title: Internalization of the chemokine receptor CCR4 can be evoked by orthosteric and allosteric receptor antagonists.
abstract: 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 β-arrestin and stimulated GTPγS binding however CCL17 did not couple to β-arrestin and only partially stimulated GTPγS 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 sites on the CCR4 receptor, and while both classes inhibited agonist ligand binding and chemotaxis, one of the allosteric sites also evoked receptor internalization. Furthermore, we also characterize an N-terminally truncated version of CCL22 which acts as a competitive antagonist at the orthosteric site, 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.
authors.affiliation: Respiratory Therapy Area, GlaxoSmithKline, Gunnels Wood Road, Stevenage, Herts SG1 2NY, UK.
authors.forename: Laura
authors.initials: L
authors.lastname: Ajram
authors.affiliation: Respiratory Therapy Area, GlaxoSmithKline, Gunnels Wood Road, Stevenage, Herts SG1 2NY, UK.
authors.forename: Malcolm
authors.initials: M
authors.lastname: Begg
authors.affiliation: Respiratory Therapy Area, GlaxoSmithKline, Gunnels Wood Road, Stevenage, Herts SG1 2NY, UK.
authors.forename: Robert
authors.initials: R
authors.lastname: Slack
authors.affiliation: Platform Technology Sciences, GlaxoSmithKline, Gunnels Wood Road, Stevenage, Herts SG1 2NY, UK.
authors.forename: Jenni
authors.initials: J
authors.lastname: Cryan
authors.affiliation: Respiratory Therapy Area, GlaxoSmithKline, Gunnels Wood Road, Stevenage, Herts SG1 2NY, UK.
authors.forename: David
authors.initials: D
authors.lastname: Hall
authors.affiliation: Respiratory Therapy Area, GlaxoSmithKline, Gunnels Wood Road, Stevenage, Herts SG1 2NY, UK.
authors.forename: Simon
authors.initials: S
authors.lastname: Hodgson
authors.affiliation: Respiratory Therapy Area, GlaxoSmithKline, Gunnels Wood Road, Stevenage, Herts SG1 2NY, UK.
authors.forename: Alison
authors.initials: A
authors.lastname: Ford
authors.affiliation: Platform Technology Sciences, GlaxoSmithKline, Gunnels Wood Road, Stevenage, Herts SG1 2NY, UK.
authors.forename: Ashley
authors.initials: A
authors.lastname: Barnes
authors.affiliation: Airway Disease Infection Section, National Heart and Lung Institute, Imperial College, Norfolk Place, London W2 1PG, UK.
authors.forename: Dawid
authors.initials: D
authors.lastname: Swieboda
authors.affiliation: Airway Disease Infection Section, National Heart and Lung Institute, Imperial College, Norfolk Place, London W2 1PG, UK.
authors.forename: Aurelie
authors.initials: A
authors.lastname: Mousnier
authors.affiliation: Respiratory Therapy Area, GlaxoSmithKline, Gunnels Wood Road, Stevenage, Herts SG1 2NY, UK; Airway Disease Infection Section, National Heart and Lung Institute, Imperial College, Norfolk Place, London W2 1PG, UK. Electronic address: r.solari@imperial.ac.uk.
authors.forename: Roberto
authors.initials: R
authors.lastname: Solari
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects / Q000502:physiology; D000818:Animals; D001491:Basophils / Q000187:drug effects / Q000378:metabolism; D016466:CHO Cells; D002478:Cells, Cultured; D054414:Chemokine CCL17 / Q000494:pharmacology; D054422:Chemokine CCL22 / Q000494:pharmacology; D002633:Chemotaxis / Q000187:drug effects / Q000502:physiology*; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D054398:Receptors, CCR4 / Q000037:antagonists & inhibitors* / Q000378:metabolism*
chemical_list: C106230:CCL17 protein, human; C515482:CCL22 protein, human; C515354:CCR4 protein, human; D054414:Chemokine CCL17; D054422:Chemokine CCL22; D054398:Receptors, CCR4
keywords: CCL17; CCL22; CCR4; Chemokine; MDC; TARC