Agonist-induced Desensitization, Internalization, and Phosphorylation of the sst2A Somatostatin Receptor

Abstract

Cellular responsiveness to the inhibitory peptide somatostatin (SRIF) or its clinically used analogs can desensitize with agonist exposure. While desensitization of other seven-transmembrane domain receptors is mediated by receptor phosphorylation and/or internalization, the mechanisms mediating SRIF receptor (sst) desensitization are unknown. Therefore, we investigated the susceptibility of the sst2A receptor isotype to ligandinduced desensitization, internalization, and phosphorylation in GH-R2 cells, a clone of pituitary tumor cells overexpressing this receptor. A 30-min exposure of cells to either SRIF or the analog SMS 201-995 (SMS) reduced both the potency and efficacy of agonist inhibition of adenylyl cyclase. Internalization of receptor-bound ligand was rapid (t1 ⁄2 ‫؍‬ 4 min) and temperature-dependent. SRIF and SMS increased the phosphorylation of the 71-kDa sst2A protein 25-fold within 15 min. Receptor phosphorylation was dependent on both the concentration and time of agonist exposure and was not affected by pertussis toxin pretreatment, indicating that receptor occupancy rather than second messenger formation was required. Receptor phosphorylation was also stimulated by phorbol 12-myristate 13-acetate activation of protein kinase C. Both ligand-stimulated and phorbol 12-myristate 13-acetate-stimulated receptor phosphorylation occurred primarily on serine. These studies are the first demonstration of agonist-dependent desensitization, internalization, and phosphorylation of the sst2A receptor and suggest that phosphorylation may mediate the homologous and heterologous regulation of this receptor.The somatostatin peptides 1 influence endocrine, exocrine, and neuronal function through binding to a family of six G protein-coupled receptors (sst1, sst2A, sst2B, sst3, sst4, and sst5) (1, 2). Within the SRIF receptor family, sst2A receptor mRNA has been detected in many tissues including the brain, pituitary, pancreas, spleen, small intestine, and stomach (1, 2), and the receptor protein has recently been shown to be widely distributed in the mammalian brain (3). Thus, this receptor isotype mediates many of the central and peripheral actions of SRIF.Early studies on the signal transduction mechanisms activated by SRIF showed that sst receptors elicited their actions predominantly via pertussis toxin-sensitive G proteins (1, 2, 4). Thus, SRIF inhibition of adenylyl cyclase and Ca 2ϩ channels, as well as SRIF stimulation of K ϩ channels, phospholipase C, serine/threonine and tyrosine phosphatases, arachidonic acid release, and mitogen-activated protein kinases are inhibited by pertussis toxin treatment (5-12). However, some actions of SRIF, such as stimulation of other tyrosine phosphatases as well as inhibition of Na/H exchange, are pertussis toxin-insensitive (13,14). The network of signaling pathways activated by individual sst receptor isotypes is largely unknown. Signaling mechanisms have been especially difficult to elucidate in the native environment of the receptors because most SRIF target cells exp...

Corticotropin-Releasing Hormone (CRH) Downregulates the Function of Its Receptor (CRF1) and Induces CRF1 Expression in Hippocampal and Cortical Regions of the Immature Rat Brain

“…The present study demonstrated a decline in CRF 1 binding capacity in neocortex, associated with increased CRF 1 mRNA expression. These findings are consistent with a mechanism of transient receptor elimination due to internalization, which leads to enhanced receptor mRNA transcription as has been shown in several other G-protein-coupled receptor systems (8,29,52). In other systems, this ligandinduced receptor internalization has been proposed to contribute to receptor desensitization via cell surface receptor downregulation (8,29), as well as by regulating transcription of mRNA (52).…”

Somatostatin displayed on filamentous phage as a receptor‐specific agonist

“…Most importantly, the fate of the bound phage may be very di erent from that of the natural ligand. After binding free somatostatin, sst 2 has recently been reported to be desensitized, internalized and phosphorylated (Hipkin et al, 1997). It has also been suggested that in some cell lines ligand binding would protect the receptor from degradation, which would increase its half life (Schonbrunn et al, 1995).…”

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).…”

“…Thus, agonist stimulation of GTPγS binding reflects receptor activation of all receptor-interacting G proteins, including the pertussis toxin sensitive G proteins that couple somatostatin receptors to adenylyl cyclase and the pertussis toxin insensitive G proteins that are at least partially responsible for coupling receptors to phospholipase C. Further, since cyclic AMP measurements were made after 15 min of agonist stimulation whereas phosphoinositide production was determined after 50 min of stimulation, the effect of receptor desensitization will differ in the two assays. We now know that desensitization can occur after few minutes of agonist stimulation for several somatostatin receptor subtypes (Beaumont et al, 1998;Elberg et al, 2002;Engstrom et al, 2006;Hipkin et al, 1997;Holliday et al, 2007;Liu et al, 2000). Thus, desensitization will influence second messenger formation in assays conducted in intact cells and the impact of desensitization on such measurements will vary with the time of incubation.…”