CYCLIC NUCLEOTIDE--RECEPTOR INTERACTIONS

环核苷酸--受体相互作用

• 批准号: 2187391
• 负责人: JOHN B SHABB
• 金额: $ 8.35万
• 依托单位: UNIVERSITY OF NORTH DAKOTA
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-07-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2187391
• 关键词: Escherichia coli; Saccharomyces cerevisiae; affinity chromatography; affinity labeling; autoradiography; chemical binding; chemical kinetics; chimeric proteins; crystallization; cyclic AMP; cyclic GMP; enzyme activity; enzyme mechanism; enzyme structure; gene deletion mutation; mutant; physical model; polymerase chain reaction; protein kinase A; protein purification; protein sequence; protein structure function; recombinant proteins; site directed mutagenesis

The long range goal of this research is to understand the molecular mechanisms by which cyclic nucleotides regulate cell function through interaction with diverse receptors. This project will establish the cAMP-binding domains of cAMP-dependent protein kinase (cAMP kinase) as model systems for examining cyclic nucleotide-receptor interactions in related proteins such as the cGMP-dependent protein kinase (cGMP kinase) and cyclic nucleotide-gated cation channels. Features of the type Ialpha regulatory subunit (RIalpha) of mammalian cAMP kinase will be identified that make it highly selective for cAMP binding as opposed to cGMP binding. The contribution of the carboxyl terminus of RIalpha to selective high affinity binding of cyclic nucleotides will be determined. An isolated cAMP-binding domain of cAMP kinase will be developed as a simple model for studying cyclic nucleotide receptor structure and function. Finally, the cAMP/cGMP-binding selectivities of the A and B domains of the yeast R subunit will be characterized. Many of the proposed experiments have been designed based on comparisons of molecular models, amino acid sequence alignments, and cyclic nucleotide-binding properties of cAMP kinase and cGMP kinase cyclic nucleotide-binding domains. Point mutations or truncations will be introduced into the RIalpha cAMP-binding domains by oligonucleotide-directed mutagenesis of the RIalpha cDNA. Recombinant R subunits will be produced in Escherichia coli, purified and characterized for their cyclic nucleotide-binding properties. specific residues in cAMP kinase (which binds cAMP with 200- fold greater affinity than cGMP) will be changed to the corresponding residues in the homologous cGMP kinase, which binds cGMP with 270-fold greater affinity than it binds cAMP, with the ultimate objective of changing cAMP kinase into an enzyme that is selectively activated by cGMP. Isolated cAMP-binding domains of RIalpha will be generated either by proteolytic digestion of the intact protein, or de novo expression of a recombinant isolated cAMP-binding domain. Wild-type and mutant yeast R subunits will be produced in bacteria. Since cAMP kinase and cGMP kinase are both involved in regulating cardiovascular function, detailed knowledge of the structural determinants that confer cyclic nucleotide selectivity to each kinase will be invaluable for the rational design of drugs that may be useful in treating cardiovascular disease.

这项研究的长期目标是了解分子 环核苷酸调节细胞功能的机制 与不同受体的相互作用。 该项目将建立 cAMP 依赖性蛋白激酶（cAMP 激酶）的 cAMP 结合域为 用于检查环核苷酸-受体相互作用的模型系统 相关蛋白质，例如 cGMP 依赖性蛋白激酶（cGMP 激酶） 和环核苷酸门控阳离子通道。 Ialpha 型的特点 哺乳动物 cAMP 激酶的调节亚基 (RIalpha) 将被鉴定 这使得它对 cAMP 结合具有高度选择性，而不是 cGMP 绑定。 RIα 羧基末端的贡献 将确定环核苷酸的选择性高亲和力结合。 cAMP 激酶的分离 cAMP 结合域将被开发为 研究环核苷酸受体结构的简单模型 功能。 最后，A 和 B 的 cAMP/cGMP 结合选择性 将表征酵母 R 亚基的结构域。 许多 所提出的实验是基于分子比较而设计的 模型、氨基酸序列比对和环核苷酸结合 cAMP 激酶和 cGMP 激酶环核苷酸结合的特性 域。 点突变或截断将被引入 RIalpha cAMP结合域通过寡核苷酸定向诱变 RIα cDNA。 重组R亚基将在大肠杆菌中生产 大肠杆菌，纯化并表征其环核苷酸结合 特性。 cAMP 激酶中的特定残基（与 200-cAMP 结合） 亲和力比 cGMP 倍大）将更改为相应的 同源 cGMP 激酶中的残基，其结合 cGMP 270 倍 比与 cAMP 结合的亲和力更大，最终目标是 将 cAMP 激酶转变为选择性激活的酶 环鸟苷酸生产规范。 RIalpha 的分离 cAMP 结合域将生成 通过完整蛋白质的蛋白水解消化，或从头表达 重组分离的 cAMP 结合域。 野生型和突变型酵母 R亚基将在细菌中产生。 由于 cAMP 激酶和 cGMP 激酶均参与调节心血管功能，详细 赋予环核苷酸的结构决定因素的知识 对每种激酶的选择性对于合理设计 可能有助于治疗心血管疾病的药物。