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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 , Chlorocebus aethiops , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Transporte Proteico , Receptores Citoplasmáticos e Nucleares/genética
2.
J Biol Chem ; 282(28): 20676-85, 2007 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-17491025

RESUMO

Mutant membrane proteins are frequently retained in the early secretory pathway by a quality control system, thereby causing disease. An example are mutants of the vasopressin V(2) receptor (V(2)R) leading to nephrogenic diabetes insipidus. Transport-defective V(2)Rs fall into two classes: those retained exclusively in the endoplasmic reticulum (ER) and those reaching post-ER compartments such as the ER/Golgi intermediate compartment. Although numerous chemical or pharmacological chaperones that rescue the transport of ER-retained membrane proteins are known, substances acting specifically in post-ER compartments have not been described as yet. Using the L62P (ER-retained) and Y205C (reaching post-ER compartments) mutants of the V(2)R as a model, we show here that the cell-penetrating peptide penetratin and its synthetic analog KLAL rescue the transport of the Y205C mutant. In contrast, the location of the L62P mutant is not influenced by either peptide because the peptides are unable to enter the ER. We also show data indicating that the peptide-mediated transport rescue is associated with an increase in cytosolic Ca(2+) concentrations. Thus, we describe a new class of substances influencing protein transport specifically in post-ER compartments.


Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Proteínas de Transporte/farmacologia , Diabetes Insípido Nefrogênico/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Receptores de Vasopressinas/metabolismo , Substituição de Aminoácidos , Cálcio/metabolismo , Sinalização do Cálcio/genética , Proteínas de Transporte/uso terapêutico , Linhagem Celular , Peptídeos Penetradores de Células , Diabetes Insípido Nefrogênico/tratamento farmacológico , Diabetes Insípido Nefrogênico/genética , Diabetes Insípido Nefrogênico/patologia , Retículo Endoplasmático/genética , Complexo de Golgi/genética , Humanos , Chaperonas Moleculares/metabolismo , Mutação de Sentido Incorreto , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Receptores de Vasopressinas/genética
3.
FEBS Lett ; 579(23): 5227-35, 2005 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-16162341

RESUMO

It is believed that the membrane-proximal C tail of the G protein-coupled receptors forms an additional alpha helix with amphipathic properties (helix 8). It was previously shown for the vasopressin V2 receptor (V2R) that a conserved dileucine motif (L(339), L(340)) in this putative helix 8 is necessary for endoplasmic reticulum (ER) to Golgi transfer of the receptor. Here, we demonstrate that the other hydrophobic residues forming the non-polar side of this helix (F(328), V(332) and L(336)) are also transport-relevant. In contrast, the multiple serine residues contributing to the more hydrophilic side (S(330), S(331), S(333), S(334), S(338)) do not influence receptor trafficking. In addition, we show unambiguously by the use of pharmacological chaperones that the hydrophobic residues of the putative helix 8 do not form a transport signal necessary for receptor sorting into ER to Golgi vesicles. Instead, they are necessary to establish a transport-competent folding state in the early secretory pathway.


Assuntos
Dobramento de Proteína , Estrutura Secundária de Proteína , Receptores de Vasopressinas/química , Receptores de Vasopressinas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Arginina Vasopressina/metabolismo , Transporte Biológico/fisiologia , Linhagem Celular , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Sinais Direcionadores de Proteínas , Receptores de Vasopressinas/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética
4.
Biochem J ; 390(Pt 2): 455-64, 2005 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-15901239

RESUMO

Approximately 5-10% of the GPCRs (G-protein-coupled receptors) contain N-terminal signal peptides that are cleaved off during receptor insertion into the ER (endoplasmic reticulum) membrane by the signal peptidases of the ER. The reason as to why only a subset of GPCRs requires these additional signal peptides is not known. We have recently shown that the signal peptide of the human ET(B)-R (endothelin B receptor) does not influence receptor expression but is necessary for the translocation of the receptor's N-tail across the ER membrane and thus for the establishment of a functional receptor [Köchl, Alken, Rutz, Krause, Oksche, Rosenthal and Schülein (2002) J. Biol. Chem. 277, 16131-16138]. In the present study, we show that the signal peptide of the rat CRF-R1 (corticotropin-releasing factor receptor 1) has a different function: a mutant of the CRF-R1 lacking the signal peptide was functional and displayed wild-type properties with respect to ligand binding and activation of adenylate cyclase. However, immunoblot analysis and confocal laser scanning microscopy revealed that the mutant receptor was expressed at 10-fold lower levels than the wild-type receptor. Northern-blot and in vitro transcription translation analyses precluded the possibility that the reduced receptor expression is due to decreased transcription or translation levels. Thus the signal peptide of the CRF-R1 promotes an early step of receptor biogenesis, such as targeting of the nascent chain to the ER membrane and/or the gating of the protein-conducting translocon of the ER membrane.


Assuntos
Sinais Direcionadores de Proteínas/fisiologia , Receptores de Hormônio Liberador da Corticotropina/genética , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Humanos , Dados de Sequência Molecular , Mutação , Transporte Proteico , Ratos , Receptores de Hormônio Liberador da Corticotropina/química , Regulação para Cima
5.
Traffic ; 5(12): 993-1005, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15522100

RESUMO

The G protein-coupled V(2) vasopressin receptor is crucially involved in water reabsorption in the renal collecting duct. Mutations in the human V(2) vasopressin receptor gene cause nephrogenic diabetes insipidus. Many of the disease-causing mutants are retained intracellularly by the quality control system of the early secretory pathway. It was previously thought that quality control system is restricted to the endoplasmic reticulum (ER). Here, we have examined the retention mechanisms of eight V(2) vasopressin receptor mutants. We show that mutants L62P, DeltaL62-R64 and S167L are trapped exclusively in the ER. In contrast, mutants R143P, Y205C, InsQ292, V226E and R337X reach the ER/Golgi intermediate compartment (ERGIC) and are rerouted to the ER. The ability of the mutant receptors to reach the ERGIC is independent of their expression levels. Instead, it is determined by their folding state. Mutant receptors in the ERGIC may be sorted into retrograde transport vesicles by an interaction of an RXR motif in the third intracellular loop with the coatomer complex I. Our data show that disease-causing mutants of a particular membrane protein may be retained in different compartments of the early secretory pathway and that the folding states of the proteins determine their retention mechanism.


Assuntos
Receptores de Vasopressinas/metabolismo , Sequência de Aminoácidos , Vesículas Citoplasmáticas/metabolismo , Retículo Endoplasmático/metabolismo , Genes Reporter , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Dobramento de Proteína , Estrutura Secundária de Proteína , Transporte Proteico/fisiologia , Receptores de Vasopressinas/genética
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