Long loop residues 33-58 in the human glycoprotein hormone common alpha 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 alpha and beta subunits that are non-covalently 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 FSH alpha (aa 33-58) were the minimal alpha-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, and 55SES57/55AAA57, were constructed by site-specific mutagenesis and expressed in insect cells. With the exception of the TML mutant, all alpha-subunit forms were produced at a level similar to that of the wild-type alpha subunit (10 micromilligram(s)). The TML mutant was not secreted. When coexpressed with the human FSH (hFSH) beta subunit the 49VQK51/49AAA51 mutant and wild-type hFSH were expressed at similar levels (1-3 micromilligram(s)). In contrast, the 55SES57/65AAA57 mutation evidenced barely detectable levels of heterodimeric hFSH, and 37YPTPL41/37APAPA41 was not detectable as heterodimer, measured in a capture enzyme-linked immunosorbent assay format that detects only heterodimeric hormone. The 49VQK51/49AAA51 mutant was devoid of receptor-binding activity, suggesting that these residues are a key alpha-subunit determinant for follitropin-receptor interaction. The 55SES57/55AAA57 mutant, 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 alpha subunit, within sequence 49VQK51. Residues within 55SES57 and 37YPTPL41 are 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.
Liu
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C
Department of Biomedical Sciences, State University of New York at Albany 12201-0509, USA.
Dias
J A
JA
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004797:Enzyme-Linked Immunosorbent Assay; D015292:Glycoprotein Hormones, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D011962:Receptors, FSH / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017421:Sequence Analysis
D015292:Glycoprotein Hormones, alpha Subunit; D011962:Receptors, FSH; D011994:Recombinant Proteins