Abstract

Activation of the G protein-coupled receptors by agonists may result in the activation of one or more G proteins and recruitment of arrestins. The extent of the activation of each of these pathways depends on the intrinsic efficacy of the ligand. Quantification of intrinsic efficacy relative to a reference compound is essential for the development of novel compounds. In the operational 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 intrinsic activity of the receptor, we developed a Michaelis-Menten based quantification of G protein activation bias that uses experimentally measured ligand affinities and provides a single measure of ligand efficacy. We used it to evaluate the signalling 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 subfamilies in response to its primary endogenous agonist, arginine vasopressin (AVP), 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 non-mammalian 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 results showed that V2R is promiscuous in its ability to engage 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 reaction, 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.