Specific binding of 4-hydroxyestradiol to mouse uterine protein: evidence of a physiological role for 4-hydroxyestradiol.

There are several indications of a possible physiological role for 4-hydroxyestradiol (4-OHE(2)) in hormone-responsive tissues. To examine a hormonal activity of 4-OHE(2), we have studied the binding of (3)H-labeled 4-OHE(2) to mouse uterine cytosolic protein. In uteri of 3-week-old mice, total binding was 319.4 +/- 13.9 fmol/mg protein. Binding in the presence of excess unlabeled 4-OHE(2) dropped to 82.1 +/- 1.7 fmol/mg protein, whereas 214.6 +/- 9.4 fmol/mg protein bound while incubating in an excess of unlabeled 17beta-estradiol (E(2)). The difference between the two binding values in the presence of excess steroid (132.5 +/- 11.1 fmol/mg protein) is taken as selective binding of 4-OHE(2) to a specific protein. In mice older than 4 weeks, the specific 4-OHE(2) binding declined: 32.0 +/- 4.0 fmol/mg protein at 8 weeks, 54.8 +/- 6.3 fmol/mg protein at 12 weeks and 54.6 +/- 5.2 fmol/mg protein at 9 months. Of other organs tested (liver, kidney, lung and whole brain) only lung showed significant selective binding of 4-OHE(2). When E(2)-binding sites are blocked, binding follows first-order kinetics, yielding a dissociation constant (K(d)) value of 11.8 +/- 2.1 nM. The specific binding of 4-OHE(2) was not inhibited by any other steroids or estrogen metabolites that were tested, except for 2-hydroxyestradiol (2-OHE(2)), which displayed competitive inhibition of 4-OHE(2) binding with an inhibition constant (K(i)) value of 98.2 +/- 12.6 nM. These results lead us to conclude that 4-OHE(2) binds to a specific binding protein, distinct and different from binding to estrogen receptors (ERalpha and ERbeta). The physiological role of this binding remains to be elucidated.