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Updated: Jan 19, 2026
Imaging Protein-protein Interactions in vivo
Published on: October 10, 2010
Flow cytometry-based FRET identifies binding intensities in PPARγ1 protein-protein interactions in living cells
Verena Trümper1, Andreas von Knethen1,2, Annegret Preuß3
1Institute of Biochemistry I - Pathobiochemistry, Faculty of Medicine, Goethe University Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt/Main, Germany.
Abstract:
PPARγ is a pharmacological target in inflammatory and metabolic diseases. Upon agonistic treatment or following antagonism, binding of co-factors is altered, which consequently affects PPARγ-dependent transactivation as well as its DNA-independent properties. Therefore, establishing techniques to characterize these interactions is an important issue in living cells. Methods: Using the FRET pair Clover/mRuby2, we set up a flow cytometry-based FRET assay by analyzing PPARγ1 binding to its heterodimerization partner RXRα. Analyses of PPARγ-reporter and co-localization studies by laser-scanning microscopy validated this system. Refining the system, we created a new readout to distinguish strong from weak interactions, focusing on PPARγ-binding to the co-repressor N-CoR2. Results: We observed high FRET in cells expressing Clover-PPARγ1 and mRuby2-RXRα, but no FRET when cells express a mRuby2-RXRα deletion mutant, lacking the PPARγ interaction domain. Focusing on the co-repressor N-CoR2, we identified in HEK293T cells the new splice variant N-CoR2-ΔID1-exon. Overexpressing this isoform tagged with mRuby2, revealed no binding to Clover-PPARγ1, nor in murine J774A.1 macrophages. In HEK293T cells, binding was even lower in comparison to N-CoR2 constructs in which domains established to mediate interaction with PPARγ binding are deleted. These data suggest a possible role of N-CoR2-ΔID1-exon as a dominant negative variant. Because binding to N-CoR2-mRuby2 was not altered following activation or antagonism of Clover-PPARγ1, we determined the effect of pharmacological treatment on FRET intensity. Therefore, we calculated flow cytometry-based FRET efficiencies based on our flow cytometry data. As with PPARγ antagonism, PPARγ agonist treatment did not prevent binding of N-CoR2. Conclusion: Our system allows the close determination of protein-protein interactions with a special focus on binding intensity, allowing this system to characterize the role of protein domains as well as the effect of pharmacological agents on protein-protein interactions.
Insights
We developed a new FRET assay to measure protein interactions in living cells, crucial for understanding inflammatory and metabolic diseases. This method quantifies binding intensity, revealing how drugs affect these interactions.
Area of Science:
- Molecular Biology
- Cellular Biology
- Pharmacology
Background:
- Peroxisome proliferator-activated receptor gamma (PPARγ) is a key target for treating inflammatory and metabolic diseases.
- PPARγ activity is modulated by co-factor binding, impacting its function in gene regulation and other cellular processes.
- Characterizing these protein-protein interactions in living cells is essential for understanding PPARγ's role and developing targeted therapies.
Purpose of the Study:
- To establish a Förster Resonance Energy Transfer (FRET) based assay for quantifying protein-protein interactions involving PPARγ in living cells.
- To analyze the binding dynamics between PPARγ1 and its heterodimerization partner RXRα.
- To investigate the interaction between PPARγ and the co-repressor N-CoR2, including a newly identified splice variant.
Main Methods:
- Development of a flow cytometry-based FRET assay utilizing the Clover/mRuby2 FRET pair.
- Validation of the FRET system through PPARγ-reporter assays and laser-scanning microscopy co-localization studies.
- Creation of a refined readout to differentiate strong from weak protein interactions, specifically focusing on PPARγ and N-CoR2 binding.
Main Results:
- High FRET signals confirmed PPARγ1 binding to RXRα, while a deletion mutant lacking the interaction domain showed no FRET.
- A novel splice variant, N-CoR2-ΔID1-exon, was identified and demonstrated no binding to PPARγ1 in HEK293T cells and macrophages.
- Pharmacological treatments (agonist or antagonist) did not alter N-CoR2 binding to PPARγ, suggesting N-CoR2-ΔID1-exon may act as a dominant-negative variant.
Conclusions:
- The developed FRET system accurately quantifies protein-protein interaction intensity in living cells.
- This assay is capable of characterizing the functional roles of specific protein domains and the impact of pharmacological agents on these interactions.
- The findings provide insights into PPARγ interactions and the potential dominant-negative function of the N-CoR2-ΔID1-exon splice variant.
