Electrophoretic analysis of large molecular weight forms of the estrogen receptor in MCF-7 human breast cancer cells

Abstract

The denatured and nondenatured forms of the estrogen receptor (ER) in MCF-7 human breast cancer cells have been characterized by gel electrophoresis using the covalently attaching ligand [ 3H]tamoxifen aziridine ([ 3H]TA). A comparison of the ER binding properties of the recently introduced commercial preparation (Amersham) of [ 3H]TA with the prototype showed no quantitative or qualitative differences, with both preparations binding efficiently and selectively at either 4°C or 37°C. The nuclear and cytosolic labeled ER, when analyzed by denaturing SDS-PAGE, migrate as 67,000 dalton proteins. [ 3H]TA labeled ER from cytosol prepared in low salt buffer without protease inhibitors migrates on 5%, 6%, or 7% nondenaturing PAGE as two discrete forms. The molecular weights of these two species, determined by Ferguson plot analysis with multiple standard protein markers, are 260,000 and 67,000 daltons, respectively. The 260 kDa form is dissociated by 0.4 M KC1 into the 67 kDa form, suggesting that the larger form is composed of one or more noncovalently attached subunit proteins. When enzyme inhibitors leupeptin and molybdate are included in cytosol prepared in low salt, two high molecular weight [ 3H]TA labeled peaks are identified, the first 600,000 and the second 430,000 daltons. These two forms migrate together on 4%, 5%, and 6% non-SDS, non-denaturing gels, and are not dissociated when cytosol is made 0.4 M in KC1. These findings indicate that the ER of these human breast cancer cells exists in one or more large discrete forms of mol. wt 430,000–600,000 daltons. These forms are not dissociated by high salt but may be digested by proteases to the subunit 67,000 dalton form. These findings support the proposal that the ER is present as a large molecular weight “holoreceptor”, and may be important for our understanding the metabolism and function of the ER in estrogen target tissues and human breast cancer.