Alcohol effect on recombinant adenylyl cyclase

Abstract

Research suggests that the cyclic AMP (cAMP) signaling pathway is implicated in the development of alcoholism. Previous work in our laboratory has demonstrated that alcohol enhances the activity of adenylyl cyclase (AC) in an isoform specific manner; human type 7 AC (AC7) is most enhanced by ethanol as measured by cAMP accumulation assay in whole cells. We hypothesize that alcohol enhances the activity of the AC protein by directly interacting with it at a specific site(s) and that alcohol effects on AC can be studied using recombinant AC expressed in bacteria. We have shown that a bacteria‐generated AC7 recombinant consisting of the C1a and C2 domains retains alcohol responsiveness. To examine whether bacteria‐generated recombinant AC proteins maintain isoform specificity of alcohol effects, we expressed in bacteria, and purified recombinant AC proteins carrying the C1a or C2 domains of AC2, AC7, and AC9. Recombinant AC7 C1a and AC7 C2 include the ethanol responsive domains identified in our previous chimeric studies. AC2 and AC9 recombinant proteins were designed to include the corresponding regions. Activity of combined AC7 C1a and AC7 C2 was enhanced by ethanol. Chimeric preparations of AC7 C1a and AC2 C2 or AC9 C1a and AC7 C2 were less responsive to ethanol exposure. This suggests that the isoform specificity of the alcohol effect is maintained in recombinant AC. Supported by NIAAA grant AA13148.