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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

PPI analyses of mutually exclusive binary PKA interactions.(A) Schematic depiction of compartmentalized PPIs of the cAMP-controlled PKA network. Besides alterations of cAMP levels, mutations in PKAc and RIa affect PKAc localizations and phosphotransferase activities. (B) Co-transfection of HEK293 cells with indicated Rluc PCA pairs followed by Rluc PCA analyses have been performed. The amount of PKI hybrid constructs have been bisected for cell transfections (representative of n = 3; SEM from triplicates). (c) Immunoblotting of F[1]-tagged PCA hybrid proteins have been performed with Rluc F[1]-specific monoclonal antibodies (Millipore, #MAB4410); representative experiment.
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f2: PPI analyses of mutually exclusive binary PKA interactions.(A) Schematic depiction of compartmentalized PPIs of the cAMP-controlled PKA network. Besides alterations of cAMP levels, mutations in PKAc and RIa affect PKAc localizations and phosphotransferase activities. (B) Co-transfection of HEK293 cells with indicated Rluc PCA pairs followed by Rluc PCA analyses have been performed. The amount of PKI hybrid constructs have been bisected for cell transfections (representative of n = 3; SEM from triplicates). (c) Immunoblotting of F[1]-tagged PCA hybrid proteins have been performed with Rluc F[1]-specific monoclonal antibodies (Millipore, #MAB4410); representative experiment.

Mentions: Here we tested the impact of distinct perturbations of proteins and/or receptor pathways (GPCRs) on defined molecular interactions using an extended PPI reporter platform. We analyzed unique properties of oncogenic bait-protein interactions with exchangeable prey proteins in cell culture and in diverse model organisms. For this purpose we constructed new Rluc PCA pairs to assess differential PPI. We selected PPI pairs of the binary and compartmentalized PKA network (Fig. 2A).


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)

PPI analyses of mutually exclusive binary PKA interactions.(A) Schematic depiction of compartmentalized PPIs of the cAMP-controlled PKA network. Besides alterations of cAMP levels, mutations in PKAc and RIa affect PKAc localizations and phosphotransferase activities. (B) Co-transfection of HEK293 cells with indicated Rluc PCA pairs followed by Rluc PCA analyses have been performed. The amount of PKI hybrid constructs have been bisected for cell transfections (representative of n = 3; SEM from triplicates). (c) Immunoblotting of F[1]-tagged PCA hybrid proteins have been performed with Rluc F[1]-specific monoclonal antibodies (Millipore, #MAB4410); representative experiment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: PPI analyses of mutually exclusive binary PKA interactions.(A) Schematic depiction of compartmentalized PPIs of the cAMP-controlled PKA network. Besides alterations of cAMP levels, mutations in PKAc and RIa affect PKAc localizations and phosphotransferase activities. (B) Co-transfection of HEK293 cells with indicated Rluc PCA pairs followed by Rluc PCA analyses have been performed. The amount of PKI hybrid constructs have been bisected for cell transfections (representative of n = 3; SEM from triplicates). (c) Immunoblotting of F[1]-tagged PCA hybrid proteins have been performed with Rluc F[1]-specific monoclonal antibodies (Millipore, #MAB4410); representative experiment.
Mentions: Here we tested the impact of distinct perturbations of proteins and/or receptor pathways (GPCRs) on defined molecular interactions using an extended PPI reporter platform. We analyzed unique properties of oncogenic bait-protein interactions with exchangeable prey proteins in cell culture and in diverse model organisms. For this purpose we constructed new Rluc PCA pairs to assess differential PPI. We selected PPI pairs of the binary and compartmentalized PKA network (Fig. 2A).

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