Site Specificity of Agonist and Second Messenger-Activated Kinases for Somatostatin Receptor Subtype 2A (Sst2A) Phosphorylation

Abstract

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...

Seven Transmembrane Receptors as Shapeshifting Proteins: The Impact of Allosteric Modulation and Functional Selectivity on New Drug Discovery

“…This can lead to differences in receptor phosphorylation "barcoding" that can go on to target receptors for different signaling pathways. Cell-type dependence is observed for phosphorylation of different residues in somatostatin type 2A receptors when studied in Chinese hamster ovary, pituitary, and GH4C1 cells (Liu et al, 2009). Likewise, different cell type-dependent phosphorylation of muscarinic m3 receptors has been observed (Torrecilla et al, 2007).…”

Pasireotide and octreotide antiproliferative effects and sst2 trafficking in human pancreatic neuroendocrine tumor cultures

“…Moreover sst 2 was more rapidly dephosphorylated and recycled after pasireotide treatment than ss14 treatment (Kao et al 2011). It has also been shown that GRK2 is required for ss14-stimulated sst 2 phosphorylation on Ser341/343 (Liu et al 2009) and that GRK2/GRK3 are required for ss14-dependent phosphorylation of sst 2 on Thr353/354 (Pöll et al 2010). Furthermore, overexpression of GRK2 increases pasireotide-dependent phosphorylation and internalization of sst 2 (Pöll et al 2010).…”

Somatostatin system: molecular mechanisms regulating anterior pituitary hormones

“…sst2 was phosphorylated and internalized after treatment with SRIF and sst2-specific agonists in stable sst2 transfectant GH4C1 cells (Hipkin et al 1997). sst2 is phosphorylated at five serine and threonine residues within the C-terminus following SRIF treatment (Liu et al 2009). Moreover, in transiently transfected GH 3 cells, both SRIF and octreotide, but not pasireotide, induced robust sst2 phosphorylation.…”