Regulation of a Cardiac Specific Effector

腺苷酸环化酶（AC）催化ATP转化为cAMP。分别与AC、G（s）和G（j）的刺激性和抑制性GTP结合蛋白偶联的神经递质和激素的受体的激活刺激或抑制AC。cAMP水平的改变调节cAMP依赖性蛋白激酶（PKA）或其他结合cAMP的蛋白质的活性，从而引发许多生物学作用。心脏中AC的主要形式是V型（ACV）和VI型（ACVI）同种型。最近，我们发现ACV的两个胞质结构域，C1和C2，可以增加分别与G（j）和G（s）偶联的受体调节AC活性的能力。此外，ACV的C2结构域作为G（s-alpha）的GT3活化蛋白（GAP）。此外，我们已经确定了一种新的蛋白质，PAM，作为AC的几种亚型的有效抑制剂。因此，本提案的总体目标是确定AC的新功能中所涉及的机制和分子相互作用，阐明PAM抑制AC所涉及的机制，并确定PAM在调节完整细胞中AC活性中的作用。本申请的具体目的是（1）阐明ACV的C2和C1结构域分别增强G（s）-和G（j）-偶联受体的GEF活性的机制。(2)确定α 3-β 5环和/或G（s-α）开关II区（与AC的C2结构域接触）上一个或多个残基的突变是否改变ACV及其C2结构域（i）作为G（s-α）-GAP和（ii）增强G（s）偶联受体的GEF活性的能力。(3)研究PAM是否与AC相互作用并抑制完整细胞中的cAMP积累。(4)确定PAM是否干扰ACV及其C2结构域作为G（s-alpha）-GAP并通过G（s）偶联受体增强信号起始的能力。cAMP水平的改变调节一系列生物学行为，从心率和收缩性到学习、长期记忆和内分泌功能。因此，阐明参与G蛋白活性的调制无环鸟苷的机制将有深远的影响，在我们的理解的各种生理和病理生理过程的调节，涉及cAMP作为第二信使。同样地，目前，可以分配给PAM的唯一生物学功能是它是AC的新的和有效的抑制剂。因此，阐明PAM在调节完整细胞AC活性中的作用及其作用机制将为AC活性如何调节提供新的见解。

Adenylyl cyclase (AC) catalyzes the conversion of ATP to cAMP. The activation of receptors for neurotransmitters and hormones which are coupled to the stimulatory and inhibitory GTP binding proteins of AC, G(s) and G(j), respectively, either stimulate or inhibit AC. The resulting alterations in cAMP levels then modulate the activity of cAMP- dependent protein kinase (PKA), or other proteins that bind cAMP, to elicit a number of biological actions. The predominant forms of AC in the heart are the type V (ACV) and type VI (ACVI) isoforms. Recently, we showed that the two cytoplasmic domains, C1 and C2, of ACV can increase the ability of receptor coupled to G(j) and G(s), respectively, to modulate AC activity. Additionally, the C2 domain of ACV acts as a GTPase activating protein (GAP) for G(s-alpha). Moreover, we have identified a novel protein, PAM, as a potent inhibitor of several isoforms of AC. Therefore, the overall objectives of this proposal are to identify the mechanisms and molecular interactions involved in the novel functions of AC, to elucidate the mechanisms involved in inhibition of AC by PAM, and to determine the role of PAM in modulating AC activity in intact cells. The specific aims of this application are (1) to elucidate the mechanisms by which the C2 and C1 domains of ACV enhance the GEF activity of G(s)- and G(j)- coupled receptors, respectively. (2) To determine whether mutations of one or more residues on alpha3-beta5 loop and/or in switch II regions of G(s- alpha), which contact the C2 domain of AC, alter the ability of ACV and its C2 domain to (i) act as G(s-alpha) -GAPs, and (ii) enhance the GEF activity of G(s) coupled receptors. (3) To investigate if PAM interacts with AC and inhibits cAMP accumulation in intact cells. (4) To determine whether PAM interferes with the ability of ACV and its C2 domain to act as a G(s-alpha)-GAP and augment the onset of signals via G(s) coupled receptors. Alterations in cAMP levels regulate an array of biological actions ranging from heart rate and contractility to learning, long-term memory and endocrine function. Therefore, the elucidation of the mechanisms involved in the modulation of G protein activity by ACV will have profound implications in our understanding of the regulation of a variety of physiological and pathophysiological processes involving cAMP as a second messenger. Likewise, presently, the only biological function that can be assigned to PAM is that it is a novel and potent inhibitor of AC. Thus the elucidation of the role of PAM in modulating AC activity in intact cells and the mechanisms of its action will provide new insights into how AC activity can be regulated.