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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

Benchmarking of PKA reporter dynamics in mouse xenografts.(A) Time- and dose-dependent effects of isoproterenol treatments on complex formation of RIIb-F[1]:PKAc-F[2] is shown (HEK293 cells stably expressing the Rluc PCA PKA reporter). Luminescent signals from each well were normalized to the last point immediately preceding the administration of isoproterenol. (B) Subcutaneously engrafted HEK293 cells stably expressing the Rluc PCA PKA reporter formed human tumor xenografts in living mice. Shown is time-dependent and non-invasive in vivo luminescence imaging of the PKA reporter in response to application of native coelenterazine (1.2 mg/kg; intra peritoneal, [i.p.]), isoproterenol (2.4 mg/kg mouse; intra venous [i.v.]) and alprenolol (8.0 mg/kg; [i.v.]). The pseudo-color scale indicates intensities of emitted luminescence signals.
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f5: Benchmarking of PKA reporter dynamics in mouse xenografts.(A) Time- and dose-dependent effects of isoproterenol treatments on complex formation of RIIb-F[1]:PKAc-F[2] is shown (HEK293 cells stably expressing the Rluc PCA PKA reporter). Luminescent signals from each well were normalized to the last point immediately preceding the administration of isoproterenol. (B) Subcutaneously engrafted HEK293 cells stably expressing the Rluc PCA PKA reporter formed human tumor xenografts in living mice. Shown is time-dependent and non-invasive in vivo luminescence imaging of the PKA reporter in response to application of native coelenterazine (1.2 mg/kg; intra peritoneal, [i.p.]), isoproterenol (2.4 mg/kg mouse; intra venous [i.v.]) and alprenolol (8.0 mg/kg; [i.v.]). The pseudo-color scale indicates intensities of emitted luminescence signals.

Mentions: We then used the Rluc PCA PKA assay as a reporter to measure activation of distinct GPCRs. For this we employed HEK293 cells that express low levels of endogenous β2ARs3842. Using a reporter cell line with stable expression of the PKA biosensor we recorded a transient dissociation of the RII:PKAc complex (within a 10 minute time interval) upon treatment with isoproterenol (Fig. 5A).


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)

Benchmarking of PKA reporter dynamics in mouse xenografts.(A) Time- and dose-dependent effects of isoproterenol treatments on complex formation of RIIb-F[1]:PKAc-F[2] is shown (HEK293 cells stably expressing the Rluc PCA PKA reporter). Luminescent signals from each well were normalized to the last point immediately preceding the administration of isoproterenol. (B) Subcutaneously engrafted HEK293 cells stably expressing the Rluc PCA PKA reporter formed human tumor xenografts in living mice. Shown is time-dependent and non-invasive in vivo luminescence imaging of the PKA reporter in response to application of native coelenterazine (1.2 mg/kg; intra peritoneal, [i.p.]), isoproterenol (2.4 mg/kg mouse; intra venous [i.v.]) and alprenolol (8.0 mg/kg; [i.v.]). The pseudo-color scale indicates intensities of emitted luminescence signals.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Benchmarking of PKA reporter dynamics in mouse xenografts.(A) Time- and dose-dependent effects of isoproterenol treatments on complex formation of RIIb-F[1]:PKAc-F[2] is shown (HEK293 cells stably expressing the Rluc PCA PKA reporter). Luminescent signals from each well were normalized to the last point immediately preceding the administration of isoproterenol. (B) Subcutaneously engrafted HEK293 cells stably expressing the Rluc PCA PKA reporter formed human tumor xenografts in living mice. Shown is time-dependent and non-invasive in vivo luminescence imaging of the PKA reporter in response to application of native coelenterazine (1.2 mg/kg; intra peritoneal, [i.p.]), isoproterenol (2.4 mg/kg mouse; intra venous [i.v.]) and alprenolol (8.0 mg/kg; [i.v.]). The pseudo-color scale indicates intensities of emitted luminescence signals.
Mentions: We then used the Rluc PCA PKA assay as a reporter to measure activation of distinct GPCRs. For this we employed HEK293 cells that express low levels of endogenous β2ARs3842. Using a reporter cell line with stable expression of the PKA biosensor we recorded a transient dissociation of the RII:PKAc complex (within a 10 minute time interval) upon treatment with isoproterenol (Fig. 5A).

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