Physical properties of estrogen receptor complexes in MCF-7 human breast cancer cells. Differences with anti-estrogen and estrogen

We have examined the binding of two high affinity radiolabeled anti-estrogens, 2-[4-(2-pyrrolidin-1-yl-ethoxy)phenyl]-2-(3-[³H]-4-hydroxyphenyl)-1-phenyl-1-nitroethene ([³H]CI628M) and 1-[4-(2-dimethylaminoethoxy)phenyl]1-(4-hydroxyphenyl)-2-(4-[³H]phenyl)-but-1(Z)-ene ([³H]trans-hydroxytamoxifen) to the estrogen receptor from MCF-7 human breast cancer cells and have used hydrodynamic methods to determine the molecular properties of estrogen and anti-estrogen receptor complexes from these cells. Saturation binding analysis indicates that each compound binds predominantly to a single class of high affinity binding sites with K(d) of 1.3 x 10^-10 M for [³H]estra-1,3,5(10)-triene-3, 17β-diol (estradiol (E₂)), 1.4 x 10^-10 M for [³H]trans-hydroxytamoxifen, and 2.2 x 10^-10 M for [³H]CI628M. Marked differences are seen in the sedimentation rate and chromatographic properties of the nuclear estrogen receptor when complexed with anti-estrogen as opposed to the estrogen, estradiol (E₂). The nuclar E₂ receptor sediments at 4.1 ± 0.03 S on high salt (0.4 M KCl) sucrose gradients; by contrast, receptor complexed with CI628M or trans-hydroxytamoxifen sediments as a 5.5 ± 0.06 S peak. Upon chromatography on Sephadex G-200 columns equilibrated with buffer containing 0.4 M KCl, the E₂ nuclear receptor complexes appear to have a Stokes radius of 4.84 ± 0.20 nm, while the nuclear CI628M and trans-hydroxytamoxifen complexes have a Stokes radius of 5.93 ± 0.20 nm. These sedimentation coefficients and Stokes radii correspond to calculated molecular weights of 83,000 for the nuclear E₂ receptor complex and 137,000 for the nuclear CI628M and trans-hydroxytamoxifen receptor complexes. By contrast, cytoplasmic estrogen receptors labeled with CI628M, trans-hydroxytamoxifen, or E₂ have similar sedimentation coefficients of 4.1 ± 0.03 S and Stokes radii of 4.39 ± 0.30 nm, corresponding to a molecular weight of 76,000. The differences in physical properties of nuclear estrogen and anti-estrogen receptor complexes are obliterated in the presence of 3 M urea where estrogen and anti-estrogen complexes of 81,000 molecular weight (3.9 S, 4.94-nm Stokes radius) are obtained. These data are consistent with the association of the nuclear anti-estrogen receptor complex with an additional protein of ~55,000 molecular weight, an association that can be reversed by 3 M urea. This anti-estrogen-promoted change in receptor association with another cellular component may be an important aspect of the estrogen-antagonist and growth-inhibiting properties of these compounds.