Estrogen receptor-independent catechol estrogen binding activity: protein binding studies in wild-type, Estrogen receptor-alpha KO, and aromatase KO mice tissues.
Primary evidence for novel estrogen signaling pathways is based upon well-documented estrogenic responses not inhibited by estrogen receptor antagonists. In addition to 17beta-E2, the catechol estrogen 4-hydroxyestradiol (4OHE2) has been shown to elicit biological responses independent of classical estrogen receptors in estrogen receptor-alpha knockout (ERalphaKO) mice. Consequently, our research was designed to biochemically characterize the protein(s) that could be mediating the biological effects of catechol estrogens using enzymatically synthesized, radiolabeled 4-hydroxyestrone (4OHE1) and 4OHE2. Scatchard analyses identified a single class of high-affinity (K(d) approximately 1.6 nM), saturable cytosolic binding sites in several ERalphaKO estrogen-responsive tissues. Specific catechol estrogen binding was competitively inhibited by unlabeled catechol estrogens, but not by 17beta-E2 or the estrogen receptor antagonist ICI 182,780. Tissue distribution studies indicated significant binding differences both within and among various tissues in wild-type, ERalphaKO, and aromatase knockout female mice. Ligand metabolism experiments revealed extensive metabolism of labeled catechol estrogen, suggesting that catechol estrogen metabolites were responsible for the specific binding. Collectively, our data provide compelling evidence for the interaction of catechol estrogen metabolites with a novel binding protein that exhibits high affinity, specificity, and selective tissue distribution. The extensive biochemical characterization of this binding protein indicates that this protein may be a receptor, and thus may mediate ERalpha/beta-independent effects of catechol estrogens and their metabolites.
Philips
Brian J
BJ
Department of Biochemistry, University of Missouri, Columbia, Missouri 65212, USA.
Ansell
Pete J
PJ
Newton
Leslie G
LG
Harada
Nobuhiro
N
Honda
Shin-Ichiro
S
Ganjam
Venkataseshu K
VK
Rottinghaus
George E
GE
Welshons
Wade V
WV
Lubahn
Dennis B
DB
D000818:Animals; D001141:Aromatase / Q000235:genetics* / Q000378:metabolism; D001667:Binding, Competitive; D004958:Estradiol / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000302:isolation & purification / Q000378:metabolism; D004965:Estrogen Antagonists / Q000378:metabolism; D047628:Estrogen Receptor alpha; D002393:Estrogens, Catechol / Q000378:metabolism*; D005260:Female; D000077267:Fulvestrant; D006894:Hydroxyestrones / Q000138:chemical synthesis / Q000302:isolation & purification / Q000378:metabolism; D007668:Kidney / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D010053:Ovary / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D011960:Receptors, Estrogen / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity; D014018:Tissue Distribution; D014599:Uterus / Q000378:metabolism
D004965:Estrogen Antagonists; D047628:Estrogen Receptor alpha; D002393:Estrogens, Catechol; D006894:Hydroxyestrones; D011506:Proteins; D011960:Receptors, Estrogen; D000077267:Fulvestrant; C026418:4-hydroxyestrone; D004958:Estradiol; C014036:4-hydroxyestradiol; D001141:Aromatase