Structure-function studies of the human thyrotropin receptor. Inhibition of binding of labeled thyrotropin (TSH) by synthetic human TSH receptor peptides.
We have probed the hormone binding regions of the entire putative extracellular domain of the human thyrotropin (TSH) receptor (hTSHr) using synthetic peptides. A series of 26 overlapping peptides comprising the complete sequence of the extracellular domain of hTSHr was synthesized. Each peptide (20 amino acid residues each) was tested for its ability to interact with TSH, as evidenced by inhibition of binding of labeled hormone to native, membrane bound TSH receptors. Four of the 26 peptides interacted with labeled TSH and inhibited its binding to thyroid membranes. The most potent of these peptides was 256-275, which inhibited 125I-bovine TSH binding with an IC50 of 31.7 +/- 1.3 microM. The remaining peptides were 16-35 (351 +/- 9.4 microM), 106-125 (282 +/- 20.5 microM), and 226-245 (951 +/- 245 microM). An additional peptide, 286-305, showed minimal activity, and the remaining 21 peptides showed no activity. Peptides 256-275, 106-125, and 16-35 also inhibited binding of 125I-human chorionic gonadotropin to ovarian membrane receptors, suggesting that those regions of the receptor are involved in binding of a common glycoprotein hormone structure such as the alpha-subunit. In contrast, peptides 226-245 and 286-305 did not inhibit human chorionic gonadotropin binding, suggesting that these two regions are involved in hormone-specific activity. Of interest is the finding that the latter two peptides are from regions of TSHr that are largely dis-homologous to the lutropin receptor, whereas the former three, with the exception of 16-35, are from regions that are largely homologous between the two receptors. The data suggest that multiple, discontinuous regions of the extracellular domain of hTSHr are involved in the binding of the hormone. Furthermore, the binding regions are localized to TSHr-specific sequences as well as to regions that are highly homologous to LHr. This suggests that homologous regions of the two receptors are likely to perform similar functions in the interaction with their specific hormone, suggesting that those regions may be involved in binding of the glycoprotein hormone common alpha-subunit.
Morris
J C
JC
Department of Medicine, Mayo Clinic and Medical School, Rochester, Minnesota 55905.
Bergert
E R
ER
McCormick
D J
DJ
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology*; D011869:Radioligand Assay; D011974:Receptors, LH / Q000737:chemistry / Q000378:metabolism; D011989:Receptors, Thyrotropin / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013552:Swine; D013961:Thyroid Gland / Q000378:metabolism; D013972:Thyrotropin / Q000037:antagonists & inhibitors / Q000378:metabolism*
D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D011974:Receptors, LH; D011989:Receptors, Thyrotropin; D013972:Thyrotropin; D066246:ErbB Receptors