Substitution of the seat-belt region of the thyroid-stimulating hormone (TSH) beta-subunit with the corresponding regions of choriogonadotropin or follitropin confers luteotropic but not follitropic activity to chimeric TSH

The region between the 10th and 12th cysteine (Cys(88)-Cys(105) in human thyroid-stimulating hormone beta-subunit (hTSH beta)) of the glycoprotein hormone beta-subunits corresponds to the disulfide-linked seat-belt region. It wraps around the common alpha-subunit and has been implicated in regulating specificity between human choriogonado-tropin (hCG) and human follicle-stimulating hormone (hFSH), but determinants of hTSH specificity are unknown, To characterize the role of this region for hTSH, we constructed hTSH chimeras in which the entire seatbelt region Cys(88)-Cys(105) or individual intercysteine segments Cys(88)-Cys(95) and Cys(95)-Cys(105) were replaced with the corresponding sequences of hCG and hFSH or alanine cassettes. Alanine cassette mutagenesis of hTSH showed that the Cys(95)-Cys(105) segment of the seat-belt was more important for TSI-I receptor binding and signal transduction than the Cys(88)-Cys(95) determinant loop region. Replacing the entire seat-belt of hTSH beta with the hCG sequence conferred full hCG receptor binding and activation to the hTSH chimera, whereas TSI-I receptor binding and activation were abolished. Conversely, introduction of the hTSH beta seat-belt sequence into hCG beta generated an hCG chimera that bound to and activated the TSH receptor but not the CG/lutropin (LH) receptor. In contrast, an hTSH chimera bearing hFSH seat-belt residues did not possess any follitropic activity, and its thyrotropic activity was only slightly reduced. This may in part be due to the fact that the net charge of the seat-belt is similar in hTSH and hFSH but different from hCG. However, exchanging other regions of charge heterogeneity between hTSH beta and hFSH beta did not confer follitropic activity to hTSH. Thus, exchanging the seatbelt region between hTSH and hCG; switches hormonal specificity in a mutually exclusive fashion, In contrast, the seat-belt appears not to discriminate between the TSH and the FSH receptors, indicating for the first time that domains outside the seat-belt region contribute to glycoprotein hormone specificity.