Multiple Roles for the C-terminal Tail of the ChemokineScavenger D6*

Received for publication, December 12, 2007, and in revised form, January 10, 2008 Published, JBC Papers in Press, January 17, 2008, DOI 10.1074/jbc.M710128200

Clare V. McCulloch‡, Valerie Morrow‡, Sandra Milasta§, Iain Comerford‡1, Graeme Milligan§, Gerard J. Graham‡,Neil W. Isaacs¶, and Robert J. B. Nibbs‡2

From the ‡Division of Immunology, Infection and Inflammation, §Faculty of Biomedical and Life Sciences, ¶Department ofChemistry, Glasgow University, Glasgow G12 8TA, Scotland, United Kingdom

D6 is a heptahelical receptor that suppresses inflammationand tumorigenesis by scavenging extracellular pro-inflamma-tory CC chemokines. Previous studies suggested this is depend-ent on constitutive trafficking of stable D6 protein to and fromthe cell surface via recycling endosomes. By internalizing che-mokine each time it transits the cell surface, D6 can, over time,remove large quantities of these inflammatory mediators. Wehave investigated the role of the conserved 58-amino acid C ter-minus of humanD6, which, unlike the rest of the protein, showsno clear homology to other heptahelical receptors. We showthat, in humanHEK293 cells, a serine cluster in this region con-trols the constitutive phosphorylation, high stability, and intra-cellular trafficking itinerary of the receptor and drives greenfluorescent protein-tagged �-arrestins to membranes at, andnear, the cell surface. Unexpectedly, however, these properties,and the last 44 aminoacids of theC terminus, are dispensable forD6 internalization and effective scavenging of the chemokineCCL3. Even in the absence of the last 58 amino acids, D6 stillinitially internalizes CCL3 but, surprisingly, exposure to ligandinhibits subsequent CCL3 uptake by this mutant. Progressivescavenging is therefore abrogated. We conclude that the hepta-helical body of D6 on its own can engage the endocytoticmachinery of HEK293 cells but that the C terminus is indispen-sable for scavenging because it prevents initial chemokineengagement of D6 from inhibiting subsequent chemokineuptake.

Inflammatory stimuli induce rapid, transient production ofmany chemokines that coordinate leukocyte recruitment bysignaling through G-protein-coupled heptahelical receptors(7TMRs)3 present on circulating leukocytes and other cells.

The human chemokine family, which contains 44 members, issubdivided into four subfamilies (CC, CXC, XC, and CX3C)based on variations of a cysteine motif, and each family is typi-cally restricted to a specific group of 7TMRs. Among CC che-mokine receptors (CCRs), CCR1 to CCR5 play prominent pro-inflammatory roles, and 19 of the 26 human CC chemokines caninteract with at least one, and oftenmore, of these receptors. Thisremarkable complexityhelps ensure robust responses to a rangeofpotential pathogens (1).Resolution is a key component of normal protective inflam-

matory responses (2). This restores tissue homeostasis and pre-vents the persistent inflammation that lies at the heart of manydestructive immunopathologies and promotes tumor forma-tion (3). Resolution is aided by D6, a 7TMR related to CCR1–5,which binds 12 of the chemokine ligands for these receptors (4,5) and is expressed, at least in humans, by lymphatic endothelialcells, trophoblasts, and some leukocyte populations (6, 7). D6null mice show prolonged exaggerated responses to cutaneousinflammatory stimuli and tumor induction (8–10), enhancedleukocyte infiltration during allergic lung inflammation (11),and increased sensitivity to experimentally induced fetalresorption (7). Conversely, epidermal D6 transgene expressionsuppresses cutaneous inflammation and tumorigenesis (10).Molecular insight has come principally from studies onD6-transfected cell lines, including human embryonic kidney(HEK) 293 cells that are widely used for 7TMR studies. In thesecell lines, D6, unlike typical chemokine receptors (e.g. CCR5),can scavenge large quantities of extracellular chemokines (12,13). Thus, a simple model emerges whereby chemokine scav-enging by D6 in vivo suppresses inflammatory leukocyterecruitment by reducing chemokine levels. Consistent with thismodel, elevated levels of bioavailable D6-binding chemokinesare observed in inflamed D6 null mice (7–11).Chemokine scavenging is thought to be dependent on rapid,

constitutive trafficking of D6 to and from the cell surface. Thisenables iterative rounds of chemokine internalization throughclathrin-coated pits without the need for signaling, with che-mokine simply associating with surface D6 molecules destined

* This work was supported by the Biotechnology and Biological SciencesResearch Council (to C. V. M., G. J. G., N. W. I., and R. J. B. N.), CancerResearch UK (to V. M., I. C., and R. J. B. N.), and The Wellcome Trust (to S. M.and G. M.). The costs of publication of this article were defrayed in part bythe payment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

1 Present address: School of Molecular and Biomedical Science, University ofAdelaide, Adelaide, 5005 South Australia, Australia.

2 To whom correspondence should be addressed: Division of Immunology, Infec-tion and Inflammation, Glasgow Biomedical Research Centre, 120 UniversityPlace, Glasgow University, Glasgow G12 8TA, Scotland, UK. Tel.: 44-141-330-3960; Fax: 44-141-330-4297; E-mail: r.nibbs@clinmed.gla.ac.uk.

3 The abbreviations used are: 7TMR, heptahelical receptor; aa, amino acids;bioCCL3, biotinylated CCL3; CCL, CC chemokine ligand; CCP, clathrin-coated pit; CCR, CC chemokine receptor; CHO, Chinese hamster ovary;

CHX, cycloheximide; Ct, C terminus; DAMGO, [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin; DAPI, 4,6-diamidino-2-phenylindole; GFP, green fluores-cent protein; HEK, human embryonic kidney; HRP, horseradish peroxidase;MEF, mouse embryo fibroblast; MFI, mean fluorescence intensity; MOR-YFP, yellow fluorescent protein-tagged �-opioid receptor; PE, phyco-erythrin; RBL-2H3, rat basophilic leukemia-2H3; RFP, red fluorescent pro-tein; S-Cy3, streptavidin-Cy3; S-PE, PE-coupled streptavidin; WT, wild type;PBS, phosphate-buffered saline.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 12, pp. 7972–7982, March 21, 2008© 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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for internalization (12–14). Consequently, �95% of D6 islocated in early and recycling endosomal compartments whereit shows remarkable stability, presumably by avoiding transit tolysosomes (12). D6 trafficking differs from signaling-competentchemokine receptors (and most other 7TMRs) that typicallyreside at the cell surface and become rapidly internalized onlyafter activation by ligand. Extensive depletion of extracellularchemokines is limited by receptor desensitization and by recep-tor down-regulation which, in the case of CXC chemokinereceptor 4 and other 7TMRs, is driven by monoubiquitinationof intracellular lysines and subsequent passage to lysosomes fordegradation (15–17).The intracellular C terminus (Ct) of 7TMRs regulates ligand-

driven internalization, often by recruiting the key 7TMR regu-lators, �-arrestins (12, 18–23). �-Arrestins block G-proteincoupling, direct 7TMRs to clathrin-coated pits, control recep-tor stability (via ubiquitination), and act as scaffolds for manykey signalingmolecules (18, 23). These functions determine thenature, magnitude, and duration of signals through 7TMRs,including several chemokine receptors (18–24), and are typi-cally dependent on ligand-driven phosphorylation of the 7TMRCt. The D6 Ct diverges considerably from other chemokinereceptors (25) and may regulate the unusual trafficking behav-ior of the receptor. However, roles for �-arrestins are unclear.Internalization of untagged D6 in HEK293 cells is not inhibitedby dominant-negative �-arrestin (12). However, red fluores-cent protein (RFP)-tagged human D6 (D6-RFP) reportedly co-localizes with green fluorescent protein (GFP)-tagged �-arres-tin-1 throughout rat basophilic leukemia (RBL)-2H3 cells, andthesubcellulardistributionofD6-RFPisdescribedas�-arrestin-dependent in mouse embryo fibroblasts (MEFs) (14).Here we show that in HEK293 cells the Ct of human D6

controls phosphorylation, recycling, and stability of the recep-tor and drives constitutive association of GFP-tagged �-ar-restins with membranes only at, and near, the cell surface. Sur-prisingly, these properties are dispensable for scavenging by D6in these cells, and in our hands, the subcellular distribution ofD6-GFP and its ability to internalize CC chemokine ligand(CCL) 3 are �-arrestin-independent in MEFs. Remarkably,even in the complete absence of the Ct, D6 still internalizesCCL3 into HEK293 cells when first exposed to the chemokine.However, progressive scavenging is not possible because expo-sure to CCL3 prevents subsequent CCL3 uptake. 14 aminoacids (aa) from the membrane-proximal domain of the D6 Ctare sufficient to restore progressive scavenging, identifying theputative “eighth helix” as a critical domain in D6 function.

EXPERIMENTAL PROCEDURES

Plasmids and Receptor Ligands—Constructs encoding D6mutants were generated by PCR using a site-directedmutagen-esis kit (Stratagene), with wild-type (WT) human D6 ormutated derivatives (in pcDNA3; Invitrogen) as template. Oli-gonucleotides used are listed in Table 1. The open readingframes of all mutants were fully sequenced prior to use. Con-structs encoding GFP-tagged wt-Rab5 or Q79L-Rab5 werefromS. Ferguson (Roberts Research Institute, London, Canada)(26) and GFP-tagged wt-Rab7 and Q67L-Rab7 were from A.Wandinger-Ness (University of New Mexico, Albuquerque)

(27). Those encoding D6-GFP, yellow fluorescent protein-tagged �-opioid receptor (MOR-YFP), and GFP-tagged �-ar-restin-1 and -2 are described elsewhere (12, 28, 29). The CCL3usedwas a nonaggregatingmutant version ofmurineCCL3 (12,30). Biotinylated and radiolabeled versions of this chemokine(bio-CCL3 and 125I-CCL3, respectively) have been describedpreviously (12, 31). [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin(DAMGO) was from Sigma.Cell Culture and Transfection—HEK293 cells were main-

tained and transfected as described previously (12), as wereWTand �-arrestin null MEFs (provided by R. Lefkowitz, HowardHughes Medical Institute, Durham, NC) (28, 32, 33). Stablytransfected HEK293 pools were used throughout because tran-siently expressed D6, or D6-GFP, accumulates in cytoplasmicdeposits in these cells causing considerable cell death. MEFtransfection also led to some cell death, apparent from the pres-ence of rounded, weakly adherent fibroblasts in the cultures.Confocal imaging of these cells showed they had unusual andinconsistent distributions of D6-GFP and MOR-YFP. Thus,images were only collected from cells that retained obviousfibroblastic morphology, and weakly adherent cells wereremoved by washing prior to flow cytometric analysis.

TABLE 1Sequences of oligonucleotides used to mutate D6To generate D6-Ala6, three sequential mutational steps were required using primerpairs 1–3. K324R and K142R were introduced individually into D6-Ala6, and theK324R primers were then used to introduce this mutation into D6-Ala6 K142R,generating D6-Ala6 K142R,K324R.D6-Ala6Pair 1Sense, 5� GGC ACT GCC CAG GCC GCT TTA GCT AGC TGTTCT GAC 3�

Antisense, 5� CTC AGA ACA GCT AGC TAA AGC GGC CTG GGCAGT GCC 3�

Pair 2Sense, 5� TCC GCT TGT GCT GAG GCT GCT ATA CTT ACTGCC CAA 3�

Antisense, 5� TTG GGC AGT AAG TAT AGC AGC CTC AGC ACAAGC GGA 3�

Pair 3Sense, 5� CCT GGC ACT GCC CAG GCC GCT TTA GCT GCTTGT GCT GAG GCT GCT 3�

Antisense, 5� AGC AGC CTC AGC ACA AGC AGC TAA AGC GGCCTG GGC AGT GCC AGG 3�

D6-340Sense, 5� GCA CCT GGC ACT GCC TGA GCC TCA TTA TCC

AGC 3�Antisense, 5� GCT GGA TAA TGA GGC TCA GGC AGT GCC AGG

TGC 3�

D6-360Sense, 5� CAA GAG GAA ATG ACT TGA ATG AAT GAC CTT

GGA 3�Antisense, 5� TCC AAG GTC ATT CAT TCA AGT CAT TTC CTC

TTG 3�

D6-326Sense, 5� TAC CTG AAG GCT TTC TGA GCT GCC GTG CTT

GGA 3�Antisense, 5� TCC AAG CAC GGC AGC TCA GAA AGC CTT CAG

GTA 3�

D6-K324RSense, 5� CCG CTT CCG CCA GTA CCT GCG GGC TTT CCT GGC

TGC CGT 3�Antisense, 5� ACG GCA GCC AGG AAA GCC CGC AGG TAC TGG

CGG AAG CGG 3�

D6-K142RSense, 5� ATG AGC CTG GAC AAG GCT CTG GAG ATC GTT

CAT 3�Antisense, 5� ATG AAC GAT CTC CAG AGC CTT GTC CAG GCT

CAT 3�

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Immunofluorescence and Confocal Microscopy—D6 immu-nofluorescence staining protocols have been described in detailpreviously (6, 12). Cy3-coupled anti-mouse IgG antibodies(Sigma) were used to detect anti-D6 antibodies. Images werecaptured using a Leica SP-2 confocal microscope configuredwith Leica confocal software, with a �40 or �63 oil immersionobjective and digital zoom. Fluorochromes were excitedsequentially with lasers at 488 nm (GFP) or 543 nm (Cy3), andwith a UV laser to excite 4,6-diamidino-2-phenylindole (DAPI)when appropriate. Images were superimposed using Leica con-focal software and assembled using ThumbsPlus (Cerious Soft-ware). In all experiments at least 10 fields of cells were exam-ined and representative images collected, with each imageshown being one from a stack of up to five serial z-sectionimages spanning the entire cell.D6 Detection (Surface and Total)—Surface and total D6

expression was determined by flow cytometry and Westernblotting, respectively, using anti-D6 antibodies as described(12). For analysis of MEFs, cells were incubated with mouse Fcblock (BD Biosciences) prior to addition of anti-D6 antibodies.In all flow cytometry experiments, control samples were pre-pared in which the primary antibody (anti-D6) was omitted.The mean fluorescence intensity (MFI) of these samples wassubtracted from the test data. Anti-D6 antibodies weredetected with phycoerythrin (PE)-coupled anti-mouse IgG(Sigma) and horseradish peroxidase (HRP)-coupled anti-mouse IgG (Amersham Biosciences) in flow cytometry andWestern blot protocols, respectively. Western blots were alsoprobedwithmouse anti-actin (Abcam) used at 1:2,000 dilution.Phosphate Labeling and D6 Immunoprecipitation—�4 �

106 cells were incubated in 8 ml of phosphate-free medium for1 h at 37 °C; radiolabeled phosphate (2.5 mCi/ml; AmershamBiosciences) was added, and the cells were incubated at 37 °Cfor a further 3 h. After washing in ice-cold PBS, cells were lysedin 500 �l of CellLytic buffer (Sigma), and the lysate was cleared(20,000 � g, 4 °C, 10 min). D6 was then immunoprecipitatedusing anti-D6 antibodies as described (34).D6 Stability Studies—Receptor stability studies were done as

described previously (12). Briefly, a series of cultures each con-taining an equivalent number of cells (usually 106) were treatedwith or without cycloheximide (CHX; Sigma) (20�g/ml). Up to24 h after CHX addition, cell lysates were prepared and ana-lyzed by Western blotting.BioCCL3 Scavenging Assay—5 � 105 cells were incubated in

1 ml of complete medium containing 50 nM bio-CCL3 at 37 °Cand 5% CO2. 20 �l samples of the medium were removed overtime, added to 20 �l of Laemmli buffer (Sigma), boiled for 5min, and analyzed by Western blotting. Blots were blockedovernight in 10% milk/PBS; bioCCL3 was detected using HRP-streptavidin (Dako) in PBS, 0.1% Tween, developed usingWestPico (Pierce), and exposed to x-ray film.BioCCL3 Tetramer Uptake—BioCCL3/streptavidin-PE (bio-

CCL3/S-PE) tetramers were prepared using 250 ng of bioCCL3and 3 �g of S-PE and used as described (12). Briefly, up to 106cells were resuspended in 50�l of HEK293mediumplus 10mMHEPES (pH 7.4) containing bio-CCL3/S-PE tetramers or 3 �gof S-PE alone, and incubated at 37 °C for 1 h with regular gentleagitation. Cells were washed in ice-cold FACS buffer (PBS plus

2% fetal calf serum) and analyzed on a FACScan flow cytometer(BD Biosciences). For each transfected cell type assessed,untransfected or mock-transfected cells, treated with bio-CCL3/S-PE tetramers or S-PE alone, were used as controls. Onoccasion, adherent cultured MEFs growing in 1 ml of mediumwere incubated for 1 h at 37 °C in situ with bioCCL3/streptavi-din-Cy3 (bioCCL3/S-Cy3) tetramers (prepared with 500 ng ofbioCCL3 and 6 �g of S-Cy3), washed, fixed, and visualized byconfocal microscopy.

125I-CCL3 Binding, Uptake, and Processing—These assayswere done as described elsewhere (12, 25, 31). Briefly, to assessCCL3 binding to receptor, cells were incubated in 6 nM 125I-CCL3 in the presence or absence of unlabeled competitorCCL3(at various concentrations) at 4 °C for 90 min. After washingwith ice-cold PBS, the amount of radiolabel associated with thecells was determined and compared with that bound to cells inthe absence of unlabeled CCL3 competitor. For uptake experi-ments, cells were surface-loaded in 12 nM 125I-CCL3 at 4 °C for1 h, washed, shifted to 37 °C for 10 min, and then washed againin either PBS or acid (0.2 M acetic acid, 0.5 M NaCl), both ice-cold, for 5 min. The proportion of radiolabel that became acid-resistant after a shift to 37 °C (indicative of internalization) wasdetermined. To analyze intracellular CCL3 processing, cellswere surface-loaded in 12 nM 125I-CCL3 at 4 °C for 1 h, washed,and shifted to 37 °C.Up to 150min later,mediumand cells wereharvested; the medium was subjected to precipitation in12.5% trichloroacetic acid at 4 °C, and the proportion ofradiolabel in the cell pellet, trichloroacetic acid-precipitable(intact 125I-CCL3 or peptide fragments thereof), and trichlo-roacetic acid-nonprecipitable (degraded) fractions wasdetermined. In all assays, each time point or condition wasperformed in triplicate.Helical Prediction—The entire intracellular C-terminal tail

of D6 (from four species) and human CCR1–5 were analyzedfor putative helical content using AGADIR software (35).Statistics—Data were analyzed using GraphPad Prism soft-

ware applying unpaired t tests.

RESULTS

�-Arrestins Localize to the Cell Periphery in D6-expressingHEK293 Cells—GFP-tagged �-arrestin-1 is reported to co-lo-calize with D6-RFP throughout transfected rat RBL-2H3 cells(14). We investigated the impact of untagged human wtD6expression on �-arrestin distribution in human HEK293 cells.In parental HEK293 cells, GFP-tagged versions of �-arrestin-1or -2 were found throughout the cytosol (Fig. 1A). In contrast,inHEK293 cells expressingwtD6, although some green fluores-cence remaineduniformly distributed throughout the cytosol, asignificant proportion was concentrated at the cell surface andassociated with vesicles predominantly at, or just beneath, theplasma membrane (Fig. 1, B–I). This was clearly visible in opti-cal sections taken through the middle (Fig. 1, B, D, and E), top(Fig. 1C), or bottom (Fig. 1, F–I) of the cells. GFP-tagged �-ar-restins showed some co-localization with D6 at these locations,although the receptorwas of very low abundance inmany of the�-arrestin� vesicles and the majority of cellular D6 remained�-arrestin-free (Fig. 1, F–I). The peripheral localization of�-ar-restins in wtD6-expressing HEK293 cells was not detectably

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altered by incubation with CCL3. Thus, in HEK293 cells, D6mediates the re-localization of�-arrestins exclusively to the cellperiphery.Loss of Constitutive D6 Phosphorylation Prevents �-Arrestin

Re-localization—Activated 7TMRs associate with �-arrestinsprincipally via phosphorylated Ct tails. The human D6 Ct har-bors a serine-rich domain that is conserved inD6 proteins fromother mammals (Fig. 2A). We previously reported that wtD6 isconstitutively phosphorylated in murine L1.2 cells (34). This isalso the case in HEK293 cells; anti-D6 immunoprecipitatesfrom 32P-labeledHEK293 cells only contained a labeled proteinof equivalent molecular weight to D6 when the cells expressedwtD6 (Fig. 2B). As in L1.2 cells (34), there was no quantitativechange in phosphorylation after CCL3 exposure (data notshown). We hypothesized that constitutive phosphorylation ofthe serine-rich motif in the D6 Ct is responsible for re-localiz-ing �-arrestins in HEK293 cells. To test this, we mutated all sixserines in the serine-rich motif to alanine (D6-Ala6) (Fig. 2A)and expressed the mutant stably in HEK293 cells. Flow cytom-etry revealed that, as with wtD6-expressing HEK293 cells,�95% of cells expressed surface D6-Ala6. However, wtD6 was�2.5-fold more abundant on the surface than D6-Ala6 (Fig.2C), and a similar difference in expression level was alsoobserved when the total cellular pool was analyzed byWesternblotting (Fig. 2D). These data indicated that the surface/cyto-plasmic ratio of D6 was not markedly affected by mutation ofthe six serines to alanines, and consistent with this, D6-Ala6,like wtD6 (12), was abundant in vesicles throughout the cyto-plasm (Fig. 2E). Phosphate labeling studies showed thatD6-Ala6 had lost the constitutive phosphorylation present onwtD6 (Fig. 2F). This is consistent with the noticeable reductionin apparent molecular weight of D6-Ala6 compared with wtD6

byWestern blotting (Fig. 2D). Significantly, D6-Ala6 was com-pletely unable to drive GFP-tagged �-arrestins to the cellperiphery (Fig. 2G), and this was not altered in the presence ofD6 ligand, CCL3.To investigate this further, two truncated D6 variants were

expressed stably in HEK293 cells. These were D6-360, lackingthe last 24 aa but retaining the serine-rich domain, andD6-340,in which the last 44 aa are removed to include the serine-richdomain (Fig. 2A). D6-360 was less well expressed thanwtD6 (tolevels equivalent to D6-Ala6), and whereas �95% of cellsexpressed surface D6-340, there was a considerable reductionin the quantity of total and surface D6-340 expressed (Fig. 2,Hand I). However, as withD6-Ala6, the surface/cytoplasmic ratiowas not dramatically affected by truncation, and the truncatedproteins were present in vesicles throughout the cytoplasm(Fig. 2J). Consistent with our D6-Ala6 analysis, D6-340 was notdetectably phosphorylated (data not shown) and was unable todrive �-arrestin-2GFP accumulation at the cell surface,whereasD6-360 behaved likewtD6 (Fig. 2K). Collectively, thesedata indicate that constitutive phosphorylation ofD6, requiringthe serine cluster in the Ct, mediates �-arrestin re-localizationto membranes at the cell periphery in HEK293 cells.The Serine-rich Domain of the D6 Ct Regulates Receptor Sta-

bility and Intracellular Trafficking Decisions—wtD6 is very sta-ble in HEK293 cells, with little change in protein level in cellstreated for 24 h with the protein synthesis inhibitor CHX (12).The mutants we had analyzed, particularly D6-340, were lesswell expressed in HEK293 cells than wtD6 so we examinedwhether receptor stability had been compromised by mutation(Fig. 3). This revealed that D6-360 had equivalent stability towtD6, but that D6-340 and D6-Ala6 were considerably less sta-ble and had nearly completely disappeared within 4 h of CHXtreatment (Fig. 3A).Loss of stability might indicate a preference for D6 mutants

to traffic to lysosomes after internalization (via early then lateendosomes), rather than passing through the early and recy-cling endosomal compartments like wtD6 (12). To investigatethis, we introduced GFP-tagged versions of wt-Rab5 and wt-Rab7 (which mark early and late endosomes, respectively) intocells expressing wtD6 or D6-Ala6, detected by immunofluores-cence (Fig. 3B). Consistent with our previous work (12),wt-Rab5-GFP showed extensive co-localization with the intra-cellular pool of wtD6. D6-Ala6, however, showedmarkedly lessco-localization with wt-Rab5-GFP. Conversely, wt-Rab7-GFPshowed minimal co-localization with wtD6 but often co-local-ized with D6-Ala6� vesicles. Next, we overexpressed constitu-tively active GFP-tagged forms of Rab5 and Rab7 (Q79L-Rab5and Q67L-Rab7) that disrupt protein passage through early orlate endosomes, respectively. Proteins that normally trafficthrough these compartments should co-localize extensivelywith these mutant Rabs. Expression of Q79L-Rab5-GFP led tothe accumulation of both wtD6 andD6-Ala6 in enlargedGFP�vesicles (Fig. 3C). However, whereas wtD6 rarely co-localizedwith Q67L-Rab7-GFP, D6-Ala6 showed extensive associationwith vesicles carrying this mutant Rab (Fig. 3C). D6-340, theother mutant with low stability, behaved like D6-Ala6, whereasD6-360 was like wtD6. Thus, the serine cluster, in addition tomediating �-arrestin localization to the cell periphery, is

FIGURE 1. wtD6 induces the re-localization of �-arrestins to membranesat and near the cell surface in HEK293 cells. Confocal images of parentalHEK293 cells (A) or HEK293 stably expressing wtD6 (B–I) transiently express-ing GFP-tagged �-arrestin-1 (D) or -2 (A–C and E–I). F–I, fixed, permeabilizedcells were stained with anti-D6 antibodies detected with Cy3-coupled anti-mouse IgG antibodies (red). Yellow fluorescence indicates D6/�-arrestin co-localization. Images shown, selected from z-section stacks, are optical sec-tions from the middle region of the cells (A, B, D, and E) or from near the cellsurface (C (top of cells); F–I, base of cell, where it attached to glass slide). E andF are magnified images from B and I, respectively. The white bars represent 20�m. Data are representative of 10 –15 images collected per experiment, andeach experiment was repeated on at least four occasions.

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required to prevent wtD6 fromentering Rab7� late endosomes,ensuring repeated recycling to thecell surface rather than degradation.The Reduced Stability of D6-Ala6

Is Dependent on Intracellular LysineResidues—Chemokine receptorsand other 7TMRs can be targeted tolysosomes by ubiquitination oflysine residues (15–17). There aretwo intracellular lysines in D6-Ala6and D6-340 that are conservedacross mammalian D6 sequences.These are at aa 324 and, interest-ingly, aa 142 within the DKYLEIVmotif unique to D6. These residuesin D6-Ala6 were mutated, individu-ally or together, to arginine to retaincharge but lose ubiquitinationpotential. Remarkably, either muta-tion alone was sufficient to reversethe low stability of D6-Ala6 (Fig.3D). Identical mutations in D6-340were equally effective at restabiliz-ing this molecule (data not shown).Although we have not been able toreproducibly detect ubiquitinationof D6-Ala6 or D6-340, our data sug-gest that preventingD6phosphoryl-ation allows ubiquitination of lysineresidues and subsequent traffickingto lysosomes for degradation.Chemokine Scavenging Does Not

Require Constitutive Phosphorylationor the Last 44 Amino Acids—Highreceptor stability, recycling, and therecruitment of �-arrestins to thecell surface might be anticipated tocontribute to effective chemokinescavenging byD6.However, the sur-face/cytoplasmic ratios of D6-Ala6and D6-340 were broadly similar towtD6 andD6-360, indicating that allmutants, like wtD6, were capable ofconstitutive internalization. Thus,we next compared the ability ofwtD6, D6-360, D6-340, and D6-Ala6 to scavenge chemokines incontinuous culture over time (Fig.4). Remarkably, despite their clearbiochemical differences, all mutantswere as effective as wtD6 at scav-enging bioCCL3, with only someslight slowing of scavenging notice-able by D6-340. Thus, in HEK293cells, residues after aa 340, includingthe serine-rich domain and thecharged extreme end of the mole-

FIGURE 2. Constitutive D6 phosphorylation controls �-arrestin re-localization in HEK293 cells. A, human D6sequence from the end of the 7th transmembrane domain (end of TM7). The positions of truncation (D6-340 andD6-360), and serines mutated to alanine in D6-Ala6, are indicated. B, autoradiograph of a dried polyacrylamide gel ofanti-D6 immunoprecipitates from HEK293 cells (expressing wtD6 or no D6) preloaded with 32P-labeled phosphate.The arrow represents the predicted location of wtD6, calculated using protein markers run on the gel. C and H,average (�S.D.) (n � 3) flow cytometric MFI values of HEK293 cells stably expressing the indicated proteins stainedwith anti-D6 antibodies (detected with PE-coupled anti-mouse IgG antibodies). D and I, autoradiographs of Westernblots of whole cell lysates of HEK293 cells stably expressing the indicated proteins, probed with anti-D6 (�-D6) oranti-actin (�-actin) (detected with HRP-coupled anti-mouse IgG antibodies). E and J, confocal images of fixed, per-meabilized HEK293 cells stably expressing the indicated proteins immunofluorescently stained using anti-D6 (�-D6)detected with Cy3-coupled anti-mouse IgG antibodies (red). In some images, nuclei are stained with DAPI (blue).F, autoradiographs of an anti-D6 Western blot (upper panel) and a dried polyacrylamide gel (lower panel) of anti-D6immunoprecipitates from HEK293 cells (expressing the indicated proteins, or no D6) preloaded with 32P-labeledphosphate. Arrowheads represent the predicted location of the D6 proteins, calculated using protein markers run onthe gels. G and K, confocal images of fixed parental HEK293 cells (No D6) or HEK293 stably expressing the indicatedD6 variants and transiently expressing GFP-tagged �-arrestin-2. Confocal images shown, selected from z-sectionstacks, are optical sections from the middle region of the cells (E, G, J, and K, upper images only), or from near the cellsurface (K, lower wtD6 and D6-360 images only). White bars in E, G, J, and K indicate 20�m. All data are representativeof experiments repeated at least three times. *, p � 0.05; **, p � 0.01.

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cule, are dispensable for D6 inter-nalization and progressive chemo-kine scavenging.GFP-tagged D6 Distributes Simi-

larly within Wild-type and �-Arres-tin Null Fibroblasts and MediatesCCL3 Uptake—The results abovesuggested that D6 trafficking is�-arrestin-independent in HEK293cells, consistent with our previousobservations using dominant-nega-tive �-arrestin constructs (12). Toinvestigate this further, we tran-siently expressed D6-GFP (D6 witha Ct GFP tag (12)) in WT and �-ar-restin null MEFs. Confocal micros-copy of fixed adherent fibroblastsrevealed that D6-GFP was predom-inantly localized inside these cells,irrespective of their genotype (Fig.5A). Also, using flow cytometry andanti-D6 antibodies, WT and �-ar-restin null MEFs expressing equiva-lent quantities of D6-GFP displayedsimilar levels of surface receptor(Fig. 5B). Thus, the characteristicdistribution of D6-GFP seen inHEK293 (12) is also seen in MEFsand is unaffected by the absence of�-arrestins. In addition, adherentWT and �-arrestin null MEFsexpressing D6-GFP were able todirect internalization of bioCCL3/S-Cy3 tetramers, visualized by opti-cal sectioning of D6-GFP� cells by

confocal microscopy (Fig. 5A). S-Cy3 alone was not internal-ized by D6-GFP-transfected or untransfected MEFs (data notshown).To quantify BioCCL3 tetramer uptake, WT and null MEFs

were transfected with D6-GFP, fed bioCCL3/S-PE tetramers orS-PE alone at 37 °C, and analyzed by flow cytometry (Fig. 5C).BioCCL3 tetramers, but not S-PE alone, were readily internal-ized into both cell types, in a manner directly proportional tothe level of expression of D6-GFP. By assessing WT and nullMEFs gated for equivalent expression of D6-GFP, it was clearthat the absence of �-arrestins did not compromise bioCCL3tetramer uptake (Fig. 5D).As controls, MEFs transiently expressing the �-arrestin-de-

pendent MOR-YFP were imaged with or without exposure toits agonist DAMGO (Fig. 5E). Unlike D6-GFP, MOR-YFP islocated principally on the surface of MEFs, independent of cellgenotype, but its activation only leads to its intracellular accu-mulation in WTMEFs.The Membrane-proximal Domain of the Ct Is Sufficient to

Allow the Heptahelical Body of D6 to Mediate ContinuousCCL3 Scavenging—D6-340 carries a short Ct that could con-ceivably coordinate trafficking events required for scavenging.Thus, we generated another D6 mutant, D6-326, which lacks

FIGURE 3. Unphosphorylated D6 mutants traffic to late endosomes and show reduced stability depend-ent on intracellular lysine residues. A and D, autoradiographs of Western blots (probed with anti-D6 anti-bodies; detected with HRP-coupled anti-mouse IgG) of cell lysates of HEK293 cells (expressing the proteinsindicated) that had been treated for 0, 2, 4, and 8 h (in A) or 0 and 8 h (in D) with 20 �g/ml CHX. B and C, confocalimages of fixed, permeabilized HEK293 cells stably expressing wtD6 or D6-Ala6 (detected with anti-D6 anti-bodies and Cy3-coupled anti-mouse IgG secondary antibodies (red)) and transiently expressing GFP-taggedRab5 or Rab7 proteins (WT or constitutively active (Q79L Rab5; Q67L Rab7)). Yellow fluorescence indicatesco-localization of D6 and Rab protein. Nuclei are stained with DAPI (blue) in most images. Images shown,selected from z-section stacks, are optical sections from the middle region of the cells. The white bars represent20 �m. Two repeat experiments yielded similar datasets.

FIGURE 4. Constitutive phosphorylation, high stability, exclusive traffick-ing through recycling endosomes, and the re-localization of �-arrestinsare dispensable for chemokine scavenging by D6. Autoradiographs ofWestern blots of aliquots of medium taken from cultures of 5 � 105 HEK293cells (stably expressing the indicated proteins) incubated in the presence of 2ml of 50 nM bioCCL3. BioCCL3 was detected using HRP-coupled streptavidin.Data are representative of three repeat experiments.

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the Ct from the point at which it diverges from other chemo-kine receptors, therefore ending with the aa KAF.Inmany respects, D6-326 behaved like D6-340 (Fig. 6). Total

and surface D6-326 expression was lower than that seen inwtD6 transfectants (but similar to cells expressing D6-340 (Fig.2, H and I)). The surface/cytoplasmic ratio seemed unaffectedby truncation indicating that constitutive trafficking wasretained (Fig. 6, A and B). Consistent with this, considerableamounts of D6-326 were inside cells (Fig. 6C). Like D6-340,D6-326 failed to drive �-arrestin-2GFP to the cell periphery(Fig. 6D), co-localized to some extent with wt-Rab7-GFP, occa-sionally with wt-Rab5-GFP, and extensively with Q79L-Rab5-GFP and Q67L-Rab7-GFP, and it was less stable than wtD6(data not shown). D6-326 had a similar affinity for CCL3 aswtD6 (according to 125I-CCL3 displacement assays performed

at 4 °C (Fig. 6E)) and was able tointernalize surface-loaded 125I-CCL3 in short term uptake assays,albeit less effectively than wtD6 butsimilar to D6-340 (Fig. 6F). Little ofthis internalized 125I-CCL3 wasreleased from cells in a trichloroace-tic acid-precipitable intact form andwas either degraded prior to releaseor retained inside the cells (Fig. 6G).Surprisingly however, despite all thesimilarities to D6-340, D6-326 wasunable to mediate progressivedepletion of bioCCL3 in continuousculture, with only some initialreduction in bioCCL3 levels beingobserved at 2 h (Fig. 6H).From these data, we hypothesized

that D6-326 was initially competentfor CCL3 uptake but that chemo-kine incubation prevented continu-ous CCL3 scavenging. To investi-gate this, we incubated wtD6 orD6-326 expressing cells with CCL3for 1 h, washed the cells extensively,and then examined their ability tomediate CCL3 tetramer uptake (Fig.6I). As we observed previously,CCL3 incubation enhances surfaceexpression of wtD6 (12) and its abil-ity to internalize bioCCL3/S-PE tet-ramers. In contrast, CCL3 incuba-tion completely abrogated theability of D6-326 to internalizeCCL3 tetramers, despite no signifi-cant change in surface receptorexpression levels. Thus, the mem-brane-proximal region (aa 326–340)of theD6Ct is sufficient to allow con-tinuous chemokine scavenging by thebody of the receptor, whereas dele-tion of the entireD6Ct creates amol-ecule whose chemokine scavenging

capabilities are limited by incubation with chemokine.

DISCUSSION

D6 scavenges extracellular pro-inflammatory CC chemo-kines and suppresses inflammation and tumorigenesis. Herewehave shown that in human HEK293 cells the Ct of human D6controls the constitutive phosphorylation, high stability, intra-cellular trafficking itinerary, and chemokine scavenging prop-erties of the receptor and drives GFP-tagged �-arrestins tomembranes at, or near, the cell surface. Surprisingly, however,the D6 Ct is only indispensable for scavenging because it pre-vents chemokine engagement of the heptahelical D6 body frominhibiting subsequent chemokine uptake.Transfected humanwtD6 is constitutively phosphorylated in

HEK293 cells, as it is in L1.2 cells (a murine B cell line) (34). In

FIGURE 5. Subcellular distribution of D6-GFP, and ability to internalize CCL3, is �-arrestin-independent.A, confocal images of adherent, fixed WT or �-arrestin-1/2 null MEFs transiently expressing D6-GFP. D6-GFPprotein is shown in the two left-hand images. Internalized bioCCL3/S-Cy3 tetramers (red) and nuclei (blue) areseen in the right two images. B, representative flow cytometric profiles of WT or �-arrestin-1/2 null MEFstransiently expressing D6-GFP. D6-GFP has been detected with anti-D6 antibodies and PE-coupled anti-mouseIgG (right two panels). In the left two panels, the anti-D6 antibody was not added. C and D, uptake in 1 h ofbioCCL3/S-PE tetramers, or S-PE alone, by WT or �-arrestin-1/2 null MEFs transiently expressing D6-GFP.C, representative flow cytometric profiles. D, average (�S.D.) (n � 4) flow cytometric MFI values for uptake ofbioCCL3/S-PE tetramers, or S-PE alone, of WT or �-arrestin-1/2 null MEFs expressing equivalent levels of D6-GFP(gated as shown in B). NS, not significant. E, confocal images of adherent, fixed WT, or �-arrestin-1/2 null MEFstransiently expressing MOR-YFP incubated with or without 10 �M DAMGO for 30 min. White bars indicate 10�m. All images shown, selected from z-section stacks, are optical sections from the middle region of the cells.Data are representative of experiments repeated on three separate occasions.

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HEK293 cells, this is dependent on a cluster of serine residues inthe Ct, which are also required to direct �-arrestins to the cellsurface. Based on precedent with many other 7TMRs (18, 23),the simplest interpretation of these data is that the serines in the

Ct cluster are themselves the targetof phosphorylation and that thispermits direct physical interactionwith �-arrestin. If so, the localiza-tion of �-arrestins is likely todirectly correlate with the localiza-tion of phosphorylated D6, i.e. at orjust under the cell surface, indicat-ing the following: (i) wtD6 under-goes transient phosphorylation as ittransits the cell surface, and (ii) onlya small fraction of the total cellularwtD6 protein is phosphorylated atany one time. It appears as though nospecific serine residue from withinthe cluster is required becausemutants inwhich only the 1st, 2nd, or3rd two serines, or the first or last fourserines, were mutated to alaninebehaved like wtD6 in all assays (datanot shown).

�-Arrestin re-localization wasnot required for D6 internalizationin HEK293 cells, but interestingly,all mutated versions of D6 unable tore-localize �-arrestins (i.e.D6-Ala6,D6-340, andD6-326) showedmark-edly reduced stability and were ableto traffic to late endosomes. Thereduced stability of these mutantswas dependent on either of twolysine residues of the intracellularsurface of D6, including the one inthe unique DKYLEmotif in the sec-ond intracellular loop. Although wehave been unable to reproduciblydemonstrate ubiquitination ofunphosphorylated mutants, ourdata are consistent with a model inwhich D6 phosphorylation and thepresence of �-arrestins at the cellsurface interferes with D6 ubiquiti-nation and subsequent trafficking tothe late endosomal compartment enroute to lysosomes. D6 may there-fore use �-arrestins to enhancereceptor stability rather than directinternalization.Constitutive phosphorylation,

high stability, and exclusive traffick-ing through recycling endosomeswere all dispensable for effectivechemokine scavenging by D6 inHEK293 cells. However, the regula-

tory potential of these phenomena may have been masked bythe very high levels of D6 transcription in our transfected celllines. The fact that D6-340 was nearly as effective as wtD6 inscavenging assays (despite being 5–6-fold less abundant) indi-

FIGURE 6. D6 lacking the entire Ct can still internalize CCL3 but is unable to mediate progressive CCL3scavenging. A, average (�S.D.) (n �3) flow cytometric MFI values of HEK293 cells stably expressing wtD6 or D6-326stained with anti-D6 antibodies (detected with PE-coupled anti-mouse IgG antibodies). B, autoradiograph of West-ern blot of whole cell lysates of HEK293 cells stably expressing wtD6, D6-326, or “No D6,” probed with anti-D6 (�-D6)or anti-actin (�-actin) (detected with HRP-coupled anti-mouse IgG antibodies). C, confocal image of fixed, perme-abilized HEK293 cells expressing D6-326 immunofluorescently stained using anti-D6 (�-D6) detected with Cy3-coupled anti-mouse IgG antibodies. D, confocal image of fixed HEK293 cells stably expressing D6-326 and tran-siently expressing GFP-tagged �-arrestin-2. Images shown in C and D, selected from z-section stacks, are opticalsections from the middle region of the cells: white bars represent 20 �m. E, radioligand (125I-CCL3) displacementcurves of HEK293 cells stably expressing wtD6 or D6-326. Each data point in the mean (�S.D.) of triplicate repeats.F, proportion of surface-loaded 125I-CCL3 internalized by HEK293 cells expressing wtD6, D6-326, or D6-340 after 10min at 37 °C, as defined by the proportion of cell-associated radiolabel that becomes resistant to acid wash after shiftto 37 °C. G, processing of internalized 125I-CCL3 by HEK293 cells expressing wtD6, D6-326, or D6-340. 125I-CCL3-loaded cells were washed, and at the times indicated, radioactivity remaining in the cells was determined (Cell pellet,black), whereas supernatant was subjected to trichloroacetic acid precipitation to determine the presence of intact(trichloroacetic acid precipitable (TCA), gray) or degraded (nontrichloroacetic acid (non-TCA)-precipitable, white)125I-CCL3. The mean (�S.D.) (n � 3) proportion of radiolabel present in each of these fractions is presented. H,autoradiographs of Western blots of aliquots of medium taken from cultures of 5�105 HEK293 cells (stably express-ing the indicated proteins) incubated in the presence of 2 ml of 50 nM bioCCL3. BioCCL3 was detected using HRP-coupled streptavidin. I, surface D6 and bioCCL3/S-PE tetramer uptake of HEK293 cells expressing wtD6 or D6-326preincubated for 1 h in 50 nM CCL3. Data are expressed relative to results obtained with cells that did not receiveCCL3 preincubation. Tetramer internalization proceeded for 1 h at 37 °C prior to analysis by flow cytometry. SurfaceD6 levels were measured by incubation with anti-D6 antibodies (detected with PE-coupled anti-mouse IgG), all at4 °C, followed by flow cytometry. The mean (�S.D.) of triplicate repeats is presented. All data shown are represent-ative of experiments done on at least three occasions. *, p � 0.05; **, p � 0.01; ***, p � 0.001; NS, not significant.

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cates that the level of receptor expression was not particularlyrate-limiting in our wtD6 transfectants. In cells expressingmuch lower levels of D6, a small change in stability may have amore profound impact on scavenging potential. Moreover, D6stability will influence the effectiveness of transcriptional regu-lation of D6.We have shown that inhibiting production of newprotein for as long as 24 h has minimal impact on phosphoryl-ated D6 levels, whereas 4 h is sufficient to nearly completelyremove unphosphorylated D6. Thus, by controlling D6 phos-phorylation in concert with its transcription, it should be pos-sible to effectively manipulate D6 protein levels to modulatescavenging. This possibility needs to be explored in cellsexpressing endogenous wtD6.Our observations contrast those of Galliera et al. (14). First,

they reported that all RFP-tagged human D6 (D6-RFP) co-lo-calized with GFP-tagged �-arrestin-1 in RBL-2H3 cells, per-haps indicating that phosphorylation of human D6 is differentin this rat basophilic leukemia cell line. However, it should benoted that in the images presented, GFP-tagged �-arrestin-1did not appear to be specifically associated with D6-RFP� ves-icles, and green fluorescence from these vesicles was equivalentto that seen in the surrounding cytosol. Second, wtD6 wasnot detectably phosphorylated in Chinese hamster ovary(CHO)-K1 cells. Cell background might influence the ability ofD6 to be phosphorylated as it transits the cell surface, and it willbe of interest to examine the stability of wtD6 in CHO-K1 cellsand its ability to drive �-arrestin re-localization. However, it isnotable that the amount of receptor successfully immunopre-cipitated was not shown (14), and low level phosphorylationmay have been missed. Third, the charged domain carried inthe last 28 aa of D6 was required for its internalization intoCHO-K1 cells (gauged by antibody uptake) (14), but we havefound that it is dispensable for internalization and scavengingin HEK293 cells. Cell type and its species of origin may beresponsible for this difference. For example, the charged end ofhuman D6 may be required for successful engagement of theendocytotic machinery of ovarian hamster cells. It may also becapable of driving endocytosis in human HEK293 cells, butother regions can clearly compensate for its absence. The sameargument could be made for D6-driven �-arrestin re-localiza-

tion; this phenomenon may havethe potential to contribute to D6endocytosis in some scenarios, butit can clearly be compensated for inHEK293 cells by membrane-proxi-mal Ct motifs or determinants inthe D6 body. This putative redun-dancy makes some sense; multiplealternative internalization mecha-nisms could conceivably provideflexibility to ensure robust chemo-kine scavenging by D6 in a range ofdifferent cellular environments.In our hands, the subcellular dis-

tribution of D6-GFP, and its capac-ity to internalize bioCCL3 tetram-ers, is independent of �-arrestins inMEFs. However, Galliera et al. (14)

reported that D6-RFPwas only found on the surface of�-arres-tin null MEFs and that �-arrestin-1-GFP could drive its redis-tribution to intracellular locations, an effect dependent on thelast 28 aa of D6. These contradictory observations are difficultto reconcile. However, itmay be significant thatwe imaged cellsafter adherence to fibronectin-coated coverslips for at least24 h, whereas Galliera et al. (14) plated cells onto glass 1 h priorto imaging. We have observed rounded MEFs with minimalfibroblastic morphology in our transfected MEF cultures, andthese cells show variable patterns ofD6-GFPdistribution, occa-sionally with strong surface expression. An alternative explana-tion is that the use of different fluorescent tags may influencethe conformation of theD6Ct to alter the specific determinantsrequired for internalization. Although these conflicting obser-vations require future resolution, it is nonetheless clear that inhuman HEK293 cells untagged human D6 can effectively scav-enge CCL3 in amanner unaffected by dominant-negative �-ar-restins (12), and independently of its ability to redistribute�-arrestins.

Despite the differences between our study and that of Gal-liera et al. (14), we agree thatWThumanD6has the potential toconstitutively drive the re-localization of �-arrestins. The pos-sible implications of this for D6 have been discussed above, butit would also seem likely that it will have implications for7TMRs co-expressed with D6. The subcellular localization of�-arrestins is of profound importance to their function as7TMR regulators and signaling scaffolds. Constitutive localiza-tion of these proteins to membranes in D6-expressing cells (beit in the cell periphery (this study) or throughout the cell (14))would be predicted to influence signaling, trafficking, and sta-bility of �-arrestin-regulated 7TMRs co-expressed with D6. Ifso, it is conceivable that the phenotypes observed in D6 nullmice may, at least to some extent, be the result of changes in7TMR behavior and not exclusively because of loss of chemo-kine scavenging.The 14-aa difference between D6-326 and D6-340 is suffi-

cient to allow effective chemokine scavenging into HEK293cells. D6-326 can internalize some CCL3 after initially encoun-tering the chemokine, but exposure to CCL3 limits subsequentchemokine scavenging without markedly affecting surface

FIGURE 7. The membrane-proximal region of the D6 Ct carries an elongated putative 8th helix with aconserved leucine-rich surface. Alignment of the membrane-proximal ends of the D6 Ct from four speciesand of human CCR1–5. The location of the predicted 8th helix is indicated in boldface in all sequences, andconserved leucines are underlined. Serine residues in the serine-rich region of D6 are italicized and underlined;the predicted end of the seventh transmembrane helix (TM7) is indicated; and the position of truncation inD6-326 and D6-340 is marked with arrows. A helical whorl of human D6 (right) reveals a tri-leucine surface. Theletters in the whorl become smaller and the lines thinner toward the C-terminal end. Despite CCR1, -4, and -5carrying two leucines, these are inappropriately spaced for alignment on a helical surface. The conserved serine(in D6) and glycine (in CCR1–5) at the N-terminal end of the helices are indicated with asterisks.

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receptor expression. On the other hand, wtD6-mediated CCL3uptake and surface wtD6 expression are consistently enhancedby CCL3 exposure (Fig. 6) (12). These data suggest the follow-ing: (i)wtD6andD6-326arebothcapableof transmittingCCL3-dependent signals that modify scavenging; (ii) the heptahelicalbody of D6, on its own, is capable of engaging the endocytoticmachinery of HEK293 cells, and (iii) the D6 Ct is only indispen-sable for scavenging because it prevents CCL3 from inhibitingsubsequent chemokine uptake. Interestingly, the membraneproximal region of the D6 Ct harbors a putative 8th helix thatwill be disrupted in D6-326 but intact in D6-340 (Fig. 7). An 8thhelix is present in the crystal structure of bovine rhodopsin,anchored by the NPXXY motif at the end of the 7th helix (36,37) and undergoes a marked shift in location after rhodopsinactivation (38, 39). The recent crystal structures of the human�2-adrenergic receptor have an 8th helix underlying the mem-brane perpendicular to the transmembrane heliceswhich is sta-bilized by a hydrophobic surface (40–43). In fact, an 8th helix isthought to be present in most, if not all, rhodopsin-like 7TMRsand play a role in signaling by directly interacting with hetero-trimeric G-protein subunits (43–49), although roles in ligandbinding have also been reported (50). The 8th helix of D6 (Fig.7) is predicted to be the following: (i) longer than that found inCCRs1–5; (ii) have a hydrophobic surface (FFY) that may asso-ciate with membrane; (iii) carry a trileucine surface, a possibleprotein-protein interaction motif; and (iv) be directly precededby a conserved serine residue (a potential site for phosphoryla-tion) rather than the glycine found in CCR1–5 and other che-mokine receptors.Wepropose that the integrity of the 8th helixis critical for progressive scavenging by D6-340, possibly byinteracting with regulatory proteins present in HEK293 cells.Futurework is required to explore the role of the 8th helix in thecontext of full-lengthD6, and to characterize the “signals” ema-nating from wtD6 and D6-326 after chemokine engagementthat modify their scavenging capabilities. These studies willprovide further molecular insight into scavenging by this indis-pensable anti-inflammatory, tumor suppressor protein.

Acknowledgments—We are grateful to S. Ferguson and A. Wand-inger-Ness for plasmids and R. Lefkowitz for MEFs. Recipient of keysupport services from Dr. A. Wilson.

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VOLUME 283 (2008) PAGES 7972–7982DOI 10.1074/jbc.A113.710128

Multiple roles for the C-terminal tail of the chemokinescavenger D6.Clare V. McCulloch, Valerie Morrow, Sandra Milasta, Iain Comerford,Graeme Milligan, Gerard J. Graham, Neil W. Isaacs, and Robert J. B. Nibbs

PAGE 7977:

The original version of Fig. 4 contained an error. The same imagewasused in the “wtD6” and “D6-Ala6” panels. We have now replaced the“D6-Ala6” panel with the correct image. This revision does not changethe results or the interpretation of the data.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 37, p. 26820, September 13, 2013© 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

26820 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288 • NUMBER 37 • SEPTEMBER 13, 2013

ADDITIONS AND CORRECTIONS

Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract) services are urged to carry notice ofthese corrections as prominently as they carried the original abstracts.

Milligan, Gerard J. Graham, Neil W. Isaacs and Robert J. B. NibbsClare V. McCulloch, Valerie Morrow, Sandra Milasta, Iain Comerford, Graeme

Multiple Roles for the C-terminal Tail of the Chemokine Scavenger D6

doi: 10.1074/jbc.M710128200 originally published online January 17, 20082008, 283:7972-7982.J. Biol. Chem. 

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