The sst2A receptor mediates many of the endocrine and neuronal actions of somatostatin and is the target of somatostatin analogs in cancer therapy. As with many G-protein coupled receptors, agonist stimulation causes sst2A receptor desensitization and internalization, events that require receptor phosphorylation. Further, heterologous receptor activation of protein kinase C (PKC) also increases sst2A receptor phosphorylation and internalization. Here we analyzed a series of sst2A receptor mutants biochemically to identify residues in the receptor carboxy terminus that were phosphorylated upon agonist stimulation and then generated four phosphorylation sensitive antibodies to those residues. Once the selectivity of each antibody for its phosphorylated and non-phosphorylated target sequence was determined, the phospho-site specific antibodies were used to demonstrate that somatostatin treatment of CHO cells expressing the wild type sst2A receptor increased phosphorylation on five residues in the receptor C-terminus: Ser341, Ser343, Ser348, Thr353, and Thr354. Phorbol-12-myristate-13-acetate (PMA) increased receptor phosphorylation only on Ser343. Inhibition of PKC blocked PMA but not somatostatin stimulation showing that different kinases catalyzed Ser343 phosphorylation. In contrast, somatostatin-stimulated sst2A receptor phosphorylation was inhibited by knockdown of G-protein coupled receptor kinase-2 (GRK2) with siRNA. Somatostatin increased sst2A receptor phosphorylation on the same five residues in GH pituitary cells as in CHO cells. However, PMA stimulated sst2A receptor phosphorylation on both Ser343 and Ser348 in GH cells. These results characterize the complex pattern of sst2A receptor phosphorylation by agonist and second messenger activated kinases for the first time and indicate that cell type specific regulation of sst2A receptor phosphorylation occurs.
Search
Reviews
[Write a review of this article]
Find related articles from these CiteULike users
