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
SN - 0014-2999
VL - 397
SP - 247
EP - 253
JO - European Journal of Pharmacology
JF - European Journal of Pharmacology
IS - 2-3
ER -