Received for publication May 19, 2004.
Revised June 30, 2004.
Accepted for publication July 1, 2004.

Molecular Basis for the Differential Agonist Affinities of Group III Metabotropic Glutamate Receptors

Abstract

Agonist stimulation of Group III metabotropic glutamate receptors (mGluRs) induces an inhibition of neurotransmitter release from neurons. The Group III mGluRs are pharmacologically defined by activation with the glutamate analog L-amino-4-phosphonobutyric acid (L-AP4). The affinities of these receptors for L-AP4 and glutamate vary over approximately a 1500-fold concentration range. The goal of this study was to elucidate the molecular basis for this dispersion of agonist affinities for the Group III receptors mGluR4, mGluR6, and mGluR7. [³H]L-AP4 binding was present in human embryonic kidney cells transfected with the high affinity mGluR4 receptor but not in cells transfected with mGluR6 or the low affinity mGluR7 receptor. Analysis of mGluR4/mGluR6 receptor chimeras revealed that replacement of the first 35 amino acids of mGluR6 with the first 50 amino acids of mGluR4 was sufficient to impart [³H]L-AP4 binding to mGluR6. Homology models of mGluR4 and mGluR7 were employed to predict amino acids that may affect ligand affinity. Mutations were made in mGluR7 to convert selected residues into the equivalent amino acids present in the high affinity mGluR4 receptor. The mGluR7 N74K mutation caused a 12-fold increase in affinity in a functional assay, while the N74K mutation in combination with mutations in residues 258 - 262, which lie outside the binding pocket, caused a 112-fold increase in affinity compared to unmutated mGluR7. Our results demonstrate that the binding site residues at position lysine 74 in mGluR4, glutamine 58 in mGluR6, and asparagine 74 in mGluR7 are key determinants of agonist affinity and that additional residues situated outside of the binding pocket, including those present in the extreme amino terminus, also contribute to agonist affinity and the pharmacological profiles of the Group III mGluRs.

Key words: Metabotropic glutamate, Structure-activity relationships and modeling, Func. analysis receptor/ion channel mutants, Fluorescence techniques, Mutagenesis/Chimeric approaches

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