BACKGROUND
the evolution of immunoglobulin superfamily  members is largely influenced by the nature of their ligands.
the extracellular part of igsf members which are predominantly expressed in the nervous system are well conserved, while the extracellular domains of members expressed by immune cells or cells involved in reproduction tend to diversify much more rapidly  <cit> .
the cea gene family, which belongs to the igsf, is a tandemly clustered multigene family.
such gene families are subject to rapid evolution due to ongoing gene duplication, deletion and mutational events  <cit> .
at present, the cea family is subdivided into two main subgroups: the cea-related cell adhesion molecule  and the pregnancy-specific glycoprotein  subgroups.
the ceacam subgroup in humans consists of  <dig> members composed of a single immunoglobulin variable -like n-terminal  domain followed by zero to six ig constant -like domains of a and b subtypes and one member which consists of two igc-like domains and two igv-like domains, one at each end of the molecule.
ceacam <dig>  ceacam <dig>  ceacam <dig>  ceacam <dig>  ceacam <dig>  ceacam <dig> and ceacam <dig> are transmembrane molecules while cea/ceacam <dig>  ceacam <dig>  ceacam <dig>  and ceacam <dig> are linked to the cell membrane via glycosylphosphatidylinositol  anchors and ceacam <dig> is most likely a secreted molecule.
for ceacam <dig>  and the recently discovered ceacam <dig>  ceacam <dig>  ceacam <dig> and ceacam <dig>  but not for other family members have orthologs been identified in rodents <cit> .
in addition to these orthologs, the transmembrane-bound ceacam <dig> , ceacam <dig>  and the secreted members ceacam <dig>  ceacam <dig>  ceacam <dig> ceacam <dig>  ceacam <dig>  ceacam <dig>  ceacam <dig>  exist in rodents <cit> .
in mice, rats and cattle, at least two ceacam <dig> alleles each have been identified, which differ considerably in their n domain sequences <cit> .
no such allelic variation has been observed in humans.
the psg, which are specifically expressed in the trophoblast, are the most abundant fetal proteins in the maternal bloodstream during late human pregnancy.
the psgs are thought to play a pivotal role in the regulation of the maternal immune response to the fetal semi-allograft <cit> .
the diversity of the ceacam/psg family of proteins is further enhanced by species-specific differential splicing of several family members <cit> .
as expected from the variable structure of the cea family members, these molecules have diverse functions.
it is well established that various cea family members play crucial roles in cell-cell adhesion, tumor development, angiogenesis, insulin metabolism, reproduction and, as more recently realized, in immunity <cit> .
ceacam <dig> functions as a natural killer cell inhibitory receptor <cit> , regulates t and b cell proliferation  <cit> , induces dendritic cell maturation <cit>  and facilitates granulocyte and monocyte survival  <cit> .
in addition, ceacam <dig>  ceacam <dig> and ceacam <dig> were found to be pivotal for the regulation of granulocyte activation in humans <cit> .
while human ceacam <dig> contains two immunoreceptor tyrosine-based inhibitory motifs  in its cytoplasmic domain, one ceacam <dig> itim is replaced by an immunoreceptor tyrosine-based switch motif  in mice and rats.
human ceacam <dig> and possibly ceacam <dig>  ceacam <dig> and ceacam <dig> harbor functional immunoreceptor tyrosine-based activation motifs  in their cytoplasmic tails  <cit> .
in contrast, except for ceacam <dig> and ceacam <dig> no such itam-containing cea family members exist in rodents.
remarkably, several pathogens namely, neisseria sp., haemophilus influenzae, and moraxella catarrhalis in humans <cit>  and mouse hepatitis virus   <cit>  use ceacams to anchor themselves to or to invade host cells.
in addition, it has been suggested that bacterial pathogens can down-regulate immune functions by binding to ceacam <dig> on cd4+ t lymphocytes and signaling via the ceacam <dig> itim motif  <cit> .
it was recently proposed that the human granulocyte-specific ceacam <dig> functions as a specifically adapted single-chain itam-dependent phagocytic receptor involved in the clearance of ceacam-binding bacteria by human granulocytes <cit> .
this indicates that fixation of genes in the human genome coding for itam-bearing ceacams can be the result of a selective pressure mediated by ceacam-binding pathogenic microbes.
in contrast, one of the two allelic variants of murine ceacam <dig> exhibits a significant lower virus receptor activity suggesting that, in mice, mutations in the ceacam <dig> gene rather than the fixation of an activating virus receptor were acquired to evade pathogen attack.
the availability of a high-quality draft genome sequence of the domestic dog   <cit>  provided the possibility to analyze the complete cea gene family in a third mammalian order.
here we show that in the dog multiple itam-bearing ceacam1-related ceacams evolved and that ceacam <dig> and ceacam <dig> have undergone concerted evolution by gene conversion.
based on sequence analyses and comparison of expression profiles we suggest that ceacam <dig> and ceacam <dig> are paired activating and inhibitory immune receptors involved in the regulation of t cell responses.
the possibility that sporadic recurrence of selective pressure mediated by species-specific pathogens was the driving force for the evolution of multiple itam-bearing ceacam1-related ceacams in the dog is discussed.
RESULTS
identification of the canine cea gene family cluster
in an effort to identify all cea gene family members in the dog genome we employed the blast program to search the genbank and ensemble databases  using cdna nucleotide sequences from all known human and mouse cea family members as query sequences.
the human and mouse cea gene families are located within the expanded leukocyte receptor complex on chromosome 19q <dig>  and chromosome  <dig>  respectively.
this approach yielded the sequences of the ceacam <dig>  ceacam <dig>  ceacam <dig>  ceacam <dig> and ceacam <dig> orthologs.
their predicted full length mrna sequences  encode proteins with domain organizations identical with their human and murine counterparts: ceacam <dig> ; ceacam <dig> ; ceacam <dig> ; ceacam <dig>  <cit> .
like their mouse and human counterparts, both ceacam <dig> and ceacam <dig> contain presumed itam consensus sequences in their cytoplasmic domains.
however, like in mouse, no ceacam <dig> cytoplasmic domain exon  <dig> present in human ceacam <dig> was found  <cit> .
as for human ceacam <dig>  no cytoplasmic domain exon could be identified due to its small size and lack of corresponding est sequences.
in a second round of search we used the blast program and cdna nucleotide sequences from the identified orthologous canine genes to screen the genbank and ensemble databases.
this strategy was employed in order to identify dog-specific genes which have evolved after the separation of the dog and primate/rodent lineage.
this approach led to the identification of  <dig> additional ceacam genes  using ceacam <dig> nucleotide sequences as query sequences.
ceacam <dig>  ceacam <dig>  ceacam <dig>  ceacam <dig>  ceacam <dig> and ceacam <dig> are arranged between xrcc <dig> and lipe, while ceacam <dig> and ceacam <dig> are located between cd79a and tgfβ <dig> on the opposite strand .
surprisingly, no genes corresponding to the psg genes were found.
the murine psg genes are located between hif3a and pglyrp <dig> spanning nearly  <dig>  mbp.
in dog, however, the distance between hif3a and pglyrp <dig> comprises only  <dig>  mbp which rules out the presence of a larger set of genes between these marker genes.
similarly, there is not enough space between xrcc <dig> and ceacam <dig>  to accommodate an expanded psg gene family as found in humans at the corresponding chromosomal locations location where the distance is approximately  <dig> mbp .
the canine ceacam gene loci on dog chromosome  <dig> exhibit a similar arrangement to that found for the human cea gene family on chromosome  <dig> but it is partially different from that of the murine cea gene family on chromosome  <dig> .
identification of exons coding for the cytoplasmic domain of ceacam1-related ceacams
in order to identify the very short cytoplasmic exons of the ceacam1-like genes which cannot be localized by direct search strategies we first localized the transmembrane exons of all ceacam genes using transmembrane exon sequences of the human and mouse ceacam genes as query sequences.
surprisingly, the  <dig> predicted transmembrane domain exons of the ceacam1-related canine genes clustered with the sequences of the transmembrane domain exons of human ceacam <dig>  ceacam <dig> and ceacam <dig> which are connected to cytoplasmic domain exons encoding a c-terminal itam motif  or a homologous truncated cytoplasmic domain with a premature stop .
the sequence of the transmembrane exon of canine ceacam <dig> clustered with transmembrane sequences from murine ceacam <dig> and ceacam <dig> and with a group of exons coding for hydrophobic gpi signal peptides from human ceacam <dig>  ceacam <dig>  ceacam <dig>  ceacam <dig>  ceacam <dig> .
ceacam5-ceacam <dig> gpi signal peptide exons are thought to have evolved from the transmembrane exon of an ancestral ceacam <dig> by introduction of a stop codon  <cit> .
ceacam1/ceacam <dig> and ceacam <dig> encode cytoplasmic domains which contain itim motifs.
taken together, this indicates that tm and cytoplasmic domain exons are exchanged together during evolution and predicts the presence of itam signaling motifs rather than itim in the cytoplasmic tails of the dog ceacam23-ceacam <dig>  ceacam <dig> and ceacam <dig> molecules.
subsequently, we screened about  <dig>  bp of nucleotide sequence downstream of the predicted canine transmembrane domain exons for the presence of the short cytoplasmic exons.
indeed, small cytoplasmic domain exons similar to the ones found in human ceacam <dig> and ceacam <dig> could be identified, which encode itam motifs close to the c-terminal end .
although homologous cytoplasmic domain exons 1– <dig> are present in ceacam <dig> the loss of the splice donor site in the cytoplasmic domain exon  <dig> leads to read through into the adjacent intron and truncation by a stop codon following after five codons.
ceacam <dig> and ceacam <dig> probably represent pseudogenes due to multiple deletions in their extracellular  and cytoplasmic domain exons .
relationship of the canine primordial ceacam genes with human and murine counterparts
to determine the degree of relatedness between the primordial canine ceacam genes with the orthologous genes in mice and man, the nucleotide and encoded amino acid sequences of the leader, the igv-type n domain, the igc-type a and b domains, the transmembrane domain and the cytoplasmic domain exons were compared .
the degree of sequence identity between corresponding extracellular ig-type domains in dog, humans and mice varies greatly at the amino acid sequence level.
the lowest value is observed for ceacam <dig> n domains , the highest for the ceacam <dig> n <dig> domain .
the nucleotide and amino acid sequences of leader , igv-like domain , igc-like domain , transmembrane domain  and combined cytoplasmic domain exons  were compared pairwise using the clustalw software .
the degree of identity is indicated in % .
cloning of full-length canine ceacam1-related cdnas and identification of splice variants
based on the genomic sequence information we designed gene-specific primers for the amplification of full length ceacam1-related cdnas from total rna, isolated either from liver or spleen.
in the published genomic dog sequence, one nucleotide is missing from the a <dig> exon of canine ceacam <dig>  cloning and sequencing of full length ceacam <dig> clones from a mix-breed dog revealed a complete  <dig> bp ceacam <dig> a <dig> exon.
in total, we could identify  <dig> different ceacam <dig> splice variants.
the largest clone contains an open reading frame of  <dig> bp coding for a  <dig> amino acid protein.
comparisons with ceacam <dig> from other species identified the cloned canine cea family member unequivocally as ceacam <dig>  it is composed of a leader sequence, four extracellular immunoglobulin-like domains , a transmembrane domain and a cytoplasmic domain .
the cytoplasmic domain contains an n-terminal itim and a c-terminal itsm motif similar to that found in the long ceacam <dig> isoforms of mouse, rat and cattle.
according to the nomenclature system for the cea family, the longest canine ceacam <dig> splice variant was designated ceacam1-4l .
the mrna encoding the splice variant with the short cytoplasmic tail ceacam1-4s  differs from ceacam1-4l mrna by the absence of the  <dig> bp nucleotide cytoplasmic domain exon <dig> that leads to a frame shift and to the use of a tga stop codon in cytoplasmic domain exon  <dig> as found for ceacam <dig> of other species.
splice variants without the a <dig> and the b domain designated ceacam1-2l  and ceacam1-2s  also exist as well as two splice variants without igc-like domains .
furthermore, we have cloned the most abundant full length ceacam <dig>  ceacam <dig>  ceacam <dig>  and ceacam <dig> cdnas.
the ceacam <dig> cdna  corresponds to the longest mrna splice variant predicted from the genomic sequence of the boxer breed and exhibits an identical nucleotide sequence.
ceacam <dig> encodes four extracellular ig-like domains, a transmembrane domain and a cytoplasmic tail which harbors a typical itam motif .
the major ceacam <dig> cdna encodes a protein with an n and a transmembrane domain .
the cytoplasmic domain does not contain the expected itam motif due to the presence of the unspliced intron between the predicted cytoplasmic domain exons  <dig> and  <dig>  the cloned ceacam <dig> splice variants also code for proteins with only one n domain followed by a transmembrane domain.
three out of four clones encode cytoplasmic domains which contain the predicted itam motif .
in one clone, the absence of the  <dig> nucleotide cytoplasmic domain exon  <dig> leads to a frame shift and the usage of an alternative stop codon located in cytoplasmic domain exon <dig> .
using supposedly ceacam28-specific primers, two products were amplified which differ in their length by  <dig> bp.
cloning and sequencing revealed that the ceacam <dig> gene codes for a protein with one n domain, two a domains, a transmembrane domain and a cytoplasmic tail which contain an itam motif .
the sequence of the cdnas cloned from the smaller pcr product was different from all genomic sequences published either for the boxer or the poodle breed and, therefore, was named ceacam <dig>  the differences are more or less randomly distributed within the whole coding region which consists of an n, a, transmembrane and cytoplasmic domain region.
two ceacam <dig> splice variants were identified, both containing an itam motif in the cytoplasmic tail.
they differ, however, in the usage of alternative splice acceptor sites of the cytoplasmic domain exon  <dig>  .
relationship of ceacam1-like genes and ceacam1
to analyze the evolutionary relationship of the newly discovered ceacam genes, multiple nucleotide sequence alignments of ig domain exons of human and dog ceacam genes were performed and phylogenetic trees constructed.
as expected, the n domain sequences of the orthologous genes  clustered pairwise, while all new canine n domain exon sequences  clustered with that from dog ceacam <dig> .
similarly, the primate-specific ceacam1-related n domain nucleotide sequences  clustered with the human ceacam <dig> n domain exon sequence except for ceacam <dig> n. the close relationship between the new dog ceacam genes and dog ceacam <dig> could also be demonstrated when the a and b domain exon nucleotide sequences were compared .
nine of the  <dig> igc-like domains of the new ceacam1-related canine molecules are of the ceacam <dig> a <dig> type and five each were of the ceacam <dig> a <dig> and b type .ceacam <dig> appears to be the only ceacam1-related gene which contains all extracellular domain exons present in ceacam <dig>  in particular three igc-like domain exons .
all other members of this subgroup either lack one of these exons or contain exons corrupted by nonsense, frame shift or splice site mutations leading to smaller predicted proteins .
assuming that the n domain is the most relevant domain for ligand interaction, the ligand specificity of ceacam <dig> seems to be most closely related to that of ceacam <dig>  the n domain amino acid sequence of ceacam <dig> differs from that of ceacam <dig> in only two positions.
these amino acid changes do not affect the center of the cfg face, a topological n domain region of ceacam members essential for ligand and pathogen interactions .
interestingly, ceacam <dig> lies next to ceacam <dig> in the ceacam <dig> gene cluster which hints to an evolutionary recent duplication event .
ceacam <dig> is the most distant ceacam <dig> relative within the group of ceacam1-related genes, sharing only 66% of its n domain exon encoded amino acid sequence .
most of the amino acid sequence differences cluster to the cfg face of the n domains, indicating a positive selection for ligand diversification.
to test this possibility, we calculated the ratio of nonsynonymous  to synonymous  nucleotide substitution rates on the branches of an n exon phylogenetic tree.
the lowest ka/ks ratio  is observed for ceacam <dig>  this indicates that the ceacam <dig> n domain is under purifying selection.
for all other canine ceacam1-related ceacams, the n domain amino acid changes appear to evolve neutrally or to be positively selected for during evolution since the corresponding ka/ks values are close to  <dig> or higher .
the amino acid  and nucleotide sequences  of igv-like domain exons were compared pairwise using the clustalw software .
the degree of identity is indicated in %.
itam-bearing ceacam1-related genes are expressed by cells of the immune system
to identify the ceacam gene expression pattern and splice variants, their nucleotide sequences were aligned, and regions of the transmembrane domain-encoding exons with sequence differences were selected to design primers that are specific for each of the ceacam1-related cdnas except for ceacam <dig> and ceacam <dig> which were amplified with a common primer pair .
the primers were used to amplify cdnas from individual ceacam by rt-pcr from rna isolated from liver, bone marrow, spleen, purified peripheral blood mononuclear cells , and granulocytes .
except ceacam <dig>  all analyzed ceacam1-related genes,  were found to be expressed in spleen.
ceacam <dig>  ceacam <dig>  ceacam <dig>  ceacam <dig> and ceacam <dig> transcripts were detected in bone marrow and ceacam <dig>  ceacam <dig>  ceacam <dig> and ceacam <dig> mrnas in pbmc.
granulocytes express in addition to different ceacam <dig> splice variants  all the itam-bearing molecules except ceacam <dig>  in liver, the presence of mainly ceacam <dig> and ceacam <dig> transcripts could be demonstrated.
ceacam <dig> and ceacam <dig> are not expected to be expressed because the sequences of essential exons appear to be corrupted .
* size of largest cloned or predicted  splice variant is shown
** ceacam <dig> cdna were also detected using the ceacam <dig> primers
cyt, cytoplasmic domain; tm, transmembrane domain; utr, untranslated region
expression of ceacam <dig> and ceacam <dig> by t cells upon activation
to further analyze the expression of ceacam1-related ceacams by immune cells, we isolated t cells from pbmc by macs sorting using cd4- and cd8-specific mabs.
this procedure resulted in a purity of the t cell populations of >97%, as determined by flow cytometry .
activation of t cells by the various stimuli was controlled by analysis of blast formation using flow cytometry .
rt-pcr analysis demonstrated that resting t cells express ceacam <dig>  ceacam <dig> and ceacam <dig> mrna simultaneously.
in addition, expression of these ceacams was also found in t cell-depleted pbmc, which are mainly composed of bcells .
stimulation of pbmc with anti-cd <dig> mab or il- <dig> had only a minor effect on ceacam <dig> expression in t cell-depleted pbmc  and t cells.
in contrast, the content of ceacam <dig> mrna seem to be reduced in t cells upon activation .
in unstimulated t cells the ceacam1/ceacam <dig> cdna ratio was <  <dig> whereas the ratio was >  <dig> and >  <dig> in t cells stimulated with il- <dig> and anti-cd <dig> mab, respectively .
the content of ceacam <dig> mrna remained unaltered in t cells upon stimulation .
ceacam <dig> and ceacam <dig> are paired immune receptors created by gene conversion
the near identity of the n domains between ceacam <dig> and ceacam <dig> is most likely due to a recent gene conversion event.
indeed, we could identify a  <dig>  bp genomic region in ceacam <dig> which shows 99% identity with the corresponding region of ceacam <dig>  the high similarity of this segment starts at position - <dig> of the 5'-flanking region and ends at position  <dig> of the intron between the ndomain and the a <dig> domain exons .
upstream of this segment, the sequence similarity between homologous regions of ceacam <dig> and ceacam <dig>  drops to 80%.
the downstream homologous region of ~ <dig>  bp demarcated by a sine element in ceacam <dig> exhibits a similarity of 92%.
the occurrence of such a regionally restricted gene conversion event is also supported by the high degree of amino acid sequence similarity of the leader  and n domain .
the a <dig> and a <dig> domains of ceacam <dig> encoded outside of the conserved genomic region share only 88% and 86% of their amino acid sequence with that of the homologous ceacam <dig> a <dig> domain .
these data suggests that the similarity between ceacam <dig> and the ceacam <dig> ancestor was ~92% before the gene conversion event occurred.
discussion
recent molecular studies have established strong evidence for four primary, superordinal mammalian clades: afrotheria, xenarthra, euarchontoglires and laurasiatheria  <cit> .
the cea gene family is well known in primates  and rodents .
however, both orders belong to the same principal lineage of placental mammals, the euarchontoglires.
since carnivores belong to the laurasiatheria clade, we expected to gain new insights into the evolution of the rapidly diverging cea family by analysis of the cea family of the dog.
first of all, identification of ceacam <dig>  ceacam <dig>  ceacam <dig>  ceacam <dig> and ceacam <dig> orthologs in the dog genome at corresponding chromosomal locations, strongly indicates that the cea gene family of the common ancestor of euarchontoglires and laurasiatheria consisted of at least five genes.
it remains to be seen whether species which represent older mammalian lineages like afrotheria, marsupials and monotremes contain the same set of primordial genes.
furthermore, the findings of this work supports our previous hypothesis that ceacam3-ceacam <dig>  ceacam <dig> and ceacam9-ceacam <dig>  ceacam <dig>  none of which is present in the dog genome , represent primate and rodent-specific genes, respectively  <cit> .
the ancestral members of the cea gene family are located in three different loci .
while ceacam <dig>  ceacam <dig> and ceacam <dig> are close together , ceacam <dig> is separated from this locus by more than  <dig> mbp and ceacam <dig> is displaced into the siglec gene cluster in humans, mice and dogs.
out of these ancestral genes, probably only ceacam <dig> ancestors gave rise to massive gene expansions in a order-specific manner  <cit> .
the ceacam1-related genes form compact clusters next to ceacam <dig> and near the cd79a gene.
in rodents even a third locus  has been encroached by amplified ceacam1-related genes.
with analysis of the cea family in a third order , the inversion event observed for the chromosomal region flanked by cd79a and lipe can now be decided to have happened in the rodent rather than in the primate lineage, since the order of genes in this locus is conserved between humans and dogs .
ceacam1-related genes can be subdivided into the psg and the ceacam subgroup genes.
the birth of the ancestral psg has been estimated to have occurred some  <dig> myr ago  <cit> , approximately the time of rodent-primate divergence <cit> .
the rapid, independent expansion of the human and mouse psg gene families occurred through further gene duplication and exon shuffling events  <cit> .
surprisingly, we did not find any psg gene in the dog genome indicating that the psg function exerted in primates and rodents is dispensable for the dog, possibly because dogs have an endotheliochorial placenta type which is different from the hemochorial placentae of primates and rodents <cit> .
semi-allogeneic fetal trophoblast cells are less invasive and concomitantly less prone to encounter the maternal immune system in endotheliochorial placentae in comparison to the hemochorial placentae where the trophoblast cells are bathed in maternal blood <cit> .
therefore, psg could be involved in the regulation of trophoblast invasion and/or modulation of the maternal immune system as has been suggest before <cit> .
alternatively, the function of the psg could have been taken over by an unrelated gene family due to functional convergence at the molecular level, similar to that found for natural killer  cell receptors, where killer cell immunoglobulin-like receptors  and c-type lectin-like receptors  are used in primates and rodents, respectively.
in contrast to the psg, the ceacam subgroup of canine ceacam1-related genes underwent a recent expansion similar to that found in primates and rodents.
in general, igsf members which are expressed by leucocytes are diverging rapidly within their extracellular domains.
this results in an average amino acid identity of about 54% between human and mouse orthologs  <cit> .
the conservation of the ceacam <dig> n domain, most relevant for pathogen and cell-cell interactions  <cit> , lies within this range, with an amino acid identity of 56%  and 42% .
this is consistent with the notion that ceacam <dig> plays a key role in various functions of the immune system <cit> .
in contrast, between mouse and human ceacam <dig>  ceacam <dig> and ceacam <dig> orthologs, the n domain amino acid identities are higher ranging from 60% to 91%, which argues for non-immunological functions, especially for ceacam <dig>  which exhibits an overall amino acid sequence identity of about 90% .
the dog ceacam1-related genes are arranged head to tail, like the kir genes in the human kir locus <cit> .
such an arrangement was found to facilitate unequal crossing over leading to gene expansion and generation of multigene families  <cit> .
furthermore, the kir gene family is subject to birth-and-death evolution, domain shuffling and mutational changes  <cit> .
our analyses indicate that the cea gene family evolved similarly.
one characteristic for this kind of evolution is the presence of multiple pseudogenes, which also exist within the canine ceacam1-like gene locus .
this type of evolution also facilitates a mechanism for qualitatively changing a receptor's signaling potential.
a number of type i transmembrane igsf members including ceacams and kirs are composed of an amino-terminal extracellular "environmental sensor" and a carboxy-terminal cytoplasmic tail which can contain either inhibitory  or activating motifs .
unequal crossing-over at such gene loci encompassing the corresponding exons could result, in a single step, in genes encoding receptors with a drastically changed signaling potential by combining the same extracellular domain with a cytoplasmic tail of opposite function.
this allows rapid adaptation to environmental changes and the creation of so-called paired immune receptors.
an example for such a mechanism is found for the kir gene kir3dl/s, where either an inhibitory variant or an activating variant encoding kir gene is found in different human individuals at the same locus <cit> .
in addition, a change in the receptor's signaling potential which may be the result of an arms race between pathogens and the host immune system has been reported recently for mice.
the murine cytomegalovirus  induces in infected cells expression of the virus-encoded gpi-anchored m <dig> protein, which binds to the activating, dap12-associating killer receptor ly49h in mcmv-resistant mice leading to nkcell-mediated cytotoxicity.
mcmv-susceptible mouse strains do not have ly49h but the extracellularly very similar itim-bearing ly49i protein which has inhibitory killer cell receptor activity.
binding of m <dig> to ly49i in such mice may inhibit nk cell cytotoxicity thus rendering the mice susceptible to mcmv infection <cit> .
it was suggested that the activating receptor gene was derived from the gene encoding the inhibitory receptor to counteract a virus that exploits the inhibitory receptor during infection and that this mechanism may apply to many other activating receptors that have inhibitory counterparts <cit> .
however, recently it was shown for the human siglec <dig> and siglec <dig> genes that the direction of gene conversion that generates paired immune receptors is not always the same.
this indicates that also other needs drive the evolution of paired receptors like the fine tuning of immune responses  <cit> .
host-pathogen interactions involving both proteins with itim or itam signaling motifs have also been reported for humans.
the primate-specific ceacam1-related protein ceacam <dig> which contains an n domain closely related to that of ceacam <dig>  and a cytoplasmic tail with an itam is exclusively expressed in granulocytes.
due to its close relatedness with the itim-bearing gonococcal receptor ceacam <dig> it can act as decoy receptor for neisserial pathogens and is able to induce clearance of gonococcal infections by granulocytes.
mutational analyses demonstrated that the cytoplasmic itam is instrumental for this process  <cit> .
to our knowledge, ceacam <dig> and ceacam <dig> represent the first paired coexpressed immune receptors which contain antagonizing itim and itam  in their respective cytoplasmic tails.
ceacam <dig> was probably formed from a ceacam <dig> ancestor by a recent gene conversion encompassing the leader and the n exon from ceacam <dig>  the latter exon encodes the region most instrumental for ligand and pathogen binding in ceacam molecules.
several findings, presented here, argue for a role of ceacam <dig> as a ceacam <dig> decoy receptor in the dog.
first, the direction of the gene conversion was most likely from the inhibitory receptor to the activating receptor, second, the ka/ks ratio of the n domain exons of these family members is indicative for a purifying selection, whereby the specificity of the putative ceacam <dig> ligand is probably conserved for ceacam <dig>  third, the amino acid changes observed for the ceacam <dig> n domain most likely have no effect on the structure of that part of the n domain shown to be the pathogen binding site in human and mouse ceacam <dig> and fourth, is expressed by immune cells , a prerequisite for a defense function against a putative pathogen.
based on a highly similar amino acid sequence and expression pattern ceacam <dig> can be envisioned to also function, like ceacam <dig>  as a decoy receptor.
however, this hypothesis raises questions about the driving force behind the fixation of the other itam-bearing ceacam1-related genes in the dog.
most likely, selective pressure by pathogens changes with time.
in times where the selective pressure exerted by pathogens on a receptor system is low, darwinian selection will act and change the specificity of a receptor now possibly recognizing an endogenous ligand.
once the receptor has gained a new function it also can change the signaling properties which may explain why older i.e.
more diverged itam-bearing receptors e.g.
like ceacam <dig> and ceacam <dig> have lost the itam or have gained new splice variants which do not contain an itam.
CONCLUSIONS
taken together, the presence of multiple ceacam1-related dog ceacams with itams could reflect a repeated arms race between species-specific pathogens and the dog immune system.
fixation and expression by effector cells of itam-containing ceacams would result from either bacterial pathogens binding to ceacam <dig> or viruses which induce the expression of ceacam <dig> ligands by infected cells as found for mcmv.
this putatively pathogen-driven evolution of the cea gene family led to the development of the ceacam1/ceacam <dig> receptor pair which could also be involved in the fine tuning of t cell responses.

 the evolution of immunoglobulin superfamily members is largely influenced by the nature of their ligands. the extracellular part of igsf members which are predominantly expressed in the nervous system are well conserved, while the extracellular domains of members expressed by immune cells or cells involved in reproduction tend to diversify much more rapidly. the cea gene family, which belongs to the igsf, is a tandemly clustered multigene family. such gene families are subject to rapid evolution due to ongoing gene duplication, deletion and mutational events. at present, the cea family is subdivided into two main subgroups: the cea-related cell adhesion molecule and the pregnancy-specific glycoprotein subgroups. the ceacam subgroup in humans consists of members composed of a single immunoglobulin variable -like n-terminal domain followed by zero to six ig constant -like domains of a and b subtypes and one member which consists of two igc-like domains and two igv-like domains, one at each end of the molecule. ceacam ceacam ceacam ceacam ceacam ceacam and ceacam are transmembrane molecules while cea/ceacam ceacam ceacam and ceacam are linked to the cell membrane via glycosylphosphatidylinositol anchors and ceacam is most likely a secreted molecule. for ceacam and the recently discovered ceacam ceacam ceacam and ceacam but not for other family members have orthologs been identified in rodents. in addition to these orthologs, the transmembrane-bound ceacam, ceacam and the secreted members ceacam ceacam ceacam ceacam ceacam ceacam ceacam exist in rodents. in mice, rats and cattle, at least two ceacam alleles each have been identified, which differ considerably in their n domain sequences. no such allelic variation has been observed in humans. the psg, which are specifically expressed in the trophoblast, are the most abundant fetal proteins in the maternal bloodstream during late human pregnancy. the psgs are thought to play a pivotal role in the regulation of the maternal immune response to the fetal semi-allograft. the diversity of the ceacam/psg family of proteins is further enhanced by species-specific differential splicing of several family members. as expected from the variable structure of the cea family members, these molecules have diverse functions. it is well established that various cea family members play crucial roles in cell-cell adhesion, tumor development, angiogenesis, insulin metabolism, reproduction and, as more recently realized, in immunity. ceacam functions as a natural killer cell inhibitory receptor, regulates t and b cell proliferation, induces dendritic cell maturation and facilitates granulocyte and monocyte survival. in addition, ceacam ceacam and ceacam were found to be pivotal for the regulation of granulocyte activation in humans. while human ceacam contains two immunoreceptor tyrosine-based inhibitory motifs in its cytoplasmic domain, one ceacam itim is replaced by an immunoreceptor tyrosine-based switch motif in mice and rats. human ceacam and possibly ceacam ceacam and ceacam harbor functional immunoreceptor tyrosine-based activation motifs in their cytoplasmic tails. in contrast, except for ceacam and ceacam no such itam-containing cea family members exist in rodents. remarkably, several pathogens namely, neisseria sp., haemophilus influenzae, and moraxella catarrhalis in humans and mouse hepatitis virus use ceacams to anchor themselves to or to invade host cells. in addition, it has been suggested that bacterial pathogens can down-regulate immune functions by binding to ceacam on cd4+ t lymphocytes and signaling via the ceacam itim motif. it was recently proposed that the human granulocyte-specific ceacam functions as a specifically adapted single-chain itam-dependent phagocytic receptor involved in the clearance of ceacam-binding bacteria by human granulocytes. this indicates that fixation of genes in the human genome coding for itam-bearing ceacams can be the result of a selective pressure mediated by ceacam-binding pathogenic microbes. in contrast, one of the two allelic variants of murine ceacam exhibits a significant lower virus receptor activity suggesting that, in mice, mutations in the ceacam gene rather than the fixation of an activating virus receptor were acquired to evade pathogen attack. the availability of a high-quality draft genome sequence of the domestic dog provided the possibility to analyze the complete cea gene family in a third mammalian order. here we show that in the dog multiple itam-bearing ceacam1-related ceacams evolved and that ceacam and ceacam have undergone concerted evolution by gene conversion. based on sequence analyses and comparison of expression profiles we suggest that ceacam and ceacam are paired activating and inhibitory immune receptors involved in the regulation of t cell responses. the possibility that sporadic recurrence of selective pressure mediated by species-specific pathogens was the driving force for the evolution of multiple itam-bearing ceacam1-related ceacams in the dog is discussed.  identification of the canine cea gene family cluster in an effort to identify all cea gene family members in the dog genome we employed the blast program to search the genbank and ensemble databases using cdna nucleotide sequences from all known human and mouse cea family members as query sequences. the human and mouse cea gene families are located within the expanded leukocyte receptor complex on chromosome 19q and chromosome respectively. this approach yielded the sequences of the ceacam ceacam ceacam ceacam and ceacam orthologs. their predicted full length mrna sequences encode proteins with domain organizations identical with their human and murine counterparts: ceacam; ceacam; ceacam; ceacam. like their mouse and human counterparts, both ceacam and ceacam contain presumed itam consensus sequences in their cytoplasmic domains. however, like in mouse, no ceacam cytoplasmic domain exon present in human ceacam was found. as for human ceacam no cytoplasmic domain exon could be identified due to its small size and lack of corresponding est sequences. in a second round of search we used the blast program and cdna nucleotide sequences from the identified orthologous canine genes to screen the genbank and ensemble databases. this strategy was employed in order to identify dog-specific genes which have evolved after the separation of the dog and primate/rodent lineage. this approach led to the identification of additional ceacam genes using ceacam nucleotide sequences as query sequences. ceacam ceacam ceacam ceacam ceacam and ceacam are arranged between xrcc and lipe, while ceacam and ceacam are located between cd79a and tgfβ on the opposite strand. surprisingly, no genes corresponding to the psg genes were found. the murine psg genes are located between hif3a and pglyrp spanning nearly mbp. in dog, however, the distance between hif3a and pglyrp comprises only mbp which rules out the presence of a larger set of genes between these marker genes. similarly, there is not enough space between xrcc and ceacam to accommodate an expanded psg gene family as found in humans at the corresponding chromosomal locations location where the distance is approximately mbp. the canine ceacam gene loci on dog chromosome exhibit a similar arrangement to that found for the human cea gene family on chromosome but it is partially different from that of the murine cea gene family on chromosome. identification of exons coding for the cytoplasmic domain of ceacam1-related ceacams in order to identify the very short cytoplasmic exons of the ceacam1-like genes which cannot be localized by direct search strategies we first localized the transmembrane exons of all ceacam genes using transmembrane exon sequences of the human and mouse ceacam genes as query sequences. surprisingly, the predicted transmembrane domain exons of the ceacam1-related canine genes clustered with the sequences of the transmembrane domain exons of human ceacam ceacam and ceacam which are connected to cytoplasmic domain exons encoding a c-terminal itam motif or a homologous truncated cytoplasmic domain with a premature stop. the sequence of the transmembrane exon of canine ceacam clustered with transmembrane sequences from murine ceacam and ceacam and with a group of exons coding for hydrophobic gpi signal peptides from human ceacam ceacam ceacam ceacam ceacam. ceacam5-ceacam gpi signal peptide exons are thought to have evolved from the transmembrane exon of an ancestral ceacam by introduction of a stop codon. ceacam1/ceacam and ceacam encode cytoplasmic domains which contain itim motifs. taken together, this indicates that tm and cytoplasmic domain exons are exchanged together during evolution and predicts the presence of itam signaling motifs rather than itim in the cytoplasmic tails of the dog ceacam23-ceacam ceacam and ceacam molecules. subsequently, we screened about bp of nucleotide sequence downstream of the predicted canine transmembrane domain exons for the presence of the short cytoplasmic exons. indeed, small cytoplasmic domain exons similar to the ones found in human ceacam and ceacam could be identified, which encode itam motifs close to the c-terminal end. although homologous cytoplasmic domain exons 1– are present in ceacam the loss of the splice donor site in the cytoplasmic domain exon leads to read through into the adjacent intron and truncation by a stop codon following after five codons. ceacam and ceacam probably represent pseudogenes due to multiple deletions in their extracellular and cytoplasmic domain exons. relationship of the canine primordial ceacam genes with human and murine counterparts to determine the degree of relatedness between the primordial canine ceacam genes with the orthologous genes in mice and man, the nucleotide and encoded amino acid sequences of the leader, the igv-type n domain, the igc-type a and b domains, the transmembrane domain and the cytoplasmic domain exons were compared. the degree of sequence identity between corresponding extracellular ig-type domains in dog, humans and mice varies greatly at the amino acid sequence level. the lowest value is observed for ceacam n domains, the highest for the ceacam n domain. the nucleotide and amino acid sequences of leader, igv-like domain, igc-like domain, transmembrane domain and combined cytoplasmic domain exons were compared pairwise using the clustalw software. the degree of identity is indicated in %. cloning of full-length canine ceacam1-related cdnas and identification of splice variants based on the genomic sequence information we designed gene-specific primers for the amplification of full length ceacam1-related cdnas from total rna, isolated either from liver or spleen. in the published genomic dog sequence, one nucleotide is missing from the a exon of canine ceacam cloning and sequencing of full length ceacam clones from a mix-breed dog revealed a complete bp ceacam a exon. in total, we could identify different ceacam splice variants. the largest clone contains an open reading frame of bp coding for a amino acid protein. comparisons with ceacam from other species identified the cloned canine cea family member unequivocally as ceacam it is composed of a leader sequence, four extracellular immunoglobulin-like domains, a transmembrane domain and a cytoplasmic domain. the cytoplasmic domain contains an n-terminal itim and a c-terminal itsm motif similar to that found in the long ceacam isoforms of mouse, rat and cattle. according to the nomenclature system for the cea family, the longest canine ceacam splice variant was designated ceacam1-4l. the mrna encoding the splice variant with the short cytoplasmic tail ceacam1-4s differs from ceacam1-4l mrna by the absence of the bp nucleotide cytoplasmic domain exon that leads to a frame shift and to the use of a tga stop codon in cytoplasmic domain exon as found for ceacam of other species. splice variants without the a and the b domain designated ceacam1-2l and ceacam1-2s also exist as well as two splice variants without igc-like domains. furthermore, we have cloned the most abundant full length ceacam ceacam ceacam and ceacam cdnas. the ceacam cdna corresponds to the longest mrna splice variant predicted from the genomic sequence of the boxer breed and exhibits an identical nucleotide sequence. ceacam encodes four extracellular ig-like domains, a transmembrane domain and a cytoplasmic tail which harbors a typical itam motif. the major ceacam cdna encodes a protein with an n and a transmembrane domain. the cytoplasmic domain does not contain the expected itam motif due to the presence of the unspliced intron between the predicted cytoplasmic domain exons and the cloned ceacam splice variants also code for proteins with only one n domain followed by a transmembrane domain. three out of four clones encode cytoplasmic domains which contain the predicted itam motif. in one clone, the absence of the nucleotide cytoplasmic domain exon leads to a frame shift and the usage of an alternative stop codon located in cytoplasmic domain exon. using supposedly ceacam28-specific primers, two products were amplified which differ in their length by bp. cloning and sequencing revealed that the ceacam gene codes for a protein with one n domain, two a domains, a transmembrane domain and a cytoplasmic tail which contain an itam motif. the sequence of the cdnas cloned from the smaller pcr product was different from all genomic sequences published either for the boxer or the poodle breed and, therefore, was named ceacam the differences are more or less randomly distributed within the whole coding region which consists of an n, a, transmembrane and cytoplasmic domain region. two ceacam splice variants were identified, both containing an itam motif in the cytoplasmic tail. they differ, however, in the usage of alternative splice acceptor sites of the cytoplasmic domain exon. relationship of ceacam1-like genes and ceacam1 to analyze the evolutionary relationship of the newly discovered ceacam genes, multiple nucleotide sequence alignments of ig domain exons of human and dog ceacam genes were performed and phylogenetic trees constructed. as expected, the n domain sequences of the orthologous genes clustered pairwise, while all new canine n domain exon sequences clustered with that from dog ceacam. similarly, the primate-specific ceacam1-related n domain nucleotide sequences clustered with the human ceacam n domain exon sequence except for ceacam n. the close relationship between the new dog ceacam genes and dog ceacam could also be demonstrated when the a and b domain exon nucleotide sequences were compared. nine of the igc-like domains of the new ceacam1-related canine molecules are of the ceacam a type and five each were of the ceacam a and b type.ceacam appears to be the only ceacam1-related gene which contains all extracellular domain exons present in ceacam in particular three igc-like domain exons. all other members of this subgroup either lack one of these exons or contain exons corrupted by nonsense, frame shift or splice site mutations leading to smaller predicted proteins. assuming that the n domain is the most relevant domain for ligand interaction, the ligand specificity of ceacam seems to be most closely related to that of ceacam the n domain amino acid sequence of ceacam differs from that of ceacam in only two positions. these amino acid changes do not affect the center of the cfg face, a topological n domain region of ceacam members essential for ligand and pathogen interactions. interestingly, ceacam lies next to ceacam in the ceacam gene cluster which hints to an evolutionary recent duplication event. ceacam is the most distant ceacam relative within the group of ceacam1-related genes, sharing only 66% of its n domain exon encoded amino acid sequence. most of the amino acid sequence differences cluster to the cfg face of the n domains, indicating a positive selection for ligand diversification. to test this possibility, we calculated the ratio of nonsynonymous to synonymous nucleotide substitution rates on the branches of an n exon phylogenetic tree. the lowest ka/ks ratio is observed for ceacam this indicates that the ceacam n domain is under purifying selection. for all other canine ceacam1-related ceacams, the n domain amino acid changes appear to evolve neutrally or to be positively selected for during evolution since the corresponding ka/ks values are close to or higher. the amino acid and nucleotide sequences of igv-like domain exons were compared pairwise using the clustalw software. the degree of identity is indicated in %. itam-bearing ceacam1-related genes are expressed by cells of the immune system to identify the ceacam gene expression pattern and splice variants, their nucleotide sequences were aligned, and regions of the transmembrane domain-encoding exons with sequence differences were selected to design primers that are specific for each of the ceacam1-related cdnas except for ceacam and ceacam which were amplified with a common primer pair. the primers were used to amplify cdnas from individual ceacam by rt-pcr from rna isolated from liver, bone marrow, spleen, purified peripheral blood mononuclear cells, and granulocytes. except ceacam all analyzed ceacam1-related genes, were found to be expressed in spleen. ceacam ceacam ceacam ceacam and ceacam transcripts were detected in bone marrow and ceacam ceacam ceacam and ceacam mrnas in pbmc. granulocytes express in addition to different ceacam splice variants all the itam-bearing molecules except ceacam in liver, the presence of mainly ceacam and ceacam transcripts could be demonstrated. ceacam and ceacam are not expected to be expressed because the sequences of essential exons appear to be corrupted. * size of largest cloned or predicted splice variant is shown ** ceacam cdna were also detected using the ceacam primers cyt, cytoplasmic domain; tm, transmembrane domain; utr, untranslated region expression of ceacam and ceacam by t cells upon activation to further analyze the expression of ceacam1-related ceacams by immune cells, we isolated t cells from pbmc by macs sorting using cd4- and cd8-specific mabs. this procedure resulted in a purity of the t cell populations of >97%, as determined by flow cytometry. activation of t cells by the various stimuli was controlled by analysis of blast formation using flow cytometry. rt-pcr analysis demonstrated that resting t cells express ceacam ceacam and ceacam mrna simultaneously. in addition, expression of these ceacams was also found in t cell-depleted pbmc, which are mainly composed of bcells. stimulation of pbmc with anti-cd mab or il- had only a minor effect on ceacam expression in t cell-depleted pbmc and t cells. in contrast, the content of ceacam mrna seem to be reduced in t cells upon activation. in unstimulated t cells the ceacam1/ceacam cdna ratio was < whereas the ratio was > and > in t cells stimulated with il- and anti-cd mab, respectively. the content of ceacam mrna remained unaltered in t cells upon stimulation. ceacam and ceacam are paired immune receptors created by gene conversion the near identity of the n domains between ceacam and ceacam is most likely due to a recent gene conversion event. indeed, we could identify a bp genomic region in ceacam which shows 99% identity with the corresponding region of ceacam the high similarity of this segment starts at position - of the 5'-flanking region and ends at position of the intron between the ndomain and the a domain exons. upstream of this segment, the sequence similarity between homologous regions of ceacam and ceacam drops to 80%. the downstream homologous region of ~ bp demarcated by a sine element in ceacam exhibits a similarity of 92%. the occurrence of such a regionally restricted gene conversion event is also supported by the high degree of amino acid sequence similarity of the leader and n domain. the a and a domains of ceacam encoded outside of the conserved genomic region share only 88% and 86% of their amino acid sequence with that of the homologous ceacam a domain. these data suggests that the similarity between ceacam and the ceacam ancestor was ~92% before the gene conversion event occurred. discussion recent molecular studies have established strong evidence for four primary, superordinal mammalian clades: afrotheria, xenarthra, euarchontoglires and laurasiatheria. the cea gene family is well known in primates and rodents. however, both orders belong to the same principal lineage of placental mammals, the euarchontoglires. since carnivores belong to the laurasiatheria clade, we expected to gain new insights into the evolution of the rapidly diverging cea family by analysis of the cea family of the dog. first of all, identification of ceacam ceacam ceacam ceacam and ceacam orthologs in the dog genome at corresponding chromosomal locations, strongly indicates that the cea gene family of the common ancestor of euarchontoglires and laurasiatheria consisted of at least five genes. it remains to be seen whether species which represent older mammalian lineages like afrotheria, marsupials and monotremes contain the same set of primordial genes. furthermore, the findings of this work supports our previous hypothesis that ceacam3-ceacam ceacam and ceacam9-ceacam ceacam none of which is present in the dog genome, represent primate and rodent-specific genes, respectively. the ancestral members of the cea gene family are located in three different loci. while ceacam ceacam and ceacam are close together, ceacam is separated from this locus by more than mbp and ceacam is displaced into the siglec gene cluster in humans, mice and dogs. out of these ancestral genes, probably only ceacam ancestors gave rise to massive gene expansions in a order-specific manner. the ceacam1-related genes form compact clusters next to ceacam and near the cd79a gene. in rodents even a third locus has been encroached by amplified ceacam1-related genes. with analysis of the cea family in a third order, the inversion event observed for the chromosomal region flanked by cd79a and lipe can now be decided to have happened in the rodent rather than in the primate lineage, since the order of genes in this locus is conserved between humans and dogs. ceacam1-related genes can be subdivided into the psg and the ceacam subgroup genes. the birth of the ancestral psg has been estimated to have occurred some myr ago, approximately the time of rodent-primate divergence. the rapid, independent expansion of the human and mouse psg gene families occurred through further gene duplication and exon shuffling events. surprisingly, we did not find any psg gene in the dog genome indicating that the psg function exerted in primates and rodents is dispensable for the dog, possibly because dogs have an endotheliochorial placenta type which is different from the hemochorial placentae of primates and rodents. semi-allogeneic fetal trophoblast cells are less invasive and concomitantly less prone to encounter the maternal immune system in endotheliochorial placentae in comparison to the hemochorial placentae where the trophoblast cells are bathed in maternal blood. therefore, psg could be involved in the regulation of trophoblast invasion and/or modulation of the maternal immune system as has been suggest before. alternatively, the function of the psg could have been taken over by an unrelated gene family due to functional convergence at the molecular level, similar to that found for natural killer cell receptors, where killer cell immunoglobulin-like receptors and c-type lectin-like receptors are used in primates and rodents, respectively. in contrast to the psg, the ceacam subgroup of canine ceacam1-related genes underwent a recent expansion similar to that found in primates and rodents. in general, igsf members which are expressed by leucocytes are diverging rapidly within their extracellular domains. this results in an average amino acid identity of about 54% between human and mouse orthologs. the conservation of the ceacam n domain, most relevant for pathogen and cell-cell interactions, lies within this range, with an amino acid identity of 56% and 42%. this is consistent with the notion that ceacam plays a key role in various functions of the immune system. in contrast, between mouse and human ceacam ceacam and ceacam orthologs, the n domain amino acid identities are higher ranging from 60% to 91%, which argues for non-immunological functions, especially for ceacam which exhibits an overall amino acid sequence identity of about 90%. the dog ceacam1-related genes are arranged head to tail, like the kir genes in the human kir locus. such an arrangement was found to facilitate unequal crossing over leading to gene expansion and generation of multigene families. furthermore, the kir gene family is subject to birth-and-death evolution, domain shuffling and mutational changes. our analyses indicate that the cea gene family evolved similarly. one characteristic for this kind of evolution is the presence of multiple pseudogenes, which also exist within the canine ceacam1-like gene locus. this type of evolution also facilitates a mechanism for qualitatively changing a receptor's signaling potential. a number of type i transmembrane igsf members including ceacams and kirs are composed of an amino-terminal extracellular "environmental sensor" and a carboxy-terminal cytoplasmic tail which can contain either inhibitory or activating motifs. unequal crossing-over at such gene loci encompassing the corresponding exons could result, in a single step, in genes encoding receptors with a drastically changed signaling potential by combining the same extracellular domain with a cytoplasmic tail of opposite function. this allows rapid adaptation to environmental changes and the creation of so-called paired immune receptors. an example for such a mechanism is found for the kir gene kir3dl/s, where either an inhibitory variant or an activating variant encoding kir gene is found in different human individuals at the same locus. in addition, a change in the receptor's signaling potential which may be the result of an arms race between pathogens and the host immune system has been reported recently for mice. the murine cytomegalovirus induces in infected cells expression of the virus-encoded gpi-anchored m protein, which binds to the activating, dap12-associating killer receptor ly49h in mcmv-resistant mice leading to nkcell-mediated cytotoxicity. mcmv-susceptible mouse strains do not have ly49h but the extracellularly very similar itim-bearing ly49i protein which has inhibitory killer cell receptor activity. binding of m to ly49i in such mice may inhibit nk cell cytotoxicity thus rendering the mice susceptible to mcmv infection. it was suggested that the activating receptor gene was derived from the gene encoding the inhibitory receptor to counteract a virus that exploits the inhibitory receptor during infection and that this mechanism may apply to many other activating receptors that have inhibitory counterparts. however, recently it was shown for the human siglec and siglec genes that the direction of gene conversion that generates paired immune receptors is not always the same. this indicates that also other needs drive the evolution of paired receptors like the fine tuning of immune responses. host-pathogen interactions involving both proteins with itim or itam signaling motifs have also been reported for humans. the primate-specific ceacam1-related protein ceacam which contains an n domain closely related to that of ceacam and a cytoplasmic tail with an itam is exclusively expressed in granulocytes. due to its close relatedness with the itim-bearing gonococcal receptor ceacam it can act as decoy receptor for neisserial pathogens and is able to induce clearance of gonococcal infections by granulocytes. mutational analyses demonstrated that the cytoplasmic itam is instrumental for this process. to our knowledge, ceacam and ceacam represent the first paired coexpressed immune receptors which contain antagonizing itim and itam in their respective cytoplasmic tails. ceacam was probably formed from a ceacam ancestor by a recent gene conversion encompassing the leader and the n exon from ceacam the latter exon encodes the region most instrumental for ligand and pathogen binding in ceacam molecules. several findings, presented here, argue for a role of ceacam as a ceacam decoy receptor in the dog. first, the direction of the gene conversion was most likely from the inhibitory receptor to the activating receptor, second, the ka/ks ratio of the n domain exons of these family members is indicative for a purifying selection, whereby the specificity of the putative ceacam ligand is probably conserved for ceacam third, the amino acid changes observed for the ceacam n domain most likely have no effect on the structure of that part of the n domain shown to be the pathogen binding site in human and mouse ceacam and fourth, is expressed by immune cells, a prerequisite for a defense function against a putative pathogen. based on a highly similar amino acid sequence and expression pattern ceacam can be envisioned to also function, like ceacam as a decoy receptor. however, this hypothesis raises questions about the driving force behind the fixation of the other itam-bearing ceacam1-related genes in the dog. most likely, selective pressure by pathogens changes with time. in times where the selective pressure exerted by pathogens on a receptor system is low, darwinian selection will act and change the specificity of a receptor now possibly recognizing an endogenous ligand. once the receptor has gained a new function it also can change the signaling properties which may explain why older i.e. more diverged itam-bearing receptors e.g. like ceacam and ceacam have lost the itam or have gained new splice variants which do not contain an itam.  taken together, the presence of multiple ceacam1-related dog ceacams with itams could reflect a repeated arms race between species-specific pathogens and the dog immune system. fixation and expression by effector cells of itam-containing ceacams would result from either bacterial pathogens binding to ceacam or viruses which induce the expression of ceacam ligands by infected cells as found for mcmv. this putatively pathogen-driven evolution of the cea gene family led to the development of the ceacam1/ceacam receptor pair which could also be involved in the fine tuning of t cell responses. 
one of these proteins, ceacam exhibits nearly complete sequence identity with the ligand-binding n domain of ceacam but antagonizing signaling motifs in the cytoplasmic tail. the direction of gene conversion accompanied by purifying selection and expression in immune cells suggests the possibility that ceacam evolved in response to selective pressure imposed by species-specific pathogens. thus, ceacam and ceacam are the first paired immune receptors identified within the cea gene family, which are expressed on t cells and are most likely involved in the fine-tuning of t cell responses. since little is known about the cea gene family in other lineages of placental mammals, we expected to gain new insights into the evolution of the rapidly diverging cea family by analyzing the cea family of the dog. comparison of nonsynonymous and synonymous substitutions indicates that the ceacam n domain is under the strongest purifying selection of all canine ceacam1-related ceacams. we found that the gene coding for the itim-bearing immunoregulatory molecule ceacam gave rise to a recent expansion of the canine cea gene family by gene duplication, similar to that previously found in humans and mice. here we describe the complete cea gene family in the dog. in addition, ceacam shows a similar expression pattern in resting immune cells and tissues as ceacam however, upon activation ceacam mrna and ceacam mrna are differentially regulated.
 although the impact of pathogens on the evolution of the mammalian immune system is still under debate, proteins, which both regulate immune responses and serve as cellular receptors for pathogens should be at the forefront of pathogen-driven host evolution. the cea gene family codes for such proteins and indeed shows tremendous species-specific variation between human and rodents. since little is known about the cea gene family in other lineages of placental mammals, we expected to gain new insights into the evolution of the rapidly diverging cea family by analyzing the cea family of the dog.  here we describe the complete cea gene family in the dog. we found that the gene coding for the itim-bearing immunoregulatory molecule ceacam gave rise to a recent expansion of the canine cea gene family by gene duplication, similar to that previously found in humans and mice. however, while the murine and human ceacams are predominantly secreted and gpi-anchored, respectively, in the dog, most of the ceacams represent itam-bearing transmembrane proteins. one of these proteins, ceacam exhibits nearly complete sequence identity with the ligand-binding n domain of ceacam but antagonizing signaling motifs in the cytoplasmic tail. comparison of nonsynonymous and synonymous substitutions indicates that the ceacam n domain is under the strongest purifying selection of all canine ceacam1-related ceacams. in addition, ceacam shows a similar expression pattern in resting immune cells and tissues as ceacam however, upon activation ceacam mrna and ceacam mrna are differentially regulated.  thus, ceacam and ceacam are the first paired immune receptors identified within the cea gene family, which are expressed on t cells and are most likely involved in the fine-tuning of t cell responses. the direction of gene conversion accompanied by purifying selection and expression in immune cells suggests the possibility that ceacam evolved in response to selective pressure imposed by species-specific pathogens. 