TY - JOUR

T1 - The role of Arg(78) in the metabotropic glutamate receptor mGlu(1) for agonist binding and selectivity

AU - Jensen, Anders A.

AU - Sheppard, P O

AU - O'Hara, P J

AU - Krogsgaard-Larsen, P

AU - Bräuner-Osborne, H

PY - 2000

Y1 - 2000

N2 - The metabotropic glutamate receptors belong to family C of the G-protein coupled receptor superfamily. These receptors all possess large extracellular amino terminal domains, where agonist binding takes place. We have previously constructed a molecular model of the amino terminal domain of the mGlu(1) receptor based on a weak amino acid sequence similarity with a family of bacterial periplasmic binding proteins (PBPs). The residues Ser(165) and Thr(188) were demonstrated to be involved in agonist binding to the receptor. Here, we report that mutation of Arg(78) in the mGlu(1b) receptor to leucine or glutamate completely knocks out [3H]quisqualic acid binding to the receptor. The constructed mutants, R78L and R78E, have also been characterized in a inositol phosphate assay. Here, the potency of (S)-glutamic acid and (S)-quisqualic acid was reduced 1000- and 100-fold, respectively, on R78L compared to the wild type (WT) receptor. (S)-Quisqualic acid was as potent on mutant R78E as it was on R78L, whereas (S)-glutamic acid was unable to activate R78E significantly at concentrations up to 10 mM. In conclusion, Arg(78) appears to be essential for agonist binding to the mGlu(1) receptor, most likely, through the formation of an ionic bond between its positively charged side chain and the distal acid group of the agonists. Furthermore, the different impact of the two mutations on (S)-glutamic acid and (S)-quisqualic acid potencies strongly indicates that while Arg(78) appears to be a common site of interaction for the agonists, the Group I subtype selectivity of (S)-quisqualic acid is probably determined by other residues in the amino terminal domain.

AB - The metabotropic glutamate receptors belong to family C of the G-protein coupled receptor superfamily. These receptors all possess large extracellular amino terminal domains, where agonist binding takes place. We have previously constructed a molecular model of the amino terminal domain of the mGlu(1) receptor based on a weak amino acid sequence similarity with a family of bacterial periplasmic binding proteins (PBPs). The residues Ser(165) and Thr(188) were demonstrated to be involved in agonist binding to the receptor. Here, we report that mutation of Arg(78) in the mGlu(1b) receptor to leucine or glutamate completely knocks out [3H]quisqualic acid binding to the receptor. The constructed mutants, R78L and R78E, have also been characterized in a inositol phosphate assay. Here, the potency of (S)-glutamic acid and (S)-quisqualic acid was reduced 1000- and 100-fold, respectively, on R78L compared to the wild type (WT) receptor. (S)-Quisqualic acid was as potent on mutant R78E as it was on R78L, whereas (S)-glutamic acid was unable to activate R78E significantly at concentrations up to 10 mM. In conclusion, Arg(78) appears to be essential for agonist binding to the mGlu(1) receptor, most likely, through the formation of an ionic bond between its positively charged side chain and the distal acid group of the agonists. Furthermore, the different impact of the two mutations on (S)-glutamic acid and (S)-quisqualic acid potencies strongly indicates that while Arg(78) appears to be a common site of interaction for the agonists, the Group I subtype selectivity of (S)-quisqualic acid is probably determined by other residues in the amino terminal domain.

KW - Amino Acid Sequence

KW - Amino Acid Substitution

KW - Arginine

KW - Binding Sites

KW - Binding, Competitive

KW - Cell Line

KW - Dose-Response Relationship, Drug

KW - Excitatory Amino Acid Agonists

KW - Glutamic Acid

KW - Humans

KW - Inositol Phosphates

KW - Models, Molecular

KW - Molecular Sequence Data

KW - Mutation

KW - Protein Structure, Tertiary

KW - Quisqualic Acid

KW - Receptors, Metabotropic Glutamate

KW - Sequence Homology, Amino Acid

M3 - Journal article

C2 - 10844121

VL - 397

SP - 247

EP - 253

JO - European Journal of Pharmacology

JF - European Journal of Pharmacology

SN - 0014-2999

IS - 2-3

ER -