Arrestin 3 Mediates Endocytosis of CCR7 following Ligation of CCL19 but Not CCL21 Melissa A. Byers, Psachal A. Calloway, Laurie Shannon, Heather D. Cunningham, Sarah Smith, Fang Li, Brian C. This information is current as Fassold and Charlotte M. Vines of September 25, 2021. J Immunol 2008; 181:4723-4732; ; doi: 10.4049/jimmunol.181.7.4723 http://www.jimmunol.org/content/181/7/4723 Downloaded from

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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 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Arrestin 3 Mediates Endocytosis of CCR7 following Ligation of CCL19 but Not CCL211

Melissa A. Byers,* Psachal A. Calloway,* Laurie Shannon,* Heather D. Cunningham,* Sarah Smith,* Fang Li,† Brian C. Fassold,* and Charlotte M. Vines2*

Internalization of ligand bound G -coupled receptors, an important cellular function that mediates receptor desensitization, takes place via distinct pathways, which are often unique for each receptor. The C-C receptor (CCR7) G protein- coupled receptor is expressed on naive T cells, dendritic cells, and NK cells and has two endogenous ligands, CCL19 and CCL21. Following binding of CCL21, 21 ؎ 4% of CCR7 is internalized in the HuT 78 human T cell lymphoma line, while 76 ؎ 8% of CCR7 is internalized upon binding to CCL19. To determine whether arrestins mediated differential internalization of CCR7/ CCL19 vs CCR7/CCL21, we used small interfering RNA (siRNA) to knock down expression of arrestin 2 or arrestin 3 in HuT 78 cells. Independent of arrestin 2 or arrestin 3 expression, CCR7/CCL21 internalized. In contrast, following depletion of arrestin 3, Downloaded from CCR7/CCL19 failed to internalize. To examine the consequence of complete loss of both arrestin 2 and arrestin 3 on CCL19/CCR7 -internalization, we examined CCR7 internalization in arrestin 2؊/؊/arrestin 3؊/؊ murine embryonic fibroblasts. Only reconsti tution with arrestin 3-GFP but not arrestin 2-GFP rescued internalization of CCR7/CCL19. Loss of arrestin 2 or arrestin 3 blocked migration to CCL19 but had no effect on migration to CCL21. Using immunofluorescence microscopy, we found that arrestins do not cluster at the membrane with CCR7 following ligand binding but cap with CCR7 during receptor internalization. These are the first studies that define a role for arrestin 3 in the internalization of a following binding of one http://www.jimmunol.org/ but not both endogenous ligands. The Journal of Immunology, 2008, 181: 4723–4732.

protein-coupled receptors (GPCRs)3 are a diverse group then internalized. Two of the four known arrestins, arrestin 2 and of seven transmembrane receptors that are activated by arrestin 3, are expressed ubiquitously in mammalian cells (8–10). G multiple sensory and chemical stimuli including light, Receptor internalization is an important immunological function odors, tastes, , and neurotransmitters (as reviewed by that controls signaling, which controls immunological responses, Ref. 1). Activation of these receptors by their ligands induces a receptor fate, and receptor desensitization (11). Yet it is unclear conformational change, which leads to activation of distinct down- how distinct receptors are shuttled to the necessary internalization by guest on September 25, 2021 stream signaling events (2–4). Following activation by agonist machinery to differentially control the signaling and fate of each binding, the GPCRs couple to heterotrimeric GTP-binding (G) internalized receptor. triggering an exchange of GDP for GTP on the G␣-sub- Arrestins can regulate GPCR internalization, degradation and/or unit and the resultant release of the ␣ subunit from the ␤/␥-dimer recycling. Arrestin binding can block association of G proteins (5). These subunits can regulate numerous effectors including ad- with phosphorylated GPCRs and thus plays an important role in ␤ enylyl cyclase and phospholipase C (6, 7). The receptor is then phosphorylation-dependent desensitization (9, 12). For instance, phosphorylated at its C terminus, which provides a substrate for arrestins mediate internalization of the ␤2 adrenergic receptor (10, ␤ high affinity binding by arrestin 2 (also known as -arrestin 1) 13). Arrestins can also serve as adaptors between the receptors and ␤ and/or arrestin 3 (also known as -arrestin 2), and the receptor is the endocytic machinery. In such cases, arrestin binding targets activated receptors to clathrin-coated pits via interactions with *Department of Microbiology, Molecular Genetics and Immunology, The University clathrin, the clathrin adaptor AP-2, and the intracellular transport of Kansas Medical Center, Kansas City, KS 66160; and †The Affiliated Hospital of ATPase N-ethylmaleimide-sensitive factor (14–16). Arrestins can Ningxia Medical College, Yinchuan, Ningxia, People’s Republic of China also regulate other signaling pathways including activating Src, Received for publication October 30, 2007. Accepted for publication July 24, 2008. ERK, and c-Jun N-terminal kinase (17–19). Alternatively, while The costs of publication of this article were defrayed in part by the payment of page the human N-formyl peptide receptor (FPR) does not require ar- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. restins to internalize, arrestins are required for FPR recycling (20). 1 This research was supported by National Institutes of Health Grants 5K22AI060815 In addition, arrestins can mediate trafficking of GPCRs to specific and 2P20RR016443-07 (to C.M.V.). The Flow Cytometry Core is supported by Na- sites in the cell, where the receptors are degraded (8, 21, 22). tional Institutes of Health Grant P20 RR016443 from the National Center for Re- CCR7 is a GPCR that is expressed on B lymphocytes, NK cells, search Resources. Confocal imaging was conducted with staff support within an Na- tional Institute of Child Health and Human Development center supported facility, the and naive and central memory T lymphocytes (23–27). CCR7 is Kansas Intellectual and Developmental Disabilities Research Center imaging core activated by two known endogenous ligands, CCL19 (MIP-3␤, (National Institute of Child Health and Human Development HD 02528). ELC, CK␤-11, Exodus-3) and CCL21 (6Ckine, SLC, TCA4, Ex- 2 Address correspondence and reprint requests to Dr. Charlotte Vines, University of Kansas Medical Center, 3901 Rainbow Boulevard, Wahl Hall West 3025, Kansas odus-2) (26, 28–31). CCL21 is expressed on the luminal side of City, KS 66160. E-mail address: [email protected] high endothelial venules, while both ligands are found within the 3 Abbreviations used in this paper: GPCR, G protein-coupled receptor; FPR, N-formyl stromal region of the T cell-rich areas (32). The peptide receptor; MEF, murine embryonic fibroblast; sf, serum free; siRNA, small CCL19 ligand is expressed on dendritic cells, and is found at lower interfering RNA. levels in the and lymph nodes than CCL21 (33). The ex- Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 pression of these ligands in different regions of the www.jimmunol.org 4724 DIFFERENTIAL INTERNALIZATION OF CCR7 implies that each ligand might have a unique role in the regulation RPMI 1640/2 mM L-glutamine) (Invitrogen) in a humidified atmosphere of Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ of this system and as a result differentially regulate signaling in T 5% CO2/air at 37°C. The arrestin2 /arrestin3 MEFs (WT and arr2 / Ϫ/Ϫ cells. Indeed, each ligand has been uniquely associated with dif- arr3 ) cell lines were a generous gift from Dr. Robert Lefkowitz (Duke University, Durham, NC). Murine CCR7 was transiently transfected into ferent autoimmune diseases. Although CCL21 has been associated the MEF arr2Ϫ/Ϫ/arr3Ϫ/Ϫ cell lines with Lipofectamine 2000 (Invitrogen) with Sjogren’s syndrome and lymphoproliferative disorders (34– according to the manufacturer’s instructions, with the following modifica- 36), CCL19 has been linked to Crohn’s (37, 38). It is unclear, tion: we halved all recommended amounts. pCDNA3.1 (Invitrogen) vector however, how each of these ligands mediates distinct effects fol- was transfected as the vector control. MEF cell lines were maintained in 10% FBS (HyClone)/90% DMEM supplemented with 2 mM L-glutamine lowing association with CCR7. at 37°C/5% CO2 in a humidified incubator. Cells were certified to be free CCR7 binds CCL19 and CCL21 with equal affinities (30), yet of mycoplasma by Charles River Laboratories. each ligand has distinct signaling pathways and modulation of cell migration. In one study, ectopic expression of CCL19 in pancreatic Flow cytometry islets resulted in the development of small infiltrates of lympho- A total of 106 HuT 78 cells were isolated from culture, rinsed twice with cytes and dendritic cells, whereas ectopic expression of CCL21 serum free (sf)RPMI 1640 and resuspended directly in 10 ␮l of PE directly resulted in larger, more organized infiltrates. From these observa- conjugated anti-human CCR7 Ab or the PE directly conjugated isotype control (R&D Systems). Cells were allowed to incubate on ice for 30 min, tions it was inferred that CCL21 might be more effective than rinsed three times with sfRPMI 1640, and analyzed immediately by flow CCL19 in promoting the recruitment of T cells (33). It was sur- cytometry on a LSRII (BD Biosciences). prising, therefore, when it was reported that only CCL19 mediates emigration of newly generated lymphocytes from the neonatal thy- RT-PCR mus (39). These observations provide further support that distinct A total of 106 HuT 78 or Jurkat e.6 cells were isolated from culture, rinsed Downloaded from signal cascades are initiated following ligand/receptor association, twice with sfRPMI 1640, and total RNA was isolated as per the manufac- turer’s instructions (Qiagen) and quantified. Then, 5 ␮g of RNA was mixed which control different cellular behaviors. The signal transduction ␮ with 50 ng of random hexamers (Invitrogen), 200 M dNTPs, ddH2O, events triggered by GPCRs are regulated both spatially and tem- Superscript II (Invitrogen) and 5ϫ First Strand buffer, 10 mM DTT, RNa- porally (4). sin (Promega). The reaction was mixed at room temperature and incubated To begin to unravel the differential regulation of the signaling at 42°C for 60 min. The Superscript II was inactivated by heating the mixture to 70°C for 15 min in the presence of NaOH. Of the resultant events between CCL19 and CCL21, we examined the internaliza- http://www.jimmunol.org/ cDNA, 5 ␮l was mixed with 10ϫ PCR buffer, 200 ␮M dNTPs, 1.5 ␮M tion of CCR7 when bound to CCL19 and CCL21. Receptor inter- ␮ Ј MgCl2, ddH2O, 0.5 M of the forward primer 5 GGAAGCTTGGGATC nalization can initiate signaling, processing and often desensitiza- GATGCCATGGACCGGGGAAACCAATGAAAAGCG-3Ј and 0.5 ␮M tion of a GPCR. After we observed differences in receptor of the reverse primer 5ЈGCGGCCGCATGGGGAGAAGGTGGTGGTGG- internalization following engagement of CCL19 and CCL21, we 3Ј. The following conditions were used: a) 95°C for 5 min (1 cycle); b) examined the role of arrestins in regulating the differential inter- 95°C for 30 s, 55°C annealing 30 s, and 72°C extension 1 min (30 cycles); c) 72°C terminal extension 10 min (1 cycle); and d) 4°C infinite hold on a nalization of CCR7 following ligand binding. To this end, we ex- PerkinElmer 9600 cycler. Products were separated by agarose gel electro- amined CCR7 internalization following depletion of arrestin 2 or phoresis; the gel was stained with ethidium bromide and imaged (Kodak arrestin 3 using small interfering RNA (siRNA) in HuT 78 human Gel Logic 1000). by guest on September 25, 2021 T cells. In addition, we examined the effect of loss of each arrestin Receptor internalization assays on migration of HuT 78 cells. Next, we examined internalization Ϫ/Ϫ Ϫ/Ϫ of CCR7 in murine embryonic fibroblasts (MEFs) that are null for MEF Arr2 /arr3 cells were transiently cotransfected with arrestin-2 arrestin expression. We report that arrestin 3 but not arrestin 2 is GFP, arrestin-3 GFP or pEGFP-N1 and muCCR7 using Lipofectamine 2000 per the manufacturer’s instructions using half of the recommended required for internalization of CCR7 when bound to CCL19. In amounts of reagents and DNA. Twenty-four hours following transfection, contrast, arrestins are not required for internalization of CCR7/ MEF Arr2Ϫ/Ϫ/arr3Ϫ/Ϫ cells were harvested from dishes by trypsinization CCL21. In addition, both arrestins control migration of HuT 78 and re-plated in larger dishes. HuT 78 suspension cells were collected from cells. Although signaling is typically associated with receptor clus- flasks, counted, and washed in PBS. Cells were re-suspended in sfRPMI 1640 (HuT 78) or sfDMEM-(MEF arr2Ϫ/Ϫ/arr3Ϫ/Ϫ)at5ϫ 106 cells per tering, we observed that arrestins do not appear to cluster at the ml, and 100 ␮l was used for each time point. Following a 10-min prein- membrane with CCR7 following ligand binding. Instead, CCR7 cubation at 37°C, cells were treated with 200 nM CCL19 or CCL21 for the forms caps following ligand association. This is the first descrip- indicated time periods. Internalization was arrested by adding 10 volumes Ϫ/Ϫ Ϫ/Ϫ tion of differential binding of arrestins that is regulated by two of ice-cold sfRPMI 1640 HuT 78 or sfDMEM MEF arr2 /arr3 .Hu- distinct endogenous chemokines, which bind to the same receptor. man ligands were used with the HuT 78 cells, while the murine ligands were used with murine CCR7 in the MEF cell lines. Receptors on the surface of the HuT 78 cells were stained with PE-conjugated anti-human Materials and Methods CCR7 Abs for 30 min on ice (as per the manufacturer’s instructions). Ϫ/Ϫ Materials FLAG-tagged murine CCR7 was detected by incubating the MEF arr2 / arr3Ϫ/Ϫ cells with anti-FLAG (M2) Ab on ice for 30 min, followed by three Recombinant human and murine CCL19 and CCL21, and PE-conjugated rinses with ice cold PBS and incubation with FITC anti-mouse secondary anti-human CCR7 Ab (FAB197P), were purchased from R&D Systems. for 30 min on ice or with CCL-19 Ig. Cells were rinsed three times in serum M2 Abs (Catalog no. F1804) were purchased from Sigma-Aldrich. Anti- free media and assayed immediately or fixed in 2% paraformaldehyde and ␤-arrestin 1/2/3 was purchased from Santa Cruz Biotechnology. PE-con- stored at 4°C for up to 4 days. Receptor levels were assayed by flow jugated anti-mouse was purchased from Jackson ImmunoResearch Labo- cytometry on a Becton Dickinson LSRII with DIVA software. Ab binding ratories. The cDNA encoding N-terminally tagged murine CCR7 was a was not inhibited by the presence of either CCL19 or CCL21 (data not generous gift from James J. Campbell (Harvard Medical School, Boston, shown). Level of receptor remaining of the surface was calculated as [mean MA). Plasmids encoding bovine arrestin2-GFP and arrestin3-GFP have channel fluorescence of PE-anti-CCR7 or FITC-anti-mouse at time-point]/ been described (20) and were generously provided by Dr. Jeffrey Benovic [mean channel fluorescence of PE-anti-CCR7 at time ϭ 0]. Statistical sig- (Thomas Jefferson University, Philadelphia, PA). The plasmid encoding nificance was determined using a paired t test. Curves or linear regressions the human FPR has been described (40) and was generously provided by were fit using Prism 4 software package (GraphPad). Dr. Eric Prossnitz (University of New Mexico Medical School, Albuquerque, NM). Receptor recycling assays Cell lines HuT 78 suspension cells were collected from flasks. Cells were counted and washed in PBS. Cells were resuspended in sf RPMI 1640 at 5ϫ106 HuT 78 and Jurkat e.6 cell lines were purchased from the American Type cells per ml and 100 ␮l was used for each time point. Following a 10-min Culture Collection, and were maintained in cRPMI 1640 (10% FBS/90% preincubation at 37°C, cells were treated with 200 nM CCL19 or CCL21 The Journal of Immunology 4725

for 20 min. Ligand was added again following 20 min of receptor inter- nalization to prevent ligand depletion. At 30 min, cells were rinsed three times in ice-cold sfRPMI 1640. Receptor recycling was initiated by addi- tion of prewarmed 37°C sfRPMI 1640. Cells were collected at the indicated time points. Recycling was arrested by addition of 10 volumes of ice-cold sfRPMI 1640. Receptors on the surface of the cells were stained with PE-conjugated anti-human CCR7 Abs for 30 min on ice (as per the man- ufacturer’s instructions). Cells were rinsed three times in sfRPMI 1640 and assayed immediately or fixed in 2% paraformaldehyde and stored at 4°C for up to 4 days. Receptor levels were assayed by flow cytometry on a Becton Dickinson LSRII with DIVA software. Level of receptor remaining FIGURE 1. CCR7 is expressed in HuT 78 cells but not in control Jurkat of the surface was calculated as [mean channel fluorescence of PE-anti- e.6 cells. A, Total mRNA was isolated from HuT 78 (CCR7ϩ) and Jurkat CCR7 at time-point]/[mean channel fluorescence of PE-anti-CCR7 at e6 (CCR7-), reverse transcribed into cDNA, and the human CCR7 message ϭ time 0]. Curves were fit using Prism 4 software package (Graphpad). was amplified by PCR using CCR7-specific primers. The products were Statistics were determined using a paired t test. separated by agarose gel electrophoresis and visualized by ethidium bro- siRNA mide staining. B, HuT 78 cells were stained with anti-CCR7-PE Ab or with the corresponding isotype control and analyzed by flow cytometry. Each FITC-labeled, double-stranded siRNA, with 19-nucleotide duplex RNA image is representative of a minimum of n ϭ 3 independent experiments. and 2-nucleotide 3Ј-dTdT over-hangs were purchased from Invitrogen. The siRNA sequences targeting human ␤-arrestin1 and ␤-arrestin2 were 5Ј- AAAGCCUUCUGCGCGGAGAAU-3Ј and 5Ј-AAGGACCGCAAAGU ϫ ␮ GUUUGUG-3Ј corresponding to positions 439–459 and 148–168 relative 90 g and resuspended in 100 l Solution R. DNA was added to an Downloaded from to the start codon, respectively. A nonsilencing RNA duplex (5Ј-AAUU Eppendorf and cells resuspended in SolutionR were layered on top. Before CUCCGAACGUGUCACGU-3Ј), as the manufacturer indicated, was used nucleofection, cells/DNA/SolutionR was transferred to an Amaxa cuvette as a control. These siRNAs have been described (41). A total of 20 pmol and cells were nucleofected on program V-01. Immediately after nucleo- ␮ of siRNAs were Nucleofected (AMAXA) using solution R (see below) or fection, 800 l of cRPMI 1640 was added and cells were transferred back transfected with Oligofectamine (Invitrogen) to introduce the siRNA. into the Eppendorf tube with the transfer pipette included in the Amaxa kit. Briefly, on the day of the transfection, cells were plated at ϳ500,000/80 ␮l Cells were then plated in complete RPMI 1640 and allowed to recover sfRPMI 1640 in a 24-well dish. A total of 1 ␮lofa20␮M stock of each overnight. Twenty-four hours following transfection, HuT 78 cells were

double-stranded siRNA was combined with 16 ␮l Opti-MEMI reduced collected, stimulated with 200 nM ligand for 4 or 8 min, and fixed with 2% http://www.jimmunol.org/ serum medium. Oligofectamine was inverted several times to mix. In a formaldehyde (30 min on ice). Cells were permeabilized with 1% Non- ϫ separate Eppendorf tube 0.8 ␮l of Oligofectamine was diluted with 2.2 ␮l idet-P 40/1 PBS for 15 min at room temperature and labeled with mouse Opti-MEMI reduced serum medium. The mixtures in tubes were incubated anti-CCR7 (1/100) (Pharmingen no.550937) in 3% BSA/PBS for 1 h. The separately at room temperature for 10 min, combined into one tube, and cells were rinsed three times and labeled with Texas red (1:250) directly allowed to incubate at room temperature for an additional 20 min to form conjugated to anti-mouse (Jackson ImmunoResearch Laboratories). Cells siRNA/Oligofectamine complexes. The 20 ␮l of complexes were added to were rinsed three times in PBS and plated on glass slides with Prolong Gold anti-fade (Molecular Probes). Immunofluorescence was visualized on the cells and incubated at 37°C in a humidified, CO2 incubator for 4 h. A total of 50 ␮l of RPMI 1640/30%FBS/L-glutamine was added to each of a Nikon 90i upright microscope via mercury fluorescent excitation then the 24 wells. Following treatment with Oligofectamine, or nucleofection confirmed via confocal scanning (Nikon C1 series confocal scan head). cells were allowed to incubate for 72 h and then assayed for internalization Lasers used for emission detection were a 488 Multi line Argon (green IR) and arrestin expression. Changes in arrestin 2 and/or arrestin 3 expression and 561 Diode laser (red IR) acquisition via the Nikon EZ-C1 series soft- by guest on September 25, 2021 levels were confirmed by Western blotting. ware (3.60). Western blot assays Results A total of 106 HuT 78 cells were harvested for each condition (cells pre- Characterization of CCR7 expression and internalization treated for 72 h with nonspecific, arrestin 2 or arrestin 3 siRNA) from in HuT 78 cells flasks, counted and washed in PBS. Cells were pelleted, and the media was replaced with 200 ␮l of RIPA lysis buffer (50 mM HEPES (pH 7.5), 150 mM To examine the expression of endogenous CCR7 in human T cells,

NaCl, 5 mM EDTA, 1 mM EGTA, 1 mM NaF, 2 mM Na3VO4, 1% NP40, we used RT-PCR (Fig. 1) to look for expression of CCR7 in two 0.1% SDS, 1% deoxycholate, and 1 mM DTT with Protease Inhibitor mixture human cell lines. In line with previous observations, HuT 78 cells from Sigma-Aldrich). Lysates were separated by SDS-PAGE on 10% acryl- expressed CCR7 mRNA, in contrast with Jurkat cells, which failed amide gels, transferred to PVDF, and probed for arrestins with a rabbit anti-␤-Arrestin-1/2/3 (H-290) Ab (Santa Cruz Biotechnology). Immune to express CCR7 (44, 45) (Fig. 1A). To verify surface localization complexes were probed with a rabbit-HRP secondary Ab (Pierce) and vi- of expressed CCR7 protein, we used FACS (Fig. 1B). We con- sualized by Super Signal West Pico Substrate (Pierce) and exposed mem- firmed that CCR7 was expressed on the surface of HuT 78 cells. branes to x-ray film or to the Fuji LAS-4000 and bands were quantified. Receptor internalization is an important cellular function that Chemotaxis assays mediates receptor desensitization (11, 46). To confirm that the HuT 78 cell line internalized and recycled in a manner similar to Chemotaxis assays performed as described (42, 43). In brief, siRNA-trans- the CCR7 expressed in peripheral blood cells (44), we used FACS fected cells were resuspended in assay buffer (Hank’s Buffered Saline So- lution (Cellgro) plus 10 mM HEPES (pH 7.4) (Invitrogen) with 0.5 g BSA based internalization and receptor recycling assays. CCR7 is acti- (A790 Sigma-Aldrich V)). Cells were diluted to 4 ϫ 106 cells/ml in com- vated by two endogenous ligands, CCL19 and CCL21 (25, 31, 47). plete RPMI 1640 and kept at room temperature during preparation of the Initially, to identify an optimal concentration of ligand for our chemotaxis chambers. Polycarbonate membranes (8-␮m pore size) were assays, we conducted a dose response (Fig. 2A) and found that 200 pre-wet with serum-free RPMI 1640. Chemokines were diluted to 200 nM nM CCL21 and 200 nM CCL19 provided maximal internalization in 1%BSA/PBS and pipetted into lower chamber of a Neuroprobe chemo- taxis chamber. Chambers were fitted with filters, gaskets, and lid. A total for CCR7. We did not observe increased internalization levels at of 50 ␮l of cells (100,000 cells/well) were loaded and cells allowed to higher concentrations, possibly due to loss of ligand solubility. migrate for2hat37°C in a 5% CO2 humidified incubator. Following Therefore, we selected 200 nM ligand to carry out further studies. incubation the membranes were removed and cells that migrated to the We compared the extent of internalization and rates of CCR7 in- lower chamber were counted. ternalization in the HuT 78 cells following binding to 200 nM Confocal microscopy CCL19 and CCL21 ligands (Fig. 2, B and C). Similar to published Ϯ A total of 2 ϫ 106 HuT 78 cells were transiently nucleofected with arrestin observations for peripheral blood leukocytes (44, 45), 76 9% of 2-GFP, arrestin 3-GFP, or pEGFP-N1 using the AMAXA R kit per the CCR7 was internalized with a t1/2 of 0.46 min following exposure manufacturer’s instructions. In brief, cells were centrifuged for 10 min at of the HuT 78 cells to 200 nM CCL19. In contrast, only 22 Ϯ 4% 4726 DIFFERENTIAL INTERNALIZATION OF CCR7

FIGURE 2. Internalization of the endogenous CCR7 GPCR in HuT 78 cells is mediated by (A, B, and D) CCL19 and (C, D) CCL21. HuT 78 cells were incubated at 37°C in the absence (t ϭ 0) or presence of 200 nM ligand for (A and D) 10 min or (B and C) the indicated time periods. Internalization was stopped by adding 10 volumes of ice-cold sfRPMI 1640. Unbound ligand was

removed by rinsing. Receptor remaining on the surface Downloaded from was stained with PE-anti-human CCR7 and quantified by FACS. Values are the mean Ϯ SE of a minimum of three independent experiments. http://www.jimmunol.org/ by guest on September 25, 2021

of CCR7 was internalized with a t1/2 of 0.28 min following expo- arrestin 2 or arrestin 3 in the HuT 78 cells (Fig. 4). We used sure of HuT 78 cells to 200 nM of CCL21. Thus, there was a more Western blots to confirm that arrestin 2 and arrestin 3 levels rapid internalization of CCR7/CCL21, although the total amount were knocked down (Fig. 4C). When normalized to the ␤-actin of CCR7/CCL19 internalization was greater. loading control, arrestin 2 siRNA reduced levels of arrestin 2 by When a receptor internalizes, the extent of this process is a 58%, and arrestin 3 siRNA reduced levels of arrestin 3 by 75%. balance between receptor internalization and recycling. Therefore, However, arrestin 3 siRNA also reduced arrestin 2 expression we hypothesized that the enhanced rate of CCR7/CCL19 internal- levels by 96%. Independent of a reduction in the expression ization was due to a reduced rate of recycling. To test this hypoth- levels of either arrestin 2 or arrestin 3, CCR7 was internalized esis, we compared CCR7 recycling following receptor internaliza- following binding to CCL21 (Fig. 4). In contrast, the extent of tion with CCL19 or CCL21. The difference in the receptor internalization of CCR7/CCL19 was significantly reduced ( p ϭ recycling, between the two ligands, was significant ( p ϭ 0.02). We 0.0013) in comparison to cells treated with nonspecific siRNA, observed the CCL19 internalized CCR7 recycled slowly (Fig. 3), following treatment with arrestin 3 siRNA. while the CCL21 internalized CCR7 recycled within 10 min (data Although these siRNA’s have been used to specifically knock- not shown). down arrestin 2 or arrestin 3 in human cells (41, 52, 53), in the HuT 78 cells used in this study, both arrestin 2 and arrestin 3 levels Role of arrestins in CCR7 internalization were reduced by the siRNA for arrestin 3. Therefore, it was un- Receptor internalization is a key cellular function that controls the clear whether loss of internalization of CCR7/CCL19 in the arres- duration of signaling from a receptor. As such, the cellular mech- tin 3 siRNA-treated cells was due to a reduction in the levels of anisms that are activated during receptor internalization play piv- arrestin 3 alone, or due to the loss of both arrestin 2 and arrestin 3 otal roles in controlling the fate of the internalized receptor and in combined. An additional consideration was that since siRNA can T cells regulate the immune response (11). Arrestins regulate in- have off target effects (54), which can knockdown the levels of ternalization of many GPCRs, such as the ␤2 adrenergic receptor, nontargeted mRNAs in the cell, it was not clear whether the ob- the V2 Vasopressin receptor, and the angiotensin II type 1A re- served effects on CCR7/CCL19 internalization were due only to ceptor, but are not required for internalization of the human FPR, depletion of arrestins. or the PAR-1 receptor (8, 20, 48–51). Thus, we questioned whether arrestin binding regulated the differential internaliza- Characterization of CCR7 internalization in MEFs tion of CCR7 following binding to either CCL19 or CCL21. For To examine internalization of CCR7/CCL19 and CCR7/CCL21 in this purpose, we used a well-characterized combination of the complete absence of arrestins, we examined internalization in siRNAs (41, 52, 53) to knock down the expression level of the MEFs, which lack arrestin (MEF arr2Ϫ/Ϫ/arr3Ϫ/Ϫ cells). This The Journal of Immunology 4727

MEF arr2Ϫ/Ϫ/arr3Ϫ/Ϫ cell line failed to internalize the ␤2 adren- Colocalization of arrestins with CCR7 ergic receptor, which is an arrestin-dependent process (8). Since Receptor internalization has multiple important functions within MEF cells lack endogenous CCR7, we transiently transfected MEF the cell. Initially, internalization moves a receptor from an area of cells with a murine N-terminally FLAG-tagged murine CCR7. To high ligand concentration on the surface of the cell to inside the facilitate detection with the M2 anti-FLAG Ab, the murine CCR7 cell. Changes in spatial temporal localization of a GPCR can con- was fused at the N terminus to the FLAG tag (28). The MEF trol signaling from these receptors. Finally, during internalization, transient transfectants were allowed to internalize in the presence a receptor is trafficked to an area within the cell where the receptor of 200 nM ligand and assayed for receptor remaining on the sur- releases its ligands and is recycled or degraded. Generally, when a face of the cells by FACS (Fig. 5, A and B). Ligand binding did not receptor binds its cognate ligand, this binding leads to a confor- affect Ab detection of the FLAG tag, as Ն100% of the receptor mational change and receptor clustering. These clusters form sig- remained detectable on cells that were labeled with Ab, on ice, in naling domains (56) and terminate further receptor stimulation by the presence of ligand. coordinating internalization. Arrestins serve as scaffolds that re- Since internalization of the CCR7/CCL19 was blocked by cruit signaling proteins to the C-terminal domains of activated knocking down arrestin 3 expression (Fig. 4), we hypothesized that GPCRs. To determine whether activation of CCR7 could induce reconstitution of expression of arrestin 3 but not arrestin 2 would coclustering with arrestins, we transiently transfected the HuT 78 restore CCR7/CCL19 internalization in MEF arr2Ϫ/Ϫ/arr3Ϫ/Ϫ cells with GFP, arrestin 3-GFP, or arrestin 2-GFP and examined cells. Therefore, we cotransfected the MEF cells with muCCR7 the clustering of CCR7 by immunofluorescence microscopy (Fig. and GFP, or muCCR7 and arrestin 2-GFP (Arr2-GFP), or 7). The FPR was used as a control for clustering of arrestins. The Downloaded from muCCR7 and arrestin 3-GFP (Arr3-GFP). This allowed us to de- cells were plated on glass coverslips, stimulated for the indicated tect and gate on only the GFP or arrestin-GFP expressing cells periods of time with either 200 nM CCL19 or CCL21. Fixed/ during FACS for the internalization assay. MEF arr2Ϫ/Ϫ/arr3Ϫ/Ϫ stained cells were examined for clustering of human CCR7 and ϭ cells internalized 23 Ϯ 4% of CCR7/CCL21 within 30 min (Fig. arrestin. In the absence of ligand, at t 0 min, we found CCR7 5B) independent of arrestin expression, while CCR7/CCL19 failed distributed evenly over the cell surface and in the cytoplasm. to internalize in the absence of arrestin. When the MEF arr2Ϫ/Ϫ/ Following treatment of HuT 78 cells with 200 nM CCL19 or http://www.jimmunol.org/ arr3Ϫ/Ϫ were reconstituted with arrestin 3, the maximum level of CCL21, in both live (data not shown) and fixed cells (Fig. 7), we failed to detect clustering of arrestins at the cell surface during CCR7/CCL19 receptor internalized was 32 Ϯ 7%. When com- receptor internalization. In addition, unlike the FPR, which we pared with internalization of CCR7/CCL19 in the presence of GFP have shown forms multiple distinct clusters at the cell membrane alone, internalization of CCR7/CCL19 was significantly increased following ligand binding (20) (Fig. 7C), we found that CCR7 in the presence of arrestin 3, ( p ϭ 0.0051), but not in the presence moved into capped structures with CCL19 or CCL21. In these of arrestin 2 ( p ϭ 0.1532). This observation suggests that the role structures, we observed that CCR7 moved to the same region of of arrestin 3 is not redundant with the role of arrestin 2 during the cell as arrestin 3 following receptor internalization with CCL19 internalization of the CCR7/CCL19 receptor/ligand complex. In at 4 and 8 min. In addition, CCR7/CCL21 and CCR7/CCL19 co- by guest on September 25, 2021 addition, this is the first observation of ligand dependent regulation localized, weakly, with arrestin 2 at 4 min, but this colocalization of internalization of a chemokine receptor by arrestin. was lost at 8 min. In the absence of arrestin over expression (Fig. Since we have shown that GPCRs can have lowered rates and 7B), CCR7/CCL21 appeared more dispersed. We did not observe extents of internalization when over-expressed in cells that do not colocalization of either CCR7 or FPR with GFP. In ϳ10% of the endogenously express the receptor (20), we questioned whether the cells we observed movement of CCR7/CCL19 to the nucleus in minimal internalization of CCR7 in the MEF cells was due to large clusters with arrestin 3-GFP (data not shown). absence arrestin 2 or other as yet unidentified factors that are nor- mally found in hematopoietic cells. To address this question, we over-expressed CCR7 in the wild-type MEF and examined inter- Discussion nalization when CCR7 was bound to CCL19 (Fig. 5C). There was This report is the first to describe differences in the mechanisms no significant difference ( p ϭ 0.1093) between levels of CCR7 that are used by T cells to internalize CCR7 following CCL19 vs internalized in the wild-type MEF when compared with the Arr3- CCL21 ligand binding. Since mice lacking either CCL19 or GFP reconstituted MEF arr2Ϫ/Ϫarr3Ϫ/Ϫ cells (Fig. 5). CCL21 have not yet been described, it is important to distinguish the physiologically distinct roles that each ligand plays to better understand the regulation of CCR7 and downstream signaling Functional role of Arrestins in CCR7/CCL19 vs CCR7/CCL21 pathways. We observed that while both CCL19 and CCL21 are migration capable of mediating internalization of CCR7, the mechanisms controlling the internalization lead to different outcomes. In this Arrestin-3 mediates chemotaxis through the CXCR4 receptor in report, we used a human T cell line, HuT 78, which like naive T HeLa and human embryonic kidney cells (55). To determine cells expresses an endogenous CCR7. Although HuT 78 internal- whether arrestins regulate migration of T cells via CCR7, we ex- ized 76% of CCR7/CCL19, only 22% of CCR7/CCL21 was inter- amined migration of HuT 78 cells following knockdown of arres- nalized (Fig. 2). In addition, we found that while CCL21 internal- tin 2 or arrestin 3. Since our maximal internalization of ligand was ized receptor is rapidly recycled, CCL19 internalized receptor at 200 nM, we selected this ligand concentration for our studies. In recycled at a slow rate in HuT 78 (Fig. 3 and data not shown). the presence of a full complement of arrestins, the nonspecific These observations are in line with previously published results siRNA-treated cells were capable of migration to CCL19 and describing CCR7 internalization and recycling in human periph- CCL21 (Fig. 6). Similar to the effects on internalization, loss of eral blood lymphocytes (44, 45). The HuT 78 cell line, however, arrestin 2 or arrestin 3 had no effect on the migration of HuT 78 provided a model that allowed us to manipulate arrestin levels and cells to 200 nM CCL21. In contrast, depletion of either arrestin 2 examine the role of arrestins in the internalization of CCR7 and in or arrestin 3 blocked migration to CCL19, when compared with the chemotaxis of T cells. Loss of arrestin 3, but not arrestin 2 cells treated with nonspecific siRNA (Fig. 6). expression blocked internalization of CCL19/CCR7 but had no 4728 DIFFERENTIAL INTERNALIZATION OF CCR7

expressed by the high endothelial venules of lymph nodes and is found within the spleen, Peyer’s patches, the thymic medulla, and the lymphatic endothelium of many tissues (62, 63). Normally, CCR7 functions to induce migration of cells into the lymph nodes (28, 64–67). Initially, CCR7 expressing cells are exposed to CCL21 in the high endothelial venules of the lymph nodes. During this exposure, if a large number of CCR7 receptors were internal- ized, this could prevent the cell from reaching the CCL19 express- ing dendritic cells. Recently, systemic application of CCL19-Ig, FIGURE 3. CCL19 bound receptors fail to recycle. HuT 78 cells were which likely internalized most available CCR7, prevented T cells allowed to internalize CCR7 in the presence of 200 nM ligand for 30 min from forming conjugates with dendritic cells in the lymph nodes, at 37°C. Cells were rinsed three times with ice-cold RPMI 1640 at 4°C and in vivo (68). In addition, whereas in the presence of a CCL19 incubated in prewarmed sfRPMI 1640 at 37°C for the indicated times to antagonist cells were able to migrate to the lymph nodes, they allow for receptor recycling. Recycled receptor was labeled with PE-anti- failed to exit (69). These studies suggested that CCL19 likely con- human CCR7 and cells were assayed by flow cytometry. Values are the mean Ϯ SE of a minimum of three independent experiments. tributes to pathways involved in T cells exiting the lymph nodes. The signaling pathway regulated by CCL19 is currently under in- vestigation in our laboratory (L. Shannon, M. Byers, and C. Vines,

effect on internalization of CCL21/CCR7. Therefore, we were sur- manuscript in preparation). In contrast, if the cell fails to encounter Downloaded from prised to find that the association of CCR7 with either arrestin 2 or a CCL19-expressing , then the low level of internal- arrestin 3 mediated chemotaxis of HuT 78 cells to CCL19 but had ization and the high rate of CCR7 recycling promoted by the no effect on chemotaxis to CCL21 (Fig. 6). Equally important, CCR7/CCL21 receptor/ligand complex (Fig. 2 and data not using confocal immunofluorescence microscopy, we found that shown) would allow the cell to continue to traffic through the both arrestin 2 and arrestin 3 polarized or capped with CCR7 at lymph node. Since stimulation with CCL19 removes ϳ80% of the some point following internalization (Fig. 7). CCR7 from the cell surface, it is not surprising the T cell becomes http://www.jimmunol.org/ The CCR7 GPCR, like the nociceptin GPCR demonstrates li- unable to respond to the CCL21 stimulus of CCR7 following en- gand-dependent differences in receptor internalization and desen- counter with CCL19 (Fig. 2 and Ref. 61). By reducing the numbers sitization (57–60). Both GPCRs differentially regulate receptor of CCR7 receptors on the surface and desensitizing the cell to fate following ligand binding (45). The differential regulation of further stimulation with CCR7 ligands (61), CCL19 may help to receptor fate suggests that ligand specific mechanisms are acti- retain the T cell in the lymph node once it has formed a T cell/ vated at the surface of the cell when each ligand binds that rapidly dendritic cell conjugate. control ligand/receptor behavior. Unlike our studies in which both CCR7 ligands that are full agonists (61) were maximally internal- Further evidence of the differential regulation of CCR7/CCL19 ized within 8 min (Fig. 2), the nociceptin receptor required differ- and CCR7/CCL21 has been revealed in studies of receptor phos- by guest on September 25, 2021 ent ligand-dependent incubation times to mediate receptor inter- phorylation in human embryonic kidney (HEK293) cells tran- nalization. This may reflect the differential functions of the siently transfected with human CCR7. In these studies, stimulation described ligands, since agonists and antagonists were internalized of CCR7 with CCL19 led to an 83% increase in phosphorylation at different rates (57–60). of the C terminus of CCR7 over basal levels in contrast with only That CCL19 and CCL21 regulate CCR7 internalization differ- an 18% increase in phosphorylation levels when activated by ently may reflect their unique functions in the immune system. CCL21 (61). Phosphorylation of GPCRs is a prerequisite for re- Although CCL19 is expressed primarily by dendritic cells and cruitment of arrestins to the C-terminal domains of the receptors. within the T cell zone of the lymph nodes, CCL21 is constitutively In our study, we find that in T cells and in MEFs, arrestins are

FIGURE 4. Internalization of CCR7/CCL19 is blocked by arrestin 3 siRNA but not arrestin 2 siRNA. HuT 78 cells were attenuated of arrestin 2 or arrestin 3 by Oligofectamine transfection of cells with siRNA in the absence of serum for 4 h. Serum was replaced, and cells were allowed to recover in the presence of siRNA for 72 h before assay. Subsequently, cells were incu- bated in the presence of 200 nM of (A) CCL19 or (B) CCL21 for the indicated periods of time, rinsed with ice-cold serum free media, and stained with PE-anti- human CCR7 or (C) lysed and assayed by Western blot for expression of arrestin 2 or arrestin 3. A, p ϭ 0.0013 for arrestin 3 vs NS siRNA. There was no significant difference any other values. Values are the mean Ϯ SE of seven independent experiments for CCL19 and four independent experiments for CCL21. The Journal of Immunology 4729

FIGURE 5. Expression of arrestin 3-GFP in arr2Ϫ/Ϫarr3Ϫ/ϪMEF (KO) cells restored internalization of CCR7/CCL19 but did not affect internalization of CCR7/CCL21. KO (A and B) or WT and KO (C) cells were transiently transfected with ␮-FLAG-tagged CCR7 and GFP alone, Arr2-GFP, or Arr3-GFP. Cells were allowed to internalize CCR7 in the presence of 200 nM (A and C) CCL19 or (B) CCL21 for 30 min at 37°C. Cells were rinsed three times and incubated on ice with M2 anti-FLAG, rinsed, and labeled with PE-anti-mouse secondary. Cells were assayed by FACS. Values are the mean Ϯ SE of three independent experiments. Downloaded from required for internalization of the CCR7/CCL19 ligand receptor The differential phosphorylation of CCR7 following engage- combination but not of CCR7/CCL21 (Figs. 4 and 5). ment of CCL19 vs CCL21 positions the receptors for binding to For ligand internalization assays and isotype controls, 104 to unique effectors, as we observed with arrestins in this study (Figs. 2 ϫ 104 cells were assayed per sample. The flow cytometry data 3–6). How the phosphorylation of CCR7/CCL19 and CCR7/ was reported as percent of total cells to be able to directly compare CCL21 are regulated, leading to homologous desensitization, is

the peaks. There were equal maximal downshifts for both CCL19 unknown. Basal levels of phosphorylation are mediated by the http://www.jimmunol.org/ and CCL21 ligands; however, a subpopulation of CCL21-treated second messenger kinase, protein kinase C (73). Other kinases, cells showed no internalization after 8 min. These results suggest however, such as the GPCR kinases, which regulate homologous that HuT 78 cells are heterogeneous in regards to CCL21 inter- desensitization of GPCRs, have not yet been identified for CCR7. nalization, which is not surprising, since HuT 78 cells, similar to Studies are currently underway in our laboratory to begin to define primary human peripheral blood cells, are not homogeneous but the GPCR kinases that differentially regulate phosphorylation derived from an heterogeneous human lymphoma (70). In addi- of CCR7. tion, these cells are at different phases of the cell cycle and may Arrestin 2 and arrestin 3 controlled chemotaxis to CCL19 (Fig. respond differently to stimulation with CCL21 depending upon 6). This was unexpected, since CCR7/CCL19 is internalized via an their state in the cell cycle. Like the HuT 78 cells, primary cells, arrestin 3-dependent, but arrestin 2-independent, pathway. How- by guest on September 25, 2021 which are heterogeneous and found at different stages of the cell ever, this was in line with previous studies that showed that inter- cycle, internalize ϳ25% of CCL21 and 60% of CCL19 in flow nalization of a GPCR is not required for chemotaxis (74). Signal- cytometry (45). While it is beyond the scope of this study to try to ing through chemokine receptors such as CCR7 can control T cell identify different subpopulations of primary or HuT 78 cells that chemotaxis (75–77) since migration of lymphocytes can be regu- behave differently in response to CCL21, it is important to note lated through integrins, heterodimeric adhesion molecules that re- that HuT 78 cells have been used to isolate several subclones that spond to signals originating at activated GPCRs (78). Therefore, it display distinct phenotypic differences from others cells in the HuT is curious that although only arrestin 3 regulates receptor internal- 78 human T cell lymphoma line (71, 72). ization, loss of arrestin 2 or arrestin 3 affects migration only to In our studies, we used well-characterized siRNAs against ar- restin 2 and arrestin 3 in HuT 78 T cells. These siRNAs have been used successfully to knockdown arrestin 2 or arrestin 3 in human embryonic kidney cells (41). Although the transfection efficiency was ϳ90%, as measured by FACS, we were unable to specifically knock down levels of arrestin 2 or arrestin 3. The inability to knockdown only arrestin 2 or arrestin 3 in the HuT 78 cells may be due to the high level of conservation between arrestin 2 and ar- restin 3 in the regions against which the siRNAs were generated. Unlike previous studies that were able to specifically deplete ar- restin 3 with arrestin 3 siRNA, we failed to see specific depletion of arrestin 3 but also observed reduced levels of arrestin 2. This made it difficult to determine whether the effects of the siRNA were due to depletion of arrestin 3 alone or due to the concomitant loss of arrestin 2. Loss of arrestin 2 and 3 did not effect CCR7/ FIGURE 6. Knockdown of expression of arrestin 2 or arrestin 3 in HuT CCL21 internalization when compared with cells treated with non- 78 inhibits migration to CCL19 but has no effect on migration to CCL21. HuT 78 cells were nucleofected with arrestin 2 siRNA, arrestin 3 siRNA, specific siRNA (Fig. 4A). Therefore, although arrestin 3 mediated or nonspecific siRNA. Cells were allowed to recover for 72 h before assay. CCR7/CCL19 internalization, neither arrestin 2 nor arrestin 3 reg- A total of 200 nM CCL19, CCL21, or carrier (1% BSA/PBS) were trans- ulated CCR7/CCL21 internalization in T cells. In support of this ferred to the lower wells of a Neuroprobe chemotaxis chamber, and cells observation, we were able to rescue CCR7/CCL19 internalization separated by a membrane were allowed to migrate for 2 h. Migrated cells Ϫ/Ϫ Ϫ/Ϫ in MEF arr2 arr3 cells by transient transfection of arrestin 2 in the lower chamber were counted. Values are the mean Ϯ SE of three but not of arrestin 3 (Fig. 5). independent experiments. 4730 DIFFERENTIAL INTERNALIZATION OF CCR7 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 7. Internalized CCR7/CCL19 colocalizes with arrestin 3. HuT 78 cells were transiently nucleofected with either (A) arrestin-3-GFP (Arr3-GFP) alone or Arr3-GFP with the human FPR (B) arrestin-2-GFP (Arr2-GFP) alone or Arr2-GFP with the human FPR GFP alone or (C) GFP with the human FPR. The cells were then either fixed with 2% paraformaldehyde before ligand stimulation (t ϭ 0) or stimulated in the presence of 200 nM CCL19 or CCL21 or 10 nM Alexa-568-formly peptide and fixed at 4 and 8 min. Cells were permeablized in 0.1% Nonidet P-40/PBS, stained with anti-human CCR7 followed by Rhodamine anti-mouse and imaged by immunofluorescence microscopy. Figures represent images from n ϭ 3 independent experiments.

CCL19 and not to CCL21. This observation could suggest some binding to intracellular signaling proteins can be better defined by redundancy of arrestin function in mediating activation of adhe- further dissecting the proteins that are recruited by arrestins fol- sion proteins that are used for migration following activation of lowing engagement of CCR7 by its ligands, CCL19 and CCL21. CCR7. Although we and others (78, 79) have shown that GPCRs As we and others have discussed, arrestin recruitment to GPCRs can regulate integrin adhesion, the roles of arrestins in the regu- can have different consequences. With the ␤2 adrenergic receptor, lation of integrin adhesion/migration has not yet been defined. In arrestins bind to the phosphorylated form to mediate recruitment of this study, we have observed distinct roles for arrestin 3 in the receptors to clathrin-coated pits (13, 20). With the V2 Vasopressin regulation of CCR7/CCL19 internalization but not CCR7/CCL21. receptor, arrestins prevent recycling by trafficking the receptor to Arrestin 2 did not regulate the internalization of CCR7/CCL19 or the lysosome (21). In contrast, while the FPR does not use arrestins CCR7/CCL21 but along with arrestin 3 controlled migration to to mediate receptor internalization (80), we have shown that the CCR7/CCL19. Since arrestins can serve as scaffolds for many dif- FPR uses arrestins to regulate receptor recycling (20) and prevent ferent effectors proteins, we anticipate that this early difference in GPCR-mediated apoptosis (81). In these studies, arrestins form The Journal of Immunology 4731 distinct small clusters with the GPCRs during internalization and 11. Marchese, A., M. M. Paing, B. R. Temple, and J. Trejo. 2008. G protein-coupled trafficking. Therefore, it was surprising to us that, although we receptor sorting to endosomes and lysosomes. Annu. Rev. Pharmacol. Toxicol. 48: 601–629. observed large areas of colocalization that resembled capping of 12. Ferguson, S. S., W. E. Downey, III, A. M. Colapietro, L. S. Barak, L. Menard, proteins (82), we failed to detect ligand-bound CCR7 in small and M. G. Caron. 1996. Role of ␤-arrestin in mediating agonist-promoted G clusters with arrestin 2-GFP or arrestin 3-GFP either in video mi- protein-coupled receptor internalization. Science 271: 363–366. 13. Goodman, O. B., Jr., J. G. Krupnick, F. Santini, V. V. Gurevich, R. B. Penn, croscopy studies (data not shown) or in fixed cells (Fig. 7). We A. W. Gagnon, J. H. Keen, and J. L. Benovic. 1996. ␤-arrestin acts as a clathrin observed that CCR7 recruits arrestin 3 only when it is in associ- adaptor in endocytosis of the ␤2-adrenergic receptor. Nature 383: 447–450. 14. Krupnick, J. G., O. B. Goodman, Jr., J. H. Keen, and J. L. Benovic. 1997. Ar- ation with CCL19 but not with CCL21. (Fig. 7A). CCR7 bound to restin/clathrin interaction: localization of the clathrin binding domain of nonvi- CCL21 fails to cluster with arrestin 3 at 4 min and only weakly at sual arrestins to the carboxy terminus. J. Biol. Chem. 272: 15011–15016. 8 min. This in contrast to CCR7 bound to CCL19, which clusters 15. Laporte, S. A., R. H. Oakley, J. Zhang, J. A. Holt, S. S. Ferguson, M. G. Caron, and L. S. Barak. 1999. The ␤2-adrenergic receptor/␤arrestin complex recruits the with arrestin 3 at both time points. Arrestin 2 forms weak clusters clathrin adaptor AP-2 during endocytosis. Proc. Natl. Acad. Sci. USA 96: with CCR7 at 4 min. These clusters are gone by 8 min. The HuT 3712–3717. 78 cells are capable of clustering arrestins with the FPR, demon- 16. McDonald, P. H., N. L. Cote, F. T. Lin, R. T. Premont, J. A. Pitcher, and R. J. Lefkowitz. 1999. Identification of NSF as a ␤-arrestin1-binding protein: strating that these cells have the machinery to tightly cluster ar- implications for ␤2-adrenergic receptor regulation. J. Biol. Chem. 274: restins with GPCRs. In contrast, we were not able to observe the 10677–10680. same strong clusters of arrestins with CCR7 in the HuT 78 cells, 17. Luttrell, L. M., S. S. Ferguson, Y. Daaka, W. E. Miller, S. Maudsley, G. J. Della Rocca, F. Lin, H. Kawakatsu, K. Owada, D. K. Luttrell, et al. 1999. suggesting that the cellular mechanisms used by CCR7 that control ␤-arrestin-dependent formation of ␤2 adrenergic receptor-Src protein kinase internalization are distinct from the mechanisms used by the FPR. complexes. Science 283: 655–661.

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