Optimization of Receptor-G Protein Coupling by Bilayer Lipid Composition II

Abstract

The visual transduction system was used as a model to investigate the effects of membrane lipid composition on receptor-G protein coupling. Rhodopsin was reconstituted into large, unilamellar phospholipid vesicles with varying acyl chain unsaturation, with and without cholesterol. The association constant (K a) for metarho-dopsin II (MII) and transducin (G t) binding was determined by monitoring MII-G t complex formation spectro-photometrically. At 20 °C, in pH 7.5 isotonic buffer, the strongest MII-G t binding was observed in 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0,22: 6PC), whereas the weakest binding was in 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (18:0,18:1PC) with 30 mol% cholesterol. Increasing acyl chain unsaturation from 18:0,18:1PC to 18:0,22:6PC resulted in a 3-fold increase in K a. The inclusion of 30 mol% cholesterol in the membrane reduced K a in both 18:0,22:6PC and 18:0,18: 1PC. These findings demonstrate that membrane compositions can alter the signaling cascade by changing protein-protein interactions occurring predominantly in the hydrophilic region of the proteins, external to the lipid bilayer. These findings, if extended to other members of the superfamily of G protein-coupled receptors, suggest that a loss in efficiency of receptor-G protein binding is a contributing factor to the loss of cognitive skills, odor and spatial discrimination, and visual function associated with n-3 fatty acid deficiency. The G protein-coupled motif is a fundamental mode of cell signaling, utilized in vision, taste, olfaction, and a variety of neurotransmitter systems. The receptors for these systems are integral membrane proteins, embedded in a lipid matrix. Neu-ronal and retinal tissues and the olfactory bulb contain high levels of the n-3 polyunsaturated acyl chain derived from do-cosahexaenoic acid (22:6n-3) 1 in their cell membrane phospho-lipids (1, 2). Approximately 50% of the acyl chains in the phospholipids of the ROS disc membrane consist of 22:6n-3 (1). The physiological significance of 22:6n-3 is demonstrated by the impaired visual response (3), learning deficits (2), loss of odor discrimination (4), and reduced spatial learning (5) associated with n-3 fatty acid deficiency. In all cases where acyl chain analysis was carried out, the 22:6n-3 content of membrane phospholipids was dramatically reduced in the n-3-deficient animals where it was replaced by 22:5n-6 (5). These findings suggest that the high levels of 22:6n-3 in membrane phospholipids play a critical role in various membrane-associated signaling pathways. A common thread in several of these processes is the ubiquitous motif of G protein-coupled signaling systems. However, molecular mechanisms linking 22:6n-3 phospholipids with essential physiological functions remain to be clarified. The study described herein aims to elucidate such mechanisms by investigating the effect of membrane lipid composition on G protein-coupled signal transduction. In G protein-coupled systems, the receptor activates an ef-fector protein through the action of a G protein (6). Receptors in this superfamily are integral membrane proteins made up of seven transmembrane helices and their respective connecting loops. In contrast, the G protein and effector proteins are generally peripheral proteins, bound to the membrane by a combination of an isoprenoid chain-lipid bilayer interactions (7, 8) and electrostatic forces (9). The receptor-binding site for the ligand is formed by the transmembrane helices and lies near the midpoint of the membrane; hence, the conformational changes accompanying receptor activation would be expected to have a dependence on the membrane lipid composition. In contrast, the interaction of the G protein with the receptor occurs primary external to the membrane bilayer (10, 11). How the lipid composition might affect the interaction between receptor and G protein external to membrane bilayer is not clear. The visual transduction system is among the best characterized G protein-coupled signaling systems (12) and is used as a model in these studies (13, 14). Light absorption results in the generation of a rapid equilibrium between MI and MII (15), and the active conformation, MII, readily associates with G t , forming the MII-G t complex, which is relatively stable in the absence of GTP (16). The interaction sites on MII involved in binding G t are composed of three cytoplasmic loops formed by the peptide sequence connecting helices III and IV, V and VI, and a putative loop formed by amino acids 310-321, anchored in the bilayer by palmitate groups esterified to Cys-322 and Cys-323 (17-19). Recent structural studies of these loops indicate a level of secondary structure in the form of-helices (20).