Fig. 4. Biased effect of I-287 on hPAR2-promoted Gα protein activation.

a–e Impact of I-287 on hPAR2-promoted G-protein activation measured by BRET². HEK293 cells co-expressing hPAR2 and the human BRET²-based α/βγ dissociation biosensors (GFP10-Gγ₁ or GFP10-Gγ₂ along with the indicated Gα-RlucII subunit), were pretreated with increasing concentrations of I-287 for 15 min followed by 1 min stimulation with an EC₈₀ concentration of hTrypsin or SLIGKV-NH₂. Results are expressed as ΔBRET in % of the response induced by EC₈₀ of respective agonists in the absence of I-287 (mean ± SEM; n = 3–6). f Schematic representation of the ebBRET-based biosensor to selectively monitor Gα_12/13 activation. Upon agonist stimulation, activated Gα₁₂ or Gα₁₃ subunits recruit their selective effector p115-RhoGEF tagged with RlucII to the plasma membrane, leading to an increase of ebBRET with the membrane-anchored rGFP-CAAX. g Dose–response curves of p115-RhoGEF recruitment at the plasma membrane induced by increasing concentrations of hTrypsin for 1 min in HEK293 cells expressing hPAR2 and the p115-RhoGEF-RlucII/rGFP-CAAX sensors in the absence (mock) or in the presence of Gα₁₂ or Gα₁₃ subunits. Results are expressed as BRET² ratio of absolute values (mean ± SEM; n = 4). h Inhibitory action of increasing concentrations of I-287 (15 min) on the G_12/13-mediated recruitment of p115-RhoGEF at plasma membrane induced by an EC₈₀ concentration of hTrypsin in HEK293 cells co-expressing hPAR2 and the human BRET²-based biosensors p115-RhoGEF-RlucII/rGFP-CAAX along with Gα₁₂ or Gα₁₃ subunits. Results are expressed as ΔBRET in % of the response induced by EC₈₀ of hTrypsin in the absence of I-287 (mean ± SEM; n = 3).