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：该提案的广泛长期目标是 了解通过循环 GMP 发出信号所涉及的机制 途径，PP5（一种蛋白丝氨酸磷酸酶）的正常生物学作用 被环 GMP 依赖性蛋白激酶磷酸化和激活 （PKG），以及四三肽重复（TPR）结构域在亚细胞中的作用 瞄准。 具体目标是： i) 确定调节机制 PP5 与心房钠尿肽 (ANP) 受体/鸟苷酸的结合 环化酶（GC-A）； PP5 针对 GC-A 并似乎介导 GC-A 去磷酸化和脱敏； ii) 确定 PP5 中的残基 被 PKG 磷酸化并测试氨基末端调节是否 PP5 的结构域具有自抑制性；测试 PKG 是否通过释放来激活 PP5 自抑制； iii) 确定 PP5 目标的结构要求 至 GC-A。 该项目的健康相关性在于获得更好的 了解 (1) 利尿钠肽的作用机制 是潜在的治疗剂，以及反应性的机制 这些肽在心血管疾病状态下会丢失； (2) PKG 信号通路，介导多种激素反应。 这 实验计划是：i) 使用代谢标记和免疫沉淀 测试完整细胞中 PP5 是否被 PKG 和 PKC 磷酸化。 到 进行免疫共沉淀实验来测试治疗是否 ANP、TPA 或 8-溴环 GMP 增加 PP5 与 GC-A 的关联。 到 使用体外膜结合测定和表面等离子共振测定 测试 PP5 磷酸化对其激酶结构域结合的影响 GC-A； ii) 使用磷酸氨基酸分析、磷酸肽图谱，以及 肽测序以确定 PP5 上被 PKG 磷酸化的位点。 使用 缺失诱变和磷酸酶测定来测试氨基酸是否 PP5的末端调节域含有自抑制序列； iii) 到 使用缺失诱变和体外结合测定来鉴定最小的 PP5 TPR 结构域与 GC-A 结合的结构要求 激酶样结构域，使用全长 PP5 的定点诱变和 GC-A 结合免疫共沉淀和脱敏测定， 测试被确定为重要的残留物的体内重要性 体外结合。