A REFINED MODEL OF THE THYROTROPIN-RELEASING-HORMONE (TRH) RECEPTOR-BINDING POCKET - EXPERIMENTAL-ANALYSIS AND ENERGY MINIMIZATION OF THE COMPLEX BETWEEN TRH AND TRH RECEPTOR

Seven transmembrane (TM) spanning, G protein-coupled receptors (GPCRs) appear to bind large glycoprotein hormones predominantly within their extracellular domains, small nonpeptidic ligands within the TM helica l bundle, and peptide ligands within the extracellular domains and TM bundle. The tripeptide thyrotropin-releasing hormone (TRH, pyroGlu-His -ProNH(2)) may bind entirely within the TM bundle of the TRH receptor (TRH-R). We have previously demonstrated direct binding contacts betwe en the pyroGlu of TRH and two residues in TM helix 3 (TM-3) of TRH-R a nd proposed a model of the binding pocket of TRH-R [Perlman, J. H., La akkonen, L., Osman, R., & Gershengorn, M. C. (1994) J. Biol. Chem. 269 , 23383-23386]. Here, we provide evidence for two additional direct in teractions between TRH and TRH-R. One interaction is between the aroma tic ring of Tyr 282 of TM-6 and His of TRH. This is based on a large i ncrease in the half-maximally effective concentration (EC(50)) of TRH for stimulation of inositol phosphate formation by Y282A TRH-R and a l oss of selectivity of this mutant receptor for TRH analogs substituted at His. We provide evidence for another interaction between Arg 306 o f TM-7 and the terminal carboxamide of TRH. Using four direct interact ions as anchors, a refined model of the TRH-R binding pocket was const ructed using geometry optimization through energy minimization. A nove l method for modeling GPCRs based on Monte Carlo and stochastic dynami cs simulations is presented in the accompanying paper.