Site-directed polyclonal antibodies inhibit binding of activated estrogen receptor to DNA.
We have generated three polyclonal antisera to the DNA-binding domain of the human estrogen receptor (hER). Antiserum AT2A was generated against a peptide spanning amino acids 231-245 of hER, while antisera AT3A and AT3B were generated against a peptide spanning amino acids 247-261 of hER. The interaction of these three antisera with ER has been characterized by sucrose density gradient analysis. The antisera bound to the unactivated (8S), salt-activated (4S), and heat transformed (5S) ER complex. All the antisera appeared to be site-specific since binding of salt-activated ER to the antisera was blocked by the presence of excess free synthetic peptides. Antisera AT3A and AT3B inhibited the binding to DNA of the KCl-activated 4S ER and the heat-transformed 5S ER. Although antiserum AT2A bound to ER, it did not inhibit DNA binding of activated ER complexes. The ability of antisera AT3A and AT3B to inhibit ER binding to DNA was concentration dependent. Once bound to the DNA, ER complexes were not significantly affected by incubation with the antisera, suggesting that binding of DNA to ER inhibits antibody ER interaction and renders that domain inaccessible to the antibodies. These results demonstrate that site-directed antibodies to ER inhibit binding of activated ER complexes to DNA in vitro.
Traish
A M
AM
Biochemistry Department, Boston University School of Medicine, MA 02118.
Kim
N
N
Wotiz
H H
HH
D000906:Antibodies / Q000378:metabolism / Q000494:pharmacology*; D000918:Antibody Specificity; D001665:Binding Sites; D002482:Cellulose / Q000031:analogs & derivatives / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D010446:Peptide Fragments / Q000276:immunology; D011189:Potassium Chloride / Q000494:pharmacology; D011960:Receptors, Estrogen / Q000276:immunology / Q000378:metabolism*
D000906:Antibodies; C033190:DNA-cellulose; D010446:Peptide Fragments; D011960:Receptors, Estrogen; D011189:Potassium Chloride; D002482:Cellulose; D004247:DNA