Regulated endocytosis and post endocytotic trafficking of somatostatin receptors

5种生长抑素受体（sst1-sst5）的过表达是奥曲肽等稳定的生长抑素类似物用于神经内分泌肿瘤诊断和治疗的分子基础。奥曲肽可控制90%患者激素分泌过多的症状。然而，在一年内，奥曲肽对50%的患者不再有效。奥曲肽主要通过sst2受体发挥作用。目前，有几种后续化合物，包括pasireotide （SOM230）、somatoprim （DG3173）和dopastatin （BIM23A760）正在临床研究中。这些进展是由这些化合物在sst2受体和其他生长抑素受体亚型中发挥奥曲肽样作用的想法驱动的。新开发的多受体生长抑素类似物主要是根据它们的结合谱来表征的。然而，到目前为止，它们激活个体生长抑素受体亚型的能力尚未得到直接评估。在本次项目建议书的准备过程中，我们成功制备了sst2受体的磷酸基特异性抗体。使用这些抗体，我们可以显示，令人惊讶的是，奥曲肽和帕西肽可以在sst2受体上诱导不同的磷酸化模式，从而刺激功能不同的信号通路。利用磷酸基特异性sst2抗体，我们还鉴定出蛋白磷酸酶1 β (pp1 β)是第一个G蛋白偶联受体磷酸酶（GRP）。我们发现pp1 β介导的sst2受体的去磷酸化始于质膜，并在抑制蛋白依赖信号的脱敏中起作用。对于sst5受体，我们也产生了磷酸化特异性抗体，并能够证明其快速磷酸化和去磷酸化分别是由G蛋白偶联受体激酶2 （GRK2）和蛋白磷酸酶1gamma （PP1gamma）介导的。sst3受体是新的泛生长抑素类似物的另一个重要药理靶点。与sst2和sst5受体不同，我们对其依赖激动剂的调控知之甚少。本项目的目的是：1)分析sst2和sst5受体不同去磷酸化的分子机制和功能后果；2)阐明sst3生长抑素受体磷酸化和内化的机制；3)确定新的泛生长抑素类似物的药理学靶点；4)确定受体内化在生长抑素受体靶向诊断和治疗中的作用。

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.