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1.
Traffic ; 15(4): 383-400, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24405750

RESUMO

The molecular mechanisms regulating G protein-coupled receptors (GPCRs) trafficking from their site of synthesis in the endoplasmic reticulum (ER) to their site of function (the cell surface) remain poorly characterized. Using a bioluminescence resonance energy transfer-based proteomic screen, we identified a novel GPCR-interacting protein; the human cornichon homologue 4 (CNIH4). This previously uncharacterized protein is localized in the early secretory pathway where it interacts with members of the 3 family of GPCRs. Both overexpression and knockdown expression of CNIH4 caused the intracellular retention of GPCRs, indicating that this ER-resident protein plays an important role in GPCR export. Overexpression of CNIH4 at low levels rescued the maturation and cell surface expression of an intracellularly retained mutant form of the ß2-adrenergic receptor, further demonstrating a positive role of CNIH4 in GPCR trafficking. Taken with the co-immunoprecipitation of CNIH4 with Sec23 and Sec24, components of the COPII coat complex responsible for ER export, these data suggest that CNIH4 acts as a cargo-sorting receptor, recruiting GPCRs into COPII vesicles.


Assuntos
Retículo Endoplasmático/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Animais , Células COS , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Transporte Proteico , Receptores Citoplasmáticos e Nucleares/genética
2.
Mol Pharmacol ; 77(5): 836-45, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20159941

RESUMO

Substitution of arginine-137 of the vasopressin type 2 receptor (V2R) for histidine (R137H-V2R) leads to nephrogenic diabetes insipidus (NDI), whereas substitution of the same residue to cysteine or leucine (R137C/L-V2R) causes the nephrogenic syndrome of inappropriate antidiuresis (NSIAD). These two diseases have opposite clinical outcomes. Still, the three mutant receptors were shown to share constitutive beta-arrestin recruitment and endocytosis, resistance to vasopressin-stimulated cAMP production and mitogen-activated protein kinase activation, and compromised cell surface targeting, raising questions about the contribution of these phenomenons to the diseases and their potential treatments. Blocking endocytosis exacerbated the elevated basal cAMP levels promoted by R137C/L-V2R but not the cAMP production elicited by R137H-V2R, demonstrating that substitution of Arg137 to Cys/Leu, but not His, leads to constitutive V2R-stimulated cAMP accumulation that most likely underlies NSIAD. The constitutively elevated endocytosis of R137C/L-V2R attenuates the signaling and most likely reduces the severity of NSIAD, whereas the elevated endocytosis of R137H-V2R probably contributes to NDI. The constitutive signaling of R137C/L-V2R was not inhibited by treatment with the V2R inverse agonist satavaptan (SR121463). In contrast, owing to its pharmacological chaperone property, SR121463 increased the R137C/L-V2R maturation and cell surface targeting, leading to a further increase in basal cAMP production, thus disqualifying it as a potential treatment for patients with R137C/L-V2R-induced NSIAD. However, vasopressin was found to promote beta-arrestin/AP-2-dependent internalization of R137H/C/L-V2R beyond their already elevated endocytosis levels, raising the possibility that vasopressin could have a therapeutic value for patients with R137C/L-V2R-induced NSIAD by reducing steady-state surface receptor levels, thus lowering basal cAMP production.


Assuntos
Diabetes Insípido Nefrogênico/genética , Síndrome de Secreção Inadequada de HAD/genética , Receptores de Vasopressinas/genética , Substituição de Aminoácidos , Arginina/genética , Arginina Vasopressina/farmacologia , Arrestinas/genética , Linhagem Celular , AMP Cíclico/metabolismo , Histidina/genética , Humanos , Rim , Microscopia de Fluorescência , Mutagênese , Mutação , Plasmídeos , Transfecção , beta-Arrestinas
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