Interaction of Three Monoclonal Antibodies with the Nonactivated and Activated Forms of the Estrogen Receptor*

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Abstract
The 9S nonactivated oligomeric estrogen receptor from fetal guinea pig uterus interacts with the H222 monoclonal antibody (whose epitope is located in the hormone-binding domain) to yield an 11S complex and with the H226 monoclonal antibody (whose epitope is located in the A/B region, just N-terminal to the DNA-binding domain) to yield a 9.4S complex. No reaction was detected with the D547 monoclonal antibody (whose epitope is located between the hormone-binding and DNA-binding domains). In high salt gradients, the 11S oligomeric receptor-H222 complex dissociates to a 8S monomer-H222 complex and the 9.4S oligomeric receptor-H226 complex to a 7S monomer-H226 complex plus the 4.5S monomer receptor not bound to the antibody. These observations suggest that the nonactivated oligomeric receptor contains more than one estradiol-binding subunit with a structure such that the H222 epitopes are fully accessible, the H226 epitopes are partially accessible, D547 epitopes are masked. The temperature-activated receptor reacts with the H226 antibody to yield two complexes that sediment at 7S and 9S in high salt gradients. The 9S complex corresponds to a receptor form complexed with more than one antibody molecule; therefore, this suggests the formation of a receptor homodimer where the two H226 epitopes are exposed. However, a single 8S peak is observed when the H222 antibody reacts with the activated receptor, suggesting that only one H222 epitope is accessible in the dimeric receptor. In addition, binding to the H222 antibody before activation prevents dimerization. Thus, the H222 appears to be close to the dimerization domain. The activated receptor reacts with the D547 antibody to yield a 8S complex that apparently contains only one antibody molecule. On the other hand, the receptor extracted from nuclei was found to be a single 5.5S form with the same immunological characteristics as the receptor activated in cytosol. In conclusion, interaction of these monoclonal antibodies with the different forms of the estrogen receptor reveals a structural transformation during activation, with a concomitant change in the exposure of the receptor functional domains.