IDENTIFICATION OF ASN289 AS A LIGAND-BINDING SITE IN THE RAT THYROTROPIN-RELEASING-HORMONE (TRH) RECEPTOR AS DETERMINED BY COMPLEMENTARY MODIFICATIONS IN THE LIGAND AND RECEPTOR - A NEW MODEL FOR TRH BINDING

To test the hypothesis that pGlu of the thyrotropin-releasing hormone (TRH, pGlu-His-ProNH(2)) binds to Asn289 in the third extracellular loop (EL3) of its receptor through a hydrogen bonding interaction, we converted Asn289 to Asp (N289D mutant) and measured the potencies of TRH and Pro(1)TRH for the wild-type and mutant receptors. TRH was 100 times less potent for the N289D receptor than for the wild-type. In contrast, Pro(1)TRH, which has a protonated proline in place of the pGlu of TRH, was 10 times more potent for the N289D receptor than for the wild-type. A similar result was obtained when Asn289 was converted to Glu, while the potency of Pro(1)TRH did not change when Asn289 was converted to Ala, confirming that the increased potency of Pro(1)TRH for the N289D receptor was due to a charge interaction between Pro(1)TRH and the mutant receptor. These findings are inconsistent with a previous model indicating a direct interaction of the pGlu of TRH with Asn110 in the third transmembrane helix of the receptor (Perlman et al. (1994) J. Biol. Chem. 269, 23383-23386). When Asn110 was converted to Asp (N110D mutant), unlike the N289D receptor, the potency of Pro(1)TRH for the N110D receptor was decreased by > 10-fold rather than increased. Therefore, a direct interaction of Asn110 with the pGlu of TRH could not be supported by our experiments. We propose a new model in which the pGlu of TRH binds to Asn289 in EL3 and conclude that, unlike catecholamines which bind completely within the transmembrane domain of their receptors, this tripeptide binds, at least in part, to the extracellular domain of its receptor.