It is proposed to investigate the hypothesis that ethanol resides at sites on the hydrophobic surface and in inter-helical regions of trans-membrane proteins where it modifies the ability of the protein to change its conformation as part of normal functioning. It is proposed that """"""""sites"""""""" of interaction may be quite common, although few are likely to provide a significant enough interaction to modulate function. The ethanol interaction partly involves hydrophobic interactions but in addition a weakening of a protein-lipid hydrogen bonding network that include water molecules at sensitive regions on the protein is proposed to play a major role. Published work from this laboratory on lipid bilayer membranes with model membrane proteins inserted indicate that ethanol significantly perturbs the phospholipid head group, acyl chain and protein-lipid interfacial regions in membranes by weakening the hydrogen bond network. In addition evidence that chronic-ethanol ingestion leads to adaptive changes in the cell membrane lipids that attenuate this (direct) effect of ethanol has been presented. In this renewal it proposed to test the hypothesis of a link between an ethanol-induced perturbation of the hydrogen bond network and effects on function in seven-transmembrane G-protein coupled receptors (GPCR) which are a large family of proteins that initiate numerous signal transduction cascades in cells. The cDNA for two representative GPCR: the vasopressin-V1A and beta2-adrenergic receptors has been inserted into baculovirus vectors and expressed in Sf9 insect cells and will be affinity purified and reconstituted into defined lipids. The reconstitution will include lipids possessing the chronic ethanol-induced modification, to test the role of perturbation the hydrogen bond network on the agonist- induced trans-membrane inter- and intra-helical conformational changes that leads to G-protein activation. Putative regions that might harbor ethanol """"""""sites"""""""" and that would be likely to lead to functional perturbation will be further delineated using point mutations.
