MLP Assay for Arrestin-AP2 Inhibitors

描述（申请人提供）：G蛋白偶联受体（GPCR）在人体生理和疾病的几乎每个方面都起着至关重要的作用。蛋白质阻滞蛋白在许多这些途径中起着至关重要的作用。阻滞蛋白与磷酸化形式的gpcr结合，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来说，阻滞蛋白作为内化和“二次”信号传导的适配分子。抑制蛋白通过一个直接与网格蛋白和适配器AP-2结合的域介导内化。抑制素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阻滞蛋白和AP-2之间的特定分子相互作用，这对某些gpcr的回收至关重要。我们还表明，破坏这种相互作用可诱导许多gpcr的快速凋亡途径。到目前为止，还没有已知的小分子可以调节阻滞素与其相互作用的蛋白质之间的相互作用。我们开发了一种涉及AP-2和荧光抑制肽的高通量流式细胞分析方法。该研究的主要目标是确定调节阻滞蛋白和AP-2之间相互作用的化合物，从而调节GPCR的运输、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在抑制蛋白依赖性调节中潜在的数百种gpcr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来代表治疗发展的基础。

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.