Hydrogen bonding interaction of thyrotropin-releasing hormone (TRH) with transmembrane tyrosine 106 of the TRH receptor

Thyrotropin-releasing hormone (TRH, pyroglutamic acid-histidine-proline- amide) binds to a seven-transmembrane-spanning, G protein-coupled receptor. We tested the hypothesis that Tyr¹⁰⁶ of the third transmembrane helix of the TRH receptor (TRH-R) binds pyroglutamyl of TRH by mutating Tyr¹⁰⁶ to Phe and replacing the ring carbonyl of the TRH pyroglutamyl moiety with a methylene group ([Pro¹]TRH). Compared to the affinity of wild-type TRH-R for TRH, the affinities of [Phe¹⁰⁶]TRH-R for TRH and of wild-type TRH-R for [Pro¹]TRH were 100,000- and 110,000-fold lower, respectively. The affinity of [Phe¹⁰⁶]TRH-R for [Pro¹]TRH was only 16-fold lower than that for TRH, demonstrating a lack of additivity of the effects of these changes in the receptor and ligand. These data provide compelling evidence that the hydroxyl group of Tyr¹⁰⁶ of the TRH-R binds the TRH pyroglutamyl carbonyl group. To our knowledge, this represents the highest affinity, non-covalent bond yet observed between single functional groups of a GPCR and ligand and is the first delineation of a direct binding interaction between a residue in the transmembrane core of a GPCR and a specific moiety of a peptide agonist.