PROTEIN STRUCTURE AND FOLDING
A Conserved Asn in Transmembrane Helix 7 Is an On/Off Switch in the Activation of the Thyrotropin Receptor*

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The thyrotropin (TSH) receptor is an interesting model to study G protein-coupled receptor activation as many point mutations can significantly increase its basal activity. Here, we identified a molecular interaction between Asp633 in transmembrane helix 6 (TM6) and Asn674 in TM7 of the TSHr that is crucial to maintain the inactive state through conformational constraint 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 in 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 would restore this link. Moreover, we show that the Asn of TM7 (conserved in most G protein-coupled receptors) is mandatory for ligand-induced cAMP accumulation, suggesting an active role of this residue in activation. In the TSHr, the conformation of this Asn residue of TM7 would be constrained, in the inactive state, by its Asp partner in TM6.

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Published, JBC Papers in Press, April 18, 2001, DOI 10.1074/jbc.M102244200

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This work was supported in part by the Belgian State, Prime Minister's office, Service for Sciences, Technology, and Culture, grants from the Fonds de la Recherche Scientifique Médicale, Fonds National de la Recherche Scientific, Association Recherche Biomédicale et Diagnostic, and BRAHMS Diagnostics, Comision Interministerial de Ciencia y Tecnologia Grant SAF99-073, Fundació La Marató TV3 Grant 0014/97, Improving Human Potential of the European Community Grant HPRI-CT-1999-00071.

Fellow of the Fonds pour la Formation à la Recherche dans l'Industrie et dans l'Agriculture.