BACKGROUND
g protein coupled receptors  are arguably the most important proteins in human evolution and medicine  <cit> .
the rhodopsin class of gpcrs alone is the most highly represented protein family in mammals  <cit> .
humans have on the order of  <dig>  <cit>  to  <dig>   <cit>  gpcrs, and they are the protein family most commonly targeted by clinical drugs.
gpcrs are transmembrane receptors that mediate the majority of extracellular signaling in mammals.
they can sense diverse types of signals, including hormones, lipids, olfactants, tastants, ions, light, and soluble and surface-anchored peptides.
despite the name, not all gpcrs signal through g proteins.
and many or most gpcrs also signal through non-g protein-mediated pathways.
the hallmark of gpcrs is that they have seven transmembrane helices.
thus, the terms seven transmembrane receptor  and gpcr are used interchangeably by some.
it has been extremely challenging to determine the evolution of the 7tmr superfamily due to the large number of 7tmrs and the existence of highly divergent subfamilies.
however, this was initiated for fully sequenced genomes from several bilateral animals, two fungi, two plants and one alveolate  <cit> .
recently-sequenced genomes  will soon begin to fill in key gaps.
notably, it is widely believed that 7tmrs do not represent a single superfamily  <cit> .
that is based on the perceived weakness, or lack of, protein sequence similarity between many 7tmrs – most importantly between the major 7tmr families of archaea, bacteria and eukaryotes.
but others claimed that analysis with psi-blast and hidden markov models suggest that bacterial, archaeal, and eukaryotic/animal 7tmrs are distantly related  <cit> .
this issue generates much attention  with regards to the evolution of vision  <cit> .
importantly, protein sequence analysis strongly suggests that bacteriorhodopsins of archaea/bacteria and rhodopsins of animals are separate 7tmr subfamilies that arose independently  <cit> .
the 7tmr subfamily named after rhodopsin contains vast numbers of non-opsin genes, and there is no indication that opsins are among the earliest members.
to our knowledge, the oldest known member of the rhodopsin subfamily is from yeast  <cit> .
the three intimate associates of gpcrs are heterotrimeric g proteins, g protein coupled receptor kinases  and arrestins.
study of those proteins could offer clues about 7tmr evolution.
phylogenetic analysis of g alpha subunits is possible due to the presence of a signature protein sequence.
in the pfam database, g alphas can be seen in protists, plants and unikonts.
grks have been reported from mammals down to nematode worms and insects  <cit> .
however, their relationship to the large family of protein kinases may complicate the determination of the phylogeny of grks; we found no reported attempt to do this.
the main focus of this work is reconstructing the arrestin phylogeny.
arrestins regulate the inactivation  <cit> , internalization  <cit> , trafficking  <cit>  and signaling  <cit>  of transmembrane receptors of the 7tm and kinase classes .
they are defined by the presence of two homologous protein sequences known as arrestin n and c domains.
arrestins selectively bind receptors that are both in an activated conformation and phosphorylated  <cit> .
this is possible because they have an autoinhibited resting state maintained by interacting polar and solvent-excluded residues in abutting arrestin-n and c domains  <cit> .
phosphates on activated receptors destabilize that "polar core" and allow binding of specific receptor residues at the fulcrum of, and on the concave faces of, the two arrestin domains.
a second feature maintaining the basal conformation is the "three-element interaction" of hydrophobic residues in beta strand i, alpha helix i, and beta strand xx in the c-terminal tail  <cit> .
release of this interaction in activated arrestin thus frees the c-terminal tail, allowing it to bind clathrin and its adaptor ap- <dig>  arrestin activities vary according to their own modifications by phosphorylation and ubiquitination, their interactions with dozens of other proteins  <cit> , and the specifics of their interactions with receptors .
until recently, when fungal arrestins were discovered  <cit> , it was believed that arrestins emerged in animals.
fruit flies and mosquitos each have two known visual arrestins and two beta arrestins  <cit> .
the worm c. elegans, which lacks vision, has one known beta arrestin  <cit> .
humans are presumed to have four arrestins: two photoreceptor-specific visual arrestins and two nearly ubiquitous beta arrestins  <cit> .
the first phylogenetic study of visual and beta arrestins was recently reported  <cit> .
however, it has not been determined how visual/beta arrestins are related to similar proteins in fungi  <cit>  and animals  <cit> .
the official gene names of the mammalian proteins are arrestin domain containing 1– <dig>  and txnip .
we refer to that subfamily as alpha arrestins – to distinguish them from their close relatives, the visual/beta arrestins, and their more distant relatives, spo0m and vps <dig> .
here we report the evolutionary history of the arrestin clan.
those findings made us consider whether visual/beta arrestin emerged in concert with opsin.
that, in turn, led us establish that opsins predated bilaterata .
we discuss what our results suggest about eye evolution and arrestin functions.
RESULTS
identification of novel members of the arrestin clan
we conducted phylogenetic analysis of arrestins and found a large tree of mostly unrecognized arrestins in eukaryotes  .
figure  <dig> is a phylogenetic tree of arrestins from select evolutionarily diverse genomes.
it shows that all the known animal arrestins  <cit>  are members of a small branch of the protein family that emerged relatively recently.
we suggest that the two subfamilies could be termed alpha and beta  class arrestins .
this applies because the alphas are the ancient/ancestral arrestins and it fits well with the historical name of the betas .
we find varied numbers of arrestins in the phyla previously known to have arrestins – fungi and animals.
for example, the yeast schizosaccharomyces pombe has three arrestins.
nematode worms and flies each have approximately twenty.
half of the worm and fly arrestins appear to have emerged in their respective lineages.
humans have a total of  <dig> arrestins.
for comparison, humans have approximately the same number of 7tmrs as caenorhabditis elegans and four times as many as drosophila  <cit> .
even protists, which are single-celled, show diverse numbers of arrestins .
for example, dictyostelium, which has approximately  <dig> 7tmrs  <cit> , has four arrestin genes, and tetrahymena has fifteen.
it is worth noting that tetrahymena have much more complex membrane and cytoskeletal architecture than animals.
their genome has a striking expansion of genes involved in membrane and cytoskeletal dynamics  <cit> .
moreover, the percent of their genome devoted to kinases is double that of fungi and animals, but they only have four annotated 7tmrs.
this hints that early arrestin function could have been associated with membrane/cytoskeletal dynamics or kinase signaling.
compared to visual and beta arrestins, alphas have conserved protein sequence and domain topology spanning almost their full lengths.
the high sequence conservation suggests their molecular functions are similar.
for example, human txnip matches arrestin n and c domains with expect scores of  <dig> e- <dig> and  <dig> e- <dig>  respectively .
we also found two domains that appear to be distantly related to arrestins, spo0m and vps <dig> .
both are clearly alpha arrestin-like, rather than beta.
this finding was independently made by investigators managing the pfam database of curated protein patterns and posted on their database .
they validate this "arrestin n-like clan" with one multiple sequence alignment of most available members of the three protein subfamilies, and provide confidence scores of 1e- <dig>  and 2e- <dig> .
spo0m proteins are present in archaea  and in diverse bacterial phyla , but not in eukaryotes.
notably, archaea and bacteria have seven transmembrane proteins  and serine/threonine/tyrosine-specific kinases, both of which were widely believed to be eukaryotic inventions until recently  <cit> .
little is known about spo0m except that it is transcriptionally regulated by sigmah and that it inhibits sporulation.
it is tempting to speculate that spo0m has a role in transmembrane signaling that links environmental status to sporulation.
the crystal structure of a mammalian vps <dig> protein revealed that it has two arrestin domains  <cit> .
however, it was concluded there is no protein sequence similarity, presumably because vps <dig> was compared to the visual/beta arrestins   <cit> .
it was also suggested that the structural similarities between the two subfamilies are mostly superficial  <cit> .
the claim is that vps <dig> lacks all of the specific functional features of visual/beta arrestins: mainly, the sites responsible for gpcr, clathrin/ap <dig>  and phosphatidylinositol phospholipid binding.
two groups disagree on the possibility that the two proteins could have similar switch mechanisms based on conformational rearrangements  <cit> .
while both proteins have polar and electrostatic interactions between the n and c domains, they are mediated by different residues on different secondary structural elements.
a caveat is that this analysis is limited by the absence of structural and functional information about alpha arrestins.
that is, vps <dig> is being compared to distantly related visual/beta arrestins, rather than the much more closely related alpha arrestins.
our results, and those of pfam, indicate vps <dig> and arrestin are true homologs.
alpha arrestins have robustly similar structure predictions to those solved for beta class arrestins and vps <dig>  <cit>  .
for example, "threading" of alpha arrestin txnip through all known crystal structures matches rod visual arrestin and vps <dig> with an identical probability value of 1e- <dig> .
its presence in plants is significant because they lack arrestin.
similar to arrestins, vps <dig> proteins very rarely have associated domains.
in addition to the two known human vps <dig> genes – vps <dig> and vps26b – we found two new ones: dscr <dig> and an un-annotated gene on 2q <dig>  .
vps26p was discovered in a genetic yeast screen for "vacuole protein sorting, vps"  <cit> .
the yeast vacuole is the equivalent of the mammalian lysosome.
vps <dig> forms part of the retromer complex that mediates protein transport from endosomes to the trans-golgi  <cit> .
it is also involved in receptor transcytosis in polarized cells as well as in wnt signaling and gradient formation  <cit> .
the retromer has two subcomplexes, one responsible for cargo-selection and the other for vesicle formation.
vps <dig>  vps <dig> and vps <dig> form the receptor cargo-binding subcomplex.
vps <dig> also has an adaptor role linking that subcomplex to the membrane-binding subcomplex.
however, the exact biochemical role of vps <dig> is not defined.
while there are four vps <dig> genes in mammals, the other retromer components are represented by single genes.
retromer function is thus reminiscent of the role of beta arrestins in receptor endocytosis.
both arrestin and vps <dig> are part of a transmembrane receptor-binding complex on the cytoplasmic face of endosomes.
arrestin further interacts with the membrane via the clathrin adaptor ap- <dig>  and vps26p does so through vps5p/vps17p .
both arrestin and vps <dig> form complexes that co-localize with key vesicular trafficking proteins such as rab <dig> and n-ethylmaleimide-sensitive factor .
nsf interacts directly with beta arrestin  <cit>  and is required for retromer endosome-to-golgi trafficking  <cit> .
it is curious that beta arrestins bind the vps <dig> partner vps <dig>  <cit> , and that vps <dig> is present in 7tmr complexes  <cit> ; however, there is no evidence those interactions are biologically relevant.
alpha arrestins were present in early eukaryotes
we used psi-blast to search for all arrestins in the genbank protein database .
we found alpha arrestins in fungi and in all multi-cellular life except plants.
mammals have six alpha and four visual/beta arrestins .
the significant protein-sequence difference between some of those alpha arrestins suggests they have more diverse functions than the more closely related visual/betas.
the single arrestin present in viruses is a vertebrate alpha arrestin, arrdc <dig>  that was horizontally transferred to canarypox virus .
we also identified arrestins in all protist groups except rhizaria, which has little sequence available.
in additional file  <dig> we describe the protist proteins.
since arrestin n and c domains are related by protein sequence, we searched for single-domain proteins that could be the ancestral arrestin domain that was duplicated.
we found that no arrestin-containing kingdom or species has exclusively single-domain proteins.
this result is consistent with the finding that the vps <dig> crystal structure reveals tandem domains  <cit> .
we believe a twin domain gene was duplicated in a bacterium or basal eukaryote and gave rise to genomes with both arrestin  and vps <dig>
we searched for arrestin-associated domains that could hint at biochemical functions.
outside of the protists, we found that these are extremely rare.
we found exactly two domains that recurred in at least two divergent phyla; they were in two distantly related orders of the fungus-like protists amoebozoa .
two dictyostelium and two entamoebidae arrestins each contain a c <dig> domain, which is a ca2+-dependent phospholipid/membrane binding element.
in one order the c <dig> domains are n-terminal in both proteins, in the other both are c-terminal.
this suggests they could have been created by independent events of exon shuffling in the two orders.
two other genes, one from each of the same two orders, contain one fyve domain in their c-terminal region.
fyve domains have exquisite specificity for phosphatidylinositol-3-phosphate p), an endosomal marker recognized by proteins involved in signaling and trafficking.
notably, vps <dig> interacts with the ptdinsp-binding protein sorting nexin  <dig>  <cit> .
the presence of associated c <dig> and fyve domains indicates some ancient arrestin functions involve plasma and endosomal membrane interactions, respectively.
this is consistent with known arrestin roles in transmembrane receptor-binding and endocytic trafficking.
difference between alpha and visual/beta arrestins
helix i of visual/beta arrestins is sequestered in the inactive conformation and is presumably released upon activation  <cit> .
this helix has hydrophobic residues on one face and basic residues on the other.
however, it is a mystery what it does, or interacts with, in the active conformation.
helix i leucine substitutions suggested a role in receptor binding  <cit> , and it was later theorized to be a membrane-docking element that permits non-specific interactions with activated receptors  <cit> .
other experiments showed that helix i is important for the formation of dense-core vesicles  <cit> .
the proposed membrane docking role of helix i remains attractive to us.
we add that such an insertion would displace membrane on the cytoplasmic leaflet of the plasma membrane.
this could induce positive curvature and promote endocytocis.
notably, helix i is absent in alpha arrestins  and is thus a major innovation in beta arrestins.
that interpretation is supported by the absence of helix i in the 3d structure of vps <dig>  <cit> , which has protein sequence similarity to alpha arrestins.
all indications suggest apha and beta arrestins have similar structural topologies, but do alphas also bind 7tmrs?
nichols and sanders-bush announced their discovery of a new mammalian arrestin  in  <dig>  <cit> .
however, to our knowledge there are no published studies of arrestin-like functions in animal alpha arrestins.
the current understanding of alpha arrestin biochemistry thus comes from fungi and yeast.
herranz, vincent and colleagues showed that fungal palf  is a bona fide arrestin by protein sequence and function  <cit> .
palf binds c-terminal sites of the activated seven transmembrane ph sensing receptor palh.
moreover, alkaline activation induces palh-dependent phosphorylation and ubiquitination of palf.
truncation of the palh cytoplasmic domain disrupts palf-binding and inhibits growth in alkaline ph.
the alpha arrestin palf thus resembles beta arrestins in its ability to bind active receptors, generate a signal and be posttranslationally modified in the process.
the function of palf, however, is in signal transduction and apparently not in inhibition of g protein signaling.
ph sensing and "vacuole protein sorting, vps" pathways are intimately associated in fungi.
herranz et al.
thus propose that the role of palf is likely to relate to endocytic trafficking.
this is notable considering that vps <dig>  an ancient arrestin relative, was discovered in a genetic screen for vps genes.
while beta arrestin tail domains have conserved clathrin-interacting motifs, alphas have py motifs.
is there evidence the latter are functional?
py motifs bind ww domains and their interactions are extensively defined by diverse biochemical and structural methods  <cit> .
the strong conservation of multiple py motifs in fungal and animal arrestins suggests they interact with ww proteins.
saccharomyces cerevisiae has three alpha arrestins, all of which have turned up in biochemical and genetic screens .
without knowing they were studying arrestins, others showed that two s. cerevisiae arrestins  bind the hect  e <dig> ubiquitin ligase rsp5p  <cit> .
moreover, they showed by mutagenesis that the interactions are mediated by two py motifs in arrestin and two ww domains in rsp5p  <cit> .
our computational analysis of the human genome identified  <dig> ww proteins that may interact with alpha arrestins .
notably nine of those genes are candidate co-orthologs of yeast rsp5p.
this suggests alpha arrestins could be involved in ligand-dependent 7tmr ubiquitination and trafficking  <cit> , which, in some yeast and mammalian cases, are mediated by hect e <dig> ligases  <cit> .
we also suspect arrestin py motifs interact with other ww proteins that are involved in cytoskeletal dynamics, endocytocis/trafficking and signaling.
using yeast two hybrid methodology, we have found that alpha arrestin tails robustly bind the ww domains of hect e <dig> ligases and of other proteins .
origins of visual/beta arrestin and rhodopsin
the fact that all known visual arrestins cluster together with beta arrestins suggested to us that this branch may have emerged together with an early opsin.
we used visual/beta arrestin and visual/beta arrestin consensus sequences to search genbank for the earliest family member we could find .
the oldest visual/beta arrestin we found is from hydra .
notably, cnidarians are the only beta arrestin-containing organisms that predate any known opsin.
we next searched for the oldest opsin genes present in all, but the trace database of genbank .
that analysis resulted in the identification of multiple hydra ests encoding partial opsin proteins .
of the known subfamilies, we only found representation of ciliary opsin.
we were able to assemble the full length open reading frame for one of these .
this cnidarian protein is strikingly similar to ciliary opsins, placing it in the same group as rag-worm ciliary opsin  and human visual opsins on the phylogenetic tree of opsins  <cit> .
cnidarian opsin genes are the first to be reported that predate the emergence of bilaterata  <cit> .
while the present study was under review, others independently reported finding cnidarian opsins  <cit> .
as we did, both of those groups found that cnidarians have ciliary opsins.
they additionally reported other highly divergent opsins that cannot be clearly classified according to the known subfamilies.
remarkably, suga, schmid and gehring identified large numbers of opsin genes in single species of cnidarians:  <dig> sea anemone,  <dig> hydra, and  <dig> jellyfish  <cit> .
they showed jellyfish opsins are expressed in multiple patterns, including in the eyes and gonads .
neither those studies nor ours  were able to find any evidence of opsins in sponges or older phyla.
this is consistent with the fact that sponges use flavin or carotenoid photopigments  <cit> .
the early evolution of vision is an open question  <cit> .
it was long known that some jellyfish have image forming eyes, but the molecular identity of their visual pigments was a mystery until now.
recently it was postulated that the absence of a master transcriptional regulator, pax <dig>  in the box jellyfish is consistent with the independent evolution of eyes in higher metazoans  <cit> .
gehring  <cit>  offered two alternative explanations to that same question: vertical evolution from a photosensitive protist and horizontal evolution through a photosynthetic cyanobacterial symbiont.
we proposed that the phylogeny of the phototransduction machinery would explain the evolution of phototransduction and vision  <cit> .
the new opsin phylogeny suggests to us that ciliary opsins are likely to be ancestral to all previously known  <cit>  visual pigments in animals.
that possibility is consistent with darwin's theory that eyes evolved once  <cit> :
how a nerve comes to be sensitive to light hardly concerns us more than how life itself originated; but i may remark that, as some of the lowest organisms in which nerves cannot be detected are capable of perceiving light, it does not seem impossible that certain sensitive elements in their sarcode should become aggregated and developed into nerves endowed with this special sensibility...
the simplest organ which can be called an eye consists of an optic nerve surrounded by pigment-cells and covered by translucent skin, but without any lens or other refractive body.
we may, however, according to m. jourdain, descend even a step lower, and find aggregates of pigment-cells, apparently serving as organs of vision, without any nerves, and resting merely on sarcodic tissue.
eyes of the above simple nature are not capable of distinct vision, and serve only to distinguish light from darkness.
the fact that both opsin and visual/beta arrestin are present in cnidaria, but not older phyla, hints that the two subfamilies could have emerged in concert.
early phototransduction offered major advantages, such as photoperiodicity and phototaxis.
presumably, the first step was the creation of photosensory opsin.
however, there must have been immense avenues for improvement after that point.
one challenge to early opsin was exposure to light intensity that varies by  <dig> orders of magnitude, the earth's daily illumination cycle.
the innovation in visual/beta arrestin could have improved the modulation of signaling gain by regulating opsin localization, endocytosis/recycling or inactivation kinetics.
gene duplication is a hugely important mechanism for evolution  <cit> .
visual phototransduction consistently uses dedicated proteins created by gene duplication .
for example, in addition to opsin 7tmrs, mammalian photoreceptor cells have their own, or nearly exclusive, g proteins, cgmp-phosphodiesterases, g protein-coupled receptor kinases, arrestins, etc.
outside the eye, a handful of each of those proteins transduces signals for the remaining hundreds of 7tmrs.
this extreme specialization illustrates the high importance of vision in animal evolution.
possible implication of pre-cambrian vision
both opsin and visual/beta arrestin are present in cnidaria, but not in sponges, the most basal metazoans .
this is interesting in light of parker's recent proposal that vision set off the cambrian explosion of life forms  <cit> .
<dig> million years ago  only three phyla existed, but, in the following  <dig> my,  <dig> phyla emerged.
no new phyla have emerged since that cambrian explosion.
parker proposed that vision triggered the cambrian explosion by creating a new world of organismal interactions  <cit> .
the key observation is that pre-cambrian phyla were soft-bodied.
however, the cambrian saw an apparently limitless diversification of hard body parts.
at the same time, the use of biological color appeared.
parker claims this scenario can be explained by the emergence of vision, which must have resulted in new behaviors such as predation.
seeing-predators would have suddenly needed rigid components to pursue, attack, and eat their prey .
their prey – which may or may not have had eyes – also had to adapt by developing hard shells or spines, camouflage or even invisibility .
parker specifically hypothesized that this "light switch" was triggered by the emergence of arthropod eyes during the cambrian.
put another way, he proposed the light switch required image perception and brain processing of the information .
we find his general argument to be compelling.
however, we believe the spark had to precede the cambrian explosion.
we propose that light perception in a pre-cambrian animal initiated new dimensions of organismal and environmental interactions.
this may have progressed steadily until a critical mass of interactions was reached, the cambrian explosion.
our findings  are clear evidence that opsin  predated the cambrian period.
this meets what we consider to be the first requirement for a visual trigger hypothesis.
if sight initiated an arms race in the biosphere, it is not surprising that some organisms refined it and others lost it .
only six of  <dig> phyla on earth have eyes, but these six seeing phyla account for >95% of all living animals.
it is not difficult to imagine that the relative success of seeing animals is due to the extraordinary advantage of vision.
CONCLUSIONS
despite the high interest in gpcr signaling, its evolution remains enigmatic.
there is some evidence that archaeal and bacterial 7tmrs are homologous to eukaryotic 7tm/gpcrs  <cit> .
however, heterotrimeric g proteins/g alpha subunits are only present in eukaryotes.
this suggests that ancestral 7tm/gpcrs signaled by mechanisms other than g protein coupling.
we found that the arrestin clan is present in archaea and bacteria, raising the possibility that spo0m could be a primordial 7tmr signaling partner.
in addition, our findings of cnidarian opsins lead us to propose that the ciliary subfamily is ancestral to all bilaterian opsins .
that is consistent with darwin's theory that eyes evolved once.
there were two major arrestin-like gene families in early eukaryotes, arrestin and vps <dig>  both protein families are well characterized and point to endocytosis/endosomal dynamics as the ancestral arrestin/vps <dig> functions.
the duplication of the arrestin domain was a critical event in the creation of ancestral arrestin/vps <dig>  this could have created autoinhibitory mechanisms , a recurrent theme in the evolution of signal transduction.
the functional similarities of beta arrestins and vps <dig> proteins lead us to speculate that the original arrestin/vps <dig> was involved in receptor internalization.
this could have had two classes of receptor effects in concert: 1) desensitization and recycling/degradation, and 2) signaling.
others have hypothesized that the original role of arrestins may have been as signaling adaptors rather than terminators  <cit> .
above we mention biochemical evidence that mammalian vps <dig> and arrestins could have overlapping roles  <cit> .
the homology of alpha and beta arrestins suggests their molecular functions may be similar.
there is evidence from fungi that the alpha arrestin palf specifically binds an activated 7tmr  <cit> .
that interaction has a positive signaling role that is not yet identified.
there are also differences between the alpha and beta classes.
beta arrestins are generally cytoplasmic in unstimulated cells, while alpha arrestins are often associated with membranes  <cit> .
only visual/betas have helix i in the n domain.
and the tails of betas contain clathrin-interacting motifs, while those of alphas have py motifs.
studies in yeast showed that alpha arrestin py motifs interact with the ww domains of the hect e <dig> ubiquitin ligase rsp5p.
we believe that a major role of alphas is to recruit ww proteins to activated receptors.
alpha and beta arrestins are widely co-expressed.
the fact that visual/beta arrestins can hetero-associate  <cit> , hints that alphas and betas may also.
given the near ubiquitious involvement of beta arrestins in 7tmr signaling, we speculate that alphas and betas may function coordinately.

 g protein coupled receptors are arguably the most important proteins in human evolution and medicine. the rhodopsin class of gpcrs alone is the most highly represented protein family in mammals. humans have on the order of to gpcrs, and they are the protein family most commonly targeted by clinical drugs. gpcrs are transmembrane receptors that mediate the majority of extracellular signaling in mammals. they can sense diverse types of signals, including hormones, lipids, olfactants, tastants, ions, light, and soluble and surface-anchored peptides. despite the name, not all gpcrs signal through g proteins. and many or most gpcrs also signal through non-g protein-mediated pathways. the hallmark of gpcrs is that they have seven transmembrane helices. thus, the terms seven transmembrane receptor and gpcr are used interchangeably by some. it has been extremely challenging to determine the evolution of the 7tmr superfamily due to the large number of 7tmrs and the existence of highly divergent subfamilies. however, this was initiated for fully sequenced genomes from several bilateral animals, two fungi, two plants and one alveolate. recently-sequenced genomes will soon begin to fill in key gaps. notably, it is widely believed that 7tmrs do not represent a single superfamily. that is based on the perceived weakness, or lack of, protein sequence similarity between many 7tmrs – most importantly between the major 7tmr families of archaea, bacteria and eukaryotes. but others claimed that analysis with psi-blast and hidden markov models suggest that bacterial, archaeal, and eukaryotic/animal 7tmrs are distantly related. this issue generates much attention with regards to the evolution of vision. importantly, protein sequence analysis strongly suggests that bacteriorhodopsins of archaea/bacteria and rhodopsins of animals are separate 7tmr subfamilies that arose independently. the 7tmr subfamily named after rhodopsin contains vast numbers of non-opsin genes, and there is no indication that opsins are among the earliest members. to our knowledge, the oldest known member of the rhodopsin subfamily is from yeast. the three intimate associates of gpcrs are heterotrimeric g proteins, g protein coupled receptor kinases and arrestins. study of those proteins could offer clues about 7tmr evolution. phylogenetic analysis of g alpha subunits is possible due to the presence of a signature protein sequence. in the pfam database, g alphas can be seen in protists, plants and unikonts. grks have been reported from mammals down to nematode worms and insects. however, their relationship to the large family of protein kinases may complicate the determination of the phylogeny of grks; we found no reported attempt to do this. the main focus of this work is reconstructing the arrestin phylogeny. arrestins regulate the inactivation, internalization, trafficking and signaling of transmembrane receptors of the 7tm and kinase classes. they are defined by the presence of two homologous protein sequences known as arrestin n and c domains. arrestins selectively bind receptors that are both in an activated conformation and phosphorylated. this is possible because they have an autoinhibited resting state maintained by interacting polar and solvent-excluded residues in abutting arrestin-n and c domains. phosphates on activated receptors destabilize that "polar core" and allow binding of specific receptor residues at the fulcrum of, and on the concave faces of, the two arrestin domains. a second feature maintaining the basal conformation is the "three-element interaction" of hydrophobic residues in beta strand i, alpha helix i, and beta strand xx in the c-terminal tail. release of this interaction in activated arrestin thus frees the c-terminal tail, allowing it to bind clathrin and its adaptor ap- arrestin activities vary according to their own modifications by phosphorylation and ubiquitination, their interactions with dozens of other proteins, and the specifics of their interactions with receptors. until recently, when fungal arrestins were discovered, it was believed that arrestins emerged in animals. fruit flies and mosquitos each have two known visual arrestins and two beta arrestins. the worm c. elegans, which lacks vision, has one known beta arrestin. humans are presumed to have four arrestins: two photoreceptor-specific visual arrestins and two nearly ubiquitous beta arrestins. the first phylogenetic study of visual and beta arrestins was recently reported. however, it has not been determined how visual/beta arrestins are related to similar proteins in fungi and animals. the official gene names of the mammalian proteins are arrestin domain containing 1– and txnip. we refer to that subfamily as alpha arrestins – to distinguish them from their close relatives, the visual/beta arrestins, and their more distant relatives, spo0m and vps. here we report the evolutionary history of the arrestin clan. those findings made us consider whether visual/beta arrestin emerged in concert with opsin. that, in turn, led us establish that opsins predated bilaterata. we discuss what our results suggest about eye evolution and arrestin functions.  identification of novel members of the arrestin clan we conducted phylogenetic analysis of arrestins and found a large tree of mostly unrecognized arrestins in eukaryotes. figure is a phylogenetic tree of arrestins from select evolutionarily diverse genomes. it shows that all the known animal arrestins are members of a small branch of the protein family that emerged relatively recently. we suggest that the two subfamilies could be termed alpha and beta class arrestins. this applies because the alphas are the ancient/ancestral arrestins and it fits well with the historical name of the betas. we find varied numbers of arrestins in the phyla previously known to have arrestins – fungi and animals. for example, the yeast schizosaccharomyces pombe has three arrestins. nematode worms and flies each have approximately twenty. half of the worm and fly arrestins appear to have emerged in their respective lineages. humans have a total of arrestins. for comparison, humans have approximately the same number of 7tmrs as caenorhabditis elegans and four times as many as drosophila. even protists, which are single-celled, show diverse numbers of arrestins. for example, dictyostelium, which has approximately 7tmrs, has four arrestin genes, and tetrahymena has fifteen. it is worth noting that tetrahymena have much more complex membrane and cytoskeletal architecture than animals. their genome has a striking expansion of genes involved in membrane and cytoskeletal dynamics. moreover, the percent of their genome devoted to kinases is double that of fungi and animals, but they only have four annotated 7tmrs. this hints that early arrestin function could have been associated with membrane/cytoskeletal dynamics or kinase signaling. compared to visual and beta arrestins, alphas have conserved protein sequence and domain topology spanning almost their full lengths. the high sequence conservation suggests their molecular functions are similar. for example, human txnip matches arrestin n and c domains with expect scores of e- and e- respectively. we also found two domains that appear to be distantly related to arrestins, spo0m and vps. both are clearly alpha arrestin-like, rather than beta. this finding was independently made by investigators managing the pfam database of curated protein patterns and posted on their database. they validate this "arrestin n-like clan" with one multiple sequence alignment of most available members of the three protein subfamilies, and provide confidence scores of 1e- and 2e-. spo0m proteins are present in archaea and in diverse bacterial phyla, but not in eukaryotes. notably, archaea and bacteria have seven transmembrane proteins and serine/threonine/tyrosine-specific kinases, both of which were widely believed to be eukaryotic inventions until recently. little is known about spo0m except that it is transcriptionally regulated by sigmah and that it inhibits sporulation. it is tempting to speculate that spo0m has a role in transmembrane signaling that links environmental status to sporulation. the crystal structure of a mammalian vps protein revealed that it has two arrestin domains. however, it was concluded there is no protein sequence similarity, presumably because vps was compared to the visual/beta arrestins. it was also suggested that the structural similarities between the two subfamilies are mostly superficial. the claim is that vps lacks all of the specific functional features of visual/beta arrestins: mainly, the sites responsible for gpcr, clathrin/ap and phosphatidylinositol phospholipid binding. two groups disagree on the possibility that the two proteins could have similar switch mechanisms based on conformational rearrangements. while both proteins have polar and electrostatic interactions between the n and c domains, they are mediated by different residues on different secondary structural elements. a caveat is that this analysis is limited by the absence of structural and functional information about alpha arrestins. that is, vps is being compared to distantly related visual/beta arrestins, rather than the much more closely related alpha arrestins. our results, and those of pfam, indicate vps and arrestin are true homologs. alpha arrestins have robustly similar structure predictions to those solved for beta class arrestins and vps. for example, "threading" of alpha arrestin txnip through all known crystal structures matches rod visual arrestin and vps with an identical probability value of 1e-. its presence in plants is significant because they lack arrestin. similar to arrestins, vps proteins very rarely have associated domains. in addition to the two known human vps genes – vps and vps26b – we found two new ones: dscr and an un-annotated gene on 2q. vps26p was discovered in a genetic yeast screen for "vacuole protein sorting, vps". the yeast vacuole is the equivalent of the mammalian lysosome. vps forms part of the retromer complex that mediates protein transport from endosomes to the trans-golgi. it is also involved in receptor transcytosis in polarized cells as well as in wnt signaling and gradient formation. the retromer has two subcomplexes, one responsible for cargo-selection and the other for vesicle formation. vps vps and vps form the receptor cargo-binding subcomplex. vps also has an adaptor role linking that subcomplex to the membrane-binding subcomplex. however, the exact biochemical role of vps is not defined. while there are four vps genes in mammals, the other retromer components are represented by single genes. retromer function is thus reminiscent of the role of beta arrestins in receptor endocytosis. both arrestin and vps are part of a transmembrane receptor-binding complex on the cytoplasmic face of endosomes. arrestin further interacts with the membrane via the clathrin adaptor ap- and vps26p does so through vps5p/vps17p. both arrestin and vps form complexes that co-localize with key vesicular trafficking proteins such as rab and n-ethylmaleimide-sensitive factor. nsf interacts directly with beta arrestin and is required for retromer endosome-to-golgi trafficking. it is curious that beta arrestins bind the vps partner vps, and that vps is present in 7tmr complexes; however, there is no evidence those interactions are biologically relevant. alpha arrestins were present in early eukaryotes we used psi-blast to search for all arrestins in the genbank protein database. we found alpha arrestins in fungi and in all multi-cellular life except plants. mammals have six alpha and four visual/beta arrestins. the significant protein-sequence difference between some of those alpha arrestins suggests they have more diverse functions than the more closely related visual/betas. the single arrestin present in viruses is a vertebrate alpha arrestin, arrdc that was horizontally transferred to canarypox virus. we also identified arrestins in all protist groups except rhizaria, which has little sequence available. in additional file we describe the protist proteins. since arrestin n and c domains are related by protein sequence, we searched for single-domain proteins that could be the ancestral arrestin domain that was duplicated. we found that no arrestin-containing kingdom or species has exclusively single-domain proteins. this result is consistent with the finding that the vps crystal structure reveals tandem domains. we believe a twin domain gene was duplicated in a bacterium or basal eukaryote and gave rise to genomes with both arrestin and vps we searched for arrestin-associated domains that could hint at biochemical functions. outside of the protists, we found that these are extremely rare. we found exactly two domains that recurred in at least two divergent phyla; they were in two distantly related orders of the fungus-like protists amoebozoa. two dictyostelium and two entamoebidae arrestins each contain a c domain, which is a ca2+-dependent phospholipid/membrane binding element. in one order the c domains are n-terminal in both proteins, in the other both are c-terminal. this suggests they could have been created by independent events of exon shuffling in the two orders. two other genes, one from each of the same two orders, contain one fyve domain in their c-terminal region. fyve domains have exquisite specificity for phosphatidylinositol-3-phosphate p), an endosomal marker recognized by proteins involved in signaling and trafficking. notably, vps interacts with the ptdinsp-binding protein sorting nexin. the presence of associated c and fyve domains indicates some ancient arrestin functions involve plasma and endosomal membrane interactions, respectively. this is consistent with known arrestin roles in transmembrane receptor-binding and endocytic trafficking. difference between alpha and visual/beta arrestins helix i of visual/beta arrestins is sequestered in the inactive conformation and is presumably released upon activation. this helix has hydrophobic residues on one face and basic residues on the other. however, it is a mystery what it does, or interacts with, in the active conformation. helix i leucine substitutions suggested a role in receptor binding, and it was later theorized to be a membrane-docking element that permits non-specific interactions with activated receptors. other experiments showed that helix i is important for the formation of dense-core vesicles. the proposed membrane docking role of helix i remains attractive to us. we add that such an insertion would displace membrane on the cytoplasmic leaflet of the plasma membrane. this could induce positive curvature and promote endocytocis. notably, helix i is absent in alpha arrestins and is thus a major innovation in beta arrestins. that interpretation is supported by the absence of helix i in the 3d structure of vps, which has protein sequence similarity to alpha arrestins. all indications suggest apha and beta arrestins have similar structural topologies, but do alphas also bind 7tmrs? nichols and sanders-bush announced their discovery of a new mammalian arrestin in. however, to our knowledge there are no published studies of arrestin-like functions in animal alpha arrestins. the current understanding of alpha arrestin biochemistry thus comes from fungi and yeast. herranz, vincent and colleagues showed that fungal palf is a bona fide arrestin by protein sequence and function. palf binds c-terminal sites of the activated seven transmembrane ph sensing receptor palh. moreover, alkaline activation induces palh-dependent phosphorylation and ubiquitination of palf. truncation of the palh cytoplasmic domain disrupts palf-binding and inhibits growth in alkaline ph. the alpha arrestin palf thus resembles beta arrestins in its ability to bind active receptors, generate a signal and be posttranslationally modified in the process. the function of palf, however, is in signal transduction and apparently not in inhibition of g protein signaling. ph sensing and "vacuole protein sorting, vps" pathways are intimately associated in fungi. herranz et al. thus propose that the role of palf is likely to relate to endocytic trafficking. this is notable considering that vps an ancient arrestin relative, was discovered in a genetic screen for vps genes. while beta arrestin tail domains have conserved clathrin-interacting motifs, alphas have py motifs. is there evidence the latter are functional? py motifs bind ww domains and their interactions are extensively defined by diverse biochemical and structural methods. the strong conservation of multiple py motifs in fungal and animal arrestins suggests they interact with ww proteins. saccharomyces cerevisiae has three alpha arrestins, all of which have turned up in biochemical and genetic screens. without knowing they were studying arrestins, others showed that two s. cerevisiae arrestins bind the hect e ubiquitin ligase rsp5p. moreover, they showed by mutagenesis that the interactions are mediated by two py motifs in arrestin and two ww domains in rsp5p. our computational analysis of the human genome identified ww proteins that may interact with alpha arrestins. notably nine of those genes are candidate co-orthologs of yeast rsp5p. this suggests alpha arrestins could be involved in ligand-dependent 7tmr ubiquitination and trafficking, which, in some yeast and mammalian cases, are mediated by hect e ligases. we also suspect arrestin py motifs interact with other ww proteins that are involved in cytoskeletal dynamics, endocytocis/trafficking and signaling. using yeast two hybrid methodology, we have found that alpha arrestin tails robustly bind the ww domains of hect e ligases and of other proteins. origins of visual/beta arrestin and rhodopsin the fact that all known visual arrestins cluster together with beta arrestins suggested to us that this branch may have emerged together with an early opsin. we used visual/beta arrestin and visual/beta arrestin consensus sequences to search genbank for the earliest family member we could find. the oldest visual/beta arrestin we found is from hydra. notably, cnidarians are the only beta arrestin-containing organisms that predate any known opsin. we next searched for the oldest opsin genes present in all, but the trace database of genbank. that analysis resulted in the identification of multiple hydra ests encoding partial opsin proteins. of the known subfamilies, we only found representation of ciliary opsin. we were able to assemble the full length open reading frame for one of these. this cnidarian protein is strikingly similar to ciliary opsins, placing it in the same group as rag-worm ciliary opsin and human visual opsins on the phylogenetic tree of opsins. cnidarian opsin genes are the first to be reported that predate the emergence of bilaterata. while the present study was under review, others independently reported finding cnidarian opsins. as we did, both of those groups found that cnidarians have ciliary opsins. they additionally reported other highly divergent opsins that cannot be clearly classified according to the known subfamilies. remarkably, suga, schmid and gehring identified large numbers of opsin genes in single species of cnidarians: sea anemone, hydra, and jellyfish. they showed jellyfish opsins are expressed in multiple patterns, including in the eyes and gonads. neither those studies nor ours were able to find any evidence of opsins in sponges or older phyla. this is consistent with the fact that sponges use flavin or carotenoid photopigments. the early evolution of vision is an open question. it was long known that some jellyfish have image forming eyes, but the molecular identity of their visual pigments was a mystery until now. recently it was postulated that the absence of a master transcriptional regulator, pax in the box jellyfish is consistent with the independent evolution of eyes in higher metazoans. gehring offered two alternative explanations to that same question: vertical evolution from a photosensitive protist and horizontal evolution through a photosynthetic cyanobacterial symbiont. we proposed that the phylogeny of the phototransduction machinery would explain the evolution of phototransduction and vision. the new opsin phylogeny suggests to us that ciliary opsins are likely to be ancestral to all previously known visual pigments in animals. that possibility is consistent with darwin's theory that eyes evolved once: how a nerve comes to be sensitive to light hardly concerns us more than how life itself originated; but i may remark that, as some of the lowest organisms in which nerves cannot be detected are capable of perceiving light, it does not seem impossible that certain sensitive elements in their sarcode should become aggregated and developed into nerves endowed with this special sensibility... the simplest organ which can be called an eye consists of an optic nerve surrounded by pigment-cells and covered by translucent skin, but without any lens or other refractive body. we may, however, according to m. jourdain, descend even a step lower, and find aggregates of pigment-cells, apparently serving as organs of vision, without any nerves, and resting merely on sarcodic tissue. eyes of the above simple nature are not capable of distinct vision, and serve only to distinguish light from darkness. the fact that both opsin and visual/beta arrestin are present in cnidaria, but not older phyla, hints that the two subfamilies could have emerged in concert. early phototransduction offered major advantages, such as photoperiodicity and phototaxis. presumably, the first step was the creation of photosensory opsin. however, there must have been immense avenues for improvement after that point. one challenge to early opsin was exposure to light intensity that varies by orders of magnitude, the earth's daily illumination cycle. the innovation in visual/beta arrestin could have improved the modulation of signaling gain by regulating opsin localization, endocytosis/recycling or inactivation kinetics. gene duplication is a hugely important mechanism for evolution. visual phototransduction consistently uses dedicated proteins created by gene duplication. for example, in addition to opsin 7tmrs, mammalian photoreceptor cells have their own, or nearly exclusive, g proteins, cgmp-phosphodiesterases, g protein-coupled receptor kinases, arrestins, etc. outside the eye, a handful of each of those proteins transduces signals for the remaining hundreds of 7tmrs. this extreme specialization illustrates the high importance of vision in animal evolution. possible implication of pre-cambrian vision both opsin and visual/beta arrestin are present in cnidaria, but not in sponges, the most basal metazoans. this is interesting in light of parker's recent proposal that vision set off the cambrian explosion of life forms. million years ago only three phyla existed, but, in the following my, phyla emerged. no new phyla have emerged since that cambrian explosion. parker proposed that vision triggered the cambrian explosion by creating a new world of organismal interactions. the key observation is that pre-cambrian phyla were soft-bodied. however, the cambrian saw an apparently limitless diversification of hard body parts. at the same time, the use of biological color appeared. parker claims this scenario can be explained by the emergence of vision, which must have resulted in new behaviors such as predation. seeing-predators would have suddenly needed rigid components to pursue, attack, and eat their prey. their prey – which may or may not have had eyes – also had to adapt by developing hard shells or spines, camouflage or even invisibility. parker specifically hypothesized that this "light switch" was triggered by the emergence of arthropod eyes during the cambrian. put another way, he proposed the light switch required image perception and brain processing of the information. we find his general argument to be compelling. however, we believe the spark had to precede the cambrian explosion. we propose that light perception in a pre-cambrian animal initiated new dimensions of organismal and environmental interactions. this may have progressed steadily until a critical mass of interactions was reached, the cambrian explosion. our findings are clear evidence that opsin predated the cambrian period. this meets what we consider to be the first requirement for a visual trigger hypothesis. if sight initiated an arms race in the biosphere, it is not surprising that some organisms refined it and others lost it. only six of phyla on earth have eyes, but these six seeing phyla account for >95% of all living animals. it is not difficult to imagine that the relative success of seeing animals is due to the extraordinary advantage of vision.  despite the high interest in gpcr signaling, its evolution remains enigmatic. there is some evidence that archaeal and bacterial 7tmrs are homologous to eukaryotic 7tm/gpcrs. however, heterotrimeric g proteins/g alpha subunits are only present in eukaryotes. this suggests that ancestral 7tm/gpcrs signaled by mechanisms other than g protein coupling. we found that the arrestin clan is present in archaea and bacteria, raising the possibility that spo0m could be a primordial 7tmr signaling partner. in addition, our findings of cnidarian opsins lead us to propose that the ciliary subfamily is ancestral to all bilaterian opsins. that is consistent with darwin's theory that eyes evolved once. there were two major arrestin-like gene families in early eukaryotes, arrestin and vps both protein families are well characterized and point to endocytosis/endosomal dynamics as the ancestral arrestin/vps functions. the duplication of the arrestin domain was a critical event in the creation of ancestral arrestin/vps this could have created autoinhibitory mechanisms, a recurrent theme in the evolution of signal transduction. the functional similarities of beta arrestins and vps proteins lead us to speculate that the original arrestin/vps was involved in receptor internalization. this could have had two classes of receptor effects in concert: 1) desensitization and recycling/degradation, and 2) signaling. others have hypothesized that the original role of arrestins may have been as signaling adaptors rather than terminators. above we mention biochemical evidence that mammalian vps and arrestins could have overlapping roles. the homology of alpha and beta arrestins suggests their molecular functions may be similar. there is evidence from fungi that the alpha arrestin palf specifically binds an activated 7tmr. that interaction has a positive signaling role that is not yet identified. there are also differences between the alpha and beta classes. beta arrestins are generally cytoplasmic in unstimulated cells, while alpha arrestins are often associated with membranes. only visual/betas have helix i in the n domain. and the tails of betas contain clathrin-interacting motifs, while those of alphas have py motifs. studies in yeast showed that alpha arrestin py motifs interact with the ww domains of the hect e ubiquitin ligase rsp5p. we believe that a major role of alphas is to recruit ww proteins to activated receptors. alpha and beta arrestins are widely co-expressed. the fact that visual/beta arrestins can hetero-associate, hints that alphas and betas may also. given the near ubiquitious involvement of beta arrestins in 7tmr signaling, we speculate that alphas and betas may function coordinately. 
others recently showed that the 3d structure of mammalian vps and the biochemical function of the yeast alpha arrestin palf are similar to those of beta arrestins. we note that only alpha arrestins have py motifs in their c-terminal tails, and only visual/betas have helix i in the arrestin n domain. g protein coupled receptors are the most numerous proteins in mammalian genomes, and the most common targets of clinical drugs. gpcrs are intimately associated with trimeric g proteins, g protein receptor kinases, and arrestins. our arrestin findings have implications on the evolution of gpcr signaling, and on the biological roles of human alpha arrestins. that finding is consistent with darwin's theory that eyes evolved once, and lends some support to parker's hypothesis that vision triggered the cambrian explosion of life forms. we identified ciliary opsins in cnidaria and propose this subfamily is ancestral to all previously known animal opsins. curiously, we identified both the oldest visual/beta arrestin and opsin genes in cnidaria. we found that the arrestin clan is comprised of the spo0m protein family in archaea and bacteria, and the arrestin and vps families in eukaryotes. the arrestin clan has human members: alphas, visual/betas, and vps genes.
 g protein coupled receptors are the most numerous proteins in mammalian genomes, and the most common targets of clinical drugs. however, their evolution remains enigmatic. gpcrs are intimately associated with trimeric g proteins, g protein receptor kinases, and arrestins. we conducted phylogenetic studies to reconstruct the history of arrestins. those findings, in turn, led us to investigate the origin of the photosensory gpcr rhodopsin.  we found that the arrestin clan is comprised of the spo0m protein family in archaea and bacteria, and the arrestin and vps families in eukaryotes. the previously known animal arrestins are members of the visual/beta subfamily, which branched from the founding "alpha" arrestins relatively recently. curiously, we identified both the oldest visual/beta arrestin and opsin genes in cnidaria. the arrestin clan has human members: alphas, visual/betas, and vps genes. others recently showed that the 3d structure of mammalian vps and the biochemical function of the yeast alpha arrestin palf are similar to those of beta arrestins. we note that only alpha arrestins have py motifs in their c-terminal tails, and only visual/betas have helix i in the arrestin n domain.  we identified ciliary opsins in cnidaria and propose this subfamily is ancestral to all previously known animal opsins. that finding is consistent with darwin's theory that eyes evolved once, and lends some support to parker's hypothesis that vision triggered the cambrian explosion of life forms. our arrestin findings have implications on the evolution of gpcr signaling, and on the biological roles of human alpha arrestins. 