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.The V1a and V2 vasopressin receptors (V1aR and V2R) are members of the G protein-coupled receptor family and both become phosphorylated upon activation by agonist (1, 2). For several receptors of this family phosphorylation and internalization have been shown to be a consequence of activation by ligand and seem to play a role in reducing the cellular responses to repeated exposure to hormones. Phosphorylation of G protein-coupled receptors enhances their ability to bind arrestin, which uncouples the receptors from G proteins, and helps recruit them to the clathrin-coated pits, which mediate the internalization process (3-7). Although phosphorylation and internalization of G protein-coupled receptors have been known to play a role in receptor desensitization for several years, the biochemical steps involved are not fully known and are still the subject of intense investigation.It has previously been observed that after removal of arginine vasopressin (AVP) from the medium and from the surface receptors with an acid wash, almost all of the internalized V1aR expressed in HEK 293 cells returns to the plasma membrane very rapidly (2), similar to what had been observed with isolated hepatocytes and smooth muscle vascular cells (8, 9). The human...