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Sample records for factor receptor interaction

  1. Macrophage Migration Inhibitory Factor-CXCR4 Receptor Interactions

    PubMed Central

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

    2016-01-01

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

  2. Diversification of Neu differentiation factor and epidermal growth factor signaling by combinatorial receptor interactions.

    PubMed Central

    Pinkas-Kramarski, R; Soussan, L; Waterman, H; Levkowitz, G; Alroy, I; Klapper, L; Lavi, S; Seger, R; Ratzkin, B J; Sela, M; Yarden, Y

    1996-01-01

    The ErbB family includes two receptors, ErbB-1 and ErbB-3, that respectively bind to epidermal growth factor and Neu differentiation factor, and an orphan receptor, ErbB-2. Unlike ErbB-1 and ErbB-2, the intrinsic tyrosine kinase of ErbB-3 is catalytically impaired. By using interleukin-3-dependent cells that ectopically express the three ErbB proteins or their combinations, we found that ErbB-3 is devoid of any biological activity but both ErbB-1 and ErbB-2 can reconstitute its extremely potent mitogenic activity. Transactivation of ErbB-3 correlates with heterodimer formation and is reflected in receptor phosphorylation and the transregulation of ligand affinity. Inter-receptor interactions enable graded proliferative and survival signals: heterodimers are more potent than homodimers, and ErbB-3-containing complexes, especially the ErbB-2/ErbB-3 heterodimer, are more active than ErbB-1 complexes. Nevertheless, ErbB-1 signaling displays dominance over ErbB-3 when the two receptors are coexpressed. Although all receptor combinations activate the mitogen-activated protein kinases ERK and c-Jun kinase, they differ in their rate of endocytosis and in coupling to intervening signaling proteins. It is conceivable that combinatorial receptor interactions diversify signal transduction and confer double regulation, in cis and in trans, of the superior mitogenic activity of the kinase-defective ErbB-3. Images PMID:8665853

  3. Molecular Docking and Interactions of Pueraria Tuberosa with Vascular Endothelial Growth Factor Receptors

    PubMed Central

    Asthana, S.; Agarwal, T.; Singothu, S.; Samal, A.; Banerjee, I.; Pal, K.; Pramanik, K.; Ray, S. S.

    2015-01-01

    Pueraria tuberosa is known for its therapeutic potentials in cardiovascular disorders, but its effect in angiogenesis has not been studied so far. In this study, a computational approach has been applied to elucidate the role of the phytochemicals in inhibition of angiogenesis through modulation of vascular endothelial growth factor receptors: Vascular endothelial growth factor receptor-1 and vascular endothelial growth factor receptor-2, major factors responsible for angiogenesis. Metabolite structures retrieved from PubChem and KNApSAcK – 3D databases, were docked using AutoDock4.2 tool. Hydrogen bond and molecular docking, absorption, distribution, metabolism and excretion and toxicity predictions were carried out using UCSF Chimera, LigPlot+ and PreADMET server, respectively. From the docking analysis, it was observed that puerarone and tuberostan had significant binding affinity for the intracellular kinase domain of vascular endothelial growth factor receptors-1 and vascular endothelial growth factor receptor-2 respectively. It is important to mention that both the phytochemicals shared similar interaction profile as that of standard inhibitors of vascular endothelial growth factor receptors. Also, both puerarone and tuberostan interacted with Lys861/Lys868 (adenosine 5’-triphosphate binding site of vascular endothelial growth factor receptors-1/vascular endothelial growth factor receptors-2), thus providing a clue that they may enforce their inhibitory effect by blocking the adenosine 5’-triphosphate binding domain of vascular endothelial growth factor receptors. Moreover, these molecules exhibited good drug-likeness, absorption, distribution, metabolism and excretion properties without any carcinogenic and toxic effects. The interaction pattern of the puerarone and tuberostan may provide a hint for a novel drug design for vascular endothelial growth factor tyrosine kinase receptors with better specificity to treat angiogenic disorders. PMID:26664060

  4. Extended Synaptotagmin Interaction with the Fibroblast Growth Factor Receptor Depends on Receptor Conformation, Not Catalytic Activity.

    PubMed

    Tremblay, Michel G; Herdman, Chelsea; Guillou, François; Mishra, Prakash K; Baril, Joëlle; Bellenfant, Sabrina; Moss, Tom

    2015-06-26

    We previously demonstrated that ESyt2 interacts specifically with the activated FGF receptor and is required for a rapid phase of receptor internalization and for functional signaling via the ERK pathway in early Xenopus embryos. ESyt2 is one of the three-member family of Extended Synaptotagmins that were recently shown to be implicated in the formation of endoplasmic reticulum (ER)-plasma membrane (PM) junctions and in the Ca(2+) dependent regulation of these junctions. Here we show that ESyt2 is directed to the ER by its putative transmembrane domain, that the ESyts hetero- and homodimerize, and that ESyt2 homodimerization in vivo requires a TM adjacent sequence but not the SMP domain. ESyt2 and ESyt3, but not ESyt1, selectively interact in vivo with activated FGFR1. In the case of ESyt2, this interaction requires a short TM adjacent sequence and is independent of receptor autophosphorylation, but dependent on receptor conformation. The data show that ESyt2 recognizes a site in the upper kinase lobe of FGFR1 that is revealed by displacement of the kinase domain activation loop during receptor activation.

  5. Structural basis of interactions between epidermal growth factor receptor and SH2 domain proteins.

    PubMed

    Sierke, S L; Longo, G M; Koland, J G

    1993-02-26

    The structural basis of the interactions between the activated epidermal growth factor (EGF) receptor and SH2 domain proteins was investigated. The c-src SH2 domain (second domain of src homology) was expressed as a recombinant fusion protein, and an in vitro assay was developed to monitor EGF receptor/SH2 domain interactions. EGF receptor tyrosine kinase domain (TKD) forms expressed in the baculovirus/insect cell system were shown to bind to the SH2 domain when phosphorylated. These TKD/SH2 domain interactions were characterized by dissociation constants of 60-320 nM. Deletion analysis indicated that the entire SH2 domain was required for recognition of the phosphorylated TKD. The binding of a highly truncated TKD protein to the SH2 domain suggested that the sites recognized by the SH2 domain included the EGF receptor autophosphorylation site, tyr992. A phosphorylated EGF receptor peptide containing tyr992 was also shown to interact with the SH2 domain. This residue may therefore mediate interactions between the EGF receptor and tyrosine kinases in the src family.

  6. Modeling the epidermal growth factor -- epidermal growth factor receptor l2 domain interaction: implications for the ligand binding process.

    PubMed

    Jorissen, Robert N; Treutlein, Herbert R; Epa, V Chandana; Burgess, Antony W

    2002-06-01

    Signaling from the epidermal growth factor (EGF) receptor is triggered by the binding of ligands such as EGF or transforming growth factor alpha (TGF-alpha) and subsequent receptor dimerization. An understanding of these processes has been hindered by the lack of structural information about the ligand-bound, dimerized EGF receptor. Using an NMR-derived structure of EGF and a homology model of the major ligand binding domain of the EGF receptor and experimental data, we modeled the binding of EGF to this EGF receptor fragment. In this low resolution model of the complex, EGF sits across the second face of the EGF receptor L2 domain and EGF residues 10-16, 36-37, 40-47 bind to this face. The structural model is largely consistent with previously published NMR data describing the residues of TGF-alpha which interact strongly with the EGF receptor. Other EGF residues implicated in receptor binding are accounted by our proposal that the ligand binding is a two-step process with the EGF binding to at least one other site of the receptor. This three-dimensional model is expected to be useful in the design of ligand-based antagonists of the receptor.

  7. The CXC Chemokine Receptor 4 Ligands Ubiquitin and Stromal Cell-derived Factor-1α Function through Distinct Receptor Interactions*

    PubMed Central

    Saini, Vikas; Staren, Daniel M.; Ziarek, Joshua J.; Nashaat, Zayd N.; Campbell, Edward M.; Volkman, Brian F.; Marchese, Adriano; Majetschak, Matthias

    2011-01-01

    Recently, we identified extracellular ubiquitin as an endogenous CXC chemokine receptor (CXCR) 4 agonist. However, the receptor selectivity and molecular basis of the CXCR4 agonist activity of ubiquitin are unknown, and functional consequences of CXCR4 activation with ubiquitin are poorly defined. Here, we provide evidence that ubiquitin and the cognate CXCR4 ligand stromal cell-derived factor (SDF)-1α do not share CXCR7 as a receptor. We further demonstrate that ubiquitin does not utilize the typical two-site binding mechanism of chemokine-receptor interactions, in which the receptor N terminus is important for ligand binding. CXCR4 activation with ubiquitin and SDF-1α lead to similar Gαi-responses and to a comparable magnitude of phosphorylation of ERK-1/2, p90 ribosomal S6 kinase-l and Akt, although phosphorylations occur more transiently after activation with ubiquitin. Despite the similarity of signal transduction events after activation of CXCR4 with both ligands, ubiquitin possesses weaker chemotactic activity than SDF-lα in cell migration assays and does not interfere with productive entry of HIV-1 into P4.R5 multinuclear activation of galactosidase indicator cells. Unlike SDF-1α, ubiquitin lacks interactions with an N-terminal CXCR4 peptide in NMR spectroscopy experiments. Binding and signaling studies in the presence of antibodies against the N terminus and extracellular loops 2/3 of CXCR4 confirm that the ubiquitin CXCR4 interaction is independent of the N-terminal receptor domain, whereas blockade of extracellular loops 2/3 prevents receptor binding and activation. Our findings define ubiquitin as a CXCR4 agonist, which does not interfere with productive cellular entry of HIV-1, and provide new mechanistic insights into interactions between CXCR4 and its natural ligands. PMID:21757744

  8. Neurosteroid dehydroepiandrosterone interacts with nerve growth factor (NGF) receptors, preventing neuronal apoptosis.

    PubMed

    Lazaridis, Iakovos; Charalampopoulos, Ioannis; Alexaki, Vassilia-Ismini; Avlonitis, Nicolaos; Pediaditakis, Iosif; Efstathopoulos, Paschalis; Calogeropoulou, Theodora; Castanas, Elias; Gravanis, Achille

    2011-04-01

    The neurosteroid dehydroepiandrosterone (DHEA), produced by neurons and glia, affects multiple processes in the brain, including neuronal survival and neurogenesis during development and in aging. We provide evidence that DHEA interacts with pro-survival TrkA and pro-death p75(NTR) membrane receptors of neurotrophin nerve growth factor (NGF), acting as a neurotrophic factor: (1) the anti-apoptotic effects of DHEA were reversed by siRNA against TrkA or by a specific TrkA inhibitor; (2) [(3)H]-DHEA binding assays showed that it bound to membranes isolated from HEK293 cells transfected with the cDNAs of TrkA and p75(NTR) receptors (K(D): 7.4 ± 1.75 nM and 5.6 ± 0.55 nM, respectively); (3) immobilized DHEA pulled down recombinant and naturally expressed TrkA and p75(NTR) receptors; (4) DHEA induced TrkA phosphorylation and NGF receptor-mediated signaling; Shc, Akt, and ERK1/2 kinases down-stream to TrkA receptors and TRAF6, RIP2, and RhoGDI interactors of p75(NTR) receptors; and (5) DHEA rescued from apoptosis TrkA receptor positive sensory neurons of dorsal root ganglia in NGF null embryos and compensated NGF in rescuing from apoptosis NGF receptor positive sympathetic neurons of embryonic superior cervical ganglia. Phylogenetic findings on the evolution of neurotrophins, their receptors, and CYP17, the enzyme responsible for DHEA biosynthesis, combined with our data support the hypothesis that DHEA served as a phylogenetically ancient neurotrophic factor.

  9. Neurosteroid Dehydroepiandrosterone Interacts with Nerve Growth Factor (NGF) Receptors, Preventing Neuronal Apoptosis

    PubMed Central

    Alexaki, Vassilia-Ismini; Avlonitis, Nicolaos; Pediaditakis, Iosif; Efstathopoulos, Paschalis; Calogeropoulou, Theodora; Castanas, Elias; Gravanis, Achille

    2011-01-01

    The neurosteroid dehydroepiandrosterone (DHEA), produced by neurons and glia, affects multiple processes in the brain, including neuronal survival and neurogenesis during development and in aging. We provide evidence that DHEA interacts with pro-survival TrkA and pro-death p75NTR membrane receptors of neurotrophin nerve growth factor (NGF), acting as a neurotrophic factor: (1) the anti-apoptotic effects of DHEA were reversed by siRNA against TrkA or by a specific TrkA inhibitor; (2) [3H]-DHEA binding assays showed that it bound to membranes isolated from HEK293 cells transfected with the cDNAs of TrkA and p75NTR receptors (KD: 7.4±1.75 nM and 5.6±0.55 nM, respectively); (3) immobilized DHEA pulled down recombinant and naturally expressed TrkA and p75NTR receptors; (4) DHEA induced TrkA phosphorylation and NGF receptor-mediated signaling; Shc, Akt, and ERK1/2 kinases down-stream to TrkA receptors and TRAF6, RIP2, and RhoGDI interactors of p75NTR receptors; and (5) DHEA rescued from apoptosis TrkA receptor positive sensory neurons of dorsal root ganglia in NGF null embryos and compensated NGF in rescuing from apoptosis NGF receptor positive sympathetic neurons of embryonic superior cervical ganglia. Phylogenetic findings on the evolution of neurotrophins, their receptors, and CYP17, the enzyme responsible for DHEA biosynthesis, combined with our data support the hypothesis that DHEA served as a phylogenetically ancient neurotrophic factor. PMID:21541365

  10. Fluorescence techniques used to measure interactions between hydroxyapatite nanoparticles and epidermal growth factor receptors.

    PubMed

    Kathawala, Mustafa H; Khoo, Stella P K; Sudhaharan, Thankiah; Zhao, Xinxin; Say Chye Loo, Joachim; Ahmed, Sohail; Woei Ng, Kee

    2015-01-01

    The potential applications of nanomaterials in therapeutics are immense and to fully explore this potential, it is important to understand the interaction of nanoparticles with cellular components. To examine the interaction between nanoparticles and cell membrane receptors, this report describes the use of advanced fluorescence techniques to measure interactions between hydroxyapatite (HA) nanoparticles and epidermal growth factor receptors (EGFRs), as a model system. FITC-labelled HA nanoparticles and monomeric red fluorescent protein (mRFP)-conjugated EGFRs expressed in Chinese hamster ovary cells (CHO-K1) were generated and their interaction measured using acceptor photobleaching-fluorescence resonance energy transfer (AP-FRET) and fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET). Results confirmed that hydroxyapatite nanoparticles not only interacted with EGFR but also attenuated downstream EGFR signalling, possibly by hindering normal dimerization of EGFR. Furthermore, the extent of signal attenuation suggested correlation with specific surface area of the nanoparticles, whereby greater specific surface area resulted in greater downstream signal attenuation. This novel demonstration establishes fluorescence techniques as a viable method to study nanoparticle interactions with proteins such as cell surface receptors. The approach described herein can be extended to study interactions between any fluorescently labelled nanoparticle-biomolecule pair.

  11. Novel Bioluminescent Binding Assays for Ligand–Receptor Interaction Studies of the Fibroblast Growth Factor Family

    PubMed Central

    Song, Ge; Shao, Xiao-Xia; Wu, Qing-Ping; Xu, Zeng-Guang; Liu, Ya-Li; Guo, Zhan-Yun

    2016-01-01

    We recently developed novel bioluminescent binding assays for several protein/peptide hormones to study their interactions with receptors using the so far brightest NanoLuc reporter. To validate the novel bioluminescent binding assay using a variety of protein/peptide hormones, in the present work we applied it to the fibroblast growth factor (FGF) family using the prototype member FGF2 as an example. A fully active recombinant FGF2 retaining a unique exposed cysteine (Cys) residue was chemically conjugated with an engineered NanoLuc carrying a unique exposed Cys residue at the C-terminus via formation of an intermolecular disulfide linkage. The NanoLuc-conjugated FGF2 (FGF2-Luc) retained high binding affinity to the overexpressed FGFR1 and the endogenous FGF receptor with the calculated dissociation constants of 161 ± 21 pM (n = 3) and 25 ± 4 pM (n = 3), respectively. In competition binding assays using FGF2-Luc as a tracer, receptor-binding potencies of wild-type or mutant FGF2s were accurately quantified. Thus, FGF2-Luc represents a novel non-radioactive tracer for the quantitative measurement of ligand–receptor interactions in the FGF family. These data suggest that the novel bioluminescent binding assay can be applied to a variety of protein/peptide hormones for ligand–receptor interaction studies. PMID:27414797

  12. Premature Ligand-Receptor Interaction during Biosynthesis Limits the Production of Growth Factor Midkine and Its Receptor LDL Receptor-related Protein 1*

    PubMed Central

    Sakamoto, Kazuma; Bu, Guojun; Chen, Sen; Takei, Yoshifumi; Hibi, Kenji; Kodera, Yasuhiro; McCormick, Lynn M.; Nakao, Akimasa; Noda, Masaharu; Muramatsu, Takashi; Kadomatsu, Kenji

    2011-01-01

    Protein production within the secretory pathway is accomplished by complex but organized processes. Here, we demonstrate that the growth factor midkine interacts with LDL receptor-related protein 1 (LRP1) at high affinity (Kd value, 2.7 nm) not only at the cell surface but also within the secretory pathway during biosynthesis. The latter premature ligand-receptor interaction resulted in aggregate formation and consequently suppressed midkine secretion and LRP1 maturation. We utilized an endoplasmic reticulum (ER) retrieval signal and an LRP1 fragment, which strongly bound to midkine and the LRP1-specialized chaperone receptor-associated protein (RAP), to construct an ER trapper. The ER trapper efficiently trapped midkine and RAP and mimicked the premature ligand-receptor interaction, i.e. suppressed maturation of the ligand and receptor. The ER trapper also diminished the inhibitory function of LRP1 on platelet-derived growth factor-mediated cell migration. Complementary to these results, an increased expression of RAP was closely associated with midkine expression in human colorectal carcinomas (33 of 39 cases examined). Our results suggest that the premature ligand-receptor interaction plays a role in protein production within the secretory pathway. PMID:21212259

  13. Interaction of the. alpha. beta. dimers of the insulin-like growth factor I receptor required for receptor autophosphorylation

    SciTech Connect

    Tollesfsen, S.E.; Stoszek, R.M.; Thompson, K. )

    1991-01-01

    The authors have recently found that association of the two {alpha}{beta} dimers of the insulin-like growth factor I (IGF I) receptor is required for formation of a high-affinity binding site for IGF I. To determine the structural requirements for IGF I activated kinase activity, they have examined the effect of dissociation of the two {alpha}{beta} dimers of the IGF I receptor on {beta} subunit autophosphorylation. The {alpha}{beta} dimers formed after treatment with 2 mM dithiothreitol (DTT) at pH 8.75 for 5 min were separated from IGF I receptor remaining as tetramers after DTT treatment by fast protein liquid chromatography on a Superose 6 gel filtration column. Purification of the {alpha}{beta} dimers was confirmed by Western blot analysis using {sup 125}I-labeled {alpha}IR-3, a monoclonal antibody to the IGF I receptor. Autophosphorylation of the IGF I receptor ({alpha}{beta}){sub 2} tetramer, treated without DTT or remaining after DTT treatment, is stimulated 1.6-2.9-fold by IGF I. In contrast, autophosporylation of the {alpha}{beta} dimers incubated in the presence or absence of IGF I (100 ng/mL) does not occur. Both IGF I receptor dimers and tetramers exhibit similar kinase activities using the synthetic substrate Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly, indicating that the failure to detect autophosphorylation of the IGF I receptor dimers does not result from inactivation of the kinase by DTT treatment. They conclude that autophosphorylation of the IGF I receptor depends upon the interaction of the two {alpha}{beta} dimers.

  14. Interactions between Type III receptor tyrosine phosphatases and growth factor receptor tyrosine kinases regulate tracheal tube formation in Drosophila.

    PubMed

    Jeon, Mili; Scott, Matthew P; Zinn, Kai

    2012-06-15

    The respiratory (tracheal) system of the Drosophila melanogaster larva is an intricate branched network of air-filled tubes. Its developmental logic is similar in some ways to that of the vertebrate vascular system. We previously described a unique embryonic tracheal tubulogenesis phenotype caused by loss of both of the Type III receptor tyrosine phosphatases (RPTPs), Ptp4E and Ptp10D. In Ptp4E Ptp10D double mutants, the linear tubes in unicellular and terminal tracheal branches are converted into bubble-like cysts that incorporate apical cell surface markers. This tube geometry phenotype is modulated by changes in the activity or expression of the epidermal growth factor receptor (Egfr) tyrosine kinase (TK). Ptp10D physically interacts with Egfr. Here we demonstrate that the Ptp4E Ptp10D phenotype is the consequence of the loss of negative regulation by the RPTPs of three growth factor receptor TKs: Egfr, Breathless and Pvr. Reducing the activity of any of the three kinases by tracheal expression of dominant-negative mutants suppresses cyst formation. By competing dominant-negative and constitutively active kinase mutants against each other, we show that the three RTKs have partially interchangeable activities, so that increasing the activity of one kinase can compensate for the effects of reducing the activity of another. This implies that SH2-domain downstream effectors that are required for the phenotype are likely to be able to interact with phosphotyrosine sites on all three receptor TKs. We also show that the phenotype involves increases in signaling through the MAP kinase and Rho GTPase pathways.

  15. Interactions between Type III receptor tyrosine phosphatases and growth factor receptor tyrosine kinases regulate tracheal tube formation in Drosophila

    PubMed Central

    Jeon, Mili; Scott, Matthew P.; Zinn, Kai

    2012-01-01

    Summary The respiratory (tracheal) system of the Drosophila melanogaster larva is an intricate branched network of air-filled tubes. Its developmental logic is similar in some ways to that of the vertebrate vascular system. We previously described a unique embryonic tracheal tubulogenesis phenotype caused by loss of both of the Type III receptor tyrosine phosphatases (RPTPs), Ptp4E and Ptp10D. In Ptp4E Ptp10D double mutants, the linear tubes in unicellular and terminal tracheal branches are converted into bubble-like cysts that incorporate apical cell surface markers. This tube geometry phenotype is modulated by changes in the activity or expression of the epidermal growth factor receptor (Egfr) tyrosine kinase (TK). Ptp10D physically interacts with Egfr. Here we demonstrate that the Ptp4E Ptp10D phenotype is the consequence of the loss of negative regulation by the RPTPs of three growth factor receptor TKs: Egfr, Breathless and Pvr. Reducing the activity of any of the three kinases by tracheal expression of dominant-negative mutants suppresses cyst formation. By competing dominant-negative and constitutively active kinase mutants against each other, we show that the three RTKs have partially interchangeable activities, so that increasing the activity of one kinase can compensate for the effects of reducing the activity of another. This implies that SH2-domain downstream effectors that are required for the phenotype are likely to be able to interact with phosphotyrosine sites on all three receptor TKs. We also show that the phenotype involves increases in signaling through the MAP kinase and Rho GTPase pathways. PMID:23213447

  16. Cytosolic glucocorticoid receptor interaction with nuclear factor-kappa B proteins in rat liver cells.

    PubMed

    Widén, Christina; Gustafsson, Jan-Ake; Wikström, Ann-Charlotte

    2003-07-01

    The glucocorticoid receptor (GR) acts as an anti-inflammatory factor. To a large extent, this activity is exerted by the interference of pro-inflammatory nuclear factor kappa B (NF-kappa B) activity. In their respective inactive forms, both GR and NF-kappa B reside in the cytoplasm and translocate to the nucleus on relevant stimulation. Previously, p65, a component of the NF-kappa B complex, and GR have been shown to interact physically in vitro, and the interaction is assumed to take place in the nucleus of cells [McKay and Cidlowski (1999) Endocrine Rev. 20, 435-459]. We have studied the interaction between GR and NF-kappa B using in vivo -like conditions. Using immunoaffinity chromatography or immunoprecipitation, combined with Western blotting, we observed that, with endogenous protein levels in cytosolic extracts of rat liver and of H4-II-E-C3 hepatoma cells and in contrast with the current belief, p65, p50 and inhibitory kappa B alpha complex interact with GR, even in the absence of glucocorticoid or an inflammatory signal. The interaction between non-liganded/non-activated GR and p65/p50 has also been verified by both p65 and p50 co-immunoprecipitations. Intracellular localization studies, using Western blotting, revealed that glucocorticoids can decrease tumour necrosis factor alpha (TNFalpha)-induced nuclear entry of p65, whereas glucocorticoid-induced GR translocation was much less affected by TNFalpha. We were also able to demonstrate a nuclear interaction of GR and p65 and p50 using in vivo -like protein concentrations. Furthermore, nuclear GR interaction with heat-shock protein 90 was enhanced distinctly by TNFalpha treatment. In conclusion, our studies suggest a strong interconnectivity between the NF-kappa B and GR-signalling pathways where also, somewhat unexpectedly, a physical interaction in the cytosol constitutes an integral part of GR-NF-kappa B cross-talk.

  17. Structural basis for the interaction of a vascular endothelial growth factor mimic peptide motif and its corresponding receptors.

    PubMed

    Giordano, Ricardo J; Anobom, Cristiane D; Cardó-Vila, Marina; Kalil, Jorge; Valente, Ana P; Pasqualini, Renata; Almeida, Fabio C L; Arap, Wadih

    2005-10-01

    Vascular endothelial growth factor (VEGF) is central to the survival and development of the vascular and nervous systems. We screened phage display libraries and built a peptide-based ligand-receptor map of binding sites within the VEGF family. We then validated a cyclic peptide, CPQPRPLC, as a VEGF-mimic that binds specifically to neuropilin-1 and VEGF receptor-1. Here, we use NMR spectroscopy to understand the structural basis of the interaction between our mimic peptide and the VEGF receptors. We show that: (1) CPQPRPLC has multiple interactive conformations; (2) receptor binding is mediated by the motif Arg-Pro-Leu; and (3) the Pro residue within Arg-Pro-Leu participates in binding to neuropilin-1 but not to VEGF receptor-1, perhaps representing an evolutionary gain-of-function. Therefore, Arg-Pro-Leu is a differential ligand motif to VEGF receptors and a candidate peptidomimetic lead for VEGF pathway modulation.

  18. Direct interaction of avermectin with epidermal growth factor receptor mediates the penetration resistance in Drosophila larvae

    PubMed Central

    Chen, Li-Ping; Wang, Pan; Sun, Ying-Jian; Wu, Yi-Jun

    2016-01-01

    With the widespread use of avermectins (AVMs) for managing parasitic and agricultural pests, the resistance of worms and insects to AVMs has emerged as a serious threat to human health and agriculture worldwide. The reduced penetration of AVMs is one of the main reasons for the development of the resistance to the chemicals. However, the detailed molecular mechanisms remain elusive. Here, we use the larvae of Drosophila melanogaster as the model organism to explore the molecular mechanisms underlying the development of penetration resistance to AVMs. We clearly show that the chitin layer is thickened and the efflux transporter P-glycoprotein (P-gp) is overexpressed in the AVM-resistant larvae epidermis. We reveal that the activation of the transcription factor Relish by the over-activated epidermal growth factor receptor (EGFR)/AKT/ERK pathway induces the overexpression of the chitin synthases DmeCHS1/2 and P-gp in the resistant larvae. Interestingly, we discover for the first time, to the best of our knowledge, that AVM directly interacts with EGFR and leads to the activation of the EGFR/AKT/ERK pathway, which activates the transcription factor Relish and induces the overexpression of DmeCHS1/2 and P-gp. These findings provide new insights into the molecular mechanisms underlying the development of penetration resistance to drugs. PMID:27249340

  19. Novel alpha1-adrenergic receptor signaling pathways: secreted factors and interactions with the extracellular matrix.

    PubMed

    Shi, Ting; Duan, Zhong-Hui; Papay, Robert; Pluskota, Elzbieta; Gaivin, Robert J; de la Motte, Carol A; Plow, Edward F; Perez, Dianne M

    2006-07-01

    alpha1-Adrenergic receptor (alpha1-ARs) subtypes (alpha1A, alpha1B, and alpha1D) regulate multiple signal pathways, such as phospholipase C, protein kinase C (PKC), and mitogen-activated protein kinases. We employed oligonucleotide microarray technology to explore the effects of both short- (1 h) and long-term (18 h) activation of the alpha1A-AR to enable RNA changes to occur downstream of earlier well characterized signaling pathways, promoting novel couplings. Polymerase chain reaction (PCR) studies confirmed that PKC was a critical regulator of alpha1A-AR-mediated gene expression, and secreted interleukin (IL)-6 also contributed to gene expression alterations. We next focused on two novel signaling pathways that might be mediated through alpha1A-AR stimulation because of the clustering of gene expression changes for cell adhesion/motility (syndecan-4 and tenascin-C) and hyaluronan (HA) signaling. We confirmed that alpha1-ARs induced adhesion in three cell types to vitronectin, an interaction that was also integrin-, FGF7-, and PKC-dependent. alpha1-AR activation also inhibited cell migration, which was integrin- and PKC-independent but still required secretion of FGF7. alpha1-AR activation also increased the expression and deposition of HA, a glycosaminoglycan, which displayed two distinct structures: pericellular coats and long cable structures, as well as increasing expression of the HA receptor, CD44. Long cable structures of HA can bind leukocytes, which this suggests that alpha1-ARs may be involved in proinflammatory responses. Our results indicate alpha1-ARs induce the secretion of factors that interact with the extracellular matrix to regulate cell adhesion, motility and proinflammatory responses through novel signaling pathways.

  20. Biochemical characterization of the molecular interaction between recombinant basic fibroblast growth factor and a recombinant soluble fibroblast growth factor receptor.

    PubMed Central

    Caccia, P; Cletini, O; Isacchi, A; Bergonzoni, L; Orsini, G

    1993-01-01

    The extracellular domain of human fibroblast growth factor receptor (XC-FGF-R) was expressed in Escherichia coli. The protein was purified to homogeneity and the interaction with basic fibroblast growth factor (bFGF), its physiological ligand, was examined. Using resins on which bFGF was reversibly bound, we analysed the characteristics of the binding between XC-FGF-R and immobilized bFGF. We also investigated the stoichiometry of the binding between XC-FGF-R and recombinant human bFGF (rhbFGF) applying non-denaturing gel electrophoresis, chemical cross-linking followed by SDS/PAGE, and gel-filtration chromatography. In cross-linking and gel-filtration chromatography experiments, a 1:1 complex between rhbFGF and XC-FGF-R was observed. The complex was separated from the non-complexed proteins using non-denaturing PAGE in the presence of 0.1% Triton X-100. The band corresponding to the complex was recognized by specific antibodies directed against bFGF and its receptor, blotted on poly(vinylidene difluoride) membranes and submitted to sequence and amino acid analysis. The data obtained from these determinations confirmed the formation of a 1:1 complex between rhbFGF and XC-FGF-R. Images Figure 1 Figure 2 Figure 3 Figure 5 PMID:8379918

  1. Thermodynamic characterization of the interaction between TRAF2 and tumor necrosis factor receptor peptides by isothermal titration calorimetry

    PubMed Central

    Ye, Hong; Wu, Hao

    2000-01-01

    The tumor necrosis factor receptor (TNFR) superfamily can induce diverse biological effects, including cell survival, proliferation, differentiation, and apoptosis. The major signal transducers for TNFRs are the family of TNF receptor associated factors (TRAFs). The direct interaction between TRAFs and the intracellular tails of TNFRs is the first step of this signal relay process. Structural studies have revealed a trimeric nature of TRAF2 and a symmetrical mode of receptor binding, suggesting the involvement of trivalent TNFR2-receptor interaction in the signal transduction. In this study, using isothermal titration calorimetry (ITC), we report thermodynamic characterization of the interaction between TRAF2 and monomeric peptide sequences from TNFR members, including TNFR2, CD40, CD30, Ox40, and 4-1BB, and the Epstein–Barr virus (EBV)-transforming protein, latent infection membrane protein-1 (LMP1). The dissociation constants of the interaction were shown to range between 40 μM and 1.9 mM, which are substantially weaker than most protein–peptide interactions. The interaction is entirely driven by exothermic enthalpy, consistent with the abundance of polar contacts. The enthalpy of the interaction has a significant temperature dependence (ΔCp = −245 cal/mol⋅K). The unfavorable entropy in the interaction and the comparison with structural energetics calculations suggest the involvement of conformational rearrangement in the interaction. The low affinity of TRAF2 to monomeric receptor peptides further supports the importance of avidity contribution in TRAF2 recruitment by these receptors upon ligand-induced trimerization or higher order oligomerization. PMID:10908665

  2. Real-time studies of the interactions between epidermal growth factor and its receptor during endocytic trafficking.

    PubMed

    Martin-Fernandez, M L; Clarke, D T; Tobin, M J; Jones, G R

    2000-09-01

    The interactions of growth factors with cell surface receptors regulate fundamental cell processes, such as growth, differentiation and transformation. Understanding the nature of these interactions at the molecular level is of fundamental importance in cell biology. This is not only from the point of view of basic science, but also because of the repercussions such knowledge might have in understanding the mode of action of drugs in cells. Receptor mediated endocytosis has been implicated in the downregulation of the mitogenic signal. However, no data are thus far available on how growth factor/receptor interactions might control endocytic trafficking. Here we show that information on modes of binding and receptor conformational changes can be obtained using time-resolved fluorescence methods. We have found that fluorescent probes bound to epidermal growth factor (EGF) show dynamic fluorescence quenching when EGF is bound to internalising EGF receptors (EGFR). We propose that this dynamic quenching takes place because EGF-bound probes interact with tryptophan residues in the extracellular domain of the EGF-EGFR complex. Real-time accumulation of fluorescent decays has also allowed us to follow the time course of a conformational change in EGFR occurring during endocytosis, and correlate this information with endosomal trafficking and EGFR recycling.

  3. The interaction between the Drosophila secreted protein argos and the epidermal growth factor receptor inhibits dimerization of the receptor and binding of secreted spitz to the receptor.

    PubMed

    Jin, M H; Sawamoto, K; Ito, M; Okano, H

    2000-03-01

    Drosophila Argos (Aos), a secreted protein with an epidermal growth factor (EGF)-like domain, has been shown to inhibit the activation of the Drosophila EGF receptor (DER). However, it has not been determined whether Aos binds directly to DER or whether regulation of the DER activation occurs through some other mechanism. Using DER-expressing cells (DER/S2) and a recombinant DER extracellular domain-Fc fusion protein (DER-Fc), we have shown that Aos binds directly to the extracellular domain of DER with its carboxyl-terminal region, including the EGF-like domain. Furthermore, Aos can block the binding of secreted Spitz (sSpi), a transforming growth factor alpha-like ligand of DER, to the extracellular domain of DER. We observed that sSpi stimulates the dimerization of both the soluble DER extracellular domain (sDER) and the intact DER in the DER/S2 cells and that Aos can block the sSpi-induced dimerization of both sDER and intact DER. Moreover, we have shown that, by directly interacting with DER, Aos and SpiAos (a chimeric protein that is composed of the N-terminal region of Spi and the C-terminal region of Aos) inhibit the dimerization and phosphorylation of DER that are induced by DER's overexpression in the absence of sSpi. These results indicate that Aos exerts its inhibitory function through dual molecular mechanisms: by blocking both the receptor dimerization and the binding of activating ligand to the receptor. This is the first description of this novel inhibitory mechanism for receptor tyrosine kinases.

  4. Splicing Factor Prp8 Interacts With NESAR and Regulates Androgen Receptor in Prostate Cancer Cells

    PubMed Central

    Wang, Dan; Nguyen, Minh M.; Masoodi, Khalid Z.; Singh, Prabhpreet; Jing, Yifeng; O'Malley, Katherine; Dar, Javid A.; Dhir, Rajiv

    2015-01-01

    Androgen receptor (AR) plays a pivotal role in the development of primary as well as advanced castration-resistant prostate cancer. Previous work in our lab identified a novel nuclear export signal (NES) (NESAR) in AR ligand-binding domain essential for AR nucleocytoplasmic trafficking. By characterizing the localization of green fluorescence protein (GFP)-tagged NESAR, we designed and executed a yeast mutagenesis screen and isolated 7 yeast mutants that failed to display the NESAR export function. One of those mutants was identified as the splicing factor pre-mRNA processing factor 8 (Prp8). We further showed that Prp8 could regulate NESAR function using short hairpin RNA knockdown of Prp8 coupled with a rapamycin export assay in mammalian cells and knockdown of Prp8 could induce nuclear accumulation of GFP-tagged AR in PC3 cells. Prp8 expression was decreased in castration-resistant LuCaP35 xenograft tumors as compared with androgen-sensitive xenografts. Laser capture microdissection and quantitative PCR showed Prp8 mRNA levels were decreased in human prostate cancer specimens with high Gleason scores. In prostate cancer cells, coimmunoprecipitation and deletion mutagenesis revealed a physical interaction between Prp8 and AR mainly mediated by NESAR. Luciferase assay with prostate specific antigen promoter-driven reporter demonstrated that Prp8 regulated AR transcription activity in prostate cancer cells. Interestingly, Prp8 knockdown also increased polyubiquitination of endogenous AR. This may be 1 possible mechanism by which it modulates AR activity. These results show that Prp8 is a novel AR cofactor that interacts with NESAR and regulates AR function in prostate cancer cells. PMID:26371515

  5. Delineating the functional map of the interaction between nimotuzumab and the epidermal growth factor receptor

    PubMed Central

    Tundidor, Yaima; García-Hernández, Claudia Patricia; Pupo, Amaury; Infante, Yanelys Cabrera; Rojas, Gertrudis

    2014-01-01

    Molecular details of epidermal growth factor receptor (EGFR) targeting by nimotuzumab, a therapeutic anti-cancer antibody, have been largely unknown. The current study delineated a functional map of their interface, based on phage display and extensive mutagenesis of both the target antigen and the Fv antibody fragment. Five residues in EGFR domain III (R353, S356, F357, T358, and H359T) and the third hypervariable region of nimotuzumab heavy chain were shown to be major functional contributors to the interaction. Fine specificity differences between nimotuzumab and other anti-EGFR antibodies were revealed. Mapping information guided the generation of a plausible in silico binding model. Knowledge about the epitope/paratope interface opens new avenues for the study of tumor sensitivity/resistance to nimotuzumab and for further engineering of its binding site. The developed mapping platform, also validated with the well-known cetuximab epitope, allows a comprehensive exploration of antigenic regions and could be expanded to map other anti-EGFR antibodies. PMID:24759767

  6. Interactions of ABCG2 (BCRP) with epidermal growth factor receptor kinase inhibitors developed for molecular imaging.

    PubMed

    Qawasmi, Israa; Shmuel, Miriam; Eyal, Sara

    2014-01-01

    The objective of this study was to investigate in vitro the interactions between novel epidermal growth factor receptor kinase inhibitors (EGFRIs) developed for positron emission tomography (PET) imaging and the major efflux transporter breast cancer resistance protein (BCRP/ABCG2). Seven compounds were evaluated, using the ATPase activity assays and Madin-Darbey canine kidney (MDCK) cells overexpressing BCRP. Five of the tested compounds activated BCRP ATPase to various extent. Overexpression of BCRP conferred resistance to ML04, ML06, methoxy-Br-ML03, and PEG6-ML05 (IC50 values for inhibition of control cell proliferation 2.1 ± 0.6, 2.2 ± 0.7, 1.8 ± 1.2, and 2.8 ± 3.1 μM, respectively, compared to >50 μM in MDCK-BCRP cells). At submicromolar concentrations, none of the EGFRIs significantly inhibited BCRP. Immunoblotting studies indicated that BCRP expression is evident in cell lines utilized for in vivo tumor grafting in small animal PET imaging studies. Thus, the intensity of EGFRIs radioactivity signals previously observed in tumor xenografts reflects an interplay between transporter-mediated distribution of the probe into tumor cells and target binding. Concomitant use of efflux transporter inhibitors may help distinguish between the contribution of efflux transport and EGFR binding to the tissue signal.

  7. Interactions of ABCG2 (BCRP) with epidermal growth factor receptor kinase inhibitors developed for molecular imaging

    PubMed Central

    Qawasmi, Israa; Shmuel, Miriam; Eyal, Sara

    2014-01-01

    The objective of this study was to investigate in vitro the interactions between novel epidermal growth factor receptor kinase inhibitors (EGFRIs) developed for positron emission tomography (PET) imaging and the major efflux transporter breast cancer resistance protein (BCRP/ABCG2). Seven compounds were evaluated, using the ATPase activity assays and Madin-Darbey canine kidney (MDCK) cells overexpressing BCRP. Five of the tested compounds activated BCRP ATPase to various extent. Overexpression of BCRP conferred resistance to ML04, ML06, methoxy-Br-ML03, and PEG6-ML05 (IC50 values for inhibition of control cell proliferation 2.1 ± 0.6, 2.2 ± 0.7, 1.8 ± 1.2, and 2.8 ± 3.1 μM, respectively, compared to >50 μM in MDCK-BCRP cells). At submicromolar concentrations, none of the EGFRIs significantly inhibited BCRP. Immunoblotting studies indicated that BCRP expression is evident in cell lines utilized for in vivo tumor grafting in small animal PET imaging studies. Thus, the intensity of EGFRIs radioactivity signals previously observed in tumor xenografts reflects an interplay between transporter-mediated distribution of the probe into tumor cells and target binding. Concomitant use of efflux transporter inhibitors may help distinguish between the contribution of efflux transport and EGFR binding to the tissue signal. PMID:25484865

  8. Delineating the functional map of the interaction between nimotuzumab and the epidermal growth factor receptor.

    PubMed

    Tundidor, Yaima; García-Hernández, Claudia Patricia; Pupo, Amaury; Cabrera Infante, Yanelys; Rojas, Gertrudis

    2014-01-01

    Molecular details of epidermal growth factor receptor (EGFR) targeting by nimotuzumab, a therapeutic anti-cancer antibody, have been largely unknown. The current study delineated a functional map of their interface, based on phage display and extensive mutagenesis of both the target antigen and the Fv antibody fragment. Five residues in EGFR domain III (R353, S356, F357, T358, and H359T) and the third hypervariable region of nimotuzumab heavy chain were shown to be major functional contributors to the interaction. Fine specificity differences between nimotuzumab and other anti-EGFR antibodies were revealed. Mapping information guided the generation of a plausible in silico binding model. Knowledge about the epitope/paratope interface opens new avenues for the study of tumor sensitivity/resistance to nimotuzumab and for further engineering of its binding site. The developed mapping platform, also validated with the well-known cetuximab epitope, allows a comprehensive exploration of antigenic regions and could be expanded to map other anti-EGFR antibodies.

  9. New androgen receptor genomic targets show an interaction with the ETS1 transcription factor.

    PubMed

    Massie, Charles E; Adryan, Boris; Barbosa-Morais, Nuno L; Lynch, Andy G; Tran, Maxine G; Neal, David E; Mills, Ian G

    2007-09-01

    The androgen receptor (AR) initiates important developmental and oncogenic transcriptional pathways. The AR is known to bind as a homodimer to 15-base pair bipartite palindromic androgen-response elements; however, few direct AR gene targets are known. To identify AR promoter targets, we used chromatin immunoprecipitation with on-chip detection of genomic fragments. We identified 1,532 potential AR-binding sites, including previously known AR gene targets. Many of the new AR target genes show altered expression in prostate cancer. Analysis of sequences underlying AR-binding sites showed that more than 50% of AR-binding sites did not contain the established 15 bp AR-binding element. Unbiased sequence analysis showed 6-bp motifs, which were significantly enriched and were bound directly by the AR in vitro. Binding sequences for the avian erythroblastosis virus E26 homologue (ETS) transcription factor family were also highly enriched, and we uncovered an interaction between the AR and ETS1 at a subset of AR promoter targets.

  10. Modeling of growth factor-receptor systems: from molecular-level protein interaction networks to whole-body compartment models

    PubMed Central

    Wu, Florence T.H.; Stefanini, Marianne O.; Mac Gabhann, Feilim; Popel, Aleksander S.

    2010-01-01

    Most physiological processes are subjected to molecular regulation by growth factors, which are secreted proteins that activate chemical signal transduction pathways through binding of specific cell-surface receptors. One particular growth factor system involved in the in vivo regulation of blood vessel growth is called the vascular endothelial growth factor (VEGF) system. Computational and numerical techniques are well-suited to handle the molecular complexity (the number of binding partners involved, including ligands, receptors, and inert binding sites) and multi-scale nature (intra-tissue vs. inter-tissue transport and local vs. systemic effects within an organism) involved in modeling growth factor system interactions and effects. This paper introduces a variety of in silico models that seek to recapitulate different aspects of VEGF system biology at various spatial and temporal scales: molecular-level kinetic models focus on VEGF ligand-receptor interactions at and near the endothelial cell surface; meso-scale single-tissue 3D models can simulate the effects of multi-cellular tissue architecture on the spatial variation in VEGF ligand production and receptor activation; compartmental modeling allows efficient prediction of average interstitial VEGF concentrations and cell-surface VEGF signaling intensities across multiple large tissue volumes, permitting the investigation of whole-body inter-tissue transport (e.g., vascular permeability and lymphatic drainage). The given examples will demonstrate the utility of computational models in aiding both basic science and clinical research on VEGF systems biology. PMID:19897104

  11. Curcumin inhibition of the functional interaction between integrin α6β4 and the epidermal growth factor receptor.

    PubMed

    Soung, Young Hwa; Chung, Jun

    2011-05-01

    The functional interaction between integrin α6β4 and growth factor receptors has been implicated in key signaling pathways important for cancer cell function. However, few attempts have been made to selectively target this interaction for therapeutic intervention. Previous studies showed that curcumin, a yellow pigment isolated from turmeric, inhibits integrin α6β4 signaling important for breast carcinoma cell motility and invasion, but the mechanism is not currently known. To address this issue, we tested the hypothesis that curcumin inhibits the functional interaction between α6β4 and the epidermal growth factor receptor (EGFR). In this study, we found that curcumin disrupts functional and physical interactions between α6β4 and EGFR, and blocks α6β4/EGFR-dependent functions of carcinoma cells expressing the signaling competent form of α6β4. We further showed that curcumin inhibits EGF-dependent mobilization of α6β4 from hemidesmosomes to the leading edges of migrating cells such as lammelipodia and filopodia, and thereby prevents α6β4 distribution to lipid rafts where functional interactions between α6β4 and EGFR occur. These data suggest a novel paradigm in which curcumin inhibits α6β4 signaling and functions by altering intracellular localization of α6β4, thus preventing its association with signaling receptors such as EGFR.

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

    PubMed Central

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

    2015-01-01

    Lysine residues are implicated in driving the ligand binding to the LDL receptor family. However, it has remained unclear how specificity is regulated. Using coagulation factor VIII as a model ligand, we now study the contribution of individual lysine residues in the interaction with the largest member of the LDL receptor family, low-density lipoprotein receptor-related protein (LRP1). Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and SPR interaction analysis on a library of lysine replacement variants as two independent approaches, we demonstrate that the interaction between factor VIII (FVIII) and LRP1 occurs over an extended surface containing multiple lysine residues. None of the individual lysine residues account completely for LRP1 binding, suggesting an additive binding model. Together with structural docking studies, our data suggest that FVIII interacts with LRP1 via an extended surface of multiple lysine residues that starts at the bottom of the C1 domain and winds around the FVIII molecule. PMID:25903134

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

    PubMed Central

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

    2015-01-01

    Protein-protein interactions can increase or decrease its therapeutic target activity and the determining factors involved, however, are largely unknown. Here, we report that tyrosine-dephosphorylation of epidermal growth factor receptor (EGFR) increases its therapeutic target activity by disrupting its interaction with estrogen receptor (ER). Protein tyrosine phosphatase H1 (PTPH1) dephosphorylates the tyrosine kinase EGFR, disrupts its interaction with the nuclear receptor ER, and increases breast cancer sensitivity to small molecule tyrosine kinase inhibitors (TKIs). These effects require PTPH1 catalytic activity and its interaction with EGFR, suggesting that the phosphatase may increase the sensitivity by dephosphorylating EGFR leading to its dissociation with ER. Consistent with this notion, a nuclear-localization defective ER has a higher EGFR-binding activity and confers the resistance to TKI-induced growth inhibition. Additional analysis show that PTPH1 stabilizes EGFR, stimulates the membranous EGFR accumulation, and enhances the growth-inhibitory activity of a combination therapy of TKIs with an anti-estrogen. Since EGFR and ER both are substrates for PTPH1 in vitro and in intact cells, these results indicate that an inhibitory EGFR-ER protein complex can be switched off through a competitive enzyme-substrate binding. Our results would have important implications for the treatment of breast cancer with targeted therapeutics. PMID:26079946

  14. Membrane contacts between endosomes and ER provide sites for PTP1B-epidermal growth factor receptor interaction.

    PubMed

    Eden, Emily R; White, Ian J; Tsapara, Anna; Futter, Clare E

    2010-03-01

    The epidermal growth factor receptor (EGFR) is a critical determinator of cell fate. Signalling from this receptor tyrosine kinase is spatially regulated by progression through the endocytic pathway, governing receptor half-life and accessibility to signalling proteins and phosphatases. Endocytosis of EGFR is required for interaction with the protein tyrosine phosphatase PTP1B (ref. 1), which localizes to the cytoplasmic face of the endoplasmic reticulum (ER), raising the question of how PTP1B comes into contact with endosomal EGFR. We show that EGFR-PTP1B interaction occurs by means of direct membrane contacts between the perimeter membrane of multivesicular bodies (MVBs) and the ER. The population of EGFR interacting with PTP1B is the same population that undergo ESCRT-mediated (endosomal sorting complex required for transport) sorting within MVBs, and PTP1B activity promotes the sequestration of EGFR on to MVB internal vesicles. Membrane contacts between endosomes and the ER form in both the presence and absence of stimulation by EGF. Thus membrane contacts between endosomes and the ER may represent a global mechanism for direct interaction between proteins on these two organelles.

  15. Interaction of Platelet Membrane Receptors with von Willebrand Factor, Ristocetin, and the Fc Region of Immunoglobulin G

    PubMed Central

    Moore, Anne; Ross, Gordon D.; Nachman, Ralph L.

    1978-01-01

    The agglutination of human platelets by ristocetin and von Willebrand factor was inhibited by aggregated immunoglobulin (Ig)G and by Fc fragments of IgG, but not by Fab, F(ab′)2 or pFc fragments of IgG. Because this inhibition occurred with formalin-fixed platelets as well as with normal platelets, a generalized aggregation of fluid membrane components by Fc fragments was not responsible for this inhibition of ristocetin and von Willebrand factor-induced agglutination. Reciprocal inhibition of platelet Fc receptors was produced by prior incubation of platelets with von Willebrand factor and ristocetin. Sucrose density gradient ultracentrifugation studies demonstrated that aggregated IgG did not form fluid-phase complexes with von Willebrand factor and ristocetin. Furthermore, passage of von Willebrand factor and ristocetin through a column of immobilized heat-aggregated IgG did not alter platelet agglutinating activity which indicates that aggregated IgG did not inactivate von Willebrand factor or ristocetin. Thus, it was likely that the IgG-mediated interference with platelet agglutination by ristocetin and von Willebrand factor did not occur in the fluid phase but at the platelet surface. These studies suggest that the platelet membrane Fc receptor may be either a part of, or sterically related to, the membrane glycoprotein I complex that interacts with von Willebrand factor, and that occupation of one of these surface components blocks the availability of the other. PMID:309473

  16. Immunotherapy of human tumour xenografts overexpressing the EGF receptor with rat antibodies that block growth factor-receptor interaction.

    PubMed Central

    Modjtahedi, H.; Eccles, S.; Box, G.; Styles, J.; Dean, C.

    1993-01-01

    Athymic mice bearing xenografts of human tumours that overexpress the receptor (EGFR) for EGF and TGF alpha have been used to evaluate the therapeutic potential of three new rat monoclonal antibodies (mAbs) directed against two distinct epitopes on the extracellular domain of the human EGFR. The antibodies, ICR16 (IgG2a), ICR62 (IgG2b) and ICR64 (IgG1), have been shown (Modjtahedi et al., 1993) to be potent inhibitors of the growth in vitro of a number of human squamous cell carcinomas because they block receptor-ligand interaction. When given i.p. at 200 micrograms dose, the three antibodies were found to induce complete regression of xenografts of the HN5 tumour if treatment with antibody commenced at the time of tumour implantation (total doses: ICR16, 3.0 mg; ICR62, 1.2 mg; ICR64, 2.2 mg). More importantly when treatment was delayed until the tumours were established (mean diam. 0.5 cm) both ICR16 and ICR62 induced complete or almost complete regression of the tumours. Furthermore, treatment with a total dose of only 0.44 mg of ICR62 was found to induce complete remission of xenografts of the breast carcinoma MDA-MB 468, but ICR16 was less effective at this dose of antibody and only 4/8 tumours regressed completely. ICR16 and ICR62 were poor inhibitors of the growth in vitro of the vulval carcinoma A431, but both induced a substantial delay in the growth of xenografts of this tumour and 4/8 tumours regressed completely in the mice treated with ICR62 (total dose 2.2 mg). Although ICR16 and ICR64 were more effective than ICR62 as growth inhibitors in vitro, ICR62 was found to be substantially better at inducing regression of the tumour xenografts due perhaps to additional activation of host immune effector functions by the IgG2b antibody. We conclude that these antibodies may be useful therapeutic agents that can be used alone without conjugation to other cytotoxic moieties. PMID:7679281

  17. NRC-interacting factor 1 is a novel cotransducer that interacts with and regulates the activity of the nuclear hormone receptor coactivator NRC.

    PubMed

    Mahajan, Muktar A; Murray, Audrey; Samuels, Herbert H

    2002-10-01

    We previously reported the cloning and characterization of a novel nuclear hormone receptor transcriptional coactivator, which we refer to as NRC. NRC is a 2,063-amino-acid nuclear protein which contains a potent N-terminal activation domain and several C-terminal modules which interact with CBP and ligand-bound nuclear hormone receptors as well as c-Fos and c-Jun. In this study we sought to clone and identify novel factors that interact with NRC to modulate its transcriptional activity. Here we describe the cloning and characterization of a novel protein we refer to as NIF-1 (NRC-interacting factor 1). NIF-1 was cloned from rat pituitary and human cell lines and was found to interact in vivo and in vitro with NRC. NIF-1 is a 1,342-amino-acid nuclear protein containing a number of conserved domains, including six Cys-2/His-2 zinc fingers, an N-terminal stretch of acidic amino acids, and a C-terminal leucine zipper-like motif. Zinc fingers 1 to 3 are potential DNA-binding BED finger domains recently proposed to play a role in altering local chromatin architecture. We mapped the interaction domains of NRC and NIF-1. Although NIF-1 does not directly interact with nuclear receptors, it markedly enhances ligand-dependent transcriptional activation by nuclear hormone receptors in vivo as well as activation by c-Fos and c-Jun. These results, and the finding that NIF-1 interacts with NRC in vivo, suggest that NIF-1 functions to regulate transcriptional activation through NRC. We suggest that NIF-1, and factors which associate with coactivators but not receptors, be referred to as cotransducers, which act in vivo either as part of a coactivator complex or downstream of a coactivator complex to modulate transcriptional activity. Our findings suggest that NIF-1 may be a functional component of an NRC complex and acts as a regulator or cotransducer of NRC function.

  18. Tumor necrosis factor alpha receptor- and Fas-associated FLASH inhibit transcriptional activity of the glucocorticoid receptor by binding to and interfering with its interaction with p160 type nuclear receptor coactivators.

    PubMed

    Kino, Tomoshige; Chrousos, George P

    2003-01-31

    Tumor necrosis factor alpha (TNF alpha) and its downstream transcription factor nuclear factor kappa B (NF-kappa B) suppress glucocorticoid action, contributing to tissue resistance to glucocorticoids in several pathologic inflammatory states. p160 nuclear receptor coactivators on the other hand, contribute to the transcriptional signal of the glucocorticoid receptor (GR) through interaction with it via LXXLL motifs in their nuclear receptor-binding (NRB) domain. To discover TNF alpha-induced factors that regulate GR activity at the coactivator level, we performed yeast two-hybrid screening using the NRB domain of the glucocorticoid receptor-interacting protein 1 (GRIP1) as bait. We found that FLICE-associated huge protein (FLASH), which transduces TNF alpha and Fas ligand signals, bound the NRB domain of GRIP1 at a region between the second and third LXXLL motifs. FLASH suppressed both GR transactivation and GRIP1 enhancement of the glucocorticoid signal and inhibited the physical interaction between GR and the GRIP1 NRB domain. Transfected green fluorescent protein-fused FLASH was located in both the cytoplasm and nucleus, while endogenous FLASH shifted its subcellular localization from the cytoplasm into the nucleus in response to TNF alpha. FLASH antisense and super-repressor I kappa B alpha inhibited the action of TNF alpha independently of each other and additively. These findings indicate that FLASH participates in TNF alpha-induced blockade of GR transactivation at the nuclear receptor coactivator level, upstream and independently of NF-kappa B.

  19. Characterization of the growth of murine fibroblasts that express human insulin receptors. II. Interaction of insulin with other growth factors

    SciTech Connect

    Randazzo, P.A.; Jarett, L. )

    1990-09-01

    The effects of insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and insulin on DNA synthesis were studied in murine fibroblasts transfected with an expression vector containing human insulin receptor cDNA (NIH 3T3/HIR) and the parental NIH 3T3 cells. In NIH 3T3/HIR cells, individual growth factors in serum-free medium stimulated DNA synthesis with the following relative efficacies: insulin greater than or equal to 10% fetal calf serum greater than PDGF greater than IGF-1 much greater than EGF. In comparison, the relative efficacies of these factors in stimulating DNA synthesis by NIH 3T3 cells were 10% fetal calf serum greater than PDGF greater than EGF much greater than IGF-1 = insulin. In NIH 3T3/HIR cells, EGF was synergistic with 1-10 ng/ml insulin but not with 100 ng/ml insulin or more. Synergy of PDGF or IGF-1 with insulin was not detected. In the parental NIH 3T3 cells, insulin and IGF-1 were found to be synergistic with EGF (1 ng/ml), PDGF (100 ng/ml), and PDGF plus EGF. In NIH 3T3/HIR cells, the lack of interaction of insulin with other growth factors was also observed when the percentage of cells synthesizing DNA was examined. Despite insulin's inducing only 60% of NIH 3T3/HIR cells to incorporate thymidine, addition of PDGF, EGF, or PDGF plus EGF had no further effect. In contrast, combinations of growth factors resulted in 95% of the parental NIH 3T3 cells synthesizing DNA. The independence of insulin-stimulated DNA synthesis from other mitogens in the NIH 3T3/HIR cells is atypical for progression factor-stimulated DNA synthesis and is thought to be partly the result of insulin receptor expression in an inappropriate context or quantity.

  20. Interaction of Tumor Necrosis Factor Receptor-associated Factor 6 (TRAF6) and Vav3 in the Receptor Activator of Nuclear Factor κB (RANK) Signaling Complex Enhances Osteoclastogenesis.

    PubMed

    Yu, Jiyeon; Yun, Hyeongseok; Shin, Bongjin; Kim, Yongjin; Park, Eui-Soon; Choi, Seunga; Yu, Jungeun; Amarasekara, Dulshara Sachini; Kim, Sumi; Inoue, Jun-Ichiro; Walsh, Matthew C; Choi, Yongwon; Takami, Masamichi; Rho, Jaerang

    2016-09-23

    The signaling pathway downstream of stimulation of receptor activator of nuclear factor κB (RANK) by RANK ligand is crucial for osteoclastogenesis. RANK recruits TNF receptor-associated factor 6 (TRAF6) to TRAF6-binding sites (T6BSs) in the RANK cytoplasmic tail (RANKcyto) to trigger downstream osteoclastogenic signaling cascades. RANKcyto harbors an additional highly conserved domain (HCR) that also activates crucial signaling during RANK-mediated osteoclastogenesis. However, the functional cross-talk between T6BSs and the HCR in the RANK signaling complex remains unclear. To characterize the cross-talk between T6BSs and the HCR, we screened TRAF6-interacting proteins using a proteomics approach. We identified Vav3 as a novel TRAF6 binding partner and evaluated the functional importance of the TRAF6-Vav3 interaction in the RANK signaling complex. We demonstrated that the coiled-coil domain of TRAF6 interacts directly with the Dbl homology domain of Vav3 to form the RANK signaling complex independent of the TRAF6 ubiquitination pathway. TRAF6 is recruited to the RANKcyto mutant, which lacks T6BSs, via the Vav3 interaction; conversely, Vav3 is recruited to the RANKcyto mutant, which lacks the IVVY motif, via the TRAF6 interaction. Finally, we determined that the TRAF6-Vav3 interaction resulting from cross-talk between T6BSs and the IVVY motif in RANKcyto enhances downstream NF-κB, MAPK, and NFATc1 activation by further strengthening TRAF6 signaling, thereby inducing RANK-mediated osteoclastogenesis. Thus, Vav3 is a novel TRAF6 interaction partner that functions in the activation of cooperative signaling between T6BSs and the IVVY motif in the RANK signaling complex. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Molecular cloning of the mouse grb2 gene: differential interaction of the Grb2 adaptor protein with epidermal growth factor and nerve growth factor receptors.

    PubMed Central

    Suen, K L; Bustelo, X R; Pawson, T; Barbacid, M

    1993-01-01

    We report the isolation and molecular characterization of the mouse grb2 gene. The product of this gene, the Grb2 protein, is highly related to the Caenorhabditis elegans sem-5 gene product and the human GRB2 protein and displays the same SH3-SH2-SH3 structural motifs. In situ hybridization studies revealed that the mouse grb2 gene is widely expressed throughout embryonic development (E9.5 to P0). However, grb2 transcripts are not uniformly distributed, and in certain tissues (e.g., thymus) they appear to be regulated during development. Recent genetic and biochemical evidence has implicated the Grb2 protein in the signaling pathways that link cell surface tyrosine kinase receptors with Ras. We have investigated the association of the Grb2 protein with epidermal growth factor (EGF) and nerve growth factor (NGF) receptors in PC12 pheochromocytoma cells. EGF treatment of PC12 cells results in the rapid association of Grb2 with the activated EGF receptors, an interaction mediated by the Grb2 SH2 domain. However, Grb2 does not bind to NGF-activated Trk receptors. Mitogenic signaling of NGF in NIH 3T3 cells ectopically expressing Trk receptors also takes place without detectable association between Grb2 and Trk. These results suggest that whereas EGF and NGF can activate the Ras signaling pathway in PC12 cells, only the EGF receptor is likely to do so through a direct interaction with Grb2. Finally, binding studies with glutathione S-transferase fusion proteins indicate that Grb2 binds two distinct subsets of proteins which are individually recognized by its SH2 and SH3 domains. These observations add further support to the concept that Grb2 is a modular adaptor protein. Images PMID:7689150

  2. p35 Regulates the CRM1-Dependent Nucleocytoplasmic Shuttling of Nuclear Hormone Receptor Coregulator-Interacting Factor 1 (NIF-1)

    PubMed Central

    Zhao, Xiao-Su; Fu, Wing-Yu; Chien, Winnie W. Y.; Li, Zhen; Fu, Amy K. Y.; Ip, Nancy Y.

    2014-01-01

    Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase, which plays critical roles in a wide spectrum of neuronal functions including neuronal survival, neurite outgrowth, and synapse development and plasticity. Cdk5 activity is controlled by its specific activators: p35 or p39. While knockout studies reveal that Cdk5/p35 is critical for neuronal migration during early brain development, functions of Cdk5/p35 have been unraveled through the identification of the interacting proteins of p35, most of which are Cdk5/p35 substrates. However, it remains unclear whether p35 can regulate neuronal functions independent of Cdk5 activity. Here, we report that a nuclear protein, nuclear hormone receptor coregulator (NRC)-interacting factor 1 (NIF-1), is a new interacting partner of p35. Interestingly, p35 regulates the functions of NIF-1 independent of Cdk5 activity. NIF-1 was initially discovered as a transcriptional regulator that enhances the transcriptional activity of nuclear hormone receptors. Our results show that p35 interacts with NIF-1 and regulates its nucleocytoplasmic trafficking via the nuclear export pathway. Furthermore, we identified a nuclear export signal on p35; mutation of this site or blockade of the CRM1/exportin-dependent nuclear export pathway resulted in the nuclear accumulation of p35. Intriguingly, blocking the nuclear export of p35 attenuated the nuclear accumulation of NIF-1. These findings reveal a new p35-dependent mechanism in transcriptional regulation that involves the nucleocytoplasmic shuttling of transcription regulators. PMID:25329792

  3. p35 regulates the CRM1-dependent nucleocytoplasmic shuttling of nuclear hormone receptor coregulator-interacting factor 1 (NIF-1).

    PubMed

    Zhao, Xiao-Su; Fu, Wing-Yu; Chien, Winnie W Y; Li, Zhen; Fu, Amy K Y; Ip, Nancy Y

    2014-01-01

    Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase, which plays critical roles in a wide spectrum of neuronal functions including neuronal survival, neurite outgrowth, and synapse development and plasticity. Cdk5 activity is controlled by its specific activators: p35 or p39. While knockout studies reveal that Cdk5/p35 is critical for neuronal migration during early brain development, functions of Cdk5/p35 have been unraveled through the identification of the interacting proteins of p35, most of which are Cdk5/p35 substrates. However, it remains unclear whether p35 can regulate neuronal functions independent of Cdk5 activity. Here, we report that a nuclear protein, nuclear hormone receptor coregulator (NRC)-interacting factor 1 (NIF-1), is a new interacting partner of p35. Interestingly, p35 regulates the functions of NIF-1 independent of Cdk5 activity. NIF-1 was initially discovered as a transcriptional regulator that enhances the transcriptional activity of nuclear hormone receptors. Our results show that p35 interacts with NIF-1 and regulates its nucleocytoplasmic trafficking via the nuclear export pathway. Furthermore, we identified a nuclear export signal on p35; mutation of this site or blockade of the CRM1/exportin-dependent nuclear export pathway resulted in the nuclear accumulation of p35. Intriguingly, blocking the nuclear export of p35 attenuated the nuclear accumulation of NIF-1. These findings reveal a new p35-dependent mechanism in transcriptional regulation that involves the nucleocytoplasmic shuttling of transcription regulators.

  4. Endoglin is an accessory protein that interacts with the signaling receptor complex of multiple members of the transforming growth factor-beta superfamily.

    PubMed

    Barbara, N P; Wrana, J L; Letarte, M

    1999-01-08

    Endoglin (CD105) is a transmembrane glycoprotein that binds transforming growth factor (TGF)-beta1 and -beta3, and coprecipitates with the Ser/Thr kinase signaling receptor complex by affinity labeling of endothelial and leukemic cells. The present study shows that in addition to TGF-beta1 and -beta3, endoglin interacts with activin-A, bone morphogenetic protein (BMP)-7, and BMP-2 but requires coexpression of the respective ligand binding kinase receptor for this association. Endoglin cannot bind ligands on its own and does not alter binding to the kinase receptors. It binds TGF-beta1 and -beta3 by associating with the TGF-beta type II receptor and interacts with activin-A and BMP-7 via activin type II receptors, ActRII and ActRIIB, regardless of which type I receptor partner is coexpressed. However, endoglin binds BMP-2 by interacting with the ligand binding type I receptors, ALK3 and ALK6. The formation of heteromeric signaling complexes was not altered by the presence of endoglin, although it was coprecipitated with these complexes. Endoglin did not interact with BMP-7 through complexes containing the BMP type II receptor, demonstrating specificity of its action. Our data suggest that endoglin is an accessory protein of multiple kinase receptor complexes of the TGF-beta superfamily.

  5. Plasticity in Interactions of Fibroblast Growth Factor 1 (FGF1) N Terminus with FGF Receptors Underlies Promiscuity of FGF1*

    PubMed Central

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

    2012-01-01

    Tissue-specific alternative splicing in the second half of Ig-like domain 3 (D3) of fibroblast growth factor receptors 1–3 (FGFR1 to -3) generates epithelial FGFR1b-FGFR3b and mesenchymal FGFR1c-FGFR3c splice isoforms. This splicing event establishes a selectivity filter to restrict the ligand binding specificity of FGFRb and FGFRc isoforms to mesenchymally and epithelially derived fibroblast growth factors (FGFs), respectively. FGF1 is termed the “universal FGFR ligand” because it overrides this specificity barrier. To elucidate the molecular basis for FGF1 cross-reactivity with the “b” and “c” splice isoforms of FGFRs, we determined the first crystal structure of FGF1 in complex with an FGFRb isoform, FGFR2b, at 2.1 Å resolution. Comparison of the FGF1-FGFR2b structure with the three previously published FGF1-FGFRc structures reveals that plasticity in the interactions of the N-terminal region of FGF1 with FGFR D3 is the main determinant of FGF1 cross-reactivity with both isoforms of FGFRs. In support of our structural data, we demonstrate that substitution of three N-terminal residues (Gly-19, His-25, and Phe-26) of FGF2 (a ligand that does not bind FGFR2b) for the corresponding residues of FGF1 (Phe-16, Asn-22, and Tyr-23) enables the FGF2 triple mutant to bind and activate FGFR2b. These findings taken together with our previous structural data on receptor binding specificity of FGF2, FGF8, and FGF10 conclusively show that sequence divergence at the N termini of FGFs is the primary regulator of the receptor binding specificity and promiscuity of FGFs. PMID:22057274

  6. Human platelet interaction with E. coli O111 promotes tissue-factor-dependent procoagulant activity, involving Toll like receptor 4.

    PubMed

    Matus, Valeria; Valenzuela, J Guillermo; Hidalgo, Patricia; Pozo, L María; Panes, Olga; Wozniak, Aniela; Mezzano, Diego; Pereira, Jaime; Sáez, Claudia G

    2017-01-01

    Platelets have a major role in clotting activation and contribute to the innate immune response during systemic infections. Human platelets contain tissue factor (TF) and express functional Toll-like receptor 4 (TLR4). However, the role of TLR4 in triggering the procoagulant properties of platelets, upon challenge with bacteria, is yet unknown. Our hypothesis is that E. coli O111-TLR4 interaction activates platelets and elicits their procoagulant activity. We demonstrated that the strain, but not ultrapure LPS, increased surface P-selectin expression, platelet dependent TF procoagulant activity (TF-PCA) and prompted a faster thrombin generation (TG). Blockade of TLR4 resulted in decreased platelet activation, TF-PCA and TG, revealing the participation of this immune receptor on the procoagulant response of platelets. Our results provide a novel mechanism by which individuals with bacterial infections would have an increased incidence of blood clots. Furthermore, the identification of platelet TF and TLR4 as regulators of the effect of E. coli O111 might represent a novel therapeutic target to reduce the devastating consequences of the hemostatic disorder during sepsis.

  7. Adenosine A1 receptor: Functional receptor-receptor interactions in the brain

    PubMed Central

    Sichardt, Kathrin

    2007-01-01

    Over the past decade, many lines of investigation have shown that receptor-mediated signaling exhibits greater diversity than previously appreciated. Signal diversity arises from numerous factors, which include the formation of receptor dimers and interplay between different receptors. Using adenosine A1 receptors as a paradigm of G protein-coupled receptors, this review focuses on how receptor-receptor interactions may contribute to regulation of the synaptic transmission within the central nervous system. The interactions with metabotropic dopamine, adenosine A2A, A3, neuropeptide Y, and purinergic P2Y1 receptors will be described in the first part. The second part deals with interactions between A1Rs and ionotropic receptors, especially GABAA, NMDA, and P2X receptors as well as ATP-sensitive K+ channels. Finally, the review will discuss new approaches towards treating neurological disorders. PMID:18404442

  8. Structural model for the interaction of a designed Ankyrin Repeat Protein with the human epidermal growth factor receptor 2.

    PubMed

    Epa, V Chandana; Dolezal, Olan; Doughty, Larissa; Xiao, Xiaowen; Jost, Christian; Plückthun, Andreas; Adams, Timothy E

    2013-01-01

    Designed Ankyrin Repeat Proteins are a class of novel binding proteins that can be selected and evolved to bind to targets with high affinity and specificity. We are interested in the DARPin H10-2-G3, which has been evolved to bind with very high affinity to the human epidermal growth factor receptor 2 (HER2). HER2 is found to be over-expressed in 30% of breast cancers, and is the target for the FDA-approved therapeutic monoclonal antibodies trastuzumab and pertuzumab and small molecule tyrosine kinase inhibitors. Here, we use computational macromolecular docking, coupled with several interface metrics such as shape complementarity, interaction energy, and electrostatic complementarity, to model the structure of the complex between the DARPin H10-2-G3 and HER2. We analyzed the interface between the two proteins and then validated the structural model by showing that selected HER2 point mutations at the putative interface with H10-2-G3 reduce the affinity of binding up to 100-fold without affecting the binding of trastuzumab. Comparisons made with a subsequently solved X-ray crystal structure of the complex yielded a backbone atom root mean square deviation of 0.84-1.14 Ångstroms. The study presented here demonstrates the capability of the computational techniques of structural bioinformatics in generating useful structural models of protein-protein interactions.

  9. Structural Model for the Interaction of a Designed Ankyrin Repeat Protein with the Human Epidermal Growth Factor Receptor 2

    PubMed Central

    Epa, V. Chandana; Dolezal, Olan; Doughty, Larissa; Xiao, Xiaowen; Jost, Christian; Plückthun, Andreas; Adams, Timothy E.

    2013-01-01

    Designed Ankyrin Repeat Proteins are a class of novel binding proteins that can be selected and evolved to bind to targets with high affinity and specificity. We are interested in the DARPin H10-2-G3, which has been evolved to bind with very high affinity to the human epidermal growth factor receptor 2 (HER2). HER2 is found to be over-expressed in 30% of breast cancers, and is the target for the FDA-approved therapeutic monoclonal antibodies trastuzumab and pertuzumab and small molecule tyrosine kinase inhibitors. Here, we use computational macromolecular docking, coupled with several interface metrics such as shape complementarity, interaction energy, and electrostatic complementarity, to model the structure of the complex between the DARPin H10-2-G3 and HER2. We analyzed the interface between the two proteins and then validated the structural model by showing that selected HER2 point mutations at the putative interface with H10-2-G3 reduce the affinity of binding up to 100-fold without affecting the binding of trastuzumab. Comparisons made with a subsequently solved X-ray crystal structure of the complex yielded a backbone atom root mean square deviation of 0.84–1.14 Ångstroms. The study presented here demonstrates the capability of the computational techniques of structural bioinformatics in generating useful structural models of protein-protein interactions. PMID:23527120

  10. Splicing Factor Prp8 Interacts With NES(AR) and Regulates Androgen Receptor in Prostate Cancer Cells.

    PubMed

    Wang, Dan; Nguyen, Minh M; Masoodi, Khalid Z; Singh, Prabhpreet; Jing, Yifeng; O'Malley, Katherine; Dar, Javid A; Dhir, Rajiv; Wang, Zhou

    2015-12-01

    Androgen receptor (AR) plays a pivotal role in the development of primary as well as advanced castration-resistant prostate cancer. Previous work in our lab identified a novel nuclear export signal (NES) (NES(AR)) in AR ligand-binding domain essential for AR nucleocytoplasmic trafficking. By characterizing the localization of green fluorescence protein (GFP)-tagged NES(AR), we designed and executed a yeast mutagenesis screen and isolated 7 yeast mutants that failed to display the NES(AR) export function. One of those mutants was identified as the splicing factor pre-mRNA processing factor 8 (Prp8). We further showed that Prp8 could regulate NES(AR) function using short hairpin RNA knockdown of Prp8 coupled with a rapamycin export assay in mammalian cells and knockdown of Prp8 could induce nuclear accumulation of GFP-tagged AR in PC3 cells. Prp8 expression was decreased in castration-resistant LuCaP35 xenograft tumors as compared with androgen-sensitive xenografts. Laser capture microdissection and quantitative PCR showed Prp8 mRNA levels were decreased in human prostate cancer specimens with high Gleason scores. In prostate cancer cells, coimmunoprecipitation and deletion mutagenesis revealed a physical interaction between Prp8 and AR mainly mediated by NES(AR). Luciferase assay with prostate specific antigen promoter-driven reporter demonstrated that Prp8 regulated AR transcription activity in prostate cancer cells. Interestingly, Prp8 knockdown also increased polyubiquitination of endogenous AR. This may be 1 possible mechanism by which it modulates AR activity. These results show that Prp8 is a novel AR cofactor that interacts with NES(AR) and regulates AR function in prostate cancer cells.

  11. Components of the CCR4-NOT complex function as nuclear hormone receptor coactivators via association with the NRC-interacting Factor NIF-1.

    PubMed

    Garapaty, Shivani; Mahajan, Muktar A; Samuels, Herbert H

    2008-03-14

    CCR4-NOT is an evolutionarily conserved, multicomponent complex known to be involved in transcription as well as mRNA degradation. Various subunits (e.g. CNOT1 and CNOT7/CAF1) have been reported to be involved in influencing nuclear hormone receptor activities. Here, we show that CCR4/CNOT6 and RCD1/CNOT9, members of the CCR4-NOT complex, potentiate nuclear receptor activity. RCD1 interacts in vivo and in vitro with NIF-1 (NRC-interacting factor), a previously characterized nuclear receptor cotransducer that activates nuclear receptors via its interaction with NRC. As with NIF-1, RCD1 and CCR4 do not directly associate with nuclear receptors; however, they enhance ligand-dependent transcriptional activation by nuclear hormone receptors. CCR4 mediates its effect through the ligand binding domain of nuclear receptors and small interference RNA-mediated silencing of endogenous CCR4 results in a marked decrease in nuclear receptor activation. Furthermore, knockdown of CCR4 results in an attenuated stimulation of RARalpha target genes (e.g. Sox9 and HoxA1) as shown by quantitative PCR assays. The silencing of endogenous NIF-1 also resulted in a comparable decrease in the RAR-mediated induction of both Sox9 and HoxA1. Furthermore, CCR4 associates in vivo with NIF-1. In addition, the CCR4-enhanced transcriptional activation by nuclear receptors is dependent on NIF-1. The small interference RNA-mediated knockdown of NIF-1 blocks the ligand-dependent potentiating effect of CCR4. Our results suggest that CCR4 plays a role in the regulation of certain endogenous RARalpha target genes and that RCD1 and CCR4 might mediate their function through their interaction with NIF-1.

  12. Interaction of nuclear factors with the upstream region of the alpha-subunit gene of chicken muscle acetylcholine receptor: variations with muscle differentiation and denervation.

    PubMed Central

    Piette, J; Klarsfeld, A; Changeux, J P

    1989-01-01

    The region lying between nucleotides (nt) -110 and -45 of chicken acetylcholine receptor alpha-subunit gene 5' upstream sequence confers developmental control of expression in primary cultures of chicken myotubes. This region interacts with several nuclear factors present in muscle cells as shown by DNase I footprinting and gel-retardation experiments. An Sp1-like factor and a guanine stretch-binding protein were found to bind to overlapping sites immediately upstream of the TATA box. Several factors interacting in the same region with a domain similar to the SV40 enhancer core appeared during in vitro differentiation of myoblasts into myotubes. The concentration of some of these factors increased also after denervation of leg muscle in newborn chickens. The specific interaction of nuclear factors with this domain may thus play a critical role in the regulation of alpha-subunit gene expression by muscle differentiation and electrical activity. Images PMID:2721497

  13. Studies on Platelet-Derived Growth Factor Beta-Receptor and Hepatocyte Growth Factor Receptor c-met in Paracrine Interactions in Human Breast Cancer

    DTIC Science & Technology

    1996-09-01

    are those of the author and are not necessarily endorsed by the US c’Where Copyrighted material is quoted, pex =ission has been o maind to use such...followed by abso- adjacent capillary endothelium of carcinoma in situ express lute ethanol and 95% ethanol , and then air dried. PDGF P3 receptor mRNA (Fig... ethanol for 5 minutes and 95% ethanol for • 15 minutes, and then air dried. Probes were diluted in prehybridization mix at 15 ng/[Ll, and 200 pA of

  14. In Vivo Estradiol, Tamoxifen and Raloxifene Modulation of Association/Dissociation Kinetics for Estrogen Receptor, Interacting Co-Factors and DNA Binding Sites

    DTIC Science & Technology

    2002-06-01

    is preferred clinically (3, 5 , 7 , 13). It is our goal to understand the molecular and cellular basis of the tissue-specific actions of these...IFinal (14 May 01 - 23 May 02) 4. TITLE AND SUBTITLE 5 . FUNDING NUMBERS In Vivo Estradiol, Tamoxifen and Raloxifene Modulation of DAMDl7-01-1-0498...Association/Dissociation Kinetics for Estrogen Receptor, Interacting Co-Factors and DNA Binding Sites 6. AUTHOR(S) Fred J. Schaufele, Ph.D. 7

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

    PubMed

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

    2010-02-19

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

  16. An ABA-increased interaction of the PYL6 ABA receptor with MYC2 Transcription Factor: A putative link of ABA and JA signaling.

    PubMed

    Aleman, Fernando; Yazaki, Junshi; Lee, Melissa; Takahashi, Yohei; Kim, Alice Y; Li, Zixing; Kinoshita, Toshinori; Ecker, Joseph R; Schroeder, Julian I

    2016-06-30

    Abscisic acid (ABA) is a plant hormone that mediates abiotic stress tolerance and regulates growth and development. ABA binds to members of the PYL/RCAR ABA receptor family that initiate signal transduction inhibiting type 2C protein phosphatases. Although crosstalk between ABA and the hormone Jasmonic Acid (JA) has been shown, the molecular entities that mediate this interaction have yet to be fully elucidated. We report a link between ABA and JA signaling through a direct interaction of the ABA receptor PYL6 (RCAR9) with the basic helix-loop-helix transcription factor MYC2. PYL6 and MYC2 interact in yeast two hybrid assays and the interaction is enhanced in the presence of ABA. PYL6 and MYC2 interact in planta based on bimolecular fluorescence complementation and co-immunoprecipitation of the proteins. Furthermore, PYL6 was able to modify transcription driven by MYC2 using JAZ6 and JAZ8 DNA promoter elements in yeast one hybrid assays. Finally, pyl6 T-DNA mutant plants show an increased sensitivity to the addition of JA along with ABA in cotyledon expansion experiments. Overall, the present study identifies a direct mechanism for transcriptional modulation mediated by an ABA receptor different from the core ABA signaling pathway, and a putative mechanistic link connecting ABA and JA signaling pathways.

  17. Familial risk for mood disorder and the personality risk factor, neuroticism, interact in their association with frontolimbic serotonin 2A receptor binding.

    PubMed

    Frokjaer, Vibe G; Vinberg, Maj; Erritzoe, David; Baaré, William; Holst, Klaus Kähler; Mortensen, Erik Lykke; Arfan, Haroon; Madsen, Jacob; Jernigan, Terry L; Kessing, Lars Vedel; Knudsen, Gitte Moos

    2010-04-01

    Life stress is a robust risk factor for later development of mood disorders, particularly for individuals at familial risk. Likewise, scoring high on the personality trait neuroticism is associated with an increased risk for mood disorders. Neuroticism partly reflects stress vulnerability and is positively correlated to frontolimbic serotonin 2A (5-HT(2A)) receptor binding. Here, we investigate whether neuroticism interacts with familial risk in relation to frontolimbic 5-HT(2A) receptor binding. Twenty-one healthy twins with a co-twin history of mood disorder and 16 healthy twins without a co-twin history of mood disorder were included. They answered self-report personality questionnaires and underwent [(18)F]altanserin positron emission tomography. We found a significant interaction between neuroticism and familial risk in predicting the frontolimbic 5-HT(2A) receptor binding (p=0.026) in an analysis adjusting for age and body mass index. Within the high-risk group only, neuroticism and frontolimbic 5-HT(2A) receptor binding was positively associated (p=0.0037). In conclusion, our data indicate that familial risk and neuroticism interact in their relation to frontolimbic 5-HT(2A) receptor binding. These findings point at a plausible neurobiological link between genetic and personality risk factors and vulnerability to developing mood disorders. It contributes to our understanding of why some people at high risk develop mood disorders while others do not. We speculate that an increased stress reactivity in individuals at high familial risk for mood disorders might enhance the effect of neuroticism in shaping the impact of potential environmental stress and thereby influence serotonergic neurotransmission.

  18. Receptor-like Molecules on Human Intestinal Epithelial Cells Interact with an Adhesion Factor from Lactobacillus reuteri.

    PubMed

    Matsuo, Yosuke; Miyoshi, Yukihiro; Okada, Sanae; Satoh, Eiichi

    2012-01-01

    A surface protein of Lactobacillus reuteri, mucus adhesion-promoting protein (MapA), is considered to be an adhesion factor. MapA is expressed in L. reuteri strains and adheres to piglet gastric mucus, collagen type I, and human intestinal epithelial cells such as Caco-2. The aim of this study was to identify molecules that mediate the attachment of MapA from L. reuteri to the intestinal epithelial cell surface by investigating the adhesion of MapA to receptor-like molecules on Caco-2 cells. MapA-binding receptor-like molecules were detected in Caco-2 cell lysates by 2D-PAGE. Two proteins, annexin A13 (ANXA13) and paralemmin (PALM), were identified by MALDI TOF-MS. The results of a pull-down assay showed that MapA bound directly to ANXA13 and PALM. Fluorescence microscopy studies confirmed that MapA binding to ANXA13 and PALM was colocalized on the Caco-2 cell membrane. To evaluate whether ANXA13 and PALM are important for MapA adhesion, ANXA13 and PALM knockdown cell lines were established. The adhesion of MapA to the abovementioned cell lines was reduced compared with that to wild-type Caco-2 cells. These knockdown experiments established the importance of these receptor-like molecules in MapA adhesion.

  19. Receptor-like Molecules on Human Intestinal Epithelial Cells Interact with an Adhesion Factor from Lactobacillus reuteri

    PubMed Central

    MATSUO, Yosuke; MIYOSHI, Yukihiro; OKADA, Sanae; SATOH, Eiichi

    2012-01-01

    A surface protein of Lactobacillus reuteri, mucus adhesion-promoting protein (MapA), is considered to be an adhesion factor. MapA is expressed in L. reuteri strains and adheres to piglet gastric mucus, collagen type I, and human intestinal epithelial cells such as Caco-2. The aim of this study was to identify molecules that mediate the attachment of MapA from L. reuteri to the intestinal epithelial cell surface by investigating the adhesion of MapA to receptor-like molecules on Caco-2 cells. MapA-binding receptor-like molecules were detected in Caco-2 cell lysates by 2D-PAGE. Two proteins, annexin A13 (ANXA13) and paralemmin (PALM), were identified by MALDI TOF-MS. The results of a pull-down assay showed that MapA bound directly to ANXA13 and PALM. Fluorescence microscopy studies confirmed that MapA binding to ANXA13 and PALM was colocalized on the Caco-2 cell membrane. To evaluate whether ANXA13 and PALM are important for MapA adhesion, ANXA13 and PALM knockdown cell lines were established. The adhesion of MapA to the abovementioned cell lines was reduced compared with that to wild-type Caco-2 cells. These knockdown experiments established the importance of these receptor-like molecules in MapA adhesion. PMID:24936355

  20. Interaction between body mass index and hormone-receptor status as a prognostic factor in lymph-node-positive breast cancer

    PubMed Central

    Chung, Il Yong; Park, Yu Rang; Min, Yul Ha; Lee, Yura; Yoon, Tae In; Sohn, Guiyun; Lee, Sae Byul; Kim, Jisun; Kim, Hee Jeong; Ko, Beom Seok; Son, Byung Ho; Ahn, Sei Hyun

    2017-01-01

    The aim of this study was to determine the relationship between the body mass index (BMI) at a breast cancer diagnosis and various factors including the hormone-receptor, menopause, and lymph-node status, and identify if there is a specific patient subgroup for which the BMI has an effect on the breast cancer prognosis. We retrospectively analyzed the data of 8,742 patients with non-metastatic invasive breast cancer from the research database of Asan Medical Center. The overall survival (OS) and breast-cancer-specific survival (BCSS) outcomes were compared among BMI groups using the Kaplan-Meier method and Cox proportional-hazards regression models with an interaction term. There was a significant interaction between BMI and hormone-receptor status for the OS (P = 0.029), and BCSS (P = 0.013) in lymph-node-positive breast cancers. Obesity in hormone-receptor-positive breast cancer showed a poorer OS (adjusted hazard ratio [HR] = 1.51, 95% confidence interval [CI] = 0.92 to 2.48) and significantly poorer BCSS (HR = 1.80, 95% CI = 1.08 to 2.99). In contrast, a high BMI in hormone-receptor-negative breast cancer revealed a better OS (HR = 0.44, 95% CI = 0.16 to 1.19) and BCSS (HR = 0.53, 95% CI = 0.19 to 1.44). Being underweight (BMI < 18.50 kg/m2) with hormone-receptor-negative breast cancer was associated with a significantly worse OS (HR = 1.98, 95% CI = 1.00–3.95) and BCSS (HR = 2.24, 95% CI = 1.12–4.47). There was no significant interaction found between the BMI and hormone-receptor status in the lymph-node-negative setting, and BMI did not interact with the menopause status in any subgroup. In conclusion, BMI interacts with the hormone-receptor status in a lymph-node-positive setting, thereby playing a role in the prognosis of breast cancer. PMID:28248981

  1. Molecular cloning, expression and functional characterization of tumor necrosis factor (TNF) receptor-associated factor (TRAF)-interacting protein (TRIP) in grass carp, Ctenopharyngodon idella.

    PubMed

    Lu, R-H; Chang, Z-G; Sun, J; Yang, F; Nie, G-X; Ji, H

    2016-10-01

    TRIP (Tumor Necrosis Factor (TNF) Receptor-Associated Factor (TRAF)-Interacting Protein), a member of the TNF superfamily, plays a crucial role in the modulation of inflammation in vertebrates. However, no information about TRIP is available in teleosts. In this study, the full-length cDNA of TRIP, containing a 5'UTR of 112 bp, an ORF of 1359 bp, and a 3'UTR of 29 bp before the poly (A) tail, was cloned from grass carp, Ctenopharyngodon idella. The TRIP gene encoded a protein of 452 amino acids with an estimated molecular mass of 51.06 KD and a predicted theoretical isoelectric point (pI) of 9.11. Quantitative real-time PCR analysis revealed that TRIP mRNA was expressed in all the tissues examined in grass carp, with the highest expression in the kidney, followed by the intestine and thymus. However, lower levels of expression were also detected in fat, spleen, liver, gonad and heart. Subcellular localization and two-hybrid analysis revealed that TRIP was located in the nucleus and that it interacted with TRAF1 and TRAF2 in HEK293T cells. Furthermore, similar to TNF-α, IL-10 and TRIP mRNA expression was upregulated in the spleen of fish fed high-fat or high-carbohydrate diets, suggesting that TRIP might be associated with the response to excessive energy intake. The mRNA relative expression of TRIP was significantly reduced (P < 0.05) after hepatocyte of C. idella was treated with 2 μg/mL lipopolysaccharide (LPS) for 4 h, while the expression levels of inflammatory cytokines TNF-α and IL-10 were significantly increased (P < 0.05). Taken together, these results indicate that TRIP might play important roles in immune defense and has the potential to be used as a anti-inflammation target in grass carp.

  2. Sorting Nexin 27 Protein Regulates Trafficking of a p21-activated Kinase (PAK) Interacting Exchange Factor (β-Pix)-G Protein-coupled Receptor Kinase Interacting Protein (GIT) Complex via a PDZ Domain Interaction*

    PubMed Central

    Valdes, Julie L.; Tang, Jingrong; McDermott, Mark I.; Kuo, Jean-Cheng; Zimmerman, Seth P.; Wincovitch, Stephen M.; Waterman, Clare M.; Milgram, Sharon L.; Playford, Martin P.

    2011-01-01

    Sorting nexin 27 (SNX27) is a 62-kDa protein localized to early endosomes and known to regulate the intracellular trafficking of ion channels and receptors. In addition to a PX domain, SNX27 is the only sorting family member that contains a PDZ domain. To identify novel SNX27-PDZ binding partners, we performed a proteomic screen in mouse principal kidney cortical collecting duct cells using a GST-SNX27 fusion construct as bait. We found that β-Pix (p21-activated kinase-interactive exchange factor), a guanine nucleotide exchange factor for the Rho family of small GTPases known to regulate cell motility directly interacted with SNX27. The association of β-Pix and SNX27 is specific for β-Pix isoforms terminating in the type-1 PDZ binding motif (ETNL). In the same screen we also identified Git1/2 as a potential SNX27 interacting protein. The interaction between SNX27 and Git1/2 is indirect and mediated by β-Pix. Furthermore, we show recruitment of the β-Pix·Git complex to endosomal sites in a SNX27-dependent manner. Finally, migration assays revealed that depletion of SNX27 from HeLa and mouse principal kidney cortical collecting duct cells significantly decreases cell motility. We propose a model by which SNX27 regulates trafficking of β-Pix to focal adhesions and thereby influences cell motility. PMID:21926430

  3. Sorting nexin 27 protein regulates trafficking of a p21-activated kinase (PAK) interacting exchange factor (β-Pix)-G protein-coupled receptor kinase interacting protein (GIT) complex via a PDZ domain interaction.

    PubMed

    Valdes, Julie L; Tang, Jingrong; McDermott, Mark I; Kuo, Jean-Cheng; Zimmerman, Seth P; Wincovitch, Stephen M; Waterman, Clare M; Milgram, Sharon L; Playford, Martin P

    2011-11-11

    Sorting nexin 27 (SNX27) is a 62-kDa protein localized to early endosomes and known to regulate the intracellular trafficking of ion channels and receptors. In addition to a PX domain, SNX27 is the only sorting family member that contains a PDZ domain. To identify novel SNX27-PDZ binding partners, we performed a proteomic screen in mouse principal kidney cortical collecting duct cells using a GST-SNX27 fusion construct as bait. We found that β-Pix (p21-activated kinase-interactive exchange factor), a guanine nucleotide exchange factor for the Rho family of small GTPases known to regulate cell motility directly interacted with SNX27. The association of β-Pix and SNX27 is specific for β-Pix isoforms terminating in the type-1 PDZ binding motif (ETNL). In the same screen we also identified Git1/2 as a potential SNX27 interacting protein. The interaction between SNX27 and Git1/2 is indirect and mediated by β-Pix. Furthermore, we show recruitment of the β-Pix·Git complex to endosomal sites in a SNX27-dependent manner. Finally, migration assays revealed that depletion of SNX27 from HeLa and mouse principal kidney cortical collecting duct cells significantly decreases cell motility. We propose a model by which SNX27 regulates trafficking of β-Pix to focal adhesions and thereby influences cell motility.

  4. Interaction between echovirus 7 and its receptor, decay-accelerating factor (CD55): evidence for a secondary cellular factor in A-particle formation.

    PubMed Central

    Powell, R M; Ward, T; Evans, D J; Almond, J W

    1997-01-01

    Soluble forms of decay-accelerating factor (DAF) (CD55), the receptor for echovirus 7, were synthesized in the yeast Pichia pastoris. Purified recombinant protein containing SCR domains 2, 3, and 4, but lacking the serine/threonine rich region, was shown to block infection of susceptible cells by echovirus 7. In contrast to the situation with poliovirus and its receptor, the neutralization of echovirus 7 by soluble DAF was completely reversible and did not lead to the formation of 135S A-particles. Binding of virus to susceptible cells, by contrast, did lead to the formation of A particles, mainly from virus that had been internalized. The data suggest that a secondary factor(s) may contribute to A-particle formation and uncoating of echovirus 7. PMID:9371589

  5. BAT3 interacts with transforming growth factor-beta (TGF-beta) receptors and enhances TGF-beta1-induced type I collagen expression in mesangial cells.

    PubMed

    Kwak, Joon Hyeok; Kim, Sung Il; Kim, Jin Kuk; Choi, Mary E

    2008-07-11

    Transforming growth factor-beta1 (TGF-beta1) plays essential roles in a wide array of cellular processes, such as in development and the pathogenesis of tissue fibrosis, including that associated with progressive kidney diseases. Tight regulation of its signaling pathways is critical, and proteins that associate with the TGF-beta receptors may exert positive or negative regulatory effects on TGF-beta signaling. In the present study we employed a yeast-based two-hybrid screening system to identify BAT3 (HLA-B-associated transcript 3) as a TGF-beta receptor-interacting protein. Analysis of endogenously expressed BAT3 in various tissues including the kidney reveals the existence of approximately 140-kDa full-length protein as well as truncated forms of BAT3 whose expression is developmentally regulated. Endogenous BAT3 protein interacts with TGF-beta receptors type I and type II in renal mesangial cells. Functional assays show that expression of full-length BAT3 results in enhancement of TGF-beta1-stimulated transcriptional activation of p3TP-Lux reporter, and these effects require the presence of functional TGF-beta signaling receptors as demonstrated in R-1B and DR-26 mutant cells. Moreover, expression of full-length BAT3, but not C-terminal truncated mutant of BAT3, enhanced TGF-beta1-induced type I collagen expression in mesangial cells, whereas knock down of BAT3 protein expression by small interfering RNA suppressed the expression of type I collagen induced by TGF-beta1. Our findings suggest that BAT3, a TGF-beta receptor-interacting protein, is capable of modulating TGF-beta signaling and acts as a positive regulator of TGF-beta1 stimulation of type I collagen expression in mesangial cells.

  6. Tumor necrosis factor (TNF) receptor-associated factor (TRAF)-interacting protein (TRIP) negatively regulates the TRAF2 ubiquitin-dependent pathway by suppressing the TRAF2-sphingosine 1-phosphate (S1P) interaction.

    PubMed

    Park, Eui-Soon; Choi, Seunga; Shin, Bongjin; Yu, Jungeun; Yu, Jiyeon; Hwang, Jung-Me; Yun, Hyeongseok; Chung, Young-Ho; Choi, Jong-Soon; Choi, Yongwon; Rho, Jaerang

    2015-04-10

    The signaling pathway downstream of TNF receptor (TNFR) is involved in the induction of a wide range of cellular processes, including cell proliferation, activation, differentiation, and apoptosis. TNFR-associated factor 2 (TRAF2) is a key adaptor molecule in TNFR signaling complexes that promotes downstream signaling cascades, such as nuclear factor-κB (NF-κB) and mitogen-activated protein kinase activation. TRAF-interacting protein (TRIP) is a known cellular binding partner of TRAF2 and inhibits TNF-induced NF-κB activation. Recent findings that TRIP plays a multifunctional role in antiviral response, cell proliferation, apoptosis, and embryonic development have increased our interest in exploring how TRIP can affect the TNFR-signaling pathway on a molecular level. In our current study, we demonstrated that TRIP is negatively involved in the TNF-induced inflammatory response through the down-regulation of proinflammatory cytokine production. Here, we demonstrated that the TRAF2-TRIP interaction inhibits Lys(63)-linked TRAF2 ubiquitination by inhibiting TRAF2 E3 ubiquitin (Ub) ligase activity. The TRAF2-TRIP interaction inhibited the binding of sphingosine 1-phosphate, which is a cofactor of TRAF2 E3 Ub ligase, to the TRAF2 RING domain. Finally, we demonstrated that TRIP functions as a negative regulator of proinflammatory cytokine production by inhibiting TNF-induced NF-κB activation. These results indicate that TRIP is an important cellular regulator of the TNF-induced inflammatory response.

  7. Tumor Necrosis Factor (TNF) Receptor-associated Factor (TRAF)-interacting Protein (TRIP) Negatively Regulates the TRAF2 Ubiquitin-dependent Pathway by Suppressing the TRAF2-Sphingosine 1-Phosphate (S1P) Interaction*

    PubMed Central

    Park, Eui-Soon; Choi, Seunga; Shin, Bongjin; Yu, Jungeun; Yu, Jiyeon; Hwang, Jung-Me; Yun, Hyeongseok; Chung, Young-Ho; Choi, Jong-Soon; Choi, Yongwon; Rho, Jaerang

    2015-01-01

    The signaling pathway downstream of TNF receptor (TNFR) is involved in the induction of a wide range of cellular processes, including cell proliferation, activation, differentiation, and apoptosis. TNFR-associated factor 2 (TRAF2) is a key adaptor molecule in TNFR signaling complexes that promotes downstream signaling cascades, such as nuclear factor-κB (NF-κB) and mitogen-activated protein kinase activation. TRAF-interacting protein (TRIP) is a known cellular binding partner of TRAF2 and inhibits TNF-induced NF-κB activation. Recent findings that TRIP plays a multifunctional role in antiviral response, cell proliferation, apoptosis, and embryonic development have increased our interest in exploring how TRIP can affect the TNFR-signaling pathway on a molecular level. In our current study, we demonstrated that TRIP is negatively involved in the TNF-induced inflammatory response through the down-regulation of proinflammatory cytokine production. Here, we demonstrated that the TRAF2-TRIP interaction inhibits Lys63-linked TRAF2 ubiquitination by inhibiting TRAF2 E3 ubiquitin (Ub) ligase activity. The TRAF2-TRIP interaction inhibited the binding of sphingosine 1-phosphate, which is a cofactor of TRAF2 E3 Ub ligase, to the TRAF2 RING domain. Finally, we demonstrated that TRIP functions as a negative regulator of proinflammatory cytokine production by inhibiting TNF-induced NF-κB activation. These results indicate that TRIP is an important cellular regulator of the TNF-induced inflammatory response. PMID:25716317

  8. Ligand-induced interaction between. alpha. - and. beta. -type platelet-derived growth factor (PDGF) receptors: Role of receptor heterodimers in kinase activation

    SciTech Connect

    Kanakaraj, P.; Raj, S.; Bishayee, S. ); Khan, S.A. )

    1991-02-19

    Two types of PDGF receptors have been cloned and sequenced. Both receptors are transmembrane glycoproteins with a ligand-stimulatable tyrosine kinase site. The authors have shown earlier that ligand-induced activation of the {beta}-type PDGF receptor is due to the conversion of the monomeric form of the receptor to the dimeric form. In the present studies, they have established the ligand-binding specificity of two receptor types and extended it further to investigate the ligand-induced association state of the {alpha}-receptor and the role of {alpha}-receptor in the activation of {beta}-receptor. These studies were conducted with cells that express one or the other type of PDGF receptor as well as with cells that express both types of receptors. Moreover, ligand-binding characteristics of the receptor were confirmed by immunoprecipitation of the receptor-{sup 125}I-PDGF covalent complex with type-specific anti-PDGF receptor antibodies. These studies revealed that all three isoforms of PDGF bind to {alpha}-receptor, and such binding leads to dimerization as well as activation of the receptor. In contrast, {beta}-receptor can be activated only by PDGF BB and not by PDGF AB or PDGF AA. However, by using antipeptide antibodies that are specific for {alpha}- or {beta}-type PDGF receptor, they demonstrated that in the presence of {alpha}-receptor, {beta}-receptor kinase can be activated by PDGF AB. They present here direct evidence that strongly suggests that such PDGF AB induced activation of {beta}-receptor is due to the formation of a noncovalently linked {alpha}-{beta} receptor heterodimer.

  9. Identification of a nuclear protein interacting with a novel site on rat androgen receptor promoter after transcription factor NFkB is displaced from adjacent site.

    PubMed

    Zaidi, Ghazala; Supakar, Prakash C

    2003-06-01

    Sequence-specific DNA-protein interactions mediate the regulation of rat androgen receptor (rAR) gene expression. Previously, DNase I footprinting revealed that nuclear factor kappa B (NFkB) binds to region -574 to -554 on rAR promoter and represses its expression. In this study, we demonstrate that when NFkB protein is removed from its site by competitor DNA in DNase I footprinting reaction, a new DNase I protected region is formed overlapping adjacently (-594 to -561). This indicates that another nuclear protein (named here as FRN, factor repressed by NFkB) binds to rAR promoter only after NFkB protein is displaced. By competitive electrophoretic mobility shift assay and mutation analysis, we confirmed the formation of FRN-DNA complex. FRN interacts with a novel sequence on rAR promoter and may play a role in regulation of rAR gene expression in concert with NFkB.

  10. Solution structure of human insulin-like growth factor II. Relationship to receptor and binding protein interactions.

    PubMed

    Torres, A M; Forbes, B E; Aplin, S E; Wallace, J C; Francis, G L; Norton, R S

    1995-04-28

    The three-dimensional structure of human insulin-like growth factor (IGF) II in aqueous solution at pH 3.1 and 300 K has been determined from nuclear magnetic resonance data and restrained molecular dynamics calculations. Structural constraints consisting of 502 NOE-derived distance constraints, 11 dihedral angle restraints, and three disulfide bridges were used as input for distance geometry calculations in DIANA and X-PLOR, followed by simulated annealing refinement and energy minimization in X-PLOR. The resulting family of 20 structures was well defined in the regions of residues 5 to 28 and 41 to 62, with an average pairwise root-mean-square deviation of 1.24 A for the backbone heavy-atoms (N, C2, C) and 1.90 A for all heavy atoms. The poorly defined regions consist of the N and C termini, part of the B-domain, and the C-domain loop. Resonances from these regions of the protein gave stronger cross peaks in two dimensional NMR spectra, consistent with significant motional averaging. The main secondary structure elements in IGF-II are alpha-helices encompassing residues 11 to 21, 42 to 49 and 53 to 59. A small anti-parallel beta-sheet is formed by residues 59 to 61 and 25 to 27, while residues 26 to 28 appear to participate in intermolecular beta-sheet formation. The structure of IGF-II in the well-defined regions is very similar to those of the corresponding regions of insulin and IGF-I. Significant differences between IGF-II and IGF-I occur near the start of the third helix, in a region known to modulate affinity for the type 2 IGF receptor, and at the C terminus. The IGF II structure is discussed in relation to its binding sites for the insulin and IGF receptors and the IGF binding proteins.

  11. Mumps Virus SH Protein Inhibits NF-κB Activation by Interacting with Tumor Necrosis Factor Receptor 1, Interleukin-1 Receptor 1, and Toll-Like Receptor 3 Complexes.

    PubMed

    Franz, Stephanie; Rennert, Paul; Woznik, Maria; Grützke, Josephine; Lüdde, Amy; Arriero Pais, Eva Maria; Finsterbusch, Tim; Geyer, Henriette; Mankertz, Annette; Friedrich, Nicole

    2017-09-15

    The mumps virus (MuV) small hydrophobic protein (SH) is a type I membrane protein expressed in infected cells. SH has been reported to interfere with innate immunity by inhibiting tumor necrosis factor alpha (TNF-α)-mediated apoptosis and NF-κB activation. To elucidate the underlying mechanism, we generated recombinant MuVs (rMuVs) expressing the SH protein with an N-terminal FLAG epitope or lacking SH expression due to the insertion of three stop codons into the SH gene. Using these viruses, we were able to show that SH reduces the phosphorylation of IKKβ, IκBα, and p65 as well as the translocation of p65 into the nucleus of infected A549 cells. Reporter gene assays revealed that SH interferes not only with TNF-α-mediated NF-κB activation but also with IL-1β- and poly(I·C)-mediated NF-κB activation, and that this inhibition occurs upstream of the NF-κB pathway components TRAF2, TRAF6, and TAK1. Since SH coimmunoprecipitated with tumor necrosis factor receptor 1 (TNFR1), RIP1, and IRAK1, we hypothesize that SH exerts its inhibitory function by interacting with TNFR1, interleukin-1 receptor type 1 (IL-1R1), and TLR3 complexes in the plasma membrane of infected cells.IMPORTANCE The MuV SH has been shown to impede TNF-α-mediated NF-κB activation and is therefore thought to contribute to viral immune evasion. However, the mechanisms by which SH mediates NF-κB inhibition remained largely unknown. In this study, we show that SH interacts with TNFR1, IL-1R1, and TLR3 complexes in infected cells. We thereby not only shed light on the mechanisms of SH-mediated NF-κB inhibition but also reveal that SH interferes with NF-κB activation induced by interleukin-1β (IL-1β) and double-stranded RNA. Copyright © 2017 American Society for Microbiology.

  12. A distinct basic fibroblast growth factor (FGF-2)/FGF receptor interaction distinguishes urokinase-type plasminogen activator induction from mitogenicity in endothelial cells.

    PubMed Central

    Rusnati, M; Dell'Era, P; Urbinati, C; Tanghetti, E; Massardi, M L; Nagamine, Y; Monti, E; Presta, M

    1996-01-01

    Basic fibroblast growth factor (FGF-2) induces cell proliferation and urokinase-type plasminogen activator (uPA) production in fetal bovine aortic endothelial GM 7373 cells. In the present paper we investigated the role of the interaction of FGF-2 with tyrosine-kinase (TK) FGF receptors (FGFRs) in mediating uPA up-regulation in these cells. The results show that FGF-2 antagonists suramin, protamine, heparin, the synthetic peptide FGF-2(112-155), and a soluble form of FGFR-1 do not inhibit FGF-2-mediated uPA up-regulation at concentrations that affect growth factor binding to cell surface receptors and mitogenic activity. In contrast, tyrosine phosphorylation inhibitors and overexpression of a dominant negative TK- mutant of FGFR-1 abolish the uPA-inducing activity of FGF-2, indicating that FGFR and its TK activity are essential in mediating uPA induction. Accordingly, FGF-2 induces uPA up-regulation in Chinese hamster ovary cells transfected with wild-type FGFR-1, -2, -3, or -4 but not with TK- FGFR-1 mutant. Small unilamellar phosphatidyl choline:cholesterol vesicles loaded with FGF-2 increased uPA production in GM 7373 cells in the absence of a mitogenic response. Liposome-encapsulated FGF-2 showed a limited but significant capacity, relative to free FGF-2, to interact with FGFR both at 4 degrees C and 37 degrees C and to be internalized within the cell. uPA up-regulation by liposome-encapsulated FGF-2 was quenched by neutralizing anti-FGF-2 antibodies, indicating that the activity of liposome-delivered FGF-2 is mediated by an extracellular action of the growth factor. Taken together, the data indicate that a distinct interaction of FGF-2 with FGFR, quantitatively and/or qualitatively different from the one that leads to mitogenicity, is responsible for the uPA-inducing activity of the growth factor. Images PMID:8868466

  13. Monomeric ß-amyloid interacts with type-1 insulin-like growth factor receptors to provide energy supply to neurons

    PubMed Central

    Giuffrida, Maria L.; Tomasello, Marianna F.; Pandini, Giuseppe; Caraci, Filippo; Battaglia, Giuseppe; Busceti, Carla; Di Pietro, Paola; Pappalardo, Giuseppe; Attanasio, Francesco; Chiechio, Santina; Bagnoli, Silvia; Nacmias, Benedetta; Sorbi, Sandro; Vigneri, Riccardo; Rizzarelli, Enrico; Nicoletti, Ferdinando; Copani, Agata

    2015-01-01

    ß-amyloid (Aß1−42) is produced by proteolytic cleavage of the transmembrane type-1 protein, amyloid precursor protein. Under pathological conditions, Aß1−42self-aggregates into oligomers, which cause synaptic dysfunction and neuronal loss, and are considered the culprit of Alzheimer's disease (AD). However, Aß1−42 is mainly monomeric at physiological concentrations, and the precise role of monomeric Aß1−42 in neuronal function is largely unknown. We report that the monomer of Aß1−42 activates type-1 insulin-like growth factor receptors and enhances glucose uptake in neurons and peripheral cells by promoting the translocation of the Glut3 glucose transporter from the cytosol to the plasma membrane. In neurons, activity-dependent glucose uptake was blunted after blocking endogenous Aß production, and re-established in the presence of cerebrospinal fluid Aß. APP-null neurons failed to enhance depolarization-stimulated glucose uptake unless exogenous monomeric Aß1−42 was added. These data suggest that Aß1−42 monomers were critical for maintaining neuronal glucose homeostasis. Accordingly, exogenous Aß1−42 monomers were able to rescue the low levels of glucose consumption observed in brain slices from AD mutant mice. PMID:26300732

  14. Steroid receptor coactivator-interacting protein (SIP) inhibits caspase-independent apoptosis by preventing apoptosis-inducing factor (AIF) from being released from mitochondria.

    PubMed

    Wang, Dandan; Liang, Jing; Zhang, Yu; Gui, Bin; Wang, Feng; Yi, Xia; Sun, Luyang; Yao, Zhi; Shang, Yongfeng

    2012-04-13

    Apoptosis-inducing factor (AIF) is a caspase-independent death effector. Normally residing in the mitochondrial intermembrane space, AIF is released and translocated to the nucleus in response to proapoptotic stimuli. Nuclear AIF binds to DNA and induces chromatin condensation and DNA fragmentation, characteristics of apoptosis. Until now, it remained to be clarified how the mitochondrial-nuclear translocation of AIF is regulated. Here we report that steroid receptor coactivator-interacting protein (SIP) interacts directly with AIF in mitochondria and specifically inhibits caspase-independent and AIF-dependent apoptosis. Challenging cells with apoptotic stimuli leads to rapid degradation of SIP, and subsequently AIF is liberated from mitochondria and translocated to the nucleus to induce apoptosis. Together, our data demonstrate that SIP is a novel regulator in caspase-independent and AIF-mediated apoptosis.

  15. Nuclear Receptor-Like Structure and Interaction of Congenital Heart Disease-Associated Factors GATA4 and NKX2-5.

    PubMed

    Kinnunen, Sini; Välimäki, Mika; Tölli, Marja; Wohlfahrt, Gerd; Darwich, Rami; Komati, Hiba; Nemer, Mona; Ruskoaho, Heikki

    2015-01-01

    Transcription factor GATA4 is a dosage sensitive regulator of heart development and alterations in its level or activity lead to congenital heart disease (CHD). GATA4 has also been implicated in cardiac regeneration and repair. GATA4 action involves combinatorial interaction with other cofactors such as NKX2-5, another critical cardiac regulator whose mutations also cause CHD. Despite its critical importance to the heart and its evolutionary conservation across species, the structural basis of the GATA4-NKX2-5 interaction remains incompletely understood. A homology model was constructed and used to identify surface amino acids important for the interaction of GATA4 and NKX2-5. These residues were subjected to site-directed mutagenesis, and the mutant proteins were characterized for their ability to bind DNA and to physically and functionally interact with NKX2-5. The studies identify 5 highly conserved amino acids in the second zinc finger (N272, R283, Q274, K299) and its C-terminal extension (R319) that are critical for physical and functional interaction with the third alpha helix of NKX2-5 homeodomain. Integration of the experimental data with computational modeling suggests that the structural arrangement of the zinc finger-homeodomain resembles the architecture of the conserved DNA binding domain of nuclear receptors. The results provide novel insight into the structural basis for protein-protein interactions between two important classes of transcription factors. The model proposed will help to elucidate the molecular basis for disease causing mutations in GATA4 and NKX2-5 and may be relevant to other members of the GATA and NK classes of transcription factors.

  16. Nuclear Receptor-Like Structure and Interaction of Congenital Heart Disease-Associated Factors GATA4 and NKX2-5

    PubMed Central

    Tölli, Marja; Wohlfahrt, Gerd; Darwich, Rami; Komati, Hiba; Nemer, Mona; Ruskoaho, Heikki

    2015-01-01

    Aims Transcription factor GATA4 is a dosage sensitive regulator of heart development and alterations in its level or activity lead to congenital heart disease (CHD). GATA4 has also been implicated in cardiac regeneration and repair. GATA4 action involves combinatorial interaction with other cofactors such as NKX2-5, another critical cardiac regulator whose mutations also cause CHD. Despite its critical importance to the heart and its evolutionary conservation across species, the structural basis of the GATA4-NKX2-5 interaction remains incompletely understood. Methods and Results A homology model was constructed and used to identify surface amino acids important for the interaction of GATA4 and NKX2-5. These residues were subjected to site-directed mutagenesis, and the mutant proteins were characterized for their ability to bind DNA and to physically and functionally interact with NKX2-5. The studies identify 5 highly conserved amino acids in the second zinc finger (N272, R283, Q274, K299) and its C-terminal extension (R319) that are critical for physical and functional interaction with the third alpha helix of NKX2-5 homeodomain. Integration of the experimental data with computational modeling suggests that the structural arrangement of the zinc finger-homeodomain resembles the architecture of the conserved DNA binding domain of nuclear receptors. Conclusions The results provide novel insight into the structural basis for protein-protein interactions between two important classes of transcription factors. The model proposed will help to elucidate the molecular basis for disease causing mutations in GATA4 and NKX2-5 and may be relevant to other members of the GATA and NK classes of transcription factors. PMID:26642209

  17. Interactions between effectors linked to serotonin receptors.

    PubMed

    Berg, K A; Maayani, S; Clarke, W P

    1998-12-15

    In general, there are two types of interactions between effector signaling pathways. "Homologous" interactions are those that occur within a receptor system to alter its own responsiveness, for example the loss of responsiveness (desensitization) that can occur upon agonist occupancy of a receptor. "Heterologous" interactions are those that occur between different receptor systems where the responsiveness of one receptor system is regulated (positively or negatively) by activation of another receptor system (i.e., "cross-talk"). Many, if not all receptors, couple to multiple cellular effector pathways and alterations in the responsiveness of a receptor system can be effector pathway-dependent which underscores the importance of studying each effector coupled to a receptor. Regulation of receptor system responsiveness, and consequently the efficacy of drugs, is a highly dynamic process. Perhaps by exploiting these interactions, new targets for pharmacotherapy may be uncovered which will provide for increased efficacy and specificity of drug action.

  18. Hepatocyte Growth Factor Modulates MET Receptor Tyrosine Kinase and β-Catenin Functional Interactions to Enhance Synapse Formation

    PubMed Central

    Xie, Zhihui; Eagleson, Kathie L.

    2016-01-01

    MET, a pleiotropic receptor tyrosine kinase implicated in autism risk, influences multiple neurodevelopmental processes. There is a knowledge gap, however, in the molecular mechanism through which MET mediates developmental events related to disorder risk. In the neocortex, MET is expressed transiently during periods of peak dendritic outgrowth and synaptogenesis, with expression enriched at developing synapses, consistent with demonstrated roles in dendritic morphogenesis, modulation of spine volume, and excitatory synapse development. In a recent coimmunoprecipitation/mass spectrometry screen, β-catenin was identified as part of the MET interactome in developing neocortical synaptosomes. Here, we investigated the influence of the MET/β-catenin complex in mouse neocortical synaptogenesis. Western blot analysis confirms that MET and β-catenin coimmunoprecipitate, but N-cadherin is not associated with the MET complex. Following stimulation with hepatocyte growth factor (HGF), β-catenin is phosphorylated at tyrosine142 (Y142) and dissociates from MET, accompanied by an increase in β-catenin/N-cadherin and MET/synapsin 1 protein complexes. In neocortical neurons in vitro, proximity ligation assays confirmed the close proximity of these proteins. Moreover, in neurons transfected with synaptophysin-GFP, HGF stimulation increases the density of synaptophysin/bassoon (a presynaptic marker) and synaptophysin/PSD-95 (a postsynaptic marker) clusters. Mutation of β-catenin at Y142 disrupts the dissociation of the MET/β-catenin complex and prevents the increase in clusters in response to HGF. The data demonstrate a new mechanism for the modulation of synapse formation, whereby MET activation induces an alignment of presynaptic and postsynaptic elements that are necessary for assembly and formation of functional synapses by subsets of neocortical neurons that express MET/β-catenin complex. PMID:27595133

  19. Study of Coxsackie B viruses interactions with Coxsackie Adenovirus receptor and Decay-Accelerating Factor using Human CaCo-2 cell line.

    PubMed

    Riabi, Samira; Harrath, Rafik; Gaaloul, Imed; Bouslama, Lamjed; Nasri, Dorsaf; Aouni, Mahjoub; Pillet, Sylvie; Pozzetto, Bruno

    2014-05-21

    Decay Accelerating Factor (DAF) and Coxsackievirus-Adenovirus Receptor (CAR) have been identified as cellular receptors for Coxsackie B viruses (CV-B). The aim of this study is to elucidate the different binding properties of CV-B serotypes and to find out if there are any amino acid changes that could be associated to the different phenotypes.Twenty clinical CV-B isolates were tested on CaCo-2 cell line using anti-DAF (BRIC216) and anti-CAR (RmcB) antibodies. CV-B3 Nancy prototype strain and a recombinant strain (Rec, CV-B3/B4) were tested in parallel. The P1 genomic region of 12 CV-B isolates from different serotypes was sequenced and the Trans-Epithelial Electrical Resistance (TEER) along with the virus growth cycle was measured. Infectivity assays revealed clear differences between CV-B isolates with regard to their interactions with DAF and CAR. All tested CV-B isolates showed an absolute requirement for CAR but varied in their binding to DAF. We also reported that for some isolates of CV-B, DAF attachment was not adapted. Genetic analysis of the P1 region detected multiple differences in the deduced amino acid sequences. Within a given serotype, variations exist in the capacity of virus isolates to bind to specific receptors, and variants with different additional ligands may arise during infection in humans as well as in tissue culture.

  20. Study of Coxsackie B viruses interactions with Coxsackie Adenovirus receptor and Decay-Accelerating Factor using Human CaCo-2 cell line

    PubMed Central

    2014-01-01

    Background Decay Accelerating Factor (DAF) and Coxsackievirus-Adenovirus Receptor (CAR) have been identified as cellular receptors for Coxsackie B viruses (CV-B). The aim of this study is to elucidate the different binding properties of CV-B serotypes and to find out if there are any amino acid changes that could be associated to the different phenotypes. Twenty clinical CV-B isolates were tested on CaCo-2 cell line using anti-DAF (BRIC216) and anti-CAR (RmcB) antibodies. CV-B3 Nancy prototype strain and a recombinant strain (Rec, CV-B3/B4) were tested in parallel. The P1 genomic region of 12 CV-B isolates from different serotypes was sequenced and the Trans-Epithelial Electrical Resistance (TEER) along with the virus growth cycle was measured. Results Infectivity assays revealed clear differences between CV-B isolates with regard to their interactions with DAF and CAR. All tested CV-B isolates showed an absolute requirement for CAR but varied in their binding to DAF. We also reported that for some isolates of CV-B, DAF attachment was not adapted. Genetic analysis of the P1 region detected multiple differences in the deduced amino acid sequences. Conclusion Within a given serotype, variations exist in the capacity of virus isolates to bind to specific receptors, and variants with different additional ligands may arise during infection in humans as well as in tissue culture. PMID:24885774

  1. Interactions Between Corticotropin-Releasing Factor and the Serotonin 1A Receptor System on Acoustic Startle Amplitude and Prepulse Inhibition of the Startle Response in Two Rat Strains

    PubMed Central

    Conti, Lisa H.

    2011-01-01

    Both the neuropeptide, corticotropin-releasing factor (CRF) and the serotonin 1A (5-HT1A) receptor systems have been implicated in anxiety disorders and there is evidence that the two systems interact with each other to affect behavior. Both systems have individually been shown to affect prepulse inhibition (PPI) of the acoustic startle response. PPI is a form of sensorimotor gating that is reduced in patients with anxiety disorders including post-traumatic stress and panic disorder. Here, we examined whether the two systems interact or counteract each other to affect acoustic startle amplitude, PPI and habituation of the startle response. In experiment 1, Brown Norway (BN) and Wistar-Kyoto (WKY) rats were administered ether an intraperitoneal (IP) injection of saline or the 5-HT1A receptor agonist, 8-OH-DPAT 10 min prior to receiving an intracerebroventricular (ICV) infusion of either saline or CRF (0.3 µg). In a second experiment, rats were administered either an IP injection of saline or the 5-HT1A receptor antagonist, WAY 100,635 10 min prior to receiving an ICV infusion of saline or CRF. Thirty min after the ICV infusion, the startle response and PPI were assessed. As we have previously shown, the dose of CRF used in these experiments reduced PPI in BN rats and had no effect on PPI in WKY rats. Administration of 8-OH-DPAT alone had no effect on PPI in either rat strain when the data from the two strains were examined separately. Administration of 8-OHDPAT added to the effect of CRF in BN rats, and the combination of 8-OH-DPAT and CRF significantly reduced PPI in WKY rats. CRF alone had no effect on baseline startle amplitude in either rat strain, but CRF enhanced the 8-OH-DPAT-induced increase in startle in both strains. Administration of WAY 100,635 did not affect the CRF-induced change in PPI and there were no interactions between CRF and WAY 100,635 on baseline startle. The results suggest that activation of the 5-HT1A receptor can potentiate the effect of

  2. Interactions between corticotropin-releasing factor and the serotonin 1A receptor system on acoustic startle amplitude and prepulse inhibition of the startle response in two rat strains.

    PubMed

    Conti, Lisa H

    2012-01-01

    Both the neuropeptide, corticotropin-releasing factor (CRF) and the serotonin 1A (5-HT(1A)) receptor systems have been implicated in anxiety disorders and there is evidence that the two systems interact with each other to affect behavior. Both systems have individually been shown to affect prepulse inhibition (PPI) of the acoustic startle response. PPI is a form of sensorimotor gating that is reduced in patients with anxiety disorders including post-traumatic stress and panic disorder. Here, we examined whether the two systems interact or counteract each other to affect acoustic startle amplitude, PPI and habituation of the startle response. In experiment 1, Brown Norway (BN) and Wistar-Kyoto (WKY) rats were administered ether an intraperitoneal (IP) injection of saline or the 5-HT(1A) receptor agonist, 8-OH-DPAT 10 min prior to receiving an intracerebroventricular (ICV) infusion of either saline or CRF (0.3 μg). In a second experiment, rats were administered either an IP injection of saline or the 5-HT(1A) receptor antagonist, WAY 100,635 10 min prior to receiving an ICV infusion of saline or CRF. Thirty min after the ICV infusion, the startle response and PPI were assessed. As we have previously shown, the dose of CRF used in these experiments reduced PPI in BN rats and had no effect on PPI in WKY rats. Administration of 8-OH-DPAT alone had no effect on PPI in either rat strain when the data from the two strains were examined separately. Administration of 8-OH-DPAT added to the effect of CRF in BN rats, and the combination of 8-OH-DPAT and CRF significantly reduced PPI in WKY rats. CRF alone had no effect on baseline startle amplitude in either rat strain, but CRF enhanced the 8-OH-DPAT-induced increase in startle in both strains. Administration of WAY 100,635 did not affect the CRF-induced change in PPI and there were no interactions between CRF and WAY 100,635 on baseline startle. The results suggest that activation of the 5-HT(1A) receptor can potentiate the

  3. A novel mechanism for regulating transforming growth factor beta (TGF-beta) signaling. Functional modulation of type III TGF-beta receptor expression through interaction with the PDZ domain protein, GIPC.

    PubMed

    Blobe, G C; Liu, X; Fang, S J; How, T; Lodish, H F

    2001-10-26

    Transforming growth factor beta (TGF-beta) mediates its biological effects through three high-affinity cell surface receptors, the TGF-beta type I, type II, and type III receptors, and the Smad family of transcription factors. Although the functions of the type II and type I receptors are well established, the precise role of the type III receptor in TGF-beta signaling remains to be established. While expression cloning signaling molecules downstream of TGF-beta, we cloned GIPC (GAIP-interacting protein, C terminus), a PDZ domain-containing protein. GIPC binds a Class I PDZ binding motif in the cytoplasmic domain of the type III receptor resulting in regulation of expression of the type III receptor at the cell surface. Increased expression of the type III receptor mediated by GIPC enhanced cellular responsiveness to TGF-beta both in terms of inhibition of proliferation and in plasminogen-activating inhibitor (PAI)-based promoter gene induction assays. In all cases, deletion of the Class I PDZ binding motif of the type III receptor prevented the type III receptor from binding to GIPC and abrogated the effects of GIPC on type III receptor expressing cells. These results establish, for the first time, a protein that interacts with the cytoplasmic domain of the type III receptor, determine that expression of the type III receptor is regulated at the protein level and that increased expression of the type III receptor is sufficient to enhance TGF-beta signaling. These results further support an essential, non-redundant role for the type III receptor in TGF-beta signaling.

  4. Differential ligand-dependent interactions between the AF-2 activating domain of nuclear receptors and the putative transcriptional intermediary factors mSUG1 and TIF1.

    PubMed Central

    vom Baur, E; Zechel, C; Heery, D; Heine, M J; Garnier, J M; Vivat, V; Le Douarin, B; Gronemeyer, H; Chambon, P; Losson, R

    1996-01-01

    Using a yeast two-hybrid system we report the isolation of a novel mouse protein, mSUG1, that interacts with retinoic acid receptor alpha (RAR alpha) both in yeast cells and in vitro in a ligand- and AF-2 activating domain (AF-2 AD)-dependent manner and show that it is a structural and functional homologue of the essential yeast protein SUG1. mSUG1 also efficiently interacts with other nuclear receptors, including oestrogen (ER), thyroid hormone (TR), Vitamin D3 (VDR) and retinoid X (RXR) receptors. By comparing the interaction properties of these receptors with mSUG1 and TIF1, we demonstrate that: (i) RXR alpha efficiently interacts with TIF1, but not with mSUG1, whereas TR alpha interacts much more efficiently with mSUG1 than with TIF1, and RAR alpha, VDR and ER efficiently interact with mSUG1 and TIF1; (ii) the amphipathic alpha-helix core of the AF-2 AD is differentially involved in interactions of RAR alpha with mSUG1 and TIF1; (iii) the AF-2 AD cores of RAR alpha and ER are similarly involved in their interaction with TIF1, but not with mSUG1. Thus, the interaction interfaces between the different receptors and either mSUG1 or TIF1 may vary depending on the nature of the receptor and the putative mediator of its AF-2 function. We discuss the possibility that mSUG1 and TIF1 may mediate the transcriptional activity of the AF-2 of nuclear receptors through different mechanisms. Images PMID:8598193

  5. An unusual cytokine:Ig-domain interaction revealed in the crystal structure of leukemia inhibitory factor (LIF) in complex with the LIF receptor

    PubMed Central

    Huyton, Trevor; Zhang, Jian-Guo; Luo, Cindy S.; Lou, Mei-Zhen; Hilton, Douglas J.; Nicola, Nicos A.; Garrett, Thomas P. J.

    2007-01-01

    Leukemia inhibitory factor (LIF) receptor is a cell surface receptor that mediates the actions of LIF and other IL-6 type cytokines through the formation of high-affinity signaling complexes with gp130. Here we present the crystal structure of a complex of mouse LIF receptor with human LIF at 4.0 Å resolution. The structure is, to date, the largest cytokine receptor fragment determined by x-ray crystallography. The binding of LIF to its receptor via the central Ig-like domain is unlike other cytokine receptor complexes that bind ligand predominantly through their cytokine-binding modules. This structure, in combination with previous crystallographic studies, also provides a structural template to understand the formation and orientation of the high-affinity signaling complex between LIF, LIF receptor, and gp130. PMID:17652170

  6. Inhibition of the focal adhesion kinase and vascular endothelial growth factor receptor-3 interaction leads to decreased survival in human neuroblastoma cell lines.

    PubMed

    Beierle, Elizabeth A; Ma, Xiaojie; Stewart, Jerry E; Megison, Michael; Cance, William G; Kurenova, Elena V

    2014-03-01

    Neuroblastoma continues to be a devastating childhood solid tumor and is responsible for over 15% of all childhood cancer-related deaths. Focal adhesion kinase (FAK) and vascular endothelial growth factor receptor-3 (VEGFR-3) are protein tyrosine kinases that are overexpressed in a number of human cancers, including neuroblastoma. These two kinases can directly interact and provide survival signals to cancer cells. In this study, we utilized siRNA to VEGFR-3 to demonstrate the biologic importance of this kinase in neuroblastoma cell survival. We also used confocal microscopy and immunoprecipitation to show that FAK and VEGFR-3 bind in neuroblastoma. Finally, employing a 12-amino-acid peptide (AV3) specific to VEGFR-3, we showed that the colocalization between FAK and VEGFR-3 could be disrupted, and that disruption resulted in decreased neuroblastoma cell survival. These studies provide insight to the FAK-VEGFR-3 interaction in neuroblastoma and demonstrate its importance in this tumor type. Focusing upon the FAK-VEGFR-3 interaction may provide a novel therapeutic target for the development of new strategies for treatment of neuroblastoma.

  7. The Epstein-Barr Virus Oncoprotein Latent Membrane Protein 1 Engages the Tumor Necrosis Factor Receptor-Associated Proteins TRADD and Receptor-Interacting Protein (RIP) but Does Not Induce Apoptosis or Require RIP for NF-κB Activation

    PubMed Central

    Izumi, Kenneth M.; McFarland, Ellen Cahir; Ting, Adrian T.; Riley, Elisabeth A.; Seed, Brian; Kieff, Elliott D.

    1999-01-01

    A site in the Epstein-Barr virus (EBV) transforming protein LMP1 that constitutively associates with the tumor necrosis factor receptor 1 (TNFR1)-associated death domain protein TRADD to mediate NF-κB and c-Jun N-terminal kinase activation is critical for long-term lymphoblastoid cell proliferation. We now find that LMP1 signaling through TRADD differs from TNFR1 signaling through TRADD. LMP1 needs only 11 amino acids to activate NF-κB or synergize with TRADD in NF-κB activation, while TNFR1 requires ∼70 residues. Further, LMP1 does not require TRADD residues 294 to 312 for NF-κB activation, while TNFR1 requires TRADD residues 296 to 302. LMP1 is partially blocked for NF-κB activation by a TRADD mutant consisting of residues 122 to 293. Unlike TNFR1, LMP1 can interact directly with receptor-interacting protein (RIP) and stably associates with RIP in EBV-transformed lymphoblastoid cell lines. Surprisingly, LMP1 does not require RIP for NF-κB activation. Despite constitutive association with TRADD or RIP, LMP1 does not induce apoptosis in EBV-negative Burkitt lymphoma or human embryonic kidney 293 cells. These results add a different perspective to the molecular interactions through which LMP1, TRADD, and RIP participate in B-lymphocyte activation and growth. PMID:10409763

  8. Negative Regulation of Grb10 Interacting GYF Protein 2 on Insulin-Like Growth Factor-1 Receptor Signaling Pathway Caused Diabetic Mice Cognitive Impairment

    PubMed Central

    Xie, Jing; Wei, Qianping; Deng, Huacong; Li, Gang; Ma, Lingli; Zeng, Hui

    2014-01-01

    Heterozygous Gigyf2+/− mice exhibits histopathological evidence of neurodegeneration such as motor dysfunction. Several lines of evidence have demonstrated the important role of insulin-like growth factor-1 receptor (IGF1R) signaling pathway in the neuropathogenic process of cognitive impairment, while decreased Grb10-Interacting GYF Protein 2 (GIGYF2) expression can alter IGF1R trafficking and its downstream signaling pathways. Growth factor receptor-bound protein 10 (Grb10), a suppressor of IGF1R pathway, has been shown to play a critical role in regulating diabetes-associated cognitive impairment. It remains unknown whether endogenous GIGYF2 expression contributes to the development of diabetes-associated cognitive impairment. Using streptozotocin (STZ)-induced diabetic mice model, we first demonstrated that a significantly increased level of GIGYF2 expression was correlated with a significant decrease in the expression of phosphorylated IGF1R as well as the phosphorylation of AKT and ERK1/2, two signaling pathways downstream of IGF1R, in the hippocampus of diabetic mice. On the contrary, in situ knockdown of GIGYF2 expression in hippocampus resulted in increased expression of phosphorylated IGF1R expression and correspondingly reversed the down-regulation of ERK1/2 phsophorylation but had no obvious effect on Grb10 expression. Functionally, knockdown of GIGYF2 expression markedly ameliorated diabetes-associated cognitive dysfunction as well as the ultrastructural pathology and abnormal neurobehavioral changes. These results suggest that increased expression of GIGYF2 might contribute to the development of diabetes-associated cognitive disorder via negatively regulating IGF1R signaling pathway. Therefore, down-regulation of GIGYF2 expression may provide a potential novel approach to treat diabetes-associated cognitive impairment caused by aberrant IGF1R signaling pathway. PMID:25268761

  9. Control of cell motility by interaction of gangliosides, tetraspanins, and epidermal growth factor receptor in A431 versus KB epidermoid tumor cells.

    PubMed

    Park, Seung-Yeol; Yoon, Seon-Joo; Freire-de-Lima, Leonardo; Kim, Jung-Hoe; Hakomori, Sen-itiroh

    2009-08-17

    Growth of epidermoid carcinoma cell lines, A431 and KB, has been known to be controlled by the interaction of epidermal growth factor (EGF) and its receptor (EGFR) with tyrosine kinase. Ganglioside GM3 was previously found to interact with EGFR and to inhibit EGFR tyrosine kinase. However, motility of these cells, controlled by EGFR and ganglioside, was not studied. The present study is focused on the control mechanism of the motility of these cells through interaction of ganglioside, tetraspanin (TSP), and EGFR. Key results are as follows: (i) The level of EGFR expressed in A431 cells is approximately 6 times higher than that expressed in KB cells, and motility of A431 cells is also much higher than that of KB cells, yet growth of A431 cells is either not affected or is inhibited by EGF. In contrast, growth of KB cells is enhanced by EGF. (ii) Levels of TSPs (CD9, CD82, and CD81) expressed in A431 cells are much higher than those expressed in KB cells, and TSPs expressed in A431 cells are reduced by treatment of cells with EtDO-P4, which inhibits the synthesis of glycosphingolipids (GSLs) and gangliosides. (iii) These TSPs are co-immunoprecipitated with EGFR in both A431 and KB cells, indicating that TSPs are closely associated with EGFR. (iv) High motility of A431 cells is greatly reduced, while low motility of KB cells is not affected, by treatment of cells with EtDO-P4. These results, taken together, suggest that there is a close correlation between high motility of A431 cells and high expression of EGFR and TSPs, and between ganglioside GM3/GM2 and TSP. A similar correlation was suggested between the low motility of KB cells and low levels of EGFR and TSP. The correlation between high motility and high level of EGFR with the ganglioside-TSP complex in A431 cells is unique. This is in contrast to our previous studies that indicate that motility of many types of tumor cells is inhibited by a high level of CD9 or CD82, together with growth factor receptors and

  10. Functional characterization of an alpha-factor-like Sordaria macrospora peptide pheromone and analysis of its interaction with its cognate receptor in Saccharomyces cerevisiae.

    PubMed

    Mayrhofer, Severine; Pöggeler, Stefanie

    2005-04-01

    The homothallic filamentous ascomycete Sordaria macrospora possesses genes which are thought to encode two pheromone precursors and two seven-transmembrane pheromone receptors. The pheromone precursor genes are termed ppg1 and ppg2. The putative products derived from the gene sequence show structural similarity to the alpha-factor precursors and a-factor precursors of the yeast Saccharomyces cerevisiae. Likewise, sequence similarity has been found between the putative products of the pheromone receptor genes pre2 and pre1 and the S. cerevisiae Ste2p alpha-factor receptor and Ste3p a-factor receptor, respectively. To investigate whether the alpha-factor-like pheromone-receptor pair of S. macrospora is functional, a heterologous yeast assay was used. Our results show that the S. macrospora alpha-factor-like pheromone precursor PPG1 is processed into an active pheromone by yeast MATalpha cells. The S. macrospora PRE2 protein was demonstrated to be a peptide pheromone receptor. In yeast MATa cells lacking the endogenous Ste2p receptor, the S. macrospora PRE2 receptor facilitated all aspects of the pheromone response. Using a synthetic peptide, we can now predict the sequence of one active form of the S. macrospora peptide pheromone. We proved that S. macrospora wild-type strains secrete an active pheromone into the culture medium and that disruption of the ppg1 gene in S. macrospora prevents pheromone production. However, loss of the ppg1 gene does not affect vegetative growth or fertility. Finally, we established the yeast assay as an easy and useful system for analyzing pheromone production in developmental mutants of S. macrospora.

  11. M10, a Caspase Cleavage Product of the Hepatocyte Growth Factor Receptor, Interacts with Smad2 and Demonstrates Anti-Fibrotic Properties in Vitro and in Vivo

    PubMed Central

    Atanelishvili, Ilia; Shirai, Yuichiro; Akter, Tanjina; Buckner, Taylor; Noguchi, Atsushi; Silver, Richard M.; Bogatkevich, Galina S.

    2016-01-01

    Hepatocyte growth factor receptor, also known as cellular mesenchymal-epithelial transition factor (c-MET, MET), is an important antifibrotic molecule that protects various tissues, including lung, from injury and fibrosis. The intracellular cytoplasmic tail of MET contains a caspase-3 recognition motif “DEVD-T” that upon cleavage by caspase-3 generates a 10 amino acid peptide, TRPASFWETS, designated as “M10”. M10 contains at its N-terminus the uncharged amino acid proline (P) directly after a cationic amino acid arginine (R) which favors the transport of the peptide through membranes. M10, when added to cell culture medium, remains in the cytoplasm and nuclei of cells for up to 24 hours. M10 effectively decreases collagen in both scleroderma and TGFβ-stimulated normal lung and skin fibroblasts. M10 interacts with the MH2 domain of Smad2 and inhibits TGFβ-induced Smad2 phosphorylation, suggesting that the antifibrotic effects of M10 are mediated in part by counteracting Smad-dependent fibrogenic pathways. In the bleomycin murine model of pulmonary fibrosis, M10 noticeably reduced lung inflammation and fibrosis. Ashcroft fibrosis scores and lung collagen content were significantly lower in bleomycin-treated mice receiving M10 as compared with bleomycin-treated mice receiving scrambled peptide. We conclude that M10 peptide interacts with Smad2 and demonstrates strong antifibrotic effects in vitro and in vivo in an animal model of lung fibrosis and should be considered as a potential therapeutic agent for systemic sclerosis and other fibrosing diseases. PMID:26772959

  12. M10, a caspase cleavage product of the hepatocyte growth factor receptor, interacts with Smad2 and demonstrates antifibrotic properties in vitro and in vivo.

    PubMed

    Atanelishvili, Ilia; Shirai, Yuichiro; Akter, Tanjina; Buckner, Taylor; Noguchi, Atsushi; Silver, Richard M; Bogatkevich, Galina S

    2016-04-01

    Hepatocyte growth factor receptor, also known as cellular mesenchymal-epithelial transition factor (c-MET, MET), is an important antifibrotic molecule that protects various tissues, including lung, from injury and fibrosis. The intracellular cytoplasmic tail of MET contains a caspase-3 recognition motif "DEVD-T" that on cleavage by caspase-3 generates a 10-amino acid peptide, TRPASFWETS, designated as "M10". M10 contains at its N-terminus the uncharged amino acid proline (P) directly after a cationic amino acid arginine (R) which favors the transport of the peptide through membranes. M10, when added to cell culture medium, remains in the cytoplasm and nuclei of cells for up to 24 hours. M10 effectively decreases collagen in both scleroderma and TGFβ-stimulated normal lung and skin fibroblasts. M10 interacts with the Mad Homology 2 domain of Smad2 and inhibits TGFβ-induced Smad2 phosphorylation, suggesting that the antifibrotic effects of M10 are mediated in part by counteracting Smad-dependent fibrogenic pathways. In the bleomycin murine model of pulmonary fibrosis, M10 noticeably reduced lung inflammation and fibrosis. Ashcroft fibrosis scores and lung collagen content were significantly lower in bleomycin-treated mice receiving M10 as compared with bleomycin-treated mice receiving scrambled peptide. We conclude that M10 peptide interacts with Smad2 and demonstrates strong antifibrotic effects in vitro and in vivo in an animal model of lung fibrosis and should be considered as a potential therapeutic agent for systemic sclerosis and other fibrosing diseases.

  13. Combinatorial and Computational Approaches to Identify Interactions of Macrophage Colony-stimulating Factor (M-CSF) and Its Receptor c-FMS*

    PubMed Central

    Rosenfeld, Lior; Shirian, Jason; Zur, Yuval; Levaot, Noam; Shifman, Julia M.; Papo, Niv

    2015-01-01

    The molecular interactions between macrophage colony-stimulating factor (M-CSF) and the tyrosine kinase receptor c-FMS play a key role in the immune response, bone metabolism, and the development of some cancers. Because no x-ray structure is available for the human M-CSF·c-FMS complex, the binding epitope for this complex is largely unknown. Our goal was to identify the residues that are essential for binding of the human M-CSF to c-FMS. For this purpose, we used a yeast surface display (YSD) approach. We expressed a combinatorial library of monomeric M-CSF (M-CSFM) single mutants and screened this library to isolate variants with reduced affinity for c-FMS using FACS. Sequencing yielded a number of single M-CSFM variants with mutations both in the direct binding interface and distant from the binding site. In addition, we used computational modeling to map the identified mutations onto the M-CSFM structure and to classify the mutations into three groups as follows: those that significantly decrease protein stability; those that destroy favorable intermolecular interactions; and those that decrease affinity through allosteric effects. To validate the YSD and computational data, M-CSFM and three variants were produced as soluble proteins; their affinity and structure were analyzed; and very good correlations with both YSD data and computational predictions were obtained. By identifying the M-CSFM residues critical for M-CSF·c-FMS interactions, we have laid down the basis for a deeper understanding of the M-CSF·c-FMS signaling mechanism and for the development of target-specific therapeutic agents with the ability to sterically occlude the M-CSF·c-FMS binding interface. PMID:26359491

  14. Direct interaction between surface β1,4-galactosyltransferase 1 and epidermal growth factor receptor (EGFR) inhibits EGFR activation in hepatocellular carcinoma

    SciTech Connect

    Tang, Wenqing; Weng, Shuqiang; Zhang, Si; Wu, Weibing; Dong, Ling; Shen, Xizhong; Zhang, Songwen; Gu, Jianxin; Xue, Ruyi

    2013-05-10

    Highlights: •β1,4GT1 interacts with EGFR both in vitro and in vivo. •β1,4GT1 co-localizes with EGFR on the cell surface. •β1,4GT1 inhibits {sup 125}I-EGF binding to EGFR. •β1,4GT1 inhibits EGF induced EGFR dimerization and phosphorylation. -- Abstract: Our previous studies showed that cell surface β1,4-galactosyltransferase 1 (β1,4GT1) negatively regulated cell survival through inhibition and modulation of the epidermal growth factor receptor (EGFR) signaling pathway in human hepatocellular carcinoma (HCC) SMMC-7721 cells. However, the underlying mechanism remains unclear. Here we demonstrated that β1,4-galactosyltransferase 1 (β1,4GT1) interacted with EGFR in vitro by GST pull-down analysis. Furthermore, we demonstrated that β1,4GT1 bound to EGFR in vivo by co-immunoprecipitation and determined the co-localization of β1,4GT1 and EGFR on the cell surface via confocal laser scanning microscopy analysis. Finally, using {sup 125}I-EGF binding experiments and Western blot analysis, we found that overexpression of β1,4GT1 inhibited {sup 125}I-EGF binding to EGFR, and consequently reduced the levels of EGFR dimerization and phosphorylation. In contrast, RNAi-mediated knockdown of β1,4GT1 increased the levels of EGFR dimerization and phosphorylation. These data suggest that cell surface β1,4GT1 interacts with EGFR and inhibits EGFR activation.

  15. Development of Nano-Liposomal Formulations of Epidermal Growth Factor Receptor Inhibitors and their Pharmacological Interactions on Drug-Sensitive and Drug-Resistant Cancer Cell Lines

    NASA Astrophysics Data System (ADS)

    Trummer, Brian J.

    A rapidly expanding understanding of molecular derangements in cancer cell function has led to the development of selective, targeted chemotherapeutic agents. Growth factor signal transduction networks are frequently activated in an aberrant fashion, particularly through the activity of receptor tyrosine kinases (RTK). This has spurred an intensive effort to develop receptor tyrosine kinase inhibitors (RTKI) that are targeted to specific receptors, or receptor subfamilies. Chapter 1 reviews the pharmacology, preclinical, and clinical aspects of RTKIs that target the epidermal growth factor receptor (EGFR). EGFR inhibitors demonstrate significant success at inhibiting phosphorylation-based signaling pathways that promote cancer cell proliferation. Additionally RTKIs have physicochemical and structural characteristics that enable them to function as inhibitors of multi-drug resistance transport proteins. Thus EGFR inhibitors and other RTKIs have both on-target and off-target activities that could be beneficial in cancer therapy. However, these agents exert a number of side effects, some of which arise from their hydrophobic nature and large in vivo volume of distribution. Side effects of the EGFR inhibitor gefitinib include skin rash, severe myelotoxicity when combined with certain chemotherapeutic agents, and impairment of the blood brain barrier to xenobiotics. Weighing the preclinical and clinical observations with the EGFR inhibitors, we developed the primary overall hypothesis of this research: that drug-carrier formulations of RTKIs such as the EGFR inhibitors could be developed based on nanoparticulate liposomal carriers. Theoretically, this carrier strategy would ameliorate toxicity and improve the biodistribution and tumor selectivity of these agents. We hypothesized specifically that liposomal formulations could shift the biodistribution of EGFR inhibitors such as gefitinib away from skin, bone marrow, and the blood brain barrier, and toward solid tumors

  16. An evolutionary conserved interaction between the Gcm transcription factor and the SF1 nuclear receptor in the female reproductive system

    PubMed Central

    Cattenoz, Pierre B.; Delaporte, Claude; Bazzi, Wael; Giangrande, Angela

    2016-01-01

    NR5A1 is essential for the development and for the function of steroid producing glands of the reproductive system. Moreover, its misregulation is associated with endometriosis, which is the first cause of infertility in women. Hr39, the Drosophila ortholog of NR5A1, is expressed and required in the secretory cells of the spermatheca, the female exocrine gland that ensures fertility by secreting substances that attract and capacitate the spermatozoids. We here identify a direct regulator of Hr39 in the spermatheca: the Gcm transcription factor. Furthermore, lack of Gcm prevents the production of the secretory cells and leads to female sterility in Drosophila. Hr39 regulation by Gcm seems conserved in mammals and involves the modification of the DNA methylation profile of mNr5a1. This study identifies a new molecular pathway in female reproductive system development and suggests a role for hGCM in the progression of reproductive tract diseases in humans. PMID:27886257

  17. Muscarinic receptor family interacting proteins: role in receptor function.

    PubMed

    Borroto-Escuela, Dasiel O; Correia, Patrícia A; Romero-Fernandez, Wilber; Narvaez, Manuel; Fuxe, Kjell; Ciruela, Francisco; Garriga, Pere

    2011-02-15

    G protein-coupled receptors constitute one of the most important families of membrane receptors through which cells respond to extracellular stimuli. Receptors of this superfamily likely function as signal transduction complexes. The identification and analysis of their components provide new insights into a better understanding of these receptors' function and regulation. We used tandem-affinity purification and mass spectrometry as a systematic approach to characterize multiprotein complexes in the acetylcholine muscarinic receptor subfamily. To overcome the limitations associated with membrane protein receptor solubilization with detergents, we developed a strategy in which receptors are co-expressed with a cytoplasmic minigene construct, encoding the third intracellular loop and the C-terminal tail tagged to the tandem-affinity-cassette of each receptor subtype. Numerous protein complexes were identified, including many new interactions in various signalling pathways. Systematic identification data set together with protein interactions reported in the literature revealed a high degree of connectivity. These allow the proposal, for the first time, of an outline of the muscarinic interactome as a network of protein complexes and a context for a more reasoned and informed approach to drug discovery and muscarinic receptor subtype specificities.

  18. A novel signal transduction cascade involving direct physical interaction of the renin/prorenin receptor with the transcription factor promyelocytic zinc finger protein.

    PubMed

    Schefe, Jan H; Menk, Mario; Reinemund, Jana; Effertz, Karin; Hobbs, Robin M; Pandolfi, Pier Paolo; Ruiz, Patricia; Unger, Thomas; Funke-Kaiser, Heiko

    2006-12-08

    A human renin/prorenin receptor (RER) has recently been cloned. To gain insight into the molecular function of the RER, we studied its signal transduction mechanisms. Initially, we found a ubiquitous and intracellular expression pattern of the human RER. Consistently, we observed several transcriptional start sites and a high promoter activity of the human RER. We could identify the transcription factor promyelocytic zinc finger (PLZF) protein as a direct protein interaction partner of the C-terminal domain of the RER by yeast 2-hybrid screening and coimmunoprecipitation. Coimmunoprecipitation experiments also indicated homodimerization of the RER. On activation of the RER by renin, PLZF is translocated into the nucleus and represses transcription of the RER itself, thereby creating a very short negative feedback loop, but activates transcription of the p85alpha subunit of the phosphatidylinositol-3 kinase (PI3K-p85alpha). Small interfering RNA against the RER abolished these effects. A PLZF cis-element in the RER promoter was identified by site-directed mutagenesis and electrophoretic mobility-shift assay. Renin stimulation caused a 6-fold recruitment of PLZF to this promoter region as shown by chromatin immunoprecipitation. Moreover, renin stimulation of rat H9c2 cardiomyoblasts induced an increase of cell number and a decrease of apoptosis. These effects were partly abolished by PI3K inhibition and completely abrogated by small interfering RNA against PLZF. Finally, experiments in PLZF knockout mice confirmed the role of PLZF as an upstream regulator of RER and PI3K-p85alpha. Our data demonstrate the existence of a novel signal transduction pathway involving the ligand renin, RER, and the transcription factor PLZF, which is of physiological and putative pathophysiological relevance.

  19. Opioid receptor trafficking and interaction in nociceptors

    PubMed Central

    Zhang, X; Bao, L; Li, S

    2015-01-01

    Opiate analgesics such as morphine are often used for pain therapy. However, antinociceptive tolerance and dependence may develop with long-term use of these drugs. It was found that μ-opioid receptors can interact with δ-opioid receptors, and morphine antinociceptive tolerance can be reduced by blocking δ-opioid receptors. Recent studies have shown that μ- and δ-opioid receptors are co-expressed in a considerable number of small neurons in the dorsal root ganglion. The interaction of μ-opioid receptors with δ-opioid receptors in the nociceptive afferents is facilitated by the stimulus-induced cell-surface expression of δ-opioid receptors, and contributes to morphine tolerance. Further analysis of the molecular, cellular and neural circuit mechanisms that regulate the trafficking and interaction of opioid receptors and related signalling molecules in the pain pathway would help to elucidate the mechanism of opiate analgesia and improve pain therapy. LINKED ARTICLES This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2 PMID:24611685

  20. Receptor dependent multidimensional QSAR for modeling drug--receptor interactions.

    PubMed

    Polanski, Jaroslaw

    2009-01-01

    Quantitative Structure Activity Relationship (QSAR) is an approach of mapping chemical structure to properties. A significant development can be observed in the last two decades in this method which originated from the Hansch analysis based on the logP data and Hammett constant towards a growing importance of the molecular descriptors derived from 3D structure including conformational dynamics and solvation scenarios. However, molecular interactions in biological systems are complex phenomena generating extremely noisy data, if simulated in silico. This decides that activity modeling and predictions are a risky business. Molecular recognition uncertainty in traditional receptor independent (RI) m-QSAR cannot be eliminated but by the inclusion of the receptor data. Modeling ligand-receptor interactions is a complex computational problem. This has limited the development of the receptor dependent (RD) m-QSAR. However, a steady increase of computational power has also improved modeling ability in chemoinformatics and novel RD QSAR methods appeared. Following the RI m-QSAR terminology this is usually classified as RD 3/6D-QSAR. However, a clear systematic m-QSAR classification can be proposed, where dimension m refers to, the static ligand representation (3D), multiple ligand representation (4D), ligand-based virtual or pseudo receptor models (5D), multiple solvation scenarios (6D) and real receptor or target-based receptor model data (7D).

  1. Endomorphins interact with tachykinin receptors.

    PubMed

    Kosson, Piotr; Bonney, Iwona; Carr, Daniel B; Lipkowski, Andrzej W

    2005-09-01

    Soon after the discovery of endomorphins several studies indicated differences between pharmacological effects of endomorphins and other MOR selective ligands, as well as differences between the effects of endomorphin I and endomorphin II. We now propose that these differences are the result of an additional non-opioid property of endomorphins, namely, their weak antagonist properties with respect to tachykinin NK1 and NK1 receptors.

  2. Plant polyphenols regulate chemokine expression and tissue repair in human keratinocytes through interaction with cytoplasmic and nuclear components of epidermal growth factor receptor system.

    PubMed

    Pastore, Saveria; Lulli, Daniela; Fidanza, Paolo; Potapovich, Alla I; Kostyuk, Vladimir A; De Luca, Chiara; Mikhal'chik, Elena; Korkina, Liudmila G

    2012-02-15

    To evaluate mechanisms underlying modulation of inflammatory chemokines in primary human keratinocytes (normal human epidermal keratinocytes) and repair-related processes in wound models by plant polyphenols (PPs) with antioxidant and superoxide scavenging properties (verbascoside [Vb], resveratrol [Rv], polydatin [Pd], quercetin [Qr], and rutin). Epidermal growth factor receptor (EGFR)-controlled chemokines CXCL8/interleukin 8 (IL-8), CCL2/monocyte chemotactic protein-1 (MCP-1), and CXCL10/interferon gamma-produced protein of 10 kDa (IP-10) were modulated by transforming growth factor alpha (TGF-α) and by the tumor necrosis factor alpha/interferon gamma combination (T/I). EGFR phosphorylation, nuclear translocation, and downstream cytoplasmic signaling pathways (extracellular regulation kinase [ERK]1/2, p38, STAT3, and PI-3K) were studied. All PPs did not affect TGF-α-induced STAT3 phosphorylation, whereas they suppressed T/I-activated NFkappaB and constitutive and T/I-induced but not TGF-α-induced ERK1/2 phosphorylation. Vb and Qr suppressed total EGFR phosphorylation, but they synergized with TGF-α to enhance nuclear accumulation of phosphorylated EGFR. Vb strongly inhibited TGF-α-induced p38 phosphorylation and T/I-induced NFkappaB and activator protein-1 (AP-1) binding to DNA. Vb was an effective inhibitor of T/I-stimulated chemokine synthesis, and it accelerated scratch wound healing in vitro. Anti-inflammatory and wound healing activities of Vb were confirmed in vivo in the full-thickness excision wound. Although Pd and Rv did not affect EGFR activation/translocation, they and Qr synergized with TGF-α and T/I in the induction of IL-8 transcription/synthesis while opposing enhanced MCP-1 and IP-10 transcription/synthesis connected with pharmacologically impaired EGFR functioning. PPs perturb the EGFR system in human keratinocytes, and this effect may be implicated in the regulation of inflammatory and repair-related processes in the skin. Anti

  3. Vitamin D3 inhibits lipopolysaccharide-induced placental inflammation through reinforcing interaction between vitamin D receptor and nuclear factor kappa B p65 subunit.

    PubMed

    Chen, Yuan-Hua; Yu, Zhen; Fu, Lin; Wang, Hua; Chen, Xue; Zhang, Cheng; Lv, Zheng-Mei; Xu, De-Xiang

    2015-06-12

    It is increasingly recognized that vitamin D3 (VitD3) has an anti-inflammatory activity. The present study investigated the effects of maternal VitD3 supplementation during pregnancy on LPS-induced placental inflammation and fetal intrauterine growth restriction (IUGR). All pregnant mice except controls were intraperitoneally injected with LPS (100 μg/kg) daily from gestational day (GD)15-17. In VitD3 + LPS group, pregnant mice were orally administered with VitD3 (25 μg/kg) before LPS injection. As expected, maternal LPS exposure caused placental inflammation and fetal IUGR. Interestingly, pretreatment with VitD3 repressed placental inflammation and protected against LPS-induced fetal IUGR. Further analysis showed that pretreatment with VitD3, which activated placental vitamin D receptor (VDR) signaling, specifically suppressed LPS-induced activation of nuclear factor kappa B (NF-κB) and significantly blocked nuclear translocation of NF-κB p65 subunit in trophoblast gaint cells of the labyrinth layer. Conversely, LPS, which activated placental NF-κB signaling, suppressed placental VDR activation and its target gene expression. Moreover, VitD3 reinforced physical interaction between placental VDR and NF-κB p65 subunit. The further study demonstrates that VitD3 inhibits placental NF-κB signaling in VDR-dependent manner. These results provide a mechanistic explanation for VitD3-mediated anti-inflammatory activity. Overall, the present study provides evidence for roles of VDR as a key regulator of placental inflammation.

  4. Cell-type specific interaction of Neu differentiation factor (NDF/heregulin) with Neu/HER-2 suggests complex ligand-receptor relationships.

    PubMed Central

    Peles, E; Ben-Levy, R; Tzahar, E; Liu, N; Wen, D; Yarden, Y

    1993-01-01

    The Neu/HER-2 receptor tyrosine kinase is overexpressed in some types of human adenocarcinomas, including tumors of the breast and the ovary. A 44 kDa glycoprotein that elevates tyrosine phosphorylation of Neu has been isolated and named Neu differentiation factor (NDF), or heregulin. Here we show that NDF affects tyrosine phosphorylation of Neu in human tumor cells of breast, colon and neuronal origin, but not in ovarian cells that overexpress the receptor. By using monoclonal antibodies (mAbs) to Neu, we found that the ovarian receptor is immunologically and biochemically similar to the mammary p185neu. Nevertheless, unlike breast-derived Neu, the ovarian protein did not display covalent cross-linking to radiolabeled NDF, and was devoid of ligand-induced association with phosphatidylinositol 3'-kinase. Direct binding analysis showed that NDF binds with high affinity (Kd approximately 10(-9) M) to mammary cells, but its weak association with ovarian cells is probably mediated by heparin-like molecules. Similar to the endogenous receptor, the ectopically overexpressed Neu of mammary cells, but not of ovarian and fibroblastic cells, exhibited elevated levels of NDF-induced phosphorylation and covalent cross-linking of the radiolabeled factor. Taken together, our results imply that NDF binding to cells requires both Neu and an additional cellular component, whose identity is still unknown, but its tissue distribution is more restricted than the expression of the neu gene. Images PMID:8096177

  5. Nck-2, a Novel Src Homology2/3-containing Adaptor Protein That Interacts with the LIM-only Protein PINCH and Components of Growth Factor Receptor Kinase-signaling Pathways

    PubMed Central

    Tu, Yizeng; Li, Fugang; Wu, Chuanyue

    1998-01-01

    Many of the protein–protein interactions that are essential for eukaryotic intracellular signal transduction are mediated by protein binding modules including SH2, SH3, and LIM domains. Nck is a SH3- and SH2-containing adaptor protein implicated in coordinating various signaling pathways, including those of growth factor receptors and cell adhesion receptors. We report here the identification, cloning, and characterization of a widely expressed, Nck-related adaptor protein termed Nck-2. Nck-2 comprises primarily three N-terminal SH3 domains and one C-terminal SH2 domain. We show that Nck-2 interacts with PINCH, a LIM-only protein implicated in integrin-linked kinase signaling. The PINCH-Nck-2 interaction is mediated by the fourth LIM domain of PINCH and the third SH3 domain of Nck-2. Furthermore, we show that Nck-2 is capable of recognizing several key components of growth factor receptor kinase-signaling pathways including EGF receptors, PDGF receptor-β, and IRS-1. The association of Nck-2 with EGF receptors was regulated by EGF stimulation and involved largely the SH2 domain of Nck-2, although the SH3 domains of Nck-2 also contributed to the complex formation. The association of Nck-2 with PDGF receptor-β was dependent on PDGF activation and was mediated solely by the SH2 domain of Nck-2. Additionally, we have detected a stable association between Nck-2 and IRS-1 that was mediated primarily via the second and third SH3 domain of Nck-2. Thus, Nck-2 associates with PINCH and components of different growth factor receptor-signaling pathways via distinct mechanisms. Finally, we provide evidence indicating that a fraction of the Nck-2 and/or Nck-1 proteins are associated with the cytoskeleton. These results identify a novel Nck-related SH2- and SH3-domain–containing protein and suggest that it may function as an adaptor protein connecting the growth factor receptor-signaling pathways with the integrin-signaling pathways. PMID:9843575

  6. Characterization of the interaction of 5'-p-fluorosulfonylbenzoyl adenosine with the epidermal growth factor receptor/protein kinase in A431 cell membranes.

    PubMed

    Buhrow, S A; Cohen, S; Garbers, D L; Staros, J V

    1983-06-25

    Treatment of membrane vesicles from A431 cells, a human epidermoid carcinoma line, with the affinity label 5'-p-fluorosulfonylbenzoyl [8-14C]adenosine (5'-p-FSO2Bz[14C]Ado) results in an inhibition of the epidermal growth factor (EGF)-stimulable protein kinase and in the modification of proteins having the same molecular weight (Mr = 170,000 and 150,000) as the receptor for EGF (Buhrow, S. A., Cohen, S., and Staros, J. V. (1982) J. Biol. Chem. 257, 4019-4022). Modification of the vesicles with 5'-p-FSO2BzAdo inhibits not only the EGF-stimulated phosphorylation of endogenous membrane proteins but also the EGF-stimulated phosphorylation of an exogenous synthetic tyrosine-containing peptide substrate. This indicates that the EGF-stimulable protein kinase is modified by 5'-p-FSO2BzAdo at a site affecting catalytic activity. Membrane vesicles were treated with 5'-p-FSO2Bz-[14C]Ado to affinity label the kinase, then the EGF receptor was purified by affinity chromatography on immobilized EGF. The EGF receptor thus purified contains the 5'-p-SO2Bz[14C]Ado moiety. These data strongly support our hypothesis that the EGF receptor and EGF-stimulable kinase are two parts of the same polypeptide chain.

  7. Androgen receptor serine 81 mediates Pin1 interaction and activity

    PubMed Central

    La Montagna, Raffaele; Caligiuri, Isabella; Maranta, Pasquale; Lucchetti, Chiara; Esposito, Luca; Paggi, Marco G.; Toffoli, Giuseppe; Rizzolio, Flavio; Giordano, Antonio

    2012-01-01

    Hormone-dependent tumors are characterized by deregulated activity of specific steroid receptors, allowing aberrant expression of many genes involved in cancer initiation, progression and metastasis. In prostate cancer, the androgen receptor (AR) protein has pivotal functions, and over the years it has been the target of different drugs. AR is a nuclear receptor whose activity is regulated by a phosphorylation mechanism controlled by hormone and growth factors. Following phosphorylation, AR interacts with many cofactors that closely control its function. Among such cofactors, Pin1 is a peptidyl-prolyl isomerase that is involved in the control of protein phosphorylation and has a prognostic value in prostate cancer. In the present study, we demonstrate that ARSer81 is involved in the interaction with Pin1, and that this interaction is important for the transcriptional activity of AR. Since Pin1 expression positively correlates with tumor grade, our results suggest that Pin1 can participate in this process by modulating AR function. PMID:22894932

  8. Induction of nerve growth factor receptors on cultured human melanocytes

    SciTech Connect

    Peacocke, M.; Yaar, M.; Mansur, C.P.; Chao, M.V.; Gilchrest, B.A. )

    1988-07-01

    Normal differentiation and malignant transformation of human melanocytes involve a complex series of interactions during which both genetic and environmental factors play roles. At present, the regulation of these processes is poorly understood. The authors have induced the expression of nerve growth factor (NGF) receptors on cultured human melanocytes with phorbol 12-tetradecanoate 13-acetate and have correlated this event with the appearance of a more differentiated, dendritic morphology. Criteria for NGF receptor expression included protein accumulation and cell-surface immunofluorescent staining with a monoclonal antibody directed against the human receptor and induction of the messenger RNA species as determined by blot-hybridization studies. The presence of the receptor could also be induced by UV irradiation or growth factor deprivation. The NGF receptor is inducible in cultured human melanocytes, and they suggest that NGF may modulate the behavior of this neural crest-derived cell in the skin.

  9. Assessment of estrogen receptor--histone interactions.

    PubMed Central

    Kallos, J; Fasy, T M; Hollander, V P

    1981-01-01

    Several different in vitro binding assays show that the estrogen receptor from rabbit uterus interacts selectively with purified histones from calf thymus. The estrogen receptor binds strongly to histones H2B and H2A, moderately to histones H3 and H4, and poorly to histone H1. In the presence of histones H2B or H2A, the position at which the estrogen receptor focuses in an isoelectric gradient is shifted to a more basic zone. Kinetic experiments show that, if histone H2B is bound to a DNA, the estrogen receptor dissociates more slowly from that DNA. The portion of the estrogen receptor molecule required for binding to histone H2B is relatively stable to tryptic digestion; in contrast, the portion of the receptor molecule responsible for DNA binding is promptly lost during limited tryptic digestion. These in vitro findings suggest that the mechanism by which the estrogen receptor selectively alters gene expression may involve specific contacts with histone molecules. PMID:6942408

  10. Cell surface interaction of annexin A2 and galectin-3 modulates epidermal growth factor receptor signaling in Her-2 negative breast cancer cells.

    PubMed

    Shetty, Praveenkumar; Bargale, Anil; Patil, Basavraj R; Mohan, Rajashekar; Dinesh, U S; Vishwanatha, Jamboor K; Gai, Pramod B; Patil, Vidya S; Amsavardani, T S

    2016-01-01

    Overexpression and activation of tyrosine kinase receptors like EGFR and Src regulate the progression and metastasis of Her-2 negative breast cancer. Recently we have reported the role of cell membrane interaction of phospholipid-binding protein annexin A2 (AnxA2) and EGFR in regulating cellular signaling in the activation of angiogenesis, matrix degradation, invasion, and cancer metastasis. Beta-galactoside-specific animal lectin galectin-3 is an apoptosis inhibitor, and cell surface-associated extracellular galectin-3 also has a role in cell migration, cancer progression, and metastasis. Similar expression pattern and membrane co-localization of these two proteins made us to hypothesize in the current study that galectin-3 and AnxA2 interaction is critical for Her-2 negative breast cancer progression. By various experimental analyses, we confirm that glycosylated AnxA2 at the membrane surface interacts with galectin-3. N-linked glycosylation inhibitor tunicamycin treatment convincingly blocked AnxA2 membrane translocation and its association with galectin-3. To analyze whether this interaction has any functional relevance, we tried to dissociate this interaction with purified plant lectin from chickpea (Cicer arietinum agglutinin). This highly specific 30 kDa plant lectin could dissociate AnxA2 from endogenous lectin galectin-3 interaction at the cell surface. This dissociation could down-regulate Bcl-2 family proteins, cell proliferation, and migration simultaneously triggering cell apoptosis. Targeting this interaction of membrane surface glycoprotein and its animal lectin in Her-2 negative breast cancer may be of therapeutic value.

  11. Functional interaction of hepatic nuclear factor-4 and peroxisome proliferator-activated receptor-gamma coactivator 1alpha in CYP7A1 regulation is inhibited by a key lipogenic activator, sterol regulatory element-binding protein-1c.

    PubMed

    Ponugoti, Bhaskar; Fang, Sungsoon; Kemper, Jongsook Kim

    2007-11-01

    Insulin inhibits transcription of cholesterol 7alpha-hydroxylase (Cyp7a1), a key gene in bile acid synthesis, and the hepatic nuclear factor-4 (HNF-4) site in the promoter was identified as a negative insulin response sequence. Using a fasting/feeding protocol in mice and insulin treatment in HepG2 cells, we explored the inhibition mechanisms. Expression of sterol regulatory element-binding protein-1c (SREBP-1c), an insulin-induced lipogenic factor, inversely correlated with Cyp7a1 expression in mouse liver. Interaction of HNF-4 with its coactivator, peroxisome proliferator-activated receptor-gamma coactivator 1alpha (PGC-1alpha), was observed in livers of fasted mice and was reduced after feeding. Conversely, HNF-4 interaction with SREBP-1c was increased after feeding. In vitro studies suggested that SREBP-1c competed with PGC-1alpha for direct interaction with the AF2 domain of HNF-4. Reporter assays showed that SREBP-1c, but not of a SREBP-1c mutant lacking the HNF-4 interacting domain, inhibited HNF-4/PGC-1alpha transactivation of Cyp7a1. SREBP-1c also inhibited PGC-1alpha-coactivation of estrogen receptor, constitutive androstane receptor, pregnane X receptor, and farnesoid X receptor, implying inhibition of HNF-4 by SREBP-1c could extend to other nuclear receptors. In chromatin immunoprecipitation studies, HNF-4 binding to the promoter was not altered, but PGC-1alpha was dissociated, SREBP-1c and histone deacetylase-2 (HDAC2) were recruited, and acetylation of histone H3 was decreased upon feeding. Adenovirus-mediated expression of a SREBP-1c dominant-negative mutant, which blocks the interaction of SREBP-1c and HNF-4, partially but significantly reversed the inhibition of Cyp7a1 after feeding. Our data show that SREBP-1c functions as a non-DNA-binding inhibitor and mediates, in part, suppression of Cyp7a1 by blocking functional interaction of HNF-4 and PGC-1alpha. This mechanism may be relevant to known repression of many other HNF-4 target genes upon

  12. High-Content Positional Biosensor Screening Assay for Compounds to Prevent or Disrupt Androgen Receptor and Transcriptional Intermediary Factor 2 Protein–Protein Interactions

    PubMed Central

    Hua, Yun; Shun, Tong Ying; Strock, Christopher J.

    2014-01-01

    Abstract The androgen receptor–transcriptional intermediary factor 2 (AR-TIF2) positional protein–protein interaction (PPI) biosensor assay described herein combines physiologically relevant cell-based assays with the specificity of binding assays by incorporating structural information of AR and TIF2 functional domains along with intracellular targeting sequences and fluorescent reporters. Expression of the AR-red fluorescent protein (RFP) “prey” and TIF2-green fluorescent protein (GFP) “bait” components of the biosensor was directed by recombinant adenovirus constructs that expressed the ligand binding and activation function 2 surface domains of AR fused to RFP with nuclear localization and nuclear export sequences, and three α-helical LXXLL motifs from TIF2 fused to GFP and an HIV Rev nucleolar targeting sequence. In unstimulated cells, AR-RFP was localized predominantly to the cytoplasm and TIF2-GFP was localized to nucleoli. Dihydrotestosterone (DHT) treatment induced AR-RFP translocation into the nucleus where the PPIs between AR and TIF2 resulted in the colocalization of both biosensors within the nucleolus. We adapted the translocation enhanced image analysis module to quantify the colocalization of the AR-RFP and TIF2-GFP biosensors in images acquired on the ImageXpress platform. DHT induced a concentration-dependent AR-TIF2 colocalization and produced a characteristic condensed punctate AR-RFP PPI nucleolar distribution pattern. The heat-shock protein 90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) and antiandrogens flutamide and bicalutamide inhibited DHT-induced AR-TIF2 PPI formation with 50% inhibition concentrations (IC50s) of 88.5±12.5 nM, 7.6±2.4 μM, and 1.6±0.4 μM, respectively. Images of the AR-RFP distribution phenotype allowed us to distinguish between 17-AAG and flutamide, which prevented AR translocation, and bicalutamide, which blocked AR-TIF2 PPIs. We screened the Library of Pharmacologically Active

  13. Interaction of the glucocorticoid receptor and the chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII): implications for the actions of glucocorticoids on glucose, lipoprotein, and xenobiotic metabolism.

    PubMed

    De Martino, Massimo U; Alesci, Salvatore; Chrousos, George P; Kino, Tomoshige

    2004-06-01

    Glucocorticoids exert their extremely diverse effects on numerous biologic activities of humans via only one protein module, the glucocorticoid receptor (GR). The GR binds to the glucocorticoid response elements located in the promoter region of target genes and regulates their transcriptional activity. In addition, GR associates with other transcription factors through direct protein-protein interactions and mutually represses or stimulates each other's transcriptional activities. The latter activity of GR may be more important than the former one, granted that mice harboring a mutant GR, which is active in terms of protein-protein interactions but inactive in terms of transactivation via DNA, survive and procreate, in contrast to mice with a deletion of the entire GR gene that die immediately after birth. We recently found that GR physically interacts with the chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII), which plays a critical role in the metabolism of glucose, cholesterol, and xenobiotics, as well as in the development of the central nervous system in fetus. GR stimulates COUP-TFII-induced transactivation by attracting cofactors via its activation function-1, while COUP-TFII represses the GR-governed transcriptional activity by tethering corepressors, such as the silencing mediator for retinoid and thyroid hormone receptors (SMRT) and the nuclear receptor corepressors (NCoRs) via its C-terminal domain. Their mutual interaction may play an important role in gluconeogenesis, lipoprotein metabolism, and enzymatic clearance of clinically important compounds and bioactive chemicals, by regulating their rate-limiting enzymes and molecules, including the phosphoenolpyruvate carboxykinase (PEPCK), the cytochrome P450 CYP3A and CYP7A, and several apolipoproteins. It appears that glucocorticoids exert their intermediary effects partly via physical interaction with COUP-TFII.

  14. The interaction between menstrual cycle, Tumour Necrosis Factor alpha receptors and sex hormones in healthy non-obese women--results from an observational study.

    PubMed

    Rzymski, Paweł; Wilczak, Maciej; Malinger, Anna; Włoszczak-Szubzda, Anna; Jarosz, Mirosław Jerzy; Opala, Tomasz

    2014-01-01

    There is growing evidence that TNF-alpha and its two receptors play an important role in hormonal regulation, metabolism, inflammation and cancer. The biological effects of TNF-alpha are mediated by two receptors, p55 and p75. The aim of this study was to analyze serum concentrations of p55 and p75 and hormonal status in healthy women during the normal menstrual cycle. Eight women aged 20-22 with regular menstrual cycles were scheduled for examination on 3(rd) , 8(th) , 14(th) and 25(th) day of their menstrual cycle. We only observed a positive correlation of p75 subunit with prolactin level (correlation coefficient 0.417; p=0.0116) and negative correlation with insulin level (correlation coefficient -0.35; p=0.032) and HOMAIR insulin resistance index correlation coefficient 0.39; p=0.0185). Furthermore, a negative correlation of p55/p75 ratio with prolactin (correlation coefficient -0.42; p=0.0101) and a positive correlations of p55/p75 ratio with insulin level (correlation coefficient 0.43; p=0.008) and HOMAIR insulin resistance factor correlation coefficient 0.45; p=0.0065) were found.

  15. Novel interactions of the mineralocorticoid receptor.

    PubMed

    Fuller, Peter J

    2015-06-15

    The mineralocorticoid receptor (MR) differs from the other steroid receptors in that it responds to two physiological ligands, aldosterone and cortisol. In epithelial tissues, aldosterone selectivity is determined by 11β-hydroxysteroid dehydrogenase type II. In other tissues cortisol is the primary ligand; in some tissues cortisol may act as an antagonist. To better target MR, an understanding of the structural determinants of tissue and ligand-specific MR activation is required. Our focus is on interactions of the ligand-binding domain (LBD) with ligand, the N-terminal domain and putative co-regulatory molecules. Molecular modelling has identified a region in the LBD of the MR and indeed other steroid receptors that critically defines ligand-specificity for aldosterone and cortisol, yet is not part of the ligand-binding pocket. An interaction between the N-terminus and LBD observed in the MR is aldosterone-dependent but is unexpectedly antagonised by cortisol. The structural basis of this interaction has been defined. We have identified proteins which interact in the presence of either aldosterone or cortisol but not both. These have been confirmed as coactivators of the full-length hMR. The structural basis of this interaction has been determined for tesmin, a ligand-discriminant coactivator of the MR. The successful identification of the structural basis of antagonism and of ligand-specific interactions of the MR may provide the basis for the development of novel MR ligands with tissue specificity. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  16. Interactions between drugs and occupied receptors.

    PubMed

    Tallarida, Ronald J

    2007-01-01

    This review has 2 parts. Part I deals with isobolographic procedures that are traditionally applied to the joint action of agonists that individually produce overtly similar effects. Special attention is directed to newer computational procedures that apply to agonists with dissimilar concentration-effect curves. These newer procedures are consistent with the isobolographic methods introduced and used by Loewe, however, the present communications provides the needed graphical and mathematical detail. A major aim is distinguishing super and sub-addictive interactions from those that are simply additive. The detection and measurement of an interaction is an important step in exploring drug mechanism and is also important clinically. Part II discusses a new use of isoboles that is applicable to a single drug or chemical whose effect is mediated by 2 or more receptor subtypes. This application produces a metric that characterizes the interaction between the receptor subtypes. The expansion of traditional isobolographic theory to this multi-receptor situation follows from the newer approaches for 2-drug combination analysis in Part I. This topic leads naturally to a re-examination of competitive antagonism and the classic Schild plot. In particular, it is shown here that the Schild plot in the multi-receptor case is not necessarily linear with unit slope. Both parts of this review emphasize the quantitative aspects rather than the many drugs that have been analyzed with isobolographic methods. The mathematical exposition is rather elementary and is further aided by several graphs. An appendix is included for the reader interested in the mathematical details.

  17. Cytoplasmic Domain Interactions of Syndecan-1 and Syndecan-4 with α6β4 Integrin Mediate Human Epidermal Growth Factor Receptor (HER1 and HER2)-dependent Motility and Survival*♦

    PubMed Central

    Wang, Haiyao; Jin, Haining; Beauvais, DeannaLee M.; Rapraeger, Alan C.

    2014-01-01

    Epithelial cells are highly dependent during wound healing and tumorigenesis on the α6β4 integrin and its association with receptor tyrosine kinases. Previous work showed that phosphorylation of the β4 subunit upon matrix engagement depends on the matrix receptor syndecan (Sdc)-1 engaging the cytoplasmic domain of the β4 integrin and coupling of the integrin to human epidermal growth factor receptor-2 (HER2). In this study, HER2-dependent migration activated by matrix engagement is compared with migration stimulated by EGF. We find that whereas HER2-dependent migration depends on Sdc1, EGF-dependent migration depends on a complex consisting of human epidermal growth factor receptor-1 (HER1, commonly known as EGFR), α6β4, and Sdc4. The two syndecans recognize distinct sites at the extreme C terminus of the β4 integrin cytoplasmic domain. The binding motif in Sdc1 is QEEXYX, composed in part by its syndecan-specific variable (V) region and in part by the second conserved (C2) region that it shares with other syndecans. A cell-penetrating peptide containing this sequence competes for HER2-dependent epithelial migration and carcinoma survival, although it is without effect on the EGFR-stimulated mechanism. β4 mutants bearing mutations specific for Sdc1 and Sdc4 recognition act as dominant negative mutants to block cell spreading or cell migration that depends on HER2 or EGFR, respectively. The interaction of the α6β4 integrin with the syndecans appears critical for it to be utilized as a signaling platform; migration depends on α3β1 integrin binding to laminin 332 (LN332; also known as laminin 5), whereas antibodies that block α6β4 binding are without effect. These findings indicate that specific syndecan family members are likely to have key roles in α6β4 integrin activation by receptor tyrosine kinases. PMID:25202019

  18. Structural and Functional Characterization of a Secreted Hookworm Macrophage Migration Inhibitory Factor (MIF) that Interacts with the Human MIF Receptor CD74

    SciTech Connect

    Cho,Y.; Jones, B.; Vermeire, J.; Leng, L.; DiFedele, L.; Harrison, L.; Xiong, H.; Kwong, Y.; Chen, Y.; et al

    2007-01-01

    Hookworms, parasitic nematodes that infect nearly one billion people worldwide, are a major cause of anemia and malnutrition. We hypothesize that hookworms actively manipulate the host immune response through the production of specific molecules designed to facilitate infection by larval stages and adult worm survival within the intestine. A full-length cDNA encoding a secreted orthologue of the human cytokine, Macrophage Migration Inhibitory Factor (MIF) has been cloned from the hookworm Ancylostoma ceylanicum. Elucidation of the three-dimensional crystal structure of recombinant AceMIF (rAceMIF) revealed an overall structural homology with significant differences in the tautomerase sites of the human and hookworm proteins. The relative bioactivities of human and hookworm MIF proteins were compared using in vitro assays of tautomerase activity, macrophage migration, and binding to MIF receptor CD74. The activity of rAceMIF was not inhibited by the ligand ISO-1, which was previously determined to be an inhibitor of the catalytic site of human MIF. These data define unique immunological, structural, and functional characteristics of AceMIF, thereby establishing the potential for selectively inhibiting the hookworm cytokine as a means of reducing parasite survival and disease pathogenesis.

  19. Systems biology of tomato fruit development: combined transcript, protein, and metabolite analysis of tomato transcription factor (nor, rin) and ethylene receptor (Nr) mutants reveals novel regulatory interactions.

    PubMed

    Osorio, Sonia; Alba, Rob; Damasceno, Cynthia M B; Lopez-Casado, Gloria; Lohse, Marc; Zanor, Maria Inés; Tohge, Takayuki; Usadel, Björn; Rose, Jocelyn K C; Fei, Zhangjun; Giovannoni, James J; Fernie, Alisdair R

    2011-09-01

    Tomato (Solanum lycopersicum) is an established model to study fleshy fruit development and ripening. Tomato ripening is regulated independently and cooperatively by ethylene and transcription factors, including nonripening (NOR) and ripening-inhibitor (RIN). Mutations of NOR, RIN, and the ethylene receptor Never-ripe (Nr), which block ethylene perception and inhibit ripening, have proven to be great tools for advancing our understanding of the developmental programs regulating ripening. In this study, we present systems analysis of nor, rin, and Nr at the transcriptomic, proteomic, and metabolomic levels during development and ripening. Metabolic profiling marked shifts in the abundance of metabolites of primary metabolism, which lead to decreases in metabolic activity during ripening. When combined with transcriptomic and proteomic data, several aspects of the regulation of metabolism during ripening were revealed. First, correlations between the expression levels of a transcript and the abundance of its corresponding protein were infrequently observed during early ripening, suggesting that posttranscriptional regulatory mechanisms play an important role in these stages; however, this correlation was much greater in later stages. Second, we observed very strong correlation between ripening-associated transcripts and specific metabolite groups, such as organic acids, sugars, and cell wall-related metabolites, underlining the importance of these metabolic pathways during fruit ripening. These results further revealed multiple ethylene-associated events during tomato ripening, providing new insights into the molecular biology of ethylene-mediated ripening regulatory networks.

  20. The Orphan Nuclear Receptor SHP Inhibits Hepatocyte Nuclear Factor 4 and Retinoid X Receptor Transactivation: Two Mechanisms for Repression

    PubMed Central

    Lee, Yoon-Kwang; Dell, Helen; Dowhan, Dennis H.; Hadzopoulou-Cladaras, Margarita; Moore, David D.

    2000-01-01

    The orphan nuclear hormone receptor SHP interacts with a number of other nuclear hormone receptors and inhibits their transcriptional activity. Several mechanisms have been suggested to account for this inhibition. Here we show that SHP inhibits transactivation by the orphan receptor hepatocyte nuclear factor 4 (HNF-4) and the retinoid X receptor (RXR) by at least two mechanisms. SHP interacts with the same HNF-4 surface recognized by transcriptional coactivators and competes with them for binding in vivo. The minimal SHP sequences previously found to be required for interaction with other receptors are sufficient for interaction with HNF-4, although deletion results indicate that additional C-terminal sequences are necessary for full binding and coactivator competition. These additional sequences include those associated with direct transcriptional repressor activity of SHP. SHP also competes with coactivators for binding to ligand-activated RXR, and based on the ligand-dependent interaction with other nuclear receptors, it is likely that coactivator competition is a general feature of SHP-mediated repression. The minimal receptor interaction domain of SHP is sufficient for full interaction with RXR, as previously described. This domain is also sufficient for full coactivator competition. Functionally, however, full inhibition of RXR transactivation requires the presence of the C-terminal repressor domain, with only weak inhibition associated with this receptor interaction domain. Overall, these results suggest that SHP represses nuclear hormone receptor-mediated transactivation via two separate steps: first by competition with coactivators and then by direct effects of its transcriptional repressor function. PMID:10594021

  1. Interaction Patches of Procaspase-1 Caspase Recruitment Domains (CARDs) Are Differently Involved in Procaspase-1 Activation and Receptor-interacting Protein 2 (RIP2)-dependent Nuclear Factor κB Signaling*

    PubMed Central

    Kersse, Kristof; Lamkanfi, Mohamed; Bertrand, Mathieu J. M.; Vanden Berghe, Tom; Vandenabeele, Peter

    2011-01-01

    Protein interaction domains belonging to the death domain-fold superfamily are six-helix bundles that mediate the assembly of large protein complexes involved in apoptotic and inflammatory signaling. Typically, death domains (DDs), a subfamily of the death domain-fold superfamily, harbor six delineated interaction patches on their surfaces that mediate three distinct and conserved types of interaction designated as types I, II, and III. Here, we show that caspase recruitment domains (CARDs), another subfamily of the death domain-fold superfamily, multimerize by employing at least two of the three reported interaction types that were identified in DDs. On the one hand, the CARD of procaspase-1 binds the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) through a type I interaction that involves a patch surrounding residue Asp-27. On the other hand, the CARD of procaspase-1 auto-oligomerizes through a type III interaction involving a patch surrounding residue Arg-45. This oligomerization allows binding of receptor-interacting protein 2 (RIP2). In addition, we show that a 1:1 interaction between ASC and procaspase-1 is sufficient for procaspase-1 to gain proteolytic activity, whereas the formation of a higher order CARD complex involving ASC, procaspase-1, and RIP2 is required for effective procaspase-1-mediated NF-κB activation. These findings indicate that the CARD of procaspase-1 is differently involved in the formation of procaspase-1 activating platforms and procaspase-1-mediated, RIP2-dependent NF-κB activation. PMID:21862576

  2. Receptor kinase signaling pathways in plant-microbe interactions.

    PubMed

    Antolín-Llovera, Meritxell; Ried, Martina K; Binder, Andreas; Parniske, Martin

    2012-01-01

    Plant receptor-like kinases (RLKs) function in diverse signaling pathways, including the responses to microbial signals in symbiosis and defense. This versatility is achieved with a common overall structure: an extracytoplasmic domain (ectodomain) and an intracellular protein kinase domain involved in downstream signal transduction. Various surfaces of the leucine-rich repeat (LRR) ectodomain superstructure are utilized for interaction with the cognate ligand in both plant and animal receptors. RLKs with lysin-motif (LysM) ectodomains confer recognitional specificity toward N-acetylglucosamine-containing signaling molecules, such as chitin, peptidoglycan (PGN), and rhizobial nodulation factor (NF), that induce immune or symbiotic responses. Signaling downstream of RLKs does not follow a single pattern; instead, the detailed analysis of brassinosteroid (BR) signaling, innate immunity, and symbiosis revealed at least three largely nonoverlapping pathways. In this review, we focus on RLKs involved in plant-microbe interactions and contrast the signaling pathways leading to symbiosis and defense.

  3. The extracellular glycoprotein SPARC interacts with platelet-derived growth factor (PDGF)-AB and -BB and inhibits the binding of PDGF to its receptors.

    PubMed Central

    Raines, E W; Lane, T F; Iruela-Arispe, M L; Ross, R; Sage, E H

    1992-01-01

    Interactions among growth factors, cells, and extracellular matrix are critical to the regulation of directed cell migration and proliferation associated with development, wound healing, and pathologic processes. Here we report the association of PDGF-AB and -BB, but not PDGF-AA, with the extracellular glycoprotein SPARC. Complexes of SPARC and 125I-labeled PDGF-BB or -AB were specifically immunoprecipitated by anti-SPARC immunoglobulins. 125I-PDGF-BB and -AB also bound specifically to SPARC that was immobilized on microtiter wells or bound to nitrocellulose after transfer from SDS/polyacrylamide gels. The binding of PDGF-BB to SPARC was pH-dependent; significant binding was detectable only above pH 6.6. The interaction of SPARC with specific dimeric forms of PDGF affected the activity of this mitogen. SPARC inhibited the binding of PDGF-BB and PDGF-AB, but not PDGF-AA, to human dermal fibroblasts in a dose-dependent manner. The expression of SPARC and PDGF was minimal in most normal adult tissues but was increased after injury. Enhanced expression of both PDGF-B chain and SPARC was seen in advanced lesions of atherosclerosis. We suggest that the coordinate expression of SPARC and PDGF-B-containing dimers following vascular injury may regulate the activity of specific dimeric forms of PDGF in vivo. Images PMID:1311092

  4. Identification of the epidermal growth factor receptor as the receptor for Salmonella Rck-dependent invasion.

    PubMed

    Wiedemann, Agnès; Mijouin, Lily; Ayoub, Mohammed Akli; Barilleau, Emilie; Canepa, Sylvie; Teixeira-Gomes, Ana Paula; Le Vern, Yves; Rosselin, Manon; Reiter, Eric; Velge, Philippe

    2016-12-01

    The Salmonella Rck outer membrane protein binds to the cell surface, which leads to bacterial internalization via a Zipper mechanism. This invasion process requires induction of cellular signals, including phosphorylation of tyrosine proteins, and activation of c-Src and PI3K, which arises as a result of an interaction with a host cell surface receptor. In this study, epidermal growth factor receptor (EGFR) was identified as the cell signaling receptor required for Rck-mediated adhesion and internalization. First, Rck-mediated adhesion and internalization were shown to be altered when EGFR expression and activity were modulated. Then, immunoprecipitations were performed to demonstrate the Rck-EGFR interaction. Furthermore, surface plasmon resonance biosensor and homogeneous time-resolved fluorescence technologies were used to demonstrate the direct interaction of Rck with the extracellular domain of human EGFR. Finally, our study strongly suggests a noncompetitive binding of Rck and EGF to EGFR. Overall, these results demonstrate that Rck is able to bind to EGFR and thereby establish a tight adherence to provide a signaling cascade, which leads to internalization of Rck-expressing bacteria.-Wiedemann, A., Mijouin, L., Ayoub, M. A., Barilleau, E., Canepa, S., Teixeira-Gomes, A. P., Le Vern, Y., Rosselin, M., Reiter, E., Velge, P. Identification of the epidermal growth factor receptor as the receptor for Salmonella Rck-dependent invasion.

  5. Lung-derived factors mediate breast cancer cell migration through CD44 receptor-ligand interactions in a novel ex vivo system for analysis of organ-specific soluble proteins.

    PubMed

    Chu, Jenny E; Xia, Ying; Chin-Yee, Benjamin; Goodale, David; Croker, Alysha K; Allan, Alison L

    2014-02-01

    Breast cancer preferentially metastasizes to lung, lymph node, liver, bone, and brain. However, it is unclear whether properties of cancer cells, properties of organ microenvironments, or a combination of both is responsible for this observed organ tropism. We hypothesized that breast cancer cells exhibit distinctive migration/growth patterns in organ microenvironments that mirror common clinical sites of breast cancer metastasis and that receptor-ligand interactions between breast cancer cells and soluble organ-derived factors mediate this behavior. Using an ex vivo model system composed of organ-conditioned media (CM), human breast cancer cells (MDA-MB-231,MDA-MB-468, SUM149, and SUM159) displayed cell line-specific and organ-specific patterns of migration/proliferation that corresponded to their in vivo metastatic behavior. Notably, exposure to lung-CM increased migration of all cell lines and increased proliferation in two of four lines (P < .05). Several cluster of differentiation (CD) 44 ligands including osteopontin (OPN) and L-selectin (SELL) were identified in lung-CM by protein arrays. Immunodepletion of SELL decreased migration of MDA-MB-231 cells, whereas depletion of OPN decreased both migration and proliferation. Pretreatment of cells with a CD44-blocking antibody abrogated migration effects (P < .05). "Stemlike" breast cancer cells with high aldehyde dehydrogenase and CD44 (ALDH(hi)CD44(+)) responded in a distinct chemotactic manner toward organ-CM, preferentially migrating toward lung-CM through CD44 receptor-ligand interactions (P < .05). In contrast, organ-specific changes in migration were not observed for ALDH(low)CD44(-) cells. Our data suggest that interactions between CD44(+) breast cancer cells and soluble factors present in the lung microenvironment may play an important role in determining organotropic metastatic behavior.

  6. CONTAMINANT INTERACTIONS WITH STEROID RECEPTORS: EVIDENCE FOR RECEPTOR BINDING.

    EPA Science Inventory

    Steroid receptors are important determinants of endocrine disrupter consequences. As the most frequently proposed mechanism of endocrine-disrupting contaminant (EDC) action, steroid receptors are not only targets of natural steroids but are also commonly sites of nonsteroidal com...

  7. The extracellular glycoprotein SPARC interacts with platelet-derived growth factor (PDGF)-AB and -BB and inhibits the binding of PDGF to its receptors

    SciTech Connect

    Raines, E.W.; Lane, T.F.; Iruela-Arispe, M.L.; Ross, R.; Sage, E.H. )

    1992-02-15

    Interactions among growth factors, cells, and extracellular matrix are critical to the regulation of directed cell migration and proliferation associated with development wound healing, and pathologic processes. Here the authors report the association of PDGF-AB and -BB, but not PDGF-AA, with the extracellular glycoprotein SPARC. Complexes of SPARC and {sup 125}I-labeled PDGF-BB or -AB were specifically immunoprecipitated by anti-SPARC immunoglobulins. {sup 125}I-PDGF-BB and -AB also bound specifically to SPARC that was immobilized on microtiter wells or bound to nitrocellulose after transfer from SDS/polyacrylamide gels. The binding of PDGF-BB to SPARC was pH-dependent; significant binding was detectable only above pH 6.6. Enhanced expression of both PDGF-B chain and SPARC was seen in advanced lesions of atherosclerosis. They suggest that the coordinate expression of SPARC and PDGF-B-containing dimers following vascular injury may regulate the activity of specific dimeric forms of PDGF in vivo.

  8. Urokinase-type plasminogen activator receptor interaction with β1 integrin is required for platelet-derived growth factor-AB-induced human mesenchymal stem/stromal cell migration.

    PubMed

    Chabot, Valérie; Dromard, Cécile; Rico, Angélique; Langonné, Alain; Gaillard, Julien; Guilloton, Fabien; Casteilla, Louis; Sensebé, Luc

    2015-09-29

    Mesenchymal stem cells (MSC) are well described for their role in tissue regeneration following injury. Migratory properties of endogenous or administrated MSC are critical for tissue repair processes. Platelet-derived growth factor (PDGF) is a chemotactic growth factor that elicits mesenchymal cell migration. However, it is yet to be elucidated if signaling pathways other than direct activation of PDGF receptor (PDGF-R) are involved in PDGF-induced cell migration. Knocking down and co-immunoprecipitation approaches were used to evaluate urokinase-type plasminogen activator receptor (uPAR) requirement and its interactions with proteins involved in migration mechanisms, in human MSC induced to migrate under PDGF-AB effect. We demonstrated that uPAR activation and its association with β1-integrin are required for PDGF-AB-induced migration. This phenomenon takes place in MSC derived from bone marrow and from adipose tissue. We showed that PDGF-AB downstream signaling requires other effector molecules in MSC such as the uPA/uPAR system and β1 integrin signaling pathway known for their role in migration. These findings provide new insights in molecular mechanisms of PDGF-AB-induced migration of human MSC that may be relevant to control MSC function and tissue remodeling after injury.

  9. Studying Haloanisoles Interaction with Olfactory Receptors.

    PubMed

    Silva Teixeira, Carla S; Silva Ferreira, António C; Cerqueira, Nuno M F S A

    2016-07-20

    In this paper, computational means were used to explain and predict the interaction of several odorant molecules, including three haloanisoles, 2,4,6-trichloroanisole (TCA), 2,4,6-tribromoanisole (TBA), and 2,4,6-trichlorophenol (TCP), with three olfactory receptors (ORs): OR1A1, OR1A2, and OR3A1. As the X-ray structure of these ORs is not known, the three-dimensional structure of each OR was modeled by homology modeling. The structures of these ORs were stabilized by molecular dynamic simulations and the complexes of the odorant molecules with each ORs were generated by molecular docking. The theoretical results have shown that each OR has distinct but well-defined binding regions for each type of odorant molecules (aldehydes and alcohols). In OR3A1, the aldehydes bind in the bottom region of the binding pocket nearby Ser257 and Thr249. In the paralogues OR1A1 and OR1A2, the aldehydes tend to interact in the top region of the binding pocket and close to a positively charged lysine. On the other hand, the alcohols interact in the bottom region of the active site and close to a negatively charged aspartate. These results indicate that when aldehydes and alcohols odorants compete in these two ORs, the aldehydes can block the access of the alcohols odorants to their specific binding site. This observation goes in line with the experimental data that reveals that when the odorant is an aldehyde, a lower quantity of ligand is needed to cause 50% of the maximum response (lower EC50), when compared with the alcohols. The theoretical results have also allowed to explain the differences in the activity of (S)-(-)-citronellol in the wild-type and mutated OR1A1. The theoretical results show that Asn109 has a preponderant role in this matter, since when it is mutated, it leads to a conformational rearrangement of the binding pocket that prevents the interaction of (S)-(-)-citronellol with Asp111 that was shown to be important for the OR activation. The good agreement between

  10. The Internal Region Leucine-rich Repeat 6 of Decorin Interacts with Low Density Lipoprotein Receptor-related Protein-1, Modulates Transforming Growth Factor (TGF)-β-dependent Signaling, and Inhibits TGF-β-dependent Fibrotic Response in Skeletal Muscles*

    PubMed Central

    Cabello-Verrugio, Claudio; Santander, Cristian; Cofré, Catalina; Acuña, Maria José; Melo, Francisco; Brandan, Enrique

    2012-01-01

    Decorin is a small proteoglycan, composed of 12 leucine-rich repeats (LRRs) that modulates the activity of transforming growth factor type β (TGF-β) and other growth factors, and thereby influences proliferation and differentiation in a wide array of physiological and pathological processes, such as fibrosis, in several tissues and organs. Previously we described two novel modulators of the TGF-β-dependent signaling pathway: LDL receptor-related protein (LRP-1) and decorin. Here we have determined the regions in decorin that are responsible for interaction with LRP-1 and are involved in TGF-β-dependent binding and signaling. Specifically, we used decorin deletion mutants, as well as peptides derived from internal LRR regions, to determine the LRRs responsible for these decorin functions. Our results indicate that LRR6 and LRR5 participate in the interaction with LRP-1 and TGF-β as well as in its dependent signaling. Furthermore, the internal region (LRR6i), composed of 11 amino acids, is responsible for decorin binding to LRP-1 and subsequent TGF-β-dependent signaling. Furthermore, using an in vivo approach, we also demonstrate that the LRR6 region of decorin can inhibit TGF-β mediated action in response to skeletal muscle injury. PMID:22203668

  11. The internal region leucine-rich repeat 6 of decorin interacts with low density lipoprotein receptor-related protein-1, modulates transforming growth factor (TGF)-β-dependent signaling, and inhibits TGF-β-dependent fibrotic response in skeletal muscles.

    PubMed

    Cabello-Verrugio, Claudio; Santander, Cristian; Cofré, Catalina; Acuña, Maria José; Melo, Francisco; Brandan, Enrique

    2012-02-24

    Decorin is a small proteoglycan, composed of 12 leucine-rich repeats (LRRs) that modulates the activity of transforming growth factor type β (TGF-β) and other growth factors, and thereby influences proliferation and differentiation in a wide array of physiological and pathological processes, such as fibrosis, in several tissues and organs. Previously we described two novel modulators of the TGF-β-dependent signaling pathway: LDL receptor-related protein (LRP-1) and decorin. Here we have determined the regions in decorin that are responsible for interaction with LRP-1 and are involved in TGF-β-dependent binding and signaling. Specifically, we used decorin deletion mutants, as well as peptides derived from internal LRR regions, to determine the LRRs responsible for these decorin functions. Our results indicate that LRR6 and LRR5 participate in the interaction with LRP-1 and TGF-β as well as in its dependent signaling. Furthermore, the internal region (LRR6i), composed of 11 amino acids, is responsible for decorin binding to LRP-1 and subsequent TGF-β-dependent signaling. Furthermore, using an in vivo approach, we also demonstrate that the LRR6 region of decorin can inhibit TGF-β mediated action in response to skeletal muscle injury.

  12. Metabotropic Glutamate Receptors and Interacting Proteins in Epileptogenesis

    PubMed Central

    Qian, Feng; Tang, Feng-Ru

    2016-01-01

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

  13. Huntingtin-Interacting Protein 1 Phosphorylation by Receptor Tyrosine Kinases

    PubMed Central

    Ames, Heather M.; Wang, Anmin A.; Coughran, Alanna; Evaul, Kristen; Huang, Sha; Graves, Chiron W.; Soyombo, Abigail A.

    2013-01-01

    Huntingtin-interacting protein 1 (HIP1) binds inositol lipids, clathrin, actin, and receptor tyrosine kinases (RTKs). HIP1 is elevated in many tumors, and its expression is prognostic in prostate cancer. HIP1 overexpression increases levels of the RTK epidermal growth factor receptor (EGFR) and transforms fibroblasts. Here we report that HIP1 is tyrosine phosphorylated in the presence of EGFR and platelet-derived growth factor β receptor (PDGFβR) as well as the oncogenic derivatives EGFRvIII, HIP1/PDGFβR (H/P), and TEL/PDGFβR (T/P). We identified a four-tyrosine “HIP1 phosphorylation motif” (HPM) in the N-terminal region of HIP1 that is required for phosphorylation mediated by both EGFR and PDGFβR but not by the oncoproteins H/P and T/P. We also identified a tyrosine residue (Y152) within the HPM motif of HIP1 that inhibits HIP1 tyrosine phosphorylation. The HPM tyrosines are conserved in HIP1's only known mammalian relative, HIP1-related protein (HIP1r), and are also required for HIP1r phosphorylation. Tyrosine-to-phenylalanine point mutations in the HPM of HIP1 result in proapoptotic activity, indicating that an intact HPM may be necessary for HIP1's role in cellular survival. These data suggest that phosphorylation of HIP1 by RTKs in an N-terminal region contributes to the promotion of cellular survival. PMID:23836884

  14. Epidermal growth factor receptor signaling in tissue

    SciTech Connect

    Shvartsman, Stanislav; Wiley, H. S.; Lauffenburger, Douglas A.

    2004-08-01

    Abstract: A peptide purified from the salivary gland of a mouse was shown few years ago to accelerate incisor eruption and eyelid opening in newborn mice, and was named epidermal growth factor (EGF). The members of this family of peptide growth factors had been identified in numerous physiological and pathological contexts. EGF binds to a cell surface EGF receptor, which induces a biochemical modification (phosphorylation) of the receptor's cytoplasmic tail. There is a growing consensus in the research community that, in addition to cellular and molecular studies, the dynamics of the EGFR network and its operation must be examined in tissues. A key challenge is to integrate the existing molecular and cellular information into a system-level description of the EGFR network at the tissue and organism level. In this paper, the two examples of EGFR signaling in tissues are described, and the recent efforts to model EGFR autocrine loops, which is a predominant mode of EGFR activation in vivo, are summarized.

  15. Nociceptin/Orphanin FQ Receptor Structure, Signaling, Ligands, Functions, and Interactions with Opioid Systems

    PubMed Central

    Bruchas, Michael R.; Calo', Girolamo; Cox, Brian M.; Zaveri, Nurulain T.

    2016-01-01

    The NOP receptor (nociceptin/orphanin FQ opioid peptide receptor) is the most recently discovered member of the opioid receptor family and, together with its endogenous ligand, N/OFQ, make up the fourth members of the opioid receptor and opioid peptide family. Because of its more recent discovery, an understanding of the cellular and behavioral actions induced by NOP receptor activation are less well developed than for the other members of the opioid receptor family. All of these factors are important because NOP receptor activation has a clear modulatory role on mu opioid receptor-mediated actions and thereby affects opioid analgesia, tolerance development, and reward. In addition to opioid modulatory actions, NOP receptor activation has important effects on motor function and other physiologic processes. This review discusses how NOP pharmacology intersects, contrasts, and interacts with the mu opioid receptor in terms of tertiary structure and mechanism of receptor activation; location of receptors in the central nervous system; mechanisms of desensitization and downregulation; cellular actions; intracellular signal transduction pathways; and behavioral actions with respect to analgesia, tolerance, dependence, and reward. This is followed by a discussion of the agonists and antagonists that have most contributed to our current knowledge. Because NOP receptors are highly expressed in brain and spinal cord and NOP receptor activation sometimes synergizes with mu receptor-mediated actions and sometimes opposes them, an understanding of NOP receptor pharmacology in the context of these interactions with the opioid receptors will be crucial to the development of novel therapeutics that engage the NOP receptor. PMID:26956246

  16. A genotypic difference in primary root length is associated with the inhibitory role of transforming growth factor-beta receptor-interacting protein-1 on root meristem size in wheat.

    PubMed

    He, Xue; Fang, Jingjing; Li, Jingjuan; Qu, Baoyuan; Ren, Yongzhe; Ma, Wenying; Zhao, Xueqiang; Li, Bin; Wang, Daowen; Li, Zhensheng; Tong, Yiping

    2014-03-01

    Previously we identified a major quantitative trait locus (QTL) qTaLRO-B1 for primary root length (PRL) in wheat. Here we compare proteomics in the roots of the qTaLRO-B1 QTL isolines 178A, with short PRL and small meristem size, and 178B, with long PRL and large meristem size. A total of 16 differentially expressed proteins were identified: one, transforming growth factor (TGF)-beta receptor-interacting protein-1 (TaTRIP1), was enriched in 178A, while various peroxidases (PODs) were more abundantly expressed in 178B. The 178A roots showed higher TaTRIP1 expression and lower levels of the unphosphorylated form of the brassinosteroid (BR) signaling component BZR1, lower expression of POD genes and reduced POD activity and accumulation of the superoxide anion O2(-) in the root elongation zone compared with the 178B roots. Low levels of 24-epibrassinolide increased POD gene expression and root meristem size, and rescued the short PRL phenotype of 178A. TaTRIP1 directly interacted with the BR receptor TaBRI1 of wheat. Moreover, overexpressing TaTRIP1 in Arabidopsis reduced the abundance of unphosphorylated BZR1 protein, altered the expression of BR-responsive genes, inhibited POD activity and accumulation of the O2(-) in the root tip and inhibited root meristem size. Our data suggested that TaTRIP1 is involved in BR signaling and inhibited root meristem size, possibly by reducing POD activity and accumulation of O2(-) in the root tip. We further demonstrated a negative correlation between the level of TaTRIP1 mRNA and PRL of landraces and modern wheat varieties, providing a valuable insight for better understanding of the molecular mechanism underlying the genotypic differences in root morphology of wheat in the future. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

  17. The C-terminal domain of the long form of cellular FLICE-inhibitory protein (c-FLIPL) inhibits the interaction of the caspase 8 prodomain with the receptor-interacting protein 1 (RIP1) death domain and regulates caspase 8-dependent nuclear factor κB (NF-κB) activation.

    PubMed

    Matsuda, Iyo; Matsuo, Kentaro; Matsushita, Yuka; Haruna, Yasushi; Niwa, Masamitsu; Kataoka, Takao

    2014-02-14

    Caspase 8 plays an essential role in the regulation of apoptotic and non-apoptotic signaling pathways. The long form of cellular FLICE-inhibitory protein (c-FLIPL) has been shown previously to regulate caspase 8-dependent nuclear factor κB (NF-κB) activation by receptor-interacting protein 1 (RIP1) and TNF receptor-associated factor 2 (TRAF2). In this study, the molecular mechanism by which c-FLIPL regulates caspase 8-dependent NF-κB activation was further explored in the human embryonic kidney cell line HEK 293 and variant cells barely expressing caspase 8. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone greatly diminished caspase 8-dependent NF-κB activation induced by Fas ligand (FasL) when c-FLIPL, but not its N-terminal fragment c-FLIP(p43), was expressed. The prodomain of caspase 8 was found to interact with the RIP1 death domain and to be sufficient to mediate NF-κB activation induced by FasL or c-FLIP(p43). The interaction of the RIP1 death domain with caspase 8 was inhibited by c-FLIPL but not c-FLIP(p43). Thus, these results reveal that the C-terminal domain of c-FLIPL specifically inhibits the interaction of the caspase 8 prodomain with the RIP1 death domain and, thereby, regulates caspase 8-dependent NF-κB activation.

  18. The C-terminal Domain of the Long Form of Cellular FLICE-inhibitory Protein (c-FLIPL) Inhibits the Interaction of the Caspase 8 Prodomain with the Receptor-interacting Protein 1 (RIP1) Death Domain and Regulates Caspase 8-dependent Nuclear Factor κB (NF-κB) Activation*

    PubMed Central

    Matsuda, Iyo; Matsuo, Kentaro; Matsushita, Yuka; Haruna, Yasushi; Niwa, Masamitsu; Kataoka, Takao

    2014-01-01

    Caspase 8 plays an essential role in the regulation of apoptotic and non-apoptotic signaling pathways. The long form of cellular FLICE-inhibitory protein (c-FLIPL) has been shown previously to regulate caspase 8-dependent nuclear factor κB (NF-κB) activation by receptor-interacting protein 1 (RIP1) and TNF receptor-associated factor 2 (TRAF2). In this study, the molecular mechanism by which c-FLIPL regulates caspase 8-dependent NF-κB activation was further explored in the human embryonic kidney cell line HEK 293 and variant cells barely expressing caspase 8. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone greatly diminished caspase 8-dependent NF-κB activation induced by Fas ligand (FasL) when c-FLIPL, but not its N-terminal fragment c-FLIP(p43), was expressed. The prodomain of caspase 8 was found to interact with the RIP1 death domain and to be sufficient to mediate NF-κB activation induced by FasL or c-FLIP(p43). The interaction of the RIP1 death domain with caspase 8 was inhibited by c-FLIPL but not c-FLIP(p43). Thus, these results reveal that the C-terminal domain of c-FLIPL specifically inhibits the interaction of the caspase 8 prodomain with the RIP1 death domain and, thereby, regulates caspase 8-dependent NF-κB activation. PMID:24398693

  19. Phosphorylation of insulin-like growth factor I receptor by insulin receptor tyrosine kinase in intact cultured skeletal muscle cells

    SciTech Connect

    Beguinot, F.; Smith, R.J.; Kahn, C.R.; Maron, R.; Moses, A.C.; White, M.F.

    1988-05-03

    The interaction between insulin and insulin-like growth factor I (IGF I) receptors was examined by determining the ability of each receptor type to phosphorylate tyrosine residues on the other receptor in intact L6 skeletal muscle cells. This was made possible through a sequential immunoprecipitation method with two different antibodies that effectively separated the phosphorylated insulin and IGF I receptors. After incubation of intact L6 cells with various concentrations of insulin or IGF I in the presence of (/sup 32/P)-orthophosphate, insulin receptors were precipitated with one of two human polyclonal anti-insulin receptor antibodies (B2 or B9). Phosphorylated IGF I receptors remained in solution and were subsequently precipitated by anti-phosphotyrosine antibodies. The identifies of the insulin and IGF I receptor ..beta..-subunits in the two immunoprecipitates were confirmed by binding affinity, by phosphopeptide mapping after trypsin digestion, and by the distinct patterns of expression of the two receptors during differentiation. Stimulated phosphorylation of the ..beta..-subunit of the insulin receptor correlated with the occupancy of the ..beta..-subunit of the insulin receptor by either insulin or IGF I as determined by affinity cross-linking. Similarly, stimulation of phosphorylation of the ..beta..-subunit of the IGF I receptor by IGF I correlated with IGF I receptor occupancy. In contrast, insulin stimulated phosphorylation of the ..beta..-subunit of the IGF I receptor at hormone concentrations that were associated with significant occupancy of the insulin receptor but negligible IGF I receptor occupancy. These findings indicate that the IGF I receptor can be a substrate for the hormone-activated insulin receptor tyrosine kinase activity in intact L6 skeletal muscle cells.

  20. Methods for studying the platelet-derived growth factor receptor

    SciTech Connect

    Bowen-Pope, D.F.; Ross, R.

    1985-01-01

    Platelet-derived growth factor (PDGF) is a basic 30,000-dalton protein circulating in normal blood sequestered within the platelet alpha granule. Radioiodinated PDGF shows saturable (e.g., 60,000-120,000 receptors per diploid human fibroblast) high affinity binding to culture PDGF-responsive cells. The apparent dissociation constant reported for this binding interaction has varied widely. This paper focuses on factors which affect (/sup 125/I)PGDF binding and on the development of a radioreceptor assay for PDGF.

  1. Mechanism of the estrogen receptor interaction with 4-hydroxytamoxifen

    SciTech Connect

    Sasson, S.; Notides, A.C.

    1988-04-01

    The binding mechanism of the estrogen receptor with 4-(/sup 3/H)hydroxytamoxifen was investigated. The equilibrium binding analysis with 4-(/sup 3/H)hydroxytamoxifen indicated a positive cooperative interaction: the Scatchard plot was convex and the Hill coefficient was 1.4-1.5. This binding appears similar to the positively cooperative interaction of the estrogen receptor with (/sup 3/H)estradiol. However, a competitive binding assay with a saturating concentration of (/sup 3/H) estradiol and variable concentrations of 4-hydroxytamoxifen produced nonparallel displacement curves indicating that the binding mechanism of the receptor with these two ligands is different. The competitive binding assay with (/sup 3/H)estradiol and 4-hydroxytamoxifen at constant molar ratios demonstrated that the receptor's affinity for estradiol was reduced and the receptor preferentially bound 4-hydroxytamoxifen. These data suggest that 4-hydroxytamoxifen interacts with the receptor differently than estradiol; it antagonizes the binding of estradiol when these two ligands are simultaneously present.

  2. Group I Metabotropic Glutamate Receptor Interacting Proteins: Fine-Tuning Receptor Functions in Health and Disease

    PubMed Central

    Kalinowska, Magdalena; Francesconi, Anna

    2016-01-01

    Group I metabotropic glutamate receptors mediate slow excitatory neurotransmission in the central nervous system and are critical to activity-dependent synaptic plasticity, a cellular substrate of learning and memory. Dysregulated receptor signaling is implicated in neuropsychiatric conditions ranging from neurodevelopmental to neurodegenerative disorders. Importantly, group I metabotropic glutamate receptor signaling functions can be modulated by interacting proteins that mediate receptor trafficking, expression and coupling efficiency to signaling effectors. These interactions afford cell- or pathway-specific modulation to fine-tune receptor function, thus representing a potential target for pharmacological interventions in pathological conditions. PMID:27296642

  3. 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

  4. Interaction of Biofunctionalized Nanoparticles with Receptors on Cell Surfaces: MC Simulations

    NASA Astrophysics Data System (ADS)

    Dormidontova, Elena; Wang, Shihu

    2015-03-01

    One of the areas of active development of modern nanomedicine is drug/gene delivery and imaging application of nanoparticles functionalized by ligands, aptamers or antibodies capable of specific interactions with cell surface receptors. Being a complex multifunctional system different structural aspects of nanoparticles affect their interactions with cell surfaces and the surface properties of cells can be different (e.g. density, distribution and mobility of receptors). Computer simulations allow a systematic investigation of the influence of multiple factors and provide a unified platform for the comparison. Using Monte Carlo simulations we investigate the influence of the nanoparticle properties (nanoparticle size, polymer tether length, polydispersity, density, ligand energy, valence and density) on nanoparticle-cell surface interactions and make predictions regarding favorable nanoparticle design for achieving multiple ligand-receptor binding. We will also discuss the implications of nanoparticle design on the selectivity of attachment to cells with high receptor density while ``ignoring'' cells with a low density of receptors.

  5. Functional Roles of the Interaction of APP and Lipoprotein Receptors

    PubMed Central

    Pohlkamp, Theresa; Wasser, Catherine R.; Herz, Joachim

    2017-01-01

    The biological fates of the key initiator of Alzheimer’s disease (AD), the amyloid precursor protein (APP), and a family of lipoprotein receptors, the low-density lipoprotein (LDL) receptor-related proteins (LRPs) and their molecular roles in the neurodegenerative disease process are inseparably interwoven. Not only does APP bind tightly to the extracellular domains (ECDs) of several members of the LRP group, their intracellular portions are also connected through scaffolds like the one established by FE65 proteins and through interactions with adaptor proteins such as X11/Mint and Dab1. Moreover, the ECDs of APP and LRPs share common ligands, most notably Reelin, a regulator of neuronal migration during embryonic development and modulator of synaptic transmission in the adult brain, and Agrin, another signaling protein which is essential for the formation and maintenance of the neuromuscular junction (NMJ) and which likely also has critical, though at this time less well defined, roles for the regulation of central synapses. Furthermore, the major independent risk factors for AD, Apolipoprotein (Apo) E and ApoJ/Clusterin, are lipoprotein ligands for LRPs. Receptors and ligands mutually influence their intracellular trafficking and thereby the functions and abilities of neurons and the blood-brain-barrier to turn over and remove the pathological product of APP, the amyloid-β peptide. This article will review and summarize the molecular mechanisms that are shared by APP and LRPs and discuss their relative contributions to AD. PMID:28298885

  6. Attenuation of loop-receptor interactions with pseudoknot formation

    PubMed Central

    Afonin, Kirill A.; Lin, Yen-Ping; Calkins, Erin R.; Jaeger, Luc

    2012-01-01

    RNA tetraloops can recognize receptors to mediate long-range interactions in stable natural RNAs. In vitro selected GNRA tetraloop/receptor interactions are usually more ‘G/C-rich’ than their ‘A/U-rich’ natural counterparts. They are not as widespread in nature despite comparable biophysical and chemical properties. Moreover, while AA, AC and GU dinucleotide platforms occur in natural GAAA/11 nt receptors, the AA platform is somewhat preferred to the others. The apparent preference for ‘A/U-rich’ GNRA/receptor interactions in nature might stem from an evolutionary adaptation to avoid folding traps at the level of the larger molecular context. To provide evidences in favor of this hypothesis, several riboswitches based on natural and artificial GNRA receptors were investigated in vitro for their ability to prevent inter-molecular GNRA/receptor interactions by trapping the receptor sequence into an alternative intra-molecular pseudoknot. Extent of attenuation determined by native gel-shift assays and co-transcriptional assembly is correlated to the G/C content of the GNRA receptor. Our results shed light on the structural evolution of natural long-range interactions and provide design principles for RNA-based attenuator devices to be used in synthetic biology and RNA nanobiotechnology. PMID:22080507

  7. Targeting Extracellular Domains D4 and D7 of Vascular Endothelial Growth Factor Receptor 2 Reveals Allosteric Receptor Regulatory Sites

    PubMed Central

    Hyde, Caroline A. C.; Giese, Alexandra; Stuttfeld, Edward; Abram Saliba, Johan; Villemagne, Denis; Schleier, Thomas; Binz, H. Kaspar

    2012-01-01

    Vascular endothelial growth factors (VEGFs) activate three receptor tyrosine kinases, VEGFR-1, -2, and -3, which regulate angiogenic and lymphangiogenic signaling. VEGFR-2 is the most prominent receptor in angiogenic signaling by VEGF ligands. The extracellular part of VEGF receptors consists of seven immunoglobulin homology domains (Ig domains). Earlier studies showed that domains 2 and 3 (D23) mediate ligand binding, while structural analysis of dimeric ligand/receptor complexes by electron microscopy and small-angle solution scattering revealed additional homotypic contacts in membrane-proximal Ig domains D4 and D7. Here we show that D4 and D7 are indispensable for receptor signaling. To confirm the essential role of these domains in signaling, we isolated VEGFR-2-inhibitory “designed ankyrin repeat proteins” (DARPins) that interact with D23, D4, or D7. DARPins that interact with D23 inhibited ligand binding, receptor dimerization, and receptor kinase activation, while DARPins specific for D4 or D7 did not prevent ligand binding or receptor dimerization but effectively blocked receptor signaling and functional output. These data show that D4 and D7 allosterically regulate VEGFR-2 activity. We propose that these extracellular-domain-specific DARPins represent a novel generation of receptor-inhibitory drugs for in vivo applications such as targeting of VEGFRs in medical diagnostics and for treating vascular pathologies. PMID:22801374

  8. Targeting extracellular domains D4 and D7 of vascular endothelial growth factor receptor 2 reveals allosteric receptor regulatory sites.

    PubMed

    Hyde, Caroline A C; Giese, Alexandra; Stuttfeld, Edward; Abram Saliba, Johan; Villemagne, Denis; Schleier, Thomas; Binz, H Kaspar; Ballmer-Hofer, Kurt

    2012-10-01

    Vascular endothelial growth factors (VEGFs) activate three receptor tyrosine kinases, VEGFR-1, -2, and -3, which regulate angiogenic and lymphangiogenic signaling. VEGFR-2 is the most prominent receptor in angiogenic signaling by VEGF ligands. The extracellular part of VEGF receptors consists of seven immunoglobulin homology domains (Ig domains). Earlier studies showed that domains 2 and 3 (D23) mediate ligand binding, while structural analysis of dimeric ligand/receptor complexes by electron microscopy and small-angle solution scattering revealed additional homotypic contacts in membrane-proximal Ig domains D4 and D7. Here we show that D4 and D7 are indispensable for receptor signaling. To confirm the essential role of these domains in signaling, we isolated VEGFR-2-inhibitory "designed ankyrin repeat proteins" (DARPins) that interact with D23, D4, or D7. DARPins that interact with D23 inhibited ligand binding, receptor dimerization, and receptor kinase activation, while DARPins specific for D4 or D7 did not prevent ligand binding or receptor dimerization but effectively blocked receptor signaling and functional output. These data show that D4 and D7 allosterically regulate VEGFR-2 activity. We propose that these extracellular-domain-specific DARPins represent a novel generation of receptor-inhibitory drugs for in vivo applications such as targeting of VEGFRs in medical diagnostics and for treating vascular pathologies.

  9. Drug interactions at GABA(A) receptors.

    PubMed

    Korpi, Esa R; Gründer, Gerhard; Lüddens, Hartmut

    2002-06-01

    Neurotransmitter receptor systems have been the focus of intensive pharmacological research for more than 20 years for basic and applied scientific reasons, but only recently has there been a better understanding of their key features. One of these systems includes the type A receptor for the gamma-aminobutyric acid (GABA), which forms an integral anion channel from a pentameric subunit assembly and mediates most of the fast inhibitory neurotransmission in the adult vertebrate central nervous system. Up to now, depending on the definition, 16-19 mammalian subunits have been cloned and localized on different genes. Their assembly into proteins in a poorly defined stoichiometry forms the basis of functional and pharmacological GABA(A) receptor diversity, i.e. the receptor subtypes. The latter has been well documented in autoradiographic studies using ligands that label some of the receptors' various binding sites, corroborated by recombinant expression studies using the same tools. Significantly less heterogeneity has been found at the physiological level in native receptors, where the subunit combinations have been difficult to dissect. This review focuses on the characteristics, use and usefulness of various ligands and their binding sites to probe GABA(A) receptor properties and to gain insight into the biological function from fish to man and into evolutionary conserved GABA(A) receptor heterogeneity. We also summarize the properties of the novel mouse models created for the study of various brain functions and review the state-of-the-art imaging of brain GABA(A) receptors in various human neuropsychiatric conditions. The data indicate that the present ligands are only partly satisfactory tools and further ligands with subtype-selective properties are needed for imaging purposes and for confirming the behavioral and functional results of the studies presently carried out in gene-targeted mice with other species, including man.

  10. Differences between disease-associated endoplasmic reticulum aminopeptidase 1 (ERAP1) isoforms in cellular expression, interactions with tumour necrosis factor receptor 1 (TNF-R1) and regulation by cytokines.

    PubMed

    Yousaf, N; Low, W Y; Onipinla, A; Mein, C; Caulfield, M; Munroe, P B; Chernajovsky, Y

    2015-05-01

    Endoplasmic reticulum aminopeptidase 1 (ERAP1) processes peptides for major histocompatibility complex (MHC) class I presentation and promotes cytokine receptor ectodomain shedding. These known functions of ERAP1 may explain its genetic association with several autoimmune inflammatory diseases. In this study, we identified four novel alternatively spliced variants of ERAP1 mRNA, designated as ΔExon-11, ΔExon-13, ΔExon-14 and ΔExon-15. We also observed a rapid and differential modulation of ERAP1 mRNA levels and spliced variants in different cell types pretreated with lipopolysaccharide (LPS). We have studied three full-length allelic forms of ERAP1 (R127-K528, P127-K528, P127-R528) and one spliced variant (ΔExon-11) and assessed their interactions with tumour necrosis factor receptor 1 (TNF-R1) in transfected cells. We observed variation in cellular expression of different ERAP1 isoforms, with R127-K528 being expressed at a much lower level. Furthermore, the cellular expression of full-length P127-K528 and ΔExon-11 spliced variant was enhanced significantly when co-transfected with TNF-R1. Isoforms P127-K528, P127-R528 and ΔExon-11 spliced variant associated with TNF-R1, and this interaction occurred in a region within the first 10 exons of ERAP1. Supernatant-derived vesicles from transfected cells contained the full-length and ectodomain form of soluble TNF-R1, as well as carrying the full-length ERAP1 isoforms. We observed marginal differences between TNF-R1 ectodomain levels when co-expressed with individual ERAP1 isoforms, and treatment of transfected cells with tumour necrosis factor (TNF), interleukin (IL)-1β and IL-10 exerted variable effects on TNF-R1 ectodomain cleavage. Our data suggest that ERAP1 isoforms may exhibit differential biological properties and inflammatory mediators could play critical roles in modulating ERAP1 expression, leading to altered functional activities of this enzyme.

  11. Interactions of Rodent Coronaviruses with Cellular Receptors

    DTIC Science & Technology

    2016-05-08

    Fluorescent-Antibody Staining to Detect Infection of Single Cells .. . ..... .......... . Challenge of BHK and MOCK Cells with MHV...absence of anti-receptor MAb-CCl..... 82 11. Fluorescent-antibody staining of OBT cells protected with anti-receptor MAb-CCl and challenged with MHV...hepatitis virus which share the same appearance in negative stains , recalling a solar corona, should be included in a group which they suggested

  12. Metabolic regulator betaKlotho interacts with fibroblast growth factor receptor 4 (FGFR4) to induce apoptosis and inhibit tumor cell proliferation.

    PubMed

    Luo, Yongde; Yang, Chaofeng; Lu, Weiqin; Xie, Rui; Jin, Chengliu; Huang, Peng; Wang, Fen; McKeehan, Wallace L

    2010-09-24

    In organs involved in metabolic homeostasis, transmembrane α and βklothos direct FGFR signaling to control of metabolic pathways. Coordinate expression of βklotho and FGFR4 is a property of mature hepatocytes. Genetic deletion of FGFR4 or βklotho in mice disrupts hepatic cholesterol/bile acid and lipid metabolism. The deletion of FGFR4 has no effect on the proliferative response of hepatocytes after liver injury. However, its absence results in accelerated progression of dimethynitrosamine-initiated hepatocellular carcinomas, indicating that FGFR4 suppresses hepatoma proliferation. The mechanism underlying the FGFR4-mediated hepatoma suppression has not been addressed. Here we show that βklotho expression is more consistently down-regulated in human and mouse hepatomas than FGFR4. Co-expression and activation by either endocrine FGF19 or cellular FGF1 of the FGFR4 kinase in a complex with βklotho restricts cell population growth through induction of apoptotic cell death in both hepatic and nonhepatic cells. The βklotho-FGFR4 partnership caused a depression of activated AKT and mammalian target of rapamycin while activating ERK1/2 that may underlie the pro-apoptotic effect. Our results show that βklotho not only interacts with heparan sulfate-FGFR4 to form a complex with high affinity for endocrine FGF19 but also impacts the quality of downstream signaling and biological end points activated by either FGF19 or canonical FGF1. Thus the same βklotho-heparan sulfate-FGFR4 partnership that mediates endocrine control of hepatic metabolism plays a role in cellular homeostasis and hepatoma suppression through negative control of cell population growth mediated by pro-apoptotic signaling.

  13. Cell-collagen interactions: the use of peptide Toolkits to investigate collagen-receptor interactions.

    PubMed

    Farndale, Richard W; Lisman, Ton; Bihan, Dominique; Hamaia, Samir; Smerling, Christiane S; Pugh, Nicholas; Konitsiotis, Antonios; Leitinger, Birgit; de Groot, Philip G; Jarvis, Gavin E; Raynal, Nicolas

    2008-04-01

    Fibrillar collagens provide the most fundamental platform in the vertebrate organism for the attachment of cells and matrix molecules. We have identified specific sites in collagens to which cells can attach, either directly or through protein intermediaries. Using Toolkits of triple-helical peptides, each peptide comprising 27 residues of collagen primary sequence and overlapping with its neighbours by nine amino acids, we have mapped the binding of receptors and other proteins on to collagens II or III. Integrin alpha2beta1 binds to several GXX'GER motifs within the collagens, the affinities of which differ sufficiently to control cell adhesion and migration independently of the cellular regulation of the integrin. The platelet receptor, Gp (glycoprotein) VI binds well to GPO (where O is hydroxyproline)-containing model peptides, but to very few Toolkit peptides, suggesting that sequence in addition to GPO triplets is important in defining GpVI binding. The Toolkits have been applied to the plasma protein vWF (von Willebrand factor), which binds to only a single sequence, identified by truncation and amino acid substitution within Toolkit peptides, as GXRGQOGVMGFO in collagens II and III. Intriguingly, the receptor tyrosine kinase, DDR2 (discoidin domain receptor 2) recognizes three sites in collagen II, including its vWF-binding site, although the amino acids that support the interaction differ slightly within this motif. Furthermore, the secreted protein BM-40 (basement membrane protein 40) also binds well to this same region. Thus the availability of extracellular collagen-binding proteins may be important in regulating and facilitating direct collagen-receptor interaction.

  14. Identification of ciliary neurotrophic factor (CNTF) residues essential for leukemia inhibitory factor receptor binding and generation of CNTF receptor antagonists.

    PubMed Central

    Di Marco, A; Gloaguen, I; Graziani, R; Paonessa, G; Saggio, I; Hudson, K R; Laufer, R

    1996-01-01

    Ciliary neurotrophic factor (CNTF) drives the sequential assembly of a receptor complex containing the ligand-specific alpha-receptor subunit (CNTFR alpha) and the signal transducers gp130 and leukemia inhibitory factor receptor-beta (LIFR). The D1 structural motif, located at the beginning of the D-helix of human CNTF, contains two amino acid residues, F152 and K155, which are conserved among all cytokines that signal through LIFR. The functional importance of these residues was assessed by alanine mutagenesis. Substitution of either F152 or K155 with alanine was found to specifically inhibit cytokine interaction with LIFR without affecting binding to CNTFR alpha or gp130. The resulting variants behaved as partial agonists with varying degrees of residual bioactivity in different cell-based assays. Simultaneous alanine substitution of both F152 and K155 totally abolished biological activity. Combining these mutations with amino acid substitutions in the D-helix, which enhance binding affinity for the CNTFR alpha, gave rise to a potent competitive CNTF receptor antagonist. This protein constitutes a new tool for studies of CNTF function in normal physiology and disease. Images Fig. 1 Fig. 6 PMID:8799186

  15. Tools and techniques to study ligand-receptor interactions and receptor activation by TNF superfamily members.

    PubMed

    Schneider, Pascal; Willen, Laure; Smulski, Cristian R

    2014-01-01

    Ligands and receptors of the TNF superfamily are therapeutically relevant targets in a wide range of human diseases. This chapter describes assays based on ELISA, immunoprecipitation, FACS, and reporter cell lines to monitor interactions of tagged receptors and ligands in both soluble and membrane-bound forms using unified detection techniques. A reporter cell assay that is sensitive to ligand oligomerization can identify ligands with high probability of being active on endogenous receptors. Several assays are also suitable to measure the activity of agonist or antagonist antibodies, or to detect interactions with proteoglycans. Finally, self-interaction of membrane-bound receptors can be evidenced using a FRET-based assay. This panel of methods provides a large degree of flexibility to address questions related to the specificity, activation, or inhibition of TNF-TNF receptor interactions in independent assay systems, but does not substitute for further tests in physiologically relevant conditions.

  16. Receptor-interacting protein kinase 2 promotes triple-negative breast cancer cell migration and invasion via activation of nuclear factor-kappaB and c-Jun N-terminal kinase pathways

    PubMed Central

    2014-01-01

    Introduction Metastasis is the main cause of breast cancer morbidity and mortality. Processes that allow for tumor cell migration and invasion are important therapeutic targets. Here we demonstrate that receptor-interacting protein kinase 2 (RIP2), a kinase known to be involved in inflammatory processes, also has novel roles in cancer cell migration and invasion. Methods A total of six breast cancer expression databases, including The Cancer Genome Atlas, were assessed for RIP2 expression among various clinical subtypes and its role as a prognostic biomarker. mRNA fluorescence in situ hybridization (FISH) for RIP2 was performed on 17 stage III breast cancers to determine if there was a correlation between RIP2 expression and lymph node involvement. RNA-interference was used to knock-down RIP2 expression in MDA-MB-231, Htb126, SUM149PT, MCF7, T47D, and HCC1428 cells. Cell migration and invasion were measured in vitro by scratch/wound healing and transwell migration assays. A xenograft mouse model was used to assess tumor growth and chemosensitivity to docetaxel in vivo in MDA-MB-231 cells with and without RIP2 small hairpin RNA knockdown. Western blot and immunofluorescence imaging were used to evaluate protein expressions. Results Interrogation of expression databases showed that RIP2 expression is significantly over-expressed in triple-negative breast cancers (TNBC: estrogen-receptor (ER) negative, progesterone-receptor (PR) negative, Her2/neu- (Her2) negative), compared to other clinical subtypes. High RIP2 expression correlates with worse progression-free survival using a combined breast cancer expression array dataset consisting of 946 patients. Multivariate analysis shows RIP2 as an independent prognostic biomarker. Knock-down of RIP2 significantly decreases migration in both scratch/wound healing and transwell migration assays in MDA-MB-231, Htb126, SUM149PT, MCF7, and T47D cells and is correlated with decreased Nuclear Factor-kappaB and c-Jun N

  17. mGlu5 Receptor Functional Interactions and Addiction

    PubMed Central

    Brown, Robyn M.; Mustafa, Sanam; Ayoub, Mohammed Akli; Dodd, Peter R.; Pfleger, Kevin D. G.; Lawrence, Andrew J.

    2012-01-01

    The idea of “receptor mosaics” is that proteins may form complex and dynamic networks with respect to time and composition. These have the potential to markedly expand the diversity and specificity of G protein-coupled receptors (GPCR) signaling, particularly in neural cells, where a few key receptors have been implicated in many neurological and psychiatric disorders, including addiction. Metabotropic glutamate type 5 receptors (mGlu5) can form complexes with other GPCRs, including adenosine A2A and dopamine D2 receptors. mGlu5-containing complexes have been reported in the striatum, a brain region critical for mediating the rewarding and incentive motivational properties of drugs of abuse. mGlu5-containing complexes and/or downstream interactions between divergent receptors may play roles in addiction–relevant behaviors. Interactions between mGlu5 receptors and other GPCRs can regulate the rewarding and conditioned effects of drugs as well as drug-seeking behaviors. mGlu5 complexes may influence striatal function, including GABAergic output of striatopallidal neurons and glutamatergic input from corticostriatal afferents. Given their discrete localization, mGlu5-[non-mGlu5] receptor interactions and/or mGlu5-containing complexes may minimize off-target effects and thus provide a novel avenue for drug discovery. The therapeutic targeting of receptor–receptor functional interactions and/or receptor mosaics in a tissue specific or temporal manner (for example, a sub-population of receptors in a “pathological state”) might reduce detrimental side effects that may otherwise impair vital brain functions. PMID:22586398

  18. FLASH interacts with p160 coactivator subtypes and differentially suppresses transcriptional activity of steroid hormone receptors.

    PubMed

    Kino, Tomoshige; Ichijo, Takamasa; Chrousos, George P

    2004-12-01

    We previously reported that tumor necrosis factor alpha receptor- and Fas-associated FLASH interacts with one of the p160 nuclear receptor coactivators, glucocorticoid receptor-interacting protein (GRIP) 1, at its nuclear receptor-binding (NRB) domain, and that inhibits the transcriptional activity of the glucocorticoid receptor (GR) by interfering with association of GR and GRIP1. Here, we further examined the specificity of FLASH suppressive effect and the physical/functional interactions between this protein and two other p160 family subtypes. The suppressive effect of FLASH on GR transactivation was observed in several cell lines and on the chromatin-integrated mouse mammary tumor virus (MMTV) promoter. FLASH strongly interacted with the NRB domain of the thyroid hormone receptor activator molecule (TRAM) 1, a member of the steroid hormone receptor coactivator (SRC) 3/nuclear receptor coactivator (N-CoA) 3 subtypes, as well as with SRC2/N-CoA2 p160 coactivator GRIP1, while its interaction with SRC1a, one of the SRC1/N-CoA1 proteins, was faint in yeast two-hybrid assays. Accordingly, FLASH strongly suppressed TRAM1- and GRIP1-induced enhancement of GR-stimulated transactivation of the MMTV promoter in HCT116 cells, while it did not affect SRC1a-induced potentiation of transcription. Furthermore, FLASH suppressed androgen- and progesterone receptor-induced transcriptional activity, but did not influence estrogen receptor-induced transactivation, possibly due to their preferential use of p160 coactivators in HCT116 and HeLa cells. Thus, FLASH differentially suppresses steroid hormone receptor-induced transcriptional activity by interfering with their association with SRC2/N-CoA2 and SRC3/N-CoA3 but not with SRC1/N-CoA1.

  19. Interaction of ethanol with opiate receptors

    SciTech Connect

    Yukhananov, R.Y.; Bujov, Y.V.; Maiskii, A.I.

    1986-04-01

    The authors study the action of ethanol on membrane-bound opiate receptors. Ethanol at 37/sup 0/C was shown to produce dose-dependent inhibition of binding of /sup 3/H-naloxone with opiate receptors. ID/sub 50/ under these conditions was 462 mM. Temperature-dependent inhibition of ligand-receptor binding suggests that ethanol does not compete for the stereospecific binding site of /sup 3/H-naloxone. Analysis of the inhibitory action of ethanol on /sup 3/H-naloxone binding in animals at different stages of experimental alcoholism revealed no differences between the control and experimental animals after 3.5 and 10 months of voluntary alcoholization.

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

    PubMed

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

    2004-11-16

    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.

  1. Physical and functional interactions between nuclear receptor LXRα and the forkhead box transcription factor FOXA2 regulate the response of the human lipoprotein lipase gene to oxysterols in hepatic cells.

    PubMed

    Kanaki, Maria; Tiniakou, Ioanna; Thymiakou, Efstathia; Kardassis, Dimitris

    2017-08-01

    Lipoprotein lipase (LPL) catalyzes the hydrolysis of triglycerides from triglyceride-rich lipoproteins such as VLDL and chylomicrons in the circulation. Mutations in LPL or its activator apolipoprotein C-II cause hypertriglyceridemia in humans and animal models. The levels of LPL in the liver are low but they can be strongly induced by a high cholesterol diet or by synthetic ligands of Liver X Receptors (LXRs). However, the mechanism by which LXRs activate the human LPL gene is unknown. In the present study we show that LXR agonists increased the mRNA and protein levels as well as the promoter activity of human LPL in HepG2 cells. A promoter deletion analysis defined the proximal -109/-28 region, which contains a functional FOXA2 element, as essential for transactivation by ligand-activated LXRα/RXRα heterodimers. Silencing of endogenous FOXA2 in HepG2 cells by siRNAs or by treatment with insulin compromised the induction of the LPL gene by LXR agonists whereas mutations in the FOXA2 site abolished the synergistic transactivation of the LPL promoter by LXRα/RXRα and FOXA2. Physical and functional interactions between LXRα and FOXA2 were established in vitro and ex vivo. In summary, the present study revealed a novel mechanism of human LPL gene induction by oxysterols in the liver with is based on physical and functional interactions between transcription factors LXRα and FOXA2. This mechanism, which may not be restricted to the LPL gene, is critically important for a better understanding of the regulation of cholesterol and triglyceride metabolism in the liver under healthy or pathological states. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Discoidin domain receptor 2 interacts with Src and Shc following its activation by type I collagen.

    PubMed

    Ikeda, Kazuo; Wang, Li-Hsien; Torres, Richard; Zhao, Hong; Olaso, Elvira; Eng, Francis J; Labrador, Pablo; Klein, Rudiger; Lovett, David; Yancopoulos, George D; Friedman, Scott L; Lin, Hsin Chieh

    2002-05-24

    Discoidin domain receptor 2 (DDR2) is an unusual receptor tyrosine kinase in that its ligand is fibrillar collagen rather than a growth factor-like peptide. We examined signal transduction pathways of DDR2. Here we show that DDR2 is also unusual in that it requires Src activity to be maximally tyrosine-phosphorylated, and that Src activity also promotes association of DDR2 with Shc. The interaction with Shc involves a portion of Shc not previously implicated in interaction with receptor tyrosine kinases. These results identify Src kinase and the adaptor protein Shc as key signaling intermediates in DDR2 signal transduction. Furthermore, Src is required for DDR2-mediated transactivation of the matrix metalloproteinase-2 promoter. The data support a model in which Src and the DDR2 receptor cooperate in a regulated fashion to direct the phosphorylation of both the receptor and its targets.

  3. Association of the Genetic Polymorphisms in Transcription Factor 7-Like 2 and Peroxisome Proliferator-Activated Receptors- γ 2 with Type 2 Diabetes Mellitus and is Interaction with Obesity Status in Emirati Population.

    PubMed

    Al-Safar, Habiba; Hassoun, Ahmed; Almazrouei, Shaikha; Kamal, Wala; Afandi, Bachar; Rais, Naushad

    2015-01-01

    Transcription factor 7-like 2 gene (TCF7L2) and peroxisome proliferator-activated receptors-γ2 (PPAR-γ2) have a profound effect on the incidence of type 2 diabetes mellitus (T2DM) and had previously been found to be associated with T2DM risk in various ppopulations. However, studies in the Arab population are inconsistent. We conducted a case control study to confirm the association of variants rs10885409 of TCF7L2 and Pro12Ala (rs1801282) of PPAR-γ2 with risk of T2DM and related complications in Emirati population of Arab origin. We also investigated the interaction of these associations with obesity status. DNA was extracted from the saliva samples of 272 T2DM patients and 216 nondiabetic Emiratis. Genotyping for rs10885409 (TCF7L2) and rs1801282 (PPAR-γ2 P12A) variants was accomplished with a TaqMan assay. The subgroups were constituted according to obesity status. In the nonobese group, the rs10885409 C allele in the recessive model was significantly associated with the incidence of T2DM (OR 1.975 [95% CI 1.127-3.461], P = 0.017), but this association was not observed in the obese group or when BMI was not considered. PPAR-γ2 risk allele Pro12 frequency (0.96) was similar in the groups tested and more than 90% population was homozygous for this allele. Our case-control study is the first of its kind in Emiratis which establishes TCF7L2 rs10885409 C allele as a T2DM risk factor in Emiratis and this association is modulated by obesity status. We also confirmed that Pro12Ala mutation in PPAR-γ2 is not associated with T2DM risk in this population.

  4. Association of the Genetic Polymorphisms in Transcription Factor 7-Like 2 and Peroxisome Proliferator-Activated Receptors-γ2 with Type 2 Diabetes Mellitus and Its Interaction with Obesity Status in Emirati Population

    PubMed Central

    Al-Safar, Habiba; Hassoun, Ahmed; Almazrouei, Shaikha; Kamal, Wala; Afandi, Bachar; Rais, Naushad

    2015-01-01

    Background. Transcription factor 7-like 2 gene (TCF7L2) and peroxisome proliferator-activated receptors-γ2 (PPAR-γ2) have a profound effect on the incidence of type 2 diabetes mellitus (T2DM) and had previously been found to be associated with T2DM risk in various ppopulations. However, studies in the Arab population are inconsistent. We conducted a case control study to confirm the association of variants rs10885409 of TCF7L2 and Pro12Ala (rs1801282) of PPAR-γ2 with risk of T2DM and related complications in Emirati population of Arab origin. We also investigated the interaction of these associations with obesity status. Methods. DNA was extracted from the saliva samples of 272 T2DM patients and 216 nondiabetic Emiratis. Genotyping for rs10885409 (TCF7L2) and rs1801282 (PPAR-γ2 P12A) variants was accomplished with a TaqMan assay. The subgroups were constituted according to obesity status. Results. In the nonobese group, the rs10885409 C allele in the recessive model was significantly associated with the incidence of T2DM (OR 1.975 [95% CI 1.127–3.461], P = 0.017), but this association was not observed in the obese group or when BMI was not considered. PPAR-γ2 risk allele Pro12 frequency (0.96) was similar in the groups tested and more than 90% population was homozygous for this allele. Conclusions. Our case-control study is the first of its kind in Emiratis which establishes TCF7L2 rs10885409 C allele as a T2DM risk factor in Emiratis and this association is modulated by obesity status. We also confirmed that Pro12Ala mutation in PPAR-γ2 is not associated with T2DM risk in this population. PMID:26273662

  5. Definition of the molecular basis for estrogen receptor-related receptor-alpha-cofactor interactions.

    PubMed

    Gaillard, Stéphanie; Dwyer, Mary A; McDonnell, Donald P

    2007-01-01

    Estrogen receptor-related receptor-alpha (ERRalpha) is an orphan nuclear receptor that does not appear to require a classical small molecule ligand to facilitate its interaction with coactivators and/or hormone response elements within target genes. Instead, the apo-receptor is capable of interacting in a constitutive manner with coactivators that stimulate transcription by acting as protein ligands. We have screened combinatorial phage libraries for peptides that selectively interact with ERRalpha to probe the architecture of the ERRalpha-coactivator pocket. In this manner, we have uncovered a fundamental difference in the mechanism by which this receptor interacts with peroxisome proliferator-activated receptor-gamma coactivator-1alpha, as compared with members of the steroid receptor coactivator subfamily of coactivators. Our findings suggest that it may be possible to develop ERRalpha ligands that exhibit different pharmacological activities as a consequence of their ability to differentially regulate coactivator recruitment. In addition, these findings have implications beyond ERRalpha because they suggest that subtle alterations in the structure of the activation function-2 pocket within any nuclear receptor may enable differential recruitment of coactivators, an observation of notable pharmaceutical importance.

  6. Catecholamime Interactions with the Cardiac Ryanodine Receptor

    NASA Astrophysics Data System (ADS)

    Klipp, Robert Carl

    The cardiac ryanodine receptor (RyR2) is a Ca2+ ion channel found in the sarcoplasmic reticulum (SR), an intracellular membranous Ca2+ storage system. It is well known that a destabilization of RyR2 can lead to a Ca2+ flux out of the SR, which results in an overload of intracellular Ca2+; this can also lead to arrhythmias and heart failure. The catecholamines play a large role in the regulation of RyR2; stimulation of the beta-adrenergic receptor on the cell membrane can lead to a hyperphosphorylation of RyR2, making it more leaky to Ca2+. We have previously shown that strong electron donors will inhibit RyR2. It is hypothesized that the catecholamines, sharing a similar structure with other proven inhibitors of RyR2, will also inhibit RyR2. Here we confirm this hypothesis and show for the first time that the catecholamines, isoproterenol and epinephrine, act as strong electron donors and inhibit RyR2 activity at the single channel level. This data suggests that the catecholamines can influence RyR2 activity at two levels. This offers promising insight into the potential development of a new class of drugs to treat heart failure and arrhythmia; ones that can both prevent the hyperphosphorylation of RyR2 by blocking the beta-adrenergic receptor, but can also directly inhibit the release of Ca2+ from RyR2.

  7. Adenosine–cannabinoid receptor interactions. Implications for striatal function

    PubMed Central

    Ferré, Sergi; Lluís, Carme; Justinova, Zuzana; Quiroz, César; Orru, Marco; Navarro, Gemma; Canela, Enric I; Franco, Rafael; Goldberg, Steven R

    2010-01-01

    Adenosine and endocannabinoids are very ubiquitous non-classical neurotransmitters that exert a modulatory role on the transmission of other more ‘classical’ neurotransmitters. In this review we will focus on their common role as modulators of dopamine and glutamate neurotransmission in the striatum, the main input structure of the basal ganglia. We will pay particular attention to the role of adenosine A2A receptors and cannabinoid CB1 receptors. Experimental results suggest that presynaptic CB1 receptors interacting with A2A receptors in cortico-striatal glutamatergic terminals that make synaptic contact with dynorphinergic medium-sized spiny neurons (MSNs) are involved in the motor-depressant and addictive effects of cannabinoids. On the other hand, postsynaptic CB1 receptors interacting with A2A and D2 receptors in the dendritic spines of enkephalinergic MSNs and postsynaptic CB1 receptors in the dendritic spines of dynorphinergic MSN are probably involved in the cataleptogenic effects of cannabinoids. These receptor interactions most probably depend on the existence of a variety of heteromers of A2A, CB1 and D2 receptors in different elements of striatal spine modules. Drugs selective for the different striatal A2A and CB1 receptor heteromers could be used for the treatment of neuropsychiatric disorders and drug addiction and they could provide effective drugs with fewer side effects than currently used drugs. This article is part of a themed issue on Cannabinoids. To view the editorial for this themed issue visit http://dx.doi.org/10.1111/j.1476-5381.2010.00831.x PMID:20590556

  8. A comprehensive pathway map of epidermal growth factor receptor signaling

    PubMed Central

    Oda, Kanae; Matsuoka, Yukiko; Funahashi, Akira; Kitano, Hiroaki

    2005-01-01

    The epidermal growth factor receptor (EGFR) signaling pathway is one of the most important pathways that regulate growth, survival, proliferation, and differentiation in mammalian cells. Reflecting this importance, it is one of the best-investigated signaling systems, both experimentally and computationally, and several computational models have been developed for dynamic analysis. A map of molecular interactions of the EGFR signaling system is a valuable resource for research in this area. In this paper, we present a comprehensive pathway map of EGFR signaling and other related pathways. The map reveals that the overall architecture of the pathway is a bow-tie (or hourglass) structure with several feedback loops. The map is created using CellDesigner software that enables us to graphically represent interactions using a well-defined and consistent graphical notation, and to store it in Systems Biology Markup Language (SBML). PMID:16729045

  9. Regulation of growth factor receptor degradation by ADP-ribosylation factor domain protein (ARD) 1.

    PubMed

    Meza-Carmen, Victor; Pacheco-Rodriguez, Gustavo; Kang, Gi Soo; Kato, Jiro; Donati, Chiara; Zhang, Chun-Yi; Vichi, Alessandro; Payne, D Michael; El-Chemaly, Souheil; Stylianou, Mario; Moss, Joel; Vaughan, Martha

    2011-06-28

    ADP-ribosylation factor domain protein 1 (ARD1) is a 64-kDa protein containing a functional ADP-ribosylation factor (GTP hydrolase, GTPase), GTPase-activating protein, and E3 ubiquitin ligase domains. ARD1 activation by the guanine nucleotide-exchange factor cytohesin-1 was known. GTPase and E3 ligase activities of ARD1 suggest roles in protein transport and turnover. To explore this hypothesis, we used mouse embryo fibroblasts (MEFs) from ARD1-/- mice stably transfected with plasmids for inducible expression of wild-type ARD1 protein (KO-WT), or ARD1 protein with inactivating mutations in E3 ligase domain (KO-E3), or containing persistently active GTP-bound (KO-GTP), or inactive GDP-bound (KO-GDP) GTPase domains. Inhibition of proteasomal proteases in mifepristone-induced KO-WT, KO-GDP, or KO-GTP MEFs resulted in accumulation of these ARD1 proteins, whereas KO-E3 accumulated without inhibitors. All data were consistent with the conclusion that ARD1 regulates its own steady-state levels in cells by autoubiquitination. Based on reported growth factor receptor-cytohesin interactions, EGF receptor (EGFR) was investigated in induced MEFs. Amounts of cell-surface and total EGFR were higher in KO-GDP and lower in KO-GTP than in KO-WT MEFs, with levels in both mutants greater (p = 0.001) after proteasomal inhibition. Significant differences among MEF lines in content of TGF-β receptor III were similar to those in EGFR, albeit not as large. Differences in amounts of insulin receptor mirrored those in EGFR, but did not reach statistical significance. Overall, the capacity of ARD1 GTPase to cycle between active and inactive forms and its autoubiquitination both appear to be necessary for the appropriate turnover of EGFR and perhaps additional growth factor receptors.

  10. Structural Analysis of Chemokine Receptor-Ligand Interactions.

    PubMed

    Arimont, Marta; Sun, Shan-Liang; Leurs, Rob; Smit, Martine; de Esch, Iwan J P; de Graaf, Chris

    2017-03-10

    This review focuses on the construction and application of structural chemokine receptor models for the elucidation of molecular determinants of chemokine receptor modulation and the structure-based discovery and design of chemokine receptor ligands. A comparative analysis of ligand binding pockets in chemokine receptors is presented, including a detailed description of the CXCR4, CCR2, CCR5, CCR9, and US28 X-ray structures, and their implication for modeling molecular interactions of chemokine receptors with small-molecule ligands, peptide ligands, and large antibodies and chemokines. These studies demonstrate how the integration of new structural information on chemokine receptors with extensive structure-activity relationship and site-directed mutagenesis data facilitates the prediction of the structure of chemokine receptor-ligand complexes that have not been crystallized. Finally, a review of structure-based ligand discovery and design studies based on chemokine receptor crystal structures and homology models illustrates the possibilities and challenges to find novel ligands for chemokine receptors.

  11. Dopamine and dopamine receptor D1 associated with decreased social interaction.

    PubMed

    Liu, Qiang; Shi, Jieyun; Lin, Rongfei; Wen, Tieqiao

    2017-02-13

    Deficits in social interaction are hallmarks of neurological and psychiatric disorders. However, its underlying mechanism is still unclear. Here, we show that the loss of dendritic cell factor 1 (Dcf1) in the nervous system of mice induces social interaction deficiency, autism-like behaviour, and influences social interaction via the dopamine system. Dopamine receptor D1 agonist rescues this social cognition phenotype, and improves short-term plasticity. Together, this study presents a new genetic mechanism that affects social interaction and may provide a new way to improve positive social interaction and treat autism spectrum disorders.

  12. [Modulation of glucocorticoid receptor interaction with non-steroidal drugs].

    PubMed

    Golikov, P P; Nikolaeva, N Iu

    1993-01-01

    The Scatchard analysis of the specific binding of triamcinolone 3H-acetonide (TA-3HA) to Type II glucocorticoid receptors of cytosol from the liver of female Wistar rats weighing 180-200 g has shown that emoxipin at concentrations of 1 and 2 mM and analgin at concentrations of 5 and 10 mM reduce the density of glucocorticoid receptors and the association constant of a hormone-receptor complex. Analgin, 5 mM, increases the dissociation velocity constant of TA-3HA 5 times the effect of unlabeled triamcinolone acetonide. Emoxipin, 1 mM, produces the same effect on the receptor dissociation velocity constant of TA-3HA as the unlabeled triamcinolone acetonide. The Berke analysis has established that emoxipin and analgin reduce glucocorticoid receptor interactions by uncompetitive inhibition.

  13. Interaction of progestins with steroid receptors in human uterus

    PubMed Central

    Kasid, Attan; Buckshee, Kamal; Hingorani, Veera; Laumas, Kesho R.

    1978-01-01

    Norethindrone (17β-hydroxy-19-nor-17α-pregn-4-en-20-yn-3-one) and norethindrone acetate (17β-acetoxy-19-nor-17α-pregn-4-en-20-yn-3-one) interfered to a varying degree, by competitive inhibition, with the binding of progesterone and oestradiol to respective cytoplasmic receptors in the human uterus. Progesterone binding to 4S macromolecule was saturable and co-specific for progestins. Competitors like norgestrel (17β-hydroxy-18-methyl-19-nor-17α-pregn-4-en-20-yn-3-one), 19-norprogesterone, medroxyprogesterone acetate (17α-acetoxy-6α-methylpregn-4-ene-3,20-dione) and compound R5020 (17,21-dimethyl-19-norpregna-4,9-diene-3,20-dione) possessed higher binding affinities for the progestin receptor. The dissociation constant (Kd) for the progesterone–receptor interaction was 0.6–1.6nm and the receptor concentration ranged between 6600 and 8200 sites/cell. Norethindrone and norethindrone acetate competed for the progesterone receptor with inhibition constants (Ki) of 6.8 and 72nm respectively. Gradient displacement and competitive-receptor assays indicated that norethindrone acetate-binding affinity for progestin receptor was approximately one-tenth that of norethindrone and progesterone. The progestins also inhibited oestradiol binding to 4.6S oestrogenic receptor by 8–12%, involving interaction at the oestradiol-binding site with a calculated Ki value of 0.5–0.8μm. The competitive interaction of progestins with steroid receptors may be of putative importance in explaining the progestin action at the target site. PMID:743257

  14. ADENOVIRUS INTERACTION WITH ITS CELLULAR RECEPTOR CAR.

    SciTech Connect

    HOWITT,J.; ANDERSON,C.W.; FREIMUTH,P.

    2001-08-01

    The mechanism of adenovirus attachment to the host cell plasma membrane has been revealed in detail by research over the past 10 years. It has long been known that receptor binding activity is associated with the viral fibers, trimeric spike proteins that protrude radially from the vertices of the icosahedral capsid (Philipson et al. 1968). In some adenovirus serotypes, fiber and other virus structural proteins are synthesized in excess and accumulate in the cell nucleus during late stages of infection. Fiber protein can be readily purified from lysates of cells infected with subgroup C viruses, for example Ad2 and Ad5 (Boulanger and Puvion 1973). Addition of purified fiber protein to virus suspensions during adsorption strongly inhibits infection, indicating that fiber and intact virus particles compete for binding sites on host cells (Philipson et al. 1968; Hautala et al. 1998). Cell binding studies using purified radiolabeled fiber demonstrated that fiber binds specifically and with high affinity to the cell plasma membrane, and that cell lines typically used for laboratory propagation of adenovirus have approximately 10{sup 4} high-affinity receptor sites per cell (Persson et al. 1985; Freimuth 1996). Similar numbers of high-affinity binding sites for radiolabeled intact virus particles also were observed (Seth et al. 1994).

  15. Cognitive enhancers (nootropics). Part 1: drugs interacting with receptors.

    PubMed

    Froestl, Wolfgang; Muhs, Andreas; Pfeifer, Andrea

    2012-01-01

    Cognitive enhancers (nootropics) are drugs to treat cognition deficits in patients suffering from Alzheimer's disease, schizophrenia, stroke, attention deficit hyperactivity disorder, or aging. Cognition refers to a capacity for information processing, applying knowledge, and changing preferences. It involves memory, attention, executive functions, perception, language, and psychomotor functions. The term nootropics was coined in 1972 when memory enhancing properties of piracetam were observed in clinical trials. In the meantime, hundreds of drugs have been evaluated in clinical trials or in preclinical experiments. To classify the compounds, a concept is proposed assigning drugs to 18 categories according to their mechanism(s) of action, in particular drugs interacting with receptors, enzymes, ion channels, nerve growth factors, re-uptake transporters, antioxidants, metal chelators, and disease-modifying drugs meaning small molecules, vaccines, and monoclonal antibodies interacting with amyloid-β and tau. For drugs, whose mechanism of action is not known, they are either classified according to structure, e.g., peptides, or their origin, e.g., natural products. The review covers the evolution of research in this field over the last 25 years.

  16. Control of leptin by metabolic state and its regulatory interactions with pituitary growth hormone and hepatic growth hormone receptors and insulin like growth factors in the tilapia (Oreochromis mossambicus).

    PubMed

    Douros, Jonathan D; Baltzegar, David A; Mankiewicz, Jamie; Taylor, Jordan; Yamaguchi, Yoko; Lerner, Darren T; Seale, Andre P; Grau, E Gordon; Breves, Jason P; Borski, Russell J

    2017-01-01

    Leptin is an important cytokine for regulating energy homeostasis, however, relatively little is known about its function and control in teleost fishes or other ectotherms, particularly with regard to interactions with the growth hormone (GH)/insulin-like growth factors (IGFs) growth regulatory axis. Here we assessed the regulation of LepA, the dominant paralog in tilapia (Oreochromis mossambicus) and other teleosts under altered nutritional state, and evaluated how LepA might alter pituitary growth hormone (GH) and hepatic insulin-like growth factors (IGFs) that are known to be disparately regulated by metabolic state. Circulating LepA, and lepa and lepr gene expression increased after 3-weeks fasting and declined to control levels 10days following refeeding. This pattern of leptin regulation by metabolic state is similar to that previously observed for pituitary GH and opposite that of hepatic GHR and/or IGF dynamics in tilapia and other fishes. We therefore evaluated if LepA might differentially regulate pituitary GH, and hepatic GH receptors (GHRs) and IGFs. Recombinant tilapia LepA (rtLepA) increased hepatic gene expression of igf-1, igf-2, ghr-1, and ghr-2 from isolated hepatocytes following 24h incubation. Intraperitoneal rtLepA injection, on the other hand, stimulated hepatic igf-1, but had little effect on hepatic igf-2, ghr1, or ghr2 mRNA abundance. LepA suppressed GH accumulation and gh mRNA in pituitaries in vitro, but had no effect on GH release. We next sought to test if abolition of pituitary GH via hypophysectomy (Hx) affects the expression of hepatic lepa and lepr. Hypophysectomy significantly increases hepatic lepa mRNA abundance, while GH replacement in Hx fish restores lepa mRNA levels to that of sham controls. Leptin receptor (lepr) mRNA was unchanged by Hx. In in vitro hepatocyte incubations, GH inhibits lepa and lepr mRNA expression at low concentrations, while higher concentration stimulates lepa expression. Taken together, these findings

  17. Role of TG-interacting factor (Tgif) in lipid metabolism.

    PubMed

    Pramfalk, Camilla; Eriksson, Mats; Parini, Paolo

    2015-01-01

    TG interacting factors (Tgifs) 1 and 2 are members of the TALE (three-amino-acid loop extension) superfamily of homeodomain proteins. These two proteins bind to the same DNA sequence and share a conserved C-terminal repression domain. Mutations in TGIF1 have been linked to holoprosencephaly, which is a human genetic disease that affects craniofacial development. As these proteins can interact with the ligand binding domain of retinoid X receptor α, a common heterodimeric partner of several nuclear receptors [e.g., liver X receptors (LXRs) and peroxisome proliferator-activated receptors (PPARs)], Tgif1 and Tgif2 might repress other transcriptional pathways activated by lipids. In line with this, Tgif1 interacts with LXRα and Tgif1 null mice have increased expression of the two Lxrα target genes apolipoproteins (Apo) c2 and a4. Also, we have recently identified Tgif1 to function as a transcriptional repressor of the cholesterol esterifying enzyme acyl-coenzyme A:cholesterol acyltransferase 2 (gene name SOAT2). As no studies yet have shown involvement of Tgif2 in the lipid metabolism, this review will focus on the role of Tgif1 in lipid and cholesterol metabolism. This article is part of a Special Issue entitled: Linking transcription to physiology in lipodomics. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Adenovirus-receptor interaction with human lymphocytes.

    PubMed

    Mentel, R; Döpping, G; Wegner, U; Seidel, W; Liebermann, H; Döhner, L

    1997-03-01

    Lymphocytes play a key role in cell-mediated immunity and are host cells for several viral and bacterial pathogens. Their importance in adenovirus (Ad) infections is not yet fully understood. The initial event, the attachment of Ad to lymphocytes and their subsets, was examined using flow cytometry. The study included analysis of stimulated T cells in binding assays with FITC-labeled Ad fiber. The results confirm that native peripheral lymphocytes express very small amounts of Ad receptors. Stimulation with PHA and interleukin 2 induced the expression. The presence of Ad DNA as a sign of internalization in stimulated cells was demonstrated using the polymerase chain reaction. The findings suggest that lymphocytes after stimulation can turn into target cells for Ad. This is particularly important if there are indications for persistence of Ad, and in the case of immunocompromised patients severe, life-threatening diseases can develop.

  19. Recombinant pigment epithelium-derived factor PEDF binds vascular endothelial growth factor receptors 1 and 2.

    PubMed

    Johnston, Erin K; Francis, Mary K; Knepper, Janice E

    2015-08-01

    Angiogenesis, or the formation of new blood vessels, is stimulated by angiogenic factors such as vascular endothelial growth factor (VEGF). Pigment epithelium-derived factor (PEDF) is a potent inhibitor of angiogenesis. To explore the mechanism by which PEDF acts, recombinant PEDF was expressed with a 6x-His tag (for purification) and a green fluorescent protein (GFP) tag. The PEDF fusion protein was confirmed to be active in inhibition of endothelial cell proliferation and migration. Direct binding of PEDF to both vascular endothelial growth factor receptor-1 (VEGFR-1) and VEGFR-2 was demonstrated in an in vitro assay similar to an enzyme-linked immunosorbent assay (ELISA). PEDF was shown by immune-confocal microscopy to be localized within treated endothelial cells. When VEGF-stimulated endothelial cells were incubated with PEDF the VEGF receptors showed intracellular localization. These data suggest that the interaction between PEDF and VEGFR-1 or VEGFR-2 may be a possible mechanism for inhibiting angiogenesis. PEDF may be binding to the VEGF receptors to promote their internalization and/or degradation to limit VEGF responses in treated cells.

  20. Interaction between the trout mineralocorticoid and glucocorticoid receptors in vitro.

    PubMed

    Kiilerich, Pia; Triqueneaux, Gérard; Christensen, Nynne Meyn; Trayer, Vincent; Terrien, Xavier; Lombès, Marc; Prunet, Patrick

    2015-08-01

    The salmonid corticosteroid receptors (CRs), glucocorticoid receptors 1 and 2 (GR1 and GR2) and the mineralocorticoid receptor (MR) share a high degree of homology with regard to structure, ligand- and DNA response element-binding, and cellular co-localization. Typically, these nuclear hormone receptors homodimerize to confer transcriptional activation of target genes, but a few studies using mammalian receptors suggest some degree of heterodimerization. We observed that the trout MR confers a several fold lower transcriptional activity compared to the trout GRs. This made us question the functional relevance of the MR when this receptor is located in the same cells as the GRs and activated by cortisol. A series of co-transfection experiments using different glucocorticoid response elements (GREs) containing promoter-reporter constructs were carried out to investigate any possible interaction between the piscine CRs. Co-transfection of the GRs with the MR significantly reduced the total transcriptional activity even at low MR levels, suggesting interaction between these receptors. Co-transfection of GR1 or GR2 with the MR did not affect the subcellular localization of the GRs, and the MR-mediated inhibition seemed to be independent of specific activation or inhibition of the MR. Site-directed mutagenesis of the DNA-binding domain and dimerization interface of the MR showed that the inhibition was dependent on DNA binding but not necessarily on dimerization ability. Thus, we suggest that the interaction between MR and the GRs may regulate the cortisol response in cell types where the receptors co-localize and propose a dominant-negative role for the MR in cortisol-mediated transcriptional activity. © 2015 Society for Endocrinology.

  1. Thermodynamic analysis of antagonist and agonist interactions with dopamine receptors.

    PubMed

    Duarte, E P; Oliveira, C R; Carvalho, A P

    1988-03-01

    The binding of [3H]spiperone to dopamine D-2 receptors and its inhibition by antagonists and agonists were examined in microsomes derived from the sheep caudate nucleus, at temperatures between 37 and 1 degree C, and the thermodynamic parameters of the binding were evaluated. The affinity of the receptor for the antagonists, spiperone and (+)-butaclamol, decreased as the incubation temperature decreased; the affinity for haloperidol did not further decrease at temperatures below 15 degrees C. The binding of the antagonists was associated with very large increases in entropy, as expected for hydrophobic interactions. The enthalpy and entropy changes associated with haloperidol binding were dependent on temperature, in contrast to those associated with spiperone and (+)-butaclamol. The magnitude of the entropy increase associated with the specific binding of the antagonists did not correlate with the degree of lipophilicity of these drugs. The data suggest that, in addition to hydrophobic forces, other forces are also involved in the antagonist-dopamine receptor interactions, and that a conformational change of the receptor could occur when the antagonist binds. Agonist binding data are consistent with a two-state model of the receptor, a high-affinity state (RH) and a low-affinity state (RL). The affinity of dopamine binding to the RH decreased with decreasing temperatures below 20 degrees C, whereas the affinity for the RL increased at low temperatures. In contrast, the affinity of apomorphine for both states of receptor decreased as the temperature decreased from 30 to 8 degrees C. A clear distinction between the energetics of high-affinity and low-affinity agonist binding was observed. The formation of the high-affinity complex was associated with larger increases in enthalpy and entropy than the interaction with the low-affinity state was. The results suggest that the interaction of the receptor with the G-proteins, induced or stabilized by the binding of

  2. Tissue Factor, Protease Activated Receptors and Pathologic Heart Remodeling

    PubMed Central

    Antoniak, Silvio; Sparkenbaugh, Erica; Pawlinski, Rafal

    2015-01-01

    Tissue factor is the primary initiator of coagulation cascade and plays an essential role in hemostasis and thrombosis. In addition, tissue factor and coagulation proteases contribute to the many cellular responses via activation of protease activated receptors. Heart is the organ demonstrating high levels of constitutive tissue factor expression. This review focuses on the role of tissue factor, coagulation proteases and protease activated receptors in heart hemostasis and the pathological heart remodeling associated with myocardial infarction, viral myocarditis and hypertension. PMID:25104210

  3. Epidermal Growth Factor Receptor Transactivation by the Cannabinoid Receptor (CB1) and Transient Receptor Potential Vanilloid 1 (TRPV1) Induces Differential Responses in Corneal Epithelial Cells

    DTIC Science & Technology

    2010-01-01

    Epidermal growth factor receptor transactivation by the cannabinoid receptor (CB1) and transient receptor potential vanilloid 1 ( TRPV1 ) induces...Available online 7 July 2010 Keywords: cannabinoid receptor 1 (CB1) transient receptor potential vanilloid 1 ( TRPV1 ) epidermal growth factor receptor (EGFR...release of endogenous metabolites that are cannabinoid receptor 1 (CB1) and transient receptor potential vanilloid 1 ( TRPV1 ) agonists. We determined

  4. The interaction of Clostridium perfringens enterotoxin with receptor claudins

    PubMed Central

    Shrestha, Archana; Uzal, Francisco A.; McClane, Bruce A.

    2016-01-01

    Clostridium perfringens enterotoxin (CPE) has significant medical importance due to its involvement in several common human gastrointestinal diseases. This 35 kDa single polypeptide toxin consists of two domains: a C-terminal domain involved in receptor binding and an N-terminal domain involved in oligomerization, membrane insertion and pore formation. The action of CPE starts with its binding to receptors, which include certain members of the claudin tight junction protein family; bound CPE then forms a series of complexes, one of which is a pore that causes the calcium influx responsible for host cell death. Recent studies have revealed that CPE binding to claudin receptors involves interactions between the C-terminal CPE domain and both the 1st and 2nd extracellular loops (ECL-1 and ECL-2) of claudin receptors. Of particular importance for this binding is the docking of ECL-2 into a pocket present in the C-terminal domain of the toxin. This increased understanding of CPE interactions with claudin receptors is now fostering the development of receptor decoy therapeutics for CPE-mediated gastrointestinal disease, reagents for cancer therapy/diagnoses and enhancers of drug delivery. PMID:27090847

  5. The interaction of Clostridium perfringens enterotoxin with receptor claudins.

    PubMed

    Shrestha, Archana; Uzal, Francisco A; McClane, Bruce A

    2016-10-01

    Clostridium perfringens enterotoxin (CPE) has significant medical importance due to its involvement in several common human gastrointestinal diseases. This 35 kDa single polypeptide toxin consists of two domains: a C-terminal domain involved in receptor binding and an N-terminal domain involved in oligomerization, membrane insertion and pore formation. The action of CPE starts with its binding to receptors, which include certain members of the claudin tight junction protein family; bound CPE then forms a series of complexes, one of which is a pore that causes the calcium influx responsible for host cell death. Recent studies have revealed that CPE binding to claudin receptors involves interactions between the C-terminal CPE domain and both the 1st and 2nd extracellular loops (ECL-1 and ECL-2) of claudin receptors. Of particular importance for this binding is the docking of ECL-2 into a pocket present in the C-terminal domain of the toxin. This increased understanding of CPE interactions with claudin receptors is now fostering the development of receptor decoy therapeutics for CPE-mediated gastrointestinal disease, reagents for cancer therapy/diagnoses and enhancers of drug delivery.

  6. Complex genomic interactions in the dynamic regulation of transcription by the glucocorticoid receptor.

    PubMed

    Miranda, Tina B; Morris, Stephanie A; Hager, Gordon L

    2013-11-05

    The glucocorticoid receptor regulates transcriptional output through complex interactions with the genome. These events require continuous remodeling of chromatin, interactions of the glucocorticoid receptor with chaperones and other accessory factors, and recycling of the receptor by the proteasome. Therefore, the cohort of factors expressed in a particular cell type can determine the physiological outcome upon treatment with glucocorticoid hormones. In addition, circadian and ultradian cycling of hormones can also affect GR response. Here we will discuss revision of the classical static model of GR binding to response elements to incorporate recent findings from single cell and genome-wide analyses of GR regulation. We will highlight how these studies have changed our views on the dynamics of GR recruitment and its modulation of gene expression.

  7. Insulin receptor: Interaction with nonreceptor glycoprotein from liver cell membranes

    PubMed Central

    Maturo, Joseph M.; Hollenberg, Morley D.

    1978-01-01

    In crude receptor preparations (either particulate or soluble) of rat liver membranes, the insulin receptor exhibits complicated binding kinetics (two binding plateaus, half-saturated at approximately 60 pM and 700 pM insulin) and an apparent chromatographic heterogeneity, suggested by the presence of two detectable, soluble insulin-binding components with apparent Stokes radii of 72 Å and 38 Å. In contrast, the insulin receptor isolated by affinity chromatography exhibits a simple binding isotherm (half-maximal saturation of binding at 700 pM insulin) without evidence for negative cooperativity and behaves as a single component (apparent Stokes radius of 38 Å) upon chromatography on Sepharose 6B. The apparent discrepancies between the properties of the unpurified insulin receptor and the affinity-purified receptor can be attributed to the presence in crude preparations of a nonreceptor constituent(s) having properties consistent with those of a membrane glycoprotein. A glycoprotein fraction from such crude soluble membrane preparations, freed from insulin receptor and subsequently partially purified using concanavalin-A-agarose, when combined with affinity-purified insulin receptor, causes both a reappearance of the complicated binding kinetics and an increase in the receptor's apparent Stokes radius from 38 Å to 72 Å. Similar results are observed for a glycoprotein fraction obtained from rat adipocyte membranes but are not observed for an identical fraction isolated from human erythrocyte membranes. We conclude that the insulin receptor in rat liver membranes can interact with another nonreceptor membrane glycoprotein that may represent either a nonrecognition moiety of the receptor oligomer or an effector molecule to the biological action of insulin. PMID:277909

  8. Tumor necrosis factor receptor- associated factor 6 (TRAF6) regulation of development, function, and homeostasis of the immune system.

    PubMed

    Walsh, Matthew C; Lee, JangEun; Choi, Yongwon

    2015-07-01

    Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is an adapter protein that mediates a wide array of protein-protein interactions via its TRAF domain and a RING finger domain that possesses non-conventional E3 ubiquitin ligase activity. First identified nearly two decades ago as a mediator of interleukin-1 receptor (IL-1R)-mediated activation of NFκB, TRAF6 has since been identified as an actor downstream of multiple receptor families with immunoregulatory functions, including members of the TNFR superfamily, the Toll-like receptor (TLR) family, tumor growth factorreceptors (TGFβR), and T-cell receptor (TCR). In addition to NFκB, TRAF6 may also direct activation of mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), and interferon regulatory factor pathways. In the context of the immune system, TRAF6-mediated signals have proven critical for the development, homeostasis, and/or activation of B cells, T cells, and myeloid cells, including macrophages, dendritic cells, and osteoclasts, as well as for organogenesis of thymic and secondary lymphoid tissues. In multiple cellular contexts, TRAF6 function is essential not only for proper activation of the immune system but also for maintaining immune tolerance, and more recent work has begun to identify mechanisms of contextual specificity for TRAF6, involving both regulatory protein interactions, and messenger RNA regulation by microRNAs.

  9. Structure-activity relationship of nuclear receptor-ligand interactions.

    PubMed

    Greschik, Holger; Moras, Dino

    2003-01-01

    Small molecules such as retinoids, steroid hormones, fatty acids, cholesterol metabolites, or xenobiotics are involved in the regulation of numerous physiological and patho-physiological processes by binding to and controlling the activity of members of the nuclear receptor (NR) superfamily of transcription factors. In addition to natural ligands, synthetic agonists or antagonists have been identified that in some cases specifically target NR isotypes, or elicit tissue-, signaling pathway-, or promoter-selective transcriptional responses. For these ligands the term "selective NR modulators" (SNRMs) has been introduced. Structure determination of apo- and holo-NR ligand-binding domains (LBDs)--some of them complexed to small coactivator or corepressor fragments--revealed the major principles of ligand-dependent NR action and determinants of (isotype-) selective ligand binding. These studies also stimulated the interpretation of tissue-specific effects of SNRMs on wild-type or mutant receptors. In contrast to the increasing knowledge on the structure-activity relationship of NRs with known SNRMs, rather basic questions remain about the regulation of orphan NRs (for which no ligands are known) or "adopted" orphan NRs (for which only recently ligands were identified). Several crystal structures of orphan NR LBDs uncovered unexpected properties, contributed to the understanding of orphan NR function, and may in the future permit the identification or design of ligands. This review will (i) focus on the current understanding of the structure-activity relationship of NR-ligand interactions, (ii) discuss recent advances in the field of "orphan" NR crystallography, and (iii) outline future challenges in NR structural biology.

  10. Binding interactions with the complementary subunit of nicotinic receptors.

    PubMed

    Blum, Angela P; Van Arnam, Ethan B; German, Laurel A; Lester, Henry A; Dougherty, Dennis A

    2013-03-08

    The agonist-binding site of nicotinic acetylcholine receptors (nAChRs) spans an interface between two subunits of the pentameric receptor. The principal component of this binding site is contributed by an α subunit, and it binds the cationic moiety of the nicotinic pharmacophore. The other part of the pharmacophore, a hydrogen bond acceptor, has recently been shown to bind to the complementary non-α subunit via the backbone NH of a conserved Leu. This interaction was predicted by studies of ACh-binding proteins and confirmed by functional studies of the neuronal (CNS) nAChR, α4β2. The ACh-binding protein structures further suggested that the hydrogen bond to the backbone NH is mediated by a water molecule and that a second hydrogen bonding interaction occurs between the water molecule and the backbone CO of a conserved Asn, also on the non-α subunit. Here, we provide new insights into the nature of the interactions between the hydrogen bond acceptor of nicotinic agonists and the complementary subunit backbone. We studied both the nAChR of the neuromuscular junction (muscle-type) and a neuronal subtype, (α4)2(β4)3. In the muscle-type receptor, both ACh and nicotine showed a strong interaction with the Leu NH, but the potent nicotine analog epibatidine did not. This interaction was much attenuated in the α4β4 receptor. Surprisingly, we found no evidence for a functionally significant interaction with the backbone carbonyl of the relevant Asn in either receptor with an array of agonists.

  11. Predicting drug-target interactions using probabilistic matrix factorization.

    PubMed

    Cobanoglu, Murat Can; Liu, Chang; Hu, Feizhuo; Oltvai, Zoltán N; Bahar, Ivet

    2013-12-23

    Quantitative analysis of known drug-target interactions emerged in recent years as a useful approach for drug repurposing and assessing side effects. In the present study, we present a method that uses probabilistic matrix factorization (PMF) for this purpose, which is particularly useful for analyzing large interaction networks. DrugBank drugs clustered based on PMF latent variables show phenotypic similarity even in the absence of 3D shape similarity. Benchmarking computations show that the method outperforms those recently introduced provided that the input data set of known interactions is sufficiently large--which is the case for enzymes and ion channels, but not for G-protein coupled receptors (GPCRs) and nuclear receptors. Runs performed on DrugBank after hiding 70% of known interactions show that, on average, 88 of the top 100 predictions hit the hidden interactions. De novo predictions permit us to identify new potential interactions. Drug-target pairs implicated in neurobiological disorders are overrepresented among de novo predictions.

  12. Structural basis of ligand interaction with atypical chemokine receptor 3

    NASA Astrophysics Data System (ADS)

    Gustavsson, Martin; Wang, Liwen; van Gils, Noortje; Stephens, Bryan S.; Zhang, Penglie; Schall, Thomas J.; Yang, Sichun; Abagyan, Ruben; Chance, Mark R.; Kufareva, Irina; Handel, Tracy M.

    2017-01-01

    Chemokines drive cell migration through their interactions with seven-transmembrane (7TM) chemokine receptors on cell surfaces. The atypical chemokine receptor 3 (ACKR3) binds chemokines CXCL11 and CXCL12 and signals exclusively through β-arrestin-mediated pathways, without activating canonical G-protein signalling. This receptor is upregulated in numerous cancers making it a potential drug target. Here we collected over 100 distinct structural probes from radiolytic footprinting, disulfide trapping, and mutagenesis to map the structures of ACKR3:CXCL12 and ACKR3:small-molecule complexes, including dynamic regions that proved unresolvable by X-ray crystallography in homologous receptors. The data are integrated with molecular modelling to produce complete and cohesive experimentally driven models that confirm and expand on the existing knowledge of the architecture of receptor:chemokine and receptor:small-molecule complexes. Additionally, we detected and characterized ligand-induced conformational changes in the transmembrane and intracellular regions of ACKR3 that elucidate fundamental structural elements of agonism in this atypical receptor.

  13. Aging-related dysregulation of dopamine and angiotensin receptor interaction.

    PubMed

    Villar-Cheda, Begoña; Dominguez-Meijide, Antonio; Valenzuela, Rita; Granado, Noelia; Moratalla, Rosario; Labandeira-Garcia, Jose L

    2014-07-01

    It is not known whether the aging-related decrease in dopaminergic function leads to the aging-related higher vulnerability of dopaminergic neurons and risk for Parkinson's disease. The renin-angiotensin system (RAS) plays a major role in the inflammatory response, neuronal oxidative stress, and dopaminergic vulnerability via type 1 (AT1) receptors. In the present study, we observed a counterregulatory interaction between dopamine and angiotensin receptors. We observed overexpression of AT1 receptors in the striatum and substantia nigra of young adult dopamine D1 and D2 receptor-deficient mice and young dopamine-depleted rats, together with compensatory overexpression of AT2 receptors or compensatory downregulation of angiotensinogen and/or angiotensin. In aged rats, we observed downregulation of dopamine and dopamine receptors and overexpression of AT1 receptors in aged rats, without compensatory changes observed in young animals. L-Dopa therapy inhibited RAS overactivity in young dopamine-depleted rats, but was ineffective in aged rats. The results suggest that dopamine may play an important role in modulating oxidative stress and inflammation in the substantia nigra and striatum via the RAS, which is impaired by aging.

  14. Structural basis of ligand interaction with atypical chemokine receptor 3

    PubMed Central

    Gustavsson, Martin; Wang, Liwen; van Gils, Noortje; Stephens, Bryan S.; Zhang, Penglie; Schall, Thomas J.; Yang, Sichun; Abagyan, Ruben; Chance, Mark R.; Kufareva, Irina; Handel, Tracy M.

    2017-01-01

    Chemokines drive cell migration through their interactions with seven-transmembrane (7TM) chemokine receptors on cell surfaces. The atypical chemokine receptor 3 (ACKR3) binds chemokines CXCL11 and CXCL12 and signals exclusively through β-arrestin-mediated pathways, without activating canonical G-protein signalling. This receptor is upregulated in numerous cancers making it a potential drug target. Here we collected over 100 distinct structural probes from radiolytic footprinting, disulfide trapping, and mutagenesis to map the structures of ACKR3:CXCL12 and ACKR3:small-molecule complexes, including dynamic regions that proved unresolvable by X-ray crystallography in homologous receptors. The data are integrated with molecular modelling to produce complete and cohesive experimentally driven models that confirm and expand on the existing knowledge of the architecture of receptor:chemokine and receptor:small-molecule complexes. Additionally, we detected and characterized ligand-induced conformational changes in the transmembrane and intracellular regions of ACKR3 that elucidate fundamental structural elements of agonism in this atypical receptor. PMID:28098154

  15. Interaction of G protein coupled receptors and cholesterol.

    PubMed

    Gimpl, Gerald

    2016-09-01

    G protein coupled receptors (GPCRs) form the largest receptor superfamily in eukaryotic cells. Owing to their seven transmembrane helices, large parts of these proteins are embedded in the cholesterol-rich plasma membrane bilayer. Thus, GPCRs are always in proximity to cholesterol. Some of them are functionally dependent on the specific presence of cholesterol. Over the last years, enormous progress on receptor structures has been achieved. While lipophilic ligands other than cholesterol have been shown to bind either inside the helix bundle or at the receptor-lipid interface, the binding site of cholesterol was either a single transmembrane helix or a groove between two or more transmembrane helices. A clear preference for one of the two membrane leaflets has not been observed. Not surprisingly, many hydrophobic residues (primarily leucine and isoleucine) were found to be involved in cholesterol binding. In most cases, the rough β-face of cholesterol contacted the transmembrane helix bundle rather than the surrounding lipid matrix. The polar hydroxy group of cholesterol was localized near the water-membrane interface with potential hydrogen bonding to residues in receptor loop regions. Although a canonical motif, designated as CCM site, was detected as a specific cholesterol binding site in case of the β2AR, this site was not found to be occupied by cholesterol in other GPCRs possessing the same motif. Cholesterol-receptor interactions can increase the compactness of the receptor structure and are able to enhance the conformational stability towards active or inactive receptor states. Overall, all current data suggest a high plasticity of cholesterol interaction sites in GPCRs.

  16. Substituted methcathinones differ in transporter and receptor interactions

    PubMed Central

    Eshleman, Amy J; Wolfrum, Katherine M; Hatfield, Meagan G; Johnson, Robert A; Murphy, Kevin V; Janowsky, Aaron

    2013-01-01

    The use of synthetic methcathinones, components of “bath salts,” is a world-wide health concern. These compounds, structurally similar to methamphetamine (METH) and 3,4-methylendioxymethamphetamine (MDMA), cause tachycardia, hallucinations and psychosis. We hypothesized that these potentially neurotoxic and abused compounds display differences in their transporter and receptor interactions as compared to amphetamine counterparts. 3,4-Methylenedioxypyrovalerone and naphyrone had high affinity for radioligand binding sites on recombinant human dopamine (hDAT), serotonin (hSERT) and norepinephrine (hNET) transporters, potently inhibited [3H]neurotransmitter uptake, and, like cocaine, did not induce transporter-mediated release. Butylone was a lower affinity uptake inhibitor. In contrast, 4-fluoromethcathinone, mephedrone and methylone had higher inhibitory potency at uptake compared to binding and generally induced release of preloaded [3H]neurotransmitter from hDAT, hSERT and hNET (highest potency at hNET), and thus are transporter substrates, similar to METH and MDMA. At hNET, 4-fluoromethcathinone was a more efficacious releaser than METH. These substituted methcathinones had low uptake inhibitory potency and low efficacy at inducing release via human vesicular monoamine transporters (hVMAT2). These compounds were low potency 1) h5-HT1A receptor partial agonists, 2) h5-HT2A receptor antagonists, 3) weak h5-HT2C receptor antagonists. This is the first report on aspects of substituted methcathinone efficacies at serotonin (5-HT) receptors and in superfusion release assays. Additionally, the drugs had no affinity for dopamine receptors, and high- mid-micromolar affinity for hSigma1 receptors. Thus, direct interactions with hVMAT2 and serotonin, dopamine, and hSigma1 receptors may not explain psychoactive effects. The primary mechanisms of action may be as inhibitors or substrates of DAT, SERT and NET. PMID:23583454

  17. The cellular response to neuregulins is governed by complex interactions of the erbB receptor family.

    PubMed Central

    Riese, D J; van Raaij, T M; Plowman, G D; Andrews, G C; Stern, D F

    1995-01-01

    Deregulated signaling by the four members of the epidermal growth factor receptor tyrosine kinase family (erbB family) is implicated in the genesis or progression of human cancers. However, efforts to analyze signaling by these receptors have been hampered by the diversity of ligands and extensive interreceptor cross talk. We have expressed the four human erbB family receptors, singly and in pairwise combinations, in a pro-B-lymphocyte cell line (Ba/F3) and investigated the range of interactions activated by the epidermal growth factor homology domain of the agonist neuregulin beta. The results provide the first comprehensive analysis of the response of this receptor family to a single peptide agonist. This peptide induced complex patterns of receptor tyrosine phosphorylation and regulation of Ba/F3 cell survival and proliferation. These data demonstrate the existence of several previously undocumented receptor interactions driven by neuregulin. PMID:7565730

  18. Modelling the interaction of steroid receptors with endocrine disrupting chemicals

    PubMed Central

    D'Ursi, Pasqualina; Salvi, Erika; Fossa, Paola; Milanesi, Luciano; Rovida, Ermanna

    2005-01-01

    Background The organic polychlorinated compounds like dichlorodiphenyltrichloroethane with its metabolites and polychlorinated biphenyls are a class of highly persistent environmental contaminants. They have been recognized to have detrimental health effects both on wildlife and humans acting as endocrine disrupters due to their ability of mimicking the action of the steroid hormones, and thus interfering with hormone response. There are several experimental evidences that they bind and activate human steroid receptors. However, despite the growing concern about the toxicological activity of endocrine disrupters, molecular data of the interaction of these compounds with biological targets are still lacking. Results We have used a flexible docking approach to characterize the molecular interaction of seven endocrine disrupting chemicals with estrogen, progesterone and androgen receptors in the ligand-binding domain. All ligands docked in the buried hydrophobic cavity corresponding to the hormone steroid pocket. The interaction was characterized by multiple hydrophobic contacts involving a different number of residues facing the binding pocket, depending on ligands orientation. The EDC ligands did not display a unique binding mode, probably due to their lipophilicity and flexibility, which conferred them a great adaptability into the hydrophobic and large binding pocket of steroid receptors. Conclusion Our results are in agreement with toxicological data on binding and allow to describe a pattern of interactions for a group of ECD to steroid receptors suggesting the requirement of a hydrophobic cavity to accommodate these chlorine carrying compounds. Although the affinity is lower than for hormones, their action can be brought about by a possible synergistic effect. PMID:16351736

  19. Modelling the interaction of steroid receptors with endocrine disrupting chemicals.

    PubMed

    D'Ursi, Pasqualina; Salvi, Erika; Fossa, Paola; Milanesi, Luciano; Rovida, Ermanna

    2005-12-01

    The organic polychlorinated compounds like dichlorodiphenyltrichloroethane with its metabolites and polychlorinated biphenyls are a class of highly persistent environmental contaminants. They have been recognized to have detrimental health effects both on wildlife and humans acting as endocrine disrupters due to their ability of mimicking the action of the steroid hormones, and thus interfering with hormone response. There are several experimental evidences that they bind and activate human steroid receptors. However, despite the growing concern about the toxicological activity of endocrine disrupters, molecular data of the interaction of these compounds with biological targets are still lacking. We have used a flexible docking approach to characterize the molecular interaction of seven endocrine disrupting chemicals with estrogen, progesterone and androgen receptors in the ligand-binding domain. All ligands docked in the buried hydrophobic cavity corresponding to the hormone steroid pocket. The interaction was characterized by multiple hydrophobic contacts involving a different number of residues facing the binding pocket, depending on ligands orientation. The EDC ligands did not display a unique binding mode, probably due to their lipophilicity and flexibility, which conferred them a great adaptability into the hydrophobic and large binding pocket of steroid receptors. Our results are in agreement with toxicological data on binding and allow to describe a pattern of interactions for a group of ECD to steroid receptors suggesting the requirement of a hydrophobic cavity to accommodate these chlorine carrying compounds. Although the affinity is lower than for hormones, their action can be brought about by a possible synergistic effect.

  20. The Two Faces of Receptor Interacting Protein Kinase-1

    PubMed Central

    Weinlich, Ricardo; Green, Douglas R.

    2014-01-01

    Receptor Interacting Protein Kinase-1 (RIPK1), a key player in inflammation and cell death, assumes opposite functions depending on the cellular context and its posttranslational modifications. Genetic evidence supported by biochemical and cellular biology approaches shed light on the circumstances in which RIPK1 promotes or inhibits these processes. PMID:25459879

  1. Mechanism of kinase activation in the receptor for colony-stimulating factor 1.

    PubMed Central

    Lee, A W; Nienhuis, A W

    1990-01-01

    Receptor tyrosine kinases remain dormant until activated by ligand binding to the extracellular domain. Two mechanisms have been proposed for kinase activation: (i) ligand binding to the external domain of a receptor monomer may induce a conformational change that is transmitted across the cell membrane (intramolecular model) or (ii) the ligand may facilitate oligomerization, thereby allowing interactions between the juxtaposed kinase domains (intermolecular model). The receptor for colony-stimulating factor 1 was used to test these models. Large insertions at the junction between the external and transmembrane domains of the receptor, introduced by site-directed mutagenesis of the cDNA, were positioned to isolate the external domain and prevent transmembrane conformational propagation while allowing for receptor oligomerization. Such mutant receptors were expressed on the cell surface, bound ligand with high affinity, exhibited ligand-stimulated autophosphorylation, and signaled mitogenesis and cellular proliferation in the presence of ligand. A second experimental strategy directly tested the intermolecular model of ligand activation. A hybrid receptor composed of the external domain of human glycophorin A and the transmembrane and cytoplasmic domains of the colony-stimulating factor 1 receptor exhibited anti-glycophorin antibody-induced kinase activity that supported mitogenesis. Our data strongly support a mechanism of receptor activation based on ligand-induced receptor oligomerization. Images PMID:2169623

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2013-05-07

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

  4. Mammary tumorigenesis induced by fibroblast growth factor receptor 1 requires activation of the epidermal growth factor receptor.

    PubMed

    Bade, Lindsey K; Goldberg, Jodi E; Dehut, Hazel A; Hall, Majken K; Schwertfeger, Kathryn L

    2011-09-15

    Fibroblast growth factor receptor 1 (FGFR1) is an oncoprotein with known involvement in mammary tumorigenesis. To understand how FGFR1 signaling promotes mammary tumorigenesis, an inducible FGFR1 (iFGFR1) system was created previously. Previous studies have demonstrated that upon iFGFR1 activation in vivo, the epidermal growth factor (EGF) ligands amphiregulin (AREG) and epiregulin (EREG) are upregulated. Both AREG and EREG interact with the EGF receptor (EGFR). Here, we investigated whether the FGFR1-induced increase in AREG and EREG expression might coordinately increase EGFR signaling to promote mammary tumorigenesis. Treatment of mouse mammary epithelial cells with either AREG or EREG conferred a greater migratory potential, increased cellular proliferation and increased extracellular regulated kinase 1/2 (ERK1/2) activation. These effects could be blocked with the EGFR-specific inhibitor erlotinib, suggesting that they are EGFR-dependent. In transgenic mice with iFGFR1 under the control of the mouse mammary tumor virus (MMTV) promoter, iFGFR1 activation also led to increased mammary epithelial cell proliferation that was inhibited with erlotinib. Taken together, these data suggest that AREG and EREG mediate tumorigenic phenotypes by activating EGFR signaling, and that the oncogenic potential of FGFR1 requires EGFR activation to promote mammary tumorigenesis.

  5. 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.

  6. Synergistic Regulation of Coregulator/Nuclear Receptor Interaction by Ligand and DNA.

    PubMed

    de Vera, Ian Mitchelle S; Zheng, Jie; Novick, Scott; Shang, Jinsai; Hughes, Travis S; Brust, Richard; Munoz-Tello, Paola; Gardner, William J; Marciano, David P; Kong, Xiangming; Griffin, Patrick R; Kojetin, Douglas J

    2017-10-03

    Nuclear receptor (NR) transcription factors bind various coreceptors, small-molecule ligands, DNA response element sequences, and transcriptional coregulator proteins to affect gene transcription. Small-molecule ligands and DNA are known to influence receptor structure, coregulator protein interaction, and function; however, little is known on the mechanism of synergy between ligand and DNA. Using quantitative biochemical, biophysical, and solution structural methods, including (13)C-detected nuclear magnetic resonance and hydrogen/deuterium exchange (HDX) mass spectrometry, we show that ligand and DNA cooperatively recruit the intrinsically disordered steroid receptor coactivator-2 (SRC-2/TIF2/GRIP1/NCoA-2) receptor interaction domain to peroxisome proliferator-activated receptor gamma-retinoid X receptor alpha (PPARγ-RXRα) heterodimer and reveal the binding determinants of the complex. Our data reveal a thermodynamic mechanism by which DNA binding propagates a conformational change in PPARγ-RXRα, stabilizes the receptor ligand binding domain dimer interface, and impacts ligand potency and cooperativity in NR coactivator recruitment. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Structure of the IGF-binding domain of the insulin-like growth factor-binding protein-5 (IGFBP-5): implications for IGF and IGF-I receptor interactions.

    PubMed Central

    Kalus, W; Zweckstetter, M; Renner, C; Sanchez, Y; Georgescu, J; Grol, M; Demuth, D; Schumacher, R; Dony, C; Lang, K; Holak, T A

    1998-01-01

    Binding proteins for insulin-like growth factors (IGFs) IGF-I and IGF-II, known as IGFBPs, control the distribution, function and activity of IGFs in various cell tissues and body fluids. Insulin-like growth factor-binding protein-5 (IGFBP-5) is known to modulate the stimulatory effects of IGFs and is the major IGF-binding protein in bone tissue. We have expressed two N-terminal fragments of IGFBP-5 in Escherichia coli; the first encodes the N-terminal domain of the protein (residues 1-104) and the second, mini-IGFBP-5, comprises residues Ala40 to Ile92. We show that the entire IGFBP-5 protein contains only one high-affinity binding site for IGFs, located in mini-IGFBP-5. The solution structure of mini-IGFBP-5, determined by nuclear magnetic resonance spectroscopy, discloses a rigid, globular structure that consists of a centrally located three-stranded anti-parallel beta-sheet. Its scaffold is stabilized further by two inside packed disulfide bridges. The binding to IGFs, which is in the nanomolar range, involves conserved Leu and Val residues localized in a hydrophobic patch on the surface of the IGFBP-5 protein. Remarkably, the IGF-I receptor binding assays of IGFBP-5 showed that IGFBP-5 inhibits the binding of IGFs to the IGF-I receptor, resulting in reduction of receptor stimulation and autophosphorylation. Compared with the full-length IGFBP-5, the smaller N-terminal fragments were less efficient inhibitors of the IGF-I receptor binding of IGFs. PMID:9822601

  8. [The Cytoskelrtal Protein Zvxin Interacts with the Hedgehog Receptor Patched].

    PubMed

    Martynova, N U; Ermolina, L V; Eroshkin, F M; Zarayskiy, A G

    2015-01-01

    Earlier, we demonstrated Zyxin influence upon Hedgehog (Hh)-signaling pathway during early patterning of the central neural system (CNS) anlage of the Xenopus laevis embryo. Now we show that Zyxin can physically interact with the transmembrane receptor of Hh, Patched2 (Ptc2). Binding of Hh by this receptor activates signaling pathway, which regulates many events, including numerous types of cell differentiation during the embryonic development. In particular, patterning of the CNS anlage. The ability of Zyxin to interact with Ptc2 have been confirmed by immunoprecipitation experiments, in which we tested mutual binding affinity of Zyxin and Ptc2, as well as mutual affinity of their deletion mutants. As a result, we have established that in Xenopus levis, Zyxin binding to Ptc2 is due to the interaction of Zyxin 2nd LIM-domain (530-590 aa) with the under-membrane region of the cytoplasmic C-terminus of Ptc2 (1159-1412 aa). We have also demonstrated that similar interaction is valid for the homologous regions of the human Zyxin and human Hh receptor, Ptc1. The data obtained allow to hypothesize existence of evolutionary conserved mechanism that modulates Hh-signaling and based on the interaction of Zyxin with Ptc.

  9. Heterodimeric coiled-coil interactions of human GABAB receptor

    PubMed Central

    Burmakina, Svetlana; Geng, Yong; Chen, Yan; Fan, Qing R.

    2014-01-01

    Metabotropic GABAB receptor is a G protein-coupled receptor that mediates inhibitory neurotransmission in the CNS. It functions as an obligatory heterodimer of GABAB receptor 1 (GBR1) and GABAB receptor 2 (GBR2) subunits. The association between GBR1 and GBR2 masks an endoplasmic reticulum (ER) retention signal in the cytoplasmic region of GBR1 and facilitates cell surface expression of both subunits. Here, we present, to our knowledge, the first crystal structure of an intracellular coiled-coil heterodimer of human GABAB receptor. We found that polar interactions buried within the hydrophobic core determine the specificity of heterodimer pairing. Disruption of the hydrophobic coiled-coil interface with single mutations in either subunit impairs surface expression of GBR1, confirming that the coiled-coil interaction is required to inactivate the adjacent ER retention signal of GBR1. The coiled-coil assembly buries an internalization motif of GBR1 at the heterodimer interface. The ER retention signal of GBR1 is not part of the core coiled-coil structure, suggesting that it is sterically shielded by GBR2 upon heterodimer formation. PMID:24778228

  10. Interaction of Plant Extracts with Central Nervous System Receptors

    PubMed Central

    Lundstrom, Kenneth; Pham, Huyen Thanh; Dinh, Long Doan

    2017-01-01

    Background: Plant extracts have been used in traditional medicine for the treatment of various maladies including neurological diseases. Several central nervous system receptors have been demonstrated to interact with plant extracts and components affecting the pharmacology and thereby potentially playing a role in human disease and treatment. For instance, extracts from Hypericum perforatum (St. John’s wort) targeted several CNS receptors. Similarly, extracts from Piper nigrum, Stephania cambodica, and Styphnolobium japonicum exerted inhibition of agonist-induced activity of the human neurokinin-1 receptor. Methods: Different methods have been established for receptor binding and functional assays based on radioactive and fluorescence-labeled ligands in cell lines and primary cell cultures. Behavioral studies of the effect of plant extracts have been conducted in rodents. Plant extracts have further been subjected to mood and cognition studies in humans. Results: Mechanisms of action at molecular and cellular levels have been elucidated for medicinal plants in support of standardization of herbal products and identification of active extract compounds. In several studies, plant extracts demonstrated affinity to a number of CNS receptors in parallel indicating the complexity of this interaction. In vivo studies showed modifications of CNS receptor affinity and behavioral responses in animal models after treatment with medicinal herbs. Certain plant extracts demonstrated neuroprotection and enhanced cognitive performance, respectively, when evaluated in humans. Noteworthy, the penetration of plant extracts and their protective effect on the blood-brain-barrier are discussed. Conclusion: The affinity of plant extracts and their isolated compounds for CNS receptors indicates an important role for medicinal plants in the treatment of neurological disorders. Moreover, studies in animal and human models have confirmed a scientific basis for the application of medicinal

  11. Evidence for Heterodimerization and Functional Interaction of the Angiotensin Type 2 Receptor and the Receptor MAS.

    PubMed

    Leonhardt, Julia; Villela, Daniel C; Teichmann, Anke; Münter, Lisa-Marie; Mayer, Magnus C; Mardahl, Maibritt; Kirsch, Sebastian; Namsolleck, Pawel; Lucht, Kristin; Benz, Verena; Alenina, Natalia; Daniell, Nicholas; Horiuchi, Masatsugu; Iwai, Masaru; Multhaup, Gerhard; Schülein, Ralf; Bader, Michael; Santos, Robson A; Unger, Thomas; Steckelings, Ulrike Muscha

    2017-06-01

    The angiotensin type 2 receptor (AT2R) and the receptor MAS are receptors of the protective arm of the renin-angiotensin system. They mediate strikingly similar actions. Moreover, in various studies, AT2R antagonists blocked the effects of MAS agonists and vice versa. Such cross-inhibition may indicate heterodimerization of these receptors. Therefore, this study investigated the molecular and functional interplay between MAS and the AT2R. Molecular interactions were assessed by fluorescence resonance energy transfer and by cross correlation spectroscopy in human embryonic kidney-293 cells transfected with vectors encoding fluorophore-tagged MAS or AT2R. Functional interaction of AT2R and MAS was studied in astrocytes with CX3C chemokine receptor-1 messenger RNA expression as readout. Coexpression of fluorophore-tagged AT2R and MAS resulted in a fluorescence resonance energy transfer efficiency of 10.8 ± 0.8%, indicating that AT2R and MAS are capable to form heterodimers. Heterodimerization was verified by competition experiments using untagged AT2R and MAS. Specificity of dimerization of AT2R and MAS was supported by lack of dimerization with the transient receptor potential cation channel, subfamily C-member 6. Dimerization of the AT2R was abolished when it was mutated at cysteine residue 35. AT2R and MAS stimulation with the respective agonists, Compound 21 or angiotensin-(1-7), significantly induced CX3C chemokine receptor-1 messenger RNA expression. Effects of each agonist were blocked by an AT2R antagonist (PD123319) and also by a MAS antagonist (A-779). Knockout of a single of these receptors made astrocytes unresponsive for both agonists. Our results suggest that MAS and the AT2R form heterodimers and that-at least in astrocytes-both receptors functionally depend on each other. © 2017 American Heart Association, Inc.

  12. Amiloride interacts with renal. cap alpha. - and. beta. -adrenergic receptors

    SciTech Connect

    Howard, M.J.; Mullen, M.D.; Insel, P.A.

    1987-07-01

    The authors have used radioligand binding techniques to assess whether amiloride and certain analogues of amiloride (ethylisopropyl amiloride and benzamil) can bind to adrenergic receptors in the kidney. They found that amiloride could compete for (/sup 3/H)rauwolscine (..cap alpha../sub 2/-adrenergic receptors), (/sup 3/H)prazosin (..cap alpha../sub 1/-adrenergic receptors), and (/sup 125/I)iodocyanopindolol (..beta..-adrenergic receptors) binding in rat renal cortical membranes with inhibitor constants of 13.6 /plus minus/ 5.7, 24.4 /plus minus/ 7.4, and 8.36 /plus minus/ 13.5 ..mu..M, respectively. Ethylisopropyl amiloride and benzamil were from 2- to 25-fold more potent than amiloride in competing for radioligand binding sites in studies with these membranes. In addition, amiloride and the two analogues competed for (/sup 3/H)prazosin sites on intact Madin-Darby canine kidney cells and amiloride blocked epinephrine-stimulated prostaglandin E/sub 2/ production in these cells. They conclude that amiloride competes for binding to several classes of renal adrenergic receptors with a rank order of potency of ..cap alpha../sub 2/ > ..cap alpha../sub 1/ > ..beta... Binding to, and antagonism of, adrenergic receptors occurs at concentrations of amiloride that are lower than previously observed nonspecific interactions of this agent.

  13. Approaches for Probing Allosteric Interactions at 7 Transmembrane Spanning Receptors

    PubMed Central

    Klein, Michael T.; Vinson, Paige N.; Niswender, Colleen M.

    2017-01-01

    orthosteric ligand–receptor interactions. In this review, we discuss the current methodologies being used to identify and characterize allosteric modulators, lending insight into the approaches that have been most successful in accurately and robustly identifying hit compounds. New label-free technologies capable of detecting phenotypic cellular changes in response to receptor activation are powerful tools well suited for assessing subtle or potentially masked cellular responses to allosteric modulation of 7TMRs. Allosteric modulator-induced receptor signal bias and the assay systems available to probe the various downstream signaling outcomes of receptor activation are also discussed. PMID:23415091

  14. The Pseudo Signal Peptide of the Corticotropin-releasing Factor Receptor Type 2A Prevents Receptor Oligomerization*

    PubMed Central

    Teichmann, Anke; Rutz, Claudia; Kreuchwig, Annika; Krause, Gerd; Wiesner, Burkhard; Schülein, Ralf

    2012-01-01

    N-terminal signal peptides mediate the interaction of native proteins with the translocon complex of the endoplasmic reticulum membrane and are cleaved off during early protein biogenesis. The corticotropin-releasing factor receptor type 2a (CRF2(a)R) possesses an N-terminal pseudo signal peptide, which represents a so far unique domain within the large protein family of G protein-coupled receptors (GPCRs). In contrast to a conventional signal peptide, the pseudo signal peptide remains uncleaved and consequently forms a hydrophobic extension at the N terminus of the receptor. The functional consequence of the presence of the pseudo signal peptide is not understood. Here, we have analyzed the significance of this domain for receptor dimerization/oligomerization in detail. To this end, we took the CRF2(a)R and the homologous corticotropin-releasing factor receptor type 1 (CRF1R) possessing a conventional cleaved signal peptide and conducted signal peptide exchange experiments. Using single cell and single molecule imaging methods (fluorescence resonance energy transfer and fluorescence cross-correlation spectroscopy, respectively) as well as biochemical experiments, we obtained two novel findings; we could show that (i) the CRF2(a)R is expressed exclusively as a monomer, and (ii) the presence of the pseudo signal peptide prevents its oligomerization. Thus, we have identified a novel functional domain within the GPCR protein family, which plays a role in receptor oligomerization and which may be useful to study the functional significance of this process in general. PMID:22689579

  15. Sperm Epidermal Growth Factor Receptor (EGFR) Mediates α7 Acetylcholine Receptor (AChR) Activation to Promote Fertilization

    PubMed Central

    Jaldety, Yael; Glick, Yair; Orr-Urtreger, Avi; Ickowicz, Debby; Gerber, Doron; Breitbart, Haim

    2012-01-01

    To attain fertilization the spermatozoon binds to the egg zona pellucida (ZP) via sperm receptor(s) and undergoes an acrosome reaction (AR). Several sperm receptors have been described in the literature; however, the identity of this receptor is not yet certain. In this study, we suggest that the α7 nicotinic acetylcholine receptor (α7nAChR) might be a sperm receptor activated by ZP to induce epidermal growth factor receptor (EGFR)-mediated AR. We found that isolated ZP or α7 agonists induced the AR in sperm from WT but not α7-null spermatozoa, and the induced AR was inhibited by α7 or EGFR antagonists. Moreover, α7-null sperm showed very little binding to the egg, and microfluidic affinity in vitro assay clearly showed that α7nAChR, as well as EGFR, interacted with ZP3. Induction of EGFR activation and the AR by an α7 agonist was inhibited by a Src family kinase (SFK) inhibitor. In conclusion we suggest that activation of α7 by ZP leads to SFK-dependent EGFR activation, Ca2+ influx, and the acrosome reaction. PMID:22577141

  16. Interaction of rat liver glucocorticoid receptor with sodium tungstate.

    PubMed

    Murakami, N; Healy, S P; Moudgil, V K

    1982-06-15

    Effects of sodium tungstate on various properties of rat liver glucocorticoid receptor were examined at pH7 and pH 8. At pH 7, [3H]triamcinolone acetonide binding in rat liver cytosol preparations was completely blocked in the presence of 10--20 mM-sodium tungstate at 4 degrees C, whereas at 37 degrees C a 30 min incubation of cytosol receptor preparation with 1 mM-sodium tungstate reduced the loss of unoccupied receptor by 50%. At pH 8.0, tungstate presence during the 37 degrees C incubation maintained the steroid-binding capacity of unoccupied glucocorticoid receptor at control (4 degrees C) levels. In addition, heat-activation of cytosolic glucocorticoid-receptor complex was blocked by 1 mM- and 10 mM-sodium tungstate at pH 7 and pH 8 respectively. The DNA-cellulose binding by activated receptor was also inhibited completely and irreversibly by 5 mM-tungstate at pH 7, whereas at pH 8 no significant effect was observed with up to 20 mM-tungstate. The entire DNA-cellulose-bound glucocorticoid-receptor complex from control samples could be extracted by incubation with 1 mM- and 20 mM-tungstate at pH 7 and pH 8 respectively, and appeared to sediment as a 4.3--4.6 S molecule, both in 0.01 M- and 0.3 M-KCl-containing sucrose gradients. Tungstate effects are, therefore, pH-dependent and appear to involve an interaction with both the non-activated and the activated forms of the glucocorticoid receptor.

  17. Argos inhibits epidermal growth factor receptor signalling by ligand sequestration.

    PubMed

    Klein, Daryl E; Nappi, Valerie M; Reeves, Gregory T; Shvartsman, Stanislav Y; Lemmon, Mark A

    2004-08-26

    The epidermal growth factor receptor (EGFR) has critical functions in development and in many human cancers. During development, the spatial extent of EGFR signalling is regulated by feedback loops comprising both well-understood activators and less well-characterized inhibitors. In Drosophila melanogaster the secreted protein Argos functions as the only known extracellular inhibitor of EGFR, with clearly identified roles in multiple stages of development. Argos is only expressed when the Drosophila EGFR (DER) is activated at high levels, and downregulates further DER signalling. Although there is ample genetic evidence that Argos inhibits DER activation, the biochemical mechanism has not been established. Here we show that Argos inhibits DER signalling without interacting directly with the receptor, but instead by sequestering the DER-activating ligand Spitz. Argos binds tightly to the EGF motif of Spitz and forms a 1:1 (Spitz:Argos) complex that does not bind DER in vitro or at the cell surface. Our results provide an insight into the mechanism of Argos function, and suggest new strategies for EGFR inhibitor design.

  18. Autophosphorylation is essential for the in vivo function of the Lotus japonicus Nod factor receptor 1 and receptor-mediated signalling in cooperation with Nod factor receptor 5.

    PubMed

    Madsen, Esben B; Antolín-Llovera, Meritxell; Grossmann, Christina; Ye, Juanying; Vieweg, Syndi; Broghammer, Angelique; Krusell, Lene; Radutoiu, Simona; Jensen, Ole N; Stougaard, Jens; Parniske, Martin

    2011-02-01

    Soil-living rhizobia secrete lipochitin oligosaccharides known as Nod factors, which in Lotus japonicus are perceived by at least two Nod-factor receptors, NFR1 and NFR5. Despite progress in identifying molecular components critical for initial legume host recognition of the microsymbiont and cloning of downstream components, little is known about the activation and signalling mechanisms of the Nod-factor receptors themselves. Here we show that both receptor proteins localize to the plasma membrane, and present evidence for heterocomplex formation initiating downstream signalling. Expression of NFR1 and NFR5 in Nicotiana benthamiana and Allium ampeloprasum (leek) cells caused a rapid cell-death response. The signalling leading to cell death was abrogated using a kinase-inactive variant of NFR1. In these surviving cells, a clear interaction between NFR1 and NFR5 was detected in vivo through bimolecular fluorescence complementation (BiFC). To analyse the inter- and intramolecular phosphorylation events of the kinase complex, the cytoplasmic part of NFR1 was assayed for in vitro kinase activity, and autophosphorylation on 24 amino acid residues, including three tyrosine residues, was found by mass spectrometry. Substitution of the phosphorylated amino acids of NFR1 identified a single phosphorylation site to be essential for NFR1 Nod-factor signalling in vivo and kinase activity in vitro. In contrast to NFR1, no in vitro kinase activity of the cytoplasmic domain of NFR5 was detected. This is further supported by the fact that a mutagenized NFR5 construct, substituting an amino acid essential for ATP binding, restored nodulation of nfr5 mutant roots. © 2010 The Authors. The Plant Journal © 2010 Blackwell Publishing Ltd.

  19. Interacting residues in an activated state of a G protein-coupled receptor.

    PubMed

    Lee, Yong-Hun; Naider, Fred; Becker, Jeffrey M

    2006-01-27

    Ste2p, the G protein-coupled receptor (GPCR) for the tridecapeptide pheromone alpha-factor of Saccharomyces cerevisiae, was used as a model GPCR to investigate the role of specific residues in the resting and activated states of the receptor. Using a series of biological and biochemical analyses of wild-type and site-directed mutant receptors, we identified Asn(205) as a potential interacting partner with the Tyr(266) residue. An N205H/Y266H double mutant showed pH-dependent functional activity, whereas the N205H receptor was non-functional and the Y266H receptor was partially active indicating that the histidine 205 and 266 residues interact in an activated state of the receptor. The introduction of N205K or Y266D mutations into the P258L/S259L constitutively active receptor suppressed the constitutive activity; in contrast, the N205K/Y266D/P258L/S259L quadruple mutant was fully constitutively active, again indicating an interaction between residues at the 205 and 206 positions in the receptor-active state. To further test this interaction, we introduced the N205C/Y266C, F204C/Y266C, and N205C/A265C double mutations into wild-type and P258L/S259L constitutively active receptors. After trypsin digestion, we found that a disulfide-cross-linked product, with the molecular weight expected for a receptor fragment with a cross-link between N205C and Y266C, formed only in the N205C/Y266C constitutively activated receptor. This study represents the first experimental demonstration of an interaction between specific residues in an active state, but not the resting state, of Ste2p. The information gained from this study should contribute to an understanding of the conformational differences between resting and active states in GPCRs.

  20. Imaging receptors and their interactions: Implications for psychiatry

    SciTech Connect

    Brodie, J.D.; Wolkin, A.; Barouche, F.; Rotrosen, J.; Angrist, B. . Dept. of Psychiatry); Fowler, J.S.; Wolf, A.P.; Dewey, S.L.; Volkow, N.D.; MacGregor, R.; Schlyer, D.J.; Bendriem, B. )

    1989-01-01

    In the past ten years various receptor ligands for dopaminergic systems have been labeled and their regional distribution and time course imaged using positron emission tomography (PET). Labeled compounds have been developed to probe the opiate, benzodiazepine and serotonin receptors among others. The interest in evaluating dopamine receptors in the human brain has clearly been related to the known anti-psychotic effect of dopamine antagonists. In the present report, we shall summarize some of our recent findings on the dopamine (DA) system which bear on the psychiatric issues of the objective determination and monitoring of adequate neuroleptic dose; whether neuroleptic non-response is due to a failure of drug delivery; and preliminary data on the cholinergic system and its putative interaction with the DA system. 6 refs., 2 figs.

  1. Solution structures and molecular interactions of selective melanocortin receptor antagonists.

    PubMed

    Lee, Chul-Jin; Yun, Ji-Hye; Lim, Sung-Kil; Lee, Weontae

    2010-12-01

    The solution structures and inter-molecular interaction of the cyclic melanocortin antagonists SHU9119, JKC363, HS014, and HS024 with receptor molecules have been determined by NMR spectroscopy and molecular modeling. While SHU9119 is known as a nonselective antagonist, JKC363, HS014, and HS024 are selective for the melanocortin subtype-4 receptor (MC4R) involved in modulation of food intake. Data from NMR and molecular dynamics suggest that the conformation of the Trp9 sidechain in the three MC4R-selective antagonists is quite different from that of SHU9119. This result strongly supports the concept that the spatial orientation of the hydrophobic aromatic residue is more important for determining selectivity than the presence of a basic, "arginine-like" moiety responsible for biological activity. We propose that the conformation of hydrophobic residues of MCR antagonists is critical for receptor-specific selectivity.

  2. Direct interaction of ligand–receptor pairs specifying stomatal patterning

    PubMed Central

    Lee, Jin Suk; Kuroha, Takeshi; Hnilova, Marketa; Khatayevich, Dmitriy; Kanaoka, Masahiro M.; McAbee, Jessica M.; Sarikaya, Mehmet; Tamerler, Candan; Torii, Keiko U.

    2012-01-01

    Valves on the plant epidermis called stomata develop according to positional cues, which likely involve putative ligands (EPIDERMAL PATTERNING FACTORS [EPFs]) and putative receptors (ERECTA family receptor kinases and TOO MANY MOUTHS [TMM]) in Arabidopsis. Here we report the direct, robust, and saturable binding of bioactive EPF peptides to the ERECTA family. In contrast, TMM exhibits negligible binding to EPF1 but binding to EPF2. The ERECTA family forms receptor homomers in vivo. On the other hand, TMM associates with the ERECTA family but not with itself. While ERECTA family receptor kinases exhibit complex redundancy, blocking ERECTA and ERECTA-LIKE1 (ERL1) signaling confers specific insensitivity to EPF2 and EPF1, respectively. Our results place the ERECTA family as the primary receptors for EPFs with TMM as a signal modulator and establish EPF2–ERECTA and EPF1–ERL1 as ligand–receptor pairs specifying two steps of stomatal development: initiation and spacing divisions. PMID:22241782

  3. Functional interaction of nuclear receptor coactivator 4 with aryl hydrocarbon receptor

    SciTech Connect

    Kollara, Alexandra; Brown, Theodore J. . E-mail: brown@mshri.on.ca

    2006-07-28

    Aryl hydrocarbon receptor (AhR) transcriptional activity is enhanced by interaction with p160 coactivators. We demonstrate here that NcoA4, a nuclear receptor coactivator, interacts with and amplifies AhR action. NcoA4-AhR and NcoA4-ARNT interactions were demonstrated by immunoprecipitation in T47D breast cancer and COS cells and was independent of ligand. Overexpression of NcoA4 enhanced AhR transcriptional activity 3.2-fold in the presence of dioxin, whereas overexpression of a splice variant, NcoA4{beta}, as well as a variant lacking the C-terminal region enhanced AhR transcriptional activity by only 1.6-fold. Enhanced AhR signaling by NcoA4 was independent of the LXXLL and FXXLF motifs or of the activation domain. NcoA4 protein localized to cytoplasm in the absence of dioxin and in both the cytoplasm and nucleus following dioxin treatment. NcoA4-facilitation of AhR activity was abolished by overexpression of androgen receptor, suggesting a potential competition of AhR and androgen receptor for NcoA4. These findings thus demonstrate a functional interaction between NcoA4 and AhR that may alter AhR activity to affect disease development and progression.

  4. CD95 death receptor and epidermal growth factor receptor (EGFR) in liver cell apoptosis and regeneration.

    PubMed

    Reinehr, Roland; Häussinger, Dieter

    2012-02-01

    Recent evidence suggests that signaling pathways towards cell proliferation and cell death are much more interconnected than previously thought. Whereas not only death receptors such as CD95 (Fas, APO-1) can couple to both, cell death and proliferation, also growth factor receptors such as the epidermal growth factor receptor (EGFR) are involved in these opposing kinds of cell fate. EGFR is briefly discussed as a growth factor receptor involved in liver cell proliferation during liver regeneration. Then the role of EGFR in activating CD95 death receptor in liver parenchymal cells (PC) and hepatic stellate cells (HSC), which represent a liver stem/progenitor cell compartment, is described summarizing different ways of CD95- and EGFR-dependent signaling in the liver. Here, depending on the hepatic cell type (PC vs. HSC) and the respective signaling context (sustained vs. transient JNK activation) CD95-/EGFR-mediated signaling ends up in either liver cell apoptosis or cell proliferation.

  5. Envelope-receptor interactions in Nipah virus pathobiology.

    PubMed

    Lee, Benhur

    2007-04-01

    Nipah (NiV) and Hendra (HeV) viruses are members of the newly defined Henipavirus genus of the Paramyxoviridae. Nipah virus (NiV) is an emergent paramyxovirus that causes fatal encephalitis in up to 70% of infected patients, and there is increasing evidence of human-to-human transmission. NiV is designated a priority pathogen in the NIAID Biodefense Research Agenda, and could be a devastating agent of agrobioterrorism if used against the pig farming industry. Endothelial syncytium is a pathognomonic feature of NiV infections, and is mediated by the fusion (F) and attachment (G) envelope glycoproteins. This review summarizes what is known about the pathophysiology of NiV infections, and documents the identification of the NiV receptor. EphrinB2, the NiV and HeV receptor, is expressed on endothelial cells and neurons, consistent with the known cellular tropism for NiV. We discuss how the identification of the henipahvirus receptor sheds light on the pathobiology of NiV infection, and how it will spur the rational development of effective therapeutics. In addition, ephrinB3, a related protein, can serve as an alternative receptor, and we suggest that differential usage of ephrinB2 versus B3 may explain the variant pathogenic profiles observed between NiV and HeV. Thus, identifying the NiV receptors opens the door for a more comprehensive analysis of the envelope-receptor interactions in NiV pathobiology. Finally, we also describe how galectin-1 (an innate immune defense lectin) can interact with specific N-glycans on the Nipah envelope fusion protein, underscoring the potential role that innate immune defense mechanisms may play against emerging pathogens.

  6. Interaction of a radiolabeled agonist with cardiac muscarinic cholinergic receptors

    SciTech Connect

    Harden, T.K.; Meeker, R.B.; Martin, M.W.

    1983-12-01

    The interaction of a radiolabeled muscarinic cholinergic receptor agonist, (methyl-/sup 3/H)oxotremorine acetate ((/sup 3/H)OXO), with a washed membrane preparation derived from rat heart, has been studied. In binding assays at 4 degrees C, the rate constants for association and dissociation of (/sup 3/H)OXO were 2 X 10(7) M-1 min-1 and 5 X 10(-3) min-1, respectively, Saturation binding isotherms indicated that binding was to a single population of sites with a Kd of approximately 300 pM. The density of (/sup 3/H)OXO binding sites (90-100 fmol/mg of protein) was approximately 75% of that determined for the radiolabeled receptor antagonist (/sup 3/H)quinuclidinyl benzilate. Both muscarinic receptor agonists and antagonists inhibited the binding of (/sup 3/H)OXO with high affinity and Hill slopes of approximately one. Guanine nucleotides completely inhibited the binding of (/sup 3/H)OXO. This effect was on the maximum binding (Bmax) of (/sup 3/H)OXO with no change occurring in the Kd; the order of potency for five nucleotides was guanosine 5'-O-(3-thio-triphosphate) greater than 5'-guanylylimidodiphosphate greater than GTP greater than or equal to guanosine/diphosphate greater than GMP. The (/sup 3/H)OXO-induced interaction of muscarinic receptors with a guanine nucleotide binding protein was stable to solubilization. That is, membrane receptors that were prelabeled with (/sup 3/H)OXO could be solubilized with digitonin, and the addition of guanine nucleotides to the soluble, (/sup 3/H)OXO-labeled complex resulted in dissociation of (/sup 3/H)OXO from the receptor. Pretreatment of membranes with relatively low concentrations of N-ethylmaleimide inhibited (/sup 3/H)OXO binding by 85% with no change in the Kd of (/sup 3/H)OXO, and with no effect on (/sup 3/H)quinuclidinyl benzilate binding.

  7. Advances in the molecular understanding of gonadotropins-receptors interactions.

    PubMed

    el Tayar, N

    1996-12-20

    The extracellular domain (ECD) of gonadotropin receptors belong to the leucine-rich repeat (LRR) protein superfamily and their transmembrane domain (TMD) is characteristic of the seven alpha-helices G-protein-coupled receptors (GPCR). The availability of the X-ray structures of porcine ribonuclease inhibitor (RI), a LRR protein, and bacteriorhodopsin (bR) allows the construction of 3D models of the ECD and the TMD of gonadotropin receptors, respectively. The predicted models are to a large extent consistent with currently available biochemical and mutational data. The models provide a reliable basis for understanding how the hormone binds and activates its receptor. The ECD, in particular the LRR region, serves as a baseball glove which efficiently catches the large hormone and optimally orient the appropriate parts of it for interaction with the seven-transmembrane-helix domain of the receptor. This in turn is expected to lead to a conformational change to be sensed by the appropriate G-protein complex leading to the stimulation of cAMP synthesis and steroids production.

  8. The structural basis of androgen receptor activation: intramolecular and intermolecular amino-carboxy interactions.

    PubMed

    Schaufele, Fred; Carbonell, Xavier; Guerbadot, Martin; Borngraeber, Sabine; Chapman, Mark S; Ma, Aye Aye K; Miner, Jeffrey N; Diamond, Marc I

    2005-07-12

    Nuclear receptors (NRs) are ligand-regulated transcription factors important in human physiology and disease. In certain NRs, including the androgen receptor (AR), ligand binding to the carboxy-terminal domain (LBD) regulates transcriptional activation functions in the LBD and amino-terminal domain (NTD). The basis for NTD-LBD communication is unknown but may involve NTD-LBD interactions either within a single receptor or between different members of an AR dimer. Here, measurement of FRET between fluorophores attached to the NTD and LBD of the AR established that agonist binding initiated an intramolecular NTD-LBD interaction in the nucleus and cytoplasm. This intramolecular folding was followed by AR self-association, which occurred preferentially in the nucleus. Rapid, ligand-induced intramolecular folding and delayed association also were observed for estrogen receptor-alpha but not for peroxisome proliferator activated receptor-gamma2. An antagonist ligand, hydroxyflutamide, blocked the NTD-LBD association within AR. NTD-LBD association also closely correlated with the transcriptional activation by heterologous ligands of AR mutants isolated from hormone-refractory prostate tumors. Intramolecular folding, but not AR-AR affinity, was disrupted by mutation of an alpha-helical ((23)FQNLF(27)) motif in the AR NTD previously described to interact with the AR LBD in vitro. This work establishes an intramolecular NTD-LBD conformational change as an initial component of ligand-regulated NR function.

  9. Interactions between fibroblast growth factors and Notch regulate neuronal differentiation.

    PubMed

    Faux, C H; Turnley, A M; Epa, R; Cappai, R; Bartlett, P F

    2001-08-01

    The differentiation of precursor cells into neurons has been shown to be influenced by both the Notch signaling pathway and growth factor stimulation. In this study, the regulation of neuronal differentiation by these mechanisms was examined in the embryonic day 10 neuroepithelial precursor (NEP) population. By downregulating Notch1 expression and by the addition of a Delta1 fusion protein (Delta Fc), it was shown that signaling via the Notch pathway inhibited neuron differentiation in the NEP cells, in vitro. The expression of two of the Notch receptor homologs, Notch1 and Notch3, and the ligand Delta1 in these NEP cells was found to be influenced by a number of different growth factors, indicating a potential interaction between growth factors and Notch signaling. Interestingly, none of the growth factors examined promoted neuron differentiation; however, the fibroblast growth factors (FGFs) 1 and 2 potently inhibited differentiation. FGF1 and FGF2 upregulated the expression of Notch and decreased expression of Delta1 in the NEP cells. In addition, the inhibitory response of the cells to the FGFs could be overcome by downregulating Notch1 expression and by disrupting Notch cleavage and signaling by the ablation of the Presenilin1 gene. These results indicate that FGF1 and FGF2 act via the Notch pathway, either directly or indirectly, to inhibit differentiation. Thus, signaling through the Notch receptor may be a common regulator of neuronal differentiation within the developing forebrain.

  10. CNTF variants with increased biological potency and receptor selectivity define a functional site of receptor interaction.

    PubMed Central

    Saggio, I; Gloaguen, I; Poiana, G; Laufer, R

    1995-01-01

    Human CNTF is a neurocytokine that elicits potent neurotrophic effects by activating a receptor complex composed of the ligand-specific alpha-receptor subunit (CNTFR alpha) and two signal transducing proteins, which together constitute a receptor for leukemia inhibitory factor (LIFR). At high concentrations, CNTF can also activate the LIFR and possibly other cross-reactive cytokine receptors in the absence of CNTFR alpha. To gain a better understanding of its structure-function relationships and to develop analogs with increased receptor specificity, the cytokine was submitted to affinity maturation using phage display technology. Variants with greatly increased CNTFR alpha affinity were selected from a phage-displayed library of CNTF variants carrying random amino acid substitutions in the putative D helix. Selected variants contained substitutions of the wild-type Gln167 residue, either alone or in combination with neighboring mutations. These results provide evidence for an important functional role of the mutagenized region in CNTFR alpha binding. Affinity enhancing mutations conferred to CNTF increased potency to trigger biological effects mediated by CNTFR alpha and enhanced neurotrophic activity on chicken ciliary neurons. In contrast, the same mutations did not potentiate the CNTFR alpha-independent receptor actions of CNTF. These CNTF analogs thus represent receptor-specific superagonists, which should help to elucidate the mechanisms underlying the pleiotropic actions of the neurocytokine. PMID:7621819

  11. Direct interactions between calcitonin-like receptor (CLR) and CGRP-receptor component protein (RCP) regulate CGRP receptor signaling.

    PubMed

    Egea, Sophie C; Dickerson, Ian M

    2012-04-01

    Calcitonin gene-related peptide (CGRP) is a neuropeptide with multiple neuroendocrine roles, including vasodilation, migraine, and pain. The receptor for CGRP is a G protein-coupled receptor (GPCR) that requires three proteins for function. CGRP binds to a heterodimer composed of the GPCR calcitonin-like receptor (CLR) and receptor activity-modifying protein (RAMP1), a single transmembrane protein required for pharmacological specificity and trafficking of the CLR/RAMP1 complex to the cell surface. In addition, the CLR/RAMP1 complex requires a third protein named CGRP-receptor component protein (RCP) for signaling. Previous studies have demonstrated that depletion of RCP from cells inhibits CLR signaling, and in vivo studies have demonstrated that expression of RCP correlates with CLR signaling and CGRP efficacy. It is not known whether RCP interacts directly with CLR to exert its effect. The current studies identified a direct interaction between RCP and an intracellular domain of CLR using yeast two-hybrid analysis and coimmunoprecipitation. When this interacting domain of CLR was expressed as a soluble fusion protein, it coimmunoprecipitated with RCP and inhibited signaling from endogenous CLR. Expression of this dominant-negative domain of CLR did not significantly inhibit trafficking of CLR to the cell surface, and thus RCP may not have a chaperone function for CLR. Instead, RCP may regulate CLR signaling in the cell membrane, and direct interaction between RCP and CLR is required for CLR activation. To date, RCP has been found to interact only with CLR and represents a novel neuroendocrine regulatory step in GPCR signaling.

  12. Direct Interactions between Calcitonin-Like Receptor (CLR) and CGRP-Receptor Component Protein (RCP) Regulate CGRP Receptor Signaling

    PubMed Central

    Egea, Sophie C.

    2012-01-01

    Calcitonin gene-related peptide (CGRP) is a neuropeptide with multiple neuroendocrine roles, including vasodilation, migraine, and pain. The receptor for CGRP is a G protein-coupled receptor (GPCR) that requires three proteins for function. CGRP binds to a heterodimer composed of the GPCR calcitonin-like receptor (CLR) and receptor activity-modifying protein (RAMP1), a single transmembrane protein required for pharmacological specificity and trafficking of the CLR/RAMP1 complex to the cell surface. In addition, the CLR/RAMP1 complex requires a third protein named CGRP-receptor component protein (RCP) for signaling. Previous studies have demonstrated that depletion of RCP from cells inhibits CLR signaling, and in vivo studies have demonstrated that expression of RCP correlates with CLR signaling and CGRP efficacy. It is not known whether RCP interacts directly with CLR to exert its effect. The current studies identified a direct interaction between RCP and an intracellular domain of CLR using yeast two-hybrid analysis and coimmunoprecipitation. When this interacting domain of CLR was expressed as a soluble fusion protein, it coimmunoprecipitated with RCP and inhibited signaling from endogenous CLR. Expression of this dominant-negative domain of CLR did not significantly inhibit trafficking of CLR to the cell surface, and thus RCP may not have a chaperone function for CLR. Instead, RCP may regulate CLR signaling in the cell membrane, and direct interaction between RCP and CLR is required for CLR activation. To date, RCP has been found to interact only with CLR and represents a novel neuroendocrine regulatory step in GPCR signaling. PMID:22315449

  13. Inhibitors for the Vitamin D Receptor-Coregulator Interaction.

    PubMed

    Teske, Kelly A; Yu, Olivia; Arnold, Leggy A

    2016-01-01

    The vitamin D receptor (VDR) belongs to the superfamily of nuclear receptors and is activated by the endogenous ligand 1,25-dihydroxyvitamin D3. The genomic effects mediated by VDR consist of the activation and repression of gene transcription, which includes the formation of multiprotein complexes with coregulator proteins. Coregulators bind many nuclear receptors and can be categorized according to their role as coactivators (gene activation) or corepressors (gene repression). Herein, different approaches to develop compounds that modulate the interaction between VDR and coregulators are summarized. This includes coregulator peptides that were identified by creating phage display libraries. Subsequent modification of these peptides including the introduction of a tether or nonhydrolyzable bonds resulted in the first direct VDR-coregulator inhibitors. Later, small molecules that inhibit VDR-coregulator inhibitors were identified using rational drug design and high-throughput screening. Early on, allosteric inhibition of VDR-coregulator interactions was achieved with VDR antagonists that change the conformation of VDR and modulate the interactions with coregulators. A detailed discussion of their dual agonist/antagonist effects is given as well as a summary of their biological effects in cell-based assays and in vivo studies.

  14. Modeling multivalent ligand-receptor interactions with steric constraints on configurations of cell surface receptor aggregates

    SciTech Connect

    Monine, Michael; Posner, Richard; Savage, Paul; Faeder, James; Hlavacek, William S

    2008-01-01

    Signal transduction generally involves multivalent protein-protein interactions, which can produce various protein complexes and post-translational modifications. The reaction networks that characterize these interactions tend to be so large as to challenge conventional simulation procedures. To address this challenge, a kinetic Monte Carlo (KMC) method has been developed that can take advantage of a model specification in terms of reaction rules for molecular interactions. A set of rules implicitly defines the reactions that can occur as a result of the interactions represented by the rules. With the rule-based KMC method, explicit generation of the underlying chemical reaction network implied by rules is avoided. Here, we apply and extend this method to characterize the interactions of a trivalent ligand with a bivalent cell-surface receptor. This system is also studied experimentally. We consider the following kinetic models: an equivalent-site model, an extension of this model, which takes into account steric constraints on the configurations of receptor aggregates, and finally, a model that accounts for cyclic receptor aggregates. Simulation results for the equivalent-site model are consistent with an equilibrium continuum model. Using these models, we investigate the effects of steric constraints and the formation of cyclic aggregates on the kinetics and equilibria of small and large aggregate formation and the percolation phase transition that occurs in this system.

  15. Nectin-like interactions between poliovirus and its receptor trigger conformational changes associated with cell entry.

    PubMed

    Strauss, Mike; Filman, David J; Belnap, David M; Cheng, Naiqian; Noel, Roane T; Hogle, James M

    2015-04-01

    Poliovirus infection is initiated by attachment to a receptor on the cell surface called Pvr or CD155. At physiological temperatures, the receptor catalyzes an irreversible expansion of the virus to form an expanded form of the capsid called the 135S particle. This expansion results in the externalization of the myristoylated capsid protein VP4 and the N-terminal extension of the capsid protein VP1, both of which become inserted into the cell membrane. Structures of the expanded forms of poliovirus and of several related viruses have recently been reported. However, until now, it has been unclear how receptor binding triggers viral expansion at physiological temperature. Here, we report poliovirus in complex with an enzymatically partially deglycosylated form of the 3-domain ectodomain of Pvr at a 4-Å resolution, as determined by cryo-electron microscopy. The interaction of the receptor with the virus in this structure is reminiscent of the interactions of Pvr with its natural ligands. At a low temperature, the receptor induces very few changes in the structure of the virus, with the largest changes occurring within the footprint of the receptor, and in a loop of the internal protein VP4. Changes in the vicinity of the receptor include the displacement of a natural lipid ligand (called "pocket factor"), demonstrating that the loss of this ligand, alone, is not sufficient to induce particle expansion. Finally, analogies with naturally occurring ligand binding in the nectin family suggest which specific structural rearrangements in the virus-receptor complex could help to trigger the irreversible expansion of the capsid. The cell-surface receptor (Pvr) catalyzes a large structural change in the virus that exposes membrane-binding protein chains. We fitted known atomic models of the virus and Pvr into three-dimensional experimental maps of the receptor-virus complex. The molecular interactions we see between poliovirus and its receptor are reminiscent of the nectin

  16. Activation of 5-HT7 receptors increases neuronal platelet-derived growth factor β receptor expression.

    PubMed

    Vasefi, Maryam S; Kruk, Jeff S; Liu, Hui; Heikkila, John J; Beazely, Michael A

    2012-03-09

    Several antipsychotics have a high affinity for 5-HT7 receptors yet despite intense interest in the 5-HT7 receptor as a potential drug target to treat psychosis, the function and signaling properties of 5-HT7 receptors in neurons remain largely uncharacterized. In primary mouse hippocampal and cortical neurons, as well as in the SH-SY5Y cell line, incubation with 5-HT, 5-carboxamidotryptamine (5-CT), or 5-HT7 receptor-selective agonists increases the expression of platelet-derived growth factor (PDGF)β receptors. The increased PDGFβ receptor expression is cyclic AMP-dependent protein kinase (PKA)-dependent, suggesting that 5-HT7 receptors couple to Gα(s) in primary neurons. Interestingly, up-regulated PDGFβ receptors display an increased basal phosphorylation state at the phospholipase Cγ-activating tyrosine 1021. This novel linkage between the 5-HT7 receptor and the PDGF system may be an important GPCR-neurotrophic factor signaling pathway in neurons.

  17. Computational studies of ligand-receptor interactions in bitter taste receptors.

    PubMed

    Miguet, Laurence; Zhang, Ziding; Grigorov, Martin G

    2006-01-01

    Phenylthiocarbamide tastes intensely bitter to some individuals, but others find it completely tasteless. Recently, it was suggested that phenylthiocarbamide elicits bitter taste by interacting with a human G protein-coupled receptor (hTAS2R38) encoded by the PTC gene. The phenylthiocarbamide nontaster trait was linked to three single nucleotide polymorphisms occurring in the PTC gene. Using the crystal structure of bovine rhodopsin as template, we generated the 3D structure of hTAS2R38 bitter taste receptor. We were able to map on the receptor structure the amino acids affected by the genetic polymorphisms and to propose molecular functions for two of them that explained the emergence of the nontaster trait. We used molecular docking simulations to find that phenylthiocarbamide exhibited a higher affinity for the target receptor than the structurally similar molecule 6-n-propylthiouracil, in line with recent experimental studies. A 3D model was constructed for the hTAS2R16 bitter taste receptor as well, by applying the same protocol. We found that the recently published experimental ligand binding affinity data for this receptor correlated well with the binding scores obtained from our molecular docking calculations.

  18. Global Quantitative Modeling of Chromatin Factor Interactions

    PubMed Central

    Zhou, Jian; Troyanskaya, Olga G.

    2014-01-01

    Chromatin is the driver of gene regulation, yet understanding the molecular interactions underlying chromatin factor combinatorial patterns (or the “chromatin codes”) remains a fundamental challenge in chromatin biology. Here we developed a global modeling framework that leverages chromatin profiling data to produce a systems-level view of the macromolecular complex of chromatin. Our model ultilizes maximum entropy modeling with regularization-based structure learning to statistically dissect dependencies between chromatin factors and produce an accurate probability distribution of chromatin code. Our unsupervised quantitative model, trained on genome-wide chromatin profiles of 73 histone marks and chromatin proteins from modENCODE, enabled making various data-driven inferences about chromatin profiles and interactions. We provided a highly accurate predictor of chromatin factor pairwise interactions validated by known experimental evidence, and for the first time enabled higher-order interaction prediction. Our predictions can thus help guide future experimental studies. The model can also serve as an inference engine for predicting unknown chromatin profiles — we demonstrated that with this approach we can leverage data from well-characterized cell types to help understand less-studied cell type or conditions. PMID:24675896

  19. The Anti-angiogenic Peptide, Loop 6, Binds Insulin-like Growth Factor-1 Receptor*

    PubMed Central

    Fernandez, Cecilia A.; Roy, Roopali; Lee, Sunyoung; Yang, Jiang; Panigrahy, Dipak; Van Vliet, Krystyn J.; Moses, Marsha A.

    2010-01-01

    Tissue inhibitors of metalloproteinases (TIMPs), the endogenous inhibitors of matrix metalloproteinases, have been shown to possess biological functions that are independent of their ability to inhibit matrix metalloproteinases. We have previously shown that the C-terminal domain of TIMP-2 and, in particular, Loop 6 inhibit capillary endothelial cell proliferation and angiogenesis both in vitro and in vivo. To elucidate the mechanism by which Loop 6 inhibits angiogenesis, we sought to determine whether its biological effects were the result of a known TIMP-2 protein-protein interaction or of a receptor-mediated event. In this study, we identify insulin-like growth factor-1 receptor as a binding partner of Loop 6/TIMP-2 and characterize this interaction on the endothelial cell surface and the consequences of this interaction on downstream receptor signaling. PMID:20940305

  20. Interaction of lipids with the neurotensin receptor 1.

    PubMed

    Bolivar, Juan H; Muñoz-García, Juan C; Castro-Dopico, Tomas; Dijkman, Patricia M; Stansfeld, Phillip J; Watts, Anthony

    2016-06-01

    Information about lipid-protein interactions for G protein-coupled receptors (GPCRs) is scarce. Here, we use electron spin resonance (ESR) and spin-labelled lipids to study lipid interactions with the rat neurotensin receptor 1 (NTS1). A fusion protein containing rat NTS1 fully able to bind its ligand neurotensin was reconstituted into phosphatidylcholine (PC) bilayers at specific lipid:protein molar ratios. The fraction of motionally restricted lipids in the range of 40:1 to 80:1 lipids per receptor suggested an oligomeric state of the protein, and the result was unaffected by increasing the hydrophobic thickness of the lipid bilayer from C-18 to C-20 or C-22 chain length PC membranes. Comparison of the ESR spectra of different spin-labelled lipids allowed direct measurement of lipid binding constants relative to PC (Kr), with spin-labelled phosphatidylethanolamine (PESL), phosphatidylserine (PSSL), stearic acid (SASL), and a spin labelled cholesterol analogue (CSL) Kr values of 1.05±0.05, 1.92±0.08, 5.20±0.51 and 0.91±0.19, respectively. The results contrast with those from rhodopsin, the only other GPCR studied this way, which has no selectivity for the lipids analysed here. Molecular dynamics simulations of NTS1 in bilayers are in agreement with the ESR data, and point to sites in the receptor where PS could interact with higher affinity. Lipid selectivity could be necessary for regulation of ligand binding, oligomerisation and/or G protein activation processes. Our results provide insight into the potential modulatory mechanisms that lipids can exert on GPCRs. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Novel Drosophila receptor that binds multiple growth factors

    SciTech Connect

    Rosner, M.R.; Thompson, K.L.; Garcia, V.; Decker, S.J.

    1986-05-01

    The authors have recently reported the identification of a novel growth factor receptor from Drosophila cell cultures that has dual binding specificity for both insulin and epidermal growth factor (EGF). This 100 kDa protein is also antigenically related to the cytoplasmic region of the mammalian EGF receptor-tyrosine kinase. They now report that this protein binds to mammalian nerve growth factor and human transforming growth factor alpha as well as insulin and EGF with apparent dissociation constants ranging from 10/sup -6/ to 10/sup -8/ M. The 100 kDa protein can be affinity-labeled with these /sup 125/I-labeled growth factors after immunoprecipitation with anti-EGF receptor antiserum. These four growth factors appear to share a common binding site, as evidenced by their ability to block affinity labelling by /sup 125/I-insulin. No significant binding to the 100 kDa protein was observed with platelet-derived growth factor, transforming growth factor beta, or glucagon. The 100 kDa Drosophila protein has a unique ligand-binding spectrum with no direct counterpart in mammalian cells and may represent an evolutionary precursor of the mammalian receptors for these growth factors.

  2. Pion form factor from a contact interaction

    SciTech Connect

    Gutierrez-Guerrero, L. X.; Bashir, A.; Cloeet, I. C.; Roberts, C. D.

    2010-06-15

    In a Poincare-covariant vector-boson-exchange theory, the pion possesses components of pseudovector origin, which materially influence its observable properties. For a range of such quantities, we explore the consequences of a momentum-independent interaction, regularized in a symmetry-preserving manner. The contact interaction, while capable of describing pion static properties, produces a form factor whose evolution for Q{sup 2}>0.17 GeV{sup 2} disagrees markedly with experiment and whose asymptotic power-law behavior conflicts strongly with perturbative QCD.

  3. Topical administration of adrenergic receptor pharmaceutics and nerve growth factor

    PubMed Central

    Steinle, Jena J

    2010-01-01

    Topical application of nerve growth factor (NGF) and adrenergic receptor pharmaceutics are currently in use for corneal ulcers and glaucoma. A recent interest in the neuroprotective abilities of NGF has led to a renewed interest in NGF as a therapeutic for retinal and choroidal diseases. NGF can promote cell proliferation through actions of the TrkA receptor or promote apoptosis through receptor p75NTR. This understanding has led to novel interest in the role of NGF for diseases of the posterior eye. The role of β-adrenergic receptor agonists and antagonists for treatments of glaucoma, diabetic retinopathy, and their potential mechanisms of action, are still under investigation. This review discusses the current knowledge and applications of topical NGF and adrenergic receptor drugs for ocular disease. PMID:20668722

  4. The epidermal growth factor receptor family: Biology driving targeted therapeutics

    PubMed Central

    Wieduwilt, M. J.; Moasser, M. M.

    2011-01-01

    The epidermal growth factor family of receptor tyrosine kinases (ErbBs) plays essential roles in regulating cell proliferation, survival, differentiation and migration. The ErbB receptors carry out both redundant and restricted functions in mammalian development and in the maintenance of tissues in the adult mammal. Loss of regulation of the ErbB receptors underlies many human diseases, most notably cancer. Our understanding of the function and complex regulation of these receptors has fueled the development of targeted therapeutic agents for human malignancies in the last 15 years. Here we review the biology of ErbB receptors, including their structure, signaling, regulation, and roles in development and disease, then briefly touch on their increasing roles as targets for cancer therapy. PMID:18259690

  5. Sex differences in opioid analgesia and addiction: interactions among opioid receptors and estrogen receptors

    PubMed Central

    2013-01-01

    Opioids are widely used as the pain reliever and also notorious for being addictive drugs. Sex differences in the opioid analgesia and addiction have been reported and investigated in human subjects and animal models. Yet, the molecular mechanism underlying the differences between males and females is still unclear. Here, we reviewed the literature describing the sex differences in analgesic responses and addiction liabilities to clinically relevant opioids. The reported interactions among opioids, estrogens, opioid receptors, and estrogen receptors are also evaluated. We postulate that the sex differences partly originated from the crosstalk among the estrogen and opioid receptors when stimulated by the exogenous opioids, possibly through common secondary messengers and the downstream gene transcriptional regulators. PMID:24010861

  6. Sex differences in opioid analgesia and addiction: interactions among opioid receptors and estrogen receptors.

    PubMed

    Lee, Cynthia Wei-Sheng; Ho, Ing-Kang

    2013-09-08

    Opioids are widely used as the pain reliever and also notorious for being addictive drugs. Sex differences in the opioid analgesia and addiction have been reported and investigated in human subjects and animal models. Yet, the molecular mechanism underlying the differences between males and females is still unclear. Here, we reviewed the literature describing the sex differences in analgesic responses and addiction liabilities to clinically relevant opioids. The reported interactions among opioids, estrogens, opioid receptors, and estrogen receptors are also evaluated. We postulate that the sex differences partly originated from the crosstalk among the estrogen and opioid receptors when stimulated by the exogenous opioids, possibly through common secondary messengers and the downstream gene transcriptional regulators.

  7. Striatal adenosine-cannabinoid receptor interactions in rats over-expressing adenosine A2A receptors.

    PubMed

    Chiodi, Valentina; Ferrante, Antonella; Ferraro, Luca; Potenza, Rosa Luisa; Armida, Monica; Beggiato, Sarah; Pèzzola, Antonella; Bader, Michael; Fuxe, Kjell; Popoli, Patrizia; Domenici, Maria Rosaria

    2016-03-01

    Adenosine A2A receptors (A2 A Rs) and cannabinoid CB1 receptors (CB1 Rs) are highly expressed in the striatum, where they functionally interact and form A2A /CB1 heteroreceptor complexes. We investigated the effects of CB1 R stimulation in a transgenic rat strain over-expressing A2 A Rs under the control of the neural-specific enolase promoter (NSEA2A rats) and in age-matched wild-type (WT) animals. The effects of the CB1 R agonist WIN 55,212-2 (WIN) were significantly lower in NSEA2A rats than in WT animals, as demonstrated by i) electrophysiological recordings of synaptic transmission in corticostriatal slices; ii) the measurement of glutamate outflow from striatal synaptosomes and iii) in vivo experiments on locomotor activity. Moreover, while the effects of WIN were modulated by both A2 A R agonist (CGS 21680) and antagonists (ZM 241385, KW-6002 and SCH-442416) in WT animals, the A2 A R antagonists failed to influence WIN-mediated effects in NSEA2A rats. The present results demonstrate that in rats with genetic neuronal over-expression of A2 A Rs, the effects mediated by CB1 R activation in the striatum are significantly reduced, suggesting a change in the stoichiometry of A2A and CB1 receptors and providing a strategy to dissect the involvement of A2 A R forming or not forming heteromers in the modulation of striatal functions. These findings add additional evidence for the existence of an interaction between striatal A2 A Rs and CB1 Rs, playing a fundamental role in the regulation of striatal functions. We studied A2A -CB1 receptor interaction in transgenic rats over-expressing adenosine A2A receptors under the control of the neuron-specific enolase promoter (NSEA2A ). In these rats, we demonstrated a reduced effect of the CB1 receptor agonist WIN 55,212-2 in the modulation of corticostriatal synaptic transmission and locomotor activity, while CB1 receptor expression level did not change with respect to WT rats. A reduction in the expression of A2A -CB1

  8. Interaction between ESRα polymorphisms and environmental factors in osteoporosis.

    PubMed

    Sonoda, Tomoko; Takada, Junichi; Iba, Kousuke; Asakura, Sumiyo; Yamashita, Toshihiko; Mori, Mitsuru

    2012-10-01

    We hypothesized that environmental factors might affect the relationship between genetic predisposition and the risk of bone mineral density (BMD) loss. Cases were 114 Japanese women with a confirmed diagnosis of postmenopausal osteoporosis and controls were 171 general Japanese women. Genetic risk of SNPs in the estrogen receptors was analyzed by a case-control study. The interaction between gene and environmental factors for osteoporosis were assessed by a case-only design. Significant increases in osteoporosis risk were observed with minor alleles of rs2077647 located in the first exon and rs2234693 located in the first intron of estrogen receptor α (ESRα). Haplotype CC at these risk SNPs was strongly associated with osteoporosis risk (odds ratio [OR] =  3.15, 95% confidence interval [CI] = 1.83-5.41). There was a statistically significant interaction between haplotype CC and alcohol drinking; moderate alcohol consumption decreased genetic risk of osteoporosis (OR  =  0.22, 95%CI  =  0.05-0.83). Copyright © 2012 Orthopaedic Research Society.

  9. Identification of TIFA as an adapter protein that links tumor necrosis factor receptor-associated factor 6 (TRAF6) to interleukin-1 (IL-1) receptor-associated kinase-1 (IRAK-1) in IL-1 receptor signaling.

    PubMed

    Takatsuna, Hiroshi; Kato, Hiroki; Gohda, Jin; Akiyama, Taishin; Moriya, Ayaka; Okamoto, Yoshinari; Yamagata, Yuriko; Otsuka, Masami; Umezawa, Kazuo; Semba, Kentaro; Inoue, Jun-Ichiro

    2003-04-04

    Tumor necrosis factor receptor-associated factor 6 (TRAF6) transduces signals from members of the Toll/interleukin-1 (IL-1) receptor family by interacting with IL-1 receptor-associated kinase-1 (IRAK-1) after IRAK-1 is released from the receptor-MyD88 complex upon IL-1 stimulation. However, the molecular mechanisms underlying regulation of the IRAK-1/TRAF6 interaction are largely unknown. We have identified TIFA, a TRAF-interacting protein with a forkhead-associated (FHA) domain. The FHA domain is a motif known to bind directly to phosphothreonine and phosphoserine. In transient transfection assays, TIFA activates NFkappaBeta and c-Jun amino-terminal kinase. However, TIFA carrying a mutation that abolishes TRAF6 binding or mutations in the FHA domain that are known to abolish FHA domain binding to phosphopeptide fails to activate NFkappaBeta and c-Jun amino-terminal kinase. TIFA, when overexpressed, binds both TRAF6 and IRAK-1 and significantly enhances the IRAK-1/TRAF6 interaction. Furthermore, analysis of endogenous proteins indicates that TIFA associates with TRAF6 constitutively, whereas it associates with IRAK-1 in an IL-1 stimulation-dependent manner in vivo. Thus, TIFA is likely to mediate IRAK-1/TRAF6 interaction upon IL-1 stimulation.

  10. MICAL-like1 mediates epidermal growth factor receptor endocytosis

    PubMed Central

    Abou-Zeid, Nancy; Pandjaitan, Rudy; Sengmanivong, Lucie; David, Violaine; Le Pavec, Gwenaelle; Salamero, Jean; Zahraoui, Ahmed

    2011-01-01

    Small GTPase Rabs are required for membrane protein sorting/delivery to precise membrane domains. Rab13 regulates epithelial tight junction assembly and polarized membrane transport. Here we report that Molecule Interacting with CasL (MICAL)-like1 (MICAL-L1) interacts with GTP-Rab13 and shares a similar domain organization with MICAL. MICAL-L1 has a calponin homology (CH), LIM, proline rich and coiled-coil domains. It is associated with late endosomes. Time-lapse video microscopy shows that green fluorescent protein–Rab7 and mcherry-MICAL-L1 are present within vesicles that move rapidly in the cytoplasm. Depletion of MICAL-L1 by short hairpin RNA does not alter the distribution of a late endosome/lysosome-associated protein but affects the trafficking of epidermal growth factor receptor (EGFR). Overexpression of MICAL-L1 leads to the accumulation of EGFR in the late endosomal compartment. In contrast, knocking down MICAL-L1 results in the distribution of internalized EGFR in vesicles spread throughout the cytoplasm and promotes its degradation. Our data suggest that the N-terminal CH domain associates with the C-terminal Rab13 binding domain (RBD) of MICAL-L1. The binding of Rab13 to RBD disrupts the CH/RBD interaction, and may induce a conformational change in MICAL-L1, promoting its activation. Our results provide novel insights into the MICAL-L1/Rab protein complex that can regulate EGFR trafficking at late endocytic pathways. PMID:21795389

  11. Alcohol action on a neuronal membrane receptor: evidence for a direct interaction with the receptor protein.

    PubMed Central

    Li, C; Peoples, R W; Weight, F F

    1994-01-01

    For almost a century, alcohols have been thought to produce their effects by actions on the membrane lipids of central nervous system neurons--the well known "lipid theory" of alcohol action. The rationale for this theory is the correlation of potency with oil/water or membrane/buffer partition coefficient. Although a number of recent studies have shown that alcohols can affect the function of certain neuronal neurotransmitter receptors, there is no evidence that the alcohols interact directly with these membrane proteins. In the present study, we report that inhibition of a neuronal neurotransmitter receptor, an ATP-gated ion channel, by a series of alcohols exhibits a distinct cutoff effect. For alcohols with a molecular volume of < or = 42.2 ml/mol, potency for inhibiting ATP-activated current was correlated with lipid solubility (order of potency: 1-propanol = trifluoroethanol > monochloroethanol > ethanol > methanol). However, despite increased lipid solubility, alcohols with a molecular volume of > or = 46.1 ml/mol (1-butanol, 1-pentanol, trichloroethanol, and dichloroethanol) were without effect on the ATP-activated current. The results suggest that alcohols inhibit the function of this neurotransmitter receptor by interacting with a small hydrophobic pocket on the receptor protein. PMID:8058780

  12. 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

  13. Astrocyte Mitogen Inhibitor Related to Epidermal Growth Factor Receptor

    NASA Astrophysics Data System (ADS)

    Nieto-Sampedro, Manuel

    1988-06-01

    Epidermal growth factor (EGF) is a well-characterized polypeptide hormone with diverse biological activities, including stimulation of astrocyte division. A soluble astrocyte mitogen inhibitor, immunologically related to the EGF receptor, is present in rat brain. Injury to the brain causes a time-dependent reduction in the levels of this inhibitor and the concomitant appearance of EGF receptor on the astrocyte surface. Intracerebral injection of antibody capable of binding the inhibitor caused the appearance of numerous reactive astrocytes. EGF receptor-related inhibitors may play a key role in the control of glial cell division in both normal and injured brain.

  14. ErbB2 resembles an autoinhibited invertebrate epidermal growth factor receptor

    SciTech Connect

    Alvarado, Diego; Klein, Daryl E.; Lemmon, Mark A.

    2009-09-25

    The orphan receptor tyrosine kinase ErbB2 (also known as HER2 or Neu) transforms cells when overexpressed, and it is an important therapeutic target in human cancer. Structural studies have suggested that the oncogenic (and ligand-independent) signalling properties of ErbB2 result from the absence of a key intramolecular 'tether' in the extracellular region that autoinhibits other human ErbB receptors, including the epidermal growth factor (EGF) receptor. Although ErbB2 is unique among the four human ErbB receptors, here we show that it is the closest structural relative of the single EGF receptor family member in Drosophila melanogaster (dEGFR). Genetic and biochemical data show that dEGFR is tightly regulated by growth factor ligands, yet a crystal structure shows that it, too, lacks the intramolecular tether seen in human EGFR, ErbB3 and ErbB4. Instead, a distinct set of autoinhibitory interdomain interactions hold unliganded dEGFR in an inactive state. All of these interactions are maintained (and even extended) in ErbB2, arguing against the suggestion that ErbB2 lacks autoinhibition. We therefore suggest that normal and pathogenic ErbB2 signalling may be regulated by ligands in the same way as dEGFR. Our findings have important implications for ErbB2 regulation in human cancer, and for developing therapeutic approaches that target novel aspects of this orphan receptor.

  15. Insights into cytokine-receptor interactions from cytokine engineering.

    PubMed

    Spangler, Jamie B; Moraga, Ignacio; Mendoza, Juan L; Garcia, K Christopher

    2015-01-01

    Cytokines exert a vast array of immunoregulatory actions critical to human biology and disease. However, the desired immunotherapeutic effects of native cytokines are often mitigated by toxicity or lack of efficacy, either of which results from cytokine receptor pleiotropy and/or undesired activation of off-target cells. As our understanding of the structural principles of cytokine-receptor interactions has advanced, mechanism-based manipulation of cytokine signaling through protein engineering has become an increasingly feasible and powerful approach. Modified cytokines, both agonists and antagonists, have been engineered with narrowed target cell specificities, and they have also yielded important mechanistic insights into cytokine biology and signaling. Here we review the theory and practice of cytokine engineering and rationalize the mechanisms of several engineered cytokines in the context of structure. We discuss specific examples of how structure-based cytokine engineering has opened new opportunities for cytokines as drugs, with a focus on the immunotherapeutic cytokines interferon, interleukin-2, and interleukin-4.

  16. Evaluation of Nod-Like Receptor (NLR) Effector Domain Interactions

    PubMed Central

    Kufer, Thomas A.; Schwarzenbacher, Robert

    2009-01-01

    Members of the Nod-like receptor (NLR) family recognize intracellular pathogens and recruit a variety of effector molecules, including pro-caspases and kinases, which in turn are implicated in cytokine processing and NF-κB activation. In order to elucidate the intricate network of NLR signaling, which is still fragmentary in molecular terms, we applied comprehensive yeast two-hybrid analysis for unbiased evaluation of physical interactions between NLRs and their adaptors (ASC, CARD8) as well as kinase RIPK2 and inflammatory caspases (C1, C2, C4, C5) under identical conditions. Our results confirmed the interaction of NOD1 and NOD2 with RIPK2, and between NLRP3 and ASC, but most importantly, our studies revealed hitherto unrecognized interactions of NOD2 with members of the NLRP subfamily. We found that NOD2 specifically and directly interacts with NLRP1, NLRP3 and NLRP12. Furthermore, we observed homodimerization of the RIPK2 CARD domains and identified residues in NOD2 critical for interaction with RIPK2. In conclusion, our work provides further evidence for the complex network of protein-protein interactions underlying NLR function. PMID:19337385

  17. How glucocorticoid receptors modulate the activity of other transcription factors: a scope beyond tethering.

    PubMed

    Ratman, Dariusz; Vanden Berghe, Wim; Dejager, Lien; Libert, Claude; Tavernier, Jan; Beck, Ilse M; De Bosscher, Karolien

    2013-11-05

    The activity of the glucocorticoid receptor (GR), a nuclear receptor transcription factor belonging to subclass 3C of the steroid/thyroid hormone receptor superfamily, is typically triggered by glucocorticoid hormones. Apart from driving gene transcription via binding onto glucocorticoid response elements in regulatory regions of particular target genes, GR can also inhibit gene expression via transrepression, a mechanism largely based on protein:protein interactions. Hereby GR can influence the activity of other transcription factors, without contacting DNA itself. GR is known to inhibit the activity of a growing list of immune-regulating transcription factors. Hence, GCs still rule the clinic for treatments of inflammatory disorders, notwithstanding concomitant deleterious side effects. Although patience is a virtue when it comes to deciphering the many mechanisms GR uses to influence various signaling pathways, the current review is testimony of the fact that groundbreaking mechanistic work has been accumulating over the past years and steadily continues to grow.

  18. Roles of tumor necrosis factor receptor associated factor 3 (TRAF3) and TRAF5 in immune cell functions

    PubMed Central

    Hildebrand, Joanne M.; Yi, Zuoan; Buchta, Claire M.; Poovassery, Jayakumar; Stunz, Laura L.; Bishop, Gail A.

    2011-01-01

    Summary A large and diverse group of receptors utilizes the family of cytoplasmic signaling proteins known as tumor necrosis factor receptor (TNFR)-associated factors (TRAFs). In recent years, there has been a resurgence of interest and exploration of the roles played by TRAF3 and TRAF5 in cellular regulation, particularly in cells of the immune system, the cell types of focus in this review. This work has revealed that TRAF3 and TRAF5 can play diverse roles for different receptors even in the same cell type, as well as distinct roles in different cell types. Evidence indicates that TRAF3 and TRAF5 play important roles beyond the TNFR-superfamily (SF) and viral mimics of its members, mediating certain innate immune receptor and cytokine receptor signals, and most recently, signals delivered by the T-cell receptor (TCR) signaling complex. Additionally, much research has demonstrated the importance of TRAF3-mediated cellular regulation via its cytoplasmic interactions with additional signaling proteins. In particular, we discuss below evidence for the participation by TRAF3 in a number of the regulatory post-translational modifications involving ubiquitin that are important in various signaling pathways. PMID:22017431

  19. Interactions between modulators of the GABAA receptor: Stiripentol and benzodiazepines

    PubMed Central

    Fisher, Janet L.

    2011-01-01

    Many patients with refractory epilepsy are treated with polytherapy, and nearly 15% of epilepsy patients receive two or more anti-convulsant agents. The anti-convulsant stiripentol is used as an add-on treatment for the childhood epilepsy syndrome known as severe myoclonic epilepsy in infancy (Dravet Syndrome). Stiripentol has multiple mechanisms of action, both enhancing GABAA receptors and reducing activity of metabolic enzymes that break down other drugs. Stiripentol is typically co-administered with other anti-convulsants such as benzodiazepines which also act through GABAA receptor modulation. Stiripentol slows the metabolism of some of these drugs through inhibition of a variety of cytochrome P450 enzymes, but could also influence their effects on GABAergic neurotransmission. Is it rational to co-administer drugs which can act through the same target? To examine the potential interaction between these modulators, we transiently transfected HEK-293T cells to produce α3β3γ2L or α3β3δ recombinant GABAA receptors. Using whole-cell patch clamp recordings, we measured the response to each benzodiazepine alone and in combination with a maximally effective concentration of stiripentol. We compared the responses to four different benzodiazepines: diazepam, clonazepam, clobazam and norclobazam. In all cases we found that these modulators were equally effective in the presence and absence of stiripentol. The δ-containing receptors were insensitive to modulation by the benzodiazepines, which did not affect potentiation by stiripentol. These data suggest that stiripentol and the benzodiazepines act independently at GABAA receptors and that polytherapy could be expected to increase the maximum effect beyond either drug alone, even without consideration of changes in metabolism. PMID:21237147

  20. Insulin receptor substrate-3, interacting with Bcl-3, enhances p50 NF-{kappa}B activity

    SciTech Connect

    Kabuta, Tomohiro; Hakuno, Fumihiko; Cho, Yoshitake; Yamanaka, Daisuke; Chida, Kazuhiro; Asano, Tomoichiro; Wada, Keiji; Takahashi, Shin-Ichiro

    2010-04-09

    The insulin receptor substrate (IRS) proteins are major substrates of both insulin receptor and insulin-like growth factor (IGF)-I receptor tyrosine kinases. Previously, we reported that IRS-3 is localized to both cytosol and nucleus, and possesses transcriptional activity. In the present study, we identified Bcl-3 as a novel binding protein to IRS-3. Bcl-3 is a nuclear protein, which forms a complex with the homodimer of p50 NF-{kappa}B, leading to enhancement of transcription through p50 NF-{kappa}B. We found that Bcl-3 interacts with the pleckstrin homology domain and the phosphotyrosine binding domain of IRS-3, and that IRS-3 interacts with the ankyrin repeat domain of Bcl-3. In addition, IRS-3 augmented the binding activity of p50 to the NF-{kappa}B DNA binding site, as well as the tumor necrosis factor (TNF)-{alpha}-induced transcriptional activity of NF-{kappa}B. Lastly, IRS-3 enhanced NF-{kappa}B-dependent anti-apoptotic gene induction and consequently inhibited TNF-{alpha}-induced cell death. This series of results proposes a novel function for IRS-3 as a transcriptional regulator in TNF-{alpha} signaling, distinct from its function as a substrate of insulin/IGF receptor kinases.

  1. Data on overlapping brain disorders and emerging drug targets in human Dopamine Receptors Interaction Network.

    PubMed

    Podder, Avijit; Latha, N

    2017-06-01

    Intercommunication of Dopamine Receptors (DRs) with their associate protein partners is crucial to maintain regular brain function in human. Majority of the brain disorders arise due to malfunctioning of such communication process. Hence, contributions of genetic factors, as well as phenotypic indications for various neurological and psychiatric disorders are often attributed as sharing in nature. In our earlier research article entitled "Human Dopamine Receptors Interaction Network (DRIN): a systems biology perspective on topology, stability and functionality of the network" (Podder et al., 2014) [1], we had depicted a holistic interaction map of human Dopamine Receptors. Given emphasis on the topological parameters, we had characterized the functionality along with the vulnerable properties of the network. In support of this, we hereby provide an additional data highlighting the genetic overlapping of various brain disorders in the network. The data indicates the sharing nature of disease genes for various neurological and psychiatric disorders in dopamine receptors connecting protein-protein interactions network. The data also indicates toward an alternative approach to prioritize proteins for overlapping brain disorders as valuable drug targets in the network.

  2. Genetic analysis of Caenorhabditis elegans glp-1 mutants suggests receptor interaction or competition.

    PubMed Central

    Pepper, Anita S-R; Killian, Darrell J; Hubbard, E Jane Albert

    2003-01-01

    glp-1 encodes a member of the highly conserved LIN-12/Notch family of receptors that mediates the mitosis/meiosis decision in the C. elegans germline. We have characterized three mutations that represent a new genetic and phenotypic class of glp-1 mutants, glp-1(Pro). The glp-1(Pro) mutants display gain-of-function germline pattern defects, most notably a proximal proliferation (Pro) phenotype. Each of three glp-1(Pro) alleles encodes a single amino acid change in the extracellular part of the receptor: two in the LIN-12/Notch repeats (LNRs) and one between the LNRs and the transmembrane domain. Unlike other previously described gain-of-function mutations that affect this region of LIN-12/Notch family receptors, the genetic behavior of glp-1(Pro) alleles is not consistent with simple hypermorphic activity. Instead, the mutant phenotype is suppressed by wild-type doses of glp-1. Moreover, a trans-heterozygous combination of two highly penetrant glp-1(Pro) mutations is mutually suppressing. These results lend support to a model for a higher-order receptor complex and/or competition among receptor proteins for limiting factors that are required for proper regulation of receptor activity. Double-mutant analysis with suppressors and enhancers of lin-12 and glp-1 further suggests that the functional defect in glp-1(Pro) mutants occurs prior to or at the level of ligand interaction. PMID:12586701

  3. Modulation of the NMDA Receptor Through Secreted Soluble Factors.

    PubMed

    Cerpa, Waldo; Ramos-Fernández, Eva; Inestrosa, Nibaldo C

    2016-01-01

    Synaptic activity is a critical determinant in the formation and development of excitatory synapses in the central nervous system (CNS). The excitatory current is produced and regulated by several ionotropic receptors, including those that respond to glutamate. These channels are in turn regulated through several secreted factors that function as synaptic organizers. Specifically, Wnt, brain-derived neurotrophic factor (BDNF), fibroblast growth factor (FGF), and transforming growth factor (TGF) particularly regulate the N-methyl-D-aspartate receptor (NMDAR) glutamatergic channel. These factors likely regulate early embryonic development and directly control key proteins in the function of important glutamatergic channels. Here, we review the secreted molecules that participate in synaptic organization and discuss the cell signaling behind of this fine regulation. Additionally, we discuss how these factors are dysregulated in some neuropathologies associated with glutamatergic synaptic transmission in the CNS.

  4. Factors affecting alum-protein interactions.

    PubMed

    Huang, Min; Wang, Wei

    2014-05-15

    Alum (or aluminum-containing) adjuvants are key components of many vaccines currently on the market. The immuno-potentiation effect of alum adjuvants is presumably due to their interaction with antigens, leading to adsorption on the alum particle surface. Understanding the mechanism of antigen adsorption/desorption and its influencing factors could provide guidance on formulation design and ensure proper in-vivo immuno-potentiation effect. In this paper, surface adsorption of several model proteins on two types of aluminum adjuvants (Alhydrogel(®) and Adjuphos(®)) are investigated to understand the underlying adsorption mechanisms, capacities, and potential influencing factors. It was found that electrostatic interactions are the major driving force for surface adsorption of all the model proteins except ovalbumin. Alhydrogel has a significantly higher adsorption capacity than Adjuphos. Several factors significantly change the adsorption capacity of both Alhydrogel and Adjuphos, including molecular weight of protein antigens, sodium chloride, phosphate buffer, denaturing agents, and size of aluminum particles. These important factors need to be carefully considered in the design of an effective protein antigen-based vaccine. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Factors That Effect Signal Transduction by the Estrogen Receptor.

    DTIC Science & Technology

    1997-10-01

    Vivat , H. Gronemeyer, R. Losson, and P. Chambon. 1996. Ligand-dependent interaction of nuclear receptors with potential transcriptional... Academy of Sciences 0027-8424/97/9410132-6S2.00/0 PNAS is available online at http://www.pnas.org. inhibitors or CDIs), kinase function (5-8). Because

  6. Induction of murine NAD(P)H:quinone oxidoreductase by 2,3,7,8-tetrachlorodibenzo-p-dioxin requires the CNC (cap 'n' collar) basic leucine zipper transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2): cross-interaction between AhR (aryl hydrocarbon receptor) and Nrf2 signal transduction.

    PubMed Central

    Ma, Qiang; Kinneer, Krista; Bi, Yongyi; Chan, Jefferson Y; Kan, Yuet Wai

    2004-01-01

    TCDD (2,3,7,8-tetrachlorodibenzo- p -dixoin) induces phase II drug-metabolizing enzyme NQO1 [NAD(P)H:quinone oxidoreductase; EC 1.6.99.2; DT-diaphorase] in a wide range of mammalian tissues and cells. Here, we analysed the molecular pathway mediating NQO1 induction by TCDD in mouse hepatoma cells. Inhibition of protein synthesis with CHX (cycloheximide) completely blocks induction of NQO1 by TCDD as well as the basal expression and induction by phenolic antioxidant tBHQ (2-t-butylbenzene-1,4-diol), implicating a labile factor in NQO1 mRNA expression. The inhibition is both time- and concentration-dependent, requires inhibition of protein synthesis, and occurs at a transcriptional level. Inhibition of NQO1 transcription by CHX correlates with a rapid reduction of the CNC bZip (cap 'n' collar basic leucine zipper) transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) through the 26 S proteasome pathway. Moreover, blocking Nrf2 degradation with proteasome inhibitor MG132 increases the amount of Nrf2 and superinduces NQO1 in the presence of TCDD or tBHQ. Finally, genetic experiments using AhR (aryl hydrocarbon receptor)-, Arnt (aryl hydrocarbon receptor nuclear translocator)- or Nrf2-deficient cells reveal that, while induction of NQO1 by TCDD depends on the presence of AhR and Arnt, the basal and inducible expression of NQO1 by either TCDD or tBHQ requires functional Nrf2. The findings demonstrate a novel role of Nrf2 in the induction of NQO1 by TCDD and provide new insights into the mechanism by which Nrf2 regulates the induction of phase II enzymes by both phenolic antioxidants and AhR ligands. PMID:14510636

  7. Platelet-activating factor: receptors and signal transduction.

    PubMed

    Chao, W; Olson, M S

    1993-06-15

    During the past two decades, studies describing the chemistry and biology of PAF have been extensive. This potent phosphoacylglycerol exhibits a wide variety of physiological and pathophysiological effects in various cells and tissues. PAF acts, through specific receptors and a variety of signal transduction systems, to elicit diverse biochemical responses. Several important future directions can be enumerated for the characterization of PAF receptors and their attendant signalling mechanisms. The recent cloning and sequence analysis of the gene for the PAF receptor will allow a number of important experimental approaches for characterizing the structure and analysing the function of the various domains of the receptor. Using molecular genetic and immunological technologies, questions relating to whether there is receptor heterogeneity, the precise mechanism(s) for the regulation of the PAF receptor, and the molecular details of the signalling mechanisms in which the PAF receptor is involved can be explored. Another area of major significance is the examination of the relationship between the signalling response(s) evoked by PAF binding to its receptor and signalling mechanisms activated by a myriad of other mediators, cytokines and growth factors. A very exciting recent development in which PAF receptors undoubtedly play a role is in the regulation of the function of various cellular adhesion molecules. Finally, there remain many incompletely characterized physiological and pathophysiological situations in which PAF and its receptor play a crucial signalling role. Our laboratory has been active in the elucidation of several tissue responses in which PAF exhibits major autocoid signalling responses, e.g. hepatic injury and inflammation, acute and chronic pancreatitis, and cerebral stimulation and/or trauma. As new experimental strategies are developed for characterizing the fine structure of the molecular mechanisms involved in tissue injury and inflammation, the

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

    PubMed

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

    2011-02-11

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

  9. Blueprints of signaling interactions between pattern recognition receptors: implications for the design of vaccine adjuvants.

    PubMed

    Timmermans, Kim; Plantinga, Theo S; Kox, Matthijs; Vaneker, Michiel; Scheffer, Gert Jan; Adema, Gosse J; Joosten, Leo A B; Netea, Mihai G

    2013-03-01

    Innate immunity activation largely depends on recognition of microorganism structures by Pattern Recognition Receptors (PRRs). PRR downstream signaling results in production of pro- and anti-inflammatory cytokines and other mediators. Moreover, PRR engagement in antigen-presenting cells initiates the activation of adaptive immunity. Recent reports suggest that for the activation of innate immune responses and initiation of adaptive immunity, synergistic effects between two or more PRRs are necessary. No systematic analysis of the interaction between the major PRR pathways were performed to date. In this study, a systematical analysis of the interactions between PRR signaling pathways was performed. PBMCs derived from 10 healthy volunteers were stimulated with either a single PRR ligand or a combination of two PRR ligands. Known ligands for the major PRR families were used: Toll-like receptors (TLRs), C-type lectin receptors (CLRs), NOD-like receptors (NLRs), and RigI-helicases. After 24 h of incubation, production of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), IL-6, and IL-10 was measured in supernatants by enzyme-linked immunosorbent assay (ELISA). The consistency of the PRR interactions (both inhibitory and synergistic) between the various individuals was assessed. A number of PRR-dependent signaling interactions were found to be consistent, both between individuals and with regard to multiple cytokines. The combinations of TLR2 and NOD2, TLR5 and NOD2, TLR5 and TLR3, and TLR5 and TLR9 acted as synergistic combinations. Surprisingly, inhibitory interactions between TLR4 and TLR2, TLR4 and Dectin-1, and TLR2 and TLR9 as well as TLR3 and TLR2 were observed. These consistent signaling interactions between PRR combinations may represent promising targets for immunomodulation and vaccine adjuvant development.

  10. Interactions between Histamine H3 and Dopamine D2 Receptors and the Implications for Striatal Function

    PubMed Central

    Ferrada, Carla; Ferré, Sergi; Casadó, Vicent; Cortés, Antonio; Justinova, Zuzana; Barnes, Chanel; Canela, Enric I.; Goldberg, Steven R.; Leurs, Rob; Lluis, Carme; Franco, Rafael

    2008-01-01

    The striatum contains a high density of histamine H3 receptors, but their role in striatal function is poorly understood. Previous studies have demonstrated antagonistic interactions between striatal H3 and dopamine D1 receptors at the biochemical level, while contradictory results have been reported about interactions between striatal H3 and dopamine D2 receptors. In the present study, by using reserpinized mice, we demonstrate the existence of behaviorally significant antagonistic postsynaptic interactions between H3 and D1 and also between H3 and dopamine D2 receptors. The selective H3 receptor agonist imetit inhibited, while the H3 receptor antagonist thioperamide potentiated locomotor activation induced by either the D1 receptor agonist SKF 38393 or the D2 receptor agonist quinpirole. High scores of locomotor activity were obtained with H3 receptor blockade plus D1 and D2 receptor co-activation, i.e., when thioperamide was co-administered with both SKF 38393 and quinpirole. Radioligand binding experiments in striatal membrane preparations showed the existence of a strong and selective H3-D2 receptor interaction at the membrane level. In agonist/antagonist competition experiments stimulation of H3 receptors with several H3 receptor agonists significantly decreased the affinity of D2 receptors for the agonist. This kind of intramembrane receptor-receptor interactions are a common biochemical property of receptor heteromers. In fact, by using Bioluminescence Resonance Energy Transfer techniques in co-transfected HEK-293 cells, H3 (but not H4) receptors were found to form heteromers with D2 receptors. The present study demonstrates an important role of postsynaptic H3 receptors in the modulation of dopaminergic transmission by means of a negative modulation of D2 receptor function. PMID:18547596

  11. Placental growth factor and vascular endothelial growth factor receptor-2 in human lung development.

    PubMed

    Janér, Joakim; Andersson, Sture; Haglund, Caj; Karikoski, Riitta; Lassus, Patrik

    2008-08-01

    We examined the pulmonary expression of 2 proangiogenic factors, namely, placental growth factor and vascular endothelial growth factor receptor-2, during lung development and acute and chronic lung injury in newborn infants. Six groups were included in an immunohistochemical study of placental growth factor and vascular endothelial growth factor receptor-2, that is, 9 fetuses, 4 preterm and 8 term infants without lung injury who died soon after birth, 5 preterm infants with respiratory distress syndrome of <2 days and 7 with respiratory distress syndrome of >10 days, and 6 with bronchopulmonary dysplasia. Placental growth factor concentrations in tracheal aspirate fluid were measured in 70 samples from 20 preterm infants during the first postnatal week. In immunohistochemical analyses, placental growth factor staining was seen in bronchial epithelium and macrophages in all groups. Distal airway epithelium positivity was observed mostly in fetuses and in preterm infants who died soon after birth. Vascular endothelial growth factor receptor-2 staining was seen in vascular endothelium in all groups and also in lymphatic endothelium in fetuses. Vascular endothelial growth factor receptor-2 staining in arterial endothelium was associated with higher and staining in venous endothelium with lower gestational age. In capillaries, less vascular endothelial growth factor receptor-2 staining was seen in bronchopulmonary dysplasia. The mean placental growth factor protein concentration in tracheal aspirate fluid during the first postnatal week was 0.64 +/- 0.42 pg/mL per IgA-secretory component unit. Concentrations during the first postnatal week were stable. Lower placental growth factor concentrations correlated with chorioamnionitis and lactosyl ceramide positivity. The vascular endothelial growth factor receptor-2 staining pattern seems to reflect ongoing differentiation and activity of different endothelia. Lower vascular endothelial growth factor receptor-2 expression

  12. The (pro)renin receptor and its interaction partners.

    PubMed

    Peters, Jörg

    2017-06-15

    The prorenin receptor was originally discovered as a receptor that binds renin and prorenin, thereby inducing pro-fibrotic intracellular signal cascades. These effects are partially mediated in vitro by angiotensin (ANG) and partially independent of ANG. Consequently, inhibitors of the interaction between the prorenin and the (pro)renin receptor (PRR) were designed hoping that they may prevent fibrotic tissue damage, for instance, in the kidney. However, this concept was challenged by the fact that overexpression of the PRR was not harmful at all, whereas depletion of the PRR was lethal or markedly detrimental. Furthermore, the high levels of prorenin needed to activate the PRR may not be reached in vivo. As it turned out, the PRR instead exhibited a variety of important functions that has nothing to do with the name given to the protein. Thus, the PRR was identified as an accessory subunit of vesicular (v)-ATPases, representing an essential chaperon for the assembly of v-ATPase subunits. In this respect, the gene encoding the PRR was also named ATP6AP2. Finally, the PRR is an essential component of the canonical and non-canonical PCP Wnt pathways. Thus, the PRR is essential for lysosomal functions, such as endocytosis, secretion, and autophagy as well as for cell division and differentiation, embryonic development, organogenesis, and stem cell biology.

  13. In vivo study of drug interaction with brain benzodiazepine receptor

    SciTech Connect

    Inoue, O.; Shinotoh, H.; Ito, T.; Suzuki, K.; Hashimoto, K.; Yamasaki, T.

    1985-05-01

    The possibility of direct estimation of in vivo Bz receptor occupancy in brain was evaluated using C-11, or H-3-flumazepil (Ro15-1788). In animal experiments, 1 ..mu..Ci of H-3-Ro15-1788 was injected at 0.5 or 20 hr after i.v. injection of various dosage of clonazepam. Then radioactivity in cerebral cortex, cerebellum and blood at 5 min. after injection of the tracer was compared. Competitive inhibition of in vivo binding was clearly observed when clonazepam was pretreated at 0.5 hr before injection of the tracer. On the other hand, brain radioactivity was increased when clonazepam was administered at 20 hr before injection of the tracer. This increase in binding of H-3-Ro15-1788 might be caused by rebound of Bz receptor function by treatment with Bz agonist, and this rebound may have an important role in physiological function. Clinical investigation concerning drug interaction with brain Bz receptor was performed in normal volunteer and patients with neurological disorders. The distribution of C-11-Ro15-1788 in the brain of patients chronically treated with clonazepam were significantly heterogeneous. However, cerebral blood flow estimated with N-13 NH3 of these patients were normal.

  14. Metal interactions with voltage- and receptor-activated ion channels.

    PubMed Central

    Vijverberg, H P; Oortgiesen, M; Leinders, T; van Kleef, R G

    1994-01-01

    Effects of Pb and several other metal ions on various distinct types of voltage-, receptor- and Ca-activated ion channels have been investigated in cultured N1E-115 mouse neuroblastoma cells. Experiments were performed using the whole-cell voltage clamp and single-channel patch clamp techniques. External superfusion of nanomolar to submillimolar concentrations of Pb causes multiple effects on ion channels. Barium current through voltage-activated Ca channels is blocked by micromolar concentrations of Pb, whereas voltage-activated Na current appears insensitive. Neuronal type nicotinic acetylcholine receptor-activated ion current is blocked by nanomolar concentrations of Pb and this block is reversed at micromolar concentrations. Serotonin 5-HT3 receptor-activated ion current is much less sensitive to Pb. In addition, external superfusion with micromolar concentrations of Pb as well as of Cd and aluminum induces inward current, associated with the direct activation of nonselective cation channels by these metal ions. In excised inside-out membrane patches of neuroblastoma cells, micromolar concentrations of Ca activate small (SK) and big (BK) Ca-activated K channels. Internally applied Pb activates SK and BK channels more potently than Ca, whereas Cd is approximately equipotent to Pb with respect to SK channel activation, but fails to activate BK channels. The results show that metal ions cause distinct, selective effects on the various types of ion channels and that metal ion interaction sites of ion channels may be highly selective for particular metal ions. PMID:7531139

  15. Tissue factor- and factor X-dependent activation of protease-activated receptor 2 by factor VIIa

    PubMed Central

    Camerer, Eric; Huang, Wei; Coughlin, Shaun R.

    2000-01-01

    Protease-activated receptor 2 (PAR2) is expressed by vascular endothelial cells and other cells in which its function and physiological activator(s) are unknown. Unlike PAR1, PAR3, and PAR4, PAR2 is not activatable by thrombin. Coagulation factors VIIa (FVIIa) and Xa (FXa) are proteases that act upstream of thrombin in the coagulation cascade and require cofactors to interact with their substrates. These proteases elicit cellular responses, but their receptor(s) have not been identified. We asked whether FVIIa and FXa might activate PARs if presented by their cofactors. Co-expression of tissue factor (TF), the cellular cofactor for FVIIa, together with PAR1, PAR2, PAR3, or PAR4 conferred TF-dependent FVIIa activation of PAR2 and, to lesser degree, PAR1. Responses to FXa were also observed but were independent of exogenous cofactor. The TF/FVIIa complex converts the inactive zymogen Factor X (FX) to FXa. Strikingly, when FX was present, low picomolar concentrations of FVIIa caused robust signaling in cells expressing TF and PAR2. Responses in keratinocytes and cytokine-treated endothelial cells suggested that PAR2 may be activated directly by TF/FVIIa and indirectly by TF/FVIIa-generated FXa at naturally occurring expression levels of TF and PAR2. These results suggest that PAR2, although not activatable by thrombin, may nonetheless function as a sensor for coagulation proteases and contribute to endothelial activation in the setting of injury and inflammation. More generally, these findings highlight the potential importance of cofactors in regulating PAR function and specificity. PMID:10805786

  16. Visualization of epidermal growth factor (EGF) receptor aggregation in plasma membranes by fluorescence resonance energy transfer. Correlation of receptor activation with aggregation.

    PubMed

    Carraway, K L; Koland, J G; Cerione, R A

    1989-05-25

    Fluorescence resonance energy transfer between epidermal growth factor (EGF) molecules, labeled with fluorescent reporter groups, was used as a monitor for EGF receptor-receptor interactions in plasma membranes isolated from human epidermoid A431 cells. Epidermal growth factor molecules labeled at the amino terminus with fluorescein isothiocyanate served as donor molecules in these energy transfer measurements, while EGF molecules labeled with eosin isothiocyanate at the amino terminus served as the energy acceptors. Both of these derivatives were shown to be active in binding to membrane receptors and in the activation of the endogenous receptor/tyrosine kinase activity. We found that membranes in the absence of added metal ion activators showed relatively little energy transfer (approximately 10% donor quenching) between the labeled growth factors. However, divalent metal ion activators of the EGF receptor/tyrosine kinase caused a significant increase in the extent of energy transfer between the labeled EGF molecules. Specifically, in the presence of 20 mM MgCl2, the extent of quenching of the donor fluorescence increased to 25% (from 10% in the absence of metal), while in the presence of 4 mM MnCl2, the extent of energy transfer was increased still further to 40-50%. The addition of an excess of EDTA resulted in the reversal of the observed energy transfer to basal levels. The increased energy transfer in the presence of these divalent cations correlated well with the ability of these metals to stimulate the EGF receptor/tyrosine kinase activity. However, the extent of receptor-receptor interactions measured by energy transfer was independent of receptor autophosphorylation. Overall, these results suggest that conditions under which the EGF receptor is primed to be active as a tyrosine kinase, within a lipid milieu, result in an increased aggregation of the receptor.

  17. A structural view of nuclear hormone receptor: endocrine disruptor interactions.

    PubMed

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

    2010-04-01

    Endocrine-disrupting chemicals (EDCs) represent a broad class of exogenous substances that cause adverse effects in the endocrine system by interfering with hormone biosynthesis, metabolism, or action. The molecular mechanisms of EDCs involve different pathways including interactions with nuclear hormone receptors (NHRs) which are primary targets of a large variety of environmental contaminants. Here, based on the crystal structures currently available in the Protein Data Bank, we review recent studies showing the many ways in which EDCs interact with NHRs and impact their signaling pathways. Like the estrogenic chemical diethylstilbestrol, some EDCs mimic the natural hormones through conserved protein-ligand contacts, while others, such as organotins, employ radically different binding mechanisms. Such structure-based knowledge, in addition to providing a better understanding of EDC activities, can be used to predict the endocrine-disrupting potential of environmental pollutants and may have applications in drug discovery.

  18. Cell and molecular biology of epidermal growth factor receptor.

    PubMed

    Ceresa, Brian P; Peterson, Joanne L

    2014-01-01

    The epidermal growth factor receptor (EGFR) has been one of the most intensely studied cell surface receptors due to its well-established roles in developmental biology, tissue homeostasis, and cancer biology. The EGFR has been critical for creating paradigms for numerous aspects of cell biology, such as ligand binding, signal transduction, and membrane trafficking. Despite this history of discovery, there is a continual stream of evidence that only the surface has been scratched. New ways of receptor regulation continue to be identified, each of which is a potential molecular target for manipulating EGFR signaling and the resultant changes in cell and tissue biology. This chapter is an update on EGFR-mediated signaling, and describes some recent developments in the regulation of receptor biology.

  19. Vascular growth factors and receptors in capillary hemangioblastomas and hemangiopericytomas.

    PubMed Central

    Hatva, E.; Böhling, T.; Jääskeläinen, J.; Persico, M. G.; Haltia, M.; Alitalo, K.

    1996-01-01

    Capillary hemangioblastomas and hemangiopericytomas are highly vascular central nervous system tumors of controversial origin. Of interest in their pathogenesis are mechanisms regulating endothelial cell growth. The endothelial cell mitogen vascular endothelial growth factor (VEGF) stimulates angiogenesis, and together with its two receptor tyrosine kinases VEGFR-1(FLT1) and VEGFR-2(KDR), is up-regulated during the malignant progression of gliomas. We have analyzed the expression of VEGF and its receptors, the related placental growth factor (PlGF) and the endothelial receptors FLT4 and Tie by in situ hybridization in capillary hemangioblastomas and hemangiopericytomas. VEGF mRNA was up-regulated in all of the hemangiopericytomas studied and highly expressed in the stromal cells of hemangioblastomas. In addition, some hemangioblastoma tumor cells expressed high levels of PlGF. Significantly elevated levels of Tie mRNA, Tie protein, VEGFR-1, and VEGFR-2 but not FLT4 mRNAs were observed in the endothelia of both tumor types. In hemangioblastomas, however, the receptors were also highly expressed by a subpopulation of stromal cells. Consistent results were obtained for a human hemangioblastoma cell line in culture. Up-regulation of the endothelial growth factors and receptors may result in autocrine or paracrine stimulation of endothelial cells and their precursors involved in the genesis of these two vascular tumors. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8774132

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

    PubMed

    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.

  1. Interaction of Pattern Recognition Receptors with Mycobacterium Tuberculosis.

    PubMed

    Mortaz, Esmaeil; Adcock, Ian M; Tabarsi, Payam; Masjedi, Mohammad Reza; Mansouri, Davood; Velayati, Ali Akbar; Casanova, Jean-Laurent; Barnes, Peter J

    2015-01-01

    Tuberculosis (TB) is considered a major worldwide health problem with 10 million new cases diagnosed each year. Our understanding of TB immunology has become greater and more refined since the identification of Mycobacterium tuberculosis (MTB) as an etiologic agent and the recognition of new signaling pathways modulating infection. Understanding the mechanisms through which the cells of the immune system recognize MTB can be an important step in designing novel therapeutic approaches, as well as improving the limited success of current vaccination strategies. A great challenge in chronic disease is to understand the complexities, mechanisms, and consequences of host interactions with pathogens. Innate immune responses along with the involvement of distinct inflammatory mediators and cells play an important role in the host defense against the MTB. Several classes of pattern recognition receptors (PRRs) are involved in the recognition of MTB including Toll-Like Receptors (TLRs), C-type lectin receptors (CLRs) and Nod-like receptors (NLRs) linked to inflammasome activation. Among the TLR family, TLR1, TLR2, TLR4, and TLR9 and their down-stream signaling proteins play critical roles in the initiation of the immune response in the pathogenesis of TB. The inflammasome pathway is associated with the coordinated release of cytokines such as IL-1β and IL-18 which also play a role in the pathogenesis of TB. Understanding the cross-talk between these signaling pathways will impact on the design of novel therapeutic strategies and in the development of vaccines and immunotherapy regimes. Abnormalities in PRR signaling pathways regulated by TB will affect disease pathogenesis and need to be elucidated. In this review we provide an update on PRR signaling during M. tuberculosis infection and indicate how greater knowledge of these pathways may lead to new therapeutic opportunities.

  2. Treponema pallidum receptor binding proteins interact with fibronectin

    PubMed Central

    1983-01-01

    Analysis of plasma proteins avidly bound to T. pallidum surfaces revealed the ability of T. pallidum to acquire numerous host macromolecules. No acquisition was evident by the avirulent spirochete, T. phagedenis biotype Reiter. Western blotting technology using hyperimmune antifibronectin serum as a probe revealed the ability of virulent treponemes to avidly bind fibronectin from a complex medium such as plasma. The specificity of the tiplike adherence of motile T. pallidum to fibronectin-coated glass surfaces and to fibronectin on HEp- 2 cells was reinforced by the observation that pretreatment of coverslips or cell monolayers with monospecific antiserum against fibronectin substantially reduced T. pallidum attachment. The stoichiometric binding of T. pallidum to fibronectin-coated coverslips and the inability of unlabeled or 35S-radiolabeled treponemes to interact with glass surfaces treated with other plasma proteins further established the specific nature of the interaction between virulent T. pallidum and fibronectin. The avid association between three outer envelope proteins of T. pallidum and fibronectin was also demonstrated. These treponemal surface proteins have been previously identified as putative receptor-binding proteins responsible for T. pallidum parasitism of host cells. The data suggest that surface fibronectin mediates tip-oriented attachment of T. pallidum to host cells via a receptor-ligand mechanism of recognition. PMID:6304227

  3. GIPC interacts with the beta1-adrenergic receptor and regulates beta1-adrenergic receptor-mediated ERK activation.

    PubMed

    Hu, Liaoyuan A; Chen, Wei; Martin, Negin P; Whalen, Erin J; Premont, Richard T; Lefkowitz, Robert J

    2003-07-11

    Beta1-adrenergic receptors, expressed at high levels in the human heart, have a carboxyl-terminal ESKV motif that can directly interact with PDZ domain-containing proteins. Using the beta1-adrenergic receptor carboxyl terminus as bait, we identified the novel beta1-adrenergic receptor-binding partner GIPC in a yeast two-hybrid screen of a human heart cDNA library. Here we demonstrate that the PDZ domain-containing protein, GIPC, co-immunoprecipitates with the beta1-adrenergic receptor in COS-7 cells. Essential for this interaction is the Ser residue of the beta1-adrenergic receptor carboxyl-terminal ESKV motif. Our data also demonstrate that beta1-adrenergic receptor stimulation activates the mitogen-activated protein kinase, ERK1/2. beta1-adrenergic receptor-mediated ERK1/2 activation was inhibited by pertussis toxin, implicating Gi, and was substantially decreased by the expression of GIPC. Expression of GIPC had no observable effect on beta1-adrenergic receptor sequestration or receptor-mediated cAMP accumulation. This GIPC effect was specific for the beta1-adrenergic receptor and was dependent on an intact PDZ binding motif. These data suggest that GIPC can regulate beta1-adrenergic receptor-stimulated, Gi-mediated, ERK activation while having no effect on receptor internalization or Gs-mediated cAMP signaling.

  4. Pharmacological Profile of Nociceptin/Orphanin FQ Receptors Interacting with G-Proteins and β-Arrestins 2

    PubMed Central

    Malfacini, D.; Ambrosio, C.; Gro’, M. C.; Sbraccia, M.; Trapella, C.; Guerrini, R.; Bonora, M.; Pinton, P.; Costa, T.; Calo’, G.

    2015-01-01

    Nociceptin/orphanin FQ (N/OFQ) controls several biological functions by selectively activating an opioid like receptor named N/OFQ peptide receptor (NOP). Biased agonism is emerging as an important and therapeutically relevant pharmacological concept in the field of G protein coupled receptors including opioids. To evaluate the relevance of this phenomenon in the NOP receptor, we used a bioluminescence resonance energy transfer technology to measure the interactions of the NOP receptor with either G proteins or β-arrestin 2 in the absence and in presence of increasing concentration of ligands. A large panel of receptor ligands was investigated by comparing their ability to promote or block NOP/G protein and NOP/arrestin interactions. In this study we report a systematic analysis of the functional selectivity of NOP receptor ligands. NOP/G protein interactions (investigated in cell membranes) allowed a precise estimation of both ligand potency and efficacy yielding data highly consistent with the known pharmacological profile of this receptor. The same panel of ligands displayed marked differences in the ability to promote NOP/β-arrestin 2 interactions (evaluated in whole cells). In particular, full agonists displayed a general lower potency and for some ligands an inverted rank order of potency was noted. Most partial agonists behaved as pure competitive antagonists of receptor/arrestin interaction. Antagonists displayed similar values of potency for NOP/Gβ1 or NOP/β-arrestin 2 interaction. Using N/OFQ as reference ligand we computed the bias factors of NOP ligands and a number of agonists with greater efficacy at G protein coupling were identified. PMID:26248189

  5. Oxidative stress effect on progesterone-induced blocking factor (PIBF) binding to PIBF-receptor in lymphocytes.

    PubMed

    de la Haba, Carlos; Palacio, José R; Palkovics, Tamas; Szekeres-Barthó, Júlia; Morros, Antoni; Martínez, Paz

    2014-01-01

    Receptor-ligand binding is an essential interaction for biological function. Oxidative stress can modify receptors and/or membrane lipid dynamics, thus altering cell physiological functions. The aim of this study is to analyze how oxidative stress may alter receptor-ligand binding and lipid domain distribution in the case of progesterone-induced blocking factor/progesterone-induced blocking factor-receptor. For membrane fluidity regionalization analysis of MEC-1 lymphocytes, two-photon microscopy was used in individual living cells. Lymphocytes were also double stained with AlexaFluor647/progesterone-induced blocking factor and Laurdan to evaluate -induced blocking factor/progesterone-induced blocking factor-receptor distribution in the different membrane domains, under oxidative stress. A new procedure has been developed which quantitatively analyzes the regionalization of a membrane receptor among the lipid domains of different fluidity in the plasma membrane. We have been able to establish a new tool which detects and evaluates lipid raft clustering from two-photon microscopy images of individual living cells. We show that binding of progesterone-induced blocking factor to progesterone-induced blocking factor-receptor causes a rigidification of plasma membrane which is related to an increase of lipid raft clustering. However, this clustering is inhibited under oxidative stress conditions. In conclusion, oxidative stress decreases membrane fluidity, impairs receptor-ligand binding and reduces lipid raft clustering. © 2013.

  6. A2A-D2 receptor-receptor interaction modulates gliotransmitter release from striatal astrocyte processes.

    PubMed

    Cervetto, Chiara; Venturini, Arianna; Passalacqua, Mario; Guidolin, Diego; Genedani, Susanna; Fuxe, Kjell; Borroto-Esquela, Dasiel O; Cortelli, Pietro; Woods, Amina; Maura, Guido; Marcoli, Manuela; Agnati, Luigi F

    2017-01-01

    Evidence for striatal A2A-D2 heterodimers has led to a new perspective on molecular mechanisms involved in schizophrenia and Parkinson's disease. Despite the increasing recognition of astrocytes' participation in neuropsychiatric disease vulnerability, involvement of striatal astrocytes in A2A and D2 receptor signal transmission has never been explored. Here, we investigated the presence of D2 and A2A receptors in isolated astrocyte processes prepared from adult rat striatum by confocal imaging; the effects of receptor activation were measured on the 4-aminopyridine-evoked release of glutamate from the processes. Confocal analysis showed that A2A and D2 receptors were co-expressed on the same astrocyte processes. Evidence for A2A-D2 receptor-receptor interactions was obtained by measuring the release of the gliotransmitter glutamate: D2 receptors inhibited the glutamate release, while activation of A2A receptors, per se ineffective, abolished the effect of D2 receptor activation. The synthetic D2 peptide VLRRRRKRVN corresponding to the receptor region involved in electrostatic interaction underlying A2A-D2 heteromerization abolished the ability of the A2A receptor to antagonize the D2 receptor-mediated effect. Together, the findings are consistent with heteromerization of native striatal astrocytic A2A-D2 receptors that via allosteric receptor-receptor interactions could play a role in the control of striatal glutamatergic transmission. These new findings suggest possible new pathogenic mechanisms and/or therapeutic approaches to neuropsychiatric disorders. © 2016 International Society for Neurochemistry.

  7. Cardiovirulent coxsackieviruses and the decay-accelerating factor (CD55) receptor.

    PubMed

    Martino, T A; Petric, M; Brown, M; Aitken, K; Gauntt, C J; Richardson, C D; Chow, L H; Liu, P P

    1998-05-10

    Group B coxsackieviruses are etiologically linked with many human diseases including acute myocarditis and associated chronic dilated cardiomyopathy. Well-established CVB3 cardiovirulent strains (CVB3c(s)) with known phenotypic difference have been used to study the pathogenesis of virus-induced heart disease. The receptor-binding characteristics of cardiovirulent CVB3 are not known, but may represent one mechanism accounting for differences in disease virulence. In this study, interactions between CVB3c(s) and the decay-accelerating factor (DAF or CD55) cell surface receptor were examined. Anti-DAF monoclonal antibodies (MAbs) blocked virus binding and infection of susceptible HeLa cells. Virus binding was significantly reduced by treatment of these cells with phosphatidylinositol phospholipase C enzyme, which rendered them DAF-deficient CVB3c(s) exhibited a differential propensity for the DAF receptor, as several cardiovirulent strains interacted more strongly than others. However, virus binding and infection was always most effectively blocked by MAbs directed against the SCR 2 and 3 domains of DAF, suggesting that binding occurs at a similar site(s) on the molecule for all strains. Virus binding and internalization were associated with DAF down-regulation at the cell surface, as monitored by flow cytometry analysis. Cardiovirulent CVB3 did not interact with molecules functionally and/or structurally related to DAF, including CD35, CD46, Factor H, or C4-binding protein. Adenovirus type 2 (Ad2) does not use the DAF receptor. However, competitive binding assays between Ad2 and CVB1-6, CVB3c(s), anti-DAF MAbs, or DAF-reduced cells indicated that DAF is associated with Ad2 receptors on the HeLa cell membrane. In summary, this study indicates that DAF is an attachment receptor for cardiovirulent CVB3 and that DAF interaction may be important in the pathogenesis of CVB-mediated heart disease.

  8. Interactions of transcription factors with chromatin.

    PubMed

    van Bakel, Harm

    2011-01-01

    Sequence-specific transcription factors (TFs) play a central role in regulating transcription initiation by directing the recruitment and activity of the general transcription machinery and accessory factors. It is now well established that many of the effects exerted by TFs in eukaryotes are mediated through interactions with a host of coregulators that modify the chromatin state, resulting in a more open (in case of activation) or closed conformation (in case of repression). The relationship between TFs and chromatin is a two-way street, however, as chromatin can in turn influence the recognition and binding of target sequences by TFs. The aim of this chapter is to highlight how this dynamic interplay between TF-directed remodelling of chromatin and chromatin-adjusted targeting of TF binding determines where and how transcription is initiated, and to what degree it is productive.

  9. Growth factor control of epidermal growth factor receptor kinase activity via an intramolecular mechanism.

    PubMed

    Koland, J G; Cerione, R A

    1988-02-15

    The mechanism by which the protein kinase activity of the epidermal growth factor (EGF) receptor is activated by binding of growth factor was investigated. Detergent-solubilized receptor in monomeric form was isolated by sucrose density gradient centrifugation and both its kinase and autophosphorylation activities monitored. In a low ionic strength medium and with MnCl2 as an activator, the activity of the monomeric receptor was EGF-independent. However, with 0.25 M ammonium sulfate present, the MnCl2-stimulated kinase activity was strikingly EGF-dependent. In contrast, the kinase activity expressed in the presence of MgCl2 showed growth factor control in the absence of added salt. Under the conditions of these experiments there was apparently little tendency for growth factor to induce aggregation of the receptor, indicating that the allosteric activation of the receptor kinase by EGF occurred via an intramolecular mechanism. Whereas detergent-solubilized receptor was the subject of these studies, the kinase activity of cell surface receptors might also be controlled by an intramolecular mechanism. These results indicate that an individual receptor molecule has the potential to function as a transmembrane signal transducer.

  10. The Under-Appreciated Promiscuity of the Epidermal Growth Factor Receptor Family

    PubMed Central

    Kennedy, Sean P.; Hastings, Jordan F.; Han, Jeremy Z. R.; Croucher, David R.

    2016-01-01

    Each member of the epidermal growth factor receptor (EGFR) family plays a key role in normal development, homeostasis, and a variety of pathophysiological conditions, most notably in cancer. According to the prevailing dogma, these four receptor tyrosine kinases (RTKs; EGFR, ERBB2, ERBB3, and ERBB4) function exclusively through the formation of homodimers and heterodimers within the EGFR family. These combinatorial receptor interactions are known to generate increased interactome diversity and therefore influence signaling output, subcellular localization and function of the heterodimer. This molecular plasticity is also thought to play a role in the development of resistance toward targeted cancer therapies aimed at these known oncogenes. Interestingly, many studies now challenge this dogma and suggest that the potential for EGFR family receptors to interact with more distantly related RTKs is much greater than currently appreciated. Here we discuss how the promiscuity of these oncogenic receptors may lead to the formation of many unexpected receptor pairings and the significant implications for the efficiency of many targeted cancer therapies. PMID:27597943

  11. The insulin-like growth factor 1 receptor in cancer: old focus, new future.

    PubMed

    Hartog, Hermien; Wesseling, Jelle; Boezen, H Marike; van der Graaf, Winette T A

    2007-09-01

    The importance of insulin-like growth factor 1 receptor (IGF-1R) signalling in malignant behaviour of tumour cells is well established. Currently, development of drugs targeting the IGF-1R as anticancer treatment is emerging. Several IGF-1R targeting strategies are being investigated in phases I and II clinical trials. Interactions of IGF-1R with insulin receptor, however, might complicate efficiency and tolerability of such drugs. This review describes mechanisms, recent developments and potential limitations of IGF-1R antibodies and tyrosine kinase inhibitors.

  12. Regulation of GABAA Receptor Dynamics by Interaction with Purinergic P2X2 Receptors*

    PubMed Central

    Shrivastava, Amulya Nidhi; Triller, Antoine; Sieghart, Werner; Sarto-Jackson, Isabella

    2011-01-01

    γ-Aminobutyric acid type A receptors (GABAARs) in the spinal cord are evolving as an important target for drug development against pain. Purinergic P2X2 receptors (P2X2Rs) are also expressed in spinal cord neurons and are known to cross-talk with GABAARs. Here, we investigated a possible “dynamic” interaction between GABAARs and P2X2Rs using co-immunoprecipitation and fluorescence resonance energy transfer (FRET) studies in human embryonic kidney (HEK) 293 cells along with co-localization and single particle tracking studies in spinal cord neurons. Our results suggest that a significant proportion of P2X2Rs forms a transient complex with GABAARs inside the cell, thus stabilizing these receptors and using them for co-trafficking to the cell surface, where P2X2Rs and GABAARs are primarily located extra-synaptically. Furthermore, agonist-induced activation of P2X2Rs results in a Ca2+-dependent as well as an apparently Ca2+-independent increase in the mobility and an enhanced degradation of GABAARs, whereas P2X2Rs are stabilized and form larger clusters. Antagonist-induced blocking of P2XRs results in co-stabilization of this receptor complex at the cell surface. These results suggest a novel mechanism where association of P2X2Rs and GABAARs could be used for specific targeting to neuronal membranes, thus providing an extrasynaptic receptor reserve that could regulate the excitability of neurons. We further conclude that blocking the excitatory activity of excessively released ATP under diseased state by P2XR antagonists could simultaneously enhance synaptic inhibition mediated by GABAARs. PMID:21343285

  13. PPAR δ agonist GW0742 interacts weakly with multiple nuclear receptors including the vitamin D receptor

    PubMed Central

    Nandhikonda, Premchendar; Yasgar, Adam; Baranowski, Athena M.; Sidhu, Preetpal S.; McCallum, Megan M.; Pawlak, Alan J.; Teske, Kelly; Feleke, Belaynesh; Yuan, Nina Y.; Kevin, Chinedum; Bikle, Daniel D.; Ayers, Steven D.; Webb, Paul; Rai, Ganesha; Simeonov, Anton; Jadhav, Ajit; Maloney, David; Arnold, Leggy A.

    2013-01-01

    A high throughput screening campaign was conducted to identify small molecules with the ability to inhibit the interaction between the vitamin D receptor (VDR) and steroid receptor coactivator 2. These inhibitors represent novel molecular probes to modulate gene regulation mediated by VDR. The peroxisome proliferator-activated receptor δ (PPARδ) agonist GW0742 was among the identified VDR-coactivator inhibitors and has been characterized herein as a pan nuclear receptor antagonist at concentrations higher than 12.1 µM. The highest antagonist activity for GW0742 was found for VDR and the androgen receptor (AR). Surprisingly, GW0742 behaved as PPAR agonist/antagonist activating transcription at lower concentration and inhibiting this effect at higher concentrations. A unique spectroscopic property of GW0742 was identified as well. In the presence of rhodamine-derived molecules, GW0742+ increased fluorescence intensity and fluorescence polarization at an excitation wavelength of 595 nm and emission wavelength of 615 nm in a dose dependent manner. The GW0742-inhibited NR-coactivator binding resulted in a reduced expression of five different NR target genes in LNCaP cells in the presence of agonist. Especially VDR target genes CYP24A1, IGFBP-3 and TRPV6 were negatively regulated by GW0742. GW0742 is the first VDR ligand inhibitor lacking the secosteroid structure of VDR ligand antagonists. Nevertheless, the VDR-meditated downstream process of cell differentiation was antagonized by GW0742 in HL-60 cells that were pretreated with the endogenous VDR agonist 1,25-dihydroxyvitamin D3. PMID:23713684

  14. The ontogeny of epidermal growth factor receptors during mouse development

    SciTech Connect

    Adamson, E.D.; Meek, J.

    1984-05-01

    In an attempt to understand the role(s) of epidermal growth factor (EGF) in vivo during murine development, we have examined the /sup 125/I-EGF binding characteristics of EGF-receptors in membrane preparations of tissues from the 12th day of gestation to parturition. Using autoradiography, the earliest time that we could detect EGF-receptors was on trophoblast cells cultured for 3 days as blastocyst outgrowths. Trophoblast eventually forms a large portion of the placenta, where EGF-receptors have long been recognized. We measured the number and affinity of EGF-receptors on tissues dissected from conceptuses from the 12th day of gestation in order to identify a stage when tissues may be most sensitive to EGF. Whereas the number of EGF receptors increases during gestation for all tissues examined, the affinity of the receptors declines for carcass and placenta and remains relatively unchanged for brain and liver. This suggests that EGF may function differently throughout development. Our hypothesis is that EGF (or its embryonic equivalent) initially stimulates proliferation in embryonic cells and then stimulates differentiation as the tissues mature. In the adult, its main role could be to stimulate tissue repair after damage.

  15. Biochemical and biological properties of the nerve growth factor receptor

    SciTech Connect

    Taniuchi, M.

    1988-01-01

    We have utilized a monoclonal antibody (192-IgG) to study the rat nerve growth factor receptor. After intraocular injection, {sup 125}I-192-IgG was retrogradely transported in sympathetic neuronal axons to the superior cervical ganglion. When the sciatic nerve was ligated to induce the accumulation of axonally transported materials, 192-IgG immunostaining was observed on both sides of the ligature, indicating that NGF receptors are transported in both orthograde and retrograde directions. By using {sup 125}I-NGF crosslinking and 192-IgG immunoprecipitation, we detected receptor molecules throughout the rat brain, thereby supporting the hypothesis that NGF is active in the central nervous system. We also discovered that sciatic nerve transection leads to a dramatic increase in the amount of NGF receptor found in the distal portion of the nerve. Immunostaining revealed that all Schwann cells in the distal axotomized nerve were expressing NGF receptors. We examined phosphorylation of NGF receptor in cultured sympathetic neurons and PC12 cells. We also examined pharmacological effects of 192-IgG. Systemic injection of 192-IgG into neonatal rats caused a permanent partial sympathectomy in a dose-dependent manner; a maximum of 50% of the cells were killed.

  16. An Integrated Model of Epidermal Growth Factor Receptor Trafficking and Signal Transduction

    PubMed Central

    Resat, Haluk; Ewald, Jonathan A.; Dixon, David A.; Wiley, H. Steven

    2003-01-01

    Endocytic trafficking of many types of receptors can have profound effects on subsequent signaling events. Quantitative models of these processes, however, have usually considered trafficking and signaling independently. Here, we present an integrated model of both the trafficking and signaling pathway of the epidermal growth factor receptor (EGFR) using a probability weighted-dynamic Monte Carlo simulation. Our model consists of hundreds of distinct endocytic compartments and ∼13,000 reactions/events that occur over a broad spatio-temporal range. By using a realistic multicompartment model, we can investigate the distribution of the receptors among cellular compartments as well as their potential signal transduction characteristics. Our new model also allows the incorporation of physiochemical aspects of ligand-receptor interactions, such as pH-dependent binding in different endosomal compartments. To determine the utility of this approach, we simulated the differential activation of the EGFR by two of its ligands, epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-α). Our simulations predict that when EGFR is activated with TGF-α, receptor activation is biased toward the cell surface whereas EGF produces a signaling bias toward the endosomal compartment. Experiments confirm these predictions from our model and simulations. Our model accurately predicts the kinetics and extent of receptor downregulation induced by either EGF or TGF-α. Our results suggest that receptor trafficking controls the compartmental bias of signal transduction, rather than simply modulating signal magnitude. Our model provides a new approach to evaluating the complex effect of receptor trafficking on signal transduction. Importantly, the stochastic and compartmental nature of the simulation allows these models to be directly tested by high-throughput approaches, such as quantitative image analysis. PMID:12885624

  17. ADP-ribosylation factor-dependent phospholipase D activation by the M3 muscarinic receptor.

    PubMed

    Mitchell, Rory; Robertson, Derek N; Holland, Pamela J; Collins, Daniel; Lutz, Eve M; Johnson, Melanie S

    2003-09-05

    G protein-coupled receptors can potentially activate phospholipase D (PLD) by a number of routes. We show here that the native M3 muscarinic receptor in 1321N1 cells and an epitope-tagged M3 receptor expressed in COS7 cells substantially utilize an ADP-ribosylation factor (ARF)-dependent route of PLD activation. This pathway is activated at the plasma membrane but appears to be largely independent of G, phospholipase C, Ca2+ q/11, protein kinase C, tyrosine kinases, and phosphatidyl inositol 3-kinase. We report instead that it involves physical association of ARF with the M3 receptor as demonstrated by co-immunoprecipitation and by in vitro interaction with a glutathione S-transferase fusion protein of the receptor's third intracellular loop domain. Experiments with mutant constructs of ARF1/6 and PLD1/2 indicate that the M3 receptor displays a major ARF1-dependent route of PLD1 activation with an additional ARF6-dependent pathway to PLD1 or PLD2. Examples of other G protein-coupled receptors assessed in comparison display alternative pathways of protein kinase C- or ARF6-dependent activation of PLD2.

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

    PubMed Central

    Landry, Mélissa; Pomerleau, Véronique; Saucier, Caroline

    2016-01-01

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

  19. Tumor necrosis factor receptor-associated factor 3 is a positive regulator of pathological cardiac hypertrophy.

    PubMed

    Jiang, Xi; Deng, Ke-Qiong; Luo, Yuxuan; Jiang, Ding-Sheng; Gao, Lu; Zhang, Xiao-Fei; Zhang, Peng; Zhao, Guang-Nian; Zhu, Xueyong; Li, Hongliang

    2015-08-01

    Cardiac hypertrophy, a common early symptom of heart failure, is regulated by numerous signaling pathways. Here, we identified tumor necrosis factor receptor-associated factor 3 (TRAF3), an adaptor protein in tumor necrosis factor-related signaling cascades, as a key regulator of cardiac hypertrophy in response to pressure overload. TRAF3 expression was upregulated in hypertrophied mice hearts and failing human hearts. Four weeks after aortic banding, cardiac-specific conditional TRAF3-knockout mice exhibited significantly reduced cardiac hypertrophy, fibrosis, and dysfunction. Conversely, transgenic mice overexpressing TRAF3 in the heart developed exaggerated cardiac hypertrophy in response to pressure overload. TRAF3 also promoted an angiotensin II- or phenylephrine-induced hypertrophic response in isolated cardiomyocytes. Mechanistically, TRAF3 directly bound to TANK-binding kinase 1 (TBK1), causing increased TBK1 phosphorylation in response to hypertrophic stimuli. This interaction between TRAF3 and TBK1 further activated AKT signaling, which ultimately promoted the development of cardiac hypertrophy. Our findings not only reveal a key role of TRAF3 in regulating the hypertrophic response but also uncover TRAF3-TBK1-AKT as a novel signaling pathway in the development of cardiac hypertrophy and heart failure. This pathway may represent a potential therapeutic target for this pathological process.

  20. Systems Biological Analysis of Epidermal Growth Factor Receptor Internalization Dynamics for Altered Receptor Levels*

    PubMed Central

    Schmidt-Glenewinkel, Hannah; Reinz, Eileen; Eils, Roland; Brady, Nathan R.

    2009-01-01

    Epidermal growth factor (EGF) receptor (EGFR) overexpression is a hallmark of many cancers. EGFR endocytosis is a critical step in signal attenuation, raising the question of how receptor expression levels affect the internalization process. Here we combined quantitative experimental and mathematical modeling approaches to investigate the role of the EGFR expression level on the rate of receptor internalization. Using tetramethylrhodamine-labeled EGF, we established assays for quantifying EGF-triggered EGFR internalization by both high resolution confocal microscopy and flow cytometry. We determined that the flow cytometry approach was more sensitive for examining large populations of cells. Mathematical modeling was used to investigate the relationship between EGF internalization kinetics, EGFR expression, and internalization machinery. We predicted that the standard parameter used to assess internalization kinetics, the temporal evolution r(t) of the ratio of internalized versus surface-located ligand·receptor complexes, does not describe a straight line, as proposed previously. Instead, a convex or concave curve occurs depending on whether initial receptor numbers or internalization adaptors are limiting the uptake reaction, respectively. To test model predictions, we measured EGF-EGFR binding and internalization in cells expressing different levels of green fluorescent protein-EGFR. As expected, surface binding of rhodamine-labeled EGF increased with green fluorescent protein-EGFR expression level. Unexpectedly, internalization of ligand· receptor complexes increased linearly with increasing receptor expression level, suggesting that receptors and not internalization adaptors were limiting the uptake in our experimental model. Finally, determining the ratio of internalized versus surface-located ligand·receptor complexes for this cell line confirmed that it follows a convex curve, supporting our model predictions. PMID:19297331

  1. A Murine Fibroblast Growth Factor (FGF) Receptor Expressed in CHO Cells is Activated by Basic FGF and Kaposi FGF

    NASA Astrophysics Data System (ADS)

    Mansukhani, Alka; Moscatelli, David; Talarico, Daniela; Levytska, Vera; Basilico, Claudio

    1990-06-01

    We have cloned a murine cDNA encoding a tyrosine kinase receptor with about 90% similarity to the chicken fibroblast growth factor (FGF) receptor and the human fms-like gene (FLG) tyrosine kinase. This mouse receptor lacks 88 amino acids in the extracellular portion, leaving only two immunoglobulin-like domains compared to three in the chicken FGF receptor. The cDNA was cloned into an expression vector and transfected into receptor-negative CHO cells. We show that cells expressing the receptor can bind both basic FGF and Kaposi FGF. Although the receptor binds basic FGF with a 15- to 20-fold higher affinity, Kaposi FGF is able to induce down-regulation of the receptor to the same extent as basic FGF. The receptor is phosphorylated upon stimulation with both FGFs, DNA synthesis is stimulated, and a proliferative response is produced in cells expressing the receptor, whereas cells expressing the cDNA in the antisense orientation show none of these responses to basic FGF or Kaposi FGF. Thus this receptor can functionally interact with two growth factors of the FGF family.

  2. Expression of endothelial cell-specific receptor tyrosine kinases and growth factors in human brain tumors.

    PubMed Central

    Hatva, E.; Kaipainen, A.; Mentula, P.; Jääskeläinen, J.; Paetau, A.; Haltia, M.; Alitalo, K.

    1995-01-01

    Key growth factor-receptor interactions involved in angiogenesis are possible targets for therapy of CNS tumors. Vascular endothelial growth factor (VEGF) is a highly specific endothelial cell mitogen that has been shown to stimulate angiogenesis, a requirement for solid tumor growth. The expression of VEGF, the closely related placental growth factor (PIGF), the newly cloned endothelial high affinity VEGF receptors KDR and FLT1, and the endothelial orphan receptors FLT4 and Tie were analyzed by in situ hybridization in normal human brain tissue and in the following CNS tumors: gliomas, grades II, III, IV; meningiomas, grades I and II; and melanoma metastases to the cerebrum. VEGF mRNA was up-regulated in the majority of low grade tumors studied and was highly expressed in cells of malignant gliomas. Significantly elevated levels of Tie, KDR, and FLT1 mRNAs, but not FLT4 mRNA, were observed in malignant tumor endothelia, as well as in endothelia of tissues directly adjacent to the tumor margin. In comparison, there was little or no receptor expression in normal brain vasculature. Our results are consistent with the hypothesis that these endothelial receptors are induced during tumor progression and may play a role in tumor angiogenesis. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7856749

  3. Therapeutic Targeting of Fibroblast Growth Factor Receptors in Gastric Cancer

    PubMed Central

    Fujimori, Yoshitaka; Otsuki, Sho; Sato, Yuya; Nakagawa, Masatoshi

    2015-01-01

    Chemotherapy has become the global standard treatment for patients with metastatic or unresectable gastric cancer (GC), although outcomes remain unfavorable. Many molecular-targeted therapies inhibiting signaling pathways of various tyrosine kinase receptors have been developed, and monoclonal antibodies targeting human epidermal growth factor receptor 2 (HER2) have become standard therapy for HER2-positive GC. An inhibitor of vascular endothelial growth factor receptor 2 or MET has also produced promising results in patients with GC. Fibroblast growth factor receptors (FGFR) play key roles in tumor growth via activated signaling pathways in GC. Genomic amplification of FGFR2 leads to the aberrant activation found in GC tumors and is related to survival in patients with GC. This review discusses the clinical relevance of FGFR in GC and examines FGFR as a potential therapeutic target in patients with GC. Preclinical studies in animal models suggest that multitargeted tyrosine kinase inhibitors (TKIs), including FGFR inhibitor, suppress tumor cell proliferation and delay tumor progression. Several TKIs are now being evaluated in clinical trials as treatment for metastatic or unresectable GC harboring FGFR2 amplification. PMID:26000013

  4. Alkylating derivative of oxotremorine interacts irreversibly with the muscarinic receptor

    SciTech Connect

    Ehlert, F.J.; Jenden, D.J.; Ringdahl, B.

    1984-03-05

    A 2-chloroethylamine derivative of oxotremorine was studied in pharmacological experiments and muscarinic receptor binding assays. The compound, N-(4-(2-chloroethylmethylamino)-2-butynyl)-2-pyrrolidone (BM 123), forms an aziridinium ion in aqueous solution at neutral pH that stimulates contractions of guinea pig ileum with a potency similar to that of oxotremorine. Following the initial stimulation, there is a long lasting period of lack of sensitivity of the guinea pig ileum to muscarinic agonists. BM 123 also produces muscarinic effects in vivo. When homogenates of the rat cerebral cortex were incubated with BM 123 and assayed subsequently in muscarinic receptor binding assays, a loss of binding capacity for the muscarinic antagonist, (/sup 3/H)N-methylscopolamine ((/sup 3/H)NMS), was noted without a change in affinity. Similar observations were made in (/sup 3/H)1-3-quinuclidinyl benzilate ((/sup 3/H)-QNB) binding assays on the forebrains of mice that had been injected with BM 123 24 hr earlier. The loss in receptor capacity for both (/sup 3/H)NMS and (/sup 3/H)-QNB was prevented by atropine treatment. Kinetic studies of the interaction of BM 123 with homogenates of the rat cerebral cortex in vitro showed that the half-time for the loss of (/sup 3/H)-QNB binding sites increased from 10 to 45 min as the concentration of BM 123 decreased from 10 to 1 ..mu..M. In contrast to the aziridinium ion, the parent 2-chloroethylamine compound and the alcoholic hydrolysis product were largely devoid of pharmacological and binding activity.

  5. Insights into Cytokine–Receptor Interactions from Cytokine Engineering

    PubMed Central

    Spangler, Jamie B.; Moraga, Ignacio; Mendoza, Juan L.; Garcia, K. Christopher

    2015-01-01

    Cytokines exert a vast array of immunoregulatory actions critical to human biology and disease. However, the desired immunotherapeutic effects of native cytokines are often mitigated by toxicity or lack of efficacy, either of which results from cytokine receptor pleiotropy and/or undesired activation of off-target cells. As our understanding of the structural principles of cytokine–receptor interactions has advanced, mechanism-based manipulation of cytokine signaling through protein engineering has become an increasingly feasible and powerful approach. Modified cytokines, both agonists and antagonists, have been engineered with narrowed target cell specificities, and they have also yielded important mechanistic insights into cytokine biology and signaling. Here we review the theory and practice of cytokine engineering and rationalize the mechanisms of several engineered cytokines in the context of structure. We discuss specific examples of how structure-based cytokine engineering has opened new opportunities for cytokines as drugs, with a focus on the immunotherapeutic cytokines interferon, interleukin-2, and interleukin-4. PMID:25493332

  6. Molecular modeling study of the differential ligand-receptor interaction at the μ, δ and κ opioid receptors

    NASA Astrophysics Data System (ADS)

    Filizola, Marta; Carteni-Farina, Maria; Perez, Juan J.

    1999-07-01

    3D models of the opioid receptors μ, δ and κ were constructed using BUNDLE, an in-house program to build de novo models of G-protein coupled receptors at the atomic level. Once the three opioid receptors were constructed and before any energy refinement, models were assessed for their compatibility with the results available from point-site mutations carried out on these receptors. In a subsequent step, three selective antagonists to each of three receptors (naltrindole, naltrexone and nor-binaltorphamine) were docked onto each of the three receptors and subsequently energy minimized. The nine resulting complexes were checked for their ability to explain known results of structure-activity studies. Once the models were validated, analysis of the distances between different residues of the receptors and the ligands were computed. This analysis permitted us to identify key residues tentatively involved in direct interaction with the ligand.

  7. New paradigm in ethylene signaling: EIN2, the central regulator of the signaling pathway, interacts directly with the upstream receptors.

    PubMed

    Bisson, Melanie M A; Groth, Georg

    2011-01-01

    The membrane protein ETHYLENE INSENSITIVE2 (EIN2), which is supposed to act between the soluble serine/threonine kinase CTR1 and the EIN3/EIL family of transcription factors, is a central and most critical element of the ethylene signaling pathway in Arabidopsis. In a recent study, we have identified that EIN2 interacts tightly with all members of the Arabidopsis ethylene receptor family - proteins that mark the starting point of the signaling pathway. Our studies show consistently that the kinase domain of the receptors is essential for the formation of the EIN2-receptor complex. Furthermore, mutational analysis demonstrates that phosphorylation is a key mechanism in controlling the interaction of EIN2 and the ethylene receptors. Interaction studies in the presence of the ethylene agonist cyanide revealed a causal link between hormone binding and complex formation. In the presence of the plant hormone agonist the auto-kinase activity of the receptors is inhibited and the non-phosphorylated kinase domain of the receptors binds tightly to the carboxyl-terminal domain of EIN2. In the absence of cyanide inhibition of the auto-kinase activity is relieved and complex formation with the phosphorylated kinase domain of the receptors is reduced. Our data suggest a novel model on the integration of EIN2 in the ethylene signaling pathway.

  8. Calsequestrin interacts directly with the cardiac ryanodine receptor luminal domain

    PubMed Central

    Handhle, Ahmed; Ormonde, Chloe E.; Thomas, N. Lowri; Bralesford, Catherine; Williams, Alan J.; Lai, F. Anthony

    2016-01-01

    ABSTRACT Cardiac muscle contraction requires sarcoplasmic reticulum (SR) Ca2+ release mediated by the quaternary complex comprising the ryanodine receptor 2 (RyR2), calsequestrin 2 (CSQ2), junctin (encoded by ASPH) and triadin. Here, we demonstrate that a direct interaction exists between RyR2 and CSQ2. Topologically, CSQ2 binding occurs at the first luminal loop of RyR2. Co-expression of RyR2 and CSQ2 in a human cell line devoid of the other quaternary complex proteins results in altered Ca2+-release dynamics compared to cells expressing RyR2 only. These findings provide a new perspective for understanding the SR luminal Ca2+ sensor and its involvement in cardiac physiology and disease. PMID:27609834

  9. Dynamic Na+-H+ exchanger regulatory factor-1 association and dissociation regulate parathyroid hormone receptor trafficking at membrane microdomains.

    PubMed

    Ardura, Juan A; Wang, Bin; Watkins, Simon C; Vilardaga, Jean-Pierre; Friedman, Peter A

    2011-10-07

    Na/H exchanger regulatory factor-1 (NHERF1) is a cytoplasmic PDZ (postsynaptic density 95/disc large/zona occludens) protein that assembles macromolecular complexes and determines the localization, trafficking, and signaling of select G protein-coupled receptors and other membrane-delimited proteins. The parathyroid hormone receptor (PTHR), which regulates mineral ion homeostasis and bone turnover, is a G protein-coupled receptor harboring a PDZ-binding motif that enables association with NHERF1 and tethering to the actin cytoskeleton. NHERF1 interactions with the PTHR modify its trafficking and signaling. Here, we characterized by live cell imaging the mechanism whereby NHERF1 coordinates the interactions of multiple proteins, as well as the fate of NHERF1 itself upon receptor activation. Upon PTHR stimulation, NHERF1 rapidly dissociates from the receptor and induces receptor aggregation in long lasting clusters that are enriched with the actin-binding protein ezrin and with clathrin. After NHERF1 dissociates from the PTHR, ezrin then directly interacts with the PTHR to stabilize the PTHR at the cell membrane. Recruitment of β-arrestins to the PTHR is delayed until NHERF1 dissociates from the receptor, which is then trafficked to clathrin for internalization. The ability of NHERF to interact dynamically with the PTHR and cognate adapter proteins regulates receptor trafficking and signaling in a spatially and temporally coordinated manner.

  10. Platelet-activating factor (PAF) receptor and genetically engineered PAF receptor mutant mice.

    PubMed

    Ishii, S; Shimizu, T

    2000-01-01

    Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a biologically active phospholipid mediator. Although PAF was initially recognized for its potential to induce platelet aggregation and secretion, intense investigations have elucidated potent biological actions of PAF in a broad range of cell types and tissues, many of which also produce the molecule. PAF acts by binding to a unique G-protein-coupled seven transmembrane receptor. PAF receptor is linked to intracellular signal transduction pathways, including turnover of phosphatidylinositol, elevation in intracellular calcium concentration, and activation of kinases, resulting in versatile bioactions. On the basis of numerous pharmacological reports, PAF is thought to have many pathophysiological and physiological functions. Recently advanced molecular technics enable us not only to clone PAF receptor cDNAs and genes, but also generate PAF receptor mutant animals, i.e., PAF receptor-overexpressing mouse and PAF receptor-deficient mouse. These mutant mice gave us a novel and specific approach for identifying the pathophysiological and physiological functions of PAF. This review also describes the phenotypes of these mutant mice and discusses them by referring to previously reported pharmacological and genetical data.

  11. Receptome: Interactions between three pain-related receptors or the "Triumvirate" of cannabinoid, opioid and TRPV1 receptors.

    PubMed

    Zádor, Ferenc; Wollemann, Maria

    2015-12-01

    A growing amount of data demonstrates the interactions between cannabinoid, opioid and the transient receptor potential (TRP) vanilloid type 1 (TRPV1) receptors. These interactions can be bidirectional, inhibitory or excitatory, acute or chronic in their nature, and arise both at the molecular level (structurally and functionally) and in physiological processes, such as pain modulation or perception. The interactions of these three pain-related receptors may also reserve important and new therapeutic applications for the treatment of chronic pain or inflammation. In this review, we summarize the main findings on the interactions between the cannabinoid, opioid and the TRPV1 receptor regarding to pain modulation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Interaction of the von Willebrand factor with platelets and thrombosis.

    PubMed

    Perutelli, P; Mori, P G

    1997-11-01

    The human von Willebrand factor (vWf) is a multimeric glycoprotein present in plasma, platelets, endothelial cells and subendothelium and synthesized in endothelial cells and megakaryocytes. vWf plays a pivotal role in the mechanisms of blood clotting and platelet thrombus formation; quantitative and qualitative abnormalities of vWf cause the most common congenital bleeding disorder in man, the von Willebrand disease. vWf stabilizes factor VIII and interacts with subendothelial components and with platelet membrane receptors. The multimeric structure of vWf provides an array of binding sites which allows multivalent interactions with its ligands, thus supporting the formation of stable platelet aggregates at the site of vascular injury, particularly under flow conditions characterized by high shear stress. In the last years, remarkable progress has been made toward understanding the structure of vWf protein and gene, and the elucidation of many structure-function relationships, which may result in improved therapeutic intervention for vWD patients, and in the development of effective strategies for antithrombotic therapy.

  13. Regulation of fibroblast growth factor receptor signalling and trafficking by Src and Eps8.

    PubMed

    Auciello, Giulio; Cunningham, Debbie L; Tatar, Tulin; Heath, John K; Rappoport, Joshua Z

    2013-01-15

    Fibroblast growth factor receptors (FGFRs) mediate a wide spectrum of cellular responses that are crucial for development and wound healing. However, aberrant FGFR activity leads to cancer. Activated growth factor receptors undergo stimulated endocytosis, but can continue to signal along the endocytic pathway. Endocytic trafficking controls the duration and intensity of signalling, and growth factor receptor signalling can lead to modifications of trafficking pathways. We have developed live-cell imaging methods for studying FGFR dynamics to investigate mechanisms that coordinate the interplay between receptor trafficking and signal transduction. Activated FGFR enters the cell following recruitment to pre-formed clathrin-coated pits (CCPs). However, FGFR activation stimulates clathrin-mediated endocytosis; FGF treatment increases the number of CCPs, including those undergoing endocytosis, and this effect is mediated by Src and its phosphorylation target Eps8. Eps8 interacts with the clathrin-mediated endocytosis machinery and depletion of Eps8 inhibits FGFR trafficking and immediate Erk signalling. Once internalized, FGFR passes through peripheral early endosomes en route to recycling and degredative compartments, through an Src- and Eps8-dependent mechanism. Thus Eps8 functions as a key coordinator in the interplay between FGFR signalling and trafficking. This work provides the first detailed mechanistic analysis of growth factor receptor clustering at the cell surface through signal transduction and endocytic trafficking. As we have characterised the Src target Eps8 as a key regulator of FGFR signalling and trafficking, and identified the early endocytic system as the site of Eps8-mediated effects, this work provides novel mechanistic insight into the reciprocal regulation of growth factor receptor signalling and trafficking.

  14. Regulation of fibroblast growth factor receptor signalling and trafficking by Src and Eps8

    PubMed Central

    Auciello, Giulio; Cunningham, Debbie L.; Tatar, Tulin; Heath, John K.; Rappoport, Joshua Z.

    2013-01-01

    Summary Fibroblast growth factor receptors (FGFRs) mediate a wide spectrum of cellular responses that are crucial for development and wound healing. However, aberrant FGFR activity leads to cancer. Activated growth factor receptors undergo stimulated endocytosis, but can continue to signal along the endocytic pathway. Endocytic trafficking controls the duration and intensity of signalling, and growth factor receptor signalling can lead to modifications of trafficking pathways. We have developed live-cell imaging methods for studying FGFR dynamics to investigate mechanisms that coordinate the interplay between receptor trafficking and signal transduction. Activated FGFR enters the cell following recruitment to pre-formed clathrin-coated pits (CCPs). However, FGFR activation stimulates clathrin-mediated endocytosis; FGF treatment increases the number of CCPs, including those undergoing endocytosis, and this effect is mediated by Src and its phosphorylation target Eps8. Eps8 interacts with the clathrin-mediated endocytosis machinery and depletion of Eps8 inhibits FGFR trafficking and immediate Erk signalling. Once internalized, FGFR passes through peripheral early endosomes en route to recycling and degredative compartments, through an Src- and Eps8-dependent mechanism. Thus Eps8 functions as a key coordinator in the interplay between FGFR signalling and trafficking. This work provides the first detailed mechanistic analysis of growth factor receptor clustering at the cell surface through signal transduction and endocytic trafficking. As we have characterised the Src target Eps8 as a key regulator of FGFR signalling and trafficking, and identified the early endocytic system as the site of Eps8-mediated effects, this work provides novel mechanistic insight into the reciprocal regulation of growth factor receptor signalling and trafficking. PMID:23203811

  15. Nuclear Receptors in Drug Metabolism, Drug Response and Drug Interactions

    PubMed Central

    Prakash, Chandra; Zuniga, Baltazar; Song, Chung Seog; Jiang, Shoulei; Cropper, Jodie; Park, Sulgi; Chatterjee, Bandana

    2016-01-01

    Orally delivered small-molecule therapeutics are metabolized in the liver and intestine by phase I and phase II drug-metabolizing enzymes (DMEs), and transport proteins coordinate drug influx (phase 0) and drug/drug-metabolite efflux (phase III). Genes involved in drug metabolism and disposition are induced by xenobiotic-activated nuclear receptors (NRs), i.e. PXR (pregnane X receptor) and CAR (constitutive androstane receptor), and by the 1α, 25-dihydroxy vitamin D3-activated vitamin D receptor (VDR), due to transactivation of xenobiotic-response elements (XREs) present in phase 0-III genes. Additional NRs, like HNF4-α, FXR, LXR-α play important roles in drug metabolism in certain settings, such as in relation to cholesterol and bile acid metabolism. The phase I enzymes CYP3A4/A5, CYP2D6, CYP2B6, CYP2C9, CYP2C19, CYP1A2, CYP2C8, CYP2A6, CYP2J2, and CYP2E1 metabolize >90% of all prescription drugs, and phase II conjugation of hydrophilic functional groups (with/without phase I modification) facilitates drug clearance. The conjugation step is mediated by broad-specificity transferases like UGTs, SULTs, GSTs. This review delves into our current understanding of PXR/CAR/VDR-mediated regulation of DME and transporter expression, as well as effects of single nucleotide polymorphism (SNP) and epigenome (specified by promoter methylation, histone modification, microRNAs, long non coding RNAs) on the expression of PXR/CAR/VDR and phase 0-III mediators, and their impacts on variable drug response. Therapeutic agents that target epigenetic regulation and the molecular basis and consequences (overdosing, underdosing, or beneficial outcome) of drug-drug/drug-food/drug-herb interactions are also discussed. Precision medicine requires understanding of a drug’s impact on DME and transporter activity and their NR-regulated expression in order to achieve optimal drug efficacy without adverse drug reactions. In future drug screening, new tools such as humanized mouse models and

  16. Nuclear Receptors in Drug Metabolism, Drug Response and Drug Interactions.

    PubMed

    Prakash, Chandra; Zuniga, Baltazar; Song, Chung Seog; Jiang, Shoulei; Cropper, Jodie; Park, Sulgi; Chatterjee, Bandana

    Orally delivered small-molecule therapeutics are metabolized in the liver and intestine by phase I and phase II drug-metabolizing enzymes (DMEs), and transport proteins coordinate drug influx (phase 0) and drug/drug-metabolite efflux (phase III). Genes involved in drug metabolism and disposition are induced by xenobiotic-activated nuclear receptors (NRs), i.e. PXR (pregnane X receptor) and CAR (constitutive androstane receptor), and by the 1α, 25-dihydroxy vitamin D3-activated vitamin D receptor (VDR), due to transactivation of xenobiotic-response elements (XREs) present in phase 0-III genes. Additional NRs, like HNF4-α, FXR, LXR-α play important roles in drug metabolism in certain settings, such as in relation to cholesterol and bile acid metabolism. The phase I enzymes CYP3A4/A5, CYP2D6, CYP2B6, CYP2C9, CYP2C19, CYP1A2, CYP2C8, CYP2A6, CYP2J2, and CYP2E1 metabolize >90% of all prescription drugs, and phase II conjugation of hydrophilic functional groups (with/without phase I modification) facilitates drug clearance. The conjugation step is mediated by broad-specificity transferases like UGTs, SULTs, GSTs. This review delves into our current understanding of PXR/CAR/VDR-mediated regulation of DME and transporter expression, as well as effects of single nucleotide polymorphism (SNP) and epigenome (specified by promoter methylation, histone modification, microRNAs, long non coding RNAs) on the expression of PXR/CAR/VDR and phase 0-III mediators, and their impacts on variable drug response. Therapeutic agents that target epigenetic regulation and the molecular basis and consequences (overdosing, underdosing, or beneficial outcome) of drug-drug/drug-food/drug-herb interactions are also discussed. Precision medicine requires understanding of a drug's impact on DME and transporter activity and their NR-regulated expression in order to achieve optimal drug efficacy without adverse drug reactions. In future drug screening, new tools such as humanized mouse models and

  17. A novel calmodulin-β-PIX interaction and its implication in receptor tyrosine kinase regulation.

    PubMed

    Singh, Vinay K; Munro, Kim; Jia, Zongchao

    2012-09-01

    Calmodulin (CaM), a ubiquitous calcium-binding protein, regulates numerous cellular processes, primarily in response to calcium flux. We have identified and characterized a novel interaction between CaM and β-p21-activated kinase interacting exchange factor (β-PIX), a putative guanine exchange factor implicated in cell signaling, using affinity pull-down assays, co-immunoprecipitation, co-localization and circular dichroism studies. Fluorescence-based titration and isothermal titration calorimetry experiments revealed a Ca(2+)-dependent binding mechanism (K(D)≤10μM). Further, we show that CaM participates in a multi-protein complex involving β-PIX and E3 ubiquitin ligase c-Cbl (casitas B-cell lymphoma), which may play a critical role in receptor tyrosine kinase regulation and downstream signaling. Copyright © 2012 Elsevier Inc. All rights reserved.

  18. Interaction between Pheromone and Its Receptor of the Fission Yeast Schizosaccharomyces pombe Examined by a Force Spectroscopy Study

    PubMed Central

    Sasuga, Shintaro; Abe, Ryohei; Nikaido, Osamu; Kiyosaki, Shoichi; Sekiguchi, Hiroshi; Ikai, Atsushi; Osada, Toshiya

    2012-01-01

    Interaction between P-factor, a peptide pheromone composed of 23 amino acid residues, and its pheromone receptor, Mam2, on the cell surface of the fission yeast Schizosaccharomyces pombe was examined by an atomic force microscope (AFM). An AFM tip was modified with P-factor derivatives to perform force curve measurements. The specific interaction force between P-factor and Mam2 was calculated to be around 120 pN at a probe speed of 1.74 μm/s. When the AFM tip was modified with truncated P-factor derivative lacking C-terminal Leu, the specific interaction between the tip and the cell surface was not observed. These results were also confirmed with an assay system using a green fluorescent protein (GFP) reporter gene to monitor the activation level of signal transduction following the interaction of Mam2 with P-factor. PMID:22500108

  19. Interaction between pheromone and its receptor of the fission yeast Schizosaccharomyces pombe examined by a force spectroscopy study.

    PubMed

    Sasuga, Shintaro; Abe, Ryohei; Nikaido, Osamu; Kiyosaki, Shoichi; Sekiguchi, Hiroshi; Ikai, Atsushi; Osada, Toshiya

    2012-01-01

    Interaction between P-factor, a peptide pheromone composed of 23 amino acid residues, and its pheromone receptor, Mam2, on the cell surface of the fission yeast Schizosaccharomyces pombe was examined by an atomic force microscope (AFM). An AFM tip was modified with P-factor derivatives to perform force curve measurements. The specific interaction force between P-factor and Mam2 was calculated to be around 120 pN at a probe speed of 1.74 μm/s. When the AFM tip was modified with truncated P-factor derivative lacking C-terminal Leu, the specific interaction between the tip and the cell surface was not observed. These results were also confirmed with an assay system using a green fluorescent protein (GFP) reporter gene to monitor the activation level of signal transduction following the interaction of Mam2 with P-factor.

  20. Memo interacts with c-Src to control Estrogen Receptor alpha sub-cellular localization.

    PubMed

    Frei, Anna; MacDonald, Gwen; Lund, Ingrid; Gustafsson, Jan-Åke; Hynes, Nancy E; Nalvarte, Ivan

    2016-08-30

    Understanding the complex interaction between growth factor and steroid hormone signaling pathways in breast cancer is key to identifying suitable therapeutic strategies to avoid progression and therapy resistance. The interaction between these two pathways is of paramount importance for the development of endocrine resistance. Nevertheless, the molecular mechanisms behind their crosstalk are still largely obscure. We previously reported that Memo is a small redox-active protein that controls heregulin-mediated migration of breast cancer cells. Here we report that Memo sits at the intersection between heregulin and estrogen signaling, and that Memo controls Estrogen Receptor alpha (ERα) sub-cellular localization, phosphorylation, and function downstream of heregulin and estrogen in breast cancer cells. Memo facilitates ERα and c-Src interaction, ERα Y537 phosphorylation, and has the ability to control ERα extra-nuclear localization. Thus, we identify Memo as an important key mediator between the heregulin and estrogen signaling pathways, which affects both breast cancer cell migration and proliferation.

  1. The interleukin 1 (IL-1) receptor accessory protein Toll/IL-1 receptor domain: analysis of putative interaction sites in vitro mutagenesis and molecular modeling.

    PubMed

    Radons, Jurgen; Dove, Stefan; Neumann, Detlef; Altmann, Reinhold; Botzki, Alexander; Martin, Michael U; Falk, Werner

    2003-12-05

    The Toll/interleukin 1 (IL-1) receptor family plays an important role in both innate and adaptive immunity. These receptors are characterized by a C-terminal homology motif called the Toll/IL-1 receptor (TIR) domain. A principal function of the TIR domain is mediating homotypic protein-protein interactions in the signal transduction pathway. To suggest interaction sites of TIR domains in the IL-1 receptor complex, we modeled the putative three-dimensional structure of the TIR domain within the co-receptor chain, IL-1 receptor accessory protein. The model was based on homology with the crystal structures of human TLR1 and TLR2. The final structure of the IL-1 receptor accessory protein TIR domain suggests the conserved regions box 1 and 2, including Pro-446, as well as box 3 within the C-terminal alpha-helix as possible protein-protein interaction sites due to their exposure and their electrostatic potential. Pro-446, corresponding to the Pro/His mutation in dominant negative TLR4, is located in the third loop at the outmost edge of the TIR domain and does not play any structural role. Inhibition of IL-1 responsiveness seen after substitution of Pro-446 by charged amino acids is due to the loss of an interaction site for other TIR domains. Amino acids 527-534 as part of the loop close to the conserved box 3 are critical for recruitment of myeloid differentiation factor 88 and to a lesser extent for IL-1 responsiveness. Modeling suggests that native folding of the TIR domain may be approached by the responsive deletion mutants delta528-534 and delta527-533, whereas the C-terminal beta-strand and/or alpha-helix is displaced in the nonresponsive mutant delta527-534.

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

    PubMed

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

    2015-05-21

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

  3. Role of fibroblast growth factor receptor signaling in kidney development.

    PubMed

    Bates, Carlton M

    2007-03-01

    Fibroblast growth factor receptors (Fgfrs) are expressed in the ureteric bud and metanephric mesenchyme of the developing kidney. Furthermore, in vitro and in vivo studies have shown that exogenous fibroblast growth factors (Fgfs) increase growth and maturation of the metanephric mesenchyme and ureteric bud. Deletion of fgf7, fgf10, and fgfr2IIIb (the receptor isoform that binds Fgf7 and Fgf10) in mice lead to smaller kidneys with fewer collecting ducts and nephrons. Overexpression of a dominant negative receptor isoform in transgenic mice has revealed more striking defects including renal aplasia or severe dysplasia. Moreover, deletion of many fgf ligands and receptors in mice results in early embryonic lethality, making it difficult to determine their roles in kidney development. Recently, conditional targeting approaches revealed that deletion of fgf8 from the metanephric mesenchyme interrupts nephron formation. Furthermore, deletion of fgfr2 from the ureteric bud resulted in both ureteric bud branching and stromal mesenchymal patterning defects. Deletion of both fgfr1 and fgfr2 in the metanephric mesenchyme resulted in renal aplasia, characterized by defects in metanephric mesenchyme formation and initial ureteric bud elongation and branching. Thus, Fgfr signaling is critical for growth and patterning of all renal lineages at early and later stages of kidney development.

  4. Immunohistochemical expression of Type IV Collagen and Autocrine Motility Factor Receptor in Odontogenic Tumours

    PubMed Central

    Sethi, Sneha

    2014-01-01

    Background: Autocrine motility factor receptor (AMFR) is a tumour motility stimulating protein secreted by tumour cells. The protein encoded by this gene is a glycosylated transmembrane protein and a receptor for autocrine motility factor. It has been known to play a role in progression of neoplastic lesions. Basement membranes are specialized extracellular matrices that serve as structural barriers as well as substrates for cellular interactions. The network of type IV collagen is thought to define the scaffold integrating other components such as laminins and perlecan into highly organized supramolecular architecture. The aim of this study was to determine and evaluate the immunohistochemical expression of Type IV Collagen and Autocrine motility factor receptor in odontogenic lesions. Materials and Methods: Immunohistochemical expression of Type IV Collagen and Autocrine motility factor receptor was evaluated in 31 odontogenic lesions, including unicystic ameloblastoma, multicystic ameloblastoma, keratocystic odontogenic tumour and ameloblastic carcinoma. Normal follicular tissue formed the control. Results: Maximum expression for Type IV Collagen was seen in multicystic ameloblastoma and minimum expression in keratocystic odontogenic tumour. The maximum expression of AMFR was seen in ameloblastic carcinoma and minimum expression in multicystic ameloblastoma. Conclusion: The results of this study suggested an association of loss of expression of type IV Collagen with progression of lesion. AMFR expression was found to be associated with the aggressive potential of tumours. PMID:25478440

  5. Solution structure of human insulin-like growth factor II; recognition sites for receptors and binding proteins.

    PubMed Central

    Terasawa, H; Kohda, D; Hatanaka, H; Nagata, K; Higashihashi, N; Fujiwara, H; Sakano, K; Inagaki, F

    1994-01-01

    The three-dimensional structure of human insulin-like growth factor II was determined at high resolution in aqueous solution by NMR and simulated annealing based calculations. The structure is quite similar to those of insulin and insulin-like growth factor I, which consists of an alpha-helix followed by a turn and a strand in the B-region and two antiparallel alpha-helices in the A-region. However, the regions of Ala1-Glu6, Pro31-Arg40 and Thr62-Glu67 are not well-defined for lack of distance constraints, possibly due to motional flexibility. Based on the resultant structure and the results of structure-activity relationships, we propose the interaction sites of insulin-like growth factor II with the type 2 insulin-like growth factor receptor and the insulin-like growth factor binding proteins. These sites partially overlap with each other at the opposite side of the putative binding surface to the insulin receptor and the type 1 insulin-like growth factor receptor. We also discuss the interaction modes of insulin-like growth factor II with the insulin receptor and the type 1 insulin-like growth factor receptor. Images PMID:7527339

  6. Expression of the estrogen receptor α, progesterone receptor and epidermal growth factor receptor in papillary thyroid carcinoma tissues

    PubMed Central

    CHEN, DAN; QI, WENJING; ZHANG, PENGXIN; GUAN, HONGWEI; WANG, LIFEN

    2015-01-01

    The present study aimed to determine the protein expression, in addition to the clinical value of the expression, of estrogen receptor α (ERα), progesterone receptor (PR) and epidermal growth factor receptor (EGFR) in papillary thyroid carcinoma (PTC). The expression of ERα, PR and EGFR was examined immunohistochemically on paraffin-embedded thyroid tissues obtained from 64 patients with PTC and 14 patients with nodular thyroid goiter (NTG). The expression level of ERα, PR and EGFR was found to be significantly elevated in the PTC tissues compared with the NTG tissues. In addition, the expression of ERα was found to be correlated with the size of PTC tumors. However, there was no significant difference between the expression levels of ERα, PR and EGFR in males and females with PTC. Thus, immunohistochemical evaluation of ERα, PR and EGFR expression in patients with PTC may aid in the prediction of the prognosis of patients with PTC. PMID:26171022

  7. The interaction between histamine H1 receptor and μ- opioid receptor in scratching behavior in ICR mice.

    PubMed

    Nakasone, Tasuku; Sugimoto, Yumi; Kamei, Chiaki

    2016-04-15

    In this study, we examined the interaction between histamine H1 receptor and μ-opioid receptor in scratching behavior in ICR mice. Both histamine and morphine caused scratching and simultaneous injection of histamine and morphine had an additive effect. Chlorpheniramine and naloxone inhibited histamine-induced scratching behavior. These two drugs also inhibited morphine-induced scratching behavior. Simultaneous injection of chlorpheniramine and naloxone caused a significant inhibition of histamine-induced scratching compared with separate injections. The same findings were also noted for morphine-induced scratching. These results strongly indicate a close relationship between histamine H1 receptor and μ-opioid receptor in scratching behavior in ICR mice.

  8. Monitoring ligand-receptor interactions by photonic force microscopy

    PubMed Central

    Jeney, Sylvia; Mor, Flavio; Koszali, Roland; Forró, László; Moy, Vincent T.

    2011-01-01

    We introduce a method for the acquisition of single molecule force measurements of ligand —receptor interactions using the photonic force microscope (PFM). Biotin-functionalized beads, manipulated with an optical trap, and a streptavidin-functionalized coverslip were used to measure the effect of different pulling forces on the lifetime of individual streptavidin-biotin complexes. By optimizing the design of the optical trap and selection of the appropriate bead size, pulling forces in excess of 50 pN were achieved. Based on the amplitude of three dimensional (3D) thermal position fluctuations of the attached bead, we were able to select for a bead-coverslip interaction that was mediated by a single streptavidin-biotin complex. Moreover, the developed experimental system was greatly accelerated by automation of data acquisition and analysis. In force-dependent kinetic measurements carried out between streptavidin and biotin, we observed that the streptavidin-biotin complex exhibited properties of a catch bond with the lifetime increasing 10 fold when the pulling force increased from 10 to 20 pN. We also show that silica beads were more appropriate than polystyrene beads for the force measurements as polystyrene tethers, longer than 200 nm, could be extracted from the beads. PMID:20516583

  9. P2X1 Receptor Antagonists Inhibit HIV-1 Fusion by Blocking Virus-Coreceptor Interactions

    PubMed Central

    Giroud, Charline; Marin, Mariana; Hammonds, Jason; Spearman, Paul

    2015-01-01

    ABSTRACT HIV-1 Env glycoprotein-mediated fusion is initiated upon sequential binding of Env to CD4 and the coreceptor CXCR4 or CCR5. Whereas these interactions are thought to be necessary and sufficient to promote HIV-1 fusion, other host factors can modulate this process. Previous studies reported potent inhibition of HIV-1 fusion by selective P2X1 receptor antagonists, including NF279, and suggested that these receptors play a role in HIV-1 entry. Here we investigated the mechanism of antiviral activity of NF279 and found that this compound does not inhibit HIV-1 fusion by preventing the activation of P2X1 channels but effectively blocks the binding of the virus to CXCR4 or CCR5. The notion of an off-target effect of NF279 on HIV-1 fusion is supported by the lack of detectable expression of P2X1 receptors in cells used in fusion experiments and by the fact that the addition of ATP or the enzymatic depletion of ATP in culture medium does not modulate viral fusion. Importantly, NF279 fails to inhibit HIV-1 fusion with cell lines and primary macrophages when added at an intermediate stage downstream of Env-CD4-coreceptor engagement. Conversely, in the presence of NF279, HIV-1 fusion is arrested downstream of CD4 binding but prior to coreceptor engagement. NF279 also antagonizes the signaling function of CCR5, CXCR4, and another chemokine receptor, as evidenced by the suppression of calcium responses elicited by specific ligands and by recombinant gp120. Collectively, our results demonstrate that NF279 is a dual HIV-1 coreceptor inhibitor that interferes with the functional engagement of CCR5 and CXCR4 by Env. IMPORTANCE Inhibition of P2X receptor activity suppresses HIV-1 fusion and replication, suggesting that P2X signaling is involved in HIV-1 entry. However, mechanistic experiments conducted in this study imply that P2X1 receptor is not expressed in target cells or involved in viral fusion. Instead, we found that inhibition of HIV-1 fusion by a specific P2X1

  10. Computer-assisted generation of a protein-interaction database for nuclear receptors.

    PubMed

    Albert, Sylvie; Gaudan, Sylvain; Knigge, Heidrun; Raetsch, Andreas; Delgado, Asuncion; Huhse, Bettina; Kirsch, Harald; Albers, Michael; Rebholz-Schuhmann, Dietrich; Koegl, Manfred

    2003-08-01

    With the increasing amount of biological data available, automated methods for information retrieval become necessary. We employed computer-assisted text mining to retrieve all protein-protein interactions for nuclear receptors from MEDLINE in a systematic way. A dictionary of protein names and of terms denoting interactions was generated, and trioccurrences of two protein names and one interaction term in one sentence were retrieved. Abstracts containing at least one such trioccurrence were manually checked by biologists to select the relevant interactions out of the automatically extracted data. In total, 4360 abstracts were retrieved containing data on protein interactions for nuclear receptors. The resulting database contains all reported protein interactions involving nuclear receptors from 1966 to September 2001. Remarkably, the annual increase in number of reported interactors for nuclear receptors has been following an exponential growth curve in the years 1991 to 2001. Apparent in the data set is the high complexity of protein interactions for nuclear receptors. The number of interactions correlates with the number of published papers for a given receptor, suggesting that the number of reported interactors is a reflection of the intensity of research dedicated to a given receptor. Indeed, comparison of the retrieved data to a systematic yeast two-hybrid-based interaction analysis suggests that most NRs are similar with respect to the number of interacting proteins. The data set obtained serves as a source for information on NR interactions, as well as a reference data set for the improvement of advanced text-mining methods.

  11. The effect of cell-ECM adhesion on signalling via the ErbB family of growth factor receptors.

    PubMed

    Alexi, Xanthippi; Berditchevski, Fedor; Odintsova, Elena

    2011-04-01

    Integrins and growth factor receptors of the ErbB family are involved in the regulation of cellular interactions with the extracellular microenvironment. Cross-talk between these two groups of transmembrane receptors is essential for cellular responses and can be regulated through the formation of multimolecular complexes. Tetraspanins as facilitators and building blocks of specialized microdomains may be involved in this process. In the present study, we demonstrated that, in contrast with previous reports, integrin-mediated adhesion did not stimulate ligand-independent activation of ErbB receptors in epithelial cells. However, integrin-dependent adhesion potentiated ligand-induced activation of EGFR (epidermal growth factor receptor) and ErbB2 and facilitated receptor homo- and hetero-dimerization. The actin cytoskeleton appeared to play a critical role in this phenomenon.

  12. Expression of androgen receptor in breast cancer & its correlation with other steroid receptors & growth factors

    PubMed Central

    Mishra, Ashwani K.; Agrawal, Usha; Negi, Shivani; Bansal, Anju; Mohil, R.; Chintamani, Chintamani; Bhatnagar, Amar; Bhatnagar, Dinesh; Saxena, Sunita

    2012-01-01

    Background & objectives: Breast cancer is the second most common malignancy in Indian women. Among the members of the steroid receptor superfamily the role of estrogen and progesterone receptors (ER and PR) is well established in breast cancer in predicting the prognosis and management of therapy, however, little is known about the clinical significance of androgen receptor (AR) in breast carcinogenesis. The present study was aimed to evaluate the expression of AR in breast cancer and to elucidate its clinical significance by correlating it with clinicopathological parameters, other steroid receptors (ER and PR) and growth factors receptors (EGFR and CD105). Methods: Expression of AR, ER, PR, epidermal growth factor receptor (EGFR) and endoglin (CD105) was studied in 100 cases of breast cancer by immunohistochemistry (IHC). Risk ratio (RR) along with 95% confidence interval (CI) was estimated to assess the strength of association between the markers and clinicopathological characteristics. Categorical principal component analysis (CATPCA) was applied to obtain new sets of linearly combined expression, for their further evaluation with clinicopathological characteristics (n=100). Results: In 31 cases presenting with locally advanced breast cancer (LABC), the expression of AR, ER, PR, EGFR and CD105 was associated with response to neoadjuvant chemotherapy (NACT). The results indicated the association of AR+ (P=0.001) and AR+/EGFR- (P=0.001) with the therapeutic response to NACT in LABC patients. The AR expression exhibited maximum sensitivity, specificity and likelihood ratio of positive and negative test. The present results showed the benefit of adding AR, EGFR and CD105 to the existing panel of markers to be able to predict response to therapy. Interpretation & conclusions: More studies on the expression profiles of AR+, AR+/CD105+ and AR+/EGFR- in larger set of breast cancer patients may possibly help in confirming their predictive role for therapeutic response

  13. Interaction between FGFR-2, STAT5, and progesterone receptors in breast cancer.

    PubMed

    Cerliani, Juan P; Guillardoy, Tomás; Giulianelli, Sebastián; Vaque, José P; Gutkind, J Silvio; Vanzulli, Silvia I; Martins, Rubén; Zeitlin, Eduardo; Lamb, Caroline A; Lanari, Claudia

    2011-05-15

    Fibroblast growth factor (FGF) receptor 2 (FGFR-2) polymorphisms have been associated with an increase in estrogen receptor and progesterone receptor (PR)-positive breast cancer risk; however, a clear mechanistic association between FGFR-2 and steroid hormone receptors remains elusive. In previous works, we have shown a cross talk between FGF2 and progestins in mouse mammary carcinomas. To investigate the mechanisms underlying these interactions and to validate our findings in a human setting, we have used T47D human breast cancer cells and human cancer tissue samples. We showed that medroxyprogesterone acetate (MPA) and FGF2 induced cell proliferation and activation of ERK, AKT, and STAT5 in T47D and in murine C4-HI cells. Nuclear interaction between PR, FGFR-2, and STAT5 after MPA and FGF2 treatment was also showed by confocal microscopy and immunoprecipitation. This effect was associated with increased transcription of PRE and/or GAS reporter genes, and of PR/STAT5-regulated genes and proteins. Two antiprogestins and the FGFR inhibitor PD173074, specifically blocked the effects induced by FGF2 or MPA respectively. The presence of PR/FGFR-2/STAT5 complexes bound to the PRE probe was corroborated by using NoShift transcription and chromatin immunoprecipitation of the MYC promoter. Additionally, we showed that T47D cells stably transfected with constitutively active FGFR-2 gave rise to invasive carcinomas when transplanted into NOD/SCID mice. Nuclear colocalization between PR and FGFR-2/STAT5 was also observed in human breast cancer tissues. This study represents the first demonstration of a nuclear interaction between FGFR-2 and STAT5, as PR coactivators at the DNA progesterone responsive elements, suggesting that FGFRs are valid therapeutic targets for human breast cancer treatment.

  14. α(2A) adrenergic receptor promotes amyloidogenesis through disrupting APP-SorLA interaction.

    PubMed

    Chen, Yunjia; Peng, Yin; Che, Pulin; Gannon, Mary; Liu, Yin; Li, Ling; Bu, Guojun; van Groen, Thomas; Jiao, Kai; Wang, Qin

    2014-12-02

    Accumulation of amyloid β (Aβ) peptides in the brain is the key pathogenic factor driving Alzheimer's disease (AD). Endocytic sorting of amyloid precursor protein (APP) mediated by the vacuolar protein sorting (Vps10) family of receptors plays a decisive role in controlling the outcome of APP proteolytic processing and Aβ generation. Here we report for the first time to our knowledge that this process is regulated by a G protein-coupled receptor, the α(2A) adrenergic receptor (α(2A)AR). Genetic deficiency of the α(2A)AR significantly reduces, whereas stimulation of this receptor enhances, Aβ generation and AD-related pathology. Activation of α(2A)AR signaling disrupts APP interaction with a Vps10 family receptor, sorting-related receptor with A repeat (SorLA), in cells and in the mouse brain. As a consequence, activation of α(2A)AR reduces Golgi localization of APP and concurrently promotes APP distribution in endosomes and cleavage by β secretase. The α(2A)AR is a key component of the brain noradrenergic system. Profound noradrenergic dysfunction occurs consistently in patients at the early stages of AD. α(2A)AR-promoted Aβ generation provides a novel mechanism underlying the connection between noradrenergic dysfunction and AD. Our study also suggests α(2A)AR as a previously unappreciated therapeutic target for AD. Significantly, pharmacological blockade of the α(2A)AR by a clinically used antagonist reduces AD-related pathology and ameliorates cognitive deficits in an AD transgenic model, suggesting that repurposing clinical α(2A)R antagonists would be an effective therapeutic strategy for AD.

  15. α2A adrenergic receptor promotes amyloidogenesis through disrupting APP-SorLA interaction

    PubMed Central

    Chen, Yunjia; Peng, Yin; Che, Pulin; Gannon, Mary; Liu, Yin; Li, Ling; Bu, Guojun; van Groen, Thomas; Jiao, Kai; Wang, Qin

    2014-01-01

    Accumulation of amyloid β (Aβ) peptides in the brain is the key pathogenic factor driving Alzheimer’s disease (AD). Endocytic sorting of amyloid precursor protein (APP) mediated by the vacuolar protein sorting (Vps10) family of receptors plays a decisive role in controlling the outcome of APP proteolytic processing and Aβ generation. Here we report for the first time to our knowledge that this process is regulated by a G protein-coupled receptor, the α2A adrenergic receptor (α2AAR). Genetic deficiency of the α2AAR significantly reduces, whereas stimulation of this receptor enhances, Aβ generation and AD-related pathology. Activation of α2AAR signaling disrupts APP interaction with a Vps10 family receptor, sorting-related receptor with A repeat (SorLA), in cells and in the mouse brain. As a consequence, activation of α2AAR reduces Golgi localization of APP and concurrently promotes APP distribution in endosomes and cleavage by β secretase. The α2AAR is a key component of the brain noradrenergic system. Profound noradrenergic dysfunction occurs consistently in patients at the early stages of AD. α2AAR-promoted Aβ generation provides a novel mechanism underlying the connection between noradrenergic dysfunction and AD. Our study also suggests α2AAR as a previously unappreciated therapeutic target for AD. Significantly, pharmacological blockade of the α2AAR by a clinically used antagonist reduces AD-related pathology and ameliorates cognitive deficits in an AD transgenic model, suggesting that repurposing clinical α2AR antagonists would be an effective therapeutic strategy for AD. PMID:25404298

  16. Ginseng pharmacology: a new paradigm based on gintonin-lysophosphatidic acid receptor interactions

    PubMed Central

    Choi, Sun-Hye; Jung, Seok-Won; Lee, Byung-Hwan; Kim, Hyeon-Joong; Hwang, Sung-Hee; Kim, Ho-Kyoung; Nah, Seung-Yeol

    2015-01-01

    Ginseng, the root of Panax ginseng, is used as a traditional medicine. Despite the long history of the use of ginseng, there is no specific scientific or clinical rationale for ginseng pharmacology besides its application as a general tonic. The ambiguous description of ginseng pharmacology might be due to the absence of a predominant active ingredient that represents ginseng pharmacology. Recent studies show that ginseng abundantly contains lysophosphatidic acids (LPAs), which are phospholipid-derived growth factor with diverse biological functions including those claimed to be exhibited by ginseng. LPAs in ginseng form a complex with ginseng proteins, which can bind and deliver LPA to its cognate receptors with a high affinity. As a first messenger, gintonin produces second messenger Ca2+ via G protein-coupled LPA receptors. Ca2+ is an intracellular mediator of gintonin and initiates a cascade of amplifications for further intercellular communications by activation of Ca2+-dependent kinases, receptors, gliotransmitter, and neurotransmitter release. Ginsenosides, which have been regarded as primary ingredients of ginseng, cannot elicit intracellular [Ca2+]i transients, since they lack specific cell surface receptor. However, ginsenosides exhibit non-specific ion channel and receptor regulations. This is the key characteristic that distinguishes gintonin from ginsenosides. Although the current discourse on ginseng pharmacology is focused on ginsenosides, gintonin can definitely provide a mode of action for ginseng pharmacology that ginsenosides cannot. This review article introduces a novel concept of ginseng ligand-LPA receptor interaction and proposes to establish a paradigm that shifts the focus from ginsenosides to gintonin as a major ingredient representing ginseng pharmacology. PMID:26578955

  17. Nuclear transportation of exogenous epidermal growth factor receptor and androgen receptor via extracellular vesicles.

    PubMed

    Read, Jolene; Ingram, Alistair; Al Saleh, Hassan A; Platko, Khrystyna; Gabriel, Kathleen; Kapoor, Anil; Pinthus, Jehonathan; Majeed, Fadwa; Qureshi, Talha; Al-Nedawi, Khalid

    2017-01-01

    Epidermal growth factor receptor (EGFR) plays a central role in the progression of several human malignancies. Although EGFR is a membrane receptor, it undergoes nuclear translocation, where it has a distinct signalling pathway. Herein, we report a novel mechanism by which cancer cells can directly transport EGFR to the nucleus of other cells via extracellular vesicles (EVs). The transported receptor is active and stimulates the nuclear EGFR pathways. Interestingly, the translocation of EGFR via EVs occurs independently of the nuclear localisation sequence that is required for nuclear translocation of endogenous EGFR. Also, we found that the mutant receptor EGFRvIII could be transported to the nucleus of other cells via EVs. To assess the role of EVs in the regulation of an actual nuclear receptor, we studied the regulation of androgen receptor (AR). We found that full-length AR and mutant variant ARv7 are secreted in EVs derived from prostate cancer cell lines and could be transported to the nucleus of AR-null cells. The EV-derived AR was able to bind the androgen-responsive promoter region of prostate specific antigen, and recruit RNA Pol II, an indication of active transcription. The nuclear-translocated AR via EVs enhanced the proliferation of acceptor cells in the absence of androgen. Finally, we provide evidence that nuclear localisation of AR could occur in vivo via orthotopically-injected EVs in male SCID mice prostate glands. To our knowledge, this is the first study showing the nuclear translocation of nuclear receptors via EVs, which significantly extends the role of EVs as paracrine transcriptional regulators. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. A hierarchical network of interreceptor interactions determines signal transduction by Neu differentiation factor/neuregulin and epidermal growth factor.

    PubMed Central

    Tzahar, E; Waterman, H; Chen, X; Levkowitz, G; Karunagaran, D; Lavi, S; Ratzkin, B J; Yarden, Y

    1996-01-01

    The ErbB family includes four homologous transmembrane tyrosine kinases. Whereas ErbB-1 binds to the epidermal growth factor (EGF), both ErbB-3 and ErbB-4 bind to the Neu differentiation factors (NDFs, or neuregulins), and ErbB-2, the most oncogenic family member, is an orphan receptor whose function is still unknown. Because previous lines of evidence indicated the existence of interreceptor interactions, we used ectopic expression of individual ErbB proteins and their combinations to analyze the details of receptor cross talks. We show that 8 of 10 possible homo-and heterodimeric complexes of ErbB proteins can be hierarchically induced by ligand binding. Although ErbB-2 binds neither ligand, even in a heterodimeric receptor complex, it is the preferred heterodimer partner of the three other members, and it favors interaction with ErbB-3. Selective receptor overexpression in human tumor cells appears to bias the hierarchical relationships. The ordered network is reflected in receptor transphosphorylation, ErbB-2-mediated enhancement of ligand affinities, and remarkable potentiation of mitogenesis by a coexpressed ErbB-2. The observed superior ability of ErbB-2 to form heterodimers, in conjunction with its uniquely high basal tyrosine kinase activity, may explain why ErbB-2 overexpression is associated with poor prognosis. PMID:8816440

  19. UV activates growth factor receptors via reactive oxygen intermediates

    PubMed Central

    1996-01-01

    Exposure of mammalian cells to UV irradiation induces rapid and transient expression of early growth response-1 gene (Egr-1) encoding a transcription factor that plays a role in cell survival. These signals from the irradiated cell surface are likely to involve more than one pathway, and we show here that an essential pathway involves activation of several growth factor receptors by reactive oxygen intermediates (ROI). UVC irradiation causes the tyrosine phosphorylation of EGF receptor (EGFR) in mouse NIH 3T3 fibroblasts and HC11 mouse mammary cells. EGFR activation by irradiation of cells is abrogated by suramin, by antioxidants, and by the presence of a dominant negative EGFR. UV induces the formation of complexes between activated EGFR and SOS, Grb2, PLC gamma, and SHC that can be precipitated with antibodies to EGFR. The activation of EGFR by UV is mimicked by H2O2, suggesting that ROI may function upstream of EGFR activation. Our observations support the hypothesis that ROI and growth factor receptors operate in the early steps of the UV signal that lead to the enhanced expression and activity of Egr-1. PMID:8601609

  20. Fibroblast growth factor receptor levels decrease during chick embryogenesis

    PubMed Central

    1990-01-01

    Two putative receptors for fibroblast growth factor (FGF) of approximately 150 and 200 kD were identified in membrane preparations from chick embryos. Specific binding (femtomoles/milligram) of 125I- aFGF to whole chick embryonic membranes was relatively constant from day 2 to 7, then decreased fivefold between days 7 and 13. Day-19 chick embryos retained 125I-aFGF binding at low levels to brain, eye, and liver tissues but not to skeletal muscle or cardiac tissues. The 200-kD FGF receptor began to decline between day 4.5 and 7 and was barely detectable by day 9, whereas the 150-kD FGF receptor began to decline by day 7 but was still detectable in day-9 embryonic membranes. It is not known whether the two FGF-binding proteins represent altered forms of one polypeptide, but it is clear that their levels undergo differential changes during development. Because endogenous chick FGF may remain bound to FGF receptor in membrane preparations, membranes were treated with acidic (pH 4.0) buffers to release bound FGF; such treatment did not affect 125I-aFGF binding and moderately increased the number of binding sites in day-7 and -19 embryos. Consequently, the observed loss of high affinity 125I-aFGF binding sites and FGF-binding polypeptides most likely represents a loss of FGF receptor protein. These experiments provide in vivo evidence to support the hypothesis that regulation of FGF receptor levels may function as a mechanism for controlling FGF-dependent processes during embryonic development. PMID:2153684

  1. Modulation of M(2) muscarinic receptor-receptor interaction by immunoglobulin G antibodies from Chagas' disease patients.

    PubMed

    Beltrame, S P; Auger, S R; Bilder, C R; Waldner, C I; Goin, J C

    2011-05-01

    Circulating immunoglobulin (Ig)G antibodies against M(2) muscarinic acetylcholine receptors (M(2) mAChR) have been implicated in Chagas' disease (ChD) pathophysiology. These antibodies bind to and activate their target receptor, displaying agonist-like activity through an unclear mechanism. This study tested the ability of serum anti-M(2) mAChR antibodies from chronic ChD patients to modulate M(2) muscarinic receptor-receptor interaction by bioluminescence resonance energy transfer (BRET). Human embryonic kidney (HEK) 293 cells co-expressing fusion proteins M(2) mAChR-Renilla luciferase (RLuc) and M(2) mAChR-yellow fluorescent protein (YFP) were exposed to the serum IgG fraction from ChD patients, and BRET between RLuc and YFP was assessed by luminometry. Unlike serum IgG from healthy subjects and conventional muscarinic ligands, ChD IgG promoted a time- and concentration-dependent increase in the BRET signal. This effect neither required cellular integrity nor occurred as a consequence of receptor activation. Enhancement of M(2) receptor-receptor interaction by ChD IgG was receptor subtype-specific and mediated by the recognition of the second extracellular loop of the M(2) mAChR. The monovalent Fab fragment derived from ChD IgG was unable to reproduce the effect of the native immunoglobulin. However, addition of ChD Fab in the presence of anti-human Fab IgG restored BRET-enhancing activity. These data suggest that the modulatory effect of ChD IgG on M(2) receptor-receptor interaction results from receptor cross-linking by bivalent antibodies.

  2. Kinetics of the Attachment of Intrinsic Factor-Bound Cobamides to Ileal Receptors

    PubMed Central

    Mathan, V. I.; Babior, Bernard M.; Donaldson, Robert M.

    1974-01-01

    was 1.3 × 106 M-1 s-1. The half-time for dissociation of intrinsic factor-bound cyanocobalamin from the ileal receptor was approximately 35 min. Free intrinsic factor inhibited the attachment of intrinsic factor-bound cyanocobalamin, but the rate of attachment of free intrinsic factor was slower than that of intrinsic factor bound to cyanocobalamin. When intrinsic factor was complexed with various analogs of cyanocobalamin, the affinities of these complexes for ileal microvillous membranes were similar to that of intrinsic factor-bound cyanocobalamin. These findings suggest that the molecular configuration of vitamin B12 is not a major determinant in the interaction between intrinsic factor-bound vitamin B12 and its ileal receptor site. PMID:4852622

  3. Characterization of the interaction of the human mineralocorticosteroid receptor with hormone response elements.

    PubMed Central

    Lombès, M; Binart, N; Oblin, M E; Joulin, V; Baulieu, E E

    1993-01-01

    Although the mineralocorticosteroid receptor (MR) belongs to the superfamily of hormone-dependent transcription factors, the molecular mechanism by which it regulates gene expression is poorly understood. Binding of the MR to target gene promoters has never been characterized, and specific mineralocorticosteroid response elements (MREs) remain to be identified. The human MR (hMR) was overexpressed in Sf21 insect cells using the baculovirus system. The high degree of similarity between the glucocorticosteroid receptor (GR) and the MR prompted us to examine the DNA-binding properties of the recombinant MR with glucocorticosteroid-regulated genes. Gel shift mobility assays demonstrated that the recombinant receptor interacted with oligonucleotides containing perfect and imperfect palindromic sequences of GRE. A monoclonal anti-hMR antibody (FD4) induced a supershift of protein-DNA complexes and identified the MR in Western blot analysis. In vitro DNAse I protection assays with the hormone-regulated murine mammary tumour virus promoter showed that recombinant hMR generated four footprints whose limits encompassed the GRE motifs. By means of these two complementary approaches, no difference between the interaction of free, agonist- or antagonist-bound MR and DNA was detected. We provide evidence that hMR functions as a sequence-specific DNA-binding protein. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8389140

  4. Physiological functions of TNF family receptor/ligand interactions in hematopoiesis and transplantation.

    PubMed

    Mizrahi, Keren; Askenasy, Nadir

    2014-07-10

    Secretion of ligands of the tumor necrosis factor (TNF) superfamily is a conserved response of parenchymal tissues to injury and inflammation that commonly perpetuates elimination of dysfunctional cellular components by apoptosis. The same signals of tissue injury that induce apoptosis in somatic cells activate stem cells and initiate the process of tissue regeneration as a coupling mechanism of injury and recovery. Hematopoietic stem and progenitor cells upregulate the TNF family receptors under stress conditions and are transduced with trophic signals. The progeny gradually acquires sensitivity to receptor-mediated apoptosis along the differentiation process, which becomes the major mechanism of negative regulation of mature proliferating hematopoietic lineages and immune homeostasis. Receptor/ligand interactions of the TNF family are physiological mechanisms transducing the need for repair, which may be harnessed in pathological conditions and transplantation. Because these interactions are physiological mechanisms of injury, neutralization of these pathways has to be carefully considered in disorders that do not involve intrinsic aberrations of excessive susceptibility to apoptosis.

  5. Interaction of lectins with membrane receptors on erythrocyte surfaces.

    PubMed

    Sung, L A; Kabat, E A; Chien, S

    1985-08-01

    The interactions of human genotype AO erythrocytes (red blood cells) (RBCs) with N-acetylgalactosamine-reactive lectins isolated from Helix pomatia (HPA) and from Dolichos biflorus (DBA) were studied. Binding curves obtained with the use of tritium-labeled lectins showed that the maximal numbers of lectin molecules capable of binding to human genotype AO RBCs were 3.8 X 10(5) and 2.7 X 10(5) molecules/RBC for HPA and DBA, respectively. The binding of one type of lectin may influence the binding of another type. HPA was found to inhibit the binding of DBA, but not vice versa. The binding of HPA was weakly inhibited by a beta-D-galactose-reactive lectin isolated from Ricinus communis (designated RCA1). Limulus polyphemus lectin (LPA), with specificity for N-acetylneuraminic acid, did not influence the binding of HPA but enhanced the binding of DBA. About 80% of LPA receptors (N-acetylneuraminic acid) were removed from RBC surfaces by neuraminidase treatment. Neuraminidase treatment of RBCs resulted in increases of binding of both HPA and DBA, but through different mechanisms. An equal number (7.6 X 10(5) of new HPA sites were generated on genotypes AO and OO RBCs by neuraminidase treatment, and these new sites accounted for the enhancement (AO cells) and appearance (OO cells) of hemagglutinability by HPA. Neuraminidase treatment did not generate new DBA sites, but increased the DBA affinity for the existing receptors; as a result, genotype AO cells increased their hemagglutinability by DBA, while OO cells remained unagglutinable. The use of RBCs of different genotypes in binding assays with 3H-labeled lectins of known specificities provides an experimental system for studying cell-cell recognition and association.

  6. Probability description of ligand-receptor interactions. Evaluation of reliability of events with small and supersmall doses. I. Kinetics of ligand-receptor interactions.

    PubMed

    Gurevich, K G; Varfolomeev, S D

    1999-09-01

    We have developed mathematical methods for describing ligand-receptor interactions (LRI) using Markov chains. Under some conditions, the mean value of ligand-receptor complexes obtained using Markov chains coincides with that obtained from the law of mass action. Using the calculated ratio of standard deviation to mean number of ligand-receptor complexes, we show that with small concentrations of ligand-receptor complexes LRI must be described using probability methods. Using data from the literature, we show that LRI description using the mass-action law under these conditions can cause significant errors in interpretation of experimental data.

  7. Insulin-Like Growth Factor 1 Receptor Is a Prognostic Factor in Classical Hodgkin Lymphoma

    PubMed Central

    Liang, Zheng; Diepstra, Arjan; Xu, Chuanhui; van Imhoff, Gustaaf; Plattel, Wouter; Van Den Berg, Anke; Visser, Lydia

    2014-01-01

    The interaction between the tumor cells in classical Hodgkin lymphoma (cHL) and the microenvironment includes aberrant activity of receptor tyrosine kinases. In this study we evaluated the expression, functionality and prognostic significance of Insulin-like growth factor-1 receptor (IGF-1R) in cHL. IGF-1R was overexpressed in 55% (44/80) of cHL patients. Phosphorylated IGF-1R was detectable in a minority of the IGF-1R positive tumor cells. The overall survival (OS, 98%) and 5-year progression-free survival (PFS, 93%) was significantly higher in IGF-1R positive cHL patients compared to IGF-1R negative patients (OS 83%, p = .029 and PFS 77%, p = .047, respectively). Three cHL cell lines showed expression of IGF-1R, with strong staining especially in the mitotic cells and expression of IGF-1. IGF-1 treatment had a prominent effect on the cell growth of L428 and L1236 cells and resulted in an increased phosphorylation of IGF1R, Akt and ERK. Inhibition of IGF-1R with cyclolignan picropodophyllin (PPP) decreased cell growth and induced a G2/M cell cycle arrest in all three cell lines. Moreover, a decrease in pCcd2 and an increase in CyclinB1 levels were observed which is consistent with the G2/M cell cycle arrest. In conclusion, IGF-1R expression in HRS cells predicts a favorable outcome, despite the oncogenic effect of IGF-1R in cHL cell lines. PMID:24489919

  8. Insulin-like growth factor 1 receptor is a prognostic factor in classical Hodgkin lymphoma.

    PubMed

    Liang, Zheng; Diepstra, Arjan; Xu, Chuanhui; van Imhoff, Gustaaf; Plattel, Wouter; Van Den Berg, Anke; Visser, Lydia

    2014-01-01

    The interaction between the tumor cells in classical Hodgkin lymphoma (cHL) and the microenvironment includes aberrant activity of receptor tyrosine kinases. In this study we evaluated the expression, functionality and prognostic significance of Insulin-like growth factor-1 receptor (IGF-1R) in cHL. IGF-1R was overexpressed in 55% (44/80) of cHL patients. Phosphorylated IGF-1R was detectable in a minority of the IGF-1R positive tumor cells. The overall survival (OS, 98%) and 5-year progression-free survival (PFS, 93%) was significantly higher in IGF-1R positive cHL patients compared to IGF-1R negative patients (OS 83%, p = .029 and PFS 77%, p = .047, respectively). Three cHL cell lines showed expression of IGF-1R, with strong staining especially in the mitotic cells and expression of IGF-1. IGF-1 treatment had a prominent effect on the cell growth of L428 and L1236 cells and resulted in an increased phosphorylation of IGF1R, Akt and ERK. Inhibition of IGF-1R with cyclolignan picropodophyllin (PPP) decreased cell growth and induced a G2/M cell cycle arrest in all three cell lines. Moreover, a decrease in pCcd2 and an increase in CyclinB1 levels were observed which is consistent with the G2/M cell cycle arrest. In conclusion, IGF-1R expression in HRS cells predicts a favorable outcome, despite the oncogenic effect of IGF-1R in cHL cell lines.

  9. Quantifying the allosteric interactions within a G-protein-coupled receptor heterodimer.

    PubMed

    Zhou, Bin; Giraldo, Jesús

    2017-07-27

    G-protein-coupled receptors are central to signal transduction and cell communication. The possibility that cells use receptor heteromerization to modulate individual receptor pathways is a surmise that cannot be precluded. Given the complexity of these processes, mathematical models contribute to understanding how receptors and their respective ligands regulate signaling. Here, a mathematical model is presented that quantifies the allosteric interactions within a receptor heterodimer. The model is based on the operational model of allosterism including constitutive receptor activity, which provides the pharmacological analysis of heteromerization with well-established and widely used modeling and fitting procedures. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Interactions between adenosine and metabotropic glutamate receptors in the rat hippocampal slice

    PubMed Central

    Shahraki, Ali; Stone, Trevor W

    2003-01-01

    We have examined excitatory postsynaptic potentials and paired-pulse interactions in rat hippocampal slices to obtain more information about the site and mechanism of interactions between metabotropic glutamate receptors and adenosine receptors. The results show that the suppression of adenosine sensitivity is explained by a selectively reduced responsiveness to A1 receptor stimulation, and does not involve any facilitation of A2A adenosine receptors, since it can be obtained in the absence of endogenous adenosine and is not prevented by the A2A receptor blocker ZM241385. The glutamate receptors involved are of the group I class since the suppression of adenosine sensitivity is produced by ACPD and the group I selective compound DHPG. Furthermore, the effects of DHPG could be prevented by LY367385, a selective antagonist at the mGlu1a subtype of group I receptors. The selective antagonist at mGlu5 receptors, SIB1893, did not prevent the suppression of adenosine sensitivity by DHPG. Blockade of the DHPG/adenosine interaction was also obtained by superfusion with the protein kinasae C inhibitor chelerythrine. Since the suppression of adenosine responses by metabotropic receptor agonists was seen in the paired-pulse paradigm, we conclude that the observed interactions occur at the level of the presynaptic terminals. The interaction with adenosine receptors is not specific, but applies also to a suppression of responses mediated by the GABAB receptor agonist baclofen. We conclude that activation of the mGlu1a subtype of receptor can suppress responses mediated via adenosine A1 receptors, probably by activating protein kinase C. Since the changes induced by metabotropic glutamate receptor agonists last for at least 60 min, the data also imply that these interactions could play an important role in changes of synaptic function long after even transient increases of glutamate release in the CNS. PMID:12684261

  11. Interleukin-1 receptor accessory protein interacts with the type II interleukin-1 receptor.

    PubMed

    Malinowsky, D; Lundkvist, J; Layé, S; Bartfai, T

    1998-06-16

    Stably transfected HEK-293 cells express on their surface the murine type II IL-1 receptor (mIL-1RII) as demonstrated by FACS analysis using the mAb 4E2, however binding of [125I]-hrIL-1beta to these cells is nearly absent. Saturable high affinity binding of [125I]-hrIL-1beta is observed when the murine IL-1 receptor accessory protein (mIL-1RAcP) is coexpressed with mIL-1RII. Binding of [125I]-hrIL-1beta to mIL-1RII-mIL-1RAcP complex can be inhibited either with antibodies to mIL-1RII (mAb 4E2), or by antibodies to mIL-1RAcP (mAb 4C5). The number of high affinity binding sites in cells stably transfected with the cDNA for mIL-1RII is dependent on the dose of cDNA for mIL-1RAcP used to transfect the cells. The high affinity complex between mIL-1RII and mIL-1RAcP is not preformed by interaction between the intracellular domains of these two transmembrane proteins, rather it appears to require the extracellular portions of mIL-1RII and mIL-1RAcP and the presence of a ligand. We suggest that in addition to its earlier described decoy receptor role, IL-1RII may modulate the responsiveness of cells to IL-1 by binding the IL-1RAcP in unproductive/non-signalling complexes and thus reducing the number of signalling IL-1RI-IL-1RAcP-agonist complexes when IL-1 is bound.

  12. Endocannabinoid receptor deficiency affects maternal care and alters the dam's hippocampal oxytocin receptor and brain-derived neurotrophic factor expression.

    PubMed

    Schechter, M; Weller, A; Pittel, Z; Gross, M; Zimmer, A; Pinhasov, A

    2013-10-01

    Maternal care is the newborn's first experience of social interaction, and this influences infant survival, development and social competences throughout life. We recently found that postpartum blocking of the endocannabinoid receptor-1 (CB1R) altered maternal behaviour. In the present study, maternal care was assessed by the time taken to retrieve pups, pups' ultrasonic vocalisations (USVs) and pup body weight, comparing CB1R deleted (CB1R KO) versus wild-type (WT) mice. After culling on postpartum day 8, hippocampal expression of oxytocin receptor (OXTR), brain-derived neurotrophic factor (BDNF) and stress-mediating factors were evaluated in CB1R KO and WT dams. Comparisons were also performed with nulliparous (NP) CB1R KO and WT mice. Compared to WT, CB1R KO dams were slower to retrieve their pups. Although the body weight of the KO pups did not differ from the weight of WT pups, they emitted fewer USVs. This impairment of the dam-pup relationship correlated with a significant reduction of OXTR mRNA and protein levels among CB1R KO dams compared to WT dams. Furthermore, WT dams exhibited elevated OXTR mRNA expression, as well as increased levels of mineralocorticoid and glucocorticoid receptors, compared to WT NP mice. By contrast, CB1R KO dams showed no such elevation of OXTR expression, alongside lower BDNF and mineralocorticoid receptors, as well as elevated corticotrophin-releasing hormone mRNA levels, when compared to CB1R KO NP. Thus, it appears that the disruption of endocannabinoid signalling by CB1R deletion alters expression of the OXTR, apparently leading to deleterious effects upon maternal behaviour.

  13. The basic amino acids in the coiled-coil domain of CIN85 regulate its interaction with c-Cbl and phosphatidic acid during epidermal growth factor receptor (EGFR) endocytosis.

    PubMed

    Zheng, Xiudan; Zhang, Jing; Liao, Kan

    2014-07-08

    During EGFR internalization CIN85 bridges EGFR-Cbl complex, endocytic machinery and fusible membrane through the interactions of CIN85 with c-Cbl, endophilins and phosphatidic acid. These protein-protein and protein-lipid interactions are mediated or regulated by the positively charged C-terminal coiled-coil domain of CIN85. However, the details of CIN85-lipid interaction remain unknown. The present study suggested a possible electric interaction between the negative charge of phosphatidic acid and the positive charge of basic amino acids in coiled-coil domain. Mutations of the basic amino acids in the coiled-coil domain, especially K645, K646, R648 and R650, into neutral amino acid alanine completely blocked the interaction of CIN85 with c-Cbl or phosphatidic acid. However, they did not affect CIN85-endophilin interaction. In addition, CIN85 was found to associate with the internalized EGFR endosomes. It interacted with several ESCRT (Endosomal Sorting Complex Required for Transport) component proteins for ESCRT assembly on endosomal membrane. Mutations in the coiled-coil domain (deletion of the coiled-coil domain or point mutations of the basic amino acids) dissociated CIN85 from endosomes. These mutants bound the ESCRT components in cytoplasm to prevent them from assembly on endosomal membrane and inhibited EGFR sorting for degradation. As an adaptor protein, CIN85 interacts with variety of partners through several domains. The positive charges of basic amino acids in the coiled-coil domain are not only involved in the interaction with phosphatidic acid, but also regulate the interaction of CIN85 with c-Cbl. CIN85 also interacts with ESCRT components for protein sorting in endosomes. These CIN85-protein and CIN85-lipid interactions enable CIN85 to link EGFR-Cbl endocytic complex with fusible membrane during EGFR endocytosis and subsequently to facilitate ESCRT formation on endosomal membrane for EGFR sorting and degradation.

  14. 5-HT2A SEROTONIN RECEPTOR BIOLOGY: Interacting proteins, kinases and paradoxical regulation

    PubMed Central

    Roth, Bryan L

    2011-01-01

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

  15. Biogenesis of lysosome-related organelles complex-1 subunit 1 (BLOS1) interacts with sorting nexin 2 and the endosomal sorting complex required for transport-I (ESCRT-I) component TSG101 to mediate the sorting of epidermal growth factor receptor into endosomal compartments.

    PubMed

    Zhang, Aili; He, Xin; Zhang, Ling; Yang, Lin; Woodman, Philip; Li, Wei

    2014-10-17

    Biogenesis of lysosome-related organelles complex-1 (BLOC-1) is a component of the molecular machinery required for the biogenesis of specialized organelles and lysosomal targeting of cargoes via the endosomal to lysosomal trafficking pathway. BLOS1, one subunit of BLOC-1, is implicated in lysosomal trafficking of membrane proteins. We found that the degradation and trafficking of epidermal growth factor receptor (EGFR) were delayed in BLOS1 knockdown cells, which were rescued through BLOS1 overexpression. A key feature to the delayed EGFR degradation is the accumulation of endolysosomes in BLOS1 knockdown cells or BLOS1 knock-out mouse embryonic fibroblasts. BLOS1 interacted with SNX2 (a retromer subunit) and TSG101 (an endosomal sorting complex required for transport subunit-I) to mediate EGFR lysosomal trafficking. These results suggest that coordination of the endolysosomal trafficking proteins is important for proper targeting of EGFR to lysosomes.

  16. Binding and regulation of cellular functions by monoclonal antibodies against human tumor necrosis factor receptors

    PubMed Central

    1990-01-01

    The present study was undertaken to further characterize the interaction of monoclonal antibodies (mAbs) against tumor necrosis factor (TNF) receptors with different targets, and to assess their ability to influence TNF effects on U937 and human endothelial cell (HEC) functions. Actions of recombinant TNF-alpha on U937 and HEC were effectively inhibited by Htr-5 and Utr-1, and to a greater extent by a combination of both mAbs. These observations indicate that TNF interaction with antigenically different components of membrane receptors (p55 and p75) represents a crucial step in transduction of signals for TNF toxicity against U937 and TNF activation of HEC functions. PMID:2172437

  17. Conformational thermostabilisation of corticotropin releasing factor receptor 1

    PubMed Central

    Kean, James; Bortolato, Andrea; Hollenstein, Kaspar; Marshall, Fiona H.; Jazayeri, Ali

    2015-01-01

    Recent technical advances have greatly facilitated G-protein coupled receptors crystallography as evidenced by the number of successful x-ray structures that have been reported recently. These technical advances include novel detergents, specialised crystallography techniques as well as protein engineering solutions such as fusions and conformational thermostabilisation. Using conformational thermostabilisation, it is possible to generate variants of GPCRs that exhibit significantly increased stability in detergent micelles whilst preferentially occupying a single conformation. In this paper we describe for the first time the application of this technique to a member of a class B GPCR, the corticotropin releasing factor receptor 1 (CRF1R). Mutational screening in the presence of the inverse agonist, CP-376395, resulted in the identification of a construct with twelve point mutations that exhibited significantly increased thermal stability in a range of detergents. We further describe the subsequent construct engineering steps that eventually yielded a crystallisation-ready construct which recently led to the solution of the first x-ray structure of a class B receptor. Finally, we have used molecular dynamic simulation to provide structural insight into CRF1R instability as well as the stabilising effects of the mutants, which may be extended to other class B receptors considering the high degree of structural conservation. PMID:26159865

  18. Vascular Endothelial Growth Factor Receptor -2 in Breast Cancer

    PubMed Central

    Guo, Shanchun; Colbert, Laronna S.; Fuller, Miles; Zhang, Yuanyuan; Gonzalez-Perez, Ruben R.

    2010-01-01

    Investigations over the last decade have established the essential role of growth factors and their receptors during angiogenesis and carcinogenesis. The vascular endothelial growth factor receptor (VEGFR) family in mammals contains three members, VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1) and VEGFR-3 (Flt-4), which are transmembrane tyrosine kinase receptors that regulate the formation of blood and lymphatic vessels. In the early 1990s, the above VEGFR were structurally characterized by cDNA cloning. Among these three receptors, VEGFR-2 is generally recognized to have a principal role in mediating VEGF-induced responses. VEGFR-2 is considered as the earliest marker for endothelial cell development. Importantly, VEGFR-2 directly regulates tumor angiogenesis. Therefore, several inhibitors of VEGFR-2 have been developed and many of them are now in clinical trials. In addition to targeting endothelial cells, the VEGF/VEGFR-2 system works as an essential autocrine/paracrine process for cancer cell proliferation and survival. Recent studies mark the continuous and increased interest in this related, but distinct, function of VEGF/VEGFR-2 in cancer cells: the autocrine/paracrine loop. Several mechanisms regulate VEGFR-2 levels and modulate its role in tumor angiogenesis and physiologic functions, i.e.: cellular localization/trafficking, regulation of cis-elements of promoter, epigenetic regulation and signaling from Notch, cytokines/growth factors and estrogen, etc. In this review, we will focus on updated information regarding VEGFR-2 research with respect to the molecular mechanisms of VEGFR-2 regulation in human breast cancer. Investigations in the activation, function, and regulation of VEGFR-2 in breast cancer will allow the development of new pharmacological strategies aimed at directly targeting cancer cell proliferation and survival. PMID:20462514

  19. The Janus Kinase (JAK) FERM and SH2 Domains: Bringing Specificity to JAK–Receptor Interactions

    PubMed Central

    Ferrao, Ryan; Lupardus, Patrick J.

    2017-01-01

    The Janus kinases (JAKs) are non-receptor tyrosine kinases essential for signaling in response to cytokines and interferons and thereby control many essential functions in growth, development, and immune regulation. JAKs are unique among tyrosine kinases for their constitutive yet non-covalent association with class I and II cytokine receptors, which upon cytokine binding bring together two JAKs to create an active signaling complex. JAK association with cytokine receptors is facilitated by N-terminal FERM and SH2 domains, both of which are classical mediators of peptide interactions. Together, the JAK FERM and SH2 domains mediate a bipartite interaction with two distinct receptor peptide motifs, the proline-rich “Box1” and hydrophobic “Box2,” which are present in the intracellular domain of cytokine receptors. While the general sidechain chemistry of Box1 and Box2 peptides is conserved between receptors, they share very weak primary sequence homology, making it impossible to posit why certain JAKs preferentially interact with and signal through specific subsets of cytokine receptors. Here, we review the structure and function of the JAK FERM and SH2 domains in light of several recent studies that reveal their atomic structure and elucidate interaction mechanisms with both the Box1 and Box2 receptor motifs. These crystal structures demonstrate how evolution has repurposed the JAK FERM and SH2 domains into a receptor-binding module that facilitates interactions with multiple receptors possessing diverse primary sequences. PMID:28458652

  20. Yeast-based reporter assays for the functional characterization of cochaperone interactions with steroid hormone receptors.

    PubMed

    Balsiger, Heather A; Cox, Marc B

    2009-01-01

    Steroid hormone receptor-mediated reporter assays in the budding yeast Saccharomyces cerevisiae have been an invaluable tool for the identification and functional characterization of steroid hormone receptor-associated chaperones and cochaperones. This chapter describes a hormone-inducible androgen receptor-mediated beta-galactosidase reporter assay in yeast. In addition, the immunophilin FKBP52 is used as a specific example of a receptor-associated cochaperone that acts as a positive regulator of receptor function. With the right combination of receptor and cochaperone expression plasmids, reporter plasmid, and ligand, the assay protocol described here could be used to functionally characterize a wide variety of nuclear receptor-cochaperone interactions. In addition to the functional characterization of receptor regulatory proteins, a modified version of this assay is currently being used to screen compound libraries for selective FKBP52 inhibitors that represent attractive therapeutic candidates for the treatment of steroid hormone receptor-associated diseases.

  1. Synthesis and Evaluation of Candidate PET Radioligands for Corticotropin-Releasing Factor Type-1 Receptors

    PubMed Central

    Lodge, Nicholas J.; Li, Yu-Wen; Chin, Frederick T.; Dischino, Douglas D.; Zoghbi, Sami S.; Deskus, Jeffrey A.; Mattson, Ronald J.; Imaizumo, Masao; Pieschl, Rick; Molski, Thaddeus F.; Fujita, Masahiro; Dulac, Heidi; Zaczek, Robert; Bronson, Joanne J.; Macor, John E.; Innis, Robert B.; Pike, Victor W.

    2014-01-01

    Introduction A radioligand for measuring the density of corticotrophin-releasing factor subtype-1 receptors (CRF1 receptors) in living animal and human brain with positron emission tomography (PET) would be a useful tool for neuropsychiatric investigations and the development of drugs intended to interact with this target. This study was aimed at discovery of such a radioligand from a group of CRF1 receptor ligands based on a core 3-(phenylamino)pyrazin-2(1H)-one scaffold. Methods CRF1 receptor ligands were selected for development as possible PET radioligands based on their binding potency at CRF receptors (displacement of [125I]CRF from rat cortical membranes), measured lipophilicity, autoradiographic binding profile in rat and rhesus monkey brain sections, rat biodistribution, and suitability for radiolabeling with carbon-11 or fluorine-18. Two identified candidates (BMS-721313 and BMS-732098) were labeled with fluorine-18. A third candidate (BMS-709460) was labeled with carbon-11 and all three radioligands were evaluated in PET experiments in rhesus monkey. CRF1 receptor density (Bmax) was assessed in rhesus brain cortical and cerebellum membranes with the CRF receptor ligand, [3H]BMS-728300. Results The three ligands selected for development showed high binding affinity (IC50 values, 0.3–8 nM) at CRF1 receptors and moderate lipophilicity (LogD, 2.8–4.4). [3H]BMS-728300 and the two 18F-labeled ligands showed region-specific binding in rat and rhesus monkey brain autoradiography, namely higher binding density in the frontal and limbic cortex, and cerebellum than in thalamus and brainstem. CRF1 receptor Bmax in rhesus brain was found to be 50–120 fmol/mg protein across cortical regions and cerebellum. PET experiments in rhesus monkey showed that the radioligands [18F]BMS-721313, [18F]BMS-732098 and [11C]BMS-709460 gave acceptably high brain radioactivity uptake but no indication of the specific binding as seen in vitro. Conclusions Candidate CRF1 receptor

  2. Calcium-Sensing Receptor: Trafficking, Endocytosis, Recycling, and Importance of Interacting Proteins.

    PubMed

    Ray, Kausik

    2015-01-01

    The cloning of the extracellular calcium-sensing receptor (CaSR) provided a new paradigm in G-protein-coupled receptor (GPCR) signaling in which principal physiological ligand is a cation, namely, extracellular calcium (Ca(o)(2+)). A wealth of information has accumulated in the past two decades about the CaSR's structure and function, its contribution to pathology in disorders of calcium in humans, and CaSR-based therapeutics. The CaSR unlike many other GPCRs must function in the presence of its ligand, thus understanding the mechanisms such as anterograde trafficking and endocytic pathways of this receptor are complex and fallen behind other classical GPCRs. Factors controlling CaSR signaling include various proteins affecting the expression of the CaSR as well as modulation of its trafficking to and from the cell surface. The dimeric cell-surface CaSR links to various heterotrimeric G-proteins (G(q/11), G(i/o), G(12/13), and G(s)) to regulate intracellular second messengers, lipid kinases, various protein kinases, and transcription factors that are part of the machinery enabling the receptor to modulate the functions of the wide variety of cells in which it is expressed. This chapter describes key features of CaSR structure and function and discusses novel mechanisms by which the level of cell-surface receptor expression can be regulated including forward trafficking during biosynthesis, desensitization, internalization and recycling from the cell surface, and degradation. These processes are impacted by its interactions with several proteins in addition to signaling molecules per se (i.e., G-proteins, protein kinases, inositol phosphates, etc.) and include small molecular weight G-proteins (Sar1, Rabs, ARF, P24A, RAMPs, filamin A, 14-3-3 proteins, calmodulin, and caveolin-1). Moreover, CaSR signaling seems compartmentalized in cell-type-specific manner, and caveolin and filamin A likely act as scaffolds that bind signaling components and other key cellular

  3. An interaction of oxytocin receptors with metabotropic glutamate receptors in hypothalamic astrocytes.

    PubMed

    Kuo, J; Hariri, O R; Micevych, P

    2009-12-01

    Hypothalamic astrocytes play a critical role in the regulation and support of many different neuroendocrine events, and are affected by oestradiol. Both nuclear and membrane oestrogen receptors (ERs) are expressed in astrocytes. Upon oestradiol activation, membrane-associated ER signals through the type 1a metabotropic glutamate receptor (mGluR1a) to induce an increase of free cytoplasmic calcium concentration ([Ca(2+)](i)). Because the expression of oxytocin receptors (OTRs) is modulated by oestradiol, we tested whether oestradiol also influences oxytocin signalling. Oxytocin at 1, 10, and 100 nm induced a [Ca(2+)](i) flux measured as a change in relative fluorescence [DeltaF Ca(2+) = 330 +/- 17 relative fluorescent units (RFU), DeltaF Ca(2+) = 331 +/- 22 RFU, and DeltaF Ca(2+) = 347 +/- 13 RFU, respectively] in primary cultures of female post-pubertal hypothalamic astrocytes. Interestingly, OTRs interacted with mGluRs. The mGluR1a antagonist, LY 367385 (20 nm), blocked the oxytocin (1 nm)-induced [Ca(2+)](i) flux (DeltaF Ca(2+) = 344 +/- 19 versus 127 +/- 11 RFU, P < 0.001). Conversely, the mGluR1a receptor agonist, (RS)-3,5-dihydroxyphenyl-glycine (100 nm), increased the oxytocin (1 nm)-induced [Ca(2+)](i) response (DeltaF Ca(2+) = 670 +/- 31 RFU) compared to either compound alone (P < 0.001). Because both oxytocin and oestradiol rapidly signal through the mGluR1a, we treated hypothalamic astrocytes sequentially with oxytocin and oestradiol to determine whether stimulation with one hormone affected the subsequent [Ca(2+)](i) response to the second hormone. Oestradiol treatment did not change the subsequent [Ca(2+)](i) flux to oxytocin (P > 0.05) and previous oxytocin exposure did not affect the [Ca(2+)](i) response to oestradiol (P > 0.05). Furthermore, simultaneous oestradiol and oxytocin stimulation failed to yield a synergistic [Ca(2+)](i) response. These results suggest that the OTR signals through the mGluR1a to release Ca(2+) from intracellular stores and

  4. Role of fibroblast growth factor receptor signaling in kidney development.

    PubMed

    Bates, Carlton M

    2011-09-01

    Fibroblast growth factor receptors (Fgfrs) are expressed throughout the developing kidney. Several early studies have shown that exogenous fibroblast growth factors (Fgfs) affect growth and maturation of the metanephric mesenchyme (MM) and ureteric bud (UB). Transgenic mice that over-express a dominant negative receptor isoform develop renal aplasia/severe dysplasia, confirming the importance of Fgfrs in renal development. Furthermore, global deletion of Fgf7, Fgf10, and Fgfr2IIIb (isoform that binds Fgf7 and Fgf10) in mice leads to small kidneys with fewer collecting ducts and nephrons. Deletion of Fgfrl1, a receptor lacking intracellular signaling domains, causes severe renal dysgenesis. Conditional targeting of Fgf8 from the MM interrupts nephron formation. Deletion of Fgfr2 from the UB results in severe ureteric branching and stromal mesenchymal defects, although loss of Frs2α (major signaling adapter for Fgfrs) in the UB causes only mild renal hypoplasia. Deletion of both Fgfr1 and Fgfr2 in the MM results in renal aplasia with defects in MM formation and initial UB elongation and branching. Loss of Fgfr2 in the MM leads to many renal and urinary tract anomalies as well as vesicoureteral reflux. Thus, Fgfr signaling is critical for patterning of virtually all renal lineages at early and later stages of development.

  5. Upregulation of epidermal growth factor receptor 4 in oral leukoplakia

    PubMed Central

    Kobayashi, Hiroshi; Kumagai, Kenichi; Gotoh, Akito; Eguchi, Takanori; Yamada, Hiroyuki; Hamada, Yoshiki; Suzuki, Satsuki; Suzuki, Ryuji

    2013-01-01

    In the present study, we investigate the expression profile of the epidermal growth factor receptor family, which comprises EGFR/ErbB1, HER2/ErbB2, HER3/ErbB3 and HER4/ErbB4 in oral leukoplakia (LP). The expression of four epidermal growth factor receptor (EGFR) family genes and their ligands were measured in LP tissues from 14 patients and compared with levels in 10 patients with oral lichen planus (OLP) and normal oral mucosa (NOM) from 14 healthy donors by real-time polymerase chain reaction (PCR) and immunohistochemistry. Synchronous mRNA coexpression of ErbB1, ErbB2, ErbB3 and ErbB4 was detected in LP lesions. Out of the receptors, only ErbB4 mRNA and protein was more highly expressed in LP compared with NOM tissues. These were strongly expressed by epithelial keratinocytes in LP lesions, as shown by immunohistochemistry. Regarding the ligands, the mRNA of Neuregulin2 and 4 were more highly expressed in OLP compared with NOM tissues. Therefore, enhanced ErbB4 on the keratinocytes and synchronous modulation of EGFR family genes may contribute to the pathogenesis and carcinogenesis of LP. PMID:23492901

  6. The importance of the adenosine A(2A) receptor-dopamine D(2) receptor interaction in drug addiction.

    PubMed

    Filip, M; Zaniewska, M; Frankowska, M; Wydra, K; Fuxe, K

    2012-01-01

    Drug addiction is a serious brain disorder with somatic, psychological, psychiatric, socio-economic and legal implications in the developed world. Illegal (e.g., psychostimulants, opioids, cannabinoids) and legal (alcohol, nicotine) drugs of abuse create a complex behavioral pattern composed of drug intake, withdrawal, seeking and relapse. One of the hallmarks of drugs that are abused by humans is that they have different mechanisms of action to increase dopamine (DA) neurotransmission within the mesolimbic circuitry of the brain and indirectly activate DA receptors. Among the DA receptors, D(2) receptors are linked to drug abuse and addiction because their function has been proven to be correlated with drug reinforcement and relapses. The recognition that D(2) receptors exist not only as homomers but also can form heteromers, such as with the adenosine (A)(2A) receptor, that are pharmacologically and functionally distinct from their constituent receptors, has significantly expanded the range of potential drug targets and provided new avenues for drug design in the search for novel drug addiction therapies. The aim of this review is to bring current focus on A(2A) receptors, their physiology and pharmacology in the central nervous system, and to discuss the therapeutic relevance of these receptors to drug addiction. We concentrate on the contribution of A(2A) receptors to the effects of different classes of drugs of abuse examined in preclinical behavioral experiments carried out with pharmacological and genetic tools. The consequences of chronic drug treatment on A(2A) receptor-assigned functions in preclinical studies are also presented. Finally, the neurochemical mechanism of the interaction between A(2A) receptors and drugs of abuse in the context of the heteromeric A(2A)-D(2) receptor complex is discussed. Taken together, a significant amount of experimental analyses provide evidence that targeting A(2A) receptors may offer innovative translational strategies

  7. A Skeletal Muscle Ryanodine Receptor Interaction Domain in Triadin

    PubMed Central

    Wium, Elize; Dulhunty, Angela F.; Beard, Nicole A.

    2012-01-01

    Excitation-contraction coupling in skeletal muscle depends, in part, on a functional interaction between the ligand-gated ryanodine receptor (RyR1) and integral membrane protein Trisk 95, localized to the sarcoplasmic reticulum membrane. Various domains on Trisk 95 can associate with RyR1, yet the domain responsible for regulating RyR1 activity has remained elusive. We explored the hypothesis that a luminal Trisk 95 KEKE motif (residues 200–232), known to promote RyR1 binding, may also form the RyR1 activation domain. Peptides corresponding to Trisk 95 residues 200–232 or 200–231 bound to RyR1 and increased the single channel activity of RyR1 by 1.49±0.11-fold and 1.8±0.15-fold respectively, when added to its luminal side. A similar increase in [3H]ryanodine binding, which reflects open probability of the channels, was also observed. This RyR1 activation is similar to activation induced by full length Trisk 95. Circular dichroism showed that both peptides were intrinsically disordered, suggesting a defined secondary structure is not necessary to mediate RyR1 activation. These data for the first time demonstrate that Trisk 95′s 200–231 region is responsible for RyR1 activation. Furthermore, it shows that no secondary structure is required to achieve this activation, the Trisk 95 residues themselves are critical for the Trisk 95-RyR1 interaction. PMID:22937102

  8. Latent Factors in Student-Teacher Interaction Factor Analysis

    ERIC Educational Resources Information Center

    Le, Thu; Bolt, Daniel; Camburn, Eric; Goff, Peter; Rohe, Karl

    2017-01-01

    Classroom interactions between students and teachers form a two-way or dyadic network. Measurements such as days absent, test scores, student ratings, or student grades can indicate the "quality" of the interaction. Together with the underlying bipartite graph, these values create a valued student-teacher dyadic interaction network. To…

  9. Optimal experimental design in an epidermal growth factor receptor signalling and down-regulation model.

    PubMed

    Casey, F P; Baird, D; Feng, Q; Gutenkunst, R N; Waterfall, J J; Myers, C R; Brown, K S; Cerione, R A; Sethna, J P

    2007-05-01

    We apply the methods of optimal experimental design to a differential equation model for epidermal growth factor receptor signalling, trafficking and down-regulation. The model incorporates the role of a recently discovered protein complex made up of the E3 ubiquitin ligase, Cbl, the guanine exchange factor (GEF), Cool-1 (beta -Pix) and the Rho family G protein Cdc42. The complex has been suggested to be important in disrupting receptor down-regulation. We demonstrate that the model interactions can accurately reproduce the experimental observations, that they can be used to make predictions with accompanying uncertainties, and that we can apply ideas of optimal experimental design to suggest new experiments that reduce the uncertainty on unmeasurable components of the system.

  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 α5β1 receptor. Other targeted membrane-based receptors include the integrins αvβ3, αvβ5, and β2 (CD18), RPTP-α/β, 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. Estrogen receptor beta interacts and colocalizes with HADHB in mitochondria.

    PubMed

    Zhou, Zhenqi; Zhou, Jianhong; Du, Yuchun

    2012-10-19

    Estrogen receptors are localized in mitochondria, but their functions in this organelle remain unclear. We previously found that ERα interacted with mitochondrial protein HADHB and affected the thiolytic cleavage activity of HADHB in β-oxidation. It is known that ERβ binds to ERα. In addition, ERβ is predominately located in mitochondria. These facts led us to speculate that ERβ may also be associated with HADHB in mitochondria. In order to test this hypothesis, we performed co-immunoprecipitation and confocal microscopy analyses with human breast cancer MCF7 cells. The results demonstrated that ERβ was indeed associated and colocalized with HADHB within mitochondria. Interestingly, in contrast to the stimulatory effect of ERα on HADHB enzyme activity observed in the previous study, silencing of ERβ enhanced the enzyme activity of HADHB in the present study, suggesting that ERβ plays an inhibitory role in HADHB enzyme activity in the breast cancer cells. Our results imply that ERα and ERβ may differentially affect cellular oxidative stress through influencing the rate of β-oxidation of fatty acids in breast cancer cells. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. Topography of human placental receptors for epidermal growth factor.

    PubMed

    Rao, C V; Ramani, N; Chegini, N; Stadig, B K; Carman, F R; Woost, P G; Schultz, G S; Cook, C L

    1985-02-10

    These studies were undertaken to determine whether term human placental microvillus plasma membranes, which are exposed to maternal blood, and basolateral plasma membranes, which are in close proximity to fetal blood capillaries, contain receptors for epidermal growth factor (EGF). These two highly purified membranes bound 125I-EGF with similar affinity (apparent dissociation constants, 0.07-0.12 nM, but the total number of available receptors was greater in microvillus (8.2 pmol/mg protein) compared to basolateral (4.9 pmol/mg protein) plasma membranes. Detailed characterization of 125I-EGF binding to these membranes revealed numerous similarities as well as differences. The two membranes contained two major (155 and 140 kDa) and at least three minor (115, 175, and 210 kDa) specific 125I-EGF binding proteins. The 115-kDa protein was only found in basolateral plasma membranes. The 155-kDa protein was predominantly labeled in microvillus, whereas the 140-kDa protein was labeled predominantly in basolateral plasma membranes. The addition of protease inhibitors did not alter the multiple 125I-EGF binding proteins pattern found in these membranes. EGF stimulated phosphorylation of 140- and 155-kDa proteins in both microvillus and basolateral plasma membranes. However, the 155-kDa protein was phosphorylated to a greater extent in microvillus, whereas both 140- and 155-kDa proteins were phosphorylated equally in basolateral plasma membranes. Light and electron microscope autoradiographic studies revealed that 125I-EGF preferentially associated with microvillus plasma membranes. The data demonstrates the presence of EGF receptors in outer cell membranes of syncytiotrophoblasts and suggests that maternal EGF may influence syncytiotrophoblast function by binding to receptors in microvillus plasma membranes, while fetal EGF may also influence syncytiotrophoblast function but via receptors in basolateral plasma membranes.

  13. Extraversion. Interaction between D2 dopamine receptor polymorphisms and parental alcoholism.

    PubMed

    Ozkaragoz, T; Noble, E P

    2000-11-01

    Both molecular genetic factors (the D2 dopamine receptor (DRD2) and the D4 dopamine receptor (DRD4) polymorphisms) and environmental influences of living in an alcoholic or nonalcoholic home on the personality traits of Extraversion and Neuroticism were assessed in drug-naive, young adolescent boys. There were no significant main effects of genetic or environmental factors on either Neuroticism or Extraversion as measured by the Junior Eysenck Personality Inventory (JEPI). However, a significant interaction between DRD2 (but not DRD4) alleles and environmental variables was observed on Extraversion. Specifically, children with the minor alleles of the DRD2 gene showed a significantly greater Extraversion score when living in an alcoholic than in a nonalcoholic home. In contrast, children with the major alleles of the DRD2 gene showed a trend in the opposite direction. Although the results are preliminary and pending replication, they nevertheless provide the first report of a specific gene-environment interaction involving a human personality trait.

  14. Inhibiting the Epidermal Growth Factor Receptor | Center for Cancer Research

    Cancer.gov

    The Epidermal Growth Factor Receptor (EGFR) is a widely distributed cell surface receptor that responds to several extracellular signaling molecules through an intracellular tyrosine kinase, which phosphorylates target enzymes to trigger a downstream molecular cascade. Since the discovery that EGFR mutations and amplifications are critical in a number of cancers, efforts have been under way to develop and use targeted EGFR inhibitors. These efforts have met with some spectacular successes, but many patients have not responded as expected, have subsequently developed drug-resistant tumors, or have suffered serious side effects from the therapies to date. CCR Investigators are studying EGFR from multiple vantage points with the goal of developing even better strategies to defeat EGFR-related cancers.

  15. Signal Transduction by Vascular Endothelial Growth Factor Receptors

    PubMed Central

    Koch, Sina; Claesson-Welsh, Lena

    2012-01-01

    Vascular endothelial growth factors (VEGFs) are master regulators of vascular development and of blood and lymphatic vessel function during health and disease in the adult. It is therefore important to understand the mechanism of action of this family of five mammalian ligands, which act through three receptor tyrosine kinases (RTKs). In addition, coreceptors like neuropilins (NRPs) and integrins associate with the ligand/receptor signaling complex and modulate the output. Therapeutics to block several of the VEGF signaling components have been developed with the aim to halt blood vessel formation, angiogenesis, in diseases that involve tissue growth and inflammation, such as cancer. In this review, we outline the current information on VEGF signal transduction in relation to blood and lymphatic vessel biology. PMID:22762016

  16. Brassinosteroid signal transduction from receptor kinases to transcription factors.

    PubMed

    Kim, Tae-Wuk; Wang, Zhi-Yong

    2010-01-01

    Brassinosteroids (BRs) are growth-promoting steroid hormones in plants. Genetic studies in Arabidopsis illustrated the essential roles of BRs in a wide range of developmental processes and helped identify many genes involved in BR biosynthesis and signal transduction. Recently, proteomic studies identified missing links. Together, these approaches established the BR signal transduction cascade, which includes BR perception by the BRI1 receptor kinase at the cell surface, activation of BRI1/BAK1 kinase complex by transphosphorylation, subsequent phosphorylation of the BSK kinases, activation of the BSU1 phosphatase, dephosphorylation and inactivation of the BIN2 kinase, and accumulation of unphosphorylated BZR transcription factors in the nucleus. Mass spectrometric analyses are providing detailed information on the phosphorylation events involved in each step of signal relay. Thus, the BR signaling pathway provides a paradigm for understanding receptor kinase-mediated signal transduction as well as tools for the genetic improvement of the productivity of crop plants.

  17. Influence of domain interactions on conformational mobility of the progesterone receptor detected by hydrogen/deuterium exchange mass spectrometry

    PubMed Central

    Goswami, Devrishi; Callaway, Celetta; Pascal, Bruce D.; Kumar, Raj; Edwards, Dean P.; Griffin, Patrick R.

    2015-01-01

    Structural and functional details of the N-terminal activation function 1 (AF1) of most nuclear receptors are poorly understood due to the highly dynamic intrinsically disordered nature of this domain. A hydrogen/deuterium exchange (HDX) mass spectrometry based investigation of TATA box binding protein (TBP) interaction with various domains of progesterone receptor (PR) demonstrate that agonist bound PR interaction with TBP via AF1 impacts the mobility of the C-terminal AF2. Results from HDX and other biophysical studies involving agonist and antagonist bound full length PR and isolated PR domains reveals the molecular mechanism underlying synergistic transcriptional activation mediated by AF1 and AF2, dominance of PR-B isoform over PR-A, and the necessity of AF2 for full AF1-mediated transcriptional activity. These results provide a comprehensive picture elaborating the underlying mechanism of PR-TBP interactions as a model for studying NR-transcription factor functional interactions. PMID:24909783

  18. Positive and Negative Cross-Talk between Lysophosphatidic Acid Receptor 1, Free Fatty Acid Receptor 4, and Epidermal Growth Factor Receptor in Human Prostate Cancer Cells.

    PubMed

    Hopkins, Mandi M; Liu, Ze; Meier, Kathryn E

    2016-10-01

    Lysophosphatidic acid (LPA) is a lipid mediator that mediates cellular effects via G protein-coupled receptors (GPCRs). Epidermal growth factor (EGF) is a peptide that acts via a receptor tyrosine kinase. LPA and EGF both induce proliferation of prostate cancer cells and can transactivate each other's receptors. The LPA receptor LPA1 is particularly important for LPA response in human prostate cancer cells. Previous work in our laboratory has demonstrated that free fatty acid 4 (FFA4), a GPCR activated by ω-3 fatty acids, inhibits responses to both LPA and EGF in these cells. One potential mechanism for the inhibition involves negative interactions between FFA4 and LPA1, thereby suppressing responses to EGF that require LPA1 In the current study, we examined the role of LPA1 in mediating EGF and FFA4 agonist responses in two human prostate cancer cell lines, DU145 and PC-3. The results show that an LPA1-selective antagonist inhibits proliferation and migration to both LPA and EGF. Knockdown of LPA1 expression, using silencing RNA, blocks responses to LPA and significantly inhibits responses to EGF. The partial response to EGF that is observed after LPA1 knockdown is not inhibited by FFA4 agonists. Finally, the role of arrestin-3, a GPCR-binding protein that mediates many actions of activated GPCRs, was tested. Knockdown of arrestin-3 completely inhibits responses to both LPA and EGF in prostate cancer cells. Taken together, these results suggest that LPA1 plays a critical role in EGF responses and that FFA4 agonists inhibit proliferation by suppressing positive cross-talk between LPA1 and the EGF receptor. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  19. TTRAP, a novel protein that associates with CD40, tumor necrosis factor (TNF) receptor-75 and TNF receptor-associated factors (TRAFs), and that inhibits nuclear factor-kappa B activation.

    PubMed

    Pype, S; Declercq, W; Ibrahimi, A; Michiels, C; Van Rietschoten, J G; Dewulf, N; de Boer, M; Vandenabeele, P; Huylebroeck, D; Remacle, J E

    2000-06-16

    CD40 belongs to the tumor necrosis factor (TNF) receptor family. CD40 signaling involves the recruitment of TNF receptor-associated factors (TRAFs) to its cytoplasmic domain. We have identified a novel intracellular CD40-binding protein termed TRAF and TNF receptor-associated protein (TTRAP) that also interacts with TNF-R75 and CD30. The region of the CD40 cytoplasmic domain that is required for TTRAP association overlaps with the TRAF6 recognition motif. Association of TTRAP with CD40 increases profoundly in response to treatment of cells with CD40L. Interestingly, TTRAP also associates with TRAFs, with the highest affinity for TRAF6. In transfected cells, TTRAP inhibits in a dose-dependent manner the transcriptional activation of a nuclear factor-kappaB (NF-kappaB)-dependent reporter mediated by CD40, TNF-R75 or Phorbol 12-myristate 13-acetate (PMA) and to a lesser extent by TRAF2, TRAF6, TNF-alpha, or interleukin-1beta (IL-1beta). TTRAP does not affect stimulation of NF-kappaB induced by overexpression of the NF-kappaB-inducing kinase (NIK), the IkappaB kinase alpha (IKKalpha), or the NF-kappaB subunit P65/RelA, suggesting it acts upstream of the latter proteins. Our results indicate that we have isolated a novel regulatory factor that is involved in signal transduction by distinct members of the TNF receptor family.

  20. Prothymosin Alpha Selectively Enhances Estrogen Receptor Transcriptional Activity by Interacting with a Repressor of Estrogen Receptor Activity

    PubMed Central

    Martini, Paolo G. V.; Delage-Mourroux, Regis; Kraichely, Dennis M.; Katzenellenbogen, Benita S.

    2000-01-01

    We find that prothymosin alpha (PTα) selectively enhances transcriptional activation by the estrogen receptor (ER) but not transcriptional activity of other nuclear hormone receptors. This selectivity for ER is explained by PTα interaction not with ER, but with a 37-kDa protein denoted REA, for repressor of estrogen receptor activity, a protein that we have previously shown binds to ER, blocking coactivator binding to ER. We isolated PTα, known to be a chromatin-remodeling protein associated with cell proliferation, using REA as bait in a yeast two-hybrid screen with a cDNA library from MCF-7 human breast cancer cells. PTα increases the magnitude of ERα transcriptional activity three- to fourfold. It shows lesser enhancement of ERβ transcriptional activity and has no influence on the transcriptional activity of other nuclear hormone receptors (progesterone receptor, glucocorticoid receptor, thyroid hormone receptor, or retinoic acid receptor) or on the basal activity of ERs. In contrast, the steroid receptor coactivator SRC-1 increases transcriptional activity of all of these receptors. Cotransfection of PTα or SRC-1 with increasing amounts of REA, as well as competitive glutathione S-transferase pulldown and mammalian two-hybrid studies, show that REA competes with PTα (or SRC-1) for regulation of ER transcriptional activity and suppresses the ER stimulation by PTα or SRC-1, indicating that REA can function as an anticoactivator in cells. Our data support a model in which PTα, which does not interact with ER, selectively enhances the transcriptional activity of the ER but not that of other nuclear receptors by recruiting the repressive REA protein away from ER, thereby allowing effective coactivation of ER with SRC-1 or other coregulators. The ability of PTα to directly interact in vitro and in vivo with REA, a selective coregulator of the ER, thereby enabling the interaction of ER with coactivators, appears to explain its ability to selectively enhance

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

    SciTech Connect

    Yuzawa,S.; Opatowsky, Y.; Zhang, Z.; Mandiyan, V.; Lax, I.; Schlessinger, J.

    2007-01-01

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

  2. Insulin receptor-insulin interaction kinetics using multiplex surface plasmon resonance.

    PubMed

    Subramanian, Kannan; Fee, Conan J; Fredericks, Rayleen; Stubbs, Richard S; Hayes, Mark T

    2013-12-01

    Type 2 diabetes affects millions of people worldwide, and measuring the kinetics of insulin receptor-insulin interactions is critical to improving our understanding of this disease. In this paper, we describe, for the first time, a rapid, real-time, multiplex surface plasmon resonance (SPR) assay for studying the interaction between insulin and the insulin receptor ectodomain, isoform A (eIR-A). We used a scaffold approach in which anti-insulin receptor monoclonal antibody 83-7 (Abcam, Cambridge, UK) was first immobilized on the SPR sensorchip by amine coupling, followed by eIR-A capture. The multiplex SPR system (ProteOn XPR36™, Bio-Rad Laboratories, Hercules, CA) enabled measurement of replicate interactions with a single, parallel set of analyte injections, whereas repeated regeneration of the scaffold between measurements caused variable loss of antibody activity. Interactions between recombinant human insulin followed a two-site binding pattern, consistent with the literature, with a high-affinity site (dissociation constant K(D1)  = 38.1 ± 0.9 nM) and a low-affinity site (K(D2)  = 166.3 ± 7.3 nM). The predominantly monomeric insulin analogue Lispro had corresponding dissociation constants K(D1)  = 73.2 ± 1.8 nM and K(D2)  = 148.9 ± 6.1 nM, but the fit to kinetic data was improved when we included a conformational change factor in which the high-affinity site was converted to the low-affinity site. The new SPR assay enables insulin-eIR-A interactions to be followed in real time and could potentially be extended to study the effects of humoral factors on the interaction, without the need for insulin labeling. Copyright © 2013 John Wiley & Sons, Ltd.

  3. Dual signaling regulated by calcyon, a D1 dopamine receptor interacting protein.

    PubMed

    Lezcano, N; Mrzljak, L; Eubanks, S; Levenson, R; Goldman-Rakic, P; Bergson, C

    2000-03-03

    The synergistic response of cells to the stimulation of multiple receptors has been ascribed to receptor cross talk; however, the specific molecules that mediate the resultant signal amplification have not been defined. Here a 24-kilodalton single transmembrane protein, designated calcyon, we functionally characterize that interacts with the D1 dopamine receptor. Calcyon localizes to dendritic spines of D1 receptor-expressing pyramidal cells in prefrontal cortex. These studies delineate a mechanism of Gq- and Gs-coupled heterotrimeric GTP-binding protein-coupled receptor cross talk by which D1 receptors can shift effector coupling to stimulate robust intracellular calcium (Ca2+i) release as a result of interaction with calcyon. The role of calcyon in potentiating Ca2+-dependent signaling should provide insight into the D1 receptor-modulated cognitive functions of prefrontal cortex.

  4. Conformational transitions and interactions underlying the function of membrane embedded receptor protein kinases.

    PubMed

    Bocharov, Eduard V; Sharonov, Georgy V; Bocharova, Olga V; Pavlov, Konstantin V

    2017-01-25

    Among membrane receptors, the single-span receptor protein kinases occupy a broad but specific functional niche determined by distinctive features of the underlying transmembrane signaling mechanisms that are briefly overviewed on the basis of some of the most representative examples, followed by a more detailed discussion of several hierarchical levels of organization and interactions involved. All these levels, including single-molecule interactions (e.g., dimerization, liganding, chemical modifications), local processes (e.g. lipid membrane perturbations, cytoskeletal interactions), and larger scale phenomena (e.g., effects of membrane surface shape or electrochemical potential gradients) appear to be closely integrated to achieve the observed diversity of the receptor functioning. Different species of receptor protein kinases meet their specific functional demands through different structural features defining their responses to stimulation, but certain common patterns exist. Signaling by receptor protein kinases is typically associated with the receptor dimerization and clustering, ligand-induced rearrangements of receptor domains through allosteric conformational transitions with involvement of lipids, release of the sequestered lipids, restriction of receptor diffusion, cytoskeleton and membrane shape remodeling. Understanding of complexity and continuity of the signaling processes can help identifying currently neglected opportunities for influencing the receptor signaling with potential therapeutic implications. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova.

  5. Heterodimeric interaction between retinoid X receptor alpha and orphan nuclear receptor OR1 reveals dimerization-induced activation as a novel mechanism of nuclear receptor activation.

    PubMed Central

    Wiebel, F F; Gustafsson, J A

    1997-01-01

    OR1 is a member of the steroid/thyroid hormone nuclear receptor superfamily which has been described to mediate transcriptional responses to retinoids and oxysterols. On a DR4 response element, an OR1 heterodimer with the nuclear receptor retinoid X receptor alpha (RXR alpha) has been described to convey transcriptional activation in both the absence and presence of the RXR ligand 9-cis retinoic acid, the mechanisms of which have remained unclear. Here, we dissect the effects of RXR alpha and OR1 ligand-binding domain interaction on transcriptional regulation and the role of the respective carboxy-terminal activation domains (AF-2s) in the absence and presence of the RXR ligand, employing chimeras of the nuclear receptors containing the heterologous GAL4 DNA-binding domain as well as natural receptors. The results show that the interaction of the RXR and OR1 ligand-binding domains unleashes a transcription activation potential that is mainly dependent on the AF-2 of OR1, indicating that interaction with RXR activates OR1. This defines dimerization-induced activation as a novel function of heterodimeric interaction and mechanism of receptor activation not previously described for nuclear receptors. Moreover, we present evidence that activation of OR1 occurs by a conformational change induced upon heterodimerization with RXR. PMID:9199332

  6. Muscarinic acetylcholine receptor-interacting proteins (mAChRIPs): targeting the receptorsome.

    PubMed

    Borroto-Escuela, D O; Agnati, Luigi F; Fuxe, Kjell; Ciruela, F

    2012-01-01

    Muscarinic acetylcholine receptors comprise a large family of G protein-coupled receptors that are involved in the regulation of many important functions of the central and peripheral nervous system. To achieve such a large range of physiological effects, these receptors interact with a large array of accessory proteins including scaffold molecules, ion channels and enzymes that operate as molecular transducers of muscarinic function in addition to the canonical heterotrimeric G proteins. Interestingly, as demonstrated for others G protein-coupled receptors, this type of receptor is also able to oligomerise, a fact that has been shown to play a critical role in their subcellular distribution, trafficking, and fine tuning of cholinergic signalling. On the other hand, the specificity of these receptor interactions may be largely determined by the occurrence of precise protein-interacting motifs, posttranslational modifications, and the differential tissue distribution and stoichiometry of the receptor-interacting proteins. Thus, the exhaustive cataloguing and documentation of muscarinic acetylcholine receptor-interacting proteins and the grasp of their specific function will explain key physiological differences in muscarinic-mediated cholinergic transmission. Overall, a better comprehension of the muscarinic receptor interactome will have a significant impact on the cholinergic pharmacology and thus provide previously unrealised opportunities to achieve greater specificity in muscarinic-related drug discovery and diagnostics.

  7. EGF receptor-ligand interaction generates extracellular hydrogen peroxide that inhibits EGFR-associated protein tyrosine phosphatases.

    PubMed

    DeYulia, Garrett J; Cárcamo, Juan M

    2005-08-19

    Hydrogen peroxide (H(2)O(2)) has been shown to be an important modulator of intracellular phosphatase activity involved in cell signaling pathways, including signaling by members of the receptor tyrosine kinase family of receptors such as the epidermal growth factor receptor (EGFR). Intracellular H(2)O(2) can be generated by mitochondria-dependent pathways, whereas we recently showed that H(2)O(2) could be generated extracellularly by receptor-ligand interaction. Here, we show that H(2)O(2) produced by EGF-EGFR interaction can modulate the activity of intracellular protein tyrosine phosphatases (PTPs). Using purified proteins, we found that EGFR-ligand interaction generates H(2)O(2) that is capable of inhibiting the activity of PTP1B in vitro. Furthermore, the addition of catalase rescued phosphatase inhibition consequent to EGF-EGFR interaction. Using cells that overexpress EGFR, we found that the addition of extracellular catalase prevented EGF-induced inhibition of EGFR-associated phosphatase activity. Our findings suggest that extracellular H(2)O(2) generated by EGFR-ligand interaction permeates the plasma membrane and inhibits EGFR-associated tyrosine phosphatase activity, thereby modulating downstream signal transduction pathways.

  8. Gastrin and D1 dopamine receptor interact to induce natriuresis and diuresis.

    PubMed

    Chen, Yue; Asico, Laureano D; Zheng, Shuo; Villar, Van Anthony M; He, Duofen; Zhou, Lin; Zeng, Chunyu; Jose, Pedro A

    2013-11-01

    Oral NaCl produces a greater natriuresis and diuresis than the intravenous infusion of the same amount of NaCl. Gastrin is the major gastrointestinal hormone taken up by renal proximal tubule (RPT) cells. We hypothesized that renal gastrin and dopamine receptors interact to synergistically increase sodium excretion, an impaired interaction of which may be involved in the pathogenesis of hypertension. In Wistar-Kyoto rats, infusion of gastrin induced natriuresis and diuresis, which was abrogated in the presence of a gastrin (cholecystokinin B receptor [CCKBR]; CI-988) or a D1-like receptor antagonist (SCH23390). Similarly, the natriuretic and diuretic effects of fenoldopam, a D1-like receptor agonist, were blocked by SCH23390, as well as by CI-988. However, the natriuretic effects of gastrin and fenoldopam were not observed in spontaneously hypertensive rats. The gastrin/D1-like receptor interaction was also confirmed in RPT cells. In RPT cells from Wistar-Kyoto but not spontaneously hypertensive rats, stimulation of either D1-like receptor or gastrin receptor inhibited Na(+)-K(+)-ATPase activity, an effect that was blocked in the presence of SCH23390 or CI-988. In RPT cells from Wistar-Kyoto and spontaneously hypertensive rats, CCKBR and D1 receptor coimmunoprecipitated, which was increased after stimulation of either D1 receptor or CCKBR in RPT cells from Wistar-Kyoto rats; stimulation of one receptor increased the RPT cell membrane expression of the other receptor, effects that were not observed in spontaneously hypertensive rats. These data suggest that there is a synergism between CCKBR and D1-like receptors to increase sodium excretion. An aberrant interaction between the renal CCK BR and D1-like receptors (eg, D1 receptor) may play a role in the pathogenesis of hypertension.

  9. Cellular interactions uncouple beta-adrenergic receptors from adenylate cyclase.

    PubMed

    Ciment, G; de Vellis, J

    1978-11-17

    C6 glioma cells and B104 neuroblastoma cells both possess adenylate cyclase activity, but only C6 cells have beta-adrenergic receptors. However, when cocultured with B104 cells, C6 cells show a marked decrease in their ability to accumulate adenosine 3', 5'-monophosphate upon stimulation with beta receptor agonists. Since both beta receptors and cholera toxin-stimulated adenylate cyclase activities are present in C6/B104 cocultures, we conclude that the beta receptor/adenylate cyclase transduction mechanism in cocultured C6 cells is uncoupled.

  10. Diversity and Bias through Receptor-Receptor Interactions in GPCR Heteroreceptor Complexes. Focus on Examples from Dopamine D2 Receptor Heteromerization.

    PubMed

    Fuxe, Kjell; Tarakanov, Alexander; Romero Fernandez, Wilber; Ferraro, Luca; Tanganelli, Sergio; Filip, Malgorzata; Agnati, Luigi F; Garriga, Pere; Diaz-Cabiale, Zaida; Borroto-Escuela, Dasiel O

    2014-01-01

    Allosteric receptor-receptor interactions in GPCR heteromers appeared to introduce an intermolecular allosteric mechanism contributing to the diversity and bias in the protomers. Examples of dopamine D2R heteromerization are given to show how such allosteric mechanisms significantly change the receptor protomer repertoire leading to diversity and biased recognition and signaling. In 1980s and 1990s, it was shown that neurotensin (NT) through selective antagonistic NTR-D2 like receptor interactions increased the diversity of DA signaling by reducing D2R-mediated dopamine signaling over D1R-mediated dopamine signaling. Furthermore, D2R protomer appeared to bias the specificity of the NTR orthosteric binding site toward neuromedin N vs. NT in the heteroreceptor complex. Complex CCK2R-D1R-D2R interactions in possible heteroreceptor complexes were also demonstrated further increasing receptor diversity. In D2R-5-HT2AR heteroreceptor complexes, the hallucinogenic 5-HT2AR agonists LSD and DOI were recently found to exert a biased agonist action on the orthosteric site of the 5-HT2AR protomer leading to the development of an active conformational state different from the one produced by 5-HT. Furthermore, as recently demonstrated allosteric A2A-D2R receptor-receptor interaction brought about not only a reduced affinity of the D2R agonist binding site but also a biased modulation of the D2R protomer signaling in A2A-D2R heteroreceptor complexes. A conformational state of the D2R was induced, which moved away from Gi/o signaling and instead favored β-arrestin2-mediated signaling. These examples on allosteric receptor-receptor interactions obtained over several decades serve to illustrate the significant increase in diversity and biased recognition and signaling that develop through such mechanisms.

  11. Structural insights into the interaction between the Cripto CFC domain and the ALK4 receptor.

    PubMed

    Calvanese, Luisa; Saporito, Angela; Oliva, Romina; D' Auria, Gabriella; Pedone, Carlo; Paolillo, Livio; Ruvo, Menotti; Marasco, Daniela; Falcigno, Lucia

    2009-03-01

    The protein Cripto is the founding member of the extra-cellular EGF-CFC growth factors, which are composed of two adjacent cysteine-rich domains: the EGF-like and the CFC. Members of the EGF-CFC family play key roles in embryonic development and are also implicated in tumourigenesis. Cripto is highly over-expressed in many tumours, while it is poorly detectable in normal tissues. Although both Cripto domains are involved in its tumourigenic activity, the CFC domain appears to play a crucial role. Indeed, through this domain, Cripto interferes with the onco-suppressive activity of Activins, either by blocking the Activin receptor ALK4 or by antagonising proteins of the TGF-beta family. We have undertaken the chemical synthesis and the structural characterisation of human CFC Cripto domain. Using a combined NMR and computational approach, supported by binding studies by SPR, we have investigated the molecular basis of the interaction between h-CFC and ALK4. Binding studies indicate that the synthetic h-CFC interacts with the ALK4 receptor with a K(D) in micro M range, whereas it does not recognise the ActRIIB receptor. The NMR study shows that the h-CFC overall topology is determined by the presence of three disulfide bridges and that residues H120 and W124 are located between the first strand and the first loop with the side chains externally exposed. A model of the CFC-ALK4 complex has also been obtained by molecular docking and shows that all residues indicated by prior mutagenesis studies can contribute to the ALK4-CFC interaction at the protein-protein interface.

  12. Tamoxifen Dependent Interaction Between the Estrogen Receptor and a Novel P21 Activated Kinase

    DTIC Science & Technology

    2002-06-01

    AD Award Number: DAMDl7-01-1-0149 TITLE: Tamoxifen Dependent Interaction Between the Estrogen Receptor and a Novel P21 Activated Kinase PRINCIPAL...Tamoxifen Dependent Interaction Between the DAMD17-00-1-0114 Estrogen Receptor and a Novel P21 Activated Kinase 6. AUTHOR(S) Steven P. Balk, M.D., Ph.D. 7...Z, Karas RH, nisms of androgen receptor activation and function. J Mendelsohn ME, Shaul PW 1999 Estrogen receptor a Steroid Biochem Mol Biol 69:307

  13. Epidermal growth factor receptors in the canine antrum

    SciTech Connect

    Zimmerman, R.P.; Gates, T.S.; Boehmer, C.G.; Mantyh, P.W.

    1988-11-01

    In this study we localized receptor binding sites for /sup 125/I-human epidermal growth factor (hEGF) in the antrum of the adult canine stomach. High levels of specific /sup 125/I-hEGF binding sites were observed over the mucosa and muscularis mucosa, whereas specific binding sites were not detectable over the submucosa, external circular and longitudinal muscle or myenteric neurons. These results are in agreement with previous studies which indicated that EGF stimulates the proliferation of cultured epithelial cells and inhibits gastric acid secretion. This suggests that EGF may be a useful therapeutic agent in the healing of gastric ulcers.

  14. Extracellular oxidation by taurine chloramine activates ERK via the epidermal growth factor receptor.

    PubMed

    Midwinter, Robyn G; Peskin, Alexander V; Vissers, Margret C M; Winterbourn, Christine C

    2004-07-30

    Taurine is present in high concentrations in neutrophils, and when the cells are stimulated taurine can react with hypochlorous acid (HOCl) to form taurine-chloramine (Tau-Cl). This compound retains oxidant activity and can affect the neutrophil itself or surrounding tissue cells. We have investigated the effects of Tau-Cl on MAPK signaling in human umbilical vein endothelial cells (HUVEC). Tau-Cl caused no loss in intracellular glutathione or inactivation of the thiol-sensitive enzyme glyceraldehyde-3-phosphate dehydrogenase, indicating that it had not entered the cells. However, stimulation of HUVEC with Tau-Cl (20-100 microM) induced the rapid activation of ERK within 10 min. This activation was abolished by inhibition of MEK by U0126, indicating that it was not because of direct oxidation of ERK. No activation of p38 was detected. These results suggest that Tau-Cl reacts with a cell membrane target that results in intracellular ERK activation. Tau-Cl over the same concentration range and time scale stimulated epidermal growth factor (EGF) receptor tyrosine phosphorylation in A431 cells and HUVEC. The EGF receptor inhibitor PD158780 significantly attenuated Tau-Cl-induced phosphorylation of both the EGF receptor and ERK. This implicates the EGF receptor in the upstream activation of ERK. The Src tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolol[3,4-d]pyrimidine had no effect on Tau-Cl-induced EGF receptor or ERK activation. We propose that Tau-Cl acts on an oxidant-sensitive target on the cell surface, this being either the EGF receptor itself or another target that can interact with the EGF receptor, with consequential activation of ERK.

  15. Epidermal growth factor and its receptors in human pancreatic carcinoma

    SciTech Connect

    Chen, Y.F.; Pan, G.Z.; Hou, X.; Liu, T.H.; Chen, J.; Yanaihara, C.; Yanaihara, N. )

    1990-05-01

    The role of epidermal growth factor (EGF) in oncogenesis and progression of malignant tumors is a subject of vast interest. In this study, radioimmunoassay and radioreceptor assay of EGF were established. EGF contents in malignant and benign pancreatic tumors, in normal pancreas tissue, and in culture media of a human pancreatic carcinoma cell line were determined. EGF receptor binding studies were performed. It was shown that EGF contents in pancreatic carcinomas were significantly higher than those in normal pancreas or benign pancreatic tumors. EGF was also detected in the culture medium of a pancreatic carcinoma cell line. The binding of 125I-EGF to the pancreatic carcinoma cells was time and temperature dependent, reversible, competitive, and specific. Scatchard analysis showed that the dissociation constant of EGF receptor was 2.1 X 10(-9) M, number of binding sites was 1.3 X 10(5) cell. These results indicate that there is an over-expression of EGF/EGF receptors in pancreatic carcinomas, and that an autocrine regulatory mechanism may exist in the growth-promoting effect of EGF on tumor cells.

  16. Gephyrin Interacts With The Glutamate Receptor Interacting Protein 1 Isoforms At GABAergic Synapses

    PubMed Central

    Yu, Wendou; Charych, Erik I.; Serwanski, David R.; Li, Rong-wen; Ali, Rashid; Bahr, Ben A.; De Blas, Angel L.

    2009-01-01

    We have previously shown that the glutamate receptor interacting protein 1 (GRIP1) splice forms GRIP1a/b and GRIP1c4-7 are present at the GABAergic postsynaptic complex. Nevertheless the role that these GRIP1 protein isoforms play at the GABAergic postsynaptic complex is not known. We are now showing that GRIP1c4-7 and GRIP1a/b interact with gephyrin, the main postsynaptic scaffold protein of GABAergic and glycinergic synapses. Gephyrin coprecipitates with GRIP1c4-7 or GRIP1a/b from rat brain extracts and from extracts of HEK293 cells that have been cotransfected with gephyrin and one of the GRIP1 protein isoforms. Moreover, purified gephyrin binds to purified GRIP1c4-7 or GRIP1a/b, indicating that gephyrin directly interacts with the common region of these GRIP1 proteins, which includes PDZ domains 4–7. An engineered deletion construct of GRIP1a/b (GRIP1a4–7), which both contains the aforementioned common region and binds to gephyrin, targets to the postsynaptic GABAergic complex of transfected cultured hippocampal neurons. In these hippocampal cultures, endogenous gephyrin colocalizes with endogenous GRIP1c4-7 and GRIP1a/b in over 90% of the GABAergic synapses. Double-labeling electron microscopy immunogold reveals that in the rat brain GRIP1c4-7 and GRIP1a/b colocalize with gephyrin at the postsynaptic complex of individual synapses. These results indicate that GRIP1c4-7 and GRIP1a/b colocalize and interact with gephyrin at the GABAergic postsynaptic complex and suggest that this interaction plays a role in GABAergic synaptic function. PMID:18315564

  17. Nicotine enhancement and reinforcer devaluation: Interaction with opioid receptors.

    PubMed

    Kirshenbaum, Ari P; Suhaka, Jesse A; Phillips, Jessie L; Voltolini de Souza Pinto, Maiary

    In rats, nicotine enhances responding maintained by non-pharmacological reinforcers, and discontinuation of nicotine devalues those same reinforcers. The goal of this study was to assess the interaction of nicotine and opioid receptors and to evaluate the degree to which nicotine enhancement and nicotine-induced devaluation are related to opioid activation. Nicotine (0.4mg/kg), or nicotine plus naloxone (0.3 or 3.0mg/kg), was delivered to rats prior to progressive ratio (PR) schedule sessions in which sucrose was used as a reinforcer. PR-schedule responding was assessed during ten daily sessions of drug delivery, and for three post-dosing days/sessions. Control groups for this investigation included a saline-only condition, and naloxone-only (0.3 or 3.0mg/kg) conditions. When administered in conjunction with nicotine, both naloxone doses attenuated nicotine enhancement of the sucrose reinforcer, and the combination of the larger dose of naloxone (3.0mg/kg) with nicotine produced significant impairments in sucrose reinforced responding. When administered alone, neither dose of naloxone (0.3 & 3.0mg/kg) significantly altered responding in comparison to saline. Furthermore, when dosing was discontinued after ten once-daily doses, all nicotine groups (nicotine-only and nicotine+naloxone combination) demonstrated significant decreases in sucrose reinforcement compared to the saline group. Although opioid antagonism attenuated reinforcement enhancement by nicotine, it did not prevent reinforcer devaluation upon discontinuation of nicotine dosing, and the higher dose of naloxone (3.0mg/kg) produced decrements upon discontinuation on its own in the absence of nicotine. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Nerve growth factor regulates the expression of bradykinin binding sites on adult sensory neurons via the neurotrophin receptor p75.

    PubMed

    Petersen, M; Segond von Banchet, G; Heppelmann, B; Koltzenburg, M

    1998-03-01

    Neurotrophins mediate specific effects on sensory neurons through tyrosine kinase receptors. Most of these neurons also co-express the neurotrophin receptor p75 (p75NTR), but its function has remained obscure. We now show that nerve growth factor but not brain-derived neurotrophic factor or neurotrophin-3 selectively increases the expression of bradykinin binding sites on cultured dorsal root ganglion neurons from adult mouse via p75NTR. This up-regulation of bradykinin binding sites did not occur in neurons from mice lacking p75NTR or in neurons from wild-type mice treated with p75NTR-blocking antibody, indicating that tyrosine kinase receptors alone are not sufficient to trigger this physiological neuronal response. Thus, the interaction of nerve growth factor with p75NTR is an important factor contributing to chronic pain conditions.

  19. Generation of monoclonal antibody targeting fibroblast growth factor receptor 3.

    PubMed

    Gorbenko, Olena; Ovcharenko, Galyna; Klymenko, Tetyana; Zhyvoloup, Olexandr; Gaman, Nadia; Volkova, Darija; Gout, Ivan; Filonenko, Valeriy

    2009-08-01

    Fibroblast growth factor receptor 3 (FGFR3) is a member of the FGFR family of receptor tyrosine kinases, whose function has been implicated in diverse biological processes, including cell proliferation, differentiation, survival, and tumorigenesis. Deregulation of FGFR3 signaling has been implicated with human pathologies, including cancer. Activating mutations in FGFR3 gene are frequently detected in bladder cancer, multiple myeloma, and noninvasive papillary urothelial cell carcinomas, while the overexpression of the receptor is observed in thyroid lymphoma and bladder cancer. The main aim of this study was to generate hybridoma clones producing antibody that could specifically recognize FGFR3/S249C mutant, but not the wild-type FGFR. To achieve this, we used for immunization bacterially expressed fragment of FGFR3 corresponding to loops II-III of the extracellular domain (GST-His/FGFR3/S249C-LII-III), which possesses oncogenic mutation at Ser249 detected in at least 50% of bladder cancers. Primary ELISA screening allowed us to isolate several hybridoma clones that showed specificity towards FGFR3/S249C, but not FGFR3wt protein. Unfortunately, these clones were not stable during single-cell cloning and expansion and lost the ability to recognize specifically FGFR3/S249C. However, this study allowed us to generate several monoclonal antibodies specific towards both FGFR3wt and FGFR3/S249C recombinant proteins. Produced hybridomas secreted MAbs that were specific in Western blotting towards bacterially expressed FGFR3wt and FGFR3/S249C, as well as the full-length receptors ectopically expressed in Sf21 and HEK293 cells. Moreover, transiently expressed wild-type and oncogenic forms of FGFR were efficiently immunoprecipitated with selected antibodies from the lysates of infected Sf21 and transiently transfected HEK293. In summary, generated antibodies should be useful as tools for examining the expression pattern and biological functions of FGFR3 in normal and

  20. PDF Receptor Expression Reveals Direct Interactions between Circadian Oscillators in Drosophila

    PubMed Central

    Im, Seol Hee; Taghert, Paul H.

    2010-01-01

    Daily rhythms of behavior are controlled by a circuit of circadian pacemaking neurons. In Drosophila, 150 pacemakers participate in this network, and recent observations suggest the network is divisible into M and E oscillators which normally interact and synchronize. Sixteen oscillator neurons (the small and large LNvs) express a neuropeptide called pigment dispersing factor (PDF) whose signaling is often equated with M oscillator output. Given the significance of PDF signaling to numerous aspects of behavioral and molecular rhythms, determining precisely where and how signaling via the PDF receptor (PDFR) occurs is now a central question in the field. Here we show that GAL4-mediated rescue of pdfr phenotypes using a UAS-PDFR transgene is insufficient to provide complete behavioral rescue. In contrast, we describe a ~70 kB PDF receptor (pdfr) transgene which does rescue the entire pdfr circadian behavioral phenotype. The transgene is widely but heterogeneously expressed among pacemakers, and also among a limited number of non-pacemakers. Our results support an important hypothesis: the small LNv cells directly target a subset of the other crucial pacemaker neurons cells. Furthermore, expression of the transgene confirms an autocrine feedback signaling by PDF back to PDF-expressing cells. Finally, the results present an unexpected PDF receptor site: the large LNv cells appear to target a population of non-neuronal cells that resides at the base of the eye. PMID:20394051

  1. PDF receptor expression reveals direct interactions between circadian oscillators in Drosophila.

    PubMed

    Im, Seol Hee; Taghert, Paul H

    2010-06-01

    Daily rhythms of behavior are controlled by a circuit of circadian pacemaking neurons. In Drosophila, 150 pacemakers participate in this network, and recent observations suggest that the network is divisible into M and E oscillators, which normally interact and synchronize. Sixteen oscillator neurons (the small and large lateral neurons [LNvs]) express a neuropeptide called pigment-dispersing factor (PDF) whose signaling is often equated with M oscillator output. Given the significance of PDF signaling to numerous aspects of behavioral and molecular rhythms, determining precisely where and how signaling via the PDF receptor (PDFR) occurs is now a central question in the field. Here we show that GAL4-mediated rescue of pdfr phenotypes using a UAS-PDFR transgene is insufficient to provide complete behavioral rescue. In contrast, we describe a approximately 70-kB PDF receptor (pdfr) transgene that does rescue the entire pdfr circadian behavioral phenotype. The transgene is widely but heterogeneously expressed among pacemakers, and also among a limited number of non-pacemakers. Our results support an important hypothesis: the small LNv cells directly target a subset of the other crucial pacemaker neurons cells. Furthermore, expression of the transgene confirms an autocrine feedback signaling by PDF back to PDF-expressing cells. Finally, the results present an unexpected PDF receptor site: the large LNv cells appear to target a population of non-neuronal cells that resides at the base of the eye. (c) 2009 Wiley-Liss, Inc.

  2. Receptor-transporter interactions of canonical ATP-binding cassette import systems in prokaryotes.

    PubMed

    Schneider, Erwin; Eckey, Viola; Weidlich, Daniela; Wiesemann, Nicole; Vahedi-Faridi, Ardeshir; Thaben, Paul; Saenger, Wolfram

    2012-04-01

    ATP-binding cassette (ABC) transport systems mediate the translocation of solutes across biological membranes at the expense of ATP. They share a common modular architecture comprising two pore-forming transmembrane domains and two nucleotide binding domains. In prokaryotes, ABC transporters are involved in the uptake of a large variety of chemicals, including nutrients, osmoprotectants and signal molecules. In pathogenic bacteria, some ABC importers are virulence factors. Canonical ABC import systems require an additional component, a substrate-specific receptor or binding protein for function. Interaction of the liganded receptor with extracytoplasmic loop regions of the transmembrane domains initiate the transport cycle. In this review we summarize the current knowledge on receptor-transporter interplay provided by crystal structures as well as by biochemical and biophysical means. In particular, we focus on the maltose/maltodextrin transporter of enterobacteria and the transporters for positively charged amino acids from the thermophile Geobacillus stearothermophilus and Salmonella enterica serovar Typhimurium. Copyright © 2011 Elsevier GmbH. All rights reserved.

  3. Distinct ErbB2 receptor populations differentially interact with beta1 integrin in breast cancer cell models

    PubMed Central

    Toscani, Andrés Martín; Sampayo, Rocío G.; Barabas, Federico Martín; Fuentes, Federico; Simian, Marina

    2017-01-01

    ErbB2 is a member of the ErbB family of tyrosine kinase receptors that plays a major role in breast cancer progression. Located at the plasma membrane, ErbB2 forms large clusters in spite of the presence of growth factors. Beta1 integrin, membrane receptor of extracellular matrix proteins, regulates adhesion, migration and invasiveness of breast cancer cells. Physical interaction between beta1 integrin and ErbB2 has been suggested although published data are contradictory. The aim of the present work was to study the interaction between ErbB2 and beta1 integrin in different scenarios of expression and activation. We determined that beta1 integrin and ErbB2 colocalization is dependent on the expression level of both receptors exclusively in adherent cells. In suspension cells, lack of focal adhesions leave integrins free to diffuse on the plasma membrane and interact with ErbB2 even at low expression levels of both receptors. In adherent cells, high expression of beta1 integrin leaves unbound receptors outside focal complexes that diffuse within the plasma membrane and interact with ErbB2 membrane domains. Superresolution imaging showed the existence of two distinct populations of ErbB2: a major population located in large clusters and a minor population outside these structures. Upon ErbB2 overexpression, receptors outside large clusters can freely diffuse at the membrane and interact with integrins. These results reveal how expression levels of beta1 integrin and ErbB2 determine their frequency of colocalization and show that extracellular matrix proteins shape membrane clusters distribution, regulating ErbB2 and beta1 integrin activity in breast cancer cells. PMID:28306722

  4. Distinct ErbB2 receptor populations differentially interact with beta1 integrin in breast cancer cell models.

    PubMed

    Toscani, Andrés Martín; Sampayo, Rocío G; Barabas, Federico Martín; Fuentes, Federico; Simian, Marina; Coluccio Leskow, Federico

    2017-01-01

    ErbB2 is a member of the ErbB family of tyrosine kinase receptors that plays a major role in breast cancer progression. Located at the plasma membrane, ErbB2 forms large clusters in spite of the presence of growth factors. Beta1 integrin, membrane receptor of extracellular matrix proteins, regulates adhesion, migration and invasiveness of breast cancer cells. Physical interaction between beta1 integrin and ErbB2 has been suggested although published data are contradictory. The aim of the present work was to study the interaction between ErbB2 and beta1 integrin in different scenarios of expression and activation. We determined that beta1 integrin and ErbB2 colocalization is dependent on the expression level of both receptors exclusively in adherent cells. In suspension cells, lack of focal adhesions leave integrins free to diffuse on the plasma membrane and interact with ErbB2 even at low expression levels of both receptors. In adherent cells, high expression of beta1 integrin leaves unbound receptors outside focal complexes that diffuse within the plasma membrane and interact with ErbB2 membrane domains. Superresolution imaging showed the existence of two distinct populations of ErbB2: a major population located in large clusters and a minor population outside these structures. Upon ErbB2 overexpression, receptors outside large clusters can freely diffuse at the membrane and interact with integrins. These results reveal how expression levels of beta1 integrin and ErbB2 determine their frequency of colocalization and show that extracellular matrix proteins shape membrane clusters distribution, regulating ErbB2 and beta1 integrin activity in breast cancer cells.

  5. Coregulation of Epidermal Growth Factor Receptor/Human Epidermal Growth Factor Receptor 2 (HER2) Levels and Locations: Quantitative Analysis of HER2 Overexpression Effects

    SciTech Connect

    Hendriks, Bart S.; Opresko, Lee; Wiley, H. S.; Lauffenburger, Douglas A.

    2003-03-01

    Elevated expression of human epidermal growth factor receptor 2 (HER2) is know to alter cell signalilng and behavioral responses implicated in tumor progression. However, multiple diverse mechanisms may be involved in these overall effects, including signaling by HER2 itself, modulation of signalilng by epidermal growth factor receptor (EGFR) and modification of trafficking dynamics for both EGFR and HER2. Continued....

  6. Cell Receptor-Basement Membrane Interactions in Health and Disease: a Kidney-Centric View

    PubMed Central

    Borza, Corina M.; Chen, Xiwu; Zent, Roy; Pozzi, Ambra

    2016-01-01

    Cell-extracellular matrix (ECM) interactions are essential for tissue development, homeostasis, and response to injury. Basement membranes (BMs) are specialized ECMs that separate epithelial or endothelial cells from stromal components and interact with cells via cellular receptors, including integrins and discoidin domain receptors. Disruption of cell-BM interactions due to either injury or genetic defects in either the ECM components or cellular receptors often lead to irreversible tissue injury and loss of organ function. Animal models that lack specific BM components or receptors either globally or in selective tissues have been used to help with our understanding of the molecular mechanisms whereby cell-BM interactions regulate organ function in physiological and pathological conditions. We review recently published work on animal models that explore how cell-BM interactions regulate kidney homeostasis in both health and disease. PMID:26610916

  7. The Interactions of Proinsulin with Insulin Receptors on the Plasma Membrane of the Liver

    PubMed Central

    Freychet, Pierre

    1974-01-01

    The interactions of proinsulin with the insulin-specific receptors were investigated in purified rat liver plasma membranes. These studies were designed to characterize the binding of proinsulin to the insulin receptors, to search for proinsulin-specific receptor sites, and to examine the possibility of proinsulin conversion at the insulin receptor site. Proinsulin was only 3-5% as potent as insulin in binding to insulin receptors. Proinsulin reacted with all of the insulin-specific receptors, and direct binding studies of [125I]porcine proinsulin and [125I]rat proinsulin did not reveal proinsulin-specific receptor sites other than the insulin receptors in rat liver membranes. Quantitative data derived from steady-state and transient-state comparative binding studies of both [125I]proinsulin and [125I]insulin indicated that a 20-fold lower association rate constant essentially accounts for the reduced affinity of proinsulin for the insulin receptors. The possibility of proinsulin conversion at the insulin receptor sites was investigated. Material recovered from the membranes upon dissociation of the proinsulin-receptor complex was intact proinsulin and did not exhibit any conversion by a variety of analytical methods. These results indicate that the lower affinity of proinsulin for the insulin receptor in the liver is an intrinsic property of the proinsulin molecule. The lower uptake of proinsulin by the insulin receptor represents, in addition to a slower degradation of the prohormone, a further mechanism by which proinsulin exerts prolonged, albeit reduced, action in vivo. PMID:4421396

  8. Hydrodynamic effects and receptor interactions of platelets and their aggregates in linear shear flow.

    PubMed Central

    Tandon, P; Diamond, S L

    1997-01-01

    We have modeled platelet aggregation in a linear shear flow by accounting for two body collision hydrodynamics, platelet activation and receptor biology. Considering platelets and their aggregates as unequal-sized spheres with DLVO interactions (psi(platelet) = -15 mV, Hamaker constant = 10(-19) J), detailed hydrodynamics provided the flow field around the colliding platelets. Trajectory calculations were performed to obtain the far upstream cross-sectional area and the particle flux through this area provided the collision frequency. Only a fraction of platelets brought together by a shearing fluid flow were held together if successfully bound by fibrinogen cross-bridging GPIIb/IIIa receptors on the platelet surfaces. This fraction was calculated by modeling receptor-mediated aggregation using the formalism of Bell (Bell, G. I. 1979. A theoretical model for adhesion between cells mediated by multivalent ligands. Cell Biophys. 1:133-147) where the forward rate of bond formation dictated aggregation during collision and was estimated from the diffusional limited rate of lateral association of receptors multiplied by an effectiveness factor, eta, to give an apparent rate. For a value of eta = 0.0178, we calculated the overall efficiency (including both receptor binding and hydrodynamics effects) for equal-sized platelets with 50,000 receptors/platelet to be 0.206 for G = 41.9 s(-1), 0.05 for G = 335 s(-1), and 0.0086 for G = 1920 s(-1), values which are in agreement with efficiencies determined from initial platelet singlet consumption rates in flow through a tube. From our analysis, we predict that bond formation proceeds at a rate of approximately 0.1925 bonds/microm2 per ms, which is approximately 50-fold slower than the diffusion limited rate of association. This value of eta is also consistent with a colloidal stability of unactivated platelets at low shear rates. Fibrinogen was calculated to mediate aggregation quite efficiently at low shear rates but not at

  9. Nicotine binding to brain receptors requires a strong cation-pi interaction.

    PubMed

    Xiu, Xinan; Puskar, Nyssa L; Shanata, Jai A P; Lester, Henry A; Dougherty, Dennis A

    2009-03-26

    Nicotine addiction begins with high-affinity binding of nicotine to acetylcholine (ACh) receptors in the brain. The end result is over 4,000,000 smoking-related deaths annually worldwide and the largest source of preventable mortality in developed countries. Stress reduction, pleasure, improved cognition and other central nervous system effects are strongly associated with smoking. However, if nicotine activated ACh receptors found in muscle as potently as it does brain ACh receptors, smoking would cause intolerable and perhaps fatal muscle contractions. Despite extensive pharmacological, functional and structural studies of ACh receptors, the basis for the differential action of nicotine on brain compared with muscle ACh receptors has not been determined. Here we show that at the alpha4beta2 brain receptors thought to underlie nicotine addiction, the high affinity for nicotine is the result of a strong cation-pi interaction to a specific aromatic amino acid of the receptor, TrpB. In contrast, the low affinity for nicotine at the muscle-type ACh receptor is largely due to the fact that this key interaction is absent, even though the immediate binding site residues, including the key amino acid TrpB, are identical in the brain and muscle receptors. At the same time a hydrogen bond from nicotine to the backbone carbonyl of TrpB is enhanced in the neuronal receptor relative to the muscle type. A point mutation near TrpB that differentiates alpha4beta2 and muscle-type receptors seems to influence the shape of the binding site, allowing nicotine to interact more strongly with TrpB in the neuronal receptor. ACh receptors are established therapeutic targets for Alzheimer's disease, schizophrenia, Parkinson's disease, smoking cessation, pain, attention-deficit hyperactivity disorder, epilepsy, autism and depression. Along with solving a chemical mystery in nicotine addiction, our results provide guidance for efforts to develop drugs that target specific types of nicotinic

  10. Surface Plasmon Resonance Study of Cooperative Interactions of Estrogen Receptor α and Specificity Protein 1 with Composite DNA Elements.

    PubMed

    Su, Xiaodi; Song, Hong Yan

    2016-01-01

    Estrogen receptor α (ERα) and Specificity protein 1 (Sp1) are transcription factors (TF) that are involved in regulating progesterone receptor (PR) gene expression through cooperative interactions with DNA. The natural composite DNA +571 ERE/Sp1 site in promoter A of the progesterone receptor contains a half-site of estrogen response elements (½ERE) upstream of two Sp1 binding sites (the proximal Sp1 (Sp1/P) and distal Sp1 (Sp1/D)) with a 4 bp spacer. Here, we have developed a protocol for studying the cooperative interaction of Sp1 and ERα with the composite DNA of +571 ERE/Sp1 site using Biacore T200, a high sensitivity surface plasmon resonance spectroscopy. With this protocol, we have concluded that Sp1 binding enhances the overall ERα binding to the composite DNA. We have also determined the optimal spacer distance between the ½ERE and Sp1/D for the best cooperative protein binding. This study is pivotal in guiding the bioinformatics simulation to yield an exact model of the spacer dependency of the transcription factor/cofactor-DNA interactions, which is important for understanding the nuclear receptor regulating activity through other coactivators.

  11. Signal transduction by the formyl peptide receptor. Studies using chimeric receptors and site-directed mutagenesis define a novel domain for interaction with G-proteins.

    PubMed

    Amatruda, T T; Dragas-Graonic, S; Holmes, R; Perez, H D

    1995-11-24

    The binding of small peptide ligands to high affinity chemoattractant receptors on the surface of neutrophils and monocytes leads to activation of heterotrimeric G-proteins, stimulation of phosphatidylinositol-phospholipase C (PI-PLC), and subsequently to the inflammatory response. It was recently shown (Amatruda, T. T., Gerard, N. P., Gerard, C., and Simon, M. I. (1993) J. Biol. Chem. 268, 10139-10144) that the receptor for the chemoattractant peptide C5a specifically interacts with G alpha 16, a G-protein alpha subunit of the Gq class, to trigger ligand-dependent stimulation of PI-PLC in transfected cells. In order to further characterize this chemoattractant peptide signal transduction pathway, we transfected cDNAs encoding the formylmethionylleucylphenylalanine receptor (fMLPR) into COS cells and measured the production of inositol phosphates. Ligand-dependent activation of PI-PLC was seen in COS cells transfected with the fMLPR and G alpha 16 and stimulated with fMLP but not in cells transfected with receptor alone or with receptor plus G alpha q. Chimeric receptors in which the N-terminal extracellular domain, the second intracellular domain, or the intracellular C-terminal tail of the fMLP receptor was replaced with C5a receptor domains (Perez, H. D., Holmes, R., Vilander, L. R., Adams, R. R., Manzana, W., Jolley, D., and Andrews, W. H. (1993) J. Biol. Chem. 268, 2292-2295) were capable of ligand-dependent activation of PI-PLC when co-transfected with G alpha 16. A chimeric receptor exchanging the first intracellular domain of the fMLPR was constitutively activated, stimulating PI-PLC in the absence of ligand. Constitutive activation of PI-PLC, to a level 233% of that seen in cells transfected with wild-type fMLP receptors, was dependent on G alpha 16. Site-directed mutagenesis of the first intracellular domain of the fMLPR (amino acids 54-62) reveals this to be a domain necessary for ligand-dependent activation of G alpha 16. These results suggest that

  12. Estrogen receptors colocalize with low-affinity nerve growth factor receptors in cholinergic neurons of the basal forebrain.

    PubMed Central

    Toran-Allerand, C D; Miranda, R C; Bentham, W D; Sohrabji, F; Brown, T J; Hochberg, R B; MacLusky, N J

    1992-01-01

    The rodent and primate basal forebrain is a target of a family of endogenous peptide signaling molecules, the neurotrophins--nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 3--and of the gonadal steroid hormone estrogen, both of which have been implicated in cholinergic function. To investigate whether or not these ligands may act on the same neurons in the developing and adult rodent basal forebrain, we combined autoradiography with 125I-labeled estrogen and either nonisotopic in situ hybridization histochemistry or immunohistochemistry. We now report colocalization of intranuclear estrogen binding sites with the mRNA and immunoreactive protein for the low-affinity nerve growth factor receptor, which binds all three neurotrophins, and for the cholinergic marker enzyme choline acetyltransferase (acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6). Colocalization of estrogen and low-affinity nerve growth factor receptors implies that their ligands may act on the same neuron, perhaps synergistically, to regulate the expression of specific genes or gene networks that may influence neuronal survival, differentiation, regeneration, and plasticity. That cholinergic neurons in brain regions subserving cognitive functions may be regulated not only by the neurotrophins but also by estrogen may have considerable relevance for the development and maintenance of neural substrates of cognition. If estrogen-neurotrophin interactions are important for survival of target neurons, then clinical conditions associated with estrogen deficiency could contribute to the atrophy or death of these neurons. These findings have implications for the subsequent decline in those differentiated neural functions associated with aging and Alzheimer disease. Images PMID:1316615

  13. Mutation of proline-1003 to glycine in the epidermal growth factor (EGF) receptor enhances responsiveness to EGF.

    PubMed Central

    Schuh, S M; Newberry, E P; Dalton, M A; Pike, L J

    1994-01-01

    We have shown previously that the epidermal growth factor (EGF) receptor is phosphorylated at Ser-1002 and that this phosphorylation is associated with desensitization of the EGF receptor. Ser-1002 is followed immediately by Pro-1003, a residue that may promote the adoption of a specific conformation at this site or severe as a recognition element for the interaction of the EGF receptor with other proteins. To examine these possibilities, we have mutated Pro-1003 of the EGF receptor to a Gly residue and have analyzed the effect of this mutation on EGF-stimulated signaling. Cells expressing the P1003G EGF receptors exhibited higher EGF-stimulated autophosphorylation and synthetic peptide phosphorylation compared to cells expressing wild-type EGF receptors. In addition, the ability of EGF to stimulate PI 3-kinase activity and mitogen-activated protein kinase activity was enhanced in cells expressing the P1003G EGF receptor. Cells expressing P1003G receptors also demonstrated an increased ability to form colonies in soft agar in response to EGF. These results indicate that mutation of Pro-1003 leads to a potentiation of the biological effects of EGF. The findings are consistent with the hypothesis that Pro-1003 plays a role in a form of regulation that normally suppresses EGF receptor function. Images PMID:7812043

  14. Identification of important residues of insulin-like peptide 5 and its receptor RXFP4 for ligand-receptor interactions.

    PubMed

    Wang, Xin-Yi; Guo, Yu-Qi; Shao, Xiao-Xia; Liu, Ya-Li; Xu, Zeng-Guang; Guo, Zhan-Yun

    2014-09-15

    Insulin-like peptide 5 (INSL5) is an insulin/relaxin superfamily peptide involved in the regulation of glucose homeostasis by activating its receptor RXFP4, which can also be activated by relaxin-3 in vitro. To determine the interaction mechanism of INSL5 with its receptor RXFP4, we studied their electrostatic interactions using a charge-exchange mutagenesis approach. First, we identified three negatively charged extracellular residues (Glu100, Asp104 and Glu182) in human RXFP4 that were important for receptor activation by wild-type INSL5. Second, we demonstrated that two positively charged B-chain Arg residues (B13Arg and B23Arg) in human INSL5 were involved in receptor binding and activation. Third, we proposed probable electrostatic interactions between INSL5 and RXFP4: the B-chain central B13Arg of INSL5 interacts with both Asp104 and Glu182 of RXFP4, meanwhile the B-chain C-terminal B23Arg of INSL5 interacts with both Glu100 and Asp104 of RXFP4. The present electrostatic interactions between INSL5 and RXFP4 were similar to our previously identified interactions between relaxin-3 and RXFP4, but had subtle differences that might be caused by the different B-chain C-terminal conformations of relaxin-3 and INSL5 because a dipeptide exchange at the B-chain C-terminus significantly decreased the activity of INSL5 and relaxin-3 to receptor RXFP4. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. Proteinase-activated receptor-2 transactivation of epidermal growth factor receptor and transforming growth factorreceptor signaling pathways contributes to renal fibrosis.

    PubMed

    Chung, Hyunjae; Ramachandran, Rithwik; Hollenberg, Morley D; Muruve, Daniel A

    2013-12-27

    Chronic kidney diseases cause significant morbidity and mortality in the population. During renal injury, kidney-localized proteinases can signal by cleaving and activating proteinase-activated receptor-2 (PAR2), a G-protein-coupled receptor involved in inflammation and fibrosis that is highly expressed in renal tubular cells. Following unilateral ureteric obstruction, PAR2-deficient mice displayed reduced renal tubular injury, fibrosis, collagen synthesis, connective tissue growth factor (CTGF), and α-smooth muscle actin gene expression at 7 days, compared with wild-type controls. In human proximal tubular epithelial cells in vitro, PAR2 stimulation with PAR2-activating peptide (PAR2-AP) alone significantly up-regulated the expression of CTGF, a potent profibrotic cytokine. The induction of CTGF by PAR2-AP was synergistically increased when combined with transforming growth factor-β (TGF-β). Consistent with these findings, treating human proximal tubular epithelial cells with PAR2-AP induced Smad2/3 phosphorylation in the canonical TGF-β signaling pathway. The Smad2 phosphorylation and CTGF induction required signaling via both the TGFβ-receptor and EGF receptor suggesting that PAR2 utilizes transactivation mechanisms to initiate fibrogenic signaling. Taken together, our data support the hypothesis that PAR2 synergizes with the TGFβ signaling pathway to contribute to renal injury and fibrosis.

  16. Galanin receptor 2-neuropeptide Y Y1 receptor interactions in the dentate gyrus are related with antidepressant-like effects.

    PubMed

    Narváez, Manuel; Borroto-Escuela, Dasiel O; Millón, Carmelo; Gago, Belén; Flores-Burgess, Antonio; Santín, Luis; Fuxe, Kjell; Narváez, José Angel; Díaz-Cabiale, Zaida

    2016-11-01

    Galanin (GAL) and the NPYY1 agonist play a role in mood regulation and both neuropeptides interact in several central functions. The present study examined the interaction between Galanin receptor 2 (GALR2) and Neuropeptide Y Y1 receptor (NPYY1R) in the dentate gyrus (DG) of the Hippocampus in relation to depression-like behavior. Using receptor autoradiography, in situ hybridization and in situ proximity ligation assay an interaction between GALR and NPYY1R was demonstrated in the DG probably involving the formation of GALR2-NPYY1R heteroreceptor complexes. These complexes were specifically observed in the polymorphic and subgranular subregions of the DG, where both receptors were found to colocalize. Moreover, this GALR2/NPYY1R interaction was linked to an enhancement of the antidepressive-like behavior mediated by NPYY1R in the forced swimming test. Specific cells populations within DG subregions may be involved in this behavioral effect since the coactivation of GALR2 and NPYY1R enhances the NPYY1R-mediated reduction in the number of c-Fos immunoreactive nuclei in the polymorphic region. These results indicate that GALR2/NPYY1R interactions can provide a novel integrative mechanism in DG in depression-related behavior and may give the basis for the development of drugs targeting GALR2/NPYY1R heteroreceptor complexes in the DG of the hippocampus for the treatment of depression.

  17. Vascular endothelial growth factor receptor 3 directly regulates murine neurogenesis

    PubMed Central

    Calvo, Charles-Félix; Fontaine, Romain H.; Soueid, Jihane; Tammela, Tuomas; Makinen, Taija; Alfaro-Cervello, Clara; Bonnaud, Fabien; Miguez, Andres; Benhaim, Lucile; Xu, Yunling; Barallobre, Maria-José; Moutkine, Imane; Lyytikkä, Johannes; Tatlisumak, Turgut; Pytowski, Bronislaw; Zalc, Bernard; Richardson, William; Kessaris, Nicoletta; Garcia-Verdugo, Jose Manuel; Alitalo, Kari; Eichmann, Anne; Thomas, Jean-Léon

    2011-01-01

    Neural stem cells (NSCs) are slowly dividing astrocytes that are intimately associated with capillary endothelial cells in the subventricular zone (SVZ) of the brain. Functionally, members of the vascular endothelial growth factor (VEGF) family can stimulate neurogenesis as well as angiogenesis, but it has been unclear whether they act directly via VEGF receptors (VEGFRs) expressed by neural cells, or indirectly via the release of growth factors from angiogenic capillaries. Here, we show that VEGFR-3, a receptor required for lymphangiogenesis, is expressed by NSCs and is directly required for neurogenesis. Vegfr3:YFP reporter mice show VEGFR-3 expression in multipotent NSCs, which are capable of self-renewal and are activated by the VEGFR-3 ligand VEGF-C in vitro. Overexpression of VEGF-C stimulates VEGFR-3-expressing NSCs and neurogenesis in the SVZ without affecting angiogenesis. Conversely, conditional deletion of Vegfr3 in neural cells, inducible deletion in subventricular astrocytes, and blocking of VEGFR-3 signaling with antibodies reduce SVZ neurogenesis. Therefore, VEGF-C/VEGFR-3 signaling acts directly on NSCs and regulates adult neurogenesis, opening potential approaches for treatment of neurodegenerative diseases. PMID:21498572

  18. Role of fibroblast growth factor receptor signaling in kidney development.

    PubMed

    Bates, Carlton M

    2011-08-01

    Fibroblast growth factor receptors (Fgfrs) consist of four signaling family members and one nonsignaling "decoy" receptor, Fgfr-like 1 (Fgfrl1), all of which are expressed in the developing kidney. Several studies have shown that exogenous fibroblast growth factors (Fgfs) affect growth and maturation of the metanephric mesenchyme (MM) and ureteric bud (UB) in cultured tissues. Transgenic and conditional knockout approaches in whole animals have shown that Fgfr1 and Fgfr2 (predominantly the IIIc isoform) in kidney mesenchyme are critical for early MM and UB formation. Conditional deletion of the ligand, Fgf8, in nephron precursors or global deletion of Fgfrl1 interrupts nephron formation. Fgfr2 (likely the IIIb isoform signaling downstream of Fgf7 and Fgf10) is critical for ureteric morphogenesis. Moreover, Fgfr2 appears to act independently of Frs2α (the major signaling adapter for Fgfrs) in regulating UB branching. Loss of Fgfr2 in the MM leads to many kidney and urinary tract anomalies, including vesicoureteral reflux. Thus Fgfr signaling is critical for patterning of virtually all renal lineages at early and later stages of development.

  19. Polychlorinated Biphenyls Disrupt Hepatic Epidermal Growth Factor Receptor Signaling.

    PubMed

    Hardesty, Josiah E; Wahlang, Banrida; Falkner, K Cameron; Clair, Heather B; Clark, Barbara J; Ceresa, Brian P; Prough, Russell A; Cave, Matthew C

    2016-07-26

    1. Polychlorinated biphenyls (PCBs) are persistent environmental pollutants that disrupt hepatic xenobiotic and intermediary metabolism, leading to metabolic syndrome and nonalcoholic steatohepatitis (NASH). 2. Since phenobarbital indirectly activates Constitutive Androstane Receptor (CAR) by antagonizing growth factor binding to the epidermal growth factor receptor (EGFR), we hypothesised that PCBs may also diminish EGFR signaling. 3. The effects of the PCB mixture Aroclor 1260 on the protein phosphorylation cascade triggered by EGFR activation were determined in murine (in vitro and in vivo) and human models (in vitro). EGFR tyrosine residue phosphorylation was decreased by PCBs in all models tested. 4. The IC50 values for Aroclor 1260 concentrations that decreased Y1173 phosphorylation of EGFR were similar in murine AML-12 and human HepG2 cells (∼2-4 μg/mL). Both dioxin and non-dioxin-like PCB congeners decreased EGFR phosphorylation in cell culture. 5. PCB treatment reduced phosphorylation of downstream EGFR effectors including Akt and mTOR, as well as other phosphoprotein targets including STAT3 and c-RAF in vivo. 6. PCBs diminish EGFR signaling in human and murine hepatocyte models and may dysregulate critical phosphoprotein regulators of energy metabolism and nutrition, providing a new mechanism of action in environmental diseases.

  20. Pregnane X receptor, constitutive androstane receptor and hepatocyte nuclear factors as emerging players in cancer precision medicine.

    PubMed

    De Mattia, Elena; Cecchin, Erika; Roncato, Rossana; Toffoli, Giuseppe

    2016-09-01

    Great research effort has been focused on elucidating the contribution of host genetic variability on pharmacological outcomes in cancer. Nuclear receptors have emerged as mediators between environmental stimuli and drug pharmacokinetics and pharmacodynamics. The pregnane X receptor, constitutive androstane receptor and hepatocyte nuclear factors have been reported to regulate transcription of genes that encode drug metabolizing enzymes and transporters. Altered nuclear receptor expression has been shown to affect the metabolism and pharmacological profile of traditional chemotherapeutics and targeted agents. Accordingly, polymorphic variants in these genes have been studied as pharmacogenetic markers of outcome variability. This review summarizes the state of knowledge about the roles played by pregnane X receptor, constitutive androstane receptor and hepatocyte nuclear factor expression and genetics as predictive markers of anticancer drug toxicity and efficacy, which can improve cancer precision medicine.

  1. Interactions between recording technique and AMPA receptor modulators.

    PubMed

    Lin, Bin; Colgin, Laura Lee; Brücher, Fernando Andres; Arai, Amy Christine; Lynch, Gary

    2002-11-15

    Whole cell recording (EPSCs) and extracellular recording (field EPSPs) were compared in hippocampal field CA1 with regard to the effects of experimental treatments that increase AMPA receptor gated currents. Cyclothiazide, which maintains AMPA receptors in the sensitized state, caused a rapid and pronounced increase in EPSCs but only minor changes in field EPSPs. This difference was evident in recordings carried out at 22 and 32 degrees C and with different solutions in the clamp pipette. The larger effect of cyclothiazide on EPSCs was unaffected by blockade of GABA and NMDA receptors. Two-dimensional current source density analyses derived from 64 recording sites were used to provide extracellular estimates of AMPA receptor mediated synaptic currents. With this method, cyclothiazide again had much smaller effects than were obtained with whole cell clamp. Differences between whole cell and extracellular recordings were present, although not as pronounced, for the ampakines, a class of drugs that slow both deactivation and desensitization of AMPA receptors. Additionally, increases in synaptic responses produced by frequency facilitation, a manipulation that enhances the number of bound receptors, were not qualitatively different between recording techniques. These results support the conclusion that the whole cell clamp technique may alter AMPA receptors in such a way as to increase the relative importance of desensitization.

  2. Structure and function of the type 1 insulin-like growth factor receptor.

    PubMed

    Adams, T E; Epa, V C; Garrett, T P; Ward, C W

    2000-07-01

    The type 1 insulin-like growth factor receptor (IGF-1R), a transmembrane tyrosine kinase, is widely expressed across many cell types in foetal and postnatal tissues. Activation of the receptor following binding of the secreted growth factor ligands IGF-1 and IGF-2 elicits a repertoire of cellular responses including proliferation, and the protection of cells from programmed cell death or apoptosis. As a result, signalling through the IGF-1R is the principal pathway responsible for somatic growth in foetal mammals, whereas somatic growth in postnatal animals is achieved through the synergistic interaction of growth hormone and the IGFs. Forced overexpression of the IGF-1R results in the malignant transformation of cultured cells: conversely, downregulation of IGF-1R levels can reverse the transformed phenotype of tumour cells, and may render them sensitive to apoptosis in vivo. Elevated levels of IGF-IR are observed in a variety of human tumour types, whereas epidemiological studies implicate the IGF-1 axis as a predisposing factor in the pathogenesis of human breast and prostate cancer. The IGF-1R has thus emerged as a therapeutic target for the development of antitumour agents. Recent progress towards the elucidation of the three-dimensional structure of the extracellular domain of the IGF-1R represents an opportunity for the rational assembly of small molecule antagonists of receptor function for clinical use.

  3. Chemokine and chemokine receptor structure and interactions: implications for therapeutic strategies

    PubMed Central

    Kufareva, Irina; Salanga, Catherina L.; Handel, Tracy M.

    2015-01-01

    The control of cell migration by chemokines involves interactions with two types of receptors: seven transmembrane chemokine-type G protein-coupled receptors and cell surface or extracellular matrix associated glycosaminoglycans. Coordinated interaction of chemokines with both types of receptors is required for directional migration of cells in numerous physiological and pathological processes. Accumulated structural information, culminating most recently in the structure of a chemokine receptor in complex with a chemokine, has led to a view where chemokine oligomers bind to glycosaminoglycans through epitopes formed when chemokine subunits come together, while chemokine monomers bind to receptors in a pseudo two-step mechanism of receptor activation. Exploitation of this structural knowledge has and will continue to provide important information for therapeutic strategies, as described in this review. PMID:25708536

  4. Can Specific Protein-Lipid Interactions Stabilize an Active State of the Beta 2 Adrenergic Receptor?

    PubMed Central

    Neale, Chris; Herce, Henry D.; Pomès, Régis; García, Angel E.

    2015-01-01

    G-protein-coupled receptors are eukaryotic membrane proteins with broad biological and pharmacological relevance. Like all membrane-embedded proteins, their location and orientation are influenced by lipids, which can also impact protein function via specific interactions. Extensive simulations totaling 0.25 ms reveal a process in which phospholipids from the membrane’s cytosolic leaflet enter the empty G-protein binding site of an activated β2 adrenergic receptor and form salt-bridge interactions that inhibit ionic lock formation and prolong active-state residency. Simulations of the receptor embedded in an anionic membrane show increased lipid binding, providing a molecular mechanism for the experimental observation that anionic lipids can enhance receptor activity. Conservation of the arginine component of the ionic lock among Rhodopsin-like G-protein-coupled receptors suggests that intracellular lipid ingression between receptor helices H6 and H7 may be a general mechanism for active-state stabilization. PMID:26488656

  5. The cytokines cardiotrophin-like cytokine/cytokine-like factor-1 (CLC/CLF) and ciliary neurotrophic factor (CNTF) differ in their receptor specificities.

    PubMed

    Tormo, Aurélie Jeanne; Letellier, Marie-Claude; Lissilaa, Rami; Batraville, Laurie-Anne; Sharma, Mukut; Ferlin, Walter; Elson, Greg; Crabé, Sandrine; Gauchat, Jean-François

    2012-12-01

    Ciliary neurotrophic factor (CNTF) and cardiotrophin-like cytokine (CLC) are two cytokines with neurotrophic and immunomodulatory activities. CNTF is a cytoplasmic factor believed to be released upon cellular damage, while CLC requires interaction with a soluble cytokine receptor, cytokine-like factor 1 (CLF), to be efficiently secreted. Both cytokines activate a receptor complex comprising the cytokine binding CNTF receptor α (CNTFRα) and two signaling chains namely, leukemia inhibitory factor receptor β (LIFRβ) and gp130. Human CNTF can recruit and activate an alternative receptor in which CNTFRα is substituted by IL-6Rα. As both CNTF and CLC have immune-regulatory activities in mice, we compared their ability to recruit mouse receptors comprising both gp130 and LIFRβ signaling chains and either IL-6Rα or IL-11Rα which, unlike CNTFRα, are expressed by immune cells. Our results indicate that 1) mouse CNTF, like its human homologue, can activate cells expressing gp130/LIFRβ with either CNTFRα or IL-6Rα and, 2) CLC/CLF is more restricted in its specificity in that it activates only the tripartite CNTFR. Several gp130 signaling cytokines influence T helper cell differentiation. We therefore investigated the effect of CNTF on CD4 T cell cytokine production. We observed that CNTF increased the number of IFN-γ producing CD4 T cells. As IFN-γ is considered a mediator of the therapeutic effect of IFN-β in multiple sclerosis, induction of IFN-γ by CNTF may contribute to the beneficial immunomodulatory effect of CNTF in mouse multiple sclerosis models. Together, our results indicate that CNTF activates the same tripartite receptors in mouse and human cells and further validate rodent models for pre-clinical investigation of CNTF and CNTF derivatives. Furthermore, CNTF and CLC/CLF differ in their receptor specificities. The receptor α chain involved in the immunomodulatory effects of CLC/CLF remains to be identified.