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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

Biosensor design principle.Design principle and features of the Rluc PCA: Indicated PKA subunits have been tagged with Rluc PCA fragments (F[1], F[2]; L, 10 aa linker). PPIs trigger folding and reconstituted activity of appended PCA-fragments. The emitted luminescence signal is a quantitative reporter of cellular PPIs. Perturbations (indicated with X) through small molecules, upstream factors, competitive PPIs, or mutations reduce protein complex formation. A selection of applications of the PPI reporter are indicated.
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f1: Biosensor design principle.Design principle and features of the Rluc PCA: Indicated PKA subunits have been tagged with Rluc PCA fragments (F[1], F[2]; L, 10 aa linker). PPIs trigger folding and reconstituted activity of appended PCA-fragments. The emitted luminescence signal is a quantitative reporter of cellular PPIs. Perturbations (indicated with X) through small molecules, upstream factors, competitive PPIs, or mutations reduce protein complex formation. A selection of applications of the PPI reporter are indicated.

Mentions: Biosensor design and preferable features of the Rluc PCA-based PPI reporter are shown in Fig. 1. Genetically encoded Rluc PCAs are applied to quantification and characterization of dynamic PPIs in real time and in vivo. The impact of perturbations of binary PPIs and upstream factors can be determined quantitatively. Specifications of PPI dynamics offer the possibility to be used for functional cellular read outs, for example of oncogenic PPI and receptor-effector interactions. It is desirable to quantify differential PPIs in distinct eukaryotic model systems to investigate physiological and pathological cell states.


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)

Biosensor design principle.Design principle and features of the Rluc PCA: Indicated PKA subunits have been tagged with Rluc PCA fragments (F[1], F[2]; L, 10 aa linker). PPIs trigger folding and reconstituted activity of appended PCA-fragments. The emitted luminescence signal is a quantitative reporter of cellular PPIs. Perturbations (indicated with X) through small molecules, upstream factors, competitive PPIs, or mutations reduce protein complex formation. A selection of applications of the PPI reporter are indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Biosensor design principle.Design principle and features of the Rluc PCA: Indicated PKA subunits have been tagged with Rluc PCA fragments (F[1], F[2]; L, 10 aa linker). PPIs trigger folding and reconstituted activity of appended PCA-fragments. The emitted luminescence signal is a quantitative reporter of cellular PPIs. Perturbations (indicated with X) through small molecules, upstream factors, competitive PPIs, or mutations reduce protein complex formation. A selection of applications of the PPI reporter are indicated.
Mentions: Biosensor design and preferable features of the Rluc PCA-based PPI reporter are shown in Fig. 1. Genetically encoded Rluc PCAs are applied to quantification and characterization of dynamic PPIs in real time and in vivo. The impact of perturbations of binary PPIs and upstream factors can be determined quantitatively. Specifications of PPI dynamics offer the possibility to be used for functional cellular read outs, for example of oncogenic PPI and receptor-effector interactions. It is desirable to quantify differential PPIs in distinct eukaryotic model systems to investigate physiological and pathological cell states.

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