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

Abstract

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 ∼11S region of the gradients. Mabs 213 and NMT-1 bound ER and formed complexes sedimenting at ∼7S and 9S, respectively. Preincubation of ER with Mab 213, followed by reincubation with Mab F9, resulted in a complex sedimenting at the ∼11S region of the gradients. Similarly, preincubation of ER with Mab NMT-1 followed by reincubation with Mab F9 also produced an ∼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.