FUNCTIONAL CHARACTERIZATION OF BETA 1D INTEGRIN

Beta 1D 整合素的功能表征

• 批准号: 2796409
• 负责人: ALEXEY M BELKIN
• 金额: $ 9.57万
• 依托单位: AMERICAN NATIONAL RED CROSS
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-15 至 2002-09-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2796409
• 关键词: affinity chromatography; antisense nucleic acid; biological signal transduction; cell adhesion; cell cell interaction; cell cycle; cell migration; cytoskeletal proteins; fibroblasts; immunoprecipitation; integrins; laboratory mouse; laboratory rabbit; mitogen activated protein kinase; muscle cells; muscle proteins; myoblasts; myotubes; protein isoforms; protein structure function; protein tyrosine kinase; receptor expression; transfection

The purpose of this project is to analyze functional properties of a novel cytoplasmic domain isoform of beta1 integrin, beta1D, that displaces the beta1A isoform in striated muscles. The major hypothesis is that beta1D integrin provides an enhanced cytoskeletal-matrix association and displays distinct signaling properties in muscle cells. The effect of beta1D integrin transfection into nonmuscle cells, on cell morphology, adhesion, spreading, contractility, cell migration and extracellular matrix assembly will be determined. Muscle cytoskeletal protein(s) interacting with beta1D integrin will be identified by coimmunoprecipitation and affinity chromatography on immobilized beta1D and beta1A cytoplasmic domain peptides or fusion proteins containing the cytoplasmic tails of beta1D and beta1A. Detected interactions of known cytoskeletal protein(s) with the cytoplasmic domain of beta1D will be studied further in vitro by solid phase binding assays with purified cytoskeletal protein(s). Antibodies will be generated against novel cytoskeletal and/or signaling protein(s) interacting with beta1D integrin in muscle cells. Signaling properties of beta1D and beta1A integrin isoforms in muscle cells, such as integrin-mediated tyrosine phosphorylation, activation of pp125 FAK, pp6 src and other protein kinases, will be studied using separated myoblasts and myotubes and adhesion of myotubes specifically via transfected human beta1D or beta1A integrin. Inhibition of beta1D synthesis in differentiating muscle cells by expression of inducible beta1D-specific antisense transcript will define the role of beta1D integrin in myodifferentiation, including myotube growth, sarcomere assembly and muscle contraction. The effect of exogenous beta1D expression on cell cycle progression in myoblasts and normal or oncogene-transformed fibroblasts will be determined. Elucidating the role of beta1D integrin in muscle cells will help to understand how these cells differentiate and fulfill their functions under normal and pathological conditions.

该项目的目的是分析A的功能特性 beta1整合素的新型细胞质结构域同工型Beta1d， 横纹肌肉中的β1a同工型移位。主要假设 是beta1d整合蛋白提供增强的细胞骨架 - 摩托蛋白 关联并在肌肉细胞中显示出不同的信号传导特性。 β1D整合素转染到非肌肉细胞的影响， 细胞形态，粘附，扩散，收缩力，细胞迁移和 将确定细胞外基质组件。 肌肉细胞骨架 与beta1d整合素相互作用的蛋白质将通过 固定的beta1d的共免疫沉淀和亲和力色谱法 和β1a细胞质结构域肽或融合蛋白含有 beta1d和beta1a的细胞质尾巴。检测到的相互作用 已知的细胞骨架蛋白（S）具有beta1d的细胞质结构域 将通过固相结合测定法进一步研究 纯化的细胞骨架蛋白（S）。将生成抗体 新型细胞骨架和/或信号传导蛋白与beta1d相互作用 肌肉细胞中整合素。 beta1d和beta1a的信号传导属性 肌肉细胞中的整合素同工型，例如整联蛋白介导的酪氨酸 磷酸化，PP125 FAK，PP6 SRC和其他蛋白质的激活 激酶将使用分离的成肌细胞和肌管进行研究 通过转染的人beta1d或 beta1a整合素。抑制beta1d合成在分化中 通过表达诱导β1D特异性反义的肌肉细胞 成绩单将定义beta1d整合素在 肌相分化，包括肌管的增长，肌膜组装和 肌肉收缩。外源性beta1d表达对细胞的影响 成肌细胞和正常或癌基因转换的循环进展 将确定成纤维细胞。阐明beta1d整合素的作用 在肌肉细胞中，将有助于了解这些细胞如何区分 并在正常和病理条件下履行其功能。