Palmitoylation of the V2 Vasopressin Receptor

Title: Palmitoylation of the V2 Vasopressin Receptor
Authors: Birnbaumer, Mariel; Sadeghi, Hamid; Innamorati, Giulio; Dagrag, Mirabelle
Publisher: Molecular Pharmacology
Date Published: January 01, 1997
Reference Number: 149
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 40kDa, 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.
The publisher has not granted permission to reproduce this article on our website.
You may, however, read this article at the Molecular Pharmacology website.
To return to this page, use your "back" key.

This translation by the NDI Foundation is to assist the lay reader. To provide a clear, accessible interpretation of the original article, we eliminated or simplified some technical detail and complicated scientific language. We concentrated our translation on those aspects of the article dealing directly with NDI. The NDI Foundation thanks the researchers for their work toward understanding and more effectively treating this disorder.
© Copyright NDI Foundation 2007 (JC)

Palmitoylation is a naturally occurring chemical process that modifies proteins in such a way that it contributes to those proteins being able to localize on the surface of cell membranes. Sadeghi, et al., investigated the vasopressin-2 receptor (V2R), a protein which must travel from inside the principal cells of the kidney collecting duct to the cell membrane surface of those cells in order to do its job: binding with the antidiuretic hormone, arginine vasopressin (AVP). The authors wanted to see if V2R undergoes the palmitoylation process in order to get to the cell surface. They also wanted to know what, if any, other roles palmitoylation plays in the structure and function of the V2R.

Picture the V2R as a beaded string. (The beads are amino acids; there are 371 of them.) When the V2R is in the cell membrane, that thin strip of tissue that encircles the cell, separating the inside of the cell from the outside, most of the V2R lies inside the membrane in seven folded clumps called transmembrane domains 1 - 7. Part of the V2R snakes outside the cell to form three curves called extracellular loops 1 - 3; part of it snakes inside the cell to form three curves called intracellular loops 1 - 3. One end of the V2R, called the amino terminus, sits outside the cell with the extracellular loops. The other end of the V2R, called the carboxy-terminus, sits inside the cell with the intracellular loops. (You can look at a diagram of a V2R here.)

To test whether the V2R is palmitoylated, it was expressed in laboratory cell cultures and tested for incorporation of (3H) palmitic acid. The tests revealed that the V2R indeed undergoes palmitoylation. Further, the authors located the site on the V2R where palmitoylation occurs, namely two cysteine amino acid residues at position 341 and 342 on the V2R. (That is, two cysteine amino acids that are the 341st and 342nd amino acids on the string of 371 amino acids that make up the V2R.) This location occurs 13 amino acids after the seventh transmembrane region. At this point the two cysteine amino acids are anchored to the cell membrane by palmitic acid.

The authors found that it did not matter whether the V2R underwent another process called glycosylation or not, it still could be palmitoylated. The authors also found that both cysteines are palmitoylated and that each one can be palmitoylated independently of the other.

By creating V2Rs that could not be palmitoylated, then comparing their ability to reach the cell membrane surface to V2Rs that were palmitoylated, the authors found a direct correlation between palmitoylation and cell surface expression: The non-palmitoylated V2Rs got to the surface 40 - 50% less than the normal V2Rs.

Normal V2Rs undergo a number of functions in order to perform their job: It must get to the cell surface to bind with AVP. Once bound, it must couple with its G-protein. When it has performed its function it must become desensitized to the AVP, go back inside the cell (internalize) and go back to its holding space (i.e. sequester itself). none of these functions were interfered with in V2Rs that were not palmitoylated. Thus, the authors conclusion was that palmitoylation enhances cell surface expression of V2R.