Regulation of a Cardiac Specific Effector

腺酰环化酶(AC)催化三磷酸腺苷(ATP)转化为cAMP。神经递质和激素受体的激活分别与AC、G(S)和G(J)的刺激性和抑制性GTP结合蛋白偶联，既可以刺激AC，也可以抑制AC。由此产生的cAMP水平的变化随后调节cAMP依赖的蛋白激酶(PKA)的活性，或其他与cAMP结合的蛋白质，从而引发一系列生物学行为。心脏中AC的主要形式是V型(ACV)和VI型(ACVI)异构体。最近，我们发现阿昔洛韦的两个胞质结构域--C_1和C_2可以增强分别与G(J)和G(S)偶联的受体调节AC活性的能力。另外，阿昔洛韦C2结构域是G(S-α)的GTP酶激活蛋白(GAP)。此外，我们还发现了一种新的蛋白质，PAM，它是几种AC异构体的有效抑制因子。因此，本研究的总体目标是确定AC的新功能所涉及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)阐明阿昔洛韦C2和C1区分别增强G(S)和G(J)偶联受体的全球环境基金活性的机制。(2)研究Aβ3-β5环和/或G(S-α)开关II区一个或多个残基与AC的C2结构域的接触突变是否会改变ACV及其C2结构域作为G(S-α)间隙的能力，以及(Ii)增强G(S)偶联受体的全球环境基金活性。(3)研究PAM是否与AC相互作用并抑制完整细胞内cAMP的积聚。(4)确定PAM是否干扰阿昔洛韦及其C2结构域作为G(S-α)间隙的能力，并通过G(S)偶联受体增强信号的起始。CAMP水平的变化调节一系列生物行为，从心率和收缩能力到学习、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多种生理和病理生理过程的调控具有深远的意义。同样，目前，PAM唯一的生物学功能是它是一种新型的、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AC活性的调控提供新的见解。

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.