BACKGROUND
immune responses are regulated by a balance of opposing signals delivered from leukocyte surface molecules  <cit> .
in the mammalian immune system, several families of activating and inhibitory receptors form an elaborated regulatory network that tightly affects all stages of immune responses.
the evolutionary history of this network is poorly understood.
while "pairing" of receptors into the inhibitory and activating forms appears to have occurred in invertebrates, there is no clear evolutionary continuity between invertebrate and vertebrate receptor systems  <cit> .
furthermore, ambiguity of relationships is often observed for paired receptors from different lineages of vertebrates  <cit> .
classical fc receptors  and killer cell immunoglobulin receptors  constitute two families that are prototypic for the paradigm of immune regulation through integration of activating and inhibitory signals.
members of each family fall into two main signaling classes.
the inhibitory receptors contain itims in their cytoplasmic tails, while the activating receptors associate with the itam-bearing transmembrane signal subunits, such as fcrγ  or dap <dig> .
fcrs are widely expressed on various leukocyte subsets.
they regulate phagocytosis, cytokines release, antibody-dependent cell mediated cytotoxicity, and antibody synthesis  <cit> .
kirs play a crucial role in regulation of human nk cell cytotoxicity via recognition of mhc class i antigens on the surface of target cells  <cit> .
during the last decade, it has been recognized that fcrs and kirs belong to large families comprised of structurally related yet highly diverse proteins.
thus far, eight human and six mouse fcr-like  genes have been described  <cit> .
two of them, designated fcrla and fcrlb according to the new nomenclature  <cit> , are intracellular proteins composed of three ig-like domains and a c-terminal mucin-like domain.
six human  and three mouse  genes code for cell surface receptors with the extracellular regions  composed of two to nine ig-like domains and intracellular regions bearing different patterns of the itim-, itsm- and itam-like motifs.
apart from the fcr-characteristic d <dig>  d <dig> and d <dig> subtypes, two new structural ig-like domain subtypes, d <dig> and d <dig>  have been identified in these proteins.
furthermore, one of the novel mouse genes, fcrls, encodes a soluble mosaic protein containing a scavenger domain  <cit> .
studies of the kir family have also revealed its considerable structural and functional heterogeneity.
human kir-like proteins  include cell surface receptors of the lilr  family as well as fcαr, gpvi, nkp <dig>  oscar, lair <dig> and lair <dig>  <cit> .
the lilr family consists of both inhibitory and activating forms: lair- <dig> is an inhibitory receptor, lair <dig> is soluble, the others are activating.
like fcrs but unlike kirs, the activating lilr receptors, as well as fcαr, oscar, nkp <dig>  and gpvi associate with the fcrγ subunit  <cit> .
however, the transmembrane regions  of activating kirls are structurally different from those of fcrs.
intriguingly, the repertoires of the fcr- and kir-related proteins are different from one species to another in higher vertebrates.
for instance, each of the six human and four mouse extracellular fcrls has a unique domain architecture  <cit> .
functional equivalents of kirs in rodents are c-type lectin receptors of the ly <dig> family  <cit> .
the mouse also lacks counterparts of fcαr and lair- <dig> and has fewer lilr homologues described as pirs  <cit> .
profound differences in the kir and fcr families have been also revealed between mammals and birds.
recent data show that the chicken genome has more than a hundred genes for kir-like paired receptors known as chirs  <cit> .
at the same time, a single fcr-related gene has been detected in this species  <cit> .
comparison of the 3-d structure of membrane-proximal domains of fcγrii and kir2d demonstrated their similar folding and prompted a suggestion that the two families may have had a common origin  <cit> .
this suggestion was made before identification of fcrls.
a later phylogenetic analysis did not provide solid support in favor of homology of five fcrl-characteristic ig-domain subtypes with the two main domain subtypes of kir-related receptors  <cit> .
nevertheless, this idea of the common ancestry of the fcr and kir families has been revived in modified form after the recent identification of a family of paired receptors called leukocyte immune-type receptors  in catfish  <cit> .
litrs are composed of several domain subtypes some of which resemble the fcr-characteristic domains d1d <dig>  whereas others are more similar to kirl domains.
such composition of ig domains has been proposed as ancestral for the tetrapod paired receptors  <cit> .
however, weak sequence similarity between litr and kirl ig domains, as well as the absence of the d <dig>  d <dig> and d <dig> type domains in litrs did not allow conclusions about definite relationships of the teleostean proteins with the higher vertebrate fcr and kir families.
the fact that all known fcr- and kir-related receptors are primarily expressed in cells of the immune system is consistent with their contribution to the immune regulation.
however, the exact functions of all fcrls and many kirls are still unknown.
the ambiguity of the structural and functional evolution of fcr- and kir-related receptors complicates our understanding of how this regulatory network is organized, which factors drives its species-specific changes and ultimately how it may be manipulated for therapeutic purposes.
to gain deeper insight into the evolution of the immunoregulation through paired receptors, we studied the fcr family in the amphibians xenopus laevis and xenopus tropicalis.
the data obtained provide evidence in favor of a common origin of the fcr and kir families and their ceaseless diversification that appears to be caused by very strong natural selection pressure.
RESULTS
the fcr family is expanded in amphibians
during our studies of the human and mouse fcr-like genes we observed that est databases contain numerous x. tropicalis and x. laevis cdnas encoding proteins structurally similar to mammalian fcrs and fcrls.
the degree of amino acid sequence identity ranged from  <dig> to 43% for different domain subtypes.
we designated these genes xfl .
the recent sequencing of the x. tropicalis genome provided an opportunity to examine the organization and structure of the xfl genes in more details.
we used in silico analysis of the version  <dig>   <dig> and  <dig> genomic sequences deposited at the jgi website.
we did not consider the consortium gene models in this survey.
direct application of gene prediction programs to genomic sequences often results in erroneous models.
to overcome this pitfall, we first identified exons coding for the xfl ec and tm domains using the tblastn search with amino acid sequences of the corresponding x. tropicalis and x. laevis est cdnas or mammalian fcr-like proteins.
the identified x. tropicalis sequences were used in the second round of the computational screening to reveal exons that might have been overlooked in the first round.
the procedure was repeated until no novel exons were identified.
the exons lacking frame-shift mutations or stop codons were examined for the presence of the ag and gt splice signals matching the phase  <dig> rule.
thereafter, the gene models were generated using both automatic and manual procedures.
the exons for the tm regions served as the gene delimiters.
this approach resulted in finding several hundred exons on  <dig> scaffolds.
of these,  <dig> scaffolds contained 1– <dig> exons that may either represent misassembled gene regions, gene fragments or pseudogenes.
the exons on  <dig> other scaffolds could be arranged in at least  <dig> xfl genes.
fig.
<dig> shows the predicted organization and exon/intron arrangement of these genes.
the exons encoding the signal peptides and cytoplasmic regions are not shown in this scheme because of poor accuracy of their prediction.
nevertheless, in certain cases, such exons could be delineated on the basis of alignment of the genomic sequences with the est cdnas.
at the time, the est databases contained x. tropicalis cdnas corresponding to  <dig> xfl genes.
the signal peptides were invariably encoded by two exons, like in the mammalian fcr-like genes.
in a fraction of xfl genes, the exons for signal peptides met the rule 30/ <dig> bp , which is characteristic of mammalian fcrl1- <dig>  fcγri and fcrlb genes.
the xfl cytoplasmic regions are encoded by one to five exons.
in the mammalian genomes, fcr-like genes are linked to the genes of the cd <dig> family.
thus, the fcrl <dig> gene is a part of a conserved syntenic group that includes the slamf <dig> , igsf <dig>  dusp <dig>  tagln <dig> and nges <dig> genes  <cit> .
we found a similar group in the scaffold  <dig> that contains  <dig> xfl genes .
the xfl gene 626_ <dig> is located between two cd2-like genes, one of which shows the greatest similarity to mammalian slamf <dig> .
xenopus homologs of the mammalian igsf <dig>  dusp <dig>  tagln <dig> and nges <dig> genes are also tightly linked.
this conserved synteny taken together with the results of the sequence comparisons  and phylogenetic analysis  strongly supports the assignment of the xfl genes as true amphibian homologs of the mammalian fcr genes.
the xfl receptors are subdivided into two classes
with a few exceptions, the predicted xfl genes code for type i cell surface receptors .
their tm regions fall into two structural types that we designated tm <dig> and tm <dig>  the characteristic feature of tm <dig> is the presence of a conserved nxxr motif at the n-termini.
tm <dig> lacks charged residues.
interestingly, tm <dig> regions are highly homologous to the tm regions of some kirls such as lilra <dig>  pir-a, ncr1/nkp <dig>  gpvi, oscar, and fcαr .
all these proteins are known to associate with the fcrγ signal subunit  <cit> .
it is important to stress that tms of classical activating fcrs are quite different, and bear a typical conserved structural motif fxxtxl  <cit> .
despite the extensive search, we did not find exons for tm regions with such a signature in the x. tropicalis genome, or in the available xenopus est cdnas.
our phylogenetic analysis supports a close relationship of the tm regions of the xfl and kirl proteins.
as shown in fig 3b, the nxxr motif-containing tms and tms of activating classical fcrs form two distinct clusters.
the tm region of dap12-associating kir2ds, a member of the human kir family of mhc class i-specific nk cell receptors, is not related to either of these groups.
comparison of the x. tropicalis genomic and est sequences showed that the tm1-containing proteins lack a cytoplasmic tail or have a very short one.
in every such case, the tm region and the tail are encoded by the same exon.
genes with a tm <dig> have longer cytoplasmic tails.
these tails contain one to three tyrosine-based motifs matching the consensus yxxl/i/v and are encoded by exons separated from tm exons.
all these structural features are compatible with the subdivision of the xfl receptors into two functional classes, activating and inhibitory.
the number of genes for each class is roughly similar and they are intermingled in the genome .
the ec regions of xfls are highly diverse
a remarkable feature of the predicted xfl proteins is an extraordinary diversity of their domain architectures.
overall  <dig> different combinations of six structural subtypes of the ig-like domains in the ec regions of xfls were found.
five subtypes were assigned to d1-d <dig> subtypes previously identified in the mammalian members of the fcr family.
a sixth subtype appears to be xenopus-specific as no close relatives were found in the protein databases.
the d <dig> subtype domain is most frequent and may be repeated up to  <dig> times in a protein.
although we cannot rule out that some of the gene predictions result from genome assembly artifacts, we found high similarity between the expressed and genomic sequences.
the est-genome comparisons showed the absence of two exons in the genomic sequences.
in both cases, close inspection demonstrated the presence of gaps in the corresponding genomic regions.
eleven of  <dig> cdnas fully matched our gene models.
this fact, together with the absence of gaps in many predicted genes and reiteration of certain domain architectures two or more times suggest a high degree of confidence in the proposed models.
among the predicted proteins there are typical pairs with identical ectodomains and distinct tm subtypes.
.
the ec regions of  <dig> xfls are composed of d <dig>  d <dig>  and d <dig> domains, like mammalian fcγri.
this is the only ec composition shared by the known mammalian and xenopus fcr-like proteins.
if we consider structural subtypes of the tm regions as distinct domains, up to  <dig> domain architectures may be distinguished among the xfl proteins, none of which are present among mammalian members of the fcr family.
lineage-specific expansion of xenopus and mammalian fcr families
while the attribution of the xfl proteins to the fcr family is unequivocal according to the reciprocal sequence comparisons and protein database analysis, it remains unclear how xenopus and mammalian proteins are related to each other.
to assess such relationships, we generated a series of phylogenetic trees with the mega <dig> software package  <cit> .
for this purpose, amino acid sequences of the ig domain subtypes from all the predicted xfls were aligned .
trees were generated using the nj and me methods.
to simplify the trees, the aligned blocks of d <dig>  d <dig> and d <dig> domains were reduced by removing redundant sequences with close association.
thereafter, the sequences for all the xfl domain subtypes were aligned together with the domain sequences of the human proteins, and their relationships were analyzed by the same procedure.
the final tree is shown on fig  <dig>  the tree topology supports subdivision of the xenopus and human ig domains into five common  and one xenopus-specific  subtypes.
most importantly, the tree shows separate clustering of the xenopus and human sequences.
this branching pattern suggests that duplications of the fcr-like genes in amphibian and mammalian lineages were lineage-specific and that separation of the mammalian genes into classical fcrs and fcrls occurred after the split of the amphibian and mammalian lineages.
the relationships among the xfl genes provide evidence for a complex pattern of family evolution in amphibians.
its detailed description is beyond the scope of the present paper and will be published separately.
what is relevant to note here, is that the strong association of the amphibian sequences with each other does not necessarily correspond to high level of sequence similarity among them.
the cumulative tree illustrates subdivision of the xfl d <dig>  d <dig> and d <dig> domains into structural variants whose relationships with each other cannot be resolved.
the d <dig>  d <dig> and d <dig> domain subtypes fall into  <dig>   <dig> and  <dig> structural groups, respectively.
the degree of sequence similarity among the group representatives  is in the range of sequence similarity between xfl and mammalian fcr domains.
in the case of d <dig>  the diversity is mainly derived from a small number of genes located in the scaffolds  <dig>   <dig>   <dig> and  <dig>  for instance, the extracellular parts of the predicted proteins 1131_ <dig> and 1131_ <dig> are composed of five d <dig> domains each.
these domains are subdivided into four groups.
each of five d <dig> domains of the xfl 1256_ <dig> represents a distinct structural variant producing a separate branch in the tree.
on the other hand, more than  <dig> proteins encoded by the genes of the other scaffolds have one to seven d <dig> domains belonging to the same group .
this group may be further subdivided into three main subgroups  <dig> ,  <dig>  and  <dig>  and a number of individual members based on the structure of the d <dig> and d <dig> domains.
for instance, the proteins encoded by the genes on the scaffold  <dig> have very similar d <dig> domains but their d <dig> and d <dig> domains fall into  <dig> structural variants with poorly resolved relationships.
this fact strongly suggests that intergenic exon recombination was a frequent event in the evolution of the xfl family.
of ten genes containing exons for the d <dig>  d <dig> or d <dig> domain subtypes, nine belong to the subgroup  <dig>  and one to the subgroup  <dig> .
interestingly subgroups  <dig>  and  <dig>  differ in patterns of amino acid replacement in the d <dig> and d <dig> domains.
the d <dig> domains of subgroup  <dig>  are characterized by extensive variation in the length and sequence of the fg loop, the equivalent of cdr <dig> of the v-type domains .
the region covering the strands c to f is well conserved.
in contrast, the d <dig> domains of subgroup  <dig>  show more variation in the region between the c' and f strands.
their f-g region is relatively conserved.
the d <dig> domains of the  <dig>  and  <dig>  subgroups also display variability at different sites  suggesting the existence of at least two classes of ligands for the group i receptors.
the d <dig> and d <dig> domains are implicated in binding to igg and ige by classical fcrs.
however, the residues known to contact the fc portion of ig are not conserved in the xfls sequences,
experimental support of xfl diversity
to gain a deeper insight into structure and expression of the xfl genes, we studied this family in x. laevis, a close relative of x. tropicalis.
in contrast to x. tropicalis that has a diploid genome, x. laevis is an allotetraploid species.
the immune system of x. laevis is one of the most thoroughly studied among lower vertebrates  <cit> .
five different x. laevis cdnas for xfl proteins were obtained from the image consortium and sequenced.
more than  <dig> cdnas,  <dig> of which were unique, were additionally cloned from several x. laevis cdna libraries using screening with an exon encoding the d <dig> domain of group  <dig> as a probe.
of  <dig> distinct cdnas,  <dig> were full-length, the others were truncated at the 5' end.
nine cdnas encoded typical cell surface proteins containing tm2-like tm regions and cytoplasmic tails of varying length with one to three tyrosine-based motifs .
the amino acid sequences of two clones had short cytoplasmic tails.
their tm regions contained the nxxr motif and were assigned to the tm <dig> subtype.
one cdna clone encoded protein lacking tm but containing a typical cytoplasmic tail with two tyrosine-based motifs.
finally, four cdnas coded for putative secreted proteins composed of the ig-like domains only.
at present, it is unclear whether or not the latter five clones represent alternative transcripts of genes encoding cell surface receptors.
as expected, the initial sequence comparisons demonstrated that most of x. laevis derived xfls may be joined into group  <dig>  their d <dig> domains shared  <dig> to 95% identical residues with each other and with the x. tropicalis d <dig> domains of the group  <dig> receptors.
these proteins were designated xfl <dig>  –  <dig> .
as in the case of x. tropicalis, the x. laevis group  <dig> proteins showed variable degree of identity  in their d <dig> and d <dig> domains.
two other proteins were designated xfl <dig> and xfl <dig>  their d <dig> domains shared 35–45% identical residues with each other and with the d <dig> domains of the group  <dig> proteins.
according to the phylogenetic analysis , the x. laevis xfl <dig> and xfl <dig> genes were most similar to the x. tropicalis 435_ <dig> and 1131_ <dig>   <dig>   <dig> genes respectively.
to estimate the genomic complexity of the xfl family in x. laevis, we performed southern blot hybridization using the d1-exon of xfl <dig> and d3-exons of the xfl <dig>  and xfl <dig> genes as probes under non-stringent conditions.
multiple  hybridizing bands were revealed on the blots probed with the d3-exon of the xfl <dig>  gene .
the xfl2- and xfl3-specific probes revealed one and three hybridizing bands, respectively.
these results demonstrated that the cloned xfl genes constitute only a part of the family and that, like in x. tropicalis, most of the x. laevis xfl genes appear to belong to the group  <dig>
x. laevis xfl genes are primarily expressed in lymphoid tissues
to assess the xfl expression pattern, we performed northern blot hybridization of total rna from various tissues of adult frogs.
the exon for d <dig> domain of the group i was used as a probe at low stringency conditions.
as expected, northern blotting revealed diffuse bands representing multiple gene transcripts.
the highest signal intensity was observed in the spleen and thymus .
to examine expression patterns of the individual genes, we designed gene-specific primers for seven different xfl cdna.
to exclude possible cross matching, the 3'-untranslated sequences were mainly used for the reverse primers.
the rt-pcr analysis demonstrated that the tissue distribution of the corresponding mrna is variable .
the transcripts of all the genes were mainly detected in lymphoid  and non-lymphoid tissues containing cells of haemopoietic origin .
expression of the xfl <dig>  xfl <dig>  and xfl <dig>  genes was detected in brain.
the tissue distribution of mrna in tadpoles was slightly broader.
in particular, all xfls tested except  <dig> , were detected in the gills which is known to be a very active immunological site owing to intense blood circulation and high exposure to antigens  <cit> .
furthermore, expression of the xfl <dig>  gene was found only in tadpole spleen.
different pattern of the xfl transcript distribution in adults and larvae suggest that the expression of at least a proportion of the xfl genes is developmentally regulated.
nt – not tested.
results are indicative of rt-pcr performed on total rna extracts from different tissue samples .
tm <dig> facilitates xfl association with the fcrγ subunit
the presence of the nxxr motif-bearing tms in many xfls suggested that, like the mammalian activating kirls, these xenopus receptors may require fcrγ chain for cell surface expression and/or signal transduction.
to examine whether this is the case, we generated a series of constructs enabling expression of xfl <dig>  xfl <dig>  and the x. laevis fcrγ subunit as recombinant epitope-tagged proteins.
xfl <dig> and xfl <dig>  were expressed in 293t cells as recombinant hemagglutinin -tagged proteins containing either their own tm <dig> regions or the tm region of pdgfr.
the x. laevis fcrγ chain was tagged with c-myc epitope.
single transfection of xfl2-ha did not induce its surface expression, and the protein accumulated intracellularly as determined by immunofluorescent microscopy of permeabilized cells.
however, the surface expression of xfl <dig> was restored when it was co-transfected with fcrγ .
in contrast to xfl <dig>  xfl <dig> -ha was targeted to the surface of the transfected cells in the absence of the adapter molecule, although its surface expression was increased two-fold in the presence of fcrγ.
both xfl <dig> and xfl <dig>  proteins were readily expressed on the cell surface when their ec regions were fused with a tm of pdgfr.
these results show that xfl molecules containing a tm <dig> region with the nxxr motif associate with the fcrγ chain.
the surface expression of xfl <dig>  is less dependent on the presence of fcrγ chain.
this may be explained by divergent structure of their tms.
such differences also have been observed among the fcrγ-associating kirls; fcrγ is critical for surface targeting of lilra <dig>  but not fcαr or oscar  <cit> .
discussion
comparative studies of mammals and chicken have revealed an unexpected structural and functional variability of the paired receptor families such as fcrls and kirls  <cit> .
studies have also indicated that the repertoires of these families have evolved in a species-specific manner.
the evolutionary factors responsible for such diversity remain poorly understood.
the recent description of litrs as putative teleost counterparts of both fcrls and kirls left many questions unanswered, since only a weak similarity of litrs to the mammalian and avian receptors has been found  <cit> .
the present study fills the gap by extending the analysis of paired receptor families to amphibians, the most primitive branch of tetrapods.
in the diploid x. tropicalis we have identified at least  <dig> genes coding for paired fcr-like cell surface receptors.
the mere fact of the family expansion is not unusual.
rapid evolutionary change of a gene content known as the "expansion-contraction" or "birth-and-death" process has been documented in many families of immunity-related receptors  <cit> .
what distinguishes the fcr family from many other paired receptor families is the extraordinary structural diversity of its members.
combinatorial joining of six ig domain subtypes generates as many as  <dig> ec architectures.
when we consider the tm subtypes as distinct domains, the number of the xfl domain architectures increases to  <dig>  none of these variants are shared by either human or mouse homologs, although five ig-domain subtypes are common for the xenopus and mammalian proteins.
overall,  <dig> different domain architectures can be defined among human, mouse, and x. tropicalis fcr-related proteins.
except for the d1- and d2-subtype domains no homologous structural elements were found between xfls and catfish litrs .
other ig-domain subtypes composing ec of litrs seem to be more similar to the kirl domains.
such mixed domain composition has been suggested to predate the kir and fcr families  <cit> .
the xfl data are consistent with the suggestions that the fcr and kir families share evolutionary roots  <cit> .
a strong argument in favor of this model is the fact that activating fcrs use a peculiar tm module  to associate with fcrγ chain that is homologous to tms of activating mammalian kirls.
tm <dig> appears to be an ancestral element of the primordial fcr/kir family that has been lost by chirs, classical fcrs and kirs, but retained by xfls as well as xenopus and mammalian kirls.
although available data do not allow inferring the structure of the kirl and fcrl ancestor, it is clear that the family evolved by inter- and intragenic recombinations in a species-specific way.
the former mechanism gave rapid change in the number of genes per family , whereas the latter was responsible for extensive domain shuffling.
the fcr-related receptors in different vertebrate species are similar in their subdivision into activating and inhibitory forms and predominant expression in lymphoid tissues.
however, the ratio of inhibitory to activating members, the cellular distribution, and the exact amount and architecture of ectodomains are unique in each examined species.
what might be the evolutionary forces responsible for this degree of diversity common among the fcr- and kir-related receptors, and xfls in particular?
we can try to answer this question by inference from the attributed function of the actual mammalian receptors, which is to regulate immune responses.
classical fcrs regulate b cell responses by binding to the igg and ige immune complexes, whereas kirs, or at least their inhibitory forms, regulate nk cell function by binding specifically to mhc class i molecules.
however, ig-binding appears to be a secondary or  specialization, since our previous  <cit>  and present data together with the definition of a chicken igy receptor as a member of the chir family  <cit>  strongly argue that classical fcrs have emerged after the separation of mammals and birds.
mhc-recognition as a potential ancient function of fcrls/kirls is more attractive.
the ability to interact with classical and non-classical mhc class i molecules is a feature of some kirls  <cit> , it has been also suggested for mammalian fcrls  <cit>  and catfish litrs  <cit> .
from this point of view the diversification of fcrl/kirls may have been driven by the necessity to match the rapid evolution of mhc loci under pathogen pressure of, as it has been suggested for the kir and ly <dig> gene clusters .
this may be the case for xfls too as there are at least  <dig> non-classical mhc class i in x. laevis  <cit> .
there are however some inconsistencies with this scenario.
first, the scope of variability of domain architectures among fcrls seems to be excessively high relative to mhc class i molecular structure.
second, the functions of mammalian kirls are not limited to mhc antigen-binding.
among ligands of these receptors, there are also collagens , iga , igg  and integrins   <cit> .
human α1-b-glycoprotein, a distant secreted relative of kirls, binds to the cysteine-rich secretory protein  <dig>  <cit> , while its opossum homolog is a snake venom metalloproteinase-neutralizing factor  <cit> .
due to the independent expansion of kir-related receptors in mammals, birds  <cit>  and amphibians  it is difficult to determine which of these functions are truly ancient or of ancestral type.
finally, mouse ly <dig> is a clear example of the self-mhc recognition served by receptors structurally different from kirls and fcrls.
an alternative explanation for the extraordinary diversity of xfls and other fcr- and kir-related receptors may be that they are directly involved in innate immunity.
combinatorial diversity is a hallmark of the immune system and it is usually associated with recognition of pathogens.
the capacity of paired receptors to directly bind to pathogens is well documented  <cit> .
in the latest of these studies it has been found that mouse pir-b, and its human relatives lilrb <dig> and lilrb <dig> recognize staphylococcus aureus and modulate tlr-mediated inflammatory responses against this bacterium.
these facts are usually interpreted in terms of adaptive coevolution of the microorganisms, which implies that the pathogen recognition is a secondary or derived function.
however, the extensive variability of the fcr/kir relatives raises the possibility that these receptors expanded primarily to fight pathogens, whereas the known immuno-regulatory functions may represent secondary acquisitions or specializations.
depending on the nature of the pathogen and the signal properties of the receptors, it is clear that pathogen-receptor interaction may be either advantageous or detrimental for the host, and as such, may rapidly change the ratio of activating versus inhibitory receptors, as well as their respective amount and specificities.
in this regard, a parallel may be drawn with the species-specific expansion of various receptor families in invertebrates that participate in innate immune responses  <cit> .
while it may be difficult to obtain direct evidence to support this scenario, it clearly deserves attention.
the elucidation of the factors responsible for the diversification of the fcr- and kir-related receptors may contribute to better understand their function and ultimately develop new therapies based on their immunoregulatory properties.
CONCLUSIONS
our study shows that in two amphibian species xenopus tropicalis and x. laevis, paired receptors have diversified into a large family of genes, xfls, preferentially expressed in lymphoid tissues.
the extracellular regions of these receptors are composed of one to eleven ig-like domains belonging to six structural subtypes.
a fraction of xfls use a tm module  to associate with fcrγ signaling subunit.
tm <dig> is highly similar to tms of activating fcrγ-associating kirls.
this fact strongly argues in favor of a common evolutionary origin of the fcr and kir families.
the variation in number and composition of distinct ig-like and tm domain subtypes generates striking diversity of domain architectures among xfls.
phylogenetic analysis shows that this diversity emerged in a lineage-specific manner.
classical fcrs and other known mammalian fcr-related proteins appear to be specific to mammals.
the continual and extensive diversification of domain architectures in the fcr and kir families indicates a strong selection pressure not completely consistent with the usual assumption that paired receptors have been primarily selected to regulate immune responses.
we propose that fcr/kir-related receptors might have primarily expanded as pathogen-recognizing components of innate immunity while their known physiological functions have been acquired later in a lineage-specific manner.

 immune responses are regulated by a balance of opposing signals delivered from leukocyte surface molecules. in the mammalian immune system, several families of activating and inhibitory receptors form an elaborated regulatory network that tightly affects all stages of immune responses. the evolutionary history of this network is poorly understood. while "pairing" of receptors into the inhibitory and activating forms appears to have occurred in invertebrates, there is no clear evolutionary continuity between invertebrate and vertebrate receptor systems. furthermore, ambiguity of relationships is often observed for paired receptors from different lineages of vertebrates. classical fc receptors and killer cell immunoglobulin receptors constitute two families that are prototypic for the paradigm of immune regulation through integration of activating and inhibitory signals. members of each family fall into two main signaling classes. the inhibitory receptors contain itims in their cytoplasmic tails, while the activating receptors associate with the itam-bearing transmembrane signal subunits, such as fcrγ or dap. fcrs are widely expressed on various leukocyte subsets. they regulate phagocytosis, cytokines release, antibody-dependent cell mediated cytotoxicity, and antibody synthesis. kirs play a crucial role in regulation of human nk cell cytotoxicity via recognition of mhc class i antigens on the surface of target cells. during the last decade, it has been recognized that fcrs and kirs belong to large families comprised of structurally related yet highly diverse proteins. thus far, eight human and six mouse fcr-like genes have been described. two of them, designated fcrla and fcrlb according to the new nomenclature, are intracellular proteins composed of three ig-like domains and a c-terminal mucin-like domain. six human and three mouse genes code for cell surface receptors with the extracellular regions composed of two to nine ig-like domains and intracellular regions bearing different patterns of the itim-, itsm- and itam-like motifs. apart from the fcr-characteristic d d and d subtypes, two new structural ig-like domain subtypes, d and d have been identified in these proteins. furthermore, one of the novel mouse genes, fcrls, encodes a soluble mosaic protein containing a scavenger domain. studies of the kir family have also revealed its considerable structural and functional heterogeneity. human kir-like proteins include cell surface receptors of the lilr family as well as fcαr, gpvi, nkp oscar, lair and lair. the lilr family consists of both inhibitory and activating forms: lair- is an inhibitory receptor, lair is soluble, the others are activating. like fcrs but unlike kirs, the activating lilr receptors, as well as fcαr, oscar, nkp and gpvi associate with the fcrγ subunit. however, the transmembrane regions of activating kirls are structurally different from those of fcrs. intriguingly, the repertoires of the fcr- and kir-related proteins are different from one species to another in higher vertebrates. for instance, each of the six human and four mouse extracellular fcrls has a unique domain architecture. functional equivalents of kirs in rodents are c-type lectin receptors of the ly family. the mouse also lacks counterparts of fcαr and lair- and has fewer lilr homologues described as pirs. profound differences in the kir and fcr families have been also revealed between mammals and birds. recent data show that the chicken genome has more than a hundred genes for kir-like paired receptors known as chirs. at the same time, a single fcr-related gene has been detected in this species. comparison of the 3-d structure of membrane-proximal domains of fcγrii and kir2d demonstrated their similar folding and prompted a suggestion that the two families may have had a common origin. this suggestion was made before identification of fcrls. a later phylogenetic analysis did not provide solid support in favor of homology of five fcrl-characteristic ig-domain subtypes with the two main domain subtypes of kir-related receptors. nevertheless, this idea of the common ancestry of the fcr and kir families has been revived in modified form after the recent identification of a family of paired receptors called leukocyte immune-type receptors in catfish. litrs are composed of several domain subtypes some of which resemble the fcr-characteristic domains d1d whereas others are more similar to kirl domains. such composition of ig domains has been proposed as ancestral for the tetrapod paired receptors. however, weak sequence similarity between litr and kirl ig domains, as well as the absence of the d d and d type domains in litrs did not allow conclusions about definite relationships of the teleostean proteins with the higher vertebrate fcr and kir families. the fact that all known fcr- and kir-related receptors are primarily expressed in cells of the immune system is consistent with their contribution to the immune regulation. however, the exact functions of all fcrls and many kirls are still unknown. the ambiguity of the structural and functional evolution of fcr- and kir-related receptors complicates our understanding of how this regulatory network is organized, which factors drives its species-specific changes and ultimately how it may be manipulated for therapeutic purposes. to gain deeper insight into the evolution of the immunoregulation through paired receptors, we studied the fcr family in the amphibians xenopus laevis and xenopus tropicalis. the data obtained provide evidence in favor of a common origin of the fcr and kir families and their ceaseless diversification that appears to be caused by very strong natural selection pressure.  the fcr family is expanded in amphibians during our studies of the human and mouse fcr-like genes we observed that est databases contain numerous x. tropicalis and x. laevis cdnas encoding proteins structurally similar to mammalian fcrs and fcrls. the degree of amino acid sequence identity ranged from to 43% for different domain subtypes. we designated these genes xfl. the recent sequencing of the x. tropicalis genome provided an opportunity to examine the organization and structure of the xfl genes in more details. we used in silico analysis of the version and genomic sequences deposited at the jgi website. we did not consider the consortium gene models in this survey. direct application of gene prediction programs to genomic sequences often results in erroneous models. to overcome this pitfall, we first identified exons coding for the xfl ec and tm domains using the tblastn search with amino acid sequences of the corresponding x. tropicalis and x. laevis est cdnas or mammalian fcr-like proteins. the identified x. tropicalis sequences were used in the second round of the computational screening to reveal exons that might have been overlooked in the first round. the procedure was repeated until no novel exons were identified. the exons lacking frame-shift mutations or stop codons were examined for the presence of the ag and gt splice signals matching the phase rule. thereafter, the gene models were generated using both automatic and manual procedures. the exons for the tm regions served as the gene delimiters. this approach resulted in finding several hundred exons on scaffolds. of these, scaffolds contained 1– exons that may either represent misassembled gene regions, gene fragments or pseudogenes. the exons on other scaffolds could be arranged in at least xfl genes. fig. shows the predicted organization and exon/intron arrangement of these genes. the exons encoding the signal peptides and cytoplasmic regions are not shown in this scheme because of poor accuracy of their prediction. nevertheless, in certain cases, such exons could be delineated on the basis of alignment of the genomic sequences with the est cdnas. at the time, the est databases contained x. tropicalis cdnas corresponding to xfl genes. the signal peptides were invariably encoded by two exons, like in the mammalian fcr-like genes. in a fraction of xfl genes, the exons for signal peptides met the rule 30/ bp, which is characteristic of mammalian fcrl1- fcγri and fcrlb genes. the xfl cytoplasmic regions are encoded by one to five exons. in the mammalian genomes, fcr-like genes are linked to the genes of the cd family. thus, the fcrl gene is a part of a conserved syntenic group that includes the slamf, igsf dusp tagln and nges genes. we found a similar group in the scaffold that contains xfl genes. the xfl gene 626_ is located between two cd2-like genes, one of which shows the greatest similarity to mammalian slamf. xenopus homologs of the mammalian igsf dusp tagln and nges genes are also tightly linked. this conserved synteny taken together with the results of the sequence comparisons and phylogenetic analysis strongly supports the assignment of the xfl genes as true amphibian homologs of the mammalian fcr genes. the xfl receptors are subdivided into two classes with a few exceptions, the predicted xfl genes code for type i cell surface receptors. their tm regions fall into two structural types that we designated tm and tm the characteristic feature of tm is the presence of a conserved nxxr motif at the n-termini. tm lacks charged residues. interestingly, tm regions are highly homologous to the tm regions of some kirls such as lilra pir-a, ncr1/nkp gpvi, oscar, and fcαr. all these proteins are known to associate with the fcrγ signal subunit. it is important to stress that tms of classical activating fcrs are quite different, and bear a typical conserved structural motif fxxtxl. despite the extensive search, we did not find exons for tm regions with such a signature in the x. tropicalis genome, or in the available xenopus est cdnas. our phylogenetic analysis supports a close relationship of the tm regions of the xfl and kirl proteins. as shown in fig 3b, the nxxr motif-containing tms and tms of activating classical fcrs form two distinct clusters. the tm region of dap12-associating kir2ds, a member of the human kir family of mhc class i-specific nk cell receptors, is not related to either of these groups. comparison of the x. tropicalis genomic and est sequences showed that the tm1-containing proteins lack a cytoplasmic tail or have a very short one. in every such case, the tm region and the tail are encoded by the same exon. genes with a tm have longer cytoplasmic tails. these tails contain one to three tyrosine-based motifs matching the consensus yxxl/i/v and are encoded by exons separated from tm exons. all these structural features are compatible with the subdivision of the xfl receptors into two functional classes, activating and inhibitory. the number of genes for each class is roughly similar and they are intermingled in the genome. the ec regions of xfls are highly diverse a remarkable feature of the predicted xfl proteins is an extraordinary diversity of their domain architectures. overall different combinations of six structural subtypes of the ig-like domains in the ec regions of xfls were found. five subtypes were assigned to d1-d subtypes previously identified in the mammalian members of the fcr family. a sixth subtype appears to be xenopus-specific as no close relatives were found in the protein databases. the d subtype domain is most frequent and may be repeated up to times in a protein. although we cannot rule out that some of the gene predictions result from genome assembly artifacts, we found high similarity between the expressed and genomic sequences. the est-genome comparisons showed the absence of two exons in the genomic sequences. in both cases, close inspection demonstrated the presence of gaps in the corresponding genomic regions. eleven of cdnas fully matched our gene models. this fact, together with the absence of gaps in many predicted genes and reiteration of certain domain architectures two or more times suggest a high degree of confidence in the proposed models. among the predicted proteins there are typical pairs with identical ectodomains and distinct tm subtypes.. the ec regions of xfls are composed of d d and d domains, like mammalian fcγri. this is the only ec composition shared by the known mammalian and xenopus fcr-like proteins. if we consider structural subtypes of the tm regions as distinct domains, up to domain architectures may be distinguished among the xfl proteins, none of which are present among mammalian members of the fcr family. lineage-specific expansion of xenopus and mammalian fcr families while the attribution of the xfl proteins to the fcr family is unequivocal according to the reciprocal sequence comparisons and protein database analysis, it remains unclear how xenopus and mammalian proteins are related to each other. to assess such relationships, we generated a series of phylogenetic trees with the mega software package. for this purpose, amino acid sequences of the ig domain subtypes from all the predicted xfls were aligned. trees were generated using the nj and me methods. to simplify the trees, the aligned blocks of d d and d domains were reduced by removing redundant sequences with close association. thereafter, the sequences for all the xfl domain subtypes were aligned together with the domain sequences of the human proteins, and their relationships were analyzed by the same procedure. the final tree is shown on fig the tree topology supports subdivision of the xenopus and human ig domains into five common and one xenopus-specific subtypes. most importantly, the tree shows separate clustering of the xenopus and human sequences. this branching pattern suggests that duplications of the fcr-like genes in amphibian and mammalian lineages were lineage-specific and that separation of the mammalian genes into classical fcrs and fcrls occurred after the split of the amphibian and mammalian lineages. the relationships among the xfl genes provide evidence for a complex pattern of family evolution in amphibians. its detailed description is beyond the scope of the present paper and will be published separately. what is relevant to note here, is that the strong association of the amphibian sequences with each other does not necessarily correspond to high level of sequence similarity among them. the cumulative tree illustrates subdivision of the xfl d d and d domains into structural variants whose relationships with each other cannot be resolved. the d d and d domain subtypes fall into and structural groups, respectively. the degree of sequence similarity among the group representatives is in the range of sequence similarity between xfl and mammalian fcr domains. in the case of d the diversity is mainly derived from a small number of genes located in the scaffolds and for instance, the extracellular parts of the predicted proteins 1131_ and 1131_ are composed of five d domains each. these domains are subdivided into four groups. each of five d domains of the xfl 1256_ represents a distinct structural variant producing a separate branch in the tree. on the other hand, more than proteins encoded by the genes of the other scaffolds have one to seven d domains belonging to the same group. this group may be further subdivided into three main subgroups, and and a number of individual members based on the structure of the d and d domains. for instance, the proteins encoded by the genes on the scaffold have very similar d domains but their d and d domains fall into structural variants with poorly resolved relationships. this fact strongly suggests that intergenic exon recombination was a frequent event in the evolution of the xfl family. of ten genes containing exons for the d d or d domain subtypes, nine belong to the subgroup and one to the subgroup. interestingly subgroups and differ in patterns of amino acid replacement in the d and d domains. the d domains of subgroup are characterized by extensive variation in the length and sequence of the fg loop, the equivalent of cdr of the v-type domains. the region covering the strands c to f is well conserved. in contrast, the d domains of subgroup show more variation in the region between the c' and f strands. their f-g region is relatively conserved. the d domains of the and subgroups also display variability at different sites suggesting the existence of at least two classes of ligands for the group i receptors. the d and d domains are implicated in binding to igg and ige by classical fcrs. however, the residues known to contact the fc portion of ig are not conserved in the xfls sequences, experimental support of xfl diversity to gain a deeper insight into structure and expression of the xfl genes, we studied this family in x. laevis, a close relative of x. tropicalis. in contrast to x. tropicalis that has a diploid genome, x. laevis is an allotetraploid species. the immune system of x. laevis is one of the most thoroughly studied among lower vertebrates. five different x. laevis cdnas for xfl proteins were obtained from the image consortium and sequenced. more than cdnas, of which were unique, were additionally cloned from several x. laevis cdna libraries using screening with an exon encoding the d domain of group as a probe. of distinct cdnas, were full-length, the others were truncated at the 5' end. nine cdnas encoded typical cell surface proteins containing tm2-like tm regions and cytoplasmic tails of varying length with one to three tyrosine-based motifs. the amino acid sequences of two clones had short cytoplasmic tails. their tm regions contained the nxxr motif and were assigned to the tm subtype. one cdna clone encoded protein lacking tm but containing a typical cytoplasmic tail with two tyrosine-based motifs. finally, four cdnas coded for putative secreted proteins composed of the ig-like domains only. at present, it is unclear whether or not the latter five clones represent alternative transcripts of genes encoding cell surface receptors. as expected, the initial sequence comparisons demonstrated that most of x. laevis derived xfls may be joined into group their d domains shared to 95% identical residues with each other and with the x. tropicalis d domains of the group receptors. these proteins were designated xfl –. as in the case of x. tropicalis, the x. laevis group proteins showed variable degree of identity in their d and d domains. two other proteins were designated xfl and xfl their d domains shared 35–45% identical residues with each other and with the d domains of the group proteins. according to the phylogenetic analysis, the x. laevis xfl and xfl genes were most similar to the x. tropicalis 435_ and 1131_ genes respectively. to estimate the genomic complexity of the xfl family in x. laevis, we performed southern blot hybridization using the d1-exon of xfl and d3-exons of the xfl and xfl genes as probes under non-stringent conditions. multiple hybridizing bands were revealed on the blots probed with the d3-exon of the xfl gene. the xfl2- and xfl3-specific probes revealed one and three hybridizing bands, respectively. these results demonstrated that the cloned xfl genes constitute only a part of the family and that, like in x. tropicalis, most of the x. laevis xfl genes appear to belong to the group x. laevis xfl genes are primarily expressed in lymphoid tissues to assess the xfl expression pattern, we performed northern blot hybridization of total rna from various tissues of adult frogs. the exon for d domain of the group i was used as a probe at low stringency conditions. as expected, northern blotting revealed diffuse bands representing multiple gene transcripts. the highest signal intensity was observed in the spleen and thymus. to examine expression patterns of the individual genes, we designed gene-specific primers for seven different xfl cdna. to exclude possible cross matching, the 3'-untranslated sequences were mainly used for the reverse primers. the rt-pcr analysis demonstrated that the tissue distribution of the corresponding mrna is variable. the transcripts of all the genes were mainly detected in lymphoid and non-lymphoid tissues containing cells of haemopoietic origin. expression of the xfl xfl and xfl genes was detected in brain. the tissue distribution of mrna in tadpoles was slightly broader. in particular, all xfls tested except, were detected in the gills which is known to be a very active immunological site owing to intense blood circulation and high exposure to antigens. furthermore, expression of the xfl gene was found only in tadpole spleen. different pattern of the xfl transcript distribution in adults and larvae suggest that the expression of at least a proportion of the xfl genes is developmentally regulated. nt – not tested. results are indicative of rt-pcr performed on total rna extracts from different tissue samples. tm facilitates xfl association with the fcrγ subunit the presence of the nxxr motif-bearing tms in many xfls suggested that, like the mammalian activating kirls, these xenopus receptors may require fcrγ chain for cell surface expression and/or signal transduction. to examine whether this is the case, we generated a series of constructs enabling expression of xfl xfl and the x. laevis fcrγ subunit as recombinant epitope-tagged proteins. xfl and xfl were expressed in 293t cells as recombinant hemagglutinin -tagged proteins containing either their own tm regions or the tm region of pdgfr. the x. laevis fcrγ chain was tagged with c-myc epitope. single transfection of xfl2-ha did not induce its surface expression, and the protein accumulated intracellularly as determined by immunofluorescent microscopy of permeabilized cells. however, the surface expression of xfl was restored when it was co-transfected with fcrγ. in contrast to xfl xfl -ha was targeted to the surface of the transfected cells in the absence of the adapter molecule, although its surface expression was increased two-fold in the presence of fcrγ. both xfl and xfl proteins were readily expressed on the cell surface when their ec regions were fused with a tm of pdgfr. these results show that xfl molecules containing a tm region with the nxxr motif associate with the fcrγ chain. the surface expression of xfl is less dependent on the presence of fcrγ chain. this may be explained by divergent structure of their tms. such differences also have been observed among the fcrγ-associating kirls; fcrγ is critical for surface targeting of lilra but not fcαr or oscar. discussion comparative studies of mammals and chicken have revealed an unexpected structural and functional variability of the paired receptor families such as fcrls and kirls. studies have also indicated that the repertoires of these families have evolved in a species-specific manner. the evolutionary factors responsible for such diversity remain poorly understood. the recent description of litrs as putative teleost counterparts of both fcrls and kirls left many questions unanswered, since only a weak similarity of litrs to the mammalian and avian receptors has been found. the present study fills the gap by extending the analysis of paired receptor families to amphibians, the most primitive branch of tetrapods. in the diploid x. tropicalis we have identified at least genes coding for paired fcr-like cell surface receptors. the mere fact of the family expansion is not unusual. rapid evolutionary change of a gene content known as the "expansion-contraction" or "birth-and-death" process has been documented in many families of immunity-related receptors. what distinguishes the fcr family from many other paired receptor families is the extraordinary structural diversity of its members. combinatorial joining of six ig domain subtypes generates as many as ec architectures. when we consider the tm subtypes as distinct domains, the number of the xfl domain architectures increases to none of these variants are shared by either human or mouse homologs, although five ig-domain subtypes are common for the xenopus and mammalian proteins. overall, different domain architectures can be defined among human, mouse, and x. tropicalis fcr-related proteins. except for the d1- and d2-subtype domains no homologous structural elements were found between xfls and catfish litrs. other ig-domain subtypes composing ec of litrs seem to be more similar to the kirl domains. such mixed domain composition has been suggested to predate the kir and fcr families. the xfl data are consistent with the suggestions that the fcr and kir families share evolutionary roots. a strong argument in favor of this model is the fact that activating fcrs use a peculiar tm module to associate with fcrγ chain that is homologous to tms of activating mammalian kirls. tm appears to be an ancestral element of the primordial fcr/kir family that has been lost by chirs, classical fcrs and kirs, but retained by xfls as well as xenopus and mammalian kirls. although available data do not allow inferring the structure of the kirl and fcrl ancestor, it is clear that the family evolved by inter- and intragenic recombinations in a species-specific way. the former mechanism gave rapid change in the number of genes per family, whereas the latter was responsible for extensive domain shuffling. the fcr-related receptors in different vertebrate species are similar in their subdivision into activating and inhibitory forms and predominant expression in lymphoid tissues. however, the ratio of inhibitory to activating members, the cellular distribution, and the exact amount and architecture of ectodomains are unique in each examined species. what might be the evolutionary forces responsible for this degree of diversity common among the fcr- and kir-related receptors, and xfls in particular? we can try to answer this question by inference from the attributed function of the actual mammalian receptors, which is to regulate immune responses. classical fcrs regulate b cell responses by binding to the igg and ige immune complexes, whereas kirs, or at least their inhibitory forms, regulate nk cell function by binding specifically to mhc class i molecules. however, ig-binding appears to be a secondary or specialization, since our previous and present data together with the definition of a chicken igy receptor as a member of the chir family strongly argue that classical fcrs have emerged after the separation of mammals and birds. mhc-recognition as a potential ancient function of fcrls/kirls is more attractive. the ability to interact with classical and non-classical mhc class i molecules is a feature of some kirls, it has been also suggested for mammalian fcrls and catfish litrs. from this point of view the diversification of fcrl/kirls may have been driven by the necessity to match the rapid evolution of mhc loci under pathogen pressure of, as it has been suggested for the kir and ly gene clusters. this may be the case for xfls too as there are at least non-classical mhc class i in x. laevis. there are however some inconsistencies with this scenario. first, the scope of variability of domain architectures among fcrls seems to be excessively high relative to mhc class i molecular structure. second, the functions of mammalian kirls are not limited to mhc antigen-binding. among ligands of these receptors, there are also collagens, iga, igg and integrins. human α1-b-glycoprotein, a distant secreted relative of kirls, binds to the cysteine-rich secretory protein, while its opossum homolog is a snake venom metalloproteinase-neutralizing factor. due to the independent expansion of kir-related receptors in mammals, birds and amphibians it is difficult to determine which of these functions are truly ancient or of ancestral type. finally, mouse ly is a clear example of the self-mhc recognition served by receptors structurally different from kirls and fcrls. an alternative explanation for the extraordinary diversity of xfls and other fcr- and kir-related receptors may be that they are directly involved in innate immunity. combinatorial diversity is a hallmark of the immune system and it is usually associated with recognition of pathogens. the capacity of paired receptors to directly bind to pathogens is well documented. in the latest of these studies it has been found that mouse pir-b, and its human relatives lilrb and lilrb recognize staphylococcus aureus and modulate tlr-mediated inflammatory responses against this bacterium. these facts are usually interpreted in terms of adaptive coevolution of the microorganisms, which implies that the pathogen recognition is a secondary or derived function. however, the extensive variability of the fcr/kir relatives raises the possibility that these receptors expanded primarily to fight pathogens, whereas the known immuno-regulatory functions may represent secondary acquisitions or specializations. depending on the nature of the pathogen and the signal properties of the receptors, it is clear that pathogen-receptor interaction may be either advantageous or detrimental for the host, and as such, may rapidly change the ratio of activating versus inhibitory receptors, as well as their respective amount and specificities. in this regard, a parallel may be drawn with the species-specific expansion of various receptor families in invertebrates that participate in innate immune responses. while it may be difficult to obtain direct evidence to support this scenario, it clearly deserves attention. the elucidation of the factors responsible for the diversification of the fcr- and kir-related receptors may contribute to better understand their function and ultimately develop new therapies based on their immunoregulatory properties.  our study shows that in two amphibian species xenopus tropicalis and x. laevis, paired receptors have diversified into a large family of genes, xfls, preferentially expressed in lymphoid tissues. the extracellular regions of these receptors are composed of one to eleven ig-like domains belonging to six structural subtypes. a fraction of xfls use a tm module to associate with fcrγ signaling subunit. tm is highly similar to tms of activating fcrγ-associating kirls. this fact strongly argues in favor of a common evolutionary origin of the fcr and kir families. the variation in number and composition of distinct ig-like and tm domain subtypes generates striking diversity of domain architectures among xfls. phylogenetic analysis shows that this diversity emerged in a lineage-specific manner. classical fcrs and other known mammalian fcr-related proteins appear to be specific to mammals. the continual and extensive diversification of domain architectures in the fcr and kir families indicates a strong selection pressure not completely consistent with the usual assumption that paired receptors have been primarily selected to regulate immune responses. we propose that fcr/kir-related receptors might have primarily expanded as pathogen-recognizing components of innate immunity while their known physiological functions have been acquired later in a lineage-specific manner. 
putative activating xfls associate with the fcrγ subunit, and their transmembrane domains are highly similar to those of activating mammalian kir-related receptors. this mode of evolution raises the possibility that the ancestral function of these paired receptors was a direct interaction with pathogens and that many physiological functions found in the mammalian receptors were secondary acquisitions or specializations. fcr- and kir-related receptors evolved through continual species-specific diversification, most likely by extensive domain shuffling and birth-and-death processes. to better understand the evolutionary history of paired receptors, we extended the study of fcr-like genes in amphibian representatives xenopus tropicalis and xenopus laevis. up to domain architectures generated by combinatorial joining of six ig-domain subtypes and two subtypes of the transmembrane regions were found in xfls. phylogenetic analysis shows that the entire repertoires of the xenopus and mammalian fcr-related proteins have emerged after the amphibian-amniotes split. the allotetraploid x. laevis displays many similar genes primarily expressed in lymphoid tissues. recent studies have revealed an unexpected diversity of domain architecture among fcr-like receptors that presumably fulfill regulatory functions in the immune system. the diploid genome of x. tropicalis contains at least genes encoding paired fcr-related receptors designated xfls. this argues in favor of a common origin for the fcr and the kir families.
 recent studies have revealed an unexpected diversity of domain architecture among fcr-like receptors that presumably fulfill regulatory functions in the immune system. different species of mammals, as well as chicken and catfish have been found to possess strikingly different sets of these receptors. to better understand the evolutionary history of paired receptors, we extended the study of fcr-like genes in amphibian representatives xenopus tropicalis and xenopus laevis.  the diploid genome of x. tropicalis contains at least genes encoding paired fcr-related receptors designated xfls. the allotetraploid x. laevis displays many similar genes primarily expressed in lymphoid tissues. up to domain architectures generated by combinatorial joining of six ig-domain subtypes and two subtypes of the transmembrane regions were found in xfls. none of these variants are shared by fcr-related proteins from other studied species. putative activating xfls associate with the fcrγ subunit, and their transmembrane domains are highly similar to those of activating mammalian kir-related receptors. this argues in favor of a common origin for the fcr and the kir families. phylogenetic analysis shows that the entire repertoires of the xenopus and mammalian fcr-related proteins have emerged after the amphibian-amniotes split.  fcr- and kir-related receptors evolved through continual species-specific diversification, most likely by extensive domain shuffling and birth-and-death processes. this mode of evolution raises the possibility that the ancestral function of these paired receptors was a direct interaction with pathogens and that many physiological functions found in the mammalian receptors were secondary acquisitions or specializations. 