Construction of a high affinity zinc binding site in the metabotropic glutamate receptor mGluR1: noncompetitive antagonism originating from the amino-terminal domain of a family C G-protein-coupled receptor
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Construction of a high affinity zinc binding site in the metabotropic glutamate receptor mGluR1 : noncompetitive antagonism originating from the amino-terminal domain of a family C G-protein-coupled receptor. / Jensen, Anders A.; Sheppard, P O; Jensen, L B; O'Hara, P J; Bräuner-Osborne, H.
In: Journal of Biological Chemistry, Vol. 276, No. 13, 2001, p. 10110-8.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Construction of a high affinity zinc binding site in the metabotropic glutamate receptor mGluR1
T2 - noncompetitive antagonism originating from the amino-terminal domain of a family C G-protein-coupled receptor
AU - Jensen, Anders A.
AU - Sheppard, P O
AU - Jensen, L B
AU - O'Hara, P J
AU - Bräuner-Osborne, H
PY - 2001
Y1 - 2001
N2 - The metabotropic glutamate receptors (mGluRs) belong to family C of the G-protein-coupled receptor (GPCR) superfamily. The receptors are characterized by having unusually long amino-terminal domains (ATDs), to which agonist binding has been shown to take place. Previously, we have constructed a molecular model of the ATD of mGluR1 based on a weak amino acid sequence similarity with a bacterial periplasmic binding protein. The ATD consists of two globular lobes, which are speculated to contract from an "open" to a "closed" conformation following agonist binding. In the present study, we have created a Zn(2+) binding site in mGluR1b by mutating the residue Lys(260) to a histidine. Zinc acts as a noncompetitive antagonist of agonist-induced IP accumulation on the K260H mutant with an IC(50) value of 2 microm. Alanine mutations of three potential "zinc coligands" in proximity to the introduced histidine in K260H knock out the ability of Zn(2+) to antagonize the agonist-induced response. Zn(2+) binding to K260H does not appear to affect the dimerization of the receptor. Instead, we propose that binding of zinc has introduced a structural constraint in the ATD lobe, preventing the formation of a "closed" conformation, and thus stabilizing a more or less inactive "open" form of the ATD. This study presents the first metal ion site constructed in a family C GPCR. Furthermore, it is the first time a metal ion site has been created in a region outside of the seven transmembrane regions of a GPCR and the loops connecting these. The findings offer valuable insight into the mechanism of ATD closure and family C receptor activation. Furthermore, the findings demonstrate that ATD regions other than those participating in agonist binding could be potential targets for new generations of ligands for this family of receptors.
AB - The metabotropic glutamate receptors (mGluRs) belong to family C of the G-protein-coupled receptor (GPCR) superfamily. The receptors are characterized by having unusually long amino-terminal domains (ATDs), to which agonist binding has been shown to take place. Previously, we have constructed a molecular model of the ATD of mGluR1 based on a weak amino acid sequence similarity with a bacterial periplasmic binding protein. The ATD consists of two globular lobes, which are speculated to contract from an "open" to a "closed" conformation following agonist binding. In the present study, we have created a Zn(2+) binding site in mGluR1b by mutating the residue Lys(260) to a histidine. Zinc acts as a noncompetitive antagonist of agonist-induced IP accumulation on the K260H mutant with an IC(50) value of 2 microm. Alanine mutations of three potential "zinc coligands" in proximity to the introduced histidine in K260H knock out the ability of Zn(2+) to antagonize the agonist-induced response. Zn(2+) binding to K260H does not appear to affect the dimerization of the receptor. Instead, we propose that binding of zinc has introduced a structural constraint in the ATD lobe, preventing the formation of a "closed" conformation, and thus stabilizing a more or less inactive "open" form of the ATD. This study presents the first metal ion site constructed in a family C GPCR. Furthermore, it is the first time a metal ion site has been created in a region outside of the seven transmembrane regions of a GPCR and the loops connecting these. The findings offer valuable insight into the mechanism of ATD closure and family C receptor activation. Furthermore, the findings demonstrate that ATD regions other than those participating in agonist binding could be potential targets for new generations of ligands for this family of receptors.
KW - Alanine
KW - Animals
KW - Binding Sites
KW - Blotting, Western
KW - CHO Cells
KW - Cell Line
KW - Cell Membrane
KW - Chlorides
KW - Cricetinae
KW - Dose-Response Relationship, Drug
KW - Glutamic Acid
KW - Histidine
KW - Humans
KW - Immunoblotting
KW - Inhibitory Concentration 50
KW - Inositol Phosphates
KW - Ions
KW - Kinetics
KW - Ligands
KW - Lysine
KW - Models, Molecular
KW - Mutation
KW - Plasmids
KW - Point Mutation
KW - Protein Binding
KW - Protein Conformation
KW - Protein Structure, Tertiary
KW - Receptors, Metabotropic Glutamate
KW - Transfection
KW - Zinc
KW - Zinc Compounds
U2 - 10.1074/jbc.M007220200
DO - 10.1074/jbc.M007220200
M3 - Journal article
C2 - 11133983
VL - 276
SP - 10110
EP - 10118
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 13
ER -
ID: 38485322