Identification of a receptor binding region on the beta subunit of human follicle-stimulating hormone

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Abstract
Mouse epidermal growth factor (mEGF) and the .beta. subunit ofhuman follicle-stimulating hormone ((hFSH) hFSH-.beta.) have been shown to inhibit binding of intact hFSH to its membrane receptor in vitro. Both hFSH-.beta. and mEGF contain the tetrapeptide sequence Thr-Arg-Asp-Leu (TRDL). Previous restuls demonstrated that synthetic TRDL inhibited binding of intact hFSH to receptor. We therefore investigated the possibility that TRDL was located on an exposed region of FSH-.beta. using a polyclonal antiserum to hFSH [NHPP anti-hFSH batch 4 (ABA)] which recognized determinants on intact hFSH and its .beta. subunit, but not the .alpha. subunit. Pituitary FSH preparations from several mammalian species produced parallel inhibition curves in a heterologous [AB4 and 125I-labeled ovine FSH (125I-oFSH)] radioimmunoassay with relative potencies similar to those observed for the same preparations assayed by radioligand receptor assay. This antiserum also competitively inhibited 125I-FSH binding to receptor. Thus, AB4 appeared to recognize antigenic determinants that are highly conserved and located at or near regions involved with horomone recognition for FSH. Synthetic TRDL inhibited 50% of 125I-hFSH binding to antiserum at a concentration of 1.36 mg/tube (9 .times. 10-3 M). Other tetrapeptides (Thr-Pro-Arg Lys and Lys-Thr-Cys Thr) had no inhibitory activity at comparable concentrations. A mixture of the free amino acids T, R, D, and inhibited radioligand binding only at significantly higher concentrations than TRDL. The presence of TRDL in a receptor contact region of FSH was further suggested by its ability to inhibit the in vitro biological response to oFSH by cultured Sertoli cells at noncytotoxic levels. The reduced potency of TRDL relative to FSH in all three assay system suggests that it represents only a portion of a larger determinant on the intact hormone and its .beta. subunit. These results support a model of hormone-receptor interaction involving multiple, discrete contact points (deterninants), some of which influence binding while others may be involved with initiation of a cellular response. Our results further suggest that TRDL is a constituent of one FSH receptor binding region.