Regulated endocytosis and post endocytotic trafficking of somatostatin receptors

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The overexpression of five somatostatin receptors (sst1-sst5) is the molecular basis for the use of stable somatostatin analogues such as octreotide in the diagnosis and therapy of neuroendocrine tumors. Octreotide controls the symptoms of hormone overproduction in 90% of patients. However, within a year, octreotide will no longer be effective in 50% of patients. Octreotide exerts its effects predominantly via the sst2 receptor. Currently, there are several successor compounds including pasireotide (SOM230), somatoprim (DG3173) and dopastatin (BIM23A760) under clinical investigation. These developments are driven by the idea that these compounds exert both octreotide-like effects at the sst2 receptor and additional effects at other somatostatin receptor subtypes. The newly developed multireceptor somatostatin analogs have primarily been characterized according to their binding profiles. However, their ability to activate individual somatostatin receptor subtypes has not been directly assessed so far. In the preparation of this project proposal, we have successfully produced phosphosite-specific antibodies for the sst2 receptor. Using these antibodies, we could show that, surprisingly, octreotide and pasireotide can induce distinct phosphorylation patterns at the sst2 receptor that lead to stimulation of functionally distinct signaling pathways. Using phosphosite-specific sst2 antibodies, we were also able to identify protein phosphatase 1beta (PP1beta) as the first G protein-coupled receptor phosphatase (GRP). We showed that the PP1beta-mediated dephosphorylation of the sst2 receptor begins at the plasma membrane and has a function in the desensitization of arrestin-dependent signaling. For the sst5 receptor, we have also generated phosphosite-specific antibodies and were able to show that its rapid phosphorylation and dephosphorylation are mediated by G protein-coupled receptor kinase 2 (GRK2) and protein phosphatase 1gama (PP1gamma), respectively. The sst3 receptor is another important pharmacological target of new pan-somatostatin analogues. In contrast to sst2 and sst5 receptors, little is known abouts its agonist-dependent regulation. The aim of this project proposal is 1) to analyze the molecular mechanisms and functional consequences of the different dephosphorylation of sst2 and sst5 receptors, 2) to elucidate the mechanisms of phosphorylation and internalization of the sst3 somatostatin receptor, 3) to identify the pharmacological targets of new pan-somatostatin analogues, and 4) to determine the role of receptor internalisation during diagnostic and therapeutic somatostatin receptor targeting.