production of lipids that, in turn, enhance PPAR?/? transcription activity. Our earlier work unveiled a cascade of events triggered by PPAR?/? that involve the oncogene Src, which promotes ultraviolet-induced skin cancer in mice via enhanced EGFR/Erk1/2 signalling and the expression of epithelial-to-mesenchymal transition (EMT) markers. Interestingly, PPAR?/? expression is correlated with the expression of SRC and EMT markers in human skin squamous cell carcinoma. Furthermore, there is a positive interaction between PPAR?/?, SRC, and TGF?1 at the transcriptional level in various human epithelial cancers. Taken together, these observations suggest the need for evaluating PPAR?/? modulators that attenuate or increase its activity, depending on the therapeutic target. PMID:26287030

  15. Profiling receptor tyrosine kinase activation by using Ab microarrays

    NASA Astrophysics Data System (ADS)

    Nielsen, Ulrik B.; Cardone, Mike H.; Sinskey, Anthony J.; MacBeath, Gavin; Sorger, Peter K.

    2003-08-01

    Signal transduction in mammalian cells is mediated by complex networks of interacting proteins. Understanding these networks at a circuit level requires devices to measure the amounts and activities of multiple proteins in a rapid and accurate manner. Ab microarrays have previously been applied to the quantification of labeled recombinant proteins and proteins in serum. The development of methods to analyze intracellular signaling molecules on microarrays would make Ab arrays widely useful in systems biology. Here we describe the fabrication of multiplex Ab arrays sensitive to the amounts and modification states of signal transduction proteins in crude cell lysates and the integration of these arrays with 96-well microtiter plate technology to create microarrays in microplates. We apply the Ab arrays to monitoring the activation, uptake, and signaling of ErbB receptor tyrosine kinases in human tumor cell lines. Data obtained from multicolor ratiometric microarrays correlate well with data obtained by using traditional approaches, but the arrays are faster and simpler to use. The integration of microplate and microarray methods for crude cell lysates should make it possible to identify and analyze small molecule inhibitors of signal transduction processes with unprecedented speed and precision. We demonstrate the future potential of this approach by characterizing the action of the epidermal growth factor receptor inhibitor PD153035 on cells by using Ab arrays; direct scale-up to array-based screening in 96- and 384-well plates should allow small molecules to be identified with specific inhibitory profiles against a signaling network.

  16. Role of Protease-Activated Receptor-1 in Glioma Growth.

    PubMed

    Xie, Qing; Bao, Xuhui; Chen, Zhan Hong; Xu, Ying; Keep, Richard F; Muraszko, Karin M; Xi, Guohua; Hua, Ya

    2016-01-01

    Activation of a thrombin receptor, protease-activated receptor-1 (PAR-1), induces angiogenesis, cell proliferation, and invasion in tumors. The present study examined the effect of host PAR-1 gene deletion on glioma growth in a mouse model. F98 glioma cells were implanted into the right caudate of either wild type (WT) or PAR-1 knockout (KO) mice. Mice underwent magnetic resonance imaging (MRI) and the brains were used for measurements of brain water content and tumor mass. Levels of hypoxia-inducible factor-1 alpha (HIF-1?) and vascular endothelial growth factor (VEGF) were also measured by ELISA (enzyme-linked immunosorbent assay). We found brain water content in the ipsilateral hemisphere and the tumor mass were significantly lower in PAR-1 KO than WT mice at day 12 after implantation of F98 cells (p?

  17. Recruitment of Dioxin Receptor to Active Transcription Sites

    PubMed Central

    Elbi, Cem; Misteli, Tom; Hager, Gordon L.

    2002-01-01

    The aryl hydrocarbon receptor (AhR or dioxin receptor) is a ligand-activated transcription factor that heterodimerizes with the AhR nuclear translocator (ARNT/HIF-1?) to form an AhR/ARNT transcription factor complex. This complex binds to specific DNA sites in the regulatory domains of numerous target genes and mediates the biological effects of exogenous ligands. Herein, we have investigated the subcellular distribution of the AhR/ARNT complex in response to ligand stimulation, by using live-cell confocal and high-resolution deconvolution microscopy. We found that unliganded AhR shows a predominantly cytoplasmic diffuse distribution in mouse hepatoma cells. On addition of ligand, AhR rapidly translocates to the nucleus and accumulates in multiple bright foci. Inhibition of transcription prevented the formation of AhR foci. Dual- and triple-immunolabeling experiments, combined with labeling of nascent RNA, showed that the foci are transcription sites, indicating that upon ligand stimulation, AhR is recruited to active transcription sites. The interaction of AhR with ARNT was both necessary and sufficient for the recruitment of AhR to transcription sites. These results indicate that AhR/ARNT complexes are recruited to specific subnuclear compartments in a ligand-dependent manner and that these foci represent the sites of AhR target genes. PMID:12058065

  18. Antitussive activity of Withania somnifera and opioid receptors.

    PubMed

    Nosálová, Gabriela; Sivová, Veronika; Ray, Bimalendu; Fra?ová, So?a; Ondrejka, Igor; Flešková, Dana

    2015-01-01

    Arabinogalactan is a polysaccharide isolated from the roots of the medicinal plant Withania somnifera L. It contains 65% arabinose and 18% galactose. The aim of the present study was to evaluate the antitussive activity of arabinogalactan in conscious, healthy adult guinea pigs and the role of the opioid pathway in the antitussive action. A polysaccharide extract was given orally in a dose of 50 mg/kg. Cough was induced by an aerosol of citric acid in a concentration 0.3 mol/L, generated by a jet nebulizer into a plethysmographic chamber. The intensity of cough response was defined as the number of cough efforts counted during a 3-min exposure to the aerosol. The major finding was that arabinogalactan clearly suppressed the cough reflex; the suppression was comparable with that of codeine that was taken as a reference drug. The involvement of the opioid system was tested with the use of a blood-brain barrier penetrable, naloxone hydrochloride, and non-penetrable, naloxone methiodide, to distinguish between the central and peripheral mu-opioid receptor pathways. Both opioid antagonists acted to reverse the arabinogalactan-induced cough suppression; the reversion was total over time with the latter antagonist. We failed to confirm the presence of a bronchodilating effect of the polysaccharide, which could be involved in its antitussive action. We conclude that the polysaccharide arabinogalactan from Withania somnifera has a distinct antitussive activity consisting of cough suppression and that this action involves the mu-opioid receptor pathways. PMID:25252908

  19. Fc? receptor pathways during active and passive immunization.

    PubMed

    Bournazos, Stylianos; Ravetch, Jeffrey V

    2015-11-01

    IgG antibodies are actively produced in response to antigenic challenge or passively administered as an effective form of immunotherapy to confer immunity against foreign antigens. Their protective activity is mediated through their bifunctional nature: a variable Fab domain mediates antigen-binding specificity, whereas the constant Fc domain engages Fc? receptors (Fc?Rs) expressed on the surface of leukocytes to mediate effector functions. While traditionally considered the invariant domain of an IgG molecule, the Fc domain displays remarkable structural heterogeneity determined primarily by differences in the amino acid sequence of the various IgG subclasses and by the composition of the complex, Fc-associated biantennary N-linked glycan. These structural determinants regulate the conformational flexibility of the IgG Fc domain and affect its capacity to interact with distinct types of Fc?Rs (type I or type II Fc?Rs). Fc?R engagement activates diverse downstream immunomodulatory pathways with pleiotropic functional consequences including cytotoxicity and phagocytosis of IgG-coated targets, differentiation and activation of antigen presenting cells, modulation of T-cell activation, plasma cell survival, and regulation of antibody responses. These functions highlight the importance of Fc?R-mediated pathways in the modulation of adaptive immune responses and suggest a central role for IgG-Fc?R interactions during active and passive immunization. PMID:26497515

  20. Trypsin activates pancreatic duct epithelial cell ion channels through proteinase-activated receptor-2

    PubMed Central

    Nguyen, Toan D.; Moody, Mark W.; Steinhoff, Martin; Okolo, Charles; Koh, Duk-Su; Bunnett, Nigel W.

    1999-01-01

    Proteinase-activated receptor-2 (PAR-2) is a G protein–coupled receptor that is cleaved by trypsin within the NH2-terminus, exposing a tethered ligand that binds and activates the receptor. We examined the secretory effects of trypsin, mediated through PAR-2, on well-differentiated nontransformed dog pancreatic duct epithelial cells (PDEC). Trypsin and activating peptide (AP or SLIGRL-NH2, corresponding to the PAR-2 tethered ligand) stimulated both an 125I– efflux inhibited by Ca2+-activated Cl– channel inhibitors and a 86Rb+ efflux inhibited by a Ca2+-activated K+ channel inhibitor. The reverse peptide (LRGILS-NH2) and inhibited trypsin were inactive. Thrombin had no effect, suggesting absence of PAR-1, PAR-3, or PAR-4. In Ussing chambers, trypsin and AP stimulated a short-circuit current from the basolateral, but not apical, surface of PDEC monolayers. In monolayers permeabilized basolaterally or apically with nystatin, AP activated apical Cl– and basolateral K+ conductances. PAR-2 agonists increased [Ca2+]i in PDEC, and the calcium chelator BAPTA inhibited the secretory effects of AP. PAR-2 expression on dog pancreatic ducts and PDEC was verified by immunofluorescence. Thus, trypsin interacts with basolateral PAR-2 to increase [Ca2+]i and activate ion channels in PDEC. In pancreatitis, when trypsinogen is prematurely activated, PAR-2–mediated ductal secretion may promote clearance of toxins and debris. PMID:9916138

  1. Muscarinic Acetylcholine Receptor M3 Modulates Odorant Receptor Activity via Inhibition of ?-Arrestin-2 Recruitment

    PubMed Central

    Jiang, Yue; Li, Yun Rose; Tian, Huikai; Ma, Minghong; Matsunami, Hiroaki

    2015-01-01

    The olfactory system in rodents serves a critical function in social, reproductive, and survival behaviors. Processing of chemosensory signals in the brain is dynamically regulated in part by an animal's physiological state. We previously reported that type 3 muscarinic acetylcholine receptors (M3-Rs) physically interact with odorant receptors (ORs) to promote odor-induced responses in a heterologous expression system. However, it is not known how M3-Rs affect the ability of olfactory sensory neurons (OSNs) to respond to odors. Here, we show that an M3-R antagonist attenuates odor-induced responses in OSNs from wild-type, but not M3-R-null mice. Using a novel molecular assay, we demonstrate that the activation of M3-Rs inhibits the recruitment of ?-arrestin-2 to ORs, resulting in a potentiation of odor-induced response in OSNs. These results suggest a role for acetylcholine in modulating olfactory processing at the initial stages of signal transduction in the olfactory system. PMID:25800153

  2. High-affinity benzodiazepine receptor ligands among benzodiazepines and betacarbolines with different intrinsic activity

    SciTech Connect

    Yliniemelae, A.; Gynther, J. ); Konschin, H.; Tylli, H. ); Rouvinen, J. )

    1989-01-01

    Structural and electrostatic features of diazepam, flumazenil, and methyl betacarboline-3-carboxylate (BCCM) have been investigated using the molecular superimposition method. These high-affinity benzodiazepine (BZ) receptor ligands are structurally unrelated and they have different intrinsic activity. These ligands are superimposed in such a way that common structural and electrostatic features essential for the high receptor binding affinity overlap. In addition to this binding pharmacophore, there are roughly three separate binding zones in the BZ receptor, one for each class of ligands. The intrinsic activity of BZ receptor ligands depends on the molecular structures and the way the ligand approaches the receptor.

  3. Protease activated receptors 1 and 4 sensitize TRPV1 in nociceptive neurones

    E-print Network

    Vellani, Vittorio; Kinsey, Anna M; Prandini, Massimiliano; Hechtfischer, Sabine C; Reeh, Peter; Magherini, Pier C; Giacomoni, Chiara; McNaughton, Peter A

    2010-09-27

    Abstract Protease-activated receptors (PAR1-4) are activated by proteases released by cell damage or blood clotting, and are known to be involved in promoting pain and hyperalgesia. Previous studies have shown that PAR2 receptors enhance activation...

  4. PTEROSTILBENE AS A NEW AGONIST FOR THE PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR ALPHA ISOFORM

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pterostilbene, a stilbenoid antioxidant found in blueberries, grapes, other small fruits, and in woody plants was shown to activate the peroxisome proliferator-activated receptor alpha (PPAR alpha) isoform. This nuclear receptor is proposed to mediate the activity of lipid-lowering drugs such as th...

  5. Distinct tyrosine phosphorylation sites in ZAP-70 mediate activation and negative regulation of antigen receptor function.

    PubMed Central

    Kong, G; Dalton, M; Wardenburg, J B; Straus, D; Kurosaki, T; Chan, A C

    1996-01-01

    Biochemical and genetic evidence has implicated two families of protein tyrosine kinases (PTKs), the Src- and Syk-PTKs, in T- and B-cell antigen receptor signaling. ZAP-70 is a member of the Syk-PTKs that associates with the T-cell antigen receptor and undergoes tyrosine phosphorylation following receptor activation. Three tyrosine residues, Tyr-292, -492, and -493, have been identified as sites of phosphorylation following T-cell antigen receptor engagement. Utilizing ZAP-70- and Syk-deficient lymphocytes (Syk-DT40 cells), we provide biochemical and functional evidence that heterologous trans-phosphorylation of Tyr-493 by a Src-PTK is required for antigen receptor-mediated activation of both the calcium and ras pathways. In contrast, cells expressing mutations at Tyr-292 or -492 demonstrate hyperactive T- and B-cell antigen receptor phenotypes. Thus, phosphorylation of ZAP-70 mediates both activation and inactivation of antigen receptor signaling. PMID:8756661

  6. Modulation of transient receptor vanilloid 1 activity by transient receptor potential ankyrin 1.

    PubMed

    Spahn, Viola; Stein, Christoph; Zöllner, Christian

    2014-02-01

    Transient receptor potential vanilloid 1 (TRPV1) is a nonselective ligand-gated cation channel responding to noxious heat, protons, and chemicals such as capsaicin. TRPV1 is expressed in sensory neurons and plays a critical role in pain associated with tissue injury, inflammation, and nerve lesions. Transient receptor potential ankyrin 1 (TRPA1) is coexpressed with TRPV1. It is activated by compounds that cause a burning sensation (e.g., mustard oil) and, indirectly, by components of the inflammatory milieu eliciting nociceptor excitation and pain hypersensitivity. Previous studies indicate an interaction of TRPV1 and TRPA1 signaling pathways. Here we sought to examine the molecular mechanisms underlying such interactions in nociceptive neurons. We first excluded physical interactions of both channels using radioligand binding studies. By microfluorimetry, electrophysiological experiments, cAMP measurements, and site-directed mutagenesis we found a sensitization of TRPV1 after TRPA1 stimulation with mustard oil in a calcium and cAMP/protein kinase A (PKA)-dependent manner. TRPA1 stimulation enhanced TRPV1 phosphorylation via the putative PKA phosphorylation site serine 116. We also detected calcium-sensitive increased TRPV1 activity after TRPA1 activation in dorsal root ganglion neurons. The inhibition of TRPA1 by HC-030031 (1,2,3,6-tetrahydro-1,3-dimethyl-N-[4-(1-methylethyl)phenyl]-2,6-dioxo-7H-purine-7-acetamide, 2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-isopropylphenyl)acetamide) after its initial stimulation (and the calcium-insensitive TRPA1 mutant D477A) still showed increased capsaicin-induced TRPV1 activity. This excludes a calcium-induced additive TRPA1 current after TRPV1 stimulation. Our study shows sensitization of TRPV1 via activation of TRPA1, which involves adenylyl cyclase, increased cAMP, subsequent translocation and activation of PKA, and phosphorylation of TRPV1 at PKA phosphorylation residues. This suggests that cross-sensitization of TRP channels contributes to enhanced pain sensitivity in inflamed tissues. PMID:24275229

  7. The Structure of the GM-CSF Receptor Complex Reveals a Distinct Mode of Cytokine Receptor Activation

    SciTech Connect

    Hansen, Guido; Hercus, Timothy R.; McClure, Barbara J.; Stomski, Frank C.; Dottore, Mara; Powell, Jason; Ramshaw, Hayley; Woodcock, Joanna M.; Xu, Yibin; Guthridge, Mark; McKinstry, William J.; Lopez, Angel F.; Parker, Michael W.

    2008-08-11

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that controls the production and function of blood cells, is deregulated in clinical conditions such as rheumatoid arthritis and leukemia, yet offers therapeutic value for other diseases. Its receptors are heterodimers consisting of a ligand-specific {alpha} subunit and a {beta}c subunit that is shared with the interleukin (IL)-3 and IL-5 receptors. How signaling is initiated remains an enigma. We report here the crystal structure of the human GM-CSF/GM-CSF receptor ternary complex and its assembly into an unexpected dodecamer or higher-order complex. Importantly, mutagenesis of the GM-CSF receptor at the dodecamer interface and functional studies reveal that dodecamer formation is required for receptor activation and signaling. This unusual form of receptor assembly likely applies also to IL-3 and IL-5 receptors, providing a structural basis for understanding their mechanism of activation and for the development of therapeutics.

  8. Diabetes or peroxisome proliferator-activated receptor alpha agonist increases mitochondrial thioesterase I activity in heart

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Peroxisome proliferator-activated receptor alpha (PPAR alpha) is a transcriptional regulator of the expression of mitochondrial thioesterase I (MTE-I) and uncoupling protein 3 (UCP3), which are induced in the heart at the mRNA level in response to diabetes. Little is known about the regulation of pr...

  9. The Chinese Herbal Medicine Sophora flavescens Activates Pregnane X Receptor

    PubMed Central

    Wang, Laiyou; Li, Feng; Lu, Jie; Li, Guodong; Li, Dan; Zhong, Xiao-bo; Guo, Grace L.

    2010-01-01

    Sophora flavescens (SF) is an herbal medicine widely used for the treatment of viral hepatitis, cancer, viral myocarditis, gastrointestinal hemorrhage, and skin diseases. It was recently reported that SF up-regulates CYP3A expression. The mechanism of SF-induced CYP3A expression is unknown. In the current study, we tested the hypothesis that SF-induced CYP3A expression is mediated by the activation of pregnane X receptor (PXR). We used two cell lines, DPX2 and HepaRG, to investigate the role of PXR in SF-induced CYP3A expression. The DPX2 cell line is derived from HepG2 cells with the stable transfection of human PXR and a luciferase reporter gene linked with a human PXR response element identified in the CYP3A4 gene promoter. In DPX2 cells, SF activated PXR in a concentration-dependent manner. We used a metabolomic approach to identify the chemical constituents in SF, which were further analyzed for their effect on PXR activation and CYP3A regulation. One chemical in SF, N-methylcytisine, was identified as an individual chemical that activated PXR. HepaRG is a highly differentiated hepatoma cell line that mimics human hepatocytes. In HepaRG cells, N-methylcytisine significantly induced CYP3A4 expression, and this induction was suppressed by the PXR antagonist sulforaphane. These results suggest that SF induces CYP3A expression via the activation of PXR. PMID:20736322

  10. Enhancer transcripts mark active estrogen receptor binding sites

    PubMed Central

    Hah, Nasun; Murakami, Shino; Nagari, Anusha; Danko, Charles G.; Kraus, W. Lee

    2013-01-01

    We have integrated and analyzed a large number of data sets from a variety of genomic assays using a novel computational pipeline to provide a global view of estrogen receptor 1 (ESR1; a.k.a. ER?) enhancers in MCF-7 human breast cancer cells. Using this approach, we have defined a class of primary transcripts (eRNAs) that are transcribed uni- or bidirectionally from estrogen receptor binding sites (ERBSs) with an average transcription unit length of ?3–5 kb. The majority are up-regulated by short treatments with estradiol (i.e., 10, 25, or 40 min) with kinetics that precede or match the induction of the target genes. The production of eRNAs at ERBSs is strongly correlated with the enrichment of a number of genomic features that are associated with enhancers (e.g., H3K4me1, H3K27ac, EP300/CREBBP, RNA polymerase II, open chromatin architecture), as well as enhancer looping to target gene promoters. In the absence of eRNA production, strong enrichment of these features is not observed, even though ESR1 binding is evident. We find that flavopiridol, a CDK9 inhibitor that blocks transcription elongation, inhibits eRNA production but does not affect other molecular indicators of enhancer activity, suggesting that eRNA production occurs after the assembly of active enhancers. Finally, we show that an enhancer transcription “signature” based on GRO-seq data can be used for de novo enhancer prediction across cell types. Together, our studies shed new light on the activity of ESR1 at its enhancer sites and provide new insights about enhancer function. PMID:23636943

  11. Characterization of the intrinsic activity for a novel class of cannabinoid receptor ligands: Indole Quinuclidine analogues

    PubMed Central

    Franks, Lirit N.; Ford, Benjamin M.; Madadi, Nikhil R.; Penthala, Narsimha R.; Crooks, Peter A.; Prather, Paul L.

    2014-01-01

    Our laboratory recently reported that a group of novel indole quinuclidine analogues bind with nanomolar affinity to cannabinoid type-1 and type-2 receptors. This study characterized the intrinsic activity of these compounds by determining whether they exhibit agonist, antagonist, or inverse agonist activity at cannabinoid type-1 and/or type-2 receptors. Cannabinoid receptors activate Gi/Go-proteins that then proceed to inhibit activity of the downstream intracellular effector adenylyl cyclase. Therefore, intrinsic activity was quantified by measuring the ability of compounds to modulate levels of intracellular cAMP in intact cells. Concerning cannabinoid type-1 receptors endogenously expressed in Neuro2A cells, a single analogue exhibited agonist activity, while eight acted as neutral antagonists and two possessed inverse agonist activity. For cannabinoid type-2 receptors stably expressed in CHO cells, all but two analogues acted as agonists; these two exceptions exhibited inverse agonist activity. Confirming specificity at cannabinoid type-1 receptors, modulation of adenylyl cyclase activity by all proposed agonists and inverse agonists was blocked by co-incubation with the neutral cannabinoid type-1 antagonist O-2050. All proposed cannabinoid type-1 receptor antagonists attenuated adenylyl cyclase modulation by cannabinoid agonist CP-55,940. Specificity at cannabinoid type-2 receptors was confirmed by failure of all compounds to modulate adenylyl cyclase activity in CHO cells devoid of cannabinoid type-2 receptors. Further characterization of select analogues demonstrated concentration-dependent modulation of adenylyl cyclase activity with potencies similar to their respective affinities for cannabinoid receptors. Therefore, indole quinuclidines are a novel structural class of compounds exhibiting high affinity and a range of intrinsic activity at cannabinoid type-1 and type-2 receptors. PMID:24858620

  12. Estradiol improves cerebellar memory formation by activating estrogen receptor beta.

    PubMed

    Andreescu, Corina E; Milojkovic, Bogdan A; Haasdijk, Elize D; Kramer, Piet; De Jong, Frank H; Krust, Andrée; De Zeeuw, Chris I; De Jeu, Marcel T G

    2007-10-01

    Learning motor skills is critical for motor abilities such as driving a car or playing piano. The speed at which we learn those skills is subject to many factors. Yet, it is not known to what extent gonadal hormones can affect the achievement of accurate movements in time and space. Here we demonstrate via different lines of evidence that estradiol promotes plasticity in the cerebellar cortex underlying motor learning. First, we show that estradiol enhances induction of long-term potentiation at the parallel fiber to Purkinje cell synapse, whereas it does not affect long-term depression; second, we show that estradiol activation of estrogen receptor beta receptors in Purkinje cells significantly improves gain-decrease adaptation of the vestibulo-ocular reflex, whereas it does not affect general eye movement performance; and third, we show that estradiol increases the density of parallel fiber to Purkinje cell synapses, whereas it does not affect the density of climbing fiber synapses. We conclude that estradiol can improve motor skills by potentiating cerebellar plasticity and synapse formation. These processes may be advantageous during periods of high estradiol levels of the estrous cycle or pregnancy. PMID:17913916

  13. Peroxisome Proliferator-Activated Receptor-? in Thyroid Autoimmunity

    PubMed Central

    Ferrari, Silvia Martina; Fallahi, Poupak; Vita, Roberto; Benvenga, Salvatore

    2015-01-01

    Peroxisome proliferator-activated receptor- (PPAR-) ? expression has been shown in thyroid tissue from patients with thyroiditis or Graves' disease and furthermore in the orbital tissue of patients with Graves' ophthalmopathy (GO), such as in extraocular muscle cells. An increasing body of evidence shows the importance of the (C-X-C motif) receptor 3 (CXCR3) and cognate chemokines (C-X-C motif) ligand (CXCL)9, CXCL10, and CXCL11, in the T helper 1 immune response and in inflammatory diseases such as thyroid autoimmune disorders. PPAR-? agonists show a strong inhibitory effect on the expression and release of CXCR3 chemokines, in vitro, in various kinds of cells, such as thyrocytes, and in orbital fibroblasts, preadipocytes, and myoblasts from patients with GO. Recently, it has been demonstrated that rosiglitazone is involved in a higher risk of heart failure, stroke, and all-cause mortality in old patients. On the contrary, pioglitazone has not shown these effects until now; this favors pioglitazone for a possible use in patients with thyroid autoimmunity. However, further studies are ongoing to explore the use of new PPAR-? agonists in the treatment of thyroid autoimmune disorders. PMID:25722716

  14. Activation of endplate nicotinic acetylcholine receptors by agonists.

    PubMed

    Auerbach, Anthony

    2015-10-15

    The interaction of a small molecule made in one cell with a large receptor made in another is the signature event of cell signaling. Understanding the structure and energy changes associated with agonist activation is important for engineering drugs, receptors and synapses. The nicotinic acetylcholine receptor (AChR) is a ?300kD ion channel that binds the neurotransmitter acetylcholine (ACh) and other cholinergic agonists to elicit electrical responses in the central and peripheral nervous systems. This mini-review is in two sections. First, general concepts of skeletal muscle AChR operation are discussed in terms of energy landscapes for conformational change. Second, adult vs. fetal AChRs are compared with regard to interaction energies between ACh and agonist-site side chains, measured by single-channel electrophysiology and molecular dynamics simulations. The five aromatic residues that form the core of each agonist binding site can be divided into two working groups, a triad (led by ?Y190) that behaves similarly at all sites and a coupled pair (led by ?W55) that has a large influence on affinity only in fetal AChRs. Each endplate AChR has 5 homologous subunits, two of ?(1) and one each of ?, ?, and either ? (fetal) or ? (adult). These nicotinic AChRs have only 2 functional agonist binding sites located in the extracellular domain, at ?? and either ?? or ?? subunit interfaces. The receptor undergoes a reversible, global isomerization between structures called C and O. The C shape does not conduct ions and has a relatively low affinity for ACh, whereas O conducts cations and has a higher affinity. When both agonist sites are empty (filled only with water) the probability of taking on the O conformation (PO) is low, <10(-6). When ACh molecules occupy the agonist sites the C?O opening rate constant and C?O gating equilibrium constant increase dramatically. Following a pulse of ACh at the nerve-muscle synapse, the endplate current rises rapidly to reach a peak that corresponds to PO ?0.96. PMID:26206191

  15. Activation of synaptic and extrasynaptic glycine receptors by taurine in preoptic hypothalamic neurons.

    PubMed

    Bhattarai, Janardhan Prasad; Park, Soo Joung; Chun, Sang Woo; Cho, Dong Hyu; Han, Seong Kyu

    2015-11-01

    Taurine is an essential amino-sulfonic acid having a fundamental function in the brain, participating in both cell volume regulation and neurotransmission. Using a whole cell voltage patch clamp technique, the taurine-activated neurotransmitter receptors in the preoptic hypothalamic area (PHA) neurons were investigated. In the first set of experiments, different concentrations of taurine were applied on PHA neurons. Taurine-induced responses were concentration-dependent. Taurine-induced currents were action potential-independent and sensitive to strychnine, suggesting the involvement of glycine receptors. In addition, taurine activated not only ?-homomeric, but also ??-heteromeric glycine receptors in PHA neurons. Interestingly, a low concentration of taurine (0.5mM) activated glycine receptors, whereas a higher concentration (3mM) activated both glycine and gamma-aminobutyric acid A (GABAA) receptors in PHA neurons. These results suggest that PHA neurons are influenced by taurine and respond via glycine and GABAA receptors. PMID:26453764

  16. Modulation of cardiac ryanodine receptor activity by ROS and RNS.

    PubMed

    Donoso, Paulina; Sanchez, Gina; Bull, Ricardo; Hidalgo, Cecilia

    2011-01-01

    Calcium release through cardiac ryanodine receptors (RyR2) triggers heart muscle contraction. Reactive oxygen/nitrogen species (ROS/RNS), normally produced in the heart, promote endogenous RyR2 S-nitrosylation and S-glutathionylation. These reversible redox modifications increase RyR2 activity in vitro, and presumably also in vivo. RyR2 S-glutathionylation increases under physiologically relevant conditions (tachycardia and exercise), suggesting that cardiac cells utilize this redox modification to increase RyR2 activity under increased demand. In contrast, in vivo changes in RyR2 S-nitrosylation in response to physiological stimuli remain uncharacterized. The number and identity of the highly reactive RyR2 cysteine residues and the nature of the redox modification they undergo are presently unknown. Likewise, the physiological sources of ROS/RNS responsible for functionally relevant RyR2 redox modifications have not been completely identified. The redox state of RyR2 is altered in heart failure leading to enhanced RyR2 activity, which presumably contributes to decrease SR calcium content and induce other calcium release abnormalities observed in heart failure. Greater understanding of RyR2 redox modulation is necessary to counteract the deleterious consequences of RyR2 activity deregulation caused by oxidative stress. PMID:21196188

  17. Analysis of the Heat Shock Response in Mouse Liver Reveals Transcriptional Dependence on the Nuclear Receptor Peroxisome Proliferator-Activated Receptor alpha ?(PPAR?)

    EPA Science Inventory

    BACKGROUND: The nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha) regulates responses to chemical or physical stress in part by altering expression of genes involved in proteome maintenance. Many of these genes are also transcriptionally regulated by h...

  18. Pathway-selective antagonism of proteinase activated receptor 2

    PubMed Central

    Suen, J Y; Cotterell, A; Lohman, R J; Lim, J; Han, A; Yau, M K; Liu, L; Cooper, M A; Vesey, D A; Fairlie, D P

    2014-01-01

    Background and Purpose Proteinase activated receptor 2 (PAR2) is a GPCR associated with inflammation, metabolism and disease. Clues to understanding how to block PAR2 signalling associated with disease without inhibiting PAR2 activation in normal physiology could be provided by studies of biased signalling. Experimental Approach PAR2 ligand GB88 was profiled for PAR2 agonist and antagonist properties by several functional assays associated with intracellular G-protein-coupled signalling in vitro in three cell types and with PAR2-induced rat paw oedema in vivo. Key Results In HT29 cells, GB88 was a PAR2 antagonist in terms of Ca2+ mobilization and PKC phosphorylation, but a PAR2 agonist in attenuating forskolin-induced cAMP accumulation, increasing ERK1/2 phosphorylation, RhoA activation, myosin phosphatase phosphorylation and actin filament rearrangement. In CHO-hPAR2 cells, GB88 inhibited Ca2+ release, but activated Gi/o and increased ERK1/2 phosphorylation. In human kidney tubule cells, GB88 inhibited cytokine secretion (IL6, IL8, GM-CSF, TNF-?) mediated by PAR2. A rat paw oedema induced by PAR2 agonists was also inhibited by orally administered GB88 and compared with effects of locally administered inhibitors of G-protein coupled pathways. Conclusions and Implications GB88 is a biased antagonist of PAR2 that selectively inhibits PAR2/Gq/11/Ca2+/PKC signalling, leading to anti-inflammatory activity in vivo, while being an agonist in activating three other PAR2-activated pathways (cAMP, ERK, Rho) in human cells. These findings highlight opportunities to design drugs to block specific PAR2-linked signalling pathways in disease, without blocking beneficial PAR2 signalling in normal physiology, and to dissect PAR2-associated mechanisms of disease in vivo. PMID:24821440

  19. Trigeminal Medullary Dorsal Horn Neurons Activated by Nasal Stimulation Coexpress AMPA, NMDA, and NK1 Receptors

    PubMed Central

    McCulloch, P. F.; DiNovo, K. M.; Westerhaus, D. J.; Vizinas, T. A.; Peevey, J. F.; Lach, M. A.; Czarnocki, P.

    2013-01-01

    Afferent information initiating the cardiorespiratory responses during nasal stimulation projects from the nasal passages to neurons within the trigeminal medullary dorsal horn (MDH) via the anterior ethmoidal nerve (AEN). Central AEN terminals are thought to release glutamate to activate the MDH neurons. This study was designed to determine which neurotransmitter receptors (AMPA, kainate, or NMDA glutamate receptor subtypes or the Substance P receptor NK1) are expressed by these activated MDH neurons. Fos was used as a neuronal marker of activated neurons, and immunohistochemistry combined with epifluorescent microscopy was used to determine which neurotransmitter receptor subunits were coexpressed by activated MDH neurons. Results indicate that, during nasal stimulation with ammonia vapors in urethane-anesthetized Sprague-Dawley rats, activated neurons within the superficial MDH coexpress the AMPA glutamate receptor subunits GluA1 (95.8%) and GluA2/3 (88.2%), the NMDA glutamate receptor subunits GluN1 (89.1%) and GluN2A (41.4%), and NK1 receptors (64.0%). It is therefore likely that during nasal stimulation the central terminals of the AEN release glutamate and substance P that then produces activation of these MDH neurons. The involvement of AMPA and NMDA receptors may mediate fast and slow neurotransmission, respectively, while NK1 receptor involvement may indicate activation of a nociceptive pathway. PMID:24967301

  20. SENESCENCE-ASSOCIATED DECLINE IN HEPATIC PEROXISOMAL ENZYME ACTIVITIES CORRESPONDS WITH DIMINISHED LEVELS OF RETINOID X RECEPTOR ALPHA, BUT NOT PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR ALPHA1

    EPA Science Inventory

    Abstract

    Aging is associated with alterations in hepatic peroxisomal metabolism and susceptibility to hepatocarcinogenecity produced by agonists of peroxisome proliferator-activated receptor alpha (PPARa). Mechanisms involved in these effects are not well understood. Howev...

  1. Contribution of regional brain melanocortin receptor subtypes to elevated activity energy expenditure in lean, active rats.

    PubMed

    Shukla, C; Koch, L G; Britton, S L; Cai, M; Hruby, V J; Bednarek, M; Novak, C M

    2015-12-01

    Physical activity and non-exercise activity thermogenesis (NEAT) are crucial factors accounting for individual differences in body weight, interacting with genetic predisposition. In the brain, a number of neuroendocrine intermediates regulate food intake and energy expenditure (EE); this includes the brain melanocortin (MC) system, consisting of MC peptides as well as their receptors (MCR). MC3R and MC4R have emerged as critical modulators of EE and food intake. To determine how variance in MC signaling may underlie individual differences in physical activity levels, we examined behavioral response to MC receptor agonists and antagonists in rats that show high and low levels of physical activity and NEAT, that is, high- and low-capacity runners (HCR, LCR), developed by artificial selection for differential intrinsic aerobic running capacity. Focusing on the hypothalamus, we identified brain region-specific elevations in expression of MCR 3, 4, and also MC5R, in the highly active, lean HCR relative to the less active and obesity-prone LCR. Further, the differences in activity and associated EE as a result of MCR activation or suppression using specific agonists and antagonists were similarly region-specific and directly corresponded to the differential MCR expression patterns. The agonists and antagonists investigated here did not significantly impact food intake at the doses used, suggesting that the differential pattern of receptor expression may by more meaningful to physical activity than to other aspects of energy balance regulation. Thus, MCR-mediated physical activity may be a key neural mechanism in distinguishing the lean phenotype and a target for enhancing physical activity and NEAT. PMID:26404873

  2. Characterization and partial purification of solubilized active opiate receptors from toad brain.

    PubMed Central

    Ruegg, U T; Cuenod, S; Hiller, J M; Gioannini, T; Howells, R D; Simon, E J

    1981-01-01

    Opiate receptors have been solubilized from toad brain membranes in active form by using digitonin. Between 40% and 50% of the stereospecific binding activity present in toad brain membranes is recoverable in the ultracentrifugal supernatant of digitonin extracts. Binding of opiates to the solubilized receptor is enhanced 4- to 5-fold by decreasing digitonin concentration to 0.1% or less prior to binding. The solubilized receptor is similar to the membrane-bound receptor in its affinity for various ligands and its sensitivity to heat, trypsin, and N-ethylmaleimide. Moreover, the sodium effect seen in membrane-bound receptor is retained in the solubilized preparation. Both membrane-bound and soluble toad receptors show weak binding of enkephalins, suggesting that they are predominantly of the mu type. The solubilized opiate receptor has an approximate molecular weight of 350,000-400,000. Purification of up to 20-fold has been achieved by gel filtration on Sepharose CL-6B. PMID:6270689

  3. Investigating real-time activation of adenosine receptors by bioluminescence resonance energy transfer technique

    NASA Astrophysics Data System (ADS)

    Huang, Yimei; Yang, Hongqin; Zheng, Liqin; Chen, Jiangxu; Wang, Yuhua; Li, Hui; Xie, Shusen

    2013-02-01

    Adenosine receptors play important roles in many physiological and pathological processes, for example regulating myocardial oxygen consumption and the release of neurotransmitters. The activations of adenosine receptors have been studied by some kinds of techniques, such as western blot, immunohistochemistry, etc. However, these techniques cannot reveal the dynamical response of adenosine receptors under stimulation. In this paper, bioluminescence resonance energy transfer technique was introduced to study the real-time activation of adenosine receptors by monitoring the dynamics of cyclic adenosine monophosphate (cAMP) level. The results showed that there were significant differences between adenosine receptors on real-time responses under stimulation. Moreover, the dynamics of cAMP level demonstrated that competition between adenosine receptors existed. Taken together, our study indicates that monitoring the dynamics of cAMP level using bioluminescence resonance energy transfer technique could be one potential approach to investigate the mechanism of competitions between adenosine receptors.

  4. Triclocarban mediates induction of xenobiotic metabolism through activation of the constitutive androstane receptor and the estrogen receptor alpha.

    PubMed

    Yueh, Mei-Fei; Li, Tao; Evans, Ronald M; Hammock, Bruce; Tukey, Robert H

    2012-01-01

    Triclocarban (3,4,4'-trichlorocarbanilide, TCC) is used as a broad-based antimicrobial agent that is commonly added to personal hygiene products. Because of its extensive use in the health care industry and resistance to degradation in sewage treatment processes, TCC has become a significant waste product that is found in numerous environmental compartments where humans and wildlife can be exposed. While TCC has been linked to a range of health and environmental effects, few studies have been conducted linking exposure to TCC and induction of xenobiotic metabolism through regulation by environmental sensors such as the nuclear xenobiotic receptors (XenoRs). To identify the ability of TCC to activate xenobiotic sensors, we monitored XenoR activities in response to TCC treatment using luciferase-based reporter assays. Among the XenoRs in the reporter screening assay, TCC promotes both constitutive androstane receptor (CAR) and estrogen receptor alpha (ER?) activities. TCC treatment to hUGT1 mice resulted in induction of the UGT1A genes in liver. This induction was dependent upon the constitutive active/androstane receptor (CAR) because no induction occurred in hUGT1Car(-/-) mice. Induction of the UGT1A genes by TCC corresponded with induction of Cyp2b10, another CAR target gene. TCC was demonstrated to be a phenobarbital-like activator of CAR in receptor-based assays. While it has been suggested that TCC be classified as an endocrine disruptor, it activates ER? leading to induction of Cyp1b1 in female ovaries as well as in promoter activity. Activation of ER? by TCC in receptor-based assays also promotes induction of human CYP2B6. These observations demonstrate that TCC activates nuclear xenobiotic receptors CAR and ER? both in vivo and in vitro and might have the potential to alter normal physiological homeostasis. Activation of these xenobiotic-sensing receptors amplifies gene expression profiles that might represent a mechanistic base for potential human health effects from exposure to TCC. PMID:22761658

  5. Triclocarban Mediates Induction of Xenobiotic Metabolism through Activation of the Constitutive Androstane Receptor and the Estrogen Receptor Alpha

    PubMed Central

    Yueh, Mei-Fei; Li, Tao; Evans, Ronald M.; Hammock, Bruce; Tukey, Robert H.

    2012-01-01

    Triclocarban (3,4,4?-trichlorocarbanilide, TCC) is used as a broad-based antimicrobial agent that is commonly added to personal hygiene products. Because of its extensive use in the health care industry and resistance to degradation in sewage treatment processes, TCC has become a significant waste product that is found in numerous environmental compartments where humans and wildlife can be exposed. While TCC has been linked to a range of health and environmental effects, few studies have been conducted linking exposure to TCC and induction of xenobiotic metabolism through regulation by environmental sensors such as the nuclear xenobiotic receptors (XenoRs). To identify the ability of TCC to activate xenobiotic sensors, we monitored XenoR activities in response to TCC treatment using luciferase-based reporter assays. Among the XenoRs in the reporter screening assay, TCC promotes both constitutive androstane receptor (CAR) and estrogen receptor alpha (ER?) activities. TCC treatment to hUGT1 mice resulted in induction of the UGT1A genes in liver. This induction was dependent upon the constitutive active/androstane receptor (CAR) because no induction occurred in hUGT1Car?/? mice. Induction of the UGT1A genes by TCC corresponded with induction of Cyp2b10, another CAR target gene. TCC was demonstrated to be a phenobarbital-like activator of CAR in receptor-based assays. While it has been suggested that TCC be classified as an endocrine disruptor, it activates ER? leading to induction of Cyp1b1 in female ovaries as well as in promoter activity. Activation of ER? by TCC in receptor-based assays also promotes induction of human CYP2B6. These observations demonstrate that TCC activates nuclear xenobiotic receptors CAR and ER? both in vivo and in vitro and might have the potential to alter normal physiological homeostasis. Activation of these xenobiotic-sensing receptors amplifies gene expression profiles that might represent a mechanistic base for potential human health effects from exposure to TCC. PMID:22761658

  6. Targeting the Urokinase Plasminogen Activator Receptor Inhibits Ovarian Cancer Metastasis

    PubMed Central

    Kenny, Hilary A.; Leonhardt, Payton; Ladanyi, Andras; Yamada, S. Diane; Montag, Anthony; Im, Hae Kyung; Jagadeeswaran, Sujatha; Shaw, David E.; Mazar, Andrew P.; Lengyel, Ernst

    2010-01-01

    Purpose To understand the functional and preclinical efficacy of targeting urokinase plasminogen activator receptor (u-PAR) in ovarian cancer. Experimental Design Expression of u-PAR was studied in 162 epithelial ovarian cancers, including 77 pairs of corresponding primary and metastatic tumors. The effect of an antibody against u-PAR (ATN-658) on proliferation, adhesion, invasion, apoptosis, and migration was assessed in three (SKOV3ip1, HeyA8, and CaOV3) ovarian cancer cell lines. The impact of the u-PAR antibody on tumor weight, number, and survival was examined in corresponding ovarian cancer xenograft models and the mechanism by which ATN-658 blocks metastasis was explored. Results Only 8% of all ovarian tumors were negative for u-PAR expression. Treatment of SKOV3ip1, HeyA8, and CaOV3 ovarian cancer cells with the u-PAR antibody inhibited cell invasion, migration and adhesion. In vivo, anti-u-PAR treatment reduced the number of tumors and tumor weight in CaOV3 and SKOV3ip1 xenografts, and reduced tumor weight and increased survival in HeyA8 xenografts. Immunostaining of CaOV3 xenograft tumors and ovarian cancer cell lines showed an increase in active-caspase 3 and TUNEL staining. Treatment with u-PAR antibody inhibited ?5-integrin and u-PAR colocalization on primary human omental ECM. Anti-u-PAR treatment also decreased the expression of urokinase, u-PAR, ?3-integrin and fibroblast growth factor receptor-1 both in vitro and in vivo. Conclusions This study shows that an antibody against u-PAR reduces metastasis, induces apoptosis, and reduces the interaction between u-PAR and ?5-integrin. This provides a rationale for targeting the u-PAR pathway in patients with ovarian cancer and for further testing of ATN-658 in this indication. PMID:21149615

  7. Activation of extrasynaptic NMDA receptors induces a PKC-dependent switch in AMPA receptor subtypes in mouse cerebellar stellate cells

    PubMed Central

    Sun, Lu; June Liu, Siqiong

    2007-01-01

    The repetitive activation of synaptic glutamate receptors can induce a lasting change in the number or subunit composition of synaptic AMPA receptors (AMPARs). However, NMDA receptors that are present extrasynaptically can also be activated by a burst of presynaptic activity, and thus may be involved in the induction of synaptic plasticity. Here we show that the physiological-like activation of extrasynaptic NMDARs induces a lasting change in the synaptic current, by changing the subunit composition of AMPARs at the parallel fibre-to-cerebellar stellate cell synapse. This extrasynaptic NMDAR-induced switch in synaptic AMPARs from GluR2-lacking (Ca2+-permeable) to GluR2-containing (Ca2+-impermeable) receptors requires the activation of protein kinase C (PKC). These results indicate that the activation of extrasynaptic NMDARs by glutamate spillover is an important mechanism that detects the pattern of afferent activity and subsequently exerts a remote regulation of AMPAR subtypes at the synapse via a PKC-dependent pathway. PMID:17584840

  8. Tocotrienol enriched palm oil prevents atherosclerosis through modulating the activities of peroxisome proliferators-activated receptors.

    PubMed

    Li, Fengjuan; Tan, Wenjuan; Kang, Zhanfang; Wong, Chi-Wai

    2010-07-01

    Palm oil is enriched in vitamin E in the form of alpha-, gamma-, and delta-tocotrienols. Dietary tocotrienol supplements have been shown to prevent atherosclerosis development in patients and preclinical animal models. However, the mechanistic basis for this health beneficial effect is not well established. Peroxisome proliferator-activated receptors alpha, gamma, and delta (PPARalpha, PPARgamma, and PPARdelta) are ligand regulated transcription factors that play essential preventive roles in the development of atherosclerosis through regulating energy metabolism and inflammation. In this study, we presented data that the tocotrienol rich fraction (TRF) of palm oil activated PPARalpha, PPARgamma, and PPARdelta in reporter based assays. Importantly, TRF attenuated the development of atherosclerosis in ApoE-/- mice through inducing PPAR target gene liver X receptor alpha (LXRalpha) and its down-stream target genes apolipoproteins and cholesterol transporters, suggesting that modulating the activities of PPARs is a key aspect of the in vivo action of tocotrienols. PMID:20138624

  9. Significance of AT1 Receptor Independent Activation of Mineralocorticoid Receptor in Murine Diabetic Cardiomyopathy

    PubMed Central

    Nagatomo, Yuji; Meguro, Tomomi; Ito, Hiroyuki; Koide, Kimi; Anzai, Toshihisa; Fukuda, Keiichi; Ogawa, Satoshi; Yoshikawa, Tsutomu

    2014-01-01

    Background Diabetes mellitus (DM) has deleterious influence on cardiac performance independent of coronary artery disease and hypertension. The objective of the present study was to investigate the role of the renin-angiotensin-aldosterone system, especially angiotensin II type 1a receptor (AT1aR) and mineralocorticoid receptor (MR) signaling, in left ventricular (LV) dysfunction induced by diabetes mellitus (DM). Methods and Results DM was induced by intraperitoneal injection of streptozotocin (200 mg/kg BW) in wild-type (WT) or AT1aR knockout (KO) male mice, and they were bred during 6 or 12 weeks. Some KO mice were administered the MR antagonist eplerenone (100 mg/kg body weight). At 6 weeks, LV diastolic function was impaired in WT-DM, but preserved in KO-DM. At that time point MR mRNA expression was upregulated, NADPH oxidase subunit (p47phox) and glutathione peroxidase (GPx1) mRNA expression were upregulated, the staining intensities of LV tissue for 4-hydroxy-2-nonenal was stronger in immunohistochemistry, the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) positive cells was increased, Bcl-2 protein expression was significantly downregulated, and the expression of SERCA2a and phosphorylated phospholamban was depressed in WT-DM, while these changes were not seen in KO-DM. At 12 weeks, however, these changes were also noted in KO-DM. Eplerenone arrested those changes. The plasma aldosterone concentration was elevated in WT-DM but not in KO-DM at 6 weeks. It showed 3.7-fold elevation at 12 weeks even in KO-DM, which suggests “aldosterone breakthrough” phenomenon. However, the aldosterone content in LV tissue was unchanged in KO-DM. Conclusions DM induced diastolic dysfunction was observed even in KO at 12 weeks, which was ameliorated by minelarocorticoid receptor antagonist, eplerenone. AT1-independent MR activation in the LV might be responsible for the pathogenesis of diabetic cardiomyopathy. PMID:24664319

  10. Hydrophobic interactions drive ligand-receptor recognition for activation and inhibition of staphylococcal quorum sensing

    PubMed Central

    Wright, Jesse S.; Lyon, Gholson J.; George, Elizabeth A.; Muir, Tom W.; Novick, Richard P.

    2004-01-01

    Two-component systems represent the most widely used signaling paradigm in living organisms. Encoding the prototypical two-component system in Gram-positive bacteria, the staphylococcal agr (accessory gene regulator) operon uses a polytopic receptor, AgrC, activated by an autoinducing peptide (AIP), to coordinate quorum sensing with the global synthesis of virulence factors. The agr locus has undergone evolutionary divergence, resulting in the formation of several distinct inter- and intraspecies specificity groups, such that most cross-group AIP-receptor interactions are mutually inhibitory. We have exploited this natural diversity by constructing and analyzing AgrC chimeras generated by exchange of intradomain segments between receptors of different agr groups. Functional chimeras fell into three general classes: receptors with broadened specificity, receptors with tightened specificity, and receptors that lack activation specificity. Testing of these chimeric receptors against a battery of AIP analogs localized the primary ligand recognition site to the receptor distal subdomain and revealed that the AIPs bind primarily to a putative hydrophobic pocket in the receptor. This binding is mediated by a highly conserved hydrophobic patch on the AIPs and is an absolute requirement for interactions in self-activation and cross-inhibition of the receptors. It is suggested that this recognition scheme provides the fundamental basis for agr activation and interference. PMID:15528279

  11. Molecular Vibration-Activity Relationship in the Agonism of Adenosine Receptors

    PubMed Central

    Chee, Hyun Keun

    2013-01-01

    The molecular vibration-activity relationship in the receptor-ligand interaction of adenosine receptors was investigated by structure similarity, molecular vibration, and hierarchical clustering in a dataset of 46 ligands of adenosine receptors. The resulting dendrogram was compared with those of another kind of fingerprint or descriptor. The dendrogram result produced by corralled intensity of molecular vibrational frequency outperformed four other analyses in the current study of adenosine receptor agonism and antagonism. The tree that was produced by clustering analysis of molecular vibration patterns showed its potential for the functional classification of adenosine receptor ligands. PMID:24465242

  12. Nkx3-1 and LEF-1 Function as Transcriptional Inhibitors of Estrogen Receptor Activity

    E-print Network

    Liu, Xiaole Shirley

    Nkx3-1 and LEF-1 Function as Transcriptional Inhibitors of Estrogen Receptor Activity Kelly A for Advanced Study, Princeton, New Jersey Abstract Estrogen receptor (ER)-associated cofactors and cooperating transcription factors are one of the primary components determining transcriptional activity of estrogen target

  13. Autocrine regulation of T-cell activation by ATP release and P2X7 receptors.

    PubMed

    Yip, Linda; Woehrle, Tobias; Corriden, Ross; Hirsh, Mark; Chen, Yu; Inoue, Yoshiaki; Ferrari, Vhe; Insel, Paul A; Junger, Wolfgang G

    2009-06-01

    T-cell activation requires the influx of extracellular calcium, although mechanistic details regarding such activation are not fully defined. Here, we show that P2X(7) receptors play a key role in calcium influx and downstream signaling events associated with the activation of T cells. By real-time PCR and immunohistochemistry, we find that Jurkat T cells and human CD4(+) T cells express abundant P2X(7) receptors. We show, using a novel fluorescent microscopy technique, that T-cell receptor (TCR) stimulation triggers the rapid release of ATP (<100 microM). This release of ATP is required for TCR-mediated calcium influx, NFAT activation, and interleukin-2 (IL-2) production. TCR activation up-regulates P2X(7) receptor gene expression. Removal of extracellular ATP by apyrase or alkaline phosphatase treatment, inhibition of ATP release with the maxi-anion channel blocker gadolinium chloride, or siRNA silencing of P2X(7) receptors blocks calcium entry and inhibits T-cell activation. Moreover, lymphocyte activation is impaired in C57BL/6 mice that express poorly functional P2X(7) receptors, compared to control BALB/c mice, which express fully functional P2X(7) receptors. We conclude that ATP release and autocrine, positive feedback through P2X(7) receptors is required for the effective activation of T cells. PMID:19211924

  14. C5a Receptor Activation GENETIC IDENTIFICATION OF CRITICAL RESIDUES IN FOUR TRANSMEMBRANE HELICES*

    E-print Network

    Lichtarge, Olivier

    , New York 10591-6705 Hormones and sensory stimuli activate serpentine re- ceptors, transmembrane. These results identify key elements of a general mechanism for the serpentine receptor switch. Serpentine activate effector enzymes and ion channels (1, 2). More than 1,000 serpentine receptors of mammals share

  15. Heterologous Activation of Protein Kinase C Stimulates Phosphorylation of -Opioid Receptor at Serine 344, Resulting

    E-print Network

    Tian, Weidong

    Heterologous Activation of Protein Kinase C Stimulates Phosphorylation of -Opioid Receptor of the current study is to investigate the effect of opioid-independent, heterologous activation of protein kinase C (PKC) on the responsiveness of opioid receptor and the underlying molecular mechanisms. Our

  16. Functional selectivity induced by mGlu4 receptor positive allosteric modulation and concomitant activation of Gq coupled receptors

    PubMed Central

    Yin, Shen; Zamorano, Rocio; Conn, P. Jeffrey; Niswender, Colleen M.

    2012-01-01

    Metabotropic glutamate receptors (mGlus) are a group of Family C Seven Transmembrane Spanning Receptors (7TMRs) that play important roles in modulating signaling transduction, particularly within the central nervous system. mGlu4 belongs to a subfamily of mGlus that is predominantly coupled to Gi/o G proteins. We now report that the ubiquitous autacoid and neuromodulator, histamine, induces substantial glutamate-activated calcium mobilization in mGlu4-expressing cells, an effect which is observed in the absence of co-expressed chimeric G proteins. This strong induction of calcium signaling downstream of glutamate activation of mGlu4 depends upon the presence of H1 histamine receptors. Interestingly, the potentiating effect of histamine activation does not extend to other mGlu4-mediated signaling events downstream of Gi/o G proteins, such as cAMP inhibition, suggesting that the presence of Gq coupled receptors such as H1 may bias normal mGlu4-mediated Gi/o signaling events. When the activity induced by small molecule positive allosteric modulators of mGlu4 is assessed, the potentiated signaling of mGlu4 is further biased by histamine toward calcium-dependent pathways. These results suggest that Gi/o-coupled mGlus may induce substantial, and potentially unexpected, calcium-mediated signaling events if stimulation occurs concomitantly with activation of Gq receptors. Additionally, our results suggest that signaling induced by small molecule positive allosteric modulators may be substantially biased when Gq receptors are co-activated. This article is part of a Special Issue entitled ‘mGluR’ PMID:22426233

  17. Activated Protein C Enhances Human Keratinocyte Barrier Integrity via Sequential Activation of Epidermal Growth Factor Receptor and Tie2*

    PubMed Central

    Xue, Meilang; Chow, Shu-Oi; Dervish, Suat; Chan, Yee-Ka Agnes; Julovi, Sohel M.; Jackson, Christopher J.

    2011-01-01

    Keratinocytes play a critical role in maintaining epidermal barrier function. Activated protein C (APC), a natural anticoagulant with anti-inflammatory and endothelial barrier protective properties, significantly increased the barrier impedance of keratinocyte monolayers, measured by electric cell substrate impedance sensing and FITC-dextran flux. In response to APC, Tie2, a tyrosine kinase receptor, was rapidly activated within 30 min, and relocated to cell-cell contacts. APC also increased junction proteins zona occludens, claudin-1 and VE-cadherin. Inhibition of Tie2 by its peptide inhibitor or small interfering RNA abolished the barrier protective effect of APC. Interestingly, APC did not activate Tie2 through its major ligand, angiopoietin-1, but instead acted by binding to endothelial protein C receptor, cleaving protease-activated receptor-1 and transactivating EGF receptor. Furthermore, when activation of Akt, but not ERK, was inhibited, the barrier protective effect of APC on keratinocytes was abolished. Thus, APC activates Tie2, via a mechanism requiring, in sequential order, the receptors, endothelial protein C receptor, protease-activated receptor-1, and EGF receptor, which selectively enhances the PI3K/Akt signaling to enhance junctional complexes and reduce keratinocyte permeability. PMID:21173154

  18. Pertussis Toxin Up-regulates Angiotensin Type 1 Receptors through Toll-like Receptor 4-mediated Rac Activation*

    PubMed Central

    Nishida, Motohiro; Suda, Reiko; Nagamatsu, Yuichi; Tanabe, Shihori; Onohara, Naoya; Nakaya, Michio; Kanaho, Yasunori; Shibata, Takahiro; Uchida, Koji; Sumimoto, Hideki; Sato, Yoji; Kurose, Hitoshi

    2010-01-01

    Pertussis toxin (PTX) is recognized as a specific tool that uncouples receptors from Gi and Go through ADP-ribosylation. During the study analyzing the effects of PTX on Ang II type 1 receptor (AT1R) function in cardiac fibroblasts, we found that PTX increases the number of AT1Rs and enhances AT1R-mediated response. Microarray analysis revealed that PTX increases the induction of interleukin (IL)-1? among cytokines. Inhibition of IL-1? suppressed the enhancement of AT1R-mediated response by PTX. PTX increased the expression of IL-1? and AT1R through NF-?B, and a small GTP-binding protein, Rac, mediated PTX-induced NF-?B activation through NADPH oxidase-dependent production of reactive oxygen species. PTX induced biphasic increases in Rac activity, and the Rac activation in a late but not an early phase was suppressed by IL-1? siRNA, suggesting that IL-1?-induced Rac activation contributes to the amplification of Rac-dependent signaling induced by PTX. Furthermore, inhibition of TLR4 (Toll-like receptor 4) abolished PTX-induced Rac activation and enhancement of AT1R function. However, ADP-ribosylation of Gi/Go by PTX was not affected by inhibition of TLR4. Thus, PTX binds to two receptors; one is TLR4, which activates Rac, and another is the binding site that is required for ADP-ribosylation of Gi/Go. PMID:20231290

  19. Cyclin D1 stimulation of estrogen receptor transcriptional activity independent of cdk4.

    PubMed Central

    Neuman, E; Ladha, M H; Lin, N; Upton, T M; Miller, S J; DiRenzo, J; Pestell, R G; Hinds, P W; Dowdy, S F; Brown, M; Ewen, M E

    1997-01-01

    Cyclin D1 plays an important role in the development of breast cancer and is required for normal breast cell proliferation and differentiation associated with pregnancy. We show that ectopic expression of cyclin D1 can stimulate the transcriptional activity of the estrogen receptor in the absence of estradiol and that this activity can be inhibited by 4-hydroxytamoxifen and ICI 182,780. Cyclin D1 can form a specific complex with the estrogen receptor. Stimulation of the estrogen receptor by cyclin D1 is independent of cyclin-dependent kinase 4 activation. Cyclin D1 may manifest its oncogenic potential in breast cancer in part through binding to the estrogen receptor and activation of the transcriptional activity of the receptor. PMID:9271411

  20. CB2 Receptor Activation Ameliorates the Proinflammatory Activity in Acute Lung Injury Induced by Paraquat

    PubMed Central

    Liu, Zhenning; Wang, Yu; Zhao, Hongyu; Zheng, Qiang; Xiao, Li; Zhao, Min

    2014-01-01

    Paraquat, a widely used herbicide, is well known to exhibit oxidative stress and lung injury. In the present study, we investigated the possible underlying mechanisms of cannabinoid receptor-2 (CB2) activation to ameliorate the proinflammatory activity induced by PQ in rats. JWH133, a CB2 agonist, was administered by intraperitoneal injection 1?h prior to PQ exposure. After PQ exposure for 4, 8, 24, and 72?h, the bronchoalveolar lavage fluid was collected to determine levels of TNF-? and IL-1?, and the arterial blood samples were collected for detection of PaO2 level. At 72?h after PQ exposure, lung tissues were collected to determine the lung wet-to-dry weight ratios, myeloperoxidase activity, lung histopathology, the protein expression level of CB2, MAPKs (ERK1/2, p38MAPK, and JNK1/2), and NF-?Bp65. After rats were pretreated with JWH133, PQ-induced lung edema and lung histopathological changes were significantly attenuated. PQ-induced TNF-? and IL-1? secretion in BALF, increases of PaO2 in arterial blood, and MPO levels in the lung tissue were significantly reduced. JWH133 could efficiently activate CB2, while inhibiting MAPKs and NF-?B activation. The results suggested that activating CB2 receptor exerted protective activity against PQ-induced ALI, and it potentially contributed to the suppression of the activation of MAPKs and NF-?B pathways. PMID:24963491

  1. THE EFFECTS OF REPEATED MORPHINE EXPOSURE ON METABOTROPIC GLUTAMATE RECEPTOR ACTIVITY IN ADOLESCENT MICE 

    E-print Network

    Seloff, Katelyn Elizabeth

    2013-02-04

    Adolescent mice exhibit marked changes in D2/D3 dopamine receptor activity following administration of certain opioids. Thus, the present study examined whether repeated exposure to morphine would also modulate the activity of metabotropic glutamate...

  2. MAPK/ERK-dependent translation factor hyperactivation and dysregulated laminin ?2 expression in oral dysplasia and squamous cell carcinoma.

    PubMed

    Degen, Martin; Natarajan, Easwar; Barron, Patricia; Widlund, Hans R; Rheinwald, James G

    2012-06-01

    Lesions displaying a variety of dysplastic changes precede invasive oral and epidermal squamous cell carcinoma (SCC); however, there are no histopathological criteria for either confirming or staging premalignancy. SCCs and dysplasias frequently contain cells that abnormally express the ?2 subunit of laminin-332. We developed cell culture models to investigate ?2 dysregulation. Normal human keratinocytes displayed density-dependent repression of ?2, whereas premalignant keratinocytes and SCC cells overexpressed ?2 and secreted laminin assembly intermediates. Neoplastic cells had hyperactive EGFR/MAPK(ERK) signaling coordinate with overexpressed ?2, and EGFR and MEK inhibitors normalized ?2 expression. Keratinocytes engineered to express HPV16 E6 or activated mutant HRAS, cRAF1, or MEK1 lost density repression of ?2 and shared with neoplastic cells signaling abnormalities downstream of ERK, including increased phosphorylation of S6 and eIF4 translation factors. Notably, qPCR results revealed that ?2 overexpression was not accompanied by increased ?2 mRNA levels, consistent with ERK-dependent, eIF4B-mediated translation initiation of the stem-looped, 5'-untranslated region of ?2 mRNA in neoplastic cells. Inhibitors of MEK, but not of TORC1/2, blocked S6 and eIF4B phosphorylation and ?2 overexpression. Immunostaining of oral dysplasias identified ?2 overexpression occurring within fields of basal cells that had elevated p-S6 levels. These results reveal a causal relationship between ERK-dependent translation factor activation and laminin ?2 dysregulation and identify new markers of preinvasive neoplastic change during progression to SCC. PMID:22546478

  3. Receptor activity of rotavirus nonstructural glycoprotein NS28.

    PubMed Central

    Au, K S; Chan, W K; Burns, J W; Estes, M K

    1989-01-01

    Rotavirus morphogenesis involves the budding of subviral particles through the rough endoplasmic reticulum (RER) membrane of infected cells. During this process, particles acquire the outer capsid proteins and a transient envelope. Previous immunocytochemical and biochemical studies have suggested that a rotavirus nonstructural glycoprotein, NS28, encoded by genome segment 10, is a transmembrane RER protein and that about 10,000 Mr of its carboxy terminus is exposed on the cytoplasmic side of the RER. We have used in vitro binding experiments to examine whether NS28 serves as a receptor that binds subviral particles and mediates the budding process. Specific binding was observed between purified simian rotavirus SA11 single-shelled particles and RER membranes from SA11-infected monkey kidney cells and from SA11 gene 10 baculovirus recombinant-infected insect cells. Membranes from insect cells synthesizing VP1, VP4, NS53, VP6, VP7, or NS26 did not possess binding activity. Comparison of the binding of single-shelled particles to microsomes from infected monkey kidney cells and from insect cells indicated that a membrane-associated component(s) from SA11-infected monkey kidney cells interfered with binding. Direct evidence showing the interaction of NS28 and its nonglycosylated 20,000-Mr precursor expressed in rabbit reticulocyte lysates and single-shelled particles was obtained by cosedimentation of preformed receptor-ligand complexes through sucrose gradients. The domain on NS28 responsible for binding also was characterized. Reduced binding of single-shelled particles to membranes was seen with membranes treated with (i) a monoclonal antibody previously shown to interact with the C terminus of NS28, (ii) proteases known to cleave the C terminus of NS28, and (iii) the Enzymobead reagent. VP6 on single-shelled particles was suggested to interact with NS28 because (i) a monoclonal antibody to the subgroup I epitope on VP6 reduced particle binding, (ii) a purified polyclonal antiserum raised against recombinant baculovirus-produced VP6 reduced ligand binding, and (iii) a monoclonal antibody to a conserved epitope on VP6 augmented ligand binding. These experimental data provide support for the hypothesized receptor role of NS28 before the budding stage of rotavirus morphogenesis. Images PMID:2552139

  4. Laminin isoforms: biological roles and effects on the intracellular distribution of nuclear proteins in intestinal epithelial cells

    SciTech Connect

    Turck, Natacha; Gross, Isabelle; Gendry, Patrick; Stutzmann, Jeanne; Freund, Jean-Noel; Kedinger, Michele; Simon-Assmann, Patricia; Launay, Jean-Francois . E-mail: Jean-Francois.Launay@inserm.u-strasbg.fr

    2005-02-15

    Laminins are structurally and functionally major components of the extracellular matrix. Four isoforms of laminins (laminin-1, -2, -5 and -10) are expressed in a specific pattern along the crypt-villus axis of the intestine. Previous works indicated that expression of these isoforms is developmentally regulated and that laminins could modulate the behaviour of intestinal cells, but the exact role of each isoform remained unclear. Here, we report the first systematic analysis of the cellular functions of the four isoforms using the human colon adenocarcinoma Caco2/TC7 cell line as a model. We compared the respective abilities of each isoform to modulate adhesion, proliferation and differentiation of intestinal epithelial cells. We found that the isoforms were functionally distinct, with laminin-10 being the most adhesive substratum, laminin-2, laminin-5 and laminin-10 enhancing cellular proliferation and at the opposite, laminin-1 stimulating intestinal cell differentiation. To begin to characterise the molecular events induced by the different isoforms, we examined by immunofluorescence the intracellular distribution of several nuclear proteins, recently highlighted by a nuclear proteomic approach. We observed clear nucleocytoplasmic redistribution of these proteins, which depended on the laminin isoform. These results provide evidence for a distinct functional role of laminins in intestinal cell functions characterised by specific localisation of nuclear proteins.

  5. The antitussive activity of delta-opioid receptor stimulation in guinea pigs.

    PubMed

    Kotzer, C J; Hay, D W; Dondio, G; Giardina, G; Petrillo, P; Underwood, D C

    2000-02-01

    In this study, the activity of the delta-opioid receptor subtype-selective agonist, SB 227122, was investigated in a guinea pig model of citric acid-induced cough. Parenteral administration of selective agonists of the delta-opioid receptor (SB 227122), mu-opioid receptor (codeine and hydrocodone), and kappa-opioid receptor (BRL 52974) produced dose-related inhibition of citric acid-induced cough with ED(50) values of 7.3, 5.2, 5.1, and 5.3 mg/kg, respectively. The nonselective opioid receptor antagonist, naloxone (3 mg/kg, i.m.), attenuated the antitussive effects of codeine or SB 227122, indicating that the antitussive activity of both compounds is opioid receptor-mediated. The delta-receptor antagonist, SB 244525 (10 mg/kg, i.p.), inhibited the antitussive effect of SB 227122 (20 mg/kg, i.p.). In contrast, combined pretreatment with beta-funaltrexamine (mu-receptor antagonist; 20 mg/kg, s.c.) and norbinaltorphimine (kappa-receptor antagonist; 20 mg/kg, s.c.), at doses that inhibited the antitussive activity of mu- and kappa-receptor agonists, respectively, was without effect on the antitussive response of SB 227122 (20 mg/kg, i.p.). The sigma-receptor antagonist rimcazole (3 mg/kg, i.p.) inhibited the antitussive effect of dextromethorphan (30 mg/kg, i.p.), a sigma-receptor agonist, but not that of SB 227122. These studies provide compelling evidence that the antitussive effects of SB 227122 in this guinea pig cough model are mediated by agonist activity at the delta-opioid receptor. PMID:10640321

  6. Nicotine Activation of ?4* Receptors: Sufficient for Reward, Tolerance, and Sensitization

    NASA Astrophysics Data System (ADS)

    Tapper, Andrew R.; McKinney, Sheri L.; Nashmi, Raad; Schwarz, Johannes; Deshpande, Purnima; Labarca, Cesar; Whiteaker, Paul; Marks, Michael J.; Collins, Allan C.; Lester, Henry A.

    2004-11-01

    The identity of nicotinic receptor subtypes sufficient to elicit both the acute and chronic effects of nicotine dependence is unknown. We engineered mutant mice with ?4 nicotinic subunits containing a single point mutation, Leu9' --> Ala9' in the pore-forming M2 domain, rendering ?4* receptors hypersensitive to nicotine. Selective activation of ?4* nicotinic acetylcholine receptors with low doses of agonist recapitulates nicotine effects thought to be important in dependence, including reinforcement in response to acute nicotine administration, as well as tolerance and sensitization elicited by chronic nicotine administration. These data indicate that activation of ?4* receptors is sufficient for nicotine-induced reward, tolerance, and sensitization.

  7. Over-expression of laminin correlates to recovery of vasogenic edema following status epilepticus.

    PubMed

    Kim, Y-J; Kim, J-Y; Ko, A-R; Kang, T-C

    2014-09-01

    In the present study, we addressed the question of whether the up-regulation of laminin expression represents the astroglio-vascular responses to status epilepticus (SE) in the rat brain to better understand the role of vasogenic edema in epileptogenic insult. In the hippocampus, vasogenic edema was observed in the hippocampus 12h after SE when astroglial degeneration was undetected. Vasogenic edema in the hippocampus was more severe in the CA1 region where astroglial loss was absent than in the dentate gyrus showing astroglial degeneration. In the piriform cortex (PC), vasogenic edema was accompanied by appearance of astroglial degeneration 12h after SE. Laminin expression in the hippocampus and the PC was increased 3 days and 4 days after SE, respectively. Laminin expression was up-regulated in the hippocampus and the PC with concomitant reduction of SMI-71 (the endothelial barrier antigen) expression. Four weeks after SE, laminin expression was reduced in vessels showing strong SMI-71 expression within vasogenic edema lesion. Inhibition of SE-induced vasogenic edema formation by BQ788 effectively prevented laminin over-expression. Therefore, our findings indicate that laminin over-expression may be one of consequences from vasogenic edema rather than astroglial loss, and that laminin over-expression may promote migration of astrocytes to damaged or newly generated vessels to repair brain-blood barrier (BBB) disruption accompanied by the reconstruction of endothelial barrier. PMID:24931765

  8. The impact of laminin on 3D neurite extension in collagen gels

    NASA Astrophysics Data System (ADS)

    Swindle-Reilly, Katelyn E.; Papke, Jason B.; Kutosky, Hannah P.; Throm, Allison; Hammer, Joshua A.; Harkins, Amy B.; Kuntz Willits, Rebecca

    2012-08-01

    The primary goal of this research was to characterize the effect of laminin on three-dimensional (3D) neurite growth. Gels were formed using type I collagen at concentrations of 0.4-2.0 mg mL-1 supplemented with laminin at concentrations of 0, 1, 10, or 100 µg mL-1. When imaged with confocal microscopy, laminin was shown to follow the collagen fibers; however, the addition of laminin had minimal effect on the stiffness of the scaffolds at any concentration of collagen. Individual neurons dissociated from E9 chick dorsal root ganglia were cultured in the gels for 24 h, and neurite lengths were measured. For collagen gels without laminin, a typical bimodal response of neurite outgrowth was observed, with increased growth at lower concentrations of collagen gel. However, alteration of the chemical nature of the collagen gel by the laminin additive shifted, or completely mitigated, the bimodal neurite growth response seen in gels without laminin. Expression of integrin subunits, ?1, ?3, ?6 and ?1, were confirmed by PCR and immunolabeling in the 3D scaffolds. These results provide insight into the interplay between mechanical and chemical environment to support neurite outgrowth in 3D. Understanding the relative impact of environmental factors on 3D nerve growth may improve biomaterial design for nerve cell regeneration.

  9. Peroxisome proliferator-activated receptors, metabolic syndrome and cardiovascular disease

    PubMed Central

    Azhar, Salman

    2011-01-01

    Metabolic syndrome (MetS) is a constellation of risk factors including insulin resistance, central obesity, dyslipidemia and hypertension that markedly increase the risk of Type 2 diabetes (T2DM) and cardiovascular disease (CVD). The peroxisome proliferators-activated receptor (PPAR) isotypes, PPAR?, PPAR?/? and PPAR? are ligand-activated nuclear transcription factors, which modulate the expression of an array of genes that play a central role in regulating glucose, lipid and cholesterol metabolism, where imbalance can lead to obesity, T2DM and CVD. They are also drug targets, and currently, PPAR? (fibrates) and PPAR? (thiazolodinediones) agonists are in clinical use for treating dyslipidemia and T2DM, respectively. These metabolic characteristics of the PPARs, coupled with their involvement in metabolic diseases, mean extensive efforts are underway worldwide to develop new and efficacious PPAR-based therapies for the treatment of additional maladies associated with the MetS. This article presents an overview of the functional characteristics of three PPAR isotypes, discusses recent advances in our understanding of the diverse biological actions of PPARs, particularly in the vascular system, and summarizes the developmental status of new single, dual, pan (multiple) and partial PPAR agonists for the clinical management of key components of MetS, T2DM and CVD. It also summarizes the clinical outcomes from various clinical trials aimed at evaluating the atheroprotective actions of currently used fibrates and thiazolodinediones. PMID:20932114

  10. Endothelial Cells Promote Pigmentation through Endothelin Receptor B Activation.

    PubMed

    Regazzetti, Claire; De Donatis, Gian Marco; Ghorbel, Houda Hammami; Cardot-Leccia, Nathalie; Ambrosetti, Damien; Bahadoran, Philippe; Chignon-Sicard, Bérengčre; Lacour, Jean-Philippe; Ballotti, Robert; Mahns, Andre; Passeron, Thierry

    2015-12-01

    Findings of increased vascularization in melasma lesions and hyperpigmentation in acquired bilateral telangiectatic macules suggested a link between pigmentation and vascularization. Using high-magnification digital epiluminescence dermatoscopy, laser confocal microscopy, and histological examination, we showed that benign vascular lesions of the skin have restricted but significant hyperpigmentation compared with the surrounding skin. We then studied the role of microvascular endothelial cells in regulating skin pigmentation using an in vitro co-culture model using endothelial cells and melanocytes. These experiments showed that endothelin 1 released by microvascular endothelial cells induces increased melanogenesis signaling, characterized by microphthalmia-associated transcription factor phosphorylation, and increased tyrosinase and dopachrome tautomerase levels. Immunostaining for endothelin 1 in vascular lesions confirmed the increased expression on the basal layer of the epidermis above small vessels compared with perilesional skin. Endothelin acts through the activation of endothelin receptor B and the mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK)1/2, and p38, to induce melanogenesis. Finally, culturing of reconstructed skin with microvascular endothelial cells led to increased skin pigmentation that could be prevented by inhibiting EDNRB. Taken together these results demonstrated the role of underlying microvascularization in skin pigmentation, a finding that could open new fields of research for regulating physiological pigmentation and for treating pigmentation disorders such as melasma. PMID:26308584

  11. Opioid receptor mechanisms at the hypoglossal motor pool and effects on tongue muscle activity in vivo

    PubMed Central

    Hajiha, Mohammad; DuBord, Marq-André; Liu, Hattie; Horner, Richard L

    2009-01-01

    Opioids can modulate breathing and predispose to respiratory depression by actions at various central nervous system sites, but the mechanisms operating at respiratory motor nuclei have not been studied. This study tests the hypotheses that (i) local delivery of the ?-opioid receptor agonist fentanyl into the hypoglossal motor nucleus (HMN) will suppress genioglossus activity in vivo, (ii) a component of this suppression is mediated by opioid-induced acetylcholine release acting at muscarinic receptors, and (iii) ?- and ?-opioid receptors also modulate genioglossus activity. Seventy-two isoflurane-anaesthetised, tracheotomised, spontaneously breathing rats were studied during microdialysis perfusion into the HMN of (i) fentanyl and naloxone (?-opioid receptor antagonist), (ii) fentanyl with and without co-application of muscarinic receptor antagonists, and (iii) ?- and ?-opioid receptor agonists and antagonists. The results showed (i) that fentanyl at the HMN caused a suppression of genioglossus activity (P < 0.001) that reversed with naloxone (P < 0.001), (ii) that neither atropine nor scopolamine affected the fentanyl-induced suppression of genioglossus activity, and (iii) that ?-, but not ?-, opioid receptor stimulation also suppressed genioglossus activity (P= 0.036 and P= 0.402 respectively). We conclude that ?-opioid receptor stimulation suppresses motor output from a central respiratory motoneuronal pool that activates genioglossus muscle, and this suppression does not involve muscarinic receptor-mediated inhibition. This ?-opioid receptor-induced suppression of tongue muscle activity by effects at the hypoglossal motor pool may underlie the clinical concern regarding adverse upper airway function with ?-opioid analgesics. The inhibitory effects of ?- and ?-opioid receptors at the HMN also indicate an influence of endogenous enkephalins and endorphins in respiratory motor control. PMID:19403616

  12. Selenoprotein W controls epidermal growth factor receptor surface expression, activation and degradation via receptor ubiquitination

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Epidermal growth factor (EGF) receptor (EGFR) is the founding member of the ErbB family of growth factor receptors that modulate a complex network of intracellular signaling pathways controlling growth, proliferation and differentiation. Selenoprotein W (SEPW1) is a diet-regulated, highly conserved...

  13. Estrogen Receptor ? Activation Rapidly Modulates Male Sexual Motivation through the Transactivation of Metabotropic Glutamate Receptor 1a.

    PubMed

    Seredynski, Aurore L; Balthazart, Jacques; Ball, Gregory F; Cornil, Charlotte A

    2015-09-23

    In addition to the transcriptional activity of their liganded nuclear receptors, estrogens, such as estradiol (E2), modulate cell functions, and consequently physiology and behavior, within minutes through membrane-initiated events. The membrane-associated receptors (mERs) underlying the acute effects of estrogens on behavior have mostly been documented in females where active estrogens are thought to be of ovarian origin. We determined here, by acute intracerebroventricular injections of specific agonists and antagonists, the type(s) of mERs that modulate rapid effects of brain-derived estrogens on sexual motivation in male Japanese quail. Brain aromatase blockade acutely inhibited sexual motivation. Diarylpropionitrile (DPN), an estrogen receptor ? (ER?)-specific agonist, and to a lesser extent 17?-estradiol, possibly acting through ER-X, prevented this effect. In contrast, drugs targeting ER? (PPT and MPP), GPR30 (G1 and G15), and the Gq-mER (STX) did not affect sexual motivation. The mGluR1a antagonist LY367385 significantly inhibited sexual motivation but mGluR2/3 and mGluR5 antagonists were ineffective. LY367385 also blocked the behavioral restoration induced by E2 or DPN, providing functional evidence that ER? interacts with metabotropic glutamate receptor 1a (mGluR1a) signaling to acutely regulate male sexual motivation. Together these results show that ER? plays a key role in sexual behavior regulation and the recently uncovered cooperation between mERs and mGluRs is functional in males where it mediates the acute effects of estrogens produced centrally in response to social stimuli. The presence of an ER-mGluR interaction in birds suggests that this mechanism emerged relatively early in vertebrate history and is well conserved. Significance statement: The membrane-associated receptors underlying the acute effects of estrogens on behavior have mostly been documented in females, where active estrogens are thought to be of ovarian origin. Using acute intracerebroventricular injections of specific agonists and antagonists following blockade of brain aromatase, we show here that brain-derived estrogens acutely facilitate male sexual motivation through the activation of estrogen receptor ? interacting with the metabotropic glutamate receptor 1a. This behavioral effect occurring within minutes provides a mechanistic explanation of how an estrogen receptor not intrinsically coupled to intracellular effectors can signal from the membrane to govern behavior in a very rapid fashion. It suggests that different subtypes of estrogen receptors could regulate the motivation versus performance aspects of behavior. PMID:26400941

  14. IP receptor-dependent activation of PPAR{gamma} by stable prostacyclin analogues

    SciTech Connect

    Falcetti, Emilia; Flavell, David M.; Staels, Bart; Tinker, Andrew; Haworth, Sheila G.; Clapp, Lucie H. . E-mail: l.clapp@ucl.ac.uk

    2007-09-07

    Stable prostacyclin analogues can signal through cell surface IP receptors or by ligand binding to nuclear peroxisome proliferator-activated receptors (PPARs). So far these agents have been reported to activate PPAR{alpha} and PPAR{delta} but not PPAR{gamma}. Given PPAR{gamma} agonists and prostacyclin analogues both inhibit cell proliferation, we postulated that the IP receptor might elicit PPAR{gamma} activation. Using a dual luciferase reporter gene assay in HEK-293 cells stably expressing the IP receptor or empty vector, we found that prostacyclin analogues only activated PPAR{gamma} in the presence of the IP receptor. Moreover, the novel IP receptor antagonist, RO1138452, but not inhibitors of the cyclic AMP pathway, prevented activation. Likewise, the anti-proliferative effects of treprostinil observed in IP receptor expressing cells, were partially inhibited by the PPAR{gamma} antagonist, GW9662. We conclude that PPAR{gamma} is activated through the IP receptor via a cyclic AMP-independent mechanism and contributes to the anti-growth effects of prostacyclin analogues.

  15. Aberrant expression of laminin-332 promotes cell proliferation and cyst growth in ARPKD.

    PubMed

    Vijayakumar, Soundarapandian; Dang, Suparna; Marinkovich, M Peter; Lazarova, Zelmira; Yoder, Bradley; Torres, Vicente E; Wallace, Darren P

    2014-03-15

    Basement membrane abnormalities have often been observed in kidney cysts of polycystic kidney disease (PKD) patients and animal models. There is an abnormal deposition of extracellular matrix molecules, including laminin-?3,?3,?2 (laminin-332), in human autosomal dominant PKD (ADPKD). Knockdown of PKD1 paralogs in zebrafish leads to dysregulated synthesis of the extracellular matrix, suggesting that altered basement membrane assembly may be a primary defect in ADPKD. In this study, we demonstrate that laminin-332 is aberrantly expressed in cysts and precystic tubules of human autosomal recessive PKD (ARPKD) kidneys as well as in the kidneys of PCK rats, an orthologous ARPKD model. There was aberrant expression of laminin-?2 as early as postnatal day 2 and elevated laminin-332 protein in postnatal day 30, coinciding with the formation and early growth of renal cysts in PCK rat kidneys. We also show that a kidney cell line derived from Oak Ridge polycystic kidney mice, another model of ARPKD, exhibited abnormal lumen-deficient and multilumen structures in Matrigel culture. These cells had increased proliferation rates and altered expression levels of laminin-332 compared with their rescued counterparts. A function-blocking polyclonal antibody to laminin-332 significantly inhibited their abnormal proliferation rates and rescued their aberrant phenotype in Matrigel culture. Furthermore, abnormal laminin-332 expression in cysts originating from collecting ducts and proximal tubules as well as in precystic tubules was observed in a human end-stage ADPKD kidney. Our results suggest that abnormal expression of laminin-332 contributes to the aberrant proliferation of cyst epithelial cells and cyst growth in genetic forms of PKD. PMID:24370592

  16. Characterization of Peroxisome Proliferator-Activated Receptor a (PPARa) -Independent Effects of PPARa Activators in the Rodent Liver: Di-(2-ethylhexyl) phthalate Also Activates the Constitutive Activated Receptor

    EPA Science Inventory

    Peroxisome proliferator chemicals (PPC) are thought to mediate their effects in rodents on hepatocyte growth and liver cancer through the nuclear receptor peroxisome proliferatoractivated receptor alpha (PPARa). Recent studies indicate that one such PPC, the plasticizer di2- et...

  17. Topical Peroxisome Proliferator Activated Receptor Activators Accelerate Postnatal Stratum Corneum Acidification

    PubMed Central

    Fluhr, Joachim W.; Man, Mao-Qiang; Hachem, Jean-Pierre; Crumrine, Debra; Mauro, Theodora M.; Elias, Peter M.; Feingold, Kenneth R.

    2015-01-01

    Previous studies have shown that pH declines from between 6 and 7 at birth to adult levels (pH 5.0–5.5) over 5–6 days in neonatal rat stratum corneum (SC). As a result, at birth, neonatal epidermis displays decreased permeability barrier homeostasis and SC integrity, improving days 5–6. We determined here whether peroxisome proliferator-activated receptor (PPAR) activators accelerate postnatal SC acidification. Topical treatment with two different PPAR? activators, clofibrate and WY14643, accelerated the postnatal decline in SC surface pH, whereas treatment with PPAR? activators did not and a PPAR?/? activator had only a modest effect. Treatment with clofibrate significantly accelerated normalization of barrier function. The morphological basis for the improvement in barrier function in PPAR?-treated animals includes accelerated secretion of lamellar bodies and enhanced, postsecretory processing of secreted lamellar body contents into mature lamellar membranes. Activity of ?-glucocerebrosidase increased after PPAR?-activator treatment. PPAR? activator also improved SC integrity, which correlated with an increase in corneodesmosome density and increased desmoglein-1 content, with a decline in serine protease activity. Topical treatment of newborn animals with a PPAR? activator increased secretory phospholipase A2 activity, which likely accounts for accelerated SC acidification. Thus, PPAR? activators accelerate neonatal SC acidification, in parallel with improved permeability homeostasis and SC integrity/cohesion. Hence, PPAR? activators might be useful to prevent or treat certain common neonatal dermatoses. PMID:18704104

  18. Transient laminin beta 1a Induction Defines the Wound Epidermis during Zebrafish Fin Regeneration.

    PubMed

    Chen, Chen-Hui; Merriman, Alexander F; Savage, Jeremiah; Willer, Jason; Wahlig, Taylor; Katsanis, Nicholas; Yin, Viravuth P; Poss, Kenneth D

    2015-08-01

    The first critical stage in salamander or teleost appendage regeneration is creation of a specialized epidermis that instructs growth from underlying stump tissue. Here, we performed a forward genetic screen for mutations that impair this process in amputated zebrafish fins. Positional cloning and complementation assays identified a temperature-sensitive allele of the ECM component laminin beta 1a (lamb1a) that blocks fin regeneration. lamb1a, but not its paralog lamb1b, is sharply induced in a subset of epithelial cells after fin amputation, where it is required to establish and maintain a polarized basal epithelial cell layer. These events facilitate expression of the morphogenetic factors shha and lef1, basolateral positioning of phosphorylated Igf1r, patterning of new osteoblasts, and regeneration of bone. By contrast, lamb1a function is dispensable for juvenile body growth, homeostatic adult tissue maintenance, repair of split fins, or renewal of genetically ablated osteoblasts. fgf20a mutations or transgenic Fgf receptor inhibition disrupt lamb1a expression, linking a central growth factor to epithelial maturation during regeneration. Our findings reveal transient induction of lamb1a in epithelial cells as a key, growth factor-guided step in formation of a signaling-competent regeneration epidermis. PMID:26305099

  19. Transient laminin beta 1a Induction Defines the Wound Epidermis during Zebrafish Fin Regeneration

    PubMed Central

    Chen, Chen-Hui; Merriman, Alexander F.; Savage, Jeremiah; Willer, Jason; Wahlig, Taylor; Katsanis, Nicholas; Yin, Viravuth P.; Poss, Kenneth D.

    2015-01-01

    The first critical stage in salamander or teleost appendage regeneration is creation of a specialized epidermis that instructs growth from underlying stump tissue. Here, we performed a forward genetic screen for mutations that impair this process in amputated zebrafish fins. Positional cloning and complementation assays identified a temperature-sensitive allele of the ECM component laminin beta 1a (lamb1a) that blocks fin regeneration. lamb1a, but not its paralog lamb1b, is sharply induced in a subset of epithelial cells after fin amputation, where it is required to establish and maintain a polarized basal epithelial cell layer. These events facilitate expression of the morphogenetic factors shha and lef1, basolateral positioning of phosphorylated Igf1r, patterning of new osteoblasts, and regeneration of bone. By contrast, lamb1a function is dispensable for juvenile body growth, homeostatic adult tissue maintenance, repair of split fins, or renewal of genetically ablated osteoblasts. fgf20a mutations or transgenic Fgf receptor inhibition disrupt lamb1a expression, linking a central growth factor to epithelial maturation during regeneration. Our findings reveal transient induction of lamb1a in epithelial cells as a key, growth factor-guided step in formation of a signaling-competent regeneration epidermis. PMID:26305099

  20. Peptide fragments of the dihydropyridine receptor can modulate cardiac ryanodine receptor channel activity and sarcoplasmic reticulum Ca2+ release.

    PubMed Central

    Dulhunty, Angela F; Curtis, Suzanne M; Cengia, Louise; Sakowska, Magdalena; Casarotto, Marco G

    2004-01-01

    We show that peptide fragments of the dihydropyridine receptor II-III loop alter cardiac RyR (ryanodine receptor) channel activity in a cytoplasmic Ca2+-dependent manner. The peptides were AC (Thr-793-Ala-812 of the cardiac dihydropyridine receptor), AS (Thr-671-Leu-690 of the skeletal dihydropyridine receptor), and a modified AS peptide [AS(D-R18)], with an extended helical structure. The peptides added to the cytoplasmic side of channels in lipid bilayers at > or = 10 nM activated channels when the cytoplasmic [Ca2+] was 100 nM, but either inhibited or did not affect channel activity when the cytoplasmic [Ca2+] was 10 or 100 microM. Both activation and inhibition were independent of bilayer potential. Activation by AS, but not by AC or AS(D-R18), was reduced at peptide concentrations >1 mM in a voltage-dependent manner (at +40 mV). In control experiments, channels were not activated by the scrambled AS sequence (ASS) or skeletal II-III loop peptide (NB). Resting Ca2+ release from cardiac sarcoplasmic reticulum was not altered by peptide AC, but Ca2+-induced Ca2+ release was depressed. Resting and Ca2+-induced Ca2+ release were enhanced by both the native and modified AS peptides. NMR revealed (i) that the structure of peptide AS(D-R18) is not influenced by [Ca2+] and (ii) that peptide AC adopts a helical structure, particularly in the region containing positively charged residues. This is the first report of specific functional interactions between dihydropyridine receptor A region peptides and cardiac RyR ion channels in lipid bilayers. PMID:14678014

  1. Antileishmanial Activity of the Estrogen Receptor Modulator Raloxifene

    PubMed Central

    Reimăo, Juliana Q.; Miguel, Danilo C.; Taniwaki, Noemi N.; Trinconi, Cristiana T.; Yokoyama-Yasunaka, Jenicer K. U.; Uliana, Silvia R. B.

    2014-01-01

    Background The treatment of leishmaniasis relies mostly on parenteral drugs with potentially serious adverse effects. Additionally, parasite resistance in the treatment of leishmaniasis has been demonstrated for the majority of drugs available, making the search for more effective and less toxic drugs and treatment regimens a priority for the control of leishmaniasis. The aims of this study were to evaluate the antileishmanial activity of raloxifene in vitro and in vivo and to investigate its mechanism of action against Leishmania amazonensis. Methodology/Principal Findings Raloxifene was shown to possess antileishmanial activity in vitro against several species with EC50 values ranging from 30.2 to 38.0 µM against promastigotes and from 8.8 to 16.2 µM against intracellular amastigotes. Raloxifene's mechanism of action was investigated through transmission electron microscopy and labeling with propidium iodide, DiSBAC2(3), rhodamine 123 and monodansylcadaverine. Microscopic examinations showed that raloxifene treated parasites displayed autophagosomes and mitochondrial damage while the plasma membrane remained continuous. Nonetheless, plasma membrane potential was rapidly altered upon raloxifene treatment with initial hyperpolarization followed by depolarization. Loss of mitochondrial membrane potential was also verified. Treatment of L. amazonensis – infected BALB/c mice with raloxifene led to significant decrease in lesion size and parasite burden. Conclusions/Significance The results of this work extend the investigation of selective estrogen receptor modulators as potential candidates for leishmaniasis treatment. The antileishmanial activity of raloxifene was demonstrated in vitro and in vivo. Raloxifene produces functional disorder on the plasma membrane of L. amazonensis promastigotes and leads to functional and morphological disruption of mitochondria, which culminate in cell death. PMID:24810565

  2. G Protein-coupled Receptor 40 (GPR40) and Peroxisome Proliferator-activated Receptor ? (PPAR?): AN INTEGRATED TWO-RECEPTOR SIGNALING PATHWAY.

    PubMed

    Wang, Shuibang; Awad, Keytam S; Elinoff, Jason M; Dougherty, Edward J; Ferreyra, Gabriela A; Wang, Jennifer Y; Cai, Rongman; Sun, Junfeng; Ptasinska, Anetta; Danner, Robert L

    2015-08-01

    Peroxisome proliferator-activated receptor ? (PPAR?) ligands have been widely used to treat type 2 diabetes mellitus. However, knowledge of PPAR? signaling remains incomplete. In addition to PPAR?, these drugs also activate G protein-coupled receptor 40 (GPR40), a G?q-coupled free fatty acid receptor linked to MAPK networks and glucose homeostasis. Notably, p38 MAPK activation has been implicated in PPAR? signaling. Here, rosiglitazone (RGZ) activation of GPR40 and p38 MAPK was found to boost PPAR?-induced gene transcription in human endothelium. Inhibition or knockdown of p38 MAPK or expression of a dominant negative (DN) p38 MAPK mutant blunted RGZ-induced PPAR? DNA binding and reporter activity in EA.hy926 human endothelial cells. GPR40 inhibition or knockdown, or expression of a DN-G?q mutant likewise blocked activation of both p38 MAPK and PPAR? reporters. Importantly, RGZ induction of PPAR? target genes in primary human pulmonary artery endothelial cells (PAECs) was suppressed by knockdown of either p38 MAPK or GPR40. GPR40/PPAR? signal transduction was dependent on p38 MAPK activation and induction of PPAR? co-activator-1 (PGC1?). Silencing of p38 MAPK or GPR40 abolished the ability of RGZ to induce phosphorylation and expression of PGC1? in PAECs. Knockdown of PGC1?, its essential activator SIRT1, or its binding partner/co-activator EP300 inhibited RGZ induction of PPAR?-regulated genes in PAECs. RGZ/GPR40/p38 MAPK signaling also led to EP300 phosphorylation, an event that enhances PPAR? target gene transcription. Thus, GPR40 and PPAR? can function as an integrated two-receptor signal transduction pathway, a finding with implications for rational drug development. PMID:26105050

  3. New insight into active muscarinic receptors with the novel radioagonist [łH]iperoxo.

    PubMed

    Schrage, Ramona; Holze, Janine; Klöckner, Jessica; Balkow, Aileen; Klause, Anne S; Schmitz, Anna-Lena; De Amici, Marco; Kostenis, Evi; Tränkle, Christian; Holzgrabe, Ulrike; Mohr, Klaus

    2014-08-01

    Activation of G protein-coupled receptors involves major conformational changes of the receptor protein ranging from the extracellular transmitter binding site to the intracellular G protein binding surface. GPCRs such as the muscarinic acetylcholine receptors are commonly probed with radioantagonists rather than radioagonists due to better physicochemical stability, higher affinity, and indifference towards receptor coupling states of the former. Here we introduce tritiated iperoxo, a superagonist at muscarinic M? receptors with very high affinity. In membrane suspensions of transfected CHO-cells, [łH]iperoxo - unlike the common radioagonists [łH]acetylcholine and [łH]oxotremorine M - allowed labelling of each of the five muscarinic receptor subtypes in radioagonist displacement and saturation binding studies. [łH]iperoxo revealed considerable differences in affinity between the even- and the odd-numbered muscarinic receptor subtypes with affinities for the M? and M? receptor in the picomolar range. Probing ternary complex formation on the M? receptor, [łH]iperoxo dissociation was not influenced by an archetypal allosteric inverse agonist, reflecting activation-related rearrangement of the extracellular loop region. At the inner side of M?, the preferred Gi protein acted as a positive allosteric modulator of [łH]iperoxo binding, whereas Gs and Gq were neutral in spite of their robust coupling to the activated receptor. In intact CHO-hM? cells, endogenous guanylnucleotides promoted receptor/G protein-dissociation resulting in low-affinity agonist binding which, nevertheless, was still reported by [łH]iperoxo. Taken together, the muscarinic superagonist [łH]iperoxo is the best tool currently available for direct probing activation-related conformational transitions of muscarinic receptors. PMID:24863257

  4. Prenatal Allospecific NK Cell Tolerance Hinges on Instructive Allorecognition through the Activating Receptor during Development.

    PubMed

    Alhajjat, Amir M; Strong, Beverly S; Lee, Amanda E; Turner, Lucas E; Wadhwani, Ram K; Ortaldo, John R; Heusel, Jonathan W; Shaaban, Aimen F

    2015-08-15

    Little is known about how the prenatal interaction between NK cells and alloantigens shapes the developing NK cell repertoire toward tolerance or immunity. Specifically, the effect on NK cell education arising from developmental corecognition of alloantigens by activating and inhibitory receptors with shared specificity is uncharacterized. Using a murine prenatal transplantation model, we examined the manner in which this seemingly conflicting input affects NK cell licensing and repertoire formation in mixed hematopoietic chimeras. We found that prenatal NK cell tolerance arose from the elimination of phenotypically hostile NK cells that express an allospecific activating receptor without coexpressing any allospecific inhibitory receptors. Importantly, the checkpoint for the system appeared to occur centrally within the bone marrow during the final stage of NK cell maturation and hinged on the instructive recognition of allogeneic ligand by the activating receptor rather than through the inhibitory receptor as classically proposed. Residual nondeleted hostile NK cells expressing only the activating receptor exhibited an immature, anergic phenotype, but retained the capacity to upregulate inhibitory receptor expression in peripheral sites. However, the potential for this adaptive change to occur was lost in developmentally mature chimeras. Collectively, these findings illuminate the intrinsic process in which developmental allorecognition through the activating receptor regulates the emergence of durable NK cell tolerance and establishes a new paradigm to fundamentally guide future investigations of prenatal NK cell-allospecific education. PMID:26136432

  5. Discovery of tripeptide-derived multifunctional ligands possessing delta/mu opioid receptor agonist and neurokinin 1 receptor antagonist activities.

    PubMed

    Nair, Padma; Yamamoto, Takashi; Cowell, Scott; Kulkarni, Vinod; Moye, Sharif; Navratilova, Edita; Davis, Peg; Ma, Shou-Wu; Vanderah, Todd W; Lai, Josephine; Porreca, Frank; Hruby, Victor J

    2015-09-01

    Several bifunctional peptides were synthesized and characterized based on the pentapeptide-derived ligand NP30 (1: Tyr-DAla-Gly-Phe-Gly-Trp-O-[3',5'-Bzl(CF3)2]). Modification and truncation of amino acid residues were performed, and the tripeptide-derived ligand NP66 (11: Dmt-DAla-Trp-NH-[3',5'-(CF3)2-Bzl]) was obtained based on the overlapping pharmacophore concept. The Trp(3) residue of ligand 11 works as a message residue for both opioid and NK1 activities. The significance lies in the observation that the approach of appropriate truncation of peptide sequence could lead to a tripeptide-derived chimeric ligand with effective binding and functional activities for both mu and delta opioid and NK1 receptors with agonist activities at mu and delta opioid and antagonist activity at NK1 receptors, respectively. PMID:26212775

  6. A bacterial tyrosine phosphatase inhibits plant pattern recognition receptor activation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Perception of pathogen-associated molecular patterns (PAMPs) by surface-localised pattern-recognition receptors (PRRs) is a key component of plant innate immunity. Most known plant PRRs are receptor kinases and initiation of PAMP-triggered immunity (PTI) signalling requires phosphorylation of the PR...

  7. Insulin receptor binding and protein kinase activity in muscles of trained rats

    SciTech Connect

    Dohm, G.L.; Sinha, M.K.; Caro, J.F.

    1987-02-01

    Exercise has been shown to increase insulin sensitivity, and muscle is quantitatively the most important tissue of insulin action. Since the first step in insulin action is the binding to a membrane receptor, the authors postulated that exercise training would change insulin receptors in muscle and in this study they have investigated this hypothesis. Female rats initially weighing approx. 100 g were trained by treadmill running for 2 h/day, 6 days/wk for 4 wk at 25 m/min (0 grade). Insulin receptors from vastus intermedius muscles were solubilized by homogenizing in a buffer containing 1% Triton X-100 and then partially purified by passing the soluble extract over a wheat germ agglutinin column. The 4 wk training regimen resulted in a 65% increase in citrate synthase activity in red vastus lateralis muscle, indicating an adaptation to exercise ( SVI). Insulin binding by the partially purified receptor preparations was approximately doubled in muscle of trained rats at all insulin concentrations, suggesting an increase in the number of receptors. Training did not alter insulin receptor structure as evidenced by electrophoretic mobility under reducing and nonreducing conditions. Basal insulin receptor protein kinase activity was higher in trained than untrained animals and this was likely due to the greater number of receptors. However, insulin stimulation of the protein kinase activity was depressed by training. These results demonstrate that endurance training does alter receptor number and function in muscle and these changes may be important in increasing insulin sensitivity after exercise training.

  8. Inhibition of androgen receptor and ?-catenin activity in prostate cancer

    PubMed Central

    Lee, Eugine; Madar, Aviv; David, Gregory; Garabedian, Michael J.; DasGupta, Ramanuj; Logan, Susan K.

    2013-01-01

    Androgen receptor (AR) is the major therapeutic target in aggressive prostate cancer. However, targeting AR alone can result in drug resistance and disease recurrence. Therefore, simultaneous targeting of multiple pathways could in principle be an effective approach to treating prostate cancer. Here we provide proof-of-concept that a small-molecule inhibitor of nuclear ?-catenin activity (called C3) can inhibit both the AR and ?-catenin–signaling pathways that are often misregulated in prostate cancer. Treatment with C3 ablated prostate cancer cell growth by disruption of both ?-catenin/T-cell factor and ?-catenin/AR protein interaction, reflecting the fact that T-cell factor and AR have overlapping binding sites on ?-catenin. Given that AR interacts with, and is transcriptionally regulated by ?-catenin, C3 treatment also resulted in decreased occupancy of ?-catenin on the AR promoter and diminished AR and AR/?-catenin target gene expression. Interestingly, C3 treatment resulted in decreased AR binding to target genes accompanied by decreased recruitment of an AR and ?-catenin cofactor, coactivator-associated arginine methyltransferase 1 (CARM1), providing insight into the unrecognized function of ?-catenin in prostate cancer. Importantly, C3 inhibited tumor growth in an in vivo xenograft model and blocked renewal of bicalutamide-resistant sphere-forming cells, indicating the therapeutic potential of this approach. PMID:24019458

  9. Activation of transforming potential of the human insulin receptor gene

    SciTech Connect

    Wang, L.H.; Lin, B.; Jong, S.M.J.; Dixon, D.; Ellis, L.; Roth, R.A.; Rutter, W.J.

    1987-08-01

    A retrovirus containing part of the human insulin receptor (hIR) gene was constructed by replacing ros sequences in the avian sarcoma virus UR2 with hIR cDNA sequences coding for 46 amino acids of the extracellular domain and the entire transmembrane and cytoplasmic domains of the ..beta.. subunit of hIR. The resulting virus, named UIR, contains the hIR sequence fused to the 5' portion of the UR2 gag gene coding for p19. UIR is capable of transforming chicken embryo fibroblasts and promoting formation of colonies in soft agar; however, it does not form tumors in vivo. A variant that arose from the parental UIR is capable of efficiently inducing sarcomas in vivo. UIR-transformed cells exhibit higher rates of glucose uptake and growth than normal cells. The 4-kilobase UIR genome codes for a membrane-associated, glycosylated gag-hIR fusion protein of 75 kDa designated P75/sup gag-hir/. P75/sup gag-hir/ contains a protein tyrosine kinase activity that is capable of undergoing autophosphorylation and of phosphorylating foreign substrates in vitro; it is phosphorylated at both serine and tyrosine residues in vivo

  10. 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.

  11. Heat and Chemical Shock Potentiation of Glucocorticoid Receptor Transactivation Requires Heat Shock Factor (HSF) Activity

    E-print Network

    Abraham, Nader G.

    Heat and Chemical Shock Potentiation of Glucocorticoid Receptor Transactivation Requires Heat Shock of Ohio, Toledo, Ohio 43614 Heat shock and other forms of stress increase glu- cocorticoid receptor (GR) activity in cells, suggesting cross-talk between the heat shock and GR signal path- ways. An unresolved

  12. Halogen bonding-enhanced electrochemical halide anion sensing by redox-active ferrocene receptors.

    PubMed

    Lim, Jason Y C; Cunningham, Matthew J; Davis, Jason J; Beer, Paul D

    2015-09-17

    The first examples of halogen bonding redox-active ferrocene receptors and their anion electrochemical sensing properties are reported. Halogen bonding was found to significantly amplify the magnitude of the receptor's metallocene redox-couple's voltammetric responses for halide sensing compared to their hydrogen bonding analogues in both acetonitrile and aqueous-acetonitrile solvent media. PMID:26289779

  13. CHARACTERIZATION OF THE PORCINE PEROXISOME PROLIFERATIVE ACTIVATED RECEPTOR GAMMA COACTIVATOR 1 (PPARGC1)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Peroxisome proliferative activated receptor gamma coactivator 1 (PPARGC1) is a coactivator of nuclear receptors with an important function in adaptive thermogenesis. It influences genes involved in regulation of body weight and composition. Therefore, PPARGC1 can be considered as a candidate gene fo...

  14. Allosteric regulation of G protein-coupled receptor activity by phospholipids.

    PubMed

    Dawaliby, Rosie; Trubbia, Cataldo; Delporte, Cédric; Masureel, Matthieu; Van Antwerpen, Pierre; Kobilka, Brian K; Govaerts, Cédric

    2016-01-01

    Lipids are emerging as key regulators of membrane protein structure and activity. These effects can be attributed either to the modification of bilayer properties (thickness, curvature and surface tension) or to the binding of specific lipids to the protein surface. For G protein-coupled receptors (GPCRs), the effects of phospholipids on receptor structure and activity remain poorly understood. Here we reconstituted purified ?2-adrenergic receptor (?2R) in high-density lipoparticles to systematically characterize the effect of biologically relevant phospholipids on receptor activity. We observed that the lipid headgroup type affected ligand binding (agonist and antagonist) and receptor activation. Specifically, phosphatidylgycerol markedly favored agonist binding and facilitated receptor activation, whereas phosphatidylethanolamine favored antagonist binding and stabilized the inactive state of the receptor. We then showed that these effects could be recapitulated with detergent-solubilized lipids, demonstrating that the functional modulation occurred in the absence of a bilayer. Our data suggest that phospholipids act as direct allosteric modulators of GPCR activity. PMID:26571351

  15. Antagonizing Peroxisome Proliferator-Activated Receptor ? Activity Selectively Enhances Th1 Immunity in Male Mice.

    PubMed

    Zhang, Monan Angela; Ahn, Jeeyoon Jennifer; Zhao, Fei Linda; Selvanantham, Thirumahal; Mallevaey, Thierry; Stock, Nick; Correa, Lucia; Clark, Ryan; Spaner, David; Dunn, Shannon E

    2015-12-01

    Females exhibit more robust Th1 responses than males. Our previous work suggested that this sex disparity is a consequence of higher activity of the androgen-induced gene peroxisome proliferator-activated receptor ? (PPAR?) in male CD4(+) T cells. The objective of this study was to elucidate the cellular and molecular mechanism of how PPAR? inhibits Th1 responses in male mice. In this study, we found that PPAR? functions within CD4(+) and CD8(+) T lymphocytes and NKT cells to negatively regulate IFN-? responses in male mice and identified Ifng as the gene target of PPAR? repression. Treatment of male CD4(+) T cells with the PPAR? agonist fenofibrate induced the recruitment of PPAR? and the nuclear receptor-interacting protein, nuclear receptor corepressor 1, to specific cis-regulatory elements in the Ifng locus. This recruitment associated with reduced histone acetylation at these sites. Knockdown of nuclear receptor corepressor 1 in primary male T cells abolished the effect of fenofibrate in reducing IFN-? production. In contrast, treatment of male T cells with IS001, a novel antagonist of PPAR?, increased Ifng gene expression and histone acetylation across the Ifng locus. Finally, we investigated the effects of IS001 on IFN-? responses in mice during infection with the Th1-associated pathogen Listeria monocytogenes and observed that IS001 enhanced IFN-? production by NKT, CD4(+), and CD8(+) T cells and improved the survival of male, but not female, mice. Our findings provide a novel mechanism of why IFN-? responses are more robust in females and introduce a small-molecule IS001 that can be used to enhance Th1 immunity in males. PMID:26491197

  16. Glucose-Sensing Receptor T1R3: A New Signaling Receptor Activated by Glucose in Pancreatic ?-Cells.

    PubMed

    Kojima, Itaru; Nakagawa, Yuko; Hamano, Kunihisa; Medina, Johan; Li, Longfei; Nagasawa, Masahiro

    2015-01-01

    Subunits of the sweet taste receptors T1R2 and T1R3 are expressed in pancreatic ?-cells. Compared with T1R3, mRNA expression of T1R2 is considerably lower. At the protein level, expression of T1R2 is undetectable in ?-cells. Accordingly, a major component of the sweet taste-sensing receptor in ?-cells may be a homodimer of T1R3 rather than a heterodimer of T1R2/T1R3. Inhibition of this receptor by gurmarin or deletion of the T1R3 gene attenuates glucose-induced insulin secretion from ?-cells. Hence the T1R3 homodimer functions as a glucose-sensing receptor (GSR) in pancreatic ?-cells. When GSR is activated by the T1R3 agonist sucralose, elevation of intracellular ATP concentration ([ATP]i) is observed. Sucralose increases [ATP]i even in the absence of ambient glucose, indicating that sucralose increases [ATP]i not simply by activating glucokinase, a rate-limiting enzyme in the glycolytic pathway. In addition, sucralose augments elevation of [ATP]i induced by methylsuccinate, suggesting that sucralose activates mitochondrial metabolism. Nonmetabolizable 3-O-methylglucose also increases [ATP]i and knockdown of T1R3 attenuates elevation of [ATP]i induced by high concentration of glucose. Collectively, these results indicate that the T1R3 homodimer functions as a GSR; this receptor is involved in glucose-induced insulin secretion by activating glucose metabolism probably in mitochondria. PMID:25947913

  17. Analytical methodology for the profiling and characterization of androgen receptor active compounds in human placenta.

    PubMed

    Indiveri, Paolo; Horwood, Julia; Abdul-Sada, Alaa; Arrebola, Juan P; Olea, Nicolas; Hill, Elizabeth M

    2014-08-01

    The exposure to endocrine disrupting chemicals during foetal development has been proposed to cause reproductive dysfunctions in the neonate or later life. In order to support such studies, an analytical method was developed to profile the receptor mediated (anti)androgenic activities present in extracts of placenta samples. Placenta samples from women giving birth to healthy male neonates were extracted and fractionated by HPLC. Fractions containing androgen receptor (AR) activity were detected using an in vitro yeast-based human androgen receptor transcription screen. GC-MS analyses of receptor active fractions resulted in detection of chemical contaminants including antimicrobial and cosmetic compounds which exhibited AR antagonist activity in the yeast screen, and endogenously derived steroids which contributed to both the agonist and antagonistic activity in the samples. The bioassay-directed fractionation methodology developed in this study revealed the potential to identify mixtures of chemical contaminants that should be investigated for potential effects on the reproductive system. PMID:24972338

  18. GABAA receptor modulation by piperine and a non-TRPV1 activating derivative.

    PubMed

    Khom, Sophia; Strommer, Barbara; Schöffmann, Angela; Hintersteiner, Juliane; Baburin, Igor; Erker, Thomas; Schwarz, Thomas; Schwarzer, Christoph; Zaugg, Janine; Hamburger, Matthias; Hering, Steffen

    2013-06-15

    The action of piperine (the pungent component of pepper) and its derivative SCT-66 ((2E,4E)-5-(1,3-benzodioxol-5-yl))-N,N-diisobutyl-2,4-pentadienamide) on different gamma-aminobutyric acid (GABA) type A (GABA(A)) receptors, transient-receptor-potential-vanilloid-1 (TRPV1) receptors and behavioural effects were investigated. GABA(A) receptor subtypes and TRPV1 receptors were expressed in Xenopus laevis oocytes. Modulation of GABA-induced chloride currents (I(GABA)) by piperine and SCT-66 and activation of TRPV1 was studied using the two-microelectrode-voltage-clamp technique and fast perfusion. Their effects on explorative behaviour, thermoregulation and seizure threshold were analysed in mice. Piperine acted with similar potency on all GABA(A) receptor subtypes (EC?? range: 42.8±7.6 ?M (????)-59.6±12.3 ?M (????). I(GABA) modulation by piperine did not require the presence of a ?(2S)-subunit, suggesting a binding site involving only ? and ? subunits. I(GABA) activation was slightly more efficacious on receptors formed from ?(2/3) subunits (maximal I(GABA) stimulation through ???? receptors: 332±64% and ????: 271±36% vs. ????: 171±22%, p<0.05) and ??-subunits (????: 375±51% vs. ????:136±22%, p<0.05). Replacing the piperidine ring by a N,N-diisobutyl residue (SCT-66) prevents interactions with TRPV1 and simultaneously increases the potency and efficiency of GABA(A) receptor modulation. SCT-66 displayed greater efficacy on GABA(A) receptors than piperine, with different subunit-dependence. Both compounds induced anxiolytic, anticonvulsant effects and reduced locomotor activity; however, SCT-66 induced stronger anxiolysis without decreasing body temperature and without the proconvulsive effects of TRPV1 activation and thus may serve as a scaffold for the development of novel GABA(A) receptor modulators. PMID:23623790

  19. Erythropoietin attenuates cardiac dysfunction in experimental sepsis in mice via activation of the ?-common receptor

    PubMed Central

    Khan, Areeg I.; Coldewey, Sina M.; Patel, Nimesh S. A.; Rogazzo, Mara; Collino, Massimo; Yaqoob, Muhammed M.; Radermacher, Peter; Kapoor, Amar; Thiemermann, Christoph

    2013-01-01

    SUMMARY There is limited evidence that the tissue-protective effects of erythropoietin are mediated by a heterocomplex of the erythropoietin receptor and the ?-common receptor (‘tissue-protective receptor’), which is pharmacologically distinct from the ‘classical’ erythropoietin receptor homodimer that is responsible for erythropoiesis. However, the role of the ?-common receptor and/or erythropoietin in sepsis-induced cardiac dysfunction (a well known, serious complication of sepsis) is unknown. Here we report for the first time that the ?-common receptor is essential for the improvements in the impaired systolic contractility afforded by erythropoietin in experimental sepsis. Cardiac function was assessed in vivo (echocardiography) and ex vivo (Langendorff-perfused heart) in wild-type and ?-common receptor knockout mice, that were subjected to lipopolysaccharide (9 mg/kg body weight; young mice) for 16–18 hours or cecal ligation and puncture (aged mice) for 24 hours. Mice received erythropoietin (1000 IU/kg body weight) 1 hour after lipopolysaccharide or cecal ligation and puncture. Erythropoietin reduced the impaired systolic contractility (in vivo and ex vivo) caused by endotoxemia or sepsis in young as well as old wild-type mice in a ?-common-receptor-dependent fashion. Activation by erythropoietin of the ?-common receptor also resulted in the activation of well-known survival pathways (Akt and endothelial nitric oxide synthase) and inhibition of pro-inflammatory pathways (glycogen synthase kinase-3?, nuclear factor-?B and interleukin-1?). All the above pleiotropic effects of erythropoietin were lost in ?-common receptor knockout mice. Erythropoietin attenuates the impaired systolic contractility associated with sepsis by activation of the ?-common receptor, which, in turn, results in activation of survival pathways and inhibition of inflammation. PMID:23519033

  20. Maturational alterations in constitutive activity of medial prefrontal cortex kappa-opioid receptors in Wistar rats.

    PubMed

    Sirohi, Sunil; Walker, Brendan M

    2015-11-01

    Opioid receptors can display spontaneous agonist-independent G-protein signaling (basal signaling/constitutive activity). While constitutive ?-opioid receptor (KOR) activity has been documented in vitro, it remains unknown if KORs are constitutively active in native systems. Using [(35) S] guanosine 5'-O-[gamma-thio] triphosphate coupling assay that measures receptor functional state, we identified the presence of medial prefrontal cortex KOR constitutive activity in young rats that declined with age. Furthermore, basal signaling showed an age-related decline and was insensitive to neutral opioid antagonist challenge. Collectively, the present data are first to demonstrate age-dependent alterations in the medial prefrontal cortex KOR constitutive activity in rats and changes in the constitutive activity of KORs can differentially impact KOR ligand efficacy. These data provide novel insights into the functional properties of the KOR system and warrant further consideration of KOR constitutive activity in normal and pathophysiological behavior. Opioid receptors exhibit agonist-independent constitutive activity; however, kappa-opioid receptor (KOR) constitutive activity has not been demonstrated in native systems. Our results confirm KOR constitutive activity in the medial prefrontal cortex (mPFC) that declines with age. With the ability to presynaptically inhibit multiple neurotransmitter systems in the mPFC, maturational or patho-logical alterations in constitutive activity could disrupt corticofugal glutamatergic pyramidal projection neurons mediating executive function. Regulation of KOR constitutive activity could serve as a therapeutic target to treat compromised executive function. PMID:26257334

  1. Alpinetin activates the ? receptor instead of the ? and ? receptor pathways to protect against rat myocardial cell apoptosis

    PubMed Central

    SUO, CHUANTAO; SUN, LIBO; YANG, SHUANG

    2014-01-01

    Alpinetin is a natural flavonoid that protects cells against fatal injury in ischemia-reperfusion. ? receptor activation protects myocardial cells from trauma; however, the mechanism is unknown. The aim of this study was to explore the function of alpinetin in ? receptor-mediated myocardial apoptosis. The myocardial cells of newly born rats were cultivated and myocardial apoptosis was induced by serum deprivation. The MTT method was used to evaluate cell viability and Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining was used to analyze apoptosis. The expression levels of opioid receptor mRNA and protein were tested using reverse transcription-polymerase reaction (RT-PCR) and western blot assays. In addition, an opioid receptor antagonist, as well as protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) inhibitors, were used to determine the inferred signaling pathway. The results showed that that alpinetin reduced the myocardial apoptosis induced by serum deprivation in a concentration-dependent manner. However, the protection conferred to the myocardial cells by alpinetin was blocked by the ? opioid receptor antagonist naltrindole, as well as by PKC and ERK inhibitors (GF109203X and U0126, respectively). In addition, it was shown that alpinetin was able to maintain the stability of the mitochondrial membrane potential, lower the level of intracytoplasmic cytochrome c and reduce Bax displacement from the cytoplasm to the mitochondria. It was concluded that alpinetin was able to activate ? receptors to induce the endogenous protection of myocardial cells via the PKC/ERK signaling pathway. PMID:24348774

  2. Modulation of nuclear receptor activity by the F domain.

    PubMed

    Patel, Shivali R; Skafar, Debra F

    2015-12-15

    The F domain located at the C-terminus of proteins is one of the least conserved regions of the estrogen receptors alpha and beta, members of the nuclear hormone receptor superfamily. Indeed, many members of the superfamily lack the F domain. However, when present, removing the F domain entirely or mutating it alters transactivation, dimerization, and the responses to agonist and antagonist ligands. This review focuses on the functions of the F domain of the estrogen receptors, particularly in relation to other members of the superfamily. PMID:26184856

  3. Phorbol ester stimulates secretory activity while inhibiting receptor-activated aminopyrine uptake by gastric glands

    SciTech Connect

    Brown, M.R.; Chew, C.S.

    1986-03-05

    Both cyclic AMP-dependent and -independent secretagogues stimulate pepsinogen release, respiration and H/sup +/ secretory activity (AP uptake) in rabbit gastric glands. 12-O-tetradecanoylphorbol-13-acetate (T), a diacyglycerol analog, activates protein kinase C (PKC) and stimulates secretion in many systems. T stimulated respiration and pepsinogen release by glands and increased AP uptake by both glands and purified parietal cells. However, T reduced AP uptake by glands stimulated with carbachol (C) or histamine (H) with an apparent IC/sub 50/ of 1 nM. Preincubation with T for 30 min produced maximum inhibition which was not reversed by removal of T. T accelerated the decline of the transient C peak while the late steady state response to H was most inhibited. H-stimulated AP uptake was also inhibited by 50 ..mu..g/ml 1-oleoyl-2-acetyl-glycerol, a reported PKC activator, but not by the inactive phorbol, 4..cap alpha..-phorbol-12,13-didecanoate. In contrast, T potentiated AP uptake by glands stimulated with submaximal doses of dibutyryl cyclic AMP. These results suggest inhibition by T is a specific effect of PKC activators. The differing effects of T on secretion indicators may result from a dual action of T on receptor and post-receptor intracellular events.

  4. Expression of Protease-Activated Receptor 1 and 2 and Anti-Tubulogenic Activity of Protease-Activated Receptor 1 in Human Endothelial Colony-Forming Cells

    PubMed Central

    Fortunato, Tiago M.; Vara, Dina S.; Wheeler-Jones, Caroline P.; Pula, Giordano

    2014-01-01

    Endothelial colony-forming cells (ECFCs) are obtained from the culture of human peripheral blood mononuclear cell (hPBMNC) fractions and are characterised by high proliferative and pro-vasculogenic potential, which makes them of great interest for cell therapy. Here, we describe the detection of protease-activated receptor (PAR) 1 and 2 amongst the surface proteins expressed in ECFCs. Both receptors are functionally coupled to extracellular signal-regulated kinase (ERK) 1 and 2, which become activated and phosphorylated in response to selective PAR1- or PAR2-activating peptides. Specific stimulation of PAR1, but not PAR2, significantly inhibits capillary-like tube formation by ECFCs in vitro, suggesting that tubulogenesis is negatively regulated by proteases able to stimulate PAR1 (e.g. thrombin). The activation of ERKs is not involved in the regulation of tubulogenesis in vitro, as suggested by use of the MEK inhibitor PD98059 and by the fact that PAR2 stimulation activates ERKs without affecting capillary tube formation. Both qPCR and immunoblotting showed a significant downregulation of vascular endothelial growth factor 2 (VEGFR2) in response to PAR1 stimulation. Moreover, the addition of VEGF (50–100 ng/ml) but not basic Fibroblast Growth Factor (FGF) (25–100 ng/ml) rescued tube formation by ECFCs treated with PAR1-activating peptide. Therefore, we propose that reduction of VEGF responsiveness resulting from down-regulation of VEGFR2 is underlying the anti-tubulogenic effect of PAR1 activation. Although the role of PAR2 remains elusive, this study sheds new light on the regulation of the vasculogenic activity of ECFCs and suggests a potential link between adult vasculogenesis and the coagulation cascade. PMID:25289673

  5. Expression of protease-activated receptor 1 and 2 and anti-tubulogenic activity of protease-activated receptor 1 in human endothelial colony-forming cells.

    PubMed

    Fortunato, Tiago M; Vara, Dina S; Wheeler-Jones, Caroline P; Pula, Giordano

    2014-01-01

    Endothelial colony-forming cells (ECFCs) are obtained from the culture of human peripheral blood mononuclear cell (hPBMNC) fractions and are characterised by high proliferative and pro-vasculogenic potential, which makes them of great interest for cell therapy. Here, we describe the detection of protease-activated receptor (PAR) 1 and 2 amongst the surface proteins expressed in ECFCs. Both receptors are functionally coupled to extracellular signal-regulated kinase (ERK) 1 and 2, which become activated and phosphorylated in response to selective PAR1- or PAR2-activating peptides. Specific stimulation of PAR1, but not PAR2, significantly inhibits capillary-like tube formation by ECFCs in vitro, suggesting that tubulogenesis is negatively regulated by proteases able to stimulate PAR1 (e.g. thrombin). The activation of ERKs is not involved in the regulation of tubulogenesis in vitro, as suggested by use of the MEK inhibitor PD98059 and by the fact that PAR2 stimulation activates ERKs without affecting capillary tube formation. Both qPCR and immunoblotting showed a significant downregulation of vascular endothelial growth factor 2 (VEGFR2) in response to PAR1 stimulation. Moreover, the addition of VEGF (50-100 ng/ml) but not basic Fibroblast Growth Factor (FGF) (25-100 ng/ml) rescued tube formation by ECFCs treated with PAR1-activating peptide. Therefore, we propose that reduction of VEGF responsiveness resulting from down-regulation of VEGFR2 is underlying the anti-tubulogenic effect of PAR1 activation. Although the role of PAR2 remains elusive, this study sheds new light on the regulation of the vasculogenic activity of ECFCs and suggests a potential link between adult vasculogenesis and the coagulation cascade. PMID:25289673

  6. Adenosine A3 receptor activation is neuroprotective against retinal neurodegeneration.

    PubMed

    Galvao, Joana; Elvas, Filipe; Martins, Tiago; Cordeiro, M Francesca; Ambrósio, António Francisco; Santiago, Ana Raquel

    2015-11-01

    Death of retinal neural cells, namely retinal ganglion cells (RGCs), is a characteristic of several retinal neurodegenerative diseases. Although the role of adenosine A3 receptor (A3R) in neuroprotection is controversial, A3R activation has been reported to afford protection against several brain insults, with few studies in the retina. In vitro models (retinal neural and organotypic cultures) and animal models [ischemia-reperfusion (I-R) and partial optic nerve transection (pONT)] were used to study the neuroprotective properties of A3R activation against retinal neurodegeneration. The A3R selective agonist (2-Cl-IB-MECA, 1 ?M) prevented apoptosis (TUNEL(+)-cells) induced by kainate and cyclothiazide (KA + CTZ) in retinal neural cultures (86.5 ± 7.4 and 37.2 ± 6.1 TUNEL(+)-cells/field, in KA + CTZ and KA + CTZ + 2-Cl-IB-MECA, respectively). In retinal organotypic cultures, 2-Cl-IB-MECA attenuated NMDA-induced cell death, assessed by TUNEL (17.3 ± 2.3 and 8.3 ± 1.2 TUNEL(+)-cells/mm(2) in NMDA and NMDA+2-Cl-IB-MECA, respectively) and PI incorporation (ratio DIV4/DIV2 3.3 ± 0.3 and 1.3 ± 0.1 in NMDA and NMDA+2-Cl-IB-MECA, respectively) assays. Intravitreal 2-Cl-IB-MECA administration afforded protection against I-R injury decreasing the number of TUNEL(+) cells by 72%, and increased RGC survival by 57%. Also, intravitreal administration of 2-Cl-IB-MECA inhibited apoptosis (from 449.4 ± 37.8 to 207.6 ± 48.9 annexin-V(+)-cells) and RGC loss (from 1.2 ± 0.6 to 8.1 ± 1.7 cells/mm) induced by pONT. This study demonstrates that 2-Cl-IB-MECA is neuroprotective to the retina, both in vitro and in vivo. Activation of A3R may have great potential in the management of retinal neurodegenerative diseases characterized by RGC death, as glaucoma and diabetic retinopathy, and ischemic diseases. PMID:26297614

  7. Neurobiology of Disease Activation of Group III Metabotropic Glutamate Receptors

    E-print Network

    Feng, Jian

    degeneration of nigral dopaminergic (DA) neurons and Parkinson's disease-like symptoms in animal models. Our rotenone-induced parkinsonism. Key words: rotenone; metabotropic glutamate receptor; microtubules; MAP kinase; -arrestin; Src; dynamin Introduction Parkinson's disease (PD) is characterized by several

  8. Fast skeletal muscle troponin I is a co-activator of estrogen receptor-related receptor {alpha}

    SciTech Connect

    Li Yuping; Chen Bin; Chen Jian; Lou Guiyu; Chen Shiuan; Zhou Dujin

    2008-05-16

    ERR{alpha} (estrogen receptor-related receptor {alpha}) is a member of the nuclear receptor superfamily. To further our understanding of the detailed molecular mechanism of transcriptional regulation by ERR{alpha}, we searched for ERR{alpha}-interacting proteins using a yeast two-hybrid system by screening a human mammary gland cDNA expression library with the ligand-binding domain (LBD) of ERR{alpha} as the 'bait'. Fast skeletal muscle troponin I (TNNI2), along with several known nuclear receptor co-activators, were isolated. We demonstrated that TNNI2 localizes to the cell nucleus and interacts with ERR{alpha} in co-immunoprecipitation experiments. GST pull-down assays also revealed that TNNI2 interacts directly with ERR{alpha}. Through luciferase reporter gene assays, TNNI2 was found to enhance the transactivity of ERR{alpha}. Combining mutagenesis and yeast two-hybrid assays, we mapped the ERR{alpha}-interacting domain on TNNI2 to a region encompassing amino acids 1-128. These findings reveal a new function for TNNI2 as a co-activator of ERR{alpha}.

  9. Alternative activation of macrophages and pulmonary fibrosis are modulated by scavenger receptor, macrophage receptor with collagenous structure.

    PubMed

    Murthy, Shubha; Larson-Casey, Jennifer L; Ryan, Alan J; He, Chao; Kobzik, Lester; Carter, A Brent

    2015-08-01

    Alternative activation of alveolar macrophages is linked to fibrosis following exposure to asbestos. The scavenger receptor, macrophage receptor with collagenous structure (MARCO), provides innate immune defense against inhaled particles and pathogens; however, a receptor for asbestos has not been identified. We hypothesized that MARCO acts as an initial signaling receptor for asbestos, polarizes macrophages to a profibrotic M2 phenotype, and is required for the development of asbestos-induced fibrosis. Compared with normal subjects, alveolar macrophages isolated from patients with asbestosis express higher amounts of MARCO and have greater profibrotic polarization. Arginase 1 (40-fold) and IL-10 (265-fold) were higher in patients. In vivo, the genetic deletion of MARCO attenuated the profibrotic environment and pulmonary fibrosis in mice exposed to chrysotile. Moreover, alveolar macrophages from MARCO(-/-) mice polarize to an M1 phenotype, whereas wild-type mice have higher Ym1 (>3.0-fold) and nearly 7-fold more active TGF-?1 in bronchoalveolar lavage (BAL) fluid (BALF). Arg(432) and Arg(434) in domain V of MARCO are required for the polarization of macrophages to a profibrotic phenotype as mutation of these residues reduced FIZZ1 expression (17-fold) compared with cells expressing MARCO. These observations demonstrate that a macrophage membrane protein regulates the fibrotic response to lung injury and suggest a novel target for therapeutic intervention. PMID:25953850

  10. G-protein Coupled Receptor 30 Interacts with Receptor Activity Modifying Protein 3 and Confers Sex-Dependent Cardioprotection

    PubMed Central

    Lenhart, Patricia M.; Broselid, Stefan; Barrick, Cordelia J.; Leeb-Lundberg, L.M. Fredrik; Caron, Kathleen M.

    2013-01-01

    Receptor activity modifying protein 3 (RAMP3) is a single pass transmembrane protein known to interact with and affect the trafficking of several G-protein coupled receptors (GPCRs). We sought to determine whether RAMP3 interacts with G-protein coupled receptor 30 (GPR30), also known as G-protein estrogen receptor 1 (GPER1). GPR30 is a GPCR that binds estradiol and has important roles in cardiovascular and endocrine physiology. Utilizing bioluminescence resonance energy transfer titration studies, co-immunoprecipitation, and confocal microscopy, we show that GPR30 and RAMP3 interact. Furthermore, the presence of GPR30 leads to increased expression of RAMP3 at the plasma membrane in HEK293 cells. In vivo, there are marked sex differences in the subcellular localization of GPR30 in cardiac cells, and the hearts of Ramp3?/? mice also show signs of GPR30 mislocalization. To determine whether this interaction might play a role in cardiovascular disease, we treated Ramp3+/+ and Ramp3?/? mice on a heart disease-prone genetic background with G-1, a specific agonist for GPR30. Importantly, this in vivo activation of GPR30 resulted in a significant reduction in cardiac hypertrophy and perivascular fibrosis that is both RAMP3- and sex-dependent. Our results demonstrate that GPR30-RAMP3 interaction has functional consequences on the localization of these proteins both in vitro and in vivo, and that RAMP3 is required for GPR30-mediated cardioprotection. PMID:23674134

  11. Endomorphin analogues with mixed ?-opioid (MOP) receptor agonism/?-opioid (DOP) receptor antagonism and lacking ?-arrestin2 recruitment activity.

    PubMed

    Cai, Jun; Song, Bowen; Cai, Yunxin; Ma, Yu; Lam, Ai-Leen; Magiera, Julia; Sekar, Sunder; Wyse, Bruce D; Ambo, Akihiro; Sasaki, Yusuke; Lazarus, Lawrence H; Smith, Maree T; Li, Tingyou

    2014-04-01

    Analogues of endomorphin (Dmt-Pro-Xaa-Xaa-NH2) modified at position 4 or at positions 4 and 3, and tripeptides (Dmt-Pro-Xaa-NH2) modified at position 3, with various phenylalanine analogues (Xaa=Trp, 1-Nal, 2-Nal, Tmp, Dmp, Dmt) were synthesized and their effects on in vitro opioid activity were investigated. Most of the peptides exhibited high ?-opioid (MOP) receptor binding affinity (KiMOP=0.13-0.81nM), modest MOP-selectivity (Ki?-opioid (DOP)/KiMOP=3.5-316), and potent functional MOP agonism (GPI, IC50=0.274-249nM) without DOP and ?-opioid (KOP) receptor agonism. Among them, compounds 7 (Dmt-Pro-Tmp-Tmp-NH2) and 9 (Dmt-Pro-1-Nal-NH2) were opioids with potent mixed MOP receptor agonism/DOP receptor antagonism and devoid of ?-arrestin2 recruitment activity. They may offer a unique template for the discovery of potent analgesics that produce less respiratory depression, less gastrointestinal dysfunction and that have a lower propensity to induce tolerance and dependence compared with morphine. PMID:24613457

  12. P2X7 receptor antagonist activity of the anti-allergic agent oxatomide.

    PubMed

    Yoshida, Kazuki; Ito, Masaaki; Matsuoka, Isao

    2015-11-15

    Activation of the P2X7 receptor by extracellular ATP is associated with various immune responses including allergic inflammation. Anti-allergic agents, such as H1-antihistamines, are known to inhibit the effects of different chemical mediators such as acetylcholine and platelet-activating factor. Therefore, we hypothesized that some anti-allergic agents might affect P2X7 receptor function. Using N18TG2 and J774 cells, which express functional P2X7 receptors, the effects of several anti-allergic agents on P2X7 receptor function were investigated by monitoring the ATP-induced increase in intracellular Ca(2+) concentrations ([Ca(2+)]i). Among the various agents tested, oxatomide significantly inhibited P2X7 receptor-mediated [Ca(2+)]i elevation in a concentration-dependent manner without affecting the P2Y2 receptor-mediated response in both N18TG2 and J774 cells. Consistently, oxatomide inhibited P2X7 receptor-mediated membrane current and downstream responses such as mitogen-activated protein kinase activation, inflammation-related gene induction, and cell death. In addition, oxatomide inhibited P2X7 receptor-mediated degranulation in mouse bone marrow-derived mast cells. Whole cell patch clamp analyses in HEK293 cells expressing human, mouse, and rat P2X7 receptors revealed that the inhibitory effect of oxatomide on ATP-induced current was most prominent for the human P2X7 receptor and almost non-existent for the rat P2X7 receptor. The potent inhibitory effects of oxatomide on human P2X7 receptor-mediated function were confirmed in RPMI8226 human B cell-like myeloma cells, which endogenously express the P2X7 receptor. Our results demonstrated that the antihistamine oxatomide also acts as a P2X7 receptor antagonist. Future studies should thus evaluate whether P2X7 receptor antagonism contributes to the anti-allergic effects of oxatomide. PMID:26463039

  13. Single-molecule view of basal activity and activation mechanisms of the G protein-coupled receptor ?2AR.

    PubMed

    Lamichhane, Rajan; Liu, Jeffrey J; Pljevaljcic, Goran; White, Kate L; van der Schans, Edwin; Katritch, Vsevolod; Stevens, Raymond C; Wüthrich, Kurt; Millar, David P

    2015-11-17

    Binding of extracellular ligands to G protein-coupled receptors (GPCRs) initiates transmembrane signaling by inducing conformational changes on the cytoplasmic receptor surface. Knowledge of this process provides a platform for the development of GPCR-targeting drugs. Here, using a site-specific Cy3 fluorescence probe in the human ?2-adrenergic receptor (?2AR), we observed that individual receptor molecules in the native-like environment of phospholipid nanodiscs undergo spontaneous transitions between two distinct conformational states. These states are assigned to inactive and active-like receptor conformations. Individual receptor molecules in the apo form repeatedly sample both conformations, with a bias toward the inactive conformation. Experiments in the presence of drug ligands show that binding of the full agonist formoterol shifts the conformational distribution in favor of the active-like conformation, whereas binding of the inverse agonist ICI-118,551 favors the inactive conformation. Analysis of single-molecule dwell-time distributions for each state reveals that formoterol increases the frequency of activation transitions, while also reducing the frequency of deactivation events. In contrast, the inverse agonist increases the frequency of deactivation transitions. Our observations account for the high level of basal activity of this receptor and provide insights that help to rationalize, on the molecular level, the widely documented variability of the pharmacological efficacies among GPCR-targeting drugs. PMID:26578769

  14. Laminin distribution during corticospinal tract development and after spinal cord injury.

    PubMed

    Sosale, A; Robson, J A; Stelzner, D J

    1988-10-01

    The glycoprotein laminin is a prominent constituent of basal laminae and has been suggested to play an important role in axonal growth. We have tested this hypothesis, by examining the temporal and spatial distribution of laminin in the rat spinal cord, relative to elongating corticospinal tract (CST) axons, during normal development and after newborn and adult spinal lesions. The distribution of laminin was demonstrated in spinal cord sections from animals ranging in age from 14 days embryonic to adult using immunocytochemistry. Anti-laminin immunolabeling was seen around blood vessels and meninges in all the animals examined. However, within the grey and white matter its distribution was age-dependent. In the normal cord, immunostaining appeared in small amounts in early embryos, but was absent from all postnatal animals even at ages when the CST was growing down the cord. Following injury, intense immunostaining was associated with lesions in both newborn and adult operates at all postoperative periods examined. Within the matrix of the lesion laminin immunostaining was especially prominent. In the intact cord it was prominent only around blood vessels near the lesion site. Our results indicate that the distribution of laminin does not closely correlate with axonal growth of the CST either during normal development or after spinal injury. PMID:3053227

  15. Synergistic effect of laminin and mesenchymal stem cells on tracheal mucosal regeneration.

    PubMed

    Lee, Doh Young; Lee, Jin Ho; Ahn, Hee-Jin; Oh, Se Heang; Kim, Tae Ho; Kim, Hee-Bok; Park, Seok-Won; Kwon, Seong Keun

    2015-03-01

    Although several studies have been successfully undertaken of tracheal reconstruction in terms of the maintaining the framework of the graft, most cases of reconstruction failure have resulted from delayed mucosal regeneration. The purposes of this study were to evaluate whether laminin-coated asymmetrically porous membrane (APM) scaffold enhances mucosal regeneration, to compare the mucosalization capability with mesenchymal stem cell (MSC) seeded APM, and to determine whether laminin coating and MSC seeding has a synergistic effect on mucosal regeneration. We reconstructed the full-thickness anterior tracheal defect of 36 New Zealand White rabbits with the APM scaffold. MSCs were isolated from the rabbit's inguinal fat. The animals were divided into 4 groups by the presence of laminin coating on APM and application of MSC [Group I, -/- (laminin/MSC); Group II, -/+; Group III, +/-; Group IV, +/+]. Endoscopy and histologic evaluation were performed and the results were compared among the groups. The results showed that ciliated columnar epithelium was regenerated earlier in groups II and III than in group I. Furthermore, the application of laminin and MSC had synergistic effects on tracheal epithelial regeneration. These results demonstrate that tracheal reconstruction by laminin-coated APM seeded with MSCs is most effective in enhancing tracheal mucosalization, and appears to be promising strategy in the regenerative treatment of tracheal defects. PMID:25617133

  16. The insulin and IGF1 receptor kinase domains are functional dimers in the activated state

    NASA Astrophysics Data System (ADS)

    Cabail, M. Zulema; Li, Shiqing; Lemmon, Eric; Bowen, Mark E.; Hubbard, Stevan R.; Miller, W. Todd

    2015-03-01

    The insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R) are highly related receptor tyrosine kinases with a disulfide-linked homodimeric architecture. Ligand binding to the receptor ectodomain triggers tyrosine autophosphorylation of the cytoplasmic domains, which stimulates catalytic activity and creates recruitment sites for downstream signalling proteins. Whether the two phosphorylated tyrosine kinase domains within the receptor dimer function independently or cooperatively to phosphorylate protein substrates is not known. Here we provide crystallographic, biophysical and biochemical evidence demonstrating that the phosphorylated kinase domains of IR and IGF1R form a specific dimeric arrangement involving an exchange of the juxtamembrane region proximal to the kinase domain. In this dimer, the active position of ?-helix C in the kinase N lobe is stabilized, which promotes downstream substrate phosphorylation. These studies afford a novel strategy for the design of small-molecule IR agonists as potential therapeutic agents for type 2 diabetes.

  17. Activation of Melatonin Receptors Reduces Relapse-Like Alcohol Consumption.

    PubMed

    Vengeliene, Valentina; Noori, Hamid R; Spanagel, Rainer

    2015-12-01

    Melatonin is an endogenous synchronizer of biological rhythms and a modulator of physiological functions and behaviors of all mammals. Reduced levels of melatonin and a delay of its nocturnal peak concentration have been found in alcohol-dependent patients and rats. Here we investigated whether the melatonergic system is a novel target to treat alcohol addiction. Male Wistar rats were subjected to long-term voluntary alcohol consumption with repeated abstinence phases. Circadian drinking rhythmicity and patterns were registered with high temporal resolution by a drinkometer system and analyzed by Fourier analysis. We examined potential antirelapse effect of the novel antidepressant drug agomelatine. Given that agomelatine is a potent MT1 and MT2 receptor agonist and a 5-HT2C antagonist we also tested the effects of melatonin itself and the 5-HT2C antagonist SB242084. All drugs reduced relapse-like drinking. Agomelatine and melatonin administered at the end of the light phase led to very similar changes on all measures of the post-abstinence drinking behavior, suggesting that effects of agomelatine on relapse-like behavior are mostly driven by its melatonergic activity. Both drugs caused a clear phase advance in the diurnal drinking pattern when compared with the control vehicle-treated group and a reduced frequency of approaches to alcohol bottles. Melatonin given at the onset of the light phase had no effect on the circadian phase and very small effects on alcohol consumption. We conclude that targeting the melatonergic system in alcohol-dependent individuals can induce a circadian phase advance, which may restore normal sleep architecture and reduce relapse behavior. PMID:25994077

  18. N-Methyl-D-Aspartate Receptor Activation May Contribute to Glufosinate Neurotoxicity

    EPA Science Inventory

    N-Methyl-D-aspartate Receptor Activation May Contribute to Glufosinate Neurotoxicity Glufosinate (GLF) at high levels in mammals causes convulsions through a mechanism that is not completely understood. The structural similarity of GLF to glutamate (GLU) implicates the glutamate...

  19. Structural basis for chemokine recognition and activation of a viral G protein–coupled receptor

    PubMed Central

    Burg, John S.; Ingram, Jessica R.; Venkatakrishnan, A. J.; Jude, Kevin M.; Dukkipati, Abhiram; Feinberg, Evan N.; Angelini, Alessandro; Waghray, Deepa; Dror, Ron O.; Ploegh, Hidde L.; Garcia, K. Christopher

    2015-01-01

    Chemokines are small proteins that function as immune modulators through activation of chemokine G protein–coupled receptors (GPCRs). Several viruses also encode chemokines and chemokine receptors to subvert the host immune response. How protein ligands activate GPCRs remains unknown. We report the crystal structure at 2.9 angstrom resolution of the human cytomegalovirus GPCR US28 in complex with the chemokine domain of human CX3CL1 (fractalkine). The globular body of CX3CL1 is perched on top of the US28 extracellular vestibule, whereas its amino terminus projects into the central core of US28. The transmembrane helices of US28 adopt an active-state–like conformation. Atomic-level simulations suggest that the agonist-independent activity of US28 may be due to an amino acid network evolved in the viral GPCR to destabilize the receptor’s inactive state. PMID:25745166

  20. Structural basis for chemokine recognition and activation of a viral G protein-coupled receptor

    SciTech Connect

    Burg, John S.; Ingram, Jessica R.; Venkatakrishnan, A.J.; Jude, Kevin M.; Dukkipati, Abhiram; Feinberg, Evan N.; Angelini, Alessandro; Waghray, Deepa; Dror, Ron O.; Ploegh, Hidde L.; Garcia, K. Christopher

    2015-03-05

    Chemokines are small proteins that function as immune modulators through activation of chemokine G protein-coupled receptors (GPCRs). Several viruses also encode chemokines and chemokine receptors to subvert the host immune response. How protein ligands activate GPCRs remains unknown. We report the crystal structure at 2.9 angstrom resolution of the human cytomegalovirus GPCR US28 in complex with the chemokine domain of human CX3CL1 (fractalkine). The globular body of CX3CL1 is perched on top of the US28 extracellular vestibule, whereas its amino terminus projects into the central core of US28. The transmembrane helices of US28 adopt an active-state-like conformation. Atomic-level simulations suggest that the agonist-independent activity of US28 may be due to an amino acid network evolved in the viral GPCR to destabilize the receptor’s inactive state.

  1. Activation of the orphan nuclear receptor steroidogenic factor 1 by?oxysterols

    PubMed Central

    Lala, Deepak S.; Syka, Peter M.; Lazarchik, Steven B.; Mangelsdorf, David J.; Parker, Keith L.; Heyman, Richard A.

    1997-01-01

    Steroidogenic factor 1 (SF-1), an orphan member of the intracellular receptor superfamily, plays an essential role in the development and function of multiple endocrine organs. It is expressed in all steroidogenic tissues where it regulates the P450 steroidogenic genes to generate physiologically active steroids. Although many of the functions of SF-1 in vivo have been defined, an unresolved question is whether a ligand modulates its transcriptional activity. Here, we show that 25-, 26-, or 27-hydroxycholesterol, known suppressors of cholesterol biosynthesis, enhance SF-1-dependent transcriptional activity. This activation is dependent upon the SF-1 activation function domain, and, is specific for SF-1 as several other receptors do not respond to these molecules. The oxysterols activate at concentrations comparable to those previously shown to inhibit cholesterol biosynthesis, and, can be derived from cholesterol by P450c27, an enzyme expressed within steroidogenic tissues. Recent studies have shown that the nuclear receptor LXR also is activated by oxysterols. We demonstrate that different oxysterols differ in their rank order potency for these two receptors, with 25-hydroxycholesterol preferentially activating SF-1 and 22(R)-hydroxycholesterol preferentially activating LXR. These results suggest that specific oxysterols may mediate transcriptional activation via different intracellular receptors. Finally, ligand-dependent transactivation of SF-1 by oxysterols may play an important role in enhancing steroidogenesis in vivo. PMID:9144161

  2. Effects of histamine H1 receptor signaling on glucocorticoid receptor activity. Role of canonical and non-canonical pathways

    PubMed Central

    Zappia, Carlos Daniel; Granja-Galeano, Gina; Fernández, Natalia; Shayo, Carina; Davio, Carlos; Fitzsimons, Carlos P.; Monczor, Federico

    2015-01-01

    Histamine H1 receptor (H1R) antagonists and glucocorticoid receptor (GR) agonists are used to treat inflammatory conditions such as allergic rhinitis, atopic dermatitis and asthma. Consistent with the high morbidity levels of such inflammatory conditions, these receptors are the targets of a vast number of approved drugs, and in many situations their ligands are co-administered. However, this drug association has no clear rationale and has arisen from clinical practice. We hypothesized that H1R signaling could affect GR-mediated activity, impacting on its transcriptional outcome. Indeed, our results show a dual regulation of GR activity by the H1R: a potentiation mediated by G-protein ?? subunits and a parallel inhibitory effect mediated by G?q-PLC pathway. Activation of the H1R by its full agonists resulted in a composite potentiating effect. Intriguingly, inactivation of the G?q-PLC pathway by H1R inverse agonists resulted also in a potentiation of GR activity. Moreover, histamine and clinically relevant antihistamines synergized with the GR agonist dexamethasone to induce gene transactivation and transrepression in a gene-specific manner. Our work provides a delineation of molecular mechanisms underlying the widespread clinical association of antihistamines and GR agonists, which may contribute to future dosage optimization and reduction of well-described side effects associated with glucocorticoid administration. PMID:26635083

  3. Effects of histamine H1 receptor signaling on glucocorticoid receptor activity. Role of canonical and non-canonical pathways.

    PubMed

    Zappia, Carlos Daniel; Granja-Galeano, Gina; Fernández, Natalia; Shayo, Carina; Davio, Carlos; Fitzsimons, Carlos P; Monczor, Federico

    2015-01-01

    Histamine H1 receptor (H1R) antagonists and glucocorticoid receptor (GR) agonists are used to treat inflammatory conditions such as allergic rhinitis, atopic dermatitis and asthma. Consistent with the high morbidity levels of such inflammatory conditions, these receptors are the targets of a vast number of approved drugs, and in many situations their ligands are co-administered. However, this drug association has no clear rationale and has arisen from clinical practice. We hypothesized that H1R signaling could affect GR-mediated activity, impacting on its transcriptional outcome. Indeed, our results show a dual regulation of GR activity by the H1R: a potentiation mediated by G-protein ?? subunits and a parallel inhibitory effect mediated by G?q-PLC pathway. Activation of the H1R by its full agonists resulted in a composite potentiating effect. Intriguingly, inactivation of the G?q-PLC pathway by H1R inverse agonists resulted also in a potentiation of GR activity. Moreover, histamine and clinically relevant antihistamines synergized with the GR agonist dexamethasone to induce gene transactivation and transrepression in a gene-specific manner. Our work provides a delineation of molecular mechanisms underlying the widespread clinical association of antihistamines and GR agonists, which may contribute to future dosage optimization and reduction of well-described side effects associated with glucocorticoid administration. PMID:26635083

  4. Autoantibody-mediated complement C3a receptor activation contributes to the pathogenesis of preeclampsia.

    PubMed

    Wang, Wei; Irani, Roxanna A; Zhang, Yujin; Ramin, Susan M; Blackwell, Sean C; Tao, Lijian; Kellems, Rodney E; Xia, Yang

    2012-09-01

    Preeclampsia (PE) is a prevalent life-threatening hypertensive disorder of pregnancy associated with increased complement activation. However, the causative factors and pathogenic role of increased complement activation in PE are largely unidentified. Here we report that a circulating maternal autoantibody, the angiotensin II type 1 receptor agonistic autoantibody, which emerged recently as a potential pathogenic contributor to PE, stimulates deposition of complement C3 in placentas and kidneys of pregnant mice via angiotensin II type 1 receptor activation. Next, we provide in vivo evidence that selectively interfering with C3a signaling by a complement C3a receptor-specific antagonist significantly reduces hypertension from 167±7 to 143±5 mm Hg and proteinuria from 223.5±7.5 to 78.8±14.0 ?g of albumin per milligram creatinine (both P<0.05) in angiotensin II type 1 receptor agonistic autoantibody-injected pregnant mice. In addition, we demonstrated that complement C3a receptor antagonist significantly inhibited autoantibody-induced circulating soluble fms-like tyrosine kinase 1, a known antiangiogenic protein associated with PE, and reduced small placental size with impaired angiogenesis and intrauterine growth restriction. Similarly, in humans, we demonstrate that C3 deposition is significantly elevated in the placentas of preeclamptic patients compared with normotensive controls. Lastly, we show that complement C3a receptor activation is a key mechanism underlying autoantibody-induced soluble fms-like tyrosine kinase 1 secretion and decreased angiogenesis in cultured human villous explants. Overall, we provide mouse and human evidence that angiotensin II type 1 receptor agonistic autoantibody-mediated activation contributes to elevated C3 and that complement C3a receptor signaling is a key mechanism underlying the pathogenesis of the disease. These studies are the first to link angiotensin II type 1 receptor agonistic autoantibody with complement activation and to provide important new opportunities for therapeutic intervention in PE. PMID:22868393

  5. Identification of Modulators of the Nuclear Receptor Peroxisome Proliferator-Activated Receptor ? (PPAR?) in a Mouse Liver Gene Expression Compendium

    PubMed Central

    Oshida, Keiyu; Vasani, Naresh; Thomas, Russell S.; Applegate, Dawn; Rosen, Mitch; Abbott, Barbara; Lau, Christopher; Guo, Grace; Aleksunes, Lauren M.; Klaassen, Curtis; Corton, J. Christopher

    2015-01-01

    The nuclear receptor family member peroxisome proliferator-activated receptor ? (PPAR?) is activated by therapeutic hypolipidemic drugs and environmentally-relevant chemicals to regulate genes involved in lipid transport and catabolism. Chronic activation of PPAR? in rodents increases liver cancer incidence, whereas suppression of PPAR? activity leads to hepatocellular steatosis. Analytical approaches were developed to identify biosets (i.e., gene expression differences between two conditions) in a genomic database in which PPAR? activity was altered. A gene expression signature of 131 PPAR?-dependent genes was built using microarray profiles from the livers of wild-type and PPAR?-null mice after exposure to three structurally diverse PPAR? activators (WY-14,643, fenofibrate and perfluorohexane sulfonate). A fold-change rank-based test (Running Fisher’s test (p-value ? 10-4)) was used to evaluate the similarity between the PPAR? signature and a test set of 48 and 31 biosets positive or negative, respectively for PPAR? activation; the test resulted in a balanced accuracy of 98%. The signature was then used to identify factors that activate or suppress PPAR? in an annotated mouse liver/primary hepatocyte gene expression compendium of ~1850 biosets. In addition to the expected activation of PPAR? by fibrate drugs, di(2-ethylhexyl) phthalate, and perfluorinated compounds, PPAR? was activated by benzofuran, galactosamine, and TCDD and suppressed by hepatotoxins acetaminophen, lipopolysaccharide, silicon dioxide nanoparticles, and trovafloxacin. Additional factors that activate (fasting, caloric restriction) or suppress (infections) PPAR? were also identified. This study 1) developed methods useful for future screening of environmental chemicals, 2) identified chemicals that activate or suppress PPAR?, and 3) identified factors including diets and infections that modulate PPAR? activity and would be hypothesized to affect chemical-induced PPAR? activity.

  6. Activation of protease-activated receptor 2 reduces glioblastoma cell apoptosis

    PubMed Central

    2014-01-01

    Background The pathogenesis of glioma is unclear. The disturbance of the apoptosis process plays a critical role in glioma growth. Factors regulating the apoptosis process are to be further understood. This study aims to investigate the role of protease activated receptor-2 (PAR2) in regulation the apoptosis process in glioma cells. Results The results showed that U87 cells and human glioma tissue expressed PAR2. Exposure to tryptase, or the PAR2 active peptide, increased STAT3 phosphorylation in the radiated U87 cells, reduced U87 cell apoptosis, suppressed the expression of p53 in U87 cells. Conclusions Activation of PAR2 can reduce the radiated U87 cell apoptosis via modulating the expression of p53. The results implicate that PAR2 may be a novel therapeutic target in the treatment of glioma. PMID:24670244

  7. Thrombin stimulates fibroblast procollagen production via proteolytic activation of protease-activated receptor 1.

    PubMed Central

    Chambers, R C; Dabbagh, K; McAnulty, R J; Gray, A J; Blanc-Brude, O P; Laurent, G J

    1998-01-01

    Thrombin is a multifunctional serine protease that has a crucial role in blood coagulation. It is also a potent mesenchymal cell mitogen and chemoattractant and might therefore have an important role in the recruitment and local proliferation of mesenchymal cells at sites of tissue injury. We hypothesized that thrombin might also affect the deposition of connective tissue proteins at these sites by directly stimulating fibroblast procollagen production. To address this hypothesis, the effect of thrombin on procollagen production and gene expression by human foetal lung fibroblasts was assessed over 48 h. Thrombin stimulated procollagen production at concentrations of 1 nM and above, with maximal increases of between 60% and 117% at 10 nM thrombin. These effects of thrombin were, at least in part, due to increased steady-state levels of alpha1(I) procollagen mRNA. They could furthermore be reproduced with thrombin receptor-activating peptides for the protease-activated receptor 1 (PAR-1) and were completely abolished when thrombin was rendered proteolytically inactive with the specific inhibitors d-Phe-Pro-ArgCH2Cl and hirudin, indicating that thrombin is mediating these effects via the proteolytic activation of PAR-1. These results suggest that thrombin might influence the deposition of connective tissue proteins during normal wound healing and the development of tissue fibrosis by stimulating fibroblast procollagen production. PMID:9639571

  8. Peroxisome proliferator-activated receptor-delta agonist ameliorated inflammasome activation in nonalcoholic fatty liver disease

    PubMed Central

    Lee, Hyun Jung; Yeon, Jong Eun; Ko, Eun Jung; Yoon, Eileen L; Suh, Sang Jun; Kang, Keunhee; Kim, Hae Rim; Kang, Seoung Hee; Yoo, Yang Jae; Je, Jihye; Lee, Beom Jae; Kim, Ji Hoon; Seo, Yeon Seok; Yim, Hyung Joon; Byun, Kwan Soo

    2015-01-01

    AIM: To evaluate the inflammasome activation and the effect of peroxisome proliferator-activated receptors (PPAR)-? agonist treatment in nonalcoholic fatty liver disease (NAFLD) models. METHODS: Male C57BL/6J mice were classified according to control or high fat diet (HFD) with or without PPAR-? agonist (GW) over period of 12 wk [control, HFD, HFD + lipopolysaccharide (LPS), HFD + LPS + GW group]. HepG2 cells were exposed to palmitic acid (PA) and/or LPS in the absence or presence of GW. RESULTS: HFD caused glucose intolerance and hepatic steatosis. In mice fed an HFD with LPS, caspase-1 and interleukin (IL)-1? in the liver were significantly increased. Treatment with GW ameliorated the steatosis and inhibited overexpression of pro-inflammatory cytokines. In HepG2 cells, PA and LPS treatment markedly increased mRNA of several nucleotide-binding and oligomerization domain-like receptor family members (NLRP3, NLRP6, and NLRP10), caspase-1 and IL-1?. PA and LPS also exaggerated reactive oxygen species production. All of the above effects of PA and LPS were reduced by GW. GW also enhanced the phosphorylation of AMPK-?. CONCLUSION: PPAR-? agonist reduces fatty acid-induced inflammation and steatosis by suppressing inflammasome activation. Targeting the inflammasome by the PPAR-? agonist may have therapeutic implication for NAFLD. PMID:26668503

  9. Activation of Transient Receptor Potential Vanilloid 4 Increases NMDA-Activated Current in Hippocampal Pyramidal Neurons

    PubMed Central

    Li, Lin; Qu, Weijun; Zhou, Libin; Lu, Zihong; Jie, Pinghui; Chen, Lei; Chen, Ling

    2013-01-01

    The glutamate excitotoxicity, mediated through N-methyl-d-aspartate receptors (NMDARs), plays an important role in cerebral ischemia injury. Transient receptor potential vanilloid 4 (TRPV4) can be activated by multiple stimuli that may happen during stroke. The present study evaluated the effect of TRPV4 activation on NMDA-activated current (INMDA) and that of blocking TRPV4 on brain injury after focal cerebral ischemia in mice. We herein report that activation of TRPV4 by 4?-PDD and hypotonic stimulation increased INMDA in hippocampal CA1 pyramidal neurons, which was sensitive to TRPV4 antagonist HC-067047 and NMDAR antagonist AP-5, indicating that TRPV4 activation potentiates NMDAR response. In addition, the increase in INMDA by hypotonicity was sensitive to the antagonist of NMDAR NR2B subunit, but not of NR2A subunit. Furthermore, antagonists of calcium/calmodulin-dependent protein kinase II (CaMKII) significantly attenuated hypotonicity-induced increase in INMDA, while antagonists of protein kinase C or casein kinase II had no such effect, indicating that phosphorylation of NR2B subunit by CaMKII is responsible for TRPV4-potentiated NMDAR response. Finally, we found that intracerebroventricular injection of HC-067047 after 60?min middle cerebral artery occlusion reduced the cerebral infarction with at least a 12?h efficacious time-window. These findings indicate that activation of TRPV4 increases NMDAR function, which may facilitate glutamate excitotoxicity. Closing TRPV4 may exert potent neuroprotection against cerebral ischemia injury through many mechanisms at least including the prevention of NMDAR-mediated glutamate excitotoxicity. PMID:23459987

  10. Dura-evoked neck muscle activity involves purinergic and N-methyl-D-aspartate receptor mechanisms.

    PubMed

    Yao, Dongyuan; Yoshida, Mitsuhiro; Sessle, Barry J

    2015-12-16

    We have previously demonstrated that noxious stimulation of craniofacial tissues including the frontal dura reflexly evokes significant increases in neck muscle electromyographic (EMG) activity. The primary aim of this study was to determine whether purinergic receptor mechanisms may be involved in these EMG effects, and whether N-methyl-D-aspartate (NMDA) receptor processes modulate the purinergic mechanisms. Application of the P2X1, P2X3 and P2X2/3 receptor agonist ?,?-methylene ATP (but not vehicle) to the dural surface evoked a significant (P<0.05) increase in ipsilateral neck EMG activity that could be suppressed by dural or intrathecal application of the selective P2X1, P2X3 and P2X2/3 receptor antagonist 2',3'-O-(2,4,6-trinitrophenyl) ATP (TNP-ATP) but not by vehicle; the intrathecal application of 2-amino-5-phosphonopentanoic acid, an NMDA receptor antagonist, also significantly reduced the neck EMG activity evoked by dural application of ?,?-methylene ATP. These data suggest that purinergic receptor mechanisms contribute to the increased neck activity that can be reflexly evoked by noxious stimulation of the frontal dura, and that NMDA as well as purinergic receptor mechanisms in the medulla may modulate these purinergic-related effects. PMID:26559728

  11. Determinants of coactivator LXXLL motif specificity in nuclear receptor transcriptional activation.

    PubMed

    McInerney, E M; Rose, D W; Flynn, S E; Westin, S; Mullen, T M; Krones, A; Inostroza, J; Torchia, J; Nolte, R T; Assa-Munt, N; Milburn, M V; Glass, C K; Rosenfeld, M G

    1998-11-01

    Ligand-dependent activation of gene transcription by nuclear receptors is dependent on the recruitment of coactivators, including a family of related NCoA/SRC factors, via a region containing three helical domains sharing an LXXLL core consensus sequence, referred to as LXDs. In this manuscript, we report receptor-specific differential utilization of LXXLL-containing motifs of the NCoA-1/SRC-1 coactivator. Whereas a single LXD is sufficient for activation by the estrogen receptor, different combinations of two, appropriately spaced, LXDs are required for actions of the thyroid hormone, retinoic acid, peroxisome proliferator-activated, or progesterone receptors. The specificity of LXD usage in the cell appears to be dictated, at least in part, by specific amino acids carboxy-terminal to the core LXXLL motif that may make differential contacts with helices 1 and 3 (or 3') in receptor ligand-binding domains. Intriguingly, distinct carboxy-terminal amino acids are required for PPARgamma activation in response to different ligands. Related LXXLL-containing motifs in NCoA-1/SRC-1 are also required for a functional interaction with CBP, potentially interacting with a hydrophobic binding pocket. Together, these data suggest that the LXXLL-containing motifs have evolved to serve overlapping roles that are likely to permit both receptor-specific and ligand-specific assembly of a coactivator complex, and that these recognition motifs underlie the recruitment of coactivator complexes required for nuclear receptor function. PMID:9808623

  12. Determinants of coactivator LXXLL motif specificity in nuclear receptor transcriptional activation

    PubMed Central

    McInerney, Eileen M.; Rose, David W.; Flynn, Sarah E.; Westin, Stefan; Mullen, Tina-Marie; Krones, Anna; Inostroza, Juan; Torchia, Joseph; Nolte, Robert T.; Assa-Munt, Nuria; Milburn, Michael V.; Glass, Christopher K.; Rosenfeld, Michael G.

    1998-01-01

    Ligand-dependent activation of gene transcription by nuclear receptors is dependent on the recruitment of coactivators, including a family of related NCoA/SRC factors, via a region containing three helical domains sharing an LXXLL core consensus sequence, referred to as LXDs. In this manuscript, we report receptor-specific differential utilization of LXXLL-containing motifs of the NCoA-1/SRC-1 coactivator. Whereas a single LXD is sufficient for activation by the estrogen receptor, different combinations of two, appropriately spaced, LXDs are required for actions of the thyroid hormone, retinoic acid, peroxisome proliferator-activated, or progesterone receptors. The specificity of LXD usage in the cell appears to be dictated, at least in part, by specific amino acids carboxy-terminal to the core LXXLL motif that may make differential contacts with helices 1 and 3 (or 3?) in receptor ligand-binding domains. Intriguingly, distinct carboxy-terminal amino acids are required for PPAR? activation in response to different ligands. Related LXXLL-containing motifs in NCoA-1/SRC-1 are also required for a functional interaction with CBP, potentially interacting with a hydrophobic binding pocket. Together, these data suggest that the LXXLL-containing motifs have evolved to serve overlapping roles that are likely to permit both receptor-specific and ligand-specific assembly of a coactivator complex, and that these recognition motifs underlie the recruitment of coactivator complexes required for nuclear receptor function. PMID:9808623

  13. Oestradiol stimulates tyrosine phosphorylation and hormone binding activity of its own receptor in a cell-free system.

    PubMed Central

    Auricchio, F; Migliaccio, A; Di Domenico, M; Nola, E

    1987-01-01

    Recent experiments have shown that calf uterus oestrogen receptor exists in a tyrosine-phosphorylated hormone binding form and in non-phosphorylated, non-hormone binding form. We report here that physiological concentrations of oestradiol in complex with the receptor stimulate the calf uterus receptor kinase that converts the non-hormone binding receptor into hormone binding receptor through phosphorylation of the receptor on tyrosine. The activity of this enzyme has been followed by reactivation of hormone binding sites and phosphorylation on tyrosine of calf uterus phosphatase-inactivated receptor. Phosphorylation of the receptor has been demonstrated by interaction of kinase 32P-phosphorylated proteins with anti-receptor antibody followed either by sucrose gradient centrifugation or SDS-PAGE of the immunoprecipitated proteins. Hormone stimulation of the kinase is inhibited by receptor occupancy of the anti-oestrogen tamoxifen. Oestradiol-receptor complex increases the affinity of the kinase for the dephosphorylated receptor. Findings of this report are consistent with the observation that several protein tyrosine kinases that are associated with peptide hormone receptors are stimulated by the binding of the hormone to the receptor. This is the first report on the activation of a tyrosine kinase by a steroid hormone. The finding that hormones can regulate their own receptor binding activity through a tyrosine kinase is also new. Images Fig. 2. Fig. 4. Fig. 5. PMID:3691476

  14. Shedding of tumor necrosis factor receptors by activated human neutrophils

    PubMed Central

    1990-01-01

    The capacity of human neutrophils (PMN) to bind tumor necrosis factor (TNF) was rapidly lost when the cells were incubated in suspension with agents that can stimulate their migratory and secretory responses. Both physiological (poly)peptides (FMLP, C5a, CSF-GM) and pharmacologic agonists (PMN, calcium ionophore A23187) induced the loss of TNF receptors (TNF-R) from the cell surface. Half-maximal loss in TNF-R ensued after only approximately 2 min with 10(-7) M FMLP at 37 degrees C, and required only 10(-9) M FMLP during a 30-min exposure. However, there were no such changes even with prolonged exposure of PMN to FMLP at 4 degrees or 16 degrees C. Scatchard analysis revealed loss of TNF- binding sites without change in their affinity (Kd approximately 0.4 nM) as measured at incompletely modulating concentrations of FMLP, C5a, PMA, or A23187. The binding of anti-TNF-R mAbs to PMN decreased in parallel, providing independent evidence for the loss of TNF-R from the cell surface. At the same time, soluble TNF-R appeared in the medium of stimulated PMN. This inference was based on the PMN- and FMLP-dependent generation of a nonsedimentable activity that could inhibit the binding of TNF to fresh human PMN or to mouse macrophages, and the ability of mAbs specific for human TNF-R to abolish inhibition by PMN-conditioned medium of binding of TNF to mouse macrophages. Soluble TNF-R activity was associated with a protein of Mr approximately 28,000 by ligand blot analysis of cell-free supernatants of FMLP-treated PMN. Thus, some portion of the FMLP-induced loss of TNF-R from human PMN is due to shedding of TNF-R. Shedding was unaffected by inhibitors of serine and thiol proteases and could not be induced with phosphatidylinositol- specific phospholipase C. Loss of TNF-R from PMN first stimulated by other agents may decrease their responsiveness to TNF. TNF-R shed by PMN may be one source of the TNF-binding proteins found in body fluids, and may blunt the actions of the cytokine on other cells. PMID:2165128

  15. Evaluation of serum procollagen aminoterminal propeptide III, laminin, and hydroxyproline as predictors of severe fibrosis in patients with chronic hepatitis C.

    PubMed

    Attallah, A M; Toson, E A; Shiha, G E; Omran, M M; Abdel-Aziz, M M; El-Dosoky, I

    2007-01-01

    In an attempt to identify biochemical analytes that could enhance the discrimination between the patients with severe liver fibrosis (F3-F4) and mild fibrosis (F1-F2) based on absolute values of biochemical markers, we measured 12 analytes, including procollagen III aminoterminal propeptide (PIIINP), laminin, proline, hydroxylproline, glycine, AST, ALT, alkaline phosphatase, albumin, total bilirubin, total protein, and prothrombin time in 252 individuals with chronic hepatitis C infection (CHC). PIIINP and laminin were determined by radio-immunoassay; the degraded amino acids were determined using high performance liquid chromatography. Statistical analyses were performed by logistic regression, and receiver operating characteristic (ROC) curves. The best linear combination of blood markers was selected by multivariate discriminant analysis (MDA) for construction of the fibrosis discriminant score (FDS). FDS, an index of five markers (PIIINP, laminin, hydroxyproline, prothrombin activity, and AST/ALT) correctly classified 82% of the patients with severe liver fibrosis at a discriminant cut-off score=-0.5 (i.e., less than -0.5 indicated severe liver fibrosis and greater than -0.5 indicated mild liver fibrosis with sensitivity (76%) and specificity (89%). This result was reproduced in a validation study with no significant difference. In conclusion, FDS is useful for identifying severe liver fibrosis in patients with CHC. PMID:17613667

  16. Genotype-Dependent Difference in 5-HT2C Receptor-Induced Hypolocomotion: Comparison with 5-HT2A Receptor Functional Activity.

    PubMed

    Bazovkina, Darya V; Kondaurova, Elena M; Naumenko, Vladimir S; Ponimaskin, Evgeni

    2015-01-01

    In the present study behavioral effects of the 5-HT2C serotonin receptor were investigated in different mouse strains. The 5-HT2C receptor agonist MK-212 applied intraperitoneally induced significant dose-dependent reduction of distance traveled in the open field test in CBA/Lac mice. This effect was receptor-specific because it was inhibited by the 5-HT2C receptor antagonist RS102221. To study the role of genotype in 5-HT2C receptor-induced hypolocomotion, locomotor activity of seven inbred mouse strains was measured after MK-212 acute treatment. We found that the 5-HT2C receptor stimulation by MK-212 decreased distance traveled in the open field test in CBA/Lac, C57Bl/6, C3H/He, and ICR mice, whereas it failed to affect locomotor activity in DBA/2J, Asn, and Balb/c mice. We also compared the interstrain differences in functional response to 5-HT2C and 5-HT2A receptors activation measured by the quantification of receptor-mediated head-twitches. These experiments revealed significant positive correlation between 5-HT2C and 5-HT2A receptors functional responses for all investigated mouse strains. Moreover, we found that 5-HT2A receptor activation with DOI did not change locomotor activity in CBA/Lac mice. Taken together, our data indicate the implication of 5-HT2C receptors in regulation of locomotor activity and suggest the shared mechanism for functional responses mediated by 5-HT2C and 5-HT2A receptors. PMID:26380122

  17. Genotype-Dependent Difference in 5-HT2C Receptor-Induced Hypolocomotion: Comparison with 5-HT2A Receptor Functional Activity

    PubMed Central

    Bazovkina, Darya V.; Kondaurova, Elena M.; Naumenko, Vladimir S.; Ponimaskin, Evgeni

    2015-01-01

    In the present study behavioral effects of the 5-HT2C serotonin receptor were investigated in different mouse strains. The 5-HT2C receptor agonist MK-212 applied intraperitoneally induced significant dose-dependent reduction of distance traveled in the open field test in CBA/Lac mice. This effect was receptor-specific because it was inhibited by the 5-HT2C receptor antagonist RS102221. To study the role of genotype in 5-HT2C receptor-induced hypolocomotion, locomotor activity of seven inbred mouse strains was measured after MK-212 acute treatment. We found that the 5-HT2C receptor stimulation by MK-212 decreased distance traveled in the open field test in CBA/Lac, C57Bl/6, C3H/He, and ICR mice, whereas it failed to affect locomotor activity in DBA/2J, Asn, and Balb/c mice. We also compared the interstrain differences in functional response to 5-HT2C and 5-HT2A receptors activation measured by the quantification of receptor-mediated head-twitches. These experiments revealed significant positive correlation between 5-HT2C and 5-HT2A receptors functional responses for all investigated mouse strains. Moreover, we found that 5-HT2A receptor activation with DOI did not change locomotor activity in CBA/Lac mice. Taken together, our data indicate the implication of 5-HT2C receptors in regulation of locomotor activity and suggest the shared mechanism for functional responses mediated by 5-HT2C and 5-HT2A receptors. PMID:26380122

  18. Structure-activity relationships of fatty acid amide ligands in activating and desensitizing G protein-coupled receptor 119.

    PubMed

    Kumar, Pritesh; Kumar, Akhilesh; Song, Zhao-Hui

    2014-01-15

    The purpose of the current study was to apply a high throughput assay to investigate the structure-activity relationships of fatty acid amides for activating and desensitizing G protein-coupled receptor 119, a promising therapeutic target for both type 2 diabetes and obesity. A cell-based, homogenous time resolved fluorescence (HTRF) method for measuring G protein-coupled receptor 119-mediated increase of cyclic adenosine monophosphate (cAMP) levels was validated and applied in this study. Using novel fatty acid amides and detailed potency and efficacy analyses, we have demonstrated that degree of saturation in acyl chain and charged head groups of fatty acid amides have profound effects on the ability of these compounds to activate G protein-coupled receptor 119. In addition, we have demonstrated for the first time that pretreatments with G protein-coupled receptor 119 agonists desensitize the receptor and the degrees of desensitization caused by fatty acid amides correlate well with their structure-activity relationships in activating the receptor. PMID:24184668

  19. Functional Selectivity and Antidepressant Activity of Serotonin 1A Receptor Ligands

    PubMed Central

    Chilmonczyk, Zdzis?aw; Bojarski, Andrzej Jacek; Pilc, Andrzej; Sylte, Ingebrigt

    2015-01-01

    Serotonin (5-HT) is a monoamine neurotransmitter that plays an important role in physiological functions. 5-HT has been implicated in sleep, feeding, sexual behavior, temperature regulation, pain, and cognition as well as in pathological states including disorders connected to mood, anxiety, psychosis and pain. 5-HT1A receptors have for a long time been considered as an interesting target for the action of antidepressant drugs. It was postulated that postsynaptic 5-HT1A agonists could form a new class of antidepressant drugs, and mixed 5-HT1A receptor ligands/serotonin transporter (SERT) inhibitors seem to possess an interesting pharmacological profile. It should, however, be noted that 5-HT1A receptors can activate several different biochemical pathways and signal through both G protein-dependent and G protein-independent pathways. The variables that affect the multiplicity of 5-HT1A receptor signaling pathways would thus result from the summation of effects specific to the host cell milieu. Moreover, receptor trafficking appears different at pre- and postsynaptic sites. It should also be noted that the 5-HT1A receptor cooperates with other signal transduction systems (like the 5-HT1B or 5-HT2A/2B/2C receptors, the GABAergic and the glutaminergic systems), which also contribute to its antidepressant and/or anxiolytic activity. Thus identifying brain specific molecular targets for 5-HT1A receptor ligands may result in a better targeting, raising a hope for more effective medicines for various pathologies. PMID:26262615

  20. Functional Selectivity and Antidepressant Activity of Serotonin 1A Receptor Ligands.

    PubMed

    Chilmonczyk, Zdzis?aw; Bojarski, Andrzej Jacek; Pilc, Andrzej; Sylte, Ingebrigt

    2015-01-01

    Serotonin (5-HT) is a monoamine neurotransmitter that plays an important role in physiological functions. 5-HT has been implicated in sleep, feeding, sexual behavior, temperature regulation, pain, and cognition as well as in pathological states including disorders connected to mood, anxiety, psychosis and pain. 5-HT1A receptors have for a long time been considered as an interesting target for the action of antidepressant drugs. It was postulated that postsynaptic 5-HT1A agonists could form a new class of antidepressant drugs, and mixed 5-HT1A receptor ligands/serotonin transporter (SERT) inhibitors seem to possess an interesting pharmacological profile. It should, however, be noted that 5-HT1A receptors can activate several different biochemical pathways and signal through both G protein-dependent and G protein-independent pathways. The variables that affect the multiplicity of 5-HT1A receptor signaling pathways would thus result from the summation of effects specific to the host cell milieu. Moreover, receptor trafficking appears different at pre- and postsynaptic sites. It should also be noted that the 5-HT1A receptor cooperates with other signal transduction systems (like the 5-HT1B or 5-HT2A/2B/2C receptors, the GABAergic and the glutaminergic systems), which also contribute to its antidepressant and/or anxiolytic activity. Thus identifying brain specific molecular targets for 5-HT1A receptor ligands may result in a better targeting, raising a hope for more effective medicines for various pathologies. PMID:26262615

  1. EPO-independent functional EPO receptor in breast cancer enhances estrogen receptor activity and promotes cell proliferation

    SciTech Connect

    Reinbothe, Susann; Larsson, Anna-Maria; Vaapil, Marica; Wigerup, Caroline; Sun, Jianmin; Jögi, Annika; Neumann, Drorit; Rönnstrand, Lars; Pĺhlman, Sven

    2014-02-28

    Highlights: • New anti-human EPOR antibody confirms full-length EPOR expression in breast cancer cells. • Proliferation of breast cancer cells is not affected by rhEPO treatment in vitro. • EPOR knockdown impairs proliferation of ERa positive breast cancer cells. • EPOR knockdown reduces AKT phosphorylation and ERa activity. - Abstract: The main function of Erythropoietin (EPO) and its receptor (EPOR) is the stimulation of erythropoiesis. Recombinant human EPO (rhEPO) is therefore used to treat anemia in cancer patients. However, clinical trials have indicated that rhEPO treatment might promote tumor progression and has a negative effect on patient survival. In addition, EPOR expression has been detected in several cancer forms. Using a newly produced anti-EPOR antibody that reliably detects the full-length isoform of the EPOR we show that breast cancer tissue and cells express the EPOR protein. rhEPO stimulation of cultured EPOR expressing breast cancer cells did not result in increased proliferation, overt activation of EPOR (receptor phosphorylation) or a consistent activation of canonical EPOR signaling pathway mediators such as JAK2, STAT3, STAT5, or AKT. However, EPOR knockdown experiments suggested functional EPO receptors in estrogen receptor positive (ER?{sup +}) breast cancer cells, as reduced EPOR expression resulted in decreased proliferation. This effect on proliferation was not seen in ER? negative cells. EPOR knockdown decreased ER? activity further supports a mechanism by which EPOR affects proliferation via ER?-mediated mechanisms. We show that EPOR protein is expressed in breast cancer cells, where it appears to promote proliferation by an EPO-independent mechanism in ER? expressing breast cancer cells.

  2. Selective Activation of G o by D2L Dopamine Receptors in NS20Y Neuroblastoma Cells

    E-print Network

    Neve, Kim A.

    Selective Activation of G o by D2L Dopamine Receptors in NS20Y Neuroblastoma Cells Val J. Watts,1 receptor was stably transfected into NS20Y neuroblastoma cells to examine inhibi- tion and sensitization

  3. Selective androgen receptor modulator activity of a steroidal antiandrogen TSAA-291 and its cofactor recruitment profile.

    PubMed

    Hikichi, Yukiko; Yamaoka, Masuo; Kusaka, Masami; Hara, Takahito

    2015-10-15

    Selective androgen receptor modulators (SARMs) specifically bind to the androgen receptor and exert agonistic or antagonistic effects on target organs. In this study, we investigated the SARM activity of TSAA-291, previously known as a steroidal antiandrogen, in mice because TSAA-291 was found to possess partial androgen receptor agonist activity in reporter assays. In addition, to clarify the mechanism underlying its tissue selectivity, we performed comprehensive cofactor recruitment analysis of androgen receptor using TSAA-291 and dihydrotestosterone (DHT), an endogenous androgen. The androgen receptor agonistic activity of TSAA-291 was more obvious in reporter assays using skeletal muscle cells than in those using prostate cells. In castrated mice, TSAA-291 increased the weight of the levator ani muscle without increasing the weight of the prostate and seminal vesicle. Comprehensive cofactor recruitment analysis via mammalian two-hybrid methods revealed that among a total of 112 cofactors, 12 cofactors including the protein inhibitor of activated STAT 1 (PIAS1) were differently recruited to androgen receptor in the presence of TSAA-291 and DHT. Prostate displayed higher PIAS1 expression than skeletal muscle. Forced expression of the PIAS1 augmented the transcriptional activity of the androgen receptor, and silencing of PIAS1 by siRNAs suppressed the secretion of prostate-specific antigen, an androgen responsive marker. Our results demonstrate that TSAA-291 has SARM activity and suggest that TSAA-291 may induce different conformational changes of the androgen receptor and recruitment profiles of cofactors such as PIAS1, compared with DHT, to exert tissue-specific activity. PMID:26335395

  4. Characterization of the single transmembrane domain of human receptor activity-modifying protein 3 in adrenomedullin receptor internalization

    SciTech Connect

    Kuwasako, Kenji; Kitamura, Kazuo; Nagata, Sayaka; Nozaki, Naomi; Kato, Johji

    2012-04-13

    Highlights: Black-Right-Pointing-Pointer RAMP3 mediates CLR internalization much less effectively than does RAMP2. Black-Right-Pointing-Pointer The RAMP3 TMD participates in the negative regulation of CLR/RAMP3 internalization. Black-Right-Pointing-Pointer A new strategy of promoting internalization and resensitization of the receptor was found. -- Abstract: Two receptor activity-modifying proteins (RAMP2 and RAMP3) enable calcitonin receptor-like receptor (CLR) to function as two heterodimeric receptors (CLR/RAMP2 and CLR/RAMP3) for adrenomedullin (AM), a potent cardiovascular protective peptide. Following AM stimulation, both receptors undergo rapid internalization through a clathrin-dependent pathway, after which CLR/RAMP3, but not CLR/RAMP2, can be recycled to the cell surface for resensitization. However, human (h)RAMP3 mediates CLR internalization much less efficiently than does hRAMP2. Therefore, the molecular basis of the single transmembrane domain (TMD) and the intracellular domain of hRAMP3 during AM receptor internalization was investigated by transiently transfecting various RAMP chimeras and mutants into HEK-293 cells stably expressing hCLR. Flow cytometric analysis revealed that substituting the RAMP3 TMD with that of RAMP2 markedly enhanced AM-induced internalization of CLR. However, this replacement did not enhance the cell surface expression of CLR, [{sup 125}I]AM binding affinity or AM-induced cAMP response. More detailed analyses showed that substituting the Thr{sup 130}-Val{sup 131} sequence in the RAMP3 TMD with the corresponding sequence (Ile{sup 157}-Pro{sup 158}) from RAMP2 significantly enhanced AM-mediated CLR internalization. In contrast, substituting the RAMP3 target sequence with Ala{sup 130}-Ala{sup 131} did not significantly affect CLR internalization. Thus, the RAMP3 TMD participates in the negative regulation of CLR/RAMP3 internalization, and the aforementioned introduction of the Ile-Pro sequence into the RAMP3 TMD may be a strategy for promoting receptor internalization/resensitization.

  5. Distribution of laminin and fibronectin isoforms in oral mucosa and oral squamous cell carcinoma

    PubMed Central

    Kosmehl, H; Berndt, A; Strassburger, S; Borsi, L; Rousselle, P; Mandel, U; Hyckel, P; Zardi, L; Katenkamp, D

    1999-01-01

    The expression of laminin and fibronectin isoforms varies with cellular maturation and differentiation and these differences may well influence cellular processes such as adhesion and motility. The basement membrane (BM) of fetal oral squamous epithelium contains the laminin chains, ?2, ?3, ?5, ?1, ?2, ?3, ?1 and ?2. The BM of adult normal oral squamous epithelium comprises the laminin chains, ?3, ?5, ?1, ?3, ?1 and ?2. A re-expression of the laminin ?2 and ?2 chains could be shown in adult hyperproliferative, dysplastic and carcinomatous lesions. In dysplasia and oral squamous cell carcinoma (OSCC), multifocal breaks of the BM are present as indicated by laminin chain antibodies. These breaks correlate to malignancy grade in their extent. Moreover, in the invasion front the ?3 and ?2 chain of laminin-5 can immunohistochemically be found outside the BM within the cytoplasm of budding carcinoma cells and in the adjacent stroma. The correlation between the morphological pattern of invasive tumour clusters and a laminin-5 immunostaining in the adjacent stroma may suggest, first, that a laminin-5 deposition outside the BM is an immunohistochemical marker for invasion and second, that OSCC invasion is guided by the laminin-5 matrix. Expression of oncofetal fibronectins (IIICS de novo glycosylated fibronectin and ED-B fibronectin) could be demonstrated throughout the stromal compartment. However, the ED-B fibronectin synthesizing cells (RNA/RNA in situ hybridization) are confined to small stroma areas and to single stroma and inflammatory cells in the invasion front. A correlation of the number of ED-B fibronectin synthesizing cells to malignancy grade could not be seen. ED-B fibronectin mRNA-positive cells seem to be concentrated in areas of fibrous stroma recruitment with a linear alignment of stromal fibro-/myofibroblasts (desmoplasia). Double staining experiments (ED-B fibronectin in situ hybridization and ?-smooth muscle actin immunohistochemistry) indicated that the stroma myofibroblasts are a preferential source of ED-B fibronectin. In conclusion, in OSCC, a fetal extracellular matrix conversion is demonstrable. Tumour cells (laminin ?2 and ?2 chain) and recruited stromal myofibroblasts (oncofetal ED-B fibronectin) contribute to the fetal extracellular matrix milieu. © 1999 Cancer Research Campaign PMID:10576667

  6. A novel steroid receptor co-activator protein (SRAP) as an alternative form of steroid receptor RNA-activator gene: expression in prostate cancer cells and enhancement of androgen receptor activity.

    PubMed Central

    Kawashima, Hidenori; Takano, Haruna; Sugita, Syozo; Takahara, Yuki; Sugimura, Kazunobu; Nakatani, Tatsuya

    2003-01-01

    We have cloned a cDNA coding for a novel steroid receptor co-activator protein termed SRAP from a rat prostate library. Although the nucleotide sequence of the SRAP has 78.2% identity to that of the human steroid receptor RNA activator (SRA), a novel RNA molecule which was reported to act as an RNA transcript without being translated into protein [Lanz, McKenna, Onate, Albrecht, Wong, Tsai, Tsai and O'Malley (1999) Cell 97, 17-27], the cDNA of SRAP is capable of generating a functional protein. Glutathione S-transferase pull-down assays showed that SRAP associates with the partial androgen receptor (AR) protein composed of a DNA-binding domain and an activation function 2. Luciferase assays demonstrated that SRAP enhances the transactivation activity of the AR, the glucocorticoid receptor and the peroxisome proliferator-activated receptor gamma(1) in a ligand-dependent manner. Using a green fluorescent protein (GFP) fusion-protein construct, we demonstrated in vivo translation of the GFP-SRAP fusion protein in HeLa cells co-transfected with pSG5AR and reporter gene in the presence of 5 alpha-dihydrotestosterone (DHT). Co-transfection of the GFP-SRAP fusion protein expression plasmid enhanced the transactivation activity of AR whereas incorporation of mutations in SRAP of the fusion protein resulted in loss of enhancement of the transactivation activity. Northern blot analysis and reverse transcriptase PCR assays showed that SRAP and SRA are expressed in rat and human prostate cancer cell lines respectively. In HeLa cells and the human prostate cancer cells line DU-145, co-transfected with SRAP, the DHT-dependent transactivation activities of AR were not completely inhibited by the anti-androgen flutamide, but the transactivation activities still remained high even in the presence of 5 microM flutamide, suggesting that SRAP may play an important role in enhancing AR activity in prostate cancer. PMID:12350225

  7. Structural Basis for Receptor Activity-Modifying Protein-Dependent Selective Peptide Recognition by a G Protein-Coupled Receptor

    PubMed Central

    Booe, Jason M.; Walker, Christopher S.; Barwell, James; Kuteyi, Gabriel; Simms, John; Jamaluddin, Muhammad A.; Warner, Margaret L.; Bill, Roslyn M.; Harris, Paul W.; Brimble, Margaret A.; Poyner, David R.; Hay, Debbie L.; Pioszak, Augen A.

    2015-01-01

    Summary Association of receptor activity-modifying proteins (RAMP1-3) with the G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) enables selective recognition of the peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) that have diverse functions in the cardiovascular and lymphatic systems. How peptides selectively bind GPCR:RAMP complexes is unknown. We report crystal structures of CGRP analog-bound CLR:RAMP1 and AM-bound CLR:RAMP2 extracellular domain heterodimers at 2.5 and 1.8 Ĺ resolutions, respectively. The peptides similarly occupy a shared binding site on CLR with conformations characterized by a ?-turn structure near their C termini rather than the ?-helical structure common to peptides that bind related GPCRs. The RAMPs augment the binding site with distinct contacts to the variable C-terminal peptide residues and elicit subtly different CLR conformations. The structures and accompanying pharmacology data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes. PMID:25982113

  8. Epoxyeicosatrienoic acid-dependent cerebral vasodilation evoked by metabotropic glutamate receptor activation in vivo

    E-print Network

    Hammock, Bruce D.

    activation in vivo Xiaoguang Liu,1 Chunyuan Li,1 Debebe Gebremedhin,2 Sung Hee Hwang,3 Bruce D. Hammock,3 receptor activation in vivo. Am J Physiol Heart Circ Physiol 301: H373­H381, 2011. First published May 20 have been shown to participate in cerebral vasodilation to neuronal activation in brain slices

  9. Two Interconvertible Folds Modulate the Activity of a DNA Aptamer Against Transferrin Receptor

    PubMed Central

    Porciani, David; Signore, Giovanni; Marchetti, Laura; Mereghetti, Paolo; Nifosě, Riccardo; Beltram, Fabio

    2014-01-01

    Thanks to their ability to recognize biomolecular targets with high affinity and specificity, nucleic acid aptamers are increasingly investigated as diagnostic and therapeutic tools, particularly when their targets are cell-surface receptors. Here, we investigate the relationship between the folding of an anti-mouse transferrin receptor DNA aptamer and its interaction with the transferrin receptor both in vitro and in living cells. We identified and purified two aptamer conformers by means of chromatographic techniques. Fluorescence-anisotropy measurements showed that only one fold is able to bind mouse transferrin receptor. Besides displaying enhanced endocytosis in living mouse fibroblasts, the purified active fold is internalized also in human pancreatic cancer cells. Starting from these observations, we rationally designed variations of the parent sequence aimed at stabilizing the active fold, and consequently increase aptamer activity. A truncated version and full-length mutants with higher affinity than the parent sequence are shown. PMID:24472870

  10. Acetylcholine release in mouse hippocampal CA1 preferentially activates inhibitory-selective interneurons via ?4?2* nicotinic receptor activation

    PubMed Central

    Bell, L. Andrew; Bell, Karen A.; McQuiston, A. Rory

    2015-01-01

    Acetylcholine (ACh) release onto nicotinic receptors directly activates subsets of inhibitory interneurons in hippocampal CA1. However, the specific interneurons activated and their effect on the hippocampal network is not completely understood. Therefore, we investigated subsets of hippocampal CA1 interneurons that respond to ACh release through the activation of nicotinic receptors and the potential downstream effects this may have on hippocampal CA1 network function. ACh was optogenetically released in mouse hippocampal slices by expressing the excitatory optogenetic protein oChIEF-tdTomato in medial septum/diagonal band of Broca cholinergic neurons using Cre recombinase-dependent adeno-associated viral mediated transfection. The actions of optogenetically released ACh were assessed on both pyramidal neurons and different interneuron subtypes via whole cell patch clamp methods. Vasoactive intestinal peptide (VIP)-expressing interneurons that selectively innervate other interneurons (VIP/IS) were excited by ACh through the activation of nicotinic receptors containing ?4 and ?2 subunits (?4?2*). ACh release onto VIP/IS was presynaptically inhibited by M2 muscarinic autoreceptors. ACh release produced spontaneous inhibitory postsynaptic current (sIPSC) barrages blocked by dihydro-?-erythroidine in interneurons but not pyramidal neurons. Optogenetic suppression of VIP interneurons did not inhibit these sIPSC barrages suggesting other interneuron-selective interneurons were also excited by ?4?2* nicotinic receptor activation. In contrast, interneurons that innervate pyramidal neuron perisomatic regions were not activated by ACh release onto nicotinic receptors. Therefore, we propose ACh release in CA1 facilitates disinhibition through activation of ?4?2* nicotinic receptors on interneuron-selective interneurons whereas interneurons that innervate pyramidal neurons are less affected by nicotinic receptor activation. PMID:25918499

  11. Effects of penicillin and erythromycin on adherence of invasive and noninvasive isolates of Streptococcus pyogenes to laminin

    PubMed Central

    Šmitran, Aleksandra; Vukovi?, Dragana; Gaji?, Ina; Marinkovi?, Jelena; Ranin, Lazar

    2015-01-01

    This study investigated the possible relationship between the invasiveness of group A Streptococcus (GAS) strains and their abilities to adhere to laminin and assessed the effects of subinhibitory concentrations of penicillin and erythromycin on the ability of GAS to adhere to laminin. The adherence of noninvasive and highly invasive isolates of GAS to laminin was significantly higher than the adherence displayed by isolates of low invasiveness. Antibiotic treatment caused significant reductions in adherence to laminin in all three groups of strains. Penicillin was more successful in reducing the adherence abilities of the tested GAS strains than erythromycin. PMID:26270594

  12. Utilizing the folate receptor for active targeting of cancer nanotherapeutics

    PubMed Central

    Zwicke, Grant L.; Mansoori, G. Ali; Jeffery, Constance J.

    2012-01-01

    The development of specialized nanoparticles for use in the detection and treatment of cancer is increasing. Methods are being proposed and tested that could target treatments more directly to cancer cells, which could lead to higher efficacy and reduced toxicity, possibly even eliminating the adverse effects of damage to the immune system and the loss of quick replicating cells. In this mini-review we focus on recent studies that employ folate nanoconjugates to target the folate receptor. Folate receptors are highly overexpressed on the surface of many tumor types. This expression can be exploited to target imaging molecules and therapeutic compounds directly to cancerous tissues. PMID:23240070

  13. Pharmacological activation of CB1 receptor modulates long term potentiation by interfering with protein synthesis.

    PubMed

    Navakkode, Sheeja; Korte, Martin

    2014-04-01

    Cognitive impairment is one of the most important side effects associated with cannabis drug abuse, as well as the serious issue concerning the therapeutic use of cannabinoids. Cognitive impairments and neuropsychiatric symptoms are caused by early synaptic dysfunctions, such as loss of synaptic connections in different brain structures including the hippocampus, a region that is believed to play an important role in certain forms of learning and memory. We report here that metaplastic priming of synapses with a cannabinoid type 1 receptor (CB1 receptor) agonist, WIN55,212-2 (WIN55), significantly impaired long-term potentiation in the apical dendrites of CA1 pyramidal neurons. Interestingly, the CB1 receptor exerts its effect by altering the balance of protein synthesis machinery towards higher protein production. Therefore the activation of CB1 receptor, prior to strong tetanization, increased the propensity to produce new proteins. In addition, WIN55 priming resulted in the expression of late-LTP in a synaptic input that would have normally expressed early-LTP, thus confirming that WIN55 priming of LTP induces new synthesis of plasticity-related proteins. Furthermore, in addition to the effects on protein translation, WIN55 also induced synaptic deficits due to the ability of CB1 receptors to inhibit the release of acetylcholine, mediated by both muscarinic and nicotinic acetylcholine receptors. Taken together this supports the notion that the modulation of cholinergic activity by CB1 receptor activation is one mechanism that regulates the synthesis of plasticity-related proteins. PMID:24412673

  14. Spontaneous and evoked glutamate release activate two populations of NMDA receptors with limited overlap

    PubMed Central

    Atasoy, Deniz; Ertunc, Mert; Moulder, Krista L.; Blackwell, Justin; Chung, ChiHye; Su, Jianzhong; Kavalali, Ege T.

    2008-01-01

    In a synapse, spontaneous and action potential-driven neurotransmitter release are assumed to activate the same set of postsynaptic receptors. Here, we tested this assumption using MK-801, a well-characterized use-dependent blocker of NMDA receptors. NMDA receptor-mediated spontaneous miniature excitatory postsynaptic currents (NMDA-mEPSCs) were substantially decreased by MK-801 within 2-minutes in a use-dependent manner. In contrast, MK-801 application at rest for 10-minutes did not significantly impair the subsequent NMDA receptor-mediated evoked EPSCs (NMDA-eEPSCs). Brief stimulation in the presence of MK-801 significantly depressed evoked NMDA-eEPSCs but only mildly affected the spontaneous NMDA-mEPSCs detected on the same cell. Optical imaging of synaptic vesicle fusion showed that spontaneous and evoked release could occur at the same synapse albeit without correlation between their kinetics. In addition, modeling glutamate diffusion and NMDA receptor activation revealed that postsynaptic densities larger than ~0.2 ?m2 can accommodate two populations of NMDA receptors with largely non-overlapping responsiveness. Collectively, these results support the premise that spontaneous and evoked neurotransmission activate distinct sets of NMDA receptors and signal independently to the postsynaptic side. PMID:18829973

  15. ACTIVATION OF DOPAMINE D3 RECEPTORS INHIBITS REWARD-RELATED LEARNING INDUCED BY COCAINE

    PubMed Central

    Kong, Han; Kuang, Wenjian; Li, Shengbin; Xu, Ming

    2010-01-01

    Memories of learned associations between the rewarding properties of drugs and environmental cues contribute to craving and relapse in humans. The mesocorticolimbic dopamine (DA) system is involved in reward-related learning induced by drugs of abuse. DA D3 receptors are preferentially expressed in mesocorticolimbic DA projection areas. Genetic and pharmacological studies have shown that DA D3 receptors suppress locomotor-stimulant effects of cocaine and reinstatement of cocaine-seeking behaviors. Activation of the extracellular signal-regulated kinase (ERK) induced by acute cocaine administration is also inhibited by D3 receptors. How D3 receptors modulate cocaine-induced reward-related learning and associated changes in cell signaling in reward circuits in the brain, however, have not been fully investigated. In the present study, we show that D3 receptor mutant mice exhibit potentiated acquisition of conditioned place preference (CPP) at low doses of cocaine compared to wild-type mice. Activation of ERK and Ca2+/calmodulin-dependent protein kinase II? (CaMKII?), but not the c-Jun N-terminal kinase and p38, in the nucleus accumbens, amygdala and prefrontal cortex is also potentiated in D3 receptor mutant mice compared to that in wild-type mice following CPP expression. These results support a model in which D3 receptors modulate reward-related learning induced by low doses of cocaine by inhibiting activation of ERK and CaMKII? in reward circuits in the brain. PMID:21168475

  16. Estrogen activation of the nuclear orphan receptor CAR (constitutive active receptor) in induction of the mouse Cyp2b10 gene.

    PubMed

    Kawamoto, T; Kakizaki, S; Yoshinari, K; Negishi, M

    2000-11-01

    The nuclear orphan receptor CAR (constitutively active receptor or constitutive androstane receptor) can be activated in response to xenochemical exposure, such as activation by phenobarbital of a response element called NR1 found in the CYP2B gene. Here various steroids were screened for potential endogenous chemicals that may activate CAR, using the NR1 enhancer and Cyp2b10 induction in transfected HepG2 cell and/or in mouse primary hepatocytes as the experimental criteria. 17beta-Estradiol and estrone activated NR1, whereas estriol, estetrol, estradiol sulfate, and the synthetic estrogen diethylstilbestrol did not. On the other hand, progesterone and androgens repressed NR1 activity in HepG2 cells, and the repressed NR1 activity was fully restored by estradiol. Moreover, estrogen treatment elicited nuclear accumulation of CAR in the mouse livers, as well as primary hepatocytes, and induced the endogenous Cyp2b10 gene. Ovariectomy did not affect either the basal or induced level of CAR in the nucleus of the female livers, while castration slightly increased the basal and greatly increased the induced levels in the liver nucleus of male mice. Thus, endogenous estrogen appears not to regulate CAR in female mice, whereas endogenous androgen may be the repressive factor in male mice. Estrogen at pharmacological levels is an effective activator of CAR in both female and male mice, suggesting a biological and/or toxicological role of this receptor in estrogen metabolism. In addition to mouse CAR, estrogens activated rat CAR, whereas human CAR did not respond well to the estrogens under the experimental conditions. PMID:11075820

  17. Structure-Activity Relationships for the Antifungal Activity of Selective Estrogen Receptor Antagonists Related to Tamoxifen

    PubMed Central

    Butts, Arielle; Martin, Jennifer A.; DiDone, Louis; Bradley, Erin K.; Mutz, Mitchell; Krysan, Damian J.

    2015-01-01

    Cryptococcosis is one of the most important invasive fungal infections and is a significant contributor to the mortality associated with HIV/AIDS. As part of our program to repurpose molecules related to the selective estrogen receptor modulator (SERM) tamoxifen as anti-cryptococcal agents, we have explored the structure-activity relationships of a set of structurally diverse SERMs and tamoxifen derivatives. Our data provide the first insights into the structural requirements for the antifungal activity of this scaffold. Three key molecular characteristics affecting anti-cryptococcal activity emerged from our studies: 1) the presence of an alkylamino group tethered to one of the aromatic rings of the triphenylethylene core; 2) an appropriately sized aliphatic substituent at the 2 position of the ethylene moiety; and 3) electronegative substituents on the aromatic rings modestly improved activity. Using a cell-based assay of calmodulin antagonism, we found that the anti-cryptococcal activity of the scaffold correlates with calmodulin inhibition. Finally, we developed a homology model of C. neoformans calmodulin and used it to rationalize the structural basis for the activity of these molecules. Taken together, these data and models provide a basis for the further optimization of this promising anti-cryptococcal scaffold. PMID:26016941

  18. Proteolytic generation of constitutive tyrosine kinase activity of the human insulin receptor.

    PubMed Central

    Hsuan, J J; Downward, J; Clark, S; Waterfield, M D

    1989-01-01

    Structural modification induced by partial digestion with trypsin has been shown to stimulate the tyrosine kinase activity of the insulin receptor both in solution and in intact cells [Tamura, Fujita-Yamaguchi & Larner (1983) J. Biol. Chem. 258, 14749-14752; Goren, White & Kahn (1987) Biochemistry 26, 2374-2382; Leef & Larner (1987) J. Biol. Chem. 262, 14837-14842]. Furthermore, experiments involving deletion of sequences encoding the extracellular domain of the insulin receptor suggest that it may function as a protooncogene in fibroblasts [Wang et al., (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5725-5729]. To further understand the structural requirements that generate this activity, the major activated fragments generated in solution following trypsin digestion have been characterized here, one of which is shown to have a similar amino acid sequence to a transforming protein. Furthermore, treatment with trypsin of intact Chinese hamster ovary cells that overexpress the human insulin receptor stimulates both autophosphorylation of the receptor and 2-deoxyglucose uptake into the cells, but does not enhance receptor internalization. Unlike digestion in solution, no proteolysis or loss of activity of the activated insulin receptor beta-subunit could be detected using intact cells, even at high trypsin concentrations, despite the existence of extracellular sites that are readily cleaved by trypsin in the solubilized receptor. These studies provide further detail of a mechanism used during trypsinization of cells in culture which mimics activation of the insulin receptor and contributes to stimulation of growth. Images Fig. 1. Fig. 3. Fig. 5. Fig. 6. PMID:2719663

  19. Cell-based systems to assess nuclear receptor activation and their use in drug development.

    PubMed

    Raucy, Judy L; Lasker, Jerome M

    2013-02-01

    The evolution of scientific information relating to the regulation of xenobiotic disposition has extended to the discovery of an intricate group of receptor systems now recognized as master regulators. These ligand-activated transcription factors are commonly designated as "nuclear receptors", and include CAR (NR1I3), PXR (NR1I2), PPAR (NR1C1, NR1C2, and NR1C3) and AhR (HLHE76). As regulators of gene expression, activation of these receptors can elicit a plethora of drug-drug interactions. The aforementioned nuclear receptors bind a wide range of structurally-unrelated ligands, such as steroid hormones, bile acids, and small drug-type molecules. A pivotal nuclear receptor with regards to regulation of drug-drug interactions is the pregnane X receptor (PXR). Gene expression profiling has demonstrated that PXR regulates over 60 human genes that are involved not only in physiological functions but also in the metabolism of xenobiotics. Moreover, chemical library screening suggests that about 10% of the compounds comprising the U. S. Food and Drug Administration 1 and 2, Sigma-Aldrich LOPAC collection, Biomol, and Tocris/TimTec bioactive collection libraries exhibit some form of PXR binding. For these reasons, efficient, rapid and economical systems have been developed to identify nuclear receptor ligands. Cell-based assays encompassing transiently and stably-transfected cells and mammalian two-hybrid systems are currently being employed by the pharmaceutical industry to screen compounds for binding to and/or activation of nuclear receptors. Overall, these systems have the ability to predict in vivo responses to receptor activation that culminate in drug-drug interactions and adverse drug effects. PMID:23330544

  20. Omigapil ameliorates the pathology of muscle dystrophy caused by laminin-alpha2 deficiency.

    PubMed

    Erb, Michael; Meinen, Sarina; Barzaghi, Patrizia; Sumanovski, Lazar T; Courdier-Früh, Isabelle; Rüegg, Markus A; Meier, Thomas

    2009-12-01

    Laminin alpha2-deficient congenital muscular dystrophy, called MDC1A, is a rare, devastating genetic disease characterized by severe neonatal hypotonia ("floppy infant syndrome"), peripheral neuropathy, inability to stand or walk, respiratory distress, and premature death in early life. Transgenic overexpression of the apoptosis inhibitor protein BCL-2, or deletion of the proapoptotic Bax gene in a mouse model for MDC1A prolongs survival and mitigates pathology, indicating that apoptotic events are involved in the pathology. Here we demonstrate that the proapoptotic glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-Siah1-CBP/p300-p53 pathway is activated in a mouse model for MDC1A. Moreover, we show that omigapil, which inhibits GAPDH-Siah1-mediated apoptosis, ameliorates several pathological hallmarks in the MDC1A mouse model. Specifically, we demonstrate that treatment with omigapil inhibits apoptosis in muscle, reduces body weight loss and skeletal deformation, increases locomotive activity, and protects from early mortality. These data qualify omigapil, which is in late phase of clinical development for human use, as a drug candidate for the treatment of MDC1A. PMID:19759319

  1. Interaction of active compounds from Aegle marmelos CORREA with histamine-1 receptor

    PubMed Central

    Nugroho, Agung Endro; Agistia, Dany Dwi; Tegar, Maulana; Purnomo, Hari

    2013-01-01

    The aim of this study is to determine the affinity of six active compounds of Aegle Marmelos Correa, they are (E, R)-Marmin, skimmianine, (S)-aegeline, aurapten, zeorin, and dustanin as antihistamines in histamine H1 receptor in comparison to cetirizin, diphenhydramine and chlorpheniramine as ligands comparison. Previously, in the in vitro study marmin obviously antagonized the histamine H1 receptor in a competitive manner. Methods: molecular docking to determine the interaction of ligand binding to its receptor. Lower docking score indicates more stable binding to that protein. Results: Marmin, skimmianine, aegeline, aurapten, zeorin, and dustanin were potential to develop as antihistamine agents, especially as histamine H1 receptor antagonists by interacting with amino acid residues, Asp107, Lys179, Lys191, Asn198, and Trp428 of histamine H1 receptor. Conclusions: Based on molecular docking, Amino acid residues involved in ligand protein interactions were Asp107, Lys179, Lys191, Asn198, and Trp428. PMID:23750086

  2. Genome-Wide Profiling of Liver X Receptor, Retinoid X Receptor, and Peroxisome Proliferator-Activated Receptor ? in Mouse Liver Reveals Extensive Sharing of Binding Sites

    PubMed Central

    Boergesen, Michael; Pedersen, Thomas Ĺskov; Gross, Barbara; van Heeringen, Simon J.; Hagenbeek, Dik; Bindesbřll, Christian; Caron, Sandrine; Lalloyer, Fanny; Steffensen, Knut R.; Nebb, Hilde I.; Gustafsson, Jan-Ĺke; Stunnenberg, Hendrik G.; Staels, Bart

    2012-01-01

    The liver X receptors (LXRs) are nuclear receptors that form permissive heterodimers with retinoid X receptor (RXR) and are important regulators of lipid metabolism in the liver. We have recently shown that RXR agonist-induced hypertriglyceridemia and hepatic steatosis in mice are dependent on LXRs and correlate with an LXR-dependent hepatic induction of lipogenic genes. To further investigate the roles of RXR and LXR in the regulation of hepatic gene expression, we have mapped the ligand-regulated genome-wide binding of these factors in mouse liver. We find that the RXR agonist bexarotene primarily increases the genomic binding of RXR, whereas the LXR agonist T0901317 greatly increases both LXR and RXR binding. Functional annotation of putative direct LXR target genes revealed a significant association with classical LXR-regulated pathways as well as peroxisome proliferator-activated receptor (PPAR) signaling pathways, and subsequent chromatin immunoprecipitation-sequencing (ChIP-seq) mapping of PPAR? binding demonstrated binding of PPAR? to 71 to 88% of the identified LXR-RXR binding sites. The combination of sequence analysis of shared binding regions and sequential ChIP on selected sites indicate that LXR-RXR and PPAR?-RXR bind to degenerate response elements in a mutually exclusive manner. Together, our findings suggest extensive and unexpected cross talk between hepatic LXR and PPAR? at the level of binding to shared genomic sites. PMID:22158963

  3. Combining Passive Sampling with Recombinant Receptor-Reporter Gene Bioassays to Assess the Receptor Activity of Victorian Rivers.

    PubMed

    Allinson, Graeme; Shiraishi, Fujio; Kamata, Ryo; Allinson, Mayumi

    2015-12-01

    This pilot study was initiated to provide new information on the 'hormonal' activity of Victorian rivers. Chemcatcher™ passive sampler systems containing Empore™ C18FF disks were deployed at eight riverine sites near Melbourne. Little estrogenic activity [<0.4-1.8 ng estradiol equivalents (EQ)/disk] and no retinoic acid activity (RAR, all samples <0.8 ng trans-retinoic acid EQ/disk) was observed. Almost all sample extracts showed aryl hydrocarbon receptor activity (from <4 to 29 ng ?-naphthoflavone EQ/disk). Overall, the disk extracts were eminently compatible with the bioassay screening technology, enabling the relative levels of 'hormonal activity' to be observed in the surface waters in and around Melbourne. From a practical perspective, the in situ sampling and pre-concentration provided by passive sampling reduces the manual handling risks associated with sample transport, and the number of laboratory operations required to obtain assay-ready solutions for analysis. PMID:26071881

  4. Ligand-mediated autophosphorylation activity of the epidermal growth factor receptor during internalization

    SciTech Connect

    Lai, W.H.; Cameron, P.H.; Doherty, J.J. II; Posner, B.I.; Bergeron, J.J. )

    1989-12-01

    The association of EGF with its receptor in endosomes isolated from rat liver homogenates was assessed biochemically by polyethylene glycol precipitation and morphologically by electron microscope radioautography. The proportion of receptor-bound ligand in endosomes at 15 min after the injection of doses of 0.1 and 1 microgram EGF/100 g body weight was 57%. This value increased to 77% for the dose of 10 micrograms EGF injected. Quantitative electron microscope radioautography carried out on endosomes isolated at 15 min after the injection of 10 micrograms 125I-EGF demonstrated that most radiolabel was over the endosomal periphery thereby indicating that ligand-receptor complexes were in the bounding membrane but not in intraluminal vesicles of the content. EGF receptor autophosphorylation activity during internalization was evaluated in plasmalemma and endosome fractions. This activity was markedly but transiently reduced on the cell surface shortly after the administration of saturating doses of EGF. The same activity, however, was augmented and prolonged in endosomes for up to 30 min after EGF injection. The transient desensitization of cell surface activity was not due to prior in vivo phosphorylation since receptor dephosphorylation in vitro failed to restore autophosphorylation activity. Transient desensitization of cell surface autophosphorylation activity coincided with a diminished capacity for endocytosis of 125I-EGF with endocytosis returning to normal after the restoration of cell surface autophosphorylation activity. The inhibition of cell surface autophosphorylation activity and the activation of endosomal autophosphorylation activity coincident with downregulation suggest that EGF receptor traffic is governed by ligand-regulated phosphorylation activity.

  5. Interleukin-1 Receptor Antagonist Has a Novel Function in the Regulation of Matrix Metalloproteinase-13 Expression

    PubMed Central

    Goto, Hisashi; Ishihara, Yuichi; Kikuchi, Takeshi; Izawa, Ario; Ozeki, Nobuaki; Okabe, Eijiro; Kamiya, Yosuke; Ozawa, Yusuke; Mizutani, Hiroki; Yamamoto, Genta; Mogi, Makio; Nakata, Kazuhiko; Maeda, Hatsuhiko; Noguchi, Toshihide; Mitani, Akio

    2015-01-01

    Interleukin-1 receptor antagonist (IL-1Ra) is an IL-1 family member, which binds to IL-1 receptors but does not induce any intracellular signaling. We addressed whether IL-1Ra has a novel function in regulation of the extracellular matrix or adhesion molecules. Polymerase chain reaction array analysis demonstrated a ~5-fold increase in matrix metalloproteinase 13 (MMP-13) mRNA expression of IL-1Ra siRNA-transfected Ca9-22 human oral squamous epithelial carcinoma cells compared with the control. In fact, MMP-13 mRNA and protein expression as well as its activity in IL-1Ra siRNA-transfected Ca9-22 cell lines were significantly higher than those in the control. IL-1Ra siRNA treatment resulted in strong elevation of MMP-13 expression, whereas addition of rhIL-1Ra (40 ng/ml) suppressed MMP-13 expression, suggesting that IL-1Ra had a specific effect on MMP-13 induction. IL-1Ra siRNA could potently suppress IL-1?. No significant difference was found between the MMP-13 mRNA expression of IL-1Ra siRNA-transfected cells and those treated with anti-IL-1? or anti-IL-1? antibodies. These results suggested that continuous supply of IL-1 had no effect on the induction of MMP-13 by IL-1Ra siRNA. Histopathological investigation of MMP-13 in periodontal tissue showed specific localization in the junctional epithelial cells of IL-1Ra knockout (KO) mice. Furthermore, infection with Aggregatibacter actinomycetemcomitans to establish an experimental periodontitis model resulted in predominant localization of MMP-13 along apical junctional epithelial cells. Laminin-5, which is degraded by MMP-13, was found in the internal basal lamina of wild-type mice, whereas the internal basal lamina of IL-1Ra KO mice did not show obvious laminin-5 localization. In particular, laminin-5 localization almost disappeared in the internal basal lamina of IL-1Ra KO mice infected with A. actinomycetemcomitans, suggesting that the suppression of IL-1Ra resulted in strong induction of MMP-13 that degraded laminin-5. In conclusion, IL-1Ra is associated with MMP-13 expression and has a novel function in such regulation without interference of the IL-1 signaling cascade. PMID:26474296

  6. Activation Biosensor for G Protein-Coupled Receptors: A FRET-Based m1 Muscarinic Activation Sensor That Regulates Gq

    PubMed Central

    Chang, Seungwoo; Ross, Elliott M.

    2012-01-01

    We describe the design, construction and validation of a fluorescence sensor to measure activation by agonist of the m1 muscarinic cholinergic receptor, a prototypical class I Gq-coupled receptor. The sensor uses an established general design in which Förster resonance energy transfer (FRET) from a circularly permuted CFP mutant to FlAsH, a selectively reactive fluorescein, is decreased 15–20% upon binding of a full agonist. Notably, the sensor displays essentially wild-type capacity to catalyze activation of G?q, and the purified and reconstituted sensor displays appropriate regulation of affinity for agonists by Gq. We describe the strategies used to increase the agonist-driven change in FRET while simultaneously maintaining regulatory interactions with G?q, in the context of the known structures of Class I G protein-coupled receptors. The approach should be generally applicable to other Class I receptors which include numerous important drug targets. PMID:23029161

  7. Activation and modulation of recombinantly expressed serotonin receptor type 3A by terpenes and pungent substances.

    PubMed

    Ziemba, Paul M; Schreiner, Benjamin S P; Flegel, Caroline; Herbrechter, Robin; Stark, Timo D; Hofmann, Thomas; Hatt, Hanns; Werner, Markus; Gisselmann, Günter

    2015-11-27

    Serotonin receptor type 3 (5-HT3 receptor) is a ligand-gated ion channel that is expressed in the central nervous system (CNS) as well as in the peripheral nervous system (PNS). The receptor plays an important role in regulating peristalsis of the gastrointestinal tract and in functions such as emesis, cognition and anxiety. Therefore, a variety of pharmacologically active substances target the 5-HT3 receptor to treat chemotherapy-induced nausea and vomiting. The 5-HT3 receptors are activated, antagonized, or modulated by a wide range of chemically different substances, such as 2-methyl-serotonin, phenylbiguanide, setrones, or cannabinoids. Whereas the action of all of these substances is well described, less is known about the effect of terpenoids or fragrances on 5-HT3A receptors. In this study, we screened a large number of natural odorous and pungent substances for their pharmacological action on recombinantly expressed human 5-HT3A receptors. The receptors were functionally expressed in Xenopus oocytes and characterized by electrophysiological recordings using the two-electrode voltage-clamp technique. A screening of two odorous mixes containing a total of 200 substances revealed that the monoterpenes, thymol and carvacrol, act as both weak partial agonists and positive modulators on the 5-HT3A receptor. In contrast, the most effective blockers were the terpenes, citronellol and geraniol, as well as the pungent substances gingerol, capsaicin and polygodial. In our study, we identified new modulators of 5-HT3A receptors out of the classes of monoterpenes and vanilloid substances that frequently occur in various plants. PMID:26456648

  8. Selective phthalate activation of naturally occurring human constitutive androstane receptor splice variants and the pregnane X receptor.

    PubMed

    DeKeyser, Joshua G; Laurenzana, Elizabeth M; Peterson, Eric C; Chen, Tao; Omiecinski, Curtis J

    2011-04-01

    Phthalates and other endocrine-disruptive chemicals are manufactured in large quantities for use as plasticizers and other commercial applications, resulting in ubiquitous human exposure and thus, concern regarding their toxicity. Innate defense against small molecule exposures is controlled in large part by the constitutive androstane receptor (CAR) and the pregnane X receptor (PXR). The human CAR gene undergoes multiple alternative splicing events resulting in the CAR2 and CAR3 variant receptors. Recent studies from our laboratory show that CAR2 is potently and specifically activated by di(2-ethylhexyl) phthalate (DEHP). We hypothesized that alternative splicing is a mechanism for increasing CAR's functional diversity, broadening the human receptors' repertoire of response to environmental xenobiotics. In these studies, we examine the interaction of alternatively spliced CARs and PXR with a range of suspected endocrine disruptors, including phthalates, bisphenol A (BPA), and 4-N-nonylphenol (NP). Transactivation and two-hybrid studies in COS-1 cells revealed differential selectivity of endocrine-disrupting chemicals for the variant CAR and PXR. Ex vivo studies showed DEHP and di-isononyl phthalate potently induced CYP2B6 and CYP3A4 expression in human hepatocytes. Mutation analysis of CAR2, in silico modeling, and ligand docking studies suggested that the SPTV amino acid insertion of CAR2 creates a unique ligand-binding pocket. Alternative gene splicing results in variant CAR receptors that selectively recognize phthalates and BPA. The interaction of phthalates with CAR and PXR suggests a xenobiotic response that is complex and biologically redundant. PMID:21227907

  9. Indirect muscarinic receptor activation by pentamidine on airway smooth muscle.

    PubMed

    Biyah, K; Molimard, M; Naline, E; Bazelly, B; Advenier, C

    1996-11-01

    1. Pentamidine is routinely used to reduce the incidence of Pneumocystis carinii pneumonia in patients infected with human immunodeficiency virus, but it has been described as inducing pulmonary adverse effects, such as cough and bronchospasm. 2. In this paper we have investigated the effects of pentamidine on guinea-pig isolated main bronchi and human isolated bronchi. Pentamidine induced a concentration-dependent contraction in both preparations with pD2 values of 9.64 +/- 0.07 (n = 8) and 9.73 +/- 0.06 (n = 8) and a maximal effect (Emax) of 40 +/- 4% and 34 +/- 5% of the response to acetylcholine (1 mM) in guinea-pig and human bronchi respectively. Atropine (0.01 to 0.1 microM) and the muscarinic M3 receptor antagonist, hexahydro-siladiphenidol (0.1 and 1 microM) inhibited pentamidine-induced concentration-responses in both preparations in a non-competitive manner, whereas only high concentrations of the M1 receptor antagonist pirenzipine (1 microM) inhibited pentamidine concentration-response curves. 3. The cholinesterase inhibitor, tacrine (1 microM), potentiated the effect of pentamidine; in contrast, morphine inhibited pentamidine-induced responses. 4. The bronchoconstrictor effect of pentamidine on guinea-pig and human isolated bronchi was not modified by the H1 histamine receptor antagonist, mepyramine, by indomethacin or by the neurokinin NK1 and NK2 receptor antagonists, CP-96,345 and SR 48969 respectively, suggesting that neither histamine receptor stimulation, arachidonic acid derivative formation, nor tachykinin release are involved in pentamidine-induced contraction of human and guinea-pig airways. 5. Our overall results suggest that pentamidine induces contraction of guinea-pig and human isolated bronchi through prejunctional cholinergic nerve stimulation. PMID:8937715

  10. Signal transduction of Helicobacter pylori during interaction with host cell protein receptors of epithelial and immune cells

    PubMed Central

    Pachathundikandi, Suneesh Kumar; Tegtmeyer, Nicole; Backert, Steffen

    2013-01-01

    Helicobacter pylori infections can induce pathologies ranging from chronic gastritis, peptic ulceration to gastric cancer. Bacterial isolates harbor numerous well-known adhesins, vacuolating cytotoxin VacA, protease HtrA, urease, peptidoglycan, and type IV secretion systems (T4SS). It appears that H. pylori targets more than 40 known host protein receptors on epithelial or immune cells. A series of T4SS components such as CagL, CagI, CagY, and CagA can bind to the integrin ?5?1 receptor. Other targeted membrane-based receptors include the integrins ?v?3, ?v?5, and ?2 (CD18), RPTP-?/?, GP130, E-cadherin, fibronectin, laminin, CD46, CD74, ICAM1/LFA1, T-cell receptor, Toll-like receptors, and receptor tyrosine kinases EGFR, ErbB2, ErbB3, and c-Met. In addition, H. pylori is able to activate the intracellular receptors NOD1, NOD2, and NLRP3 with important roles in innate immunity. Here we review the interplay of various bacterial factors with host protein receptors. The contribution of these interactions to signal transduction and pathogenesis is discussed. PMID:24280762

  11. Laminin Peptide-Immobilized Hydrogels Modulate Valve Endothelial Cell Hemostatic Regulation

    PubMed Central

    Balaoing, Liezl Rae; Post, Allison Davis; Lin, Adam Yuh; Tseng, Hubert; Moake, Joel L.; Grande-Allen, K. Jane

    2015-01-01

    Valve endothelial cells (VEC) have unique phenotypic responses relative to other types of vascular endothelial cells and have highly sensitive hemostatic functions affected by changes in valve tissues. Furthermore, effects of environmental factors on VEC hemostatic function has not been characterized. This work used a poly(ethylene glycol) diacrylate (PEGDA) hydrogel platform to evaluate the effects of substrate stiffness and cell adhesive ligands on VEC phenotype and expression of hemostatic genes. Hydrogels of molecular weights (MWs) 3.4, 8, and 20 kDa were polymerized into platforms of different rigidities and thiol-modified cell adhesive peptides were covalently bound to acrylate groups on the hydrogel surfaces. The peptide RKRLQVQLSIRT (RKR) is a syndecan-1 binding ligand derived from laminin, a trimeric protein and a basement membrane matrix component. Conversely, RGDS is an integrin binding peptide found in many extracellular matrix (ECM) proteins including fibronectin, fibrinogen, and von Willebrand factor (VWF). VECs adhered to and formed a stable monolayer on all RKR-coated hydrogel-MW combinations. RGDS-coated platforms supported VEC adhesion and growth on RGDS-3.4 kDa and RGDS-8 kDa hydrogels. VECs cultured on the softer RKR-8 kDa and RKR-20 kDa hydrogel platforms had significantly higher gene expression for all anti-thrombotic (ADAMTS-13, tissue factor pathway inhibitor, and tissue plasminogen activator) and thrombotic (VWF, tissue factor, and P-selectin) proteins than VECs cultured on RGDS-coated hydrogels and tissue culture polystyrene controls. Stimulated VECs promoted greater platelet adhesion than non-stimulated VECs on their respective culture condition; yet stimulated VECs on RGDS-3.4 kDa gels were not as responsive to stimulation relative to the RKR-gel groups. Thus, the syndecan binding, laminin-derived peptide promoted stable VEC adhesion on the softer hydrogels and maintained VEC phenotype and natural hemostatic function. In conclusion, utilization of non-integrin adhesive peptide sequences derived from basement membrane ECM may recapitulate balanced VEC function and may benefit endothelialization of valve implants. PMID:26090873

  12. Evidence for Activation of Toll-Like Receptor and Receptor for Advanced Glycation End Products in Preterm Birth

    PubMed Central

    Noguchi, Taketoshi; Sado, Toshiyuki; Naruse, Katsuhiko; Shigetomi, Hiroshi; Onogi, Akira; Haruta, Shoji; Kawaguchi, Ryuji; Nagai, Akira; Tanase, Yasuhito; Yoshida, Shozo; Kitanaka, Takashi; Oi, Hidekazu; Kobayashi, Hiroshi

    2010-01-01

    Objective. Individuals with inflammation have a myriad of pregnancy aberrations including increasing their preterm birth risk. Toll-like receptors (TLRs) and receptor for advanced glycation end products (RAGE) and their ligands were all found to play a key role in inflammation. In the present study, we reviewed TLR and RAGE expression, their ligands, and signaling in preterm birth. Research Design and Methods. A systematic search was performed in the electronic databases PubMed and ScienceDirect up to July 2010, combining the keywords “preterm birth,” “TLR”, “RAGE”, “danger signal”, “alarmin”, “genomewide,” “microarray,” and “proteomics” with specific expression profiles of genes and proteins. Results. This paper provides data on TLR and RAGE levels and critical downstream signaling events including NF-kappaB-dependent proinflammatory cytokine expression in preterm birth. About half of the genes and proteins specifically present in preterm birth have the properties of endogenous ligands “alarmin” for receptor activation. The interactions between the TLR-mediated acute inflammation and RAGE-mediated chronic inflammation have clear implications for preterm birth via the TLR and RAGE system, which may be acting collectively. Conclusions. TLR and RAGE expression and their ligands, signaling, and functional activation are increased in preterm birth and may contribute to the proinflammatory state. PMID:21127710

  13. G-protein-coupled receptor 30 interacts with receptor activity-modifying protein 3 and confers sex-dependent cardioprotection.

    PubMed

    Lenhart, Patricia M; Broselid, Stefan; Barrick, Cordelia J; Leeb-Lundberg, L M Fredrik; Caron, Kathleen M

    2013-01-01

    Receptor activity-modifying protein 3 (RAMP3) is a single-pass transmembrane protein known to interact with and affect the trafficking of several G-protein-coupled receptors (GPCRs). We sought to determine whether RAMP3 interacts with GPR30, also known as G-protein-coupled estrogen receptor 1. GPR30 is a GPCR that binds estradiol and has important roles in cardiovascular and endocrine physiology. Using bioluminescence resonance energy transfer titration studies, co-immunoprecipitation, and confocal microscopy, we show that GPR30 and RAMP3 interact. Furthermore, the presence of GPR30 leads to increased expression of RAMP3 at the plasma membrane in HEK293 cells. In vivo, there are marked sex differences in the subcellular localization of GPR30 in cardiac cells, and the hearts of Ramp3(-/-) mice also show signs of GPR30 mislocalization. To determine whether this interaction might play a role in cardiovascular disease, we treated Ramp3(+)(/)(+) and Ramp3(-/-) mice on a heart disease-prone genetic background with G-1, a specific agonist for GPR30. Importantly, this in vivo activation of GPR30 resulted in a significant reduction in cardiac hypertrophy and perivascular fibrosis that is both RAMP3 and sex dependent. Our results demonstrate that GPR30-RAMP3 interaction has functional consequences on the localization of these proteins both in vitro and in vivo and that RAMP3 is required for GPR30-mediated cardioprotection. PMID:23674134

  14. Class A scavenger receptor promotes osteoclast differentiation via the enhanced expression of receptor activator of NF-{kappa}B (RANK)

    SciTech Connect

    Takemura, Kenichi; Department of Orthopaedic and Neuro-Musculoskeletal Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto ; Sakashita, Naomi; Fujiwara, Yukio; Komohara, Yoshihiro; Lei, XiaoFeng; Ohnishi, Koji; Suzuki, Hiroshi; Kodama, Tatsuhiko; Mizuta, Hiroshi; Takeya, Motohiro

    2010-01-22

    Osteoclasts originate from bone marrow monocyte/macrophage lineage cells, and their differentiation depends on macrophage colony-stimulating factor (M-CSF) and receptor activator nuclear factor kappa B (RANK) ligand. Class A scavenger receptor (SR-A) is one of the principal functional molecules of macrophages, and its level of expression declines during osteoclast differentiation. To investigate the role of SR-A in osteoclastogenesis, we examined pathological changes in femoral bone and the expression levels of osteoclastogenesis-related molecules in SR-A{sup -/-} mice. The femoral osseous density of SR-A{sup -/-} mice was higher than that of SR-A{sup +/+} mice, and the number of multinucleated osteoclasts was significantly decreased. An in vitro differentiation assay revealed that the differentiation of multinucleated osteoclasts from bone marrow-derived progenitor cells is impaired in SR-A{sup -/-} mice. Elimination of SR-A did not alter the expression level of the M-CSF receptor, c-fms; however, the expression levels of RANK and RANK-related osteoclast-differentiation molecules such as nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1) and microphthalmia-associated transcription factor (MITF) significantly decreased. Furthermore, acetylated low-density lipoprotein (AcLDL), an SR-A ligand, significantly increased the expression level of RANK and MITF during osteoclast differentiation. These data indicate that SR-A promotes osteoclastogenesis via augmentation of the expression level of RANK and its related molecules.

  15. Peroxisome proliferator-activated receptor {alpha} agonist-induced down-regulation of hepatic glucocorticoid receptor expression in SD rats

    SciTech Connect

    Chen Xiang; Li Ming; Sun Weiping; Bi Yan; Cai Mengyin; Liang Hua; Yu Qiuqiong; He Xiaoying; Weng Jianping

    2008-04-18

    It was reported that glucocorticoid production was inhibited by fenofibrate through suppression of type-1 11{beta}-hydroxysteroid dehydrogenase gene expression in liver. The inhibition might be a negative-feedback regulation of glucocorticoid receptor (GR) activity by peroxisome proliferator-activated receptor alpha (PPAR{alpha}), which is quickly induced by glucocorticoid in the liver. However, it is not clear if GR expression is changed by fenofibrate-induced PPAR{alpha} activation. In this study, we tested this possibility in the liver of Sprague-Dawley rats. GR expression was reduced by fenofibrate in a time- and does-dependent manner. The inhibition was observed in liver, but not in fat and muscle. The corticosterone level in the blood was increased significantly by fenofibrate. These effects of fenofibrate were abolished by PPAR{alpha} inhibitor MK886, suggesting that fenofibrate activated through PPAR{alpha}. In conclusion, inhibition of GR expression may represent a new molecular mechanism for the negative feedback regulation of GR activity by PPAR{alpha}.

  16. Activation of peroxisome proliferator-activated receptor ? ameliorates monocrotaline-induced pulmonary arterial hypertension in rats

    PubMed Central

    XIE, XINMING; WANG, GUIZUO; ZHANG, DEXIN; ZHANG, YONGHONG; ZHU, YANTING; LI, FANGWEI; LI, SHAOJUN; LI, MANXIANG

    2015-01-01

    Activation of peroxisome proliferator-activated receptor ? (PPAR?) suppresses the proliferation of pulmonary artery smooth muscle cells (PASMCs) and vascular remodeling in rats and humans, and therefore improves the development of pulmonary arterial hypertension (PAH). However, molecular mechanisms underlying these effects have not been completely understood. In the present study, the effects of PPAR? activation in monocrotaline (MCT)-induced pulmonary artery remodeling in rats were investigated. Eighteen Sprague-Dawley (SD) rats were randomly assigned into three groups (n=6): Control (Con), PAH and PAH treated with rosiglitazone (MCT + Rosi). The right ventricular systolic pressure (RVSP), the ratio of the right to left ventricle plus septum weight [RV/(LV + S)], the percentage of medial wall thickness (%MT) and wall area (%WA) were used to evaluate the development of PAH. Tissue morphology was measured using hematoxylin and eosin staining. The protein levels of the phosphatase and tensin homologue deleted on chromosome ten (PTEN), Akt (ser473) phosphorylation (p-Akt) and total Akt in intrapulmonary arteries were determined by western blot analysis. MCT treatment significantly increased the RVSP, which was reduced by rosiglitazone treatment. The ratio of RV/(LV + S), %MT and %WA induced by MCT were similarly inhibited, which was associated with the increase of PTEN expression and the inhibition of Akt phosphorylation levels by rosiglitazone. In conclusion, activation of PPAR? ameliorates the proliferation of PASMCs and vascular remodeling by regulating the PTEN/PI3K/Akt pathway, suggesting that the activation of PPAR? has potential benefits for PAH. PMID:26171162

  17. Activity-dependent upregulation of presynaptic kainate receptors at immature CA3-CA1 synapses.

    PubMed

    Clarke, Vernon R J; Molchanova, Svetlana M; Hirvonen, Teemu; Taira, Tomi; Lauri, Sari E

    2014-12-10

    Presynaptic kainate-type glutamate receptors (KARs) regulate glutamate release probability and short-term plasticity in various areas of the brain. Here we show that long-term depression (LTD) in the area CA1 of neonatal rodent hippocampus is associated with an upregulation of tonic inhibitory KAR activity, which contributes to synaptic depression and causes a pronounced increase in short-term facilitation of transmission. This increased KAR function was mediated by high-affinity receptors and required activation of NMDA receptors, nitric oxide (NO) synthetase, and postsynaptic calcium signaling. In contrast, KAR activity was irreversibly downregulated in response to induction of long-term potentiation in a manner that depended on activation of the TrkB-receptor of BDNF. Both tonic KAR activity and its plasticity were restricted to early stages of synapse development and were lost in parallel with maturation of the network due to ongoing BDNF-TrkB signaling. These data show that presynaptic KARs are targets for activity-dependent modulation via diffusible messengers NO and BDNF, which enhance and depress tonic KAR activity at immature synapses, respectively. The plasticity of presynaptic KARs in the developing network allows nascent synapses to shape their response to incoming activity. In particular, upregulation of KAR function after LTD allows the synapse to preferentially pass high-frequency afferent activity. This can provide a potential rescue from synapse elimination by uncorrelated activity and also increase the computational dynamics of the developing CA3-CA1 circuitry. PMID:25505341

  18. Regulated and constitutive activation of specific signalling pathways by the human S1P5 receptor

    PubMed Central

    Niedernberg, Anke; Blaukat, Andree; Schöneberg, Torsten; Kostenis, Evi

    2003-01-01

    We tested the hypothesis, whether G Protein-coupled receptors (GPCRs) may differentially regulate specific signalling pathways by constitutive and agonist-induced activation using the human sphingosine 1-phosphate receptor S1P5 as a model. S1P5 receptor-expressing HEK293 cells exhibited a high degree of basal activity for both inhibition of adenylyl cyclase and extracellular signal regulated kinase (ERK) when cultured in serum, which contains high levels of sphingosine 1-phosphate (S1P). However, basal activity was independent of the presence of S1P: (i) constitutive activity remained when cells were cultured in delipidated serum, (ii) addition of S1P to delipidated serum did not increase basal S1P5 receptor signalling. Conversely, constitutive inhibition of forskolin-stimulated adenylyl cyclase was further enhanced by S1P in S1P5-HEK293 cells. Transfection of several mammalian cell lines (CHO-K1, HEK293, NIH-3T3, RH7777) with the S1P5 receptor induced cell rounding, which was more pronounced in the presence of S1P-containing serum. Rounded cell morphology did not correlate with apoptotic cell death, but led to detachment of cells. Cell surface ELISA assays showed that a fraction of plasma membrane S1P5 receptors were dose-dependently internalized with S1P. These data reveal that intrinsic inhibition of unstimulated adenylyl cyclase or ERK activity by the S1P5 receptor is insensitive to ligand modulation. Conversely, effects on forskolin-stimulated adenylyl cyclase, cell morphology and internalization can be further augmented with S1P. Our results suggest that different signal transduction pathways are not equally activated through constitutively active GPCRs with promiscuous signalling characteristics. PMID:12569073

  19. Activation of peroxisome proliferator-activated receptor-{alpha} enhances fatty acid oxidation in human adipocytes

    SciTech Connect

    Lee, Joo-Young; Hashizaki, Hikari; Goto, Tsuyoshi; Sakamoto, Tomoya; Takahashi, Nobuyuki; Kawada, Teruo

    2011-04-22

    Highlights: {yields} PPAR{alpha} activation increased mRNA expression levels of adipocyte differentiation marker genes and GPDH activity in human adipocytes. {yields} PPAR{alpha} activation also increased insulin-dependent glucose uptake in human adipocytes. {yields} PPAR{alpha} activation did not affect lipid accumulation in human adipocytes. {yields} PPAR{alpha} activation increased fatty acid oxidation through induction of fatty acid oxidation-related genes in human adipocytes. -- Abstract: Peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}) is a key regulator for maintaining whole-body energy balance. However, the physiological functions of PPAR{alpha} in adipocytes have been unclarified. We examined the functions of PPAR{alpha} using human multipotent adipose tissue-derived stem cells as a human adipocyte model. Activation of PPAR{alpha} by GW7647, a potent PPAR{alpha} agonist, increased the mRNA expression levels of adipocyte differentiation marker genes such as PPAR{gamma}, adipocyte-specific fatty acid-binding protein, and lipoprotein lipase and increased both GPDH activity and insulin-dependent glucose uptake level. The findings indicate that PPAR{alpha} activation stimulates adipocyte differentiation. However, lipid accumulation was not changed, which is usually observed when PPAR{gamma} is activated. On the other hand, PPAR{alpha} activation by GW7647 treatment induced the mRNA expression of fatty acid oxidation-related genes such as CPT-1B and AOX in a PPAR{alpha}-dependent manner. Moreover, PPAR{alpha} activation increased the production of CO{sub 2} and acid soluble metabolites, which are products of fatty acid oxidation, and increased oxygen consumption rate in human adipocytes. The data indicate that activation of PPAR{alpha} stimulates both adipocyte differentiation and fatty acid oxidation in human adipocytes, suggesting that PPAR{alpha} agonists could improve insulin resistance without lipid accumulation in adipocytes. The expected effects of PPAR{alpha} activation are very valuable for managing diabetic conditions accompanied by obesity, because PPAR{gamma} agonists, usually used as antidiabetic drugs, induce excessive lipid accumulation in adipocytes in addition to improvement of insulin resistance.

  20. Adhesion of membranes via receptor-ligand complexes: Domain formation, binding cooperativity, and active processes

    E-print Network

    Thomas R. Weikl; Mesfin Asfaw; Heinrich Krobath; Bartosz Rozycki; Reinhard Lipowsky

    2009-06-09

    Cell membranes interact via anchored receptor and ligand molecules. Central questions on cell adhesion concern the binding affinity of these membrane-anchored molecules, the mechanisms leading to the receptor-ligand domains observed during adhesion, and the role of cytoskeletal and other active processes. In this review, these questions are addressed from a theoretical perspective. We focus on models in which the membranes are described as elastic sheets, and the receptors and ligands as anchored molecules. In these models, the thermal membrane roughness on the nanometer scale leads to a cooperative binding of anchored receptor and ligand molecules, since the receptor-ligand binding smoothens out the membranes and facilitates the formation of additional bonds. Patterns of receptor domains observed in Monte Carlo simulations point towards a joint role of spontaneous and active processes in cell adhesion. The interactions mediated by the receptors and ligand molecules can be characterized by effective membrane adhesion potentials that depend on the concentrations and binding energies of the molecules.

  1. Small-molecule nociceptin receptor agonist ameliorates mast cell activation and pain in sickle mice.

    PubMed

    Vang, Derek; Paul, Jinny A; Nguyen, Julia; Tran, Huy; Vincent, Lucile; Yasuda, Dennis; Zaveri, Nurulain T; Gupta, Kalpna

    2015-12-01

    Treatment of pain with morphine and its congeners in sickle cell anemia is suboptimal, warranting the need for analgesics devoid of side effects, addiction and tolerance liability. Small-molecule nociceptin opioid receptor ligands show analgesic efficacy in acute and chronic pain models. We show that AT-200, a high affinity nociceptin opioid receptor agonist with low efficacy at the mu opioid receptor, ameliorated chronic and hypoxia/reoxygenation-induced mechanical, thermal and deep tissue/musculoskeletal hyperalgesia in HbSS-BERK sickle mice. The antinociceptive effect of AT-200 was antagonized by SB-612111, a nociceptin opioid receptor antagonist, but not naloxone, a non-selective mu opioid receptor antagonist. Daily 7-day treatment with AT-200 did not develop tolerance and showed a sustained anti-nociceptive effect, which improved over time and led to reduced plasma serum amyloid protein, neuropeptides, inflammatory cytokines and mast cell activation in the periphery. These data suggest that AT-200 ameliorates pain in sickle mice via the nociceptin opioid receptor by reducing inflammation and mast cell activation without causing tolerance. Thus, nociceptin opioid receptor agonists are promising drugs for treating pain in sickle cell anemia. PMID:26294734

  2. Small-molecule nociceptin receptor agonist ameliorates mast cell activation and pain in sickle mice

    PubMed Central

    Vang, Derek; Paul, Jinny A.; Nguyen, Julia; Tran, Huy; Vincent, Lucile; Yasuda, Dennis; Zaveri, Nurulain T.; Gupta, Kalpna

    2015-01-01

    Treatment of pain with morphine and its congeners in sickle cell anemia is suboptimal, warranting the need for analgesics devoid of side effects, addiction and tolerance liability. Small-molecule nociceptin opioid receptor ligands show analgesic efficacy in acute and chronic pain models. We show that AT-200, a high affinity nociceptin opioid receptor agonist with low efficacy at the mu opioid receptor, ameliorated chronic and hypoxia/reoxygenation-induced mechanical, thermal and deep tissue/musculoskeletal hyperalgesia in HbSS-BERK sickle mice. The antinociceptive effect of AT-200 was antagonized by SB-612111, a nociceptin opioid receptor antagonist, but not naloxone, a non-selective mu opioid receptor antagonist. Daily 7-day treatment with AT-200 did not develop tolerance and showed a sustained anti-nociceptive effect, which improved over time and led to reduced plasma serum amyloid protein, neuropeptides, inflammatory cytokines and mast cell activation in the periphery. These data suggest that AT-200 ameliorates pain in sickle mice via the nociceptin opioid receptor by reducing inflammation and mast cell activation without causing tolerance. Thus, nociceptin opioid receptor agonists are promising drugs for treating pain in sickle cell anemia. PMID:26294734

  3. The subcellular localization of the receptor for platelet-activating factor in neutrophils affects signaling and activation characteristics.

    PubMed

    Andréasson, Emil; Önnheim, Karin; Forsman, Huamei

    2013-01-01

    The localization in neutrophils, of the receptor for platelet-activating factor (PAFR), has been determined using subcellular fractionation and a receptor mobilization protocol. We show that the PAFR is expressed primarily in the plasma membrane. Although activation of neutrophils by PAF induces responses typical also of agonists that bind the formyl peptide receptors (FPR), known to be stored in mobilizable organelles, some quantitative as well as qualitative differences were observed when neutrophils were activated through these receptors. PAF is equipotent to fMLF (high affinity agonist for FPR1) to cleave off L-selectin and to induce granule/vesicle secretion but is more potent than fMLF to induce a rise in intracellular Ca(2+). Similar to fMLF, PAF induced also a robust release of reactive oxygen species, but with higher EC50 value and was less sensitive to a PI3K inhibitor compared to the fMLF response. Despite the lack of a granule localized storage pool of receptors, the PAF-induced superoxide production could be primed; receptor mobilization was, thus, not required for priming of the PAF response. The desensitized PAFR could not be reactivated, suggesting that distinct signaling pathways are utilized for termination of the responses triggered through FPR1 and PAFR. PMID:24069041

  4. Evidence for the existence of a specific g?protein-coupled receptor activated by guanosine.

    PubMed

    Volpini, Rosaria; Marucci, Gabriella; Buccioni, Michela; Dal Ben, Diego; Lambertucci, Catia; Lammi, Carmen; Mishra, Ram C; Thomas, Ajiroghene; Cristalli, Gloria

    2011-06-01

    Guanosine, released extracellularly from neurons and glial cells, plays important roles in the central nervous system, including neuroprotection. The innovative DELFIA Eu-GTP binding assay was optimized for characterization of the putative guanosine receptor binding site at rat brain membranes by using a series of novel and known guanosine derivatives. These nucleosides were prepared by modifying the purine and sugar moieties of guanosine at the 6- and 5'-positions, respectively. Results of these experiments prove that guanosine, 6-thioguanosine, and their derivatives activate a G?protein-coupled receptor that is different from the well-characterized adenosine receptors. PMID:21500353

  5. How does high-frequency sound or vibration activate vestibular receptors?

    PubMed

    Curthoys, I S; Grant, J W

    2015-03-01

    The mechanism by which vestibular neural phase locking occurs and how it relates to classical otolith mechanics is unclear. Here, we put forward the hypothesis that sound and vibration both cause fluid pressure waves in the inner ear and that it is these pressure waves which displace the hair bundles on vestibular receptor hair cells and result in activation of type I receptor hair cells and phase locking of the action potentials in the irregular vestibular afferents, which synapse on type I receptors. This idea has been suggested since the early neural recordings and recent results give it greater credibility. PMID:25567092

  6. Ligand Activation of Overexpressed Epidermal Growth Factor Receptors Transforms NIH 3T3 Mouse Fibroblasts

    NASA Astrophysics Data System (ADS)

    Riedel, Heimo; Massoglia, Sharon; Schlessinger, Joseph; Ullrich, Axel

    1988-03-01

    The cell surface receptor for the mitogenic peptide epidermal growth factor (EGF) is involved in control of normal cell growth and may play a role in the genesis of human neoplasia such as squamous carcinoma and glioblastoma. Soft-agar growth and focus-formation experiments with NIH 3T3 mouse fibroblasts transfected with an expression plasmid demonstrated the ligand-dependent transforming potential of the human EGF receptor without structural alterations. Activation of overexpressed normal receptor alone appears to be sufficient for transformation of NIH 3T3 cells in vitro.

  7. Enhanced AMPA receptor activity increases operant alcohol self-administration and cue-induced reinstatement.

    PubMed

    Cannady, Reginald; Fisher, Kristen R; Durant, Brandon; Besheer, Joyce; Hodge, Clyde W

    2013-01-01

    Long-term alcohol exposure produces neuroadaptations that contribute to the progression of alcohol abuse disorders. Chronic alcohol consumption results in strengthened excitatory neurotransmission and increased ?-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPA) receptor signaling in animal models. However, the mechanistic role of enhanced AMPA receptor activity in alcohol-reinforcement and alcohol-seeking behavior remains unclear. This study examined the role of enhanced AMPA receptor function using the selective positive allosteric modulator, aniracetam, in modulating operant alcohol self-administration and cue-induced reinstatement. Male alcohol-preferring (P-) rats, trained to self-administer alcohol (15%, v/v) versus water were pre-treated with aniracetam to assess effects on maintenance of alcohol self-administration. To determine reinforcer specificity, P-rats were trained to self-administer sucrose (0.8%, w/v) versus water, and effects of aniracetam were tested. The role of aniracetam in modulating relapse of alcohol-seeking was assessed using a response contingent cue-induced reinstatement procedure in P-rats trained to self-administer 15% alcohol. Aniracetam pre-treatment significantly increased alcohol-reinforced responses relative to vehicle treatment. This increase was not attributed to aniracetam-induced hyperactivity as aniracetam pre-treatment did not alter locomotor activity. AMPA receptor involvement was confirmed because 6,7-dinitroquinoxaline-2,3-dione (AMPA receptor antagonist) blocked the aniracetam-induced increase in alcohol self-administration. Aniracetam did not alter sucrose-reinforced responses in sucrose-trained P-rats, suggesting that enhanced AMPA receptor activity is selective in modulating the reinforcing function of alcohol. Finally, aniracetam pre-treatment potentiated cue-induced reinstatement of alcohol-seeking behavior versus vehicle-treated P-rats. These data suggest that enhanced glutamate activity at AMPA receptors may be key in facilitating alcohol consumption and seeking behavior, which could ultimately contribute to the development of alcohol abuse disorders. PMID:23126443

  8. EGF receptor transactivation and MAP kinase mediate proteinase-activated receptor-2-induced chloride secretion in intestinal epithelial cells.

    PubMed

    van der Merwe, Jacques Q; Hollenberg, Morley D; MacNaughton, Wallace K

    2008-02-01

    We examined the stimulus-secretion pathways whereby proteinase-activated receptor 2 (PAR-2) stimulates Cl(-) secretion in intestinal epithelial cells. SCBN and T84 epithelial monolayers grown on Snapwell supports and mounted in modified Ussing chambers were activated by the PAR-2-activating peptides SLIGRL-NH(2) and 2-furoyl-LIGRLO-NH(2). Short-circuit current (I(sc)) was used as a measure of net electrogenic ion transport. Basolateral, but not apical, application of SLIGRL-NH(2) or 2-furoyl-LIGRLO-NH(2) caused a concentration-dependent change in I(sc) that was significantly reduced in Cl(-)-free buffer and by the intracellular Ca(2+) blockers thapsigargin and BAPTA-AM, but not by the Ca(2+) channel blocker verapamil. Inhibitors of PKA (H-89) and CFTR (glibenclamide) also significantly reduced PAR-2-stimulated Cl(-) transport. PAR-2 activation was associated with increases in cAMP and intracellular Ca(2+). Immunoblot analysis revealed increases in phosphorylation of epidermal growth factor (EGF) receptor (EGFR) tyrosine kinase, Src, Pyk2, cRaf, and ERK1/2 in response to PAR-2 activation. Pretreatment with inhibitors of cyclooxygenases (indomethacin), tyrosine kinases (genistein), EGFR (PD-153035), MEK (PD-98059 or U-0126), and Src (PP1) inhibited SLIGRL-NH(2)-induced increases in I(sc). Inhibition of Src, but not matrix metalloproteinases, reduced EGFR phosphorylation. Reduced EGFR phosphorylation paralleled the reduction in PAR-2-stimulated I(sc). We conclude that activation of basolateral, but not apical, PAR-2 induces epithelial Cl(-) secretion via cAMP- and Ca(2+)-dependent mechanisms. The secretory effect involves EGFR transactivation by Src, leading to subsequent ERK1/2 activation and increased cyclooxygenase activity. PMID:18032480

  9. Synergistic activation of estrogen receptor with combinations of environmental chemicals

    SciTech Connect

    Arnold, S.F.; Klotz, D.M.; Collins, B.M.

    1996-06-07

    Certain chemicals in the environment are estrogenic. The low potencies of the compounds, when studied singly, suggest that they may have little effect on biological systems. The estrogenic potencies of combinations of such chemicals were screened in a simple yeast estrogen potencies of combination of such chemicals were screened in a simple yeast estrogen systems (YES) containing human estrogen receptor (hER). Combinations of two weak environmental estrogens, such as dieldrin, endosulfan, or toxaphene, were 100 times as potent in hER-mediated transactivation as any chemical alone. Hydroxylated polychlorinated biphenyls shown previously to synergistically alter sexual development in turtles also synergized in the YES. The synergistic interaction of chemical mixtures with the estrogen receptor may have profound environmental implications. These results may represent a previously uncharacterized level of regulation of estrogen-associated responses. 32 refs., 3 figs., 3 tabs.

  10. Activation of NTS A(1) adenosine receptors inhibits regional sympathetic responses evoked by activation of cardiopulmonary chemoreflex.

    PubMed

    Ichinose, Tomoko K; Minic, Zeljka; Li, Cailian; O'Leary, Donal S; Scislo, Tadeusz J

    2012-09-01

    Previously we have shown that adenosine operating via the A(1) receptor subtype may inhibit glutamatergic transmission in the baroreflex arc within the nucleus of the solitary tract (NTS) and differentially increase renal (RSNA), preganglionic adrenal (pre-ASNA), and lumbar (LSNA) sympathetic nerve activity (ASNA>RSNA?LSNA). Since the cardiopulmonary chemoreflex and the arterial baroreflex are mediated via similar medullary pathways, and glutamate is a primary transmitter in both pathways, it is likely that adenosine operating via A(1) receptors in the NTS may differentially inhibit regional sympathetic responses evoked by activation of cardiopulmonary chemoreceptors. Therefore, in urethane-chloralose-anesthetized rats (n = 37) we compared regional sympathoinhibition evoked by the cardiopulmonary chemoreflex (activated with right atrial injections of serotonin 5HT(3) receptor agonist phenylbiguanide, PBG, 1-8 ?g/kg) before and after selective stimulation of NTS A(1) adenosine receptors [microinjections of N(6)-cyclopentyl adenosine (CPA), 0.033-330 pmol/50 nl]. Activation of cardiopulmonary chemoreceptors evoked differential, dose-dependent sympathoinhibition (RSNA>ASNA>LSNA), and decreases in arterial pressure and heart rate. These differential sympathetic responses were uniformly attenuated in dose-dependent manner by microinjections of CPA into the NTS. Volume control (n = 11) and blockade of adenosine receptor subtypes in the NTS via 8-(p-sulfophenyl)theophylline (8-SPT, 1 nmol in 100 nl) (n = 9) did not affect the reflex responses. We conclude that activation of NTS A(1) adenosine receptors uniformly inhibits neural and cardiovascular cardiopulmonary chemoreflex responses. A(1) adenosine receptors have no tonic modulatory effect on this reflex under normal conditions. However, when adenosine is released into the NTS (i.e., during stress or severe hypotension/ischemia), it may serve as negative feedback regulator for depressor and sympathoinhibitory reflexes integrated in the NTS. PMID:22814665

  11. Structural plasticity of GABAergic axons is regulated by network activity and GABAA receptor activation

    PubMed Central

    Schuemann, Anne; Klawiter, Agnieszka; Bonhoeffer, Tobias; Wierenga, Corette J.

    2013-01-01

    Coordinated changes at excitatory and inhibitory synapses are essential for normal brain development and function. It is well established that excitatory neurons undergo structural changes, but our knowledge about inhibitory structural plasticity is rather scarce. Here we present a quantitative analysis of the dynamics of GABAergic boutons in the dendritic region of the hippocampal CA1 area using time-lapse two-photon imaging in organotypic hippocampal cultures from GAD65-GFP mice. We show that ~20% of inhibitory boutons are not stable. They are appearing, disappearing and reappearing at specific locations along the inhibitory axon and reflect immature or incomplete synapses. Furthermore, we observed that persistent boutons show large volume fluctuations over several hours, suggesting that presynaptic content of inhibitory synapses is not constant. Our data show that inhibitory boutons are highly dynamic structures and suggest that inhibitory axons are continuously probing potential locations for inhibitory synapse formation by redistributing presynaptic material along the axon. In addition, we found that neuronal activity affects the exploratory dynamics of inhibitory axons. Blocking network activity rapidly reduces the number of transient boutons, whereas enhancing activity reduces the number of persistent inhibitory boutons, possibly reflecting enhanced competition between boutons along the axon. The latter effect requires signaling through GABAA receptors. We propose that activity-dependent regulation of bouton dynamics contributes to inhibitory synaptic plasticity. PMID:23805077

  12. Laminin-?2 Chain-Deficient Congenital Muscular Dystrophy: Pathophysiology and Development of Treatment.

    PubMed

    Durbeej, Madeleine

    2015-01-01

    Laminin-211 is a major constituent of the skeletal muscle basement membrane. It stabilizes skeletal muscle and influences signal transduction events from the myomatrix to the muscle cell. Mutations in the gene encoding the ?2 chain of laminin-211 lead to congenital muscular dystrophy type 1A (MDC1A), a life-threatening disease characterized by severe hypotonia, progressive muscle weakness, and joint contractures. Common complications include severely impaired motor ability, respiratory failure, and feeding difficulties. Several adequate animal models for laminin-?2 chain deficiency exist and analyses of different MDC1A mouse models have led to a significant improvement in our understanding of MDC1A pathogenesis. Importantly, the animal models have been indispensable tools for the preclinical development of new therapeutic approaches for laminin-?2 chain deficiency, highlighting a number of important disease driving mechanisms that can be targeted by pharmacological approaches. In this chapter, I will describe laminin-211 and discuss the cellular and molecular pathophysiology of MDC1A as well as progression toward development of treatment. PMID:26610911

  13. T-cell receptor-induced JNK activation requires proteolytic inactivation of CYLD by MALT1

    PubMed Central

    Staal, Jens; Driege, Yasmine; Bekaert, Tine; Demeyer, Annelies; Muyllaert, David; Van Damme, Petra; Gevaert, Kris; Beyaert, Rudi

    2011-01-01

    The paracaspase mucosa-associated lymphoid tissue 1 (MALT1) is central to lymphocyte activation and lymphomagenesis. MALT1 mediates antigen receptor signalling to NF-?B by acting as a scaffold protein. Furthermore, MALT1 has proteolytic activity that contributes to optimal NF-?B activation by cleaving the NF-?B inhibitor A20. Whether MALT1 protease activity is involved in other signalling pathways, and the identity of the relevant substrates, is unknown. Here, we show that T-cell receptors (TCR) activation, as well as overexpression of the oncogenic API2–MALT1 fusion protein, results in proteolytic inactivation of CYLD by MALT1, which is specifically required for c-jun N-terminal kinase (JNK) activation and the inducible expression of a subset of genes. These results indicate a novel role for MALT1 proteolytic activity in TCR-induced JNK activation and reveal CYLD cleavage as the underlying mechanism. PMID:21448133

  14. Novel Oxazolidinone-Based Peroxisome Proliferator Activated Receptor Agonists: Molecular Modeling, Synthesis, and Biological Evaluation.

    PubMed

    Fresno, N; Macías-González, M; Torres-Zaguirre, A; Romero-Cuevas, M; Sanz-Camacho, P; Elguero, J; Pavón, F J; Rodríguez de Fonseca, F; Goya, P; Pérez-Fernández, R

    2015-08-27

    A series of new peroxisome proliferator activated receptors (PPARs) chiral ligands have been designed following the accepted three-module structure comprising a polar head, linker, and hydrophobic tail. The majority of the ligands incorporate the oxazolidinone moiety as a novel polar head, and the nature of the hydrophobic tail has also been varied. Docking studies using the crystal structure of an agonist bound to the ligand binding domain of the PPAR? receptor have been performed as a tool for their design. Suitable synthetic procedures have been developed, and compounds with different stereochemistries have been prepared. Evaluation of basal and ligand-induced activity proved that several compounds showed agonist activity at the PPAR? receptor, thus validating the oxazolidinone template for PPAR activity. In addition, two compounds, 2 and 4, showed dual PPAR?/PPAR? agonism and interesting food intake reduction in rats. PMID:26226490

  15. Selective ligand behaviors provide new insights into agonist activation of nicotinic acetylcholine receptors.

    PubMed

    Marotta, Christopher B; Rreza, Iva; Lester, Henry A; Dougherty, Dennis A

    2014-05-16

    Nicotinic acetylcholine receptors are a diverse set of ion channels that are essential to everyday brain function. Contemporary research studies selective activation of individual subtypes of receptors, with the hope of increasing our understanding of behavioral responses and neurodegenerative diseases. Here, we aim to expand current binding models to help explain the specificity seen among three activators of ?4?2 receptors: sazetidine-A, cytisine, and NS9283. Through mutational analysis, we can interchange the activation profiles of the stoichiometry-selective compounds sazetidine-A and cytisine. In addition, mutations render NS9283--currently identified as a positive allosteric modulator--into an agonist. These results lead to two conclusions: (1) occupation at each primary face of an ? subunit is needed to activate the channel and (2) the complementary face of the adjacent subunit dictates the binding ability of the agonist. PMID:24564429

  16. Effects of LIF dose and laminin plus fibronectin on axotomized sciatic nerves.

    PubMed

    Dowsing, B J; Hayes, A; Bennett, T M; Morrison, W A; Messina, A

    2000-09-01

    Leukemia inhibitory factor (LIF), a cytokine which has neurotrophic and myotrophic activities, has been shown to enhance nerve regeneration and consequent return of muscle function in the entubulation model of sciatic nerve repair. Fibronectin (FN) and laminin (LN) are two extracellular matrix (ECM) components that, when combined, promote axon growth in the entubulation model. The aim of this study was to determine the optimal LIF dose and the efficacy of FN plus LN administered either alone or simultaneously with the optimal LIF dose. We found that at 12 weeks following nerve repair, a single 10 ng LIF dose produced the largest medial gastrocnemius (MG) muscle mass (P < 0.0001) and maximum force contraction (P < 0.001). The diameter of the axons in the FN plus LN group were significantly greater than for saline (P < 0.001) and the LIF dose groups (P < 0.01). When 10 ng LIF was combined with FN plus LN, the MG muscle mass was significantly greater than the optimal LIF dose (P < 0.05), suggesting an additive effect. Our findings support the view that combinations of factors, which perhaps act on complementary mechanisms for nerve regeneration, will be required to maximally potentiate nerve regeneration and return of muscle function after nerve injury. PMID:10951438

  17. 2012LandesBioscience.Donotdistribute. Study of streptococcal hemoprotein receptor

    E-print Network

    Nizet, Victor

    acquisition and virulence of M1T1 group A streptococcus Samira Dahesh,1 Victor Nizet1,2,3 and Jason N. Cole1: hemoprotein, iron acquisition, iron starvation, laminin-binding, Shr, Streptococcus pyogenes, virulence, whole blood Abbreviations: GAS, group A streptococcus; Shr, streptococcal hemoprotein receptor; Sia

  18. Identification of functionally important residues in the silkmoth pheromone biosynthesis-activating neuropeptide receptor, an insect ortholog of the vertebrate Neuromedin U Receptor

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The biosynthesis of sex pheromone components in many lepidopteran insects is regulated by interactions between pheromone biosynthesis-activating neuropeptide (PBAN) and the PBAN receptor (PBANR), a class-A G-protein-coupled receptor (GPCR). To identify functionally important amino acid residues in t...

  19. Asymmetric Cleavage of B-Carotene Yields a Transcriptional Repressor of Retinoid X Receptor and Peroxisome Proliferator-Activated Receptor Responses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    B-Carotene and its metabolites exert a broad range of effects, in part by regulating transcriptional responses through specific nuclear receptor activation. Symmetric cleavage of B-carotene can yield 9-cis retinoic acid (9-cisRA), the natural ligand for the nuclear receptor RXR, the obligate heterod...

  20. Human scavenger receptor class B type 1 is regulated by activators of peroxisome proliferators-activated receptor-gamma in hepatocytes.

    PubMed

    Ahmed, Rania Abdel Muneem; Murao, Koji; Imachi, Hitomi; Yu, Xiao; Li, Junhun; Wong, Norman C W; Ishida, Toshihiko

    2009-04-01

    High-density lipoprotein (HDL) particles play a critical role in cholesterol metabolism. The hepatic scavenger receptor class B type I (SR-B1) binds HDL particles for mediating reverse cholesterol transport (RCT), thus lowering the risk of atherosclerosis. Thiazolidinediones (TZDs), known to have potent enhancing effects on insulin sensitivity, have been developed for the treatment of non-insulin-dependent diabetes mellitus. They are a high-affinity ligand for the peroxisome proliferator-activated receptor gamma (PPAR-gamma), which belongs to a nuclear receptor superfamily. In this study, we examined the effects of thiazolidinedione PPAR-gamma on hepatic SR-B1 gene expression in human hepatoma G2 cell-line (HepG2). Results showed that hepatic SR-B1 mRNA and protein were increased on exposure to thiazolidinediones. Transcriptional activity of human SR-B1 (hSR-B1) gene paralleled the endogenous expression of the gene and was dependent on the dose of thiazolidinediones. We investigated the influence on the promoter activity of vector expressing PPAR and retinoid X receptor (RXR), cotransfected into the HepG2 cells along with SR-B1 promoter-reporter gene constructs. PPAR-gamma and RXR sufficiently induced the SR-B1 promoter activity in the HepG2 cells. Chromatin immunoprecipitation (ChIP) assay confirmed the binding of the PPAR-gamma to the SR-B1 promoter region. The mutagenesis of this binding site abolished the ability of the thiazolidinediones or PPARs to stimulate promoter activity. Together, these results indicate that the stimulation of SR-B1 expression in the liver is mediated in part by activation of the PPAR-gamma and RXR, and raise the possibility that this stimulation using thiazolidinediones conditions provides a protective mechanism for accelerated atherosclerosis in diabetes mellitus. PMID:19156545

  1. Induction of Direct Antimicrobial Activity Through Mammalian Toll-Like Receptors

    NASA Astrophysics Data System (ADS)

    Thoma-Uszynski, Sybille; Stenger, Steffen; Takeuchi, Osamu; Ochoa, Maria Teresa; Engele, Matthias; Sieling, Peter A.; Barnes, Peter F.; Röllinghoff, Martin; Bölcskei, Pal L.; Wagner, Manfred; Akira, Shizuo; Norgard, Michael V.; Belisle, John T.; Godowski, Paul J.; Bloom, Barry R.; Modlin, Robert L.

    2001-02-01

    The mammalian innate immune system retains from Drosophila a family of homologous Toll-like receptors (TLRs) that mediate responses to microbial ligands. Here, we show that TLR2 activation leads to killing of intracellular Mycobacterium tuberculosis in both mouse and human macrophages, through distinct mechanisms. In mouse macrophages, bacterial lipoprotein activation of TLR2 leads to a nitric oxide-dependent killing of intracellular tubercle bacilli, but in human monocytes and alveolar macrophages, this pathway was nitric oxide-independent. Thus, mammalian TLRs respond (as Drosophila Toll receptors do) to microbial ligands and also have the ability to activate antimicrobial effector pathways at the site of infection.

  2. Molecular Mechanisms of Antiseizure Drug Activity at GABAA Receptors

    PubMed Central

    Greenfield, L. John

    2013-01-01

    The GABAA receptor (GABAAR) is a major target of antiseizure drugs (ASDs). A variety of agents that act at GABAARs s are used to terminate or prevent seizures. Many act at distinct receptor sites determined by the subunit composition of the holoreceptor. For the benzodiazepines, barbiturates, and loreclezole, actions at the GABAAR are the primary or only known mechanism of antiseizure action. For topiramate, felbamate, retigabine, losigamone and stiripentol, GABAAR modulation is one of several possible antiseizure mechanisms. Allopregnanolone, a progesterone metabolite that enhances GABAAR function, led to the development of ganaxolone. Other agents modulate GABAergic “tone” by regulating the synthesis, transport or breakdown of GABA. GABAAR efficacy is also affected by the transmembrane chloride gradient, which changes during development and in chronic epilepsy. This may provide an additional target for “GABAergic” ASDs. GABAAR subunit changes occur both acutely during status epilepticus and in chronic epilepsy, which alter both intrinsic GABAAR function and the response to GABAAR-acting ASDs. Manipulation of subunit expression patterns or novel ASDs targeting the altered receptors may provide a novel approach for seizure prevention. PMID:23683707

  3. Carbonyl stress and NMDA receptor activation contribute to methylglyoxal neurotoxicity.

    PubMed

    de Arriba, Susana Garcia; Krügel, Ute; Regenthal, Ralf; Vissiennon, Zacharie; Verdaguer, Esther; Lewerenz, Anne; García-Jordá, Elvira; Pallas, Mercé; Camins, Antoni; Münch, Gerald; Nieber, Karen; Allgaier, Clemens

    2006-03-01

    Methylglyoxal (MG) is a reactive alpha-ketoaldehyde physiologically generated as a by-product of glycolysis. MG that is able to form protein adducts resulting in advanced glycation end products accumulates under conditions associated with neurodegeneration such as impaired glucose metabolism or oxidative stress. In the present study, short-term exposure of human neuroblastoma SH-SY5Y cells to MG was associated with an early depolarization of the plasma membrane, glutamate release, and formation of reactive oxygen species. In addition, long-term exposure (24 h) of SH-SY5Y cells to MG caused a decrease in cell viability, intracellular ATP, and rhodamine 123 (Rh-123) fluorescence. ATP depletion and the decrease in Rh-123 fluorescence were prevented by carbonyl scavengers, the nitric oxide synthase inhibitor L-NAME, and N-methyl-d-aspartate (NMDA) receptor antagonists. Furthermore, the MG-induced glutamate release and the loss in cell viability were prevented by NMDA receptor antagonists. Therefore, MG renders cells more vulnerable to excitotoxicity. In conclusion, carbonyl scavengers as well as NMDA receptor antagonists may represent effective therapeutic tools to reduce the risk of pathophysiological changes associated with carbonyl stress in neurodegenerative diseases. PMID:16520230

  4. Melittin modulates keratinocyte function through P2 receptor-dependent ADAM activation.

    PubMed

    Sommer, Anselm; Fries, Anja; Cornelsen, Isabell; Speck, Nancy; Koch-Nolte, Friedrich; Gimpl, Gerald; Andrä, Jörg; Bhakdi, Sucharit; Reiss, Karina

    2012-07-01

    Melittin, the major component of the bee venom, is an amphipathic, cationic peptide with a wide spectrum of biological properties that is being considered as an anti-inflammatory and anti-cancer agent. It modulates multiple cellular functions but the underlying mechanisms are not clearly understood. Here, we report that melittin activates disintegrin-like metalloproteases (ADAMs) and that downstream events likely contribute to the biological effects evoked by the peptide. Melittin stimulated the proteolysis of ADAM10 and ADAM17 substrates in human neutrophil granulocytes, endothelial cells and murine fibroblasts. In human HaCaT keratinocytes, melittin induced shedding of the adhesion molecule E-cadherin and release of TGF-?, which was accompanied by transactivation of the EGF receptor and ERK1/2 phosphorylation. This was followed by functional consequences such as increased keratinocyte proliferation and enhanced cell migration. Evidence is provided that ATP release and activation of purinergic P2 receptors are involved in melittin-induced ADAM activation. E-cadherin shedding and EGFR phosphorylation were dose-dependently reduced in the presence of ATPases or P2 receptor antagonists. The involvement of P2 receptors was underscored in experiments with HEK cells, which lack the P2X7 receptor and showed strikingly increased response to melittin stimulation after transfection with this receptor. Our study provides new insight into the mechanism of melittin function which should be of interest particularly in the context of its potential use as an anti-inflammatory or anti-cancer agent. PMID:22613720

  5. Agonists and protein kinase C-activation induce phosphorylation and internalization of FFA1 receptors.

    PubMed

    Sosa-Alvarado, Carla; Hernández-Méndez, Aurelio; Romero-Ávila, M Teresa; Sánchez-Reyes, Omar B; Takei, Yoshinori; Tsujimoto, Gozoh; Hirasawa, Akira; García-Sáinz, J Adolfo

    2015-12-01

    FFA1 (previously known as GPR40) is a free fatty acid receptor involved in the regulation of inflammatory processes and insulin secretion. The cellular actions resulting from FFA1 activation have received considerable attention. However, little is known on the regulation of the receptor function. In the present work, using cells transfected with this receptor, docosahexaenoic acid and ?-linolenic acid increased intracellular calcium concentration and ERK 1/2 phosphorylation. It was also observed that FFA1 is a phosphoprotein whose phosphorylation state was increased (2- to 3-fold) by agonists (i.e., free fatty acids) and also by phorbol myristate acetate. Agonist- and phorbol ester-mediated FFA1 phosphorylation was markedly reduced by inhibitors of protein kinase C. Receptor stimulation by free fatty acids and protein kinase C activation also induced receptor internalization as evidenced by confocal microscopy. In summary, our data show that FFA1 is a phosphoprotein whose phosphorylation state is modulated by agonists and protein kinase C activation; such covalent modification is associated with receptor internalization. PMID:26526350

  6. Copyright 2001 by the Genetics Society of America Genes Affecting the Activity of Nicotinic Receptors Involved in

    E-print Network

    Schafer, William R.

    Copyright © 2001 by the Genetics Society of America Genes Affecting the Activity of Nicotinic of nicotinic acetylcholine receptors such as nicotine and levamisole stimulate egg laying; however, the genetic of levamisole-sensitive nicotinic receptors in nematodes. Seven of these genes, including the nicotinic receptor

  7. Activation of myeloid dendritic cells by deoxynucleic acids from Cordyceps sinensis via a Toll-like receptor 9-dependent pathway

    E-print Network

    Huang, Ching-Tsan

    -derived dendritic cells) IL-12p40 TNF- CD40(endocytosis)chloroquinbafilomycin ADNADNA TLR9(toll-like receptor 9 [2] [3] Toll-like receptor TLR9 DNA[4] [4] DNA [4] DNA (1)DNA (2)DNA (3)DNA TLR9 Cp. Kawakami, Toll-like receptor 9-dependent activation of myeloid dendritic cells by deoxynucleic acids from

  8. Constitutive Activity of a Chimeric D2/D1 Dopamine Receptor LAURA B. KOZELL and KIM A. NEVE

    E-print Network

    Neve, Kim A.

    Constitutive Activity of a Chimeric D2/D1 Dopamine Receptor LAURA B. KOZELL and KIM A. NEVE nonselective dopamine antagonists and had affinities for most of the selec- tive antagonists that were intermediate between those of the parent receptors. D2[1­ 4,7] and D2[1­5,7] mediated dopamine receptor agonist

  9. Endothelin-1 activates ETA receptors to cause reflex scratching in BALB/c mice

    PubMed Central

    McQueen, D S; Noble, M A H; Bond, S M

    2007-01-01

    Background and purpose: Endothelin-1 (ET-1) is present in murine and human skin and causes itch (pruritus) when injected in humans. This behavioural study examined the scratch reflex evoked by ET-1 in mice. Experimental approach: An automated detector was used to determine whether ET-1 causes reflex scratching, the behavioural correlate of itching, in BALB/c mice. Selective agonists and antagonists were used to probe the ET receptor(s) involved. Key results: ET-1 evoked dose-related reflex scratching lasting up to 20?min following intradermal injection (0.1-100?ng; 0.04-40?pmol). The ED50 for ET-1 induced scratching was 2.1?ng and desensitization occurred with cumulative dosing. High doses of the ETB receptor agonist IRL1620 (10??g; 5.5?nmol), also caused scratching (ED50 1.3 ?g, 0.7 nmol). The ETA receptor antagonist BQ123 significantly reduced scratching evoked by ET-1 and IRL 1620, suggesting that both agonists caused scratching via an ETA receptor-dependent mechanism. The ETB receptor antagonist BQ788 significantly reduced scratching evoked by IRL1620 but had no effect on scratching evoked by ET-1. This indicated that activation of ETB receptors by high doses of ETB agonist, but not ET-1, can trigger scratching. Conclusion and implications: ET-1 is a potent endogenous activator of reflex scratching (itch). Mechanisms for ET-induced scratching are considered, including direct action of ET-1 on pruriceptive nerve endings and indirect actions via release of endogenous mediators such as histamine from mast cells. ET-1 and ETA receptors, possibly also ETB receptors, are potential targets for developing specific anti-pruritic drugs to treat pruritic skin disorders such as atopic dermatitis. PMID:17351652

  10. An Fc-receptor activity of plasma membranes from guinea-pig peritoneal polymorphonuclear leucocytes.

    PubMed Central

    Tsunawaki, S; Mizuno, D; Kakinuma, K; Kasahara, M

    1982-01-01

    Plasma membranes prepared from guinea-pig peritoneal polymorphonuclear leucocytes showed an immune-complex-binding activity that corresponded well with the activity in intact cells. The characteristics of this activity were reversible binding, dependence on the Fc portion of antigen-complexed IgG (immunoglobulin G), competition with aggregated IgG and independence from energy metabolism. These results support the conclusion that the binding activity found in the isolated plasma membranes is an Fc-receptor activity of guinea-pig peritoneal polymorphonuclear leucocytes. The activity showed Kd = 6.7 x 10(-8) M-IgG and maximum binding of 17 pmol of IgG/mg of membrane protein when measured with an immune complex of alpha-amylase and homologous guinea-pig anti-(alpha-amylase) IgG. Inhibition of the Fc-receptor activity by a series of various salts indicated the contribution of the hydrophobic interaction to the binding. Inhibitory effects of salts or metal-chelating reagents on the Fc-receptor activity were also observed on superoxide generation by these cells induced by the immune complex, suggesting a role of the Fc receptor as the immune-complex-binding site responsible for the initiation of superoxide generation. PMID:6289800

  11. Developmental toxicity of perfluorononanoic acid is dependent on peroxisome proliferator activated receptor-alpha.

    EPA Science Inventory

    Perfluorononanoic acid (PFNA) is one of the predominant perfluoroalkyl acids in the environment and in tissues of humans and wildlife. PFNA strongly activates the mouse and human peroxisome proliferator-activated receptor-alpha (PPAR?) in vitro and negatively impacts development ...

  12. Novel indole and azaindole (pyrrolopyridine) cannabinoid (CB) receptor agonists: design, synthesis, structure-activity relationships, physicochemical properties and biological activity.

    PubMed

    Blaazer, Antoni R; Lange, Jos H M; van der Neut, Martina A W; Mulder, Arie; den Boon, Femke S; Werkman, Taco R; Kruse, Chris G; Wadman, Wytse J

    2011-10-01

    The discovery, synthesis and structure-activity relationship (SAR) of a novel series of cannabinoid 1 (CB(1)) and cannabinoid 2 (CB(2)) receptor ligands are reported. Based on the aminoalkylindole class of cannabinoid receptor agonists, a biphenyl moiety was introduced as novel lipophilic indole 3-acyl substituent in 11-16. Furthermore, the 3-carbonyl tether was replaced with a carboxamide linker in 17-20 and the azaindole (pyrrolopyridine) nucleus was designed as indole bioisostere with improved physicochemical properties in 21-25. Through these SAR efforts, several high affinity CB(1)/CB(2) dual cannabinoid receptor ligands were identified. Indole-3-carboxamide 17 displayed single-digit nanomolar affinity and ~80 fold selectivity for CB(1) over the CB(2) receptor. The azaindoles displayed substantially improved physicochemical properties (lipophilicity; aqueous solubility). Azaindole 21 elicited potent cannabinoid activity. Cannabinoid receptor agonists 17 and 21 potently modulated excitatory synaptic transmission in an acute rat brain slice model of cannabinoid receptor-modulated neurotransmission. PMID:21885167

  13. ALK receptor activation, ligands and therapeutic targeting in glioblastoma and in other cancers

    PubMed Central

    Wellstein, Anton

    2012-01-01

    The intracellular anaplastic lymphoma kinase (ALK) fragment shows striking homology with members of the insulin receptor family and was initially identified as an oncogenic fusion protein resulting from a translocation in lymphoma and more recently in a range of cancers. The full-length ALK transmembrane receptor of ~220 kDa was identified based on this initial work. This tyrosine kinase receptor and its ligands, the growth factors pleiotrophin (PTN) and midkine (MK) are highly expressed during development of the nervous system and other organs. Each of these genes has been implicated in malignant progression of different tumor types and shown to alter phenotypes as well as signal transduction in cultured normal and tumor cells. Beyond its role in cancer, the ALK receptor pathway is thought to contribute to nervous system development, function, and repair, as well as metabolic homeostasis and the maintenance of tissue regeneration. ALK receptor activity in cancer can be up-regulated by amplification, overexpression, ligand binding, mutations in the intracellular domain of the receptor and by activity of the receptor tyrosine phosphatase PTPRz. Here we discuss the evidence for ligand control of ALK activity as well as the potential prognostic and therapeutic implications from gene expression and functional studies. An analysis of 18 published gene expression data sets from different cancers shows that overexpression of ALK, its smaller homolog LTK (leukocyte tyrosine kinase) and the ligands PTN and MK in cancer tissues from patients correlate significantly with worse course and outcome of the disease. This observation together with preclinical functional studies suggests that this pathway could be a valid therapeutic target for which complementary targeting strategies with small molecule kinase inhibitors as well as antibodies to ligands or the receptors may be used. PMID:23267434

  14. Engagement of signaling pathways of protease-activated receptor 2 and ?-opioid receptor in bone cancer pain and morphine tolerance.

    PubMed

    Bao, Yanju; Gao, Yebo; Hou, Wei; Yang, Liping; Kong, Xiangying; Zheng, Honggang; Li, Conghuang; Hua, Baojin

    2015-09-15

    Pain is one of the most common and distressing symptoms suffered by patients with progression of cancer. Using a rat model of bone cancer, recent findings suggest that proteinase-activated receptor 2 (PAR2) signaling pathways contribute to neuropathic pain and blocking PAR2 amplifies antinociceptive effects of systemic morphine. The purpose of our study was to examine the underlying mechanisms responsible for the role of PAR2 in regulating bone cancer-evoked pain and the tolerance of systemic morphine. Breast sarcocarcinoma Walker 256 cells were implanted into the tibia bone cavity of rats and this evoked significant mechanical and thermal hyperalgesia. Our results showed that the protein expression of PAR2 and its downstream pathways (protein kinases namely, PKC? and PKA) and transient receptor potential vanilloid 1 (TRPV1) were amplified in the dorsal horn of the spinal cord of bone cancer rats compared to control rats. Blocking spinal PAR2 by using FSLLRY-NH2 significantly attenuated the activities of PKC?/PKA signaling pathways and TRPV1 expression as well as mechanical and thermal hyperalgesia. Also, inhibition of PKC?/PKA and TRPV1 significantly diminished the hyperalgesia observed in bone cancer rats. Additionally, blocking PAR2 enhanced the attenuations of PKC?/PKA and cyclic adenosine monophosphate induced by morphine and further extended analgesia of morphine via ?-opioid receptor (MOR). Our data revealed specific signaling pathways, leading to bone cancer pain, including the activation of PAR2, downstream PKC?/PKA, TRPV1 and resultant sensitization of MOR. Targeting one or more of these signaling molecules may present new opportunities for treatment and management of bone cancer pain often observed in clinics. PMID:25708385

  15. Review of the expression of Peroxisome Proliferator Activated Receptors alpha (PPAR?), beta (PPAR ?), and gamma (PPAR() in rodent and human development.

    EPA Science Inventory

    The peroxisome proliferator-activated receptors (PPAR) belong to the nuclear hormone receptor superfamily and there are three primary isotypes, PPAR?, ?, and (. These receptors regulate important physiological processes that impact lipid homeostasis, inflammation, adipogenesis, r...

  16. Exercise reduces adipose tissue via cannabinoid receptor type 1 which is regulated by peroxisome proliferator-activated receptor-{delta}

    SciTech Connect

    Yan Zhencheng; Liu Daoyan; Zhang Lili; Shen Chenyi; Ma Qunli; Cao Tingbing; Wang Lijuan; Nie Hai; Zidek, Walter; Tepel, Martin; Zhu Zhiming . E-mail: zhuzm@yahoo.com

    2007-03-09

    Obesity is one major cardiovascular risk factor. We tested effects of endurance exercise on cannabinoid receptor type 1 (CB1) and peroxisome proliferator-activated receptor-{delta} (PPAR-{delta})-dependent pathways in adipose tissue. Male Wistar rats were randomly assigned to standard laboratory chow or a high-fat diet without and with regular endurance exercise. Exercise in rats on high-fat diet significantly reduced visceral fat mass, blood pressure, and adipocyte size (each p < 0.05). Adipocyte hypertrophy induced by high-fat diet was accompanied by increased CB1 expression in adipose tissue, whereas exercise significantly reduced CB1 expression (each p < 0.05). CB1 receptor expression and adipocyte differentiation were directly regulated by PPAR-{delta}. Adipocyte hypertrophy induced by high-fat diet was accompanied by reduced PPAR-{delta}. Furthermore, selective silencing of PPAR-{delta} by RNA interference in 3T3-L1-preadipocyte cells significantly increased CB1 expression from 1.00 {+-} 0.06 (n = 3) to 1.91 {+-} 0.06 (n = 3; p < 0.01) and increased adipocyte differentiation, whereas adenovirus-mediated overexpression of PPAR-{delta} significantly reduced CB1 expression to 0.39 {+-} 0.03 (n = 3; p < 0.01) and reduced adipocyte differentiation. In the presence of the CB1 antagonist rimonabant adipocyte differentiation in stimulated 3T3 L1 preadipocyte cells was significantly reduced. The study indicates that high-fat diet-induced hypertrophy of adipocytes is associated with increased CB1 receptor expression which is directly regulated by PPAR-{delta}. Both CB1 and PPAR-{delta} are intimately involved in therapeutic interventions against a most important cardiovascular risk factor.

  17. Propofol depresses cerebellar Purkinje cell activity via activation of GABAA and glycine receptors in vivo in mice.

    PubMed

    Jin, Ri; Liu, Heng; Jin, Wen-Zhe; Shi, Jin-Di; Jin, Qing-Hua; Chu, Chun-Ping; Qiu, De-Lai

    2015-10-01

    Propofol is an intravenous sedative-hypnotic agen, which causes rapid and reliable loss of consciousness. Under in vitro conditions, propofol activates GABAA and glycine receptors in spinal cord, hippocampus and hypothalamus neurons. However, the effects of propofol on the cerebellar neuronal activity under in vivo conditions are currently unclear. In the present study, we examined the effects of propofol on the spontaneous activity of Purkinje cells (PCs) in urethane-anesthetized mice by cell-attached recording and pharmacological methods. Our results showed that cerebellar surface perfusion of propofol (10-1000?M) induced depression of the PC simple spike (SS) firing rate in a dose-dependent manner, but without significantly changing the properties of complex spikes (CS). The IC50 of propofol for inhibiting SS firing of PCs was 144.5?M. Application of GABAA receptor antagonist, SR95531 (40?M) or GABAB receptor antagonist, saclofen (20?M), as well as glycine receptor antagonist, strychnine (10?M) alone failed to prevent the propofol-induced inhibition of PCs spontaneous activity. However, application the mixture of SR95531 (40?M) and strychnine (10?M) completely blocked the propofol-induced inhibition of PC SS firing. These data indicated that cerebellar surface application of propofol depressed PC SS firing rate via facilitation of GABAA and functional glycine receptors activity in adult cerebellar PCs under in vivo conditions. Our present results provide a new insight of the anesthetic action of propofol in cerebellar cortex, suggesting that propofol depresses the SS outputs of cerebellar PCs which is involved in both GABAA and glycine receptors activity. PMID:26142083

  18. Toll-like Receptor 2 Activation Promotes Tumor Dendritic Cell Dysfunction by Regulating IL-6 and IL-10 Receptor Signaling.

    PubMed

    Tang, Michael; Diao, Jun; Gu, Hongtao; Khatri, Ismat; Zhao, Jun; Cattral, Mark S

    2015-12-29

    Although dendritic cell (DC) dysfunction in cancer is a well-recognized consequence of cancer-associated inflammation that contributes to immune evasion, the mechanisms that drive this process remain elusive. Here, we show the critical importance of tumor-derived TLR2 ligands in the generation of immunosuppressive IL-10-producing human and mouse DCs. TLR2 ligation induced two parallel synergistic processes that converged to activate STAT3: stimulation of autocrine IL-6 and IL-10 and upregulation of their respective cell surface receptors, which lowered the STAT3 activation threshold. We identified versican as a soluble tumor-derived factor that activates TLR2 in DCs. TLR2 blockade in vivo improved intra-tumor DC immunogenicity and enhanced the efficacy of immunotherapy. Our findings provide a basis for understanding the molecular mechanisms of DC dysfunction in cancer and identify TLR2 as a relevant therapeutic target to improve cancer immunotherapy. PMID:26711349

  19. Fatty acylated caveolin-2 is a substrate of insulin receptor tyrosine kinase for insulin receptor substrate-1-directed signaling activation.

    PubMed

    Kwon, Hayeong; Lee, Jaewoong; Jeong, Kyuho; Jang, Donghwan; Pak, Yunbae

    2015-05-01

    Here, we demonstrate that insulin receptor (IR) tyrosine kinase catalyzes Tyr-19 and Tyr-27 phosphorylation of caveolin-2 (cav-2), leading to stimulation of signaling proteins downstream of IR, and that the catalysis is dependent on fatty acylation status of cav-2, promoting its interaction with IR. Cav-2 is myristoylated at Gly-2 and palmitoylated at Cys-109, Cys-122, and Cys-145. The fatty acylation deficient mutants are unable to localize in the plasma membrane and not phosphorylated by IR tyrosine kinase. IR interacts with the C-terminal domain of cav-2 containing the cysteines for palmitoylation. IR mutants, Y999F and K1057A, but not W1220S, fail interaction with cav-2. Insulin receptor substrate-1 (IRS-1) is recruited to interact with the IR-catalyzed phospho-tyrosine cav-2, which facilitates IRS-1 association with and activation by IR to initiate IRS-1-mediated downstream signaling. Cav-2 fatty acylation and tyrosine phosphorylation are necessary for the IRS-1-dependent PI3K-Akt and ERK activations responsible for glucose uptake and cell survival and proliferation. In conclusion, fatty acylated cav-2 is a new substrate of IR tyrosine kinase, and the fatty acylation and phosphorylation of cav-2 present novel mechanisms by which insulin signaling is activated. PMID:25667086

  20. Hepatic-specific activation of peroxisome proliferator-activated receptor ? coactivator-1? protects against steatohepatitis.

    PubMed

    Bellafante, Elena; Murzilli, Stefania; Salvatore, Lorena; Latorre, Dominga; Villani, Gaetano; Moschetta, Antonio

    2013-04-01

    Development of hepatic steatosis and its progression to steatohepatitis may be the consequence of dysfunction of several metabolic pathways, such as triglyceride synthesis, very low-density lipoprotein (VLDL) secretion, and fatty acid ?-oxidation. Peroxisome proliferator-activated receptor ? coactivator-1? (PGC-1?) is a master regulator of mitochondrial biogenesis and oxidative metabolism, lipogenesis, and triglyceride (TG) secretion. Here we generated a novel mouse model with constitutive hepatic activation of PGC-1? and studied the role of this transcriptional coactivator in dietary-induced steatosis and steatohepatitis. Selective activation of PGC-1? within hepatocytes is able to protect the liver from lipid overload and from progression to fibrosis. The protective function exerted by PGC-1? is due to its ability to induce mitochondrial oxidative phosphorylation, fatty acid ?-oxidation, and citrate cycle, as well as to decrease oxidative stress and promote TG secretion in the blood stream. These findings bolster the concept that a combined hepatic specific action of PGC-1? on lipid synthesis and secretion, as well as on mitochondrial biogenesis and function, could protect against steatohepatitis. PMID:23299802

  1. Buprenorphine-elicited alteration of adenylate cyclase activity in human embryonic kidney 293 cells coexpressing ?-, ?-opioid and nociceptin receptors

    PubMed Central

    Wang, Pei-Chen; Ho, Ing-Kang; Lee, Cynthia Wei-Sheng

    2015-01-01

    Buprenorphine, a maintenance drug for heroin addicts, exerts its pharmacological function via ?- (KOP), ?-opioid (MOP) and nociceptin/opioid receptor-like 1 (NOP) receptors. Previously, we investigated its effects in an in vitro model expressing human MOP and NOP receptors individually or simultaneously (MOP, NOP, and MOP+NOP) in human embryonic kidney 293 cells. Here, we expanded this cell model by expressing human KOP, MOP and NOP receptors individually or simultaneously (KOP, KOP+MOP, KOP+NOP and KOP+MOP+NOP). Radioligand binding with tritium-labelled diprenorphine confirmed the expression of KOP receptors. Immunoblotting and immunocytochemistry indicated that the expressed KOP, MOP and NOP receptors are N-linked glycoproteins and colocalized in cytoplasmic compartments. Acute application of the opioid receptor agonists— U-69593, DAMGO and nociceptin— inhibited adenylate cyclase (AC) activity in cells expressing KOP, MOP and NOP receptors respectively. Buprenorphine, when applied acutely, inhibited AC activity to ~90% in cells expressing KOP+MOP+NOP receptors. Chronic exposure to buprenorphine induced concentration-dependent AC superactivation in cells expressing KOP+NOP receptors, and the level of this superactivation was even higher in KOP+MOP+NOP-expressing cells. Our study demonstrated that MOP receptor could enhance AC regulation in the presence of coexpressed KOP and NOP receptors, and NOP receptor is essential for concentration-dependent AC superactivation elicited by chronic buprenorphine exposure. PMID:26153065

  2. Buprenorphine-elicited alteration of adenylate cyclase activity in human embryonic kidney 293 cells coexpressing ?-, ?-opioid and nociceptin receptors.

    PubMed

    Wang, Pei-Chen; Ho, Ing-Kang; Lee, Cynthia Wei-Sheng

    2015-11-01

    Buprenorphine, a maintenance drug for heroin addicts, exerts its pharmacological function via ?- (KOP), ?-opioid (MOP) and nociceptin/opioid receptor-like 1 (NOP) receptors. Previously, we investigated its effects in an in vitro model expressing human MOP and NOP receptors individually or simultaneously (MOP, NOP, and MOP+NOP) in human embryonic kidney 293 cells. Here, we expanded this cell model by expressing human KOP, MOP and NOP receptors individually or simultaneously (KOP, KOP+MOP, KOP+NOP and KOP+MOP+NOP). Radioligand binding with tritium-labelled diprenorphine confirmed the expression of KOP receptors. Immunoblotting and immunocytochemistry indicated that the expressed KOP, MOP and NOP receptors are N-linked glycoproteins and colocalized in cytoplasmic compartments. Acute application of the opioid receptor agonists- U-69593, DAMGO and nociceptin- inhibited adenylate cyclase (AC) activity in cells expressing KOP, MOP and NOP receptors respectively. Buprenorphine, when applied acutely, inhibited AC activity to ~90% in cells expressing KOP+MOP+NOP receptors. Chronic exposure to buprenorphine induced concentration-dependent AC superactivation in cells expressing KOP+NOP receptors, and the level of this superactivation was even higher in KOP+MOP+NOP-expressing cells. Our study demonstrated that MOP receptor could enhance AC regulation in the presence of coexpressed KOP and NOP receptors, and NOP receptor is essential for concentration-dependent AC superactivation elicited by chronic buprenorphine exposure. PMID:26153065

  3. TOPICAL APPLICATION OF LAMININ-322 TO DIABETIC MOUSE WOUNDS

    PubMed Central

    Sullivan, Stephen R.; Underwood, Robert A.; Sigle, Randall O.; Fukano, Yuko; Muffley, Lara A.; Usui, Marcia L.; Gibran, Nicole S.; Antezana, Marcos A.; Carter, William G.; Olerud, John E.

    2007-01-01

    Background Keratinocyte migration is essential for wound healing and diabetic wound keratinocytes migrate poorly. Keratinocyte migration and anchorage appears to be mediated by laminin-332 (LM-332). Impaired diabetic wound healing may be due to defective LM-332 mediated keratinocyte migration. Objective To evaluate LM-332 expression in diabetic (db/db) and control (db/-) mice and to test LM-332 wound healing effects when applied to mouse wounds. Methods LM-332 expression in mouse wounds was evaluated using immunohistochemistry. LM-332 wound healing effects were evaluated by directly applying soluble LM-332, a LM-332 biomaterial, or a control to mouse wounds. Percent wound closure and histology score, based on healing extent, were measured. Results Precursor LM-332 expression was markedly reduced in db/db when compared to db/- mice. In vitro, soluble LM-332 and LM-332 biomaterial demonstrated significant keratinocyte adhesion. In vivo, soluble LM-332 treated wounds had the highest histology score, but significant differences were not found between wound treatments (p>0.05). No differences in percentage wound closure between treatment and control wounds were found (p>0.05). Conclusion The db/db wounds express less precursor LM-332 when compared to db/-. However, LM-332 application did not improve db/db wound healing. LM-332 purified from keratinocytes was primarily physiologically cleaved LM-332 and may not regulate keratinocyte migration. Application of precursor LM-332 rather than cleaved LM-332 may be necessary to improve wound healing, but this isoform is not currently available in quantities sufficient for testing. PMID:17719208

  4. Tubulointerstitial nephritis antigen interacts with laminin and type IV collagen and promotes cell adhesion.

    PubMed

    Kalfa, T A; Thull, J D; Butkowski, R J; Charonis, A S

    1994-01-21

    Tubulointerstitial nephritis (TIN) antigen has been recently identified as a novel basement membrane macromolecule. It consists of a single chain of 58 kDa and exhibits a restricted distribution. The interaction between TIN antigen and laminin or type IV collagen has been studied using solid-phase binding assays and found to be for both macromolecules specific, saturable, and with an affinity in the low micromolar range. In similar assays, TIN antigen did not interact with heparin. In turbidimetry assays, it was found that the presence of TIN antigen did not affect the polymerization of type IV collagen but had a concentration-dependent inhibitory effect on laminin polymerization and on preformed laminin polymers. TIN antigen was able to promote adhesion of epithelial cells derived from kidney tubules and of endothelial cells derived from aorta. The data suggest that TIN antigen may be a macromolecule of importance both for basement membrane ultrastructure and cellular adhesion. PMID:8294412

  5. Selective estrogen-receptor modulators suppress microglial activation and neuronal cell death via an estrogen receptor-dependent pathway.

    PubMed

    Ishihara, Yasuhiro; Itoh, Kouichi; Ishida, Atsuhiko; Yamazaki, Takeshi

    2015-01-01

    Growing evidence shows that steroid hormones, especially 17?-estradiol (E2), protect neuronal cells by attenuating excess activation of microglia. However, the use of E2 in the clinic is controversial because of its peripheral actions in reproductive organs and its potential to increase risk for endometrial cancer and breast cancer. Selective estrogen-receptor modulators (SERMs) bind to estrogen receptors (ERs), but their effects as ER agonists or antagonists are dependent on the target tissue. SERMs pose very little cancer risk as a result of their anti-estrogen action in reproductive organs, but their action in the brain is not well understood. In this study, we investigated the effects of SERMs tamoxifen (Tam) and raloxifene (Rlx) on microglial activation and subsequent neuronal injury. Tam and Rlx suppressed the increases in proinflammatory cytokines and chemokine expression that were induced by lipopolysaccharide (LPS) in rat primary microglia cultures. The microglial-conditioned media pretreated with Tam or Rlx significantly attenuated cellular injury in SH-SY5Y cells elicited by microglial-conditioned media treated with LPS alone. Rat primary microglia expressed ER? and ER? primarily in the nucleus, and thus we examined the involvement of ERs in the suppressive action of Tam and Rlx on microglial activation using a pure ER antagonist, ICI182,780. Pretreatment with ICI182,780 abolished the suppressive effects of SERMs on microglial activation, as well as their protective action on SH-SY5Y cells. A luciferase assay using a vector with three estrogen response elements (EREs) revealed that Tam and Rlx activated ERE-mediated transcription in rat primary microglia. Taken together, these results suggest that Tam and Rlx suppress microglial activation and subsequent neuronal cell death via an ER-mediated transcription pathway. SERMs could represent a novel therapeutic strategy for disorders of the central nervous system based on their ability to suppress neuroinflammation. PMID:25305410

  6. Expression of fibrinogen receptors during activation and subsequent desensitization of human platelets by epinephrine.

    PubMed

    Shattil, S J; Motulsky, H J; Insel, P A; Flaherty, L; Brass, L F

    1986-12-01

    Epinephrine causes platelet aggregation and secretion by interacting with alpha 2-adrenergic receptors on the platelet surface. Platelet aggregation requires the binding of fibrinogen to a specific receptor on the membrane glycoprotein IIb-IIIa complex. Although the IIb-IIIa complex is identifiable on the surface of resting platelets, the fibrinogen receptor is expressed only after platelet activation. The current studies were designed to examine the effect of occupancy of platelet alpha 2-adrenergic receptors by epinephrine on the expression of fibrinogen receptors and on the aggregation of platelets. The ability of epinephrine to induce the expression of fibrinogen receptors was studied under two different conditions: acute stimulation (less than 1 min) and prolonged stimulation (50 to 90 min), the latter of which is associated with a reduction or "desensitization" of the platelet aggregation response. Expression of the fibrinogen receptor was monitored with 125I-fibrinogen as well as with 125I-PAC-1 (PAC-1), a monoclonal antibody that binds to the glycoprotein IIb-IIIa complex only after platelets are activated. Epinephrine caused an immediate increase in PAC-1 and fibrinogen binding that was dependent on occupancy of the alpha 2-receptor by epinephrine and on the presence of extracellular free Ca (KCa = 30 mumol/L). By itself, 1 mmol/L Mg was unable to support induction of the fibrinogen receptor by epinephrine. However, it did decrease the Ca requirement by about two orders of magnitude. Prolonged stimulation of unstirred platelets by epinephrine led to a 70% decrease in the aggregation response when the platelets were subsequently stirred. Despite their decreased aggregation response, desensitized platelets bound PAC-1 and fibrinogen normally, indicating that the loss of aggregation was not due simply to a decrease in fibrinogen receptor expression. Although desensitization was not affected by pretreatment of the platelets with aspirin, it was partially prevented when extracellular Ca was chelated by EDTA during the long incubation with epinephrine. These studies demonstrate that once platelet alpha 2-adrenergic receptors are occupied by epinephrine, extracellular Ca is involved in initiating the aggregation response by supporting the induction of the fibrinogen receptor and the binding of fibrinogen. Furthermore. Ca-dependent reactions subsequent to fibrinogen binding may be necessary for maximal platelet aggregation and are impaired when platelets become desensitized to epinephrine. PMID:3779100

  7. Classical and atypical agonists activate M1 muscarinic acetylcholine receptors through common mechanisms.

    PubMed

    Randáková, Alena; Dolejší, Eva; Rudajev, Vladimír; Zim?ík, Pavel; Doležal, Vladimír; El-Fakahany, Esam E; Jakubík, Jan

    2015-07-01

    We mutated key amino acids of the human variant of the M1 muscarinic receptor that target ligand binding, receptor activation, and receptor-G protein interaction. We compared the effects of these mutations on the action of two atypical M1 functionally preferring agonists (N-desmethylclozapine and xanomeline) and two classical non-selective orthosteric agonists (carbachol and oxotremorine). Mutations of D105 in the orthosteric binding site and mutation of D99 located out of the orthosteric binding site decreased affinity of all tested agonists that was translated as a decrease in potency in accumulation of inositol phosphates and intracellular calcium mobilization. Mutation of D105 decreased the potency of the atypical agonist xanomeline more than that of the classical agonists carbachol and oxotremorine. Mutation of the residues involved in receptor activation (D71) and coupling to G-proteins (R123) completely abolished the functional responses to both classical and atypical agonists. Our data show that both classical and atypical agonists activate hM1 receptors by the same molecular switch that involves D71 in the second transmembrane helix. The principal difference among the studied agonists is rather in the way they interact with D105 in the orthosteric binding site. Furthermore, our data demonstrate a key role of D105 in xanomeline wash-resistant binding and persistent activation of hM1 by wash-resistant xanomeline. PMID:25882246

  8. A peptide ligand of the human thrombin receptor antagonizes alpha-thrombin and partially activates platelets.

    PubMed

    Rasmussen, U B; Gachet, C; Schlesinger, Y; Hanau, D; Ohlmann, P; Van Obberghen-Schilling, E; Pouysségur, J; Cazenave, J P; Pavirani, A

    1993-07-01

    The peptide YFLLRNP antagonizes the aggregation of human platelets when induced by low concentrations of alpha-thrombin or the thrombin receptor agonist peptide (SFLLRNP), demonstrating that it interacts specifically with the thrombin receptor. Platelets exposed to YFLLRNP show immediate shape change (pseudopod formation) and potentiation of the ADP and platelet-activating factor response, but no Ca2+ mobilization, P47 (pleckstrin) phosphorylation, secretion, or aggregation. Thus, YFLLRNP induces a state of partial activation of the platelets. Furthermore, with platelets prestimulated with adrenalin (10 microM), YFLLRNP induces aggregation, but no secretion, and only in the presence of added fibrinogen. We also found that prostacyclin inhibits the YFLLRNP-induced shape change; but EDTA, aspirin, and apyrase (ADP scavenger) do not. Thus, the thrombin receptor in platelets may communicate, independently of Ca2+ mobilization and P47 phosphorylation (protein kinase C activation), with intracellular signaling mechanisms that 1) modulate the cytoskeleton structure, 2) potentiate other platelet responses, and 3) stimulate coupling between the thrombin receptor and fibrinogen binding (the glycoprotein IIb-IIIa complex). YFLLRNP may be useful for differentiating between several possible activation states of the platelet thrombin receptor. PMID:8390990

  9. Regulation of GPR119 receptor activity with endocannabinoid-like lipids.

    PubMed

    Syed, Samreen K; Bui, Hai Hoang; Beavers, Lisa S; Farb, Thomas B; Ficorilli, James; Chesterfield, Amy K; Kuo, Ming-Shang; Bokvist, Krister; Barrett, David G; Efanov, Alexander M

    2012-12-15

    The GPR119 receptor plays an important role in the secretion of incretin hormones in response to nutrient consumption. We have studied the ability of an array of naturally occurring endocannabinoid-like lipids to activate GPR119 and have identified several lipid receptor agonists. The most potent receptor agonists identified were three N-acylethanolamines: oleoylethanolamine (OEA), palmitoleoylethanolamine, and linoleylethanolamine (LEA), all of which displayed similar potency in activating GPR119. Another lipid, 2-oleoylglycerol (2-OG), also activated GPR119 receptor but with significantly lower potency. Endogenous levels of endocannabinoid-like lipids were measured in intestine in fasted and refed mice. Of the lipid GPR119 agonists studied, the intestinal levels of only OEA, LEA, and 2-OG increased significantly upon refeeding. Intestinal levels of OEA and LEA in the fasted mice were low. In the fed state, OEA levels only moderately increased, whereas LEA levels rose drastically. 2-OG was the most abundant of the three GPR119 agonists in intestine, and its levels were radically elevated in fed mice. Our data suggest that, in lean mice, 2-OG and LEA may serve as physiologically relevant endogenous GPR119 agonists that mediate receptor activation upon nutrient uptake. PMID:23074242

  10. Indole and Tryptophan Metabolism: Endogenous and Dietary Routes to Ah Receptor Activation.

    PubMed

    Hubbard, Troy D; Murray, Iain A; Perdew, Gary H

    2015-10-01

    The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor recognized for its role in xenobiotic metabolism. The physiologic function of AHR has expanded to include roles in immune regulation, organogenesis, mucosal barrier function, and the cell cycle. These functions are likely dependent upon ligand-mediated activation of the receptor. High-affinity ligands of AHR have been classically defined as xenobiotics, such as polychlorinated biphenyls and dioxins. Identification of endogenous AHR ligands is key to understanding the physiologic functions of this enigmatic receptor. Metabolic pathways targeting the amino acid tryptophan and indole can lead to a myriad of metabolites, some of which are AHR ligands. Many of these ligands exhibit species selective preferential binding to AHR. The discovery of specific tryptophan metabolites as AHR ligands may provide insight concerning where AHR is activated in an organism, such as at the site of inflammation and within the intestinal tract. PMID:26041783

  11. Loss of constitutive activity of the growth hormone secretagogue receptor in familial short stature

    PubMed Central

    Pantel, Jacques; Legendre, Marie; Cabrol, Sylvie; Hilal, Latifa; Hajaji, Yassir; Morisset, Séverine; Nivot, Sylvie; Vie-Luton, Marie-Pierre; Grouselle, Dominique; de Kerdanet, Marc; Kadiri, Abdelkrim; Epelbaum, Jacques; Le Bouc, Yves; Amselem, Serge

    2006-01-01

    The growth hormone (GH) secretagogue receptor (GHSR) was cloned as the target of a family of synthetic molecules endowed with GH release properties. As shown recently through in vitro means, this receptor displays a constitutive activity whose clinical relevance is unknown. Although pharmacological studies have demonstrated that its endogenous ligand — ghrelin — stimulates, through the GHSR, GH secretion and appetite, the physiological importance of the GHSR-dependent pathways remains an open question that gives rise to much controversy. We report the identification of a GHSR missense mutation that segregates with short stature within 2 unrelated families. This mutation, which results in decreased cell-surface expression of the receptor, selectively impairs the constitutive activity of the GHSR, while preserving its ability to respond to ghrelin. This first description, to our knowledge, of a functionally significant GHSR mutation, which unveils the critical importance of the GHSR-associated constitutive activity, discloses an unusual pathogenic mechanism of growth failure in humans. PMID:16511605

  12. Castor oil induces laxation and uterus contraction via ricinoleic acid activating prostaglandin EP3 receptors

    PubMed Central

    Tunaru, Sorin; Althoff, Till F.; Nüsing, Rolf M.; Diener, Martin; Offermanns, Stefan

    2012-01-01

    Castor oil is one of the oldest drugs. When given orally, it has a laxative effect and induces labor in pregnant females. The effects of castor oil are mediated by ricinoleic acid, a hydroxylated fatty acid released from castor oil by intestinal lipases. Despite the wide-spread use of castor oil in conventional and folk medicine, the molecular mechanism by which ricinoleic acid acts remains unknown. Here we show that the EP3 prostanoid receptor is specifically activated by ricinoleic acid and that it mediates the pharmacological effects of castor oil. In mice lacking EP3 receptors, the laxative effect and the uterus contraction induced via ricinoleic acid are absent. Although a conditional deletion of the EP3 receptor gene in intestinal epithelial cells did not affect castor oil-induced diarrhea, mice lacking EP3 receptors only in smooth-muscle cells were unresponsive to this drug. Thus, the castor oil metabolite ricinoleic acid activates intestinal and uterine smooth-muscle cells via EP3 prostanoid receptors. These findings identify the cellular and molecular mechanism underlying the pharmacological effects of castor oil and indicate a role of the EP3 receptor as a target to induce laxative effects. PMID:22615395

  13. Castor oil induces laxation and uterus contraction via ricinoleic acid activating prostaglandin EP3 receptors.

    PubMed

    Tunaru, Sorin; Althoff, Till F; Nüsing, Rolf M; Diener, Martin; Offermanns, Stefan

    2012-06-01

    Castor oil is one of the oldest drugs. When given orally, it has a laxative effect and induces labor in pregnant females. The effects of castor oil are mediated by ricinoleic acid, a hydroxylated fatty acid released from castor oil by intestinal lipases. Despite the wide-spread use of castor oil in conventional and folk medicine, the molecular mechanism by which ricinoleic acid acts remains unknown. Here we show that the EP(3) prostanoid receptor is specifically activated by ricinoleic acid and that it mediates the pharmacological effects of castor oil. In mice lacking EP(3) receptors, the laxative effect and the uterus contraction induced via ricinoleic acid are absent. Although a conditional deletion of the EP(3) receptor gene in intestinal epithelial cells did not affect castor oil-induced diarrhea, mice lacking EP(3) receptors only in smooth-muscle cells were unresponsive to this drug. Thus, the castor oil metabolite ricinoleic acid activates intestinal and uterine smooth-muscle cells via EP(3) prostanoid receptors. These findings identify the cellular and molecular mechanism underlying the pharmacological effects of castor oil and indicate a role of the EP(3) receptor as a target to induce laxative effects. PMID:22615395

  14. A novel oxyiminoalkanoic acid derivative, TAK-559, activates human peroxisome proliferator-activated receptor subtypes.

    PubMed

    Sakamoto, Junichi; Kimura, Hiroyuki; Moriyama, Shinji; Imoto, Hiroshi; Momose, Yu; Odaka, Hiroyuki; Sawada, Hidekazu

    2004-07-01

    A novel oxyiminoalkanoic acid derivative, TAK-559, (E)-4-[4-[(5-methyl-2-phenyl-1, 3-oxazol-4-yl)methoxy]benzyloxyimino]-4-phenylbutyric acid, was synthesized as a candidate of a new type of insulin-sensitizing agent. We report here activation of human peroxisome proliferator-activated receptor (hPPAR) subtypes by TAK-559. In a transient transactivation assay, TAK-559 was a potent hPPARgamma1 and hPPARalpha agonist with EC50 values of 31 and 67 nM, respectively. Furthermore, TAK-559 was a partial agonist for hPPARgamma1 with about 68% of maximal activation obtained with rosiglitazone (5-(4-(2-(methyl(2-pyridinyl)amino)ethoxy) benzyl)-1,3-thiazolidine-2,4-dione), a thiazolidinedione derivative, which is known as a PPARgamma agonist. PPARdelta was significantly activated at a high concentration (10 microM) of TAK-559. Competition-binding assays using radiolabeled ligand indicated that the transactivation of all hPPAR subtypes by TAK-559 was due to direct binding of TAK-559 to each subtype. We also demonstrated that TAK-559 acts to recruit the coactivator SRC-1 to each of hPPARgamma1 and hPPARalpha, and to dissociate the corepressor NCoR from each of hPPARgamma1 and hPPARalpha. Taken together, we conclude that TAK-559 is a dual agonist for hPPARgamma1 and hPPARalpha with nearly equal EC50 values, a partial agonist for hPPARgamma1, and has a rather slight agonist activity for hPPARdelta. PMID:15219816

  15. Activation of Protease Activated Receptor 2 by Exogenous Agonist Exacerbates Early Radiation Injury in Rat Intestine

    SciTech Connect

    Wang Junru; Boerma, Marjan; Kulkarni, Ashwini; Hollenberg, Morley D.; Hauer-Jensen, Martin

    2010-07-15

    Purpose: Protease-activated receptor-2 (PAR{sub 2}) is highly expressed throughout the gut and regulates the inflammatory, mitogenic, fibroproliferative, and nociceptive responses to injury. PAR{sub 2} is strikingly upregulated and exhibits increased activation in response to intestinal irradiation. We examined the mechanistic significance of radiation enteropathy development by assessing the effect of exogenous PAR{sub 2} activation. Methods and Materials: Rat small bowel was exposed to localized single-dose radiation (16.5 Gy). The PAR{sub 2} agonist (2-furoyl-LIGRLO-NH{sub 2}) or vehicle was injected intraperitoneally daily for 3 days before irradiation (before), for 7 days after irradiation (after), or both 3 days before and 7 days after irradiation (before-after). Early and delayed radiation enteropathy was assessed at 2 and 26 weeks after irradiation using quantitative histologic examination, morphometry, and immunohistochemical analysis. Results: The PAR{sub 2} agonist did not elicit changes in the unirradiated (shielded) intestine. In contrast, in the irradiated intestine procured 2 weeks after irradiation, administration of the PAR{sub 2} agonist was associated with more severe mucosal injury and increased intestinal wall thickness in all three treatment groups (p <.05) compared with the vehicle-treated controls. The PAR{sub 2} agonist also exacerbated the radiation injury score, serosal thickening, and mucosal inflammation (p <.05) in the before and before-after groups. The short-term exogenous activation of PAR{sub 2} did not affect radiation-induced intestinal injury at 26 weeks. Conclusion: The results of the present study support a role for PAR{sub 2} activation in the pathogenesis of early radiation-induced intestinal injury. Pharmacologic PAR{sub 2} antagonists might have the potential to reduce the intestinal side effects of radiotherapy and/or as countermeasures in radiologic accidents or terrorism scenarios.

  16. Enhanced AMPA Receptor Activity Increases Operant Alcohol Self-administration and Cue-Induced Reinstatement

    PubMed Central

    Cannady, Reginald; Fisher, Kristen R.; Durant, Brandon; Besheer, Joyce; Hodge, Clyde W.

    2012-01-01

    Long-term alcohol exposure produces neuroadaptations that contribute to the progression of alcohol abuse disorders. Chronic alcohol consumption results in strengthened excitatory neurotransmission and increased AMPA receptor signaling in animal models. However, the mechanistic role of enhanced AMPA receptor activity in alcohol reinforcement and alcohol-seeking behavior remains unclear. This study examined the role of enhanced AMPA receptor function using the selective positive allosteric modulator, aniracetam, in modulating operant alcohol self-administration and cue-induced reinstatement. Male alcohol-preferring (P-) rats, trained to self-administer alcohol (15%, v/v) versus water were pretreated with aniracetam to assess effects on maintenance of alcohol self-administration. To determine reinforcer specificity, P-rats were trained to self-administer sucrose (0.8%, w/v) versus water, and effects of aniracetam were tested. The role of aniracetam in modulating relapse of alcohol-seeking was assessed using a response-contingent cue-induced reinstatement procedure in P-rats trained to self-administer 15% alcohol. Aniracetam pretreatment significantly increased alcohol-reinforced responses relative to vehicle treatment. This increase was not attributed to aniracetam-induced hyperactivity as aniracetam pretreatment did not alter locomotor activity. AMPA receptor involvement was confirmed because DNQX (AMPA receptor antagonist) blocked the aniracetam-induced increase in alcohol self-administration. Aniracetam did not alter sucrose-reinforced responses in sucrose-trained P-rats, suggesting that enhanced AMPA receptor activity is selective in modulating the reinforcing function of alcohol. Finally, aniracetam pretreatment potentiated cue-induced reinstatement of alcohol-seeking behavior versus vehicle treated-P-rats. These data suggest that enhanced glutamate activity at AMPA receptors may be key in facilitating alcohol consumption and seeking behavior which could ultimately contribute to the development of alcohol abuse disorders. PMID:23126443

  17. Communication over the Network of Binary Switches Regulates the Activation of A2A Adenosine Receptor

    PubMed Central

    Lee, Yoonji; Choi, Sun; Hyeon, Changbong

    2015-01-01

    Dynamics and functions of G-protein coupled receptors (GPCRs) are accurately regulated by the type of ligands that bind to the orthosteric or allosteric binding sites. To glean the structural and dynamical origin of ligand-dependent modulation of GPCR activity, we performed total ~ 5 ?sec molecular dynamics simulations of A2A adenosine receptor (A2AAR) in its apo, antagonist-bound, and agonist-bound forms in an explicit water and membrane environment, and examined the corresponding dynamics and correlation between the 10 key structural motifs that serve as the allosteric hotspots in intramolecular signaling network. We dubbed these 10 structural motifs “binary switches” as they display molecular interactions that switch between two distinct states. By projecting the receptor dynamics on these binary switches that yield 210 microstates, we show that (i) the receptors in apo, antagonist-bound, and agonist-bound states explore vastly different conformational space; (ii) among the three receptor states the apo state explores the broadest range of microstates; (iii) in the presence of the agonist, the active conformation is maintained through coherent couplings among the binary switches; and (iv) to be most specific, our analysis shows that W246, located deep inside the binding cleft, can serve as both an agonist sensor and actuator of ensuing intramolecular signaling for the receptor activation. Finally, our analysis of multiple trajectories generated by inserting an agonist to the apo state underscores that the transition of the receptor from inactive to active form requires the disruption of ionic-lock in the DRY motif. PMID:25664580

  18. Prostaglandin E2 regulates angiogenesis via activation of fibroblast growth factor receptor-1.

    PubMed

    Finetti, Federica; Solito, Raffaella; Morbidelli, Lucia; Giachetti, Antonio; Ziche, Marina; Donnini, Sandra

    2008-01-25

    Prostaglandin E(2) (PGE(2)) behaves as a mitogen in epithelial tumor cells as well as in many other cell types. We investigated the actions of PGE(2) on microvascular endothelial cells (capillary venular endothelial cells) with the purpose of delineating the signaling pathway leading to the acquisition of the angiogenic phenotype and to new vessel formation. PGE(2) (100 nM) produced activation of the fibroblast growth factor receptor 1 (FGFR-1), as measured by its phosphorylation, but not of vascular endothelial growth factor receptor 2. PGE(2) stimulated the EP3 subtype receptor, as deduced by abrogation of EP3 Galpha(i) subunit activity through pertussis toxin. Consistent with this result, in human umbilical venular endothelial cells missing the EP3 receptor, PGE(2) did not phosphorylate FGFR-1. Upon binding to its receptor, PGE(2) initiated an autocrine/paracrine signaling cascade involving the intracellular activation of c-Src, activation of matrix metalloproteinase (predominantly MMP2), which in turn caused the mobilization of membrane-anchored fibroblast growth factor-2 (FGF-2). In fact, in cells unable to release FGF-2 the transfection with both FGFR-1 and EP3 did not result in FGFR-1 phosphorylation in response to PGE(2). Relevance for the FGF2-FGFR-1 system was highlighted by confocal analysis, showing receptor internalization after cell exposure to the prostanoid. ERK1/2 appeared to be the distal signal involved, its phosphorylation being sensitive to either cSrc inhibitor or FGFR-1 blocker. Finally, PGE(2) stimulated cell migration and capillary formation in aortic rings, which were severely reduced by inhibitors of signaling molecules or by receptor antagonist. In conclusion, this study provides evidence for the involvement of FGFR-1 through FGF2 in eliciting PGE(2) angiogenic responses. This signaling pattern is similar to the autocrine-paracrine mechanism which operates in endothelial cells to support neovascular growth. PMID:18042549

  19. Nidogen-1 regulates laminin-1-dependent mammary-specific gene expression

    SciTech Connect

    Pujuguet, Philippe; Simian, Marina; Liaw, Jane; Timpl, Rupert; Werb, Zena; Bissell, Mina J..

    2000-02-01

    Nidogen-1 (entactin) acts as a bridge between the extracellular matrix molecules laminin-1 and type IV collagen, and thus participates in the assembly of basement membranes. To investigate the role of nidogen-1 in regulating cell-type-specific gene expression in mammary epithelium, we designed a culture microecosystem in which each component, including epithelial cells, mesenchymal cells, lactogenic hormones and extracellular matrix, could be controlled. We found that primary and established mesenchymal and myoepithelial cells synthesized and secreted nidogen-1, whereas expression was absent in primary and established epithelial cells. In an epithelial cell line containing mesenchymal cells, nidogen-1 was produced by the mesenchymal cells but deposited between the epithelial cells. In this mixed culture, mammary epithelial cells express b-casein in the presence of lactogenic hormones. Addition of either laminin-1 plus nidogen-1, or laminin-1 alone to mammary epithelial cells induced b- casein production. We asked whether recombinant nidogen-1 alone could signal directly for b-casein. Nidogen-1 did not induce b-casein synthesis in epithelial cells, but it augmented the inductive capacity of laminin-1. These data suggest that nidogen-1 can cooperate with laminin-1 to regulate b-casein expression. Addition of full length nidogen-1 to the mixed cultures had no effect on b-casein gene expression; however, a nidogen-1 fragment containing the laminin-1 binding domain, but lacking the type IV collagen-binding domain, had a dominant negative effect on b-casein expression. These data point to a physiological role for nidogen-1 in the basement membrane-induced gene expression by epithelial cells.

  20. A novel signaling pathway of tissue kallikrein in promoting keratinocyte migration: Activation of proteinase-activated receptor 1 and epidermal growth factor receptor

    SciTech Connect

    Gao, Lin; Chao, Lee; Chao, Julie

    2010-02-01

    Biological functions of tissue kallikrein (TK, KLK1) are mainly mediated by kinin generation and subsequent kinin B2 receptor activation. In this study, we investigated the potential role of TK and its signaling pathways in cultured human keratinocyte migration and in a rat skin wound healing model. Herein, we show that TK promoted cell migration and proliferation in a concentration- and time-dependent manner. Inactive TK or kinin had no significant effect on cell migration. Interestingly, cell migration induced by active TK was not blocked by icatibant or L-NAME, indicating an event independent of kinin B2 receptor and nitric oxide formation. TK's stimulatory effect on cell migration was inhibited by small interfering RNA for proteinase-activated receptor 1 (PAR{sub 1}), and by PAR{sub 1} inhibitor. TK-induced migration was associated with increased phosphorylation of epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK), which was blocked by inhibition of protein kinase C (PKC), Src, EGFR and ERK. TK-induced cell migration and EGFR phosphorylation were blocked by metalloproteinase (MMP) inhibitor, heparin, and antibodies against EGFR external domain, heparin-binding EGF-like growth factor (HB-EGF) and amphiregulin (AR). Local application of TK promoted skin wound healing in rats, whereas icatibant and EGFR inhibitor blocked TK's effect. Skin wound healing was further delayed by aprotinin and neutralizing TK antibody. This study demonstrates a novel role of TK in skin wound healing and uncovers new signaling pathways mediated by TK in promoting keratinocyte migration through activation of the PAR{sub 1}-PKC-Src-MMP pathway and HB-EGF/AR shedding-dependent EGFR transactivation.

  1. Quipazine reduces food intake in the rat by activation of 5-HT2-receptors.

    PubMed Central

    Hewson, G.; Leighton, G. E.; Hill, R. G.; Hughes, J.

    1988-01-01

    1. To determine which subtype(s) of 5-hydroxytryptamine (5-HT) receptor are involved in the anorectic action of quipazine, the ability of selective antagonists at 5-HT2- and 5-HT3-receptors, and an antagonist at 5-HT1-like receptors, to block this response were investigated in non-deprived rats, trained to eat a palatable diet. 2. Quipazine (0.5-8 mg kg-1, i.p.) produced a dose-related reduction in the intake of palatable diet. 3. The anorectic effect of 4 mg kg-1 quipazine was antagonized by the nonselective 5-HT-receptor antagonist methysergide (5 mg kg-1, i.p.) and by the selective 5-HT2-receptor antagonists ketanserin (1 mg kg-1 and 2.5 mg kg-1, i.p.) and ritanserin (0.5 mg kg-1 and 1 mg kg-1, i.p.). The selective 5-HT3-receptor antagonist GR38032F (1 mg kg-1, i.p.) and (-)-pindolol (4 mg kg-1, i.p.), which blocks some of the effects mediated at 5-HT1-like receptors, did not block the reduction in food intake produced by this dose of quipazine. 4. None of the 5-HT-receptor antagonists had any effect on food intake when they were administered alone, suggesting that endogenous 5-HT is not involved in the tonic control of food intake under the conditions of these experiments. 5. It is concluded that the anorectic action of quipazine is mediated, at least in part, by activation of 5-HT2-receptors. PMID:2906561

  2. Site Specificity of Agonist and Second Messenger-Activated Kinases for Somatostatin Receptor Subtype 2A (Sst2A) Phosphorylation

    PubMed Central

    Liu, Qisheng; Bee, Mark S.; Schonbrunn, Agnes

    2009-01-01

    Somatostatin receptor subtype 2A (sst2A) mediates many of the endocrine and neuronal actions of somatostatin and is the target of somatostatin analogs in cancer therapy. As with many G-protein-coupled receptors, agonist stimulation causes sst2A receptor desensitization and internalization, events that require receptor phosphorylation. Furthermore, heterologous receptor activation of protein kinase C (PKC) also increases sst2A receptor phosphorylation and internalization. Here we analyzed a series of sst2A receptor mutants biochemically to identify residues in the receptor carboxyl terminus that were phosphorylated upon agonist stimulation, and we then generated four phosphorylation-sensitive antibodies to those residues. Once the selectivity of each antibody for its phosphorylated and nonphosphorylated target sequence was determined, the phospho-site-specific antibodies were used to demonstrate that somatostatin treatment of Chinese hamster ovary (CHO) cells expressing the wild type sst2A receptor increased phosphorylation on five residues in the receptor C terminus: Ser341, Ser343, Ser348, Thr353, and Thr354. Phorbol 12-myristate 13-acetate (PMA) increased receptor phosphorylation only on Ser343. Inhibition of PKC blocked PMA but not somatostatin stimulation, showing that different kinases catalyzed Ser343 phosphorylation. In contrast, somatostatin-stimulated sst2A receptor phosphorylation was inhibited by knockdown of G-protein coupled receptor kinase-2 with siRNA. Somatostatin increased sst2A receptor phosphorylation on the same five residues in GH4C1 pituitary cells as in CHO cells. However, PMA stimulated sst2A receptor phosphorylation on both Ser343 and Ser348 in GH4C1 cells. These results characterize the complex pattern of sst2A receptor phosphorylation by agonist and second messenger-activated kinases for the first time and indicate that cell type-specific regulation of sst2A receptor phosphorylation occurs. PMID:19389921

  3. Investigation of the fate of type I angiotensin receptor after biased activation.

    PubMed

    Szakadáti, Gyöngyi; Tóth, András D; Oláh, Ilona; Erdélyi, László Sándor; Balla, Tamas; Várnai, Péter; Hunyady, László; Balla, András

    2015-06-01

    Biased agonism on the type I angiotensin receptor (AT1-R) can achieve different outcomes via activation of G protein-dependent and -independent cellular responses. In this study, we investigated whether the biased activation of AT1-R can lead to different regulation and intracellular processing of the receptor. We analyzed ?-arrestin binding, endocytosis, and subsequent trafficking steps, such as early and late phases of recycling of AT1-R in human embryonic kidney 293 cells expressing wild-type or biased mutant receptors in response to different ligands. We used Renilla luciferase-tagged receptors and yellow fluorescent protein-tagged ?-arrestin2, Rab5, Rab7, and Rab11 proteins in bioluminescence resonance energy transfer measurements to follow the fate of the receptor after stimulation. We found that not only is the signaling of the receptor different upon using selective ligands, but the fate within the cells is also determined by the type of the stimulation. ?-arrestin binding and the internalization kinetics of the angiotensin II-stimulated AT1-R differed from those stimulated by the biased agonists. Similarly, angiotensin II-stimulated wild-type AT1-R showed differences compared with a biased mutant AT1-R (DRY/AAY AT1-R) with regards to ?-arrestin binding and endocytosis. We found that the differences in the internalization kinetics of the receptor in response to biased agonist stimulation are due to the differences in plasma membrane phosphatidylinositol 4,5-bisphosphate depletion. Moreover, the stability of the ?-arrestin binding is a major determinant of the later fate of the internalized AT1-R receptor. PMID:25804845

  4. NK cell function triggered by multiple activating receptors is negatively regulated by glycogen synthase kinase-3?.

    PubMed

    Kwon, Hyung-Joon; Kwon, Soon Jae; Lee, Heejae; Park, Hye-Ran; Choi, Go-Eun; Kang, Sang-Wook; Kwon, Seog Woon; Kim, Nacksung; Lee, Soo Young; Ryu, Sangryeol; Kim, Sun Chang; Kim, Hun Sik

    2015-09-01

    Activation of NK cells is triggered by combined signals from multiple activating receptors that belong to different families. Several NK cell activating receptors have been identified, but their role in the regulation of effector functions is primarily understood in the context of their individual engagement. Therefore, little is known about the signaling pathways broadly implicated by the multiple NK cell activation cues. Here we provide evidence pointing to glycogen synthase kinase (GSK)-3? as a negative regulator of multiple NK cell activating signals. Using an activation model that combines NKG2D and 2B4 and tests different signaling molecules, we found that GSK-3 undergoes inhibitory phosphorylation at regulatory serine residues by the engagement of NKG2D and 2B4, either individually or in combination. The extent of such phosphorylation was closely correlated with the degree of NK cell activation. NK cell functions, such as cytokine production and cytotoxicity, were consistently enhanced by the knockdown of GSK-3? or its inhibition with different pharmacological inhibitors, whereas inhibition of the GSK-3? isoform had no effect. In addition, NK cell function was augmented by the overexpression of a catalytically inactive form of GSK-3?. Importantly, the regulation of NK cell function by GSK-3? was common to diverse activating receptors that signal through both ITAM and non-ITAM pathways. Thus, our results suggest that GSK-3? negatively regulates NK cell activation and that modulation of GSK-3? function could be used to enhance NK cell activation. PMID:26022178

  5. Luciferase activity under direct ligand-dependent control of a muscarinic acetylcholine receptor

    PubMed Central

    Thor, Doreen; Le Duc, Diana; Strotmann, Rainer; Schöneberg, Torsten

    2009-01-01

    Background Controlling enzyme activity by ligand binding to a regulatory domain of choice may have many applications e.g. as biosensors and as tools in regulating cellular functions. However, until now only a small number of ligand-binding domains have been successfully linked to enzyme activity. G protein-coupled receptors (GPCR) are capable of recognizing an extraordinary structural variety of extracellular signals including inorganic and organic molecules. Ligand binding to GPCR results in conformational changes involving the transmembrane helices. Here, we assessed whether ligand-induced conformational changes within the GPCR helix bundle can be utilized to control the activity of an integrated enzyme. Results As a proof of principle, we inserted the luciferase amino acid sequence into the third intracellular loop of the M3 muscarinic acetylcholine receptor. This fusion protein retained both receptor and enzyme function. Receptor blockers slightly but significantly reduced enzyme activity. By successive deletion mutagenesis the enzyme activity was optimally coupled to ligand-induced conformational helix movements. Conclusion Our results demonstrate that in engineered GPCR-enzyme chimeras, intracellular enzyme activity can be directly controlled by a GPCR serving as the extracellular ligand-binding domain. PMID:19450256

  6. Activating Killer Cell Ig-Like Receptors in Health and Disease

    PubMed Central

    Ivarsson, Martin A.; Michaëlsson, Jakob; Fauriat, Cyril

    2014-01-01

    Expression of non-rearranged HLA class I-binding receptors characterizes human and mouse NK cells. The postulation of the missing-self hypothesis some 30?years ago triggered the subsequent search and discovery of inhibitory MHC-receptors, both in humans and mice. These receptors have two functions: (i) to control the threshold for NK cell activation, a process termed “licensing” or “education,” and (ii) to inhibit NK cell activation during interactions with healthy HLA class I-expressing cells. The discovery of activating forms of KIRs (aKIR) challenged the concept of NK cell tolerance in steady state, as well as during immune challenge: what is the biological role of the activating KIR, in particular when NK cells express aKIRs in the absence of inhibitory receptors? Recently it was shown that aKIRs also participate in the education of NK cells. However, instead of lowering the threshold of activation like iKIRs, the expression of aKIRs has the opposite effect, i.e., rendering NK cells hyporesponsive. These findings may have consequences during NK cell response to viral infection, in cancer development, and in the initial stages of pregnancy. Here we review the current knowledge of activating KIRs, including the biological concept of aKIR-dependent NK cell education, and their impact in health and disease. PMID:24795726

  7. Serotonin stimulates lateral habenula via activation of the post-synaptic serotonin 2/3 receptors and transient receptor potential channels.

    PubMed

    Zuo, Wanhong; Zhang, Yong; Xie, Guiqin; Gregor, Danielle; Bekker, Alex; Ye, Jiang-Hong

    2016-02-01

    There is growing interest on the role of the lateral habenula (LHb) in depression, because it closely and bilaterally connects with the serotoninergic raphe nuclei. The LHb sends glutamate efferents to the raphe nuclei, while it receives serotoninergic afferents, and expresses a high density of serotonin (5-HT) receptors. Recent studies suggest that 5-HT receptors exist both in the presynaptic and postsynaptic sites of LHb neurons, and activation of these receptors may have different effects on the activity of LHb neurons. The current study focused on the effect of 5-HT on the postsynaptic membrane. We found that 5-HT initiated a depolarizing inward current (I(5-HTi)) and accelerated spontaneous firing in ?80% of LHb neurons in rat brain slices. I(5-HTi) was also induced by the 5-HT uptake blocker citalopram, indicating activity of endogenous 5-HT. I(5-HTi) was diminished by 5-HT2/3 receptor antagonists (ritanserin, SB-200646 or ondansetron), and activated by the selective 5-HT2/3 agonists 1-(3-Chlorophenyl) piperazine hydrochloride or 1-(3-Chlorophenyl) biguanide hydrochloride. Furthermore, I(5-HTi) was attenuated by 2-Aminoethyl diphenylborinate, a blocker of transient receptor potential channels, and an IP3 receptor inhibitor, indicating the involvement of transient receptor potential channels. These results demonstrate that the reciprocal connection between the LHb and the 5-HT system highlights a key role for 5-HT stimulation of LHb neurons that may be important in the pathogenesis of depression. PMID:26471419

  8. Analysis of Nidogen-1/Laminin ?1 Interaction by Cross-Linking, Mass Spectrometry, and Computational Modeling Reveals Multiple Binding Modes

    PubMed Central

    Lössl, Philip; Kölbel, Knut; Tänzler, Dirk; Nannemann, David; Ihling, Christian H.; Keller, Manuel V.; Schneider, Marian; Zaucke, Frank; Meiler, Jens; Sinz, Andrea

    2014-01-01

    We describe the detailed structural investigation of nidogen-1/laminin ?1 complexes using full-length nidogen-1 and a number of laminin ?1 variants. The interactions of nidogen-1 with laminin variants ?1 LEb2–4, ?1 LEb2–4 N836D, ?1 short arm, and ?1 short arm N836D were investigated by applying a combination of (photo-)chemical cross-linking, high-resolution mass spectrometry, and computational modeling. In addition, surface plasmon resonance and ELISA studies were used to determine kinetic constants of the nidogen-1/laminin ?1 interaction. Two complementary cross-linking strategies were pursued to analyze solution structures of laminin ?1 variants and nidogen-1. The majority of distance information was obtained with the homobifunctional amine-reactive cross-linker bis(sulfosuccinimidyl)glutarate. In a second approach, UV-induced cross-linking was performed after incorporation of the diazirine-containing unnatural amino acids photo-leucine and photo-methionine into laminin ?1 LEb2–4, laminin ?1 short arm, and nidogen-1. Our results indicate that Asn-836 within laminin ?1 LEb3 domain is not essential for complex formation. Cross-links between laminin ?1 short arm and nidogen-1 were found in all protein regions, evidencing several additional contact regions apart from the known interaction site. Computational modeling based on the cross-linking constraints indicates the existence of a conformational ensemble of both the individual proteins and the nidogen-1/laminin ?1 complex. This finding implies different modes of interaction resulting in several distinct protein-protein interfaces. PMID:25387007

  9. Detection of the Phosphorylation of the Estrogen Receptor ? as an Outcome of GPR30 Activation.

    PubMed

    Clark, Sara; Pollard, Kevin; Rainville, Jennifer; Vasudevan, Nandini

    2016-01-01

    Phosphorylation of the serine residues in estrogen receptor (ER) ? is important in transcriptional activation. Hence, methods to detect such posttranslational modification events are valuable. We describe, in detail, the analysis of the phosphorylated ER? by electrophoretic separation of proteins and subsequent immuno-blotting techniques. In particular, phosphorylation of the ER? is one possible outcome of activation of the putative membrane estrogen receptor (mER), GPR30. Hence, phosphorylation represents a cross talk event between GPR30 and ER? and may be important in estrogen-regulated physiology. PMID:26585157

  10. Allosteric Binding Site and Activation Mechanism of Class C G-Protein Coupled Receptors: Metabotropic Glutamate Receptor Family.

    PubMed

    Feng, Zhiwei; Ma, Shifan; Hu, Guanxing; Xie, Xiang-Qun

    2015-05-01

    Metabotropic glutamate receptors (mGluR) are mainly expressed in the central nervous system (CNS) and contain eight receptor subtypes, named mGluR1 to mGluR8. The crystal structures of mGluR1 and mGluR5 that are bound with the negative allosteric modulator (NAM) were reported recently. These structures provide a basic model for all class C of G-protein coupled receptors (GPCRs) and may aid in the design of new allosteric modulators for the treatment of CNS disorders. However, these structures are only combined with NAMs in the previous reports. The conformations that are bound with positive allosteric modulator (PAM) or agonist of mGluR1/5 remain unknown. Moreover, the structural information of the other six mGluRs and the comparisons of the mGluRs family have not been explored in terms of their binding pockets, the binding modes of different compounds, and important binding residues. With these crystal structures as the starting point, we built 3D structural models for six mGluRs by using homology modeling and molecular dynamics (MD) simulations. We systematically compared their allosteric binding sites/pockets, the important residues, and the selective residues by using a series of comparable dockings with both the NAM and the PAM. Our results show that several residues played important roles for the receptors' selectivity. The observations of detailed interactions between compounds and their correspondent receptors are congruent with the specificity and potency of derivatives or compounds bioassayed in vitro. We then carried out 100 ns MD simulations of mGluR5 (residue 26-832, formed by Venus Flytrap domain, a so-called cysteine-rich domain, and 7 trans-membrane domains) bound with antagonist/NAM and with agonist/PAM. Our results show that both the NAM and the PAM seemed stable in class C GPCRs during the MD. However, the movements of "ionic lock," of trans-membrane domains, and of some activation-related residues in 7 trans-membrane domains of mGluR5 were congruent with the findings in class A GPCRs. Finally, we selected nine representative bound structures to perform 30 ns MD simulations for validating the stabilities of interactions, respectively. All these bound structures kept stable during the MD simulations, indicating that the binding poses in this present work are reasonable. We provided new insight into better understanding of the structural and functional roles of the mGluRs family and facilitated the future structure-based design of novel ligands of mGluRs family with therapeutic potential. PMID:25762450

  11. NTB-A Receptor Crystal Structure: Insights into Homophilic Interactions in the Signaling Lymphocytic Activation Molecule Receptor Family

    SciTech Connect

    Cao,E.; Ramagopal, U.; Fedorov, A.; Fedorov, E.; Yan, Q.; Lary, J.; Cole, J.; Nathenson, S.; Almo, S.

    2006-01-01

    The signaling lymphocytic activation molecule (SLAM) family includes homophilic and heterophilic receptors that regulate both innate and adaptive immunity. The ectodomains of most SLAM family members are composed of an N-terminal IgV domain and a C-terminal IgC2 domain. NK-T-B-antigen (NTB-A) is a homophilic receptor that stimulates cytotoxicity in natural killer (NK) cells, regulates bactericidal activities in neutrophils, and potentiates T helper 2 (Th2) responses. The 3.0 {angstrom} crystal structure of the complete NTB-A ectodomain revealed a rod-like monomer that self-associates to form a highly kinked dimer spanning an end-to-end distance of {approx}100 {angstrom}. The NTB-A homophilic and CD2-CD58 heterophilic dimers show overall structural similarities but differ in detailed organization and physicochemical properties of their respective interfaces. The NTB-A structure suggests a mechanism responsible for binding specificity within the SLAM family and imposes physical constraints relevant to the colocalization of SLAM-family proteins with other signaling molecules in the immunological synapse.

  12. Activation of histamine H3-receptors inhibits carrier-mediated norepinephrine release during protracted myocardial ischemia. Comparison with adenosine A1-receptors and alpha2-adrenoceptors.

    PubMed

    Imamura, M; Lander, H M; Levi, R

    1996-03-01

    We previously showed that prejunctional histamine H3-receptors downregulate norepinephrine exocytosis, which is markedly enhanced in early myocardial ischemia. In the present study, we investigated whether H3-receptors modulate nonexocytotic norepinephrine release during protracted myocardial ischemia. In this setting, decreased pH(i) in sympathetic nerve endings sequentially leads to a compensatory activation of the Na+-H+ antiporter (NHE), accumulation of intracellular Na+, reversal of the neuronal uptake of norepinephrine, and thus carrier-mediated release of norepinephrine. Accordingly, norepinephrine overflow from isolated guinea pig hearts undergoing 20-minute global ischemia and 45-minute reperfusion was attenuated approximately 80% by desipramine (10 nmol/L) and 70% by 5-(N-ethyl-N-isopropyl)-amiloride (EIPA, 10 micromol/L), inhibitors of norepinephrine uptake and NHE, respectively. The H3-receptor agonist imetit (0.1 micromol/L) decreased carrier-mediated norepinephrine release by approximately 50%. This effect was blocked by the H3-receptor antagonist thioperamide (0.3 micromol/L), indicating that H-receptor activation inhibits carrier-mediated norepinephrine release. At lower concentrations, imetit (10 nmol/L) or EIPA (3 micromol/L) did not inhibit carrier-mediated norepinephrine release. However, a 25% inhibition occurred with imetit (10 nmol/L) and EIPA (3 micromol/L) combined. This synergism suggests an association between H-receptors and NHE. Conceivably, activation of H-receptors may lead to inhibition of NHE. In fact, alpha2-adrenoceptor activation, which is known to stimulate NHE, enhanced norepinephrine release, whereas alpha2-adrenoceptor blockade attenuated it. Furthermore, activation of adenosine A1-receptors markedly attenuated norepinephrine release, whereas their inhibition potentiated it. Because norepinephrine directly correlated with the severity of reperfusion arrhythmia and imetit reduced the incidence of ventricular fibrillation by 50%, our findings with H-receptor agonists may further the development of novel pharmacological means to reduce reperfusion arrhythmias in the clinical setting. PMID:8593706

  13. Phasic dopamine release drives rapid activation of striatal D2-receptors

    PubMed Central

    Marcott, Pamela F; Mamaligas, Aphroditi A; Ford, Christopher P

    2014-01-01

    Summary Striatal dopamine transmission underlies numerous goal-directed behaviors. Medium spiny neurons (MSNs) are a major target of dopamine in the striatum. However, as dopamine does not directly evoke a synaptic event in MSNs, the time course of dopamine signaling in these cells remains unclear. To examine how dopamine release activates D2-receptors on MSNs, G-protein activated inwardly rectifying potassium (GIRK2; Kir 3.2) channels were virally overexpressed in the striatum and the resulting outward currents were used as a sensor of D2-receptor activation. Electrical and optogenetic stimulation of dopamine terminals evoked robust D2-receptor inhibitory post-synaptic currents (IPSCs) in GIRK2-expressing MSNs that occurred in under a second. Evoked D2-IPSCs could be driven by repetitive stimulation and were not occluded by background dopamine tone. Together, the results indicate that D2-receptors on MSNs exhibit functional low affinity and suggest that striatal D2-receptors can encode both tonic and phasic dopamine signals. PMID:25242218

  14. Oral administration of protease inhibits enterotoxigenic Escherichia coli receptor activity in piglet small intestine.

    PubMed Central

    Mynott, T L; Luke, R K; Chandler, D S

    1996-01-01

    The virulence of enterotoxigenic Escherichia coli (ETEC) is attributed to their ability to adhere via fimbrial adhesins to specific receptors located on the intestinal mucosa. A novel approach to preventing ETEC induced diarrhoea would be to prevent attachment of ETEC to intestine by proteolytically modifying the receptor attachment sites. This study aimed to examine the effect of bromelain, a proteolytic extract obtained from pineapple stems, on ETEC receptor activity in porcine small intestine. Bromelain was administered orally to piglets and K88+ ETEC attachment to small intestine was measured at 50 cm intervals using an enzyme immunoassay. K88+ ETEC attachment to intestinal sections that were not treated with bromelain varied appreciably between sampling sites. Variability in receptor activity along the intestinal surface is though to be caused by the localised effects of endogenous proteases. Oral administration of exogenous protease inhibited K88+ ETEC attachment to pig small intestine in a dose dependent manner (p < 0.05). Attachment of K88+ ETEC was negligible after treatment, resembling the levels of attachment of K88 to piglets of the genetically determined non-adhesive phenotype, which are resistant to K88+ ETEC infection. Serum biochemical analysis and histopathological examination of treated piglets showed no adverse effects of the bromelain treatment. It is concluded that administration of bromelain can inhibit ETEC receptor activity in vivo and may therefore be useful for prevention of K88+ ETEC induced diarrhoea. PMID:8566855

  15. Telmisartan ameliorates glutamate-induced neurotoxicity: roles of AT1 receptor blockade and PPAR? activation

    PubMed Central

    Wang, Juan; Pang, Tao; Hafko, Roman; Benicky, Julius; Sanchez-Lemus, Enrique; Saavedra, Juan M.

    2014-01-01

    Sartans (Angiotensin II AT1 Receptor Blockers, ARBs) are powerful neuroprotective agents in vivo and protect against IL-1? neurotoxicity in vitro. The purpose of this research was to determine the extent of sartan neuroprotection against glutamate excitotoxicity, a common cause of neuronal injury and apoptosis. The results show that sartans are neuroprotective, significantly reducing glutamate-induced neuronal injury and apoptosis in cultured rat primary cerebellar granule cells (CGCs). Telmisartan was the most potent sartan studied, with an order of potency telmisartan > candesartan > losartan > valsartan. Mechanisms involved reduction of pro-apoptotic caspase-3 activation, protection of the survival PI3K/Akt/GSK-3? pathway, and prevention of glutamate-induced ERK1/2 activation. NMDA receptor stimulation was essential for glutamate-induced cell injury and apoptosis. Participation of AT1A receptor was supported by glutamate-induced upregulation of AT1A gene expression and AT1 receptor binding. Conversely, AT1B or AT2 receptor played no role. Glutamate-induced neuronal injury and the neuroprotective effect of telmisartan were decreased, but not abolished, in CGCs obtained from AT1A knock-out mice. This indicates that although AT1 receptors are necessary for glutamate to exert its full neurotoxic potential, part of the neuroprotective effect of telmisartan is independent of AT1 receptor blockade. PPAR? activation was also involved in the neuroprotective effects of telmisartan, as telmisartan enhanced PPAR? nuclear translocation, and the PPAR? antagonist GW9662 partially reversed the neuroprotective effects of telmisartan. The present results substantiate the therapeutic use of sartans, in particular telmisartan, in neurodegenerative diseases and traumatic brain disorders where glutamate neurotoxicity plays a significant role. PMID:24316465

  16. Antiseizure Activity of Midazolam in Mice Lacking ?-Subunit Extrasynaptic GABA(A) Receptors.

    PubMed

    Reddy, Sandesh D; Younus, Iyan; Clossen, Bryan L; Reddy, Doodipala Samba

    2015-06-01

    Midazolam is a benzodiazepine anticonvulsant with rapid onset and short duration of action. Midazolam is the current drug of choice for acute seizures and status epilepticus, including those caused by organophosphate nerve agents. The antiseizure activity of midazolam is thought to result from its allosteric potentiation of synaptic GABA(A) receptors in the brain. However, there are indications that benzodiazepines promote neurosteroid synthesis via the 18-kDa cholesterol transporter protein (TSPO). Therefore, we investigated the role of neurosteroids and their extrasynaptic GABA(A) receptor targets in the antiseizure activity of midazolam. Here, we used ?-subunit knockout (DKO) mice bearing a targeted deletion of the extrasynaptic receptors to investigate the contribution of the extrasynaptic receptors to the antiseizure activity of midazolam using the 6-Hz and hippocampus kindling seizure models. In both models, midazolam produced rapid and dose-dependent protection against seizures (ED50, 0.4 mg/kg). Moreover, the antiseizure potency of midazolam was undiminished in DKO mice compared with control mice. Pretreatment with PK11195 [1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide], a TSPO blocker, or finasteride, a 5?-reductase neurosteroid inhibitor, did not affect the antiseizure effect of midazolam. The antiseizure activity of midazolam was significantly reversed by pretreatment with flumazenil, a benzodiazepine antagonist. Plasma and brain levels of the neurosteroid allopregnanolone were not significantly greater in midazolam-treated animals. These studies therefore provide strong evidence that neurosteroids and extrasynaptic GABA(A) receptors are not involved in the antiseizure activity of midazolam, which mainly occurs through synaptic GABA(A) receptors via direct binding to benzodiazepine sites. This study reaffirms midazolam's use for controlling acute seizures and status epilepticus. PMID:25784648

  17. Molecular cloning of an orphan G-protein-coupled receptor that constitutively activates adenylate cyclase.

    PubMed Central

    Eggerickx, D; Denef, J F; Labbe, O; Hayashi, Y; Refetoff, S; Vassart, G; Parmentier, M; Libert, F

    1995-01-01

    A human gene encoding an orphan G-protein-coupled receptor named ACCA (adenylate cyclase constitutive activator) was isolated from a genomic library using as a probe a DNA fragment obtained by low-stringency PCR. Human ACCA (hACCA) is a protein of 330 amino acids that exhibits all the structural hallmarks of the main family of G-protein-coupled receptors. Expression of hACCA resulted in a dramatic stimulation of adenylate cyclase, similar in amplitude to that obtained with other Gs-coupled receptors fully activated by their respective ligands. This stimulation was obtained in a large variety of stable cell lines derived from various organs, and originating from different mammalian species. hACCA was found to be the human homologue of a recently reported mouse orphan receptor (GPCR21). The mouse ACCA (mACCA) was therefore recloned by PCR, and expression of mACCA in Cos-7 cells demonstrated that the mouse receptor behaved similarly as a constitutive activator of adenylate cyclase. It is not known presently whether the stimulation of adenylate cyclase is the result of a true constitutive activity of the receptor or, alternatively, is the consequence of a permanent stimulation by a ubiquitous ligand. The tissue distribution of mACCA was determined by RNase protection assay. Abundant transcripts were found in the brain, whereas lower amounts were detected in testis, ovary and eye. Various hypotheses concerning the constitutive activity of ACCA and their potential biological significance are discussed. Images Figure 4 Figure 5 PMID:7639700

  18. Chronic AT2 receptor activation attenuates renal AT1 receptor function and blood pressure in obese Zucker rats

    PubMed Central

    Ali, Quaisar; Wu, Yonnie; Hussain, Tahir

    2013-01-01

    Abnormal regulation of the renin angiotensin system such as enhanced renal AT1R function and reduced ACE2 activity contributes to obesity-related hypertension. Here we tested whether long-term AT2R activation affects renal function in obesity using lean and obese Zucker rats treated with the AT2R agonist CGP42112A for 2-weeks. This caused blood pressure to decrease by 13 mmHg which was associated with increased urinary sodium excretion in the obese rats. Cortical ACE2 expression and activity, the Mas receptor (MasR), and its ligand angiotensin-(1-7) were all increased in CGP-treated obese compared with control rats. Candesartan-induced natriuresis, a measure of AT1R function, was reduced but cortical AT1R expression and angiotensin II levels were similar in CGP-treated obese compared to control rats. Renin and AT2R expression in obese rats was not affected by CGP-treatment. In HK-2 cells in-vitro, CGP-treatment caused increased ACE2 activity and MasR levels but decreased AT1R levels and renin activity. Thus, long-term AT2R activation shifts the opposing arms of renin angiotensin system and contributes to natriuresis and blood pressure reduction in obese animals. Our study highlights the importance of AT2R as a target for treating obesity related hypertension. PMID:23823602

  19. Pharmacological properties of FK886, a new, centrally active neurokinin-1 receptor antagonist.

    PubMed

    Yoshino-Furukawa, Takako; Maeda, Yasue; Kikuchi, Aya; Sakuma, Hiroyuki; Imazumi, Katsunori; Yamakuni, Hisashi; Sogabe, Hajime; Matsuo, Masahiko; Manda, Toshitaka; Uchida, Wataru

    2013-01-01

    The pharmacological properties of the novel neurokinin-1 (NK(1)) receptor antagonist FK886, ([3,5-bis(trifluoromethyl)phenyl][(2R)-2-(3-hydroxy-4-methylbenzyl)-4-{2-[(2S)-2-(methoxymethyl)morpholin-4-yl]ethyl}piperazin-1-yl]methanone dihydrochloride), were studied. FK886 potently inhibited the binding of [(125)I]Bolton-Hunter-labeled substance P ([(125)I]BH-SP; 100?pM) to human NK(1) receptors expressed in Chinese hamster ovary (CHO) cells (IC(50)=0.70?nM). It also possessed high affinities for dog, ferret, gerbil and guinea pig NK(1) receptors, but not for rat NK(1) receptor. FK886 was highly selective for the NK(1) receptor, with 250- and >20000-fold selectivity for human NK(1) over NK(2) and NK(3), respectively. Further, it did not inhibit radioligand binding at 54 different sites, including receptors, ion channels and transporters. FK886 inhibited substance P (3.2?nM)-induced inositol phosphate formation in human NK(1) receptor-expressing CHO cells (IC(50)=1.4?nM) without stimulating NK(1) receptors. The antagonism exerted by FK886 against human NK(1) receptor was insurmountable in saturation binding experiments, with both the affinity and B(max) of [(125)I]BH-SP being significantly reduced. After intravenous administration, FK886 (0.01-0.1?mg/kg) dose-dependently inhibited the foot-tapping behavior induced by intracerebroventricular administration of a selective NK(1) receptor agonist, GR73632 (10?pmol), in gerbils, with significant inhibition being observed at doses of 0.032-0.1?mg/kg, indicating excellent brain penetration. The brain penetration of FK886 was further demonstrated by the cerebral distribution of radioactivity after intravenous injection of radiolabeled FK886. Taken together, these results demonstrate that FK886 is a potent, highly selective and centrally active, insurmountable antagonist of the NK(1) receptor, and suggest that FK886 antagonizes various NK(1) receptor-mediated biological effects in the central nervous system. PMID:23302639

  20. Identification of chemical modulators of the constitutive activated receptor (CAR) in a gene expression compendium

    PubMed Central

    Oshida, Keiyu; Vasani, Naresh; Jones, Carlton; Moore, Tanya; Hester, Susan; Nesnow, Stephen; Auerbach, Scott; Geter, David R.; Aleksunes, Lauren M.; Thomas, Russell S.; Applegate, Dawn; Klaassen, Curtis D.; Corton, J. Christopher

    2015-01-01

    The nuclear receptor family member constitutive activated receptor (CAR) is activated by structurally diverse drugs and environmentally-relevant chemicals leading to transcriptional regulation of genes involved in xenobiotic metabolism and transport. Chronic activation of CAR increases liver cancer incidence in rodents, whereas suppression of CAR can lead to steatosis and insulin insensitivity. Here, analytical methods were developed to screen for chemical treatments in a gene expression compendium that lead to alteration of CAR activity. A gene expression biomarker signature of 83 CAR-dependent genes was identified using microarray profiles from the livers of wild-type and CAR-null mice after exposure to three structurally-diverse CAR activators (CITCO, phenobarbital, TCPOBOP). A rank-based algorithm (Running Fisher’s algorithm (p-value ? 10-4)) was used to evaluate the similarity between the CAR biomarker signature and a test set of 28 and 32 comparisons positive or negative, respectively, for CAR activation; the test resulted in a balanced accuracy of 97%. The biomarker signature was used to identify chemicals that activate or suppress CAR in an annotated mouse liver/primary hepatocyte gene expression database of ~1850 comparisons. CAR was activated by 1) activators of the aryl hydrocarbon receptor (AhR) in wild-type but not AhR-null mice, 2) pregnane X receptor (PXR) activators in wild-type and to lesser extents in PXR-null mice, and 3) activators of PPAR? in wild-type and PPAR?-null mice. CAR was consistently activated by five conazole fungicides and four perfluorinated compounds. Comparison of effects in wild-type and CAR-null mice showed that the fungicide propiconazole increased liver weight and hepatocyte proliferation in a CAR-dependent manner, whereas the perfluorinated compound perfluorooctanoic acid (PFOA) increased these endpoints in a CAR-independent manner. A number of compounds suppressed CAR coincident with increases in markers of inflammation including acetaminophen, concanavalin A, lipopolysaccharide, and 300 nm silica particles. In conclusion, we have shown that a CAR biomarker signature coupled with a rank-based similarity method accurately predicts CAR activation. This analytical approach, when applied to a gene expression compendium, increased the universe of known chemicals that directly or indirectly activate CAR, highlighting the promiscuous nature of CAR activation and signaling through activation of other xenobiotic-activated receptors. PMID:25949234

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

    PubMed Central

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

    2015-01-01

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

  2. Spatio-temporally precise activation of engineered receptor tyrosine kinases by light

    PubMed Central

    Grusch, Michael; Schelch, Karin; Riedler, Robert; Reichhart, Eva; Differ, Christopher; Berger, Walter; Inglés-Prieto, Álvaro; Janovjak, Harald

    2014-01-01

    Receptor tyrosine kinases (RTKs) are a large family of cell surface receptors that sense growth factors and hormones and regulate a variety of cell behaviours in health and disease. Contactless activation of RTKs with spatial and temporal precision is currently not feasible. Here, we generated RTKs that are insensitive to endogenous ligands but can be selectively activated by low-intensity blue light. We screened light-oxygen-voltage (LOV)-sensing domains for their ability to activate RTKs by light-activated dimerization. Incorporation of LOV domains found in aureochrome photoreceptors of stramenopiles resulted in robust activation of the fibroblast growth factor receptor 1 (FGFR1), epidermal growth factor receptor (EGFR) and rearranged during transfection (RET). In human cancer and endothelial cells, light induced cellular signalling with spatial and temporal precision. Furthermore, light faithfully mimicked complex mitogenic and morphogenic cell behaviour induced by growth factors. RTKs under optical control (Opto-RTKs) provide a powerful optogenetic approach to actuate cellular signals and manipulate cell behaviour. PMID:24986882

  3. Interhelical interaction and receptor phosphorylation regulate the activation kinetics of different human ?1-adrenoceptor variants.

    PubMed

    Ahles, Andrea; Rodewald, Fabian; Rochais, Francesca; Bünemann, Moritz; Engelhardt, Stefan

    2015-01-16

    G protein-coupled receptors represent the largest class of drug targets, but genetic variation within G protein-coupled receptors leads to variable drug responses and, thereby, compromises their therapeutic application. One of the most intensely studied examples is a hyperfunctional variant of the human ?1-adrenoceptor that carries an arginine at position 389 in helix 8 (Arg-389-ADRB1). However, the mechanism underlying the higher efficacy of the Arg-389 variant remained unclear to date. Despite its hyperfunctionality, we found the Arg-389 variant of ADRB1 to be hyperphosphorylated upon continuous stimulation with norepinephrine compared with the Gly-389 variant. Using ADRB1 sensors to monitor activation kinetics by fluorescence resonance energy transfer, Arg-389-ADRB1 exerted faster activation speed and arrestin recruitment than the Gly-389 variant. Both activation speed and arrestin recruitment depended on phosphorylation of the receptor, as shown by knockdown of G protein-coupled receptor kinases and phosphorylation-deficient ADRB1 mutants. Structural modeling of the human ?1-adrenoceptor suggested interaction of the side chain of Arg-389 with opposing amino acid residues in helix 1. Site-directed mutagenesis of Lys-85 and Thr-86 in helix 1 revealed that this interaction indeed determined ADRB1 activation kinetics. Taken together, these findings indicate that differences in interhelical interaction regulate the different activation speed and efficacy of ADRB1 variants. PMID:25451930

  4. Tissue plasminogen activator inhibits NMDA-receptor-mediated increases in calcium levels in cultured hippocampal neurons

    PubMed Central

    Robinson, Samuel D.; Lee, Tet Woo; Christie, David L.; Birch, Nigel P.

    2015-01-01

    NMDA receptors (NMDARs) play a critical role in neurotransmission, acting as essential mediators of many forms of synaptic plasticity, and also modulating aspects of development, synaptic transmission and cell death. NMDAR-induced responses are dependent on a range of factors including subunit composition and receptor location. Tissue-type plasminogen activator (tPA) is a serine protease that has been reported to interact with NMDARs and modulate NMDAR activity. In this study we report that tPA inhibits NMDAR-mediated changes in intracellular calcium levels in cultures of primary hippocampal neurons stimulated by low (5 ?M) but not high (50 ?M) concentrations of NMDA. tPA also inhibited changes in calcium levels stimulated by presynaptic release of glutamate following treatment with bicucculine/4-aminopyridine (4-AP). Inhibition was dependent on the proteolytic activity of tPA but was unaffected by ?2-antiplasmin, an inhibitor of the tPA substrate plasmin, and receptor-associated protein (RAP), a pan-ligand blocker of the low-density lipoprotein receptor, two proteins previously reported to modulate NMDAR activity. These findings suggest that tPA can modulate changes in intracellular calcium levels in a subset of NMDARs expressed in cultured embryonic hippocampal neurons through a mechanism that involves the proteolytic activity of tPA and synaptic NMDARs. PMID:26500501

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

    DOE PAGESBeta

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

    2015-07-24

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

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

    SciTech Connect

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

    2015-07-24

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

  7. Activation of the G protein-coupled estrogen receptor, but not estrogen receptor ? or ?, rapidly enhances social learning.

    PubMed

    Ervin, Kelsy Sharice Jean; Mulvale, Erin; Gallagher, Nicola; Roussel, Véronique; Choleris, Elena

    2015-08-01

    Social learning is a highly adaptive process by which an animal acquires information from a conspecific. While estrogens are known to modulate learning and memory, much of this research focuses on individual learning. Estrogens have been shown to enhance social learning on a long-term time scale, likely via genomic mechanisms. Estrogens have also been shown to affect individual learning on a rapid time scale through cell-signaling cascades, rather than via genomic effects, suggesting they may also rapidly influence social learning. We therefore investigated the effects of 17?-estradiol and involvement of the estrogen receptors (ERs) using the ER? agonist propyl pyrazole triol, the ER? agonist diarylpropionitrile, and the G protein-coupled ER 1 (GPER1) agonist G1 on the social transmission of food preferences (STFP) task, within a time scale that focused on the rapid effects of estrogens. General ER activation with 17?-estradiol resulted in a modest facilitation of social learning, with mice showing a preference up to 30min of testing. Specific activation of the GPER1 also rapidly enhanced social learning, with mice showing a socially learned preference up to 2h of testing. ER? activation instead shortened the expression of a socially learned food preference, while ER? activation had little to no effects. Thus, rapid estrogenic modulation of social learning in the STFP may be the outcome of competing action at the three main receptors. Hence, estrogens' rapid effects on social learning likely depend on the specific ERs present in brain regions recruited during social learning. PMID:25957002

  8. The metabotropic glutamate receptor 4 is internalized and desensitized upon protein kinase C activation

    PubMed Central

    Mathiesen, Jesper Mosolff; Ramirez, M Teresa

    2006-01-01

    The metabotropic glutamate receptor 4 (mGluR4) is a G?i-coupled receptor that modulates glutamatergic neurotransmission. As mGluR4 expression and activation have been implicated in a number of pathological conditions and because the internalization and desensitization properties of this receptor are poorly understood, studies were designed to investigate these aspects of mGluR4 biology. Neither agonist activation by L-(+)-2-amino-4-phosphonobutyric acid (L-AP4) nor L-glutamate caused mGluR4 internalization when cmyc-tagged mGluR4 was expressed in a human embryonic kidney 293 cell line as assessed by cell surface enzyme-linked immunosorbent and immunostaining assays. Instead, a modest increase in mGluR4 surface expression was observed and found to be receptor specific as the competitive antagonist ?-cyclopropyl-4-phosphonophenylglycine (CPPG) blocked this effect. In contrast, mGluR4 internalized when the protein kinase C (PKC) pathway was activated either by phorbol-12-myristate-13-acetate (PMA) or by the activation of the G?q-coupled, neurokinin 3 receptor (NK3R) when co-expressed. This process was PKC-dependent as the specific PKC inhibitor GF 109203X inhibited PMA and NK3R-mediated internalization. PKC activation by PMA caused desensitization of mGluR4 as measured by forskolin-stimulated cAMP inhibition, whereas agonist activation had no effect on desensitization. When mGluR4's coupling was redirected from adenylyl cyclase to phospholipase C by coexpression of a chimeric G?qo5 protein, mGluR4 both internalized and desensitized in response to its agonists. These findings demonstrate that mGluR4 internalization and desensitization are agonist-independent unless pathways leading to the activation of PKC are induced. PMID:16582932

  9. Activation of Neuropeptide Y Receptors Modulates Retinal Ganglion Cell Physiology and Exerts Neuroprotective Actions In Vitro

    PubMed Central

    Martins, Joăo; Elvas, Filipe; Brudzewsky, Dan; Martins, Tânia; Kolomiets, Bogdan; Tralhăo, Pedro; Gřtzsche, Casper R.; Cavadas, Cláudia; Castelo-Branco, Miguel; Woldbye, David P. D.; Picaud, Serge; Santiago, Ana R.

    2015-01-01

    Neuropeptide Y (NPY) is expressed in mammalian retina but the location and potential modulatory effects of NPY receptor activation remain largely unknown. Retinal ganglion cell (RGC) death is a hallmark of several retinal degenerative diseases, particularly glaucoma. Using purified RGCs and ex vivo rat retinal preparations, we have measured RGC intracellular free calcium concentration ([Ca2+]i) and RGC spiking activity, respectively. We found that NPY attenuated the increase in the [Ca2+]i triggered by glutamate mainly via Y1 receptor activation. Moreover, (Leu31, Pro34)?NPY, a Y1/Y5 receptor agonist, increased the initial burst response of OFF-type RGCs, although no effect was observed on RGC spontaneous spiking activity. The Y1 receptor activation was also able to directly modulate RGC responses by attenuating the NMDA-induced increase in RGC spiking activity. These results suggest that Y1 receptor activation, at the level of inner or outer plexiform layers, leads to modulation of RGC receptive field properties. Using in vitro cultures of rat retinal explants exposed to NMDA, we found that NPY pretreatment prevented NMDA-induced cell death. However, in an animal model of retinal ischemia-reperfusion injury, pretreatment with NPY or (Leu31, Pro34)?NPY was not able to prevent apoptosis or rescue RGCs. In conclusion, we found modulatory effects of NPY application that for the first time were detected at the level of RGCs. However, further studies are needed to evaluate whether NPY neuroprotective actions detected in retinal explants can be translated into animal models of retinal degenerative diseases. PMID:26311075

  10. Increased Dopamine Receptor Activity in the Nucleus Accumbens Shell Ameliorates Anxiety during Drug Withdrawal

    PubMed Central

    Radke, Anna K; Gewirtz, Jonathan C

    2012-01-01

    A number of lines of evidence suggest that negative emotional symptoms of withdrawal involve reduced activity in the mesolimbic dopamine system. This study examined the contribution of dopaminergic signaling in structures downstream of the ventral tegmental area to withdrawal from acute morphine exposure, measured as potentiation of the acoustic startle reflex. Systemic administration of the general dopamine receptor agonist apomorphine or a cocktail of the D1-like receptor agonist SKF82958 and the D2-like receptor agonist quinpirole attenuated potentiated startle during morphine withdrawal. This effect was replicated by apomorphine infusion into the nucleus accumbens shell. Finally, apomorphine injection was shown to relieve startle potentiation during nicotine withdrawal and conditioned place aversion to morphine withdrawal. These results suggest that transient activation of the ventral tegmental area mesolimbic dopamine system triggers the expression of anxiety and aversion during withdrawal from multiple classes of abused drugs. PMID:22692565

  11. Critical roles of