HYDROGEN-BONDING INTERACTION OF THYROTROPIN-RELEASING-HORMONE (TRH) WITH TRANSMEMBRANE TYROSINE-106 OF THE TRH RECEPTOR

Thyrotropin-releasing hormone (TRH, pyroglutamic acid-histidine-prolin e-amide) binds to a seven-transmembrane-spanning, G protein-coupled re ceptor. We tested the hypothesis that Tyr106 of the third transmembran e helix of the TRH receptor (TRH-R) binds pyroglutamyl of TRH by mutat ing Tyr106 to Phe and replacing the ring carbonyl of the TRH pyrogluta myl moiety with a methylene group ([Pro1]TRH). Compared to the affinit y 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 lo wer, respectively. The affinity of [Phe106]TRH-R for [Pro1]TRH was onl y 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, thi s represents the highest affinity, non-covalent bond yet observed betw een single functional groups of a GPCR and ligand and is the first del ineation of a direct binding interaction between a residue in the tran smembrane core of a GPCR and a specific moiety of a peptide agonist.