The PR-A and PR-B isoforms of progesterone receptors (PR) have different physiological functions, and their ratio varies widely in breast cancers. To determine whether the two PR regulate different genes, we used human breast cancer cell lines engineered to express one or the other isoform. Cells were treated with progesterone in triplicate, time-separated experiments, allowing statistical analyses of microarray gene expression data. Of 94 progesterone-regulated genes, 65 are uniquely regulated by PR-B, 4 uniquely by PR-A, and only 25 by both. Almost half the genes encode proteins that are membrane-bound or involved in membraneinitiated signaling. We also find an important set of progesterone-regulated genes involved in mammary gland development and/or implicated in breast cancer. This first, large scale study of PR gene regulation has important implications for the measurement of PR in breast cancers and for the many clinical uses of synthetic progestins. It suggests that it is important to distinguish between the two isoforms in breast cancers and that isoform-specific genes can be used to screen for ligands that selectively modulate the activity of PR-A or PR-B. Additionally, use of natural target genes, rather than "consensus" response elements, for transcription studies should improve our understanding of steroid hormone action.

The hsp90 chaperoning pathway is a multiprotein system that is required for the production or activation of many cell regulatory proteins, including the progesterone receptor (PR). We report here the identity of GCUNC-45 as a novel modulator of PR chaperoning by hsp90. GCUNC-45, previously implicated in the activities of myosins, can interact in vivo and in vitro with both PR-A and PR-B and with hsp90. Overexpression and knockdown experiments show GCUNC-45 to be a positive factor in promoting PR function in the cell. GCUNC-45 binds to the ATP-binding domain of hsp90 to prevent the activation of its ATPase activity by the cochaperone Aha1. This effect limits PR chaperoning by hsp90, but this can be reversed by FKBP52, a cochaperone that is thought to act later in the pathway. These findings reveal a new cochaperone binding site near the N terminus of hsp90, add insight on the role of FKBP52, and identify GCUNC-45 as a novel regulator of the PR signaling pathway.The activities of the progesterone receptor (PR) are intimately linked to its associations with other proteins that are essential for normal progesterone action. Unliganded PR is associated with a multifunctional complex of proteins which includes the heat shock proteins hsp70 and hsp90 plus several cochaperone proteins. This heterocomplex is responsible for correct assembly and folding of the PR as well as preventing its degradation (36). Recent studies indicate additional roles for molecular chaperones in receptor trafficking and in the maintenance of receptor function in the nucleus (11,12). Hormone binding promotes conformational changes in the PR and its release from the chaperone complex. The resulting PR dimer is then able to associate with specific coactivators and general transcription factors as well as progestin response elements in the promoters of target genes (13,19,27,44,50).Cell-free systems using the purified proteins have been of crucial importance in dissecting the ordered pathway that leads to the hormone-responsive state of the receptor (36,39,52). This hsp90-dependent chaperoning pathway is initiated by hsp40 and hsp70 binding to PR, followed by the binding of Hop-hsp90 to hsp70 (16). This intermediate complex is then modified by loss of hsp70 and Hop and recruitment of p23, resulting in a receptor able to bind hormone. In the cell, this last step also incorporates the cochaperone FKBP51, FKBP52, or Cyp40, but this step has eluded dissection in vitro.Although the overall mechanism might be as described above, details and dynamics of this process are still unclear. Furthermore, several additional proteins have been discovered recently that interact with hsp90 or hsp70, such as Chip, Bag1, TPR2, and Aha1 (23,31,36,39,52). These are all likely to have important roles in some aspect of hsp70/hsp90 chaperoning to provide a more intricate and versatile process than that described in the current model. There are also a number of proteins that interact with the PR as transcriptional coregulators or to relate PR functions with other cell signa...

Twenty‐six peptide analogs of the Saccharomyces cerevisiaeα‐factor, a tridecapeptide mating pheromone (W1H2W3L4Q5L6K7P8G9Q10p11M12Y13) with either l‐ or D‐alanine replacement of each amino acid residue (Ala‐scanned) and with the isosteric replacement of methionine at position 12 by norleucine, were synthesized, purified to homogeneity and assayed for biological activity and receptor binding. Two new and effective antagonists. [D‐Ala3,Nle12]α‐factor and [D‐Ala4,Nle12]α‐factor, were found among the series, and the [D‐Ala10,Nle12]α‐factor demonstrated a marked ability to increase the biological activity of [Nle12]α‐factor without having any effect by itself. One analog, the [L‐Ala1α‐factor, showed a 3‐fold increase in bioactivity over the [Nle12]α‐factor, although its binding to the α‐factor receptor was about 70‐fold less than [Nle12]α‐factor. Residues near the carboxyl terminus contributed more strongly to receptor binding than other residues, whereas those near the amine terminus of the α‐factor played an important role in signal transduction. The effect of insertion of D‐Ala residues at positions 7, 8, 9 and 10 on bioactivity and receptor binding of the peptide suggested a specific positioning role of the central loop in establishing optimal contacts between the receptor and the ends of the pheromone. We conclude that the α‐factor may be divided into segments with dominant roles in forming the biologically active pheromone conformation, in receptor binding and in initiating signal transduction. The discovery of such relationships was made possible by the systematic variation of each residue in the peptide and by the testing of each analog in highly defined biological and binding assays.

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