RESUMO
The regulation of cell function by fibroblast growth factors (FGFs) classically occurs through a dual receptor system of a tyrosine kinase receptor (FGFR) and a heparan sulfate proteoglycan co-receptor. Mutations in some consensus N-glycosylation sites in human FGFR result in skeletal disorders and craniosynostosis syndromes, and biophysical studies in vitro suggest that N-glycosylation of FGFR alters ligand and heparan sulfate binding properties. The evolutionarily conserved FGFR signaling system of Caenorhabditis elegans has been used to assess the role of N-glycosylation in the regulation of FGFR signaling in vivo. The C. elegans FGF receptor, EGL-15, is N-glycosylated in vivo, and genetic substitution of specific consensus N-glycosylation sites leads to defects in the maintenance of fluid homeostasis and differentiation of sex muscles, both of which are phenotypes previously associated with hyperactive EGL-15 signaling. These phenotypes are suppressed by hypoactive mutations in EGL-15 downstream signaling components or activating mutations in the phosphatidylinositol 3-kinase pathway, respectively. The results show that N-glycans negatively regulate FGFR activity in vivo supporting the notion that mutation of N-glycosylation sites in human FGFR may lead to inappropriate activation of the receptor.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Sítios de Ligação/genética , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Diferenciação Celular , Feminino , Glicosilação , Humanos , Masculino , Microscopia de Fluorescência , Dados de Sequência Molecular , Músculo Liso/citologia , Músculo Liso/metabolismo , Mutagênese Sítio-Dirigida , Mutação , Mioblastos/citologia , Mioblastos/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/genética , Homologia de Sequência de AminoácidosRESUMO
G-protein modulation of neuronal nicotinic acetylcholine receptor (nAChR) channels in rat intrinsic cardiac ganglia was examined using dialyzed whole-cell and excised membrane patch-recording configurations. Cell dialysis with GTPgammaS increased the agonist affinity of nAChRs, resulting in a potentiation of nicotine-evoked whole-cell currents at low concentrations. ACh- and nicotine-evoked current amplitudes were increased approximately twofold in the presence of GTPgammaS. In inside-out membrane patches, the open probability (NP(o)) of nAChR-mediated unitary currents was reversibly increased fourfold after bath application of 0.2 mm GTPgammaS relative to control but was unchanged in the presence of GDPbetaS. The modulation of nAChR-mediated whole-cell currents was agonist specific; currents evoked by the cholinergic agonists ACh, nicotine, and 1,1-dimethyl-4-phenylpiperazinium iodide, but not cytisine or choline, were potentiated in the presence of GTPgammaS. The direct interaction between G-protein subunits and nAChRs was examined by bath application of either G(o)alpha or Gbetagamma subunits to inside-out membrane patches and in glutathione S-transferase pull-down and coimmunoprecipitation experiments. Bath application of 50 nm Gbetagamma increased the open probability of ACh-activated single-channel currents fivefold, whereas G(o)alpha (50 nm) produced no significant increase in NP(o). Neuronal nAChR subunits alpha3-alpha5 and beta2 exhibited a positive interaction with G(o)alpha and Gbetagamma, whereas beta4 and alpha7 failed to interact with either of the G-protein subunits. These results provide evidence for a direct interaction between nAChR and G-protein subunits, underlying the increased open probability of ACh-activated single-channel currents and potentiation of nAChR-mediated whole-cell currents in parasympathetic neurons of rat intrinsic cardiac ganglia.
Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Neurônios/fisiologia , Receptores Nicotínicos/fisiologia , Animais , Animais Recém-Nascidos , Western Blotting/métodos , Células Cultivadas , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Estimulação Elétrica/métodos , Gânglios Parassimpáticos/citologia , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacologia , Guanosina Trifosfato/farmacologia , Imunoprecipitação/métodos , Ativação do Canal Iônico/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Potenciais da Membrana/efeitos da radiação , Biologia Molecular/métodos , Neurônios/efeitos dos fármacos , Neurônios/efeitos da radiação , Nicotina/farmacologia , Técnicas de Patch-Clamp/métodos , Probabilidade , Ratos , Tionucleotídeos/farmacologia , Fatores de TempoRESUMO
Nuclear transport proteins such as CSE1, NUP93 and Importin-alpha have recently been shown to be chromatin-associated proteins in yeast, which have unexpected functions in gene regulation. Here we report interactions between the mammalian histone acetyltransferase CBP with nuclear transport proteins CAS (a CSE1 homologue) and Importin-alpha (Impalpha) and NUP93. CAS was found to bind the SRC1 interaction domain (SID) of CBP via a leucine-rich motif in the N-terminus of the protein, that is conserved in other SID-binding proteins. Coimmunoprecipitation experiments also revealed that CBP and Impalpha proteins form a complex. As Impalpha is a known acetylation target of CBP/p300, and is recycled to the cytoplasm via the exportin CAS, we investigated whether HDAC inhibitors would alter the subcellular localization of these proteins. Treatment of COS-1 cells with the HDAC inhibitors trichostatin A or sodium butyrate resulted in sequestration of Impalpha in the nuclear envelope, accumulation of CAS in nuclear aggregates, and an increased number of CBP-containing PML bodies per cell. In addition, HDACi treatment appeared to enhance the association of Impalpha and CBP in coimmunoprecipitation experiments. Our results provide evidence for novel functional interactions between the chromatin modification enzyme CBP and nuclear transport proteins in mammalian cells.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Proteína de Ligação a CREB/metabolismo , Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , Histona Desacetilases/metabolismo , Proteínas Nucleares/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Motivos de Aminoácidos/efeitos dos fármacos , Motivos de Aminoácidos/genética , Animais , Células COS , Proteína de Ligação a CREB/genética , Proteínas de Transporte/genética , Núcleo Celular/efeitos dos fármacos , Chlorocebus aethiops , Proteína Substrato Associada a Crk/genética , Proteína Substrato Associada a Crk/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores de Histona Desacetilases , Histona Desacetilases/genética , Humanos , Substâncias Macromoleculares/metabolismo , Membrana Nuclear/efeitos dos fármacos , Membrana Nuclear/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/genética , Estrutura Terciária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/genética , alfa Carioferinas/genética , alfa Carioferinas/metabolismoRESUMO
CREB-binding protein (CBP) and p300 contain modular domains that mediate protein-protein interactions with a wide variety of nuclear factors. A C-terminal domain of CBP (referred to as the SID) is responsible for interaction with the alpha-helical AD1 domain of p160 coactivators such as the steroid receptor coactivator (SRC1), and also other transcriptional regulators such as E1A, Ets-2, IRF3, and p53. Here we show that the pointed (PNT) domain of Ets-2 mediates its interaction with the CBP SID, and describe the effects of mutations in the SID on binding of Ets-2, E1A, and SRC1. In vitro binding studies indicate that SRC1, Ets-2 and E1A display mutually exclusive binding to the CBP SID. Consistent with this, we observed negative cross-talk between ERalpha/SRC1, Ets-2, and E1A proteins in reporter assays in transiently transfected cells. Transcriptional inhibition of Ets-2 or GAL4-AD1 activity by E1A was rescued by co-transfection with a CBP expression plasmid, consistent with the hypothesis that the observed inhibition was due to competition for CBP in vivo. Sequence comparisons revealed that SID-binding proteins contain a leucine-rich motif similar to the alpha-helix Aalpha1 of the SRC1 AD1 domain. Deletion mutants of E1A and Ets-2 lacking the conserved motif were unable to bind the CBP SID. Moreover, a peptide corresponding to this sequence competed the binding of full-length SRC1, Ets-2, and E1A proteins to the CBP SID. Thus, a leucine-rich amphipathic alpha-helix mediates mutually exclusive interactions of functionally diverse nuclear proteins with CBP.