insights into the GPCR system at the early divergence of animals

16 Pages·2015·3.22 MB·English

by  Arunkumar Krishnan

Krishnanetal.BMCEvolutionaryBiology (2014) 14:270
DOI10.1186/s12862-014-0270-4
RESEARCH ARTICLE Open Access
The GPCR repertoire in the demosponge
Amphimedon queenslandica: insights into the
GPCR system at the early divergence of animals
Arunkumar Krishnan1†, Rohit Dnyansagar1,2†, Markus Sällman Almén1, Michael J Williams1, Robert Fredriksson1,
Narayanan Manoj2 and Helgi B Schiöth1*
Abstract
Background: Gprotein-coupledreceptors (GPCRs) play a central rolein eukaryotic signal transduction.However,
theGPCR component of this signalling system,atthe early origins ofmetazoans is notfully understood.Here we
aim to identify and classify GPCRs inAmphimedonqueenslandica(sponge),a member ofan earliest diverging
metazoan lineage (Porifera). Furthermore, phylogenetic comparisons ofspongeGPCRs with eumetazoan and
bilaterian GPCRs will be essential to our understanding ofthe GPCR system at therootsof metazoan evolution.
Results: We present a curated list of 220GPCRs inthesponge genome after excluding incomplete sequences and
false positivesfrom our initial dataset of 282 predicted GPCR sequences obtained using Pfam search. Phylogenetic
analysis reveals that thesponge genome contains members belonging to four ofthefive majorGRAFS families
including Glutamate(33), Rhodopsin (126), Adhesion (40) and Frizzled (3). Interestingly, thesponge Rhodopsin family
sequences lack orthologous relationships with those found ineumetazoan and bilaterian lineages, sincethey
clustered separately to form spongespecific groups in the phylogenetic analysis. Thissuggests that sponge
Rhodopsins diverged considerably from that found inother basal metazoans. A few sponge Adhesionsclustered
basal to Adhesion subfamilies commonly found inmost vertebrates, suggesting some Adhesion subfamilies may
have diverged prior to theemergenceof Bilateria. Furthermore, atleast eight of the sponge Adhesionmembers
have a hormone binding motif (HRM domain) in theirN-termini, although hormones have yet to be identified in
sponges. We also phylogenetically clarified thatsponge has homologs of metabotropic glutamate (mGluRs) and
GABAreceptors.
Conclusion: Our phylogenetic comparisons of sponge GPCRs withother metazoangenomes suggest that sponge
contains a significantly diversified set of GPCRs. This is evident atthefamily/subfamily levelcomparisons for most
GPCR families, in particular for theRhodopsin family of GPCRs. Insummary, this study provides a framework to
perform future experimental and comparative studies to further verify and understand the roles of GPCRs that
predates the divergence of bilaterian and eumetazoan lineages.
Keywords: Neurotransmitters, Gprotein-coupledreceptors, Adhesion,Signaltransduction,Porifera,Eumetazoa
*Correspondence:[email protected]
†Equalcontributors
1DepartmentofNeuroscience,FunctionalPharmacology,UppsalaUniversity,
BiomedicalCenter,Box593,75124Uppsala,Sweden
Fulllistofauthorinformationisavailableattheendofthearticle
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Dedicationwaiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle,
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Krishnanetal.BMCEvolutionaryBiology (2014) 14:270 Page2of16
Background repertoire with 890 and 420 GPCR coding genes, re-
The G protein-coupled receptor (GPCR) superfamily is spectively[17,18].
one of the largest families of integral transmembrane Although several studies including genome-wide ana-
proteins in vertebrates and plays a dominant role in sig- lysis of the sponge demonstrated the presence of several
nal transduction in most eukaryotes. GPCRs, which me- GPCRs, a comprehensive overview of sponge GPCR
diate most of the cellular responses through hormones, families is still lacking and their relationship to the ver-
neurotransmitters and environmental stimulants are thus sions found in eumetazoans and bilaterians is largely
major drug targets, with approximately 36% of current unknown. This is important because sponges and the
clinical drugs targeting these receptors [1,2]. In humans, eumetazoans (Nematostella and Trichoplax) are known
there are around 800 genes coding for GPCRs, and we to lack most of cell types found in bilaterians. For ex-
earlierclassifiedthemintofivemainGRAFSfamilies:Glu- ample,spongesaresimpleporebearinganimalsthatlack
tamate, Rhodopsin, Adhesion, Frizzled and Secretin [3,4]. gut, a nervous system and muscle, but constitute an in-
Subsequent GPCR mining studies have suggested that ternalnetworkofcanalsandciliatedchoanocytechambers
GRAFS families are present in most bilaterian species that pump water to extract food [19-21]. Placozoans
[5-8]. In addition, our earlier studies demonstrated that (Trichoplax) are flat animals consisting of a lower and
fourofthefiveGRAFSfamilies(excludingSecretin,which upper epithelium, which sandwich layers of multinucle-
evolved after the divergence of cnidarians) are found in atedfibre cells [17,22]. Similarly, nerves, sensorycells and
basalfungi,indicatingthattheGlutamate,Rhodopsin,Ad- muscle cells are apparently absent in placozoans. In con-
hesion,andFrizzledfamiliesevolvedbeforethedivergence trast, Nematostella is regarded as one of the first animals
ofmetazoanlineages[9]. possessinganervoussystem.InNematostella,anectoder-
Although the four GRAFS families first evolved in the mal and endodermal nerve net constituting of a simple
basal fungi, only a few sequences were unambiguous ho- and diffuse nervous system runs throughout the animal’s
mologs of metazoan representatives [9]. For example, body[23,24].
only a few homologues of the Rhodopsin family were In order to better understand the components of the
found in basal fungi and Rhodopsin GPCRs were not GPCR system and its evolution at the early origins of
found in choanoflagellates (Monosiga brevicollis and Sal- Metazoa, we aimed to curate a complete set of GPCRs
pingoeca rosetta) and filasterean Capsaspora owczarzaki in sponge, as well as provide a comparative analysis with
[9,10]. Moreover, these closest metazoan relatives are GPCRsfound ineumetazoans (NematostellaandTricho-
limited to only a few genes coding for Adhesion and plax) and bilaterians (humans and sea urchin; Strongylo-
GlutamateGPCRfamilies.Theseobservationsclearlyin- centrotus purpuratus). Utilising the sponge genome, we
dicate that the first large expansions of Rhodopsin sought to answer questions such as, 1) does one of the
GPCRs, as well as other families of GPCRs, occurred at mostancientmetazoanlineagehaveorthologsofmamma-
the early origins of metazoans. This model is well sup- lian GPCRs, 2) do sponges hold mammalian-like subfam-
ported by the recent genome release of Amphimedon ily level classifications for each major GRAFS families, 3)
queenslandica (hereafter referred to as sponge), which are sponge GPCRs orthologous to those found in cnidar-
belongs to one of the earliest diverging and oldest sur- iansandotherpre-bilaterianlineages.
viving phyletic branches of Metazoa. The draft genome
as well as the transcriptome profiling of sponge indi- Results
cated the presence of several Rhodopsin-like GPCRs and IdentificationandclassificationofGPCRsinsponge
anoverallcountofmorethan200GPCRs,includingAd- In order to generate a complete set of sponge GPCRs,
hesion and Glutamate family GPCRs [11,12]. Additional we aligned the sponge proteome with Hidden Markov
studies on some specific subsets of sponge GPCRs such Models (HMM) of the 14831 families contained within
as Glutamate [13] and Frizzled [14] provided further in- the Pfam database (version 27). We retrieved all se-
sights into the GPCR component in sponge. Taken to- quencesthatcontainedthePfam domains corresponding
gether this suggests that the last common ancestor of to the various GPCR families (see Methods). This initial
metazoans possessed a complex GPCR system, perhaps screen identified 282 GPCR sequences belonging to the
withexpansionswithintheRhodopsinfamilyincompari- GPCR_A Pfam clan (CL0192). These numbers are simi-
son to pre-metazoan lineages like Choanoflagellata [15] lar and comparable with previous studies where sponge
and Filasterea [16]. Furthermore, genome data of species GPCR sequences were identified [10-12]. However, this
that diverged after sponges provided valuable insights initial list of GPCRs included fragments and possibly
into the evolution of the GPCR superfamily. Previous some false positives, and thus had to be refined before
mining of GPCRs in a cnidarian, Nematostella vectensis performing phylogenetic analysis to obtain stable and
and a placozoan, Trichoplax adhaerens revealed that consistent topologies. Therefore, we examined these 282
these pre-bilaterian metazoans contained a large GPCR GPCRs for the presence of seven transmembrane (TM)
Krishnanetal.BMCEvolutionaryBiology (2014) 14:270 Page3of16
helices using HMMTOP and Phobius servers. To re- Amphimedon, which may provide better resolution of the
move fragments and false positives we excluded the se- fragmentary sequences/regions. To avoid possible confu-
quences having less than five or more than eight TM sion in subsequent paragraphs, a whole family is denoted
regions from the final dataset. To cross verify the list of usingthecorrespondingfamilynameinitalicswithanini-
sponge GPCRs, we aligned each putative sponge GPCR tial capital letter (Rhodopsin), while the homologs/mem-
sequence with our tagged human GPCRs using the stan- bers of a particular family are denoted as Rhodopsins or
dalone BLASTP program (data not shown). Such step- AdhesionsorAdhesion-likereceptors.
wise processes led to the verification and categorisation
of a final dataset containing 220 GPCRs. A majority of PhylogeneticverificationofGRAFStopological
these were categorised into four of the five main GRAFS classification
families, including 126 Rhodopsin (7tm_1), 40 Adhesion The human GPCR repertoire can be classified into five
(7tm_2), 33 Glutamate (7tm_3) and three Frizzled re- main groups (Glutamate, Rhodopsin, Adhesion, Frizzled
ceptors. However, we did not find Secretin family recep- and Secretin; GRAFS) based on phylogenetic analysis [4].
tors in the sponge genome. It must be mentioned that Subsequent comparative phylogenetic studies in several
an earlier report suggested that sponge has Secretin fam- vertebrate and invertebrate species have supported this
ily GPCRs [12] possibly due to the presence of the HRM classification system and established that GPCRs indeed
(hormone receptor motif) domain in their N-termini, formed distinct clusters corresponding to its five main
which is a usual characteristic of Secretin GPCRs [4,25]. families[6,8,28].ToinvestigatewhethertheGPCRsidenti-
However, Secretin family GPCRs are mostly activated by fied in sponge also exhibit distinct phylogenetic clusters
peptide hormones, which to date have not been identi- corresponding to the GRAF classes (excluding Secretin,
fied in the sponge genome [11]. Our finding that the which is absent in sponges), we performed a Bayesian
sponge genome lack Secretins is also consistent with phylogenetic analysis using all sponge GPCR sequences.
earlier studies which proposed that Secretin family des- Totesttherobustnessofthetopologyaswellastoresolve
cended from the Adhesion family after the split of the the orthology relationships between the members in
cnidarians from other bilaterians and that the Secretin sponge and other bilaterians and pre-bilaterians, we ex-
GPCRs are a bilaterian innovation [26,27]. Considering panded the dataset to include a few representative se-
that Adhesion and Secretin families belong to the same quences from the Trichoplax, Nematostella and sea
class of GPCRs (Class B) and encode a 7tm_2 trans- urchin GPCRs. This set contained equal proportion of
membrane domain, it is sometimes difficult to distin- representatives from Rhodopsin, Glutamate and Adhesion
guish between the families and they can be wrongly GPCR families. The overall unrooted topology indicated
assigned. Since Adhesion is a parent family to Secretin thatthe Rhodopsin, Glutamate and Adhesion GPCRs des-
GPCRs and due to the lack of experimental support for cend into separate and distinct clusters, whereas Frizzled
the presence of Secretin GPCR activity in sponges we GPCRs were placed basal to the Adhesion GPCR node
here label these class B (7tm_2) receptors as Adhesion (Figure1).
family receptors. Nevertheless, the presence of an HRM
domain in these Adhesion GPCRs is intriguing and Rhodopsinreceptorfamily
should prompt further experimental verifications to pro- The human Rhodopsin family GPCRs are classified into
vide evidence for GPCR mediated hormonal activity in four major groups termed α-, β-, γ-, and δ that are di-
sponges. vided into 13 major subfamilies. Some of these 13 sub-
Interestingly, in addition to the GRAFS families, we families like amine and peptide binding Rhodopsin
identified 14 cyclic AMP-like receptors (Dicty_CAR; family receptors are present and seem fairly conserved
PF05462), two intimal thickness-related-like receptors in most of the analysed bilaterians [4,6-8,28]. Similarly,
(PF06814), and one lung-7TM-like receptor (PF06814) in order to categorize sponge Rhodopsins and explore
in the sponge genome. Moreover, we identified a puta- their similarity to those found in other species, we
tive homolog of GPR143 (PF02101), which in humans aligned each sponge Rhodopsin family sequence against
and other mammals is associated with ocular albinism. a database of GPCRs containing complete repertories
In summary, the proportion of sponge GPCRs to the from human, sea urchin, Trichoplax and Nematostella.
genome size is comparable to several other metazoans The entire list of blast hits are provided in Additional
and that it also constitutes a large expansion within the file2.Furthermore,weperformedBLASTsearchesagainst
Rhodopsin family [11,13]. The complete set of sponge manually annotated and reviewed Rhodopsin (7tm_1)
GPCR sequences identified in this study is available in GPCRs obtained from the Swiss-Prot database (available
FASTA format (see Additional file 1). It must be noted in Additional file 3). This reviewed list of Rhodopsin
herethatthenumbersprovidedinthisstudymayvaryfrom GPCRs included most of the GPCRs from well charac-
futurepredictionsusingsubsequentgenomeassembliesof terised vertebrates, as well as from several well-known
Krishnanetal.BMCEvolutionaryBiology (2014) 14:270 Page4of16
Figure1PhylogeneticrelationshipsofGPCRsidentifiedinthespongegenome.Thetreetopologyshowsdistinctphylogeneticclusters
belongingtotheGlutamate,Rhodopsin,Adhesion,andFrizzledfamiliesoftheGRAFSclassificationsystem.Thetreealsoincludesafew
representativesofGPCRfamiliesfromothergenomes.Theedgesarecoloredaccordingtothefamilies,whiletheaccessionIDsarecolored
differentlyforeachspecies.TheillustrationshowsputativeAdhesionlikeGPCRsthatlackstheconventionallongN-termini.However,these
AdhesionGPCRscontainthecharacteristic7tm_2domainandplacedbasaltothemajornodethatcontainsalltheotherAdhesionsincludedin
thephylogenetictreemaking.SpongespecificRhodopsinclusters(AqRho-AtoAqRho-E)withinthemajorRhodopsinclusterarehighlighted.
invertebrate model organisms. From BLASTP search human and sponge GPCR repertoires was within the
results we were unable to classify most of the sponge Rhodopsin family. Although a few of the spongeRhodop-
Rhodopsin family sequences into any of the 13 known sins were placed in the same branch containing human
Rhodopsin-likeGPCRssubfamilies.Thisisbecausesponge Rhodopsins, they lacked reliable confidence value sup-
Rhodopsins failed to satisfy our classification criteria portfrom both treemakingmethods(ML andBayesian).
that at least four of the first five hits must be from the Taken together, our results from the phylogenetic ana-
same subfamily. However, a few sponge Rhodopsins had lysis suggest that the sponge Rhodopsins lacked unam-
their best aligned hits (E-values ranging from e-10 to e- biguous orthologous relationships to any of the known
20) to beta-adrenergic receptors, serotonin and opsin human subfamilies (see Figure 2). This is consistent with
family receptors, among others (see Additional file 3). the BLAST results that could not classify sponge Rho-
These BLASTP results were subsequently verified using dopsins into subfamilies. Moreover, it must be men-
Bayesian and Maximum Likelihood (ML) based phylo- tioned here that a few of the sponge Rhodopsins had
genetic analysis. their top hits (see Additional file 3) as amine and opsin-
Phylogenetic analysis using Bayesian and ML methods like receptors in the blast search. However, these se-
was performed to resolve the relationships between hu- quences failed to form a coherent group with the human
man and sponge Rhodopsin GPCR family sequences Rhodopsin homologs. Instead, they clustered separately
(Figure 2). The results obtained from both tree building and are found scattered within the major node that
methods indicated that the largest differences between grouped the sponge Rhodopsins (see Figure 2). This
Krishnanetal.BMCEvolutionaryBiology (2014) 14:270 Page5of16
Figure2(Seelegendonnextpage.)
Krishnanetal.BMCEvolutionaryBiology (2014) 14:270 Page6of16
(Seefigureonpreviouspage.)
Figure2PhylogenetictreeshowingrelationshipsbetweenRhodopsinfamilyGPCRsinspongeandhumangenomes.Thetreetopology
wasinferredfromBayesiananalysiswithagammacorrectionusingMrBayessoftware.Thephylogenetictreeisbasedonlyonthetransmembrane
region.TheMCMCanalysiswasusedtotesttherobustnessofthenodesandwassupportedbyanon-parametricbootstrapanalysiswith500replicates.
TheedgescorrespondingtohumanRhodopsinfamilyGPCRsarehighlightedingreen.EdgescontainingspongeRhodopsinsarehighlightedinblue.
SpongespecificclustersandarelabeledasAqRho-A–AqRho-E(whereAqstandsforAmphimedonqueenslandica,RhoforRhodopsinlikeGPCRsandA
toErepresentthedistinctclustersinthephylogenetictree).ThevaluesindicatedatmajorbranchesareposteriorprobabilityvaluesfromBayesian
analysisandpercentagebootstrapvaluesfromMaximumlikelihoodanalysis.Redasterisksymboldenotessequencesthathaveatleastfourofthetop
fivehitsasbeta-adrenergic,serotoninandopsinfamilyreceptors(seeAdditionalfile3)inourblastsearch.However,theydidnotseemtoforma
coherentgroupwiththehumanRhodopsins.Scalebarsindicatephylogeneticdistanceasnumberofsubstitutionspersite.Phylogeneticrelationships
betweenspongeRhodopsins,theeumetazoans(NematostellaandTrichoplax)andsea-urchingenomesareprovidedinAdditionalfile4.
distinctiverepertoire ofspongeRhodopsinshadrelatively human Group I and Group II Adhesions. This implies
high similarity between them and form five observable that these sequences are putative ancestral representa-
clusters (Figure 2). Here, we putatively labelled these tives of Groups I/II (Figure 3). A closer examination of
spongespecific clusters asAqRho AtoE. the phylogenetic relationships showed that there were
Since the phylogenetic distance between the GPCR several Adhesions from sponge, Trichoplax and Nema-
dataset representing human and sponge was large, we tostella placed in a major node that contained human
wanted to investigate whether similar phylogenetic rela- Adhesions from groups VI and VII (Figure 3). The re-
tionships existed between sponge and other closely re- maining sponge Adhesions are most likely sponge spe-
lated species. Therefore, we extended our study to three cific, since they clustered separately from any known
additional species having completely sequenced ge- Adhesion groups. This observation was consistent with
nomes. This included two non-bilaterian animals from other analysed metazoans, where most of the Adhesions
the eumetazoan lineage, the placozoan Trichoplax and from sea urchin and other genomes clustered separately
the cnidarian Nematostella, as well as the deuterostome from thehuman counterparts(see Figure3).
bilaterian, sea urchin (Additional file 4). The phylogen- Another noteworthy observation was that some of the
etic trees indicated a similar topology wherein sponge sponge specific Adhesions have short N-termini and lack
Rhodopsins lackorthologousrelationshipstothosefound GPCR proteolytic site (GPS). However, these protein
in Nematostella, Trichoplax and sea urchin (Additional transcripts contained the core 7tm_2 domain region,
file4). characteristic to all Adhesion GPCRs. It is possible that
these Adhesions were incompletely modelled at the
Adhesionreceptorfamily N-termini due to sequencing errors. An alternative ex-
The human genome contains 33 Adhesion receptors that planation is that these sponge Adhesions may truly lack a
phylogenetically cluster into eight main groups (I-VIII), GPS site and the N-terminal domains, since the diver-
with VLGR1 placed as an out-group. Earlier studies gence is also reflected in the transmembrane helices that
demonstrated that potential homologs of genes belong- were utilized for phylogenetic tree making. These se-
ing to families I, III, IV, V, VIII and VLGR1 are present quences clustered separately from rest of the sponge
in most invertebrates, whereas families II,VI and VII are Adhesion GPCRs (see Figure 3). However, future experi-
more likely to be vertebrate innovations [6-8,26]. To ex- mental verification, as well as mining of Adhesions in
plore whether sponge Adhesions show homologous rela- other sponge genomes, is required to confirm these attri-
tionships to any known Adhesion GPCR groups, we butesofspongeAdhesions.Thismightalsoclarifywhether
included 33 human Adhesions and all identified sponge the short N-termini are more prevalent in ancient Adhe-
Adhesions for phylogenetic analysis. Furthermore, we in- sion GPCRs and the long N-termini was later gained due
cluded Adhesions from other metazoans to explore their toadditionandshufflingofkeydomainsduringthecourse
relationship with sponge Adhesions. Phylogenetic ana- ofmetazoanevolution.
lysis revealed that a few sponge Adhesions were placed Although, a few sponge Adhesions lack a GPS site and
basal to the node that contained human Adhesions be- N-terminal domains, the rest show diverse domain
longing to family VIII (Figure 3). This tree topology was architecture similar to that observed in other metazoan
bettersupportedwhentheanalysiswasrestricted toonly Adhesions.TheGPS domain, acommon cleavagesitefor
human and sponge Adhesions (Additional file 5). Also, many members of this family is present in 28 out of 40
the sponge Adhesion sequence Aq715659 clustered basal sponge Adhesions. Another interesting feature was the
to the node containing human Group I and Group II presence of a HRM domain in at least eight of the
Adhesion sequences (Figure 3). Additionally, two more sponge Adhesions although hormones have not been re-
sequences from sea urchin (Sp00392) and Nematostella ported in sponges (Figure 4). Intriguingly, we could also
(Nv24490) were placed in the same node containing identify sponge Adhesions (Aq712029 and Aq715659)
Krishnanetal.BMCEvolutionaryBiology (2014) 14:270 Page7of16
Figure3PhylogeneticrelationshipsbetweenAdhesionfamilyGPCRsinspongeandothergenomes.Thecolorschemeforthebranchesis
accordingtospeciesused.Theposteriorprobabilityvalues>0.95;0.9-0.95and0.7to0.9arehighlightedinfilledgreen,orangeandgreycircles,
respectively.AccessionnumbersformostoftheseaurchinAdhesionswereremovedfromthefinalrepresentationfordisplayreasons.Human
AdhesionGPCRGroupsItoVIIIareheightedingrey.
that harbour multiple repeats of the Scavenger receptor Glutamatereceptorfamily
cysteine-rich protein (SRCR) domain (Figure 4). To the Glutamate receptors (GLRs) are crucial modulators of
best of our knowledge identification of SRCR repeats is neurotransmission, and in humans there are 22 receptors
unique to Adhesion GPCRs and it is worth mentioning consisting of eight metabotropic glutamate receptors
that SRCR repeats are often associated with immune (GRMs), two GABABRs, the calcium-sensing receptor
systemfunctions invertebrates[29,30]. (CASR),thesweetandumamitastereceptors(TAS1R1–3),
Krishnanetal.BMCEvolutionaryBiology (2014) 14:270 Page8of16
Figure4N-terminaldomainarchitectureofaselectionofspongeAdhesionGPCRs.Thedomainswereidentifiedbyaligningsponge
AdhesionstothelatestversionofPfamlibrary.FewAdhesionGPCRsequencesthatlackN-terminaldomainsarenotshown.Thedomainsshown
inthefigureinclude;7TM:seven-transmembranedomain,DUF:Domainofunknownfunction,EGF:epidermalgrowthfactor-likedomain,fn3:
fibronectintypeIIIdomain,GPS:GPCRproteolyticsitedomain,HRM:Hormonereceptordomain,IG/IG_2/IG_3:immunoglobulindomains,I-set:
ImmunoglobulinI-setdomain,SNARE:solubleN-ethylmaleimide-sensitivefactor(NSF)attachmentprotein(SNAP)receptordomain,SRCR:Scavenger
receptorcysteine-richdomain,V-set:ImmunoglobulinV-setdomain.
GPRC6A and seven orphan receptors [2,4]. Phylogenetic demonstratedthatamongthe33indentifiedspongeGLRs,
analysis of the Glutamate family members from human only seven are homologous to GRMs, while the rest were
and sponge (Figure 5) revealed that sponge had seven sponge specificreceptors(Additional file 6). However, the
GLRshomologoustohumanmetabotropicGlutamatere- spongeGLRsthathadsimilaritytoGABABandtwoother
ceptors (GRMs). In addition, phylogenetic analysis re- orphans (GPR158 and GPR179) failed to give stable or
vealed that two sponge GLRs clustered with human consistenttopologyinalargerdatasetandclusteredsepar-
GABAB receptors and another three were placed on the ately from the known Glutamate receptors, suggesting
same node containing GPR158 and GPR179, but with a theyaredivergentfromothermetazoancounterparts.
low posterior probability support (Figure 5). To test the
robustness of these relationships, we included Glutamate Frizzledreceptorfamily
GPCRs from Nematostella, Trichoplax and sea urchin. The sponge proteome dataset contained three full length
An overall unrooted tree obtained from a large dataset members of the Frizzled GPCR family. A comparative
Krishnanetal.BMCEvolutionaryBiology (2014) 14:270 Page9of16
Figure5PhylogenetictreeshowingrelationshipbetweenGlutamatefamilyGPCRsinspongeandhuman.Theedgescontaininghuman
GlutamatefamilyGPCRsarehighlightedingreen,whiletheedgescontainingspongeGlutamatearehighlightedinblue.Phylogeneticrelationships
betweenspongeGlutamate,theeumetazoans(NematostellaandTrichoplax)andsea-urchingenomesareprovidedinAdditionalfile6.
phylogenetic analysis with Frizzled receptors from sponge models that contained less than 4 TM regions. Similarly,
and other metazoan genomes was performed. For the an earlier study identified eight Frizzled- like GPCRs in
phylogenetic tree construction, we also included the the sponge genome[14].However, a few ofthese seem to
closely related smoothened GPCR family members from be incompletely modeled and were not included in the
human and other metazoans. Phylogenetic relationships finalspongeGPCRdatasetforbetterhandlingoftheMSA
revealed that sponge Frizzled GPCRs are fast evolving or (Multiplesequencealignment)dataforsubsequentphylo-
divergent from other metazoan counterparts (as indicated geneticstudies.
by Long Branch lengths) (see Additional file 7). Two
sponge Frizzled receptors were placed basal to the node OtherGPCRfamilies
that contained human FZD9, FZD10 receptors. Also, one Our analysis revealed that the sponge proteome dataset
Frizzledreceptoreachfromotheranalysedmetazoanswas also contains members of other GPCR families that do
placed in the same node with human FZD9, FZD10 re- notbelongtotheGRAFS classification system.Thesein-
ceptors. Interestingly, one sponge Frizzled-like receptor cluded cAMP-like, intimal thickness-related receptor
was placed basal to the smoothened receptor cluster like (ITR-like), lung 7TM receptor-like and ocular albin-
(Additional file 7). This finding was consistent with a re- ism like (GPR143) receptors. Subsequent cross-genome
cent study that identified a smoothened receptor in phylogenetic analysis was performed on these GPCR
sponges [31]. It must be mentioned here that our initial families with the corresponding family members obtained
screenforFrizzledGPCRsidentified9Frizzled-likeGPCR from other species (Additional file 8). Protein sequences
sequences,ofwhichsixwereremovedduetofragmentary belonging to these GPCR families were obtained from
Krishnanetal.BMCEvolutionaryBiology (2014) 14:270 Page10of16
Nematostella, Trichoplax, and sea urchin using Pfam where phylogenetic analysis revealed that most members
HMM profile based searches. Corresponding sequences of the Adhesion and Glutamate families grouped into
from human wereobtained from the Swiss-Prot database. sponge-specificclusters.Insummary,thestudydescribes
Overall phylogenetic tree topology indicated the presence the sponge GPCR gene families in detail and our phylo-
of GPR143-like, lung 7TM-like and intimal thickness- genetic comparisons postulates a significantly diversified
relatedreceptorsinsponge.Thesespongesequencesclus- subsetofGPCRsinsponge.
tered with their corresponding family sequences obtained
from otherspecies(Additional file 8).They formedsepar- SpongeRhodopsins
ate clusters in the phylogenetic analysis with high confi- Comparative phylogenetic analysis demonstrates that
dencesupport.Phylogeneticanalysisalsorevealedthatthe sponge Rhodopsin family GPCRs do not share ortholo-
14 cAMP-like receptors identified in the sponge genome gous relationship with those found in eumetazoans and
form a separate cluster with high confidence support other bilaterians (see Figure 2 and Additional file 4). In
(Additional file 8). These 14 cAMP-like sequences con- addition, sponge Rhodopsin-like GPCRs form five distinct
tainedthe coreregionencodedbyaPfamdomain(Dicty_ clusters that are most likely sponge specific (Figure 2).
CAR; PF05462) characteristic to the Dictyostelium cAMP Here, we putatively labelled these sponge specific clusters
GPCR family. Similarly, pairwise similarity search per- as AqRho-A to E. It must be mentioned here that several
formed using these sequences as queries clearly demon- Rhodopsinsbelongingtothesespongespecificclustersare
strated that cAMP family sequences are among the top contained in the same contig region and located adjacent
hits. However, sponge cAMP-likereceptorsclustered sep- toeach other.Severaloftheflankingsequences arefound
arately from the Dictyostelium cAMP GPCR sequences, as many as a cluster of 2 to 8 sequences and share rela-
suggestingthattheyarequitedivergentorfastevolving.It tively high pairwise protein sequence identities ranging
wouldthusbeinterestingtoexperimentallyverifywhether from 51% to 74%. This suggests that the expansions of
the cAMP-like receptors in the sponge genome have spongeRhodopsinsarepossiblydrivenbygeneduplication
analogous roles to the previously known functions of events and it seems most likely true for other pre-
DictyosteliumcAMPGPCRfamily. bilaterianmetazoansas well. However, tofurther examine
whether these sequences can be classified into any of the
Discussion known13Rhodopsinsubfamilies,weperformedaBLASTP
The draft genome, as well as the transcriptome of search against the Swiss-Prot database. The results ob-
Amphimedon queenslandica (sponge), revealed the gen- tained from the BLAST search showed that a few
etic complexity ofthis primitive animalindetail andcat- sponge Rhodopsin-like GPCRs had their top hits as ad-
alogued the presence of several crucial gene families, renergic, serotonin, dopamine, and opsin-like receptors
including GPCRs and other signaling system compo- (see Additional file 3). This is in line with the draft
nents [11,12]. However, a detailed curation of sponge genome report of sponge (Amphimedon), which demon-
GPCR families/subfamilies and phylogenetic compari- strated the presence of serotonin and dopamine-like re-
sons with those versions found in eumetazoans and bila- ceptors [11]. This is also consistent with a recent study
terians needs to be performed to better understand the that identified adrenergic-like receptors in sponges [31].
GPCR component in sponges from an evolutionary per- Althoughthepairwisesimilaritysearchresultssuggestthe
spective. In this study, we curated GPCRs in the sponge presence of these putative receptors, our phylogenetic
genome and have phylogenetically compared the recep- analysis was unable to reveal any clear orthologous rela-
tors to those found in other metazoans. Our HMM tionshipsofthespongeRhodopsinstothebilateriancoun-
basedsearchapproachandphylogeneticanalysisdemon- terparts. A possible explanation could be that sponge
strates that sponge contains four of the five main Rhodopsins have diverged considerably, possibly based on
GRAFS families, namely, Rhodopsin, Adhesion, Glutam- sponge specific physiology and behavior [19,20]. This hy-
ate and Frizzled. It is noteworthy that the sponge gen- pothesis is plausible because species such as Trichoplax
ome encodes one of the most ancient metazoan lineage and Nematostella, belonging to the eumetazoan lineage
specificexpansionsoftheRhodopsinfamilyofGPCRs[11]. and diverged from sponges later in the metazoan species
Moreover, our phylogenetic analysis with pre-bilaterian tree, contain Rhodopsin-like GPCRs more similar to bila-
metazoans homologs clearly reveals that the Rhodopsin terians than sponges. In fact, earlier studies provided evi-
family has undergone significant diversifications in these dence that eumetazoans do contain putative orthologues
pre-bilaterian metazoans. Possible explanations could be for some of the amine and peptide binding receptors
that they diversified due to the evolution of diverse mor- [34-37]. The recent genome release of Mnemiopsis leidyi
phological characteristics and adaptations of these spe- suggests that ctenophores are the sister group to the rest
cies during the course of the early metazoan evolution oftheextantanimals,includingsponges,andthatcompo-
[23,32,33]. This is also evident in other GPCR families, nents of neuronal signaling were already present in an