In situ photolabelling of the human androgen receptor.
In situ photoaffinity labelling of the human androgen receptor has been performed in the LNCaP (Lymph Node Carcinoma of the Prostate) cell line. The covalently labelled receptors were identified by SDS-PAGE. Intact LNCaP cells, incubated with [3H]-R1881 and subsequently irradiated with u.v. light and directly solubilized in SDS-buffer, revealed two photolabelled protein bands at 110 and 50 kDa. Irradiation of intact cells and subsequent isolation of nuclei followed by extraction with 0.5 M NaCl resulted in one major photolabelled protein band at 110 kDa. The labelling of this band could be completely suppressed by a 100-fold molar excess of non-radioactive R1881. Photolabelling of androgen receptors in a cytosolic preparation of LNCaP cells after anion exchange chromatography resulted in a much lower labelling efficiency compared with the in situ labelling procedure, although the androgen receptor was purified 100-fold. The steroid binding domain of the human androgen receptor has been partially mapped with chymotrypsin and S. aureus V8 protease digestion. Proteolytic digestion with chymotrypsin of purified photoaffinity-labelled 110 kDa human androgen receptor resulted in the generation of a 15 kDa peptide which still contains the covalently linked hormone. It is concluded that the in situ photoaffinity labelling technique can be applied successfully for characterization of the steroid binding domain of androgen receptors in prostate cancer cells and in other androgen target cells. Furthermore, it was demonstrated that the human androgen receptor is a monomer with a molecular mass of 110 kDa, of which the steroid binding site is confined to a 15 kDa domain.
Brinkmann
A O
AO
Department of Biochemistry II, Erasmus University Rotterdam, The Netherlands.
Kuiper
G G
GG
Bolt-de Vries
J
J
Mulder
E
E
D000345:Affinity Labels; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D002918:Chymotrypsin; D004963:Estrenes / Q000378:metabolism; D006801:Humans; D008207:Lymphatic Metastasis; D008297:Male; D015741:Metribolone; D010446:Peptide Fragments / Q000032:analysis; D011471:Prostatic Neoplasms; D011944:Receptors, Androgen / Q000302:isolation & purification / Q000378:metabolism*; D012697:Serine Endopeptidases; D045165:Testosterone Congeners / Q000378:metabolism
D000345:Affinity Labels; D004963:Estrenes; D010446:Peptide Fragments; D011944:Receptors, Androgen; D045165:Testosterone Congeners; D015741:Metribolone; D012697:Serine Endopeptidases; D002918:Chymotrypsin; C028344:glutamyl endopeptidase