Characterization of polyclonal antibodies to preselected domains of the human estrogen receptor.
We have synthesized three peptides with sequences identical to the DNA-binding domain of the human estrogen receptor (ER). These peptides correspond to sequences from amino acids 201-215, 231-245, and 247-261. We have used these peptides to develop polyclonal antibodies to the DNA-binding domain of ER. Six positive antisera were obtained against these peptides, as determined by enzyme-linked immunosorbent assay. Three of these antisera recognized the functional form of ER, as determined by sucrose density gradient analysis. The antisera that recognized the native form of ER were then tested for their ability to cross-react with other steroid receptors and with ER from other species. No cross-reaction with the native progesterone, glucocorticoid, or androgen receptors was observed. The antisera cross-reacted with cytosolic ER from calf and rat uterine tissues as well as human breast cancer tissue. The antisera recognized ER in its monomeric (4S), dimeric (5S), and multimeric (8S) forms. These antisera were site specific, since the free peptides displaced ER binding to the antibodies. The antibodies also recognized the unoccupied ER, as demonstrated by sucrose density gradients and postlabeling analysis. Thus, we have obtained three site-specific antibodies that recognize the DNA-binding region of the ER. These antibodies should prove useful as structural probes for the analysis of receptor-DNA interactions and elucidation of the functional domains of ER.
Traish
A
A
Department of Biochemistry, Boston University, School of Medicine, Massachusetts 02118.
Kim
N
N
Wotiz
H H
HH
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D002417:Cattle; D004247:DNA / Q000235:genetics; D005260:Female; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D008297:Male; D010449:Peptide Mapping; D010455:Peptides / Q000276:immunology; D051381:Rats; D011919:Rats, Inbred Strains; D011960:Receptors, Estrogen / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
D000906:Antibodies; D007106:Immune Sera; D010455:Peptides; D011960:Receptors, Estrogen; D004247:DNA