Arrestin interactions with non-receptor binding partners

描述(申请人提供)：G蛋白偶联受体(GPCRs)的信号通过受体磷酸化和arrestin与活性磷酸受体结合来调节。Arrestin结合终止G蛋白介导的信号转导，标记GPCRs进行内化，并经常通过c-Src、ERK1/2和JNK3激活级联将信号重定向到G蛋白非依赖的通路。受体结合和游离形式的arrestin都调节功能并影响多个信号分子在细胞内的定位。这一建议的主要目的是阐明arrestin作为细胞内多蛋白信号复合体的组织者的结构基础。我们建议确定这两种非视觉抑制蛋白的游离态和受体结合态与非受体信号分子c-Src、ERK2、JNK3、上游激酶和泛素连接酶MDM2相互作用的arrestin元件。为此，我们建议使用定点突变、直接结合试验、阻滞素的定点自旋标记和EPR光谱分析，以及活细胞内的转运试验。为了阐明哪些蛋白质直接与arrestin结合，以及Src、ERK2和JNK3与arrestin受体复合体相互作用的功能结果，我们还建议从纯化的组分中重建含有arrestin的“信号小体”，即arrestin-受体复合体。根据arrestin分子的大小及其非受体相互作用伙伴的数量，我们假设这些激酶中的一些(如果不是全部的话)将相互竞争arrestin的结合。我们建议检验这一假设，并测量Src、ERK2和JNK3对游离和受体结合的arrestin的亲和力。识别非受体伙伴的arrestin结合位点将使我们能够构建具有选择性增强或禁用的相互作用位点的arrestin突变体。利用这些突变体进行有针对性的实验操作，我们建议研究这些相互作用在细胞中的生理意义。这些信息将为设计可用作实验和治疗工具的相互作用表面的多肽和小分子模拟物奠定基础。

DESCRIPTION (provided by applicant): The signaling by G protein-coupled receptors (GPCRs) is regulated by receptor phosphorylation and arrestin binding to active phosphoreceptor. Arrestin binding terminates G protein-mediated signaling, tags GPCRs for internalization, and often redirects the signaling to G-protein-independent pathways via c-Src and ERK1/2 and JNK3 activation cascades. Both receptor-bound and free forms of arrestin regulate the function and affect the intracellular localization of multiple signaling molecules. The main objective of this proposal is to elucidate the structural basis of arrestin function as an organizer of multi-protein signaling complexes in the cell. We propose to identify arrestin elements involved in the interactions of both non-visual arrestins in their free and receptor-bound state with non-receptor signaling molecules c-Src, ERK2, JNK3, upstream kinases, and ubiquitin ligase Mdm2. To this end, we propose to use site-directed mutagenesis, direct binding assay, site-directed spin labeling of arrestins and EPR spectroscopy, as well as trafficking assays in living cells. We also propose to reconstruct arrestin-containing "signalosomes", i.e., the arrestin-receptor complexes with protein kinases, from purified components in order to elucidate which proteins bind arrestin directly and what are the functional consequences of the interaction of Src, ERK2, and JNK3 with arrestin-receptor complex. Based on the size of the arrestin molecule and the number of its non-receptor interaction partners, we hypothesize that some (if not all) of these kinases will compete with each other for arrestin binding. We propose to test this hypothesis and measure the affinity of Src, ERK2, and JNK3 for free and receptor-bound arrestin. Identification of arrestin binding sites for non-receptor partners will allow us to construct arrestin mutants with selectively enhanced or disabled interaction sites. Using these mutants for targeted experimental manipulation we propose to study the physiological significance of these interactions in the cell. This information will set the stage for designing peptide and small molecule mimics of the interaction surfaces that can be used as experimental and therapeutic tools.