EMR4, a Novel Epidermal Growth Factor (EGF)-TM7 MoleculeUp-regulated in Activated Mouse Macrophages, Binds to a PutativeCellular Ligand on B Lymphoma Cell Line A20*

Received for publication, May 2, 2002Published, JBC Papers in Press, May 21, 2002, DOI 10.1074/jbc.M204306200

Martin Stacey‡§¶, Gin-Wen Chang‡�, Stephanie L. Sanos‡**, Laura R. Chittenden‡‡§§,Lisa Stubbs‡‡§§, Siamon Gordon‡**, and Hsi-Hsien Lin‡§¶¶

From the ‡Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE,United Kingdom and ‡‡Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory,Livermore, California 94550

A novel member of the EGF-TM7 family, mEMR4, wasidentified and characterized. The full-length mouseEMR4 cDNA encodes a predicted 689-amino acid pro-tein containing two epidermal growth factor (EGF)-like modules, a mucin-like spacer domain, and a seven-transmembrane domain with a cytoplasmic tail.Genetic mapping established that mEMR4 is localizedin the distal region of mouse chromosome 17 in closeproximity to another EGF-TM7 gene, F4/80 (Emr1).Similar to F4/80, mEMR4 is predominantly expressedon resident macrophages. However, a much lower ex-pression level was also detected in thioglycollate-elicited peritoneal neutrophils and bone marrow-derived dendritic cells. The expression of mEMR4 isup-regulated following macrophage activation in Bio-gel and thioglycollate-elicited peritoneal macro-phages. Similarly, mEMR4 is over-expressed in TNF-�-treated resident peritoneal macrophages, whereasinterleukin-4 and -10 dramatically reduce the expres-sion. mEMR4 was found to undergo proteolytic proc-essing within the extracellular stalk region resultingin two protein subunits associated noncovalently as aheterodimer. The proteolytic cleavage site was identi-fied by N-terminal amino acid sequencing and locatedat the conserved GPCR (G protein-coupled receptor)proteolytic site in the extracellular region. Using mul-tivalent biotinylated mEMR4-mFc fusion proteins as aprobe, a putative cell surface ligand was identified ona B lymphoma cell line, A20, in a cell-binding assay.The mEMR4-ligand interaction is Ca2�-independentand is mediated predominantly by the second EGF-likemodule. mEMR4 is the first EGF-TM7 receptor knownto mediate the cellular interaction between myeloidcells and B cells.

The G protein-coupled receptors (GPCRs),1 with more than1000 members identified to date, constitute one of the largestprotein superfamilies in nature (1, 2). By coupling heterotrim-eric G proteins to their characteristic seven-transmembrane(TM7) and cytoplasmic regions, GPCRs mediate the signaltransduction of an extensive array of exogenous stimuli includ-ing hormones, cytokines, peptides, amino acid derivatives, ions,neurotransmitters, light, taste, and odors (1, 3). A total of fiveclasses of GPCRs are categorized based upon the similarity oftheir TM7 sequences (4). In recent years, a rapidly growingsubfamily of class-B GPCRs with an unusual long N-terminalextracellular region has been identified (LNB-TM7; for reviewsee Refs. 5 and 6). Among them, the epidermal growth factor(EGF)-TM7 receptor subfamily has been studied with greatinterest by us and others (for review see Refs. 7 and 8).

At present, eight members of the EGF-TM7 family includinghuman EGF module-containing mucin-like hormone receptor 1(EMR1), F4/80 (mouse Emr1), EMR2, EMR3, human and ratEGF-TM7-latrophilin-related protein (ETL), and human andmouse CD97 have been reported (9–18). The EGF-TM7 mole-cules are distinguished by a novel hybrid structure that con-tains various numbers of N-terminal EGF-like modules con-nected to a TM7 domain by a mucin-like spacer (7, 8).Characterized by a set of 6 conserved cysteine residues typi-cally disulfide bond in a 1–3, 2–4, 5–6 arrangement, the tan-demly arrayed EGF-like modules of the EGF-TM7 moleculesbelong to the class I EGF-like modules often found in connec-tive tissue proteins such as fibrillin-1 and -2 and fibulins (19).The majority of these EGF-like modules also contain a consen-sus sequence associated with calcium binding (cb), (D/N)X(D/N)(E/Q)Xm(D/N*)Xn(Y/F), where m and n are variable and *indicates possible �-hydroxylation (19, 20). Calcium performs akey role in the orientation of cbEGF pairs by restricting con-

* The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

The nucleotide sequence(s) reported in this paper has been submittedto the GenBankTM/EBI Data Bank with accession number(s) AY032690.

§ These authors contributed equally to this work.¶ Supported by a Goodger scholarship.� Supported by a University Challenge Seed Fund award from the

University of Oxford.** Supported by grants from the Medical Research Council, UK.§§ The work of these authors was performed under the auspices of the

United States Department of Energy, Office of Biological and Environ-mental Research, at the University of California, Lawrence LivermoreNational Laboratory, under Contract No. W-7405-Eng-48.

¶¶ Supported by a research grant from Celltech R&D. To whom corre-spondence should be addressed: Sir William Dunn School of Pathology,University of Oxford, South Parks Road, Oxford, OX1 3RE, UK. Tel.:44-1865-275532; Fax: 44-1865-275515; E-mail: hlin@molbiol.ox.ac.uk.

1 The abbreviations used are: GPCR, G protein-coupled receptor;EGF, epidermal growth factor; TM7, seven transmembrane; mEMR4,mouse epidermal growth factor module-containing mucin-like receptor4; cbEGF, calcium-binding EGF module; GPS, GPCR proteolytic site;ETL, EGF-TM7-latrophilin-related; RACE, rapid amplification ofcDNA ends; RT-PCR, reverse transcription-polymerase chain reaction;M�, macrophages; PMN, polymorphonuclear cells; DC, dendritic cells;RPM�, resident peritoneal macrophages; BioM�, Biogel-elicited perito-neal macrophages; ThioM�, thioglycollate-elicited peritoneal macro-phages; ThioPMN, thioglycollate-elicited neutrophils; BMM�, bonemarrow-derived macrophages; BMDC, bone marrow-derived dendriticcells; mAb, monoclonal antibodies; Fc, crystallizable fragment; mFc,mouse Fc; FACS, fluorescence-activated cell sorting; HRP, horseradishperoxidase; Chr, chromosome; IL, interleukin; TNF-�, tumor necrosisfactor-�; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)pro-pane-1,3-diol; MES, 4-morpholineethanesulfonic acid; HBSS, Hanks’balanced salt solution; BSA, bovine serum albumin; contig, group ofoverlapping clones; FIRE, F4/80-like-receptor.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 32, Issue of August 9, pp. 29283–29293, 2002Printed in U.S.A.

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formational flexibility of the interdomain linkage that is im-portant in presenting a specific surface for protein-protein in-teraction. Genetic mutations causing amino acid changes in thecbEGF modules have been found in diseases such as familialhypercholesterolemia, hemophilia B, protein S deficiency, andthe Marfan syndrome (21–24).

Following the EGF-like modules, a Ser/Thr-rich, mucin-likespacer region of �200–250 amino acids precedes the TM7region. Within this region, a highly conserved Cys-rich domainlocated immediately before the first TM segment has beenidentified in all EGF-TM7 proteins. This domain, termed theGPCR proteolytic site (GPS) domain, is characterized by fourinvariant Cys, one Gly, and two conserved Trp residues spacedin restricted positions (25). The GPS domain was initially iden-tified in a sperm receptor for egg jelly from sea urchins(Strongylocentrotus purpuratus) (26) and later found in a num-ber of LNB-TM7 receptors including HE6 (27), GPR56 (28, 29),BAI1 (30), CL1–3 (25, 31–33), Celsr1 (34, 35), Ig-Hepta (36),and the flamingo (fmi) gene product from Drosophila (37, 38).Several of these proteins, notably CL1 (calcium-independentreceptor for latrotoxin (CIRL)/latrophilin), ETL, and CD97,have been shown to be proteolytically cleaved within this do-main, resulting in two subunits that remain noncovalentlyassociated as a heterodimer (14, 16, 25). Although its signifi-cance is still unknown, this unusual yet highly conserved post-translational protein modification is likely to have a physiolog-ical function for the EGF-TM7 receptors.

The EGF-TM7 receptors are predominantly expressed inmyeloid cells (F4/80, EMR1, EMR2, EMR3, and CD97) andsmooth muscle cells (ETL and CD97) (9–16, 39–41). Basedupon their unique protein structure and restricted expressionpatterns, it has been suggested that the EGF-TM7 moleculesmay play a role in the cellular functions of myeloid leukocytesand cardiac muscle differentiation by interacting with othercell surface proteins or extracellular matrix proteins, leading tointracellular signaling. Indeed, the presence of cellular ligandshas been demonstrated for CD97 (42), EMR3 (13), and EMR2.2

CD97 is the first EGF-TM7 molecule shown to interact with adefined cellular ligand, CD55 (decay-accelerating factor; DAF)(42, 43). We have recently further demonstrated that CD97interacts with CD55 solely by the EGF-like modules and thatthe interaction is characterized by a low affinity (86 �M) andrapid off-rate (�0.6 s�1) (44). Likewise, by employing solublemultivalent EMR3 probes in a sensitive cell-binding assay, aputative EMR3 ligand has been found on the surface of humanmonocyte-derived macrophages and activated human neutro-phils (13). However, it is currently not known whether theseprotein-protein interactions can lead to signal transduction viathe TM7 moiety of these receptors.

In addition to the strong structural-functional similarity, theEGF-TM7 molecules are also closely linked at the genomiclevel. With the exception of ETL, which maps to human chro-mosome (Chr) 1, all other human EGF-TM7 members are lo-cated within Chr 19p13 region with EMR1 on 19p13.3, andCD97, EMR2, and EMR3 on 19p13.1 (9, 12, 13, 15). Further-more, the mouse homologues of EMR1 (F4/80) and CD97 weremapped to the corresponding syntenic regions of mouse Chr 17and Chr 8, respectively (11, 45, 46). More significantly, thegenomic organization of CD97 and EMR2 was found to beidentical in that each EGF-like module is encoded by a singleexon and the TM7 region by a total of five exons (12, 47). Thesefindings strongly indicate that the EGF-TM7 genes are derivedfrom a common ancestral gene through gene duplication andare highly conserved among vertebrate species. It is therefore

of great interest to decipher how individual EGF-TM7 genesevolve and function.

Herein, we describe the molecular and functional character-ization of a new EGF-TM7 molecule, mouse EMR4. ThemEMR4 gene maps to the distal region of mouse Chr 17 in closeproximity to Emr1 and encodes a cell-surface receptor restrict-edly expressed in macrophages (M�), neutrophils (PMN), anddendritic cells (DC). The regulation of mRNA expression, thebiochemical-structural characteristics, and the ligand-bindingproperties of mEMR4 are discussed.

EXPERIMENTAL PROCEDURES

Materials—All chemicals and reagents were obtained from Sigmaunless otherwise specified. Cell culture media and supplements werepurchased from Invitrogen. Cell lines were provided by the cell bank atthe Sir William Dunn School of Pathology, University of Oxford. Lab-oratory-bred mice were housed in and provided by the animal facility atthe Sir William Dunn School of Pathology under standard pathogen-free conditions with access to food and water ad lib. Recombinantmurine IL-4, IL-10, TNF-�, and interferon-� were obtained from R&DSystems.

Molecular Cloning of the mEMR4 cDNA—The mEMR4 full-lengthcDNA was obtained by rapid amplification of cDNA ends (RACE)-polymerase chain reaction (PCR) using a mouse spleen MarathonReady cDNA library (CLONTECH). Two rounds of PCR reactions wereconducted to amplify visible mEMR4 cDNA fragments. Marathonadapter primers, AP-1 (5�-CCATCCTAATACGACTCACTATAGGGC-3�) and AP-2 (5�-ACTCACTATAGGGCTCGAGCGGC-3�) were pairedindividually with nested mEMR4-specific primers, 5�-1 (5�-TTGAGAG-AAGAAAGTTTGCTTCTCAA-3�) and 5�-2 (5�-CGCTGGCCCCAAGAA-GCTCCAGATGAA-3�) as well as 3�-1 (5�-CTGGAAGGGCTACATCTT-TTTCTCACT-3�) and 3�-2 (5�-GGCAGATTCAAGAAGAGGTTCATGTA-T-3�) to generate 5�-RACE and 3�-RACE fragments, respectively.mEMR4 cDNA fragments were separated on a 1% agarose gel, purified,subcloned, and sequenced using standard molecular techniques.

Sequence Analysis—All cDNA fragments and expression constructswere subjected to DNA sequencing of both strands to confirm theirintegrity. DNA sequencing reactions were performed using the Big-Dye™ Terminator DNA sequencing kit (PE Applied Biosystems). Sam-ples were electrophoresed on an ABI 373A DNA Sequencer and ana-lyzed by ABI Prism Model Version 2.1.1 software (PE AppliedBiosystems). An homologous DNA sequence search was carried outusing BLAST algorithm against DNA sequences in GenBankTM/EMBLdata bases. Protein alignment and alignment of consensus sequenceswere analyzed using ClustalW software.

Chromosome Mapping Analysis—To determine the chromosomal po-sition of the mEMR4 gene, the segregation of sequences detected by afull-length mEMR4 cDNA probe was followed in 118 progeny of a Musmusculus � Mus spretus interspecific back-cross: (C3Hf/R1-Mg-fSl-ENURg/� � M. spretus) � C3Hf/R1. The mEMR4 cDNA probe hybrid-ized to fragments of �4.8, 4.5, 4.38, 4.2, 3.9, 1.9, 1.8, and 0.85 kb inHincII-digested M. spretus (S) DNA and fragments of �4.8, 4.5, 4.38,4.2, 3.9, 3.45, 3.05, 1.55, and 0.8 kb in HincII-digested C3Hf inbredmouse (M) DNA. Other variant fragments were described recently (48)and were traced as follows: Nfya, BamHI (S) 1.6 kb (M) 3.2 kb; C3, PvuII(S) 4.6, 6.2 kb (M) 3.3 kb; Mllt1, EcoRI (S) 1.3 kb (M) 0.8 kb; Emr1,BamHI (S) 7.7, 5.2 kb (M) 15.0, 9.8 kb; Vav, TaqI (S) 5.0, 5.5 kb (M) 5.6kb; Rfx2, TaqI (S) 2.7 kb (M) 3.6 kb. Gene linkage, order, and intergenicdistances with associated standard errors were calculated according tostandard statistical methods with the aid of the Map Manager dataanalysis programs (49, 50).

Cell Culture—All culture media were supplemented with 10% heatinactivated fetal calf serum, 2 mM L-glutamine, 50 IU/ml penicillin, and50 �g/ml streptomycin. All cells were incubated at 37 °C in a 5% CO2,95% humidity incubator. A20 cell line was cultured in RPMI 1640medium. HEK293T cells were grown in Dulbecco’s modified Eagle’smedium. Mouse primary cells were obtained from 8–10-week-old C57/Bl6 or BALB/c mice. Mouse bone marrow-derived M� (BMM�) werecultured in RPMI 1640 medium supplemented with 15% L-cell-condi-tioned medium as described previously (51). Mouse resident peritonealM� (RPM�), Biogel-elicited peritoneal M� (BioM�), thioglycollate-elic-ited peritoneal M� (ThioM�), and thioglycollate-elicited peritoneal neu-trophils (ThioPMN) were obtained using standard protocols describedpreviously (52, 53). For the cytokine treatment of RPM�, cells werecultured for 1 h for adherence, washed, and then treated with orwithout IL-4 (20 ng/ml), IL-10 (10 ng/ml), TNF-� (10 ng/ml), and inter-2 Lin et al., manuscript in preparation.

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feron-� (250 u/ml) for 36 h prior to the isolation of total RNA. Mousespleen B and T cells were isolated from a single cell suspension bymagnetic separation of B220 mAb-coated Dynabeads (Dynal A.S., Oslo,Norway) and by nylon wool purification, respectively (54). Mouse bone-marrow-derived dendritic cells (BMDC) were obtained by incubatingbone marrow cells in the presence of granulocyte/macrophage colony-stimulating factor as described previously (55).

RNA Blot and RT-PCR Analysis—Total RNA was prepared frommouse primary cells and cell lines using the acid guanidinium thiocy-anate-phenol-chloroform method (56). Total RNA (10 �g) was electro-phoresed on a 1% formaldehyde-agarose gel, blotted onto a nylon mem-brane (Duralon-UV, Stratagene), and hybridized with gene-specificprobes as previously describe (11). Similarly, a commercially availablemouse multiple tissue Northern blot (OriGene Technologies, Inc) washybridized with a full-length mEMR4 cDNA probe according to themanufacturer’s instructions. Hybridized blots were washed in 0.5�SSC (0.15 M NaCl and 0.015 M sodium citrate), 1% SDS at 55 °C for 30min and exposed to x-ray films (X-Omat, Kodak) at �80 °C for 2–4 days.Radioactive probes were stripped from RNA blots by washing in 0.1�SSC, 1% SDS for 15 min at 95 °C, and the blots were rehybridized witha mouse �-actin cDNA probe to compare the amount of RNA loaded ineach lane. RT-PCR analysis was conducted using RNA isolated frommouse spleens to study whether mEMR4 is alternatively spliced. Prim-ers used in the analysis include 5�-57TTCCCAGAATGTTGATGGGAG-CAACTAGA85 and 5�-1090CACACCATCCTCCTCATGGGGTAGAGCC-A1062 for the 5�-end extracellular domain and 5�-875TCTGAACCTGTA-CTCCTGACTTTACAA901 and 5�-2134TCAATCACTAATAGTTCTGCTC-CAGTA2108 for the 3�-end TM7 domain. The PCR reactions were carriedout using the Advantage HF PCR kit (CLONTECH) for 30 cycles of94 °C for 30 s, 65 °C for 30 s, and 72 °C for 1.5 min.

Construction of Expression Vectors—All mEMR4 expression vectorswere constructed on pcDNA3.1(�) or pcDNA3.1-V5-His (Invitrogen).For the construction of V5-His-tagged mEMR4 vector, pmEMR4-V5-His, the entire mEMR4 open reading frame was amplified by PCR using5�-Hind (5�-CTTAAGCTTCCCAGAATGTTGATGGGAGCAACTAGA)and 3�-BamH (5�-TGGATCCTTGGGCCCATCACTAATAGTTCTGCTC-CAGTA) and subcloned into the pcDNA3.1-V5-His vector via HindIIIand BamHI sites. For the construction of vectors expressing mouse Fc(mFc) fusion proteins, two mEMR4 DNA fragments containing eitherthe entire extracellular domain (1–341 amino acids) or the EGF-likemodules (1–147 amino acids) were generated by PCR using 5�-Hind �341BamH (5�-AAGGATCCACACCATCCTCCTCATGGGGTAGAGC-CA) and 5�-Hind � 147EcoR (5�-TGAATTCACTCAGTACTCCCAAAG-TA), respectively. A truncated mFc DNA fragment containing the hingeand constant regions (CH2 � 3) of a mouse �2b immunoglobulin heavychain was generated by PCR using 5�-AGGATCCTGAATTCGCGGCC-GCAGAGCCCAGCGGGCCT and 5�-TCTTCTAGATTTACCCGGAGAC-CGGGA primers and the PSV-169/mIgG2b-322 plasmid as a template(57, 58). A DNA fragment encoding the consensus peptide sequence,DPNSGSLHHILDAQKMVWNHR*, recognized by the Escherichia colibiotin holoenzyme synthetase BirA (59), was generated by PCR using5�-Bio (5�-AATCTAGAGATCCAAATTCCGGA-3�) and 3�-Bio (5�-TAGT-AGGGGCCCTTAACGATGATTCCACACC-3�) primers and HLA A2plasmid as a template (60). The mFc fragment and the biotinylationsignal sequence were subcloned immediately downstream of either theentire extracellular domain (1–341 amino acids) or the EGF-like mod-ules (1–147 amino acids) of mEMR4 in pcDNA3.1(�) via theappropriate restriction sites (underlined). The final constructs thereforecontain two EGF-like modules with or without the full-length mucin-like stalk region followed by an mFc fragment and a biotinylation signaland are called pmEMR4-341mFc-Bio and pmEMR4-147mFc-Bio, respec-tively. Similarly, a construct coding for EMR2 (EGF1,2)-mFc-Bio fusionprotein was generated by subcloning the first and second EGF-likemodules of EMR2 into the pmFc-Bio vector. The domain-swapping mFc-Bio chimeras containing the first EGF-like module of EMR2 and thesecond EGF-like module of mEMR4 (pEMR2(1)/4(2)-mFc-Bio) or viceversa (pEMR4(1)/2(2)-mFc-Bio) were engineered by the SOE (splicingby overlapping extension)-PCR technique (61) using 5�-GCTAAGCTT-GAACCATGGGAGGCCGCGTCTTTCTCGT, 5�-TAGACACTCATTAA-TATCGTCACAAGTCTCCATGGGGGT, 5�-ACCCCCATGGAGACTTG-TGACGATATTAATGAGTGTCTA, and 5�-TGAATTCACTCAGTACTC-CCAAAGTA primers for pEMR2 (1)/4 (2)-mFc-Bio and 5�-Hind, 5�-TG-CACACTCGTTGATGTCTTGACATTTCTCATGGGG, 5�-GAGCCCCA-TGAGAAATGTCAAGACATCAACGAGTGTGCA and 5�-TAGGATCCA-AGCCTGGCTTGTAGTCTCT primers for pEMR4 (1)/2(2)-mFc-Bio. AnmFc-Bio expression construct encoding only the mFc-Bio fusion protein,pSec-mFc-Bio, was generated for use as a control by subcloning the mFcand biotinylation signal fragments into the pSecTag2 vector (Invitro-

gen) immediately after the leader peptide of the immunoglobulin�-chain.

Transient Transfection of Cells and Protein Analysis—The mEMR4expression constructs were transfected into HEK293T cells using Lipo-fectAMINE (Invitrogen) according to the manufacturer’s protocol. Con-ditioned medium and whole cell lysates in cell lysis buffer (20 mM

Tris-HCl, pH 7.4, 5 mM MgCl2, 100 mM NaCl, 1 mM sodium orthovana-date, 1 mM AEBSF (4-(2-aminoethyl)benzensulfonyl fluoride), 5 mM

levamisole, 1� Complete™ protease inhibitors (Roche Molecular Bio-chemicals)) were collected 48 h post-transfection, and protein concen-trations were determined by a Dc Protein Analysis Kit (Bio-Rad). Stand-ard SDS-PAGE and Western blot analysis were carried out as describedpreviously (62) using 8% gels and an anti-V5 monoclonal antibody(mAb) (Invitrogen) or an anti-mouse Fc-specific mAb (Sigma) followedby appropriate secondary Abs for ECL detection (Amersham Bio-sciences). For immunoprecipitation, cells were biotinylated with EZ-Link sulfo-NHS-LC-biotin (Pierce) at room temperature for 30 min,washed extensively with phosphate-buffered saline, and lysed with celllysis buffer. Cell lysates were precleared with protein G-Sepharosebeads prior to the incubation with an anti-V5 mAb at 4 °C for 1 hfollowed by incubation with protein G-Sepharose beads. After extensivewashing, immunoprecipitated complexes were resuspended in electro-phoresis sample buffer and subjected to standard gel electrophoresisand Western blotting using Extravidin-HRP (1:5000) for detection. Forthe production of soluble mEMR4-mFc fusion proteins, HEK293T cellswere transfected with 40 �g of DNA/175-cm2 flask using calcium phos-phate precipitation as described previously (13, 44). The medium wasreplaced with 25 ml of serum-free Opti-MEM I 16–18 h post transfec-tion and incubated for a further 72 h. Conditioned medium was col-lected, spun, and passed through a 0.45-�m filter followed by proteinA-Sepharose 4 Fast Flow (Amersham Biosciences) column purificationaccording to the manufacturer’s protocols. For N-glycosidase F treat-ment, 5 �g of the purified mEMR4-341mFc fusion protein was incubatedwith or without 3 units of N-glycosidase F (Roche Molecular Biochemi-cals) in 20 mM sodium phosphate buffer, pH 7.0, at 37 °C for 20 h.Samples were then reduced, run on an 8% SDS-PAGE, and stained withCoomassie Brilliant Blue. For N-terminal amino acid sequencing, thepurified mEMR4-341mFc fusion protein was reduced and run on a 10%Novex bis-Tris NuPAGE precast gel (Invitrogen) at 200 mA/gel usingMES buffer. The gel was electroblotted to a Novex 0.2 �m polyvinyli-dene difluoride membrane (Invitrogen) and stained with CoomassieBrilliant Blue. The desired �40-kDa band was excised, washed exten-sively with 10% methanol, and subjected to sequencing on an AppliedBiosystems 494A Procise protein sequencer (PerkinElmer Life Sciences,Applied Biosystems Division, Warrington, UK) using standard sequenc-ing cycles (63).

In Vitro Biotinylation of mEMR4-mFc Fusion Proteins—PurifiedmFc fusion proteins were buffered in 10 mM Tris-HCl, pH 8.0, bydialysis and incubated with 1 �l of BirA enzyme (Avidity, Denver, CO)overnight at room temperature. Excess biotin was subsequently re-moved by dialysis with 10 mM Tris-HCl, pH 7.3, containing 10 mM CaCl2and 100 mM NaCl. Following the confirmation of integrity of the bioti-nylated proteins using Western blotting probed with Extravidin-HRP(Sigma), the proteins were quantified by dot-blot analysis using myelinbasic protein-biotin (Avidity, Denver, CO) as standard and stored at�80 °C.

Cellular Ligand-binding Assay—To search for the putative cellularligand of mEMR4, various mouse cell lines were subjected to a FACS-based cell-binding analysis using a well established method for detect-ing protein-protein interaction (44, 59). In brief, 20 �l of avidin-coatedfluorescent beads (Spherotech, Inc., Libertyville, IL) were washed twiceand added to 2 �g of biotinylated protein in Hanks’ balanced saltsolution (HBSS) containing 0.5% bovine serum albumin (BSA) (HBSS/BSA) in a total volume of 50 �l. The bead-protein mixture was sonicatedat 20% power for 1 min (Heat Systems Sonicator) and then incubated at4 °C for 1 h. Nonbinding proteins were removed by washing twice withHBSS/BSA and the beads were resuspended in 50 �l of HBSS/BSA. Thebead-protein complex was sonicated again immediately before its addi-tion to single cell suspensions in a 96-well plate (1 � 106 cells/50 �lbeads/well). The cell-bead mixture was spun at 250 � g at 4 °C for 20min, incubated for a further 40 min at 4 °C, and subsequently resus-pended in 500 �l of HBSS for FACS analysis. Where necessary, 10 mM

EGTA was added in the reaction.

RESULTS

mEMR4 Is a New Member of the EGF-TM7 Family—In aneffort to identify the murine homologues of human EMR2 and

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EMR3 genes previously characterized by us (12, 13), a homol-ogy search in the data bases was carried out, and two mouseEST clones (GenBankTM accession numbers BG080641 andAA823656) containing sequences homologous to the EGF-TM7genes were identified. Using oligonucleotide primers derivedfrom these clones, 5�-end and 3�-end RACE reactions wereperformed to obtain the full-length cDNA, which was similarbut distinct from EMR2 and -3 and was therefore designatedmEMR4. The composite 2177-bp mEMR4 RACE product con-tains an open reading frame of 2067 bp encoding a 689 aminoacid protein (Fig. 1A). A Kyte-Doolittle hydropathy profile ofthe predicted protein revealed a hydrophobic signal peptide atthe N terminus and seven hydrophobic segments at the Cterminus (data not shown). A predicted signal peptide cleavagesite at residue Met31 of the precursor protein indicated that theresulting mature mEMR4 protein is a cell surface moleculecontaining a long N-terminal extracellular region of 310 aminoacids, a 7TM region of 254 amino acids, and a cytoplasmic tailof 94 amino acids (Fig. 1, A and B). The estimated molecularmass for the mature mEMR4 protein is 74 kDa.

At the most N-terminal portion of the mature EMR4 protein,two tandemly arrayed EGF-like modules were identified. Pro-tein sequence analysis indicated that the first EGF-like moduleis a non Ca2�-binding EGF (cbEGF) domain, whereas the sec-ond is a cbEGF domain (Fig. 2). Immediately C-terminal to theEGF-like modules is a Ser/Thr-rich, mucin-like spacer regionwithin which a highly conserved Cys-rich GPS domain wasidentified right before the first TM region (Fig. 2) (5, 6, 64). TheTM7 region of the mEMR4 is most similar to those of EMR3and EMR2 (60 and 59% identical, respectively) and hence couldbe categorized as a class-B GPCR. Following the TM7 region,mEMR4 has a relatively long cytoplasmic tail with one poten-tial protein kinase C-phosphorylation site and one casein ki-nase II phosphorylation site (Fig. 1A). Two other potentialprotein kinase C phosphorylation sites and one casein kinase IIphosphorylation site were also predicted in the intracellularloop regions (Fig. 1A). There are a total of eight potentialN-glycosylation sites in the extracellular region of mEMR4,with three on the first EGF module, four on the mucin-likestalk, and one on the first extracellular loop of the TM7 region,suggesting that the mEMR4 protein is highly glycosylated(Fig. 1B).

mEMR4 Maps to Mouse Chromosome 17—To date, everyknown human member of the EGF-TM7 family, with the ex-ception of ETL, was located on human Chr 19p13 region, sug-gesting a strong evolutionary link among these molecules (9,12, 13, 15). To determine the genetic map of the mEMR4 gene,the segregation pattern of sequences detected by a mEMR4full-length cDNA probe was followed in a M. musculus � M.spretus interspecific back-cross (48). Three variant HincII-di-gested DNA fragments co-segregated in M. spretus were iden-tified. Interspecific back-cross analysis clearly established link-age between mEMR4 and several other genes known to belocated on mouse Chr 17, between Nfya and the tightly linkedmarkers of C3, Mllt1, Emr1, Vav, and Rfx2 (Fig. 3). The loca-tion of mEMR4 was mapped at �10.43 (� 2.85) centimorgansand 0.86 (� 0.86) centimorgans from those flanking genes,respectively (Fig. 3). The C3, Emr1, Vav, and Rfx2 region ofmurine Chr 17, as determined by physical and comparativemapping, is known to be syntenic to the human Chr 19p13.3locus (48). Interestingly, a search in the human genome database identified a genomic contig of human Chr 19p13.3 (acces-sion no. NT011169.4) containing the human EMR1 gene andanother incomplete EGF-TM7 gene highly homologous tomEMR4 (82% identical on existing sequence; data not shown).This putative human homologue of mEMR4 is �20 kb proximal

to EMR1, comparable with the genetic map of Emr1 andmEMR4 in the mouse genome, indicating that this region of thegenome is highly conserved among species.

mEMR4 Is Expressed Predominantly in Resident Macro-phages and Is Up-regulated following Macrophage Activation—The majority of the EGF-TM7 genes are expressed highly andrestrictedly in myeloid cells including monocytes, macro-phages, and granulocytes (7, 8). Northern blot analysis wascarried out to examine the expression pattern of mEMR4 usingRNA samples from multiple mouse tissues and various primarycells. Fig. 4A shows that a major mEMR4 transcript of approx-imate 3.3 kb was expressed abundantly in spleen and liver,whereas a low level of expression was found in lung and kidneyand weakly in thymus. No signal was detected in brain, heart,skeletal muscle, skin, small intestine, stomach, or testis. Theexpression pattern is similar to those of other EGF-TM7 mem-bers and is consistent with that of subpopulations of residentmacrophages. Indeed, among various primary cells tested, theexpression of mEMR4 was strongly detected in resident peri-toneal macrophages (RPM�), BioM�, and ThioM� (Fig. 4B).BMDC and ThioPMN showed lower levels of expression, al-though we could not rule out the possibility of M� contamina-tion in ThioPMN preparation. Splenic B220�-B cells showed avery weak signal, and no signal was detected in splenic T cellsand BMM� (Fig. 4B).

Interestingly, the expression was found to be up-regulated inBioM� and even more so in ThioM�, suggesting an induction ofgene expression following macrophage activation (Fig. 4B). Toinvestigate this further, RPM� were cultured with or withoutvarious pro- and anti-inflammatory cytokines for 36 h. In con-cert with the up-regulation of mEMR4 in primed/activated M�

such as BioM� and ThioM�, TNF-�-treated RPM� expressed ahigher level of expression in comparison to that of untreatedcells, whereas interferon-� did not seem to induce further ex-pression (Fig. 4C). In contrast, IL-10 treatment substantiallyreduced mEMR4 expression in RPM� and IL-4 seemed to abol-ish the expression (Fig. 4C). The expression of mEMR4 inRPM� therefore appeared to be highly regulated.

The majority of the EGF-TM7 genes are also characterizedby extensive alternative splicing of mRNA. Alternative splicingoccurs predominantly at the 5�-end of the transcripts resultingin multiple protein isoforms that contain different numbersand/or combinations of EGF-like domains (12, 16–18). RT-PCRanalysis using different primer sets that cover the 5�-end and3�-end regions of the gene was employed to determine whethermEMR4 also expressed alternatively spliced transcripts. Fig.4D shows that both 5�-end and 3�-end primer sets generate onlyone band of PCR product of expected sizes indicating that,unlike other EGF-TM7 members, mEMR4 is not alternativelyspliced. Hence, only one protein species is predicted.

mEMR4 Is a Heterodimeric Glycoprotein Resulted from theProteolytic Cleavage in the Extracellular Domain—To charac-terize the biochemical properties of the mEMR4 protein, thefull-length mEMR4 open reading frame was subcloned into anexpression vector and tagged with a V5-His epitope at theC-terminal end (see “Experimental Procedures”). Total cell ly-sates from HEK293T cells transiently transfected with or with-out this vector were analyzed by Western blotting using ananti-V5 mAb (Fig. 5A). Two specific bands of �85–100 and 34kDa were observed in the vector-transfected cells but not inmock-transfected cells. The 85–100 kDa band appeared to bedim and diffuse and might represent unprocessed mature pro-tein species (see below). Alternatively, it might represent im-mature protein precursor as the sample contained total celllysates. The smeared appearance also suggested that mEMR4is glycosylated. The 34-kDa band is very intense, specific, but

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FIG. 1. Primary amino acid sequence and predicted structure of mEMR4. A, the deduced amino acid sequence of mEMR4. The putativesignal peptide is shown in boldface letters, and the first and second EGF-like module as well as the TM7 region are highlighted in yellow, orange,and gray backgrounds, respectively. The potential N-linked glycosylation sites are enclosed in boxes, and the GPS domain is underlined in green.The potential protein kinase C phosphorylation sites and casein kinase II phosphorylation sites are underlined once and twice, respectively. B,schematic structure of mEMR4. The EGF-like modules are represented by numbered and color-coded triangles, the GPS domain is shown as a greenbox, and the TM7 region is represented by membrane-spanning cylinders. Filled circles denote potential N-linked glycosylation sites.

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much smaller than the expected mature mEMR4 protein. How-ever, it is consistent with the predicted size of the TM7 andcytoplasmic tail regions. In light of the presence of the GPSdomain in the stalk region of mEMR4, the possibility of pro-

teolytic cleavage was investigated further using biotinylationof cell surface proteins followed by immunoprecipitation by an�-V5 mAb. Immunoprecipitated proteins were separated in anSDS-PAGE and detected by Extravidin-HRP by Western blot-

FIG. 2. Amino acid sequence alignments of mEMR4, hEMR3, and rETL. The amino acid sequences of mEMR4, hEMR3, and rETL proteinsare aligned for maximal homology. Identical and similar residues are shaded in red and gray backgrounds, respectively. The EGF-like modules,the GPS domain, and the transmembrane regions are indicated individually. Asterisks indicate the cysteine, glycine, and tyrosine residuesconserved in the GPS domain.

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ting. Fig. 5B shows that a major band of �50 kDa was detectedalong with a minor band of �90–100 kDa. The latter wasprobably the uncleaved form of the receptor, whereas the ear-lier band is the cleaved extracellular domain of mEMR4. Novisible bands were detected in mock-transfected samples. Theproteolytic cleavage of mEMR4 was finally confirmed using afusion protein containing the full-length mEMR4 extracellulardomain (1–341 amino acids) and an mFc fragment, mEMR4-

341mFc (see “Experimental Procedures”). Fig. 5C shows thatwhen conditioned media collected from cells transfected withmEMR4-341mFc and a control mFc construct were probed withan anti-mFc mAb, both samples produced a band of approxi-mately 40 kDa, which correlates to the size of the mFc protein.A very faint band of �80–100 kDa was also detected in themEMR4-341mFc protein sample, which is likely to be the un-cleaved full-length fusion protein. No signal was found in sam-ples from mock-transfected cells. This result indicates that themajority of mEMR4-341mFc fusion protein is proteolyticallycleaved within the extracellular stalk region very close to the Cterminus of the GPS domain, thus generating a cleaved productthe same size as the control mFc protein. Furthermore, itshows that the TM7 region is not required for the cleavageactivity, as the fusion protein contains only the extracellularregion. To characterize the proteolytic process further, we pu-rified the mFc fusion protein by protein A affinity columnchromatography and resolved it in an 8% SDS-PAGE (Fig. 5D).Three protein bands of approximate 80–100, 46–60, and 40kDa were visible after Coomassie Brilliant Blue staining, rep-resenting, respectively, the unprocessed mature protein (�80–100 kDa), the cleaved extracellular domain (�46–60 kDa), andthe mFc fragment (�40 kDa). When the same protein samplewas treated with N-glycosidase F, three protein bands of re-duced sizes (�65–75, �40, and �37 kDa) were identified, in-dicating that mEMR4 is indeed a glycoprotein. Moreover, it

FIG. 3. Genetic mapping of mEMR4. The linkage of mEMR4 toNfya and Vav genes on mouse chromosome 17 was determined bySouthern blot hybridization of HincII-digested DNA from 118 interspe-cific back-cross progeny. Black boxes represent the inheritance of aC3Hf allele for a particular set of loci, and white boxes represent theM. spretus allele. Gene linkage, order, and intergenic distance withassociated standard errors were determined using Map Manager dataanalysis software.

FIG. 4. The expression profile of mEMR4. A, Northern blots containing equal amounts (2 �g) of poly(A)� RNA from the indicated mousetissues were subsequently hybridized with 32P-labeled probes specific for mEMR4 and E-actin. An approximate 3.30-kb mEMR4 transcript isindicated. B and C, total RNA (10 �g/lane) from various mouse primary cells and cytokine-treated RPM� was analyzed by Northern blotting asdescribed under “Experimental Procedures.” The numbers indicate the positions of molecular weight markers. Ethidium bromide staining of thegels in the lower panels shows the equal loading and integrity of the RNA samples. D, RT-PCR analysis of mEMR4 using primer sets covering the5�-end extracellular region and the 3�-end TM7 region shows that only a single band of PCR product is generated from the tissues examined. Thenumbers indicate the sizes of the PCR products.

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shows clearly that the two cleaved protein subunits remainassociated after proteolysis. To determine the cleavage site, the�40-kDa mFc band was excised for N-terminal amino acidsequencing. The resulting sequence, SSFAVLMALP, matchesprecisely to the mEMR4 stalk sequence (residues 327–336) andis consistent with the conserved GPS cleavage site (Fig. 5E).The cleavage therefore occurred at the peptide bond betweenLeu326 and Ser327.

mEMR4 Binds to a Putative Cellular Ligand on A20 Cells viathe Second EGF-like Module—To examine whether mEMR4can mediate protein-protein interaction and to search for itspotential cellular ligand, a previously described cell-bindingassay was modified and employed (13, 44). The EGF-like mod-ules of mEMR4 were fused with a truncated Fc region of amouse immunoglobulin (IgG2b subtype) and a biotinylationsignal to generate pmEMR4-147mFc (see “Experimental Proce-dures”) (Fig. 6A). The mFc region enabled us to purify largeamounts of soluble proteins from transfected mammalian cells,whereas the biotinylation signal allowed efficient in vitro bioti-nylation of the purified proteins (Fig. 6B). By coupling thesoluble biotinylated mEMR4-mFc fusion proteins to avidin-coated fluorescent beads, a multivalent probe with increasedbinding abilities and sensitivities are generated. A FACS-basedcell-binding assay was then performed to screen for the pres-ence of the mEMR4 ligand (see “Experimental Procedures”).

From a total of eight cell lines (J774, NIH3T3, RAW, P388D1,Wehi-231, Daudi, BW-1547, EL-4, and A20) tested, A20, amouse B lymphoma cell line, showed a strong fluorescencesignal, indicating that it expresses a putative mEMR4 ligandon the cell surface (Fig. 6C). The interaction between the mul-tivalent mEMR4 probe and A20 cells is specific because nobinding signal was found when a similar EMR2 (1,2)-mFcfusion protein was used in the same assay. Moreover, theinteraction was shown to be Ca2�-independent because theaddition of EGTA did not affect binding (Fig. 6C). The Ca2�-independent interaction suggested that the first EGF-like mod-ule might be involved in the binding because it is predicted tobe a non-cbEGF (Figs. 1 and 2). To locate the ligand-bindingdomain, the first and second EGF-like modules of mEMR4 andEMR2 were exchanged by genetic engineering to generate chi-meric protein probes (Fig. 6A). FACS analysis using the do-main-swapping chimeras surprisingly but unequivocallyshowed that the interaction is mediated predominantly by thesecond EGF-like module of mEMR4 (Fig. 6D).

DISCUSSION

With two N-terminal EGF-like modules, a mucin-like spacerregion and a class-B GPCR-related TM7 domain, mEMR4 isthe ninth member of the EGF-TM7 family to be identified todate (Figs. 1 and 2). As with the majority of the EGF-TM7

FIG. 5. Biochemical analysis of the mEMR4 protein. A, Western blot analysis of total cell lysates from HEK293T cells transientlytransfected with (lane 2) or without (lane 1) pmEMR4-V5-His vectors. Samples were separated in a 8% polyacrylamide-SDS gel under reducingconditions, blotted, and probed with an anti-V5 mAb. The arrow indicates a nonspecific band observed in both samples. B, Western blotanalysis of biotinylated cell surface proteins immunoprecipitated with anti-V5 mAb and probed with Extravidin-HRP. Lanes 1 and 2 representsamples from mock- and pmEMR4-V5-His vector-transfected cells, respectively. C, Western blot analysis of conditioned medium fromHEK293T cells transiently transfected with (lane 3) or without (lane 1) pmEMR4-341mFc vectors. Lane 2 contains conditioned medium fromHEK293T cells transiently transfected with a positive control pSec-mFc-Bio vector. The blot was probed with an anti-mFc mAb. Numbers onthe right indicate the positions of molecular weight markers. D, Coomassie Brilliant Blue staining of the protein A column-purifiedmEMR4-341mFc protein treated with (lane 2) or without (lane 1) N-glycosidase F. E, comparison of the amino acid sequences of the GPS domainamong indicated LNB-TM7 proteins. The amino acid sequences obtained from N-terminal sequencing of the cleaved mFc protein are boxed.The arrow indicates the cleavage site.

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genes, mEMR4 is expressed predominantly in myeloid cellsincluding RPM�, BioM�, ThioM�, and BMDC (Fig. 4), suggest-ing that it may play a functional role in these professionalphagocytes and antigen-presenting cells. The up-regulation ofmEMR4 expression in BioM�, ThioM�, and TNF-�-treatedRPM� further suggested that mEMR4 might participate insituations involving activated M� such as inflammation andinfection. The regulated mEMR4 gene expression by pro- andanti-inflammatory cytokines indicates that the mEMR4 gene ishighly controlled at the transcriptional level (Fig. 4C). Similartightly regulated gene expression has also been found for otherEGF-TM7 genes. For example, CD97, a T-cell activationmarker, is rapidly up-regulated at a very early stage upon Tcell activation, whereas F4/80 has been shown to be diminishedfrom F4/80�ve Langerhans cells to F4/80�ve interdigitatingDCs in response to local antigen exposure (15, 39, 65). Under-standing the regulatory mechanisms governing the expressionprofiles of individual EGF-TM7 genes in the future will shedlight on their temporal and lineage-specific expression patternsand possibly their specific functions.

The proteolytic cleavage of mEMR4 at the GPS site, as re-vealed here by Western blot analysis and N-terminal aminoacid sequencing (Fig. 5), clearly shows that mEMR4 is proteo-lytically processed from a precursor polypeptide resulting in anextracellular domain subunit and a TM7 subunit. In addition,the immunoprecipitation of biotinylated cell surface proteinsby anti-V5 mAb further indicates that the cleaved extracellulardomain subunit is associated with the TM7 domain subunit(Fig. 5B). The same protein modification has been demon-

strated for other LNB-TM7 molecules and is possibly a commonfeature for all the GPS domain-containing proteins. Althoughthe functional significance of this modification is still unclear,several modes of actions regarding intracellular signaling couldbe envisioned as a consequence of this well conserved modifi-cation. The tethered extracellular domain could be shed follow-ing ligand binding or cellular activation leading to the activa-tion of the “unoccupied” TM7 receptor domain. Alternatively,the resulting ‘unoccupied’ TM7 receptor domain may becomeavailable to other peptide hormone-like ligands similar to thoseof the classical class B-GPCRs. In contrast, the shedding of theextracellular domain could possibly be a mechanism for recep-tor internalization/down-regulation leading to the terminationof signaling. It is also possible that the proteolytic cleavage atthe GPS site is prerequisite for the maturation and function ofreceptors, which may acquire certain conformational changesdue to the cleavage. The detailed relationship between proteo-lytic processing and receptor functions awaits further investi-gation. In addition, the consensus GPS domain and proteolyticcleavage site found in the LNB-TM7 molecules strongly sug-gested the presence of a functionally conserved protease(s), thetrue identity of which is also of particular interest.

In concert with the similarity in structure, expression, andprotein modification, the genetic mapping of mEMR4 to thedistal region of mouse Chr 17 (Fig. 3) also points to a wellconserved gene family located mainly in human Chr 19p13region (and the syntenic mouse Chr regions) (9, 11–13, 15, 45,46). Interestingly, the Chr 19p13 EGF-TM7 genes can be fur-ther subdivided into two distinct loci, with EMR1 and -4 on

FIG. 6. Cell surface ligand-binding analysis. A, the schematic representation of the biotinylated mFc fusion proteins. The EGF-like modulesare represented by numbered and colored triangles. The two gray circles represent the mFc region, and the small green circle indicates thebiotinylation signal. B, Western blot analysis of biotinylated EMR4/EMR2-mFc fusion proteins. Extravidin-HRP was used to detect biotinylatedmFc protein (lane 1), EMR2(1,2)-mFc-Bio (lane 2), EMR4(1,2)-mFc-Bio (lane 3), EMR2(1)/4(2)-mFc-Bio (lane 4), and EMR4(1)/2(2)-mFc-Bio (lane5) proteins. Numbers on the left indicate the positions of molecular weight markers. C, the FACS profile shows that A20 cells interact withfluorescent beads coated with multivalent EMR4-mFc probes but not with EMR2-mFc probes; the interaction is not inhibited in the presence of10 mM EGTA. D, the ligand-binding domain is mapped to the second EGF-like module of EMR4 using the domain-swapping chimeras.

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human Chr 19p13.3 (mouse Chr 17) and CD97, EMR2, andEMR3 on human Chr 19p13.1 (mouse Chr 8). Moreover, mem-bers of other gene families were also found closely linked withthe EGF-TM7 genes within these two loci. For example, tran-scription factors RFX1 and RFX2, two closely related site-specific DNA-binding proteins important for immune systemfunctions (66, 67), are tightly linked with CD97 and EMR1,respectively, both in human and mouse genomes (11, 46, 48).These findings have given rise to the notion that both sets ofgene families were derived from an ancient gene duplicationevent encompassing their predecessors and subsequently sep-arated during evolution (46). The identification and mapping ofmEMR4 as well as the detailed genomic sequence informationgradually emerging from the human and mouse genomeprojects will no doubt further delineate the extent of geneduplication and details of their evolution.

Although the significance of the interaction between mEMR4and A20 cells (Fig. 6) has yet to be established, the predomi-nant myeloid cell expression profile and the up-regulation ofmEMR4 in activated M� suggested that the interaction couldplay an important role in modulating immune and inflamma-tory responses by allowing “cross-talk” between myeloid cellsand the ligand-bearing cells. The ligation of the mEMR4 recep-tor could potentially activate M� via the TM7 moiety to amplifyimmune and inflammatory responses. On the other hand, thesignaling could occur in the opposite direction to activate theligand-bearing cells. A20 is a B cell lymphoma line derivedfrom a spontaneous reticulum cell neoplasm (68). At present, itis not known whether the putative mEMR4 ligand is restrictedonly to B cell populations or is shared also by other cell lin-eages. Our preliminary results show that it is not detectable insingle spleen cell suspension from naive mice but is present inthe white pulp B cell zones of infected mice.3 If the ligand isindeed restricted to certain B cell subpopulations, the mEMR4-ligand interaction could potentially stimulate the proliferationor differentiation of the ligand-bearing B cells. The exact na-ture of the mEMR4 ligand is currently under investigation.

The EGF-like module is one of the most common structuralmotifs used by cell surface and extracellular matrix proteinsand has been identified in proteins involved in cellular adhe-sion, cell fate determination, extracellular matrix structure,receptor-ligand interactions, and blood coagulation (20). Recentprotein structure studies have revealed a major double-stranded �-sheet conformation for the EGF-like modules (19,69), which mediate protein-protein interaction either via theintramolecular loops on the surface or via the Ca2� ion chelatedby their calcium-binding domains (20, 70). The finding that theinteraction between mEMR4 and its ligand on A20 cells isCa2�-independent but mediated predominantly by the Ca2�-binding second EGF-like module (Fig. 6) may reflect the factthat mEMR4 contains only one non-cbEGF and one cb-EGFmodule (Fig. 1). It is possible that in this setting calciumbinding is not critical in maintaining the surface structure forprotein-protein interaction. In this regard, it is of interest tonote that EMR3, similarly containing only one non-cbEGF andone cbEGF module, also binds to its ligand in a Ca2�-independ-ent manner.3 It is therefore reasonable to suggest that themEMR4 ligand-binding site might be located in the variableloop regions of the second EGF-like module.

At the last phase of preparation of this manuscript, a veryrecent paper describing the molecular cloning of a F4/80-like-receptor (FIRE), which is identical to mEMR4, was publishedby Caminschi et al. (71). FIRE was identified based upon itsdifferential expression patterns between CD8� and CD8� DC

subsets. Although the reported expression pattern of FIRE intissues and primary cells is consistent with our finding, FIREwas found to be down-regulated upon DC and M� activation, indirect conflict with our results (Fig. 4). A possible cause for thisdiscrepancy may be the different analysis systems used in thestudies. The down-regulation of FIRE was detected by FACSanalysis of cell surface protein expression, whereas the up-regulation of mEMR4 was determined by Northern blot anal-ysis of RNA expression. It remains possible that followingcellular activation, mEMR4 RNA was up-regulated, leading tothe production of more receptor molecules, but due to theprotein cleavage and possible subsequent shedding of the ex-tracellular domain, the overall level of cell surface receptorproteins detected is reduced. It is also possible that the epitoperecognized by the anti-FIRE mAb in question is regulated/modulated by the modification of the receptor protein such asglycosylation in relation to the activation status of cells. It ishoped that the functional and biochemical characterization ofmEMR4 presented here including glycosylation, proteolyticcleavage, and ligand interaction, along with the use of addi-tional reagents and experiments in the future, such as mAbsspecific to the intracellular region of mEMR4 or the epitopemapping of FIRE mAbs, can help in the exploration of thesepossibilities.

The unique hybrid structure of the EGF-TM7 receptors issuggestive of a dual function in which the EGF-like modulescarry out cellular adhesion/interaction while the TM7 moietytransmits intracellular signaling messages (7, 8). The recentdemonstration of the presence of cell surface ligands for CD97,EMR3, EMR2,2 and mEMR4 herein has further strengthenedthis hypothesis. As the first step toward definitively provingthis hypothesis, the sensitive ligand-binding assay describedabove is currently been employed to identify molecularly thecognate cell surface ligands for the individual EGF-TM7 recep-tors. Once identified, future demonstrations of the signalingpathway of the EGF-TM7 receptors and the linkage betweenthe dual adhesion-signaling function will be the keys to unrav-eling the cellular importance of these unusual TM7 receptors.

Acknowledgments—We thank Antony Willis (Department of Bio-chemistry, Oxford University) for performing N-terminal amino acidsequencing. BMDC and PSV-169 mIgG2b-322 plasmids were kindlyprovided by Drs. T.-C. Chen and L. Martinez-Pomares, respectively.

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EGF-TM7 Molecule mEMR4 Binds to a Cellular Ligand on A20 Cells 29293

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Siamon Gordon and Hsi-Hsien LinMartin Stacey, Gin-Wen Chang, Stephanie L. Sanos, Laura R. Chittenden, Lisa Stubbs,

Cell Line A20LymphomaActivated Mouse Macrophages, Binds to a Putative Cellular Ligand on B

EMR4, a Novel Epidermal Growth Factor (EGF)-TM7 Molecule Up-regulated in

doi: 10.1074/jbc.M204306200 originally published online May 21, 20022002, 277:29283-29293.J. Biol. Chem. 

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