Human Epidermal Growth Factor (EGF) Module-containing Mucin-like Hormone Receptor 3 Is a New Member of the EGF-TM7 Family That Recognizes a Ligand on Human Macrophages and Activated Neutrophils*

Martin StaceyDagger, Hsi-Hsien Lin§, Katherine L. Hilyard, Siamon Gordon||, and Andrew J. McKnight||**

From the Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE,  Roche Discovery Welwyn, 40 Broadwater Road, Welwyn Garden City AL7 3AY, and the ** Department of Clinical Sciences, Institute of Liver Studies, King's College Hospital, Bessemer Road, London SE5 9PJ, United Kingdom

Received for publication, February 6, 2001, and in revised form, March 8, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The epidermal growth factor (EGF)-TM7 subgroup of G-protein-coupled receptors is composed predominantly of leukocyte-restricted glycoproteins defined by their unique hybrid structure, in which extracellular EGF-like domains are coupled to a seven-span transmembrane moiety via a mucin-like stalk. The EGF-TM7 group comprises mouse F4/80, human EGF module-containing mucin-like hormone receptor (EMR) 1, human EMR2, and human and mouse CD97, the genes for which map to human chromosome 19p13 and the syntenic regions of the mouse genome. In this study we describe the cloning and characterization of EMR3, a novel human EGF-TM7 molecule, and show the existence of its cellular ligand. The EMR3 gene maps closely to the existing members of the EGF-TM7 family on human chromosome 19p13.1 and, in common with other EGF-TM7 genes, is capable of generating different protein isoforms through alternative splicing. Two alternative splice forms have been isolated: one encoding a 652-amino acid cell surface protein consisting of two EGF-like domains, a mucin stalk, and a putative G-protein-coupled receptor domain and the other encoding a truncated soluble form containing only two EGF-like domains. As with other members of the EGF-TM7 family, EMR3 mRNA displays a predominantly leukocyte-restricted expression pattern, with highest levels in neutrophils, monocytes, and macrophages. Through the use of soluble EMR3 multivalent probes we have shown the presence of a ligand at the surface of monocyte-derived macrophages and activated human neutrophils. These interactions suggest a potential role for EMR3 in myeloid-myeloid interactions during immune and inflammatory responses.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Professional phagocytic cells such as neutrophils and macrophages play essential roles in a wide range of both immune and homeostatic functions. Many crucial processes such as chemotaxis and migration, pathogen recognition and phagocytosis, inflammation, and microbial killing are mediated in part by the vast repertoire of leukocyte cell surface receptors. One rapidly growing group of receptors that may be involved in such processes is that of the epidermal growth factor (EGF)1-TM7 family (1, 2).

At present the EGF-TM7 family comprises human and mouse CD97, mouse F4/80, human EMR1 (EGF module-containing mucin-like hormone receptor 1), and human EMR2 (3-8). These predominantly leukocyte-restricted glycoproteins are defined by their unique chimeric structures, which consist of varying numbers of N-terminal EGF-like repeats coupled to a family B G-protein-coupled receptor (GPCR)-related moiety via a mucin-like stalk (Fig. 1). The founding member of the EGF-TM7 family, F4/80, comprises seven EGF-like domains, a spacer region, and a hydrophobic seven-span transmembrane (TM7) region. As a result of its macrophage-restricted expression, F4/80 has long been used as an excellent marker for populations of mouse tissue macrophages. EMR1, the likely human homologue of F4/80, possesses six EGF-like domains and shares 68% overall amino acid identity with its murine counterpart. CD97, a B and T cell activation antigen, is expressed constitutively by human granulocytes and monocytes and at low levels by resting T and B cells and is markedly and rapidly up-regulated upon T and B cell activation. CD97 occurs on the cell surface as three major isoforms resulting from alternative RNA splicing. All CD97 isoforms have been shown to bind to a membrane protein of the complement system, CD55 (DAF); however, the isoform containing three EGF-like domains binds with higher affinity to CD55 than the other isoforms. More recently, human EMR2 has been cloned that contains EGF-like domains that are highly homologous to those of CD97. Similarly, EMR2 RNA undergoes alternative splicing; however, the isoforms appear to be more myeloid-restricted and are known not to interact with CD55.


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Fig. 1.   The EGF-TM7 family comprises mouse F4/80 (GenBankTM accession number X93328), human EMR1 (GenBankTM accession number X81479), human EMR2 (GenBankTM accession number AF114491), human EMR3 (GenBankTM accession number AF239764), and human and mouse CD97 (GenBankTM accession numbers U76764 and Y18365). Triangles represent EGF-like domains, filled circles denote potential sites of N-linked glycosylation, and membrane-spanning cylinders show transmembrane domains of the putative GPCR moieties. The largest known isoform of each molecule is depicted.

The EGF-like domain, like those found in EGF-TM7 proteins, is a common structural module often occurring as tandem repeats in extracellular proteins involved in cellular adhesion, receptor-ligand interactions, and blood coagulation. EGF-like domains consist of ~50 amino acids and are defined by six conserved cysteine residues that form three intra-domain disulfide bonds. A subset of EGF-like domains contains a Ca2+-binding consensus sequence, (D/N)X(D/N)(E/Q)Xm(D/N*)Xn(Y/F) (where m and n are variable, and * indicates possible beta -hydroxylation) (9). Chelated Ca2+ performs a key role in the orientation of EGF-like pairs by restricting flexibility of the interdomain linkage, thus allowing the presentation of specific surfaces for protein-protein interactions to occur (10). The importance of EGF-like domains is demonstrated by natural mutations in the genes encoding fibrillin-1, low density lipoprotein receptor, factor IX, and protein S, which are responsible for Marfan syndrome, familial hypercholesterolemia, hemophilia B, and protein S deficiency, respectively (11-14).

The transmembrane regions of the EGF-TM7 molecules show the greatest degree of sequence homology to GPCR B family. GPCRs constitute the largest gene superfamily within the genome and have been shown to mediate a range of cellular responses to an extensive array of stimuli including peptides, amino acid derivatives, lipid analogues, ions, and external sensory stimuli such as light, taste, and odors through intracellular signaling (15).

Based upon the restricted expression and hybrid structure of EGF-TM7 family members, it has been suggested that these cell surface proteins may play a significant role within the immune system, mediating adhesion through their EGF-like repeats with concomitant intracellular signaling via the GPCR moiety. Although signaling in these molecules has yet to be demonstrated, CD97 has been shown to bind to a membrane protein of the complement system, CD55 (DAF), demonstrating its role in cell-cell interactions (16).

In this study we describe the cloning and characterization of human EMR3, a new member of the EGF-TM7 family. The EMR3 gene maps closely to other members of the EGF-TM7 group on human chromosome 19p13.1 and encodes a leukocyte-restricted protein expressed predominantly on neutrophils, monocytes, and macrophages. Multivalent EMR3 probes were used to demonstrate the presence of a cell surface ligand for EMR3 on both monocyte-derived macrophages and activated human neutrophils, suggesting a potential role for EMR3 in myeloid cell interactions.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- All chemicals and reagents were obtained from Sigma unless otherwise specified. Cell culture media and supplements were purchased from Life Technologies, Inc. X-Vivo 10 medium was from BioWhittaker (Walkersville, MD). Human buffy coats were purchased from the National Blood Service, Bristol, UK. Cell lines were provided by the cell bank at the Sir William Dunn School of Pathology, University of Oxford.

Cloning and Sequence Analysis-- Oligonucleotide primers were designed from the sequence of a human chromosome 19 cosmid (GenBankTM accession number AC004262, clone R29368) that showed homology with the TM1 and membrane proximal region of F4/80 and CD97. 5' and 3' EMR3 cDNA fragments were amplified from human spleen Marathon Ready cDNA by rapid amplification of cDNA ends (RACE) under conditions recommended by the manufacturer (CLONTECH). In brief, 5'-RACE products amplified from primer E1 (5'-CTGGTGTTCTGGATGGCTTTACACAGGAG) and the anchor-specific primer AP-1 of the manufacturer were used in a secondary nested PCR reaction using E2 (5'-GCACGGGATCCTCCTGCACATCGTAGTGG) and the anchor-specific AP-2 primer. The 5'-proximal cDNA product was then subcloned and sequenced using standard molecular techniques. The 3'-proximal cDNA fragment EMR3 cDNA was obtained using E3 (5'-TGTGTCTACTGGAAGAGCACAGGGCA) and E4 (5'-TGATACACGTGAACAAGAGTCAC) with AP-1 and AP-2 primers in two rounds of PCR as described above. Full-length EMR3 cDNA was amplified from human spleen cDNA using primers E5 (5'-TTTCTTGAGCTAGGAAAGGTGGTGGTT) and E6 (5'-TTGAAGAGAAATGCAATTTACCACACA) and HF-polymerase under conditions recommended by the manufacturer (CLONTECH). The PCR products were subcloned into pGEM-T (Promega, Madison, WI), sequenced, and subsequently excised and ligated into pcDNA3 (Invitrogen) using NotI and ApaI restriction enzymes.

RNA Blot Analysis and RT-PCR-- Total RNA was prepared from various cell lines and human peripheral blood mononuclear cells, polymorphonuclear cells, monocytes, lymphocytes, as well as day 1 and day 7 monocyte-derived macrophages using the acid guanidium thiocyanate-phenol-chloroform technique (17). Total RNA (10 µg) was electrophoresed on a 1% formaldehyde-agarose gel, blotted onto a nylon membrane (Stratagene), and then hybridized with gene-specific probes as previously described (18). Likewise, commercially available human multiple tissue Northern blots (CLONTECH) were hybridized with a full-length EMR3 cDNA probe according to the instructions of the manufacturer. Hybridized blots were washed in 0.5× SSC (0.15 M NaCl and 0.015 M sodium citrate) containing 1% SDS at 55 °C for 30 min and exposed to x-ray film (Kodak Biomax MR) 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 with a human beta -actin cDNA probe to compare the amount of RNA loaded in each lane. RT-PCR data using multiple tissue cDNA panels (CLONTECH) and RNA templates from various cell lines were also used to study the EMR3 expression profile. Advantage polymerase (CLONTECH) and primers E5 (5'-CACAAATGCAGGGACCATTGCT) and E6 (5'-GGATGTCCATTGAGTTCATTTG) were used to amplify EMR3-specific cDNA fragments of 700 base pairs under conditions recommended by the manufacturer. beta 2-microglobulin-specific primers F1 (5'-GATTCAGGTTTACTCAGG) and R1 (5'-CCATGATGCTGCTTACATG) were also used in both RT-positive and RT-negative PCR reactions to compare cDNA levels and show a lack of genomic DNA contamination within each PCR reaction.

Production of Biotinylated EMR3 Recombinant Protein-- A cDNA fragment encoding the peptide sequence NSGSLHHILDAQKMVWNHR*, recognized by the Escherichia coli biotin holoenzyme synthetase BirA, was generated by PCR using Bio5 (5'-TAGTAGGGATCCGAATTCCGGATCACTGCATCATATT-3') and Bio3 (5'-TAGTAG- GGGCCCTTAACGATGATTCCACACC-3') primers and an HLA-A2 plasmid construct, containing the peptide coding sequence as template (19). Following BamHI and ApaI digestion, the cDNA fragment was subcloned immediately downstream of the stalk region of EMR3 in pcDNA3. Soluble biotinylated EMR3 protein comprising the EGF-like domains and the stalk region was produced as described previously (19). Briefly, conditioned medium collected from transfected 293T cells was concentrated to ~0.5 ml using a 30-kDa cutoff Centriprep filtration unit (Millipore), dialyzed against 10 mM Tris-HCl (pH 8) buffer, and incubated with 1 µl of BirA enzyme and manufacturer substrates (Avidity) overnight at room temperature. Excess biotin was subsequently removed by dialysis against 10 mM Tris-HCl (pH 7.3) buffer containing 10 mM CaCl2 and 150 mM NaCl. The biotinylated proteins were then aliquoted and stored at -80 °C after quantification by dot-blot analysis using biotinylated maltose-binding protein (Avidity) as standard.

Isolation of Blood Cells-- 10 ml of heparinized blood from a healthy donor was overlaid on a PolymorphprepTM gradient and centrifuged as directed by the instructions of the manufacturer (Nycomed Pharma AS, Oslo, Norway). The erythrocyte, mononuclear, and polymorphonuclear cell fractions were collected separately, washed once in 0.5× RPMI and twice in RPMI, and then resuspended in RPMI medium. Purified neutrophils were activated by incubation at 37 °C for 30 min with 100 nM formyl-Met-Leu-Phe.

Cell Binding Assay Using Biotinylated Protein-coupled Fluorescent Beads-- Cell binding assays using fluorescent beads coupled to biotinylated proteins were performed as previously described (19). In brief, 10-µl avidin-coated fluorescent beads (Spherotech Inc.) were washed twice with phosphate-buffered saline containing 1% bovine serum albumin (phosphate-buffered saline/bovine serum albumin) and added to 1 µg of biotinylated protein in a total volume of 50 µl. The bead and biotinylated protein mixture was sonicated for 1 min and then incubated at 4 °C for 1 h. Non-binding proteins were removed by washing twice with phosphate-buffered saline/bovine serum albumin, and the beads were then resuspended in 50 µl of RPMI, 10% fetal calf serum. The bead-protein complex was sonicated again immediately before adding to various cell types in a 96-well plate (5 × 105 cells/50 µl of RPMI, 10% fetal calf serum/well). The cell-bead mixture was spun at 1000 × g at 4 °C for 20 min, incubated for a further 40 min at 4 °C, and finally resuspended in 500 µl of phosphate-buffered saline for FACS analysis.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In a search for novel members of the EGF-TM7 group of leukocyte receptors, DNA sequence data bases were screened for expressed sequence tags or genomic clones that displayed sequence homology with existing EGF-TM7 family members. A cosmid clone (R29368) derived from human chromosome 19 DNA (GenBankTM accession number AC004262) demonstrated ~50% amino acid identity to the first transmembrane domain and membrane proximal region of F4/80, EMR1, and CD97. The cosmid sequence was therefore used for primer design (E1-4) (see "Experimental Procedures") in subsequent 5'- and 3'-RACE reactions. Using human spleen cDNA template and primers E1-4 plus AP1 and AP2 anchor sequences, two novel EGF-TM7 molecules designated EMR2 (8) and EMR3 were independently isolated. The EMR2 DNA sequence was found to match the R29368 cosmid sequence, whereas the EMR3 DNA sequence suggests that the gene-specific primers misprimed, resulting in the amplification of a closely related, yet independent cDNA species.

The full-length EMR3 cDNA (Fig. 2) is comprised of 2247 nucleotides encoding a predicted 652-amino acid polypeptide. Based on the protein analysis programs from the Expasy server, the EMR3 sequence is predicted to contain a 21-amino acid signal peptide (residues 1-21) and two EGF-like domains (residues 25-66 and 67-118), with the N-terminal EGF-like domain being a non-Ca2+-binding module and the second EGF-like domain containing a Ca2+-binding consensus sequence. The EGF-like domains are followed by a spacer region (residues 118-350) containing several potential N- and O-linked glycosylation sites, a putative GPCR proteolytic site (20), a family B GPCR-related TM7 region (residues 350-602), and a short cytoplasmic tail (residues 602-652).


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Fig. 2.   Full-length cDNA and predicted amino acid sequence of EMR3. Bold type represents the predicted signal peptide (amino acids 1-21), shaded areas represent the two EGF-like domains (amino acids 22-118), and the broken underlined sequence shows the nucleotide deletion (nucleotides 439-479) found in the EMR3 soluble alternate splice form. Boxes show potential N-linked glycosylation sites, dotted underlining shows the consensus GPCR proteolytic site sequence (amino acids 320-345), and double underlined sequences show predicted transmembrane domains. The cDNA sequence has been submitted to the GenBankTM/EBI Data Bank with accession number AF239764.

As well as this membrane-bound form, other cDNA clones contained a 40-nucleotide deletion (nucleotides 439-479). This deletion, which corresponds to the splicing out of a single exon (data obtained from a partial genomic clone; GenBankTM accession number AC0022156), results in a frameshift leading to a premature stop codon and the production of a truncated soluble form of EMR3 protein that comprises the two EGF-like domains of the molecule. Multiple sequence alignment using the ClustalW software shows EMR3 to be most closely related to EMR2, followed by other members of the EGF-TM7 group of molecules: F4/80, EMR1, and CD97 (Fig. 3). The TM7 region of EMR3 shows ~90% sequence identity to EMR2, whereas the sequence identity in the stalk regions and EGF-like domains is ~35 and 50%, respectively.


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Fig. 3.   Amino acid sequence alignment of existing human EGF-TM7 proteins. EMR3, EMR2, CD97, and EMR1 sequences are aligned using ClustalW software for maximal homology. Residues displaying identity in >= 75% of the sequence are shaded.

RT-PCR and Northern blot analysis performed on various cell lines and tissues showed EMR3 mRNA to have a leukocyte-restricted expression profile resembling that of other known EGF-TM7 molecules (Fig. 4). Northern blot analysis of EMR3 identified two major transcripts at 2.4 and 4 kilobases expressed at high levels in immune tissues (spleen, bone marrow, and peripheral blood leukocytes) and at much lower levels in the placenta and lung (Fig. 4a). RT-PCR was performed using primers from the 5' untranslated region (sense) and the stalk region (antisense) of EMR3 and gave similar results to the Northern blot data (Fig. 4b). Highest levels of expression were again observed in the spleen, peripheral blood leukocytes, and lung, followed by intermediate levels in the placenta and bone marrow, with low detected levels in heart, kidney, liver, pancreas, skeletal muscle, lymph node, thymus, tonsil, and fetal liver. EMR3 expression was not detected in the brain.


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Fig. 4.   EMR3 expression is restricted predominantly to immune tissues. a, Northern blot showing EMR3 mRNA expression profile in various human tissues. b, RT-PCR results of a cDNA panel from various human tissues using a primer pair from the 5' untranslated region and stalk region of EMR3. c, Northern blot of total RNA from fractionated human blood cell populations probed with an EMR3-specific cDNA fragment. d, RNA from various human hemopoietic and non-hemopoietic cells was used as a template for RT-PCR using EMR3-specific primers and beta 2-microglobulin (beta 2M)-specific primers. kb, kilobases; PMN, polymorphonuclear cells; PBMC, peripheral blood mononuclear cells.

To dissect the expression profile of EMR3 mRNA in peripheral blood cells further, RT-PCR was performed on RNA samples from eight hemopoietic cell lines and, as negative controls, two human fibroblast cell lines (Fig. 4d). Following 30 cycles of PCR amplification, EMR3 expression was undetectable in all of the cell lines; however, after 40 cycles of amplification EMR3 expression was detected in two monocyte/macrophage cell lines (HL60 and U937), two B cell lines (Daudi and H9), and one T cell line (Jurkat). No signal was detected in the other cell lines, including the non-hemopoietic cell line HepG2 and 293 cells. To allow further examination of the expression profile in peripheral blood leukocytes, RNA was extracted from fractionated blood including polymorphonuclear cells, peripheral blood mononuclear cells, and lymphocytes from healthy donors and used in subsequent Northern blot analysis (Fig. 4c). The blot shows that the vast majority of the EMR3 hybridization signal is derived from the polymorphonuclear cell fraction, which contains mainly neutrophils, with a small number of eosinophils and basophils. Much lower levels of EMR3 expression were detected in freshly isolated monocytes and cultured macrophages, whereas no expression was observed in the RNA isolated from lymphocytes.

It has been well documented that many of the cell-cell interactions within the immune system are of low affinity and transient in nature (21). As such, when screening for such interactions, it is often necessary to use multivalent probes to increase the avidity of the molecular association to allow detection. In a search for the cellular ligand(s) of EMR3, a previously established sensitive assay system was utilized for screening cells that may express potential ligand(s). Recombinant soluble EMR3 containing a biotinylation sequence recognized by the E. Coli enzyme BirA (Fig. 5a) was biotinylated in vitro and coupled to avidin-coated fluorescent beads (22). The multivalent fluorescent bead-biotinylated EMR3 complex (Fig. 5b) was then used as a ligand probe in a FACS-based assay. Fluorescent beads coated with CD97 (EGF-1,2,5 isoform) and EMR2 (EGF-1,2 isoform) were also produced and served as binding and non-binding controls, respectively. Because of the high level of EMR3 expression in peripheral blood leukocytes, as measured by RNA analysis, various cell types were isolated from healthy donors by PolymorphprepTM separation and subsequently screened for interactions with the multivalent EMR3 probe. Following the incubation of human erythrocytes with CD97-multivalent fluorescent beads, a predicted shift in fluorescence was observed (Fig. 5c). This indicated that the CD97-coated fluorescent beads effectively bound to the CD97 cellular ligand CD55, which is known to be expressed at high levels on erythrocytes and other cells in the blood. In contrast, the FACS profiles of the EMR3- and the EMR2-coated fluorescent beads showed no shift in fluorescence, indicating no detectable interactions with human erythrocyte cell surface molecules (Fig. 5c). Similarly, no shift in fluorescence was detected when EMR3-coated fluorescent beads were incubated with either resting polymorphonuclear cells or peripheral blood mononuclear cells (Fig. 5, d and f). However, a significant shift in fluorescence was observed when EMR3-coated fluorescent beads were incubated with monocyte-derived macrophages and activated neutrophils following their exposure to 100 nM formyl-Met-Leu-Phe for 30 min (Fig. 5, e and d). These results indicate the presence of a ligand for EMR3 that is up-regulated on monocyte-derived macrophages and human neutrophils following activation. Both the monocyte-derived macrophages and the activated neutrophils failed to interact with EMR2-coated beads, proving the specificity of the interaction between EMR3 and a molecule on the surface of the myeloid cells.


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Fig. 5.   Demonstration of an EMR3 ligand on human macrophages and activated neutrophils. a, soluble EMR3 protein containing an amino acid consensus sequence recognized by the BirA enzyme allowing in vitro biotinylation of the lysine residue (*). b, multivalent EMR3 probes produced by coupling soluble biotinylated proteins to avidin-coated fluorescent beads. c-f, FACS profiles of fluorescent beads coated with EMR3, CD97 (EGF-1,2,5), or EMR2 (EGF-1,2) incubated with erythrocytes, resting and formyl-Met-Leu-Phe-activated neutrophils, monocyte-derived macrophages, and lymphocytes, respectively.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The data presented show EMR3 to possess the typical characteristics displayed by other EGF-TM7 proteins. The EMR3 gene is located on chromosome 19 and is capable of encoding different EMR3 protein isoforms through alternative RNA splicing. This results in the production of a predominantly leukocyte-restricted cell surface protein comprised of two EGF-like domains coupled to a seven-span transmembrane moiety via a mucin-like spacer region as well as its soluble secreted counterpart lacking both the transmembrane and stalk regions. In addition to these data, a sensitive FACS-based assay system using multivalent EMR3 fluorescent beads as a ligand probe demonstrated the presence of an EMR3 ligand on both human monocyte-derived macrophages and activated human neutrophils.

As with existing members of the EGF-TM7 family, RT-PCR data and Northern blot analysis show EMR3 to be leukocyte-restricted (Fig. 4). In Northern analysis of fractionated human blood, the majority of EMR3 signal is derived from polymorphonuclear cells (Fig. 4c). The main cellular components of this isolated fraction are neutrophils; however, a signal from the lower number of eosinophils and basophils cannot be ruled out. Lower levels of EMR3 mRNA were also detected in monocytes and culture-derived macrophages, suggesting that EMR3 is a myeloid-restricted molecule. The future development of EMR3-specific antibodies will greatly facilitate further characterization of expression within these cells. Once such antibodies are available, it would be interesting to show whether EMR3 is present in intracellular vesicles such as storage granules or mainly at the cell surface and to determine whether the activation state of polymorphonuclear cells, macrophages, or monocytes affects the levels or distribution of EMR3.

The significance of the interaction between EMR3 and monocyte-derived macrophages and activated neutrophils detected by FACS has yet to be established. However, because the majority of EMR3 is shown to be expressed by myeloid cells such as neutrophils and macrophages (Fig. 4), the interaction could play an important role in modulating immune and inflammatory responses by allowing "cross-talk" between myeloid cells. The ligation of EMR3 with a ligand on macrophages and activated neutrophils could potentially activate or deactivate cellular responses. Resting neutrophils and macrophages might be activated via signal transduction through the GPCR moiety of EMR3 following ligand binding, thereby amplifying immune and inflammatory responses, or signaling could possibly occur in the opposite direction. The binding of the EMR3 ligand to EMR3 could result in the modulation of macrophages and activated neutrophils, thereby reducing inflammatory cell-mediated tissue damage as a result of negative signaling.

The exact nature of the EMR3 ligand is not yet known; however, a related member of the EGF-TM7 family, CD97, is known to bind CD55 (DAF), which belongs to the regulators of complement activation family of proteins that are involved in the prevention of complement-mediated lysis of healthy cells. Although EMR3 does not bind CD55, other candidate proteins containing similar short consensus repeat domains include CD46 (membrane cofactor protein), CD35 (CR1), and CD21 (CR2) (23). The further biochemical characterization of EMR3 ligand is underway.

Because the multivalent EMR3 probes were produced with soluble EMR3, it raises the question whether membrane anchoring of EMR3 is necessary for ligand binding in vivo. Indeed, an EMR3 cDNA clone containing a 40-nucleotide deletion has also been isolated that is predicted to encode a soluble form of EMR3 lacking the transmembrane domains and the mucin-like region. The presence of a putative GPCR proteolytic site in EMR3 (residues 320-345) as defined by Krasnoperov et al. (27) gives further weight to the possibility that a soluble form of EMR3 mediates receptor ligation. The GPCR proteolytic site sequence is believed to be defined by a highly conserved pattern of Cys residues and a Leu-Thr site that was initially identified in a sperm receptor for egg jelly in sea urchins (Strongylocentrotus purpuratus) (24). More recently, the site has been shown to be involved in the cleavage of the extracellular domains of CD97 and latrophilin (CL1) (25-27), resulting in the formation of membrane-associated heterodimers in which the transmembrane domains are non-covalently associated with the extracellular domains. This has led to the suggestion that the subsequent release of the extracellular domains could then allow interaction with ligands on neighboring cells and at the same time allow the binding of other ligands such as peptide hormones to the GPCR moiety of the EGF-TM7 proteins.

The cluster of closely related EGF-TM7 genes on human chromosome 19p13 and on the syntenic regions of the mouse genome suggests an ancient gene family that has arisen through gene duplication and exon shuffling (28). Indeed, the serendipitous discovery of EMR3 during the initial cloning of EMR2 was due to the fact that there is a greater than 90% identity within the exons encoding the transmembrane domains of EMR2 (exons 14-19) and EMR3 (8). However, despite the very high degree of sequence identity shared by EMR3 and EMR2 within their transmembrane domains, the two molecules display much lower levels of identity within their mucin-like stalk regions and EGF-like domains (35 and 50%, respectively). This leads to the possibility of related proteins having different ligand binding properties, mediated through their extracellular regions, yet similar intracellular signaling, mediated via their GPCR moieties. The converse could also be postulated for two other EGF-TM7 proteins. Human CD97 and human EMR2 contain extremely similar EGF-like domains (differing by only six amino acids within the five EGF-like domains) yet possess much less similar transmembrane domain sequences. The temporal and spatial regulation of such receptors could allow differential intracellular signaling via the transmembrane domain to occur by the same ligand binding to the extracellular domain.

With the complete sequencing and publication of the human genome in sight and the sequencing of the mouse genome well under way, it is interesting to speculate whether more EGF-TM7 genes reside on chromosome 19 or within the mouse genome. If more do exist, the cloning and characterization of these proteins and their ligands will facilitate our understanding of the physiological importance of the EGF-TM7 proteins within the immune system.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF239764.

Dagger Funded through a BBSRC/Roche CASE studentship and a Goodger Scholarship. To whom correspondence should be addressed. Tel.: 44 1865 275532; Fax: 44 1865 275515; E-mail: mstacey@molbiol.ox.ac.uk.

§ Supported by the Wellcome Trust Initiative for Cardiovascular Research.

|| Supported by grants from the Medical Research Council.

Published, JBC Papers in Press, March 14, 2001, DOI 10.1074/jbc.M101147200

    ABBREVIATIONS

The abbreviations used are: EGF, epidermal growth factor; EMR, EGF module-containing mucin-like hormone receptor; GPCR, G-protein-coupled receptor; DAF, decay-accelerating factor; RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction; RT-PCR, reverse transcription-PCR; FACS, fluorescence-activated cell sorting.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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