REGULATION OF ADENYLYL CYCLASE SIGNALING PATHWAYS

腺苷酸环化酶信号通路的调节

• 批准号: 6606897
• 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/6606897
• 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（根据申请人的摘要改编）：营地的生产是 在许多层面上控制，特别是在其合成水平上 调节腺苷环酶（AC），催化转化的酶 ATP到营地。 AC的激素调节是由受体催化的 依次调节AC活性的异三聚体G蛋白的激活。这 异三聚体G蛋白GS的α亚基刺激所有九种同工型 AC。许多其他监管影响也受到这些影响 酶。例如，AC的心脏同工型被PKC刺激，并且是 被钙，PKA和Gi-Alpha抑制。因此，ACS担任关键作用 各种投入的集成商。交流活动也可以通过新的 欣赏的蛋白质家族，指定的RGS（G蛋白的调节剂 信号）。家庭成员的特征是他们抑制 G蛋白α亚基的活性。没有已知的RGS家庭成员可以调节 GS-Alpha的活性，尽管RGS蛋白可以抑制CAMP的产生 当体内表达时。交流的两个细胞质结构域创造了一个漂亮的 对称酶，在这些结构域的界面上形成催化位点。 申请人已经开发了一个系统，这两个细胞质结构域是 每个人在大肠杆菌中独立表达。简单混合可溶性蛋白 重构全交流活动。最初研究抑制的数据 Gi-Alpha的可溶性AC导致了以下假设。 AC引起构象变化，从而降低了两者的亲和力 彼此的结构域又降低了催化活性。许多 AC的调节剂，包括RGS蛋白，可能会通过 影响两个域之间界面的构象。这 提案将检验这些假设，并确定几种机制 抑制调节器。具体目的是（1）确定动力学机制 Gi-α的介导的AC抑制作用。 （2）检查的结构特征 gi-alpha-ac综合体。 （3）确定RGS抑制AC的机制 蛋白质。