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个方面都起着关键作用。蛋白质arrestin在许多这些途径中起着至关重要的作用。Arrestin与GPCR的磷酸化形式结合，并阻止受体与G蛋白结合并激活G蛋白，导致脱敏。此外，对于许多GPCR，抑制蛋白作为衔接分子用于内化以及“次级”信号传导。抑制蛋白通过直接结合网格蛋白和衔接子AP-2的结构域介导内化。抑制蛋白还与激酶和其他信号蛋白结合。我们最近的研究结果已经确定了arrestin和AP-2之间的特定分子相互作用，这对于回收某些GPCR至关重要。我们还表明，破坏这种相互作用会诱导许多GPCR的快速凋亡途径。迄今为止，还没有已知的小分子调节arrestin与其相互作用蛋白之间的相互作用。我们已经开发了一种高通量流式细胞术检测涉及AP-2和荧光抑制肽。拟议研究的主要目标是确定调节arrestin和AP-2之间相互作用的化合物，从而调节GPCR运输，信号传导和细胞凋亡。抑制这种相互作用的小分子将是评估AP-2在潜在数百种GPCR的抑制蛋白依赖性调节中的作用的有价值的工具，并且可以通过其诱导疾病细胞凋亡的能力代表治疗开发的基础。 公共卫生相关性：受体在人类生物学和疾病的几乎每个方面都发挥着至关重要的作用。受体活性和功能由大量相互作用的蛋白质介导。最大的一类受体是G蛋白偶联受体（GPCR）家族。蛋白抑制蛋白在抑制和激活GPCR信号传导中起关键作用。我们已经确定了arrestin和细胞蛋白AP-2之间的特定相互作用。迄今为止，还没有已知的小分子调节arrestin与其相互作用蛋白之间的相互作用。这项研究的目的是确定抑制或刺激arrestin和AP-2之间相互作用的化合物，以提供一种有价值的工具来评估这种相互作用在数百种GPCR调控中的作用。这样的分子将对该领域的研究人员有很大的好处，并且可以在其诱导过度刺激的疾病细胞如癌细胞凋亡的能力中发现治疗效用。

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.