Fig. 7.

Gαq overexpression enhances cAMP response of endogenous β₂-AR. a cAMP accumulation in HEK293T cells either overexpressing Gαq (mCer-tag) or an equivalent number of untransfected control cells following stimulation with increasing concentration of fenoterol (0.3 nM to 100 μM) in the presence of 2 μM forskolin (see Methods). The increase in cAMP with fenoterol concentration was fit to a four-parameter logistic function to estimate EC₅₀ and E_max (Methods). Values are mean ± SD from six experiments with at least three repeats per experiment (n ≥ 6). Statistically significant differences in the EC₅₀ and E_max values were assessed by Students’ t-test. b cAMP accumulation in HEK293T cells either overexpressing Gαq (mCer-tag) or equivalent number of untransfected control cells, following stimulation with fenoterol (10 μM) and forskolin (2 μM), in the presence of PKC inhibitors Bim1 (1 μM) or Gö6983 (1 μM), or DMSO. Cells overexpressing Gαq had accumulated greater cAMP even in the presence of PKC inhibitors. The drug treatments do not influence the cAMP response across the three untransfected or the three transfected conditions. Box-and-whisker plots: center line is median, box ends are upper and lower quartiles, whisker ends are 1.5 × IQR (interquartile range) from four independent experiments with three technical replicates per experiment (n ≥ 4). Statistically significant differences were assessed by two-way ANOVA followed by Tukey’s post-hoc test. Significance of p < 0.05 is indicated by *. c Western blot analysis of lysates from equivalent cell numbers of Gαq overexpression ( + ) and untransfected cells ( − ). The blottings were probed with the indicated antibodies with anti-vinculin serving as loading control. d Effect of Gαq overexpression on the levels of indicated proteins is expressed as the ratio of band intensity in the Gαq overexpression lysate to the untransfected lysate