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

Abstract

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 β-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 β-arrestin. This residue is relatively conserved among class A GPCRs, and analogous mutations within other GPCRs similarly impaired β-arrestin recruitment while maintaining G protein signaling. To investigate the mechanism of this signaling bias, we used an active-state structure of the β2-adrenergic receptor (β2R) to build β2R-WT and β2R-Y1995.38A models in complex with the full β2R agonist BI-167107 for molecular dynamics simulations. These analyses identified conformational rearrangements in β2R-Y1995.38A that propagated from the extracellular ligand binding site to the intracellular surface, resulting in a modified orientation of the second intracellular loop in β2R-Y1995.38A, which is predicted to affect its interactions with β-arrestin. Our findings provide a structural basis for how ligand binding site alterations can allosterically affect GPCR-transducer interactions and result in biased signaling.

Ligand with Two Modes of Interaction with the Dopamine D2 Receptor–An Induced-Fit Mechanism of Insurmountable Antagonism

“…Direct protein–protein interaction between D 2 R and beta-arrestin2 was analyzed using BRET assays where the interaction of the receptor (donor) with beta-arrestin2 (acceptor) results in a shift of the emission spectra of the Rluc8 tag (after incubation with coelenterazine-h) from 485 to 525 nm as previously described by our laboratory. 37 The resulting luminescence was read and quantified on a Flexstation III multiplate reader (Molecular Devices, San Jose, CA). Results were expressed as a normalized ratio of emission observed with control agonist alone.…”

Structure of a D2 dopamine receptor–G-protein complex in a lipid membrane

“…Residues Phe189 5.38 and Ile184 ECL2 play an important role in this conformational change, since the swivel of the Phe189 5.38 phenyl group makes room for a downward shift of the Ile184 ECL2 sidechain. Recent studies have implicated these two residues’ movement in DRD2 activation, as well as in variable interaction with arrestin-biased agonists 32 , 33 . Repacking of sidechains in these upper TM segments ( Fig.…”

Allosteric coupling and biased agonism in G protein‐coupled receptors

“…Moreover, compound 2 shows less interactions with S193 5.42 at the bottom of the binding pocket which is an important trigger of Ga i -protein coupling. MLS1547 is a G i/o -biased ligand [88,89] and shows also strong interactions with a hydrophobic pocket in the extracellular domains comprising I184 and F189 5.38 at the extracellular part of TM5. In contrast to the two b-arrestin-biased ligands, the interaction of MLS1547 with F189 5.38 has been suggested to be responsible for impaired b-arrestin recruitment.…”