Abstract
After we identified pGlu-βGlu-Pro-NH2 as the first functional antagonist of the cholinergic central actions of the thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH2), we became interested in finding the receptor-associated mechanism responsible for this antagonism. By utilizing a human TRH receptor (hTRH-R) homology model, we first refined the active binding site within the transmembrane bundle of this receptor to enhance TRH’s binding affinity. However, this binding site did not accommodate the TRH antagonist. This directed us to consider a potential allosteric binding site in the extracellular domain (ECD). Searches for ECD binding pockets prompted the remodeling of the extracellular loops and the N-terminus. We found that different trajectories of ECDs produced novel binding cavities that were then systematically probed with TRH, as well as its antagonist. This led us to establish not only a surface-recognition binding site for TRH, but also an allosteric site that exhibited a selective and high-affinity binding for pGlu-βGlu-Pro-NH2. The allosteric binding of this TRH antagonist is more robust than TRH’s binding to its own active site. The findings reported here may shed light on the mechanisms and the multimodal roles by which the ECD of a TRH receptor is involved in agonist and/or antagonist actions.
Highlights
Two forms of G protein-coupled receptors (GPCRs) have been identified for thyrotropinreleasing hormone (TRH, pGlu-His-Pro-NH2, Figure 1a) in the rodent brain: TRH-R1 and TRH-R2 [1]
When evaluating a recent homology model of TRH’s cognate receptor [9], the peptide arbitrarily docked within the human TRH receptor (hTRH-R) binding pocket resulted in a free energy change (∆G)
Of −7.0 kcal/mol and a binding affinity that was estimated in the mM range [17,18]. These initial findings are in agreement with a recently published study using a different hTRH-R
Summary
Two forms of G protein-coupled receptors (GPCRs) have been identified for thyrotropinreleasing hormone (TRH, pGlu-His-Pro-NH2 , Figure 1a) in the rodent brain: TRH-R1 and TRH-R2 [1]. An experimentally determined structure of any TRH receptor remains to be seen, highly conserved Class A GPCR features have aided the development of a homology model for the hTRH-R based on the neuropeptide Y1 receptor [9]. In this model, the seven transmembrane regions are linked with three extracellular loops (ECLs) and three intracellular loops.
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