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In-vivo detection of binary PKA network interactions upon activation of endogenous GPCRs.

Röck R, Bachmann V, Bhang HE, Malleshaiah M, Raffeiner P, Mayrhofer JE, Tschaikner PM, Bister K, Aanstad P, Pomper MG, Michnick SW, Stefan E - Sci Rep (2015)

Bottom Line: Membrane receptor-sensed input signals affect and modulate intracellular protein-protein interactions (PPIs).Consequent changes occur to the compositions of protein complexes, protein localization and intermolecular binding affinities.This extends the application spectrum of Rluc PCA for the quantification of PPI-based receptor-effector relationships in physiological and pathological model systems.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry and Center for Molecular Biosciences, University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria.

ABSTRACT
Membrane receptor-sensed input signals affect and modulate intracellular protein-protein interactions (PPIs). Consequent changes occur to the compositions of protein complexes, protein localization and intermolecular binding affinities. Alterations of compartmentalized PPIs emanating from certain deregulated kinases are implicated in the manifestation of diseases such as cancer. Here we describe the application of a genetically encoded Protein-fragment Complementation Assay (PCA) based on the Renilla Luciferase (Rluc) enzyme to compare binary PPIs of the spatially and temporally controlled protein kinase A (PKA) network in diverse eukaryotic model systems. The simplicity and sensitivity of this cell-based reporter allows for real-time recordings of mutually exclusive PPIs of PKA upon activation of selected endogenous G protein-coupled receptors (GPCRs) in cancer cells, xenografts of mice, budding yeast, and zebrafish embryos. This extends the application spectrum of Rluc PCA for the quantification of PPI-based receptor-effector relationships in physiological and pathological model systems.

No MeSH data available.


Related in: MedlinePlus

Analyses of PKA reporter sensitivity and its miniaturization using human cells.(a) Time-dependent evaluation of complex formation of RIIb-F[1]:PKAc-F[2] hours post transfection (1 h, 2 h and 4 h). The effect of forskolin (50 μM; 10 min) on PKA complex formation has been determined. (b) Fold increase of luminescence signals originating from the complemented Rluc PCA based PKA reporter following transient overexpression in HEK293 cells for indicated time-frames is shown. Immunoblotting shows expression levels of endogenous and overexpressed PKA subunits. The same membrane has been probed first with RIIb and then with PKAc antibodies (BD Biosciences; #610626, #610981). (c) Dose-dependent effects of forskolin exposure on PPI-mediated luminescence measured simultaneously in 1536-well plate format. HEK293 cells transiently expressing the PKA reporter were treated for 5 min with 50 μM forskolin, transferred to a 1536 well plate and luminescence in the presence of benzyl-coelenterazine (CTZ) was captured for 30 sec on the Fusion imaging platform (Biorad). The pseudo-color scale indicates intensities of emitted luminescence signals. The quantification summarizes the effect of forskolin exposure on RIIb:PKAc interaction.
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f4: Analyses of PKA reporter sensitivity and its miniaturization using human cells.(a) Time-dependent evaluation of complex formation of RIIb-F[1]:PKAc-F[2] hours post transfection (1 h, 2 h and 4 h). The effect of forskolin (50 μM; 10 min) on PKA complex formation has been determined. (b) Fold increase of luminescence signals originating from the complemented Rluc PCA based PKA reporter following transient overexpression in HEK293 cells for indicated time-frames is shown. Immunoblotting shows expression levels of endogenous and overexpressed PKA subunits. The same membrane has been probed first with RIIb and then with PKAc antibodies (BD Biosciences; #610626, #610981). (c) Dose-dependent effects of forskolin exposure on PPI-mediated luminescence measured simultaneously in 1536-well plate format. HEK293 cells transiently expressing the PKA reporter were treated for 5 min with 50 μM forskolin, transferred to a 1536 well plate and luminescence in the presence of benzyl-coelenterazine (CTZ) was captured for 30 sec on the Fusion imaging platform (Biorad). The pseudo-color scale indicates intensities of emitted luminescence signals. The quantification summarizes the effect of forskolin exposure on RIIb:PKAc interaction.

Mentions: Following analyses of PKA dynamics upon activation of G-alpha-s coupled receptor cascades we benchmarked features of the Rluc PCA, first in intact cells and subsequently in model organisms. We decided to focus our efforts on cytoplasmatic localized RIIb:PKAc interactions33. Genetically encoded RIIb-F[1] and PKAc-F[2] hybrid-constructs were transiently over-expressed in HEK293 cells. Within two hours of transfection we detected luminescence resulting from the PPI reporter. Addition of forskolin triggered dissociation of the PKA complex, which resulted in a disruption of the RIIb:PKAc complex and spontaneous unfolding of the Rluc PCA reporter, and thus a decreased luminescence signal (Fig. 4A). Next, we quantitatively measured PPIs in a time-dependent manner. Four hours of transient over-expression of the reporter was sufficient to detect dynamic PPIs (Fig. 4A). However, at this stage, expression of the Rluc PCA could not be tracked using immuno-blotting (Fig. 4B). Six-fold prolongation of the expression-time caused a 417-fold increase of the PPI signal (24 h). Consequently, quantification of PKA Rluc PCA reporter (henceforth, the PKA reporter) was possible at varying expression levels of the reporter, when compared with those of the endogenous proteins, RIIb and PKAc respectively (Fig. 4B). Next we performed quantitative, dose-dependent measurements of PPIs with intact HEK293 cells in a 1536-well plate format to show that we could adapt this system for high throughput screenings (HTS). We illustrate that the simplicity and sensitivity of the Rluc PCA allows simultaneous recordings of dose-dependent effects of forskolin on the PKA reporter in a feasible short time-frame (less than 10 min) for treatment and measurements (Fig. 4C).


In-vivo detection of binary PKA network interactions upon activation of endogenous GPCRs.

Röck R, Bachmann V, Bhang HE, Malleshaiah M, Raffeiner P, Mayrhofer JE, Tschaikner PM, Bister K, Aanstad P, Pomper MG, Michnick SW, Stefan E - Sci Rep (2015)

Analyses of PKA reporter sensitivity and its miniaturization using human cells.(a) Time-dependent evaluation of complex formation of RIIb-F[1]:PKAc-F[2] hours post transfection (1 h, 2 h and 4 h). The effect of forskolin (50 μM; 10 min) on PKA complex formation has been determined. (b) Fold increase of luminescence signals originating from the complemented Rluc PCA based PKA reporter following transient overexpression in HEK293 cells for indicated time-frames is shown. Immunoblotting shows expression levels of endogenous and overexpressed PKA subunits. The same membrane has been probed first with RIIb and then with PKAc antibodies (BD Biosciences; #610626, #610981). (c) Dose-dependent effects of forskolin exposure on PPI-mediated luminescence measured simultaneously in 1536-well plate format. HEK293 cells transiently expressing the PKA reporter were treated for 5 min with 50 μM forskolin, transferred to a 1536 well plate and luminescence in the presence of benzyl-coelenterazine (CTZ) was captured for 30 sec on the Fusion imaging platform (Biorad). The pseudo-color scale indicates intensities of emitted luminescence signals. The quantification summarizes the effect of forskolin exposure on RIIb:PKAc interaction.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4477410&req=5

f4: Analyses of PKA reporter sensitivity and its miniaturization using human cells.(a) Time-dependent evaluation of complex formation of RIIb-F[1]:PKAc-F[2] hours post transfection (1 h, 2 h and 4 h). The effect of forskolin (50 μM; 10 min) on PKA complex formation has been determined. (b) Fold increase of luminescence signals originating from the complemented Rluc PCA based PKA reporter following transient overexpression in HEK293 cells for indicated time-frames is shown. Immunoblotting shows expression levels of endogenous and overexpressed PKA subunits. The same membrane has been probed first with RIIb and then with PKAc antibodies (BD Biosciences; #610626, #610981). (c) Dose-dependent effects of forskolin exposure on PPI-mediated luminescence measured simultaneously in 1536-well plate format. HEK293 cells transiently expressing the PKA reporter were treated for 5 min with 50 μM forskolin, transferred to a 1536 well plate and luminescence in the presence of benzyl-coelenterazine (CTZ) was captured for 30 sec on the Fusion imaging platform (Biorad). The pseudo-color scale indicates intensities of emitted luminescence signals. The quantification summarizes the effect of forskolin exposure on RIIb:PKAc interaction.
Mentions: Following analyses of PKA dynamics upon activation of G-alpha-s coupled receptor cascades we benchmarked features of the Rluc PCA, first in intact cells and subsequently in model organisms. We decided to focus our efforts on cytoplasmatic localized RIIb:PKAc interactions33. Genetically encoded RIIb-F[1] and PKAc-F[2] hybrid-constructs were transiently over-expressed in HEK293 cells. Within two hours of transfection we detected luminescence resulting from the PPI reporter. Addition of forskolin triggered dissociation of the PKA complex, which resulted in a disruption of the RIIb:PKAc complex and spontaneous unfolding of the Rluc PCA reporter, and thus a decreased luminescence signal (Fig. 4A). Next, we quantitatively measured PPIs in a time-dependent manner. Four hours of transient over-expression of the reporter was sufficient to detect dynamic PPIs (Fig. 4A). However, at this stage, expression of the Rluc PCA could not be tracked using immuno-blotting (Fig. 4B). Six-fold prolongation of the expression-time caused a 417-fold increase of the PPI signal (24 h). Consequently, quantification of PKA Rluc PCA reporter (henceforth, the PKA reporter) was possible at varying expression levels of the reporter, when compared with those of the endogenous proteins, RIIb and PKAc respectively (Fig. 4B). Next we performed quantitative, dose-dependent measurements of PPIs with intact HEK293 cells in a 1536-well plate format to show that we could adapt this system for high throughput screenings (HTS). We illustrate that the simplicity and sensitivity of the Rluc PCA allows simultaneous recordings of dose-dependent effects of forskolin on the PKA reporter in a feasible short time-frame (less than 10 min) for treatment and measurements (Fig. 4C).

Bottom Line: Membrane receptor-sensed input signals affect and modulate intracellular protein-protein interactions (PPIs).Consequent changes occur to the compositions of protein complexes, protein localization and intermolecular binding affinities.This extends the application spectrum of Rluc PCA for the quantification of PPI-based receptor-effector relationships in physiological and pathological model systems.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry and Center for Molecular Biosciences, University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria.

ABSTRACT
Membrane receptor-sensed input signals affect and modulate intracellular protein-protein interactions (PPIs). Consequent changes occur to the compositions of protein complexes, protein localization and intermolecular binding affinities. Alterations of compartmentalized PPIs emanating from certain deregulated kinases are implicated in the manifestation of diseases such as cancer. Here we describe the application of a genetically encoded Protein-fragment Complementation Assay (PCA) based on the Renilla Luciferase (Rluc) enzyme to compare binary PPIs of the spatially and temporally controlled protein kinase A (PKA) network in diverse eukaryotic model systems. The simplicity and sensitivity of this cell-based reporter allows for real-time recordings of mutually exclusive PPIs of PKA upon activation of selected endogenous G protein-coupled receptors (GPCRs) in cancer cells, xenografts of mice, budding yeast, and zebrafish embryos. This extends the application spectrum of Rluc PCA for the quantification of PPI-based receptor-effector relationships in physiological and pathological model systems.

No MeSH data available.


Related in: MedlinePlus