Hamid Sadeghi

Phosphorylation of the V2 Vasopressin Receptor

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 desensitization 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.

Arrestin effects on internalization of vasopressin receptors

Arrestins have been shown to facilitate the recruitment of G protein-coupled receptors to the clathrin-coated vesicles that mediate their internalization. After (8)Arg-vasopressin-induced internalization, the human V2 vasopressin receptor failed to recycle to the cell surface, whereas the vasopressin type 1a receptor (V1a) subtype did. The possibility that the lack of recycling could identify a novel role for arrestins was investigated by examining the effect of coexpressing wild-type and dominant negative arrestins on the recycling of wild-type and mutant V2 and V1a receptors. Coexpression of the V1a or V2 receptors with the last 100 amino acids of arrestin reduced significantly their internalization, whereas coexpression of wild-type and mutant arrestins had diverse effects on internalization. Arrestin3 but not arrestin2 increased the internalization of the V1aR without altering its recycling pattern. Both nonvisual arrestins enhanced vasopressin type 2 receptor (V2R) internalization, inducing the appearance of a pool of recycling receptor in addition to the nonrecycling pool. The effect of arrestins on the internalization of the chimeric V1a/V2 receptor and its reciprocal chimera was specified by the identity of the carboxyl-terminal segment. The S363A mutation that confers recycling to the V2R did not alter its interaction with arrestins. Truncation of the carboxyl-terminal segment of the V2R impaired ligand-induced internalization that could be fully restored by wild-type arrestins. Internalization of the V2 and V1a receptors required dynamin GTPase activity.

Maturation of receptor proteins in eukaryotic expression systems.

Transient and stable expression in eukaryotic cells is commonly used to examine receptor function. Characterization of the V2 vasopressin receptor synthesized in transiently transfected cells revealed the presence of large quantities of immature protein and a small fraction of fully mature protein. The immature protein was characterized by its sensitivity to endoglycosidase H treatment, abnormal migration in SDS PAGE, and a tendency to form aggregates. Prevention of protein glycosylation by mutagenesis increased the fraction of mature protein produced, but did not eliminate the need for the maturation step. On the other hand, stably transfected cells produce almost exclusively mature receptor protein with a t1/2 of 6 h, while the immature form has a t1/2 of 20 min. In the absence of N-linked glycosylation the t1/2 of the mature V2 receptor in stably transfected cells was reduced to 4.5 h. In transient expression experiments the immature receptor proteins exhibited a prolonged t1/2 of about 8 h. Comparison of the half life of the immature form of the wild type and the R137H mutant V2 receptor did not reveal differences despite the lower amounts of mutant mature receptor detected by binding.

PHOSPHORYLATION AND RECYCLING KINETICS OF G PROTEIN-COUPLED RECEPTORS

The rate of ligand-induced phosphorylation of the V2 and V1a vasopressin receptors was characterized in HEK 293 cells. Both receptors were phosphorylated predominantly by GRKs, and the V1a receptor was also phosphorylated by protein kinase C regardless of the presence or absence of ligand. Phosphorylation of the V1aR catalyzed by GRKs reached maximal values at the shortest measured time: 15 seconds, and decayed rapidly with a t1/2 of 6 min in the continuous presence of AVP. In agreement with the hypothesis that dephosphorylation must precede receptor recycling to the cell surface, the V1aR returned rapidly to the cell surface after removal of the hormone from the medium. Phosphate incorporation into the V2R proceeded at a slower pace, and the internalized phosphorylated receptor failed to recycle to the cell surface and retained its phosphate for a long time in the presence or absence of ligand. A single mutation in the carboxy terminus of the V2R accelerated de-phosphorylation of the protein and conferred recycling properties to the V2R. These experiments provided molecular evidence for the hypothesis that internalization is required for de-phosphorylation and recycling of reactivated G protein coupled receptors to the cell surface.

Processing and Ligand-Induced Modifications of the V2 Vasopressin Receptor

Synthesis, processing and agonist-induced modifications of the V2 vasopressin receptor were examined in stably or transiently transfected HEK293 cells. Metabolic labeling with S methionine for 30 min revealed a predominant precursor protein which subsequently gave rise to the mature receptor on the cell surface. Maturation of the receptor was unrelated to glycosylation suggesting that it was the consequence of protein refolding. In addition to monomeric forms of V2 receptor protein, oligomers of the precursor protein were also detected in SDS-PAGE. These oligomers seemed to be dimers and tetrameres, and were more apparent in transiently transfected cells that produced higher quantities of protein then stably transfected cells. No oligomers of the mature receptor were detected, and co-transfection of the wild type with a mutant V2 receptor lacking G-protein coupling activity did not alter the function of the wild type receptor. These results indicated that the formation of oligomeric was most likely a consequence of overproduction of the protein and not a required step for receptor function. Addition of vasopressin promoted phosphorylation and sequestration of the wild type receptor, and of the R137H mutant receptor which lacks coupling to G proteins. Activation of protein kinases A or C did not result in phosphorylation of un-occupied receptor. Phosphate incorporated into the protein was stable in the continuous presence of the ligand despite sequestration of the receptor protein. Deletion of the last 14 amino acids abolished receptor phosphorylation but not sequestration and desensitization, indicating that these two processes are not dependent on protein phosphorylation. Additionally, phosphorylation and sequestration of the R137H mutant receptor revealed that phosphorylation and sequestration does not require coupling to Gs. The wild type V2 vasopressin receptor was found to be palmitoylated at two cysteines at the carboxyl terminus. Either cysteine could be palmitoylated independently of each other and the presence of at least one was required to obtain receptor expression similar to the wild type. The turnover of the palmitic acid incorporated into the receptor was not altered by the addition of vasopressin demonstrating that this post-translational modification of the receptor was not altered by the ligand-promoted phosphorylation of the protein.