Palmitoylation of the V2 Vasopressin Receptor
- Department of Anesthesiology and Molecular Biology Institute, University of California, Los Angeles School of Medicine, Los Angeles, California 90095
Abstract
Palmitoylation of the V2 vasopressin receptor (V2R) and its functional role were investigated in transfected cells. Palmitoylation was assessed by incubating transfected cells with [3H]palmitic acid and immunoprecipitating the receptor with an antibody raised against a portion of the third intracellular loop of V2R. Wild-type and nonglycosylated V2R yielded tritium signals at 45–55 and 40 kDa, respectively, demonstrating that the V2R is palmitoylated and that receptor palmitoylation is independent of glycosylation. Substitution of CC341/342 for serines eliminated receptor palmitoylation, whereas replacement of a single amino acid, C341S or C342S, restored partial palmitoylation. Saturation binding assays revealed decreased cell surface expression of the nonpalmitoylated receptor compared with the wild-type; this effect was more pronounced when a truncated form of V2R (G345ter) was studied. The presence of either cysteine residue (C341S or C342S) elevated receptor expression to normal levels, most likely due to the partial restoration of palmitoylation. Ligand binding affinity, hormone-induced stimulation of adenylyl cyclase activity, receptor internalization, and desensitization were not affected by the absence of palmitoylation. No increase but rather a slight decrease in the extent of receptor palmitoylation was detected after exposure to vasopressin. It was concluded that the V2R is palmitoylated in both cysteines, each cysteine is palmitoylated independently from the other, and palmitoylation enhances cell surface expression of the V2R.
Footnotes
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Send reprint requests to: Mariel Birnbaumer, Ph.D., UCLA School of Medicine, Dept. of Anesthesiology, 10833 Le Conte Avenue, Box 951778, Los Angeles, CA 90095. E-mail:marielb{at}cvmail.anes.ucla.edu
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This work was supported in part by NIH Grant DK 41–244 to MB.
- Abbreviations:
- LH
- luteinizing hormone
- hCG
- human chorionic gonadotropin
- DMEM
- Dulbecco’s modified Eagle’s medium
- HBSS
- Hanks’ balanced salt solution
- D-PBS
- Dulbecco’s phosphate-buffered saline
- FBS
- fetal bovine serum
- AVP
- arginine vasopressin
- Iso
- isoproterenol
- VIP
- vasoactive intestinal peptide
- HEK
- human embryonic kidney
- V2R
- V2 vasopressin receptor
- RIPA
- radioimmunoprecipitation assay
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- Received September 30, 1996.
- Accepted March 31, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
