Properties of the human arginine vasopressin V2 receptor after site-directed mutagenesis of its putative palmitoylation site.
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.
Schülein
R
R
Rudolf-Buchheim-Institut für Pharmackologie, Justus-Liebig-Universität Giessen, Federal Republic of Germany.
Liebenhoff
U
U
Müller
H
H
Birnbaumer
M
M
Rosenthal
W
W
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001127:Arginine Vasopressin / Q000378:metabolism; D001483:Base Sequence; D002460:Cell Line; D018076:DNA, Complementary; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism*; D011485:Protein Binding; D017483:Receptors, Vasopressin / Q000819:agonists / Q000235:genetics / Q000378:metabolism*
D018076:DNA, Complementary; D010169:Palmitic Acids; D017483:Receptors, Vasopressin; D001127:Arginine Vasopressin; D019308:Palmitic Acid; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases