REGULATION OF A CARDIAC SPECIFIC EFFECTOR

环磷酸腺苷（cAMP）作为第二信使在细胞内起着关键作用。 调节各种生物事件。 在心脏的变化， cAMP水平调节收缩性和心率。酶 腺苷酸环化酶（AC）负责cAMP的合成， 由GTP结合调节蛋白G和G I调节， 分别刺激和抑制其活性。 目前，9种形式 已克隆并鉴定了哺乳动物AC。 其中一些 同种型以组织特异性方式表达。 V AC类型为 在心脏中表达的主要形式。 虽然各种AC 同种型受Ca 2+或Ca 2 +/钙调蛋白的不同调节， 蛋白激酶，所有亚型都受到G蛋白α亚基的刺激， S，G是α。 虽然已经进行了几项研究来确定 G的α区域对AC的刺激很重要 活性，AC上与G的α相互作用的区域仍然存在 未知 此外，我们实验室和其他实验室的最新数据表明， 表明AC分子内分子间相互作用 催化活性所必需的。因此， 该应用程序是为了识别VAC型（心脏）上的区域 特异性），其参与分子内相互作用， 与G的α相互作用 这一目标将通过以下方式实现： 追求 以下具体目标：（I）阐明ACV的区域， 通过采用多种方法与G的α相关联，包括（a） 酵母双杂交测定，（B）重组的、纯化的亚结构域 和活性（可溶性以及全长）形式的ACV，和（c） （II）为了鉴定C1和C2结构域中的区域， （三）相互作用的ACV; ACV上参与分子间作用的区域的重要性 与G的α相互作用以及分子内相互作用， 酶活性的调节。 除了描述AC刺激所涉及的机制之外， 通过G的α，本申请中的研究将提供信息 关于心脏特异性V型的结构组织 AC. 此外，这些发现将在以下方面具有潜在意义： 设计替代的、新的和特定的药理学干预措施 用于治疗心血管异常， 由于心脏收缩力和输出量增加而产生的压力 这是由于心脏中cAMP水平的大幅升高。

Cyclic AMP (cAMP) plays a pivotal role as a second messenger in modulating a variety of biological events. In the heart alterations in cAMP levels modulate contractility and heart rate. The enzyme responsible for synthesis of cAMP, adenylyl cyclase (AC) is intricately regulated by GTP binding regulatory proteins, G's and G I which stimulate and inhibit its activity, respectively. Currently, nine forms of mammalian AC have been cloned and characterized. Some of these isoforms are expressed in a tissue specific manner. The type V AC is a predominant form expressed in the heart. Although the various AC isoforms are differentially regulated by Ca 2+, or Ca2+/calmodulin and protein kinases, all isoforms are stimulated by the alpha-subunit of G s, G's alpha. While several studies have been performed to determine the regions on G's alpha which are important for stimulation of AC activity, the regions on AC which interact with G's alpha remain unknown. Moreover, recent data from our laboratory and those of others suggest that intramolecular interactions within the AC molecule are necessary for catalytic activity. Therefore, the overall objective of this application is to identify regions on thetype V AC (cardiac specific) which are involved in intramolecular interaactions and interactions with G's alpha. This objective will be achieved by pursuing the following specific aims: (I) To elucidate the regions of ACV which associate with G's alpha by employing multiple approaches including (a) the yeast two-hybrid assay, (b) the recombinant, purified, subdomains and active (soluble as well as full-length) forms of ACV, and ( c ) mutagenesis; (II) To identify the regions in the C1 and C2 domains of ACV which interact with each other, (III) To determine the functional importance of the region(s) on ACV involved in intemolecular interactions with G's alpha and in the intramolecular interactions in the modulation of enzyme activity. In addition to delineating the mechanism(s) involved in AC stimulation by G's alpha, studies in this application will provide information concerning the structural organization of the cardiac specific type V AC. Moreover, the findings will be of potential significance in designing alternate, novel, and specific pharmacological interventions for treatment of cardiovascular abnormalities such as high blood pressure which result from increased cardiac contractility and output due to large elevations in cAMP levels in the heart.