REGULATION OF ADENYLYL CYCLASE SIGNALING PATHWAYS

描述(改编自申请者摘要)：CAMP的制作是 在许多水平上进行控制，特别是在其合成的水平上 腺酰环化酶(AC)的调节，该酶催化 三磷酸腺苷进入营地。AC的激素调节是由受体催化的 激活异源三聚体G蛋白，进而调节AC活性。这个 异源三聚体G蛋白Gs的α亚基刺激所有九种异构体 交流电。许多其他监管影响也对这些公司产生了影响。 酵素。例如，AC的心脏亚型受到PKC的刺激，并且是 被钙、PKA和GI-α抑制。因此，ACS的关键作用是 不同输入的积分器。交流活动也可以由一个新的 被认可的蛋白质家族，被命名为RGS(G蛋白的调节因子 信令)。家庭成员的特点是他们有能力抑制 G蛋白α亚基的活性。目前还没有已知的RGS家族成员来调节 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 对称的酶，在这些结构域的界面上形成一个催化位置。 申请人已经开发了一种系统，由此这两个细胞质结构域是 每种基因都在大肠杆菌中独立表达。可溶性蛋白质的简单混合 重新构建完整的AC活动。检查抑制作用的初始数据 Gi-α的可溶性AC导致了Gi-α结合的假说 TO 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的调节剂，包括RGS蛋白，可能通过以下方式改变催化活性 影响两个结构域之间界面的构象。这 提案将检验这些假设，并确定以下几个机制 抑制性调节器。具体目标是：(1)确定动力学机制 Gi-α介导的AC抑制作用。(2)考察其结构特点 胃肠-α-交感神经复合体。(3)确定RGS对AC的抑制机制 蛋白质。

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.