7-ALPHA-17-ALPHA-DIMETHYL-19-NORTESTOSTERONE (MIBOLERONE) INDUCES CONFORMATIONAL-CHANGES IN PROGESTERONE RECEPTORS DISTINCT FORM THOSE INDUCED BY ORG-2058

Using synthetic peptides with sequences derived from specific regions of human estrogen (ER) and progesterone (PR) receptors, we have develo ped site-directed monoclonal and polyclonal antibodies to specific dom ains of these receptors. These antibodies interact specifically with t he native (nondenatured) receptors and detect changes in the conformat ion of these proteins. Monoclonal antibody PR-AT 4.14 bound more tight ly to PR-ORG 2058 complexes than to PR-7 alpha,17 alpha-dimethyl-19-no r-testoster one (7 alpha,17 alpha, DMNT; mibolerone) complexes, sugges ting possible ligand-induced conformational changes in PR. In the abse nce of the antibody, PR-[H-3]ORG 2058 complexes sedimented as 4S-5S en tity on sucrose density gradients (SDG) containing 0.4 M KCI. In the p resence of the antibody, PR-[H-3]ORG 2058 complexes sedimented as 7-8S complexes. In contrast, at the same concentrations of antibody, PR-[H -3]7 alpha,17 alpha, DMNT complexes sedimented at 4S-5S legion in the absence of the antibody and as two populations in the presence of the antibody, suggesting that the antibody did not recognize all of the PR -7 alpha,17 alpha, DMNT complexes. To exclude the possibility that the inability of the antibody to recognize receptor-[H-3]7 alpha,17 alpha , DMNT complexes was die to its binding to androgen receptors, unlabel ed 5 alpha-dihydrotestostel one (5 alpha-DHT) (50 nM) was added to the incubation to inhibit 7 alpha,17 alpha, DMNT binding to androgen rece ptors. While PR-[H-3]ORG 2058 complexes were immunoprecipitated in the presence of the antibody, PR-[H-3]7 alpha,17 alpha, DMNT complexes we re only partially immunoprecipitated, further confirming the results o btained with SDG. Exchange of 7 alpha,17 alpha, DMNT bound to PR with [H-3]ORG 2058 resulted in a complex with tight binding of PR to the an tibody, while exchange of ORG 2058 bound to PR with PR-[H-3]7 alpha,17 alpha, DMNT reduced the binding of PR to the antibody, suggesting the steroid-induced conformational changes in PR were reversible. Increas ing the concentration of the antibody in the incubation resulted in a greater proportion of the PR-7 alpha,17 alpha, DMNT sedimenting in the 7-8S region of SDG, suggesting lower antibody affinity for this compl ex and higher affinity for PR-ORG 2058 complexes. The monoclonal antib ody recognized the unoccupied PR and PR-R U 486 complexes, as determin ed by SDG analysis and post-labeling with [H-3]ORG 2058. These observa tions suggest that 7 alpha,17 alpha, DMNT binding to PR induced confor mational changes which altered the epitope on the receptor, thereby re ducing or eliminating the antibody binding to the receptor.