Human progesterone receptor complexed with the antagonist RU 486 binds to hormone response elements in a structurally altered form.

Structural and functional properties of human progesterone receptors (PR) bound with the antiprogestin, RU 486, and the progestin agonist, R5020, were compared in order to identify receptor mechanisms responsible for the inability of RU 486 to activate the transcriptional capacity of receptors. RU 486 interaction with human PR did not inhibit receptor transformation as assessed by dissociation of nontransformed 8-10S oligomeric receptors (in vitro and in vivo) and by tight binding of PR to nuclei/chromatin in whole cells. Assays based on immunoprecipitation of PR-DNA complexes with an antibody to human PR and gel retardation were used to analyze the effect of RU 486 on receptor binding to the hormone response element (HRE) of the mouse mammary tumor virus (MMTV). RU 486 did not impair PR recognition of the MMTV HRE. Quantitative affinity constants and kinetic parameters of PR binding to these specific DNA sites were similar for receptors complexed with either agonist or antagonist. However, PR-RU 486 complexes exhibited an altered sedimentation rate on sucrose gradients and a faster mobility when bound to the MMTV HRE as assessed by gel retardation. These results indicate that human PR transformed by RU 486 exhibit no impairment in binding to specific DNA sites of target genes, but when bound to DNA assumes a structural form different from that of the receptor-agonist complexes to activate transcription results from this structural alteration in PR, which does not permit protein-protein interactions required for receptor-mediated induction of gene transcription.

[1]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[2]  D. Edwards,et al.  A quantitative comparison of dual control of a hormone response element by progestins and glucocorticoids in the same cell line. , 1989, Molecular endocrinology.

[3]  B. O’Malley,et al.  Cooperative binding of steroid hormone receptors contributes to transcriptional synergism at target enhancer elements , 1989, Cell.

[4]  E. Baulieu,et al.  The antiprogesterone RU486 stabilizes the heterooligomeric, non-DNA-binding, 8S-form of the rabbit uterus cytosol progesterone receptor , 1989, Steroids.

[5]  M. Schauer,et al.  Binding of hormone accelerates the kinetics of glucocorticoid and progesterone receptor binding to DNA. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[6]  D. Edwards,et al.  Human progesterone receptor binding to mouse mammary tumor virus deoxyribonucleic acid: dependence on hormone and nonreceptor nuclear factor(s). , 1989, Molecular endocrinology.

[7]  M. Perricaudet,et al.  Receptors bound to antiprogestin form abortive complexes with hormone responsive elements , 1988, Nature.

[8]  K. Horwitz,et al.  Human progesterone receptor transformation and nuclear down-regulation are independent of phosphorylation. , 1988, Molecular endocrinology.

[9]  D. Edwards,et al.  Steroid hormone-dependent interaction of human progesterone receptor with its target enhancer element. , 1988, Molecular endocrinology.

[10]  D. Edwards,et al.  Immunocytochemical localization of progesterone receptors in breast cancer with anti-human receptor monoclonal antibodies. , 1988, Cancer research.

[11]  B. O’Malley,et al.  Molecular interactions of steroid hormone receptor with its enhancer element: Evidence for receptor dimer formation , 1988, Cell.

[12]  M. Ptashne.  How eukaryotic transcriptional activators work , 1988, Nature.

[13]  P. Chambon,et al.  The estrogen receptor binds tightly to its responsive element as a ligand-induced homodimer , 1988, Cell.

[14]  L. Poellinger,et al.  Requirement of hormone for thermal conversion of the glucocorticoid receptor to a DNA-binding state , 1988, Nature.

[15]  B. Katzenellenbogen,et al.  Ligand-modulated regulation of progesterone receptor messenger ribonucleic acid and protein in human breast cancer cell lines. , 1988, Molecular endocrinology.

[16]  E. Baulieu.  Steroid hormone antagonists at the receptor level: A role for the heat‐shock protein MW 90,000 (hsp 90) , 1987, Journal of cellular biochemistry.

[17]  D. Edwards,et al.  Immunologic analysis of human breast cancer progesterone receptors. 1. Immunoaffinity purification of transformed receptors and production of monoclonal antibodies. , 1987, Biochemistry.

[18]  V. Moudgil,et al.  Transformation of calf uterine progesterone receptor: analysis of the process when receptor is bound to progesterone and RU38486. , 1987, Biochemistry.

[19]  E. Baulieu,et al.  Antiglucocorticosteroid effects suggest why steroid hormone is required for receptors to bind DNA in vivo but not in vitro , 1987, Nature.

[20]  J. Martial,et al.  Glucocorticoid receptors bound to the antagonist RU486 are not downregulated despite their capacity to interact in vitro with defined gene regions. , 1987, Journal of steroid biochemistry.

[21]  D. Fowlkes,et al.  A rapid, sensitive, and inexpensive assay for chloramphenicol acetyltransferase. , 1987, DNA.

[22]  M. Beato,et al.  Steroid-free glucocorticoid receptor binds specifically to mouse mammary tumour virus DNA , 1986, Nature.

[23]  C. Le Page,et al.  Sequence‐specific DNA binding of the progesterone receptor to the uteroglobin gene: effects of hormone, antihormone and receptor phosphorylation. , 1986, The EMBO journal.

[24]  T. Schmidt.  In vitro activation and DNA binding affinity of human lymphoid (CEM-C7) cytoplasmic receptors labeled with the antiglucocorticoid RU 38486. , 1986, Journal of steroid biochemistry.

[25]  B. Katzenellenbogen,et al.  Antiprogestin-receptor complexes: differences in the interaction of the antiprogestin RU38,486 and the progestin R5020 with the progesterone receptor of human breast cancer cells. , 1986, Biochemical and biophysical research communications.

[26]  V. Moudgil,et al.  Activation of rat liver glucocorticoid receptor bound to the antiglucocorticoid RU38486. , 1985, Biochemical and biophysical research communications.

[27]  E. Milgrom,et al.  Mechanism of action of an antiprogesterone, RU486, in the rabbit endometrium. Effects of RU486 on the progesterone receptor and on the expression of the uteroglobin gene. , 1985, European journal of biochemistry.

[28]  E. Baulieu,et al.  DNA binding properties of glucocorticosteroid receptors bound to the steroid antagonist RU‐486. , 1984, The EMBO journal.

[29]  D. Philibert.  RU 38486: An Original Multifaceted Antihormone in Vivo , 1984 .

[30]  E. Baulieu,et al.  Inhibition of glucocorticosteroid action in cultured L-929 mouse fibroblasts by RU 486, a new anti-glucocorticosteroid of high affinity for the glucocorticosteroid receptor. , 1983, Experimental cell research.

[31]  M. Wigler,et al.  DNA-mediated transfer of the adenine phosphoribosyltransferase locus into mammalian cells. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[32]  E. Baulieu,et al.  Stimulation of specific transcription and DNA binding studies suggest that in vitro transformed RU 486-glucocorticosteroid receptor complexes display agonist activity. , 1988, Journal of steroid biochemistry.

[33]  K. Horwitz,et al.  Multiple human progesterone receptor messenger ribonucleic acids and their autoregulation by progestin agonists and antagonists in breast cancer cells. , 1988, Molecular endocrinology.

[34]  D. Philibert,et al.  RU 38486: potent antiglucocorticoid activity correlated with strong binding to the cytosolic glucocorticoid receptor followed by an impaired activation. , 1984, Journal of steroid biochemistry.