10.6084/m9.figshare.5124865.v1 Sato T. Sato T. Iwase M. Iwase M. Miyama M. Miyama M. Komai M. Komai M. Ohshima E. Ohshima E. Asai A. Asai A. Yano H. Yano H. Miki I. Miki I. Supplementary Material for: Internalization of CCR4 and Inhibition of Chemotaxis by K777, a Potent and Selective CCR4 Antagonist Karger Publishers 2013 Receptor antagonists Chemotaxis Anti-asthmatic drugs 2013-06-05 00:00:00 Dataset https://karger.figshare.com/articles/dataset/Supplementary_Material_for_Internalization_of_CCR4_and_Inhibition_of_Chemotaxis_by_K777_a_Potent_and_Selective_CCR4_Antagonist/5124865 CC chemokine receptor 4 (CCR4) is a G protein-coupled receptor that regulates the chemotaxis of Th2 lymphocytes, which are key players in allergic diseases. K777 is a small compound identified in a binding assay using a CCR4 ligand, CCL17. K777 inhibited both CCL17 binding and CCL17-induced chemotaxis in Hut78 cells (IC<sub>50</sub>: 57 and 8.9 nmol/l, respectively). The K777-mediated inhibition of chemotaxis was potent even in the presence of a 10-fold higher concentration of CCL17. The imaging and flow cytometric analyses revealed that K777 induced CCR4 internalization, with a ∼50% reduction of cell surface CCR4. K777 did not inhibit CXCR4-induced chemotaxis or internalization and did not bring about Ca<sup>2+</sup> mobilization by itself. A Scatchard plot analysis of the binding assay using radiolabeled K777 revealed a single high-affinity binding site on the CCR4 molecule. These results indicate that K777 is a selective CCR4 antagonist featuring the potent chemotaxis inhibition, to which the internalization-inducible ability of K777 to hide a part of cell surface CCR4 may contribute.