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 sequence is located between the DNA-binding domain and the hormone-binding domain in the region within the sequences required for stability of the 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 dissociated by salt treatment, the antibody recognized the resulting 4S form of PR. This would suggest that the epitope is masked in the 8S form of PR and exposed in the 4S form. To study whether a similar complex exists in vivo, we used the antibody for immunohistochemistry. Two different fixation techniques were employed, freeze-drying-vapor fixation and liquid fixation. In the animals not treated with progesterone, intensive nuclear staining was detected independent of the fixation technique. When receptor from similarly treated animals was analyzed by sucrose gradient, all of the receptor molecules were in the oligomeric complex (8S). Ligand binding is known to promote a dissociation of this complex. Thus progesterone treatment should lead to an increased immunodetection of the epitope; however, progesterone treatment decreased the intensity of PR immunostaining. These results suggest that the oligomeric complex (8S), present in tissue extracts, does not exist in intact cell nuclei. They also call into question the proposed role of hsp90 in regulating progesterone receptor function.
Pekki
A
A
Department of Biomedical Sciences, University of Tampere, Finland.
Ylikomi
T
T
Syvälä
H
H
Tuohimaa
P
P
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002467:Cell Nucleus / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002645:Chickens; D000939:Epitopes / Q000032:analysis; D015151:Immunoblotting; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D010057:Oviducts / Q000378:metabolism; D010455:Peptides / Q000276:immunology / Q000378:metabolism*; D011233:Precipitin Tests; D011374:Progesterone / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011980:Receptors, Progesterone / Q000276:immunology / Q000378:metabolism*; D013997:Time Factors; D014157:Transcription Factors / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010455:Peptides; D011980:Receptors, Progesterone; D014157:Transcription Factors; D011374:Progesterone