CYCLIC NUCLEOTIDE--RECEPTOR INTERACTIONS

这项研究的长期目标是了解分子 环核苷酸通过以下途径调节细胞功能的机制 与不同的受体相互作用。该项目将建立 CAMP依赖蛋白激酶(cAMP激酶)的cAMP结合域AS 研究环核苷酸-受体相互作用的模型系统 相关蛋白，如cGMP依赖的蛋白激酶(cGMP激酶) 和环核苷酸门控阳离子通道。Ialpha型的特征 将确定哺乳动物cAMP激酶的调节亚单位(RIpha) 这使得它对cAMP结合具有高度的选择性，而不是cGMP 有约束力的。RIpha的羧基末端对蛋白质的贡献 将确定环核苷酸的选择性高亲和力结合。 一个分离的cAMP结合结构域将作为一种 研究环核苷酸受体结构和结构的简单模型 功能。最后，A和B的cAMP/cGMP结合选择性 将描述酵母R亚基的结构域。许多人 建议的实验是基于对分子的比较而设计的 模型、氨基酸序列比对和环核苷酸结合 CAMP-K和cGMP-K环核苷酸结合特性 域名。点突变或截断将被引入到 RIpha cAMP结合区的寡核苷酸诱变 RIpha基因的c DNA。重组R亚基将在大肠杆菌中产生 Coli，纯化并鉴定其环核苷酸结合特性 属性。CAMP激酶中的特定残基(结合cAMP与200- 比cGMP更强的亲和力)将更改为相应的 同源cGMP激酶中的残基，它与cGMP结合270倍 比它绑定cAMP更大的亲和力，最终目标是 将cAMP激酶转变为一种选择性激活的酶 CGMP。分离的RIpha cAMP结合结构域将产生 通过对完整蛋白的蛋白水解性消化，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和cGMP 两者都参与调节心血管功能，详细说明 关于赋予环核苷酸的结构决定因素的知识 对每一种酶的选择性对于合理设计 可能对治疗心血管疾病有用的药物。

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.