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.
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