IDENTIFICATION OF THE SITES OF N-LINKED GLYCOSYLATION ON THE FOLLICLE-STIMULATING-HORMONE (FSH) RECEPTOR AND ASSESSMENT OF THEIR ROLE IN FSH RECEPTOR FUNCTION

The FSH receptor (FSHR) contains a large extracellular domain in which exist three potential sites for N-linked glycosylation. A truncated f orm of the FSHR representing only the extracellular domain was created and expressed in mammalian cells. We show that this truncated recepto r is glycosylated, though the carbohydrates are not as fully processed as those of the full-length receptor. This truncated receptor, which remains intracellular, binds FSH with an affinity comparable to that o f the full-length FSHR. Therefore, although other regions of the FSHR may contribute to hormone binding, the extracellular domain alone can confer high affinity binding. The above results suggest that N-linked FSHR carbohydrates may, in some way, be required for FSH binding. Ther efore, further experiments, done in the context of the full-length rec eptor, were performed to determine the actual sites of glycosylation i n the FSHR as well as to elucidate their role in the functions of the FSHR. Site-directed mutagenesis was done to individually or collective ly disrupt the potential sites for N-linked glycosylation. Western blo t analyses of the wild type vs. mutant receptors demonstrate that, of the three potential sites for N-linked glycosylation, Asn's 174 and 27 6 are actually glycosylated. Binding assays demonstrate that these two N-linked FSHR carbohydrates are redundant in function since carbohydr ate at either Asn174 or Asn276 allows the receptor to be expressed on the cell surface and to bind FSH with normal affinity. However, FSH bi nding activity is not observed with nonglycosylated mutant receptors w here both sites have been collectively disrupted. Similarly, when cell s expressing the wild type FSHR were treated with tunicamycin to preve nt N-linked glycosylation, the resulting nonglycosylated FSHR was not able to bind FSH. In contrast, normal high affinity binding of FSH was maintained when N-linked carbohydrates were enzymatically removed fro m wild type receptors. Our results demonstrate that while N-linked car bohydrates on the FSH receptor are not required directly for the bindi ng of hormone, a carbohydrate at either Asn174 or Asn276 is required f or the efficient folding of the nascent receptor protein into a confor mation that allows high affinity binding of hormone.