Binding of site-directed monoclonal antibodies to an epitope located in the A/B region (amino acids 140-154) of human estrogen receptor-induced conformational changes in an epitope in the DNA-binding domain.
The interactions of estrogen receptor (ER) with monoclonal antibody (Mab) F9, developed against a synthetic 30-mer hybrid oligopeptide, were determined in the presence or absence of Mab NMT-1, raised against 15-mer peptide from the N-terminal A/B region (amino acids 140- 154) or Mab 213, raised to a peptide AT3 in the DNA-binding domain (amino acids 247-263). Mab F9 bound ER and formed a complex sedimenting at the approximately 11S region of the gradients. Mabs 213 and NMT-1 bound ER and formed complexes sedimenting at approximately 7S and 9S, respectively. Preincubation of ER with Mab 213, followed by reincubation with Mab F9, resulted in a complex sedimenting at the approximately 11S region of the gradients. Similarly, preincubation of ER with Mab NMT-1 followed by reincubation with Mab F9 also produced an approximately 11S complex on the gradients. These observations suggest that binding of Mab F9 to ER induced conformational changes causing the release of Mab 213 and Mab NMT-1 from ER. Furthermore, binding of Mab NMT-1 to the A/B region of ER also produced conformational changes causing the release of Mab 213 from its epitope in the DNA-binding region. These results indicate that binding of Mab F9 and Mab NMT-1, with epitopes located within amino acids 140-154 of the A/B region of ER, induced conformational changes in the DNA-binding domain, as determined by the inability of Mab 213 to remain bound to its epitope. These data further suggest that the DNA-binding region is sensitive to conformational changes induced in the native protein.
Traish
A M
AM
Department of Biochemistry, Boston University School of Medicine, MA 02118, USA.
Pavao
M
M
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism*; D001360:Avidin / Q000378:metabolism; D002417:Cattle; D004247:DNA / Q000378:metabolism*; D000939:Epitopes / Q000378:metabolism*; D005260:Female; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D011487:Protein Conformation; D011960:Receptors, Estrogen / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D011960:Receptors, Estrogen; D001360:Avidin; D004247:DNA