Characterization of the DNA-binding domain of the mouse uterine estrogen receptor using site-specific polyclonal antibodies.
The DNA-binding domain of the mouse uterine estrogen receptor (ER) was characterized using site-specific polyclonal antibodies. The peptides used as antigens have sequences corresponding to amino acids 185-199 and 227-245, the two zinc finger regions of the DNA-binding domain of the human ER, and produced anti-sera designated A-1542 and A-1554, respectively. Mouse uterine nuclear ER and salt-activated 4 S cytosol receptor, as well as 8 S untransformed cytosol receptor, were observed to react with the antisera by Western blot and sucrose density gradient centrifugation analyses indicating that the DNA-binding domain of the 8 S cytosol receptor is not completely masked by heat shock protein 90 or other proteins. Only A-1554 detected a nuclear-specific doublet form of the ER on Western blot analysis. In a gel shift assay, neither antisera altered the pattern of the nuclear ER interaction with the vitellogenin A2 estrogen response element (VRE). In contrast, antiserum A-1554 partially shifted the 8 S cytosol receptor-VRE complex. This concurs with mutational analysis and x-ray crystallography studies with the human ER that have shown that the second finger is not in contact with the DNA. The results of the gel shift assay were confirmed by sucrose density gradient analysis using the same buffer conditions. The nuclear receptor-VRE complex did not react with either antisera, suggesting that when the dimeric nuclear receptor form binds the VRE, the specific receptor epitopes involved with the DNA binding may be blocked and unable to bind the antisera. The cytosol receptor-VRE complex reacted only partially with the second finger antisera A-1554, suggesting that on receptor monomers the second finger epitope is not completely blocked by DNA binding or dimer formation.
Ikeda
M
M
Receptor Biology Section, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709.
Ogata
F
F
Curtis
S W
SW
Lubahn
D B
DB
French
F S
FS
Wilson
E M
EM
Korach
K S
KS
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000302:isolation & purification / Q000378:metabolism*; D001666:Binding Sites, Antibody; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D003600:Cytosol / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D004958:Estradiol / Q000378:metabolism; D005260:Female; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011960:Receptors, Estrogen / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D014599:Uterus / Q000378:metabolism*; D016335:Zinc Fingers / Q000502:physiology
D000906:Antibodies; D004268:DNA-Binding Proteins; D011960:Receptors, Estrogen; D004958:Estradiol