Rab11-Family Interacting Protein 2 and Myosin Vb Are Required for CXCR2 Recycling and Receptor-mediated Chemotaxis

Abstract

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 ...

On the move: endocytic trafficking in cell migration

“…Noteworthy, overexpressing a dominant negative mutant of Rab11 or truncation mutants of Rab11-family interacting protein 2 (Rab11-FIP2) and myosin Vb significantly impairs CXCR2 recycling and, more importantly, hampers its re-sensitization, consequently leading to a less efficient migration towards its ligand in vitro [22]. Moreover, a CXCR2 mutant lacking the C-terminal LLKIL motif responsible for binding to AP-2 partially loses its internalization capacity upon chemokine binding and shows reduced migration towards CXCL8 [23].…”

Powering membrane traffic in endocytosis and recycling

“…2). Strong evidence from various cell types links myosin Vb with the recycling of ATPbinding cassette (ABC) transporters 107 and the receptors for transferrin 108 , chemokines 109 and acetylcholine 110 . In all cases, motor activity requires RAB11, as recently supported by a permeabilized cell-transport assay 111 and the RAB11-family-interacting protein-2 (FIP2) 112 .…”

“…In all cases, motor activity requires RAB11, as recently supported by a permeabilized cell-transport assay 111 and the RAB11-family-interacting protein-2 (FIP2) 112 . For example, the chemokine receptor CXCR2 interacts with both myosin Vb and the RAB11-FIP2 protein in RAB11a-positive vesicles in a manner that is ligand-dependent, and myosin Vb seems important for receptor recycling to the surface 109 . The same complex of myosin Vb and FIP2 might ensure correct inheritance of RAB11a recycling endosomes during cell division 113 .…”

Mechanoenzymatic Characterization of Human Myosin Vb

“…The overexpression of the myosin Vb tail retarded the transferrin recycling and caused concentration of transferrin receptors in pericentrosomal vesicles in HeLa cells (24). Myosin Vb also regulates the recycling of chemokine receptor CXCR2 (25) and the M4 muscarinic acetylcholine receptor (26). Moreover, the inhibition of myosin Vb activity attenuates the accumulation of transferrin in the cytoplasm and increases the level of the plasma membrane transferrin receptor (27).…”