LOCALIZATION OF THE ESTROGEN-RECEPTOR IN UTERINE CELLS BY AFFINITY LABELING WITH [H-3] TAMOXIFEN AZIRIDINE

The possibility that estrogen receptors may exist in uterine plasma membranes was investigated by covalent labeling of estrogen receptors in mouse uterine cells with [H-3]tamoxifen aziridine (TA). Isolated epithelial and stromal cells of immature mice were incubated with [H-3]TA in the presence or absence of unlabeled tamoxifen, homogenized and separated into nuclear, cytosolic and microsomal fractions by differential centrifugation. These fractions were subjected to SDS-polyacrylamide gel electrophoresis and the proteins labeled covalently with TA were visualized by autoradiography. Proteins labeled specifically with [H-3]TA were observed almost exclusively in the nuclear fraction of both epithelial and stromal cells. In contrast, very little labeled protein was detected in the cytosolic or microsomal fraction. Although these data do not preclude the possibility that estrogen binding sites are present in plasma membranes of uterine cells, this cellular fraction is definitely not labeled to a significant extent by [H-3]TA. Thus, if membrane estrogen binding sites exist, their structural conformations may be different from that of nuclear estrogen receptors.