Agonist-induced Phosphorylation of Somatostatin Receptor Subtype 1 (Sst1)

Abstract

The sst1 somatostatin (SRIF) receptor subtype is widely expressed in the endocrine, gastrointestinal, and neuronal systems as well as in hormone-sensitive tumors, yet little is known about its regulation. Here we investigated the desensitization, internalization, and phosphorylation of sst1 expressed in CHO-K1 cells. Treatment of cells with 100 nM SRIF for 30 min reduced maximal SRIF inhibition of adenylyl cyclase from 40 to 10%. This desensitization was rapid (t1 ⁄2 < 2 min) and dependent on agonist concentration (EC 50 ‫؍‬ 2 nM). However, internalization of receptor-bound ligand occurred slowly (t1 ⁄2 > 180 min). Incubation of cells with SRIF also caused a rapid (t1 ⁄2 < 2 min) increase in sst1 receptor phosphorylation in a dose-dependent manner (EC 50 ‫؍‬ 1.3 nM), as determined in a mobility shift phosphorylation assay. Receptor phosphorylation was not affected by pertussis toxin, indicating a requirement for receptor occupancy rather than signaling. The protein kinase C activator, phorbol 12-myristate 13-acetate also stimulated sst1 receptor phosphorylation whereas forskolin did not. Both agonist-and phorbol 12-myristate 13-acetate-stimulated receptor phosphorylation occurred mainly on serine. These studies are the first to demonstrate phosphorylation of the sst1 receptor and suggest that phosphorylation mediated uncoupling, rather than sequestration, leads to its desensitization.The two physiologically active somatostatin (SRIF) 1 peptides, SRIF14 and SRIF28, potently regulate numerous endocrine, exocrine, and neuronal functions by interacting with a family of six G protein-coupled receptors (sst1, sst2A, sst2B, sst3, sst4, and sst5) (1-4). The cellular changes induced by sst receptors include inhibition of secretion, modulation of neuronal transmission, and smooth muscle contraction, as well as inhibition of proliferation and stimulation of apoptosis. The sst1 receptor subtype is particularly widely distributed in the gastrointestinal tract (5, 6) and the brain (7, 8) as well as being expressed in several other normal tissues (9, 10). Additionally, sst1 is found in neuroendocrine, prostate, and mammary tumors (11-13). Thus, this receptor subtype is believed to mediate many of the central and peripheral actions of SRIF as well as to present a potential target for cancer therapy and diagnosis (1-4, 14).The sst1 receptor, like other members of the somatostatin receptor family, is now known to inhibit adenylyl cyclase by interacting with pertussis toxin-sensitive G i /G o proteins (15-17). Other signaling mechanisms potently regulated by the sst1 receptor in a pertussis toxin-sensitive manner include inhibition of Ca 2ϩ influx, membrane hyperpolarization, activation of protein tyrosine phosphatases, and stimulation of the mitogenactivated protein kinase cascade (18 -20). However, the sst1 receptor has also been shown to act via pertussis toxin-insensitive mechanisms. Both activation of Na ϩ /H ϩ exchange and potentiation of ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainate current responses to gluta...

Selective agonism in somatostatin receptor signaling and regulation

“…Somatostatin binding is known to be rapidly followed by phosphorylation of sst1, sst2A and sst3 receptors by G protein coupled receptor kinases or GRKs (Elberg et al, 2002;Hipkin et al, 1997;Liu and Schonbrunn, 2001;Roth et al, 1997). The GRK family consists of six serine-threonine kinases that specifically bind to and phosphorylate agonist activated GPCRs (Moore et al, 2007;Reiter and Lefkowitz, 2006).…”

Expression, pharmacology, and functional role of somatostatin receptor subtypes 1 and 2 in human macrophages

“…Of all SRIF receptor subtypes, sst 2 is the one that was found to be the most efficiently internalized by SRIF analogs [36,55,65,66], including L-779,976 [65,67]. In contrast, the internalization of sst 1 was suggested to be speciesspecific [68], although the cloned human sst 1 seems to internalize poorly [69,70]. Taken together, our results do not support the hypothesis that the lack of effects of SRIF-14 (and cortistatin-14) on macrophage viability can be ascribable to the more efficient receptor down-regulation by SRIF-14 as compared with its synthetic analogs.…”

Distinct Phosphorylation Sites in the SST2A Somatostatin Receptor Control Internalization, Desensitization, and Arrestin Binding

“…After centrifugation, cells were solubilized in lysis buffer (HEPES-buffered saline containing 4 mg/ml dodecyl-␤-maltoside and PPI) for 60 min at 4°C, and they were centrifuged again at 100,000g for 30 min. Solubilized, radiolabeled receptors were subjected to a two-step purification consisting of lectin affinity chromatography followed by immunoprecipitation with mouse monoclonal HA antibody (Hipkin et al, 1997;Liu and Schonbrunn, 2001). For lectin affinity chromatography, the cell lysate was incubated at 4°C for 90 min or overnight with 100 l (packed volume) of washed wheat germ agglutininagarose.…”

“…Metabolic labeling of cells and subsequent purification of the sst2A receptor was carried out essentially as described previously (Hipkin et al, 1997;Liu and Schonbrunn, 2001). In brief, CHO cells expressing HAtagged sst2A receptors were incubated for 3 h with […”