Influence Of Protein/lipid Interactions On Signal Transduction

Alcohol Abuse And Alcoholism

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Abstract

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. Four projects were initiated this year; 1) Determination of the membrane requirements for rhodopsin to form dimers 2) The effects of membrane phosphlipid composition in determining alcohol partition coefficients 3)An investigation of possible structural specificity in the interaction of rhodopsin and alcohols 4) The ability of a wide variety to support normal rhodopsin structural stability and conformation changes[unreadable] [unreadable] Dimerization and oligomerization of G protein-coupled receptor (GPCR) has become a new paradigm of signal transduction. Rhodopsin is a prototypical member of the GPCR family and often serves as a template in structural and functional studies of other GPCRs. Early studies depicted rhodopsin as rapidly diffusing monomers, while recent studies describe rhodopsin as highly ordered dimers in native disk membranes. The discrepancy between the early monomeric studies and the recent dimeric results is not fully reconciled. Our hypothesis is that the hydrophobic environment, which differed among these studies, modulates the organization of rhodopsin. We investigated the thermal denaturation of rhodopsin in native disk membranes, reconstituted vesicles, and detergent micelles using differential scanning calorimetry (DSC). The transition temperatures and thermal denaturation profiles of rhodopsin differed significantly among these samples. The transition peak was much narrower in native disk membranes than that in reconstituted vesicles or detergent micelles, indicating different levels of cooperativity among these samples. Equilibrium and kinetic thermodynamic analysis determined dimeric structures in native disk membranes and monomeric structures in reconstituted vesicles or detergent micelles. These results suggest a weak association among rhodopsin molecules, which is readily disrupted when it is isolated in detergents or reconstituted in lipid vesicles as seen previously. Our findings could explain most of the seemingly contradictory results in the field. Recent studies have shown that monomeric rhodopsin is capable of interacting with the G protein transducin, and the functional advantage of a rhodopsin dimer has yet to be demonstrated. Future functional studies will be carried out to clarify the functional roles of GPCR dimers/oligomers. [unreadable] [unreadable] Using isothermal titration calorimetry we have established that the membrane-water partition coefficient for alcohol varies with phospholipid composition. Partitioning into the membrane is particularly sensitive to phospholipid headgroup composition. Alcohol partitioning is 4 to 5 times higher in a PC/PS membrane than in a PC/PE membrane and 2 to 3 times than in a pure PC membrane. We have begun to investigate the effects of phospholipid acyl chain composition and find that the presence of DHA acyl chains increases the partition coefficient by a factor of 2 in a PC membrane. We will extend this study to investigate alcohol partitioning in more complex mixtures, with particular emphasis on the changes in membrane composition associated with chronic ethanol exposure, such as increased cholesterol content and reduced levels of polyunsaturated acyl chains.[unreadable] [unreadable] We have used 5 structural isomers of butanol to examine possible structural specificity in the interaction of alcohols with rhodopsin. R and S stereoisomers of 1-butanol have identical effects on both the activating conformation change of rhodopsin and rhodopsins thermal stability. The most structurally compact isomer, tert-butanol, had the smallest effect on these processes, while the most extended isomer, 1-butanol, had the greatest effect on both processes. Selected variation of membrane and alcohol concentrations strongly implies that all effects on protein conformation change and stability are due to alcohol molecules acting at the lipid-protein interface.[unreadable] [unreadable] Due to the continuing challenge of reconstituting purified membrane proteins in quantities sufficient for many for many biophysical techniques, there is increasing use of membrane proteins solubilized in detergents. We analyzed the effects of anionic, cationic, zwitterionic and non-ionic detergents on the conformation changes involved in rhodopsin activation and deactivation, which occur over time scales from microseconds to hundreds of seconds. The fastest conformation changes, associated with formation of the G protein-binding conformation, were significantly distorted by all detergents. The slowest conformation changes, associated with release of ligand, proceeded with native-like kinetics in detergents with a high micelle molecular weight, regardless of detergent ionic character. The results will provide membrane biophysicists with useful guidelines for determining when it is appropriate to study solubilized membrane proteins and which detergents may provide reasonably native conditions.