BRET data analyses and coupling efficiency evaluation

All BRET ratios were standardized using the equation below and represented as universal BRET (uBRET) values: uBRET = ((BRET ratio – A)/(B-A)) * 10,000. Constants A and B correspond to the following values:

A = pre-established BRET ratio obtained from transfection of negative control (vector coding for RlucII alone).

B = pre-established BRET ratio obtained from transfection of positive control (vector coding for a GFP10-RlucII fusion protein).

For a given signaling pathway, uBRET values at each agonist concentration were normalized as the % of the response obtained in the absence of agonist (vehicle) and concentration-response curves were fitted in GraphPad Prism 8.3 software using a four-parameter logistic nonlinear regression model. Results are expressed as mean ± SEM of at least three independent experiments.

A ligand-promoted response was considered real when the E_max value was ≥to the mean + 2*SD of the response obtained in vehicle condition and that a pEC₅₀ value could be determined in the agonist concentration range used to stimulate the receptor. Consequently, a score of 0 or 1 was assigned to each signaling pathway depending on an agonist’s ability to activate the tested pathway (0 = no activation; 1 = activation). In the case were responses associated to endogenous receptor were detectable, we considered as ‘distorted’ and exclude all the responses observed in the presence of transfected receptor for which E_max was ≤to 2*mean of the E_max value obtained with endogenous receptors or pEC₅₀ was ≥to 2*mean of the pEC₅₀ value obtained with endogenous receptors. Consequently, a score of 0 was assigned for these distorted responses in radial graph representation (Figure 3—figure supplement 1) and concentration-response curves were placed on a gray background in signaling signature profile panels (Supplementary file 3). Whenever transfected receptors produced an increase in E_max or a left-shift in pEC₅₀ values compared to endogenous receptors, responses were considered ‘true’ and were assigned with a score of 1 for radial graph representation (Figure 3—figure supplement 1) and concentration-response curves were placed on a yellow background in signaling signature profile panels to indicate a partial contribution of endogenous receptors (Supplementary file 3).

We used a double normalization of E_max and pEC₅₀ values to compare the signaling efficiency obtained for the 100 GPCRs across all receptors and pathways. E_max and pEC₅₀ values deduced from concentration-response curves were first normalized between 0 and 1 across receptors by ranking the receptors as a function of the receptor that most efficiently activate a given pathway and then using the activation value for the pathway (including G protein and βarrestin subtypes) that a given receptor most efficiently activate as a reference for the other pathways that can be activated by this receptor. This double normalization can be translated in the following formalized equation:

STEP1: For each receptor and for each pathway:

Emax GPCRxEmax GPCRRef_{Pathway A} = Pathway specific normalized score for GPCR_x on pathway A ([PSNS GPCR_x]_{Pathway A})

where: GPCR_x is receptor being analyzed, GPCR_Ref is the receptor giving greatest E_max on pathway A of all receptors studied (i.e. reference receptor for pathway A). A PSNS was determined for every receptor and every pathway coupled to that receptor.

STEP2: For any given receptor:

PSNS GPCRxPathway APSNS GPCRxRef pathway= Normalized pathway A coupling score for GPCR_x

where: [PSNS GPCR_x] _{Pathway A} is the pathway specific normalized score for GPCR_x on pathway A, and [PSNS GPCR_x] _{Ref pathway} is the pathway specific normalized score for the pathway giving the highest PSNS for GPCR_x (i.e., reference pathway for GPCR_x).

For the safety target panel ligand screen using the combined G_z/G₁₅ sensor, the fold ligand-induced stimulation was calculated for each receptor by dividing the BRET ratio after ligand addition (measured at 10 min post stimulation) by the basal BRET ratio prior to receptor stimulation. Activation thresholds were defined as the mean + 2*SD of the ligand-stimulated response obtained with receptor-null cells expressing only the combined G_z/G₁₅ sensor.