PROGESTERONE-RECEPTOR DOES NOT FORM OLIGOMERIC (8S), NON-DNA-BINDING COMPLEX IN INTACT CELL-NUCLEI

We raised a polyclonal antibody, alpha D, against a synthetic peptide (amino acids 522-535) of chicken progesterone receptor (PR). The seque nce is located between the DNA-binding domain and the hormone-binding domain in the region within the sequences required for stability of th e oligomeric form of PR. In the immunoblot, alpha D reacted with both A and B forms of PR. In the sucrose gradient and dot-blot the antibody did not recognize the so-called 8S form of PR, which is an oligomeric complex of PR and other proteins. When the oligomeric complex was dis sociated by salt treatment, the antibody recognized the resulting 4S f orm of PR. This would suggest that the epitope is masked in the 8S for m of PR and exposed in the 4S form. To study whether a similar complex exists in vivo, we used the antibody for immunohistochemistry. Two di fferent fixation techniques were employed, freeze-drying-vapor fixatio n and liquid fixation. In the animals not treated with progesterone, i ntensive nuclear staining was detected independent of the fixation tec hnique. When receptor from similarly treated animals was analyzed by s ucrose gradient, all of the receptor molecules were in the oligomeric complex (8S). Ligand binding is known to promote a dissociation of thi s complex. Thus progesterone treatment should lead to an increased imm unodetection of the epitope; however, progesterone treatment decreased the intensity of PR immunostaining. These results suggest that the ol igomeric complex (8S), present in tissue extracts, does not exist in i ntact cell nuclei. They also call into question the proposed role of h sp90 in regulating progesterone receptor function. (C) 1995 Wiley-Liss , Inc.