Subcellular Organization of Signaling in Smooth Muscle

描述(申请人提供)：这是一项新申请的第二次修订，总体目标是研究调节血管收缩能力的信号通路在个体分化的平滑肌细胞中协调的机制。这一应用将集中于检验两个重要的蛋白激酶PKC和ERK1/2的靶向和激活需要直接或间接地与三种潜在的支架蛋白相互作用的普遍假设：小窝蛋白(Cav)、钙粘蛋白(Cap)和新发现的蛋白质--平滑肌绒毛蛋白(SmAV)。初步和已发表的数据表明，这3个蛋白质参与调节：(1)收缩性，(2.)信令和(3)皮质细胞靶向。其具体目的是验证假设：1)CAP、SmAV和CAV定义离散的皮质细胞结构域；2)SmAV在靶向和激活ERK1/2的过程中作为支架蛋白在平滑肌中发挥作用；3)PKC的磷酸化和激活需要与CAV、CAP和SmAV相互作用；以及4)这种细胞类型中不同的钙依赖和非钙依赖的ERK1/2通路是支架特异性调控的结果。该提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去卷积显微镜、原位光亲和交联、FRET、膜分离、反义和小鼠基因敲除模型、诱饵多肽、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人员或合作者的实验室建立的。预计结果将大大促进我们对支架蛋白在一个集成的相关系统中如何工作的理解。通过对分化的血管细胞进行研究，结果将与心血管疾病直接相关。此外，还将获得与信令社区广泛相关的目标机制方面的新信息。

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.