The somatostatin subtype 2A (sst2A) receptor, a member of the G protein-coupled receptor superfamily, mediates many of the neuroendocrine and neuromodulatory actions of somatostatin, and it 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 carboxyl terminus. Therefore, we generated mutant receptors in which serine (SerϪ), threonine (ThrϪ), or both (SerϪ/ThrϪ) residues in these regions were mutated to alanine. In contrast to the wild-type receptor, somatostatin treatment did not stimulate the phosphorylation of the SerϪ/ThrϪ mutant, and it did not produce desensitization. Furthermore, internalization of the SerϪ/ ThrϪ mutant occurred 5 times more slowly than with the wildtype 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 it did not affect receptor desensitization. In both the wild-type and SerϪ receptors, agonist binding produced a stable arrestin-receptor complex that was maintained during receptor trafficking, whereas arrestin was not recruited to either the ThrϪ or the SerϪ/ThrϪ 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.

A homology model of the M 1 muscarinic acetylcholine receptor, based on the X-ray structure of bovine rhodopsin, has been used to interpret the results of scanning and point mutagenesis studies on the receptor's transmembrane (TM) domain. Potential intramolecular interactions that are important for the stability of the protein fold have been identified. The residues contributing to the binding site for the antagonist, N-methyl scopolamine, and the agonist, acetylcholine, have been mapped. The positively charged headgroups of these ligands probably bind in a charge-stabilized aromatic cage formed by amino acid side chains in TM helices TM3, TM6 and TM7, while residues in TM4 may participate as part of a peripheral docking site. Closure of the cage around the headgroup of acetylcholine may be part of the mechanism for transducing binding energy into receptor activation, probably by disrupting a set of Van der Waals interactions between residues lying beneath the binding site that help to constrain the receptor to the inactive state, in the absence of agonist. This may trigger the reorganization of a hydrogen-bonding network between highly conserved residues in the core of the receptor, whose integrity is crucial for achievement of the activated state.

Somatostatin receptor subtype 2A (sst2A) 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. Furthermore, 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 carboxyl terminus that were phosphorylated upon agonist stimulation, and we then generated four phosphorylation-sensitive antibodies to those residues. Once the selectivity of each antibody for its phosphorylated and nonphosphorylated target sequence was determined, the phospho-site-specific antibodies were used to demonstrate that somatostatin treatment of Chinese hamster ovary (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 with siRNA. Somatostatin increased sst2A receptor phosphorylation on the same five residues in GH4C1 pituitary cells as in CHO cells. However, PMA stimulated sst2A receptor phosphorylation on both Ser343 and Ser348 in GH4C1 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.G protein-coupled receptors (GPCRs), which constitute the largest family of membrane receptors, respond to ligand stimulation by activating heterotrimeric G proteins that regulate the activity of effector enzymes and ion channels, producing changes in the concentration of specific intracellular second messengers. GPCR action is regulated, in turn, by feedback inhibitory processes that reduce receptor responses to prolonged or repetitive ligand stimulation, a process called desensitization. The first step in GPCR desensitization involves receptor phosphorylation by two types of serine/threonine protein kinases. G protein-coupled receptor kinases (GRKs) specifically phosphorylate the agonist-occupied receptor and thereby trigger homologous receptor desensitization, whereas second messenger-activated kinases can phosphorylate GPCRs in either the ligand-bound or the unoccupied state (Reiter and Lefkowitz, 2006;Moore et al., 2007). Because GPCR phosphorylation by second messengeractivated kinases does not require receptor occupancy by agonist, these kinases can produce heterologous, as well as hom...