Folding and Cell Surface Expression of the Vasopressin V2 Receptor: Requirement of the Intracellular C-terminus
| Title: | Folding and Cell Surface Expression of the Vasopressin V2 Receptor: Requirement of the Intracellular C-terminus |
|---|---|
| Authors: | Schulein, Ralf; Oksche, Alexander; Rosenthal, Walter; Dehe, Marcel; Wiesner, Burkhard |
| Publisher: | FEBS Letters |
| Date Published: | March 06, 1998 |
| Reference Number: | 234 |
We characterized truncations of the human vasopressin V2 receptor to determine the role of the intracellular C-terminus (comprising about 44 amino acids) in receptor function and cell surface expression. In contrast to the wild-type receptor, the naturally occurring mutant R337X failed to confer specific [3H]AVP binding to transfected cells. In addition, no vasopressin-sensitive adenylyl cyclase was detectable in membrane preparations of these cells. Laser scanning microscopy revealed that c-myc epitope- or green fluorescent protein-tagged R337X mutant receptors were retained within the endoplasmic reticulum. Increasing the number of C-terminal residues (truncations after codons 348, 354 and 356) restored G protein coupling, but revealed a length-dependent reduction of cell surface expression. Replacement of positively charged residues within the C-terminus by glutamine residues also decreased cell surface expression. A chimeric V2 receptor with the C-terminus replaced by that of the beta2-adrenergic receptor did not bind [3H]AVP and was retained within the cell. These data suggest that residues in the N-terminal part of the C-terminus are necessary for correct folding and that C-terminal residues are important for efficient cell surface expression.
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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 acid residues. (An amino acid loses a water molecule when it links to another amino acid. The majority of the amino acid which remains is called a residue.)
The majority of the residues lie within the cell membrane forming seven coils called transmembrane domains 1 - 7. Part of the V2R snakes outside the cell membrane forming three curves called extracellular loops 1, 2 and 3. Part of the V2R snakes inside the cell to form three curves called intracellular loops 1, 2 and 3. One end, called the amino terminus, sits outside the cell with the extracellular loops. The other end, called the carboxy terminus (C-terminus), lies inside the cell with the intracellular loops. (You can look at a diagram of V2R here.)
The C-terminus has 44 amino acid residues. Oksche et al. knew that one mutant V2R, mutant R337X, only had the first 9 of the 44 amino acids of the C-terminus. People bearing the mutant X-linked nephrogenic diabetes insipidus (NDI), a disorder characterized by the V2R's inability to perform its function of binding with the antidiuretic hormone, arginine vasopressin (AVP). Normally, AVP binds to the V2R and this initiates a molecular sequence which allows the kidney to concentrate urine and reabsorb water flowing through the kidney collecting duct.
In addition to testing with the naturally occurring R337X, the authors created four V2R mutations, all of which lacked some part of the C-terminus:
- Mutation L339, which only had the first 11 of the 44 amino residues.
- Mutation P349, which only had the first 21 of the 44 amino residues.
- Mutation D355X, which had the first 27.
- Mutation S357X, which had the first 29.
The authors then transfected cDNA's encoding the wild-type or the various mutant V2Rs into separate cell cultures. They analyzed the cultures looking for evidence to show whether or not each mutant performed several of the functions of a healthy, intact V2R. That evidence would be the ability to:
- travel from the ER to the cell surface,
- bind with AVP,
- couple to a G protein.
In doing this, the authors hoped to get a clearer picture of how much and what parts of the C-terminus are necessary for proper function.
They found that mutations R337X and L339X could travel to the cell curface and subsequently not bind to AVP or couple with G protein (remaining trapped in the ER). These two mutant V2Rs could not exit the ER because they could not fold themselves into the proper shape to exit.
Mutations P349X, D355X and S357 were capable of binding with AVP, though none was transported to the cell surface as the WT. The mutation that had the longest C-terminus (S357) was transported by, most efficiently, the next longest mutation (D355X) the next most efficiently, and the least longest mutation (P349X) the next most efficiently. Thus, the length of the C-terminus influences the extent to which the respective V2Rs are able to transport to the cell surface (where binding with AVP takes place).
The authors also created mutants where the positively charged residues in the C-terminus were replaced by residues with a negative charge. This too, produced V2Rs with reduced cell surface expression. The authors also created a V2R with a C-terminus from another type of receptor. This V2R could neither bind with AVP, stimulate the G-protein, or transport itself to the cell surface.
All this evidence suggests that the section of the C-terminus furthest from the seventh transmembrane domain contains specific residues required for a V2R capable of transporting itself to the cell surface and that part of the C-terminus nearest to the seventh transmembrane domain are required for proper folding.