Estrogens exert fast non-genomic actions in their target tissues which may involve the participation of receptors located at the cell membrane. Studies were performed to identify and characterize membrane-associated 17β- estradiol binding proteins in rabbit uterus. Specific and saturable [3H]17β-estradiol binding sites of high affinity (K(d) = 0.36 nM) were detected in uterine microsomes at higher concentration than in cytosol (370 ± 98 vs. 270 ± 87 fmol/mg protein, respectively). Various other steroid hormones, the stereoisomer 17α-estradiol and the antiestrogen tamoxifen were significantly less effective than 17β-estradiol to compete with the radioactive ligand for binding to the membranes. The microsome binding sites were trypsin-sensitive and could be extracted to a great extent (80-90%) with 0.4/0.6 M KCl. Assays of the marker enzyme glucose-6-P dehydrogenase excluded membrane contamination with cytosolic soluble components. Immunoblot analysis of particulate and soluble fractions using monoclonal antibodies against the transactivation, heat shock protein recognition, and steroid binding domains of the nuclear estrogen receptor (ER; 67 kDa), revealed lower concentrations of the ER in membranes and the presence of five additional immunoreactive proteins of 57, 50, 32, 28, and 11 kDa which were absent in cytosol. Moreover, the antibody against the steroid binding domain was as effective as an inhibitor for cytosolic and membrane specific radioligand binding. Extraction of microsomes with the nondenaturing detergent CHAPS allowed a 2- fold enrichment of ER-like binding proteins as shown by antibody labeling and [3H]17β-estradiol binding analysis. The results of this work are consistent with the existence of novel 17β-estradiol membrane binding proteins structurally related to the intracellular ER. Future studies should investigate whether any of these proteins are involved in the primary events (e.g. receptor function) mediating nongenomic estrogen effects.
ASJC Scopus subject areas
- Endocrinology, Diabetes and Metabolism