The use of androgen receptor amino/carboxyl-terminal interaction assays to investigate androgen receptor gene mutations in subjects with varying degrees of androgen insensitivity.
Five mutations in the ligand-binding domain (LBD) of the human androgen receptor (hAR) found in patients with varying degrees of androgen insensitivity syndrome (AIS) were investigated for their effects on receptor dynamics. These were Arg(871)Gly (mild), Ser(814)Asn (partial), Glu(772)Ala (partial), Val(866)Met (complete), and Arg(774)Cys (complete). Previous analysis showed that the mutant receptors exhibited near-normal kinetics, except Arg(774)Cys, which had severely reduced androgen binding, and Val(866)Met, which showed increased equilibrium dissociation constant (K(d)) and elevated dissociation rate (k) values. Ser(814)Asn exhibited ligand-selective k values, i.e. increased for dihydrotestosterone and mibolerone, but normal for methyltrenolene. Using mammalian two-hybrid assays, hAR amino/carboxyl (N/C)-terminal interactions of the mutant receptors were analyzed in the presence and absence of the hAR coactivator transcription intermediary factor 2 (TIF2). The mutations conferred decreased hAR N/C-terminal interaction, i.e. mild (approximately 1.5-fold), partial (2-fold), and complete (10-fold), that mirrored the degree of AIS. All mutant LBDs showed a 2- to 3-fold increase in N/C-terminal interactions when TIF2 was cotransfected, although of a magnitude still less than that of wild-type LBD with TIF2. The ligand-selective properties of the Ser(814)Asn mutant were also clearly reflected by the N/C-terminal interactions. Thus, measurement of N/C-terminal interactions may assist in the molecular analysis of mutant hARs associated with AIS.
Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, and Department of Human Genetics, McGill University, Montréal, Québec, Canada.
Shereen A
SA
Ghali
Bruce
B
Gottlieb
Rose
R
Lumbroso
Lenore K
LK
Beitel
Youssef
Y
Elhaji
Jian
J
Wu
Leonard
L
Pinsky
Mark A
MA
Trifiro
D000293:Adolescent; D000328:Adult; D013734:Androgen-Insensitivity Syndrome / Q000235:genetics*; D000728:Androgens / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002460:Cell Line; D002478:Cells, Cultured; D002648:Child; D002675:Child, Preschool; D013196:Dihydrotestosterone / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008297:Male; D008958:Models, Molecular; D009154:Mutation; D009277:Nandrolone / Q000031:analogs & derivatives* / Q000378:metabolism; D051958:Nuclear Receptor Coactivator 2; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D017433:Protein Structure, Secondary; D011944:Receptors, Androgen / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000235:genetics / Q000494:pharmacology; D015533:Transcriptional Activation; D014162:Transfection
D000728:Androgens; C498001:NCOA2 protein, human; D051958:Nuclear Receptor Coactivator 2; D010446:Peptide Fragments; D011944:Receptors, Androgen; D014157:Transcription Factors; D013196:Dihydrotestosterone; D009277:Nandrolone; C100075:mibolerone