Abstract
The region between the 10th and 12th cysteine (Cys88-Cys105 in human thyroid-stimulating hormone beta-subunit (hTSHbeta)) 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 choriogonadotropin (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 seat-belt region Cys88-Cys105 or individual intercysteine segments Cys88-Cys95 and Cys95-Cys105 were replaced with the corresponding sequences of hCG and hFSH or alanine cassettes. Alanine cassette mutagenesis of hTSH showed that the Cys95-Cys105 segment of the seat-belt was more important for TSH receptor binding and signal transduction than the Cys88-Cys95 determinant loop region. Replacing the entire seat-belt of hTSHbeta with the hCG sequence conferred full hCG receptor binding and activation to the hTSH chimera, whereas TSH receptor binding and activation were abolished. Conversely, introduction of the hTSHbeta seat-belt sequence into hCGbeta 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 hTSHbeta and hFSHbeta did not confer follitropic activity to hTSH. Thus, exchanging the seat-belt 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.
Highlights
From the Laboratory of Molecular Endocrinology, Department of Medicine, University of Maryland School of Medicine and the Institute of Human Virology, Medical Biotechnology Center, Baltimore, Maryland 21201, the §Molecular and Cellular Endocrinology Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, the ¶Wadsworth Center, New York State Department of Health, Albany, New York 12201, and the ʈDepartment of Molecular Biology, University of Wyoming, Laramie, Wyoming 82071
This seat-belt consists of two intercysteine segments, a surface-exposed hydrophilic loop between the 10th and 11th cysteine (Cys88-Cys95 in hTSH) and a carboxyl-terminal segment between the 11th and 12th cysteine (Cys95-Cys105 in hTSH), and is in close proximity to ␣-subunit domains important for the structural integrity and
The human choriogonadotropin (hCG)/hTSH chimera 94TSH109 was obtained by introduction of the hTSH seat-belt residues into the hCG -subunit (Fig. 2)
Summary
Vol 272, No 24, Issue of June 13, pp. 15532–15540, 1997 Printed in U.S.A. Substitution of the Seat-belt Region of the Thyroid-stimulating Hormone (TSH) -Subunit with the Corresponding Regions of Choriogonadotropin or Follitropin Confers Luteotropic but Not Follitropic Activity to Chimeric TSH*. Thyrotropin (thyroid-stimulating hormone (TSH)) choriogonadotropin (CG), follitropin (follicle-stimulating hormone (FSH)), and lutropin (luteinizing hormone (LH)) are structurally related heterodimers that together form the glycoprotein hormone family [1] These hormones belong to the superfamily of cystine-knot growth factors [2, 3] and activate specific Gprotein-coupled receptors notable for large extracellular domains containing multiple leucine-rich motifs [4]. In the crystal structure of hCG [2, 3], this region corresponds to the “seat-belt” region (Cys88-Cys105 in hTSH), so-called because it wraps around the ␣-subunit and orients it in the heterodimer while remaining covalently bonded to the -subunit through disulfide linkages between Cys9-Cys and Cys26-Cys110 This seat-belt consists of two intercysteine segments, a surface-exposed hydrophilic loop between the 10th and 11th cysteine (Cys88-Cys in hTSH) and a carboxyl-terminal segment between the 11th and 12th cysteine (Cys95-Cys105 in hTSH), and is in close proximity to ␣-subunit domains important for the structural integrity and. Our findings reveal previously unrecognized differences in the regulation of specificity among the glycoprotein hormones
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