Abstract
Radiosequence analysis of peptide fragments of the estrogen receptor (ER) from MCF-7 human breast cancer cells has been used to identify cysteine 530 as the site of covalent attachment of an estrogenic affinity label, ketononestrol aziridine (KNA), and an antiestrogenic affinity label, tamoxifen aziridine (TAZ). ER from MCF-7 cells was covalently labeled with [3H]TAZ or [3H]KNA and purified to greater than 95% homogeneity by immunoadsorbent chromatography. Limit digest peptide fragments, generated by prolonged exposure of the labeled receptor to trypsin, cyanogen bromide, or Staphylococcus aureus V8 protease, were purified to homogeneity by high performance liquid chromatography (HPLC), and the position of the labeled residue was determined by sequential Edman degradation. With both aziridines, the labeled residue was at position 1 in the tryptic peptide, position 2 in the cyanogen bromide peptide, and position 7 in the V8 protease peptide. This localizes the site of labeling to a single cysteine at position 530 in the receptor sequence. The identity of cysteine as the site of labeling was confirmed by HPLC comparison of the TAZ-labeled amino acid (as the phenylthiohydantoin and phenylthiocarbamyl derivatives) and the KNA-labeled amino acid (as the phenylthiocarbamyl derivative) with authentic standards prepared by total synthesis. Cysteine 530 is located in the hormone binding domain of the receptor, near its carboxyl terminus. This location is consistent with earlier studies using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to analyze the size of the proteolytic fragments containing the covalent labeling sites for TAZ and KNA and the antigen recognition sites for monoclonal antibodies. The fact that both the estrogenic and antiestrogenic affinity labeling agents react covalently with the same cysteine indicates that differences in receptor-agonist and receptor-antagonist complexes do not result in differential covalent labeling of amino acid residues in the hormone binding domain.
Highlights
Radiosequence analysis of peptide fragments of the The estrogen receptor (ER)’ is an intracellular protein that estrogen receptor (ER) froMmCF-7 human breast can- mediates the actionsof estrogens in targectells such as those cer cells has been used to identify cysteine530 as the of the mammary gland, uterus, and pituitary
Limit digest pep- modulate gene transcription.Theactions of estrogensare tide fragments, generated by prolonged exposure of antagonizedby antiestrogens, compoundswhich compete with the labeled receptor to trypsin, cyanogen bromide,or estrogens for binding to the receptor, but fail to effectively
Cysteine 530 is indicated that the hormone binding domain is located in located inthe hormone binding domainof the receptor, the carboxyl-terminal portionof the receptor
Summary
Radiosequence analysis of peptide fragments of the The estrogen receptor (ER)’ is an intracellular protein that estrogen receptor (ER) froMmCF-7 human breast can- mediates the actionsof estrogens in targectells such as those cer cells has been used to identify cysteine530 as the of the mammary gland, uterus, and pituitary. The availability of the chemically synthesized PTH-deriv- receptor sequence around Cys-530 and the cleavage sites for ative of TAZ-Cys permitted the analysiosf the cycle fractions trypsin, V8 protease, and CNBr are shown iFnig.
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