Long Loop Residues 33–58 in the Human Glycoprotein Hormone Common α Subunit Contain Structural Components for Subunit Heterodimerization and Human Follitropin-Receptor Binding

The family of human glycoprotein hormones that includes follitropin (FSH) are heterodimeric proteins, each composed of single α and β subunits that are noncovalently linked but tightly associated. Previous studies by this laboratory, which used a synthetic peptide approach, suggested that residues 51–58 of the long loop of FSHα (aa 33–58) were the minimal α-subunit contact area between the subunits. Since carbohydrate at N52 is important for receptor activation but not for receptor binding, a link between receptor activation and heterodimer assembly was established. To address this issue, four composite alanine substitution mutants, 37YPTPL41/37APAPA41, 46TML48/46AAA48,49VQK51/49AAA51, and55SES57/55AAA57, were constructed by site-specific mutagenesis and expressed in insect cells. With the exception of the TML mutant, all α-subunit forms were produced at a level similar to that of the wild-type α subunit (10 μg/ml). The TML mutant was not secreted. When coexpressed with the human FSH (hFSH) β subunit the49VQK51/49AAA51mutant and wild-type hFSH were expressed at similar levels (1–3 μg/ml). In contrast, the55SES57/55AAA57mutation evidenced barely detectable levels of heterodimeric hFSH, and37YPTPL41/37APAPA41was not detectable as heterodimer, measured in a capture enzyme-linked immunosorbent assay format that detects only heterodimeric hormone. The49VQK51/49AAA51mutant was devoid of receptor-binding activity, suggesting that these residues are a key α-subunit determinant for follitropin–receptor interaction. The55SES57/55AAA57mutant, though scarcely made, retained receptor-binding activity comparable to the wild-type hormone. This work demonstrates for the first time a receptor-binding region in the FSH α subunit, within sequence49VQK51. Residues within55SES57and37YPTPL41are involved in subunit assembly. Homology modeling of FSH, based on the human chorionic gonadotropin crystal structure, revealed that the FSH receptor-binding site is composed of residues from both subunits assembled through subunit association.