REGULATION OF ADENYLYL CYCLASES

描述：许多激素的基本机制， 神经递质、气味物质和趋化因子控制着 健康和疾病是所有真核细胞的基本反应： 这些细胞-细胞信号分子的受体激活异源三聚体 调节效应物的G蛋白，包括腺苷酸环化酶。 成员 腺苷酸环化酶的超家族，包括九种哺乳动物亚型， 以及D. discoideum酶，ACA，共享一个十二跨膜，双 胞质结构域拓扑 我认为酶的活性是 由胞质结构域的“异二聚体”的形成控制。 的 ACA在D. 盘状突细胞已被用于开发一种强大的遗传系统， 分析这些重要信号通路的调节。 随机 诱变和表型筛选将用于分离未偶联的， ACA和哺乳动物II型腺苷酸的组成型活性突变 将选择等位基因特异性第二位点抑制子， 研究腺苷酸环化酶内的分子内相互作用 ”异二聚体。“这些突变将被映射到 二聚体来了解酶是如何被调节的。 一系列相关研究旨在揭示受体（例如 这些趋化因子与G-α（i）（G-α-q）连接，而不是与G-α（i）（G-α-q）连接， G-α激活腺苷酸环化酶。 化学引诱剂和G-α-β - 介导的ACA激活需要快速易位， 来自胞质溶胶的含普列克底物蛋白同源（PH）结构域的蛋白质，CRAC 到细胞膜上。 重新本地化所需的CRAC域 对于ACA的激活，将通过定点突变来描述 在活细胞中使用HA和GFP标记的构建体。 膜结合 将确定CRAC的位点，并通过化学引诱物和 将研究G蛋白。 总体而言，拟议的研究将 提供了深入了解PH结构域在体内的功能以及 腺苷酸环化酶的激活和调节机制。

DESCRIPTION: The basic mechanisms by which many hormones, neurotransmitters, odorants, and chemokines control physiological events in health and disease ar fundamental responses of all eukaryotic cells: The receptors for these cell-cell signaling molecules activate heterotrimeric G-proteins that regulate effectors, including adenylyl cyclases. Members of the superfamily of adenylyl cyclases, including nine mammalian subtypes as well as the D. discoideum enzyme, ACA, share a twelve transmembrane, dual cytoplasmic domain topology. I is proposed that enzyme activity is controlled by formation of a "heterodimer' of the cytoplasmic domains. The critical role that ACA plays in aggregation and differentiation of D. discoideum cells has been exploited to develop a powerful system for genetic analysis of the regulation of these important signaling pathways. Random mutagenesis and phenotypic screening will be used to isolate uncoupled and constitutively active mutations in ACA and mammalian Type II adenylyl cyclases Allele-specific second-site suppressors will be selected to investigate intramolecular interactions within the adenylyl cyclase "heterodimer.' The mutations will be mapped onto structural models of the dimer to understand how the enzymes are regulated. A related series of studies are designed to reveal how receptors, such as thos for chemokines, that are linked to G-alpha(i) (G-alpha-q) rather than G-alpha-activate adenylyl cyclases. Chemoattractant and G-alpha-beta -mediated activation of ACA requires the rapid translocation of the pleckstrin homology (PH)-domain containing protein, CRAC, from the cytosol to the plasma membrane. The domains of CRAC required for its relocalization and for activation of ACA will be delineated by site directed mutagenesis using HA- and GFP-tagged constructs in living cells. The membrane binding site for CRAC will be determined and its regulation by chemoattractants and G-proteins will be investigated. Taken together, proposed studies will provide insights into the functions of PH-domains in vivo as well as into the mechanisms of activation and regulation of adenylyl cyclases.