Appropriate polarization following pharmacological rescue of V2 vasopressin receptors encoded by X-linked nephrogenic diabetes insipidus alleles involves a conformation of the receptor that also attains mature glycosylation.
To understand the mechanisms of G protein-coupled receptor delivery and steady state localization, we examined the trafficking itineraries of wild type (WT) and mutant V2 vasopressin receptors (V2Rs) in polarized Madin-Darby canine kidney II (MDCK II) cells and in COS M6 cells; the mutant V2Rs represent selected alleles responsible for X-linked nephrogenic diabetes insipidus. The WT V2R is localized on the plasma membrane and mediates arginine vasopressin (AVP)-stimulated cAMP accumulation, whereas the clinically relevant V2R mutants, L292P V2R, Delta V278 V2R, and R337X V2R, are retained intracellularly, are insensitive to extracellularly added AVP, and are not processed beyond initial immature glycosylation, manifest by their endoglycosidase H sensitivity. Reduced temperature and pharmacological, but not chemical, strategies rescue mutant V2Rs to the cell surface of COS M6 cells; surface rescue of L292P V2R and R337X V2R, but not of Delta V278 V2R, parallels acquisition of AVP-stimulated cAMP production. Pharmacological rescue of the L292P or R337X V2R by incubation with the membrane-permeant V2R antagonist, SR121463B, leads to a mature glycosylated form of the receptor that achieves localization on the basolateral surface of polarized MDCK II cells indistinguishable from that of the WT V2R. Surprisingly, however, the immature form of the mutant L292P V2R escapes to the apical, but not basolateral, surface of polarized MDCK II cells, even in the absence of SR121463B. These findings are consistent with the interpretation that the receptor conformation that allows appropriate processing through the N-linked glycosylation pathway is also essential for V2R targeting to the appropriate surface of polarized epithelial cells.
Tan
Christopher M
CM
Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-6600, USA.
Nickols
Hilary Highfield
HH
Limbird
Lee E
LE
D019943:Amino Acid Substitution; D000818:Animals; D001127:Arginine Vasopressin / Q000494:pharmacology; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D003919:Diabetes Insipidus / Q000235:genetics*; D004285:Dogs; D006031:Glycosylation; D006801:Humans; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017483:Receptors, Vasopressin / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection; D014960:X Chromosome
D017483:Receptors, Vasopressin; D011994:Recombinant Proteins; D001127:Arginine Vasopressin