Stimulation Upon Stromal Cell-Derived Factor-1 Vesicles +

Gα13 and Rho Mediate Endosomal Trafficking of CXCR4 into Rab11 + Vesicles upon Stromal Cell-Derived Factor-1 Stimulation This information is current as of September 29, 2021. Ashok Kumar, Kimberly N. Kremer, Daniel Dominguez, Madhavi Tadi and Karen E. Hedin J Immunol 2011; 186:951-958; Prepublished online 8 December 2010; doi: 10.4049/jimmunol.1002019 Downloaded from http://www.jimmunol.org/content/186/2/951

References This article cites 45 articles, 22 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/186/2/951.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Ga13 and Rho Mediate Endosomal Trafﬁcking of CXCR4 into Rab11+ Vesicles upon Stromal Cell-Derived Factor-1 Stimulation

Ashok Kumar,1 Kimberly N. Kremer, Daniel Dominguez,2 Madhavi Tadi,3 and Karen E. Hedin

CXCR4, like other G protein-coupled receptors, signals via heterotrimeric guanine nucleotide-binding proteins (G proteins) to regulate gene transcription, migration, development, growth, and transformation. We describe a formerly uncharacterized function of a G protein: a role in receptor trafﬁcking. We previously showed that CXCR4 and the TCR physically associate and form a heterodimer upon stromal cell-derived factor-1 or CXCL12 (SDF-1) stimulation in human T cells to prolong ERK activation and,

thereby, lead to gene upregulation and cytokine secretion. The CXCR4–TCR heterodimers occur on the cell surface and in an Downloaded from intracellular compartment in response to SDF-1. Neither the intracellular compartment to which the CXCR4–TCR heterodimers localize nor the mechanism for localization has been elucidated. In this article, we characterize molecular mechanisms required for postendocytic trafficking of CXCR4. Upon SDF-1 stimulation, CXCR4 localizes to Rab11+ vesicles, a recycling compartment near the microtubule organizing center and Golgi apparatus. This trafficking requires the CXCR4 C-terminal tail domain but not the CXCR4 ubiquitination sites. The TCR also constitutively localizes to this Rab11+ compartment. Trafficking of CXCR4 into the + Rab11 , TCR-containing endosomes requires actin polymerization. Furthermore, inhibiting Rho activation or depleting Ga13 http://www.jimmunol.org/ prevented trafficking of CXCR4 into the Rab11+ endosomes without hindering the ability of CXCR4 to endocytose. These results indicated that, upon SDF-1 treatment, Ga13 and Rho mediate the actin polymerization necessary for trafficking CXCR4 into the Rab11+, recycling endosomal compartment, which also contains constitutively recycling TCR and, thus, CXCR4–TCR heterodimers. To our knowledge, this is the first report of Ga13 as a mediator of receptor trafficking. The Journal of Immunology, 2011, 186: 951–958.

XCR4 is a ubiquitously expressed G protein-coupled intracellular trafficking, and recycling, as well as gene expression. receptor (GPCR), a chemokine receptor whose sole li- CXCR4 endocytosis, upon ligand binding, regulates its signal by guest on September 29, 2021 C gand is the chemokine, stromal cell-derived factor-1 transduction, inhibits HIV-1 infection, and may be responsible for (SDF-1). CXCR4 signals to regulate T cell development (1), mi- the abnormal immunity of patients with WHIM syndrome ex- gration (2), and cytokine gene expression and secretion, and it pressing truncated CXCR4 (6, 7). Many tumor cells display ab- costimulates T lymphocyte immune activation (2–5). CXCR4 me- normally elevated cell-surface CXCR4 that contributes to metastasis diates HIV-1 infection (6) and forms a complex with the T lym- and proliferation (6). Therefore, understanding the molecular phocyte Ag receptor (TCR) in response to SDF-1 (3). CXCR4 mechanisms that CXCR4 uses to regulate its endocytic trafficking also regulates the functions of other immune and nonimmune cells and cell-surface levels is critical to understanding the physiological and enhances the proliferation and/or metastasis of many tumor and disease functions of CXCR4. types (6). All functions of CXCR4 critically depend on its cell- Two relatively well-studied processes that regulate the levels surface expression, which is regulated by CXCR4 endocytosis, of cell-surface CXCR4 include ligand-dependent receptor endocytosis and the degradation of endocytosed receptors. CXCR4 in- Department of Immunology, Mayo Clinic College of Medicine, Mayo Clinic, Ro- ternalization requires phosphorylation of the CXCR4 C-terminal chester, MN 55905 tail domain, which provokes endocytosis via b-arrestin (6, 8). In 1Current address: KARD Scientific, Beverly, MA. some cell types, CXCR4 internalization also depends on Hip (9), 2Current address: Department of Pharmacology, University of North Carolina School myosin IIA (10), cortactin (11), Rab5 (12, 13), and/or the dileucine of Medicine, Chapel Hill, NC. motif within the C-terminal tail domain of CXCR4 (14). The C- 3Current address: Memorial Sloan-Kettering Cancer Center, New York, NY. terminal tail domain also mediates the degradation of endocytosed Received for publication June 17, 2010. Accepted for publication November 6, 2010. CXCR4, via its ubiquitination at three sites together with AIP4, This work was supported in part by the Joanne G. and Gary N. Owen Fund in b-arrestin 2 (15–17), Hrs, Vps4 (14), and the ESCRT machinery Immunology Research, the Alma B. Stevenson Endowment Fund for Medical Re- (18). Much less is understood about the molecular signaling mech- search, and by National Institutes of Heath RO1 Grant GM59763 (to K.E.H.). anisms responsible for regulating CXCR4 trafficking into non- Address correspondence and reprint requests to Dr. Karen E. Hedin, Mayo Clinic, lysosomal compartments including recycling endosomes. CXCR4 Guggenheim Building, Third Floor, 200 First Street Southwest, Rochester, MN + 55905. E-mail address: [email protected] localizes to early and recycling endosomes in CD34 cells (19), and Abbreviations used in this article: EGF, epidermal growth factor; GPCR, G protein- CXCR4 recycling involves cortactin in HEK293 cells (11) and coupled receptor; MTOC, microtubule organizing center; PO, propylene oxide; PIM1 in T cells (20). Several Rho GTPases that regulate actin RhoGEF, Rho guanine nucleotide exchange factor; SDF-1, stromal cell-derived factor-1; polymerization are localized to endosomes. Rho B delays the shRNA, short hairpin RNA. trafficking of the epidermal growth factor (EGF) receptor to late Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 endosomes (21) and enhances the lysosomal trafficking of CXCR2, www.jimmunol.org/cgi/doi/10.4049/jimmunol.1002019 952 Ga13-RHO–ACTIN–SIGNALING PATHWAY MEDIATES CXCR4 TRAFFICKING which is required for the migration of HEK293 cells toward skin gelatin, and 0.04% sodium azide (pH 7.2) and then stained with CXCR2 ligands (22). Although the roles of Rho GTPases in me- CXCR4 mAb (MAB173; R&D Systems), followed by goat anti-mouse Fc- diating postendocytic CXCR4 trafficking have not been charac- FITC (Jackson ImmunoResearch, West Grove, PA), with or without Hoechst dye #33342 (Invitrogen, San Diego, CA). terized, CXCR4 activates Rho in response to SDF-1 treatment, and this is required for migration (23, 24). Transient transfections and plasmids CXCR4 signaling activates heterotrimeric GTP-binding G pro- Transient transfections were performed as described (3); transfection effi- teins composed of a and bg subunits, including members of the ciencies were 60–70% for Jurkat T cells and 25–50% for PBMC T cells. Gi, Gq, and G12/13 subfamilies (23, 25). Although Ga13 has not Plasmids encoding fluorescent fusion proteins of CXCR4 (CXCR4-YFP, been shown to regulate receptor trafficking, it is clear that it has CXCR4-RFP) and TCR-CD3-z (TCR-CD3-z-YFP) are described in Ref. 3. Plasmids encoding mutant CXCR4 fluorescent fusion proteins CXCR4-Dc- many critical physiological roles. Ga13-deficient mice die in utero ter (truncated after CXCR4 aa residue 321) and CXCR4-KKK/AAA (alanine (26, 27). Mouse T lymphocytes doubly deficient in Ga12 and instead of lysine at CXCR4 aa residues 327, 331, and 333), were created via Ga13, but not cells only deficient in Ga12, provoke lymphade- site-directed mutagenesis of CXCR4-WT-YFP. Plasmids encoding Rab11- nopathy and increased susceptibility to T cell-mediated diseases RFP and GalT-CFP were gifts of R. Pagano (Mayo Clinic), Centrin-2–GFP (28). GTP-bound Ga12 and Ga13 stimulate Rho guanine nucleo- was a gift from J. Salisbury (Mayo Clinic), and RhoN19 was a gift from D. Billadeau (Mayo Clinic). The Ga13 short hairpin RNA (shRNA)-encoding tide exchange factors (RhoGEFs), thereby activating Rho GTPases plasmid was made by annealing and ligating the DNA oligonucleotides 59- (29). Ga13-mediated Rho activation is required for cell migration GATCCCCGAAGATCGACTGACCAATCTTCAAGAGAGATTGGTC- and shape changes (23, 26, 29). Ga13 also signals via other ef- AGTCGATCTTCTTTTTGGAA-39 and 59-AGCTTTTCCAAAAAGAA- fectors (29) and integrins (30). GATCGACTGACCAATCTCTCTTGAAATTGGTCAGTCGATCTTCG- GG-39 into pCMS3.H1p.eGFP (31) or pCMS4.H1p.mCherry (gift from D. We previously described a CXCR4–TCR heterodimer that forms Billadeau), which is a derivative of pCMS4.H1p.eGFP (31) and encodes Downloaded from in response to SDF-1 treatment of T cells and that mediates the mCherry instead of eGFP driven by a separate promoter. Ga13 protein levels distinctly prolonged ERK MAPK activation that is necessary for were determined by immunoblotting with anti-Ga13 (Santa Cruz Biotech- gene transcription and cytokine production in response to SDF-1 nology, Santa Cruz, CA) and actin (Novus Biologicals, Littleton, CO). (3, 4). Interestingly, CXCR4–TCR heterodimers can be detected on Confocal imaging of fluorescent fusion proteins transiently the cell surface and intracellularly, suggesting that postendocytic expressed in live cells receptor trafficking may influence the functions of this heterodimer. http://www.jimmunol.org/ However, neither the intracellular compartment to which the Jurkat or PBMC T cells were transiently transfected with plasmids and analyzed 16–24 h later, with the exception of the Ga13-shRNA-plasmid– CXCR4–TCR heterodimer localizes nor the mechanism respon- transfected cells, which were analyzed after 48 h. Cells were immobilized sible for CXCR4 localization to this compartment has been de- on 0.17 mm D T dishes (Bioptechs, Butler, PA) coated with 20 mg/ml scribed. In this study, we investigated the molecular mechanisms human fibronectin (BD Biosciences, San Jose, CA). The same, live cells 3 that regulate the postendocytic trafficking of CXCR4 in response were imaged using a Plan-Apochromat 100/1.4 NA oil-immersion ob- jective with or without SDF-1 for 15 and 30 min on a 37˚C stage. CFP and to SDF-1 stimulation. Our results define the subcellular compart- YFP were visualized using an LSM 5 Live laser scanning confocal mi- ment that contains CXCR4–TCR heterodimers and describe a new croscope (Carl Zeiss, Oberkochen, Germany) with laser/emission filters of role for G13 in regulating CXCR4 endosomal trafficking. 440/455–525 nm for CFP and 532/560–675 nm for YFP. YFP and RFP

were visualized using an LSM 510 laser scanning confocal microscope by guest on September 29, 2021 (Carl Zeiss) with laser/emission filters of 488/505–550 nm for YFP and Materials and Methods 543/560–615 or 568/585–615 nm for RFP or mCherry. Three-dimensional Cells quantitation of confocal images was performed using KS400 Image Analysis Software (Carl Zeiss). The analysis region used for quantifying Human PBMC T cells were isolated from the peripheral blood platelet- the trafficking of CXCR4 into clustered endosomes was defined as a circle pheresis residue of healthy volunteers using RosetteSep T cell-enrichment of radius r/4, where r is the radius of a circle with an area equivalent to the mixture (Stem Cell Technologies). Blood was obtained and used with maximal cross-sectional area of the cell. informed consent and approval by the Mayo Institutional Review Board. Jurkat and PBMC T cells were maintained in Medium A (RPMI 1640 Electron microscopy supplemented with 10% FCS, 10 mM HEPES [pH 7.4], 2 mM L-glutamine, and 2 mM 2-ME) at ,106 cells/ml. Cells were treated with SDF-1 for 20 min, washed in PBS, fixed in Trump’s fixative (4% paraformaldehyde, 1% glutaraldehyde), and resuspended in Stimulation conditions, reagents, flow-cytometric measurement 2% agar. Specimens were incubated for 30 min in PBS, 60 min in 1% of CXCR4 cell-surface levels and recycling, and statistical osmic acid, 30 min in H2O, 30 min in 2% uranyl acetate at 55˚C, 10 min analysis each in 60, 70, 80, 95, and 100% EtOH, 20 min in propylene oxide (PO), 60 min in PO with 50% Spurr’s resin (32), 60 min in PO with 25% Spurr’s Stimulations were performed at 37˚C with 5 3 1028 M SDF-1a (R&D resin, and 16 h in 100% Spurr’s resin. Specimens were embedded and Systems, Minneapolis, MN). Where indicated, cells were pretreated for 60 allowed to polymerize for 16 h at 60˚C. Sections (0.1 mm) were stained min with 20 mM cytochalasin D (Calbiochem, San Diego, CA) or vehicle with 2% uranyl acetate/lead citrate and visualized using a Technai G212 (DMSO). CXCR4 mAb conjugated to PE or allophycocyanin was used for Transmission Electron Microscope (FEI, Hillsboro, OR). Where indicated, flow cytometry (R&D Systems). To assay CXCR4 surface expression, endogenous cell-surface CXCR4 on live Jurkat cells was labeled with Ab Jurkat cells were transfected or pretreated as indicated and incubated with to CXCR4 (R&D Systems), followed by immunogold-goat-anti-mouse or without SDF-1 for 20 min to induce CXCR4 endocytosis. To assess IgG + IgM (GE Healthcare, Piscataway, NJ). Cells were treated with ve- CXCR4 recycling back to the cell surface, we used the method described hicle or SDF-1 for 30 min at 37˚C and then fixed and processed as above. previously (20), which allows for sufficient time for recycling but not gene transcription of CXCR4. Cells were treated with SDF-1 for 20 min, Rho-activation assay washed three times with PBS, and stained immediately for CXCR4 or Levels of active, GTP-bound Rho were compared using a kit, according to incubated for 1 h in culture media and then stained for CXCR4 sur- the manufacturer’s instructions (Millipore, Billerica, MA). face levels. Statistical analysis was performed via the two-tailed t test (Microsoft Excel). The means of two distributions were considered significantly different at p , 0.05. Results CXCR4 endocytosis and trafficking into a clustered endosomal Immunohistochemistry compartment in response to SDF-1 require the CXCR4 PBMC T cells on cover slips coated with poly-L-lysine (Sigma, St. Louis, C-terminal tail domain but not the CXCR4 ubiquitination sites MO) were stimulated with SDF-1 and then fixed by incubation in 5% paraformaldehyde/PBS, followed by 0.2% Tween-20/PBS. Fixed cells We previously detected CXCR4–TCR heterodimers on the cell were blocked in 5% normal goat serum, 1% glycerol, 0.1% BSA, 0.1% fish surface and in intracellular vesicles following SDF-1 stimulation. The Journal of Immunology 953

To determine the type and location of the CXCR4–TCR–con- nor the intracellular levels (Fig. 1D; p . 0.05) of CXCR4-Dc-ter- taining vesicles, as well as the signals that stimulate CXCR4 YFP changed detectably in response to SDF-1 stimulation. These trafficking into these vesicles, we first analyzed the endocytosis results are consistent with the results of previous studies that showed and intracellular trafficking of endogenous CXCR4 in normal that CXCR4 with a C-terminal tail truncation does not internalize human peripheral blood T cells (PBMC T cells). PBMC T cells in response to SDF-1 (6), whereas CXCR4-KKK/AAA does (33). were treated with SDF-1, and CXCR4 was detected by immuno- Therefore, we used three-dimensional confocal image quantitation histochemistry. As expected, SDF-1 treatment induced CXCR4 to assay CXCR4-YFP construct trafficking into the clustered endocytosis from the plasma membrane (Fig. 1A). Interestingly, endosome compartment. Our results showed that SDF-1 treatment the endocytosed CXCR4 of SDF-1–treated cells appeared within significantly increased the trafficking of CXCR4-WT-YFP and endosomes that were remarkably tightly clustered. Nuclear CXCR4-KKK/AAA-YFP into clustered endosomes. More than staining with Hoechst dye showed that the clustered CXCR4- 24% of total cellular CXCR4-WT-YFP and CXCR4-KKK/AAA- containing endosomes were not located within the nucleus. Sim- YFP (equivalent to .50% of all intracellular CXCR4-YFP) local- ilar results were seen using Jurkat T cells (data not shown). ized into a single, small intracellular analysis volume upon SDF-1 Therefore, we next analyzed the endocytosis and intracellular traf- treatment (Fig. 1E; p , 0.05). Moreover, CXCR4-KKK/AAA-YFP ficking of CXCR4 fluorescent fusion proteins in living cells via trafficked into clustered endosomes in a manner indistinguishable confocal microscopy. Prior to stimulation, the wild-type CXCR4 from CXCR4-WT-YFP, indicating that ubiquitination is dispens- fluorescent fusion protein (CXCR4-WT-YFP) was primarily lo- able for this trafficking step (p . 0.05). The CXCR4-Dc-ter-YFP cated on the cell surface of a live Jurkat T cell (Fig. 1B, left construct, which serves as a control for this quantitation because column). The same individual live cell was imaged again after 15 it does not endocytose, displayed no detectable increase in in- Downloaded from min of stimulation with SDF-1 (Fig. 1B, right column). SDF-1 tracellular clustering in response to SDF-1 (Fig. 1E; p . 0.05). treatment induced CXCR4-WT-YFP endocytosis and trafficking Changes in CXCR4 trafficking in response to SDF-1 were largely into tightly clustered endosomes. Jurkat cells expressing fluores- complete after 15 min, because no significant differences in the cent fusion proteins of truncated CXCR4 lacking the C-terminal mean distribution of CXCR4-WT-YFP molecules among the anal- tail domain (CXCR4-Dc-ter-YFP) or CXCR4 mutated at all three ysis regions were detected between 15 and 30 min of SDF-1 treat-

ubiquitination sites (CXCR4-KKK/AAA-YFP) (33) were imaged ment (n = 15; p . 0.05; data not shown). Thus, CXCR4 endocytosis http://www.jimmunol.org/ before and after SDF-1 stimulation. Similar to CXCR4-WT-YFP, and trafficking into a clustered endosomal compartment in response the mutant CXCR4 constructs were localized to the plasma mem- to SDF-1 requires the CXCR4 C-terminal tail domain but not the branes of untreated cells (Fig. 1B, left panel). CXCR4-Dc-ter-YFP CXCR4 ubiquitination sites. remained at the membrane and failed to internalize in response to SDF-1 (Fig. 1B, right panel), whereas like CXCR4-WT-YFP, The CXCR4-containing endosomes cluster near the CXCR4-KKK/AAA-YFP localized to clustered endosomes in microtubule organizing center and Golgi response to SDF-1 treatment (Fig. 1B, right column). Three- Endosomes move via actin remodeling and microtubules attached dimensional fluorescence image quantitation of multiple cells to the microtubule organizing center (MTOC) (34). Therefore, we confirmed these results, showing that 15 min of SDF-1 treatment investigated the relationship between the MTOC and the CXCR4- by guest on September 29, 2021 significantly decreased membrane levels of CXCR4-WT-YFP and containing clustered endosome compartment. Labeling the MTOC CXCR4-KKK/AAA-YFP (Fig. 1C; p , 0.05) while significantly with a fluorescent MTOC-specific protein (Centrin-2) revealed increasing the intracellular levels of these molecules (Fig. 1D; p , that, following SDF-1 treatment, wild-type CXCR4 endocytosed 0.05). In contrast, neither the membrane levels (Fig. 1C; p . 0.05) from the plasma membrane and trafficked into endosomes clus-

FIGURE 1. CXCR4 endocytosis and trafficking into a clustered endosomal compartment in response to SDF-1 requires the CXCR4 C-terminal tail domain but not the CXCR4 ubiquitination sites. A,PBMC T cells were stimulated with or without SDF-1 and then fixed and stained for endogenous CXCR4 using anti– CXCR4-FITC (green). Hoechst staining (blue) and white outline show the nucleus and plasma membrane, respectively. Representative z-slice confocal images of 12 individual cells are shown. B, Jurkat T cells were transiently transfected with plasmids encoding fluorescent fusion proteins of CXCR4-WT, CXCR4- Dc-ter, or CXCR4-KKK/AAA. Confocal microscopy was used to image the same individual live cells before and after 15 min of SDF-1 stimulation. A three- dimensional (3D) image reconstruction or individual z-slice confocal images of representative cells are shown. Arrows indicate CXCR4-YFP that trafficked into the clustered endosomal compartment. Results of three-dimensional image quantitation of multiple experiments, as in B, showing for each CXCR4 construct the fraction of total cellular CXCR4-YFP fluorescence present on the plasma membrane (C), the intracellular region (D), or only within clustered endosomes (E). Bars denote mean values 6 SEM before and after 15 min of SDF-1 stimulation. *p , 0.05; significantly different from unstimulated samples. 954 Ga13-RHO–ACTIN–SIGNALING PATHWAY MEDIATES CXCR4 TRAFFICKING Downloaded from

FIGURE 2. The CXCR4-containing endosomes cluster near the MTOC and Golgi. A, Representative z-slice confocal images of a live Jurkat T cell expressing a fluorescent fusion protein of CXCR4-WT (CXCR4-WT-RFP; red) and the MTOC marker Centrin-2 (Centrin-GFP; green), before and after SDF-1 treatment (n = 7). B, Representative z-slice confocal images of a live Jurkat T cell expressing CXCR4-WT-YFP (green) and the Golgi marker GalT- CFP (blue) with and without SDF-1 treatment (n = 12). C, Jurkat T cells were stimulated with SDF-1 and then fixed and analyzed by electron microscopy http://www.jimmunol.org/ (n = 22). Arrows, Golgi; M, mitochondria; N, nucleus; PM, plasma membrane. D, Endogenous cell-surface CXCR4 on live Jurkat T cells was labeled with anti–CXCR4-immunogold, and cells were treated with SDF-1 for 30 min and then analyzed by electron microscopy. White arrows, Golgi; black arrows, immunogold-CXCR4 (n = 6). tered tightly around the MTOC (Fig. 2A). Confocal microscopy resulting in an 87% decrease in cell-surface levels of CXCR4 (Fig. further revealed that the clustered endosomes containing endo- 3C). Cells that were treated with SDF-1 and then washed to cytosed CXCR4 were also located near membranes identified as remove the SDF-1 and allowed to recycle endocytosed CXCR4

the Golgi complex by a fluorescent Golgi marker protein (Fig. back to the cell surface restored CXCR4 cell-surface levels back by guest on September 29, 2021 2B). Electron microscopy more precisely located the MTOC and to 50% of the levels seen on unstimulated cells (Fig. 3C). Thus, Golgi membranes and revealed that, as in other cell types (35), about one half of internalized CXCR4 is recycled back to the cell Golgi membranes are located near the MTOC in Jurkat T cells surface, consistent with previous findings (20). Finally, we con- (Fig. 2C). Finally, electron microscopy following immunogold firmed that, like wild-type CXCR4, the CXCR4-KKK/AAA-YFP labeling of endogenously expressed cell-surface CXCR4 con- mutant previously analyzed in Fig. 1A also traffics to Rab11+ firmed that endogenous CXCR4 responds to SDF-1 treatment by recycling endosomes (Fig. 3D). Thus, ubiquitination is not re- endocytosing and trafficking into endosomal compartments lo- quired for CXCR4 internalization or trafficking into Rab11+ re- cated near the MTOC and Golgi (Fig. 2D). cycling endosomes. Together, these results indicated that SDF-1 stimulates CXCR4 endocytosis and trafficking into the same + The CXCR4-containing endosomes are Rab11+ endosomes clustered Rab11 recycling endosomes through which the TCR through which the TCR constitutively recycles constitutively recycles. To further characterize the endosomal compartment containing Cytochalasin D inhibits postendocytic CXCR4 trafficking into endocytosed CXCR4, we determined whether the clustered + CXCR4-containing vesicles contained Rab11, a marker of recy- the clustered Rab11 endosomal compartment cling endosomes that mediates receptor recycling back to the cell Receptor trafficking can also require actin remodeling. Therefore, surface (36). A Rab11 fluorescent fusion protein (Rab11-RFP) we examined the role of actin polymerization in mediating CXCR4 constitutively localized within clustered vesicles in PBMC endocytosis and trafficking into the clustered Rab11+ endosomes. T cells and Jurkat T cells (Fig. 3A,3B, red). SDF-1 treatment Compared with cells pretreated with vehicle (DMSO) alone, induced the endocytosis and trafficking of CXCR4-WT-YFP into pretreatment with cytochalasin D, a drug that inhibits actin po- the same Rab11+ endosomes (Fig. 3A, green). Because the TCR lymerization, did not impair SDF-1–dependent endocytosis of constitutively recycles (37), we next determined whether TCR endogenous CXCR4 (Fig. 4A). Cytochalasin D pretreatment also subunits were also present within the clustered Rab11+ CXCR4- did not detectably inhibit the loss of CXCR4 fluorescent fusion containing endosomes of SDF-1–treated cells. A fluorescent fu- protein from the cell surface in response to SDF-1 (Fig. 4B,4C). sion protein of the TCR-CD3-z subunit (TCR-CD3-z-YFP) was In contrast to its lack of effect on CXCR4 endocytosis, cytocha- detected on the cell surface and within the tightly clustered lasin D pretreatment significantly reduced the fraction of total Rab11-containing endosomes in PBMC and Jurkat T cells (Fig. CXCR4-WT-YFP that trafficked into clustered endosomes (from 3B). Because CXCR4 internalizes and traffics to Rab11+ recycling 0.33 6 0.11 to 0.10 6 0.03, a reduction of 74%; p , 0.05) (Fig. endosomes in response to SDF-1, we confirmed that CXCR4 4B,4D). Fig. 4E confirms that cytochalasin D pretreatment recycles back to the cell surface following internalization. Treat- interferes with the trafficking of internalized CXCR4-WT-YFP ing Jurkat T cells with SDF-1 provoked CXCR4 endocytosis, into Rab11-RFP–containing endosomes. Moreover, in contrast The Journal of Immunology 955

FIGURE 3. The CXCR4-containing endosomes are Rab11+ endosomes through which the TCR constitutively recycles. A, B, and D, Representative z-slice confocal images of the same live PBMC T cells or Jurkat T cells expressing the indicated ﬂuorescent fusion proteins (green) and Rab11 (Rab11-RFP; red), with or without SDF-1 treatment (n = 6–13). C, Jurkat cells were treated with or without SDF-1 for 20 min. Cells were stained for CXCR4 surface levels immediately or washed to remove SDF-1 and incubated for 1 h to allow CXCR4 to recycle back to the cell surface before staining for CXCR4 surface levels. Bars denote the mean results for each condition 6 SEM (n = 3). *p , 0.05; signiﬁcantly different from SDF-1– treated cells without recycling. Downloaded from

to cells treated with vehicle alone, cells pretreated with cytocha- regulating CXCR4 trafficking has not been determined. Rho is http://www.jimmunol.org/ lasin D displayed a significantly reduced ability to recycle CXCR4 activated in response to SDF-1 stimulation in Jurkat T cells (Fig. back onto the cell surface after SDF-1–induced endocytosis (from 5A); therefore, we investigated the role of Rho activation in reg- 58% to 27%; p , 0.05) (Fig. 4F). Thus, actin polymerization is ulating the internalization and endosomal trafficking of CXCR4. required for the postendocytic trafficking of endocytosed CXCR4 To inhibit Rho activation, we used a plasmid encoding RhoN19, into Rab11+-recycling endosomes in response to SDF-1, whereas which inhibits the abilities of RhoGEFs to activate Rho (38). it is dispensable for CXCR4 endocytosis in response to SDF-1. Similar to cytochalasin D, RhoN19 did not inhibit the endocytosis of endogenous CXCR4 (Fig. 5B) or the loss of CXCR4-WT-YFP Rho activation is required for postendocytic trafficking of from the cell membrane in response to SDF-1 treatment (Fig. 5C, by guest on September 29, 2021 CXCR4 into clustered endosomes 5D). However, RhoN19 expression significantly inhibited the Rho GTPases can mediate receptor endocytosis, actin polymeri- trafficking of endocytosed CXCR4 into the clustered endosome zation, and endosomal dynamics (21); however, the role of Rho in compartment (from 0.20 6 0.03 to 0.10 6 0.03; p , 0.05)

FIGURE 4. Cytochalasin D inhibits postendocytic CXCR4 trafficking into the clustered Rab11+ endosomal compartment. A, Jurkat T cells were pretreated with cytochalasin D (Cyto D) or vehicle (DMSO), and the endocytosis of endogenous CXCR4 was stimulated by treating cells with SDF-1 for 20 min. Cells were stained with fluorescently conjugated CXCR4 mAb and analyzed by flow cytometry (n = 3). B, Jurkat T cells expressing CXCR4-WT-YFP (green) were pretreated with Cyto D or vehicle and then assayed as in Fig. 1B for CXCR4-WT-YFP endocytosis and trafficking in response to SDF-1. Representative z-slice confocal images are shown. C and D, Results of multiple experiments as in B, quantitated as in Fig. 1C and 1E. Bars denote the means 6 SEM of four to seven experiments. *p , 0.05; significantly different from SDF-1–treated control (DMSO) samples. E, Jurkat T cells expressing CXCR4-WT-YFP and Rab11-RFP, assayed as in B. F, Jurkat T cells were pretreated with Cyto. D or vehicle (DMSO) and assayed for their ability to recycle endocytosed CXCR4 back to the cell surface, as in Fig. 3C.*p , 0.05; significantly different from control (DMSO) samples (n = 3). 956 Ga13-RHO–ACTIN–SIGNALING PATHWAY MEDIATES CXCR4 TRAFFICKING

FIGURE 5. Rho activation is required for postendocytic trafficking of CXCR4 into clustered endosomes. A, Jurkat T cells were stimulated for 2 min with SDF-1. Active GTP-bound Rho was affinity purified and detected by SDS-PAGE and immunoblotting (upper gel). Whole-cell lysates were analyzed separately as a control (lower gel)(n = 3). B, Jurkat T cells were transfected with a plasmid encoding RhoN19 or a control vector, together with a plasmid Downloaded from encoding eGFP. GFP+ cells were assayed for CXCR4 cell-surface levels, as in Fig. 4A (n = 3). C, Jurkat T cells were transiently transfected with a plasmid encoding CXCR4-WT-YFP, together with a control plasmid or a plasmid encoding RhoN19, and then assayed as in Fig. 1B for CXCR4-WT-YFP endocytosis and trafficking in response to SDF-1. Representative z-slice confocal images are shown. D and E, Results of multiple experiments as in C, quantitated as in Fig. 1C and 1E. Bars denote the means 6 SEM of eight experiments. *p , 0.05; significantly different from SDF-1–treated control plasmid-transfected cells. http://www.jimmunol.org/ (Fig. 5C,5E). RhoN19 expression also decreased the level of Ga13 is required for postendocytic trafficking of CXCR4 into endogenous basal cell-surface CXCR4 on unstimulated cells by 4– the Rab11+ recycling endosomal compartment in response to 7-fold (Fig. 5B), an expected outcome of chronically inhibiting SDF-1 treatment + CXCR4 constitutive trafficking into Rab11 endosomes, which Ga13, a heterotrimeric G protein a subunit, is required for Rho typically mediate receptor recycling back to the cell surface. Thus, activation in T cells in response to SDF-1 stimulation (23). Be- Rho activation, in addition to actin polymerization, is required for cause we showed that Rho activation is required for the trafficking the trafficking of endocytosed CXCR4 into recycling endosomes of CXCR4 into the clustered recycling endosomes, we asked in response to SDF-1 treatment. whether Ga13 mediates endocytosis and/or trafficking of CXCR4. by guest on September 29, 2021

FIGURE 6. Ga13 is required for postendocytic trafficking of CXCR4 into the clustered Rab11+, recycling endosomal compartment in response to SDF-1 treatment. A, Jurkat T cells were transiently transfected with a plasmid encoding Ga13 shRNA and mCherry or a control plasmid encoding mCherry. Whole-cell lysates were immunoblotted to assay Ga13 protein levels compared with actin (control). B, Jurkat T cells were transiently- transfected as in A, with the exception that cells were additionally cotransfected with a plasmid encoding CXCR4-WT-YFP (green). Cells were assayedasinFig.1B for CXCR4-WT-YFP endocytosis and trafficking in response to SDF-1. C and D, Results of multiple experiments as in B, quantitated as in Fig. 1C and 1E. Bars denote the means 6 SEM of eight experiments. *p , 0.05; significantly different from SDF-1–treated control cells. E, Jurkat cells were transfected with a plasmid encoding Ga13 shRNA as in A, and mCherry+ cells were assayed for endogenous CXCR4 cell- surface levels and SDF-1–dependent CXCR4 endocytosis as in Fig. 4A (n =3).F, Jurkat cells were transiently transfected with a plasmid encoding Ga13 shRNA and eGFP or a control plasmid encoding eGFP. eGFP+ cells were assayed for recycling of endocytosed CXCR4, as in Fig. 3C (n =3). *Significantly different from control vector-transfected cells. The Journal of Immunology 957

Jurkat T cells were transiently transfected with a plasmid en- endocytosis was not detectably impaired by inhibiting Rho acti- coding shRNA directed against Ga13 or a control plasmid. The vation or actin polymerization. The SDF-1–dependent trafficking shRNA depleted Ga13 protein levels by 65–80% (Fig. 6A). of CXCR4 into the Rab11+ recycling endosomes also required the Therefore, we analyzed CXCR4-WT-YFP trafficking in living G protein a subunit Ga13. Consistent with these results, inhibition Ga13-deficient cells via confocal microscopy. The Ga13 shRNA- of actin polymerization or depletion of Ga13 inhibited the ability expressing plasmid and the control plasmid express the mCherry of endocytosed CXCR4 to recycle back to the cell surface. Be- fluorescent protein via a separate promoter, permitting the de- cause CXCR4 signaling induces actin polymerization via Ga13- termination of transfected cells. In cells expressing the control mediated activation of Rho (23), these results indicated that vector encoding only mCherry, CXCR4-WT-YFP endocytosed CXCR4 regulates its own trafficking into Rab11+ recycling endo- and trafficked into clustered endosomes in a normal manner (Fig. somes via activation of a G13-Rho-actin–signaling pathway. In 6B, upper panels). In contrast, in cells transfected with the addition, depletion of Ga13 decreased the levels of cell-surface mCherry-Ga13 shRNA vector, CXCR4-WT-YFP endocytosed but CXCR4 on unstimulated cells, suggesting that Ga13 also reg- did not traffic normally into the tightly clustered recycling endo- ulates the constitutive recycling of CXCR4. somal compartment (Fig. 6B, lower panels). Three-dimensional These results are important for several reasons. First, G13 has quantitative image analysis of multiple experiments confirmed not been shown to regulate receptor trafficking, and neither actin that although CXCR4-WT-YFP endocytosed in a nearly normal polymerization nor Rho activation has been shown to regulate manner in Ga13 shRNA-transfected cells (Fig. 6C), endocytosed trafficking of CXCR4. Therefore, these results describe a new role CXCR4-WT-YFP failed to traffic into the clustered endosome for the G13-Rho-actin pathway in regulating CXCR4 expression compartment (Fig. 6D). CXCR4-containing endosomes were in- and subcellular trafficking in T lymphoid cell types. Second, because Downloaded from stead randomly dispersed in Ga13-deficient cells (a y-axis fraction many other GPCRs signal by activating G13, these receptors may of 0.1 indicates the random intracellular dispersal of endocytosed also use the G13-Rho-actin pathway to regulate their endosomal CXCR4-WT-YFP) (Fig. 6D). Interestingly, although Ga13 protein trafficking and cell-surface levels. This idea is supported by the depletion did not greatly affect the endocytosis of endogenous embryonic lethality of Ga13-deficient mice, as well as the multi- CXCR4, Ga13 protein depletion decreased basal cell-surface ple defects seen with a number of Ga13 and/or Ga12 conditional

levels of CXCR4 by 5–7-fold (Fig. 6E). Qualitatively similar to knockout mouse models (26, 27). Third, because genetic deletion of http://www.jimmunol.org/ the results of chronic RhoN19 overexpression (Fig. 5B), this result CXCR4, Ga13, or RhoA expression affects the same approximate is consistent with Ga13 depletion chronically inhibiting the con- stage of T cell development in the thymus (1, 40–43), our results stitutive trafficking of CXCR4 into the Rab11+ recycling endo- suggested that Ga13 and Rho may primarily function to promote some compartment. Further consistent with this result, Ga13 thymocyte development via their effects on CXCR4 trafficking protein depletion significantly inhibited the recycling of CXCR4 and cell-surface levels. Finally, the Ga13-Rho-actin–signaling path- back to the cell surface after its endocytosis in response to SDF-1 way described in this article may also regulate CXCR4 trafficking in (from 49% in vector control-transfected cells to 30% in Ga13 many cancer cells (44) and, thereby, contribute to oncogenesis. In- shRNA vector-transfected cells; p , 0.05) (Fig. 6F). Together, deed, Ga13 and CXCR4 dysregulation have been associated with these results indicate that, upon SDF-1 treatment, CXCR4 sig- the metastasis and hyperplasia of many tumor types (6, 26, 29, 45). by guest on September 29, 2021 naling via Ga13 directs the trafficking of endocytosed CXCR4 In addition to CXCR4, which enters this endosomal compart- into the clustered Rab11+ endosome compartment that also con- ment upon SDF-1 treatment, we found that a fraction of the TCR tains TCR subunits. subunits are constitutively localized within these same clustered Rab11+ recycling endosomes. This result is consistent with previous reports that the TCR constitutively and rapidly recycles such Discussion that at any one time, one third of total cellular TCR is located Although much is known about the mechanisms responsible for the within the cell (37). We previously showed that CXCR4 forms internalization and degradation of GPCRs (13), relatively little is a heterodimer with the TCR in response to SDF-1 that is important understood about the signaling pathways that regulate the traffick- for signal transduction and that some CXCR4–TCR heterodimers ing of GPCRs into nonlysosomal compartments, such as recycling reside within clustered endosomes (3). Therefore, our results endosomes that return the receptors to the surface. Yet endosomal characterize the subcellular location of the intracellular CXCR4– trafficking regulates the levels of receptors on cell surfaces and, TCR heterodimers as Rab11+-recycling endosomes clustered near consequently, has significant effects on ligand-dependent signal- the MTOC and Golgi. By virtue of their location within recycling ing and cellular responses. Our results describe novel molecular endosomes, it is possible that intracellular CXCR4–TCR hetero- mechanisms that permit SDF-1 to stimulate the trafficking of dimers can recycle back to the cell surface, thus altering the CXCR4 specifically into Rab11+ recycling endosomes. fraction of cell-surface TCR and CXCR4 molecules existing as We showed in this study that CXCR4 endocytoses and localizes heterodimers and, consequently, affect CXCR4-mediated signal into Rab11+ recycling endosomes clustered near the MTOC and transduction. In addition, it is possible that the CXCR4–TCR Golgi in response to SDF-1 stimulation. Moreover, approximately heterodimers in Rab11+ endosomes clustered near the Golgi might one half of the internalized CXCR4 recycled back to the cell signal from that location. Indeed, we recently showed that N-Ras surface, which is consistent with previous findings in T cells (20). is activated on the Golgi in response to SDF-1 stimulation (K.N. In contrast, endocytosis and ubiquitination lead to degradation of Kremer, A. Kumar, and K.E. Hedin, submitted for publication). CXCR4 in epithelial cells (33). Receptor ubiquitination can reg- The trafficking of GPCRs, like CXCR4, and their hetero- ulate the internalization and endosomal trafficking of other dimerization with other cell-surface receptors significantly regulate receptors (39); however, our study showed that CXCR4 trafficking the outcomes of ligand-dependent signal transduction. Our results into Rab11+ recycling endosomes was not impaired by point- represent a significant advance in understanding the molecular mutation of CXCR4’s ubiquitination sites. Interestingly, Rho ac- mechanisms that regulate the subcellular location of CXCR4 and tivation and actin polymerization were specifically required for CXCR4–TCR heterodimers in T cells. Additional studies are CXCR4 trafficking into the Rab11+ recycling endosomes. In needed to explain how the subcellular locations of these receptors contrast, the prior trafficking step of SDF-1–dependent CXCR4 regulate their signal transduction and downstream cellular effects. 958 Ga13-RHO–ACTIN–SIGNALING PATHWAY MEDIATES CXCR4 TRAFFICKING

Acknowledgments 22. Neel, N. F., L. A. Lapierre, J. R. Goldenring, and A. Richmond. 2007. RhoB plays an essential role in CXCR2 sorting decisions. J. Cell Sci. 120: 1559–1571. We thank Dr. R. Pagano for the GalT-CFP and Rab11-RFP constructs, 23. Tan, W., D. Martin, and J. S. Gutkind. 2006. The Galpha13-Rho signaling axis is Dr. J. Salisbury for the Centrin-2-GFP, and Dr. D. Billadeau for the required for SDF-1-induced migration through CXCR4. J. Biol. Chem. 281: RhoN19, pCMS3.eGFP.H1p, and pCMS4.mCherry.H1p constructs. 39542–39549. 24. Vicente-Manzanares, M., J. R. Cabrero, M. Rey, M. Pe´rez-Martıńez, A. Ursa, K. Itoh, and F. Sańchez-Madrid. 2002. A role for the Rho-p160 Rho coiled-coil Disclosures kinase axis in the chemokine stromal cell-derived factor-1alpha- The authors have no financial conflicts of interest. induced lymphocyte actomyosin and microtubular organization and chemotaxis. J. Immunol. 168: 400–410. 25. Soede, R. D., I. S. Zeelenberg, Y. M. Wijnands, M. Kamp, and E. Roos. 2001. Stromal cell-derived factor-1-induced LFA-1 activation during in vivo migration References of T cell hybridoma cells requires Gq/11, RhoA, and myosin, as well as Gi and 1. Trampont, P. C., A. C. Tosello-Trampont, Y. Shen, A. K. Duley, A. E. Sutherland, Cdc42. J. Immunol. 166: 4293–4301. T. P. Bender, D. R. Littman, and K. S. Ravichandran. 2010. CXCR4 acts as 26. Worzfeld, T., N. Wettschureck, and S. Offermanns. 2008. G(12)/G(13)-mediated a costimulator during thymic beta-selection. Nat. Immunol. 11: 162–170. signalling in mammalian physiology and disease. Trends Pharmacol. Sci. 29: 2. Patrussi, L., and C. T. Baldari. 2008. Intracellular mediators of CXCR4- 582–589. dependent signaling in T cells. Immunol. Lett. 115: 75–82. 27. Offermanns, S., V. Mancino, J. P. Revel, and M. I. Simon. 1997. Vascular system 3. Kumar, A., T. D. Humphreys, K. N. Kremer, P. S. Bramati, L. Bradfield, defects and impaired cell chemokinesis as a result of Galpha13 deficiency. C. E. Edgar, and K. E. Hedin. 2006. CXCR4 physically associates with the T cell Science 275: 533–536. receptor to signal in T cells. Immunity 25: 213–224. 28. Herroeder, S., P. Reichardt, A. Sassmann, B. Zimmermann, D. Jaeneke, 4. Kremer, K. N., A. Kumar, and K. E. Hedin. 2007. Haplotype-independent co- J. Hoeckner, M. W. Hollmann, K. D. Fischer, S. Vogt, R. Grosse, et al. 2009. stimulation of IL-10 secretion by SDF-1/CXCL12 proceeds via AP-1 binding to Guanine nucleotide-binding proteins of the G12 family shape immune functions the human IL-10 promoter. J. Immunol. 178: 1581–1588. by controlling CD4+ T cell adhesiveness and motility. Immunity 30: 708–720. 5. Contento, R. L., B. Molon, C. Boularan, T. Pozzan, S. Manes, S. Marullo, and 29. Kelly, P., P. J. Casey, and T. E. Meigs. 2007. Biologic functions of the G12 Downloaded from A. Viola. 2008. CXCR4-CCR5: a couple modulating T cell functions. Proc. Natl. subfamily of heterotrimeric g proteins: growth, migration, and metastasis. Bio- Acad. Sci. USA 105: 10101–10106. chemistry 46: 6677–6687. 6. Busillo, J. M., and J. L. Benovic. 2007. Regulation of CXCR4 signaling. Bio- 30. Gong, H., B. Shen, P. Flevaris, C. Chow, S. C. Lam, T. A. Voyno-Yasenetskaya, chim. Biophys. Acta 1768: 952–963. T. Kozasa, and X. Du. 2010. G protein subunit Galpha13 binds to integrin 7. Alkhatib, G. 2009. The biology of CCR5 and CXCR4. Curr. Opin. HIV AIDS 4: alphaIIbbeta3 and mediates integrin “outside-in” signaling. Science 327: 340– 96–103. 343. 8. Busillo, J. M., S. Armando, R. Sengupta, O. Meucci, M. Bouvier, and 31. Gomez, T. S., S. D. McCarney, E. Carrizosa, C. M. Labno, E. O. Comiskey, J. L. Benovic. 2010. Site-specific phosphorylation of CXCR4 is dynamically J. C. Nolz, P. Zhu, B. D. Freedman, M. R. Clark, D. J. Rawlings, et al. 2006. HS1 regulated by multiple kinases and results in differential modulation of CXCR4 functions as an essential actin-regulatory adaptor protein at the immune synapse. http://www.jimmunol.org/ signaling. J. Biol. Chem. 285: 7805–7817. Immunity 24: 741–752. 9. Fan, G. H., W. Yang, J. Sai, and A. Richmond. 2002. Hsc/Hsp70 interacting 32. Spurr, A. R. 1969. A low-viscosity epoxy resin embedding medium for electron protein (hip) associates with CXCR2 and regulates the receptor signaling and microscopy. J. Ultrastruct. Res. 26: 31–43. trafficking. J. Biol. Chem. 277: 6590–6597. 33. Marchese, A., and J. L. Benovic. 2001. Agonist-promoted ubiquitination of the 10. Rey, M., A. Valenzuela-Fernańdez, A. Urzainqui, M. Yań˜ez-Mo´,M.Pe´rez- G protein-coupled receptor CXCR4 mediates lysosomal sorting. J. Biol. Chem. Martıńez, P. Penela, F. Mayor, Jr., and F. Sańchez-Madrid. 2007. Myosin IIA is 276: 45509–45512. involved in the endocytosis of CXCR4 induced by SDF-1alpha. J. Cell Sci. 120: 34. Grimes, M. L., and H. M. Miettinen. 2003. Receptor tyrosine kinase and G- 1126–1133. protein coupled receptor signaling and sorting within endosomes. J. Neurochem. 11. Luo, C., H. Pan, M. Mines, K. Watson, J. Zhang, and G. H. Fan. 2006. CXCL12 84: 905–918. induces tyrosine phosphorylation of cortactin, which plays a role in CXC che- 35. Donaldson, J. G., and J. Lippincott-Schwartz. 2000. Sorting and signaling at the mokine receptor 4-mediated extracellular signal-regulated kinase activation and Golgi complex. Cell 101: 693–696. chemotaxis. J. Biol. Chem. 281: 30081–30093. 36. Jones, M. C., P. T. Caswell, and J. C. Norman. 2006. Endocytic recycling by guest on September 29, 2021 12. Venkatesan, S., J. J. Rose, R. Lodge, P. M. Murphy, and J. F. Foley. 2003. pathways: emerging regulators of cell migration. Curr. Opin. Cell Biol. 18: 549– Distinct mechanisms of agonist-induced endocytosis for human chemokine 557. receptors CCR5 and CXCR4. Mol. Biol. Cell 14: 3305–3324. 37. Liu, H., M. Rhodes, D. L. Wiest, and D. A. Vignali. 2000. On the dynamics of 13. Neel, N. F., E. Schutyser, J. Sai, G. H. Fan, and A. Richmond. 2005. Chemokine TCR:CD3 complex cell surface expression and downmodulation. Immunity 13: receptor internalization and intracellular trafficking. Cytokine Growth Factor 665–675. Rev. 16: 637–658. 38. Coso, O. A., M. Chiariello, J. C. Yu, H. Teramoto, P. Crespo, N. Xu, T. Miki, and 14. Moore, C. A., S. K. Milano, and J. L. Benovic. 2007. Regulation of receptor J. S. Gutkind. 1995. The small GTP-binding proteins Rac1 and Cdc42 regulate trafficking by GRKs and arrestins. Annu. Rev. Physiol. 69: 451–482. the activity of the JNK/SAPK signaling pathway. Cell 81: 1137–1146. 15. Marchese, A., C. Raiborg, F. Santini, J. H. Keen, H. Stenmark, and J. L. Benovic. 39. Murphy, J. E., B. E. Padilla, B. Hasdemir, G. S. Cottrell, and N. W. Bunnett. 2003. The E3 ubiquitin ligase AIP4 mediates ubiquitination and sorting of the G 2009. Endosomes: a legitimate platform for the signaling train. Proc. Natl. Acad. protein-coupled receptor CXCR4. Dev. Cell 5: 709–722. Sci. USA 106: 17615–17622. 16. Bhandari, D., J. Trejo, J. L. Benovic, and A. Marchese. 2007. Arrestin-2 interacts 40. Cleverley, S., S. Henning, and D. Cantrell. 1999. Inhibition of Rho at different with the ubiquitin-protein isopeptide ligase atrophin-interacting protein 4 and stages of thymocyte development gives different perspectives on Rho function. mediates endosomal sorting of the chemokine receptor CXCR4. J. Biol. Chem. Curr. Biol. 9: 657–660. 282: 36971–36979. 41. Coffield, V. M., W. S. Helms, Q. Jiang, and L. Su. 2004. Galpha13 mediates 17. Bhandari, D., S. L. Robia, and A. Marchese. 2009. The E3 ubiquitin ligase a signal that is essential for proliferation and survival of thymocyte progenitors. atrophin interacting protein 4 binds directly to the chemokine receptor CXCR4 J. Exp. Med. 200: 1315–1324. via a novel WW domain-mediated interaction. Mol. Biol. Cell 20: 1324–1339. 42. Gu, Y., H. D. Chae, J. E. Siefring, A. C. Jasti, D. A. Hildeman, and 18. Hanyaloglu, A. C., and M. von Zastrow. 2008. Regulation of GPCRs by endo- D. A. Williams. 2006. RhoH GTPase recruits and activates Zap70 required for cytic membrane trafficking and its potential implications. Annu. Rev. Pharmacol. T cell receptor signaling and thymocyte development. Nat. Immunol. 7: 1182– Toxicol. 48: 537–568. 1190. 19. Zhang, Y., A. Foudi, J. F. Geay, M. Berthebaud, D. Buet, P. Jarrier, A. Jalil, 43. Dorn, T., U. Kuhn, G. Bungartz, S. Stiller, M. Bauer, J. Ellwart, T. Peters, W. Vainchenker, and F. Louache. 2004. Intracellular localization and constitutive K. Scharffetter-Kochanek, M. Semmrich, M. Laschinger, et al. 2007. RhoH is endocytosis of CXCR4 in human CD34+ hematopoietic progenitor cells. Stem important for positive thymocyte selection and T-cell receptor signaling. Blood Cells 22: 1015–1029. 109: 2346–2355. 20. Grundler, R., L. Brault, C. Gasser, A. N. Bullock, T. Dechow, S. Woetzel, 44. Alsayed, Y., H. Ngo, J. Runnels, X. Leleu, U. K. Singha, C. M. Pitsillides, V. Pogacic, A. Villa, S. Ehret, G. Berridge, et al. 2009. Dissection of PIM serine/ J. A. Spencer, T. Kimlinger, J. M. Ghobrial, X. Jia, et al. 2007. Mechanisms of threonine kinases in FLT3-ITD-induced leukemogenesis reveals PIM1 as regu- regulation of CXCR4/SDF-1 (CXCL12)-dependent migration and homing in lator of CXCL12-CXCR4-mediated homing and migration. J. Exp. Med. 206: multiple myeloma. Blood 109: 2708–2717. 1957–1970. 45. Aittaleb, M., C. A. Boguth, and J. J. Tesmer. 2010. Structure and function of 21. Ridley, A. J. 2006. Rho GTPases and actin dynamics in membrane protrusions heterotrimeric G protein-regulated Rho guanine nucleotide exchange factors. and vesicle trafficking. Trends Cell Biol. 16: 522–529. Mol. Pharmacol. 77: 111–125.