FORSKOLIN AND GS ALPHA-SENSITIVE SOLUBLE ADENYL CYCLASE

环磷酸腺苷(CAMP)是一个关键的第二信使，控制多种 现象，包括新陈代谢、基因转录、嗅觉、心脏 速率和内存。环磷酸腺苷是由腺酰环化酶合成的。这个 细胞内cAMP浓度的调节主要是在 综合。许多腺酰环化酶的活性是受控制的。 动态地由各种激素、神经递质和其他 调节分子。这条途径的基本组成部分是 受体、异三聚体G蛋白和腺苷环化酶。自.以来 参与调节cAMP浓度的成分存在缺陷 与几种人类疾病有关，包括甲状腺和脑下垂体 对于肿瘤来说，了解更多关于cAMP的调节是很重要的。 哺乳动物的腺酰环化酶有一个共同的结构，大致包括两个 40 kDa的细胞质结构域，由两个强烈疏水的 伸展一下。计划进行生化和遗传分析，以确定 腺酰环化酶的调节域(S)。我已经证明了一个 含两个连接的腺酰环化酶胞质结构域的构建 (IC/1IIC/2)可以在大肠杆菌中以可溶性酶的形式表达，并且可以 解救缺乏腺苷酰环化酶菌株的分解代谢缺陷 (Delta Cya)。可溶性腺酰环化酶IC/1IIC/2可受 G/S阿尔法和福斯科林。 可溶性腺酰环化酶将作为模型体系来研究 参与G/Sα或相互作用的腺酰环化酶位点(S) 福斯科林。E.Coli Delta CyA分解代谢缺陷的互补 将用于筛选随机突变的IC/1IIC/2表达 获得腺酰环化酶活性或调节的突变体文库 作者：G/S阿尔法或福斯科林。E.Coli Delta的互补失败 CyA将用于筛选失去腺苷的IC/1IIC/2突变体 环化酶活性或由G/Sα或Forsklin调节。敏感度 这些突变酶对G/Sα和Forsklin的影响将被评估并 有趣的突变将通过DNA测序来定位。这个 可溶性腺酰环化酶将被提纯至均一。纯净的 将酶与Forsklin类似物进行光亲和标记，以确定 Forskolin结合部位。腺酰环化酶部位的测定 与G/S Alpha交互将允许操纵 G/Sα与腺酰环化酶某些亚型的相互作用 从而为研究G/Sα的生理作用提供了一种策略 通过给定类型的腺苷传递的中介信号 循环酶。腺酰环化酶Forskolin结合部位的测定 将提供关键信息，以帮助设计更具体和 有效的Forsklin类似物可激活腺酰环化酶。

Cyclic AMP (cAMP) is a key second messenger that controls diverse phenomena, including metabolism, gene transcription, olfaction, heart rate, and memory. Cyclic AMP is synthesized by adenylyl cyclases. The modulation of intracellular cAMP concentration is largely at the level of synthesis. The activities of many adenylyl cyclases are controlled dynamically by a variety of hormones, neurotransmitters, and other regulatory molecules. The fundamental components of this pathway are receptors, heterotrimeric G proteins, and adenylyl cyclases. Since defects in the components involved in regulating cAMP concentration are associated with several human diseases, including thyroid and pituitary tumors, it is important to learn more about the regulation of cAMP. Mammalian adenylyl cyclases have a common structure, including two roughly 40 kDa cytoplasmic domains, punctuated by two intensely hydrophobic stretches. Biochemical and genetic analyses are planned to define the regulatory domain(s) of adenylyl cyclase. I have demonstrated that a construct containing two ligated cytoplasmic domains of adenylyl cyclase (IC/1IIC/2) can be expressed as a soluble enzyme in E. coli and it can rescue the catabolic defect of E. coli strains that lack adenylyl cyclase (delta cya). The soluble adenylyl cyclase, IC/1IIC/2, can be regulated by G/s alpha and forskolin. Soluble adenylyl cyclase will be used as a model system to study the adenylyl cyclase site(s) involved in interactions with G/s alpha or forskolin. Complementation of the catabolic defects of E. coli delta cya will be used to screen the randomly mutagenized IC/1IIC/2 expression library for the mutants that gain adenylyl cyclase activity or regulation by G/s alpha or forskolin. Failure of complementation of E. coli delta cya will be used to screen IC/1IIC/2 mutants that lose the adenylyl cyclase activity or regulation by G/s alpha or forskolin. The sensitivity of these mutant enzymes to G/s alpha and forskolin will be assessed and the interesting mutations will be localized by DNA sequencing. The soluble adenylyl cyclases will be purified to homogeneity. The purified enzyme will be labelled with photoaffinity forskolin analogs to determine the forskolin binding site. Determination of the site of adenylyl cyclase that interacts with G/s alpha will allow for manipulation of the interaction between G/s alpha and certain subtypes of adenylyl cyclase, thus providing a strategy to study the physiological role of the G/s alpha mediated signal that is transmitted through a given type of adenylyl cyclase. Determination of the forskolin binding site of adenylyl cyclase will provide crucial information to aid in the design of more specific and potent forskolin analogs to activate adenylyl cyclases.