17α-(Haloacetamidoalkyl)estradiols alkylate the human estrogen receptor at cysteine residues 417 and 530

Results obtained in a previous study suggested that cysteine residues in the estrogen receptor were covalent attachment sites for four 17α-(haloacetamidoalkyl)estradiols (halo, bromo or iodo; alkyl, methyl, ethyl, or propyl). To identify the putative concerned cysteines, we expressed wild-type and various cysteine → alanine mutants of the human estrogen receptor in COS cells and determined their ability to be alkylated by the four electrophiles. The quadruple mutant, in which all the cysteines (residues 381, 417, 447, and 530) of the hormone-binding site were changed to alanines, showed very little electrophile labeling, whereas the four single mutants (C381A, C417A, C447A, and C530A) were alkylated as efficiently as the wild-type receptor. These results (i) demonstrate that cysteine residues were covalent attachment sites of electrophiles and (ii) indicate that more than one cysteine residue could be alkylated. Analysis of three double mutants (C381A/C530A, C417A/C530A, and C447A/C530A) provided strong e...