Biochem. J. (2014) 457, 313–322 (Printed in Great Britain) doi:10.1042/BJ20130827 313 Identification of a binding element for the cytoplasmic regulator FROUNT in the membrane-proximal C-terminal region of chemokine receptors CCR2 and CCR5 Etsuko TODA*, Yuya TERASHIMA* 1 , Kaori ESAKI†, Sosuke YOSHINAGA†, Minoru SUGIHARA‡, Yutaka KOFUKU§, Ichio SHIMADA§, Makiko SUWA‡‖, Shiro KANEGASAKI¶, Hiroaki TERASAWA† and Kouji MATSUSHIMA* *Department of Molecular Preventive Medicine, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan †Faculty of Life Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto 862-0973, Japan ‡National Institute of Advanced Industrial Science and Technology (AIST), Computational Biology Research Center (CBRC), Koutou-ku, Tokyo 135-0064, Japan §Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan ‖The Department of Chemistry and Biological Science, Aoyama Gakuin University, 5-10-1 Fuchinobe, Chuo-ku, Sagamihara-shi, Kanagawa 252-5258, Japan ¶YU-ECI Research Center for Medical Science, Yeungnam University, Gyeongsan 712-749, Korea Chemokine receptors mediate the migration of leucocytes during inflammation. The cytoplasmic protein FROUNT binds to chemokine receptors CCR2 [chemokine (C-C motif) receptor 2] and CCR5, and amplifies chemotactic signals in leucocytes. Although the interaction between FROUNT and chemokine receptors is important for accurate chemotaxis, the interaction mechanism has not been elucidated. In the present study we identified a 16-amino-acid sequence responsible for high-affinity binding of FROUNT at the membrane-proximal C-terminal intracellular region of CCR2 (CCR2 Pro-C) by yeast two-hybrid analysis. Synthesized peptides corresponding to the CCR2 Pro-C sequence directly interacted with FROUNT in vitro. CCR2 Pro-C was predicted to form an amphipathic helix structure. Residues on the hydrophobic side are completely conserved among FROUNT-binding receptors, suggesting that the hydrophobic side is the responsible element for FROUNT binding. The L316T mutation to the hydrophobic side of the predicted helix decreased the affinity for FROUNT. Co- immunoprecipitation assays revealed that the CCR2 L316T mutation diminished the interaction between FROUNT and full- length CCR2 in cells. Furthermore, this mutation impaired the ability of the receptor to mediate chemotaxis. These findings provide the first description of the functional binding element in helix 8 of CCR2 for the cytosolic regulator FROUNT that mediates chemotactic signalling. Key words: chemokine (C-C motif) receptor 2 (CCR2), chemo- taxis, FROUNT, G-protein-coupled receptor (GPCR), helix 8. INTRODUCTION The chemokine receptor family comprises 19 GPCRs (G-protein- coupled receptors). The C-terminal intracellular domain of chemoattractant receptors is indispensable for chemotaxis [1,2] and the subsequent receptor endocytosis [3,4]. In particular, the membrane-proximal region of chemokine receptors seems to play an essential role in chemotaxis [5–10]. The cytoplasmic protein FNT [FROUNT; also known as NUP85 (nuclear pore complex protein Nup85)] directly interacts with the C-terminal intracellular domain of activated CCR2 [chemokine (C-C motif) receptor 2] and amplifies the chemokine- elicited PI3K (phosphoinositide 3-kinase)–Rac–lamellipodium protrusion cascade, promoting subsequent chemotaxis. If the interaction between FNT and CCR2 is blocked, the chemotactic response is prevented [11], suggesting that the interaction between FNT and CCR2 plays a crucial role in CCR2-mediated chemotaxis. We reported previously that FNT is a common regulator of CCR2 and another chemokine receptor CCR5 [12]. Although the functions of CCR2 and CCR5 differ owing to different chemokine usage, these receptors mediate the migration and infiltration of monocyte/macrophages and are involved in the pathogenesis and progression of cancer and inflammatory diseases, such as arteriosclerosis, multiple sclerosis and graft versus host disease [13–16]. Thus the interaction between CCR2/CCR5 and FNT is a promising drug target for inflammatory diseases; however, the molecular mechanism underlying the binding of FNT to CCR2/CCR5 has not been elucidated. To investigate the molecular mechanism of the interaction between CCR2 and FNT, in the present study we have identified a short sequence in the membrane-proximal C-terminal region of CCR2 (CCR2 Pro-C) responsible for efficient binding to FNT. This sequence is predicted to form a helical structure, and we have identified residues (Leu 316 and His 323 ) crucial for FNT binding on the hydrophobic side of the predicted helix. Interestingly, the residues on the hydrophobic side of CCR2 are identical to those of the other chemokine receptor CCR5 to which FNT binds. Thus in the present study we define the hydrophobic side of a 16-amino- acid putative helix of CCR2/CCR5 Pro-C as an element for high- affinity binding to FNT. Furthermore, co-immunoprecipitation assays revealed that a low-affinity L316T mutation in this binding element impairs the interaction between full-length CCR2 and FNT in cultured cells. This low-affinity mutation correlated with the impaired chemotaxis mediated by CCR2. EXPERIMENTAL Proteins and peptides Full-length human FNT was expressed in Escherichia coli cells as a soluble protein, as described by Esaki et al. [17]. The C-terminal Abbreviations: CCR, chemokine (C-C motif) receptor; CXCR, chemokine (C-X-C motif) receptor; Em, emission; FNT, FROUNT; GPCR, G-protein-coupled receptor; HEK, human embryonic kidney; HTRF, homogeneous time-resolved fluorescence; LASP-1, LIM and Src homology 3 domain protein-1; ONPG, o-nitrophenyl β-D-galactopyranoside; PI3K, phosphoinositide 3-kinase; SPR, surface plasmon resonance; Y2H, yeast two-hybrid. 1 To whom correspondence should be addressed (email tera@m.u-tokyo.ac.jp). c  The Authors Journal compilation c  2014 Biochemical Society Biochemical Journal www.biochemj.org