title: A new structural model for the thyrotropin (TSH) receptor, as determined by covalent cross-linking of TSH to the recombinant receptor in intact cells: evidence for a single polypeptide chain.
abstract: The most widely held model for the human TSH receptor is of holoreceptor of 80 kDa with two subunits of approximately 50 and 30 kDa linked by disulfide bridges, with the former subunit containing the major hormone-binding site. We reexamined this model by covalently cross-linking radiolabeled TSH to the recombinant human TSH receptor stably expressed in Chinese hamster ovary (CHO) cells. When cross-linking was performed after the preparation of CHO membranes, analysis of hormone-receptor complexes under reducing and nonreducing conditions provided results supporting the two-subunit TSH receptor model. In contrast, however, cross-linking of TSH to the TSH receptor in intact CHO cells before membrane preparation revealed, even under reducing conditions, an approximately 100-kDa receptor as well as an approximately 54-kDa hormone-binding subunit. The approximately 100-kDa holoreceptor size is consistent with the size of the TSH receptor, as predicted from its derived amino acid sequence. The proportions of the approximately 100-kDa TSH receptor and the 54-kDa fragment varied in different experiments, suggesting the occurrence of proteolytic cleavage. Cross-linking of radiolabeled TSH to intact cells expressing a mutant TSH receptor (TSHR-D1) lacking amino acids 317-366 localized the proteolytic cleavage site to just up-stream of amino acid residue 317. In summary, the present data obtained by cross-linking TSH to recombinant human TSH receptors in intact cells provides evidence that the receptor exists in vivo as an approximately 100-kDa glycoprotein with a single polypeptide chain with intramolecular disulfide bridges.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Thyroid Molecular Biology Unit, Veterans Administration Medical Center, University of California, San Francisco 94121.
authors.forename: D
authors.initials: D
authors.lastname: Russo
authors.forename: G D
authors.initials: GD
authors.lastname: Chazenbalk
authors.forename: Y
authors.initials: Y
authors.lastname: Nagayama
authors.forename: H L
authors.initials: HL
authors.lastname: Wadsworth
authors.forename: P
authors.initials: P
authors.lastname: Seto
authors.forename: B
authors.initials: B
authors.lastname: Rapoport
mesh_terms: D000818:Animals; D001345:Autoradiography; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D006224:Cricetinae; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007457:Iodine Radioisotopes; D008961:Models, Structural; D011487:Protein Conformation; D011989:Receptors, Thyrotropin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013972:Thyrotropin / Q000378:metabolism*
chemical_list: D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; D011989:Receptors, Thyrotropin; D011994:Recombinant Proteins; D013972:Thyrotropin