A structural basis for how ligand binding site changes can allosterically regulate GPCR signaling and engender functional selectivity.

Signaling bias is the propensity for some agonists to preferentially stimulate G protein-coupled receptor (GPCR) signaling through one intracellular pathway versus another. We previously identified a G protein-biased agonist of the D2 dopamine receptor (D2R) that results in impaired beta-arrestin recruitment. This signaling bias was predicted to arise from unique interactions of the ligand with a hydrophobic pocket at the interface of the second extracellular loop and fifth transmembrane segment of the D2R. Here, we showed that residue Phe189 within this pocket (position 5.38 using Ballesteros-Weinstein numbering) functions as a microswitch for regulating receptor interactions with beta-arrestin. This residue is relatively conserved among class A GPCRs, and analogous mutations within other GPCRs similarly impaired beta-arrestin recruitment while maintaining G protein signaling. To investigate the mechanism of this signaling bias, we used an active-state structure of the beta(2)-adrenergic receptor (beta 2R) to build beta 2R-WT and beta 2R-Y199(5.38A) models in complex with the full beta 2R agonist BI-167107 for molecular dynamics simulations. These analyses identified conformational rearrangements in beta 2R-Y199(5.38A) that propagated from the extracellular ligand binding site to the intracellular surface, resulting in a modified orientation of the second intracellular loop in beta 2R-Y199(5.38A), which is predicted to affect its interactions with beta-arrestin. Our findings provide a structural basis for how ligand binding site alterations can allosterically affect GPCR-transducer interactions and result in biased signaling.