Quantification of dynamic protein complexes using Renilla luciferase fragment complementation applied to protein kinase A activities in vivo

Abstract

The G protein-coupled receptor (GPCR) superfamily represents the most important class of pharmaceutical targets. Therefore, the characterization of receptor cascades and their ligands is a prerequisite to discovering novel drugs. Quantification of agonist-induced second messengers and downstream-coupled kinase activities is central to characterization of GPCRs or other pathways that converge on GPCR-mediated signaling. Furthermore, there is a need for simple, cell-based assays that would report on direct or indirect actions on GPCR-mediated effectors of signaling. More generally, there is a demand for sensitive assays to quantify alterations of protein complexes in vivo. We describe the development of a Renilla luciferase (Rluc)-based protein fragment complementation assay (PCA) that was designed specifically to investigate dynamic protein complexes. We demonstrate these features for GPCR-induced disassembly of protein kinase A (PKA) regulatory and catalytic subunits, a key effector of GPCR signaling. Taken together, our observations show that the PCA allows for direct and accurate measurements of live changes of absolute values of protein complex assembly and disassembly as well as cellular imaging and dynamic localization of protein complexes. Moreover, the Rluc-PCA has a sufficiently high signal-to-background ratio to identify endogenously expressed Galpha(s) protein-coupled receptors. We provide pharmacological evidence that the phosphodiesterase-4 family selectively down-regulates constitutive beta-2 adrenergic- but not vasopressin-2 receptor-mediated PKA activities. Our results show that the sensitivity of the Rluc-PCA simplifies the recording of pharmacological profiles of GPCR-based candidate drugs and could be extended to high-throughput screens to identify novel direct modulators of PKA or upstream components of GPCR signaling cascades.

Fig. 1.

Schematic representation of the PCA strategy using Rluc fragments to study the dynamic complex of the PKA heterodimer [regulatory (R) and catalytic (C) PKA subunits] in vivo. Cellular cAMP levels are controlled directly by adenylyl cyclases (AC, production) and PDE (degradation). cAMP elevation and association with the R subunit of PKA induces dissociation of R and C subunits, resulting in decreasing Rluc-PCA activity.

Fig. 2.

A dynamic PKA activity sensor based on Rluc-PCA. (A) Schematic representation for the design of a PCA-based PKA reporter. The regulatory (Reg) and catalytic (Cat) PKA subunits were fused to fragment 1 [F(1)] or fragment 2 [F(2)] of Rluc. (B) The Rluc-PCA was detected from transiently transfected HEK293T cells in suspension aliquoted to 96-well microtiter plates. Immunoblot analysis verifies expression of PCA-tagged proteins (representative experiment ± SD of triplicates). RLU, relative luminescence units. (C) Effect of forskolin (100 μM) and 3-isobutyl-1-methylxanthine (100 μM) treatment on Reg-F(1):Cat-F(2) and Reg-F(1):Reg-F(2). The Rluc-PCA was detected from transiently transfected HEK293T cells in suspension and aliquoted to 96-well microtiter plates (mean ± SD from three independent experiments). (D) HEK293T cells coexpressing Reg-F(1):Cat-F(2) were treated for the indicated times with forskolin (100 μM) and were subjected to immunoprecipitation and Western blotting using anti-Rluc antibodies. (E) The Rluc-PCA was detected from untreated and forskolin-treated (100 μM, 15 min) HEK293T cells stably expressing Reg-F(1):Cat-F(2) in suspension (± SD from three independent samples). Immunoblot analysis shows the expression of endogenously expressed and overexpressed PKA subunits.

Fig. 3.

Cellular imaging of bioluminescence of transiently transfected HEK293T cells expressing full-length Rluc and Rluc-PCA. (A) The Rluc-PCA was detected from HEK293T cells expressing indicated PCA fusion proteins (10 seconds) or full-length Rluc (1-second integration time) grown on 96-well microtiter plates (mean ± SD from triplicates). (B and C) Visualization of Rluc bioluminescence of HEK293T cells. By using a CCD camera and integration time of 30 seconds we imaged the bioluminescence (shown in gray scale) of full-length Rluc (B) and localized bioluminescence of HEK293T cells expressing Reg-F(1):Cat-F(2) (120 seconds) in PBS supplemented with 5 μM benzyl-coelenterazine (C). (D) Effect of 30 min of forskolin (100 μM) pretreatment on the bioluminescence of Reg-F(1):Cat-F(2) (120 seconds). Cotransfection of the red fluorescent protein (RFP) serves as control for transfection. C, cytoplasm; N, nucleus. (Scale bars: 5 μm.)

Fig. 4.

Effect of GPCR agonists, antagonists, and PDE inhibition on PKA activities. The Rluc-PCA was detected from transiently transfected HEK293T cells grown on white-walled 96-well microtiter plates. (A) Effect of combinations of alprenolol (10 μM, 60 min) pretreatment and isoproterenol (10 μM, 15 min) treatment of stable HEK293 clones expressing the β₂AR on Reg-F(1):Cat-F(2) or Reg-F(1):Reg-F(2) (mean ± SD from triplicates). (B) Effect of SR121463B (10 μM, 60 min) pretreatment and AVP (100 nM, 15 min) treatment of stable HEK293 clones expressing the V₂R on Reg-F(1):Cat-F(2) or Reg-F(1):Reg-F(2) (mean ± SD triplicates). (C) Stable V₂R-HEK293 and β₂AR-HEK293 cells were pretreated for indicated times with milrinone (10 μM) and increasing concentrations of rolipram (μM) followed by plate reader analysis of the effect on the Reg-F(1):Cat-F(2) (mean ± SD from three independent experiments). (D) Stable β₂AR-HEK293 cells were pretreated for 30 min with the selective β₂AR-antagonist ICI118,551 (1 μM) and isoproterenol (1 μM, 15 min) or increasing concentrations of rolipram (μM, 15 min) followed by Rluc-PCA analysis of Reg-F(1):Cat-F(2) (mean ± SD from three independent experiments).

Fig. 5.

Identification of endogenously expressed GPCR cascades using the Rluc-PCA PKA reporter. (A) The Rluc-PCA was detected from attached HEK293T, HeLa, COS7, or U2OS cells grown on white-walled 96-well microtiter plates. Shown is the effect of combinations of alprenolol (10 μM, 60 min) pretreatment and isoproterenol (10 μM, 15 min) treatment and combinations of SR121463B (1 μM, 60 min) pretreatment and AVP (100 nM, 15 min) treatment on Reg-F(1):Cat-F(2) (mean ± SD from three independent experiments). (B) The Rluc-PCA was detected from U2OS cells stably expressing Reg-F(1):Cat-F(2) in the presence and absence of isoproterenol (1 μM) (Left). Immunoblot analysis verifies the expression of endogenously expressed and overexpressed regulatory and catalytic PKA subunits. EC₅₀ values for isoproterenol-mediated β-adrenergic activation were obtained by using increasing concentrations of the ligand measured as changes of Reg-F(1):Cat-F(2) (Right; mean ± SD from three independent experiments). (C) Real-time kinetics (normalized on the control experiment) in response to isoproterenol (1 μM) of Reg-F(1):Cat-F(2) recorded with attached U2OS cells (stable clone 1; three independent samples, representative experiment). Washing steps were performed four times with PBS after 10 min of first isoproterenol treatment. (D) Real-time kinetics (normalized on the control experiment) of changes of Reg-F(1):Cat-F(2) recorded with attached U2OS cells (stable clone 1) when treated with combinations of ICI118,551, alprenolol (both 10 μM, pretreatment 60 min), and isoproterenol (1 μM). Half-times of PKA dissociation were calculated from three independent experiments (mean ± SD). Calculation of EC₅₀ and t_1/2 values and curve fittings were done with Prism 3.01 (GraphPad) (mean ± SD from at least three independent samples).