Received for publication May 30, 2007.
Revised November 1, 2007.
Accepted for publication November 2, 2007.

Distinct phosphorylation sites in the sst2 somatostatin receptor control internalization, desensitization, and arrestin binding

Abstract

The sst2A receptor, a member of the G-protein coupled receptor superfamily, mediates many of the neuroendocrine and neuromodulatory actions of somatostatin and represents the primary target for somatostatin analogs used in cancer therapy and tumor localization. Agonist stimulation leads to the rapid phosphorylation, endocytosis and desensitization of the sst2A receptor, however little is known about the role of phosphorylation in sst2A regulation. Sst2A phosphorylation occurs on serine and threonine residues in the third intracellular loop and carboxy-terminus. Therefore, we generated mutant receptors in which serine (Ser-), threonine (Thr-) or both (Ser-/Thr-) residues in these regions were mutated to alanine. In contrast to the wild type receptor, SS14 treatment did not stimulate the phosphorylation of the Ser-/Thr- mutant and did not produce desensitization. Further, internalization of the Ser-/Thr- mutant occurred 5-times more slowly than with the wild type receptor. Mutating only the Ser residues did not inhibit either internalization or desensitization. In contrast, mutating only the Thr residues inhibited receptor endocytosis to the same extent as in the full mutant but did not affect receptor desensitization. In both the wild type and Ser- receptors, agonist binding produced a stable arrestin-receptor complex that was maintained during receptor trafficking whereas arrestin was not recruited to either the Thr- or the Ser-/Thr- receptors. These results demonstrate that agonist stimulated receptor phosphorylation is necessary for both desensitization and rapid internalization of the sst2A receptor. However, sst2A receptor internalization and uncoupling can occur independently, involve different receptor phosphorylation sites and exhibit different requirements for stable arrestin association.

Key words: Neuropeptides, Somatostatin, Gi family, Adenylyl cyclases, cAMP, Desensitization/uncoupling, GRKs, barrestins, Phosphorylation/Dephosphorylation, Immunocytochemistry, Mutagenesis/Chimeric approaches