G_i proteins, caveolae and GRK2 regulate cytoplasmic but not nuclear PKA activation in response to β₂-ARs stimulation. Average time course of the YFP/CFP ratio upon β₂-AR stimulation in ARVMs expressing AKAR3-NES (A, C, E, and G) or AKAR3-NLS (B, D, F, and H). (A and B) ARVMs treated or not with PTX (1.5 µg/ml, 2 h) were exposed to 10 nM Iso plus 100 nM CGP to stimulate β₂-ARs. In all other protocols, β₂-ARs were stimulated with 30 nM Iso plus 100 nM CGP. (C and D) ARVMs were treated or not with 2 mM MβCD for 1 h. (E) ARVMs were co-transduced with Ad.AKAR3-NES (MOI 200) and Ad.β-Galactosidase (β-Gal, MOI 2000) or Ad.AKAR3-NES and an adenovirus encoding a dominant-negative Cav3 mutant (Ad. Cav3DN, MOI 2000) for 48 h. (F) ARVMs were co-transduced with Ad.AKAR3-NLS and Ad. β-Gal or Ad.AKAR3-NLS and Ad.Cav3DN for 48 h. (G) ARVMs were co-transduced with Ad.AKAR3-NES (MOI 200) and Ad.β-Gal (MOI 1000) or Ad.AKAR3-NES (MOI 200) and Ad.βARK-ct (MOI 1000) for 48 h. (H) ARVMs were co-transduced with Ad.AKAR3-NLS (MOI 200) and Ad.β-Gal (MOI 1000) or Ad.AKAR3-NLS (MOI 200) and Ad.βARK-ct (MOI 1000) for 48 h. In each panel, the number of cells/animals is indicated in brackets for the different experimental conditions. Statistical significance is indicated as *P < 0.05; **P , 0.01; ***P < 0.001 by nested ANOVA with Tukey’s post-hoc test.