Biased activation of the vasopressin V2 receptor probed by molecular dynamics simulations, NMR and pharmacological studies.

G protein-coupled receptors (GPCRs) control critical cell signaling. Their response to extracellular stimuli involves conformational changes to convey signals to intracellular effectors, among which the most important are G proteins and β-arrestins (βArrs). Biased activation of one pathway is a field of intense research in GPCR pharmacology. Combining NMR, site-directed mutagenesis, molecular pharmacology, and molecular dynamics (MD) simulations, we studied 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 partial Gs protein-biased agonist. A double-labeling NMR scheme was developed to study the receptor conformational changes and ligand binding: V2R was subjected to lysine CH methylation for complementary NMR studies, whereas the agonists were tagged with a paramagnetic probe. Paramagnetic relaxation enhancements and site-directed mutagenesis validated the ligand binding modes in the MD simulations. We found that the bias for the Gs protein over the βArr pathway involves interactions between the conserved NPxxY motif in the transmembrane helix 7 (TM7) and TM3, compacting helix 8 (H8) toward TM1 and likely inhibiting βArr signaling. A similar mechanism was elicited for the pathogenic mutation I130N, which constitutively activates the Gs proteins without concomitant βArr recruitment. The findings suggest common patterns of biased signaling in class A GPCRs, as well as a rationale for the design of G protein-biased V2R agonists.