CYTOSKELETAL PROTEINS

细胞骨架蛋白

• 批准号: 2189029
• 负责人: MARY C. BECKERLE
• 金额: $ 24.61万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 1998-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2189029
• 关键词: antibody; antisense nucleic acid; biochemical evolution; biological signal transduction; cell adhesion; cytoskeletal proteins; gene expression; immunocytochemistry; laboratory mouse; laboratory rabbit; microinjections; molecular cloning; nuclear magnetic resonance spectroscopy; protein sequence; protein structure function; site directed mutagenesis; tissue /cell culture; zinc

Cell interactions with extracellular matrix components play important roles in a number of biological processes such as embryonic development, wound healing, and malignant transformation. Our long-term goal is to how the associations of cells with extracellular matrix molecules can stimulate changes in cell morphology, locomotion, growth properties, and differentiation state. The mechanism by which receptor engagement at an adhesive membrane triggers behavioral responses by cells is not understood. However, it is likely that at least some of the proteins involved in transmission of signals to the interior of the cells are concentrated at the cytoplasmic face of the adhesive membrane. Two interactive proteins that have emerged as candidates for participation in signal transduction events at sites of membrane-substratum contact are zyxin and the cysteine rich protein, CRP. Both zyxin and CRP exhibit LIM domains, zinc-binding sequences that are also found in a number of transcription factors and a protooncogene product. We postulate that the LIM domain serves as a protein-binding interface and that the cytoskeletal LIM proteins mediate the assembly of a signal transduction machine at adhesive membranes. Biophysical, biochemical, molecular and cell biological strategies will be employed to probe the structure and function of zyxin and CRP. Spectroscopic approaches and site-directed mutagenesis will be utilized to examine the structural features of the LIM domain. The amino acid residues that participate in zinc coordination will be defined and the importance of the metal for the structure and function of the LIM domain will be evaluated. The results from these experiments will have general impact on our understanding of the properties of the LIM domain, a sequence motif found in a number of proteins involved in signal transduction. Mapping studies will be performed to characterize the associations between zyxin and its two known protein partners, alpha-actinin and CRP and to define the sequences in zyxin and CRP that target them for association with the adhesion plaque and the actin cytoskeleton. An interaction cloning approach will be employed in an effort to define the full complement of zyxin-binding partners. To probe the functions of zyxin and CRP in living cells, the effects of elimination or over-expression of zyxin or CRP sequences on cell behavior will be examined. Finally, an evolutionary comparison of avian, murine and Drosophila zyxin and CRP sequences will highlight the highly conserved and, by implication, functionally important regions of the two proteins. These studies will also lay the foundation for future molecular and genetic analyses of zyxin and CRP function. The broad-based approach proposed in this application will provide insight into the structure and function of the LIM domain as well as the two cytoskeletal proteins that display this zinc-binding motif.

细胞与细胞外基质组件的相互作用很重要 在许多生物学过程中的作用，例如胚胎发育， 伤口愈合和恶性转化。我们的长期目标是如何 细胞与细胞外基质分子的关联可以 刺激细胞形态，运动，生长特性和 分化状态。 受体参与粘合膜的机制 细胞触发行为反应尚不清楚。但是，是 至少有一些涉及传播的蛋白质 通往细胞内部的信号集中在细胞质上 粘合膜的脸部。出现的两个互动蛋白 作为参与信号转导事件的候选人 膜 - 肌肉接触是Zyxin和富含半胱氨酸的蛋白，CRP。 Zyxin和CRP都表现出LIM结构域，锌结合序列是 也发现了许多转录因子和原子量 产品。我们假设LIM结构域用作蛋白质结合 界面和细胞骨架LIM蛋白介导了组装 粘合膜的信号转导机。 生物物理，生化，分子和细胞生物学策略将是 用于探测Zyxin和CRP的结构和功能。 光谱方法和定向诱变将用于 检查LIM域的结构特征。氨基酸残基 参与锌协调的情况将被定义和重要性 金属用于LIM域的结构和功能 评估。这些实验的结果将对 我们对LIM域的性质的理解，一个序列基序 在许多参与信号转导的蛋白质中发现。映射 将进行研究以表征Zyxin之间的关联 及其两个已知的蛋白质伴侣，α-肌动蛋白和CRP，并定义 Zyxin和CRP中的序列将它们靶向与 粘附斑块和肌动蛋白细胞骨架。相互作用克隆 将采用方法来定义完整的补充 Zyxin结合伙伴。探测Zyxin和CRP在生活中的功能 细胞，消除或过表达Zyxin或CRP的影响 将检查有关细胞行为的序列。最后，进化 禽，鼠和果蝇Zyxin和CRP序列的比较将 突出显示高度保守的，并暗示着功能很重要 两种蛋白质的区域。这些研究也将奠定基础 用于Zyxin和CRP功能的未来分子和遗传分析。这 本应用程序中提出的基于广泛的方法将提供洞察力 进入LIM域的结构和功能以及两个 显示此锌结合基序的细胞骨架蛋白。