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 () in the TSH receptor (TSHR) hinge region (residues 277-418). TSHR287-366, 287-371, 287-376, and 287-384 progressively lost their response to TSH stimulation of cAMP generation in intact cells, consistent with a progressive loss of TSH binding. The longest deletion (TSHR287-384), 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 several fold, 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 TSHR287-371.

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 and 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.