Abstract
The role of membrane composition in modulating the rate of G protein-receptor complex formation was examined using rhodopsin and transducin (G(t)) as a model system. Metarhodopsin II (MII) and MII-G(t) complex formation rates were measured, in the absence of GTP, via flash photolysis for rhodopsin reconstituted in 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (18:0,18:1PC) and 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0,22:6PC) bilayers, with and without 30 mol% cholesterol. Variation in bilayer lipid composition altered the lifetime of MII-G(t) formation to a greater extent than the lifetime of MII. MII-G(t) formation was fastest in 18:0,22:6PC and slowest in 18:0,18:1PC/30 mol% cholesterol. At 37 degrees C and a G(t) to photolyzed rhodopsin ratio of 1:1 in 18:0,22:6PC bilayers, MII-G(t) formed with a lifetime of 0.6 +/- 0.06 ms, which was not significantly different from the lifetime for MII formation. Incorporation of 30 mol% cholesterol slowed the rate of MII-G(t) complex formation by about 400% in 18:0,18:1PC, but by less than 25% in 18:0,22:6PC bilayers. In 18:0,22:6PC, with or without cholesterol, MII-G(t) formed rapidly after MII formed. In contrast, cholesterol in 18:0,18:1PC induced a considerable lag time in MII-G(t) formation after MII formed. These results demonstrate that membrane composition is a critical factor in determining the temporal response of a G protein-coupled signaling system.
Highlights
From the Section of Fluorescence Studies, Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, Rockville, Maryland 20852
For samples containing Gt, it was necessary to separate the rate of Metarhodopsin II (MII) formation from that of the MII-Gt complex by characterizing the kinetics of MII formation observed in the absence of Gt
The increase in absorbance corresponding to the formation of MII was analyzed using the two microscopic photoreaction models shown in Fig. 1, which characterize the portion of the rhodopsin photoreaction cascade from the decay of lumirhodopsin to the formation of MII
Summary
Transducin; 22:6n-3, docosahexaenoic acid, or DHA; 22:5n-6, docosapentaenoic acid, or DPA; 18:0,22:6PC, 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine; 18:0,18:1PC, 1-steof G protein activation. The rate of MII-Gt complex formation is governed by a two-dimensional, diffusion-controlled search in the plane of the disc membrane of Gt for photoactivated rhodopsin, in its MII conformational state [6] Previous studies in this laboratory [16] and others [17, 18] demonstrate that the equilibrium concentration of MII is increased by the presence of phospholipids with one or more 22:6n-3 acyl chains. An important aspect of visual signal transduction is rapid response, and studies on rhesus monkeys show that electroretinagram b-wave implicit times [19] and a-wave aroyl-2-oleoyl-sn-glycero-3-phosphocholine; MI, metarhodopsin I; MII, metarhodopsin II; ROS, rod outer segment; ͗͘MII, average lifetime of metarhodopsin II formation; ͗͘MII-Gt, average lifetime mation; KeϪqG, MI-MII equilibrium constant in the of MII-Gt absence complex forof Gt; KeϩqG, apparent MI-MII equilibrium constant in the presence of Gt; TBS, Trisbuffered saline; GTP␥S, guanosine 5Ј-3-O-(thio)triphosphate. The latter process represents the first stage in signal amplification in the visual transduction pathway
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