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

Biochem J. 2014 Jan 15;457(2):313-22. doi: 10.1042/BJ20130827.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Cell Membrane / genetics
  • Cell Membrane / metabolism*
  • Conserved Sequence
  • Cytoplasm / metabolism*
  • Humans
  • Jurkat Cells
  • Molecular Sequence Data
  • Nuclear Pore Complex Proteins / genetics
  • Nuclear Pore Complex Proteins / metabolism*
  • Protein Binding / physiology
  • Random Allocation
  • Receptors, CCR2 / genetics
  • Receptors, CCR2 / metabolism*
  • Receptors, CCR5 / genetics
  • Receptors, CCR5 / metabolism*

Substances

  • CCR2 protein, human
  • CCR5 protein, human
  • NUP85 protein, human
  • Nuclear Pore Complex Proteins
  • Receptors, CCR2
  • Receptors, CCR5