REGULATION OF A CARDIAC SPECIFIC EFFECTOR

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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.