Introduction Chemokine receptors are seven-transmembrane G-proteincoupled receptors (GPCR) implicated in immune and inflammatory responses that regulate crucial processes, such as the activation and migration of leukocytes, development of immune cells (Rossi and Zlotnick, 2000; Rottman, 1999), and angiogenesis (Salcedo and Oppenheim, 2003). The binding of chemokines to their receptors triggers diverse signaling cascades, including activation of G proteins and the PI3-K, Jak/STAT, Rho-p160 ROCK and MAPK pathways (Ganju et al., 1998; Mellado et al., 1998; Vicente-Manzanares et al., 2002; Vicente-Manzanares et al., 1999; Vicente-Manzanares et al., 2003; Wu et al., 1993). The activation of these signaling pathways is often accompanied by the internalization of chemokine receptors and their trafficking back to the plasma membrane. This intracellular turnover determines the leukocyte responsiveness to chemokines (Fan et al., 2004). CXCR4 is a chemokine receptor that plays an important role in homeostasis, cell migration, inflammation, B lymphocyte development and tumor metastasis (Müller et al., 2001; Proudfoot et al., 1999; Wells et al., 1998). It binds to SDF-1 and is one of the most important co-receptors for HIV (Berger et al., 1999; Bleul et al., 1996a; Bleul et al., 1996b). It is well established that both SDF-1 and phorbol esters promote a rapid endocytosis of CXCR4 (Signoret et al., 1997), but only the internalization mediated by SDF-1 uses an arrestindependent pathway (Orsini et al., 1999). Nevertheless, the molecular mechanisms involved in CXCR4 internalization are still poorly understood. It is known that ligand-stimulated chemokine receptor internalization occurs through clathrincoated pits that fuse to early endosomes, in which chemokine receptor is dephosphorylated by protein phosphatase 2A, and delivered to either recycling endosomes or late endosomes/ lysosomes for its degradation, as demonstrated for CXCR2 (Fan et al., 2004). Several mechanisms for the regulation of CXCR4 internalization, both dependent and independent of its phosphorylation, have been described (Fernandis et al., 2002). The C-terminal domain of CXCR4, rich in serine/threonine residues, is crucial for receptor internalization, because these residues are phosphorylated by GPCR kinases (GRKs) (Haribabu et al., 1997). This promotes the binding of arrestins, which induces the dissociation of receptor from G proteins, thus switching off signaling pathways (Orsini et al., 1999). Arrestins also act as adaptors between the receptor and the endocytic machinery, which includes molecules such as clathrin and its adaptor AP-2. Furthermore, ubiquitylation is also involved in the endocytosis and fate of CXCR4 mediated by -arrestin. In this regard, a degradation motif in CXCR4 has been identified, and it is also well established that the proteasome pathway plays a major role in its downmodulation (Fernandis et al., 2002; Marchese et al., 2003). Nonmuscle myosin II is a molecular motor and a member of the myosin superfamily of proteins that comprise at least three distinct isoforms: MIIA, MIIB and MIIC (Simons et al., 1991). MIIA and MIIB are present in all cell types (Simons et Endocytosis of chemokine receptors regulates signal transduction initiated by chemokines, but the molecular mechanisms underlying this process are not fully defined. In this work, we assessed the involvement of the motor protein nonmuscle myosin heavy chain IIA (MIIA) in the endocytosis of CXCR4 induced by SDF-1 (also known as CXCL12) in T lymphocytes. Overexpression of the Cterminal half of MIIA inhibited the ligand-induced endocytosis of CXCR4, but not that of transferrin receptor. Targeting MIIA either by silencing its expression with small interfering RNA (siRNA) or by blebbistatin treatment also inhibited endocytosis of CXCR4. Inhibition of endocytosis of CXCR4 by targeting endogenous MIIA resulted in an increased migration of T cells induced by SDF-1 , and in the inhibition of the HIV-1-Env antifusogenic activity of this chemokine. Coimmunoprecipitation and protein-protein binding studies demonstrated that MIIA interacts with both the cytoplasmic tail of CXCR4 and -arrestin. Moreover, SDF1 promotes a rapid MIIA-arrestin dissociation. Our data reveal a novel role for MIIA in CXCR4 endocytosis, which involves its dynamic association with -arrestin and highlights the role of endogenous MIIA as a regulator of CXCR4 internalization and, therefore, the onset of SDF-1 signaling.