ANALYSIS OF THE INTERACTION BETWEEN GLUCOCORTICOID AND PROGESTERONE RECEPTORS IN LIVING CELLS BY FLUORESCENCE CROSS-CORRELATION

MELISA, M; OGARA, MF; STORTZ, M; LEVI, V; PECCI, A

Bariloche

Simposio; 3rd South American Symposium in Signal Transduction and Molecular Medicine; 2015

Glucocorticoid receptor (GR) and progesterone receptor (PR) are closely related members of the steroid receptor family of transcription factors. They share many similar structural and functional characteristics, including DNA sequence recognition specificity. Despite their similarity, the cognate hormones display a very distinct spectrum of physiological actions. In the mammary gland, the activation of PR is associated with cell proliferation and tumor progression while activated GR was demonstrated to favor cell differentiation. Thus, the relative abundance of both receptors may modulate the proliferative and differentiation response of the mammary epithelium. In view of these precedents, the objective of this work was to test the ability of both receptors to be part of the same complex. To assess if PR and GR have the potential to form complexes in vivo, mammary tumor epithelial T47D cells were transfected with expression vectors encoding both receptors fused to fluorescent proteins (eGFP-PR and mCherry-GR) and incubated with Dexamethasone (DEX) or the progestin R5020. Then, Number and Brightness analysis (N&B) was performed in living cells. This novel technique, based on moment-analysis, provides the average number of moving fluorescent molecules and their brightness at every pixel in the images. The molecular brightness can be measured by analyzing frame to frame the intensity fluctuations in the confocal volume. To evaluate mCherry-GR and eGFP-PR associations, cross correlation analysis of the intensity fluctuations was performed. When eGFP-PR and mCherry-GR particles were analyzed, a symmetric cross correlation (brightness cross correlation [Bcc]) centered on zero was observed indicating an absence of interaction. However, mCherry-GR and eGFP-PR showed an asymmetric, positive Bcc value in cells treated with R5020 or DEX (Bcc = 0.009 + 0.004; Bcc = 0.007 + 0.003, respectively), indicating that both receptors are present in the same complex. Even more, when cells were treated with R5020+Dex the positive Bcc value significantly increased (Bcc = 0.042 + 0.004), suggesting an increase in the number of complexes containing both activated receptors. Together these results showed that PR and GR can be part of the same protein complex upon hormonal stimulation, supporting the idea that they can be recruited to shared regions as heterodimers.