Functional characterization of naturally occurring mutant androgen receptors from subjects with complete androgen insensitivity

Terry R. Brown, Dennis B. Lubahn, Elizabeth M. Wilson, Frank S. French, Claude J. Migeon, Jeffry Lynn Corden

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)1759-1772
Number of pages14
JournalMolecular Endocrinology
Volume4
Issue number12
StatePublished - 1990

Fingerprint

Androgen Receptors
Androgens
Arginine
Fibroblasts
Steroids
Sex Differentiation
Messenger RNA
Skin
Mutation

ASJC Scopus subject areas

  • Molecular Biology
  • Endocrinology, Diabetes and Metabolism

Cite this

Functional characterization of naturally occurring mutant androgen receptors from subjects with complete androgen insensitivity. / Brown, Terry R.; Lubahn, Dennis B.; Wilson, Elizabeth M.; French, Frank S.; Migeon, Claude J.; Corden, Jeffry Lynn.

In: Molecular Endocrinology, Vol. 4, No. 12, 1990, p. 1759-1772.

Research output: Contribution to journalArticle

Brown, Terry R. ; Lubahn, Dennis B. ; Wilson, Elizabeth M. ; French, Frank S. ; Migeon, Claude J. ; Corden, Jeffry Lynn. / Functional characterization of naturally occurring mutant androgen receptors from subjects with complete androgen insensitivity. In: Molecular Endocrinology. 1990 ; Vol. 4, No. 12. pp. 1759-1772.
@article{3e002383d93746c3a9000dec360d6dd2,
title = "Functional characterization of naturally occurring mutant androgen receptors from subjects with complete androgen insensitivity",
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.",
author = "Brown, {Terry R.} and Lubahn, {Dennis B.} and Wilson, {Elizabeth M.} and French, {Frank S.} and Migeon, {Claude J.} and Corden, {Jeffry Lynn}",
year = "1990",
language = "English (US)",
volume = "4",
pages = "1759--1772",
journal = "Molecular Endocrinology",
issn = "0888-8809",
publisher = "The Endocrine Society",
number = "12",

}

TY - JOUR

T1 - Functional characterization of naturally occurring mutant androgen receptors from subjects with complete androgen insensitivity

AU - Brown, Terry R.

AU - Lubahn, Dennis B.

AU - Wilson, Elizabeth M.

AU - French, Frank S.

AU - Migeon, Claude J.

AU - Corden, Jeffry Lynn

PY - 1990

Y1 - 1990

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0025613044&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0025613044&partnerID=8YFLogxK

M3 - Article

C2 - 2082179

AN - SCOPUS:0025613044

VL - 4

SP - 1759

EP - 1772

JO - Molecular Endocrinology

JF - Molecular Endocrinology

SN - 0888-8809

IS - 12

ER -