Laboratory of Receptor Biology and Gene Expression

Hormone Action and Oncogenesis Section

Progesterone Receptor

Green Fluorescent Protein Chimeras Reveal Differences
Between the A- and B- Forms of the Human Progesterone Receptor

Subcellular localization and transcriptional activity of green fluorescent protein-progesterone receptor A and B chimeras (GFP-PRA and GFP-PRB) were examined in living mammalian cells. Both GFP-PRA and B chimeras were found to be similar in transcriptional activity compared to their non-GFP counterparts. GFP-PRA and PRA were both weakly active, while GFP-PRB and PRB gave a 20-40 fold induction using a reporter gene containing the full length MMTV LTR linked to the luciferase gene (pLTRluc). Using fluorescence microscopy, nuclear/cytoplasmic distributions for the unliganded and hormone activated forms of GFP-PRA and GFP-PRB were characterized. The two forms of the receptor were found to have distinct intracellular distributions; GFP-PRA was found to be more nuclear than GFP-PRB in four cell lines examined. This finding is particularly interesting given the fact that PRA and PRB contain the same known nuclear localization signals, and that PRA is merely a 165 amino acid truncated version of PRB. Using confocal microscopy and a live cell chamber, nuclear translocation of GFP-PRA and B using agonist (R5020) and antagonist (RU486) were also tested. GFP-PR chimeras will serve as useful tools for future colocalization studies with other transcription factors (using blue or yellow fluorescent protein tags).

Progesterone Receptor Trafficking
This is a blank space


Return to Top of Page	This is a blank cell

Live-cell Microscopy: GFP-PRA and GFP-PRB Constructs, with Agonist (R5020) and Antagonist (RU486)

un-induced	un-induced RU 5020
GFP-PRA
GFP-PRB
un-induced	un-induced RU 486
GFP-PRA
GFP-PRB

Methods: 1471.1 cells ( mouse mammary tumor derived line) were transfected with either pGFP-PRA or pGFP-PRB by electroporation and plated onto glass cover slips. 24 hours later, coverslips were placed into a live cell chamber and imaged using confocal microscopy. For hormone inductions, cells were induced with R5020 (a synthetic progestin) for 6 hours prior to visualization.

Return to Top of Page

This is a blank cell

Cells Transfected with Either GFP-PRA or GFP-PRB Were Analyzed in Terms of Their Nuclear/Cytoplasmic Distribution


Return to Top of Page	This is a blank cell

Conclusions

This study demonstrates, for the first time, subcellular localization of the human progesterone receptor in living cells. In the cell lines tested so far (1471.1, 3134, HeLa), PRA tends to be more nuclear than PRB. PRA is known to be a repressor of PRB in some systems.
Previously, localization studies on the A form of the PR have been extremely limited due to the lack of a good anti-PRA antibody, used for immunolocalization. Use of GFP chimeras eliminates the need for anti-PR antibodies.
Green fluorescent protein-progesterone receptor chimeras behave similarly to their non-GFP counterparts in terms of transcriptional activity and responsiveness to agonists and antagonists.
Overall, GFP chimeras are useful tools for observing subcellular trafficking and localization of steroid hormone receptors and may applications for other nuclear receptors as well.
Studying the molecular mechanisms of steroid hormone receptor activity may ultimately lead to the identification of better therapeutic targets (for birth control, reproductive disorders, inflammatory diseases) or improved treatment of steroid-responsive cancers.