G protein-coupled receptors (GPCR) are ubiquitous components of signal transduction pathways, including taste, smell, vision, and many neurotransmitter systems. GPCRs are also targets of a great many pharmaceutical drugs. This project is designed to assess the role of membrane lipid composition, especially polyunsaturated phospholipids, in modulating GPCR signal transduction and to elucidate the mechanism of action of ethanol in these systems. The visual transduction pathway of the retinal rod photoreceptor is the best characterized member of this receptor superfamily and is being used as a model system in these studies. System properties under study include: 1. the kinetics and extent of formation of metarhodopsin II (MII), the G protein activating form of activated rhodopsin; 2. MII/G protein complex formation; 3. the rate of G protein activation; 4. cGMP phosphodiesterase (PDE) activation; and 5. the GTPase activity of the G protein. Both functional measures in the transduction pathway and lipid bilayer physical properties are being investigated. Current studies demonstrate that the kinetics and extent of formation of the MII-G protein complex are dependent on both acyl chain formation and cholesterol content. In particular the kinetic of MII-G protein formation is slowed by 50% in 18:0,18:1PC relative to 18:0,22:6PC. The addition of cholesterol doubles the lag time in complex formation in 18:0,18:1PC, whereas the lag time is essentially unchanged upon the addition of cholesterol to 18:0,22:6PC. In other experiments, it is shown that the PDE activity, a measure of the integrated visual pathway function, is also dependent upon acyl chain composition. Here again, 22:6n-3 phospholipids yield the highest levels of activity. A novel method of varying ROS disk membrane cholesterol content was developed which allowed studying the initial steps in the visual signaling pathway over the range of 5 to 45 mole percent cholesterol. Here again, both the kinetics and level of MII and MII-G complex formation were found to be dependent on cholesterol content. Taken together, these studies indicate that 22:6n-3 containing phospholipids enhance the efficiency of a G protein-coupled signaling system. In a recent collaboration, various steps in the visual signaling pathway were studied in ROS from rats raised on n-3 adequate and deficient diets. Under these conditions, 22:6n-3 is replaced by 22:5n-6. We have found that the ROS from n-3 deficient rats were less sensitive to light stimulation than were the ROS from n-3 adequate rats. These studies provide a basis for understanding the visual and cognitive deficits associated with a nutritional deficiency of n-3 fatty acids.
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