Subcellular Organization of Signaling in Smooth Muscle

平滑肌信号传导的亚细胞组织

• 批准号: 7141512
• 负责人: KATHLEEN G MORGAN
• 金额: $ 49.75万
• 依托单位: BOSTON BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7141512
• 关键词: biological signal transduction; calcium flux; calponin; caveolins; confocal scanning microscopy; enzyme activity; ferrets; fluorescence resonance energy transfer; genetically modified animals; intracellular; laboratory mouse; membrane proteins; mitogen activated protein kinase; organ culture; phosphorylation; protein kinase C; protein protein interaction; protein structure function; recombinant proteins; vascular smooth muscle

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.

描述（由申请人提供）：这是新申请的第二次修订，总体目标是研究调节血管收缩性的信号通路在个体分化的平滑肌细胞中协调的机制。该申请将重点测试一般假设，即两种重要蛋白激酶 PKC 和 ERK1/2 的靶向和激活需要与三种潜在支架蛋白直接或间接相互作用：小窝蛋白 (CaV)、钙调蛋白 (CaP) 和新发现的蛋白质，平滑肌 Archvillin (SmAV)。初步和已发表的数据表明这 3 种蛋白质参与调节：(1.) 收缩性、(2.) 信号传导和 (3.) 皮质细胞靶向。具体目的是测试以下假设：1）CaP、SmAV 和 CaV 定义离散的皮质细胞域； 2) SmAV 在平滑肌中 ERK1/2 的靶向和激活中充当支架蛋白； 3) PKC 磷酸化和激活需要与 CaV、CaP 和 SmAV 相互作用； 4) 该细胞类型中独特的 Ca 依赖性和 Ca 非依赖性 ERK1/2 途径是支架特异性调节的结果。该提案将使用新鲜分离的细胞、器官培养物和重组蛋白。技术包括定量共焦和反卷积显微镜、原位光亲和交联、FRET、膜分级分离、反义和小鼠敲除模型、诱饵肽、一系列标准生化、生理和分子技术，所有这些技术都是在主要研究者或合作者的实验室中建立的。预计结果将显着增进我们对支架蛋白如何在集成的相关系统中发挥作用的理解。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，还将获得有关与信号传递界广泛相关的靶向机制的新信息。