Differential B cell expression of mouse Fc receptor homologs

Randall S. Davis1,2,3, Robert P. Stephan1,3, Ching-Cheng Chen1,6,9, Glynn Dennis, Jr.8 and Max D. Cooper1,3,4,5,6,7

1 Division of Developmental and Clinical Immunology, 2 Division of Hematology/Oncology, 3 Department of Medicine, 4 Department of Pediatrics, 5 Department of Pathology and 6 Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294-3300, USA
7 Howard Hughes Medical Institute, Birmingham, AL 35294-3300, USA
8 Laboratory of Immunopathogenesis and Bioinformatics, Science Applications International Corporation-Frederick, Building 550, Room 204 FCRDC PO Box B, Boyles Street, Frederick, MD 21702–1201, USA
9 Present address: Department of Pathology, Stanford University, 269 Campus Drive, Room CCSR 3250, Stanford, CA 94305–5176, USA

Correspondence to: M. D. Cooper; E-mail: max.cooper{at}ccc.uab.edu


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Five Fc receptor homologs (FcRH1–5) possessing inhibitory and/or activating signaling motifs are differentially expressed during B cell differentiation in humans. In this analysis we describe their three mouse orthologs, moFcRH1, moFcRH2 and moFcRH3. The moFcRH genes are located in a chromosome 3 region that is syntenic with the FcRH locus on human chromosome 1. They encode proteins with 2–5 Ig-like domains that share 20–61% extracellular identity with their human counterparts. One moFcRH1 isoform lacks a transmembrane domain as do both moFcRH2 isoforms. The other moFcRH1 isoform and two moFcRH3 isoforms have transmembrane domains and cytoplasmic ITIM and ITAM-like consensus sequences implying their inhibitory or activating signaling potential. Whereas the moFcRH1 and moFcRH3 orthologs are preferentially expressed at different stages in B cell differentiation, the structurally novel moFcRH2 gene is expressed in non-lymphoid tissues. The highly restricted pattern of moFcRH3 expression suggests this member of the phylogenetically conserved FcRH family may have an important immunoregulatory role in marginal zone B cells.

Keywords: B cell differentiation, Fc receptors, immunoglobulin superfamily, immunoreceptor tyrosine-based motifs, phylogeny


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Five members of a family of human Fc receptor homologs, huFcRH1–5, have been identified on the basis of their sequence homology with the classical Fc receptors for IgG, Fc{gamma}RI, Fc{gamma}RII and Fc{gamma}RIII, and are located in the midst of the classical FcR locus at 1q21-23 (1,2). They have also been identified as immunoglobulin superfamily receptor translocation associated (IRTA) genes (3,4), SH-2 domain-containing phosphatase anchor proteins (SPAP) (5) and IgSF-FcR-Gp42 (IFGP) genes (6). Interestingly, the huFcRH1-5 family members are differentially expressed by B cells, wherein they encode type I transmembrane proteins with 3–9 extracellular domains and cytoplasmic tails with consensus tyrosine-based signaling motifs. Amino acid sequence analysis indicates that these receptors utilize five Ig domain subtypes, some of which are shared by their FcR relatives, Fc{gamma}RI, Fc{gamma}RII, Fc{gamma}RIII and Fc{varepsilon}R{alpha} (1,2,7). Some of the Ig domain subtypes are also shared by two more recently defined Fc receptor like molecules, the Fc receptor-like homolog expressed in B cells (FcRL/FREB/FcRX) (810) and a mouse Fc{gamma}RIII ortholog (FcRL3/CD16-2) (7,11). Although most of the FcRHs, including huFcRH2, huFcRH3, huFcRH4, huFcRH5 and FcRX, possess sequence homology with Fc binding sites of the classical Fc receptors, experimental evidence for Fc receptor function has not yet been reported.

Members of the human FcRH1–5 subfamily belong to the network of receptors that possess immunoreceptor tyrosine-based activating motifs (ITAM), inhibition motifs (ITIM) or both (12). ITAMs are characterized by two repeats of the consensus sequence Y-X-X-L/I separated by 6–8 amino acids (E/D)-X-X-Y-X-X-(L/I)-X6–8-Y-X-X-(L/I), while ITIMs characteristically have a six amino acid consensus sequence (I/V/L/S)-X-Y-X-X-(L/V) (1315). The FcRH ITIM sequence motifs closely match the defined consensus, whereas most FcRH ITAM candidate motifs correspond less precisely to the consensus sequence, and some of the sequences ambiguously resemble either part of an ITAM or an ITIM. Like the Fc{gamma}RII receptors, the presence of these potential immunoregulatory motifs in the FcRH cytoplasmic tails suggests autonomous signaling potential. In contrast, the other activating classical FcRs require co-association with the FcR common {gamma}-chain (FcR{gamma}c) or other adaptor subunits for signaling competence (1619).

The classical Fc receptors in mice have been shown to positively or negatively modulate antibody mediated responses of lymphocytes and inflammatory cells (20). The locus for the mouse Fc receptor genes is split between mouse chromosome 3, where the moFc{gamma}RI resides, and mouse chromosome 1, where moFc{varepsilon}R{alpha}, moFc{gamma}RII and moFc{gamma}RIII are located (2124). There are a limited number of mouse FcR genes relative to their more highly diversified human counterparts. This feature has facilitated their targeted disruption to enable clarification of their biological roles as activating and inhibitory receptors. Mice deficient in the Fc receptors have profoundly altered humoral immune responses, immediate hypersensitivity, cytotoxic inflammatory responses and immune complex mediated inflammation (2530). These findings emphasize the important pathophysiologic roles of Fc receptors in autoimmunity, allergy and inflammation.

The immunoregulatory potential of the huFcRH1–5 family members and their preferential B cell expression raise interesting issues regarding their biological importance. Their identification also reveals that this 1q21-23 region contains an unexpectedly large number of genes with potential Ig binding function and signaling capacity. As a first step towards gaining greater insight into the diversity of this gene family and the functional potential of the individual family members, we sought to identify and characterize the FcRH relatives in mice. Using amino acid sequences of Ig-like domains specific to the huFcRH1–5 subfamily to search the available databases, three potential mouse orthologs (moFcRH1–3) were identified. In this report we describe their chromosomal location, genomic configuration, sequence and cellular expression patterns. In addition, these mouse Fc receptor homologs are compared with their human counterparts.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Isolation of moFcRH1–3 cDNA clones
Rapid amplification of cDNA ends (RACE)–PCR was performed using a Marathon-Ready mouse spleen and SMART RACE PBL cDNA libraries according to the manufacturer's instructions (Clontech, Palo Alto, CA). Gene-specific primers were as follows: moFcRH1, forward 5'-GAGGCTCACCCCCAATCTTCTACCAGT-3' and reverse 5'-CTGCTGTTAGTGAAGGTTGTGTCTTTG-3'; moFcRH2, forward 5'-GAGGTGGTAACGCTCAATGTCACAG-3' and reverse 5'-GGAAGGCTGATGCTCAACCAGTCGGAC-3'; and moFcRH3, forward 5'-CTCTCTGGAGGAGGAGCATACTTC-3' and reverse 5'-GCAGCACCAGAAAATCTTCAGAAAACTC-3'. RACE amplification products were subjected to a second round of nested PCR and visualized following agarose gel electrophoresis by ethidium bromide staining.

Generation of full-length moFcRH1–3 cDNAs
Primers used in end-to-end amplification to generate full-length cDNAs were as follows: moFcRH1, forward 5'-GTGAGAGGCACCTTCAAGTTACCAT-3' and reverse 5'-GGTGAAGGACTCATCTAATGAACCG-3'; moFcRH2, forward 5'-CCACAGTGTTCTATCCCAGATCCGT-3' and reverse 5'-GAGGCCCAGTGCAGAAAGTAGGAG-3'; and moFcRH3, forward 5'-GTGAGTGACTACCATTGCGAGCAAG-3' and reverse 5'-CAGGCCCAGTAGAAGCATCGG-3'. Each amplification reaction underwent initial denaturation at 94°C for 30 s followed by 30 cycles of denaturation at 94°C for 5 s and annealing at 68°C for 4 min, and final extension at 72°C for 6 min.

DNA sequence analysis
PCR products were ligated into the pCR2.1 TOPO T/A vector (Invitrogen, Carlsbad, CA). Inserts were sequenced on both strands by the dideoxy chain termination method using SequiTherm EXCEL II (Epicentre Technologies, Madison, WI) and an automated sequencer (LiCor, Lincoln, NE). Nucleotide and amino acid sequence alignment was analyzed with a DNASTAR (Madison, WI) software package and homology searches were performed using the Basic Local Alignment Search Tool (BLAST) (31).

RNA blot analysis
A mouse tissue northern blot (Ambion, Austin, TX) was hybridized with the following PCR amplified and [{alpha}-32P]dCTP labeled gene specific probes from respective moFcRH cDNAs: a 549 bp fragment (569–1117) corresponding to D2–3'UTR of moFcRH1, a 198 bp fragment (48–245) corresponding to the S2–D1 region of moFcRH2 and a 440 bp fragment (289–728) corresponding to D1–D3 of moFcRH3. According to the manufacturer's instructions, membranes were hybridized overnight at 65°C, washed and exposed to X-ray film.

For northern blots of mouse cell lines, RNA was extracted from freshly grown cells with the RNeasy kit (Qiagen, Valencia, CA). Ten micrograms of total RNA were electrophoresed through a 1% formaldehyde/agarose gel and blotted onto Nytran membrane following the manufacturer's instructions (Schleicher & Schuell, Keene, NH). The membrane was hybridized with [{alpha}-32P]dCTP labeled gene specific probes (as described above) overnight at 65°C, in 0.25 M NaH2PO4 (pH 7.2), 1 mM EDTA, 5% SDS, 0.1% sodium pyrophosphate (Na4P2O7.10H2O) and 100 µg/ml of total yeast RNA. Washes were performed at 65°C: twice in 0.125 M NaH2PO4 (pH 7.2), 0.05 mM EDTA and 2.5% SDS and twice in 0.025 M NaH2PO4 (pH 7.2), 0.01 mM EDTA and 0.5% SDS. Membranes were exposed to film for 1–7 days at –80°C.

Immunofluorescence and cell sorting
Bone marrow cell suspensions prepared from BALB/cJ mice (Jackson Laboratory, Bar Harbor, ME) were stained with APC-conjugated anti-CD19, PE-conjugated anti-CD43 (both from BD Biosciences, San Diego, CA) and FITC-conjugated goat anti-µ heavy chain (Southern Biotechnology Associates, Birmingham, AL) and cells representative of the different stages in B cell differentiation (32) were separated using a MoFlo flow cytometer (Cytomation, Fort Collins, CO). Spleen cells from BALB/cJ mice were stained with FITC-conjugated anti-CD19, PE-conjugated CD23 (BD Biosciences) and Cy5-conjugated anti-CD21 (a kind gift of Dr John Kearney), APC-conjugated anti-CD4 and PE-conjugated anti-CD8 (BD Biosciences), or PE-conjugated DX5 and FITC-conjugated anti-CD11c (BD Biosciences) and the cells were differentially sorted using a MoFlo flow cytometer. Newly-formed (NF) B cells were isolated as CD19+CD21CD23, follicular (FO) B cells were isolated as CD19+CD21+CD23+, marginal zone (MZ) B cells were isolated as CD19+CD21hiCD23lo (33), NK cells were isolated as DX5+CD11c, and dendritic cells were isolated as DX5CD11c+. Peritoneal lavage cells (PLC) from BALB/cJ mice were obtained by saline lavage of the peritoneal cavity. PLC cells were stained with APC-conjugated anti-CD19, PE-conjugated anti-CD5 and FITC-conjugated anti-CD11b (all from BD Biosciences), and separated with a MoFlow flow cytometer. Sorted cells were >98% pure and real-time PCR was performed at least three times from two independent sorts.

Reverse transcription (RT)–PCR
Total cellular RNA extracted from mouse cell lines with the RNeasy kit (Qiagen) was primed with random hexamers and oligo dT primers and reverse transcribed with SuperScript III (Life Technologies, Carlsbad, CA) into single-stranded cDNA. Gene specific primers for RT–PCR were moFcRH1 forward 5'-GCATCATCCTGGGGAACAGTTCAGCAC-3' and reverse 5'-CTCATCTAATGAACCGCAGTG-3'; moFcRH2 5'-GGCAACGACCCAGCTACGCTA-3' and reverse 5'-CGCCACATCTCCGATGAAG-3'; and moFcRH3 forward 5'-AGGTGAACATCAGTGACGC-3' and reverse 5'-TGGTTGAGTTCTCCGTACTTCT-3'. Each amplification reaction underwent initial denaturation at 94°C for 5 min followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 1 min and final extension at 72°C for 7 min. Amplified products were visualized in 1% agarose gels containing ethidium bromide and documented with the Bio-Rad Fluor-S Imager (Hercules, CA).

Real-time PCR
RNA from sorted cells, mouse tissues and cell lines was isolated using the RNeasy Mini kit with on column DNase digestion as recommended by the manufacturer (Qiagen). Total cellular RNA was primed with random hexamers and oligo dT primers and reverse transcribed with SuperScript III (Life Technologies) into single-stranded cDNA. Gene specific primers for real-time PCR were as follows: moFcRH1 forward 5'-GAACCTGCTGGAATCTCTGATGT-3' and reverse 5'-TCCCTCCATCACCCATCCT-3'; moFcRH2 forward 5'-TGACTGCCTCTCGCAGTGTCT-3' and reverse 5'-TGACTTGAGATACAGGGATCCTCTCTA-3'; and moFcRH3 forward 5'-GCCAAGCCGACAGCTTACTTC-3' and reverse 5'-ACAGCAGGTGGAGCTTGCA-3'; ß-actin forward 5'-GCTCTGGCTCCTAGCACCAT-3' and reverse 5'-GCCACCGATCCACACAGAGT-3'. The real-time PCR reactions were set up per manufacturer's instructions with 2x Syber Green PCR Master Mix (Applied Biosystems, Warrington, UK) and run on a 7900HT Sequence Detection System (Applied Biosystems).

Cell lines
Mouse cell lines included RAW8.1 and SCID7 pro-B cell lines, the 70Z/3 pre-B cell line, WEHI-231 and WEHI-279 immature-B cell lines, the CH-12, A20 and X16C8.5 B cell lines, the EL4 T cell line and the myeloid cell line WEHI-3.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Identification of moFcRH1, moFcRH2 and moFcRH3
Amino acid sequences of three huFcRH Ig domain subtypes, the D3, D4 and D5 of huFcRH3, were employed in this search of NCBI and Celera mouse expressed sequence tag (EST) and protein databases. Through use of the BLASTP and TBLASTN algorithms we identified a previously defined protein sequence (AAG28775) and multiple ESTs representative of two other cDNAs (31). Contig assembly and translation of their open reading frames (ORF) led to the identification of three huFcRH counterparts that we provisionally term moFcRH1, moFcRH2 and moFcRH3. Of these, moFcRH1 and moFcRH2 have been identified previously as mIFGP1 and mIFGP2 (6). Further analysis of genomic sequences indicates that the moFcRH locus is located in a chromosome 3 region ~250 kb telomeric of CD1d and ~10 Mb 5' of moFc{gamma}RI (45.2 cM) according to the Jackson Laboratory Mouse Genome Informatics database (Fig. 1A). Bacterial artificial chromosomes (BAC) that span the locus were analyzed by PCR to verify the database predicted moFcRH1–3 gene placement (data not shown) and the d3Mit187 microsatellite marker at 42.4 cM was found to fall within the moFcRH2 gene.



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Fig. 1. Chromosomal location, protein structure and sequence diversity of moFcRH1, moFcRH2 and moFcRH3. (A) Organization of the mouse FcRH locus on chromosome 3. The relative FcRH and neighboring gene locations and transcriptional orientations were defined by their position in NCBI, Ensembl and Celera sequence databases and the Mouse Genome Informatics Database. BAC clones (RP23-135C10 and RP23-228P24) that span the locus are displayed in relation to the genes that they contain. Note the position of the d3Mit187 microsatellite marker within FcRH2. (B) Schematic representation of moFcRH isoforms. Their corresponding cDNAs encode potentially secreted and type I transmembrane proteins with similar amino terminal extracellular regions, but different carboxy termini. The predicted mature proteins have extracellular (EC) regions with variable numbers of Ig and/or SRCR domains and potential sites of N-linked glycosylation ({intimmdxh137fx1_lw}). Transmembrane (TM) regions are uncharged. MoFcRH1 has one consensus ITAM (green boxes), whereas moFcRH3 has a consensus ITIM (red box) and an ITAM-like sequence. Both moFcRH2 isoforms are likely secreted and the SRCR domain is highlighted orange. The amino acid length of each region is indicated. (C) Multiple alignment comparison of moFcRH1–3 aa sequences (one-letter code) based on moFcRH3. Amino acid identity is represented by dots (.) and gaps are indicated by dashes (-). Potential N-linked glycosylation sites are underlined. Consensus ITIM (red) and ITAM or ITAM-like (green) sequences are indicated. Predicted structural domains are labeled: SP, signal peptide; EC, extracellular; MP-TM, membrane-proximal transmembrane; and CY, cytoplasmic regions. Amino acid lengths are indicated in parentheses.

 
RACE–PCR was used to define the 5' and 3' ends of moFcRH1–3 transcripts in blood mononuclear cell and splenocyte cDNA libraries. Primers designed from these sequences were used to isolate the corresponding full-length cDNAs. Analysis of the full-length cDNA sequences identified two moFcRH1 isoforms, moFcRH1S and moFcRH1L with ORFs of 903 bp and 1032 bp, two moFcRH2 isoforms, moFcRH2Ig and moFcRH2sc with ORFs of 1204 bp and 1530 bp, and two moFcRH3 isoforms, moFcRH3S andmoFcRH3L with ORFs of 1524 bp and 1788 bp. Single genes were identified for moFcRH1–3 through analysis of genomic sequences in the NCBI and Celera databases, suggesting that each of the two isoforms of moFcRH1moFcRH3 result from alternative splicing (described below). MoFcRH1S, moFcRH1L, moFcRH2Ig, moFcRH2sc, moFcRH3S and moFcRH3L are predicted to encode a family of potentially secreted and type I transmembrane receptors of 300 aa, 343 aa, 400 aa, 509 aa, 505 aa and 595 aa, respectively (34) (Fig. 1B and C). Based on the predicted signal peptide cleavage sites (35,36) these proteins would have core molecular masses of 30 699 Da, 35 398 Da, 42 234 Da, 54 005 Da, 53 486 Da and 63 823 Da, respectively. The moFcRH1–3 protein structures would include 2–5 extracellular Ig domains with 5–6 potential N-linked glycosylation sites (Fig. 1B).

The two moFcRH1 isoforms differ by alternative splicing of the transmembrane exon. The short isoform, moFcRH1S, lacks the sequence encoded by this exon and is presumably a secreted protein. The longer isoform, moFcRH1L, is a type I transmembrane protein with an uncharged transmembrane region and a cytoplasmic tail that contains a potential ITAM.

Both moFcRH2 isoforms, moFcRH2Ig and moFcRH2sc, have four extracellular Ig-like domains and lack transmembrane regions. The carboxy terminus of the moFcRH2sc isoform contains in addition a single type B scavenger receptor cysteine-rich (SRCR) domain with eight cysteines (37). This unusual chimeric protein, not found among other presently characterized human and mouse proteins, shares 56% amino acid identity with the amino terminal domain of the product of its closest neighboring gene, moSp{alpha}/CD5L/Api6 (38).

The two moFcRH3 isoforms identified in this analysis are type I transmembrane proteins that differ by alternative splicing of the first of five Ig-like domains. The short isoform, moFcRH3S, lacks the sequence encoding the first Ig domain, but the rest of the sequence is identical to moFcRH3L which contains all five Ig domains. Their cytoplasmic tails contain a consensus ITIM and an ITAM-like sequence with a glutamic acid at the +3 position relative to the second tandem tyrosine. This analysis suggests the mouse has only two FcRH members that closely resemble their human FcRH1–5 counterparts. Despite the conserved extracellular regions for these FcRH family members, only moFcRH1 and moFcRH3 may possess immunoregulatory tyrosine-based signaling potential.

MoFcRH1, moFcRH2 and moFcRH3 genomic organization
Genomic sequences of the moFcRH locus were identified by BLASTN searches of the Ensembl, Celera and NCBI databases using the moFcRH cDNA sequences. Exon/intron boundaries were determined by comparing moFcRH cDNAs with genomic DNA sequences using the AG/GT rule. A C57BL/6 derived BAC clone, RP23 135C10, corresponding to the 5' end of the locus was found to contain moFcRH2 and moFcRH1, but not moFcRH3 (Fig. 1A). The position of moFcRH3 was confirmed by PCR verification of its position on a partially overlapping BAC clone, RP23 228P24, which lies just telomeric of RP23 135C10 (data not shown). The moFcRH locus thus spans ~215 kb and is organizationally more complex than the human syntenic region. The 5' end of the locus is demarcated by the 3' end of the moFcRH2 gene, which lies in the reverse transcriptional orientation relative to moFcRH1 and moFcRH3.

MoFcRH1 consists of 10 exons that span ~15.2 kb. All exon/intron boundaries follow the phase 1 splicing pattern, in that splicing occurs after the first nucleotide of the triplet codon except for the CY1/CY2 boundary, which follows a phase 2 pattern, and the CY4/CY5-3'UTR boundary, which follows the phase 0 pattern (Fig. 2). Like other members of the extended FcR family, the first exon 5'UT/S1 encodes the 5' untranslated region, the ATG translation initiation codon, and the beginning of a split signal peptide. The second exon, S2, encodes the second half of the split signal peptide and is only 21 bp in length, a characteristic feature of all FcR and FcRH relatives except for FcRX (1,2,10,22,24,3942). The Ig-like extracellular domain is encoded by two exons, D1 and D2, separated by an intron of only 167 bp. Exon 5 encodes the membrane-proximal, hydrophobic/uncharged transmembrane domain and the beginning of the cytoplasmic tail. The presumably secreted moFcRH1S isoform splices out this transmembrane encoding exon and maintains the same ORF with the remaining exons that encode the cytoplasmic region of the moFcRH1L isoform. The cytoplasmic tail is encoded by five exons, CY1–CY5, the last of which also encodes the translation termination codon and the 3'UT. MoFcRH1 and moFcRH3 are separated by ~44 kb and, unlike moFcRH2, both are transcriptionally oriented towards the telomere.



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Fig. 2. Genomic organization of the mouse FcRH family members. Exon–intron boundaries were determined by comparing the full-length cDNAs with the BAC clone genomic sequences that span the region and the AG/GT rule. Exons are numbered and labeled as squares with translated (closed) and untranslated (open) regions. Domains are listed as: UT, untranslated; S, signal peptide; D, extracellular domain; TM, transmembrane; and CY, cytoplasmic regions.

 
The moFcRH2 gene spans ~12.8 kb, consists of eight exons that follow the phase 1 splicing pattern, and contains a split signal peptide encoded by two exons: 5'UT/S1 encodes the 5' untranslated region, translation initiation codon and first half of the split signal peptide. The microsatellite marker d3Mit187 is located in the ~1.2 kb intron separating exons 1 and 2. S2 encodes the second half of the split signal peptide and is 21 bp long. D1–4 generate individual Ig-like domains and in the case of the short moFcRH2Ig splice isoform, D4 also encodes four additional amino acids (NVT-GRTI), the translation stop codon and a 3'UT. The most unusual feature of this gene is that in addition to the Ig encoding D1–D4 exons, in the long moFcRH2sc isoform, a D5 exon encodes a type B scavenger receptor cysteine-rich domain and D6/3'UT encodes the last seven amino acids of the predicted protein, including the translation stop codon and the 3'UT. The Sp{alpha}/CD5L/Api6 scavenger receptor lies ~94 kb directly telomeric of moFcRH2. Sp{alpha} is ~13.1 kb in length, transcriptionally oriented towards the telomere and located ~5.5 kb 5' of moFcRH1. The tandem positioning of Sp{alpha} and moFcRH1 in mice mimics the positioning of these two genes in humans.

MoFcRH3 contains 13 exons that span ~32 kb. All of its exon/intron boundaries follow the phase 1 splicing pattern, except for the CY1/CY2 boundary which is phase 2. Like other family members, moFcRH3 has a split signal peptide encoded by two exons, the second of which is also 21 bp. The extracellular Ig domains, D1–D5, are encoded by five exons clustered within a ~6.4 kb region. The first of these, D1, is spliced out in the moFcRH3S isoform while maintaining the ORF with the remaining 3' encoding exons. These are followed by an ~6.1 kb intron and exon 8 that encodes the membrane-proximal, hydrophobic/uncharged transmembrane region and proximal portion of the cytoplasmic domain. The CY1–CY4 regions of the cytoplasmic tail are encoded by four exons, the last of which also encodes the translation termination codon and the beginning of the 3'UT. Unlike moFcRH1 and moFcRH2, moFcRH3 has a second 3'UT transcribed exon that is ~9 kb from CY4.

Relationship of mouse FcRH1–3 with other FcR/FcRH family members
The Ig domains present in members of the extended human FcR/FcRH family can be divided into five subtypes on the basis of their respective amino acid identities in a CLUSTAL driven analysis (1,7,43). Comparison of the moFcRH1–3 domain relationships with the mouse FcRs and FcRHs in an unrooted pairwise analysis indicates that the extracellular regions of the mouse FcR/FcRH family members consist of different combinations of the five Ig-like domain subtypes (Fig. 3A and B). The ordering of the membrane-proximal to membrane-distal Ig domains for the mouse FcRH and FcR family members resembles that observed for their human relatives. Greater amino acid sequence relatedness is usually seen for membrane-proximal domains, while more membrane-distal Ig domains characteristically exhibit greater diversity.



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Fig. 3. Analysis of the extracellular homology among the moFcRH and extended mouse FcR family members. (A) Inferred phylogenetic relatedness among mouse FcRH and FcR Ig-like domains. Amino acid sequences corresponding to individual Ig-like domains were aligned using CLUSTAL_X with BLOSSUM substitution matrices (59). Un-rooted tree topology was estimated with the weighted pair group method using arithmetic averages (60) available in the TreeCon software package (61). Branch values represent percentage bootstrap support after 1000 replicates and values <50% are not shown. Five major nodes supporting previously defined domain designations are colored in accordance with the extracellular domains depicted in B. (B) Pairwise analysis of individual Ig-like subunits was performed with the CLUSTAL method algorithm using moFcRH3 as the index of comparison. Individual homologous domains are color-coded to indicate relatedness. Percent amino acid identities for related domains are indicated and aligned in relation to the comparative moFcRH3 subunit. Comparisons that are not applicable are left blank. (C). Extracellular region identities of human and mouse FcRHs. Amino acid sequences were derived from moFc{gamma}RI (NP_034316), moFc{gamma}RII (NP_034317), moFc{gamma}RIII (NP_034318), moFc{varepsilon}RI{alpha} (AAA37600), FcRL3 (NP_653142) and moFcRX (AAM97592), huFcRH1 (AAK91777), huFcRH2 (AAK91778), huFcRH3 (AAK91779), huFcRH4 (AAK93970) and huFcRH5 (AAK93971).

 
Using moFcRH3 as the index molecule for comparison and the CLUSTAL algorithm, which does not penalize for missing sequence, full-length amino acid identity with moFcRH1–2 was limited to 27–28%, and ranged from 20–24% when the analysis included moFc{gamma}RI, moFc{gamma}RII, moFc{gamma}RIII, moFc{varepsilon}R{alpha} and the more recently identified moFcR homologs, FcRL3/CD16-2 and moFcRX (43) (Fig. 3B). Extracellular moFcRH3 comparisons yielded slightly higher identity values of 39% and 32% for moFcRH1 and moFcRH2. Identity values were more limited when moFcRH3 was compared with other mouse relatives, 22–25% for moFc{gamma}RII, moFc{gamma}RIII, moFc{varepsilon}R{alpha} and FcRL3/CD16-2 on chromosome 1, and 29% extracellular identity with its moFc{gamma}RI neighbor on chromosome 3.

An interspecies comparison of mouse and human FcRH orthologs indicates that moFcRH1 has greatest extracellular identity (61%) with huFcRH1 (Fig. 3C). The Ig domains of moFcRH2 have 46% identity with both huFcRH1 and huFcRH2. Although moFcRH3 exhibits 45% extracellular identity with huFcRH2 and 40% with huFcRH1, huFcRH3 and huFcRH5, the domain composition of moFcRH3 is most similar to huFcRH3. Comparisons of the cytoplasmic domains indicate 43% identity between huFcRH1 and moFcRH1 and 48% identity between moFcRH3 and huFcRH5 (data not shown).

Pairwise Ig domain comparisons of FcR/FcRH family members indicate that moFcRH3 possesses the D1 and D2 Ig binding domain subtypes employed by the FcRs. To extend this analysis, we threaded the amino acid sequence of moFcRH3 D1 and D2 into the Position Specific Scoring Matrix (PSSM) database to compare this sequence with currently solved protein folds in the library (44). This analysis indicates that huFc{gamma}RIIB is the closest recognizable structure to the linear and predicted secondary structure of moFcRH3 D1 and D2. A significance value of 2.26e-05 indicates that moFcRH3 has a high probability of forming a similar structural fold to that of huFc{gamma}RIIB.

Tissue distribution of moFcRH1, moFcRH2 and moFcRH3 expression
MoFcRH1 transcripts of ~1.6 kb were easily identified by RNA blot analysis in spleen and thymus and at lower abundance in kidney and lung (Fig. 4A). MoFcRH2 was more widely expressed, with transcripts of ~2.4 kb being detected in whole embryo (14 day), lung, ovary, brain, testes, thymus, heart and kidney, albeit in trace levels in the latter four tissues. Notably, moFcRH2 transcripts were not seen in spleen. In contrast, moFcRH3 transcripts were found only in the spleen, where a faint band of ~2.5 kb was sometimes detected with gene specific probes (data not shown). The inconsistency of this finding suggested that northern blot analysis is insufficiently sensitive for accurate detection of moFcRH3 expression in tissues, perhaps because moFcRH3 is expressed at relatively low levels or by a minor subpopulation of cells.



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Fig. 4. Analysis of moFcRH expression in mouse tissues (A) and cell lines (B). RNA blots were hybridized with [{alpha}-32P]dCTP-labeled PCR generated probes from respective moFcRH cDNAs as described in Methods. Relative mRNA abundance is estimated by hybridization with a ß-actin probe.

 
When moFcRH1–3 expression was examined by northern blot analysis of eight myeloid and lymphoid cell lines, six of which represent different stages of B cell differentiation ranging from pro-B (RAW8.1) to mature isotype-switched B cells (A20), transcripts were detected for moFcRH1 and moFcRH3, but not for moFcRH2 (Fig. 4B). A moFcRH1 probe hybridized with prominent transcripts of ~1.7 kb and ~2.0 kb in the A20 B cell line, and in lesser abundance in the IgM+ WEHI-231 and CH-12 B cell lines, but not in pro-B, pre-B, the X16C8.5 B, T and myeloid cell lines. Sequence analysis of the two types of moFcRH1 transcripts identified by RT–PCR amplification of the full-length moFcRH1 cDNA from mouse splenocytes indicated that these isoforms differ by the presence (moFcRH1L) or absence (moFcRH1S) of the transmembrane domain encoding exon. RT–PCR analysis of the above cell lines, together with additional pro-B (SCID7), B (WEHI-279) and plasmacytoid (Ag8) cell lines, demonstrated the presence of moFcRH1 transcripts in all of the B cell lines tested, but not in the plasmacytoid, T and myeloid cell lines (data not shown). MoFcRH3 transcripts of ~2.5 kb were abundant in the WEHI-231 B cell line and not in the other B lineage cell lines. However, moFcRH3 transcripts could be detected in three of five B cell lines tested by RT–PCR, but not in pro-B, pre-B, plasmacytoid, myeloid and T cell lines. By contrast, moFcRH2 transcripts could not be identified in any of the lymphoid or myeloid cell lines by either northern or RT–PCR analysis. These findings indicate that moFcRH1 and moFcRH3 are preferentially expressed at different stages in B cell differentiation, while moFcRH2 is expressed in non-lymphoid tissues.

MoFcRH expression in primary B lineage cells
The patterns of moFcRH1–3 expression were examined during normal B lineage differentiation by isolating subpopulations of B lineage cells via their defining cell surface antigens from bone marrow, spleen and peritoneal lavage samples. Gene specific primers were then employed in a real-time PCR analysis of moFcRH1–3 transcripts. Whereas moFcRH1 transcripts were barely detectable in early B lineage cells of bone marrow origin, they were present in higher levels in the newly-formed, follicular and marginal zone subpopulations of splenic B cells (Fig. 5). Minimal moFcRH1 expression was evident in sorted populations of splenic T cells, dendritic cells and NK cells. In peritoneal lavage samples, moFcRH1 expression was detectable in each of the B cell subsets, B1a, B1b and B2, but not in macrophages (data not shown). MoFcRH2 transcripts were not detectable in bone marrow and splenic B lineage cells, or in other types of splenocytes, but were surprisingly abundant in peritoneal macrophages (Fig. 5; and data not shown). In contrast, moFcRH3 expression was most prominent in marginal zone B cells, much less evident in the newly-formed and follicular splenic B cell subpopulations, and not seen in macrophages, DC, NK and T cells from either spleen or the peritoneal cavity. These results indicate a relatively broad pattern of expression for moFcRH1 during B cell differentiation, while moFcRH3 is preferentially expressed by marginal zone B cells.



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Fig. 5. MoFcRH1, moFcRH2 and moFcRH3 expression in sorted cells from primary and secondary lymphoid tissues. Real-time PCR was used to evaluate the relative expression of moFcRH1 (top), moFcRH2 (middle), or moFcRH3 (bottom) in the indicated subpopulations. Except for the peritoneal lavage cells (PLC), each subpopulation was FACS-purified from the indicated tissue based on the cell-surface phenotypes as described in Methods. B cell subpopulations are abbreviated as follows: Imm B (immature), Mat B (mature), NF B (newly-formed), FO B (follicular) and MZ B (marginal zone). The expression of ß-actin in each subpopulation is used to control for the mRNA levels in each sample.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
This analysis indicates there are only three mouse gene relatives of the five human Fc receptor homologs, huFcRH1–5, and that significant differences exist between these human and mouse counterparts. Unlike the uninterrupted human FcR/FcRH locus in the chromosome 1q21-23 region, the mouse FcR family is split between chromosomes 1 and 3. The moFcRH1–3 genes are located on chromosome 3 near their Fc{gamma}RI relative (22). The corresponding mouse FcRH proteins share similar Ig domain subtypes with their human counterparts, but only two of them, FcRH1 and 3, closely resemble the human FcRH1–5 family members. The moFcRH1 and moFcRH3 transmembrane receptors have consensus ITAMs and/or ITIMs in their cytoplasmic tails, and these features suggest signaling capabilities similar to those of their human relatives (45,46). In addition, the expression patterns for moFcRH1 and moFcRH3 mimic the restricted B cell lineage and stage-specific patterns previously observed for huFcRH1–5 expression (1,4). In striking contrast, moFcRH2 encodes two very different, presumably secreted isoforms that are expressed primarily in non-lymphoid tissues. These findings indicate a remarkable degree of variation in the FcRH locus during mammalian speciation.

Our identification of huFcRH1–5 derived from an informatics based search using a consensus sequence generated from the Fc binding regions of Fc{gamma}RI, Fc{gamma}RII and Fc{gamma}RIII (1). The related extracellular composition, gene structure and signaling potential of these receptors suggests a common phylogenetic origin of the FcR and FcRH families (17). The validity of this interpretation is strengthened by the characterization of the mouse FcRH1–3 gene family here and in a previous report describing ESTs for two of these family members, moFcRH1/mIFGP1 and moFcRH2/mIFGP2 (6). As for the other FcR/FcRH family members, with the exception of moFcRX and huFcRX, the moFcRH1–3 genes share structural similarity that includes a trademark 21 bp S2 exon encoding the second portion of a split signal peptide. MoFcRH proximity to the most closely related FcR family member, moFc{gamma}RI, on chromosome 3 also attests to the evolutionary relationship between the FcR and FcRH gene families.

The FcR and FcRH genes comprise a larger, more diverse family in humans and mice than formerly appreciated. The FcR gene family members, like the FcRH family members, are composed of multiple genetic units that reflect significant duplication and diversification over the ~90 million years since rodents and primates shared a common ancestor (39,47). While the location of moFcRH1–3 near moFc{gamma}RI, CD1 and Sp{alpha} is not surprising, given the relative positions of these genes in humans, it is noteworthy that the FcR family cluster is divided between chromosomes 1 and 3 in mice. A high recombinatorial rate for this chromosomal region may account for the inverted orientation of moFcRH2, the inclusion of an SRCR domain encoding exon which may have its origin in the Sp{alpha} gene, and the apparent loss of moFcRH2 lymphoid specific promoter elements. Involvement of the IRTA1/FcRH4 gene in a chromosomal translocation with the human Ig locus accords with other evidence indicating that the FcR/FcRH region is a recombinatorial hotspot (3).

The mammalian FcRH and FcR genes appear to represent an ancient gene family with members of the FcRH gene family being conserved in chickens, frogs and bony fish (4850). Remarkably, at least 20 FcRH family members have recently been identified in Xenopus (50). Additional phylogenetic analysis of the FcR and FcRH families is needed to clarify the evolution of the FcR/FcRH locus, and this endeavor may also yield informative clues to the functions of these Ig-like receptors. In this regard, the closest phylogenetic relative of the huFc{gamma}RIII (CD16) gene has only recently been identified in the mouse, FcRL3/CD16-2 (11).

The tandem order of the different subtypes of Ig domains in the extracellular regions of FcRH and FcR family members (Fig. 3) is highly conserved. The Ig Fc binding sites are located in the FcR D1 (red in Fig. 3B) linker regions and the neighboring D2 (dark blue) domains. These Ig domain subtypes are seen less frequently in the moFcRHs than in huFcRH family members, although moFcRH3 has D1 and D2 domains that may have Ig-binding potential. This possibility is supported by a three dimensional position specific scoring matrix (3D-PSSM) analysis indicating a high degree of structural similarity between these two moFcRH3 domains and the related huFcR{gamma}IIB domains. In contrast, the more membrane-proximal Ig domains of moFcRH3 and moFcRH1 (light blue and green in Fig. 3B) are FcRH family specific. The absence of FcR-like Ig domains in moFcRH1 suggests that, like the huFcRH1, it may not have Fc binding potential, but rather a different ligand(s).

The tyrosine-based motifs in the cytoplasmic regions of the transmembrane moFcRH1 and moFcRH3 molecules resemble those of the huFcRHs. Like its nearest human relative, huFcRH1, moFcRH1 has a canonical ITAM in its cytoplasmic tail, suggesting the potential for cellular activation. A different function would be predicted for the moFcRH1S isoform that lacks a transmembrane region; such alternatively spliced isoforms have similarly been observed for moFc{gamma}RII (51). MoFcRH3 differs in that it possesses an ITIM and an ITAM-like sequence, while its closest human relative, huFcRH5, has two consensus ITIMs and an ITAM-like sequence. The presence of ITIM and ITAM-like motifs in moFcRH3 and several members of the human FcRH family suggests they may have either inhibitory or activating potential depending upon the signaling context. A precedent for this type of dual functionality has been demonstrated for CD22, another transmembrane molecule on B cells (52,53).

MoFcRH2 differs from all of the human FcRHs in its preferential expression by non-lymphoid lineage cells. Neither of the two predicted moFcRH2 isoforms has a transmembrane region, and both are therefore likely to be secreted. The Ig/SRCR domain-containing moFcRH2sc isoform is an unusual chimeric protein. The exon encoding the SRCR domain could have been derived from a recombination event involving moSp{alpha}, its chromosome 3 neighbor, given the high degree of identity (56%) with the Sp{alpha} N-terminal SRCR domain (38,54). Moreover, in contrast with the lymphoid tissue-specific expression of mouse and human Sp{alpha}, moFcRH2 is primarily expressed in non-lymphoid tissues.

Like their human counterparts, the moFcRH1 and moFcRH3 genes are selectively expressed by B lineage cells at different stages in differentiation. MoFcRH1 is expressed by many of the mature B cell subpopulations, including newly formed, follicular and marginal zone B cells. MoFcRH1 may thus serve an activating role on mature B cells, as appears to be the case for its human counterpart, huFcRH1 (46). The most remarkable feature of moFcRH3 is that it is preferentially expressed by B cells in the marginal zone subpopulation. Marginal zone B cells are typified by their topographic location, pre-activated state and distinct receptor repertoire, characteristics that enable them to participate in early responses to T independent blood-borne antigens (55,56). However, it may be noteworthy that memory B cells are also present in the marginal zone (57), especially since huFcRH4/IRTA1 has been shown to be preferentially expressed by memory B cells, where it may serve an important regulatory role in antibody responses (45,58). It will therefore be important to determine which type(s) of marginal zone B cells account for the moFcRH3 expression that we have observed.

The true functions of moFcRH1 and moFcRH3 on B lineage cells will only become clear with further investigation, but differences in their structure and expression patterns suggest potential immunoregulatory roles. MoFcRH1 possesses an ITAM-like sequence in its cytoplasmic tail, whereas moFcRH3 has both an ITIM and ITAM-like motif. Given the presence of domain subtypes (D1 and D2) that resemble the Fc binding domains of classical FcRs, it is possible that moFcRH3 may bind Ig or antigen-antibody complexes on marginal zone B cells. Since these cells also express Fc{gamma}RIIB, a competitive or cooperative relationship could exist between these receptors. The resulting impact on activation or inhibition is difficult to predict, especially given it may depend upon the Ig isotype and other microenvironmental factors. MoFcRH1 is more likely to have an activating function on the B cells that express it. Our studies of human FcRH1 indicate broad expression among mature B cell populations, where it can serve a role as an activation co-receptor (C.-M. Leu, R. S. Davis, L. A. Gartland, W. D. Fine, and M. D. Cooper, submitted for publication). In conclusion, the differential B lineage expression and signaling potential of moFcRH1 and moFcRH3 suggest that these receptors will differentially modulate B cell activation and differentiation.


    Acknowledgements
 
We thank Dr Peter D. Burrows for helpful discussion and critical review of the manuscript, E. Ann Brookshire for help in manuscript preparation, Dr Larry G. Gartland for help with flow cytometry and Drs Zeev Pancer and Yoshiki Kubagawa for advice and sequencing assistance. We are grateful to Drs Ute Laessing, Martin Bachmann and John F. Kearney for sharing information about the short isoform of moFcRH3. This work was supported in part by NIH grants AI39816 and AI48098. R.S.D. was a Leukemia Lymphoma Society Special Fellow and is supported in part by a K08 award AI55638 from NIH/NIAID. M.D.C. is a Howard Hughes Medical Institute Investigator.


    Notes
 
NCBI accession nos: moFcRH1L, AY506554; moFcRH1S, AY506555; moFcRH2sc, AY506556; moFcRH2Lg, AY506557; moFcRH3, AY506558.

Transmitting editor: T. Watanabe

Received 6 May 2004, accepted 30 June 2004.


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