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.

Reassessment of sst(5) somatostatin receptor expression in normal and neoplastic human tissues using the novel rabbit monoclonal antibody UMB-4.

OBJECTIVE: The frequent overexpression of somatostatin receptors (sst) in neuroendocrine tumors provides the molecular basis for the diagnostic and therapeutic application of stable somatostatin analogs. Whereas octreotide acts mainly via the sst(2) receptor, the novel pan-somatostatin analog pasireotide exhibits particular high affinity for the sst(5) receptor. To determine whether a patient is a candidate for octreotide or pasireotide therapy, it is important to evaluate the somatostatin receptor status. However, so far highly specific rabbit monoclonal antibodies have been developed for the sst(2) receptor only (clone UMB-1). METHODS: Here, we have extensively characterized a novel rabbit monoclonal antibody for the human sst(5) receptor (clone UMB-4). In a comparative immunohistochemical study, the expression of sst(5) and sst(2) receptors was assessed using UMB-4 and UMB-1, respectively. RESULTS: Western blot experiments unequivocally demonstrated that UMB-4 selectively detected its cognate sst(5) receptor and did not cross-react with other proteins present in crude tissue homogenates. UMB-4 yielded a highly effective immunostaining of distinct cell populations in formalin-fixed, paraffin-embedded human tissues with a predominance of plasma membrane staining. In the pituitary, sst(5) was present on all growth hormone (GH)- and adrenocorticotropin hormone (ACTH)-producing cells whereas sst(2) was only observed on a subpopulation of GH-positive cells. Consequently, sst(5) was detectable on the majority of GH and ACTH adenomas. In contrast, sst(2) was only seen on GH but not on ACTH adenomas. CONCLUSIONS: The rabbit monoclonal antibodies UMB-4 and UMB-1 will facilitate the assessment of the somatostatin receptor status of human tumors during routine histopathological examinations.