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Properties of the human arginine vasopressin V2 receptor after site-directed mutagenesis of its putative palmitoylation site

Item Type:Article
Title:Properties of the human arginine vasopressin V2 receptor after site-directed mutagenesis of its putative palmitoylation site
Creators Name:Schuelein, R. and Liebenhoff, U. and Mueller, H. and Birnbaumer, M. and Rosenthal, W.
Abstract:Most G-protein-coupled receptors have conserved cysteine residues in their C-terminal cytoplasmic domain that appear to be generally palmitoylated. An example is the human arginine vasopressin V2 receptor with cysteine residues at positions 341 and 342. Site-directed mutagenesis of the putative palmitoylation site was used to study the significance of palmitoylation for the V2 receptor. A multifunctional expression plasmid was constructed by cloning the V2 receptor cDNA into the vector pCDNAI.Neo. The resulting plasmid allowed site-directed mutagenesis experiments without subcloning, and stable and transient expression of the V2 receptor in Ltk- and COS.M6 cells respectively. The conserved cysteine residues Cys-341 and Cys-342 were placed by serine residues, yielding the single mutants C-341S and C-342S and the double mutant C-341S/C-342S. Functional expression in stably transfected Ltk- cells showed that the affinity of the three mutant receptors for arginine vasopressin was not altered. In contrast with the activation of adenylate cyclase through beta 2 adrenergic receptors, arginine vasopressin stimulated adenylate cyclase to the same extent and with similar EC50 values in both wild-type and mutant receptors. Transient expression of the C-341S/C-342S mutant receptor in COS.M6 cells confirmed an unaltered affinity of the mutant receptor for arginine vasopressin. However, the number of arginine vasopressin-binding sites on the cell surface was reduced by 30%, suggesting that the transport of the mutant receptor to the cell surface was impaired. In addition, the decrease in detectable arginine vasopressin-binding sites on the cell surface following pre-exposure to hormone was reduced, indicating that the sequestration/internalization of the mutant receptor on the cell surface was affected. The present data indicate that palmitoylation of the V2 receptor is important for intracellular trafficking and/or sequestration/internalization but not for agonist binding or activation of the Gs/adenylate cyclase system.
Keywords:Adenylate Cyclase, Arginine Vasopressin, Base Sequence, Cell Line, Complementary DNA, GTP-Binding Proteins, Molecular Sequence Data, Site-Directed Mutagenesis, Palmitic Acid, Palmitic Acids, Protein Binding, Vasopressin Receptors, Animals
Source:Biochemical Journal
Publisher:Portland Press
Page Range:611-616
Date:15 January 1996
Official Publication:http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=8573100
PubMed:View item in PubMed

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