G protein-coupled receptors are ubiquitous components of signal transduction systems. This project is designed to assess the role of polyunsaturated phospholipids in modulating G protein-coupled signal transduction and to elucidate of the mechanism of action of ethanol in these systems. The visual transduction pathway in the rod photoreceptor, a prototypical G protein-coupled system, is being used as a model system. The effect of alcohols and lipid composition on: the kinetics and extent of formation of metarhodopsin II (MII), the G protein activating form of rhodopsin; MII/G protein complex formation; the rate of G protein activation; cGMP phosphodiesterase activation; and the GTPase activity of the G protein are being studied. Along with the functional measures in the transduction pathway, acyl chain packing properties of the phospholipid bilayer are being determined by use of time-resolved fluorescence spectroscopy. The isolation and reconstitution of rhodopsin into bilayers of defined lipid composition has allowed an evaluation of the role of phospholipid acyl chain composition. Short chain alcohols, such as ethanol, promote MII formation, while longer chain alcohols, such as decanol, are inhibitory. Intermediate length alcohols show a smooth transition from excitatory to inhibitory. Phospholipid bilayers containing 22:6n3 acyl chains are more sensitive to the effect of alcohols than are more saturated acyl chain phospholipids. The effects of ethanol, acyl chain composition and cholesterol are well correlated with changes in phospholipid acyl chain packing free volume, as characterized by the time-resolved fluorescence anisotropy behavior of the membrane probe, diphenylhexatriene (DPH). These results demonstrate that 22:6n3 containing phosphoplids are the best promoters of rhodopsin activation and provide unique structural features to the bilayer in the form of lateral domain formation. In addition, bilayers rich in 22:6n3 are least effected by the acyl chain ordering effects of cholesterol, which are found to be inhibitory with respect to rhodopsin activation. The studies described here strongly support a lipid-mediated component in the mechanism of action for alcohols in modulating the activation of a G protein-coupled receptor. Our observations are best explained by a novel phospholipid acyl chain packing model developed in this laboratory, in which the presence of phospholipids like those found in the retina and synaptosomes (e.g., polyunsaturated acyl chains, in mixed saturated-unsaturated and dipolyunsaturated acyl chain phospholipids) leads to the formation of lateral domains or clusters in the surface of the membrane. It would appear that these structural features play an important role in mediating receptor function in membranes containing long chain polyunsaturated phospholipids.
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