Discovery and mapping of an intracellular antagonist binding site at the chemokine receptor CCR2
Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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Discovery and mapping of an intracellular antagonist binding site at the chemokine receptor CCR2. / Zweemer, Annelien J M; Bunnik, Julia; Veenhuizen, Margo; Miraglia, Fabiana; Lenselink, Eelke B; Vilums, Maris; de Vries, Henk; Gibert, Arthur; Thiele, Stefanie; Rosenkilde, Mette M; IJzerman, Adriaan P; Heitman, Laura H.
I: Molecular Pharmacology, Bind 86, Nr. 4, 2014, s. 358-68.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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TY - JOUR
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
N1 - Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.
PY - 2014
Y1 - 2014
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 Y(7.53) and phenylalanine F(8.50) of the NPxxYx(5,6)F motif, as well as V(6.36) at the bottom of TM-VI and K(8.49) in 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 Y(7.53) and phenylalanine F(8.50) of the NPxxYx(5,6)F motif, as well as V(6.36) at the bottom of TM-VI and K(8.49) in 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
VL - 86
SP - 358
EP - 368
JO - Molecular Pharmacology
JF - Molecular Pharmacology
SN - 0026-895X
IS - 4
ER -
ID: 137817839