A conserved Asn in transmembrane helix 7 is an on/off switch in the activation of the thyrotropin receptor

The thyrotropin (TSH) receptor is an interesting model to study G protein-c oupled receptor activation as many point mutations can significantly increa se its ba sal activity. Here, we identified a molecular interaction between Asp(633) in transmembrane helix 6 (TM6) and Asn(674) in TM7 of the TSHr th at is crucial to maintain the inactive state through conformational constra int of the Asn. We show that these residues are perfectly conserved in the glycohormone receptor family, except in one case, where they are exchanged, suggesting a direct interaction. Molecular modeling of the TSHr, based on the high resolution structure of rhodopsin, strongly favors this hypothesis . Our approach combining site-directed mutagenesis with molecular modeling shows that mutations disrupting this interaction, like the D633A mutation i n TM6, lead to high constitutive activation. The strongly activating N674D (TM7) mutation, which in our modeling breaks the TM6-TM7 link, is reverted to wild type-like behavior by an additional D633N mutation (TM6), which wou ld restore this link. Moreover, we show that the Asn of TM7 (conserved in m ost G protein-coupled receptors) is mandatory for ligand-induced cAMP accum ulation, suggesting an active role of this residue in activation. In the TS Hr, the conformation of this Asn residue of TM7 would be constrained, in th e inactive state, by its Asp partner in TM6.