Proc. Natl. Acad. Sci. USAVol. 92, pp. 7381-7385, August 1995Immunology

Determinants of the phagocytic signal mediated by the type IIIAFcy receptor, FcyRIIIA: Sequence requirements and interactionwith protein-tyrosine kinasesJONG-GU PARK* AND ALAN D. SCHREIBER7 Silverstein, University of Pennsylvania School of Medicine, 3400 Spruce Street, Philadelphia, PA 19104

Communicated by Robert Austrian, University of Pennsylvania Medical Center, Philadelphia, PA, April 17, 1995 (received for review February15, 1995)

ABSTRACT The Fcy receptor-associated y and C sub-units contain a conserved cytoplasmic motif, termed theimmunoglobulin gene tyrosine activation motif, which con-tains a pair ofYXXL sequences. The tyrosine residues withinthese YXXL sequences have been shown to be required fortransduction of a phagocytic signal. We have previouslyreported that the y subunit of the type IIIA Fcy receptor(Fc'yRIIIA) is -6 times more efficient in mediating phago-cytosis than the C subunit of FcyRIIIA. By exchanging regionsof the cytoplasmic domains of the homologous y and T chains,we observed that the cytoplasmic area of the y chain bearinga pair of the conserved YXXL sequences is important inphagocytic signaling. Further specificity of phagocytic signal-ing is largely determined by the two internal XX amino acidsin the YXXL sequences. In contrast, the flanking amino acidsof the YXXL sequences including the seven intervening aminoacids between the two YXXL sequences do not significantlyaffect the phagocytic signal. Furthermore, the protein-tyrosine kinase Syk, but not the related kinase ZAP-70,stimulated FcyRIIIA-mediated phagocytosis. ZAP-70, how-ever, increased phagocytosis when coexpressed with the Srcfamily kinase Fyn. These data demonstrate the importance ofthe two specific amino acids within the y subunit YXXLcytoplasmic sequences in phagocytic signaling and explain thedifference in phagocytic efficiency of the y and {chains. Theseresults indicate the importance of Syk in FcyRIIIA-mediatedphagocytosis and demonstrate that ZAP-70 and Syk differ intheir requirement for a Src-related kinase in signal transduc-tion.

Receptors for IgG are involved in diverse immune responses,including phagocytosis, immune complex clearance, and anti-body-dependent cellular cytotoxicity (1, 2). These effectorfunctions are preceded by a cascade of biochemical eventsinitiated by the crosslinking of Fc receptors on the surface ofphagocytic cells (3, 4). Since Fc receptors do not possessintrinsic tyrosine kinase activity, they require nonreceptortyrosine kinases for signal transduction. Crosslinking of Fcreceptors leads to tyrosine phosphorylation of the receptor oran associated subunit and activation of signal transductionmolecules, including protein-tyrosine kinases (5-10).The type IIIA Fc-y receptor (FcyRIIIA) complex requires its

,y or ; subunit both for cell surface expression and for signaltransduction (11, 12). The 'y and ; subunits as well as severalreceptors of the immunoglobulin gene superfamily contain aconserved motif, termed ITAM (for immunoglobulin genetyrosine activation motif or ARH1/ARAM for antigen re-ceptor homology 1) (13-15). The salient feature of this motifis the sequence YXXL-X7-YXXL. The tyrosine residues of theconserved YXXL sequences appear to be required for signal

transduction and are phosphorylated upon receptor crosslink-ing (16-18).

In experiments employing COS-1 cells transfected with Fc'yreceptors, we demonstrated that FcyRIIIA induces the phago-cytosis of IgG-sensitized cells and that both its y and ; subunitscan induce the phagocytic signal (12, 18, 19). The y chain,however, is -6 times more efficient than the ; chain (18). Todetermine the cytoplasmic region of the fy chain responsible forthis difference, we constructed exchange mutants between 'yand ; for cotransfection with Fc-yRIIIA-a. These mutants werealso studied in the presence of the Syk family of kinases. Ourfindings indicate that the specific amino acids within theYXXL sequences and the Syk kinase determine the signal forphagocytosis.

MATERIALS AND METHODSConstruction of Recombinant Plasmids. The coding se-

quence of the FcyRIIIA-a cDNA (kindly provided by BicePerussia, Jefferson College of Medicine, Philadelphia) wasligated into the Xba I and Sac I cloning sites of the simian virus40-driven eukaryotic expression vector pSVL (PharmaciaLKB). The cDNA for the human FcsRI/FcyRIIIA 'y subunit(kindly provided by Jean-Pierre Kinet, National Institutes ofHealth, Bethesda) was cloned into and expressed from thesame vector. T-cell antigen receptor/FcyRIIIA ; subunit(kindly provided by Richard D. Klausner, National Institutesof Health) was cloned into the Xba I and BamHI cloning sitesof pSVL. The truncation mutant of the ; subunit was con-structed [GAG (nucleotides 351-353) to TAG] by using PCR.Various exchange and substitution mutants of ; and y wereconstructed by overlap PCR (20). Five mutant clones with acorrect-size insert were selected, and the sequence was con-firmed. One of the clones with the correct DNA sequence wasemployed for each mutant for transient transfection experi-ments. The eukaryotic expression plasmid containing the SykcDNA (p72SYk/pME18S) was kindly provided by ClementCouture (La Jolla Institute for Allergy and Immunology, LaJolla, CA). The eukaryotic expression plasmid containing thehuman ZAP-70 cDNA (psV7d-ZAP-70) was kindly providedby Andrew Chan (Howard Hughes Medical Institute, Wash-ington University School of Medicine, St. Louis).

Cell Culture and Transfection of COS-1 Cells. COS-1 cellswere cultured in high-glucose Dulbecco's modified Eagle'smedium (DMEM) supplemented with glutamine (25 mg/ml),penicillin (100 units/ml), streptomycin (100 ,ug/ml), and 10%(vol/vol) heat-inactivated fetal bovine serum. Cells were main-tained in the tissue culture incubator at 37°C with 5% C02/95% air. Transfections with cDNAs were carried out asdescribed (12). Briefly, 5 x 105 COS-1 cells were seeded on

Abbreviations: FcyRIIIA, type IIIA Fcy receptor; EA, IgG-coatederythrocytes; PI, phagocytic index; SH2, Src homology 2; ITAM,immunoglobulin gene tyrosine activation motif.*To whom reprint requests should be addressed.

7381

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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35-mm well plates 24 hr prior to transfection. Plates werewashed twice and incubated for 30 min withDMEM (GIBCO/BRL) without serum. Transfection was carried out for 4 hr at37°C followed by 10% (vol/vol) dimethyl sulfoxide shock. Cellswere studied 48 hr after transfection.

Flow Cytometry. Anti-FcyRIII monoclonal antibody 3G8(21) was used to assess protein expression on the cell surfaceof transfected COS-1 cells. Cells were gently removed fromplates, counted, and resuspended in 50 ,ul of staining buffer(lx PBS without Ca2+/Mg2+, containing 0.02% sodium azideand 0.1% bovine serum albumin) with 5 ,ul of primary antibodyand placed on ice for 45 min. After two washes, fluoresceinisothiocyanate-labeled F(ab')2 goat anti-mouse IgG wasadded, and incubation was continued at 4°C for 30 min. Isotypecontrols were employed for all reactions. Fluorescence wasmeasured on a FACStar (Becton Dickinson). For all samples,10,000 events were recorded on a logarithmic fluorescencescale, and mean fluorescence intensity data and histogramswere generated.

Binding and Phagocytosis of IgG-Sensitized Erythrocytes.IgG-coated sheep erythrocytes (EA) were prepared as de-scribed (12). COS-1 cells were incubated with EA at 37°C for35 min. Unbound EA were removed by extensive washing, andCOS-1 cells were stained with Wright-Giemsa to determinethe number of cells with bound EA. For the analysis ofphagocytosis, COS-1 cells bound with EA (after three wash-ings) were subjected briefly (35 sec) to hypotonic PBS toremove surface-bound EA prior to staining. Phagocytosedcells were stained with Wright-Giemsa to score ingestederythrocytes. Electron microscopy has demonstrated that in-gested EA are within defined intracellular phagocytic vacuoles(12, 18). Results were expressed as phagocytic index (PI), thenumber of ingested erythrocytes per 100 COS-1 cells positivefor Fc receptor expression.

RESULTSDifference in Phagocytic Signaling Between FcyRIIIA-'y

and FcyRIIIA-CI. Although the y chain and the ; chain ofFc-yRIIIA induce comparable cell surface expression of theFc,yRIIIA complex in COS-1 transfectants, the 'y subunit is

A - FC-. -TM -I-

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considerably more active in mediating a phagocytic signalwhen compared with the ; subunit (-6-fold) (12). Thissignificant difference in phagocytic signaling between the twohomologous subunits provided a mechanism by which toexamine the regions of the y and ; subunits responsible for thisdifference in signaling.The -y subunit contains only one pair of the conserved

cytoplasmic YXXL sequences [the putative Src homology 2(SH2) binding site], but the ; subunit contains three pairs. Tosimplify the comparative structure-function analysis betweenthese two FcyRIIIA subunits, we engineered a truncationmutant of C, C1. The C1 mutant contained only one pair of thetransmembrane proximal YXXL sequences (Fig. 1A). C1, y,and wild-type ; were individually cotransfected withFcyRIIIA-a to examine cell surface expression of theFcyRIIIA receptor complex and phagocytic signaling. Asshown by flow cytometry and EA binding, Fcy receptorexpression in COS-1 transfectants was comparable with thethree different subunits (Fig. 1B and Table 1). WhereasFc,yRIIIA-y induced efficient phagocytosis, both FcyRIIIA-Cand -C1 mediated markedly reduced levels of phagocytosiscompared to that of y (Table 1). The decreased phagocyticsignaling of C1 allowed us to utilize C1 as a template for theconstruction of several exchange mutants between 'y and 1.The Two Internal XX Amino Acids of the YXXL Pair

Determine Efficiency of Phagocytic Signaling. To delineatethe region of the y cytoplasmic tail responsible for efficientphagocytic signaling, three chimeric exchange mutants, Cy,CyC, and 'yCy, were engineered (Fig. 2). In Cy, the C-terminalportion of the C1 cytoplasmic domain including the YXXL pairwas replaced by the equivalent C-terminal y sequence. Cyshowed a significant increase in phagocytic efficiency (PI from28 to 212), recovering most of the phagocytic capacity ofwild-type y (PI = 270 + 79; Table 2). In CyC, the first ;YXXLand following C-terminal seven amino acids were replacedwith the y sequence. CyC mediated modestly increased phago-cytosis compared to that of C1. In yCy, the first YXXL regionof wild-type 'y was replaced with the C1 sequence. Thisexchange increased the phagocytic efficiency from a PI of 28for C1 to a PI of 170. These data suggested that the cytoplasmic

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FIG. 1. Schematic diagram of human cDNA of FcyRIIIA subunits and flow cytometric analysis of COS-1 transfectants. (A) Fc-yRIIIA-a, -y,

-C, and -C1, a truncation mutant of C, are denoted at the left side of the schematic diagram. Shown above the diagram are the extracellular domain(EC), the transmembrane domain (TM), and the cytoplasmic domain (CY). Above y is the abbreviated nucleotide sequence of the conserved ITAMcontaining two YXXL sequences. In C1, the C-terminal sequence containing two pairs of the YXXL sequences was removed by introducing a stopcodon, leaving only the transmembrane-proximal YXXL pair. (B) Fluorescence histograms of COS-1 cells expressing the FcyRIIIA-a and the 'y,

C, or C1 subunit. The dotted lines represent cells stained with an IgG isotype control, whereas the solid lines represent cells stained withanti-Fc-yRIIIA monoclonal antibody 3G8. (Left) Transfection with Fc-yRIIIA-a + y subunit. (Center) Transfection with FcyRIIIA-a + wild-typeC subunit. (Right) Transfection with FcyRIIIA-a + C1 subunit.

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Table 1. Phagocytosis and Fc receptor expression by COS-1 cellstransfected with FcyRIIIA-'y, -C, or -C1

Phagocytosis Expression

FcyRIIIA PI + cells,* % MFI EA binding, %a 0 0 15 3 ± 1a +y (wt) 270 ± 79 7.8 ± 1.7 187 ± 59 40 ± 4a + (wt) 46 ± 7 2.5 ± 0.7 154 ± 13 39 ± 8a + c1 28 ± 9 2.0 ± 1.0 268 ± 30 34 ± 5

Transfection efficiency was determined by flow cytometry. At leastthree separate experiments were performed for each clone. For eachexperiment, 900-1000 cells were microscopically examined (x 1000) ina random fashion to determine the PI (19). wt, wild type; MFI, meanfluorescence intensity. Results are expressed as the mean ± SEM.*Percentage of cells with phagocytosed erythrocytes.

region containing the YXXL pair of the y and g subunitsinfluenced their phagocytic signaling efficiency.We next examined specifically the role of the two XX amino

acids within each YXXL sequence. Three substitution mu-tants, denoted C((yN-YXXL), C( yC-YXXL), and C(,yD-YXXL), were constructed with C1 as a template. One substi-tution mutant, denoted 'y(MD-YXXL), was constructed with yas a template (Fig. 2). In C(yN-YXXL), replacing the XX in theN-terminal YXXL of ; with the corresponding amino acids of,y increased only slightly the phagocytic index (Table 2). With(yC-YXXL), however, replacing the XX in the C-terminalYXXL with the corresponding -y chain amino acids increasedthe PI from 28 to 154 and, in C(yD-YXXL), replacement of

.-TM -S-- CYYY TG L-X7-Y Er L

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FIG. 2. Schematic diagram of exchange mutants between -y and 1.

The nucleotide sequences of the conserved ITAM containing twoYXXL sequences are shown above y and C. Vertical dashes are shownto aid identifying the positions of exchanged amino acids. The first linerepresents -y, and the second line represents C1, the truncation mutantof C. In Cy, the C-terminal sequence of C1 starting at the N-terminalYXXL was replaced with the corresponding sequence of -y. In CyC, theC-terminal sequence of Cy from the C-terminal YXXL was replacedwith the sequence of C1. In yCy, the cytoplasmic sequence of y

including the N-terminal YXXL and the seven intervening amino acidsbetween the two YXXL sequences were replaced with those of C1. InC(yN-YXXL), the XX sequence of the C1 N-terminal YXXL wasreplaced with the corresponding y sequence. In C(yC-YXXL), the XXsequence of the C1 C-terminal YXXL was replaced with the ysequence. In C(-yD-YXXL), both XX sequences of the C1 YXXLsequences were replaced with the 'y sequences. In 'y(CD-YXXL), bothXX sequences of the y YXXL sequences were replaced with the C1sequences. TM, transmembrane domain; CY, cytoplasmic domain.

Table 2. Phagocytosis and Fc receptor expression by COS-1 cellstransfected with FcyRIIIA-a and exchange mutants between yand C1

Phagocytosis Expression

Fc-yRIIIA PI + cells, % MFI EA binding, %a + y (wt) 270 ± 79 7.8 ± 1.7 187 ± 59 40 ± 4a + c1 28 ± 09 2.0 ± 1.0 268 ± 30 34 ± 5a + y 212 ± 32 8.3 ± 0.6 240 ± 66 34 ± 1a + CyA 79 ± 26 3.0 ± 1.0 236 ± 60 38 ± 4a + y;/y 170 ± 37 6.0 ± 1.2 194 ± 57 35 ± 7a + {(-yN-YXXL) 59 ± 34 1.5 ± 0.6 141 ± 09 39 ± 4a + (^C-YXXL) 154 ± 28 4.3 ± 0.5 168 ± 41 39 ± 5a + C(yD-YXXL) 251 ± 36 6.0 ± 1.0 171 ± 48 35 ± 1a +y(-D-YXXL) 79 ± 25 3.3 ± 1.0 175 ± 68 37 ± 3

The details and abbreviations are as given in Table 1.

both N- and C-terminal XX amino acids further increased thephagocytic index to a level similar to that of the wild-type 'ychain. In contrast, when the y chain XX sequences werereplaced with the corresponding ; sequences in y(MD-YXXL),the phagocytic index decreased from 270 to 79. These resultsindicate the importance of the specific amino acids within eachYXXL in determining phagocytic signaling. In addition, thetwo XX sequences in the YXXL sequences of the 'y chainITAM provide phagocytic potency in an additive manner,demonstrating the necessity for a pair of YXXL for efficientphagocytic signaling.A Similar Sequence Requirement for a Phagocytic Signal Is

Realized by the Tyrosine Kinase Syk We have recently shownthat the protein-tyrosine kinase Syk increases both FcyRIIIA-and Fc-yRI-mediated phagocytosis -3- to 6-fold in COS-1transfectants (19). The exchange and substitution mutantsdescribed above were employed to study the effect of Syk inphagocytic signaling. Syk increased the phagocytic activity ofcells with wild-type and mutant y and ; chains 2- to 8-fold (Fig.3). Substitution of both XX sequences in the ; chain YXXLsequences with those of the 'y chain in C(,yD-YXXL) resultedin the most efficient phagocytosis among the mutant receptors.Although Syk increased the effect on phagocytosis of the ;chain, the interaction of Syk with the 'y chain resulted in highphagocytic efficiency. Taken together with our previous ob-servations (19), the data indicate that Syk is important for the

1400

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800-

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200-

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FIG. 3. Effect of Syk on phagocytosis mediated by FcyRIIIA-acotransfected with the wild-type or the mutant y or subunit. PI wasdetermined in COS-1 cells with or without the protein-tyrosine kinaseSyk. The bar graphs represent the mean ± SEM from at least threeindependent experiments. Detailed descriptions of the exchange mu-tants are shown in the legend of Fig. 2.

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450-

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Os150-

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FIG. 4. Effect of ZAP-70 and Fyn on phagocytosis mediated byFc-yRIIIA-a cotransfected with -y, ;, or C1. Phagocytosis was examinedfor COS-1 cells transfected with Fc-yRIIIA in the presence or absenceof the protein-tyrosine kinases ZAP-70 and Fyn. The graphs representthe mean ± SEM from at least three independent experiments.

phagocytic signal induced by the -y chain and that the specific,y chain amino acids within the YXXL cytoplasmic sequences

are also important for the extent of phagocytosis induced bySyk.ZAP-70, a Syk Homologue, Increases FcyRIIIA-Mediated

Phagocytosis in the Presence of Fyn. There are two membersof the Syk kinase family, Syk and ZAP-70. The Syk homologueZAP-70 was studied in cotransfection experiments withFcyRIIIA-y, -C, and -C1. In marked contrast to Syk enhance-ment of phagocytic signaling, ZAP-70 did not increase phago-cytosis transduced by either y, C, or C1 (Fig. 4). ZAP-70 hasbeen observed to be dependent on Src family kinases for itsefficient association with the ; chain (22). One such Src familykinase is Fyn. We examined, therefore, whether co-transfection of ZAP-70 with Fyn leads to induction of phago-cytosis. When ZAP-70 was cotransfected with the Src familykinase Fyn, ZAP-70 increased phagocytosis 1.4-fold with the,y chain and 5-fold with the ; chain, demonstrating functionaldependence of ZAP-70 on Fyn. Fyn alone did not increaseFc-yRIIIA-y- or -c-mediated phagocytosis, and Fyn did notincrease phagocytosis induced by the y chain in the presenceof Syk (data not shown). These observations suggest that thetwo related protein-tyrosine kinases Syk and ZAP-70 differ intheir requirement for signal transduction.

DISCUSSIONIn this study, we examined the molecular basis of intracellularsignal transduction employed by Fcy receptor subunits as wellas protein-tyrosine kinases. Although the tyrosine and leucineresidues are conserved in the ITAM of several receptors of theimmunoglobulin gene superfamily (13), the XX amino acids ofYXXLs as well as the flanking amino acids differ. Thisdivergence in amino acid sequence might account for thefunctional differences of the receptors. Our data obtainedfrom the exchange mutants suggest that functional differencesin phagocytosis between the two related subunits -y and ; are

primarily accounted for by the internal amino acids of theYXXL sequence and their paired presence in the ITAM.Among the mutant C1 chains, the C-terminal XX exchange

mutants had a greater effect on phagocytic signaling than theN-terminal XX exchange mutants. The double exchange mu-tants of C1 mediated significantly enhanced phagocytosis butdid not completely restore the phagocytic efficiency obtainedwith wild-type y. This small difference in phagocytic efficiencymay be due to flanking amino acids of the ITAM or a possiblestructural change present in the mutant.

Syk protein-tyrosine kinase contains two SH2 domains. Thisstructure is in contrast to that of Src protein-tyrosine kinases,which contain only one SH2 domain (23). In our previousreport, the stimulatory effect of Syk on Fc-yRIIIA-y-mediatedphagocytosis was shown to require both of the conservedtyrosine residues of -y (19). Binding ofboth SH2 domains to thetwo phosphorylated YXXL motifs may thus be necessary forstable association between y and Syk. That phagocytic signal-ing specificity is contributed by each YXXL in an additivemanner suggests dual roles played by the YXXL motif pair,both signaling specificity as well as maintaining stable associ-ation between y and Syk.

Recently a phosphopeptide library was used to determinethe sequence specificity of the peptide binding sites of SH2domains. These studies showed that an individual SH2 domainselected a unique internal sequence (24, 25). The C-terminalSH2 domain of Syk was shown to favor a phosphopeptide ofpY(Q/T/E)(E/Q)L (pY = phosphotyrosine). The C- andN-terminal YXXL motifs of y are YTGL and YETL, respec-tively, which are not identical to pY(Q/T/E)(E/Q)L. Thesedifferences may be explained by the following observations.First, phosphorylated threonine was not included in the phos-phopeptide library. It has been reported that threonine resi-dues of the y chain are constitutively phosphorylated (5).Phosphorylated threonine residues might render a distinctmicroenvironment with their charge and structure. Second,only the C-terminal SH2 domain of Syk was used for phos-phopeptide library selection. It is possible that the bindingpreference of each SH2 domain differs when present as a pair.There also may be other differences not observed in peptidebinding studies but necessary for intracellular function.The Src family tyrosine kinases exhibit functional promis-

cuity (26), and the SH2 domains of Src kinases have almostidentical sequence selection for synthetic phosphopeptides(24). The Syk kinase family does not appear to share suchredundancy in function. ZAP-70 is homologous to Syk, andZAP-70 has been shown to associate with the C chain in theT-cell antigen receptor-CD3 complex (22). In contrast to Sykkinase, however, ZAP-70 does not increase phagocytosis me-diated by Fc-yRIIIA-y or -g: It is possible that the molecularassociation by the two SH2 domains of Syk or ZAP-70 providesspecificity for their substrates. In a recent report, Syk andZAP-70 were appended to the extracellular domain of CD16(Fc-yRIIIA-a) and were examined for their ability to signal forintracellular Ca2+ and cytotoxicity. Both the Syk and ZAP-70chimeras were able to mobilize intracellular Ca2+, but only theSyk chimera induced cytotoxicity (27). These results are inagreement with our data (phagocytosis and cytotoxicity areboth effector functions), indicating the specificity of Syk andZAP-70 function.

In summary, we have used an in vitro functional model toevaluate the signaling activity of the Fc-y receptor subunits -yand ; with the Syk kinase. The induction of phagocytosis ismost efficient with the y chain and depends on specific aminoacids within the YXXL sequences in the y-chain cytoplasmicdomain. The protein-tyrosine kinase Syk, rather than ZAP-70,is effective in stimulating phagocytosis.

We thank Drs.. Zena Indik and Sharon Hunter for review anddiscussion of this manuscript. This work was -supported by GrantsHL-28207 and HL-27068 from the National Institutes of Health andby CorBec Pharmaceuticals, Inc.

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