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Hinge and Amino-Terminal Sequences Contribute to Solution Dimerization of Human Progesterone Receptor

Department of Pathology and Program in Molecular Biology (M.J.T., T.L., D.P.E.) University of Colorado Health Sciences Center Denver, Colorado 80262
Department of Physiology (S.J., P.C., D.F.S.) Wayne State University School of Medicine, Detroit, Michigan 48201

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.

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