A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein

Abstract

G protein-coupled receptors (GPCRs) respond to a diverse array of ligands, mediating cellular responses to hormones and neurotransmitters, as well as the senses of smell and taste. The structures of the GPCR rhodopsin and several G proteins have been determined by x-ray crystallography, yet the organization of the signaling complex between GPCRs and G proteins is poorly understood. The observations that some GPCRs are obligate heterodimers, and that many GPCRs form both homo-and heterodimers, has led to speculation that GPCR dimers may be required for efficient activation of G proteins. However, technical limitations have precluded a definitive analysis of G protein coupling to monomeric GPCRs in a biochemically defined and membrane-bound system. Here we demonstrate that a prototypical GPCR, the ␤2-adrenergic receptor (␤2AR), can be incorporated into a reconstituted highdensity lipoprotein (rHDL) phospholipid bilayer particle together with the stimulatory heterotrimeric G protein, Gs. Single-molecule fluorescence imaging and FRET analysis demonstrate that a single ␤2AR is incorporated per rHDL particle. The monomeric ␤2AR efficiently activates Gs and displays GTP-sensitive allosteric ligandbinding properties. These data suggest that a monomeric receptor in a lipid bilayer is the minimal functional unit necessary for signaling, and that the cooperativity of agonist binding is due to G protein association with a receptor monomer and not receptor oligomerization.␤2-adrenergic receptor ͉ single-molecule spectroscopy ͉ oligomerization

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].…”

Useful pharmacological parameters for G-protein-coupled receptor homodimers obtained from competition experiments. Agonist–antagonist binding modulation

“…they are not in equilibrium and they cannot be converted into each other. This is possible in artificial sytems such as that described by Whorton et al, [3] but there is evidence that it is not likely to happen in cells.…”

The effect of ligand efficacy on the formation and stability of a GPCR-G protein complex

“…␤ 2 AR requires a lipid bilayer to efficiently couple to Gs. Whorton et al recently showed that purified ␤ 2 AR can be reconstituted into recombinant HDL particles (rHDL) as monomers, and that monomeric ␤ 2 AR couples efficiently to Gs (31). Thus, mB-␤ 2 AR was reconstituted into rHDL, and the response to the agonist isoproterenol (ISO) was determined.…”