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受体的激活，要么刺激AC，要么抑制AC。由此产生的cAMP水平的改变，然后调节cAMP依赖蛋白激酶（PKA）的活性，或其他结合cAMP的蛋白，引发一系列生物作用。心脏中AC的主要形式是V型（ACV）和VI型（ACVI）亚型。最近，我们发现ACV的两个细胞质结构域C1和C2可以分别增加受体偶联G(j)和G(s)调节AC活性的能力。此外，ACV的C2结构域作为G（s- α）的GTPase激活蛋白（GAP）。此外，我们已经发现了一种新的蛋白PAM，它是几种AC亚型的有效抑制剂。因此，本研究的总体目标是确定AC新功能的机制和分子相互作用，阐明PAM抑制AC的机制，并确定PAM在完整细胞中调节AC活性的作用。本应用的具体目的是：(1)阐明ACV的C2和C1结构域分别增强G(s)-和G(j)-偶联受体GEF活性的机制。(2)确定α 3- β 5环和/或G（s- α）的开关II区（与AC的C2结构域接触）上的一个或多个残基的突变是否会改变ACV及其C2结构域(i)作为G(s- α) - gap的能力，以及（II）增强G(s)偶联受体的GEF活性。(3)研究PAM是否与AC相互作用并抑制完整细胞中cAMP的积累。(4)确定PAM是否干扰ACV及其C2结构域作为G(s- α)-GAP的能力，并通过G(s)偶联受体增强信号的启动。cAMP水平的改变调节着一系列生物活动，从心率和收缩力到学习、长期记忆和内分泌功能。因此，阐明ACV调节G蛋白活性的机制将对我们理解cAMP作为第二信使参与的各种生理和病理生理过程的调节具有深远的意义。同样，目前，PAM唯一的生物学功能是它是一种新的有效的AC抑制剂。因此，阐明PAM在完整细胞中调节AC活性的作用及其作用机制将为如何调节AC活性提供新的见解。

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.