REGULATION OF ADENYLYL CYCLASES

描述：许多荷尔蒙， 神经递质、气味和趋化因子控制着生理事件 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 调节效应器的G蛋白，包括腺苷环化酶。成员： 腺酰环化酶超家族，包括哺乳动物的9个亚型 和盘状芽孢杆菌的酶，ACA一样，共用一个十二跨膜， 胞质结构域拓扑结构。I提出，酶的活性是 由细胞质结构域的“异源二聚体”的形成控制。 ACA在石杉聚集分化过程中起着重要作用。 盘状软骨细胞已经被用来开发一种强大的遗传系统 对这些重要信号通路的调控进行分析。随机 将使用诱变和表型筛选来分离解偶联和 ACA和哺乳动物II型腺苷酸的结构性活性突变 将选择周期酶等位基因特异的第二位点抑制子来 研究腺酰环化酶中的分子内相互作用 这些突变将被映射到结构模型上。 二聚体，以了解酶是如何调节的。 一系列相关的研究旨在揭示受体，如 趋化因子，与G-α(I)(G-α-Q)相连，而不是 G-α-激活腺酰环化酶。化学诱导剂与G-α-β -介导的ACA激活需要快速移位 胞浆中含有Pleckstrin同源(PH)结构域的蛋白CRAC 到质膜上。CRAC重新本地化所需的领域 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标记的结构。膜结合 将确定CRAC的位置，并通过化学诱导剂和 将对G蛋白进行研究。总而言之，拟议的研究将 提供对PH结构域在体内的功能的洞察以及对 腺酰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.