Michaelis-Menten Quantification of Ligand Signaling Bias Applied to the Promiscuous Vasopressin V2 Receptor

Activation of G protein-coupled receptors by agonists may re-sult in the activation of one or more G proteins and recruitment of arrestins. The extent of the activation of each of these path-ways depends on the intrinsic efficacy of the ligand. Quantifica-tion of intrinsic efficacy relative to a reference compound is essential for the development of novel compounds. In the oper-ational model, changes in efficacy can be compensated by changes in the "functional" affinity, resulting in poorly defined values. To separate the effects of ligand affinity from the intrin-sic activity of the receptor, we developed a Michaelis-Menten based quantification of G protein activation bias that uses ex-perimentally measured ligand affinities and provides a single measure of ligand efficacy. We used it to evaluate the signaling of a promiscuous model receptor, the Vasopressin V2 receptor (V2R). Using BRET-based biosensors, we show that the V2R engages many different G proteins across all G protein subfa-milies in response to its primary endogenous agonist, arginine vasopressin, including Gs and members of the Gi/o and G12/13 families. These signaling pathways are also activated by the synthetic peptide desmopressin, oxytocin, and the nonmam-malian hormone vasotocin. We compared bias quantification using the operational model with Michaelis-Menten based quantification; the latter accurately quantified ligand efficacies despite large difference in ligand affinities. Together, these re-sults showed that the V2R is promiscuous in its ability to en-gage several G proteins and that its' signaling profile is biased by small structural changes in the ligand. SIGNIFICANCE STATEMENT By modelling the G protein activation as Michaelis-Menten re-action, we developed a novel way of quantifying signalling bias. V2R activates, or at least engages, G proteins from all G protein subfamilies, including Gi2, Gz, Gq, G12, and G13. Their relative activation may explain its Gs-independent signalling.