REGULATION OF ADENYLYL CYCLASE

腺苷酸环化酶的调节

基本信息

批准号：
2140687
负责人：
Roger A Johnson
金额：
$ 25.51万
依托单位：
STATE UNIVERSITY NEW YORK STONY BROOK
依托单位国家：
美国
项目类别：
财政年份：
1986
资助国家：
美国
起止时间：
1986-09-01 至 1995-03-31
项目状态：
已结题

项目摘要

Adenylyl cyclase is a family of enzymes that catalyze the formation of cAMP from ATP. Membrane-bound forms of the mammalian enzyme are stimulated or inhibited by hormones via G-protein-linked cell-surface receptors and are inhibited via a cytoplasmic domain ("P"-site) by specific analogs of adenosine. The proposed investigations will use biochemical and physiological approaches to focus on the regulation of adenylyl cyclase via the "P"-site. Experiments will identify the structural requirements for an the locus of "P"-site-specific ligands synthesized by this laboratory will be determined. b) Radioactive affinity probes, 2'5'dd3'FSB[3H]Ado and 2'5'dd3'A[125I]PPA, will be used to tag the "P"-site domain of adenylyl cyclase. c) Labeled peptides will be isolated and sequenced and sequences will be compared with known structures of adenylyl cyclases and other membrane proteins, e.g. transporters or channels, that may have "P"-site consensus sequences. d) Antibodies to these and other selected peptides will be prepared. e) These antibodies will be used to evaluate cloned and expressed enzymes. And f) additional radioactive and fluorescent probes for the "p"-site and catalytic domains will be developed and used to measure interactions and distances between the two binding domains. One of the most important questions with regard to "P"-site-mediated inhibition of adenylyl cyclase is its physiological relevance. Inhibition of adenylyl cyclase that is coordinated with gene expression, RNA processing, or other aspects of nucleic acid metabolism, are attractive possibilities. To ascertain whether and to what extent inhibition of adenylyl cyclase via the "p"-site is regulated physiologically, investigations will: a) determine whether there are naturally occurring "P"-site agonists and/or antagonists more potent than the nucleic acid metabolites 2'd3'AMP and 3'AMP; b) explore whether, under what conditions, and to what extent the levels of 2'd3'AMP and 3"AMP may change in cells (e.g. in response to hormones and to changes in cell growth conditions, especially with conditions known to lower cellular cAMP content or to inhibit adenylyl cyclase); and c) isolate and characterize the enzyme catalyzing formation and degradation of 2'd3'AMP and 3'AMP. The biochemical approaches proposed are doable and will provide a unique handle on adenylyl cyclase. The highest priority will be to identify the sites(s) of inactivation by 2'5'dd3'FSBAdo. It is hoped that a new class of regulator of adenylyl cyclase will be defined, its source, fate, and binding site identified, and that a link will also have been established between regulation of adenylyl cyclase and regulation of nucleic acid metabolism.

腺苷酸环化酶是催化cAMP形成的酶家族来自ATP。刺激哺乳动物酶的膜结合形式或通过G蛋白连接的细胞表面受体抑制激素，为通过细胞质结构域（“ p” - 位）抑制腺苷。拟议的调查将使用生化和通过生理方法专注于调节腺苷环酶的调节 “ P” - 部位。实验将确定一个结构要求该实验室合成的“ p” - 特定于特定的配体的基因座可以确定。 b）放射性亲和力探针，2'5'ddd3'fsb [3h] ado和 2'5'dd3'a [125i] ppa，将用于标记adenylyl的“ p” - 点域循环酶。 c）将分离和测序标记的肽以及序列将与已知的腺苷循环酶和其他已知结构进行比较膜蛋白，例如运输商或通道，可能具有“ p”位置共识序列。 d）这些和其他选定肽的抗体会做好准备。 e）这些抗体将用于评估克隆和表达的酶。 f）其他放射性和荧光探针对于“ P” - 将开发和用于催化域测量两个结合域之间的相互作用和距离。之一关于“ p” - 部位介导的抑制作用的最重要问题腺苷酸环化酶是其生理相关性。抑制adenylyl 与基因表达，RNA加工或其他相协调的环化酶核酸代谢的各个方面是有吸引力的可能性。到确定是否以及在何种程度上抑制腺苷环酶通过 “ P” - 部位受生理调节，调查将：a）确定是否存在天然存在的“ p” - 部位激动剂和/或拮抗剂比核酸代谢产物2'd3'Amp和3'amp更有效； b）探索是否，在什么条件下以及在何种程度上 2'd3'amp和3英寸放大器可能会改变细胞（例如，响应激素和细胞生长条件的变化，特别是在已知的条件下较低的细胞cAMP含量或抑制腺苷酸环化酶）；和c）分离并表征酶催化的形成和降解 2'd3'amp和3'amp。提出的生化方法是可行的，并且将为腺苷循环酶提供独特的手柄。最高优先级将通过2'5'ddd3'fsbado来识别失活的地点。这是希望将定义一类新的adenylyl环酶调节剂它的来源，命运和约束地点已确定，并且链接也将在调节腺苷酸环化酶和调节之间已经建立了核酸代谢。