Protein/lipid Interactions and Signal Transduction

Alcohol Abuse And Alcoholism

Investigators

Linked publications & trials

Abstract

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. In a recent collaboration, various steps in the visual signaling pathway were studied in retinal rod outer segment (ROS) from rats raised on n-3 adequate and deficient diets. Under these conditions, the n-3 acyl chain, 22:6n-3, is replaced by n-6 acyl chain, 22:5n-6, in membrane phospholipids. We have 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 the PDE activity. In addition we have 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 one double bond at carbon nineteen. The observed differences 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. Collaborative efforts employing atomic force microscopy (AFM) have 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.