Human follitropin heterodimerization and receptor binding structural motifs: identification and analysis by a combination of synthetic peptide and mutagenesis approaches.

The family of human glycoprotein hormones, including follitropin (FSH), are heterodimeric proteins, each composed of single alpha- and beta-subunits that are tightly associated but non-covalently linked. To study structure and function relationships of FSH, synthetic peptides were used to inhibit subunit association, to map epitopes of FSH antibodies and as antigens to generate site specific antipeptide antibodies which could be used for topographic analysis. Interpretation of such results are generally more straightforward than when peptides are used with radioreceptor assays or in cell cultures which are complex systems. The data we collected using the synthetic peptide approach suggested that FSH residues homologous to human chorionic gonadotropin (hCG) loops L3 beta and L2 alpha are involved in subunit contact. FSH residues homologous to hCG loops L2 beta and L3 alpha seemed involved in receptor binding. Loop L2 beta also seemed involved in subunit contact. Those data provided a rationale for extensive mutagenesis of the four regions of hFSH. Mutagenesis data provided additional information and higher resolution of function when combined with the three dimensional structure of hCG. In the aggregate, this information has provided a reasonable model of the receptor binding site of hFSH. Our current model of the FSH receptor site is that of a discontinuous functional epitope including L3 beta, L2 alpha and L3 alpha. The juxtaposition of residues beta D93, alpha K5 1, alpha Y88 and of alpha Y89 in the 'binding-facet' of hFSH suggest the feasibility of designing a synthetic peptide mimetic of FSH. Additional residues of the alpha-subunit are involved, along this facet of the molecule. The data collected studying hFSH therefore demonstrates that the alpha-subunit features prominently in the mechanism of FSH binding to and stabilizing the interaction with its receptor. In contrast, the beta-subunit determinant loop serves as discriminator in addition to stabilizing the binding interaction whereas mutagenesis data indicates that L2 beta does neither. Instead, L2 beta appears to stabilize FSH conformation, possibly, the alpha-subunit, required for competent binding. In this regard, synthetic peptides provided data which were a useful guide to plan mutagenesis studies and which contributed to the process of understanding the structure and function of the gonadotropins.