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Vol. 15, Issue 5, 2456-2469, May 2004
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Department of Veterans Affairs, Nashville, Tennessee 37212-2637;
* Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37212-2175;
Department of Surgery, Vanderbilt University School of Medicine, Nashville, Tennessee 37212-2175; and
# Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37212-2175
Submitted September 29, 2003;
Revised February 17, 2004;
Accepted February 23, 2004
Monitoring Editor: Guido Guidotti
Agonist-stimulated internalization followed by recycling to the cell membrane play an important role in fine-tuning the activity of chemokine receptors. Because the recycling of chemokine receptors is critical for the reestablishment of the cellular responsiveness to ligand, it is crucial to understand the mechanisms underlying the receptor recycling and resensitization. In the present study, we have demonstrated that the chemokine receptor CXCR2 associated with myosin Vb and Rab11-family interacting protein 2 (FIP2) in a ligand-dependent manner. Truncation of the C-terminal domain of the receptor did not affect the association, suggesting that the interactions occur upstream of the C terminus of CXCR2. After ligand stimulation, the internalized CXCR2 colocalized with myosin Vb and Rab11-FIP2 in Rab11a-positive vesicles. The colocalization lasted for 
2 h, and little colocalization was observed after 4 h of ligand stimulation. CXCR2 also colocalized with myosin Vb tail or Rab11-FIP2 (129–512), the N-terminal–truncated mutants of myosin Vb and Rab11-FIP2, respectively, but in a highly condensed manner. Expression of the enhanced green fluorescent protein-tagged myosin Vb tail significantly retarded the recycling and resensitization of CXCR2. CXCR2 recycling was also reduced by the expression Rab11-FIP2 (129–512). Moreover, expression of the myosin Vb tail reduced CXCR2- and CXCR4-mediated chemotaxis. These data indicate that Rab11-FIP2 and myosin Vb regulate CXCR2 recycling and receptor-mediated chemotaxis and that passage of internalized CXCR2 through Rab11a-positive recycling system is critical for physiological response to a chemokine.
Abbreviations used: FACS, fluorescence-activated cell sorting; GPCR, G protein-coupled receptor; HEK, human embryonic kidney; Rab11-FIP2, Rab11-family interacting protein 2; Rab11-FIP1, Rab11-family interacting protein 1; Rab11-FIP3, Rab11-family interacting protein 3; RBL, rat basophilic leukemia.
Corresponding author. E-mail address: ann.richmond{at}vanderbilt.edu.
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