REGULATION OF THE ANP RECEPTOR/GUANYLYL CYCLASE

ANP 受体/鸟苷酸环化酶的调节

• 批准号: 6030628
• 负责人: MICHAEL DAVID CHINKERS
• 金额: $ 23.2万
• 依托单位: UNIVERSITY OF SOUTH ALABAMA
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6030628
• 关键词: atrial natriuretic peptide; biological signal transduction; cGMP dependent protein kinase; cell line; chemical binding; conformation; cyclic GMP; enzyme activity; enzyme structure; gene expression; guanylate cyclase; hormone receptor; hormone regulation /control mechanism; immunoprecipitation; laboratory rabbit; molecular site; phosphoprotein phosphatase; phosphorylation; protein kinase C; protein sequence; receptor binding; surface plasmon resonance; transfection

DESCRIPTION: The broad long-term objectives of this proposal are to understand the mechanisms involved in signaling through the cyclic GMP pathway, the normal biological role of PP5, a protein-serine phosphatase that is phosphorylated and activated by cyclic GMP-dependent protein kinase (PKG), and the role of tetratricopeptide repeat (TPR) domains in subcellular targeting. The specific aims are to: i) identify the mechanisms regulating the binding of PP5 to the atrial natriuretic peptide (ANP) receptor/guanylyl cyclase (GC-A); PP5 is targeted to GC-A and appears to mediate GC-A dephosphorylation and desensitization; ii) identify the residues in PP5 that are phosphorylated by PKG and test whether the amino-terminal regulatory domain of PP5 is autoinhibitory; test whether PKG activates PP5 by relieving autoinhibition; iii) determine the structural requirements for PP5 targeting to GC-A. The health relatedness of the project lies in gaining a better understanding of (1) the mechanisms of action of natriuretic peptides that are potential therapeutic agents, and the mechanisms by which responsiveness to these peptides is lost in cardiovascular disease states; (2) of the PKG signaling pathway, which mediates diverse hormonal responses. The experimental plan is: i) to use metabolic labeling and immunoprecipitation to test whether PP5 is phosphorylated by PKG and PKC in intact cells. To perform co-immunoprecipitation experiments to test whether treatments with ANP, TPA, or 8-bromo cyclic GMP increase association of PP5 with GC-A. To use in vitro membrane-binding assays and surface plasmon resonance assays to test effects of PP5 phosphorylation on its binding to the kinase-domain of GC-A; ii) to use phosphoamino acid analysis, phosphopeptide mapping, and peptide sequencing to identify sites on PP5 phosphorylated by PKG. To use deletion mutagenesis and phosphatase assays to test whether the amino terminal regulatory domain of PP5 contains autoinhibitory sequences; iii) to use deletion mutagenesis and in vitro binding assays to identify the minimal structural requirements for binding of the PP5 TPR domain to the GC-A kinase-like domain, to use site directed mutagenesis of full-length PP5 and GC-A, in conjunction with co-immunoprecipitation and desensitization assays, to test the in vivo importance of residues identified as important for binding in vitro.

描述：该提议的广泛长期目标是 了解通过环状GMP信令涉及的机制 途径，PP5的正常生物学作用，一种蛋白质链磷酸酶 通过环状GMP依赖性蛋白激酶激活磷酸化并激活 （PKG），以及四肽重复（TPR）域在亚细胞中的作用 定位。 具体目的是：i）确定调节的机制 PP5与心房NATRIARITE肽（ANP）受体/Guanylyl的结合 环化酶（GC-A）; PP5针对GC-A，似乎介导GC-A 去磷酸化和脱敏； ii）确定PP5中的残基 通过PKG磷酸化并测试是否氨基末端调节 PP5的域是自身抑制性的；测试PKG是否通过缓解来激活PP5 自身抑制； iii）确定PP5靶向的结构要求 到GC-A。 该项目的健康相关性在于获得更好的 理解（1）纳特里尿肽作用机理 是潜在的治疗剂，以及响应能力的机制 这些肽在心血管疾病状态下丢失。 （2）PKG 信号通路，介导各种激素反应。 这 实验计划是：i）使用代谢标签和免疫沉淀 测试PP5是否被完整细胞中的PKG和PKC磷酸化。 到 进行共免疫沉淀实验，以测试是否处理 ANP，TPA或8-Bromo Cyclic GMP与GC-A的关联增加。 到 使用体外膜结合测定法和表面等离子体共振测定 PP5磷酸化对其与激酶结构域结合的测试作用 GC-A; ii）使用磷酸氨基酸分析，磷酸肽图和 肽测序以识别PPG磷酸化的PP5上的位点。 使用 缺失诱变和磷酸酶测定法以测试氨基是否是否 PP5的末端调节结构域包含自抑制序列； iii）to 使用缺失诱变和体外结合测定来识别最小 PP5 TPR域与GC-A结合的结构要求 类似激酶的结构域，使用位点的全长PP5的诱变和 GC-A与共免疫沉淀和脱敏测定法一起 测试在体内重要的残基的重要性 体外结合。