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Functional Characterization of Naturally Occurring Mutant Androgen Receptors from Subjects with Complete Androgen Insensitivity

Department of Pediatrics, Johns Hopkins University School of Medicine Baltimore, Maryland 21205
Department of Howard Hughes Medical Institute, Johns Hopkins University School of Medicine Baltimore, Maryland 21205
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine Baltimore, Maryland 21205
Laboratories for Reproductive Biology and Departments of Biochemistry, University of North Carolina Chapel Hill, North Carolina 27599
Laboratories for Reproductive Biology and Departments of pathology University of North Carolina Chapel Hill, North Carolina 27599
Laboratories for Reproductive Biology and Departments of Pediatrics, University of North Carolina Chapel Hill, North Carolina 27599

Address requests for reprints to: Dr. Terry R. Brown, Division of Pediatric Endocrinology, CMSC 3-110, Johns Hopkins Hospital, 600 North Wolfe Street, Baltimore, Maryland 21205.

Abstract

Mutations in the androgen receptor (AR) are thought to cause complete androgen insensitivity (CAIS) in 46,XY human subjects who have a female phenotype despite normal adult male concentrations of plasma testosterone. Assays of AR binding in cultured skin fibroblasts from subjects with CAIS show either an apparent absence of AR (AR–) or normal levels of AR (AR+) binding. In several subjects with CAIS, AR–, no gross AR mutation was detected by Southern blot analyses of genomic DNA and normal sized 10 kilobase mRNA was present on Northern blots of poly(A⁺) RNA from cultured genital skin fibroblasts. We have used the polymerase chain reaction to amplify individual exons within the human AR gene of subjects with CAIS and have identified point mutations in three subjects. In one AR– subject (R774C), amino acid 774 was changed from arginine (CGC) to cysteine (TGC), in another AR– subject (R831Q), arginine (CGA) was changed to glutamine (CAA) at position 831, and in an AR+ subject (V866M) a methionine (ATG) was substituted for valine (GTG) at position 866.

Transfection of wild type and mutant AR cDNA clones into COS cells results in detection of AR protein by immunoblotting. AR ligand binding activity is absent in cells transfected with AR mutants R774C and R831Q, but present with AR mutant V866M. Androgen binding in cells transfected with AR mutant V866M has a 6-fold lower apparent binding affinity than that of wild-type AR. Transcriptional activation of the MMTV-CAT reporter gene was androgen dependent and specific and nearly maximal at physiological concentrations (10^–10 M) of androgen when wild-type AR was transfected into cells, whereas neither AR mutants R774C nor R831Q were able to stimulate CAT activity even at 10^–8 M androgen. AR mutant V866M was able to stimulate CAT activity but the androgen dose dependency was shifted toward pharmacological concentrations of steroid that exceed in vivo levels.

The molecular basis of CAIS in humans exhibits genetic heterogeneity. Our study shows that some cases of CAIS are explained by an inability to form a functional AR-steroid complex and hence, the AR is unable to activate transcription of genes essential for male sex differentiation during fetal development.

FOOTNOTES

This work was supported by NIH Grant DK-00180, the NICHHD Center for Population Research, and the Howard Hughes Medical Institute.

This work was presented in part at the Workshop Conference on Androgen Therapy: Biological and Clinical Consequences, 1990, Marco Island, FL and the 72nd Annual Meeting of The Endocrine Society, 1990, Atlanta, GA (Abstract 861).

Received for publication July 30, 1990. Revision received September 14, 1990. Accepted for publication September 14, 1990.

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