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 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, 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 transmembrane helix 3 (3.37) and Arg-306 in transmembrane helix 7 (7.39), were important for midazolam binding but another, Tyr-282 in transmembrane helix 6 (6.51), was not. A highly conserved residue, Trp-279 in transmembrane helix 6 (6.48), which was reported to be critical in stabilizing TRH-R1 in an inactive state but not for TRH binding, was critical for midazolam binding. We used our previous model of the unoccupied TRH-R1 to generate a model of the TRH-R1/midazolam complex. The experimental results and the molecular model of the complex suggest that midazolam binds to TRH-R1 within a transmembrane helical pocket that partially overlaps the TRH binding pocket. This result is consistent with the competitive antagonism of midazolam binding. We suggest that the mechanism of inverse agonism effected by midazolam involves its direct interaction with Trp-279, which contributes to the stabilization of the inactive conformation of TRH-R1.Seven transmembrane-spanning receptors (7TMRs) comprise one of the largest protein families in mammalian genomes that transduce a variety of signals across cell-surface membranes to activate numerous cellular responses. Because of the involvement of the 7TMRs in many physiological processes, they have been selected as targets for many therapeutic drugs. All 7TMRs share a putative topological structure, but the molecular details of receptor conformational change upon agonist and inverse agonist binding that lead to activation and inactivation, respectively, have not yet been fully understood. For the last decade, intensive research has been focused on identifying receptor agonist binding sites and the dynamic conformational changes induced upon agonist binding (Osman et al., 1999). However, data about the binding of inverse agonists and the inactivation mechanism of 7TMRs are more limited. Interpretations of the results of these studies have been made more complex because the three-dimensional structure of only a single 7TMR, bovine rhodopsin, has been solved by X-ray crystallography (Palczewski et al., 2000).Thyrotropin-releasing hormone (TRH) is a tripeptide pyroGlu-His-Pro-amide that is synthesized and released from the hypothalamus to regulate pituitary hormone levels. TRH functions as a neurotransmitter/neuromodulator in the central and peripheral nervous systems also. TRH binds to specific 7TMRs, TRH receptors (TRH-Rs), and induces a cellular response through G-protein-related signaling pathways. The three-dimensional structure of TRH receptor subtype-1 (TRH-R1) a...

Synthesis, Receptor Binding, and CNS Pharmacological Studies of New Thyrotropin‐Releasing Hormone (TRH) Analogues

“…The agonist behavior of the analogues was tested in HEK 293EM cells stably expressing TRH-R1 or TRH-R2 by incubating the cells with various doses of the analogues as described. [21, 37] The extent of agonist behavior was then determined by measuring signaling through a reporter gene, and the data are reported as EC 50 values (μM) in Table 1.…”

Intravenous and Intratracheal Thyrotropin Releasing Hormone and Its Analog Taltirelin Reverse Opioid-Induced Respiratory Depression in Isoflurane Anesthetized Rats

“…interesting to determine if there are differences in the breathing effects of intravenous TRH relative to agents that increase endogenous TRH levels through blockade of TRH proteolysis (Scalabrino et al, 2007), or following leptin administration, which induces endogenous TRH expression (Burgueno et al, 2007) and gates TRH responsivity (Rogers et al, 2009). Finally, benzodiazepines (e.g., chlordiazepoxide and midazolam) are antagonists/inverse agonists of the TRH receptor and antagonize some physiologic responses to TRH administration (e.g., TSH and prolactin secretion) (Roussel et al, 1986;Lu et al, 2004). By themselves, benzodiazepines cause minimal respiratory depression.…”

Computational Modeling Approaches to Structure−Function Analysis of G Protein-Coupled Receptors

“…Indeed, the only helix bend correctly predicted by Baldwin's study was the kink at P267(6.50). The results of Baldwin's studies 10,11 and the electron density 2D and 3D maps of rhodopsin 9,12 have been milestones and invaluable sources of information for GPCR modeling in the years that preceded, − and even in those that followed, the release of the crystal structure of rhodopsin. −

9 Cartoon representation of the seven-helix bundle and of H8 of the rhodopsin structure1U19 (yellow-green) 54 superimposed on the computational models by Baldwin and co-workers (left side, in blue), by Herzyk and Hubbard (central panel, in orange), and by Pogozheva and co-workers (right side, in violet) . The helix bundles are seen from the intracellular side in a direction perpendicular to the membrane surface.

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