The long-term goal of this research is to elucidate the molecular and cellular mechanisms underlying the effects of ethanol on the cyclic adenosine monophosphate (cAMP) signaling pathway in the central nervous system. The activity of adenylyl cyclase (AC), the enzyme that generates cAMP, is enhanced by pharmacologically relevant concentrations of ethanol in an AC isoform-specific manner. This selectivity indicates that within a cAMP generating system, AC is a primary target of ethanol's action. With previous support from this grant, we have identified three discrete regions of type7 AC (AC7) important for the effect of ethanol on its activity (ethanol responsive domains), as well as the amino acid residues within these regions that are potentially responsible for the enhancing effect of ethanol. We now propose to continue this project by testing the hypothesis that ethanol enhances AC activity by directly interacting with AC molecules at specific binding site(s).
In Specific Aim 1, using a series of mutant AC7s in the cAMP accumulation assay, we will identify crucial amino acid residues in the ethanol responsive domains that are responsible for the effect of ethanol and determine which physicochemical properties of each residue are important.
In Specific Aim 2, we will design and produce recombinant AC7 proteins using a bacterial expression system. We will determine the three dimensional structure of the catalytic domains of AC7 including the ethanol responsive domains, identify key amino acid residues involved in the interaction with ethanol, and examine the effect of ethanol on the conformation of AC7 using NMR spectroscopy. Studies proposed in the two Specific Aims will complement each other to answer the following questions: 1) Which amino acid residues in the ethanol responsive domains are important for ethanol's effect, and what are the locations of these residues in the three dimensional structure of the protein? 2) What are the key residues involved in binding to ethanol, and what are their functional contributions? 3) Does ethanol change the structure and dynamics of the ethanol responsive domains? If so, which amino acid residues are involved in ethanol-induced conformational change(s), and what is the functional contribution of those residues? The knowledge we will obtain is crucial for elucidating the mechanism by which ethanol modulates the activity of AC. The proposed research will provide a rational basis for future drug development targeting AC molecules. The approach employed in this research can also be adapted to the study of other proteins important in the alcohol research field.
Cyclic adenosine monophosphate (cAMP) signal transduction has been postulated to be a contributing factor to the development of and predisposition to alcoholism in humans. The information we will obtain through the proposed research will help us to understand how alcohol acts on biological systems at the molecular level. This type of research is very important for rational drug development for the intervention of alcohol abuse and alcoholism.
|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|
|Yoshimura, Masami; Pearson, Susan; Kadota, Yoichi et al. (2006) Identification of ethanol responsive domains of adenylyl cyclase. Alcohol Clin Exp Res 30:1824-32|
|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|
|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|