CYCLIC NUCLEOTIDE--RECEPTOR INTERACTIONS

环核苷酸--受体相互作用

• 批准号: 2187390
• 负责人: JOHN B SHABB
• 金额: $ 8.87万
• 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/2187390
• 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.

这项研究的远距离目标是了解分子 环状核苷酸调节细胞功能的机制 与不同受体的相互作用。 这个项目将建立 依赖营地蛋白激酶（营地激酶）的营地结合域 用于检查环状核苷酸受体相互作用的模型系统 相关蛋白，例如CGMP依赖性蛋白激酶（CGMP激酶） 和环状核苷酸门控阳离子通道。 类型ialpha的功能 将确定哺乳动物营地激酶的调节亚基（Rialpha） 这使得与CGMP相比具有高度选择性 结合。 Rialpha的羧基末端对 将确定循环核苷酸的选择性高亲和力结合。 将开发一个孤立的营地激酶的营地结合域作为一个 用于研究环状核苷酸受体结构和 功能。 最后，A和B的营地/CGMP结合选择性 酵母亚基的域将被表征。 许多 提出的实验是根据分子的比较设计的 模型，氨基酸序列比对和环状核苷酸结合 CAMP激酶和CGMP激酶环状核苷酸结合的特性 域。 点突变或截断将被引入 Rialpha营地结合域通过寡核苷酸指导的诱变 rialpha cDNA。 重组r亚基将在大肠杆菌中产生 大肠杆菌的纯化和特征是其环状核苷酸结合 特性。 营地激酶中的特定残基（该残基与营地与200-结合 折叠比CGMP更大的亲和力将更改为相应的 同源CGMP激酶中的残基，该残基与CGMP结合270倍 比它更大的亲和力使训练营的最终目标 将营地激酶变成被选择性激活的酶 CGMP。 将生成Rialpha的孤立营地结合域 通过完整蛋白的蛋白水解消化或从头表达 重组孤立的营地结合域。 野生型和突变酵母 R亚基将在细菌中产生。 由于营地激酶和CGMP 激酶都参与调节心血管功能，详细 了解赋予环状核苷酸的结构决定因素 对每个激酶的选择性对于合理设计将是无价的 可能可用于治疗心血管疾病的药物。