Adenosine is a hormone with localized effects in most, if not all, organ systems. It is released under stress of low energy levels in a cell, resulting in decreased oxygen demand and increased oxygen delivery. The receptors for adenosine(AR) are members of the G protein-coupled receptor (GPCR) superfamily, but little firm biochemical information is available concerning the amino acids that comprise the ligand-binding pocket of the four subtypes of AR, and it is anticipated the the unique structure of adenosine will require a binding pocket distinct from that for monoamines. The widespread effects of adenosine agonists and antagonists serve to illustrate the potential importance of developing subtype-specific agonists and antagonists of AR. The goal of this program is to generate firm biochemical data that can be utilized to develop reliable molecular models of the AR which will facilitate receptor-based design of subtype specific agonists and antagonists of AR. The present studies aim to establish, by the Substituted Cysteine Accessibility Methods (SCAM), which amino acids of the transmembrane spans (TM) of the A1AR are accessible to the aqueous-milieu and, therefore, are positioned within the ligand-binding crevice of the A1AR. This strategy entails substituting cysteines for individual amino acids and determining the reactivity of the cysteines with hydrophilic, lipophobic, cysteine-specific reagents. If the cysteine-specific reagents irreversibly inhibit ligand binding and the presence of agonists and/or antagonists retard the rate of inactivation of the receptor, the cysteine must be positioned within the ligand binding pocket. The periodicity of reactive cysteines will provide insights concerning the structural nature of the TM. Similar methodology will be employed to determine if agonists and xanthine-type antagonists occupy the same binding site, and determine the relative orientations of adenosine and xanthines within the binding site. In a third phase of this program, studies are designed to delineate the three-dimensional arrangement of the TM of the A1AR by determining some of the contact points between the seven TM by combining expression of non-overlapping fragments of the A1AR and cysteine-scanning mutagenesis with disulfide crosslinking to reveal these contact points. These studies should provide insights concerning the mechanism of signal transduction through the receptor protein and in conjunction with data generated from the SCAM studies will allow discrimination between clockwise and counterclockwise bundling of the seven TM. Thus, these studies should provide data that will test currently available computational models of GPCR and allow the molecular modeling of the A1AR based on firm biochemical data that establishes the gross three-dimensional arrangement of the membrane spanning portions of the receptor and the amino acids accessible from the aqueous environment of the ligand binding pocket.
| Dawson, E S; Wells, J N (2001) Determination of amino acid residues that are accessible from the ligand binding crevice in the seventh transmembrane-spanning region of the human A(1) adenosine receptor. Mol Pharmacol 59:1187-95 |
| Scholl, D J; Wells, J N (2000) Serine and alanine mutagenesis of the nine native cysteine residues of the human A(1) adenosine receptor. Biochem Pharmacol 60:1647-54 |
