Rhodopsin-G-protein interactions monitored by resonance energy transfer

Abstract

Resonance energy transfer measurements were implemented to monitor the specific interactions between G-protein and rhodopsin in phospholipid vesicles reconstituted with the purified proteins. Fluorescently labeled G-protein was extracted from bleached rod outer segments (ROS) reacted with several sulfhydryl reagents: N-(1-pyrenyl)maleimide (P), monobromobimane (B), 7-(diethylamino)-3-(4-maleimidylphenyl)-4-methylcoumarin (C), and N-(4-anilino-1-naphthyl)maleimide (A). Limited labeling of ROS, resulting in the modification of less than a single -SH residue per G-protein molecule and less than 0.2 residue per rhodopsin, did not impair the specific in situ interactions between rhodopsin and G-protein. This was demonstrated by preservation of their light-activated tight association and Gpp(NH)p binding and their fast dissociation with excess GTP. The distribution of fluorescent label among the three subunits of G-protein revealed a highly reactive -SH group in the gamma subunit accessible to labeling when G-protein was bound specifically to bleached rhodopsin. Recombination of purified fluorescent derivatives of G-protein with purified rhodopsin reconstituted in lipid vesicles restored the light-activated Gpp(NH)p binding to a level comparable to that measured with unlabeled G-protein. Similar observations were obtained with ROS depleted of peripheral proteins. Likewise, modification of up to two -SH groups per rhodopsin molecule with the fluorescent reagents did not affect the functional recombination of G-protein with rhodopsin in reconstituted lipid vesicles or in depleted ROS. Interactions between rhodopsin and G-protein were monitored by resonance energy transfer measurements, with the following fluorescent conjugates as donor/acceptor couples: P-rhodopsin/C-G-protein, P-rhodopsin/B-G-protein, and P-G-protein/C-rhodopsin.(ABSTRACT TRUNCATED AT 250 WORDS)

Curvature and Hydrophobic Forces Drive Oligomerization and Modulate Activity of Rhodopsin in Membranes

“…FRET has been previously used to study rhodopsin interactions (32)(33)(34), as well as interactions of rhodopsin with transducin (35). For the FRET experiments, we labeled rhodopsin (Rho) at Cys 316 either with the donor Alexa 488 ( Fig.…”

Subunit interactions of GTP‐binding proteins

“…This might reflect immobilization of the fluorescent receptor in liposomes. Borochov-Neori and Montal (1989), who have measured energy transfer between N-(l-pyreny1)maleimidelabelled rhodopsin (or transducin) and monobromobimanetransducin or 7-(diethylamino)-3-(4-maleimidylphenyl)-4-methylcoumarin-labelled transducin (or rhodopsin) in rod outer segment membrane vesicles, have assigned an important role in energy transfer to trapping and immobilization of rhodopsin by lipids.…”

“…However, quantitative information on interactions of subunits with each other and with receptors is scarce. Until now, such interactions have been analyzed, with few exceptions (see Borochov-Neori and Montal, 1989), on the basis of changes in hydrodynamic parameters determined by sucrose gradient centrifugation or biotinylation (Codina et al, 1984;Kohnken and Hildebrandt, 1989). Association -dissociation of G-protein subunits were also inferred indirectly from binding to matrix-bound subunits (Linder et al, 1991).…”

Structural heterogeneity of membrane receptors and GTP‐binding proteins and its functional consequences for signal transduction

“…Recently, fluorescence energy transfer was applied in a study of rhodopsin -G-protein interaction (Borochov-Neori and Montal, 1989). This is also being used in our laboratory (unpublished results) for the study of interactions of G-protein subunits with the fl-adrenoceptor using liposomes reconstituted with pure separate receptor and a-and py-subunits.…”

“…Among them are receptors for catecholamines and cholinergic ligands and biogenic amines such as serotonin and dopamine, receptors for substances K (Masu et al, 1987) andP (Yokota et al, 1989) and luteinizing hormone (Loosfelt et al, 1989;McFarland et a]., 1989) as well as rhodopsin, the light receptor in visual signal transmission (Stryer, 1986) and the receptors for the mating factor of yeast (Dietzel and Kurjan, 1987;Herskowitz and Marsh, 1987) and for the chemotactic CAMP in Dictyostelium discoideum (van Haastert, 1987). This list is incomplete (see also Barnard, 1988;Boulay et al, 1990). Birnbaumer et al (1990) have listed about 80 receptors activated by G-proteins.…”