Hinge and amino-terminal sequences contribute to solution dimerization of human progesterone receptor.

We and others have shown previously that progesterone receptors (PR) form homodimers in solution in the absence of DNA and that dimers are the preferential form of receptor that binds with high affinity to target DNA. To determine the sequence regions involved in solution homodimerization, wild type PR and truncated PR proteins were expressed in an insect baculovirus system. The expression constructs included the ligand-binding domain [LBD, amino acids (aa) 688-933], the LBD plus hinge (hLBD, aa 634-933), the hLBD plus the DNA-binding domain (DhLBD, aa 538-933), and the full- length A and B isoforms of PR. PR-PR interactions were detected by three methods, coimmunoprecipitation of the PR fragments with full-length PR-A, pull-down of PR-polypeptides with polyhistidine-tagged versions of the same polypeptides immobilized to metal affinity columns and cooperative ligand-binding assays (Hill coefficient, n(H) > 1 indicating PR-PR interaction). By all three assays, the LBD alone was not sufficient to mediate protein-protein interaction. However, the LBD did exhibit other properties ascribed to this domain, including binding to steroids with a relatively good affinity and specificity, ligand-induced conformational changes at the carboxyl terminus tail and binding of heat shock protein 90 and its dissociation in response to hormone. Thus, failure of the expressed LBD to mediate dimerization does not appear to be due to an extensively misfolded or unstable polypeptide. The minimal carboxyl-terminal fragment capable of mediating PR-PR interaction was the hLBD construct. However, by immobilized metal affinity chromatography assay, self-association of PR-A was 3.5-fold more efficient than that of either the DhLBD or hLBD constructs. An expressed amino-terminal domain (aa 165-535) lacking the DNA-binding domain, hinge, and LBD was found to physically associate with PR-A or with another amino-terminal fragment lacking the LBD, but retaining the DNA-binding domain. These results provide evidence for direct amino-terminal interactions in the more efficient PR-PR interaction exhibited by wild-type PR-A, as compared with DhLBD and hLBD constructs. The overall results of this paper are consistent with the conclusion that the carboxyl-terminal LBD is not sufficient for mediating PR dimerization and that multiple regions, including the hinge and amino-terminal sequences, contribute either directly or indirectly to homodimerization of PR.

[1]  K. Yamamoto,et al.  Crystallographic analysis of the interaction of the glucocorticoid receptor with DNA , 2003, Nature.

[2]  K. Yamamoto,et al.  Modular Structure of Glucocorticoid Receptor Domains Is Not Equivalent to Functional Independence , 1996, The Journal of Biological Chemistry.

[3]  D. Pappin,et al.  Biochemical characterization and novel isolation of pure estrogen receptor hormone-binding domain , 1996, The Journal of Steroid Biochemistry and Molecular Biology.

[4]  D. Edwards,et al.  Binding of [3H]progesterone to the human progesterone receptor: differences between individual and mixed isoforms. , 1996, Endocrinology.

[5]  E. Baulieu,et al.  Glucocorticosteroid receptor dimerization investigated by analysis of receptor binding to glucocorticosteroid responsive elements using a monomer-dimer equilibrium model. , 1996, Biochemistry.

[6]  J. Léger,et al.  Putative steroid binding domain of the human mineralocorticoid receptor, expressed in E. coli in the presence of heat shock proteins shows typical native receptor characteristics , 1996, The Journal of Steroid Biochemistry and Molecular Biology.

[7]  B. Katzenellenbogen,et al.  Ligand-dependent, transcriptionally productive association of the amino- and carboxyl-terminal regions of a steroid hormone nuclear receptor. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[8]  E. Langley,et al.  Evidence for an Anti-parallel Orientation of the Ligand-activated Human Androgen Receptor Dimer (*) , 1995, The Journal of Biological Chemistry.

[9]  K. Umesono,et al.  The nuclear receptor superfamily: The second decade , 1995, Cell.

[10]  Thorsten Heinzel,et al.  Ligand-independent repression by the thyroid hormone receptor mediated by a nuclear receptor co-repressor , 1995, Nature.

[11]  R. Evans,et al.  A transcriptional co-repressor that interacts with nuclear hormone receptors , 1995, Nature.

[12]  S. F. Arnold,et al.  An antiestrogen: a phosphotyrosyl peptide that blocks dimerization of the human estrogen receptor. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[13]  J. Katzenellenbogen,et al.  Molecular characterization by mass spectrometry of the human estrogen receptor ligand-binding domain expressed in Escherichia coli. , 1995, Molecular endocrinology.

[14]  B. Lefebvre,et al.  Structural determinants of the ligand-binding site of the human retinoic acid receptor alpha. , 1995, Biochemistry.

[15]  K. Yamamoto,et al.  Structure refinement of the glucocorticoid receptor-DNA binding domain from NMR data by relaxation matrix calculations. , 1995, Journal of molecular biology.

[16]  V. Giguère,et al.  The nonconserved hinge region and distinct amino-terminal domains of the ROR alpha orphan nuclear receptor isoforms are required for proper DNA bending and ROR alpha-DNA interactions , 1995, Molecular and cellular biology.

[17]  L. Nilsson,et al.  Modulation of DNA‐binding specificity within the nuclear receptor family by substitutions at a single amino acid position , 1995, Proteins.

[18]  X. Jacq,et al.  Human TAFII30 is present in a distinct TFIID complex and is required for transcriptional activation by the estrogen receptor , 1994, Cell.

[19]  T. Nemoto,et al.  Dimerization characteristics of the DNA- and steroid-binding domains of the androgen receptor , 1994, The Journal of Steroid Biochemistry and Molecular Biology.

[20]  C. Glass.  Differential recognition of target genes by nuclear receptor monomers, dimers, and heterodimers. , 1994, Endocrine reviews.

[21]  D. Edwards,et al.  The DNA-bending protein HMG-1 enhances progesterone receptor binding to its target DNA sequences , 1994, Molecular and cellular biology.

[22]  O. Jänne,et al.  Analysis of androgen receptor-DNA interactions with receptor proteins produced in insect cells. , 1994, The Journal of biological chemistry.

[23]  B. O’Malley,et al.  The human estrogen receptor hormone binding domain dimerizes independently of ligand activation , 1994, The Journal of Steroid Biochemistry and Molecular Biology.

[24]  H. Thole.  The side chains responsible for the dimerization of the estradiol receptor by ionic bonds are lost in a 17 kDa fragment extending downstream from aa 303 , 1994, The Journal of Steroid Biochemistry and Molecular Biology.

[25]  B. O’Malley,et al.  Human progesterone receptor A form is a cell- and promoter-specific repressor of human progesterone receptor B function. , 1993, Molecular endocrinology.

[26]  E. Wilson,et al.  Steroid requirement for androgen receptor dimerization and DNA binding. Modulation by intramolecular interactions between the NH2-terminal and steroid-binding domains. , 1993, The Journal of biological chemistry.

[27]  M. V. Van Dyke,et al.  Protein-protein interaction studies using immobilized oligohistidine fusion proteins. , 1993, Analytical biochemistry.

[28]  M. Beato,et al.  Steroid hormone receptors: interaction with deoxyribonucleic acid and transcription factors. , 1993, Endocrine reviews.

[29]  K. Yamamoto,et al.  Evidence that the hormone binding domain of steroid receptors confers hormonal control on chimeric proteins by determining their hormone-regulated binding to heat-shock protein 90. , 1993, Biochemistry.

[30]  E. Milgrom,et al.  Specific binding of progesterone receptor to progesterone-responsive elements does not require prior dimerization. , 1993, European journal of biochemistry.

[31]  K. Dahlman-Wright,et al.  Protein-protein interactions between the DNA-binding domains of nuclear receptors: Influence on DNA-binding , 1993, The Journal of Steroid Biochemistry and Molecular Biology.

[32]  D. Edwards,et al.  Effects of the steroid antagonist RU486 on dimerization of the human progesterone receptor. , 1992, Biochemistry.

[33]  D. Edwards,et al.  Ligands induce conformational changes in the carboxyl-terminus of progesterone receptors which are detected by a site-directed antipeptide monoclonal antibody. , 1992, Molecular endocrinology.

[34]  P. Chambon,et al.  Cooperation of proto‐signals for nuclear accumulation of estrogen and progesterone receptors. , 1992, The EMBO journal.

[35]  D. Edwards,et al.  Hormone and antihormone induce distinct conformational changes which are central to steroid receptor activation. , 1992, The Journal of biological chemistry.

[36]  K. Dahlman-Wright,et al.  Determinants of high-affinity DNA binding by the glucocorticoid receptor: evaluation of receptor domains outside the DNA-binding domain. , 1992, Biochemistry.

[37]  W. Herr,et al.  Ethidium bromide provides a simple tool for identifying genuine DNA-independent protein associations. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[38]  J. Drouin,et al.  Homodimer formation is rate-limiting for high affinity DNA binding by glucocorticoid receptor. , 1992, Molecular endocrinology.

[39]  B. Katzenellenbogen,et al.  Structural requirements for high affinity ligand binding by estrogen receptors: a comparative analysis of truncated and full length estrogen receptors expressed in bacteria, yeast, and mammalian cells. , 1992, Molecular endocrinology.

[40]  H. Bujard,et al.  Physical interaction between heat shock proteins DnaK, DnaJ, and GrpE and the bacterial heat shock transcription factor σ 32 , 1992, Cell.

[41]  D. F. Skafar.  Differences in the binding mechanism of RU486 and progesterone to the progesterone receptor. , 1991, Biochemistry.

[42]  D. Edwards,et al.  Characterization and functional properties of the A and B forms of human progesterone receptors synthesized in a baculovirus system. , 1991, Molecular endocrinology.

[43]  D. F. Skafar.  Differential DNA binding by calf uterine estrogen and progesterone receptors results from differences in oligomeric states. , 1991, Biochemistry.

[44]  L. Poellinger,et al.  Assembly of a glucocorticoid receptor complex prior to DNA binding enhances its specific interaction with a glucocorticoid response element. , 1991, The Journal of biological chemistry.

[45]  G. Perdew,et al.  Evidence that the 90-kDa heat shock protein (HSP90) exists in cytosol in heteromeric complexes containing HSP70 and three other proteins with Mr of 63,000, 56,000, and 50,000. , 1991, The Journal of biological chemistry.

[46]  H. Akil,et al.  Localization of the 90-kDa heat shock protein-binding site within the hormone-binding domain of the glucocorticoid receptor by peptide competition. , 1991, The Journal of biological chemistry.

[47]  B. O’Malley,et al.  Dimerization of the chicken progesterone receptor in vitro can occur in the absence of hormone and DNA. , 1990, Molecular endocrinology.

[48]  K. Dahlman-Wright,et al.  The steroid-binding properties of recombinant glucocorticoid receptor: A putative role for heat shock protein hsp90 , 1990, The Journal of Steroid Biochemistry and Molecular Biology.

[49]  J. Schwabe,et al.  Solution structure of the DNA-binding domain of the oestrogen receptor. , 1990, Nature.

[50]  P. Sharp,et al.  Human estrogen receptor forms multiple protein-DNA complexes. , 1990, The Journal of biological chemistry.

[51]  P Chambon,et al.  Two distinct estrogen‐regulated promoters generate transcripts encoding the two functionally different human progesterone receptor forms A and B. , 1990, The EMBO journal.

[52]  T. Mantle,et al.  Glutathione S-transferases. , 1990, Biochemical Society transactions.

[53]  S. Fawell,et al.  Characterization and colocalization of steroid binding and dimerization activities in the mouse estrogen receptor , 1990, Cell.

[54]  P. Eriksson,et al.  Protein-protein contacts in the glucocorticoid receptor homodimer influence its DNA binding properties. , 1990, The Journal of biological chemistry.

[55]  D. Edwards,et al.  Human progesterone receptor complexed with the antagonist RU 486 binds to hormone response elements in a structurally altered form. , 1989, Molecular endocrinology.

[56]  W. Welch,et al.  Binding of heat shock proteins to the avian progesterone receptor , 1989, Molecular and cellular biology.

[57]  E. Milgrom,et al.  Mechanisms of nuclear localization of the progesterone receptor: Evidence for interaction between monomers , 1989, Cell.

[58]  T. Perlmann,et al.  The purified activated glucocorticoid receptor is a homodimer. , 1989, The Journal of biological chemistry.

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

[60]  R. Evans,et al.  The steroid and thyroid hormone receptor superfamily. , 1988, Science.

[61]  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.

[62]  G. Theofan,et al.  Characterization of the calf uterine progesterone receptor and its stabilization by nucleic acids. , 1984, Endocrinology.

[63]  W B Jakoby,et al.  Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. , 1974, The Journal of biological chemistry.

[64]  G. Scatchard,et al.  THE ATTRACTIONS OF PROTEINS FOR SMALL MOLECULES AND IONS , 1949 .

[65]  D. Edwards,et al.  Dimerization of mammalian progesterone receptors occurs in the absence of DNA and is related to the release of the 90-kDa heat shock protein. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[66]  S. L. Le Grice,et al.  Rapid purification of homodimer and heterodimer HIV-1 reverse transcriptase by metal chelate affinity chromatography. , 1990, European journal of biochemistry.

[67]  R. Evans,et al.  Identification of receptors for retinoids as members of the steroid and thyroid hormone receptor family. , 1990, Methods in enzymology.

[68]  H. Samuels,et al.  Characterization of the domain structure of chick c-erbA by deletion mutation: in vitro translation and cell transfection studies. , 1989, Molecular endocrinology.

[69]  D. Koshland.  7 The Molecular Basis for Enzyme Regulation , 1970 .