The long-term goal of this research is to elucidate the molecular and cellular mechanisms underlying effects of ethanol on the cAMP signaling pathway in the central nervous system. cAMP signal transduction has been postulated to play an important role in an animal?s physiological and behavioral responses to ethanol and the development of alcoholism in humans. The activity of adenylyl cyclase (AC), the enzyme which generates cAMP, is enhanced by pharmacologically relevant concentrations of ethanol, and the enhancing effect of ethanol on AC activity is AC isoform-specific. This indicates that within a cAMP generating system, which consists of G protein-coupled receptors, heterotrimeric G proteins, and AC?s, AC is the target of action of ethanol. If this is the case, an ethanol-sensitive AC molecule has a structural element that is a target of action of ethanol. The hypothesis to be tested is that ethanol-sensitive isoforms of AC contain a domain that is responsible for the effect of ethanol on the activity of this enzyme. This structural element will be termed an """"""""ethanol-responsive domain."""""""" To identify the ethanol-responsive domain of AC, chimeras between ethanol-sensitive and ethanol-insensitive isoforms of AC will be generated using a bacterial in vivo recombination system and also site-directed chimera formation based on polymerase chain reaction. Out of these chimeric mutants, only enzymatically active chimera will be isolated using genetic screening with an Escherichia coli strain defective in its AC gene. The effect of ethanol on the activity of a series of the chimeric AC mutants will be examined in mammalian cells to locate the ethanol-responsive domain. To identify important amino acid residues necessary for the effect of ethanol on the activity of AC, mutations will be introduced in this ethanol-responsive domain of the ethanol-sensitive isoform of AC by site-directed mutagenesis, and the effect of ethanol on the mutants will be analyzed. The knowledge we will obtain is crucial for elucidation of the mechanism by which ethanol modulates the activity of AC. The series of mutants of AC that will be generated and analyzed in the proposed study will be a valuable resource for future studies of the structure/function relationships of AC molecules.
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