Identification of a binding element for the cytoplasmic regulator FROUNT in the membrane-proximal carboxy-terminal region of chemokine receptors CCR2 and CCR5

Chemokine receptors mediate the migration of leukocytes during inflammation. The cytoplasmic protein FROUNT binds to chemokine receptors CCR2 and CCR5, and amplifies chemotactic signals in leukocytes. Although the interaction between FROUNT and chemokine receptors is important for accurate chemotaxis, the interaction mechanism has not been elucidated. Here we identified a 16-amino-acid sequence responsible for high-affinity binding of FROUNT at the membrane-proximal carboxy (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. Leu316Thr mutation to the hydrophobic side of the predicted helix decreased affinity for FROUNT. Co-immunoprecipitation assay revealed that CCR2-Leu316Thr 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 signaling.

Source: BJ Cell - October 15, 2013 Category: Biochemistry Authors: E Toda, Y Terashima, K Esaki, S Yoshinaga, M Sugihara, Y Kofuku, I Shimada, M Suwa, S Kanegasaki, H Terasawa, K Matsushima Tags: BJ Cell Source Type: research