Follitropin Conformational Stability Mediated by Loop 2β Effects Follitropin−Receptor Interaction

Abstract

Follicle-stimulating hormone (FSH) is in the family of pituitary/placental glycoprotein hormones which also includes luteinizing hormone (LH), chorionic gonadotropin (hCG), and thyroid-stimulating hormone. These hormones are heterodimers composed of common α- and similar but unique β-subunits. The 21 amino acid loop between Y33 and F53 of the FSH β-subunit (L2β) can be switched into L2β of hCGβ without a loss of receptor binding, yet mutation of hFSHβ ³⁷LVY³⁹ to ³⁷AAA³⁹ was antecedent to a 20-fold reduction in receptor binding (based on ID₅₀). A mutation in the LHβ gene, which causes Q54 to be R, causes hypogonadism. This residue is conserved in the glycoprotein hormones and corresponds to Q48 in hFSHβ. Mutation of hFSHβ ⁴⁸QKTCT⁵² to ⁴⁸AAACA⁵² resulted in a failure of heterodimer formation. In the current study single mutations were made to pinpoint which of the seven hFSHβ residues in the ³⁷LVY³⁹ to ³⁷AAA³⁹ and the ⁴⁸QKTCT⁵² to ⁴⁸AAACA⁵² mutants were responsible for the observed phenotypes. A single mutation of T52 to alanine was sufficient to cause a reduction in expression of heterodimeric hormone. Single mutants Q48A, T50A, V38A, Y39A, and, to a lesser extent, T52A formed heterodimer. However, these hFSH mutants were markedly unstable at pH 2.0. Thus, acid dissociation can be used to reveal metastable forms of this protein. Mutant hFSHβ Q48A was also 8-fold less active than wild-type hFSH when assayed for binding to hFSH receptors. hFSHβ V38A and Y39A mutants affected receptor binding; however, neither mutation alone caused greater than a 2-fold decrease in receptor binding activity. In summary, these results identify single important residues in the long loop (between Y33 and F53) of the hFSH β-subunit which are required for proper subunit interactions that provide conformational stability which in turn is necessary for FSH−receptor interaction.