RECEPTOR-BINDING AND SIGNAL-TRANSDUCTION ARE DISSOCIABLE FUNCTIONS REQUIRING DIFFERENT SITES ON FOLLICLE-STIMULATING-HORMONE

Separate sites for glycoprotein hormone receptor binding and signal tr ansduction have yet to be elucidated. In general, certain peptide regi ons are thought to be critical for receptor binding, whereas the oligo saccharides are thought to be important for signal transduction. Using site-directed mutagenesis of FSH, we made selective amino acid substi tutions and oligosaccharide alterations to try and identify specific s ites mediating receptor binding distinct from signal transduction and vice versa. We substituted Lys or Asp for beta Arg(35) in the purporte d receptor binding loop between cysteine-32 and -51, and we substitute d Gln for alpha Asn(52), alpha Asn(78), beta Asn(7), or beta Asn(24), the attachment sites for each of the oligosaccharide side-chains. We d etermined the binding and signal-transducing activity of wild-type and mutant human FSH at the human FSH receptor, as recent data suggest th at glycoprotein hormone-receptor interactions are species specific. Th e binding activities of FSH with Lys or Asp substituted for beta Arg(3 5) were reduced 71% and 98%, respectively, but their signal transducti on, at equivalent levels of binding activity, was unaffected. The bind ing activity of FSH lacking the oligosaccharide at alpha Asn(52) was e nhanced 2- to 3-fold, but its signal-transducing activity, at equivale nt levels of receptor binding, was decreased 72%. In contrast, the bin ding and signal-transducing activities of FSH lacking the alpha Asn(78 ), beta Asn(7), or beta Asn(24) oligosaccharide were unaffected. Thus, a specific amino acid (beta Arg(35)) is important for high affinity b inding, but is not involved in signal transduction, whereas a specific oligosaccharide (alpha Asn(52)) is important for signal transduction, but is not required for high affinity binding. Therefore, receptor bi nding and signal transduction are dissociable functions involving diff erent sites on the FSH glycoprotein.