MLP Assay for Arrestin-AP2 Inhibitors

描述(由申请人提供)：G蛋白偶联受体(GPCR)在人类生理和疾病的几乎每一个方面都发挥着关键作用。Arrestin蛋白在许多这些途径中起着至关重要的作用。Arrestin与GPCRs的磷酸化形式结合，阻止受体与G蛋白结合并激活，导致脱敏。此外，对许多GPCRs来说，arrestin作为内化和“二级”信号转导的适配分子发挥作用。Arrestin通过直接与笼状蛋白和接头AP-2结合的结构域来调节内化。抑制素还可以与激活酶和其他信号蛋白结合。我们最近的结果已经确定了arrestin和AP-2之间的特定分子相互作用，这对于回收某些GPCRs是至关重要的。我们还表明，破坏这种相互作用会导致许多GPCR的快速凋亡途径。到目前为止，还没有已知的小分子来调节arrestin及其相互作用的蛋白质之间的相互作用。我们已经建立了一种高通量的流式细胞术分析AP-2和一个荧光花青素多肽。这项拟议研究的主要目标是确定调控Arrestin和AP-2之间相互作用的化合物，从而调节GPCR运输、信号传递和细胞凋亡。抑制这种相互作用的小分子将是一个有价值的工具，以评估AP-2在可能的数百个GPCR的arrestin依赖的调节中的作用，并可能通过其诱导疾病细胞凋亡的能力代表治疗开发的基础。 与公共卫生相关：受体在人类生物学和疾病的几乎每一个方面都发挥着至关重要的作用。受体的活性和功能是由大量相互作用的蛋白质介导的。最大的一类受体是G蛋白偶联受体(GPCR)家族。Arrestin蛋白在抑制和激活GPCRs信号转导过程中起着关键作用。我们已经确定了arrestin和细胞蛋白AP-2之间的特定相互作用。到目前为止，还没有已知的小分子来调节arrestin及其相互作用的蛋白质之间的相互作用。拟议研究的目标是确定抑制或刺激Arrestin和AP-2之间相互作用的化合物，以提供一个有价值的工具来评估这种相互作用在调节数百个GPCRs中的作用。这种分子将对该领域的研究人员大有裨益，并能够在过度刺激的疾病细胞(如癌细胞)中诱导凋亡，从而找到治疗用途。

DESCRIPTION (provided by applicant): G protein-coupled receptors (GPCR) play a critical role in almost every aspect of human physiology and disease. The protein arrestin plays a vital role in many of these pathways. Arrestin binds to the phosphorylated form of GPCRs and prevents the receptor from binding to and activating G proteins, resulting in desensitization. In addition, for many GPCRs, arrestin functions as an adapter molecule for internalization as well as "secondary" signaling. Arrestin mediates internalization via a domain that binds directly to clathrin and the adapter AP-2. Arrestins also bind to kinases and other signaling proteins. Our recent results have identified a specific molecular interaction between arrestin and AP-2 that is critical for the recycling certain GPCRs. We have also shown that disrupting this interaction induces a rapid apoptotic pathway for many GPCRs. To date there are no small molecules known that regulate interactions between arrestin and its interacting proteins. We have developed a high throughput flow cytometric assay involving AP-2 and a fluorescent arrestin peptide. The primary goal of the proposed research is to identify compounds that regulate the interaction between arrestin and AP-2, resulting in modulation of GPCR trafficking, signaling and apoptosis. A small molecule that inhibits this interaction will be a valuable tool to assess the role of AP-2 in the arrestin-dependent regulation of potentially hundreds of GPCRs and could represent the basis for therapeutic development through its ability to induce apoptosis in disease cells. PUBLIC HEALTH RELEVANCE: Receptors play a vital role in almost every aspect of human biology and disease. Receptor activity and function are mediated by a large collection of interacting proteins. The largest class of receptors is the G protein-coupled receptor (GPCR) family. The protein arrestin plays a critical role in both inhibiting and activating signaling by GPCRs. We have identified a specific interaction between arrestin and the cellular protein AP-2. To date there are no small molecules known that regulate interactions between arrestin and its interacting proteins. The goal of the proposed research is to identify compounds that inhibit or stimulate the interaction between arrestin and AP-2 to provide a valuable tool to assess the role of this interaction in the regulation of hundreds of GPCRs. Such a molecule would be of great benefit for researchers in the field and could find therapeutic utility in its ability to induce apoptosis in hyper stimulated disease cells such as cancer cells.