The Antagonist pGlu-βGlu-Pro-NH2 Binds to an Allosteric Site of the Thyrotropin-Releasing Hormone Receptor

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.

[1]  L. Prokai,et al.  [β-Glu2]TRH Is a Functional Antagonist of Thyrotropin-Releasing Hormone (TRH) in the Rodent Brain , 2021, International journal of molecular sciences.

[2]  Marco Biasini,et al.  ProMod3—A versatile homology modelling toolbox , 2021, PLoS Comput. Biol..

[3]  Milot Mirdita,et al.  HH-suite3 for fast remote homology detection and deep protein annotation , 2019, BMC Bioinformatics.

[4]  D. Larhammar,et al.  Structural basis of ligand binding modes at the neuropeptide Y Y1 receptor , 2018, Nature.

[5]  Alex C. Conner,et al.  Understanding the common themes and diverse roles of the second extracellular loop (ECL2) of the GPCR super-family , 2017, Molecular and Cellular Endocrinology.

[6]  Torsten Schwede,et al.  The SWISS-MODEL Repository—new features and functionality , 2016, Nucleic Acids Res..

[7]  A. Sangamwar,et al.  Synthesis and biology of ring-modified l-Histidine containing thyrotropin-releasing hormone (TRH) analogues. , 2016, European journal of medicinal chemistry.

[8]  Q. Du,et al.  The multiple roles of histidine in protein interactions , 2013, Chemistry Central Journal.

[9]  Cheng Zhang,et al.  High-resolution Crystal Structure of Human Protease-activated Receptor 1 Bound to the Antagonist Vorapaxar Hhs Public Access , 2022 .

[10]  Daniel Kuhn,et al.  DoGSiteScorer: a web server for automatic binding site prediction, analysis and druggability assessment , 2012, Bioinform..

[11]  Bryan L. Roth,et al.  Structure of the human kappa opioid receptor in complex with JDTic , 2012, Nature.

[12]  J. Simms,et al.  Lifting the lid on GPCRs: the role of extracellular loops , 2011, British journal of pharmacology.

[13]  Matthias Rarey,et al.  Analyzing the Topology of Active Sites: On the Prediction of Pockets and Subpockets , 2010, J. Chem. Inf. Model..

[14]  Ning Ma,et al.  BLAST+: architecture and applications , 2009, BMC Bioinformatics.

[15]  Arthur J. Olson,et al.  AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading , 2009, J. Comput. Chem..

[16]  H. Schiöth,et al.  The G protein-coupled receptor subset of the rat genome , 2007, BMC Genomics.

[17]  M. Gershengorn,et al.  Thyrotropin-releasing hormone and its receptors--a hypothesis for binding and receptor activation. , 2007, Pharmacology & therapeutics.

[18]  H. Schiöth,et al.  The Repertoire of G-Protein–Coupled Receptors in Fully Sequenced Genomes , 2005, Molecular Pharmacology.

[19]  R. Osman,et al.  Agonist-induced conformational changes in thyrotropin-releasing hormone receptor type I: disulfide cross-linking and molecular modeling approaches. , 2005, Biochemistry.

[20]  Kurt Kristiansen,et al.  Molecular mechanisms of ligand binding, signaling, and regulation within the superfamily of G-protein-coupled receptors: molecular modeling and mutagenesis approaches to receptor structure and function. , 2004, Pharmacology & therapeutics.

[21]  M. Gershengorn,et al.  Thyrotropin-releasing hormone receptors -- similarities and differences. , 2003, Journal of molecular endocrinology.

[22]  Peter L. Freddolino,et al.  Prediction of structure and function of G protein-coupled receptors , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[23]  P. M. Hinkle,et al.  Role of TRH receptors as possible mediators of analeptic actions of TRH-like peptides , 2002, Brain Research.

[24]  J. Ballesteros,et al.  Structural mimicry in G protein-coupled receptors: implications of the high-resolution structure of rhodopsin for structure-function analysis of rhodopsin-like receptors. , 2001, Molecular pharmacology.

[25]  M. Schäfer,et al.  Expression of thyrotropin‐releasing hormone receptor 2 (TRH‐R2) in the central nervous system of rats , 2000, The Journal of comparative neurology.

[26]  K. Palczewski,et al.  Crystal Structure of Rhodopsin: A G‐Protein‐Coupled Receptor , 2002, Chembiochem : a European journal of chemical biology.

[27]  A. Smolyar,et al.  Static and dynamic roles of extracellular loops in G-protein-coupled receptors: a mechanism for sequential binding of thyrotropin-releasing hormone to its receptor. , 1998, Biophysical journal.

[28]  R. Osman,et al.  Role of the extracellular loops of the thyrotropin-releasing hormone receptor: evidence for an initial interaction with thyrotropin-releasing hormone. , 1997, Biochemistry.

[29]  Thomas Lengauer,et al.  A fast flexible docking method using an incremental construction algorithm. , 1996, Journal of molecular biology.

[30]  F Guarnieri,et al.  A refined model of the thyrotropin-releasing hormone (TRH) receptor binding pocket. Experimental analysis and energy minimization of the complex between TRH and TRH receptor. , 1996, Biochemistry.

[31]  G. Yarbrough.  Thyrotropin releasing hormone and CNS cholinergic neurons , 1983 .

[32]  R. Stevens,et al.  Structure of the human k-opioid receptor in complex with JDTic , 2012 .

[33]  Matthias Rarey,et al.  A consistent description of HYdrogen bond and DEhydration energies in protein–ligand complexes: methods behind the HYDE scoring function , 2012, Journal of Computer-Aided Molecular Design.

[34]  Ľ. Stárka,et al.  The computer modelling of human TRH receptor, TRH and TRH-like peptides. , 2005, Physiological Research.

[35]  R. Osman,et al.  Minireview: Insights into G protein-coupled receptor function using molecular models. , 2001, Endocrinology.

[36]  R. Osman,et al.  Minireview: Insights into G Protein-Coupled Receptor Function Using Molecular Models* * The work from our laboratories was supported by USPHS Grant DK-43036. , 2001, Endocrinology.

[37]  A. Sali,et al.  Modeling of loops in protein structures , 2000, Protein science : a publication of the Protein Society.

[38]  B. O'dowd,et al.  TRH-R2 exhibits similar binding and acute signaling but distinct regulation and anatomic distribution compared with TRH-R1. , 2000, Molecular endocrinology.