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The Journal of Immunology, 1999, 163: 6371-6377.
Copyright © 1999 by The American Association of Immunologists

Characterization of Paired Ig-Like Receptors in Rats1 ,2

Glynn Dennis, Jr.*,{dagger}, Robert P. Stephan*, Hiromi Kubagawa*,{ddagger} and Max D. Cooper3,*,{dagger},§,¶,||

* Division of Developmental and Clinical Immunology and Departments of {dagger} Microbiology, {ddagger} Pathology, § Pediatrics, and Medicine and || Howard Hughes Medical Institute, University of Alabama, Birmingham, AL 35294


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To explore the phylogenetic history of the murine paired Ig-like receptors of activating (PIR-A) and inhibitory (PIR-B) types, we isolated PIR homologues from a rat splenocyte cDNA library. The rat (ra) PIR-A and raPIR-B cDNA sequences predict transmembrane proteins with six highly conserved extracellular Ig-like domains and distinctive membrane proximal, transmembrane, and cytoplasmic regions. The raPIR-B cytoplasmic region contains prototypic inhibitory motifs, whereas raPIR-A features a charged transmembrane region and a short cytoplasmic tail. Southern blot analysis predicts the presence of multiple Pira genes and a single Pirb gene in the rat genome. Although raPIR-A and raPIR-B are coordinately expressed by myeloid cells, analysis of mRNA detected unpaired expression of raPIR-A by B cells and raPIR-B by NK cells. Collectively, these findings indicate that the structural hallmarks of the Pir gene family are conserved in rats and mice, yet suggest divergence of PIR regulatory elements during rodent speciation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The mouse paired Ig-like receptor (PIR)4-B/p91 belongs to an emerging family of type I transmembrane proteins that possess extracellular (EC) Ig-like domains and cytoplasmic regions containing one or more immunoregulatory tyrosine-based inhibitory motifs (ITIM) (1, 2, 3). These ITIM-bearing receptors often share EC domain homology with activating receptor counterparts that have distinctive transmembrane and cytoplasmic regions. The cytoplasmic tail of PIR-B contains three ITIM units that can mediate inhibitory signals via interaction with the src-homology phosphatase SHP-1 (1, 2, 4, 5, 6, 7, 8). PIR-B shares greater than 90% homology with the EC domain of its PIR-A counterparts that have charged arginine residues in their transmembrane segments and short cytoplasmic tails with no known functional motifs (1, 9). PIR-A proteins associate with dimeric Fc receptor {gamma} chains (FcR{gamma}) containing immunoregulatory tyrosine-based activation motifs (ITAM) to form activation receptors (7, 10, 11).

The genes encoding PIR-A and PIR-B map to the proximal end of mouse chromosome 7 (1, 9). Sequence comparison indicates that the mouse (mo) PIRs share significant homology with several leukocyte-specific Ig-like receptor genes that are tightly clustered in a syntenic region of human chromosome 19 (12, 13, 14, 15). This leukocyte receptor complex (LRC) on chromosome 19q13.4 includes a mulitgene family of Ig-like receptors that have been described as follows: Ig-like transcripts (ILT) (12); leukocyte Ig-like receptors (LIR) (13); monocyte/macrophage Ig-like receptors (MIR) (14); human monocyte cDNA 18 (HM18) (16); human PIR (huPIR) (17); and will subsequently be referred to as the human (hu)ILT/LIR/MIR multigene family. The moPIR and huILT/LIR/MIR families both contain activating and inhibitory members with similar EC regions. However, the respective mouse and human EC regions are quite distinct in that moPIRs have six Ig-like domains, four C2-type, and two V-like, whereas huILT/LIR/MIR have either four or two Ig-like domains, all of which are of C2-type (1, 12, 13, 14). Another distinction is that mice possess one inhibitory Pirb gene and approximately eight activating Pira genes, while humans have multiple inhibitory gene members, and relatively few activating receptor genes (13, 15, 18). The hallmark paired expression of PIR-A and PIR-B in mouse myeloid, B, and dendritic cell lineages is not a general rule for their human counterparts, where the cellular distribution of huILT/LIR/MIR expression extends beyond the myeloid, B, and dendritic cell lineages to include subpopulations of NK cells and T cells (12, 13, 14).

The evolutionary history of moPIR and huILT/LIR/MIR seems complex in that many structural features are conserved, yet fundamental differences are evident in their genomic organization and cellular distribution. In this report, we characterize rat PIR cDNAs and examine the phylogenetic relationships among rat, mouse, and human relatives.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation of cDNA clones

A splenic cDNA {lambda}gt11 library derived from a Sprague Dawley rat (Clontech, Palo Alto, CA) was screened as previously described (1). In brief, 5 x 105 phage plaques were lifted onto duplicate nitrocellulose filters and probed overnight with an [{alpha}-32P]dCTP-labeled, EcoRI-digested cDNA fragment (1.5 kb) of the mouse PIR-B extracellular domain. Posthybridization filters were washed twice in 1x SSC and 0.1% SDS at 55°C and exposed to x-ray film for 48 h. Hybridizing phage cDNA clones were purified with two subsequent rounds of screening followed by proteinase K digestion, phenol-chloroform extraction, and ethanol precipitation.

Sequence analysis

DNA sequencing was performed using an automated sequencer (Li-Cor, Lincoln, NE). Each clone was sequenced three or more times on the same or opposite strand by the dideoxy chain-termination method using Thermo Sequenase (Amersham Pharmacia Biotech, Amersham, Arlington Heights, IL) or SequiTherm Excel (Epicentre Technologies, Madison, WI) cycle sequencing kits. Nucleotide and amino acid sequences were analyzed using the DNASTAR (Madison, WI), BLAST (19), PHYLIP (20), and GENEDOC (21) programs.

Generation of full-length raPIR-A and raPIR-B cDNAs

mRNA was purified from a Lewis rat spleen using FastTrack mRNA isolation kits (Invitrogen, Carlsbad, CA), and full-length raPIR cDNAs were generated following the Marathon cDNA amplification kit protocol (Clontech). RaPIR specific reverse primers used in 5'-rapid amplification of cDNA ends (RACE) were 5'-CTCCTGTGACCACTAGCTCCA-3' and 5'-GAGCTTCTGAGGTCCTTCTACAGTA-3'. RaPIR-specific forward primers used in 3'-RACE were 5'-GAGGCCAGTAACAGATGCTATGGTG-3' and 5'-CCTCTCAGTGATGCCCAACTCC-3'. Primers used in end-to-end PCR to generate full-length PIR-A were forward 5'-CCTTCACCTTCACAGCCCT-3' and reverse 5'-GCAGAGTGTAGAAACATTGAAACC-3'. Primers used in end-to-end PCR to generate full-length PIR-B were forward 5'-CCTTCACCTTCACAGCCCT-3' and reverse 5'-GTTACATTACTTAGTGCAGTCAGG-3'. The resulting amplification products were cloned into pCR2.1 (Invitrogen).

Abs, cell sorting, and cell lines

mAbs HIS24-PE (IgG2b{kappa}; anti-CD45R), HIS48-FITC (IgM{kappa}; anti-granulocyte), and 10/78-PE (IgG1{kappa}; anti-NKR-P1A) were obtained from PharMingen (San Diego, CA). B and NK cells were isolated from the spleens of 4- to 6-mo-old female Lewis rats by immunofluorescent staining and flow cytometry. B cells were identified as CD45R+/CD3-. NK cells were identified as NKR-P1A+/CD3-. Granulocytes were isolated from bone marrow of the same rats as HIS48+. Cells were double sorted to greater than 99% purity. B (YB2/0), T (C58(NT)D), myeloid (RBL-2H3), and alveolar macrophage (NR8383) cell lines were cultured at 37°C and 7.5% CO2 according to the distributors recommendations (American Type Culture Collection (ATCC), Manassas, VA), with the exception of the T cell line, which was grown in media supplemented with 10% FCS in place of horse serum.

DNA and RNA blotting

High m.w. genomic DNA was extracted from Lewis rat kidneys and digested to completion with BamHI, EcoRI, HindIII, PstI, and XbaI restriction endonucleases. DNA digests were resolved in 0.8% agarose gels and transferred to nylon filters. Total RNA was obtained from rat tissues, resolved in 1.7% agarose/formaldehyde gels, and transferred to nylon filters. Employing [{alpha}-32P]dCTP-labeled probes, filters were hybridized overnight and washed twice in 2x SSC/0.1% SDS at room temperature for 15 min, twice under high stringency (0.5x SSC/0.1% SDS at 65°C) for 30 min, and exposed to x-ray film. The following probes were used: 1) a PCR-generated 1.5-kb insert of rat cDNA clone R16 as a common extracellular probe; and 2) a PCR-generated PIR-B-specific cytoplasmic probe of 228 bp.

RT-PCR

Total RNA was reverse transcribed into sscDNA using random primed SuperScriptII (Life Technologies, Rockville, MD). Common forward primers 5'-CCTCTCAGTGATGCCTAACTCCA-3', PIR-A-specific reverse primers 5'-CCATCCTGATGATTCTCCG-3', and PIR-B-specific reverse primers 5'-CTGGGCATAGGTCACATCC-3' amplified products of 366 bp and 820 bp, respectively. Control ß-actin forward 5'-TACAACCTCCTTGCAGCTCC-3' and reverse 5'-GGATCTTCATGAGGTAGTCAGTC-3' primers were used. Each amplification reaction underwent an initial denaturation at 94°C for 2 min followed by 35 cycles of denaturation at 94°C for 5 s, annealing at 66°C for 15 s, and extension at 72°C for 30 s, followed by a 2-min final extension. Amplified products were visualized in 2% agarose gels containing ethidium bromide and documented with the Bio-Rad Fluor-S Imager (Hercules, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Isolation of rat PIR-A and PIR-B

In experiments aimed toward identifying homologues of the mouse Pir genes, analysis of EcoRI-digested genomic DNA from humans, monkeys, rats, mice, dogs, cows, rabbits, chickens, and yeast indicated cross-hybridization of a moPIR-B EC domain probe to all vertebrate DNA samples, with the highest reactivity to rat DNA (Ref. 1 and data not shown). Screening of a rat splenic cDNA library with a mouse PIR-B extracellular region probe yielded three hybridizing clones (R16, R29, and R34). Sequence analysis indicated that clone R16 was a partial cDNA (1.5 kb) that encoded four Ig-like domains with homology to moPIR. Similarly, analysis of clones R29 and R34 indicated that these were incompletely spliced cDNAs encoding Ig-like domains similar to moPIR. RACE extension of clone R16 in the 5' and 3' directions yielded invariant 5'-RACE products, but distinct PIR-A-like and PIR-B-like 3'-RACE products. Full-length raPIR-A and raPIR-B candidates were then generated by end-to-end PCR using common 5' primers complementary to the signal sequence and distinct 3' primers for the PIR-A and PIR-B 3'-untranslated regions. As depicted in Fig. 1GoA, the full-length raPIR-B cDNA (2.5 kb) is predicted to encode a type I transmembrane protein with six extracellular Ig-like domains, a short EC membrane proximal domain, an uncharged transmembrane segment, and a long cytoplasmic tail with four putative ITIM motifs. The full-length raPIR-A cDNA (2.3 kb) likewise is predicted to encode a type I transmembrane protein with six extracellular Ig-like domains homologous to those of raPIR-B, a distinct EC membrane proximal domain, a charged transmembrane segment, and a short cytoplasmic tail devoid of ITIM. Southern blot analysis of rat genomic DNA employing a common raPIR extracellular probe derived from cDNA clone R16 resulted in multiple (6, 7, 8, 9, 10) hybridizing restriction fragments (Fig. 1GoB). Conversely, a specific probe spanning the cytoplasmic tail of raPIR-B resulted in a relatively simple hybridization pattern of one or two predominant hybridizing fragments. This analysis suggests that raPIR-B, like its moPIR-B relative (1), may be encoded by a single gene that shares extracellular region homology with multiple raPIR-A genes.



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  		FIGURE 1. A, Schematic representation of the raPIR-A and raPIR-B cell surface proteins. Their extracellular regions have six Ig-like domains, D1 through D6, the second and fourth of which are V-like whereas all others are C2-type. RaPIR-A is predicted to encode a short cytoplasmic region and a transmembrane segment with a charged arginine (R) residue. The predicted raPIR-B protein encodes a long cytoplasmic tail with four putative ITIM motifs (outlined boxes) and an uncharged transmembrane segment. B, Southern blot analysis of the Pir gene family. DNA from Lewis rat kidneys was digested with BamHI (B), EcoRI (E), HindIII (H), PstI (P), XbaI (X), and analyzed with probes corresponding to the common extracellular region (left panel) or to the PIR-B specific cytoplasmic region (right panel).

 
As shown in Fig. 2Go, comparison of the extracellular amino acid sequences of raPIR-B, moPIR-B (1), huILT-1 (12), huMIR-10 (14), and huLIR-1 (13) indicates that the signal sequences of raPIR-B and moPIR-B are highly conserved, whereas the huILT/LIR/MIR signal sequences are relatively distinct. Alignment of the amino-terminal (D1) through the most membrane proximal (D6) Ig-like domains of raPIR-B indicates high similarity for all corresponding moPIR-B domains. Notably, domains 1 and 2 of raPIR-B and moPIR-B are not shared with the huILT/LIR/MIRs, which possess only four domains. The longest single stretch of amino acid identity between rat, mouse, and human sequences is found within raPIR-B D5, where an 18-aa stretch surrounding the second cysteine of raPIR-B D5 contains only two substitutions and a conserved N-linked glycosylation site. These findings indicate that the extracellular domains of rat and mouse PIRs are highly conserved, possess six Ig-like domains, and are distinct from the four Ig-like domain structure of their human counterparts.



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  		FIGURE 2. Multiple alignment of rat PIR-B (AF16936), mouse PIR-B (AF038149), human ILT-1 (U82275), MIR-10 (AF004231), and LIR-1 (AF009220) extracellular regions. Amino acids are numbered with reference to the start of the signal sequence for each sequence, and one Ig-like domain is aligned per block of amino acid sequence. The top line represents the extracellular portion of raPIR-B; conserved residues are represented as dots (.) and gaps are indicated by dashes (-). Cysteine residues likely to be involved in intradomain disulfide bond formation are highlighted in bold, and potential N-linked glycosylation sites are underlined.

 
The raPIR-B cytoplasmic region contains five tyrosine residues (Fig. 3GoA), four of which are embedded in the amino acid sequences SLYASV, EKYAQV, VTYAQL, and SVYAAL (single letter amino acid code) that resemble the consensus for ITIM motifs (I/L/V/S-x-Y-xx-L/V) (22, 23). Amino acid sequence alignments of the cytoplasmic tails of raPIR-B, moPIR-B (1), mogp49-B (24), and human inhibitory LRC members (25, 26, 27) illustrate perfect conservation of the third raPIR-B ITIM. This particular ITIM unit, which contributes to the inhibitory function of moPIR-B via the binding of SHP-1 (4, 5, 6), may therefore contribute to an inhibitory function predicted for raPIR-B.



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  		FIGURE 3. Multiple alignment of transmembrane and cytoplasmic amino acid sequences for activating and inhibitory relatives of rat PIR-A and PIR-B. A, Alignment of rat PIR-B (AF16936), mouse PIR-B (AF038149), mouse gp49-B (2997305), human ILT-2 (U82279), KIR (AF022049), and LAIR-1 (AF013249). Amino acids are numbered with reference to the start of each predicted transmembrane domain, which is underlined. The four putative ITIM units are highlighted in bold, and the tyrosines are indicated with asterisks. B, Alignment of the transmembrane and cytoplasmic amino acid sequences of rat PIR-A (AF169637), rat KILR-1 (AF082533), mouse PIR-A (U96682), mouse MAR-1 (AJ223765), human ILT-1 (U82275), human NKp46 (AJ001383), human Fc{alpha}R (U4774), and bovine Fc{gamma}2R (2136749). Amino acids are numbered starting with the conserved aspartic acid, and the predicted transmembrane domain is underlined. The proposed transmembrane motif is highlighted in bold, and the conserved arginine is highlighted by an asterisk. Conserved residues are represented as dots (.) and gaps are indicated by dashes (-).

 
The raPIR-A transmembrane domain (Fig. 3GoB) shares the presence of a charged arginine residue with moPIR-A (1), rat killer cell Ig-like receptor (KILR)-1 (AF082533), mouse activation receptor (MAR)-1 (28), bovine Fc receptor for IgG2 (boFc{gamma}2R) (29), huILT-1 (12), human Fc receptor for IgA (Fc{alpha}R) (30), and the human NK cell activation receptor (NKp46) (31). Amino acid sequence comparison of transmembrane segments indicates a conserved motif of six amino acids (F/N-L-I/L-R-M/I-G/A) surrounding the arginine residue of these activating receptor relatives. The positively charged arginine residue in the transmembrane segments of the moPIR-A and Fc{alpha}R {alpha}-chain molecules promotes their association with the negatively charged transmembrane segment of the ITAM-containing FcR{gamma} chain (10, 11, 32, 33), thereby suggesting that raPIR-A may also associate with an ITAM-containing element.

Tissue distribution of raPIR expression

RNA blot analysis of various rat tissues resulted in the hybridization of an EC probe with ~3.0-kb transcripts in spleen and bone marrow cells (Fig. 4GoA). Hybridizing transcripts were less abundant in the thymus, ovary, and lung, whereas raPIR transcripts were not seen in other tissues. When the expression pattern was examined by RT-PCR (Fig. 4GoB), raPIR-A transcripts were detected in B, myeloid, and macrophage cell lines, whereas raPIR-B transcripts were detected only in myeloid and macrophage cell lines. When normal B cells (CD45R+/CD3-), NK cells (NKR-P1A+/CD3-), and granulocytes (HIS48+) were isolated and examined, raPIR-A transcripts were seen in B cells and granulocytes, whereas raPIR-B transcripts were found in NK cells and granulocytes. Thus, while raPIR-A and raPIR-B are coordinately transcribed in myeloid lineage cells, raPIR-A and raPIR-B transcripts are differentially expressed in B cells vs NK cells.



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  		FIGURE 4. Tissue distribution of raPIR. A, (top panel) RNA blot probed with a PCR-generated probe encoding the insert of cDNA clone R16; (bottom panel) relative loading control using ß-actin as a probe. The migration of the 28S (4.7 kb) and 18S (1.9 kb) ribosomal bands is indicated on the right side. B, RT-PCR analysis of cell lines (B cell, T cell, myeloid, and macrophage) and normal CD45R+ (B220) B cells, NKR-P1A+ NK cells, and HIS48+ granulocytes. Full-length raPIR-A and raPIR-B plasmids were employed as positive control templates to demonstrate primer pair specificity. Amplification products were electrophoresed, stained with ethidium bromide, and visualized with the Bio-Rad Fluora-S gel documentation system.

 
Phylogenetic analysis of raPIR

When the EC domains of raPIR-B, moPIR-B (1), raKILR-1 (AF082533), mogp49-B (2), moMAR-1 (28), and human LRC members were compared using the PHYLIP and CLUSTAL software packages with amino acid sequences, the implied relationships in the derivative trees largely conform to their percentages of identity (Fig. 5Go). Results from this phylogenetic analysis suggest that raPIR, moPIR, and mogp49 form a group that is closely related to the huILT/LIR/MIR family.



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  		FIGURE 5. Predicted extracellular regions, percentage identity, and dendogram of implied divergence for raPIR and related proteins. Amino acid sequences were analyzed for raPIR-B, raKILR-1, moPIR-B, mogp49-B, huILT-1, huMIR-10, huLIR-1, huKIR, huLAIR-1, huNKp46, huFc{alpha}R, boFc{gamma}2R, and bovine signal regulatory protein {alpha} (boSIRP{alpha}) (EMBL accession number Y11045). boSIRP{alpha} was included as an outlier to root the tree, and the numerical scale indicates relative substitution number. The dendogram was produced by the PAM100 distance matrix and Clustal programs.

 
These results also indicate that huILT/LIR/MIRs are more like their rat and mouse counterparts than other human LRC members. The six-domain EC regions of rat and mouse PIRs, however, are distinct from the four-domain EC structure of most huILT/LIR/MIR members. This raises the question as to which EC domains of rat and mouse PIRs are shared with huILT/LIR/MIRs. Pairwise amino acid alignments for each Ig-like domain of raPIR-B, moPIR-B, and huLIR-1 (Fig. 6GoA) indicate that the domain organization of rat and mouse (murine) PIRs do not correspond directly (implied by highest percentage similarity) with the huLIR-1 domain organization. For instance, while the membrane proximal domains of murine PIRs (D5 and D6) correspond to the membrane proximal human domains (D3 and D4), the membrane distal murine domain (D1) is more similar to murine D3 than to the membrane distal human domain (D1). Likewise, murine D4 is more similar to murine D2 than to any human domain. As depicted in Fig. 6GoB, this pairwise analysis suggests that the membrane proximal domains (D5 and D6) of murine PIRs correspond to the membrane proximal domains (D3 and D4) of huLIR-1, whereas the membrane distal murine domains (D1 through D4) may have undergone expansion and diversification relative to the membrane distal domains (D1 and D2) of their human counterparts.



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  		FIGURE 6. Pairwise comparisons between individual Ig-like domains of raPIR-B, moPIR-B, and huLIR-1. A, Table of percentage similarities among all domains analyzed. The highest pairwise homology is indicated by black boxes. Outlined boxes highlight rat and mouse domains that do not have obvious human correspondents based on their percentages of similarity. B, Model of the raPIR-B and huLIR-1 EC regions depicting the domains between raPIR-B and huLIR-1 with the highest percentages of similarity. Solid arrows indicate corresponding domains between raPIR-B and huLIR-1. Dashed arrows indicate the raPIR-B domains that have highest percentages of similarity to other raPIR-B domains (outlined boxes in A). EC domains are numbered from amino-terminal (D1) to membrane proximal (D6) for rat and mouse and D1-D4 for human. Percentage similarities were obtained using the CLUSTAL program.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The rat homologues of mouse PIR-A and PIR-B have been identified in the present study as type I transmembrane proteins that have six highly similar extracellular Ig-like domains and distinctive transmembrane and cytoplasmic regions. The name paired Ig-like receptor was coined in part to designate the coordinate expression of PIR-A and PIR-B transcripts by mouse B cells and myeloid cells (1). Despite the remarkable conservation of structural features for the rat and mouse PIR family members, rat PIR-A and PIR-B do not maintain the hallmark paired expression of activating and inhibitory receptors. Instead, differential, or unpaired, expression of raPIRA and raPIR-B was observed for B and NK cells, respectively. The expression of raPIR-B by NK cells more closely resembles the picture in humans, where NK cells may selectively express inhibitory members of the huILT/LIR/MIR multigene family (13). Unpaired expression of raPIR-A by B cells, however, is more unique. The expression pattern in rats thereby resembles that in humans where activating and inhibitory members of the PIR family are coordinately expressed in myeloid cells, yet may be differentially regulated in lymphoid and dendritic cell lineages. The rat may thus prove to be a useful model for the dissection of opposing signaling pathways employed by these activating and inhibitory receptors.

Inhibitory receptors represent a diverse group of recently recognized cell surface receptors that can modulate cellular activation via the interaction of their ITIM-containing cytoplasmic tails with the src-homology phosphatases SHP-1, SHP-2, and SHIP-1 (reviewed in Ref. 3). The predicted cytoplasmic tail of raPIR-B contains four potential ITIM motifs that are conserved in moPIR-B and the huILT/LIR/MIR inhibitory receptors. The SHP-1-binding ITIM unit, VTYAQL, is identical in rats, mice, and humans, thereby suggesting that raPIR-B will also bind SHP-1 and serve as an inhibitory receptor.

Many inhibitory receptors, including PIR-B, have a high level of EC domain homology with proteins that have distinct transmembrane and cytoplasmic regions. These noninhibitory counterparts commonly have an arginine residue in their transmembrane segments and have short cytoplasmic tails without recognizable signaling motifs. RaPIR-A is similar to raPIR-B in its EC region, but has a transmembrane segment endowed with an arginine residue. The presence of an arginine in the transmembrane segments of moPIR-As promotes their association with ITAM-containing FcR{gamma} chain dimers to form activating signaling complexes (7, 10, 11). Thus, the raPIR-As, like their mouse counterparts, are likely to form activating receptor complexes in conjunction with ITAM-containing coreceptors.

Human chromosome 19q13.4 encodes a large cluster of Ig-superfamily receptors, the majority of which have not been found to be conserved in mice (12, 13, 14, 15, 26, 27, 31, 34). Recent cloning of NKp46 in humans, rats, and mice represents the first example of an LRC member that is conserved in rodents. The huNKp46 homologues KILR-1 (raNKp46) and MAR-1 (moNKp46) have been mapped respectively to syntenic regions of rat chromosome 1 and mouse chromosome 7. Recently, a rat ITIM-bearing receptor, neutrophil Ig-like receptor (NILR)-1, has also been mapped to rat chromosome 1 within 1.4 cM of raNKp46, thereby indicating the existence of a leukocyte receptor complex on rat chromosome 1 (35). The mouse Pira and Pirb genes are located in the same region of mouse chromosome 7 as moNKp46, (1, 28), thereby predicting that raPIR should map within the leukocyte receptor complex of rat chromosome 1. Monoallelic expression of paternal alleles encoding the Pir genes was demonstrated recently in mice (36), extending the number of genes that are genomically imprinted in this region of mouse chromosome 7 (37). It is noteworthy that huILT/LIR/MIR are also in close proximity to paternally imprinted genes on chromosome 19q13.4 (38), raising the interesting possibility that genomic imprinting may be a conserved feature of these syntenic multigene families.

The identification of PIR homologues in rats clearly suggests a shared evolutionary history for the raPIR, moPIR, and huILT/LIR/MIR families. However, several distinctive features complicate attempts to understand their phylogenetic relationships. Individual domain comparisons for the rat, mouse, and human family members indicate that the murine PIRs are highly conserved and, unlike the huILT/LIR/MIRs, share EC regions that possess two extra Ig-like domains at the N-terminal portion, possibly resulting from exon duplication/recombination events. On the other hand, unpaired expression of PIR-A and PIR-B transcripts in rat B and NK cells more closely resembles the expression pattern of huILT/LIR/MIR. Hence, raPIRs combine characteristics of both their mouse and human relatives, suggesting that PIR and ILT/LIR/MIR share a common ancestor that has maintained a high degree of genetic plasticity during mammalian evolution. Discovery of PIR and ILT/LIR/MIR homologues in additional species should shed further light on the evolutionary history of this diverse group of cell surface receptors.


   Acknowledgments
 
We thank Ann Brookshire for helping in preparing the manuscript, and Pete Burrows, Chen-lo Chen, Vincent Hurez, Stephanie Blandin, and Gregory Ippolito for helpful advice and criticism.


   Footnotes
 
1 This work was supported in part by National Institutes of Health Grants AI42127 and AI39816. M.D.C. is a Howard Hughes Medical Institute investigator. Back

2 The cDNA sequences reported in this manuscript have been submitted to GenBank under accession numbers AF16936 and AF169637. Back

3 Address correspondence and reprint requests to Dr. Max D. Cooper, Division of Developmental and Clinical Immunology, 378 WTI, University of Alabama, Birmingham, AL 35294. Back

4 Abbreviations used in this paper: PIR, paired Ig-like receptor; bo, bovine; D, domain; EC, extracellular Ig-like domains; Fc{alpha}R, Fc receptor for IgA; Fc{gamma}2R, Fc receptor for IgG2; FcR{gamma}, Fc receptor common {gamma} chain; gp49, glycoprotein 49; hu, human; ILT, Ig-like transcript; ITAM, immunoregulatory tyrosine-based activation motif; ITIM, immunoregulatory tyrosine-based inhibitory motif; KILR-1, killer cell Ig-like receptor-1; KIR, killer cell-inhibitory receptor; LAIR-1, leukocyte-associated Ig-like receptor-1; LIR, leukocyte Ig-like receptor; LRC, leukocyte receptor complex; MAR-1, mouse activation receptor-1; MIR, monocyte/macrophage Ig-like receptor; mo, mouse; NKp46, NK cell protein 46; ra, rat; SHP-1, src-homology-2 phosphatase-1; RACE, rapid amplification of cDNA ends. Back

Received for publication August 6, 1999. Accepted for publication September 22, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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