Disulfide bond mutations in follicle-stimulating hormone result in uncoupling of biological activity from intracellular behavior

The crystal structure of human CG reveals that each subunit is a member of the superfamily of cystine-knot growth factors. Although the distribution o f the cysteine residues in all the beta -subunits is conserved, the conform ation of the human FSH dimer differs from that of the CG/LH dimers. This su ggests that the function of the cystine bonded loops in the human FSH beta -subunit may differ from that in the CGP-subunit. To address this issue, we deleted two disulfide bonds in the FSH beta domain: cys 20-104 and cys 28- 82, which correspond to the disulfide bonds 26-110 and 34-88, respectively, in the CGP-subunit. The cys 26-110 bond is associated with the "seat-belt" region and cys 54-88 is a bond in the cystine knot. Coexpression of the wi ld-type alpha -subunit with the FSH beta cysteine mutants in CHO cells reve aled no detectable heterodimer. The FSH beta mutants were then incorporated into a single chain where the beta -subunit is genetically fused to the al pha -subunit. In such a model the rate-limiting subunit assembly step is by -passed and mutations that otherwise block heterodimer formation can be eva luated in terms of biological activity. Compared with the nonmutated single chain, the single-chain 28-82 mutant is secreted more slowly and its recov ery is substantially reduced, whereas secretion and recovery of the 20-104 mutant was not significantly affected. The receptor binding affinity of the cys 28-82 mutant did not differ from wild-type and binding of the cys 20-1 04 mutant was decreased only 2-fold. The signal transduction data parallel the binding affinities, although the maximal accumulation of cAMP is less f or the cys 20-104 mutant than that seen for cys 28-82 and nonmutated single -chains variants. These data support the hypothesis that the determinants f or intracellular behavior and bioactivity of the gonadatropins are not the same, and that the cystine knot is a critical determinant for the formation of a stable, assembly-competent subunit. In addition, the data imply that the "seat-belt" conformation does not play a prominent role in the bioactiv ity of FSH.