TRANSIENT PHOSPHORYLATION OF THE V1A VASOPRESSIN RECEPTOR

The V1a arginine vasopressin receptor (V1aR) expressed in HEK 293 cell s was phosphorylated after binding to arginine vasopressin (AVP). The phosphate was incorporated very rapidly into the protein but remained attached for a very short time despite the continuous presence of horm one. The extent of phosphorylation depended upon the concentration of AVP suggesting the involvement of G-protein-coupled receptor kinases. Protein kinase C (PKC) contributed to V1aR phosphorylation as demonstr ated by the fact that inhibition of the kinase decreased the amount of phosphate incorporated into the receptor. However, PKC activity was n ot responsible for the transient nature of V1aR phosphorylation. The h ormone-free receptor could be phosphorylated by phorbol ester-activate d PKC. Although the phosphorylation was transient, the phosphate group s incorporated remained on the receptor protein longer than those inco rporated after AVP treatment. PRC phosphorylation of unoccupied V1aR w as not sufficient to promote sequestration. Vasopressin also promoted sequestration of about 80% of the surface receptor, but measurements o f the rate of accumulation of inositol phosphates in the sustained pre sence of the ligand did not reveal a significant desensitization of co upling to phospholipase C activity. The addition of a V1aR antagonist inhibited the sustained accumulation of inositol phosphates establishi ng that the sustained stimulation of PLC was mediated by receptors loc ated on the cell surface. The transient character of V1aR phosphorylat ion seemed intrinsic to the receptor protein rather than a consequence of signaling within the cell, and receptor sequestration appeared to be responsible for the desensitization observed in HEK 293 cells.