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Pivotal function for cytoplasmic protein FROUNT in CCR2-mediated monocyte chemotaxis

Abstract

Ligation of the chemokine receptor CCR2 on monocytes and macrophages with its ligand CCL2 results in activation of the cascade consisting of phosphatidylinositol-3-OH kinase (PI(3)K), the small G protein Rac and lamellipodium protrusion. We show here that a unique clathrin heavy-chain repeat homology protein, FROUNT, directly bound activated CCR2 and formed clusters at the cell front during chemotaxis. Overexpression of FROUNT amplified the chemokine-elicited PI(3)K–Rac–lamellipodium protrusion cascade and subsequent chemotaxis. Blocking FROUNT function by using a truncated mutant or antisense strategy substantially diminished signaling via CCR2. In a mouse peritonitis model, suppression of endogenous FROUNT markedly prevented macrophage infiltration. Thus, FROUNT links activated CCR2 to the PI(3)K–Rac–lamellipodium protrusion cascade and could be a therapeutic target in chronic inflammatory immune diseases associated with macrophage infiltration.

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Figure 1: FROUNT specifically binds to CCR2.
Figure 2: Subcellular distribution of CCR2 and FROUNT.
Figure 3: Requirement for FROUNT for CCR2 cluster formation.
Figure 4: Activation of the PI(3)K–Rac–lamellipodium protrusion cascade in FROUNT-transduced cells.
Figure 5: Suppression of FROUNT function diminishes chemotactic activity.
Figure 6: FROUNT in macrophage infiltration in vivo.

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Acknowledgements

We thank S. Nagata and C. Vestergaard for review of the manuscript; G. Nolan for Phoenix cells; and K. Kodaira, H. Yasukawa, K. Yamashita, Y. Masamune, M. Naito, K. Yokoyama, H. Nomiyama, S. Tomita, K. Kuno, T. Yokomizo, T. Tojo, A. Yamauchi, T. Tamatani, Y. Goshoh, M. Haino, S. Hashimoto, Y. Zhang, Y. Wang, H. Shibuya, C. Matsushima, N. Toyoda, S. Fujita and all the members of our laboratory for reagents and discussions. Supported in part by Solution Oriented Research for Science and Technology, Japan Science and Technology Corporation.

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Correspondence to Kouji Matsushima.

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Competing interests

Y.T., M.E. and S.K. are employed by Effector Cell Institute, which holds the patent on FROUNT, as does K.M.

Supplementary information

Supplementary Fig. 1

Structure of FROUNT and its binding site in CCR2. (PDF 246 kb)

Supplementary Fig. 2

Endogenous FNT binds to endogenous CCR2 in THP-1 cells. (PDF 1725 kb)

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Terashima, Y., Onai, N., Murai, M. et al. Pivotal function for cytoplasmic protein FROUNT in CCR2-mediated monocyte chemotaxis. Nat Immunol 6, 827–835 (2005). https://doi.org/10.1038/ni1222

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