Allosteric coupling from G protein to the agonist-binding pocket in GPCRs.

G-protein-coupled receptors (GPCRs) remain the primary conduit by which cells detect environmental stimuli and communicate with each other. Upon activation by extracellular agonists, these seven-transmembrane-domain-containing receptors interact with heterotrimeric G proteins to regulate downstream second messenger and/or protein kinase cascades. Crystallographic evidence from a prototypic GPCR, the ß2-adrenergic receptor (ß2AR), in complex with its cognate G protein, Gs, has provided a model for how agonist binding promotes conformational changes that propagate through the GPCR and into the nucleotide-binding pocket of the G protein α-subunit to catalyse GDP release, the key step required for GTP binding and activation of G proteins. The structure also offers hints about how G-protein binding may, in turn, allosterically influence ligand binding. Here we provide functional evidence that G-protein coupling to the ß2AR stabilizes a 'closed' receptor conformation characterized by restricted access to and egress from the hormone-binding site. Surprisingly, the effects of G protein on the hormone-binding site can be observed in the absence of a bound agonist, where G-protein coupling driven by basal receptor activity impedes the association of agonists, partial agonists, antagonists and inverse agonists. The ability of bound ligands to dissociate from the receptor is also hindered, providing a structural explanation for the G-protein-mediated enhancement of agonist affinity, which has been observed for many GPCR­G-protein pairs. Our data also indicate that, in contrast to agonist binding alone, coupling of a G protein in the absence of an agonist stabilizes large structural changes in a GPCR. The effects of nucleotide-free G protein on ligand-binding kinetics are shared by other members of the superfamily of GPCRs, suggesting that a common mechanism may underlie G-protein-mediated enhancement of agonist affinity.

Reconstitution of G protein-coupled receptors into a model bilayer system: reconstituted high-density lipoprotein particles.

Reconstituted high-density lipoprotein particles (rHDL) are powerful platforms used as a model phospholipid bilayer system to study membrane proteins. They consist of a discoidal-shaped planar bilayer of phospholipids that is surrounded by a dimer of apolipoprotein A-I (apoA-I). The amphipathic nature of apoA-1 shields the hydrophobic acyl chains of the lipids from solvent and keeps the particles soluble in aqueous environments. These monodispersed, nanoscale discoidal HDL particles are approximately 10-11 nm in diameter with a thickness that is dependent on the length of the phospholipid acyl chain. Reconstituted HDL particles can be assembled in vitro using purified apoA-1 and purified lipids. Investigators have utilized this model bilayer system to co-reconstitute membrane proteins, and take advantage of the small size and its monodispersion. Our laboratory and others have utilized the rHDL approach to study the behavior of G protein-coupled receptors. In this chapter, we describe strategies for the preparation of rHDL particles containing GPCRs in their monomeric form and discuss various methodologies used to analyze the reconstituted receptor function.

Ligand-regulated oligomerization of beta(2)-adrenoceptors in a model lipid bilayer.

The beta(2)-adrenoceptor (beta(2)AR) was one of the first Family A G protein-coupled receptors (GPCRs) shown to form oligomers in cellular membranes, yet we still know little about the number and arrangement of protomers in oligomers, the influence of ligands on the organization or stability of oligomers, or the requirement for other proteins to promote oligomerization. We used fluorescence resonance energy transfer (FRET) to characterize the oligomerization of purified beta(2)AR site-specifically labelled at three different positions with fluorophores and reconstituted into a model lipid bilayer. Our results suggest that the beta(2)AR is predominantly tetrameric following reconstitution into phospholipid vesicles. Agonists and antagonists have little effect on the relative orientation of protomers in oligomeric complexes. In contrast, binding of inverse agonists leads to significant increases in FRET efficiencies for most labelling pairs, suggesting that this class of ligand promotes tighter packing of protomers and/or the formation of more complex oligomers by reducing conformational fluctuations in individual protomers. The results provide new structural insights into beta(2)AR oligomerization and suggest a possible mechanism for the functional effects of inverse agonists.