Arrestin interactions with non-receptor binding partners

描述（由申请人提供）：G蛋白偶联受体（gpcr）的信号传导受受体磷酸化和抑制蛋白与活性磷受体结合的调节。阻滞蛋白结合终止G蛋白介导的信号通路，标记gpcr内化，并通过c-Src、ERK1/2和JNK3激活级联将信号重定向到G蛋白非依赖性通路。受体结合和自由形式的阻滞蛋白调节功能并影响多种信号分子的细胞内定位。本提案的主要目的是阐明作为细胞中多蛋白信号复合物组织者的抑制蛋白功能的结构基础。我们建议确定在自由和受体结合状态下与非受体信号分子c-Src、ERK2、JNK3、上游激酶和泛素连接酶Mdm2相互作用的非视觉抑制素元件。为此，我们建议在活细胞中使用定点诱变、直接结合实验、定点自旋标记阻滞和EPR光谱，以及运输实验。我们还建议从纯化的组分中重建含有抑制素的“信号体”，即与蛋白激酶结合的抑制素受体复合物，以阐明哪些蛋白质直接结合抑制素，以及Src、ERK2和JNK3与抑制素受体复合物相互作用的功能后果。根据阻滞蛋白分子的大小和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）将相互竞争以结合阻滞蛋白。我们建议验证这一假设，并测量Src、ERK2和JNK3对自由和受体结合阻滞的亲和力。鉴定非受体伙伴的阻滞蛋白结合位点将使我们能够构建具有选择性增强或禁用相互作用位点的阻滞蛋白突变体。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。这一信息将为设计可作为实验和治疗工具的相互作用表面的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.