A SERINE CLUSTER PREVENTS RECYCLING OF THE V2 VASOPRESSIN RECEPTOR

Receptor recycling plays a critical role in the regulation of cellular responsiveness to environmental stimuli, Agonist-promoted phosphoryla tion of G protein-coupled receptors has been related to their desensit ization, internalization, and sequestration, Dephosphorylation of inte rnalized G protein-coupled receptors by cytoplasmic phosphatases has b een shown to be pa-dependent, and it has been postulated to be necessa ry for receptors to recycle to the cell surface, The internalized V2 v asopressin receptor (V2R) expressed in HEK 293 cells is an exception t o this hypothesis because it does not recycle to the plasma membrane f or hours after removal of the ligand, Because this receptor is phospho rylated only by G protein-coupled receptor kinases (GRKs), the relatio nship between recycling and GRK-mediated phosphorylation was examined, A nonphosphorylated V2R, truncated upstream of the GRK phosphorylatio n sites, rapidly returned to the cell surface after removal of vasopre ssin, Less-drastic truncations of V2R revealed the presence of multipl e 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 s tability of phosphate groups incorporated into the recycling S363A mut ant V2Rs revealed that the recycling receptor was dephosphorylated aft er hormone withdrawal, whereas the wild-type V2R was not, providing mo lecular evidence for the hypothesis that GRK sites must be dephosphory lated prior to receptor recycling, These experiments uncovered a role for GRK phosphorylation in intracellular sorting and revealed a GRK-de pendent anchoring domain that blocks V2R recycling.