Actin Filaments Are Involved in the Regulation of Trafficking of Two Closely Related Chemokine Receptors, CXCR1 and CXCR2

Abstract

The ligand-induced internalization and recycling of chemokine receptors play a significant role in their regulation. In this study, we analyzed the involvement of actin filaments and of microtubules in the control of ligand-induced internalization and recycling of CXC chemokine receptor (CXCR)1 and CXCR2, two closely related G protein-coupled receptors that mediate ELR-expressing CXC chemokine-induced cellular responses. Nocodazole, a microtubule-disrupting agent, did not affect the IL-8-induced reduction in cell surface expression of CXCR1 and CXCR2, nor did it affect the recycling of these receptors following ligand removal and cell recovery at 37°C. In contrast, cytochalasin D, an actin filament depolymerizing agent, promoted the IL-8-induced reduction in cell surface expression of both CXCR1 and CXCR2. Cytochalasin D significantly inhibited the recycling of both CXCR1 and CXCR2 following IL-8-induced internalization, the inhibition being more pronounced for CXCR2 than for CXCR1. Potent inhibition of recycling was observed also when internalization of CXCR2 was induced by another ELR-expressing CXC chemokine, granulocyte chemotactic protein-2. By the use of carboxyl terminus-truncated CXCR1 and CXCR2 it was observed that the carboxyl terminus domains of CXCR1 and CXCR2 were partially involved in the regulation of the actin-mediated process of receptor recycling. The cytochalasin D-mediated inhibition of CXCR2 recycling had a functional relevance because it impaired the ability of CXCR2-expressing cells to mediate cellular responses. These results suggest that actin filaments, but not microtubules, are involved in the regulation of the intracellular trafficking of CXCR1 and CXCR2, and that actin filaments may be required to enable cellular resensitization following a desensitized refractory period.

Inhibitory effects of cytoskeleton disrupting drugs and GDP-locked Rab mutants on bradykinin B2 receptor cycling

“…5). The alternate tubulin ligand nocodazole failed to inhibit the interleukin-8-induced endocytosis of CXCR1 and -2 in another study [9], consistent with current findings, that therefore limit the role of microtubules in GPCR cycling to intracellular progression. Overexpression of the dominant negative (GDP-locked) version of Rab 5 fully reproduced the effect of tubulin ligands on B 2 R-GFP cycling (Fig.…”

“…The actin cytoskeleton is currently considered as the substrate for this form of vesicular translocation [6]. Thus, cytochalasin D inhibited the recycling of agoniststimulated CXCR1 and -1B -adrenoceptors [9,23]. Cytoskeletal elements that support Rab4-mediated cargo translocation are not defined as well as those related to Rab11 or Rab5.…”

“…On the other hand, receptors sorted for recycling could travel along with Rab11 (as shown for the cannabinoid CB 2 receptor 2 -adrenoceptors) [7,8] along the actin cytoskeleton [6]. Thus, the rapid recycling of the interleukin-8 receptors CXCR1 and CXCR2 2 -adrenoceptors are inhibited by cytochalasin D, an actin filament-disrupting drug [8,9]. Rab4 has also a role in the recycling of internalized cargo to the cell surface (tra 2 -adrenoceptor) [10,11].…”

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

“…Vesicle recycling of chemokine receptors, including CXCR2, has been implicated in chemotaxis (Perez et al, 1986(Perez et al, , 1987(Perez et al, , 1989Zaslaver et al, 2001). In this study, we demonstrate that expression of a C-terminal fragment of myosin Vb (myosin Vb tail) does not affect the random cell migration (our unpublished data) but significantly reduces the chemotactic response to CXCL1 of the CXCR2-expressing cells, suggesting the specific involvement of myosin Vb in the chemokine receptor-mediated chemotaxis.…”

“…The dephosphorylated receptors are either delivered to recycling endosomes or to late endosomes/lysosomes for degradation, depending upon the presence of the extracellular ligands (Fan et al, 2003). The recycling and degradation rate may vary among different chemokine receptors (Chuntharapai and Kim, 1995;Zaslaver et al, 2001). Although mechanisms underlying ligand-induced receptor endocytosis have been extensively studied, the mechanisms for the recycling of chemokine receptors are still poorly understood.…”