FOLLITROPIN CONFORMATIONAL STABILITY MEDIATED BY LOOP 2-BETA EFFECTS FOLLITROPIN-RECEPTOR INTERACTION

Follicle-stimulating hormone (FSH) is in the family of pituitary/place ntal glycoprotein hormones which also includes luteinizing hormone (LH ), chorionic gonadotropin (hCG), and thyroid-stimulating hormone. Thes e hormones are heterodimers composed of common alpha- and similar but unique beta-subunits. The 21 amino acid loop between Y33 and F53 of th e FSH beta-subunit (L2 beta) can be switched into L2 beta of hCG beta without a loss of receptor binding, yet mutation of hFSH beta (37)LVY( 39) to (37)AAA(39) was antecedent to a 20-fold reduction in receptor b inding (based on ID50). A mutation in the LH beta gene, which causes Q 54 to be R, causes hypogonadism. This residue is conserved in the glyc oprotein hormones and corresponds to Q48 in hFSH beta. Mutation of hFS H beta (48)QKTCT(52) to (48)AAACA(52) resulted in a failure of heterod imer formation. In the current study single mutations were made to pin point which of the seven hFSH beta residues in the (37)LVY(39) to (37) AAA(39) and the (48)QKTCT(52) to (48)AAACA(52) mutants were responsibl e for the observed phenotypes. A single mutation of T52 to alanine was sufficient to cause a reduction in expression of heterodimeric hormon e. Single mutants Q48A, T50A, V38A, Y39A, and, to a lesser extent, T52 A formed heterodimer. However, these hFSH mutants were markedly unstab le at pH 2.0. Thus, acid dissociation can be used to reveal metastable forms of this protein. Mutant hFSH beta Q48A was also 8-fold less act ive than wild-type hFSH when assayed for binding to hFSH receptors. hF SH beta V38A and Y39A mutants affected receptor binding; however, neit her mutation alone caused greater than a 2-fold decrease in receptor b inding activity. In summary, these results identify single important r esidues in the long loop (between Y33 and F53) of the hFSH beta-subuni t which are required for proper subunit interactions that provide conf ormational stability which in turn is necessary for FSH-receptor inter action.