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

Anders A. Jensen; P O Sheppard; L B Jensen; P J O'Hara; H Bräuner-Osborne

doi:10.1074/jbc.M007220200

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

Anders A. Jensen, P O Sheppard, L B Jensen, P J O'Hara, H Bräuner-Osborne

11 Citationer (Scopus)

Abstract

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.

Originalsprog	Engelsk
Tidsskrift	Journal of Biological Chemistry
Vol/bind	276
Udgave nummer	13
Sider (fra-til)	10110-8
Antal sider	9
ISSN	0021-9258
DOI	https://doi.org/10.1074/jbc.M007220200
Status	Udgivet - 2001

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10.1074/jbc.M007220200

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Citationsformater

Jensen, A. A., Sheppard, P. O., Jensen, L. B., O'Hara, P. J., & Bräuner-Osborne, H. (2001). 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. Journal of Biological Chemistry, 276(13), 10110-8. https://doi.org/10.1074/jbc.M007220200

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 et al.

I: Journal of Biological Chemistry, Bind 276, Nr. 13, 2001, s. 10110-8.

Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review

Jensen, AA, Sheppard, PO, Jensen, LB, O'Hara, PJ & Bräuner-Osborne, H 2001, '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', Journal of Biological Chemistry, bind 276, nr. 13, s. 10110-8. https://doi.org/10.1074/jbc.M007220200

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title = "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",

abstract = "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.",

keywords = "Alanine, Animals, Binding Sites, Blotting, Western, CHO Cells, Cell Line, Cell Membrane, Chlorides, Cricetinae, Dose-Response Relationship, Drug, Glutamic Acid, Histidine, Humans, Immunoblotting, Inhibitory Concentration 50, Inositol Phosphates, Ions, Kinetics, Ligands, Lysine, Models, Molecular, Mutation, Plasmids, Point Mutation, Protein Binding, Protein Conformation, Protein Structure, Tertiary, Receptors, Metabotropic Glutamate, Transfection, Zinc, Zinc Compounds",

author = "Jensen, {Anders A.} and Sheppard, {P O} and Jensen, {L B} and O'Hara, {P J} and H Br{\"a}uner-Osborne",

year = "2001",

doi = "10.1074/jbc.M007220200",

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pages = "10110--8",

journal = "Journal of Biological Chemistry",

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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

SN - 0021-9258

VL - 276

SP - 10110

EP - 10118

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 13

ER -