Schramm, Andrea and Mueller-Thuemen, Philip and Littmann, Timo and Harloff, Manuela and Ozawa, Takeaki and Schlossmann, Jens (2018) Establishing a Split Luciferase Assay for Proteinkinase G (PKG) Interaction Studies. INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, 19 (4): 1180. ISSN 1422-0067,
Full text not available from this repository. (Request a copy)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 PKGI alpha (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.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | KINASE-I-ALPHA; VASCULAR SMOOTH-MUSCLE; FRAGMENT COMPLEMENTATION ASSAYS; GREEN FLUORESCENT PROTEIN; HUMAN PLATELETS; CGMP; CAMP; BETA; ACTIVATION; CELLS; PKG; cGK; RGS2; cGMP; PCA; luciferase; protein-protein interaction |
| Subjects: | 600 Technology > 615 Pharmacy |
| Divisions: | Chemistry and Pharmacy > Institute of Pharmacy > Pharmacology and Toxicology (Prof. Schlossmann, formerly Prof. Seifert) |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 20 Mar 2020 08:17 |
| Last Modified: | 20 Mar 2020 08:17 |
| URI: | https://pred.uni-regensburg.de/id/eprint/14806 |
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