Definition of a Negative Modulation Domain in the Human Progesterone Receptor

Abstract

The progesterone receptor (PR) occurs in two major forms, the full-length PRB and the amino-truncated PRA, which lacks 164 amino-terminal residues. PRB functions as a strong transcriptional activator of progesterone-responsive genes, whereas PRA is inactive in several cell types where it may even act as a trans-dominant repressor of PRB and other steroid receptors, like the glucocorticoid receptor or, reportedly, the estrogen receptor. We initially observed that a PR deleted of its entire amino domain (PR538-C) is incapable of trans-repressing PRB or glucocorticoid receptor, suggesting that a negative modulation domain must be contained in the region between position 165 and 538. After testing progressive deletion mutants and chimeras, we demonstrate that this negative modulating domain is confined within 120 residues in the amino-terminal region and that it contains a subdomain of 40 residues that is crucial for intermolecular transrepression. Duplication, deletion, and transplantation of the negative modulation domain show that the negative modulation domain has only a limited functional autonomy. In our hands, transrepression of estrogen receptor could not be substantiated, and, under our conditions, at least an equimolar concentration of PRA expression plasmid is required for transrepression. Our deletion studies reveal domains that correlate with strong homology patches between the amino-terminal domains of mammalian and avian PR.

The Inhibitory Function in Human Progesterone Receptor N Termini Binds SUMO-1 Protein to Regulate Autoinhibition and Transrepression

“…The expressed proteins were resolved on SDS-PAGE, and conjugated protein was detected by immunoblotting with AB-52 or an anti-GFP polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA). (6,(37)(38)(39) have identified at least three different inhibitory properties of PRs. Autoinhibition refers to an IF located in the PR N terminus whose deletion increases the activity of PR-A at least 6-fold from a tandem progesterone response element (PRE 2 )-containing promoter.…”

“…However, based on complete dose-response data from a comprehensive series of deletion mutants constructed in NTA, NTB, and full-length receptors, it is now clear that the repressor activity requires both the N-terminal SUMO-1-binding site and the C-terminally liganded HBD. Huse et al (38) used a systematic set of N-terminal PR deletions to map autoinhibition to residues 387-427. However, they mapped the transrepression region to residues 307-347, using GR as the target.…”

“…We (37) and others (38,39) have identified an inhibitory function (IF) in the N-terminal 165-456 amino acids between AF3 and AF1 common to both PR isoforms. Deletion of all or part of this autoinhibitory region in PR-A increases their transcriptional activity.…”

“…A transrepressor activity, distinct from autoinhibition, also maps to the IF domain of PR-A. This activity is defined by the ability of PR-A to inhibit the transcriptional activity of PR-B, ER, or GR (38,39). The mechanisms that distinguish autoinhibition from transrepression, both of which originate from the IF domain, are unclear.…”

“…The mechanisms that distinguish autoinhibition from transrepression, both of which originate from the IF domain, are unclear. Recent attempts to precisely map these N-terminal repressor functions have led to ambiguous conclusions, identifying multiple, nonoverlapping regions (38,39).…”