Pattern recognition receptors are crucial in initiating and shaping innate and adaptive immune responses and often belong to families of structurally and evolutionarily related proteins. The human C-type lectin-like receptors encoded in the DECTIN-1 cluster within the NK gene complex contain prominent receptors with pattern recognition function, such as DECTIN-1 and LOX-1. All members of this cluster share significant homology and are considered to have arisen from subsequent gene duplications. Recent developments in sequencing and the availability of comprehensive sequence data comprising many species showed that the receptors of the DECTIN-1 cluster are not only homologous to each other but also highly conserved between species. Even in Caenorhabditis elegans, genes displaying homology to the mammalian C-type lectin-like receptors have been detected. In this paper, we conduct a comprehensive phylogenetic survey and give an up-to-date overview of the currently available data on the evolutionary emergence of the DECTIN-1 cluster genes.

Some leukocyte receptors come in groups of two or more where the partners share ligand(s) but transmit opposite signals. Some of the ligands, such as MHC class I, are fast evolving, raising the problem of how paired opposing receptors manage to change in step with respect to ligand binding properties and at the same time conserve opposite signaling functions. An example is the KLRC (NKG2) family, where opposing variants have been conserved in both rodents and primates. Phylogenetic analyses of the KLRC receptors within and between the two orders show that the opposing partners have been subject to post-speciation gene homogenization restricted mainly to the parts of the genes that encode the ligand binding domains. Concerted evolution similarly restricted is demonstrated also for the KLRI, KLRB (NKR-P1), KLRA (Ly49), and PIR receptor families. We propose the term merohomogenization for this phenomenon and discuss its significance for the evolution of immune receptors.

Ly49 receptors in rodents, like killer cell immunoglobulin-like receptors in humans, regulate natural killer (NK) cell activity. Although inhibitory Ly49 receptors clearly recognize classical major histocompatibility complex class I (MHC-I) molecules, the role for the activating Ly49 receptors has been less well understood. Here, we discuss recent data from a rat model for listeriosis. Rats depleted of NK cells, or more specifically the Ly49 receptor-bearing cells, showed increased bacterial loads in their spleen. Athymic nude rats with no functional T cells but increased numbers of Ly49-expressing NK cells were more resistant to infection, indicating a central role of NK cells in early immune defense against Listeria in this species. Listeria infection of macrophages or enteric epithelial cells led to upregulation of MHC-I, including nonclassical (Ib) molecules not regularly recognized by T cells. We have shown that activating Ly49 receptors are more efficiently stimulated when binding to upregulated class Ib antigens on infected cells. From this we postulate that activating Ly49 receptors may have a sentinel function in the early immune response against Listeria in detecting diseased cells 'flagged' by increased MHC-Ib expression.

The natural killer gene complex (NKC) encodes several dozens of C-type lectin-like receptors that, in various ways, tune the reactivity of NK cells and other cytotoxic lymphocytes depending on the cellular environment. Among these are C-type lectin-like receptors such as NKG2D, CD94/NKG2A and the murine Ly49 receptors that bind to cell surface glycoproteins of the major histocompatibility complex (MHC) class I family and thereby facilitate detection of stressed cells or cells exhibiting aberrant MHC class I expression. In contrast, NKRP1 receptors including the prototypic NK1.1 do not engage ligands with an MHC class-I-like fold, but rather interact with the likewise C-type lectin-like CLEC2 glycoproteins. Notably, CLEC2 and NKRP1 molecules not only share the same fold, but are also genetically linked in the NKC. Recent research efforts began to systematically elucidate the expression and function of the numerous NKRP1 and CLEC2 family members in rodents and revealed previously unnoticed corresponding receptor/ligand pairs in humans. Here, we provide a snapshot of the current knowledge on receptors of the NKRP1 family and their genetically linked CLEC2 ligands in mouse and man.

Two clusters of rat Nkrp1 genes can be distinguished based on phylogenetic relationships and functional characteristics. The proximal (centromeric) cluster encodes the well-studied NKR-P1A and NKR-P1B receptors and the distal cluster, the largely uncharacterized, NKR-P1F and NKR-P1G receptors. The inhibitory NKR-P1G receptor is expressed only by the Ly49s3(+) NK cell subset as detected by RT-PCR, while the activating NKR-P1F receptor is detected in both Ly49s3(+) and NKR-P1B(+) NK cells. The mouse NKR-P1G ortholog is expressed by both NKR-P1D(-) and NKR-P1D(+) NK cells in C57BL/6 mice. The rat and mouse NKR-P1F and NKR-P1G receptors demonstrate a striking, cross-species conservation of specificity for Clr ligands. NKR-P1F and NKR-P1G reporter cells reacted with overlapping panels of tumour cell lines and with cells transiently transfected with rat Clr2, Clr3, Clr4, Clr6 and Clr7 and mouse Clrc, Clrf, Clrg and Clrd/x, but not with Clr11 or Clrb, which serve as ligands for NKR-P1 from the proximal cluster. These data suggest that the conserved NKR-P1F and NKR-P1G receptors function as promiscuous receptors for a rapidly evolving family of Clr ligands in rodent NK cells.

We here report the molecular cloning of a novel family of killer-cell lectin-like (KLR) receptors in the rat and the mouse, termed KLRI. In both species, there are two members, KLRI1 and KLRI2. While the extracellular lectin-like domains of KLRI1 and KLRI2 are similar [74%(rat) and 83%(mouse) amino acid identity], they differ intracellularly. KLRI1 has two immunoreceptor tyrosine-based inhibition motifs (ITIMs) in the cytoplasmic domain, suggesting an inhibitory function. KLRI2 has no ITIM, but a positively charged lysine residue in the transmembrane region, suggesting association with activating adapter molecules. Klri1 and Klri2 are localized within the natural killer (NK) cell gene complex on rat chromosome 4 and mouse chromosome 6. By RT-PCR and Northern blot analysis KLRI1 and KLRI2 were selectively expressed by NK cells in both rat and mouse. Epitope-tagged expression constructs of rat KLRI1 and rat KLRI2 induced surface expression of a nondisulphide-linked protein of M(r) 36,000/39,000 and M(r) 34,000, respectively.

Signaling by the CD94/NKG2 heterodimeric NK cell receptor family has been well characterized in the human but has remained unclear in the mouse and rat. In the human, the activating receptor CD94/NKG2C associates with DAP12 by an ionic bond between oppositely charged residues within the transmembrane regions of NKG2C and DAP12. The lysine residue responsible for DAP12 association is absent in rat and mouse NKG2C and -E, raising questions about signaling mechanisms in these species. As a possible substitute, rat and mouse NKG2C and -E contain an arginine residue in the transition between the transmembrane and stalk regions. In this article, we demonstrate that, similar to their human orthologs, NKG2A inhibits, whereas NKG2C activates, rat NK cells. Redirected lysis assays using NK cells transfected with a mutated NKG2C construct indicated that the activating function of CD94/NKG2C did not depend on the transmembrane/stalk region arginine residue. Flow cytometry and biochemical analysis demonstrated that both DAP12 and DAP10 can associate with rat CD94/NKG2C. Surprisingly, DAP12 and DAP10 did not associate with NKG2C but instead with CD94. These associations depended on a transmembrane lysine residue in CD94 that is unique to rodents. Thus, in the mouse and rat, the ability to bind activating adaptor proteins has been transferred from NKG2C/E to the CD94 chain as a result of mutation events in both chains. Remarkable from a phylogenetic perspective, this sheds new light on the evolution and function of the CD94/NKG2 receptor family.

Natural killer (NK) cells discriminate between normal syngeneic cells and infected, neoplastic or MHC-disparate allogeneic cells. The reactivity of NK cells appears to be regulated by a balance between activating receptors that recognize non-self or altered self, and inhibitory receptors recognizing normal, self-encoded MHC class I molecules. Subfamilies of NK receptors undergo rapid evolution, and appear to co-evolve with the MHC. We here review present views on the evolution and function of NK cell receptors, with an emphasis on knowledge gained in cattle and rodents.

NK cells identify infected, neoplastic, or MHC-disparate target cells via several different receptors. The NK cell receptor KLRE1 lacks known signaling motifs but has nevertheless been shown to regulate NK cell-mediated cytotoxicity. Here we demonstrate that KLRE1 forms functional heterodimers with either KLRI1 or KLRI2. Cotransfection with KLRE1 was necessary for surface expression of the NK cell receptor chains KLRI1 and KLRI2 in 293T cells. Moreover, KLRE1 can be coimmunoprecipitated with KLRI1 or KLRI2 from transfected NK cell lines. By flow cytometry, KLRE1 and KLRI1 showed colinear expression on NK cells, suggesting surface expression as heterodimers. Unlike other killer cell lectin-like receptors, KLRE1/KLRI1 and KLRE1/KLRI2 heterodimers predominantly migrated as single chains in SDS-PAGE, indicating noncovalent association. KLRI1 was coimmunoprecipitated with the tyrosine phosphatase Src homology region 2 domain-containing phosphatase 1. In accordance with an inhibitory function, anti-HA Ab induced reduced killing of FcR-bearing targets by KLRI1-HA-transfected NK cell lines in a redirected cytotoxicity assay. Reciprocally, KLRI2-HA transfectants displayed increased killing in this assay. Finally, Ab to KLRE1 induced inhibition in KLRI1-transfected cells but increased cytotoxicity in KLRI2 transfectants, demonstrating that KLRE/I1 is a functional inhibitory heterodimer in NK cells, whereas KLRE/I2 is an activating heterodimeric receptor.

We report the molecular cloning of a KIR3DL1 receptor in the mouse and the rat, between 37.4 and 45.4% identical with primate killer cell Ig-like receptors (KIRs/CD158). Both mouse and rat molecules contain a pair of immunoreceptor tyrosine-based inhibition motifs in their cytoplasmic regions, suggesting an inhibitory function. Southern blot analysis indicated a single KIR gene in the rat, whereas the mouse genome contains more than one KIR-related element. The rat Kir3dl1 locus was mapped to the leukocyte receptor gene complex on chromosome 1, whereas mouse Kir3dl1 was localized to the X chromosome. RT-PCR demonstrated that KIR3DL1 was selectively expressed by NK cells in both rat and mouse. An epitope-tagged expression construct of mouse KIR3DL1 transfected into 293T cells induced expression of a approximately 55-kDa protein. Our data indicate that KIR receptors may contribute to the NK cell receptor repertoire in rodents, alongside the Ly-49 family.

Some leukocyte receptors come in groups of two or more where the partners share ligand(s) but transmit opposite signals. Some of the ligands, such as MHC class I, are fast evolving, raising the problem of how paired opposing receptors manage to change in step with respect to ligand binding properties and at the same time conserve opposite signaling functions. An example is the KLRC (NKG2) family, where opposing variants have been conserved in both rodents and primates. Phylogenetic analyses of the KLRC receptors within and between the two orders show that the opposing partners have been subject to post-speciation gene homogenization restricted mainly to the parts of the genes that encode the ligand binding domains. Concerted evolution similarly restricted is demonstrated also for the KLRI, KLRB (NKR-P1), KLRA (Ly49), and PIR receptor families. We propose the term merohomogenization for this phenomenon and discuss its significance for the evolution of immune receptors.

A major subset of non-alloreactive NK cells in PVG strain rats is generally low in Ly49 receptors, but expresses the rat NKR-P1B(PVG) receptor (previously termed NKR-P1C). The NKR-P1B(+) NK subset is inhibited by a non-polymorphic target cell ligand, which we have shown here to be a C-type lectin-related molecule (Clr). Clr11 ligates two divergent NKR-P1B alleles as judged by an NFAT-driven reporter assay, and inhibits NK-cell cytotoxicity of NKR-P1B(+) NK cells. Clr11 also interacts with the prototypic NKR-P1A receptor and exerts a stimulatory influence on NK lysis. NKR-P1A and B are encoded by adjacent genes in the proximal part of the NK gene complex and show close sequence homology in their extracellular region. They diverge from another pair, NKR-P1F and -G, which is encoded by a second, distal Nkrp1 gene cluster. NKR-P1F and -G bind an overlapping panel of Clr ligands, but not Clr11. Rat Clr molecules appear to be constitutively expressed by hematopoietic cells; expression in tumor cell lines is more variable. The data show the existence of two phylogenetic groups of NKR-P1 molecules, which demonstrate conservation of ligand-binding properties independent of signaling function.

We report the molecular cloning of two novel single-member receptor families with homology to LILR/CD85, PIR, and gp49: LILRC1 in the rat and the mouse, and LILRC2 in the rat. LILRC1 and LILRC2 both have two extracellular Ig-like domains and a cytoplasmic tail devoid of any known signaling motifs. The transmembrane regions of LILRC1 and LILRC2 contain an arginine residue, a common feature in receptors that associate with activating adaptor proteins. Rat and mouse LILRC1 are orthologs sharing 81.5% amino acid identity. LILRC2 represents a distinct receptor family, 47.9% identical to LILRC1. No murine LILRC2 ortholog was detected in genome or expressed sequence tag sequence databases. By radiation hybrid mapping, the rat Lilrc1 and Lilrc2 loci were localized to the leukocyte receptor gene complex (LRC) on chromosome 1, and the mouse Lilrc1 locus was mapped to the LRC on chromosome 7. Moreover, the mouse and rat Lilrc1 loci were localized to similar positions within the LRC. As shown by RT-PCR, rat LILRC1 was expressed by B cells, neutrophils, and a macrophage cell line. Transcription of LILRC2 was detected in T cells, B cells, neutrophils, and macrophages.

Mouse gp49B is a member of the leukocyte immunoglobulin-like receptor family. It is constitutively expressed by mast cells and certain myeloid cells, and expression can be induced on natural killer (NK) cells and T cells. We have cloned several rat cDNA, 78% identical to mouse gp49B at the amino acid level, that represent the rat orthologue to mouse gp49B. A mouse monoclonal antibody (WEN29) against rat gp49B was generated. By flow cytometry and Northern blot analysis, gp49B was found to be expressed by neutrophils and monocytes, but not NK cells (primary or IL-2-activated), T cells (resting or concanavalin A-stimulated) or peritoneal mast cells. Following pervanadate treatment, the tyrosine phosphatase SHP-1 was co-immunoprecipitated with gp49B in the macrophage cell line R2. In glutathione S-transferase pull-down experiments, the cytoplasmic tail of rat gp49B associated with the SH2 domains of both SHP-1 and SHP-2, dependent on intact and phosphorylated immunoreceptor tyrosine-based inhibition motifs (ITIM). Compared to mouse, the cytoplasmic domain of rat gp49B contains a third ITIM-like sequence (YLYASV) that was phosphorylated by several Src family tyrosine kinases, enhanced the phosphorylation of other ITIM, and bound to the SH2 domains of SHP-2, suggesting a role in the recruitment of downstream phosphatases.

We here report the cDNA sequences of 11 new rat Ly49 genes with full and three with incomplete open reading frames. Although obtained from different inbred rat strains, these as well as six previously published cDNA represent non-allelic genes matching different loci in the Brown Norway (BN) rat genome, which is predicted to contain 34 Ly49 loci distributed over the distal part of the NK cell gene complex. Some of the cloned genes appear to be mutated to non-function in the BN genome, which harbors additional genes with full open reading frames, suggesting at least 26 non-allelic functional Ly49 genes in the rat. Of the encoded receptors, 13 are predicted to be inhibitory, eight to be activating, whereas five may be both ('bifunctional'). Phylogenetic analysis bears evidence of a highly dynamic genetic region, in which only the most distally localized Ly49 gene has a clear-cut mouse ortholog. In phylograms, the majority of the genes cluster into three subgroups with the genes mapping together, defining three chromosomal regions that seem to have undergone recent expansions. When comparing the lectin-like domains, the receptors form smaller subgroups, most containing at least one inhibitory and one activating or 'bifunctional' receptor, where close sequence similarities suggest recent homogenization events.

Within the mammalian immune system, natural killer (NK) cells contribute to the first line of defence against infectious agents and tumours. Their activity is regulated, in part, by cell surface NK cell receptors. NK receptors can be divided into two unrelated, but functionally analogous superfamilies based on the structure of their extracellular ligand-binding domains. Receptors belonging to the C-type lectin superfamily are predominantly encoded in the natural killer complex (NKC), while receptors belonging to the immunoglobulin superfamily are predominantly encoded in the leukocyte receptor complex (LRC). Natural killer cell receptors are emerging as a rapidly evolving gene family which can display significant intra- and interspecific variation. To date, most studies have focused on eutherian mammals, with significantly less known about the evolution of these receptors in marsupials. Here, we describe the identification of 43 immunoglobulin domain-containing LRC genes in the genome of the Tasmanian devil (Sarcophilus harrisii), the largest remaining marsupial carnivore and only the second marsupial species to be studied. We also identify orthologs of NKC genes KLRK1, CD69, CLEC4E, CLEC1B, CLEC1A and an ortholog of an opossum NKC receptor. Characterisation of these regions in a second, distantly related marsupial provides new insights into the dynamic evolutionary histories of these receptors in mammals. Understanding the functional role of these genes is also important for the development of therapeutic agents against Devil Facial Tumour Disease, a contagious cancer that threatens the Tasmanian devil with extinction.

Receptors on natural killer (NK) cells are classified as C-type lectins or as Ig-like molecules, and many of them are encoded by two genomic clusters designated natural killer gene complex (NKC) and leukocyte receptor complex, respectively. Here, we describe the analysis of an NKC-encoded chicken C-type lectin, previously annotated as homologue to CD94 and NKG2 and thus designated chicken CD94/NKG2. To further elucidate its potential function on NK cells, we produced a specific mab by immunizing with stably transfected HEK293 cells expressing this lectin. Staining of various chicken tissues revealed minimal reactivity with bursal, or thymus cells. In peripheral blood mononuclear cell and spleen, however, the mab reacted with virtually all thrombocytes, whereas most NK cells in organs such as embryonic spleen, lung and intestine were found to be negative. These findings indicate that the gene may not resemble CD94/NKG2, but rather a CLEC-2 homologue, a claim further supported by sequence features such as an additional extracellular cysteine residue and the presence of a cytoplasmic motif known as a hem immunoreceptor tyrosine-based activation motif, found in C-type lectins such as Dectin-1, CLEC-2, but not CD94/NKG2. The biochemical analyses demonstrated that CLEC-2 is present on the cell surface as heavily glycosylated homodimer, which upon mab crosslinking induced thrombocyte activation, as measured by CD107 expression. These analyses reveal that the chicken NKC may not encode NK cell receptor genes, in particular not CD94 or NKG2 genes, and identifies a chicken CLEC-2 homologue.

The myeloid cluster within the natural killer (NK) gene complex comprises several C-type lectin-like receptor genes of diverse and highly important functions in the immune system such as LOX-1 and DECTIN-1. Based on sequences that have become available by whole genome sequencing, we conducted a comparison of the human, chimpanzee, mouse and rat NK gene complex to better characterize this gene family and additional genes of this region in regard of their phylogenetic relationship and evolution within the complex. We found that the arrangement of genes within the primate cluster differs from the order and orientation of the corresponding genes in the rodent complex which can be explained by evolutionary duplication and inversion events. Analysis of individual genes revealed a high sequence conservation supporting the prime importance of the encoded proteins. Expression analyses of the more recently described CLEC12B and CLEC9A genes displayed not only mRNA expression in monocytic and dendritic cells, but in contrast to other members of the family also in lymphocytes. Further, two additional genes were identified, which do not encode proteins with lectin-like domain structure and seem to be widely expressed.

Natural killer (NK) cell receptors belong to two unrelated, but functionally analogous gene families: the immunoglobulin superfamily, situated in the leukocyte receptor complex (LRC) and the C-type lectin superfamily, located in the natural killer complex (NKC). Here, we describe the largest NK receptor gene expansion seen to date. We identified 213 putative C-type lectin NK receptor homologs in the genome of the platypus. Many have arisen as the result of a lineage-specific expansion. Orthologs of OLR1, CD69, KLRE, CLEC12B, and CLEC16p genes were also identified. The NKC is split into at least two regions of the genome: 34 genes map to chromosome 7, two map to a small autosome, and the remainder are unanchored in the current genome assembly. No NK receptor genes from the LRC were identified. The massive C-type lectin expansion and lack of Ig-domain-containing NK receptors represents the most extreme polarization of NK receptors found to date. We have used this new data from platypus to trace the possible evolutionary history of the NK receptor clusters.

The human natural killer gene complex (NKC) encodes for numerous C-type lectin-like receptors (CTLR), which are expressed on various immune cells including natural killer (NK) cells and myeloid cells. Certain activation-induced, NKC-encoded CTLR are grouped into the C-type lectin domain family 2 (CLEC2 family) which, in humans, comprises AICL (CLEC2B), CD69 (CLEC2C), and LLT1 (CLEC2D). In this paper, we characterize a novel member of the CLEC2 family, the human orphan gene CLEC2A. The C-type lectin-like domain (CTLD) of CLEC2A is most similar to the CTLD of LLT1 (~60% similarity). Like mouse CLEC2 family members Clr-b and Clr-g, CLEC2A lacks two highly conserved cysteines (Cys4 and Cys5), which form an intramolecular bond in the CTLD of most CTLR. Alternative splicing of exon 2 and of two distinct terminal exons (exon 5A/B), respectively, gives rise to four CLEC2A variants differing in the usage of the transmembrane domain and/or in the carboxyterminal portion of the CTLD. CLEC2A transcripts were detected primarily in myeloid cell lines, but not in epithelial cell lines. In tissues, CLEC2A is selectively expressed in the skin and, at lower abundance, in hematopoietic and gonadal tissues. Finally, we show that the CLEC2A1 variant is readily expressed at the cell surface, where it may serve as a ligand for NKC-encoded NK receptors.

In mammals, natural killer (NK) cell C-type lectin receptors were encoded in a gene cluster called natural killer gene complex (NKC). The NKC is not reported in chicken yet. Instead, NK receptor genes were found in the major histocompatibility complex. In this study, two novel chicken C-type lectin-like receptor genes were identified in a region on chromosome 1 that is syntenic to mammalian NKC region. The chromosomal locations were validated with fluorescent in situ hybridization. Based on 3D structure modeling, sequence homology, chromosomal location, and phylogenetic analysis, one receptor is the orthologue of mammalian cluster of differentiation 69 (CD69), and the other is highly homologous to CD94 and NKG2. Like CD94/NKG2 gene found in teleostean fishes, chicken CD94/NKG2 has the features of both human CD94 and NKG2A. Unlike mammalian NKC, these two chicken C-type lectin receptors are not closely linked but separated by 42 million base pairs according to the chicken draft genome sequence. The arrangement of several other genes that are located outside the mammalian NKC is conserved among chicken, human, and mouse. The chicken NK C-type lectin-like receptors in the NKC syntenic region indicate that this chromosomal region existed before the divergence between mammals and aves.

The functions of natural killer (NK) cells are clearly regulated by major histocompatibility complex (MHC) class I molecules on their cellular targets. In mice, this is due to the action of MHC-specific inhibitory receptors belonging to the Ly49 family oflectin-like molecules. The Ly49 receptors are encoded in the NK gene complex (NKC) that contains clusters of genes for other lectin-like receptors on NK cells and other hematopoietic cells. Interestingly, recent studies have shown that some of these lectin-like receptors, belonging to the Nkrpl family, can recognize other lectin-like molecules, termed Clr, also encoded in the NKC. These genetically linked loci for receptor-ligand pairs suggest a genetic strategy to preserve this interaction and show several other contrasts with Ly49-MHC interactions. In this review, we discuss these issues and summarize recent developments concerning this non-MHC-dependent regulation of NK cell function.

Many receptors on natural killer (NK) cells recognize major histocompatibility complex class I molecules in order to monitor unhealthy tissues, such as cells infected with viruses, and some tumors. Genes encoding families of NK receptors and related sequences are organized into two main clusters in humans: the natural killer complex on Chromosome 12p13.1, which encodes C-type lectin molecules, and the leukocyte receptor complex on Chromosome 19q13.4, which encodes immunoglobulin superfamily molecules. The composition of these gene clusters differs markedly between closely related species, providing evidence for rapid, lineage-specific expansions or contractions of sets of loci. The choice of NK receptor genes is polarized in the two species most studied, mouse and human. In mouse, the C-type lectin-related Ly49 gene family predominates. Conversely, the single Ly49 sequence is a pseudogene in humans, and the immunoglobulin superfamily KIR gene family is extensive. These different gene sets encode proteins that are comparable in function and genetic diversity, even though they have undergone species-specific expansions. Understanding the biological significance of this curious situation may be aided by studying which NK receptor genes are used in other vertebrates, especially in relation to species-specific differences in genes for major histocompatibility complex class I molecules.

In mammals, the cell surface receptors encoded by the leukocyte receptor complex (LRC) regulate the activity of T lymphocytes and B lymphocytes, as well as that of natural killer cells, and thus provide protection against pathogens and parasites. The chicken genome encodes many Ig-like receptors that are homologous to the LRC receptors. The chicken Ig-like receptor (CHIR) genes are members of a large monophyletic gene family and are organized into genomic clusters, which are in conserved synteny with the mammalian LRC. One-third of CHIR genes encode polypeptide molecules that contain both activating and inhibitory motifs. These genes are present in different phylogenetic groups, suggesting that the primordial CHIR gene could have encoded both types of motifs in a single molecule. In contrast to the mammalian LRC genes, the CHIR genes with similar function (inhibition or activation) are evolutionarily closely related. We propose that, in addition to recombination, single nucleotide substitutions played an important role in the generation of receptors with different functions. Structural models and amino acid analyses of the CHIR proteins reveal the presence of different types of Ig-like domains in the same phylogenetic groups, as well as sharing of conserved residues and conserved changes of residues between different CHIR groups and between CHIRs and LRCs. Our data support the notion that the CHIR gene clusters are regions homologous to the mammalian LRC gene cluster and favor a model of evolution by repeated processes of birth and death (expansion-contraction) of the Ig-like receptor genes.

We here report the cDNA sequences of 11 new rat Ly49 genes with full and three with incomplete open reading frames. Although obtained from different inbred rat strains, these as well as six previously published cDNA represent non-allelic genes matching different loci in the Brown Norway (BN) rat genome, which is predicted to contain 34 Ly49 loci distributed over the distal part of the NK cell gene complex. Some of the cloned genes appear to be mutated to non-function in the BN genome, which harbors additional genes with full open reading frames, suggesting at least 26 non-allelic functional Ly49 genes in the rat. Of the encoded receptors, 13 are predicted to be inhibitory, eight to be activating, whereas five may be both ('bifunctional'). Phylogenetic analysis bears evidence of a highly dynamic genetic region, in which only the most distally localized Ly49 gene has a clear-cut mouse ortholog. In phylograms, the majority of the genes cluster into three subgroups with the genes mapping together, defining three chromosomal regions that seem to have undergone recent expansions. When comparing the lectin-like domains, the receptors form smaller subgroups, most containing at least one inhibitory and one activating or 'bifunctional' receptor, where close sequence similarities suggest recent homogenization events.