R. Schulein et al., PROPERTIES OF THE HUMAN ARGININE-VASOPRESSIN V2 RECEPTOR AFTER SITE-DIRECTED MUTAGENESIS OF ITS PUTATIVE PALMITOYLATION SITE, Biochemical journal, 313, 1996, pp. 611-616
Most G-protein-coupled receptors have conserved cysteine residues in t
heir C-terminal cytoplasmic domain that appear to be generally palmito
ylated. 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 p
lasmid was constructed by cloning the V2 receptor cDNA into the vector
pCDNAI.Neo. The resulting plasmid allowed site-directed mutagenesis e
xperiments 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 replaced by serine residue
s, yielding the single mutants C-341S and C-342S and the double mutant
C-341S/C-342S. Functional expression in stably transfected Ltk(-) cel
ls showed that the affinity of the three mutant receptors for arginine
vasopressin was not altered. In contrast with the activation of adeny
late cyclase through beta(2) adrenergic receptors, arginine vasopressi
n stimulated adenylate cyclase to the same extent and with similar EC(
50) values in both wild-type and mutant receptors. Transient expressio
n of the C-341S/C-342S mutant receptor in COS.M6 cells confirmed an un
altered affinity of the mutant receptor for arginine vasopressin. Howe
ver, the number of arginine vasopressin-binding sites on the cell surf
ace was reduced by 30%, suggesting that the transport of the mutant re
ceptor to the cell surface was impaired. In addition, the decrease in
detectable arginine vasopressin-binding sites on the cell surface foll
owing pre-exposure to hormone was reduced, indicating that the sequest
ration/internalization of the mutant receptor on the cell surface was
affected. The present data indicate that palmitoylation of the V2 rece
ptor is important for intracellular trafficking and/or sequestration/i
nternalization but not for agonist binding or activation of the G(s)/a
denylate cyclase system.