High efficiency covalent radiolabeling of the human androgen receptor. Studies in cultured fibroblasts using dihydrotestosterone 17β-bromoacetate

W. J. Kovacs, M. K. Turney

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Analysis of mutations affecting the androgen receptor protein in human cells has been limited because of the low abundance and lability of these proteins in target tissues. All methods used to date have been based on the noncovalent interaction of radiolabeled androgens with the receptor's ligand binding site. We report here synthesis and use of the electrophilic affinity label dihydrotestosterone 17β-bromoacetate. This ligand, prepared as a radioactive compound of high specific activity, rapidly and covalently binds to a protein of 58,000 daltons in cytosol from normal genital skin fibroblasts. This protein is a high affinity, saturable specific binding site for the ligand and was not detectable in cultured cells from a subject with androgen resistance or in receptor-negative nongenital fibroblasts. The efficiency of incorporation of the covalent radiolabel into the 58-kD protein is greater than 80% based on estimates of receptor content using noncovalent ligands in intact cell assays. These studies demonstrate that dihydrotestosterone 17β-bromoacetate is useful for high efficiency covalent labeling of the human androgen receptor in crude cytosolic extracts from cultured cells.

Original languageEnglish (US)
Pages (from-to)342-348
Number of pages7
JournalJournal of Clinical Investigation
Volume81
Issue number2
DOIs
StatePublished - Jan 1 1988

Fingerprint

Fibroblasts
Ligands
Androgen Receptors
Proteins
Cultured Cells
Binding Sites
Affinity Labels
Complex Mixtures
Cytosol
Androgens
human AR protein
dihydrotestosterone 17-bromoacetate
Skin
Mutation

All Science Journal Classification (ASJC) codes

  • Medicine(all)

Cite this

@article{a3ba99ff9a8d4265a1a907053f35b381,
title = "High efficiency covalent radiolabeling of the human androgen receptor. Studies in cultured fibroblasts using dihydrotestosterone 17β-bromoacetate",
abstract = "Analysis of mutations affecting the androgen receptor protein in human cells has been limited because of the low abundance and lability of these proteins in target tissues. All methods used to date have been based on the noncovalent interaction of radiolabeled androgens with the receptor's ligand binding site. We report here synthesis and use of the electrophilic affinity label dihydrotestosterone 17β-bromoacetate. This ligand, prepared as a radioactive compound of high specific activity, rapidly and covalently binds to a protein of 58,000 daltons in cytosol from normal genital skin fibroblasts. This protein is a high affinity, saturable specific binding site for the ligand and was not detectable in cultured cells from a subject with androgen resistance or in receptor-negative nongenital fibroblasts. The efficiency of incorporation of the covalent radiolabel into the 58-kD protein is greater than 80{\%} based on estimates of receptor content using noncovalent ligands in intact cell assays. These studies demonstrate that dihydrotestosterone 17β-bromoacetate is useful for high efficiency covalent labeling of the human androgen receptor in crude cytosolic extracts from cultured cells.",
author = "Kovacs, {W. J.} and Turney, {M. K.}",
year = "1988",
month = "1",
day = "1",
doi = "10.1172/JCI113326",
language = "English (US)",
volume = "81",
pages = "342--348",
journal = "Journal of Clinical Investigation",
issn = "0021-9738",
publisher = "The American Society for Clinical Investigation",
number = "2",

}

TY - JOUR

T1 - High efficiency covalent radiolabeling of the human androgen receptor. Studies in cultured fibroblasts using dihydrotestosterone 17β-bromoacetate

AU - Kovacs, W. J.

AU - Turney, M. K.

PY - 1988/1/1

Y1 - 1988/1/1

N2 - Analysis of mutations affecting the androgen receptor protein in human cells has been limited because of the low abundance and lability of these proteins in target tissues. All methods used to date have been based on the noncovalent interaction of radiolabeled androgens with the receptor's ligand binding site. We report here synthesis and use of the electrophilic affinity label dihydrotestosterone 17β-bromoacetate. This ligand, prepared as a radioactive compound of high specific activity, rapidly and covalently binds to a protein of 58,000 daltons in cytosol from normal genital skin fibroblasts. This protein is a high affinity, saturable specific binding site for the ligand and was not detectable in cultured cells from a subject with androgen resistance or in receptor-negative nongenital fibroblasts. The efficiency of incorporation of the covalent radiolabel into the 58-kD protein is greater than 80% based on estimates of receptor content using noncovalent ligands in intact cell assays. These studies demonstrate that dihydrotestosterone 17β-bromoacetate is useful for high efficiency covalent labeling of the human androgen receptor in crude cytosolic extracts from cultured cells.

AB - Analysis of mutations affecting the androgen receptor protein in human cells has been limited because of the low abundance and lability of these proteins in target tissues. All methods used to date have been based on the noncovalent interaction of radiolabeled androgens with the receptor's ligand binding site. We report here synthesis and use of the electrophilic affinity label dihydrotestosterone 17β-bromoacetate. This ligand, prepared as a radioactive compound of high specific activity, rapidly and covalently binds to a protein of 58,000 daltons in cytosol from normal genital skin fibroblasts. This protein is a high affinity, saturable specific binding site for the ligand and was not detectable in cultured cells from a subject with androgen resistance or in receptor-negative nongenital fibroblasts. The efficiency of incorporation of the covalent radiolabel into the 58-kD protein is greater than 80% based on estimates of receptor content using noncovalent ligands in intact cell assays. These studies demonstrate that dihydrotestosterone 17β-bromoacetate is useful for high efficiency covalent labeling of the human androgen receptor in crude cytosolic extracts from cultured cells.

UR - http://www.scopus.com/inward/record.url?scp=0023836805&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0023836805&partnerID=8YFLogxK

U2 - 10.1172/JCI113326

DO - 10.1172/JCI113326

M3 - Article

C2 - 3339123

AN - SCOPUS:0023836805

VL - 81

SP - 342

EP - 348

JO - Journal of Clinical Investigation

JF - Journal of Clinical Investigation

SN - 0021-9738

IS - 2

ER -