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.