Use of monoclonal antibodies to subunits of human chorionic gonadotropin to examine the orientation of the hormone in its complex with receptor.

Abstract

Monoclonal antibodies were prepared against the a and f8 subunits of human chorionic gonadotropin (hCG). Although all were selected on the basis of their ability to bind the intact hormone, each also bound one of the two subunits but not both. Using a solid phase double antibody system to measure the relative binding to sites on the surface of hCG, we observed that four ofthe five antibodies bound to different sites on the molecule. This information was correlated with the ability of each antibody to inhibit the biological activity of hCG. Of the five antibodies tested for their ability to inhibit hCG-induced stimulation of rat testes steroidogenesis in vitro, two proved to be potent inhibitors, whereas the other three had almost no effect. This inhibition of steroidogenesis was highly correlated with the ability of the antibodies to inhibit hCG binding to testes homogenates. Thus, we have begun to derive a scheme that describes the relative binding positions of individual monoclonal antibodies and receptor on hCG. The purified monoclonal antibodies were iodinated and employed to evaluate which antigenic sites on hCG remained free in hCG-receptor complexes. The data indicated that portions of the g subunit in hCG-receptor complexes were buried (i.e., failed to bind radiolabeled antibody), whereas other portions remained exposed (i.e., they bound radiolabeled antibody). Those antibodies that interacted with portions of hCG that became inaccessible in the receptor complex also blocked the biological actions of hCG, whereas those that interacted with exposed sites had little or no effect on activity. Although we did not find antibodies to the a subunit that would bind to the hormone-receptor complex, we found that one of the two antibodies specific to a subunit epitopes blocked the actions of the hormone. Both antigenic determinants on the at subunit appeared to be lost after the hCG-receptor complex had formed. These studies suggest that each hCG subunit participates in the hormone-receptor complex and that portions of the ,B subunit project from the surface of the receptor.Human chorionic gonadotropin (hCG) is a glycoprotein hormone, composed of a and ,B subunits, which interacts with gonadal receptors to stimulate steroid synthesis (1). The isolated subunits exhibit less than 0.1% of the activity of the intact hormone and, within the limits of highest purity available, are thought to lack biological activity (1-3). As the subunits recombine, they reacquire biological activity (4), and spectral studies indicate that the conformation of the subunits is altered when they are recombined (5), suggesting that unique structural properties of the intact hormone may be needed for activity. Because neither subunit is active alone, the relative roles of the subunits in the actions of the hormone have been difficult to assess. Conceivably, only one of the subunits comes into direct contact with the receptor.Although the binding of hCG and related gonadotropins to Leydig cells has been studied at length (1), data con...

Influence of Subunit Interactions on Lutropin Specificity

“…Hormone and analog heterodimers released into the medium were quantified using a sandwich immunoassay similar to one we reported (29) except that different antibodies and standards were used, depending on the hormone or analog being measured. To quantify heterodimers consisting of human/bovine ␣-subunit chimeras and hCG ␤-subunits, we captured the analogs with an anti-␤-subunit antibody (B112) having high affinity for an hCG ␤-subunit epitope that included Asn 77 (28).…”

Molecular characterization, modeling, in silico analysis of equine pituitary gonadotropin alpha subunit and docking interaction studies with ganirelix

“…It was precisely shown that which amino-acids of the alpha-subunit are implicated in the interaction with gonadotropin receptors. Some earlier studies based on photo affinity labelling (Hong et al 1999;Sohn et al 2002) immunological studies (Moyle et al 1982) and mutational studies (Yoo et al 1991;Zeng et al 1995;Arnold et al 1998) demonstrated that the alpha subunit also interacts with the receptor. However, we report for the first time, the predicted three dimensional structures of the mature alpha subunit of eCG and its molecular docking with ganirelix which may play role in elaborating the responsibility played by alpha subunit of eCG in the process of reproduction and maintenance of pregnancy in equines as well as other species.…”

Human Lutropin (hLH) and Choriogonadotropin (CG) Are Assembled by Different Pathways

“…They were transfected into COS-7 cells obtained from ATCC (Manassas, VA) using a calcium phosphate procedure (13). Secreted analogs were harvested 3 days after transfection and analyzed in monoclonal antibody sandwich immunoassays (14) employing antibodies A113 and B401, obtained from Dr. William Munroe (Hybritech Inc., a division of Beckman Coulter, Inc., San Diego, CA) and B110 produced as described (15). Antibody A113 recognizes conformation-dependent ␣-subunit epitopes in the heterodimer.…”