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Int. J. Mol. Sci. 2018, 19(4), 1180; https://doi.org/10.3390/ijms19041180

Establishing a Split Luciferase Assay for Proteinkinase G (PKG) Interaction Studies

1
Department of Pharmacology and Toxicology, University of Regensburg, 93053 Regensburg, Germany
2
Department of Biosystems Science and Engineering, ETH Zurich, 4058 Basel, Switzerland
3
Department of Pharmaceutical and Medicinal Chemistry II, University of Regensburg, 93053 Regensburg, Germany
4
Department of chemistry, School of Science, University of Tokyo, Tokyo 113-0033, Japan
*
Author to whom correspondence should be addressed.
Received: 19 March 2018 / Revised: 4 April 2018 / Accepted: 4 April 2018 / Published: 12 April 2018
(This article belongs to the Special Issue cGMP-Signalling in Cells: Molecular and Functional Features)
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Abstract

Nitric oxide (NO/cyclic guanosine monophosphate (cGMP)-regulated cellular mechanisms are involved in a variety of (patho-) physiological processes. One of the main effector molecules in this system, proteinkinase G (PKG), serves as a molecular switch by phosphorylating different target proteins and thereby turning them on or off. To date, only a few interaction partners of PKG have been described although the identification of protein–protein interactions (PPI) is indispensable for the understanding of cellular processes and diseases. Conventionally used methods to detect PPIs exhibit several disadvantages, e.g., co-immunoprecipitations, which depend on suitable high-affinity antibodies. Therefore, we established a cell-based protein-fragment complementation assay (PCA) for the identification of PKG target proteins. Here, a reporter protein (click beetle luciferase) is split into two fragments and fused to two different possible interaction partners. If interaction occurs, the reporter protein is functionally complemented and the catalyzed reaction can then be quantitatively measured. By using this technique, we confirmed the regulator of G-Protein signaling 2 (RGS2) as an interaction partner of PKGIα (a PKG-isoform) following stimulation with 8-Br-cGMP and 8-pCPT-cGMP. Hence, our results support the conclusion that the established approach could serve as a novel tool for the rapid, easy and cost-efficient detection of novel PKG target proteins. View Full-Text
Keywords: PKG; cGK; RGS2; cGMP; PCA; luciferase; protein–protein interaction PKG; cGK; RGS2; cGMP; PCA; luciferase; protein–protein interaction
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).

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Schramm, A.; Mueller-Thuemen, P.; Littmann, T.; Harloff, M.; Ozawa, T.; Schlossmann, J. Establishing a Split Luciferase Assay for Proteinkinase G (PKG) Interaction Studies. Int. J. Mol. Sci. 2018, 19, 1180.

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