This project is designed to assess the role of membrane lipid composition, especially polyunsaturated phospholipids, in modulating G protein-coupled receptor (GPCR) signal transduction and to elucidate the mechanism of action of ethanol in these systems. GPCRs 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. 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. This year we completed an examination of various steps in the visual signaling pathway in retinal rod outer segments (ROS) from rats raised on either an n-3 adequate or an n-3 deficient diet. Under these dietary conditions, the n-3 acyl chain, 22:6n-3, is replaced by an n-6 acyl chain, 22:5n-6, in membrane phospholipids. We found that the ROS from n-3 deficient rats have reduced levels of rhodopsin activation, slower receptor G protein complex formation and a 3-fold reduction in sensitivity of the signaling pathway, as measured by PDE activity. In addition we demonstrated that the 22:5n-6 acyl chains are packed in a more ordered array than those of 22:6n-3. The exquisite coupling of membrane physical properties and membrane protein function is demonstrated by the fact that 22:5n-6 and 22:6n-3 differ by only a double bond at carbon nineteen that is present in 22:6n-3, but absent in 22:5n-6. The observed differences in the initial stages of visual signal transduction provide an explanation of the functional changes observed in the electroretinograms of n-3 deficient animals and non-human primates. These studies provide a basis for understanding the visual and cognitive deficits associated with a nutritional deficiency of n-3 fatty acids. In a second study completed this year we extensively examined the role of rhodopsin packing density on bilayer properties and receptor function. Rhodopsin was reconstituted into 18:0,22:6n-3PC bilayers, which mimics the high content of polyunsaturated n-3 acyl chains present in phospholipids in disk membranes, at lipid to protein ratios ranging from 40:1 to 422:1. Increased rhodopsin packing density resulted in: 1) slower motion of the fluorescent membrane probe DPH as indicated by an increased rotational correlation time and decreased rotational diffusion coefficient; 2) altered equilibrium DPH distribution in the lipid bilayer, which resulted in a reduction of membrane acyl chain packing free volume; 3) no detectable change of rhodopsin denaturation transition temperature; 4) reduced level of MII formation is response to light. These results demonstrate that although rhodopsin packing density has a direct influence on the dynamics and packing of lipid bilayers that consequently alter rhodopsin function. They also demonstrate that the thermal stability of rhodopsin is determined by the layer lipids adjacent to the protein, rather than by the acyl chain packing properties of the bulk phospholipids. A continuing collaborative study employing atomic force microscopy (AFM) has led to the observation of lateral domains in reconstituted lipid-rhodopsin bilayers. Using AFM, individual rhodopsin molecules were resolved in native disk membranes. In this study, the presence and role of lateral domains in the visual signaling pathway is being investigated. The dependence of these domains on cholesterol content is also being investigated.
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