Identification of phosphorylation sites regulating sst3 somatostatin receptor trafficking.

The human somatostatin receptor 3 (sst(3)) is expressed in about 50% of all neuroendocrine tumors and hence a promising target for multireceptor somatostatin analogs. The sst(3) receptor is unique among ssts in that it exhibits a very long intracellular C-terminal tail containing a huge number of potential phosphate acceptor sites. Consequently, our knowledge about the functional role of the C-terminal tail in sst(3) receptor regulation is very limited. Here, we have generated a series of phosphorylation-deficient mutants that enabled us to determine crucial sites for its agonist-induced beta-arrestin mobilization, internalization, and down-regulation. Based on this information, we generated phosphosite-specific antibodies for C-terminal Ser(337)/Thr(341), Thr(348), and Ser(361) that enabled us to investigate the temporal patterns of sst(3) phosphorylation and dephosphorylation. We found that the endogenous ligand somatostatin induced a rapid and robust phosphorylation that was completely blocked by the sst(3) antagonist NVP-ACQ090. The stable somatostatin analogs pasireotide and octreotide promoted clearly less phosphorylation compared with somatostatin. We also show that sst(3) phosphorylation occurred within seconds to minutes, whereas dephosphorylation of the sst(3) receptor occurred at a considerable slower rate. In addition, we also identified G protein-coupled receptor kinases 2 and 3 and protein phosphatase 1 alpha and 1 beta as key regulators of sst(3) phosphorylation and dephosphorylation, respectively. Thus, we here define the C-terminal phosphorylation motif of the human sst(3) receptor that regulates its agonist-promoted phosphorylation, beta-arrestin recruitment, and internalization of this clinically relevant receptor.