Integrin-Transglutaminase Associated and Cell Adhesion

整合素转谷氨酰胺酶相关和细胞粘附

• 批准号: 6913996
• 负责人: ALEXEY M BELKIN
• 金额: $ 4.28万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-02-01 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6913996
• 关键词: SDS polyacrylamide gel electrophoresis; affinity chromatography; binding sites; biological signal transduction; cell adhesion; cell cell interaction; cell membrane; crosslink; cytoskeleton; enzyme activity; extracellular matrix; fibroblasts; fibronectins; flow cytometry; gene mutation; guanosinetriphosphatases; integrins; laboratory rabbit; myosins; protein binding; protein glutamine gamma glutamyltransferase; western blottings

DESCRIPTION (provided by applicant): Interactions of cells with their neighbors and the surrounding extracellular matrix are critically important for normal physiological and pathological processes. The major goal of this project is to characterize the role of the newly identified integrin-binding adhesion coreceptor, tissue transglutaminase (tTG), in cell adhesion and adhesion-dependent cellular functions, including signaling and assembly of fibronectin (Fn) matrix. A number of deletion and point mutants, as well as chimeric constructs containing swaps with the a subunit of Factor XIII, a structural homologue of tTG which does not interact with integrins or Fn, will be employed to map the integrin- and Fn-binding sites on tTG. Dominant negative tTG mutants which retain integrin binding, but fail to interact with Fn, will be used in various assays designed to analyze the adhesive function of tTG. We will quantitate an increase in adhesion strength generated by integrin-associated surface tTG. A proposed bridging function of tTG in cell adhesion as a mediator of integrin-Fn interaction will be defined. We will test the ability of surface tTG to alter the overall pattern of cell-ECM recognition. The effects of tTG on integrin activation, clustering and cytoskeletal association will be studied. Since our initial data indicate the ability of tTG to alter activation of Rho and Rac GTPases, we will separate and define the roles of cell surface and cytoplasmic tTG in integrin-mediated and direct activation of Rho and Rac GTPases. Then, the contributions of Rho, Rac and their common downstream target, myosin II, to the unique phenotype of cells that express high levels of surface tTG, will be determined. We will also continue to analyze mechanisms of stimulation of Fn assembly by integrin-bound tTG. A hypothesis will be tested that binding of Fn to tTG or dual interaction of Fn with integrins and tTG exposes a cryptic self-assembly site in modules I9III1 of Fn, and thereby stimulates fibril formation. Finally, the effect of association with integrins on the ability of tTG to crosslink Fn will be examined. Together, the proposed experiments will provide novel insights into the role of tTG in cell adhesion, adhesion-mediated signaling and Fn matrix formation. Furthermore, our studies will define how adhesive and signaling functions of integrins are regulated by their association with tTG. Ultimately, this acquired knowledge will advance our understanding of basic mechanisms of cell-matrix interactions.

描述（由申请人提供）：细胞与邻居和周围细胞外基质的相互作用对于正常的生理和病理过程至关重要。该项目的主要目的是表征新鉴定的整联蛋白结合粘附的共受体，组织转谷氨酰胺酶（TTG），在细胞粘附和粘附依赖性细胞功能中的作用，包括信号传导和纤连蛋白（FN）基质的组装。许多缺失和点突变体以及包含与因子XIII亚基的掉期的嵌合构建体，这是TTG的结构同源物，不与整合素或FN相互作用，用于映射TTG上的整合素和FN结合位点。保留整联蛋白结合但无法与FN相互作用的显性负TTG突变体将用于分析TTG的粘附功能的各种测定中。我们将定量通过整联蛋白相关的表面TTG产生的粘附强度的提高。将定义TTG在细胞粘附中提出的桥接功能作为整合素 - FN相互作用的介体。我们将测试表面TTG改变细胞ECM识别的整体模式的能力。将研究TTG对整联蛋白激活，聚类和细胞骨架关联的影响。由于我们的初始数据表明TTG改变了RHO和RAC GTPases的激活的能力，因此我们将分离并定义细胞表面和细胞质TTG在整联蛋白介导的Rho和RAC GTPase中的作用。然后，将确定RHO，RAC及其常见的下游靶标对表达高水平表面TTG的独特表型的贡献。我们还将继续分析整联蛋白结合的TTG刺激FN组装的机制。将检验一个假设，即FN与FN和TTG的FN与TTG的结合或双重相互作用，在FN的模块I9III1中暴露了一个隐秘的自组装位点，从而刺激原纤维形成。最后，将检查与整合素的关联对TTG交联FN能力的影响。共同的实验将提供有关TTG在细胞粘附，粘附介导的信号传导和FN基质形成的作用的新见解。此外，我们的研究将定义整合素的粘合剂和信号传导如何通过其与TTG的关联来调节。最终，这一获得的知识将促进我们对细胞 - 玛底相互作用的基本机制的理解。