The internalization and intracellular trafficking of chemokine receptors have important implications for the cellular responses elicited by chemokine receptors. The major pathway by which chemokine receptors internalize is the clathrin-mediated pathway, but some receptors may utilize lipid rafts/ caveolae-dependent internalization routes. This review discusses the current knowledge and controversies regarding these two different routes of endocytosis. The functional consequences of internalization and the regulation of chemokine receptor recycling will also be addressed. Modifications of chemokine receptors, such as palmitoylation, ubiquitination, glycosylation, and sulfation, may also impact trafficking, chemotaxis and signaling. Finally, this review will cover the internalization and trafficking of viral and decoy chemokine receptors.

The homing of proinflammatory (M1) and the "alternatively activated" anti-inflammatory (M2) macrophages plays a different role in the process of inflammation. Chemokines are the major mediators of macrophage chemotaxis, but how they differentially regulate M1 and M2 macrophages remains largely unclear. In the present study, we attempted to screen chemokines that differentially induce chemotaxis of M1 and M2 macrophages and to explore the underlying mechanism. Among the 41 chemokines that specifically bind to 20 chemokine receptors, CCL19, CCL21, CCL24, CCL25, CXCL8, CXCL10, and XCL2 specifically induced M1 macrophage chemotaxis, whereas CCL7 induced chemotaxis of both M1 and M2 macrophages. Whereas the differential effects of these chemokines on M1/M2 macrophage chemotaxis could be attributable to the predominant expression of their cognate receptors on the macrophage subsets, CCR7, the receptor for CCL19/CCL21, appeared to be an exception. Immunoblot analysis indicated an equivalent level of CCR7 in the whole cell lysate of M1 and M2 macrophages, but CCL19 and CCL21 only induced M1 macrophage chemotaxis. Both immunoblot and confocal microscopy analyses demonstrated that CCR7 was predominantly expressed on the cell surface of M1 but in the cytosol of M2 macrophages before ligand stimulation. As a result, CCL19 or CCL21 induced activation of both MEK1-ERK1/2 and PI3K-AKT cascades in M1 but not in M2 macrophages. Intriguingly, CCL19/CCL21-mediated M1 macrophage chemotaxis was blocked by specific inhibition of PI3K rather than MEK1. Together, these findings suggest that recruitment of M1 and M2 macrophages is fine tuned by different chemokines with the involvement of specific signaling pathways.

Agonist treatment of cells expressing the chemokine receptor, CXCR2, induces receptor phosphorylation and internalization through a dynamin-dependent mechanism. In the present study, we demonstrate that a carboxyl terminus-truncated mutant of CXCR2 (331T), which no longer undergoes agonist-induced phosphorylation, continues to undergo ligand-induced internalization in HEK293 cells. This mutant receptor exhibits reduced association with β-arrestin 1 but continues to exhibit association with adaptin 2 α and β subunits. Replacing Leu320-321 and/or Ile323-Leu324 with Ala (LL320,321AA, IL323,324AA, and LLIL320,321,323,324AAAA) in wild-type CXCR2 or 331T causes little change in ligand binding and signaling through Ca 2+ mobilization but greatly impairs the agonist-induced receptor sequestration and ligand-mediated chemotaxis. The LL320,321AA, IL323,324AA, and LLIL320,321,323,324AAAA mutants of CXCR2 exhibit normal binding to β-arrestin 1 but exhibit decreased binding to adaptin 2α and β. These data demonstrate a role for the LLKIL motif in the carboxyl terminus of CXCR2 in receptor internalization and cell chemotaxis and imply a role for adaptin 2 in the endocytosis of CXCR2.The chemokine receptor, CXCR2, 1 is a member of a superfamily of G protein-coupled seventransmembrane receptors (GPCRs) that transduce intracellular signals via heterotrimeric guanine nucleotide-binding proteins (G proteins). Upon stimulation by agonists, such as interleukin 8 (IL-8) or melanoma growth-stimulatory activity (MGSA)/growth-regulatory protein (GRO), CXCR2 activates a series of G protein-mediated events, including phosphatidylinositide hydrolysis, to generate inositol 1,4,5-trisphosphate and diacylglycerol, as well as mobilization of intracellular free Ca 2+ to initiate a series of cellular responses (1). In addition, CXCR2 mediates cell chemotaxis, a distinct function of chemokine receptors (2). Like many other types of GPCRs, CXCR2 undergoes a dynamic trafficking between the cell surface and the intracellular compartments (3). Such trafficking may be involved in both transmission and termination of the receptor signals and may play an important role in mediating cell chemotaxis. For CXCR2, the most remarkable trafficking process is agonistinduced receptor internalization. 1 Abbreviations: CXCR2, receptor for CXC chemokines formerly defined as interleukin 8 receptor B; IL-8, interleukin 8; MGSA/GRO, melanoma growth-stimulatory activity/growth-regulatory protein; GPCRs, G protein-coupled receptors; G proteins, guanine nucleotidebinding proteins; β2-AR, β2-adrenergic receptor; AP-2, adaptin 2; HEK293 cells, human embryonic kidney 293 cells; RBL-2H3 cells, rabbit basophilic leukemia cells; DSP, dithiobis(succinimido propionate); GRKs, G protein-coupled receptor kinases; FITC, fluorescein isothiocyanate; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PBS, phosphatebuffered saline; SDS, sodium dodecyl sulfate. Agonist-induced phosphorylation of the carboxyl terminus by G protein-couple...

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 ...

Intracellular trafficking of chemokine receptors plays an important role in finetuning the functional responses of neutrophils and lymphocytes in the inflammatory process and HIV infection. Although many chemokine receptors internalize through clathrin-coated pits, regulation of the receptor trafficking is not fully understood. The present study demonstrated that CXCR2 was colocalized with transferrin and low-density lipoprotein (LDL) after agonist treatment for different periods of time, suggesting 2 intracellular trafficking pathways for this receptor. CXCR2 was colocalized with Rab5 and Rab11a, which are localized in early and recycling endosomes, respectively, in response to agonist stimulation for a short period of time, suggesting a recycling pathway for the receptor trafficking. However, overexpression of a dominant-negative Rab5-S34N mutant significantly attenuated CXCR2 sequestration. The internalized CXCR2 was recycled back to the cell surface after removal of the agonist and recovery of the cells, but receptor recycling was inhibited by overexpression of a dominant-negative Rab11a-S25N mutant. After prolonged (4-hour) agonist treatment, CXCR2 exhibited significantly increased colocalization with Rab7, which is localized in late endosomes. The colocalization of CXCR2 with LDL and LAMP-1 suggests that CXCR2 is targeted to lysosomes for degradation after prolonged ligand treatment. However, the colocalization of CXCR2 with Lamp1 was blocked by the overexpression of a dominantnegative Rab7-T22N mutant. In cells overexpressing Rab7-T22N, CXCR2 was retained in the Rab5-and Rab11a-positive endosomes after prolonged (4-hour) agonist treatment. Our data suggest that the intracellular trafficking of CXCR2 is differentially regulated by Rab proteins. IntroductionChemokines are a family of small proteins that regulate leukocyte trafficking and are therefore important in lymphoid organogenesis, inflammation, and host defense. Based on the presence of the conserved cysteines near the N-terminus of chemokines, chemokines can be classified into CC, CXC, CX3C, and C subfamilies. [1][2][3][4] The effects of chemokines on target cells are mediated by a family of G-protein-coupled 7-transmembrane receptors (GPCRs). Chemokine receptors are expressed predominantly in neutrophils and lymphocytes. To date, 19 chemokine receptors have been identified, including 11 CC chemokine receptors (CCR1-11), 6 CXC chemokine receptors (CXCR1-6), and one for each C and CX3C chemokine receptor. 4 These receptors play important roles in inflammatory response, chemotaxis, immune cell development, leukocyte homing, and angiogenesis. 5,6 Some of the chemokine receptors (eg, CCR5 and CXCR4) act as coreceptors in HIV infection. 7 Agonist occupancy of chemokine receptors, like other types of GPCRs, activates the coupled heterotrimeric G proteins that mobilize intracellular second messengers such as inositol triphosphates, cyclic adenosine monophosphate, and intracellular free calcium. 8 The receptors are rapidly phosphorylated by G-proteincouple...

Chemokine receptor-initiated signaling plays critical roles in cell differentiation, proliferation, and migration. However, the regulation of chemokine receptor signaling under physiological and pathological conditions is not fully understood. In the present study, we demonstrate that the CXC chemokine receptor 4 (CXCR4) formed a complex with ferritin heavy chain (FHC) in a ligand-dependent manner. Our in vitro binding assays revealed that purified FHC associated with both the glutathione S-transferase-conjugated N-terminal and C-terminal domains of CXCR4, thereby suggesting the presence of more than one FHC binding site in the protein sequence of CXCR4. Using confocal microscopy, we observed that stimulation with CXCL12, the receptor ligand, induced colocalization of the internalized CXCR4 with FHC into internal vesicles. Furthermore, after CXCL12 treatment, FHC underwent time-dependent nuclear translocation and phosphorylation at serine residues. By contrast, a mutant form of FHC in which serine 178 was replaced by alanine (S178A) failed to undergo phosphorylation, suggesting that serine 178 is the major phosphorylation site. Compared with the wild type FHC, the FHC-S178A mutant exhibited reduced association with CXCR4 and constitutive nuclear translocation. We also found that CXCR4-mediated extracellular signal-regulated kinase 1/2 (ERK1/2) activation and chemotaxis were inhibited by overexpression of wild type FHC but not FHC-S178A mutant, and were prolonged by FHC knockdown. In addition to CXCR4, other chemokine receptor-initiated signaling appeared to be similarly regulated by FHC, because CXCR2-mediated ERK1/2 activation was also inhibited by FHC overexpression and prolonged by FHC knockdown. Altogether, our data provide strong evidence for an important role of FHC in chemokine receptor signaling and receptor-mediated cell migration.Chemokines comprise a large family of low molecular mass (8 -10 kDa) proteins with chemotactic and proactivatory effects on different leukocyte lineages. Approximately 40 chemokines have been identified and several studies have established their central role in a number of physiological situations, including T helper cell responses, hematopoiesis, homeostasis, and angiogenesis (1). Chemokines mediate their biological effects by binding to specific seven-transmembrane domain G protein-coupled receptor subtypes, designated CXCR1 through CXCR6, CCR1 through CCR11, XCR1, and CX3CR1, based on their specific preference for certain chemokines (2, 3). Among these chemokine receptors, CXCR4 has received a great deal of attention because this receptor subtype plays a role not only in leukocyte homing but also in human immunodeficiency virus infection, development of the immune and central nervous systems, and cancer metastasis (4 -9). Its ligand, CXCL12, which also binds to RDC1 that is proposed to rename as CXCR7 (10), has been found to induce migration of peripheral blood lymphocytes (11), CD34ϩ progenitor cells (4), and pre-and pro-B cell lines (12). Mice embryos lacking either the C...

Learning and memory have been suggested to be important in the development of opiate addiction. Based on the recent findings that calcium/calmodulin-dependent protein kinase II (CaMKII) is essential in learning and memory processes, and morphine treatment increases CaMKII activity in hippocampus, the present study was undertaken to examine whether inhibition of hippocampal CaMKII prevents morphine tolerance and dependence. Here, we report that inhibition of CaMKII by intrahippocampal dentate gyrus administration of the specific inhibitors KN-62 and KN-93 to rats significantly attenuated the tolerance to the analgesic effect of morphine and the abstinence syndrome precipitated by opiate antagonist naloxone. In contrast, both KN-04 and KN-92, the inactive structural analogs of KN-62 and KN-93, failed to attenuate morphine tolerance and dependence, indicating that the observed effects of KN-62 and KN-93 are mediated through inhibition of CaMKII. Furthermore, administration of CaMKII antisense oligonucleotide into rat hippocampal dentate gyrus, which decreased the expression of CaMKII specifically, also attenuated morphine tolerance and dependence, while the corresponding sense oligonucleotide of CaMKII did not exhibit such inhibitory effect. Moreover, the KN-62 treatment abolished the rewarding properties of morphine as measured by the conditioned place preference. These results suggest that hippocampal CaMKII is critically involved in the development of morphine tolerance and dependence, and inhibition of this kinase may have some therapeutic benefit in the treatment of opiate tolerance and dependence.

Signals that induce phosphorylation of IB proteins can also cause phosphorylation of NF-B proteins. For example, tumor necrosis factor-␣ induces phosphorylation of RelA at serine 536 by IKK (18) and at serine 529 through casein kinase II (19). In vitro studies have suggested that phosphorylation of NF-B transcription factors, including RelA and p50, enhances DNA binding ability (20,21). In vivo, the inducible phosphorylation on NF-B is correlated with dimerization, release from IB, nuclear translocation, or activation of the transcription function of 22). Inhibition of interleukin-1-stimulated RelA phosphorylation by mesalamine is accompanied by decreased transcriptional activity of NF-B (23).The phosphorylation of NF-B transcription factors is regulated not only by protein serine/threonine kinases, but also by protein serine/threonine phosphatases. Compared with the well established mechanisms for the phosphorylation of NF-B transcription factors, the mechanisms underlying the dephosphorylation of these transcription factors are still poorly understood. Four major classes of protein phosphatases have been described, include PP1, PP2A, PP2B (calcineurin), and PP2C. PP2B is calcium-dependent, and PP2C is magnesium-dependent, whereas PP1 and PP2A are not dependent upon divalent cations. PP1 and PP2A are widely expressed in mammalian cells and are involved in the regulation of signaling pathways by a mechanism of phosphorylation/dephosphorylation with a variety of protein kinases (24). The predominant form of PP2A in cells is a heterotrimeric holoenzyme. The core components (core enzyme) of all trimeric forms are the 36-kDa catalytic subunit (PP2Ac) and a 65-kDa regulatory subunit (PP2Aa or PR65). In addition, there are several associated variable regulatory subunits (B subunits) that bind to the core enzyme and confer substrate specificity to its dephosphorylating activity (24). PP2A has been shown to form a complex with calcium/ calmodulin-dependent protein kinase IV (25); casein kinase (26); p21-activated kinase-1 and -3 and p70 S6 kinase (27); and certain G-protein-coupled receptors such as the ␤ 2 -adrenergic receptor and CXCR2 (28, 29). However, a previous study demonstrated that the SV40 small t antigen, which associates with