SITE-DIRECTED MUTAGENESIS DEFINES THE INDIVIDUAL ROLES OF THE GLYCOSYLATION SITES ON FOLLICLE-STIMULATING-HORMONE

To determine the specific role of each follicle stimulating hormone (F SH) oligosaccharide, we mutated Asn to Gln at each glycosylation site (alpha Gln(52), alpha Gln(78), alpha Gln(52-78), beta Gln(7), beta Gln (24), and beta Gln(7-24)) to selectively inhibit oligosaccharide attac hment. For wild-type and mutant FSH, we determined the binding affinit y to homogenized rat Sertoli cells and the signal-transducing activity in cultured rat granulosa cells. The binding affinity of FSH lacking any one of the oligosaccharides was increased over wild-type FSH, whil e the signal-transducing activity of FSH lacking the oligosaccharide a t alpha Asn(52) (alpha Gln(52) FSH) was markedly reduced, and that of FSH lacking either beta oligosaccharide (beta Gln(7) and beta Gln(24) FSH) was slightly reduced. At each FSH beta glycosylation site, we mad e a second amino acid substitution to inhibit glycosylation (beta Tyr( 9) and beta Tyr(26)) and an amino acid substitution that preserved gly cosylation (beta Ser(9) and beta Ser(26)). The amino acid sequence of the second p subunit glycosylation site was important for signal trans duction, regardless of the presense or absence of the oligosaccharide. Thus, while each FSH oligosaccharide has a similar impact on binding affinity, the alpha(52) oligosaccharide has a disproportionate role in signal transduction, and the amino acid sequence at beta Asn(24) func tions in both binding and signal transduction.