Characterization of normal and point-mutated human androgen receptors expressed in the baculovirus system.
The baculovirus system is able to generate large amounts of a protein, permitting detailed analysis of structure-function relations. We have used this system to overexpress and characterize normal human androgen receptors (hAR) and mutant hARs from humans with complete or partial androgen insensitivity. Maximum specific binding of [3H]mibolerone (MB) in recombinant baculovirus-infected Spodoptera frugiperda (Sf9) cells varied from 15 to 40 pmol/mg protein, about 1000-fold higher than in genital skin fibroblasts, and peaked 48-72 h after infection. In contrast, Coomassie blue staining and Western blotting revealed maximum accumulation of 100-120 kDa hAR proteins 96 h post-infection. Normal and mutant hARs were specifically photo-affinity-labeled with [3H]methyltrienolone (MT), and had normal steroid-binding selectivity: the order of competition was androgen > estrogen > progestin > glucocorticoid. Normal hAR was phosphorylated in Sf9 cells, reacted with antibodies against phosphoserine and phosphothreonine after purification using testosterone-biotin, and transactivated a transfected androgen response element-luciferase reporter in infected Sf9 cells. Two mutant hARs had increased rates of dissociation from MB and MT that were in accord with the associated degree of clinical androgen insensitivity: complete, Pro903Ser > partial, Leu820Val; the third, Ile663Asn, was not abnormal. Our data extend the characterization of normal hAR produced by baculovirus-infected Sf9 cells, and demonstrate, for the first time, that point-mutated hARs so produced can display distinctive biochemical phenotypes.
Beitel
L K
LK
Lady Davis Institute for Medical Research, Sir Mortimer B Davis-Jewish General Hospital, Montreal, Quebec, Canada.
Sabbaghian
N
N
Alarifi
A
A
Alvarado
C
C
Pinsky
L
L
Trifiro
M
M
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D001667:Binding, Competitive; D015153:Blotting, Western; D002460:Cell Line; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine; D006801:Humans; D007700:Kinetics; D008297:Male; D015741:Metribolone / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009277:Nandrolone / Q000031:analogs & derivatives / Q000378:metabolism; D010766:Phosphorylation; D017354:Point Mutation; D011944:Receptors, Androgen / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D016324:Sequence Tagged Sites; D012867:Skin / Q000378:metabolism; D018411:Spodoptera; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D045165:Testosterone Congeners / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection
D011944:Receptors, Androgen; D011994:Recombinant Proteins; D045165:Testosterone Congeners; D015741:Metribolone; D006639:Histidine; D009277:Nandrolone; C100075:mibolerone