Received for publication March 17, 2004.
Revised August 5, 2004.
Accepted for publication August 10, 2004.

A Model of Inverse Agonist Action at Thyrotropin-releasing Hormone Receptor Type 1: Role of a Conserved Tryptophan in Helix 6

Abstract

A binding pocket for thyrotropin-releasing hormone (TRH) within the transmembrane helices (TMHs) of the TRH receptor type 1 (TRH-R1) has been identified based on experimental evidence and computer simulations. To determine the binding site for a competitive inverse agonist, midazolam (MID), three of the four residues that directly contact TRH and other residues that restrain TRH-R1 in an inactive conformation were screened by mutagenesis and binding assays. We found that two residues that directly contact TRH, Asn-110 in TMH3 (3.37) and Arg-306 in TMH7 (7.39), were important for MID binding but another, Tyr-282 in TMH6 (6.51), was not. A highly conserved residue Trp-279 in TMH6 (6.48), which was reported to be critical in stabilizing TRH-R1 in an inactive state but not for TRH binding, was critical for MID binding. We used our previous model of the unoccupied TRH-R1 to generate a model of the TRH-R1/MID complex. The experimental results and the molecular model of the complex suggest that MID binds to TRH-R1 within a TMH pocket that partially overlaps the TRH binding pocket. This result is consistent with the competitive antagonism of MID binding. We suggest that the mechanism of inverse agonism effected by MID involves its direct interaction with Trp-279 that contributes to the stabilization of the inactive conformation of TRH-R1.

Key words: Thyrotropin/TRH, Structure-activity relationships and modeling, Benzodiazepines

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