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, DL62-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. The neurohypophysial hormone 8-arginine vasopressin (AVP, or antidiuretic hormone) regulates antidiuresis via the G protein-coupled V 2 vasopressin receptor (V 2 R) (1). The receptor is expressed in the basolateral membrane of principal cells, the main cell type of the renal collecting duct. Upon AVP binding, the receptor activates the G s / adenylyl cyclase system. The subsequent rise in intracellular cAMP levels leads to the activation of the protein kinase A and to the fusion of aquaporin 2 (AQP2)-bearing vesicles with the apical and basolateral membrane by an as yet incompletely understood mechanism (AQP2 shuttle) (2). The redistribution of AQP2 in the plasma membrane increases the water permeability of the epithelium and thereby greatly facilitates water reabsorption.Mutations in the V 2 R gene cause X-linked nephrogenic diabetes insipidus (NDI), a disease characterized by the kidney's inability to respond to AVP (3); the consequence is a profound diuresis. To date, more than 150 distinct NDI-causing mutations in the V 2 R gene have been described (3, see also
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.
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