Efficient and highly selective covalent labeling of the estrogen receptor with [³H]tamoxifen aziridine

Tamoxifen aziridine, (Z)-(1-[4-(2-[N-aziridinyl] ethoxy)phenyl])-1,2-diphenyl-1-butene, an electrophilic analog of the antiestrogen tamoxifen, has been prepared in tritium-labeled form by catalytic tritium-halogen exchange on a brominated precursor. The radiolabeled material is obtained in a state of high chemical and radiochemical purity and has a specific activity of 20 Ci/mmol. [³H]Tamoxifen aziridine reacts covalently with a species that appears to be the estrogen receptor in cell-free cytosol preparations from rat uterus and in intact MCF-7 human breast cancer cells. The labeling of receptor in unfractionated cytosol is quite rapid at 25°C, and is very efficient and selective, so that nearly complete labeling can be achieved using only a fewfold excess of reagent, with minimal labeling of other nonreceptor proteins that are present in the unfractionated cytosol. The labeling is estrogen specific in that it is blocked only by estrogens and anti-estrogens, and is not observed in nontarget tissues and cells lacking the estrogen receptor. Covalent labeling of the estrogen receptor by [³H]tamoxifen aziridine is slower at 0 than at 25°C, but is equally complete; at 37°C, however, receptor or aziridine instability limits the extent of labeling. The covalent link to the receptor is quite stable, being unaffected by hot solvents, detergents, and organic acids. The estrogen receptor in MCF-7 human breast cancer cells can be labeled efficiently and selectively by exposure of whole cells to [³H]tamoxifen aziridine at 37°C, labeled receptor is found mainly in the salt extract of the nuclear fraction. Receptor-negative MDA-MB-231 human breast cancer cells show no estrogen-specific labeling. Rat uterine estrogen receptor, labeled covalently with [³H]tamoxifen aziridine, can be analyzed directly by sodium dodecyl sulfate-polyacrylamide gel electrophoresis where it appears as a major component with a M(r)=60,000-66,000, together with minor amounts of a smaller species (M(r) ~ 50,000). The estrogen receptor covalently labeled with [³H]tamoxifen aziridine displays sedimentation behavior on high and low salt sucrose gradients equivalent to that of receptor labeled reversibly with [³H]estradiol. Efficient and selective covalent labeling agents for the estrogen receptor, such as [³H]tamoxifen aziridine, should be of wide utility in studies of receptor structure and function.