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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 zebrafish embryos.(A) Illustration of developmental stages of fertilized zebrafish embryos with specification of elapsed hours post fertilization (hpf). Morphological analyses of reporter construct injection at 24 hpf. (B) Normalized quantification of PPI of RIIb-F[1]:PKAc-F[2] 3 hpf, 6 hpf and 9.5 hpf (zebrafish embryos; representative experiment). (C) Impact of time-dependent forskolin (50 μM) and isoproterenol (10 μM) exposure of de-chorionated zebrafish embryos on complex formation of overexpressed Rluc PCA based PKA reporter at the 8 hpf stage (representative of n = 3; SEM from independent measurements).
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f7: Benchmarking of PKA reporter dynamics in zebrafish embryos.(A) Illustration of developmental stages of fertilized zebrafish embryos with specification of elapsed hours post fertilization (hpf). Morphological analyses of reporter construct injection at 24 hpf. (B) Normalized quantification of PPI of RIIb-F[1]:PKAc-F[2] 3 hpf, 6 hpf and 9.5 hpf (zebrafish embryos; representative experiment). (C) Impact of time-dependent forskolin (50 μM) and isoproterenol (10 μM) exposure of de-chorionated zebrafish embryos on complex formation of overexpressed Rluc PCA based PKA reporter at the 8 hpf stage (representative of n = 3; SEM from independent measurements).

Mentions: Finally, we tested the Rluc PCA PKA reporter in zebrafish (Danio rerio) embryos, a widely used vertebrate developmental model system, which has recently also been adapted for drug discovery purposes, particularly to screen for molecules that act on the common GPCR family of receptors 101149505152,. GPCR pathways play critical roles at different stages of development (e.g. Wnt signaling, Hedgehog, and chemokine signaling)910535455. We attempted to determine if embryos respond to external activation of βARs and if direct activation of adenylyl cyclases (ACs) is detectable using our reporter system. Over the course of a 24 h time frame, a variety of developmental stages of fertilized zebrafish embryos can be distinguished (Fig. 7A). First, we showed that injection of equal amounts of mRNA encoding the Rluc PCA-based PKA reporter constructs (RII-F[1]:PKAc-F[2]) did not affect embryo development by morphological criteria (Fig. 7A). We focused on stages between 3 and 24 hours post fertilization (hpf). After removing the chorion, embryos were subjected to Rluc PCA measurements. We observed PKA originating Rluc PCA signal decrease with time (Fig. 7B). After removing the chorion we treated embryos either with forskolin or isoproterenol for five and 30 min followed by Rluc PCA analyses. In comparison to the controls, we observed changes in PKA reporter signal. Both forskolin and isoproterenol treatment triggered PKA activation in the embryos at the 8 hpf stage (Fig. 7C). Also after 24 h hpf it was possible to analyze dynamic PPIs (Supplementary Figure S4). These data indicate that for elaborated PPI studies in the zebrafish model system it will be necessary to generate transgenic animals. These experiments indicate that Rluc PCA-based reporters are suitable for analysis of dynamic PPIs at different embryological stages of zebrafish embryos. This can be also useful for real-time recordings of defined receptor-regulated PPIs in and between developmental stages using either wild type or specific disease-relevant mutant strains. The PKA activity status is controlled through a variety of cAMP linked GPCRs which are prime targets for drug discovery10113953545556. Therefore the PKA reporter can be adapted to studies on pharmaceutically relevant and endogenously expressed GPCRs in the zebrafish model. It is therefore suitable to test small-molecules acting on GPCRs in this model through both Gαs- and Gαi-coupled receptors that exist in zebrafish. Several small molecules act on these receptors and their discovery have resulted in preclinical and clinical trials10. One major advantage of the zebrafish embryos is that the efficacies of small molecules targeting PKA-linked pathways can be assessed. Simultaneously a phenotypic toxicology profile can be applied, potentially accelerating drug discovery.


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 zebrafish embryos.(A) Illustration of developmental stages of fertilized zebrafish embryos with specification of elapsed hours post fertilization (hpf). Morphological analyses of reporter construct injection at 24 hpf. (B) Normalized quantification of PPI of RIIb-F[1]:PKAc-F[2] 3 hpf, 6 hpf and 9.5 hpf (zebrafish embryos; representative experiment). (C) Impact of time-dependent forskolin (50 μM) and isoproterenol (10 μM) exposure of de-chorionated zebrafish embryos on complex formation of overexpressed Rluc PCA based PKA reporter at the 8 hpf stage (representative of n = 3; SEM from independent measurements).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: Benchmarking of PKA reporter dynamics in zebrafish embryos.(A) Illustration of developmental stages of fertilized zebrafish embryos with specification of elapsed hours post fertilization (hpf). Morphological analyses of reporter construct injection at 24 hpf. (B) Normalized quantification of PPI of RIIb-F[1]:PKAc-F[2] 3 hpf, 6 hpf and 9.5 hpf (zebrafish embryos; representative experiment). (C) Impact of time-dependent forskolin (50 μM) and isoproterenol (10 μM) exposure of de-chorionated zebrafish embryos on complex formation of overexpressed Rluc PCA based PKA reporter at the 8 hpf stage (representative of n = 3; SEM from independent measurements).
Mentions: Finally, we tested the Rluc PCA PKA reporter in zebrafish (Danio rerio) embryos, a widely used vertebrate developmental model system, which has recently also been adapted for drug discovery purposes, particularly to screen for molecules that act on the common GPCR family of receptors 101149505152,. GPCR pathways play critical roles at different stages of development (e.g. Wnt signaling, Hedgehog, and chemokine signaling)910535455. We attempted to determine if embryos respond to external activation of βARs and if direct activation of adenylyl cyclases (ACs) is detectable using our reporter system. Over the course of a 24 h time frame, a variety of developmental stages of fertilized zebrafish embryos can be distinguished (Fig. 7A). First, we showed that injection of equal amounts of mRNA encoding the Rluc PCA-based PKA reporter constructs (RII-F[1]:PKAc-F[2]) did not affect embryo development by morphological criteria (Fig. 7A). We focused on stages between 3 and 24 hours post fertilization (hpf). After removing the chorion, embryos were subjected to Rluc PCA measurements. We observed PKA originating Rluc PCA signal decrease with time (Fig. 7B). After removing the chorion we treated embryos either with forskolin or isoproterenol for five and 30 min followed by Rluc PCA analyses. In comparison to the controls, we observed changes in PKA reporter signal. Both forskolin and isoproterenol treatment triggered PKA activation in the embryos at the 8 hpf stage (Fig. 7C). Also after 24 h hpf it was possible to analyze dynamic PPIs (Supplementary Figure S4). These data indicate that for elaborated PPI studies in the zebrafish model system it will be necessary to generate transgenic animals. These experiments indicate that Rluc PCA-based reporters are suitable for analysis of dynamic PPIs at different embryological stages of zebrafish embryos. This can be also useful for real-time recordings of defined receptor-regulated PPIs in and between developmental stages using either wild type or specific disease-relevant mutant strains. The PKA activity status is controlled through a variety of cAMP linked GPCRs which are prime targets for drug discovery10113953545556. Therefore the PKA reporter can be adapted to studies on pharmaceutically relevant and endogenously expressed GPCRs in the zebrafish model. It is therefore suitable to test small-molecules acting on GPCRs in this model through both Gαs- and Gαi-coupled receptors that exist in zebrafish. Several small molecules act on these receptors and their discovery have resulted in preclinical and clinical trials10. One major advantage of the zebrafish embryos is that the efficacies of small molecules targeting PKA-linked pathways can be assessed. Simultaneously a phenotypic toxicology profile can be applied, potentially accelerating drug discovery.

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