Subcellular Organization of Signaling in Smooth Muscle

描述（由申请人提供）：这是一个新申请的第二次修订，其总体目标是研究调节血管收缩的信号通路在个体分化平滑肌细胞中协调的机制。该应用程序将重点测试两个重要蛋白激酶PKC和ERK1/2的靶向和激活的一般假设，需要直接或间接地与三种潜在的支架蛋白相互作用：小窝蛋白（CaV），钙钙蛋白（CaP）和新发现的蛋白平滑肌弓绒毛蛋白（SmAV）。初步和已发表的数据表明，这3种蛋白参与调节：(1)收缩性，(2)信号传导和(3)皮质细胞靶向。具体目的是验证以下假设：1)CaP、SmAV和CaV定义了离散的皮质细胞结构域；2) SmAV作为支架蛋白在平滑肌ERK1/2的靶向和激活中起作用；3) PKC的磷酸化和激活需要与CaV、CaP和SmAV相互作用；4)这种细胞类型中不同的ca依赖性和ca非依赖性ERK1/2通路是支架特异性调控的结果。该方案将使用新鲜分离的细胞、器官培养和重组蛋白。技术包括定量共聚焦和反褶积显微镜，原位光亲和交联，FRET，膜分离，反义和小鼠敲除模型，诱饵肽，一系列标准的生化，生理和分子技术，所有这些都是在主要研究者或合作者的实验室建立的。结果有望显著推进我们对支架蛋白如何在一个完整的相关系统中起作用的理解。通过研究分化的血管细胞，结果将与心血管疾病直接相关。此外，将获得与信号学界广泛相关的靶向机制的新信息。

DESCRIPTION (provided by applicant): This is a second revision of a new application with the general goal of investigating the mechanisms by which signaling pathways that regulate vascular contractility are coordinated in individual differentiated smooth muscle cells. The application will focus on testing the general hypothesis that the targeting and activation of two important protein kinases, PKC and ERK1/2, require interaction, directly or indirectly, with three potential scaffold proteins: caveolin (CaV), calponin (CaP) and a newly identified protein, smooth muscle archvillin (SmAV). Preliminary and published data demonstrate the involvement of these 3 proteins in the regulation of: (1.) contractility, (2.) signaling and (3.) cortical cell targeting. The specific aims are to test the hypotheses: 1) that CaP, SmAV and CaV define discrete cortical cellular domains; 2) that SmAV functions as a scaffolding protein in the targeting and activation of ERK1/2 in smooth muscle; 3) that PKC phosphorylation and activation require interaction with CaV, CaP and SmAV; and, 4) that the distinct Ca-dependent and Ca-independent ERK1/2 pathways in this cell type are the result of scaffold-specific regulation. The proposal will use freshly dissociated cells, organ culture and recombinant proteins. Techniques include quantitative confocal and deconvolution microscopy, in situ photoaffinity crosslinking, FRET, membrane fractionation, antisense and murine knockout models, decoy peptides, a range of standard biochemical, physiological and molecular techniques, and all are established in the principal investigator's or collaborator's laboratories. Results are expected to significantly advance our understanding of how scaffold proteins work in an integrated, relevant system. By working on differentiated vascular cells, the results will have direct relevance to cardiovascular disease. Additionally, novel information will be gained on targeting mechanisms that will be of broad relevance to the signaling community.