Disease‐causing V₂ Vasopressin Receptors are Retained in Different Compartments of the Early Secretory Pathway

Abstract

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

Rescue of a Nephrogenic Diabetes Insipidus-causing Vasopressin V2 Receptor Mutant by Cell-penetrating Peptides

“…In contrast, the Y205C mutant accumulated in a perinuclear region. A substantial colocalization with the ERGIC marker and a weaker colocalization with the Golgi marker were observed, again in agreement with previous results (14).…”

“…18 h after transfection, the cell culture medium was supplemented with bafilomycin A 1 (1 M) or vehicle and incubated for another 4 h. The immunofluorescence procedure using anti-ERGIC-53 or anti-58-kDa Golgi protein antibody was carried out as described (14).…”

“…Plasmid Constructions-Plasmids encoding the C-terminally GFP-tagged V 2 R mutants L62P and Y205C (14) and wild-type V 2 R (25) have been described. The corresponding cyan fluorescent protein (CFP) constructs of the wild-type receptor, L62P, and Y205C were generated by cloning the receptor moieties of the wild-type V 2 R-GFP, L62P-GFP, and Y205C-GFP plasmids on a BamHI/SacI fragment into the pECFP-N1 vector.…”

“…It has also been demonstrated that the QCS is not restricted to the ER but involves post-ER compartments such as the ER/Golgi intermediate compartment (ERGIC) (12,13). For the V 2 R, we have shown recently that transport-defective mutant receptors fall into two classes: mutants like L62P are retained exclusively in the ER, whereas others like Y205C escape to the ERGIC (14). In the ERGIC, they are recognized by an as yet incompletely understood mechanism.…”

Cystic Fibrosis Transmembrane Conductance Regulator Trafficking Is Mediated by the COPI Coat in Epithelial Cells

“…The potassium channel KCNK3 harbors a di-basic motif that is recognized by the COPI subunit ␤-COP (32). Similarly, upon misfolding of the G protein-coupled V2 vasopressin receptor, the ␤-COP subunit of COPI interacts with the RXR motif present in the cytosolic loop 3 of the receptor, resulting in the retention of the protein in the ER (33). Thus, the association of these proteins with COPI vesicles prevents the delivery of misfolded proteins to the PM.…”