Abstract
Our previous work has shown that the progesterone receptor (PR) can exist in two distinct functional states in mammary adenocarcinoma cells. The differences in function included the ability to activate a promoter in organized chromatin, sensitivity to ligand, and ligand-independent activation. To determine whether these functional differences were because of altered cellular processing, we carried out biochemical analyses of the functionally distinct PRs. Although the majority of PR is localized to the nucleus, biochemical partitioning resulted in a loosely bound (cytosolic) fraction, and a tightly bound (nuclear) fraction. In the absence of progestins, the functionally distinct PRs differed significantly in partitioning between the two fractions. To characterize these fractions further, we analyzed interactions of unliganded PR with chaperones by coimmunoprecipitation. We determined that PR in the cytosolic fraction associated with hsp90 and p23. In contrast, PR in the nuclear fraction consisted of complexes containing hsp90, p23, and FKBP51 as well as PR that was dimerized and highly phosphorylated. Hormone treatment significantly reduced the formation of all PR-chaperone complexes. The hsp90 inhibitor, geldanamycin, similarly blocked transcriptional activity of both functionally distinct receptors. However, the two forms of the PR differed in their ability to associate with the mouse mammary tumor virus promoter in organized chromatin. These findings provide new information about the composition and distribution of mature progesterone receptor complexes in mammary adenocarcinoma cells, and suggest that differences in receptor subcellular distribution have a significant impact on their function. These findings also reveal that transiently expressed steroid receptors may not always be processed like their endogenous counterparts.
Highlights
Progesterone receptor (PR)1 functions in the development of lobular alveolar structures in the normal mammary gland [1, 2]
Our previous studies detailed profound functional differences between two forms of the PR in mammary adenocarcinoma cells, one transiently expressed and one constitutively expressed. These functionally distinct PRs differed in their ability to activate a target gene requiring chromatin remodeling, their sensitivity to progestins, and their ability to be activated by other signaling pathways in a ligand-independent fashion
We have characterized the two forms of PR in terms of their cellular distribution and nuclear binding properties, their interactions with chaperone proteins, and their dimerization status. These results provide new information about the diversity and cellular localization of specific chaperone-receptor complexes and the nature of the unliganded receptor in mammary adenocarcinoma cells
Summary
Vol 279, No 15, Issue of April 9, pp. 15231–15239, 2004 Printed in U.S.A. Progesterone Receptor Deficient in Chromatin Binding Has an Altered Cellular State*. The two forms of the PR differed in their ability to associate with the mouse mammary tumor virus promoter in organized chromatin These findings provide new information about the composition and distribution of mature progesterone receptor complexes in mammary adenocarcinoma cells, and suggest that differences in receptor subcellular distribution have a significant impact on their function. This cell-free system revealed that maintenance of progestin binding of the PR required hsp, Hip, Hop, hsp, and p23 [16] These findings suggested a functionally significant role for PR processing in its ability to activate transcription. Despite its increased nuclear binding, we observed that tPR did not associate with the stably replicating MMTV promoter even upon addition of ligand These results indicate that unliganded PR complexes are not uniform and can be differentially distributed. Our findings are of high relevance to studies of steroid receptor function because transiently expressed receptors in cultured cells are often used as models for in vivo steroid receptor function
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