CYCLIC NUCLEOTIDE--RECEPTOR INTERACTIONS

这项研究的长期目标是了解 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结合结构域， 用于检查环核苷酸-受体相互作用的模型系统 相关蛋白，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 将鉴定哺乳动物cAMP激酶的调节亚基（RI α 这使得它对cAMP结合具有高度选择性， 约束力 RI α的羧基末端对 将测定环核苷酸的选择性高亲和性结合。 将开发一种分离的cAMP激酶的cAMP结合结构域， 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 建议的实验已经设计的基础上比较的分子 模型、氨基酸序列比对和环核苷酸结合 cAMP激酶和cGMP激酶环核苷酸结合特性 域. 点突变或截短将被引入到 RIalpha cAMP结合结构域的体外诱变 RI α cDNA。 重组R亚基将在大肠杆菌中产生 大肠杆菌，纯化并表征其环核苷酸结合 特性. cAMP激酶中的特异性残基（其与200- 亲和力比cGMP大1倍）将被改变为相应的 同源cGMP激酶中的残基，其与cGMP结合270倍 比它结合cAMP的亲和力更大，最终目的是 将cAMP激酶改变为被选择性激活的酶， cGMP。 分离的RIalpha cAMP结合结构域将通过以下方式产生： 通过完整蛋白质的蛋白水解消化，或 重组分离的cAMP结合结构域。 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 激酶都参与调节心血管功能，详细 赋予环核苷酸的结构决定簇的知识 对每种激酶的选择性对于合理设计 治疗心血管疾病的药物。

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.