Structural and functional characterization of the low affinity estrogen receptor from pregnant rat uteri

Abstract

The purpose of this project was to identify a rich source of Type II estrogen receptor (Type II ER), purify the receptor to homogeneity and determine its physical and pharmacological properties. Our results showed that pregnant rat uterus is highly enriched in the Type II ER; therefore this tissue has been used to study this receptor. The investigation demonstrated that the crude low affinity Type II ER (Kd {dollar}\sim{dollar}30nM) is significantly elevated during pregnancy and becomes the major {dollar}(\ge{dollar}88%) form of ER in rat uteri, while the high affinity Type I estrogen receptor (Type I ER, K{dollar}\sb{lcub}\rm D{rcub}{dollar} {dollar}\sim{dollar}0.10nM) form remained unchanged. This result may suggest a possible role for the low affinity ER in pregnancy. The Type II ER has been purified to homogeneity from uteri obtained from pregnant rats. The purified receptor {dollar}(>{dollar}97% purity) has an apparent estradiol binding affinity of 24 nM with a Bmax of 13 nmol/mg protein. HPLC analysis indicates that the purified Type II receptor has a native molecular weight of 63 kDa while the denatured molecular weight on SDS-PAGE is about 58 kDa. The purified receptor has a steroid specificity for estradiol {dollar}\ge{dollar} quercetin {dollar}\ge{dollar} diethylstilbestrol {dollar}\ge{dollar} tamoxifen, = dihydrotestosterone. The receptor appears not to contain any DNA binding activity and lacks sigmoidal steroid binding activity. However the steroid binding of the Type II ER is sensitive to sulfhydryl group reagents. This study has also identified a ERE DNA binding activity that copurified with Type II ER during the initial stages of purification. However, upon further purification by ion exchange chromatography the DNA binding activity (ERE Binding) was separated from the Type II ER. This ERE binding protein eluted from a Q-sepharose column at a conductivity corresponding to 250 mM NaCl. The ERE binding activity appears to be a novel protein in that it formed a discrete, rapidly migrating complex with the ERE oligonucleotide. In contrast the high affinity Type I ER formed a heterogeneous mixture of multiple ER-ERE complexes. Immunological analysis demonstrate that the purified low affinity Type II ER was not recognized by antibodies directed against the DNA binding domain of the recombinant Type I ER or by anti-tyrosinase antibody. The results indicate that the Type II ER shares no sequence homology with tyrosinase or the Type I ER.