A. Pekki et al., PROGESTERONE-RECEPTOR DOES NOT FORM OLIGOMERIC (8S), NON-DNA-BINDING COMPLEX IN INTACT CELL-NUCLEI, Journal of cellular biochemistry, 58(1), 1995, pp. 95-104
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.