Phosphorylation of the V2 Vasopressin Receptor
| Title: | Phosphorylation of the V2 Vasopressin Receptor |
|---|---|
| Authors: | Sadeghi, Hamid; Birnbaumer, Mariel; Innamorati, Giulio; Eberle, Alex N. |
| Publisher: | The Journal of Biological Chemistry |
| Date Published: | January 04, 1997 |
| Reference Number: | 148 |
The V2 vasopressin receptor undergoes ligand-induced sequestration and desensitization (Birnbaumer, M., Antaramian, A., Themmen, A.P.N., and Gilbert, S. (1992) J. Biol. Chem. 267, 11783-11788). The V2 receptor expressed in transfected cells labeled with [32P] orthophosphate was phosphorylated following the addition of 100 nM arginine vasopressin (AVP). Phosphorylation was complete 5 min. after addition of AVP, and was not stimulated by increased levels of Ca2+ or cAMP. The half-maximal dose of AVP that stimulated phosphorylation was 2.4 ± 0.4 nM, similar to the receptor KD of 4.5 ± 0.4 nM. The role of phosphorylation on receptor desensitization was investigated by studying two vasopressin receptors 14 and 27 amino acids shorter than the wild type receptor. The missing segments were not needed for normal ligand binding or coupling to GS, but the last 14 amino acids were required for phosphorylation. The truncated receptors exposed to 100 nM AVP were sequestered and desensitized. The R137H V2R mutant receptor that binds vasopressin with wild type-like affinity and does not couple to GS (Rosenthal, W., Antaramian, A., Gilbert, S., and Birnbaumer, M. (1993) J. Biol. Chem. 268, 13030-13033) was phosphorylated and subjected to ligand-induced sequestration. These results established that phosphorylation is not essential for sequestration and desensitation of the V2 vasopressin receptor. Furthermore, they revealed that the conformation acquired after ligand occupancy is necessary for receptor phosphorylation and sequestration, while coupling to GS is not.
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- The GPCR must be sequestered, i.e., removed from the cell membrane to the inside of the cell.
- Those points of the receptor that extend into the inside of the cell must undergo phosphorylation (a metabolic process that introduces a phosphate group into the receptor). Sequestration reduces the number of receptors available to extracellular messengers (for the focus of this research, the number of V2 receptors (V2Rs) available to arginine vasopressin (AVP)). Phosphorylation is thought to reduce the level of activity of the receptor.
The human V2 vasopressin receptor (V2R), a member of the GPCR family, is activated by a specific extracellular messenger, the antidiuretic hormone, arginine vasopressin (AVP). Once AVP binds with V2R, the V2R stimulates adenylyl cyclase activity which in turn elevates an important metabolic regulator called cAMP. This induces the movement of a water-transporting protein called aquaporin-2 (AQP2) to shuttle to specific kidney cell membranes to make them more water permeable, thus facilitating water reabsorption, the intended biological function of the AVP/V2R coupling.
In this study, Innamorati, et al., examined the role of phosphorylation in V2 receptor desensitization. To do so, they studied the activity of two lines of V2 receptors manipulated to be 14 and 27 amino acids shorter, respectively, than the normal V2 receptor to which they were compared. Since the manipulated V2Rs were shorter because they were missing their tail ends, they are referred to as truncated receptors.
Their research revealed that both the normal V2R and the V2R manipulated so as not to carry a sugar molecule underwent the phosphorylation process when exposed to AVP. Thus, the absence of sugar did not interfere with the incorporation of phosphate into the receptor.
The V2R lives in specific cell membranes. If you imagine the receptor as a string, the greater part of it lies within the cell membrane (that thin band that encircles the cell, separating the cell from its environment) in seven folded clumps called transmembrane helices. Part of the V2R snakes outside the cell to form three curves called extracellular loops 1, 2, and 3; part snakes inside the cell to form three curves called intracellular loops 1, 2, and 3. The head of the V2R, called the amino-terminal, is outside the cell, and the tail, or carboxyl-terminal, lies inside the cell. (Look at diagram of V2R).
The authors searched for the location on the V2R where phosphorylation took place. They found it in the carboxyl-terminal. Their mutated V2Rs did not have their full carboxyl-terminals. The V2R line missing 27 amino acids had even less of a tail than the line missing 14 amino acids. The researchers found that the truncations did not alter the binding affinity of the mutant receptors for AVP. Neither did it reduce the V2R's ability to stimulate the adenylyl cyclase system. However, the truncated receptors could not incorporate phosphate, which means the carboxyl-terminal is required for the phosphorylation of the V2R. Interestingly, deleting the last 14 amino acids from the V2R impaired phosphorylation of the V2R to a greater extent than deleting the last 27 amino acids.
Next, the authors experimented with the truncated V2R's ability to be desensitized by AVP. (Remember, desensitization is necessary to end the metabolic process initiated by the extracellular messenger. In this case the V2R must be temporarily desensitized to AVP, once the process it has set in motion -- facilitating water reabsorption by the kidney -- begins.) They found the truncated receptor's ability to be desensitized was impaired, perhaps due to the absence of phosphorylation.
The authors then examined the role of phosphorylation in the sequestration of the V2R. They did this by comparing the extent of sequestration (i.e., the extent to which the V2R will leave the membrane and go inside the cell where it will be unavailable to bind with AVP) in the truncated V2Rs with normal V2Rs. They found that the truncated V2Rs disappeared from the cell surface when exposed to AVP, as did the normal V2Rs. Since the truncated receptors were not capable of phosphorylation, yet they could still be sequestered, the authors' experiment showed that phosphorylation was not required for receptor sequestration. Furthermore, both the normal and truncated receptors returned to the cell surface within the same time frame.
Innamorati, et al., also found that the binding of AVP was a requirement for V2R phosphorylation, and that it is the shape the V2R takes after binding with AVP that allows it to undergo phosphorylation and sequestration.