A Serine Cluster Prevents Recycling of the V2 Vasopressin Receptor
| Title: | A Serine Cluster Prevents Recycling of the V2 Vasopressin Receptor |
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| Authors: | Innamorati, Giulio; Sadeghi, Hamid; Birnbaumer, Mariel; Tran, N. T. |
| Publisher: | Proceedings of the National Academy of Sciences of the United States of America |
| Date Published: | March 01, 1998 |
| Reference Number: | 187 |
Receptor recycling plays a critical role in the regulation of cellular responsiveness to environmental stimuli. Agonist-promoted phosphorylation of G protein-coupled receptors has been related to their desensitization, internalization, and sequestration. Dephosphorylation of internalized G protein-coupled receptors by cytoplasmic phosphatases has been shown to be pH-dependent, and it has been postulated to be necessary for receptors to recycle to the cell surface. The internalized V2 vasopressin receptor (V2R) expressed in HEK 293 cells is an exception to this hypothesis because it does not recycle to the plasma membrane for hours after removal of the ligand. Because this receptor is phosphorylated only by G protein-coupled receptor kinases (GRKs), the relationship between recycling and GRK-mediated phosphorylation was examined. A nonphosphorylated V2R, truncated upstream of the GRK phosphorylation sites, rapidly returned to the cell surface after removal of vasopressin. Less-drastic truncations of V2R revealed the presence of multiple phosphorylation sites and suggested a key role for a serine cluster present at the C terminus. Replacement of any one of Ser-362, Ser-363, or Ser-364 with Ala allowed quantitative recycling of full-length V2R without affecting the extent of internalization. Examination of the stability of phosphate groups incorporated into the recycling S363A mutant V2Rs revealed that the recycling receptor was dephosphorylated after hormone withdrawal, whereas the wild-type V2R was not, providing molecular evidence for the hypothesis that GRK sites must be dephosphorylated prior to receptor recycling. These experiments uncovered a role for GRK phosphorylation in intracellular sorting and revealed a GRK-dependent anchoring domain that blocks V2R recycling.
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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 movement of the V2R back to the inside of the cell involves three steps: the desensitization, internalization, and the sequestration (the prolonged retention inside the cell) of the V2R. Unlike some other receptors that recycle to the cell surface after the removal of the hormone to which they bind, V2R takes hours to do so.
Phosphorylation is a metabolic process that introduces a phosphate group into an organic molecule, in this case, the V2R. Phosphorylation helps make possible the V2Rs' movement from the cell surface to inside the cell. G protein-coupled receptor kinases (GRK) catalyze the phosphorylation of V2R. In this article, Innamorati, et al., examine the relationship between V2R recycling and GRK-mediated phosphorylation. To do so, they designed experiments using laboratory cell cultures that would highlight the AVP promoted internalization of the V2R.
To visualize what a V2R looks like, imagine a beaded string (the beads are amino acids). The majority of the V2R lies in seven folded clumps, called transmembrane domains 1 - 7, inside the cell membrane, the thin strip of tissue that separates the inside of the cell from the outside. 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, is outside the cell with the extracellular loops. The other end, called the carboxy terminus, is inside the cell with the intracellular loops. (Please look at a diagram of V2R.)
The V2R's carboxy terminus, once it has been phosphorylated by GRKs, plays an important role in the V2R's internalization (movement back to the inside of the cell). In order to be phosphorylated, the carboxy terminus must have places on it called acceptor sites that will accept the GRKs. All the phosphorylation acceptor sites seem to be located within the last 15 amino acids of the V2R's carboxy terminus.
The authors experimented on the V2Rs' behavior in laboratory cell cultures by shortening V2R carboxy termini to eliminate the GRK acceptor sites. The result was that the truncated V2Rs could rapidly return to the cell surface, a behavior they did not normally exhibit when possessed of full-length carboxy termini.
By creating V2Rs with progressively less extreme truncations (i.e., more of their carboxy termini left on) and monitoring the time it took for them to return to the cell surface, the authors were able to determine that normal V2Rs have nine GRK acceptor sites (also known as GRK phosphorylation sites) in their carboxy termini. They also identified three non-essential amino acids naturally occurring in V2R carboxy termini (serines at positions 362, 363 and 364) that are necessary to prevent the V2R from returning to the cell surface once it has been internalized. When the authors substituted the non-essential amino acid, alanine, for the serines at 362, 363 or 364, they found the V2R could move inside the cell and also return to the surface of the cell much more quickly than when the serine was in place.
The authors observed that the phosphorylation of serines at 362, 363 and 364 in the carboxy terminus blocks the dephosphorylation of the internalized V2R and thus its return to the cell surface. This finding provides molecular evidence for the theory that the V2R must be dephosphorylated before it can return to the cell surface.