title: The thyrotropin receptor hinge region is not simply a scaffold for the leucine-rich domain but contributes to ligand binding and signal transduction.
abstract: The glycoprotein hormone receptor hinge region connects the leucine-rich and transmembrane domains. The prevalent concept is that the hinge does not play a significant role in ligand binding and signal transduction. Portions of the hinge are redundant and can be deleted by mutagenesis or are absent in certain species. A minimal hinge will be more amenable to future investigation of its structure and function. We, therefore, combined and progressively extended previous deletions (Delta) in the TSH receptor (TSHR) hinge region (residues 277-418). TSHRDelta287-366, Delta287-371, Delta287-376, and Delta287-384 progressively lost their response to TSH stimulation of cAMP generation in intact cells, consistent with a progressive loss of TSH binding. The longest deletion (TSHRDelta287-384), reducing the hinge region from 141 to 43 amino acids, totally lost both functions. Surprisingly, however, with deletions extending from residues 371-384, constitutive (ligand-independent) activity increased severalfold, reversing the suppressive (inverse agonist) effect of the TSHR extracellular domain. TSHR-activating point mutations I486F and I568T in the first and second extracellular loops (especially the former) had reduced activity on a background of TSHRDelta287-371. In summary, our data support the concept that the TSHR hinge contributes significantly to ligand binding affinity and signal transduction. Residues within the hinge, particularly between positions 371-384, appear involved in ectodomain inverse agonist activity. In addition, the hinge is necessary for functionality of activating mutations in the first and second extracellular loops. Rather than being an inert linker between the leucine-rich and transmembrane domains, the TSHR hinge is a signaling-specificity domain.
authors.affiliation: Autoimmune Disease Unit, Cedars-Sinai Research Institute, University of California, Los Angeles, California 90048, USA.
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Mizutori
authors.forename: Chun-Rong
authors.initials: CR
authors.lastname: Chen
authors.forename: Sandra M
authors.initials: SM
authors.lastname: McLachlan
authors.forename: Basil
authors.initials: B
authors.lastname: Rapoport
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D002522:Chlorocebus aethiops; D005434:Flow Cytometry; D007930:Leucine / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D008954:Models, Biological; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011989:Receptors, Thyrotropin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*
chemical_list: D008024:Ligands; D011989:Receptors, Thyrotropin; D007930:Leucine