Hormone-induced dissociation of the androgen receptor-heat-shock protein complex: use of a new monoclonal antibody to distinguish transformed from nontransformed receptors.
The hormone-induced transformation process of the androgen receptor in the androgen-responsive human prostatic carcinoma cell line LNCaP was studied. Immunoprecipitation of the nontransformed cytosolic receptor (8S on sucrose gradients) with a specific monoclonal antibody (F39.4.1) resulted in coprecipitation of three heat-shock proteins (hsp90, hsp70, and hsp56). Upon incubation of the cells with the synthetic androgen R1881, the sedimentation value of the receptor complex decreased to an intermediate form of 6S, and an almost complete loss of coprecipitating heat-shock proteins was observed. After a 2-h incubation, the receptor was recovered in considerable part from the nuclear fraction (extraction with high salt; 4.6S form). By use of the bifunctional cross-linker dimethyl pimelimidate, dissociation of the 8S complex, but not of the 6S complex, was blocked. A newly developed monoclonal antibody (F52.24.4), directed against the C-terminal part of the DNA-binding domain of the androgen receptor, specifically recognized both the 4.6S and the 6S forms of the receptor but did not react with the nontransformed 8S form. It is concluded that the unoccupied androgen receptor is associated with several heat-shock proteins and that transformation of the receptor to the tight nuclear-binding form is a multistep process that involves the dissociation of heat-shock proteins from the receptor.
Veldscholte
J
J
Department of Endocrinology & Reproduction, Erasmus University of Rotterdam, The Netherlands.
Berrevoets
C A
CA
Zegers
N D
ND
van der Kwast
T H
TH
Grootegoed
J A
JA
Mulder
E
E
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D002461:Cell Line, Transformed; D002467:Cell Nucleus / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D003429:Cross Reactions; D003600:Cytosol / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D003907:Dexamethasone / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D008163:Luminescent Measurements; D008297:Male; D015741:Metribolone / Q000378:metabolism / Q000494:pharmacology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D011397:Promegestone / Q000378:metabolism / Q000494:pharmacology*; D011471:Prostatic Neoplasms; D011944:Receptors, Androgen / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D011987:Receptors, Steroid / Q000276:immunology
D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D010455:Peptides; D011944:Receptors, Androgen; D011987:Receptors, Steroid; D015741:Metribolone; D003907:Dexamethasone; D011397:Promegestone