Profiling the Signaling Repertoire of One Hundred Therapeutically‐Relevant Human GPCRs

Abstract

It is now recognized that a given GPCR can engage multiple signaling pathways and that specific ligands can selectively promote the engagement of different subsets of these pathways. To fully explore the potential of this functional selectivity phenomenon in drug discovery and development, it becomes essential to have an exhaustive description of the effectors that can be engaged by a given receptor, providing receptor‐ and ultimately ligand‐specific signaling signatures. Here, we describe a central resource for interrogating the signaling profiles of over 100 therapeutically relevant human GPCRs in response to their endogenous ligands. We used 15 pathway‐selective bioluminescence resonance energy transfer (BRET) biosensors monitoring the activation of each Gα protein, as well as βarrestins (−/+ co‐expressed GRK2). The G protein sensors used in the study represent a new generation of BRET‐based sensors that monitor the recruitment of G protein effectors to the plasma membrane with no need for modifying the G proteins or the receptors. Over 1,500 concentration‐responses curves were generated and revealed a great diversity in the coupling selectivity of the studied receptors. Our data highlight that the Gi□family displayed the highest general coupling while Gs and G12/13 families were less frequently engaged by the receptors tested. Some GPCRs showed greater selectivity, with activation restricted to a single G protein subtype or members of the same G protein family (22% of tested GPCRs). Others showed pleiotropic activation profiles engaging G proteins belonging to multiple families (38% and 31% of tested receptors coupled to members of two and three G protein families, respectively) and, in some cases, all tested G proteins. Somewhat surprisingly, some receptors showed G protein subtype selectivity among the members of the same family. This underappreciated level of receptor coupling selectivity raises fundamental questions on the structural determinants of selectivity among closely related G proteins. A similar receptor‐specific profile of activation was observed for barrestins with 74% of GPCRs recruiting βarrestin‐2, and co‐expression of GRK2 favoring this recruitment. This difference in functional selectivity for different GPCRs highlights the importance of testing the multiple pathways coupled to a receptor. Interestingly, our analysis reveals that monitoring the activation of Gz and G15 allowed to detect the activation for the vast majority of the 100 GPCRs tested. Consequently, combining Gz and G15 sensors provides a practical tool to detect the activity of almost any GPCR. Our BRET‐based biosensors also allow the detection of receptor constitutive activity, which can be applied for GPCR deorphanization or to identify new drugs targeting constitutively activated GPCRs overexpressed in cancer. Hence, in addition to describing and validating an exhaustive G protein activity sensor platform, the study provides an exhaustive description of the signaling repertoire of 100 therapeutically relevant GPCRs that may underlie their therapeutic efficacy/safety profiles.