Properties of the Human Arginine Vasopressin V2 Receptor after Site-Directed Mutagenesis of its Putative Palmitoylation Site
|Title:||Properties of the Human Arginine Vasopressin V2 Receptor after Site-Directed Mutagenesis of its Putative Palmitoylation Site|
|Authors:||Birnbaumer, Mariel; Liebenhoff, Ursula; Schulein, Ralf; Rosenthal, Walter; Muller, Helmut|
|Date Published:||January 15, 1996|
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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)
The V2R is a string of 371 amino acids. The amino acids are called residues because when an amino acid links with another amino acid, it loses a water molecule. The majority of the amino acid remains and is called a residue.
The model of the V2R developed by researchers describes the V2R in the following way: Part of the V2R is coiled within the cell membrane in seven coils called transmembrane domains 1 - 7. Part of the V2R snakes out the cell membrane into the extracellular environment forming 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 (N-terminus), sits outside the cell with the extracellular loops. The other end, called the carboxy-terminus (C-terminus), sits inside the cell with the intracellular loops. Researchers study the different parts of the V2R to see what function they perform to enable the V2R to do its job. (You can view a diagram of V2R here.)
Schulein, et al. focused on the last 44 residues that make up the C-terminus. They knew that two cysteine residues that were the 341st and 342nd residues of the V2R were sites where the acyl radical of palmitic acid (a fatty acid) attached itself to the V2R. The attachment process is called palmitoylation. The authors wanted to study the significance of palmitoylation for the V2R. Did it affect the V2R's ability to function?
To find out, they created mutant V2Rs where cysteine residues 341 and 342 were removed and replaced by serine residues, which cannot allow palmitoylation to occur. They created three different mutant V2Rs:
- one with both cysteines removed and replaced with serines, a double mutant called C-3415/C-342,
- one with only cysteine 341 removed and replaced with a serine, C-341S,
- one with only cysteine 342 removed and replaced with a serine, C-342S.
They injected each mutant into laboratory cell cultures infused with AVP then analyzed the results, comparing them to the results of injecting a normal V2R into the cell culture.
The authors found that all the mutants bound with AVP in amounts equal to normal V2Rs. This indicates that palmitoylation of the V2R is not necessary for the V2R to bind with AVP. Nor is palmitoylation of the V2R required for the V2R to be able to couple with the G protein and adenylyl cyclase system. This was determined by measuring the ability of the different V2Rs to stimulate AdC.
However, the researchers found that the mutated V2Rs could not transport themselves to the cell surface as efficiently as normal V2Rs. Nor could they reenter the cell and sequester themselves as efficiently. The authors concluded that palmitoylation does not influence the V2Rs' ability to bind with AVP or stimulate the G protein/AdC system. But it does influence its ability to transport itself to the cell surface and its ability to internalize and sequester itself after it becomes desensitized to AVP. A possible reason for this is that palmitoyls help signal the vesicles that carry V2R to and from the cell surface. Or perhaps the palmitoyls help shape the C-terminus in a way that allows it to interact with other components of the vesicular transport systems.