REGULATION OF ADENYLYL CYCLASE SIGNALING PATHWAYS

描述（改编自申请人的摘要）：cAMP的产生是 控制在许多层面上，特别是在其合成的水平， 腺苷酸环化酶（AC）的调节，该酶催化 ATP到cAMP。AC的激素调节通过受体催化的 激活异源三聚体G蛋白，进而调节AC活性。的 异源三聚体G蛋白Gs的α亚基刺激所有九种亚型 的AC。许多其他监管影响也带来了对这些 内切酶例如，AC的心脏同种型被PKC刺激，并被激活。 被钙PKA和Gi-α抑制因此，AC发挥着关键作用， 不同输入的集成商。AC活性也可以通过一种新的 一个受欢迎的蛋白质家族，命名为RGS（G蛋白调节因子 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。没有已知的RGS家族成员调节 尽管RGS蛋白可以抑制cAMP的产生， 当在体内表达时。AC的两个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形成催化位点。 本申请人已经开发了一种系统，其中这两个胞质结构域是 各自在E.杆菌简单混合可溶性蛋白质 完全恢复AC活动。检查抑制的初始数据 可溶性AC与Gi-alpha的结合导致了这样的假设，即Gi-alpha的结合 AC诱导构象变化，降低两者的亲和力 结构域相互作用，反过来降低催化活性。许多 AC的调节剂，包括RGS蛋白，可以通过以下方式改变催化活性： 从而影响两个畴之间的界面的构象。这 该提案将测试这些假设，并确定几个机制 抑制调节剂。具体目标是：（1）确定动力学机制 Gi-alpha介导的AC抑制。(2)检查的结构特征 Gi-alpha-AC复合物(3)确定RGS抑制AC的机制 proteins.

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.