Use Of Polyclonal And Monoclonal Antibodies To Study Hcg-receptor Interactions (gonadotropin, Glycoprotein Hormones).

Abstract

Although the glycoprotein hormones lutropin (LH), follitropin (FSH), and thyrotropin (TSH) bind to different receptors, each contains an identical alpha subunit. Specificity is somehow endowed by the beta subunits which are distinct for each hormone. Human choriogonadotropin (hCG) is a natural LH analog that contains a beta subunit nearly identical to that of LH. The roles of these subunits in the recognition and high affinity binding of hCG to receptor was examined. Polyclonal and monoclonal antibodies specific for the individual subunits of hCG were used to probe the hormone-receptor interaction. Conformation-specific and sequence-specific antibodies were examined for their abilities to bind Triton X-100-solubilized ('125)I-hCG-receptor complex and to inhibit hormone binding to crude rat ovarian membranes containing receptor. Immunoreactive sites on the alpha subunit were masked in the detergent solubilized hCG-receptor complex indicating that this subunit is in direct contact with the receptor. This conclusion is supported by the observation that all of these antibodies were able to inhibit hormone binding to receptor. The antibody combining sites on the beta subunit were largely unaffected by association with the receptor, showing that the epitopes are not directly involved in receptor binding. Even though the immunoreactive sites are not located on the receptor binding surface of the beta subunit, most, but not all, of these polyclonal and monoclonal antibodies were able to inhibit ('125)I-hCG binding to receptor. Although the inhibition of binding may be due to steric interference due to the size of the antibody molecules, a two-step model for hCG binding to receptor is presented that also explains these results. In this model, the beta subunit initially binds with the receptor with a highly specific but low affinity interaction. This activates a site for the high affinity binding of the alpha subunit and stabilization of the complex. This is an attractive model as it may be applied to other glycoprotein hormones sharing an alpha subunit.