Mutation-induced Quisqualic Acid and Ibotenic Acid Affinity at the Metabotropic Glutamate Receptor Subtype 4

Abstract

The metabotropic glutamate receptors (mGluRs) are key modulators of excitatory neurotransmission in the central nervous system. The eight mGluR subtypes are seven trans-membrane-spanning proteins that possess a large extracellular amino-terminal domain in which the endogenous ligand binding pocket resides. In this study, we have identified four non-conserved amino acid residues that are essential for differentiating mGluR1 from mGluR4. Our approach has been to increase the affinity of the classic mGluR1 agonists, quisqualic acid and ibotenic acid, at mGluR4 by making various point mutations that mimicked mGluR1 residues. Based on ligand docking to homology models, the non-conserved residues, Lys-74, Glu-287, Ser-313, and Lys-317, were chosen for the mutational studies and all of the mutations proved capable of partially or completely restoring the affinities of the ligands. In particular, the mutations K74Y and K317R induced dramatic triple-order-of-magnitude increases in the affinity of ibotenic acid at mGluR4, making the affinity equivalent to that of mGluR1. Furthermore, the affinity of quisqualic acid at mGluR4 was increased to the same level as mGluR1 by the two double mutations, K74Y/K317R and K74Y/E287G. Advanced analysis of ligand conformation and docking procedures were used for the interpretation of these results. The study shows that mGluR subtype selectivity results from a complex interplay of residues shaping the binding pocket, rather than being attributable to a single specific ligand-receptor interaction.The excitatory neurotransmitter glutamate elicits and modulates synaptic responses in the central nervous systems by activating two families of receptors: ligand-gated cation channels termed ionotropic glutamate receptors (iGluRs) 1 and Gprotein-coupled receptors termed metabotropic glutamate receptors (mGluRs). To date, the use of cloning techniques has led to the identification of eight mGluR subtypes and multiple splice variants, which have been subdivided into three groups (I-III) based on their sequence homology, signal transduction mechanisms, and pharmacological properties (1). The stimulatory group I receptors (mGluR1 and mGluR5) give rise to postsynaptic increases in cytoplasmic calcium and/or presynaptic increases in neurotransmitter release, whereas groups II (mGlu2 and mGlu3) and III (mGlu4, mGlu6, mGlu7, and mGlu8) are primarily localized presynaptically and typically inhibit neurotransmission as autoreceptors either at glutamatergic synapses or at other neurotransmitter-releasing synapses (2). mGluRs are seven trans-membrane-spanning proteins that possess a large extracellular amino-terminal domain (ATD), characteristic of the family C G-protein coupled receptors to which they belong. The binding site of the endogenous ligand is contained within the ATD (3, 4), which is composed of two lobes connected by a hinge region, giving rise to a closed and an open form. The two lobes are thought to close upon binding the agonist such as a Venus flytrap when touched by an insect (5, 6). Recen...

Selectivity and Evolutionary Divergence of Metabotropic Glutamate Receptors for Endogenous Ligands and G Proteins Coupled to Phospholipase C or TRP Channels

“…3). A role for this residue in ligand recognition is consistent with previous reports that the mGluR7 mutant N74K decreased the EC 50 of the group III-specific synthetic agonist L-AP4 by about 10-fold as compared with wild type mGluR7 (41), and that a K74Y mutation in mGluR4 enhanced its affinity for the mGluR1-specific synthetic agonist ibotenic acid (42). However, the evolutionary and functional significance of this residue can only be understood in the context of the endogenous ligands with which the receptors have been challenged over the course of evolution.…”

Targeting the Metabotropic Glutamate Receptor mGluR4 for the Treatment of Diseases of the Central Nervous System

“…Recently, amino acid residues that are responsible for the pharmacological selectivity of group I relative to group III metabotropic have been identified [15]. Mutation of a few non-conserved amino acids in the mGluR4 receptor to those in mGluR1 conferred the ability to bind the group I selective agonists quisqualate and ibotenic acid on the mGluR4 receptor [15]. These residues in mGluR4 were in both lobe 1; Lys74, and lobe 2; Glu287, Ser313 and Lys317.…”

“…Several of the key residues for agonist binding in mGluR4, Arg78, Ser159, Thr182, and Lys405, are highly conserved residues in all of the mGluRs and are implicated in agonist binding in the mGluR1 LBD structure [6]. Recently, amino acid residues that are responsible for the pharmacological selectivity of group I relative to group III metabotropic have been identified [15]. Mutation of a few non-conserved amino acids in the mGluR4 receptor to those in mGluR1 conferred the ability to bind the group I selective agonists quisqualate and ibotenic acid on the mGluR4 receptor [15].…”

An Introduction to Molecular Modeling of G-Protein Coupled Receptors

“…In another modeling study on the same subgroup of mGluRs, it was shown that the pharmacological profile of the Group III receptor, mGluR4, could be completely switched via mutagenesis to a receptor that responded to the Group I specific agonists quisqualic acid and ibotenic acid [78]. Based on the homology model, residues K74, E287, S313 and K317 in mGluR4 were mutated to the corresponding residues in mGluR1.…”