Discovery and mapping of an intracellular antagonist binding site at the chemokine receptor CCR2

Annelien J M Zweemer; Julia Bunnik; Margo Veenhuizen; Fabiana Miraglia; Eelke B Lenselink; Maris Vilums; Henk de Vries; Arthur Gibert; Stefanie Thiele; Mette M Rosenkilde; Adriaan P IJzerman; Laura H Heitman

doi:10.1124/mol.114.093328

Discovery and mapping of an intracellular antagonist binding site at the chemokine receptor CCR2

Annelien J M Zweemer, Julia Bunnik, Margo Veenhuizen, Fabiana Miraglia, Eelke B Lenselink, Maris Vilums, Henk de Vries, Arthur Gibert, Stefanie Thiele, Mette M Rosenkilde, Adriaan P IJzerman, Laura H Heitman

22 Citations (Scopus)

Abstract

The chemokine receptor CCR2 is a G protein-coupled receptor that is involved in many diseases characterized by chronic inflammation, and therefore a large variety of CCR2 small molecule antagonists has been developed. On the basis of their chemical structures these antagonists can roughly be divided into two groups with most likely two topographically distinct binding sites. The aim of the current study was to identify the binding site of one such group of ligands, exemplified by three allosteric antagonists, CCR2-RA-[R], JNJ-27141491, and SD-24. We first used a chimeric CCR2/CCR5 receptor approach to obtain insight into the binding site of the allosteric antagonists and additionally introduced eight single point mutations in CCR2 to further characterize the putative binding pocket. All constructs were studied in radioligand binding and/or functional IP turnover assays, providing evidence for an intracellular binding site for CCR2-RA-[R], JNJ-27141491, and SD-24. For CCR2-RA-[R] the most important residues for binding were found to be the highly conserved tyrosine Y^7.53and phenylalanine F^8.50of the NPxxYx_(5,6)F motif, as well as V^6.36at the bottom of TM-VI and K^8.49in helix-VIII. These findings demonstrate for the first time the presence of an allosteric intracellular binding site for CCR2 antagonists. This contributes to an increased understanding of the interactions of diverse ligands at CCR2 and may allow for a more rational design of future allosteric antagonists.

Original language	English
Journal	Molecular Pharmacology
Volume	86
Issue number	4
Pages (from-to)	358-68
Number of pages	11
ISSN	0026-895X
DOIs	https://doi.org/10.1124/mol.114.093328
Publication status	Published - 1 Oct 2014

Keywords

Allosteric Site
Amino Acid Motifs
Amino Acid Sequence
Animals
CHO Cells
COS Cells
Cell Line, Tumor
Cercopithecus aethiops
Cricetinae
Cricetulus
Humans
Imidazoles
Ligands
Molecular Sequence Data
Point Mutation
Protein Binding
Pyrrolidines
Receptors, CCR2
Sulfonamides

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title = "Discovery and mapping of an intracellular antagonist binding site at the chemokine receptor CCR2",

abstract = "The chemokine receptor CCR2 is a G protein-coupled receptor that is involved in many diseases characterized by chronic inflammation, and therefore a large variety of CCR2 small molecule antagonists has been developed. On the basis of their chemical structures these antagonists can roughly be divided into two groups with most likely two topographically distinct binding sites. The aim of the current study was to identify the binding site of one such group of ligands, exemplified by three allosteric antagonists, CCR2-RA-[R], JNJ-27141491, and SD-24. We first used a chimeric CCR2/CCR5 receptor approach to obtain insight into the binding site of the allosteric antagonists and additionally introduced eight single point mutations in CCR2 to further characterize the putative binding pocket. All constructs were studied in radioligand binding and/or functional IP turnover assays, providing evidence for an intracellular binding site for CCR2-RA-[R], JNJ-27141491, and SD-24. For CCR2-RA-[R] the most important residues for binding were found to be the highly conserved tyrosine Y7.53and phenylalanine F8.50of the NPxxYx(5,6)F motif, as well as V6.36at the bottom of TM-VI and K8.49in helix-VIII. These findings demonstrate for the first time the presence of an allosteric intracellular binding site for CCR2 antagonists. This contributes to an increased understanding of the interactions of diverse ligands at CCR2 and may allow for a more rational design of future allosteric antagonists.",

keywords = "Allosteric Site, Amino Acid Motifs, Amino Acid Sequence, Animals, CHO Cells, COS Cells, Cell Line, Tumor, Cercopithecus aethiops, Cricetinae, Cricetulus, Humans, Imidazoles, Ligands, Molecular Sequence Data, Point Mutation, Protein Binding, Pyrrolidines, Receptors, CCR2, Sulfonamides",

author = "Zweemer, {Annelien J M} and Julia Bunnik and Margo Veenhuizen and Fabiana Miraglia and Lenselink, {Eelke B} and Maris Vilums and {de Vries}, Henk and Arthur Gibert and Stefanie Thiele and Rosenkilde, {Mette M} and IJzerman, {Adriaan P} and Heitman, {Laura H}",

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doi = "10.1124/mol.114.093328",

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T1 - Discovery and mapping of an intracellular antagonist binding site at the chemokine receptor CCR2

AU - Zweemer, Annelien J M

AU - Bunnik, Julia

AU - Veenhuizen, Margo

AU - Miraglia, Fabiana

AU - Lenselink, Eelke B

AU - Vilums, Maris

AU - de Vries, Henk

AU - Gibert, Arthur

AU - Thiele, Stefanie

AU - Rosenkilde, Mette M

AU - IJzerman, Adriaan P

AU - Heitman, Laura H

PY - 2014/10/1

Y1 - 2014/10/1

N2 - The chemokine receptor CCR2 is a G protein-coupled receptor that is involved in many diseases characterized by chronic inflammation, and therefore a large variety of CCR2 small molecule antagonists has been developed. On the basis of their chemical structures these antagonists can roughly be divided into two groups with most likely two topographically distinct binding sites. The aim of the current study was to identify the binding site of one such group of ligands, exemplified by three allosteric antagonists, CCR2-RA-[R], JNJ-27141491, and SD-24. We first used a chimeric CCR2/CCR5 receptor approach to obtain insight into the binding site of the allosteric antagonists and additionally introduced eight single point mutations in CCR2 to further characterize the putative binding pocket. All constructs were studied in radioligand binding and/or functional IP turnover assays, providing evidence for an intracellular binding site for CCR2-RA-[R], JNJ-27141491, and SD-24. For CCR2-RA-[R] the most important residues for binding were found to be the highly conserved tyrosine Y7.53and phenylalanine F8.50of the NPxxYx(5,6)F motif, as well as V6.36at the bottom of TM-VI and K8.49in helix-VIII. These findings demonstrate for the first time the presence of an allosteric intracellular binding site for CCR2 antagonists. This contributes to an increased understanding of the interactions of diverse ligands at CCR2 and may allow for a more rational design of future allosteric antagonists.

AB - The chemokine receptor CCR2 is a G protein-coupled receptor that is involved in many diseases characterized by chronic inflammation, and therefore a large variety of CCR2 small molecule antagonists has been developed. On the basis of their chemical structures these antagonists can roughly be divided into two groups with most likely two topographically distinct binding sites. The aim of the current study was to identify the binding site of one such group of ligands, exemplified by three allosteric antagonists, CCR2-RA-[R], JNJ-27141491, and SD-24. We first used a chimeric CCR2/CCR5 receptor approach to obtain insight into the binding site of the allosteric antagonists and additionally introduced eight single point mutations in CCR2 to further characterize the putative binding pocket. All constructs were studied in radioligand binding and/or functional IP turnover assays, providing evidence for an intracellular binding site for CCR2-RA-[R], JNJ-27141491, and SD-24. For CCR2-RA-[R] the most important residues for binding were found to be the highly conserved tyrosine Y7.53and phenylalanine F8.50of the NPxxYx(5,6)F motif, as well as V6.36at the bottom of TM-VI and K8.49in helix-VIII. These findings demonstrate for the first time the presence of an allosteric intracellular binding site for CCR2 antagonists. This contributes to an increased understanding of the interactions of diverse ligands at CCR2 and may allow for a more rational design of future allosteric antagonists.

KW - Allosteric Site

KW - Amino Acid Motifs

KW - Amino Acid Sequence

KW - Animals

KW - CHO Cells

KW - COS Cells

KW - Cell Line, Tumor

KW - Cercopithecus aethiops

KW - Cricetinae

KW - Cricetulus

KW - Humans

KW - Imidazoles

KW - Ligands

KW - Molecular Sequence Data

KW - Point Mutation

KW - Protein Binding

KW - Pyrrolidines

KW - Receptors, CCR2

KW - Sulfonamides

U2 - 10.1124/mol.114.093328

DO - 10.1124/mol.114.093328

M3 - Journal article

C2 - 25024169

SN - 0026-895X

VL - 86

SP - 358

EP - 368

JO - Molecular Pharmacology

JF - Molecular Pharmacology

IS - 4

ER -

Discovery and mapping of an intracellular antagonist binding site at the chemokine receptor CCR2

Abstract

Keywords

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