Monomeric G protein-coupled receptor rhodopsin in solution activates its G protein transducin at the diffusion limit

Abstract

G protein-coupled receptors mediate biological signals by stimulating nucleotide exchange in heterotrimeric G proteins (G␣␤␥).Receptor dimers have been proposed as the functional unit responsible for catalytic interaction with G␣␤␥. To investigate whether a G protein-coupled receptor monomer can activate G␣␤␥, we used the retinal photoreceptor rhodopsin and its cognate G protein transducin (G t) to determine the stoichiometry of rhodopsin/Gt binding and the rate of catalyzed nucleotide exchange in G t. Purified rhodopsin was prepared in dodecyl maltoside detergent solution. Rhodopsin was monomeric as concluded from fluorescence resonance energy transfer, copurification studies with fluorescent labeled and unlabeled rhodopsin, size exclusion chromatography, and multiangle laser light scattering. A 1:1 complex between light-activated rhodopsin and G t was found in the elution profiles, and one molecule of GDP was released upon complex formation. Analysis of the speed of catalytic rhodopsin/G t interaction yielded a maximum of Ϸ50 Gt molecules per second and molecule of activated rhodopsin. The bimolecular rate constant is close to the diffusion limit in the diluted system. The results show that the interaction of Gt with an activated rhodopsin monomer is sufficient for fully functional Gt activation. Although the activation rate in solution is at the physically possible limit, the rate in the native membrane is still 10-fold higher. This is likely attributable to the precise orientation of the G protein to the membrane surface, which enables a fast docking process preceding the actual activation step. Whether docking in membranes involves the formation of rhodopsin dimers or oligomers remains to be elucidated. enzyme catalysis ͉ nucleotide exchange ͉ light scattering

High-resolution distance mapping in rhodopsin reveals the pattern of helix movement due to activation

“…As in most previous SDSL studies, results presented here were obtained for rhodopsin in solutions of DM, a detergent that apparently retains the structure and functionality of R and R* in the native membrane (14,15,35,36). Fig.…”

Light on the structure of thromboxane A2 receptor heterodimers

“…Nonetheless, the mutant TPs that reached the plasma membrane bound the antagonist [ 3 H]-SQ29,548 with normal affinity, and could be activated by the stable TXA 2 agonist U46619 as efficiently as the wild-type receptors at equal level of expression. These data are consistent with the observation that monomeric GPCRs are the minimal functional unit in G protein activation and that dimerization/oligomerization is not absolutely required for this process [9][10][11][12][13][14]43]. However, the U46619 agonist, stimulated TM1 mutant TPa and TPb with a marked reduction in potency, thus suggesting that dimer formation favors a more efficient signaling complex.…”

“…In spite of the evidence that rhodopsin and the b 2 -adrenergic receptor (b2-AR) activate their cognate G protein in the monomeric form [9][10][11][12] and that supramolecular organizations of rhodopsin [9], neurotensin 1 receptor [13] and leukotriene B 4 receptor (BLT 2 ) [14] reduce G protein coupling, formation of GPCR oligomers in living cells has been widely demonstrated [15][16][17]. Indeed, it was recently inferred that the b 2 -AR exists in dynamic equilibrium between monomeric and higher-order oligomers, with the average size of the oligomer being a tetramer and with inverse agonists promoting higher order oligomerization [15].…”

Single-molecule analysis of fluorescently labeled G-protein–coupled receptors reveals complexes with distinct dynamics and organization

“…Whereas monomeric GPCRs, including rhodopsin (19,20) and the β 2 AR (21), efficiently activate G proteins, several studies suggest that GPCRs can form di-/oligomers (6-10). The exact size and stability of such complexes are largely unknown.…”

“…Similarly, β 1 ARs have been suggested to form either stable (15) or transient interactions (16). Moreover, whereas the di-/oligomerization of family-A GPCRs has been proposed to play roles in receptor trafficking (17) and/or signaling (18), it is apparently not required for receptor function (19)(20)(21). The current uncertainty on these topics calls for new methods capable of directly monitoring the size and stability of GPCR supramolecular complexes at physiological expression levels in living cells.…”