STRUCTURAL DETERMINANTS OF CFTR/SYNTAXIN INTERACTIONS

CFTR/突触融合蛋白相互作用的结构决定因素

• 批准号: 6354719
• 负责人: KEVIN L KIRK
• 金额: $ 15.34万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6354719
• 关键词: Xenopus oocyte; apical membrane; binding proteins; cellular polarity; chimeric proteins; chloride channels; cystic fibrosis; enzyme activity; enzyme induction /repression; fluorescent dye /probe; gastrointestinal epithelium; immunoprecipitation; intermolecular interaction; mutant; protein kinase A; protein kinase C; protein sequence; protein structure function; receptor binding; syntaxin; tissue /cell culture; transfection; western blottings

The broad goal of this application is to define the physiological relevance and the structural basis of the regulation of CFTR Cl- channels by syntaxin 1A. Syntaxin 1A is a membrane protein that is expressed in colonic epithelial cells where it localizes at or near the apical cell surface. Syntaxin 1A physically interacts with CFTR and negatively modulates CFTR C1- currents when these molecules are co- expressed in Xenopus oocytes. Our unpublished results indicate that the physical interaction between syntaxin 1A and CFTR is inhibited by PKA phosphorylation and by n-Sec1, a syntaxin-binding protein that reverses the negative modulation of CFTR by syntaxin 1A. We hypothesize that syntaxin 1A fine tunes CFTR C1- current activity in response to physiological clues (e.g., PKA activation). The specific aims of this proposal are as follows. First, we will test the hypothesis that the CFTR-syntaxin 1A interaction is regulated by physiologically relevant factors; namely PKA, PKC and n-Sec1. PKC is of interest because it phosphorylates n-Sec1 and inhibits its ability to bind syntaxin 1A; thus, PKC activation may release syntaxin 1A from n-Sec1 and thereby control the availability of syntaxin 1A for CFTR. Second, we will define the minimal domains of CFTR and syntxin 1A that are required for binding and test the functional activities of mutants that are defective at binding. One of our long-term goals is to generate functionally active CFTR mutants that cannot be negatively modulated by syntaxin 1A in vivo. Third, we will test the hypothesis that CFTR and syntaxin 1A co- reside in a molecular complex at the apical surfaces of colonic epithelial cells. We will determine if syntaxin 1A co-immunoprecipitates with surface-labeled CFTR. We will also determine if CFTR resides with syntaxin 1A in a multimeric complex that includes additional candidate regulators of C1- current activity. The results of our studies should provide new insights into the regulation of CFTR function in epithelial tissues.

该应用的广泛目标是定义生理 相关性和调节CFTR Cl-的结构基础 通过语法1a的通道。 语法1a是一种膜蛋白，是 在结肠上皮细胞中表达，该细胞位于或附近 顶部细胞表面。 语法1a与CFTR物理相互作用，并且 当这些分子是共同的时，负面调节CFTR C1-电流 在爪蟾卵母细胞中表达。 我们未发表的结果表明 语法1a和CFTR之间的物理相互作用受到抑制 PKA磷酸化和通过N-SEC1（一种语法结合蛋白）N-SEC1 通过语法1a逆转CFTR的负调制。 我们 假设语法1A微调CFTR C1-当前活动 对生理线索的反应（例如PKA激活）。 具体目标 该提案如下。 首先，我们将检验以下假设 CFTR-Syntaxin 1a相互作用受生理相关的调节 因素；即PKA，PKC和N-SEC1。 PKC很感兴趣，因为它 磷酸化N-SEC1并抑制其结合语法1a的能力；因此， PKC激活可以从N-SEC1释放语法1a，从而控制 CFTR的语法1a的可用性。 第二，我们将定义 结合所需的CFTR和Syntxin 1a的最小域 并测试有缺陷的突变体的功能活动 结合。 我们的长期目标之一是产生功能活跃 CFTR突变体不能被语法1a负面调节 体内。 第三，我们将检验CFTR和语法1A共同的假设 驻留在结肠上皮的顶端表面的分子复合物中 细胞。 我们将确定语法1A是否共免疫沉淀 表面标记的CFTR。 我们还将确定CFTR是否驻留在 多聚体综合体中的语法1a，包括其他候选者 C1-电流活动的调节剂。 我们的研究结果应该 提供有关上皮中CFTR功能调节的新见解 组织。