A single-base substitution in exon 6 of the androgen receptor gene causing complete androgen insensitivity: the mutated receptor fails to transactivate but binds to DNA in vitro

Abstract

A single-base substitution in the coding region of the androgen receptor (AR) gene caused complete androgen insensitivity in a patient with 46,XY karyotype. The mutation was a T-to-G transition in exon 6 and changed the codon 807 from ATG (methionine) to AGG (arginine) in the hormone-binding domain of the protein. The mutation was inserted into the wild-type human AR cDNA and the resulting cDNA expressed in CV-1 cells. Native and mutated AR proteins synthesized in recipient cells had identical molecular masses. Ligand-binding activity of the mutant receptor was less than 5% of that of the wild-type AR. The mutant's interaction with an androgen-response element in vitro was identical to that of the native aporeceptor; however, it did not transactivate a reporter gene construct in transfected CV-1 cells. Androgen insensitivity in our patient was thus due to altered structure of the receptor's steroid-binding region, which prevented the mutated AR from gaining a transcriptionally active form in vivo.

Interaction of Androgen Receptors with Androgen Response Element in Intact Cells

“…However, our present data and those of Kuil and Mulder (12) demonstrate that specific DNA binding in living cells is not achieved without a ligand-induced allosteric change in the LBD. The findings that hARM807R binds well to AREs in vitro (21) and is localized in nuclei of transfected cells (this study) are interpreted to mean that the steroid-induced change in LBD conformation is primarily required for the release of AR from associated inhibitory protein complexes, rather than for generation of a receptor form capable of ARE recognition.…”

“…The hARM807R mutant is incapable of hormone binding, totally inactive in transactivation assays, and the reason for complete androgen insensitivity of a patient (21). It could be speculated that inappropriate folding of this mutant's LBD may render it free of intracellular chaperones such as heat shock proteins (32), thereby permitting its binding to AREs even in vivo.…”

“…For transcriptional activation experiments, the reporters pARE 2 -tk-CAT and pMMTV-CAT were used (20). Expression vectors pSGrAR, pSGhAR, pSGrARC562G, pSGhARM807R, pSGrAR⌬46 -408, pSGrAR⌬46 -408/C562G, and pSGrAR⌬641-902 2 have been described previously (5,8,21,22). pG5SV40CAT containing five GAL4-binding elements upstream of the SV40 promoter and pSV40CAT control vector were gifts from Dr. N. Lehming (Max-Delbrü ck Laboratory, Max-Plack Institute, Cologne, Germany).…”

“…rARC562G with a Cys 3 Gly substitution at codon 562 does not bind to DNA in vitro and is transcriptionally inactive (22). hARM807R is incapable of hormone binding because of a Met 3 Arg substitution at codon 807 (21). In electrophoretic mobility shift assays under cell-free conditions, hARM807R bound to ARE with approximately the same affinity as the wild-type receptor in the absence of androgen (21), but exposure to androgen did not elicit a conformational change in the LBD of hARM807R in a fashion similar to that in the wild-type LBD (21,27).…”

“…hARM807R is incapable of hormone binding because of a Met 3 Arg substitution at codon 807 (21). In electrophoretic mobility shift assays under cell-free conditions, hARM807R bound to ARE with approximately the same affinity as the wild-type receptor in the absence of androgen (21), but exposure to androgen did not elicit a conformational change in the LBD of hARM807R in a fashion similar to that in the wild-type LBD (21,27). rARC562G and hARM807R mutants did not interfere with pCMV-ARE 2 -CAT activity in a steroid-dependent fashion, but they exhibited weak hormone-independent interference, essentially indistinguishable from that of the wild-type apo-AR (Fig.…”