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 ϳ2 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.
INTRODUCTIONChemokine receptors belong to the large family of seventransmembrane G protein-coupled receptors (GPCRs) that function in immune and inflammatory response by regulating the activation and migration of leukocytes, immune cell development, and angiogenesis (Nagasawa et al., 1996;Luster 1998;Belperio et al., 2000;Murphy et al., 2000;Zlotnik and Yoshie, 2000). Some of them (e.g., CCR5 and CXCR4) participate in HIV infection of CD4 ϩ T lymphocytes as coreceptors (Berger et al., 1999). Ligand binding to the chemokine receptors triggers various signaling cascades, including activation of G proteins, phosphotidylinositol 3-kinase, Janus kinase/signal transducers and activators of transcription proteins, the Rho-p160 ROCK axis, and the MAPK pathway (Wu et al., 1993;Ganju et al., 1998;Mellado et al., 1998;Vicente-Manzanares et al., 1999;Vicente-Manzanares et al., 2002). Chemokine activation of these intracellular signals is often accompanied by chemokine receptor internalization and trafficking back to the cell membrane. The intracellular trafficking of chemokine receptors controls their activities, and the balance between the chemokine receptor recycling and degradation dictates the leukocyte responsiveness to chemokines (Sabroe et al., 1997;Asagoe et al., 1998;Khandaker et al., 1998;Mack et al., 1998).Ligand stimulated chemokine receptor internalization can be accomplished ...