Figure 6.

PKN-recruitment based biosensor to monitor receptor-induced activation of Rho signalling for TPαR and β₁AR. (a) Schematic representation of the PKN-recruitment based biosensor. (b) Schematic representation of the mode of action of p115-RGS-CAAX construct (P115-CAAX) as a dominant negative construct for G protein signalling. HEK 293 cells were transfected with PKN-RlucII and rGFP-CAAX along with either (c–f) HA-TPαR or (g,h) β₁AR, in the presence or absence of (d) constitutively active RhoA mutant (Q63L), (e) Gα₁₂, Gα₁₃ or their constitutively active (CAM) versions (Gα₁₂CA Q231L and Gα₁₃CA Q226L) or (f-h) p115-CAAX. (c) Treatment with the TP antagonist SQ 29,548 inhibits PKN recruitment following TPαR activation by U46619. 0 ng represents the activity of endogenously expressed receptor. Data are expressed as ΔBRET signal and are the mean ± SEM (n = 3). Statistical comparisons for antagonistic effect were done using two-way ANOVA followed by post-hoc comparison with Sidak’s test. (d,e) PKN recruitment to the plasma membrane upon U46619-induced TPαR activation in the presence or absence of (d) constitutively active RhoA mutant (Q63L) or (e) Gα₁₂, Gα₁₃ or their CAM versions. Data are expressed as BRET signal and are mean ± SEM ((d) n = 6, (e) n = 3). Statistical comparisons were done using two-way ANOVA followed by post-hoc comparison with Tukey’s test. #### p < 0.0001 compared to (d) Q63L or (e) TPαR Vehicle Mock. (f) Inhibition of PKN recruitment upon U46619-induced TPαR activation in the presence of the dominant negative p115-CAAX construct or the Gq inhibitor YM-254890. Data expression and statistical comparisons were done as in (d,e). n = 4, #### p < 0.0001 compared to -p115-CAAX. (g) Kinetics of PKN recruitment upon isoproterenol-induced β₁AR activation in the presence of the dominant negative p115-CAAX (representative of n = 3). (h) β₁AR-mediated PKN recruitment in the presence of p115-CAAX. Data are expressed as the area under the curve (AUC), calculated from 30 sec kinetics of 1 µM isoproterenol stimulation, and are the mean ± SEM (n = 4). #### p < 0.0001 compared to -p115-CAAX. ns: non-significant.