IMPORTANCE OF EXTRACELLULAR DOMAINS FOR LIGAND-BINDING IN THE THYROTROPIN-RELEASING-HORMONE RECEPTOR

The role of putative extracellular sequences for ligand binding in the TRH receptor was examined using deletion or substitution mutations. Each mutant receptor was transiently expressed in TRH receptor-minus GH(1)2C(1)b rat pituitary cells, and binding of 4 nM [H-3]pGlu-N(-)MeHis-Pro-NH2, ([H-3]MeTRH) was measured. When binding was not detected, signal transduction at 10 mu M MeTRH was measured to assess receptor expression. Deletion of most of the N-terminal sequences (Glu(2)-Leu(22)), including two potential glycosylation sites, had no effect on the affinity of the receptor for MeTRH. Segmental deletions or simultaneous substitution of multiple amino acid residues in the first, second, or third extracellular loop (EL1, EL2, or EL3) resulted, however, in total loss of [H-3]MeTRH binding, suggesting important roles for the loop sequences in either receptor expression or ligand binding. Individual substitutions were made to test further the role of the specific extracellular loop sequences in TRH binding. In EL1, conversion of Tyr(93) to Ala resulted in a more than 20-fold decrease in affinity for MeTRH. In EL2 and the top portion of the fifth transmembrane helix, conversion of Tyr(181) to phe, Tyr(188) to Ala, and Phe(199) to Ala resulted in a large (>100-fold) decrease in affinity for MeTRH, and conversion of Tyr(188) to phe and phe(198) to Ala caused an agonist-specific 4- to 5-fold decrease in affinity. In EL3, conversion of Asn(289) to Ala and of Ser(290) to Ala caused a large (>100-fold) decrease in affinity for MeTRH. These results suggest important roles for the extracellular loops in high affinity TRH binding and lead us to propose a model in which TRH binds to the extracellular domain of its receptor.