(A) Schematic of the modified AKAR-18RBS FRET reporter used in these studies. The PKA RII binding site common to all AKAP18 isoforms was fused to the N-terminus of AKAR2 to create a FRET-based kinase activity sensor that anchors type II PKA holoenzyme. Phosphorylation of a consensus site Thr by PKA induces recruitment of the adjacent Forkhead homology-associated (FHA) phospho-Thr binding domain. Subsequent rearrangement brings together the ECFP and YFP (citrine) moieties to produce an increase in FRET signal as readout of kinase activity. (B) Co-immunoprecipitation of AKAR-18RBS-PKA complexes with holoenzymes composed of three different RII forms. AKAR-18RBS was immunoprecipitated with anti-GFP antibodies and bound PKA subunits were detected by western blotting. (C) Lysates from cells transfected with the AKAR-18RBS reporter and each of the three RIIαforms were immunoblotted to confirm similar expression levels in each cohort. (D) Basal (unstimulated) raw FRET signals from cells expressing RIIα (gray), RIIα Δ44–86 (green) and RIIα ZeChimera (orange). (E and G) Time course of AKAR-18RBS activation in response to the β-adrenergic agonist isoproterenol in cells co-expressing the FRET reporter with (E) RIIα wild type, (F) RIIα Δ44–86, or (G) RIIα ZeChimera. Isoproterenol (Iso, 10 μM) was added at t = 100 s and FRET was recorded for 5 min post-stimulation. Warmer colors indicate increasing phosphorylation as shown in the pseudo-color scale. (H) Amalgamated traces of the Iso stimulated changes in FRET in each cohort (0–400 s). Data are normalized to unstimulated basal FRET level for each respective RIIα form. Changes in the AKAR-18RBS normalized FRET ratio are shown from cells expressing RIIα wild type (black), RIIα Δ44–86 (green) and RIIα ZeChimera (orange). Mutation of the phosphoacceptor threonine in the FRET reporter to create AKAR-18RBS–T/A blocks phosphorylation and abolishes FRET in response to Iso treatment (Inset). (I) Scatter plot representation of peak FRET signals from all cells at 200 s. This plot indicates that AKAR-18RBS-PKA complexes formed with RIIα wild type (black) or the RIIα ZeChimera (orange) had similar peak responses, while complexes formed with RIIα Δ44–86 displayed significantly greater peak FRET responses (p<0.001). This plot also shows that in all cases, some cells fail to respond entirely; these non-responders are included in the amalgamated data analysis presented in (H). (J) C subunit of PKA association with AKAR-18RBS, AKAR-18RBS–pro or AKAR-18RBS–T/A complexes. AKAR-18RBS and AKAR-18RBS–T/A co-precipitate RIIα and endogenous PKA catalytic subunit; AKAR-18RBS–pro, a mutant that cannot bind RII subunits, fails to co-precipitate PKA complexes (top panels). Iso treatment (1 μM, 5 min) does not cause dissociation of PKAc from the AKAR-18RBS or AKAR-18RBS–T/A complexes (top panel, lanes 1–2 and 5–6). Control immunoblots show equivalent levels of AKAR-18RBS, AKAR-18RBS–pro and AKAR-18RBS–T/A in immunoprecipitates as well as RIIα and PKAc expression in cell lysates (middle panels). Immunoblotting for phospho-PKA substrates (R-X-X-pS/T motif) confirms that Iso treatment activates endogenous β-ARs and initiates downstream phosphorylation events (bottom panel). (K) Immunoprecipitation of full-length AKAP18 complexes following Iso treatment. Cells expressing AKAP18γ and RIIα variants were treated with vehicle or Iso (1 μM, 5 min) and AKAP18γ complexes were immunoprecipitated. Immunoblotting shows that Iso has no effect on the amount of PKA catalytic subunit in complexes formed with RIIα wild type, RIIα Δ44–86, or RIIα ZeChimera.