REGULATION OF ADENYLYL CYCLASE SIGNALING PATHWAYS

腺苷酸环化酶信号通路的调节

• 批准号: 6769990
• 负责人: Carmen W. Dessauer
• 金额: $ 23.92万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6769990
• 关键词: G protein; Sf9 cell line; X ray crystallography; active sites; adenylate cyclase; biological signal transduction; cyclic AMP; enzyme complex; enzyme inhibitors; enzyme mechanism; hormone regulation /control mechanism; isozymes; protein kinase A; protein kinase C

DESCRIPTION (adapted from applicant's abstract): The production of cAMP is controlled at many levels, particularly at the level of its synthesis by regulation of adenylyl cyclase (AC), the enzyme catalyzing the conversion of ATP to cAMP. Hormonal regulation of AC occurs by the receptor-catalyzed activation of heterotrimeric G proteins that in turn regulate AC activity. The alpha subunit of the heterotrimeric G protein Gs stimulates all nine isoforms of AC. Many other regulatory influences are also brought to bear on these enzymes. For example, the cardiac isoforms of AC are stimulated by PKC and are inhibited by calcium, PKA, and Gi-alpha. ACs thus serve critical roles as integrators of diverse inputs. AC activity can also be regulated by a newly appreciated family of proteins, designated RGS (regulators of G protein signaling). Family members are characterized by their ability to dampen the activity of G protein alpha subunits. No known RGS family member regulates the activity of Gs-alpha, although RGS proteins can inhibit the production of cAMP when expressed in vivo. The two cytoplasmic domains of AC create a beautifully symmetrical enzyme, forming a catalytic site at the interface of these domains. The applicant has developed a system whereby these two cytoplasmic domains are each expressed independently in E. coli. Simple mixing of the soluble proteins reconstitutes full AC activity. Initial data examining the inhibition of soluble AC by Gi-alpha has led to the hypothesis that the binding of Gi-alpha to AC induces a conformational change that decreases the affinity of the two domains for each other and in turn decreases catalytic activity. Many regulators of AC, including RGS proteins, may alter catalytic activity by influencing the conformation of the interface between the two domains. This proposal will test these hypotheses and determine the mechanism of several inhibitory regulators. Specific aims are to (1) Determine the kinetic mechanism of Gi-alpha mediated inhibition of AC. (2) Examine the structural features of the Gi-alpha-AC complex. (3) Determine the mechanism of inhibition of AC by RGS proteins.

描述（改编自申请人的摘要）：cAMP 的生产是 在许多层面上进行控制，特别是在其合成层面上 腺苷酸环化酶 (AC) 的调节，该酶催化腺苷酸环化酶的转化 ATP 转化为 cAMP。 AC 的激素调节是由受体催化的 异源三聚体 G 蛋白的激活进而调节 AC 活性。这 异源三聚体 G 蛋白 Gs 的 α 亚基刺激所有九种亚型 交流电。许多其他监管影响也对这些产生影响 酶。例如，AC 的心脏亚型受 PKC 刺激，并且 受钙、PKA 和 Gi-alpha 抑制。因此，AC 发挥着关键作用： 不同输入的集成商。 AC 活动也可以通过新的监管 受赞赏的蛋白质家族，称为 RGS（G 蛋白调节因子） 信号）。家庭成员的特点是有能力抑制 G蛋白α亚基的活性。没有已知的 RGS 家族成员负责监管 Gs-α 的活性，尽管 RGS 蛋白可以抑制 cAMP 的产生 当在体内表达时。 AC 的两个细胞质结构域创造了一个美丽的 对称酶，在这些结构域的界面处形成催化位点。 申请人开发了一种系统，通过该系统这两个细胞质结构域 每个在大肠杆菌中独立表达。可溶性蛋白质的简单混合 重建完整的 AC 活动。检查抑制作用的初始数据 Gi-alpha 可溶性 AC 导致了这样的假设：Gi-alpha 的结合 AC 会引起构象变化，从而降低两者的亲和力 结构域相互影响，进而降低催化活性。许多 AC 的调节因子，包括 RGS 蛋白，可能通过以下方式改变催化活性： 影响两个域之间界面的构象。这 提案将测试这些假设并确定几个机制 抑制性调节剂。具体目标是（1）确定动力学机制 Gi-α 介导的 AC 抑制。 (2) 考察结构特点 Gi-α-AC复合物。 (3)确定RGS抑制AC的机制 蛋白质。