Definition of an Fc receptor-related gene (FcRX) expressed in human and mouse B cells
Randall S. Davis1,3,
Haitao Li2,
Ching-Cheng Chen2,
Yui-Hsi Wang2,
Max D. Cooper2,7 and
Peter D. Burrows2,6
Divisions of 1 Hematology/Oncology, and 2 Developmental and Clinical Immunology; Departments of 3 Medicine, 4 Pediatrics, 5 Pathology and 6 Microbiology; and 7 Howard Hughes Medical Institute, University of Alabama, Birmingham, AL 35294-3300, USA
Correspondence to: P. D. Burrows, WTI 378, 1530 3rd Avenue South, Birmingham, AL 35294-3300, UDA. E-mail: peterb{at}uab.edu
Transmitting editor: T. Watanabe
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Abstract
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The recent identification of five human Fc receptor (FcR) homologs, hFcRH15, has extended the known FcR family and identified an unanticipated richness of the chromosome 1q region in genes encoding potential Ig-binding proteins. In a database search for additional relatives of this family we identified expressed sequence tag representatives of a new FcR-related molecule (hFcRX) and its mouse ortholog (mFcRX). The FcRX cDNAs were cloned from human lymph node and mouse spleen cDNA libraries. hFcRX is located centromeric of Fc
RII and Fc
RIII at 1q23, and its mouse ortholog resides in a syntenic region of chromosome 1. The genes encode proteins with 67% interspecies identity that lack both N-linked glycosylation sites and transmembrane regions. Two of the four FcRX domains are Ig-like, and share characteristics similar to Fc
RI domains 2 and 3, having 28% overall extracellular identity with hFc
RI and 27% identity with mFc
RI respectively. FcRX transcripts are found primarily in secondary lymphoid tissues, where they are expressed by B lineage cells. FcRX thus may function as a secreted or intracellular protein in normal and neoplastic B cells.
Keywords: B cell-specific gene, chromosome 1, Fc receptor
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Introduction
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Receptors for the Fc portion (FcR) of Ig, Fc
RI, Fc
RII, Fc
RIII and Fc
RI, are widely expressed on cells of the immune system where they function to modulate cellular and humoral immunity (1,2). These FcR family members are related on the basis of their ligand-binding Ig-like extracellular regions that maintain common subunits. Individual FcR mediate antibody isotype and cell-type-specific regulatory roles, which include adjustment of humoral antibody concentrations, initiation of cellular cytotoxicity and involvement in hypersensitivity responses (3). Given the vital function of FcR in establishing homeostasis of the adaptive immune system, subversion of the normal physiologic role of these receptors may lead to autoimmunity and malignancy (4,59).
In parallel with recognition of the large Fc
R-related gene family located within the leukocyte receptor gene complex on human chromosome 19q13, an increased number of Ig-like FcR-related genes has been recognized on human chromosome 1q2123 (1017). Five FcR-related genes, the human Fc receptor homologs (hFcRH15), have been identified on the basis of their Ig-like domain homology with the classical FcR (18,19) and on the basis of the translocation of one of these genes into the Ig locus in a myeloma cell line (20,21). Like the classical FcR, the hFcRH15 reside in the human chromosome 1q21 region and encode type I transmembrane proteins that share similar extracellular Ig-like domains and cytoplasmic regions with consensus motifs, that suggest an inhibitory or activating signaling function.
Using consensus sequence motifs conserved among Fc
RI and hFcRH15, we performed a database search for new members of this family. Our analysis led to the identification of multiple expressed sequence tags (ests) and the isolation of cDNA clones that encode a new FcR relative that we term hFcRX. We report here the genomic location, sequence, predicted structure and cellular expression pattern for hFcRX and its mouse ortholog, mFcRX.
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Methods
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Isolation of hFcRX and mFcRX cDNA clones
RACE-PCR was performed using Marathon-Ready human lymph node and mouse spleen cDNA libraries (Clontech, Palo Alto, CA). Gene-specific primers were as follows: hFcRX, forward 5'-CAC CTC CCA CAT TGA ATC CAG CTC CT-3' and reverse 5'-TTC AGC AGT AGC CTT TGT GGT CCC AG-3'; mFcRX, forward 5'-CAC TCT GGC TCC TAC TGG TGT GAG GC-3' and reverse 5'-AAG CGG TTG GTT TCT GAA GAG CCT GT-3'. RACE amplification products were subjected to a second round of nested PCR and visualized by agarose gel electrophoresis and ethidium bromide staining. PCR products were ligated into the pCR2.1 TOPO T/A vector (Invitrogen, Carlsbad, CA). The 5' end of the human cDNA was originally identified by searching the NCBI est database. Several homologous overlapping est sequences (accession nos BM471887, BF305905 and BF303959) were obtained and found to align to the genomic sequence upstream of the hFcRX gene. RT-PCR was used to confirm that the 5' end predicted by these ests and genomic sequences could be detected in hFcRX mRNA.
Sequence analysis
Inserts were DNA sequenced on both strands by the dideoxy chain termination method using Thermo Sequenase (Amersham Pharmacia Biotech, Arlington Heights, IL) and an automated sequencer (LiCor, Lincoln, NE). Nucleotide and amino acid sequence alignment was performed using the DNASTAR (Madison, WI) software package and homology searches were performed using BLAST (22).
RNA and DNA blot analysis
Northern blots (Clontech) were hybridized with [
-32P]dCTP labeled probes: a 924-bp PCR product (bp 1461069 from the first ATG) corresponding to the D1D4 region of hFcRX cDNA and a 905-bp PCR product (bp 2061110 from the first ATG) corresponding to D1D4 including the proximal 3' untranslated region (UTR) of the mFcRX cDNA. Membranes were hybridized for 1 h at 65°C, washed and exposed to X-ray film (23). A multispecies zoo blot containing EcoRI-digested DNA (Seegene, Seoul, South Korea) was hybridized with the same mouse probe and washed at low stringency (2 x SSC/0.1% SDS at 37°C) before exposure to X-ray film.
Human and mouse tissues
Human bone marrow samples were obtained from ribs resected from renal transplant donors and long bones of 13- to 19-week-old pre-viable fetuses in accordance with policies established by the Institutional Review Board of the University of Alabama at Birmingham. Tonsillar tissue was also obtained with Institutional Review Board approval. Mononuclear cells were isolated from the bone marrow and tonsillar samples by Ficoll-Hypaque gradient centrifugation. Mononuclear spleen cell suspensions were prepared from BALB/c mice.
Antibodies
Immunofluorescence assays employed the following mAb: FITC-labeled anti-CD38 (AMAC, Westbrook, ME), FITC-labeled goat antibodies to human IgM, phycoerythrin (PE)-labeled anti-CD19 and anti-IgD, allophycocyanin-labeled anti-CD34 (BD PharMingen, San Diego, CA) and biotinylated mouse anti-human
/
L chain mAb (a generous gift of Dr Hiromi Kubagawa, University of Alabama at Birmingham).
Immunofluorescence and cell sorting and RNA isolation
Human bone marrow mononuclear cells were incubated with PE-labeled anti-CD19 and counterstained with allophycocyanin-labeled anti-CD34 mAb or biotin-labeled F(ab')2 goat anti-human
H chain antibody or anti-human
/
L chains mAb revealed by streptavidin APC. Human tonsillar cells were separated into CD19+ and CD19 subpopulations by magnetic cell sorting (Miltenyi Biotech, Auburn, CA). Viable CD19+ cells were stained with FITC-labeled anti-CD38 (Immunotech, Westbrook, ME) and PE-labeled anti-IgD mAb (Southern Biotechnology, Birmingham, AL) before sorting cells with a FACStar Plus (Becton Dickinson, Mountain View, CA) or MoFlo (Cytomation, Fort Collins, CO) instrument. Cells were sorted into TRIzol reagent (Life Technologies, Grand Island, NY) for RNA isolation. Total cellular RNA was primed with random hexamers and oligo(dT) primers, and reverse transcribed with SuperScript II (Life Technologies) into single-stranded cDNA.
RT-PCR
RT-PCR was performed for bone marrow and tonsil samples with Taq polymerase (Life Technologies) and for cell lines with AccuPrime Supermix I (Invitrogen). Amplified products were visualized in 1% agarose gels containing ethidium bromide and documented with the Fluor-S Imager (Biorad, Hercules, CA). Gene-specific primers used in RT-PCR were as follows: hFcRX, forward 5'-CGC TGC AGT GTG AGG GAC CTG-3' and reverse 5'-TGA GCA GGT GAC CGA GGA GGA-3'; mFcRX, forward 5'-ACT TCC AGT TCA AGG GCT ACA C-3' and reverse 5'-GAG CTG GAA TTA TTG CGG TGG-3'.
Cell lines
Human cell lines included REH, Nalm 16 and 207 pro-B cell lines (23); 697 and OB5 pre-B cell lines (25,26); Ramos, Daudi and Raji B cell lines (2729); THP-1 and U937 monocytoid cell lines, HL-60 promyelocytic and KG-1 myelocytic cell lines, Jurkat T cell line and the K562 erythroid cell line (ATCC, Rockville, MD). Mouse cell lines included RAW8.1 (ATCC, Rockville, MD) and SCID7 (30) pro-B cell lines; 70Z/3 and BC76 (a kind gift from Paul Kincade) pre-B cell lines; WEHI-231 and WEHI-279 immature B cell lines (ATCC); A20 and X16C8.5 B cell lines (ATCC); the EL4 T cell line (ATCC); NK-T cell lines NKT (DN3A4-1-4) and 2C12 (N382C12) [gifts from Mitchell Kronenberg (31,32)]; the myeloid cell line WEHI-3 (ATCC); and the fibroblast cell line 3T3 (ATCC).
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Results
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Identification of hFcRX and mFcRX
The amino acid sequence of the third Ig-like domain of hFc
RI (CD64), a subdomain that has common identity among hFcRH15 extracellular domains, was used in a database search for additional relatives of the FcR and FcRH family. A query performed on 9 August 2001 of the National Center for Biotechnology Information (NCBI) protein and est databases using the BLASTP and TBLASTN algorithms (21) yielded a hypothetical protein (MGC4595) and multiple ests (accession nos W03476, BE271199, AL560266 and N29316) whose translated open reading frames (ORF) had homology with hFc
RI. When the putative Ig-like protein, provisionally termed hFcRX, was submitted to the mouse est database, an orthologous set of related sequences designated mFcRX was identified. Further analysis of high-throughput genome sequences in the NCBI and Celera databases located hFcRX in a 1q23 region centromeric of hFc
RII. MoFcRX mapped to a syntenic region on mouse chromosome 1.
To determine if hFcRX is expressed in lymphocytes, a search of the Lymphochip est database (33) was performed using the BLASTN and TBLASTN algorithms that identified the MGC4595 est sequence. According to the Lymphochip microarray data, hFcRX est expression is relatively abundant in normal germinal center B cells and centroblasts, but not in activated or resting B or T cells. HuFcRX was also found to be differentially expressed among the lymphoid B lineage malignancies. Of 46 diffuse large B cell lymphomas approximately 14 samples displayed above-average hFcRX expression. Furthermore, five of nine follicular lymphoma and five of 11 B cell chronic lymphocytic leukemia (CLL) samples exhibited hFcRX overexpression.
hFcRX and mFcRX cDNAs were isolated from human lymph node and mouse spleen cDNA libraries by RACE-PCR in the 5' and 3' directions. Sequence analysis of multiple overlapping cDNA clones enabled the construction of full-length cDNAs (Fig. 1).


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Fig. 1. Human (A) and mouse (B) FcRX cDNA sequences. The longest cDNA sequences obtained are illustrated. Predicted ATG start codons are boxed. The human sequence contains multiple potential start codons in the predicted ORF, but the one indicated best matches the Kozak consensus sequence (ACCATGG) for translation initiation (55). Stop codons and potential poly(A) addition signals (AATAAA) are underlined. Accession numbers: hFcRX AF531423; mFcRX AF531424.
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hFcRX and mFcRX have ORF of 1080 and 1062 bp, encoding proteins of 359 and 353 amino acids. Based on predicted consensus signal sequence cleavage sites (35,36), the mature proteins have estimated mol. wt = 35849 and 35225 Da. Both genes are predicted to have multiple potential translation start sites, four for hFcRX and two for mFcRX, that would initiate translation of proteins consisting of four domains (D1D4) (Fig. 2). D1 encodes a region predicted to be part of the mature protein that is partially Ig-like, but lacks a second cysteine residue for disulfide bonding. D2 and D3 are Ig-like loops with a high degree of interspecies identity. D4 is longer in hFcRX than its mouse ortholog, but both are leucine and proline rich. The predicted hFcRX and mFcRX are acidic proteins with pI of 5.0 and 5.2 respectively. Notably, both mouse and human FcRX lack potential N-linked glycosylation sites or transmembrane regions (37,38), suggesting they may be either secreted or intracellular proteins.

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Fig. 2. Alignment of hFcRX and mFcRX amino acid sequences (one letter code) based on the hFcRX sequence. Amino acid identity is represented by dots (.) and gaps, for optimal alignment, are indicated by dashes (). Potential translational initiation sites are underlined and the one predicted by the Kozak consensus sequence is in bold. Conserved cysteine residues are also indicated in bold. Putative structural domains are labeled: SP, signal peptide; D1D4, domains. Amino acid lengths are indicated in parentheses.
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Two versions of the mFcRX cDNA were obtained in these studies, one with two alanines, beginning at nucleotide position 108, and one with three. Both sequences were derived from BALB/c mice, ruling out a genetic polymorphism to account for the additional amino acid. Moreover, two C57BL/6-derived est sequences (accession numbers BB638258 and BB637253) have a similar sequence dichotomy. Analysis of the genomic structure of the mFcRX gene (see below) indicates that the two sequences derive from a micro alternative splicing event involving exon 1 and the very 5' end of exon 2. The two predicted splice variants are illustrated below, with exonic sequence in capital letters and splice donor/acceptor signals in underlined lower case:
Exon 1Exon 2
Ggt-----agCAGCTGCC
GCAGCTGCC
AlaAlaAla
Ggt-----agcagCTGCC
GCTGCC
AlaAla
Whether the difference in numbers of alanines at this position results in a change in protein function or stability is unknown.
Tissue distribution of hFcRX and mFcRX
RNA blot analysis of 16 human tissues including six primary or secondary lymphoid tissues was performed using hFcRX and hß-actin control probes (Fig. 3A). The hFcRX probe hybridized with a transcript of
2.5 kb in all lymphoid tissues examined, but the greatest intensity by far was observed in spleen and lymph node. The hFcRX transcripts were undetectable in non-lymphoid tissues, indicating immune cell-specific expression in humans. Analysis of eight mouse tissues with mFcRX and ß-actin probes identified several hybridizing transcripts of
1.62.3 kb that were most abundantly expressed in spleen (Fig. 3B). The smaller transcript was also detected in testes. Lung and liver showed weak hybridization with the mFcRX probe, but transcripts were undetectable in the other tissues examined. Since our database searches and DNA blot analysis (see below) indicate that FcRX is a single-copy gene, the presence of multiple transcripts in mouse but not human tissues suggests differential splicing of the primary mFcRX transcript. These results indicate that both human and mouse FcRX are preferentially expressed in lymphoid tissues.

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Fig. 3. Analysis of hFcRX (A) and mFcRX (B) expression in different tissues. RNA blots were analyzed with discriminating [ -32P]dCTP-labeled probes generated from the respective FcRX cDNAs. Relative mRNA abundance is indicated by hybridization with a ß-actin probe.
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Examination of a panel of cell lines of human and mouse origin by RT-PCR was performed to determine which hematopoietic cell types expressed FcRX (Table 1). Transcripts for hFcRX and mFcRX were amplified primarily in mature B cells, although expression was also noted in a few human cell lines of the pro-B or pre-B cell phenotype. There was no evidence of expression by non-B cell lines indicating that FcRX is a B lineage-specific gene.
Analysis of FcRX expression in primary lymphoid tissues
To explore the expression of hFcRX in early B lineage cells, bone marrow samples were obtained and distinct subpopulations of B cells were isolated according to their light scatter characteristics and differential expression of the CD34, CD19 and IgM cell surface markers (39). The stages of early B lymphocyte differentiation can be defined as: common lymphoid progenitor/stem cell (CD34+/CD19), pro-B cell (CD34+/CD19+), pre-B cell (CD19+/IgM+/) and B cell (CD19+/IgM+). RT-PCR analysis demonstrated abundant expression of hFcRX in bone marrow B cells, whereas only low levels of transcript amplification were seen in earlier B lineage cells. To determine its expression in secondary lymphoid tissues, isolated tonsillar subpopulations were analyzed for hFcRX transcription. The five discrete subpopulations of CD19+ B lineage cells that can be distinguished by their differential expression of cell surface IgD and CD38 represent different stages in B cell differentiation: follicular mantle (IgD+CD38), pre-germinal center (IgD+CD38+), germinal center (IgDCD38+), memory (IgDCD38) and mature plasma cells (CD38++) (18,40). RT-PCR analysis indicated expression of hFcRX in all subpopulations, although a greater relative amplification was noted in pre-germinal and germinal center B cells. In mouse spleen, mFcRX was expressed in CD19+ B cells, but not in the CD4+ or CD8+ T cell subpopulations.
Homology-based Ig-like domain analysis of the FcR-related gene family
Comparison of the amino acid sequences of human and mouse FcRX indicates >65% interspecies identity. Of the classical FcR, Fc
RI is the most similar to hFcRX with 32% identity in its extracellular region. When compared with the FcRH15 family, the degree of identity to hFcRX is much more limited (1424%). To evaluate the relatedness of the different Ig domains among the FcR family members, an analysis of individual subunits was performed (Fig. 4). Ig-like domains that cluster together in a phylogenetic tree were color coded to illustrate the relationship between the domains of hFcRX and its FcR relatives. The amino acid sequence identity of comparable mFcRX and hFcRX domains, D2 (dark blue) and D3 (yellow), is 80 and 73% respectively. Among the classical FcR, the FcRX D2 and D3 domain types are conserved in Fc
RI, with 33 and 52% identity. Fc
RIIB, Fc
RIII and Fc
RI notably lack the D3 domain common to FcRX and Fc
RI. However, the hFcRX D2 domain is present with higher identity to Fc
RIIB and Fc
RI (36 and 37% respectively) than to Fc
RIII (31%). The modularity of shared domains is also apparent when hFcRX is compared with the FcRH15 family members. FcRH1 lacks the FcRX D2 domain subtype and has the most limited identity among the FcRH to the hFcRX D3 at 26%. However, like Fc
RI, the similarity of the hFcRX D2 and D3 Ig-like subunits is maintained among FcRH25 with 2630 and 3438% identity respectively. This analysis thus suggests a common ancestry for all members of this family with variable usage of the different subtypes of Ig domains.
hFcRX and mFcRX genomic structure
To establish the genomic structures for hFcRX and mFcRX, cDNA sequences were used to search the NCBI and Celera genome sequence databases. For hFcRX, a BAC clone, AL359541, was identified that contains the uninterrupted hFcRX gene sequence. Exonintron boundaries were characterized by comparing the cDNA and est sequences, and by applying the AG/GT rule for splice donor/acceptors (Fig. 5). The hFcRX gene consists of five exons and four introns that span
7.4 kb. The first exon, 5'UT/S, contains the 5' UTR and four potential translation initiation sites, the second and third of which contain the most conserved Kozak consensus sequence (34). Given its characteristic hydrophobicity this exon could encode a splice site for a signal peptide, as is predicted by analysis of this region (35,36). The second exon, D1, is preceded by a long intron of
3.4 kb. In comparison with the classical FcR related proteins, the structure of the region encoded by D1 resembles that of a partial Ig domain lacking a second cysteine that would enable disulfide bonding. The third exon encodes a bona fide Ig-like domain, D2, as does exon four, D3. The last exon, D4/3'UT, encodes a leucine and proline rich region, the stop codon and the 3' UTR. Based on our cDNA sequence analysis, the human gene contains three potential polyadenlyation sites in the 3'UTR located 39, 143 and 945 bp downstream of the translation stop codon. Sites 2 and 3 are functional since we isolated RACE-PCR clones corresponding to polyadenlyation downstream of these sites. The longer 3'UTR has two more AU-rich elements than the shorter one, suggesting a potential role in regulating hFcRX mRNA stability.

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Fig. 5. Genomic organization of hFcRX and mFcRX. Genomic structure was determined by comparing cDNA clones with the BAC clones that overlap the region. Exonintron boundaries were characterized by sequence comparisons and the AG/GT rule. Exons are numbered and indicated by rectangles: untranslated (open) and translated (closed). Domains are listed as: UT, untranslated; S, signal peptide; D1D4, domains.
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The mFcRX gene structure is analogous to that of its human counterpart. According to the Celera genomic database (mCG8652), mFcRX is also comprised of five exons and four introns. Although there is a gap of unknown length in the sequence of the first intron, the full length of the gene is at least 8 kb. The first exon, 5'UT/S, contains the 5'UTR as well as two potential translation initiation sites. Similar to hFcRX, this region has hydrophobic character and a potential splice site for a signal peptide (35,36). The first intron is at least 4.4 kb in length. Exon two, D1, encodes a partial Ig-like region. The third and fourth exons encode Ig-like domains, D2 and D3 respectively. The last exon of mFcRX encodes a leucine/proline-rich region and the 3'UTR, D4/3'UT. Only one polyadenlyation site, located 132 bp downstream of the stop codon, was identified in our mFcRX cDNA clones.
Phylogeny of FcRX
The highly conserved nature of FcRX in mice and humans indicates the existence of a common ancestral FcRX gene more than 80 million years ago. When a multispecies zoo blot was hybridized at low stringency with the mouse FcRX probe, a single hybridizing band was observed in mouse DNA, consistent with the DNA sequence database analysis indicating that FcRX is a single-copy gene. Weakly cross-hybridizing bands were seen in rat, dog, cow and pig DNA samples, but not in rabbit, chicken, frog, nematode or yeast. This analysis suggests that the FcRX gene is conserved among many, but not all mammalian radiations.
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Discussion
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The present studies define another member of the extended FcR family of genes, FcRX, in humans and mice on the basis of its relatedness to hFc
RI and hFcRH15. The hFcRX gene resides near hFc
RII and hFc
RIII at 1q23, and the mFcRX maps to a syntenic locus on mouse chromosome 1. The predicted FcRX molecules share significant homology with the classic FcR and their FcRH relatives. However, despite having two Ig-like subunits in common, the overall structure of the human and mouse FcRX molecules is significantly different from the classical FcR and FcRH, thereby suggesting a novel role as secreted or intracellular proteins. Like FcRH15, FcRX is expressed primarily by mature B cells in secondary lymphoid tissues.
The phylogenetic relatedness of FcRX to the FcR gene family raises the question of its Ig binding potential. Mutation and X-ray crystallographic analysis have identified the D1/D2 linker and D2 N-terminal cysteine region as the ligand binding interface for the Fc portion of Ig by the classical FcR (4146). Our phylogenetic analysis indicates the conservation of the D2 subunit in FcRX. Moreover, a CLUSTAL-based alignment of amino acid sequences for these receptors indicates the D1/D2 linker region is also maintained, suggesting that FcRX could bind Ig (43). However, the predicted FcRX protein lacks a D1 domain (red subunit in Fig. 4) that is a feature of all the classical FcR family members. The modular organization of Ig domains, first noted among the Fc
R (48), is maintained with the addition of the new family member, FcRX (Figs 4 and 5). At a genomic level, the exons that encode these Ig domains are thus likely to maintain conserved features that promote their duplication and recombination through evolution (48).
The early recognition of the classical FcR genes in the 1q2123 region and the more recent discovery of their FcRH15 neighbors has identified the 1q region as one that is rich in genes with the potential to bind Ig, although Ig binding has been demonstrated unequivocally only for the classical FcR at present. The genomic location of the FcRX genes closely links them with hFc
RII and hFc
RIII at human chromosome 1q23 and in a syntenic region on mouse chromosome 1. This proximity provides further evidence of FcRX relatedness to the FcR and FcRH gene family. One feature of the genomic structure that is highly conserved among FcR-related family members is a characteristic 21 bp S2 exon that encodes the second half of the split signal peptide for all of these genes in mice and in humans (4952). Although FcRX is clearly a phylogenic relative of these genes by virtue of its genomic location and Ig-like domain homology, it lacks the S2 exon. The signal peptide predicted in this portion of the FcRX molecule is instead encoded by the S and D1 exons. The predicted absence of a hydrophobic transmembrane region indicates that FcRX may be secreted or reside intracellularly. Indeed, during the preparation of this manuscript, two groups of investigators have independently identified genes identical to FcRX (named FcRL and FREB) and have used immunohistochemistry to demonstrate intracellular protein expression in tonsillar centroblasts (53,54).
The analysis of FcRX expression in primary cells and cell lines representative of different hematopoietic lineages indicates that FcRX is expressed predominantly by the mature B cells that reside in secondary lymphoid tissues and this analysis is in accord with the recent analysis of the FcRL/FREB expression pattern (51,52). Although trace levels of FcRX transcripts were amplified in sorted precursor B cell populations derived from human bone marrow, FcRX was amplified in greatest abundance in the mature B cells. Its expression was noted to some degree in all subpopulations of tonsillar B cells, but was particularly prominent in cells involved in the germinal center reaction. FcRX/FcRL/FREB thus joins the previously identified Fc
RIIB, FcRH15 and Fc
/µR as the most recent FcR-related gene known to be preferentially expressed in B cells. Its discrete expression in mature B cells, and its 67% conservation in humans and mice, suggest that it may play an important role in mature B cell development and regulation.
The identification in the Lymphochip (33) database of an est corresponding to a alternatively spliced form of hFcRX lacking the D1 region provides additional insight into the differential expression of hFcRX in malignancies of B cell lineage. A subset of diffuse large B cell lymphomas, follicular lymphomas and B cell CLL all exhibited hFcRX overexpression. The discrete expression pattern of this gene during normal B cell differentiation and its differential expression in B lineage malignancies suggest a potential role in the transformation process. FcRX could also serve as a marker of certain B cell malignancies that may or may not correlate with clinical prognosis.
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Acknowledgements
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The authors would like to thank Dr Glynn Dennis for helpful discussion, Dr Larry Gartland for cell sorting and Ms Ann Brookshire for editorial assistance. This work has been supported in part by NIH grants AI39816 and AI48098. R. S. D. was supported by an NIH Hematology Training Grant, DK07488. M. D. C. is a Howard Hughes Medical Institute Investigator.
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Abbreviations
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Ddomain
CLLchronic lymphocytic leukemia
estexpressed sequence tag
FcRFc receptor
FcRHFc receptor homolog
NCBINational Center for Biotechnology Information
ORFopen reading frame
PEphycoerythrin
UTRuntranslated region
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