Phosphorylation of threonine 333 regulates trafficking of the human sst5 somatostatin receptor.

The frequent overexpression of the somatostatin receptors sst2 and sst5 in neuroendocrine tumors provides the molecular basis for therapeutic application of novel multireceptor somatostatin analogs. Although the phosphorylation of the carboxyl-terminal region of the sst2 receptor has been studied in detail, little is known about the agonist-induced regulation of the human sst5 receptor. Here, we have generated phosphosite-specific antibodies for the carboxyl-terminal threonines 333 (T333) and 347 (T347), which enabled us to selectively detect either the T333-phosphorylated or the T347-phosphorylated form of sst5. We show that agonist-mediated phosphorylation occurs at T333, whereas T347 is constitutively phosphorylated in the absence of agonist. We further demonstrate that the multireceptor somatostatin analog pasireotide and the sst5-selective ligand L-817,818 but not octreotide or KE108 were able to promote a detectable T333 phosphorylation. Interestingly, BIM-23268 was the only sst5 agonist that was able to stimulate T333 phosphorylation to the same extent as natural somatostatin. Agonist-induced T333 phosphorylation was dose-dependent and selectively mediated by G protein-coupled receptor kinase 2. Similar to that observed for the sst2 receptor, phosphorylation of sst5 occurred within seconds. However, unlike that seen for the sst2 receptor, dephosphorylation and recycling of sst5 were rapidly completed within minutes. We also identify protein phosphatase 1γ as G protein-coupled receptor phosphatase for the sst5 receptor. Together, we provide direct evidence for agonist-selective phosphorylation of carboxyl-terminal T333. In addition, we identify G protein-coupled receptor kinase 2-mediated phosphorylation and protein phosphatase 1γ-mediated dephosphorylation of T333 as key regulators of rapid internalization and recycling of the human sst5 receptor.

A transplantable phosphorylation probe for direct assessment of G protein-coupled receptor activation.

The newly developed multireceptor somatostatin analogs pasireotide (SOM230), octreotide and somatoprim (DG3173) have primarily been characterized according to their binding profiles. However, their ability to activate individual somatostatin receptor subtypes (sst) has not been directly assessed so far. Here, we transplanted the carboxyl-terminal phosphorylation motif of the sst(2) receptor to other somatostatin receptors and assessed receptor activation using a set of three phosphosite-specific antibodies. Our comparative analysis revealed unexpected efficacy profiles for pasireotide, octreotide and somatoprim. Pasireotide was able to activate sst(3) and sst(5) receptors but was only a partial agonist at the sst(2) receptor. Octreotide exhibited potent agonistic properties at the sst(2) receptor but produced very little sst(5) receptor activation. Like octreotide, somatoprim was a full agonist at the sst(2) receptor. Unlike octreotide, somatoprim was also a potent agonist at the sst(5) receptor. Together, we propose the application of a phosphorylation probe for direct assessment of G protein-coupled receptor activation and demonstrate its utility in the pharmacological characterization of novel somatostatin analogs.

Deciphering µ-opioid receptor phosphorylation and dephosphorylation in HEK293 cells.

BACKGROUND AND PURPOSE: The molecular basis of agonist-selective signalling at the µ-opioid receptor is poorly understood. We have recently shown that full agonists such as [D-Ala(2)-MePhe(4)-Gly-ol]enkephalin (DAMGO) stimulate the phosphorylation of a number of carboxyl-terminal phosphate acceptor sites including threonine 370 (Thr(370)) and serine 375 (Ser(375)), and that is followed by a robust receptor internalization. In contrast, morphine promotes a selective phosphorylation of Ser(375) without causing rapid receptor internalization. EXPERIMENTAL APPROACH: Here, we identify kinases and phosphatases that mediate agonist-dependent phosphorylation and dephosphorylation of the µ-opioid receptor using a combination of phosphosite-specific antibodies and siRNA knock-down screening in HEK293 cells. KEY RESULTS: We found that DAMGO-driven phosphorylation of Thr(370) and Ser(375) was preferentially catalysed by G-protein-coupled receptor kinases (GRKs) 2 and 3, whereas morphine-driven Ser(375) phosphorylation was preferentially catalysed by GRK5. On the functional level, inhibition of GRK expression resulted in enhanced µ-opioid receptor signalling and reduced receptor internalization. Analysis of GRK5-deficient mice revealed that GRK5 selectively contributes to morphine-induced Ser(375) phosphorylation in brain tissue. We also identified protein phosphatase 1γ as a µ-opioid receptor phosphatase that catalysed Thr(370) and Ser(375) dephosphorylation at or near the plasma membrane within minutes after agonist removal, which in turn facilitates receptor recycling. CONCLUSIONS AND IMPLICATIONS: Together, the morphine-activated µ-opioid receptor is a good substrate for phosphorylation by GRK5 but a poor substrate for GRK2/3. GRK5 phosphorylates µ-opioid receptors selectively on Ser(375), which is not sufficient to drive significant receptor internalization.

Rapid dephosphorylation of G protein-coupled receptors by protein phosphatase 1ß is required for termination of ß-arrestin-dependent signaling.

Termination of signaling of activated G protein-coupled receptors (GPCRs) is essential for maintenance of cellular homeostasis. It is well established that ß-arrestin redistributes to phosphorylated GPCRs and thereby facilitates desensitization of classical G protein-dependent signaling. ß-Arrestin in turn serves as a scaffold to initiate a second wave of signaling. Here, we report a molecular mechanism that regulates the termination of unconventional ß-arrestin-dependent GPCR signaling. We identify protein phosphatase 1ß (PP1ß) as a phosphatase for the cluster of phosphorylated threonines ((353)TTETQRT(359)) within the sst(2A) somatostatin receptor carboxyl terminus that mediates ß-arrestin binding using siRNA knock-down screening. We show that PP1ß-mediated sst(2A) dephosphorylation is initiated directly after receptor activation at or near the plasma membrane. As a functional consequence of diminished PP1ß activity, we find that somatostatin- and substance P-induced but not epidermal growth factor-induced ERK activation was aberrantly enhanced and prolonged. Thus, we demonstrate a novel mechanism for fine tuning unconventional ß-arrestin-dependent GPCR signaling in that recruitment of PP1ß to activated GPCRs facilitates GPCR dephosphorylation and, hence, leads to disruption of the ß-arrestin-GPCR complex.

Agonist-selective patterns of µ-opioid receptor phosphorylation revealed by phosphosite-specific antibodies.

BACKGROUND AND PURPOSE: Morphine activates the µ-opioid receptor without causing its rapid endocytosis. In contrast, full agonists such as [d-Ala(2) -MePhe(4) -Gly-ol]enkephalin (DAMGO) or etonitazene stimulate a rapid and profound internalization. However, the detailed molecular events underlying the differential regulation of receptor trafficking by distinct opioid agonists remain incompletely understood. EXPERIMENTAL APPROACH: Here, we have generated phosphosite-specific antibodies for the carboxyl-terminal residues serine 363 (Ser363), threonine 370 (Thr370) and serine 375 (Ser375), which enabled us to selectively detect either the Ser363-, Thr370- or Ser375-phosphorylated form of the receptor. KEY RESULTS: We showed that agonist-induced phosphorylation occurs at Thr370 and Ser375, whereas Ser363 is constitutively phosphorylated in the absence of agonist. We further demonstated that DAMGO and etonitazene stimulated the phosphorylation of both Thr370 and Ser375. In contrast, morphine promoted the phosphorylation of Ser375, but failed to stimulate Thr370 phosphorylation. In the presence of DAMGO, Ser375 phosphorylation occurred at a faster rate than phosphorylation of Thr370, indicating that Ser375 is the primary site of agonist-dependent phosphorylation. Activation of PKC by phorbol 12-myristate 13-acetate increased receptor phosphorylation only on Thr370, but not on Ser375, indicating that Thr370 can also undergo heterologous PKC-mediated phosphorylation. We also showed that µ receptor dephosphorylation can occur within minutes at or near the plasma membrane, and that agonist removal is a major prerequisite for Thr370 and Ser375 dephosphorylation. CONCLUSIONS AND IMPLICATIONS: Together, we showed for the first time that distinct agonists stimulate site-specific patterns of phosphorylation, which are intimately related to their ability to elicit µ-opioid receptor sequestration. LINKED ARTICLE: This article is commented on by Kelly, pp. 294-297 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01387.x.

Structural determinants of agonist-selective signaling at the sst(2A) somatostatin receptor.

The clinically used somatostatin (SS-14) analogs octreotide and pasireotide (SOM230) stimulate distinct species-specific patterns of sst(2A) somatostatin receptor phosphorylation and internalization. Like SS-14, octreotide promotes the phosphorylation of at least six carboxyl-terminal serine and threonine residues, namely S341, S343, T353, T354, T356, and T359, which in turn leads to a robust endocytosis of both rat and human sst(2A) receptors. Unlike SS-14, pasireotide fails to induce any substantial phosphorylation or internalization of the rat sst(2A) receptor. Nevertheless, pasireotide is able to stimulate a selective phosphorylation of S341 and S343 of the human sst(2A) receptor followed by a clearly detectable receptor sequestration. Here, we show that transplantation of amino acids 1-180 of the human sst(2A) receptor to the rat sst(2A) receptor facilitates pasireotide-induced internalization. Conversely, construction of a rat-human sst(2A) chimera conferred resistance to pasireotide-induced internalization. We then created a series of site-directed mutants leading to the identification of amino acids 27, 30, 163, and 164 that when exchanged to their human counterparts facilitated pasireotide-driven S341/S343 phosphorylation and internalization of the rat sst(2A) receptor. Exchange of these amino acids to their rat counterparts completely blocked the pasireotide-mediated internalization of the human sst(2A) receptor. Notably, octreotide and SS-14 stimulated a full phosphorylation and internalization of all mutant sst(2A) receptors tested. Together, these findings suggest that pasireotide activates the sst(2A) receptor via a molecular switch that is structurally and functionally distinct from that turned on during octreotide-driven sst(2A) activation.

Pasireotide and octreotide stimulate distinct patterns of sst2A somatostatin receptor phosphorylation.

Pasireotide (SOM230) is currently under clinical evaluation as a successor compound to octreotide for the treatment of acromegaly, Cushing's disease, and carcinoid tumors. Whereas octreotide acts primarily via the sst(2A) somatostatin receptor, pasireotide was designed to exhibit octreotide-like sst(2A) activity combined with enhanced binding to other somatostatin receptor subtypes. In the present study, we used phophosite-specific antibodies to examine agonist-induced phosphorylation of the rat sst(2A) receptor. We show that somatostatin and octreotide stimulate the complete phosphorylation of a cluster of four threonine residues within the cytoplasmic (353)TTETQRT(359) motif in a variety of cultured cell lines in vitro as well as in intact animals in vivo. This phosphorylation was mediated by G protein-coupled receptor kinases (GRK) 2 and 3 and followed by rapid cointernalization of the receptor and ss-arrestin into the same endocytic vesicles. In contrast, pasireotide failed to promote substantial phosphorylation and internalization of the rat sst(2A) receptor. In the presence of octreotide or SS-14, SOM230 showed partial agonist behavior, inhibiting phosphorylation, and internalization of sst(2A). Upon overexpression of GRK2 or GRK3, pasireotide stimulated selective phosphorylation of Thr356 and Thr359 but not of Thr353 or Thr354 within the (353)TTETQRT(359) motif. Pasireotide-mediated phosphorylation led to the formation of relatively unstable beta-arrestin-sst(2A) complexes that dissociated at or near the plasma membrane. Thus, octreotide and pasireotide are equally active in inducing classical G protein-dependent signaling via the sst(2A) somatostatin receptor. Yet, we find that they promote strikingly different patterns of sst(2A) receptor phosphorylation and, hence, stimulate functionally distinct pools of beta-arrestin.