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The Use of Androgen Receptor Amino/Carboxyl-Terminal Interaction Assays to Investigate Androgen Receptor Gene Mutations in Subjects with Varying Degrees of Androgen Insensitivity

Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital (S.A.G., B.G., R.L., L.K.B., Y.E., L.P., M.A.T.); and Departments of Human Genetics (S.A.G., L.P., M.A.T.), Oncology (J.W.), Biology (L.P.), Medicine (L.P., M.A.T.), and Pediatrics (L.P.), McGill University, Montréal, Québec, Canada H3T 1E2

Address all correspondence and requests for reprints to: B. Gottlieb, Ph.D., Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, 3755 Cote Ste. Catherine Road, Montreal, Quebec, Canada H3T 1E2. E-mail: bruce.gottlieb{at}mcgill.ca.

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 Arg⁸⁷¹Gly (mild), Ser⁸¹⁴Asn (partial), Glu⁷⁷²Ala (partial), Val⁸⁶⁶Met (complete), and Arg⁷⁷⁴Cys (complete). Previous analysis showed that the mutant receptors exhibited near-normal kinetics, except Arg⁷⁷⁴Cys, which had severely reduced androgen binding, and Val⁸⁶⁶Met, which showed increased equilibrium dissociation constant (K_d) and elevated dissociation rate (k) values. Ser⁸¹⁴Asn 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 (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 Ser⁸¹⁴Asn 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.

Abbreviations: aa, Amino acids; AF, activation function; AIS, androgen insensitivity syndrome; CAIS, complete androgen insensitivity syndrome; DBD, DNA-binding domain; DHT, dihydrotestosterone; GAL, galactosidase; GRIP1, glucocorticoid receptor interacting protein 1; GSF, genital skin fibroblast; hAR, human androgen receptor; LBD, ligand-binding domain; Luc, luciferase; MAIS, mild androgen insensitivity syndrome; MB, mibolerone; MT, methyltrenolene; NTD, N-terminal domain; pCMV, cytomegalovirus promoter; pMMTV, mouse mammary tumor virus promoter; PR, progesterone receptor; pVP16, p virus transcriptional regulator protein 16; T, testosterone; SRC, steroid receptor coactivator; TIF2, transcription intermediary factor 2.

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