Structural Basis for Allosteric Coupling Between G Protein and the Agonist‐Binding Pocket in GPCRs

G protein‐coupled receptors (GPCRs) translate extracellular ligand binding into intracellular signaling responses via allosteric communication between two distant binding sites. For many GPCRs, allosteric communication between agonists and G proteins has been observed in the form of G protein‐mediated enhancement of agonist affinity. However, the mechanism by which G proteins allosterically regulate a receptor's orthosteric ligand binding site has remained unclear. Based on recent crystal structures of the β 2 ‐adrenergic receptor (β 2 AR), we hypothesized that stabilization of an active receptor conformation by nucleotide‐free G protein leads to closing of the receptor's orthosteric site around a bound agonist, thus slowing agonist dissociation and enhancing affinity. Using purified receptors reconstituted into high‐density lipoprotein (rHDL) particles, we performed equilibrium and kinetic radioligand binding assays to characterize the allosteric effects of G protein and G protein‐mimetic nanobodies on the ligand‐binding properties of multiple GPCRs. We also investigated the contribution of individual residues within β 2 AR in mediating high‐affinity agonist binding. When β 2 AR was bound to nucleotide‐free Gs heterotrimer, we observed slowed association of the antagonist [ 3 H]DHAP, which was reversible by GDP or GTPγS. A Gs‐mimetic nanobody (Nb80), like the nucleotide‐free G protein, slowed [ 3 H]DHAP association but also slowed [ 3 H]DHAP dissociation. However, this effect was not antagonist‐specific, as Nb80 also inhibited association of the full agonist [ 3 H]formoterol, the partial agonist [ 3 H]CGP‐12177, and the inverse agonist [ 3 H]carvedilol. Mutation of tyrosine 308 to alanine in β 2 AR abolished the rate‐slowing effects of Nb80 on antagonist and agonist association. Together, these data suggest that transit into or out of the orthosteric site in the active β 2 AR conformation (stabilized by either Gs or Nb80) is restricted due to closing of the orthosteric site, and that Tyr308 of β 2 AR is an important residue for controlling access to the orthosteric site. Interestingly, the M2 muscarinic acetylcholine receptor and mu opioid receptor behaved similarly to β 2 AR when bound to nucleotide‐free G protein or G protein‐mimetic nanobodies, suggesting that G protein‐mediated closing of the orthosteric site may extend to GPCRs beyond β 2 AR and the amine receptor family. Support or Funding Information This work was supported by AHA 13PRE17110027 and NIH GM083118.