A free carboxylate oxygen in the side chain of position 674 in transmembrane domain 7 is necessary for TSH receptor activation

A specific H-bonding network formed between the central regions of transmem brane domain 6 and transmembrane domain 7 has been proposed to be critical for stabilizing the inactive state of glycoprotein hormone receptors. Many different constitutively activating TSH receptor point mutations have been identified in hyperfunctioning thyroid adenomas in the lower portion of tra nsmembrane domain 6. Position D633 in transmembrane domain 6 of the human T SH receptor is the only one in which four different constitutively activati ng amino acid exchanges have been identified. Further in vitro substitution s led to constitutive activation of the TSH receptor (D633Y, F, C) as well as to the first inactivating TSH receptor mutation in transmembrane domain 6 without changes of membrane expression or TSH binding (D633R). Molecular modeling of this inactivating TSH receptor mutation revealed potential inte raction partners of R633 in transmembrane domain 3 and/or transmembrane dom ain 7, presumably via hydrogen bonds that could be responsible for locking the TSH receptor in a completely inactive state. To further elucidate the H -bond network that most likely maintains the inactive state of the TSH rece ptor, we investigated these potential interactions by generating TSH recept or double mutants designed to break up possible H bonds. We excluded S508 i n transmembrane domain 3 as a possible interaction partner of R633. In cont rast, a partial response to TSH stimulation was rescued in a receptor const ruct with the double-substitution D633R/N674D. Our results therefore confir m the H bond between position 633 in transmembrane domain 6 and 674 in tran smembrane domain 7 suggested by molecular modeling of the inactivating muta tion D633R. Moreover, the mutagenesis results, together with a three-dimens ional structure model, indicate that for TSH receptor activation and G prot ein-coupled signaling, at least one free available carboxylate oxygen is re quired as a hydrogen acceptor atom at position 674 in transmembrane domain 7.