PT - JOURNAL ARTICLE AU - Annelien J.M. Zweemer AU - Julia Bunnik AU - Margo Veenhuizen AU - Fabiana Miraglia AU - Eelke B. Lenselink AU - Maris Vilums AU - Henk de Vries AU - Arthur Gibert AU - Stefanie Thiele AU - Mette M. Rosenkilde AU - Adriaan P. IJzerman AU - Laura H. Heitman TI - Discovery and Mapping of an Intracellular Antagonist Binding Site at the Chemokine Receptor CCR2 AID - 10.1124/mol.114.093328 DP - 2014 Jul 14 TA - Molecular Pharmacology PG - mol.114.093328 4099 - http://molpharm.aspetjournals.org/content/early/2014/07/14/mol.114.093328.short 4100 - http://molpharm.aspetjournals.org/content/early/2014/07/14/mol.114.093328.full 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 be roughly 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 Y7.53 and phenylalanine F8.50 of the NPxxYX(5,6)F motif, as well as V6.36 at the bottom of TM-VI and K8.49 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.