IDENTIFICATION OF AMINO-ACIDS IN THE C-TERMINAL REGION OF HUMAN FOLLICLE-STIMULATING-HORMONE (FSH) BETA-SUBUNIT INVOLVED IN BINDING TO HUMAN FSH RECEPTOR

Recent analyses of human FSH (hFSH) using antipeptide antibodies, monoclonal antibodies, and chimeric constructions of hCG/hFSH strongly suggest that the C-terminal region, including residues 81-100 of the hFSH beta-subunit, is involved in subunit association as well as hFSH heterodimer binding and/or activation of receptor. To test this hypothesis, site-directed mutagenesis was used to generate five triple alanine mutants of the C-terminal region of hFSH beta: Q81, H83, G85; K86, D88, S89; D90, S91, T92; D93, T95, V96; and R97, G98, L99. The baculovirus-infected insect cell system was used for expression. High Five(R) cells were infected with virus harboring either Delta hFSH beta complementary DNA (cDNA) or wild-type hFSH beta (hFSH beta(wt)) cDNA and coinfected with virus containing hFSH alpha cDNA. After infections, media were assayed for FSH using a heterodimer-specific enzyme-linked immunosorbent capture assay. All Delta hFSH beta subunits formed heterodimers with hFSH alpha(wt) subunit and were secreted in the medium. These results suggest, for all five mutants, that sidechains of amino acids substituted with alanine had no significant role in subunit association. The FSHs Delta hFSH and hFSH(wt) were tested in a RRA, using cell lines that express the hFSH receptor, to determine if there were any changes in binding activity. Similarly, Delta hFSH and hFSH(wt) were compared for receptor activation by measuring the levels of progesterone production in an in vitro FSH bioassay. Delta hFSH-(93-96) exhibited minimal binding activity and no detectable steroidogenic activity. Delta hFSH-(97-99) showed reduced binding affinity compared with that of hFSH(wt), whereas the binding potency and bioactivity of the remaining Delta hFSH were comparable to those of hFSH(wt). These data demonstrate that within the hFSH beta-(81-99) region, FSH receptor-binding sites are contained within the sequence 93-99.