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.

Agency
National Institute of Health (NIH)
Institute
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Type
Research Project (R01)
Project #
5R01AA013148-02
Application #
6622055
Study Section
Alcohol and Toxicology Subcommittee 4 (ALTX)
Program Officer
Sorensen, Roger
Project Start
2002-03-01
Project End
2007-02-28
Budget Start
2003-03-01
Budget End
2004-02-29
Support Year
2
Fiscal Year
2003
Total Cost
$228,250
Indirect Cost
Name
University of Colorado Denver
Department
Pharmacology
Type
Schools of Medicine
DUNS #
041096314
City
Aurora
State
CO
Country
United States
Zip Code
80045
Qualls-Creekmore, Emily; Gupta, Ratna; Yoshimura, Masami (2017) The effect of alcohol on recombinant proteins derived from mammalian adenylyl cyclase. Biochem Biophys Rep 10:157-164
Al-Bagdadi, Fakhri; Young, Matthew J; Geaghan, James P et al. (2016) Observation on the ultrastructure morphology of HeLa cells treated with ethanol: Statistical analysis. Ultrastruct Pathol 40:324-332
Hill, Rebecca A; Xu, Wu; Yoshimura, Masami (2016) Role of an adenylyl cyclase isoform in ethanol's effect on cAMP regulated gene expression in NIH 3T3 cells. Biochem Biophys Rep 8:162-167
Gupta, Ratna; Qualls-Creekmore, Emily; Yoshimura, Masami (2013) Real-time monitoring of intracellular cAMP during acute ethanol exposure. Alcohol Clin Exp Res 37:1456-65
Dokphrom, Usa; Qualls-Creekmore, Emily; Yoshimura, Masami (2011) Effects of alcohols on recombinant adenylyl cyclase type 7 expressed in bacteria. Alcohol Clin Exp Res 35:1915-22
Hasanuzzaman, Mohammad; Yoshimura, Masami (2010) Effects of straight chain alcohols on specific isoforms of adenylyl cyclase. Alcohol Clin Exp Res 34:743-9
Kou, Jinghong; Yoshimura, Masami (2007) Isoform-specific enhancement of adenylyl cyclase activity by n-alkanols. Alcohol Clin Exp Res 31:1467-72
Hines, Lisa M; Hoffman, Paula L; Bhave, Sanjiv et al. (2006) A sex-specific role of type VII adenylyl cyclase in depression. J Neurosci 26:12609-19
Yoshimura, Masami; Pearson, Susan; Kadota, Yoichi et al. (2006) Identification of ethanol responsive domains of adenylyl cyclase. Alcohol Clin Exp Res 30:1824-32
Nelson, Eric J; Hellevuo, Kaisa; Yoshimura, Masami et al. (2003) Ethanol-induced phosphorylation and potentiation of the activity of type 7 adenylyl cyclase. Involvement of protein kinase C delta. J Biol Chem 278:4552-60