Internalization of the chemokine receptor CCR4 can be evoked by orthosteric and allosteric receptor antagonists

The chemokine receptor CCR4 has at least two natural agonist ligands, MDC (CCL22) and TARC (CCL17) which bind to the same orthosteric site with a similar affinity. Both ligands are known to evoke chemotaxis of CCR4-bearing T cells and also elicit CCR4 receptor internalization. A series of small molecule allosteric antagonists have been described which displace the agonist ligand, and inhibit chemotaxis. The aim of this study was to determine which cellular coupling pathways are involved in internalization, and if antagonists binding to the CCR4 receptor could themselves evoke receptor internalization. CCL22 binding coupled CCR4 efficiently to beta-arrestin and stimulated GTP gamma S binding however CCL17 did nor couple to beta-arrestin and only partially stimulated GTP gamma S binding. CCL22 potently induced internalization of almost all cell surface CCR4, while CCL17 showed only weak effects. We describe four small molecule antagonists that were demonstrated to bind to two distinct allosteric sires on the CCR4 receptor, and while both classes inhibited agonist ligand binding and chemotaxis, one of the allosteric sires also evoked receptor internalization. Furthermore, we also characterize an N-terminally truncated version of CCL22 which acts as a competitive antagonist at the orthosteric sire, and surprisingly also evokes receptor internalization without demonstrating any agonist activity. Collectively this study demonstrates that orthosteric and allosteric antagonists of the CCR4 receptor are capable of evoking receptor internalization, providing a novel strategy for drug discovery against this class of target. (C) 2014 The Authors. Published by Elsevier EN. This is an open access article under the CC BY-NC-ND license (http://creatvecommons.org/licenses/by-nc-nd/3.0/).