The Non-competitive Antagonists 2-Methyl-6-(phenylethynyl)pyridine and 7-Hydroxyiminocyclopropan[b]chromen-1a-carboxylic Acid Ethyl Ester Interact with Overlapping Binding Pockets in the Transmembrane Region of Group I Metabotropic Glutamate Receptors*

We have investigated the mechanism of inhibition and site of action of the novel human metabotropic glutamate receptor 5 (hmGluR5) antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP), which is structurally unrelated to classical metabotropic glutamate receptor (mGluR) ligands. Schild analysis indicated that MPEP acts in a non-competitive manner. MPEP also inhibited to a large extent constitutive receptor activity in cells transiently overexpressing rat mGluR5, suggesting that MPEP acts as an inverse agonist. To investigate the molecular determinants that govern selective ligand binding, a mutagenesis study was performed using chimeras and single amino acid substitutions of hmGluR1 and hmGluR5. The mutants were tested for binding of the novel mGluR5 radioligand [3H]2-methyl-6-(3-methoxyphenyl)ethynyl pyridine (M-MPEP), a close analog of MPEP. Replacement of Ala-810 in transmembrane (TM) VII or Pro-655 and Ser-658 in TMIII with the homologous residues of hmGluR1 abolished radioligand binding. In contrast, the reciprocal hmGluR1 mutant bearing these three residues of hmGluR5 showed high affinity for [3H]M-MPEP. Radioligand binding to these mutants was also inhibited by 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester (CPCCOEt), a structurally unrelated non-competitive mGluR1 antagonist previously shown to interact with residues Thr-815 and Ala-818 in TMVII of hmGluR1. These results indicate that MPEP and CPCCOEt bind to overlapping binding pockets in the TM region of group I mGluRs but interact with different non-conserved residues.

[1]  M. Al-Moslih,et al.  The kinetics of DEAE-dextran-induced cell sensitization to transfection. , 1973, The Journal of general virology.

[2]  J. Dubuisson,et al.  Proteins of bovine herpesvirus type 4 released into the culture medium of productively infected cells: identification of a 135K glycoprotein involved in viral attachment. , 1992, The Journal of general virology.

[3]  S. Nakanishi,et al.  Role of the large extracellular domain of metabotropic glutamate receptors in agonist selectivity determination. , 1993, The Journal of biological chemistry.

[4]  Terri L. Gilbert,et al.  The ligand-binding domain in metabotropic glutamate receptors is related to bacterial periplasmic binding proteins , 1993, Neuron.

[5]  B. Conklin,et al.  Substitution of three amino acids switches receptor specificity of Gqα to that of Giα , 1993, Nature.

[6]  T. Knöpfel,et al.  Metabotropic glutamate receptors: novel targets for drug development. , 1995, Journal of medicinal chemistry.

[7]  A. Young,et al.  Molecular and functional characterization of recombinant human metabotropic glutamate receptor subtype 5 , 1995, Neuropharmacology.

[8]  T. Knöpfel,et al.  The agonist selectivity of a class III metabotropic glutamate receptor, human mGluR4a, is determined by the N-terminal extracellular domain , 1995, Neuroreport.

[9]  J. Bockaert,et al.  Molecular, functional, and pharmacological characterization of the metabotropic glutamate receptor type 5 splice variants: comparison with mGluR1 , 1995, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[10]  Atsuko Fukunaga,et al.  A novel class of antagonists for metabotropic glutamate receptors, 7-(Hydroxyimino)cyclopropa[b]chromen-1a-carboxylates , 1996 .

[11]  L. Prézeau,et al.  Changes in the carboxyl-terminal domain of metabotropic glutamate receptor 1 by alternative splicing generate receptors with differing agonist-independent activity. , 1996, Molecular pharmacology.

[12]  J. Neale,et al.  N‐Acetylaspartylglutamate Selectively Activates mGluR3 Receptors in Transfected Cells , 1997, Journal of neurochemistry.

[13]  J. Baldwin,et al.  An alpha-carbon template for the transmembrane helices in the rhodopsin family of G-protein-coupled receptors. , 1997, Journal of molecular biology.

[14]  T. Knöpfel,et al.  Expression and Coupling to Polyphosphoinositide Hydrolysis of Group I Metabotropic Glutamate Receptors in Early Postnatal and Adult Rat Brain , 1997, The European journal of neuroscience.

[15]  J. Pin,et al.  Pharmacology and functions of metabotropic glutamate receptors. , 1997, Annual review of pharmacology and toxicology.

[16]  L. Prézeau,et al.  A Cluster of Basic Residues in the Carboxyl-terminal Tail of the Short Metabotropic Glutamate Receptor 1 Variants Impairs Their Coupling to Phospholipase C* , 1998, The Journal of Biological Chemistry.

[17]  J. Bockaert,et al.  The G protein-coupling profile of metabotropic glutamate receptors, as determined with exogenous G proteins, is independent of their ligand recognition domain. , 1998, Molecular pharmacology.

[18]  J. Bockaert,et al.  Comparative effect of l-CCG-I, DCG-IV and γ-carboxy-l-glutamate on all cloned metabotropic glutamate receptor subtypes , 1998, Neuropharmacology.

[19]  S. Moro,et al.  Human P2Y1 receptor: molecular modeling and site-directed mutagenesis as tools to identify agonist and antagonist recognition sites. , 1998, Journal of medicinal chemistry.

[20]  R. Challiss,et al.  Reversible and non-competitive antagonist profile of CPCCOEt at the human type 1α metabotropic glutamate receptor , 1998, Neuropharmacology.

[21]  S. Nakanishi,et al.  Expression and Purification of the Extracellular Ligand Binding Region of Metabotropic Glutamate Receptor Subtype 1* , 1998, The Journal of Biological Chemistry.

[22]  F. Gasparini,et al.  CPCCOEt, a noncompetitive metabotropic glutamate receptor 1 antagonist, inhibits receptor signaling without affecting glutamate binding. , 1999, Molecular pharmacology.

[23]  D. Jane,et al.  Pharmacological agents acting at subtypes of metabotropic glutamate receptors , 1999, Neuropharmacology.

[24]  Roland Heckendorn,et al.  2-Methyl-6-(phenylethynyl)-pyridine (MPEP), a potent, selective and systemically active mGlu5 receptor antagonist , 1999, Neuropharmacology.

[25]  G. Battaglia,et al.  Selective blockade of metabotropic glutamate receptor subtype 5 is neuroprotective , 2000, Neuropharmacology.

[26]  B. Meldrum,et al.  Anticonvulsant activity of two metabotropic glutamate Group I antagonists selective for the mGlu5 receptor: 2-methyl-6-(phenylethynyl)-pyridine (MPEP), and (E)-6-methyl-2-styryl-pyridine (SIB 1893) , 2000, Neuropharmacology.