title: Point mutation in the ligand-binding domain of the progesterone receptor generates a transdominant negative phenotype.
abstract: A short conserved alpha-helix in the carboxyl-terminal activation function of the ligand-binding domain of steroid hormone receptors, called AF2, is important for ligand-dependent transactivation of inducible genes. We have generated two AF2 mutants of the B isoform of human progesterone receptor (PRB): a point mutant, PRBE911A, and a short deletion, PRB delta907-913R. The two mutants are expressed at levels comparable to the wild type receptor in transfected cells. The PRBE911A mutant showed similar hormone- and DNA- binding affinities as the wild type receptor, whereas the PRB delta907-913R mutant was defective in hormone and DNA binding. Both mutants were inactive when transiently transfected in CV-1 cells, which do not express endogenous PR. However, the point mutant, but not the deletion mutant, inhibited transactivation by cotransfected wild type PRB in a hormone-dependent fashion. The activity of endogenous PR in T47D cells or of endogenous glucocorticoid receptor in HeLa cells was also inhibited by the PRBE911A, but not by the deletion mutant. The point mutant was less active when introduced into an N-terminal truncated form of PR, where it gave rise to proteins that formed homodimers with poor affinity for DNA, but were able to form heterodimers with PRB. The negative dominant phenotype of the PRBE911A mutant likely originates from competition with wild type receptors for binding to DNA and will be useful for mechanistic studies of receptor function.
authors.affiliation: Institut für Molekularbiologie und Tumorforschung, Philipps Universität, Marburg, Germany.
authors.forename: W
authors.initials: W
authors.lastname: Gong
authors.forename: S
authors.initials: S
authors.lastname: Chávez
authors.forename: M
authors.initials: M
authors.lastname: Beato
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D017353:Gene Deletion; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D017354:Point Mutation; D011374:Progesterone / Q000378:metabolism*; D011980:Receptors, Progesterone / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics*; D015533:Transcriptional Activation
chemical_list: D011980:Receptors, Progesterone; D011374:Progesterone