Abstract
G protein-coupled receptors (GPCRs) control critical intercellular communications by responding to extracellular stimuli and undertaking conformational changes to convey disparate signals to intracellular effectors. We combined NMR, molecular pharmacology, and enhanced sampling molecular dynamics (MD) simulations to study the conformational diversity of the vasopressin V2 receptor (V2R) bound to different types of ligands: the antagonist tolvaptan, the endogenous unbiased agonist arginine-vasopressin, and MCF14, a Gs-protein biased agonist. We developed a doublelabeling NMR scheme to study the conformational dynamics: V2R was subjected to lysine 13 CH3 methylation, whereas the agonists were tagged with a paramagnetic probe. Paramagnetic relaxation enhancements were used to validate the ligand binding poses in the MD simulations. We found that the bias for the Gs protein over the -arrestin pathway involves interactions between the conserved NPxxY motif in the transmembrane helix (TM) 7 and a central hydrophobic patch in TM3, which constrains TM7 and likely inhibits β-arrestin signaling. A similar mechanism was observed for the naturally occurring V2R mutation, I130 3.43 N, which constitutively activates the Gs protein without concomitant-arrestin recruitment. This mechanism resembles previous findings on the µ-opioid receptor and indicate common patterns in class A GPCRs.