BACKGROUND
g protein-coupled receptors  are one of the largest superfamilies of cellular receptor proteins, generally consisting of seven transmembrane helices  connected by three extracellular and three cytoplasmic loops of varying lengths.
different gpcrs respond to a wide variety of different external stimuli  and activate a number of different gtp binding proteins , there by initiating a wide spectrum of intracellular responses.
gpcrs play important roles in cellular signaling networks involving such processes as neurotransmission, taste, smell, vision, cellular metabolism, differentiation and growth, inflammatory and immune responses and secretion.
abnormalities of signaling by gpcrs are the root cause of disorders that affect most tissues and organs in our body, such as color blindness, thrombosis, restenosis, atherosclerosis, hyper functioning thyroid adenoma and nephrogenic diabetes insipidus and precocious puberty.
gpcrs are of major importance to the pharmaceutical industry since they play major roles in the pathogenesis of human diseases and are targets for more than half of the current therapeutic agents on the market  <cit> .
despite the importance of gpcrs in physiology and diseases, only one high-resolution structure has been solved, that of bovine rhodopsin  <cit> .
a majority of the identified gpcrs are with no known ligand specificity , which presents a challenge for identifying their native ligands and defining their function.
characterizing the role of any gpcr involves the identification of both the activating ligand and the activated g protein.
a diverse range of procedures have led to the identification of ligands for orphan receptors:  identifying relationship between receptor and ligand expression patterns  <cit> ,  testing tissue extracts in receptor-based functional assays and  testing ligands for identified gpcrs on orphan gpcrs with high sequence identity  <cit>  and in some cases randomly evaluating orphan gpcrs against arrayed families of known ligands.
the physiological role of these receptors can be well understood by the identification of natural ligands, which further advance the design of pharmacologically active surrogate activators or inhibitors of the gpcrs that have defined native ligands.
strategies described above will be facilitated by better prediction of ligand structure or chemical class of orphan gpcrs.
proteins similar in sequence often exhibit similar functions.
therefore, sequence homology can be used as a primary criterion for functional screening.
this powerful principle can be extended to proteins that are homologous in different species.
this has led to the identification of many new novel gpcrs across different species  <cit> .
many orphan gpcrs are conserved among different species suggesting that they should be active and thus bind novel ligands.
this led to the idea that orphan gpcrs could be used as targets to identify their natural ligands and consequently led to the discovery of novel transmitters  <cit> .
those orphan receptors that share more than  <dig> percent of sequence identity with the gpcrs with known ligands are very likely to also share common ligands  <cit> .
often, the direct association of ligand class to orphan receptors is non-trivial by simple blast searches even at high sequence identity  <cit> .
the top ranking hits constitute gpcrs from diverse ligand classes  and may not suggest a consensus on possible ligand class to be inferred directly.
however, if the sequence identity is below the twilight zone , predictions using direct sequence search methods often fail.
phylogenetic tree building has shown that receptors that respond to the same, or similar, agonists often cluster together, even with low sequence identity.
for example, most members of the prostanoid receptor subfamily share less than  <dig> percent amino acid identity, yet these receptors are more like one another than any other gpcr  <cit> .
phylogenetic clustering methods were used to elucidate the chemical nature of receptor ligands, which led to the identification of natural ligands for many orphan receptors  <cit> .
gpcrs were previously classified into distinct families by different groups  <cit> .
the classifications would include rhodopsin-like receptors, secretin receptor-like receptors, metabotropic glutamate-like receptors, adhesion-like receptors and frizzled/smoothened-like receptors as proposed by fredriksson and coworkers  <cit> ; in addition, other groups have proposed two more classes, viz., the fungal pheromone receptor like family and cyclic amp receptors family  <cit> .
these classification schemes were generated mostly from individual genome studies  <cit> .
studies in model organisms and cross-genome comparisons have provided major insights in the general understanding of numerous genes and pathways involved in a wide variety of physiological processes and human diseases  <cit> .
drosophila is a very good model organism owing to the simplicity in the genetic system and a short lifespan enabling the screening of large individuals to identify mutations in new candidate genes that may have human counterparts involved in cellular physiology and diseases  <cit> .
despite disparity in morphology or phenotype, drosophila shows similarity with humans in developmental and cellular processes like core aspects of cell cycle, signaling pathways, apoptosis, neuronal signaling, cytoskeleton and core proteome   <cit> .
we, therefore, sought out to adopt drosophila gpcrs to study human gene function using comparative genomics  <cit> .
a large number of drosophila gpcrs have no characterized ligands.
on the other hand, many human gpcrs are well characterized in their physiology and pharmacology.
in this study, we collected a large set of gpcr sequences from human and drosophila genomes and performed cross-genome multiple phylogenetic analyses.
further analysis reveals unexpected levels of similarity between gpcrs of these two species and phylogenetic association could be employed to predict ligands  for many of drosophila and human orphan receptors.
RESULTS
cross genome phylogenetic analysis of human and drosophila non-olfactory receptors resulted in eight major groups.
they are i) peptide receptors, ii) chemokine receptors, iii) nucleotide and lipid receptors iv) biogenic amine receptors v) secretin receptors vi) glutamate receptors vii) cell adhesion receptors and viii) frizzled receptors.
these were further classified into  <dig> clusters  with eleven clusters of peptide receptors, two clusters of chemokine receptors, six clusters of nucleotide and lipid receptors, five clusters of biogenic amine receptors, two clusters of secretin receptors, four clusters of glutamate receptors and one cluster each of cell adhesion and frizzled receptors .
about thirty one gpcr sequences could not be assigned to any of these clusters; these are discussed separately below as unassociated gpcrs.
our method sometimes resulted in clusters with members whose ligands belong to different chemical structure or classes and these results are discussed in detail below.
peptide receptors
clusters  <dig> to  <dig> comprise of peptide receptors .
the size of peptide ligands can vary from two amino acids to as many as  <dig>  some of the natural peptide ligands include apelin, bombesin, calcitonin, endothelin, galanin, gastrin, ghrelin, neurotensin, neuropeptide b, w, y, orexin, oxytocin, relaxin, somatostatin, urocortins, etc.
these receptors are involved in many human diseases including chronic inflammatory diseases, degenerative diseases, autoimmune diseases, cancer, cardiovascular diseases etc, thus they could be of new therapeutic targets  <cit> .
receptors with known ligands in cluster  <dig> binds to galanins or kisspeptins or cyclic peptides.
drosophila allostatin receptors   are very closely related to galanin receptors  <cit> .
receptors, q969v1_hum and q96s47_hum, are closely related to gp24_hum receptor that bind to melanin-concentrating hormone and may have similar cyclic peptides as their ligands.
as the name suggests, orphan receptor, sapr_hum, does not bind to somatostatins and angiotensins  <cit>  since it is distantly related to gp24_hum and ur2r_hum receptors in this tree.
instead, this receptor may bind to similar cyclic peptides.
cluster  <dig> consists of receptors for opioid, somatostatin and neuropeptide  ligands forming different branches.
opioids and somatostatins are obtained from preprocessing of larger precursor peptides.
it is known that gpr7_hum and gpr8_hum bind to npb/w ligands  <cit> .
drosophila orphan receptors, q8isj9_dro and q8i943_dro branch is close to somatostatin receptors and might bind to ligands similar to somatostatins.
small peptide  receptors comprise of cluster  <dig>  the human orphan receptors encoded by gpr15_hum, gpr25_hum and q8ngz8_hum are related to apj_hum and show significant amino acid identity suggesting these might bind to small peptide endogenous ligands.
cluster  <dig> comprises of endothelin and bombesin receptors with known ligands , the neuromedin b receptor  and bombesin receptor ).
drosophila orphan receptors, q9v9k3_dro and q9v858_dro, share the branch with bombesin, grpr and nmbr receptors.
they share many conserved amino acids, known to be important for high affinity binding of gastrin-releasing peptide  and bombesin to grpr and nmb binding to nmb-r  <cit>  .
this suggests q9v9k3_dro and q9v858_dro might bind to similar neuropeptide for its activation.
human orphan receptor gpr37_hum is closely related to etb2_hum suggesting it may bind to endothelin-like peptides.
q8tdv0_hum is sequentially similar to both galanin  and bombesin receptors but sub-clustering of peptide receptors by maximum likelihood method has placed it in this cluster suggesting closer association of these two clusters.
cluster  <dig> is composed of receptors for neurotensin , neuromedin u , motilin, growth hormone secretagogue, thyrotropin-releasing hormone and some of prx-amide peptides.
gpr39_hum is closely related to nt receptors and might bind to neurotensin ligands.
drosophila receptors, q8itc7_dro, q9vfw5_dro, q9vfw6_dro, q8itc9_dro and q9vp15_dro form a separate branch, which are closely related to vertebrate neuromedin receptors and they bind to prxa pyrokinins or fxprxamide or cap2b-like peptides  or ecdysis triggering hormones  .
q9vdc4_dro forms a distinct branch and is sequentially close to ghsr_hum, trfr_hum, q8itc7_dro and q9vfw5_dro and might bind to neuropeptides.
drosophila orphan receptors, q9w4h3_dro, q9vt27_dro, q8swr3_dro, q9v5t1_dro, q9w025_dro and q9w027_dro, branch out from that of trfr_hum and might form a separate family of receptors binding to novel neuropeptide ligands.
supporting our analysis, q9w025_dro and q9w027_dro were reported as first receptors specific for drosophila myosuppressins   <cit>  and q9w4h3_dro was reported as neuropeptide proctolin binding receptor  <cit> .
q9vt27_dro is very closely related to q9w4h3_dro and might bind to proctolin or similar neuropeptide ligands for its activation.
cluster  <dig> consists of peptide hormone receptors binding arginine vasopressin  or growth hormone releasing hormone or oxytocin or gonadotropin-releasing hormone ii or crustacean cardioactive peptide  or corazonin or adipokinetic hormone  .
gp19_hum is related to drosophila ccap receptor  that is activated by ccap and akh, but not by avp.
thus, ccap and akh might as well bind to gp19_hum for its activation.
drosophila gonadotropin-releasing hormone and/or corazonin receptor  and putative corazonin  receptor clusters well with human counterparts  suggesting early evolution of grhr receptors.
q8ngu9_hum forms a separate branch, but shares sequence similarity with avp receptors and might bind to similar neuropeptide ligands.
cluster  <dig> comprises leucine-rich repeat-containing g protein-coupled receptors  like glycoprotein receptors, follicle stimulating hormone receptor , thyroid-stimulating hormone receptor , luteinizing hormone receptor  and receptors binding to relaxin.
these are unique in having a large n-terminal extracellular  domain containing leucine-rich repeats important for interaction with the glycoprotein ligands and are classified into three sub-groups  <cit> .
our analysis also shows that there are three lgr subfamilies:  the glycoprotein hormone receptors lshr_hum, fshr_hum, tshr_hum, q8sx01_dro and q9ndi1_dro  lgr4_hum lgr5_hum and lgr6_hum  lgr5_hum, lgr7_hum and lgr8_hum, q9vbp0_dro, and q9vyg0_dro.
drosophila orphan receptors q8sx01_dro and q9ndi1_dro are closely related to human glycoprotein hormone receptors and might bind to glycoprotein hormones.
q9vbp0_dro and q9vyg0_dro are very similar in their overall domain architecture to lgrs with long n-termini, but their similar relationship in extracellular domain arrangements are also evident from this phylogenetic analysis without considering the n and c termini.
cluster  <dig> consists of peptide receptors with known ligands such as gastrin , cholecystokinin , orexin  and neuropeptide ff  or morphine modulating peptides.
gpr103_hum  is closely related to neuropeptide ff receptors, as predicted by our phylogenetic analysis and previous prediction on human gpcrs  <cit> .
subsequently, gpr <dig> was characterized and a novel rf-amide peptide, p <dig> was shown to be its ligand  <cit> .
drosophila orphan receptors, q9vwr3_dro  and q9vwq9_dro , are related to each other and branch off from the cholecystokinin  receptors and might have cholecystokinin as its natural ligand.
q14439_hum branch off orexin receptors that bind to two novel neuropeptides, orexin-a and b, derived from a common prepro-orexin precursor by proteolytic processing  <cit> .
the receptors with known ligands binding to chemotactic substances  and anaphylactic complement factors) are part of cluster  <dig>  these ligands are structurally very diverse but functionally related peptides.
human orphan receptors, gp32_hum and q8nga4_hum branch out early from fml receptors and may probably bind to smaller hydrophilic peptides.
l4r1_hum, l4r2_hum and q8tdt2_hum form a separate branch distant from other chemotactic peptide receptors with out bootstrap support.
cml1_hum and gpr1_hum form a separate branch distinct from the other branches, and also gpr44_hum forming an individual branch.
prediction of ligands for these receptors is not possible using this phylogenetic tree, but these receptors may be activated by chemotactic substances  <cit> .
mas proto-oncogene, mas-related genes  and sensory neuron-specific g protein-coupled receptors  form cluster  <dig>  angiotensin  has been identified as an endogenous ligand for the g protein-coupled receptor mas  <cit> .
snsrs are activated by proenkephalin a peptide fragments, like bovine adrenal medulla peptide  <dig> .
some mrgs and snsrs are expressed in nociceptive sensory neurons suggesting that they could be involved in pain sensation or its modulation.
previous studies also suggest that ligands for mrg receptors may include neuropeptides that modulate pain sensitivity  <cit> .
human orphan receptor q8ngk7_hum is closely related to mrg receptor.
all receptors with known ligands in cluster  <dig> are neuropeptide receptors.
drosophila tachykinin-like peptide receptors  and human neurokinin receptors  form a closely-knit branch.
pkr1_hum  and pkr2_hum  form a separate branch of receptors that bind to prokineticins  <cit> .
q9vrm0_dro is closely related to drosophila receptor nyr_dro that bind to neuropeptide y. q9vrm0_dro might probably bind to similar neuropeptides.
neuropeptide y binding receptors ) form a separate branch.
the human prolactin-releasing peptide  binding gpr10_hum forms a separate branch in this phylogenetic tree  <cit> .
drosophila orphan receptors, q9vw75_dro and q8sz35_dro constitute a separate branch close to other neuropeptide receptors that might functionally be activated by neuropeptides.
similarly, orphan receptor gp72_hum forms a new branch.
drosophila orphan receptor q9w189_dro is a very distantly related member and was only grouped into this cluster by blastp results.
chemokine receptors
chemokine receptors are phylogenetically represented by two clusters  <dig> and  <dig> .
chemokines are important molecules in inflammatory responses, as immunomodulators and they also have critical functions in lymphopoiesis  <cit> .
there are no drosophila members belong to this group of receptors suggesting these receptors might be recent in evolutionary origin.
they have been divided into two subfamilies on the basis of the arrangement of the two disulphide-bond forming n-terminal cysteine residues, cxc and cc.
many human cxc chemokines that mainly act on neutrophils are clustered at chromosome 4q12– <dig>  while many cc chemokines that mainly act on monocytes are located in another cluster at chromosome 17q <dig> .
our phylogenetic analysis has also divided chemokine receptors into two major clusters, concurrent with that of chemokine classes, suggesting co-evolution of receptors and ligands  <cit> .
cluster  <dig> consists of receptors associated with cc type chemokines.
as reported previously through earlier approach  <cit>  o75307_hum  might bind to cc-type chemokine ligand.
cluster  <dig> consists of both cxc and cc-type receptors.
admr_hum and q8ne10_hum  form a branch whereas duff antigen and q96ch1_hum are distantly related to cml2_hum.
these two branches are associated to chemokine receptors based on blastp similarity at an e-value significance of 5e- <dig> and 7e- <dig>  respectively, with other members of this cluster.
nucleotide and lipid receptors
nucleotide and lipid receptors consists of six clusters , except for cluster  <dig>  and cluster  <dig>  there are no counter parts from drosophila.
opsins are included in cluster  <dig> that are activated by isoprenoid ligands.
drosophila opsins show significantly high homology to human opsins.
there is strong conservation of the retinal binding site and other regions suggesting that they are derived from a common ancestor and diverged thereafter retaining structural and functional features  <cit> .
drosophila receptor q9vtu7_dro is closely related to ops3–5_dro receptors, which are localized in the inner-cells of the drosophila eye .
this suggests q9vtu7_dro might be localized in the inner cells of drosophila eye.
receptors for pyramidine or purine nucleotides, cysteinyl leukotriene, nicotinic acid  and short, medium and long chain fatty acids make up cluster  <dig>  q9bxc0_hum , q8tds5_hum and gp31_hum share the branch with closely related nicotinic acid  receptor  <cit>  and might have similar carboxylic acids as their ligands.
q8tdq8_hum and q96p68_hum are related to each other as well as to p2y receptors and may bind to p2y nucleotides.
gp17_hum and gp82_hum receptors are distantly related to other members in this cluster and might represent potential new subfamilies binding to nucleotide or lipids.
cluster  <dig> is a heterogeneous group of receptors binding to lipids, nucleotides, modified nucleotides and platelet activating factor .
orphan receptor q8tdu7_hum  is closely related to platelet adp-binding receptor .
q96jz8_hum  is closely related to udp-glucose receptor  and might bind to a modified nucleotide ligand.
gpr34_hum forms a separate branch which is distantly related to pafr_hum.
no prediction of ligands is possible for gpr34_hum with this phylogenetic tree.
cluster  <dig> consists of lipid receptors ) and exceptionally some of the peptide receptors  are represented in different branches.
although they bind to different ligands, they identify each other during sequence searches and display 23–29% sequence identity.
the functionally important motifs are fairly conserved  <cit>  .
indeed, this unusual branching including peptide and lipid receptors has been noted earlier by methner's and fredicksson's groups  <cit> .
cluster  <dig> is composed of receptors binding to prostaglandins, prostacyclins and thromboxanes.
all these ligands are derivatives of arachidonic acid , which serves as the precursor via the cyclooxygenase  pathway.
drosophila orphan receptor q9vvj1_dro within this tree might bind to ligands derived from aa by the action of cox.
cluster  <dig> is also a heterogeneous group of receptors consisting of protease-activated receptors, psychosine receptors, lysophosphatidylcholine and sphingosylphosphorylcholine.
ovarian cancer g-protein-coupled receptor  <dig> , previously described as a receptor for sphingosylphosphorylcholine, acts as a proton-sensing receptor stimulating inositol phosphate formation  <cit> , whereas gpr <dig> is also involved in ph homeostasis, but elicits cyclic amp formation  <cit> .
ogr <dig>  and gpr <dig> are different from other sphingosylphosphorylcholine binding endothelial differentiation gene  receptors.
orphan p2y receptors in this cluster are misnomers since they do not cluster with the classical neuropeptide receptors  and instead appear to be co-clustered with members of this heterogeneous cluster.
either they may have uncommon nucleotide as natural ligand or despite their structural similarity to the p2y family they may not be nucleotide receptors  <cit> .
gp35_hum and q8n580_hum, ebi2_hum and gp18_hum and gp20_hum cluster as separate branches and are distantly related to members of other branches but probably bind to lipids as their natural ligands.
biogenic amine receptors
biogenic amine receptors consists of five clusters mainly consisting of trace amine; melatonin; serotonin receptors; histamines, muscarinic acetylcholine, adenosine and histamine; dopamine, octopamine and adrenaline receptors .
in these clusters fairly good intermixing of human and drosophila receptors is observed.
this suggests biogenic amine receptors have ancient evolutionary origin as they are observed in invertebrates to higher vertebrates.
cluster  <dig> is represented mainly by trace amine  receptors .
trace amines binding these receptors are believed to play an important role in human disorders such as depression, attention deficit disorder, schizophrenia and parkinson's disease  <cit> .
they form a subfamily of gpcrs, distinct from, but related to serotonin , norepinephrine  and dopamine  receptors.
drosophila orphan receptors q9vg54_dro and q9vcz3_dro are closely related to 5h4_hum.
q9p1p4_hum  and q9p1p5_hum  are closely related to q96rj0_hum .
similarly o14804_hum, a putative neurotransmitter receptor  is closely related to trace amine  receptors.
cluster  <dig> consists of melatonin receptors  and other related orphan receptors .
melatonin receptors bind to and are activated by biogenic amine 5-methoxy-n-acetyltryptamine .
the melatonin-related receptor , despite sharing considerable amino acid sequence identity with other melatonin receptors, does not bind melatonin  <cit> .
the receptors in this cluster show considerable sequence similarity to neuropeptide y  receptors than other biogenic amine receptors and were previously grouped along with npy receptors  <cit> .
all receptors with known ligands of cluster  <dig> consist of serotonin receptors.
these are structurally distinct from serotonin receptors in cluster  <dig>  drosophila orphan receptors q9veg1_dro and q9veg2_dro form a separate branch but are closely related to other serotonin receptors in this tree and might have similar ligand  for its activation.
q8tdv2_hum and q16538_hum , however, are distantly related to other receptors in this tree and were placed only based on blastp similarity.
receptors of biogenic amines  and many orphan receptors are all placed in different branches in cluster  <dig>  drosophila orphan receptor q9vhw1_dro branch out along with muscarinic acetylcholine and histamine receptors in this tree and might bind to acetylcholine or histamines for its activation.
q9vaa2_dro is closely related to that of adenosine receptors.
super conserved receptors expressed in brain  from vertebrate species form a separate branch and might represent potential novel subfamily of gpcrs binding to undiscovered endogenous biogenic amine ligands  <cit> .
high-affinity lysophosphatidic acid  receptor homologs o43898_hum and gpr63_hum form a distinct branch.
similarly, orphan receptors gp21_hum and gp51_hum, gpr62_hum and q8tdv4_hum, q8ndv2_hum  and q8ngv3_hum and q9vmi4_dro form a distinct branch, suggesting only distant relationship with other members of the cluster.
receptors of biogenic amines , few serotonergic receptors and many orphan receptors are represented in different branches in cluster  <dig>  drosophila dopamine 2-like receptor , q8is45_dro, groups well with the human counterparts suggesting that their evolution extends much before drosophila.
interestingly, dop2_dro is grouped with the adrenaline receptors instead with dopaminergic receptors and shows similar sequence identity  with vertebrate alpha 1-, and beta-adrenergic, and d1-like, d2-like dopaminergic and serotonergic receptors.
this drosophila receptor has been discussed as a novel structural class of dopamine receptors  <cit> .
drosophila octopamine receptor isoforms in mushroom bodies   branch out with human alpha  <dig> adrenergic  _hum) receptors since they share high sequence identity  in tm regions with alpha  <dig> adrenergic receptors  <cit> .
q9ve32_dro branches out from human alpha  <dig> adrenergic receptors and may have adrenaline as its ligand for activation.
orphan striatum-specific g protein-coupled receptor , though grouped with biogenic amine receptors, may represent a novel subtype of gpcr due to the lack of conservation of key functional residues  <cit> .
orphan receptors, q9w3v5_dro and q8tdv5_hum, q96p66_hum and q8n6u8_hum, q9vhp6_dro and q9vbg4_dro form their own branch sharing distant relationship with other receptors in this tree and might represent potential novel subfamilies of biogenic amine gpcrs.
class b  receptors
class b receptors are represented by two clusters  consisting of classical hormone receptors and drosophila methuselah  like proteins .
the ligands for receptors of cluster  <dig> are structurally related polypeptide hormones of 27– <dig> amino-acid residues , secretin, calcitonin, corticotropin-releasing factor , urocortins, growth-hormone-releasing hormone , vasoactive intestinal peptide , glucagon, glucagon-like peptides  and glucose-dependent insulinotropic polypeptide .
drosophila orphan receptors, q9v716_dro and q9v6c7_dro are closely related to the human receptor for corticotropin releasing factor receptor  which binds to urocortins.
q9v6n4_dro, q9vyh9_dro and q9nef7_dro are related to calcitonin  and calcitonin gene-related peptide type  <dig> receptors .
three small accessory proteins, called receptor activity-modifying proteins , interact with these calcitonin receptors and can generate six pharmacologically distinct receptors.
if this phenomenon of ramp-enabled receptor diversity exists in other receptors, then it will further complicate the ligand-receptor interactions of gpcrs, assuming they still bind to structurally similar ligands.
human orphan receptor, q8nhb4_hum, is very closely related to ptrr_hum receptor binding to parathyroid hormone and parathyroid hormone-related protein .
methuselah receptors and its paralogs of drosophila solely represent cluster  <dig>  the drosophila mutant methuselah  was identified from a screen for single gene mutations that extended average lifespan of an organism and also increased resistance to several forms of stress, including starvation, heat, and oxidative damage  <cit> .
there are no obvious homologues of these receptors within human or c. elegans genomes.
drosophila receptors, q8inm0_dro, q8ipd0_dro and q95nu7_dro, are closely related to previously identified mth members and may be new paralogs of these receptors.
cell adhesion receptors
large number of gpcrs with long extracellular n-termini, containing gpcr proteolytic site  domain, are represented in cluster  <dig> .
several of these receptors also have one or many functional domains such as epidermal growth factor , leucine rich repeat , hormone-binding domain  and immunoglobulin  domains  <cit> .
these form several distantly related branches.
except cd97_hum, all the receptors in this cluster are orphans with no known ligands  <cit> .
there are only four drosophila sequences representing these receptors.
class c  receptors
receptors of class c are divided mainly into four clusters : metabotropic glutamate receptors , γ-aminobutryic acid  receptors, calcium-sensing receptors  and retinoic acid-inducible g-protein-coupled receptors  .
cluster  <dig> consists of human and drosophila mgrs.
human mgrs are sub-grouped into three different branches: first contains mgr1_hum and mgr5_hum and second contains mgr2_hum and mgr3_hum.
the third branch, including mgr4_hum, 6– <dig> and drosophila mgrs represent a separate subgroup  <cit> .
drosophila orphan receptor q9v4u4_dro is closely related to mgr_dro and might bind to glutamate for its activation.
calcium-sensing receptor  forms cluster  <dig> along with a set of orphan receptors .
these orphan receptors either may have ligands and/or function similar to that of casr_hum or they may act as pheromone/olfactory receptors.
phylogenetic tree of most members  of family  <dig> gpcrs across different genomes , caenorhabditis elegans, drosophila melanogaster, japanese pufferfish , goldfish , human , mouse , rat  and salmon ) have shown casr_hum forms a separate branch part of pheromone/olfactory cluster of class c gpcrs  <cit> .
to note that olfactory and gustatory/taste receptors are not considered in this work.
cluster  <dig> consists of retinoic acid-inducible g-protein-coupled receptors .
raigs have short  extracellular amino-terminal domains  as opposed to the other receptors currently assigned to class c. boss_dro also has short atd and branch out very early with the members of raigs and may represent new single member subfamily of class c receptors.
the gabab receptors are present in cluster  <dig>  it is represented by four sub-branches, of which three are gababr1-3_hum type receptors and fourth sub-branch of drosophila orphan receptors  related to that of gaba receptors.
gabab <dig> is exclusively present in drosophila as separate branch whose function is not yet known.
previous results have only been able to functionally characterize d-gababr <dig> and r <dig> when the two subtypes are co-expressed either in xenopus laevis oocytes or mammalian cell lines, whilst d-gababr <dig> was inactive in any combination.
this suggests d-gababr <dig> requires a counterpart other than d-gababr <dig> and r <dig> to form a functional heterodimer  <cit> .
thus the current clustering approach suggests that q9vka4_dro or q9vr40_dro may interact with d-gababr <dig> and form a functional heterodimer.
frizzled/smoothened receptors
cluster  <dig> comprises receptors with a long  n-terminus and conserved cysteine rich domains  which are likely to participate in wnt ligand binding .
these receptors control the specification of cell fate, cell adhesion, migration, polarity and proliferation  <cit> .
this cluster is represented by ten human  and four drosophila  frizzled receptors together with smoothened  receptors.
the topology of the phylogenetic tree shows one smoothened and four frizzled branches.
frz1_dro is closely related to human fzd3_hum and fzd6_hum.
frz2_dro is related to fzd5_hum and fzd8_hum, whereas frz3_hum and frz4_hum form separate branches distantly related to other receptors.
unassociated gpcrs
thirty one gpcr sequences could not be included in any cluster with appreciable bootstrap values or blastp similarity.
this can either be viewed as members of single member clusters with certain atypical parts of their sequences that could be a result of chimeric origin of the receptors or due to evolutionary pressure not shared by their closest phylogenetic neighbors  <cit> .
we have therefore placed these receptors separately as unassociated gpcrs, although these receptors clearly do not belong to the same group .
most of the unassociated receptors remain as orphan receptors.
CONCLUSIONS
the phylogenetic analyses performed using human and drosophila gpcrs suggest that the sequences can be divided into  <dig> clusters and reveals unexpected level of similarity between human and drosophila gpcrs.
<dig> clusters group drosophila and human gpcrs together suggesting high evolutionary conservation across species for gpcr sequences.
there are  <dig> clusters, four of nucleotide-lipid receptors three clusters of peptide receptors and two clusters of chemokine and one cluster of glutamate receptors that do not contain any representation from drosophila gpcrs in our current dataset of sequences considered.
perhaps the immune-related receptors, such as the chemokine ones, are not either recognized yet or not present in lower organisms such as drosophila.
if there is a clear absence of such classes of receptors, this might also suggest that immune defense is regulated by proteins other than gpcrs in drosophila.
interestingly, there is one cluster of secretin drosophila receptors where there is no human representation.
these proteins are involved in aging in drosophila.
furthermore, in this analysis, we also notice that out of the  <dig> clusters that co-cluster human and drosophila gpcrs, drosophila gpcrs remain isolated sub-clusters in  <dig> of them leaving behind only nine clusters that allow easy inter-mixing of the two sets of sequences.
this includes  <dig> clusters each of peptide and biogenic amine receptors and one cluster each of class b, c and frizzled receptors.
the current clustering analysis provides ligand class association to  <dig> drosophila  and  <dig> human orphan receptors could be associated with probable ligand classes using co-clustering principles as earlier observed within human gpcr sequences alone  <cit> .
further, similar cellular localizations have been suggested for drosophila orphan receptors that belong to the opsin family .
gpcrs with similar extracellular domain architecture also co-cluster suggesting this similarity is encoded even within the gpcr domain.
further this analysis also suggests dimerizing partner  for d-gababr <dig> that might form a functional heterodimer.
we have determined the relationship of the receptors within subgroups of the large gpcr superfamily by means of a cross-genome phylogenetic clustering approach.
these studies also revealed a higher-level phylogenetic organization in which clusters with common ligand structure or chemistry, or a shared function, are evident across genomes.
we hope that this approach proves valuable for identifying the natural ligands of drosophila and human orphan receptors.

 g protein-coupled receptors are one of the largest superfamilies of cellular receptor proteins, generally consisting of seven transmembrane helices connected by three extracellular and three cytoplasmic loops of varying lengths. different gpcrs respond to a wide variety of different external stimuli and activate a number of different gtp binding proteins, there by initiating a wide spectrum of intracellular responses. gpcrs play important roles in cellular signaling networks involving such processes as neurotransmission, taste, smell, vision, cellular metabolism, differentiation and growth, inflammatory and immune responses and secretion. abnormalities of signaling by gpcrs are the root cause of disorders that affect most tissues and organs in our body, such as color blindness, thrombosis, restenosis, atherosclerosis, hyper functioning thyroid adenoma and nephrogenic diabetes insipidus and precocious puberty. gpcrs are of major importance to the pharmaceutical industry since they play major roles in the pathogenesis of human diseases and are targets for more than half of the current therapeutic agents on the market. despite the importance of gpcrs in physiology and diseases, only one high-resolution structure has been solved, that of bovine rhodopsin. a majority of the identified gpcrs are with no known ligand specificity, which presents a challenge for identifying their native ligands and defining their function. characterizing the role of any gpcr involves the identification of both the activating ligand and the activated g protein. a diverse range of procedures have led to the identification of ligands for orphan receptors: identifying relationship between receptor and ligand expression patterns, testing tissue extracts in receptor-based functional assays and testing ligands for identified gpcrs on orphan gpcrs with high sequence identity and in some cases randomly evaluating orphan gpcrs against arrayed families of known ligands. the physiological role of these receptors can be well understood by the identification of natural ligands, which further advance the design of pharmacologically active surrogate activators or inhibitors of the gpcrs that have defined native ligands. strategies described above will be facilitated by better prediction of ligand structure or chemical class of orphan gpcrs. proteins similar in sequence often exhibit similar functions. therefore, sequence homology can be used as a primary criterion for functional screening. this powerful principle can be extended to proteins that are homologous in different species. this has led to the identification of many new novel gpcrs across different species. many orphan gpcrs are conserved among different species suggesting that they should be active and thus bind novel ligands. this led to the idea that orphan gpcrs could be used as targets to identify their natural ligands and consequently led to the discovery of novel transmitters. those orphan receptors that share more than percent of sequence identity with the gpcrs with known ligands are very likely to also share common ligands. often, the direct association of ligand class to orphan receptors is non-trivial by simple blast searches even at high sequence identity. the top ranking hits constitute gpcrs from diverse ligand classes and may not suggest a consensus on possible ligand class to be inferred directly. however, if the sequence identity is below the twilight zone, predictions using direct sequence search methods often fail. phylogenetic tree building has shown that receptors that respond to the same, or similar, agonists often cluster together, even with low sequence identity. for example, most members of the prostanoid receptor subfamily share less than percent amino acid identity, yet these receptors are more like one another than any other gpcr. phylogenetic clustering methods were used to elucidate the chemical nature of receptor ligands, which led to the identification of natural ligands for many orphan receptors. gpcrs were previously classified into distinct families by different groups. the classifications would include rhodopsin-like receptors, secretin receptor-like receptors, metabotropic glutamate-like receptors, adhesion-like receptors and frizzled/smoothened-like receptors as proposed by fredriksson and coworkers; in addition, other groups have proposed two more classes, viz., the fungal pheromone receptor like family and cyclic amp receptors family. these classification schemes were generated mostly from individual genome studies. studies in model organisms and cross-genome comparisons have provided major insights in the general understanding of numerous genes and pathways involved in a wide variety of physiological processes and human diseases. drosophila is a very good model organism owing to the simplicity in the genetic system and a short lifespan enabling the screening of large individuals to identify mutations in new candidate genes that may have human counterparts involved in cellular physiology and diseases. despite disparity in morphology or phenotype, drosophila shows similarity with humans in developmental and cellular processes like core aspects of cell cycle, signaling pathways, apoptosis, neuronal signaling, cytoskeleton and core proteome. we, therefore, sought out to adopt drosophila gpcrs to study human gene function using comparative genomics. a large number of drosophila gpcrs have no characterized ligands. on the other hand, many human gpcrs are well characterized in their physiology and pharmacology. in this study, we collected a large set of gpcr sequences from human and drosophila genomes and performed cross-genome multiple phylogenetic analyses. further analysis reveals unexpected levels of similarity between gpcrs of these two species and phylogenetic association could be employed to predict ligands for many of drosophila and human orphan receptors.  cross genome phylogenetic analysis of human and drosophila non-olfactory receptors resulted in eight major groups. they are i) peptide receptors, ii) chemokine receptors, iii) nucleotide and lipid receptors iv) biogenic amine receptors v) secretin receptors vi) glutamate receptors vii) cell adhesion receptors and viii) frizzled receptors. these were further classified into clusters with eleven clusters of peptide receptors, two clusters of chemokine receptors, six clusters of nucleotide and lipid receptors, five clusters of biogenic amine receptors, two clusters of secretin receptors, four clusters of glutamate receptors and one cluster each of cell adhesion and frizzled receptors. about thirty one gpcr sequences could not be assigned to any of these clusters; these are discussed separately below as unassociated gpcrs. our method sometimes resulted in clusters with members whose ligands belong to different chemical structure or classes and these results are discussed in detail below. peptide receptors clusters to comprise of peptide receptors. the size of peptide ligands can vary from two amino acids to as many as some of the natural peptide ligands include apelin, bombesin, calcitonin, endothelin, galanin, gastrin, ghrelin, neurotensin, neuropeptide b, w, y, orexin, oxytocin, relaxin, somatostatin, urocortins, etc. these receptors are involved in many human diseases including chronic inflammatory diseases, degenerative diseases, autoimmune diseases, cancer, cardiovascular diseases etc, thus they could be of new therapeutic targets. receptors with known ligands in cluster binds to galanins or kisspeptins or cyclic peptides. drosophila allostatin receptors are very closely related to galanin receptors. receptors, q969v1_hum and q96s47_hum, are closely related to gp24_hum receptor that bind to melanin-concentrating hormone and may have similar cyclic peptides as their ligands. as the name suggests, orphan receptor, sapr_hum, does not bind to somatostatins and angiotensins since it is distantly related to gp24_hum and ur2r_hum receptors in this tree. instead, this receptor may bind to similar cyclic peptides. cluster consists of receptors for opioid, somatostatin and neuropeptide ligands forming different branches. opioids and somatostatins are obtained from preprocessing of larger precursor peptides. it is known that gpr7_hum and gpr8_hum bind to npb/w ligands. drosophila orphan receptors, q8isj9_dro and q8i943_dro branch is close to somatostatin receptors and might bind to ligands similar to somatostatins. small peptide receptors comprise of cluster the human orphan receptors encoded by gpr15_hum, gpr25_hum and q8ngz8_hum are related to apj_hum and show significant amino acid identity suggesting these might bind to small peptide endogenous ligands. cluster comprises of endothelin and bombesin receptors with known ligands, the neuromedin b receptor and bombesin receptor ). drosophila orphan receptors, q9v9k3_dro and q9v858_dro, share the branch with bombesin, grpr and nmbr receptors. they share many conserved amino acids, known to be important for high affinity binding of gastrin-releasing peptide and bombesin to grpr and nmb binding to nmb-r. this suggests q9v9k3_dro and q9v858_dro might bind to similar neuropeptide for its activation. human orphan receptor gpr37_hum is closely related to etb2_hum suggesting it may bind to endothelin-like peptides. q8tdv0_hum is sequentially similar to both galanin and bombesin receptors but sub-clustering of peptide receptors by maximum likelihood method has placed it in this cluster suggesting closer association of these two clusters. cluster is composed of receptors for neurotensin, neuromedin u, motilin, growth hormone secretagogue, thyrotropin-releasing hormone and some of prx-amide peptides. gpr39_hum is closely related to nt receptors and might bind to neurotensin ligands. drosophila receptors, q8itc7_dro, q9vfw5_dro, q9vfw6_dro, q8itc9_dro and q9vp15_dro form a separate branch, which are closely related to vertebrate neuromedin receptors and they bind to prxa pyrokinins or fxprxamide or cap2b-like peptides or ecdysis triggering hormones. q9vdc4_dro forms a distinct branch and is sequentially close to ghsr_hum, trfr_hum, q8itc7_dro and q9vfw5_dro and might bind to neuropeptides. drosophila orphan receptors, q9w4h3_dro, q9vt27_dro, q8swr3_dro, q9v5t1_dro, q9w025_dro and q9w027_dro, branch out from that of trfr_hum and might form a separate family of receptors binding to novel neuropeptide ligands. supporting our analysis, q9w025_dro and q9w027_dro were reported as first receptors specific for drosophila myosuppressins and q9w4h3_dro was reported as neuropeptide proctolin binding receptor. q9vt27_dro is very closely related to q9w4h3_dro and might bind to proctolin or similar neuropeptide ligands for its activation. cluster consists of peptide hormone receptors binding arginine vasopressin or growth hormone releasing hormone or oxytocin or gonadotropin-releasing hormone ii or crustacean cardioactive peptide or corazonin or adipokinetic hormone. gp19_hum is related to drosophila ccap receptor that is activated by ccap and akh, but not by avp. thus, ccap and akh might as well bind to gp19_hum for its activation. drosophila gonadotropin-releasing hormone and/or corazonin receptor and putative corazonin receptor clusters well with human counterparts suggesting early evolution of grhr receptors. q8ngu9_hum forms a separate branch, but shares sequence similarity with avp receptors and might bind to similar neuropeptide ligands. cluster comprises leucine-rich repeat-containing g protein-coupled receptors like glycoprotein receptors, follicle stimulating hormone receptor, thyroid-stimulating hormone receptor, luteinizing hormone receptor and receptors binding to relaxin. these are unique in having a large n-terminal extracellular domain containing leucine-rich repeats important for interaction with the glycoprotein ligands and are classified into three sub-groups. our analysis also shows that there are three lgr subfamilies: the glycoprotein hormone receptors lshr_hum, fshr_hum, tshr_hum, q8sx01_dro and q9ndi1_dro lgr4_hum lgr5_hum and lgr6_hum lgr5_hum, lgr7_hum and lgr8_hum, q9vbp0_dro, and q9vyg0_dro. drosophila orphan receptors q8sx01_dro and q9ndi1_dro are closely related to human glycoprotein hormone receptors and might bind to glycoprotein hormones. q9vbp0_dro and q9vyg0_dro are very similar in their overall domain architecture to lgrs with long n-termini, but their similar relationship in extracellular domain arrangements are also evident from this phylogenetic analysis without considering the n and c termini. cluster consists of peptide receptors with known ligands such as gastrin, cholecystokinin, orexin and neuropeptide ff or morphine modulating peptides. gpr103_hum is closely related to neuropeptide ff receptors, as predicted by our phylogenetic analysis and previous prediction on human gpcrs. subsequently, gpr was characterized and a novel rf-amide peptide, p was shown to be its ligand. drosophila orphan receptors, q9vwr3_dro and q9vwq9_dro, are related to each other and branch off from the cholecystokinin receptors and might have cholecystokinin as its natural ligand. q14439_hum branch off orexin receptors that bind to two novel neuropeptides, orexin-a and b, derived from a common prepro-orexin precursor by proteolytic processing. the receptors with known ligands binding to chemotactic substances and anaphylactic complement factors) are part of cluster these ligands are structurally very diverse but functionally related peptides. human orphan receptors, gp32_hum and q8nga4_hum branch out early from fml receptors and may probably bind to smaller hydrophilic peptides. l4r1_hum, l4r2_hum and q8tdt2_hum form a separate branch distant from other chemotactic peptide receptors with out bootstrap support. cml1_hum and gpr1_hum form a separate branch distinct from the other branches, and also gpr44_hum forming an individual branch. prediction of ligands for these receptors is not possible using this phylogenetic tree, but these receptors may be activated by chemotactic substances. mas proto-oncogene, mas-related genes and sensory neuron-specific g protein-coupled receptors form cluster angiotensin has been identified as an endogenous ligand for the g protein-coupled receptor mas. snsrs are activated by proenkephalin a peptide fragments, like bovine adrenal medulla peptide. some mrgs and snsrs are expressed in nociceptive sensory neurons suggesting that they could be involved in pain sensation or its modulation. previous studies also suggest that ligands for mrg receptors may include neuropeptides that modulate pain sensitivity. human orphan receptor q8ngk7_hum is closely related to mrg receptor. all receptors with known ligands in cluster are neuropeptide receptors. drosophila tachykinin-like peptide receptors and human neurokinin receptors form a closely-knit branch. pkr1_hum and pkr2_hum form a separate branch of receptors that bind to prokineticins. q9vrm0_dro is closely related to drosophila receptor nyr_dro that bind to neuropeptide y. q9vrm0_dro might probably bind to similar neuropeptides. neuropeptide y binding receptors ) form a separate branch. the human prolactin-releasing peptide binding gpr10_hum forms a separate branch in this phylogenetic tree. drosophila orphan receptors, q9vw75_dro and q8sz35_dro constitute a separate branch close to other neuropeptide receptors that might functionally be activated by neuropeptides. similarly, orphan receptor gp72_hum forms a new branch. drosophila orphan receptor q9w189_dro is a very distantly related member and was only grouped into this cluster by blastp results. chemokine receptors chemokine receptors are phylogenetically represented by two clusters and. chemokines are important molecules in inflammatory responses, as immunomodulators and they also have critical functions in lymphopoiesis. there are no drosophila members belong to this group of receptors suggesting these receptors might be recent in evolutionary origin. they have been divided into two subfamilies on the basis of the arrangement of the two disulphide-bond forming n-terminal cysteine residues, cxc and cc. many human cxc chemokines that mainly act on neutrophils are clustered at chromosome 4q12– while many cc chemokines that mainly act on monocytes are located in another cluster at chromosome 17q. our phylogenetic analysis has also divided chemokine receptors into two major clusters, concurrent with that of chemokine classes, suggesting co-evolution of receptors and ligands. cluster consists of receptors associated with cc type chemokines. as reported previously through earlier approach o75307_hum might bind to cc-type chemokine ligand. cluster consists of both cxc and cc-type receptors. admr_hum and q8ne10_hum form a branch whereas duff antigen and q96ch1_hum are distantly related to cml2_hum. these two branches are associated to chemokine receptors based on blastp similarity at an e-value significance of 5e- and 7e- respectively, with other members of this cluster. nucleotide and lipid receptors nucleotide and lipid receptors consists of six clusters, except for cluster and cluster there are no counter parts from drosophila. opsins are included in cluster that are activated by isoprenoid ligands. drosophila opsins show significantly high homology to human opsins. there is strong conservation of the retinal binding site and other regions suggesting that they are derived from a common ancestor and diverged thereafter retaining structural and functional features. drosophila receptor q9vtu7_dro is closely related to ops3–5_dro receptors, which are localized in the inner-cells of the drosophila eye. this suggests q9vtu7_dro might be localized in the inner cells of drosophila eye. receptors for pyramidine or purine nucleotides, cysteinyl leukotriene, nicotinic acid and short, medium and long chain fatty acids make up cluster q9bxc0_hum, q8tds5_hum and gp31_hum share the branch with closely related nicotinic acid receptor and might have similar carboxylic acids as their ligands. q8tdq8_hum and q96p68_hum are related to each other as well as to p2y receptors and may bind to p2y nucleotides. gp17_hum and gp82_hum receptors are distantly related to other members in this cluster and might represent potential new subfamilies binding to nucleotide or lipids. cluster is a heterogeneous group of receptors binding to lipids, nucleotides, modified nucleotides and platelet activating factor. orphan receptor q8tdu7_hum is closely related to platelet adp-binding receptor. q96jz8_hum is closely related to udp-glucose receptor and might bind to a modified nucleotide ligand. gpr34_hum forms a separate branch which is distantly related to pafr_hum. no prediction of ligands is possible for gpr34_hum with this phylogenetic tree. cluster consists of lipid receptors ) and exceptionally some of the peptide receptors are represented in different branches. although they bind to different ligands, they identify each other during sequence searches and display 23–29% sequence identity. the functionally important motifs are fairly conserved. indeed, this unusual branching including peptide and lipid receptors has been noted earlier by methner's and fredicksson's groups. cluster is composed of receptors binding to prostaglandins, prostacyclins and thromboxanes. all these ligands are derivatives of arachidonic acid, which serves as the precursor via the cyclooxygenase pathway. drosophila orphan receptor q9vvj1_dro within this tree might bind to ligands derived from aa by the action of cox. cluster is also a heterogeneous group of receptors consisting of protease-activated receptors, psychosine receptors, lysophosphatidylcholine and sphingosylphosphorylcholine. ovarian cancer g-protein-coupled receptor, previously described as a receptor for sphingosylphosphorylcholine, acts as a proton-sensing receptor stimulating inositol phosphate formation, whereas gpr is also involved in ph homeostasis, but elicits cyclic amp formation. ogr and gpr are different from other sphingosylphosphorylcholine binding endothelial differentiation gene receptors. orphan p2y receptors in this cluster are misnomers since they do not cluster with the classical neuropeptide receptors and instead appear to be co-clustered with members of this heterogeneous cluster. either they may have uncommon nucleotide as natural ligand or despite their structural similarity to the p2y family they may not be nucleotide receptors. gp35_hum and q8n580_hum, ebi2_hum and gp18_hum and gp20_hum cluster as separate branches and are distantly related to members of other branches but probably bind to lipids as their natural ligands. biogenic amine receptors biogenic amine receptors consists of five clusters mainly consisting of trace amine; melatonin; serotonin receptors; histamines, muscarinic acetylcholine, adenosine and histamine; dopamine, octopamine and adrenaline receptors. in these clusters fairly good intermixing of human and drosophila receptors is observed. this suggests biogenic amine receptors have ancient evolutionary origin as they are observed in invertebrates to higher vertebrates. cluster is represented mainly by trace amine receptors. trace amines binding these receptors are believed to play an important role in human disorders such as depression, attention deficit disorder, schizophrenia and parkinson's disease. they form a subfamily of gpcrs, distinct from, but related to serotonin, norepinephrine and dopamine receptors. drosophila orphan receptors q9vg54_dro and q9vcz3_dro are closely related to 5h4_hum. q9p1p4_hum and q9p1p5_hum are closely related to q96rj0_hum. similarly o14804_hum, a putative neurotransmitter receptor is closely related to trace amine receptors. cluster consists of melatonin receptors and other related orphan receptors. melatonin receptors bind to and are activated by biogenic amine 5-methoxy-n-acetyltryptamine. the melatonin-related receptor, despite sharing considerable amino acid sequence identity with other melatonin receptors, does not bind melatonin. the receptors in this cluster show considerable sequence similarity to neuropeptide y receptors than other biogenic amine receptors and were previously grouped along with npy receptors. all receptors with known ligands of cluster consist of serotonin receptors. these are structurally distinct from serotonin receptors in cluster drosophila orphan receptors q9veg1_dro and q9veg2_dro form a separate branch but are closely related to other serotonin receptors in this tree and might have similar ligand for its activation. q8tdv2_hum and q16538_hum, however, are distantly related to other receptors in this tree and were placed only based on blastp similarity. receptors of biogenic amines and many orphan receptors are all placed in different branches in cluster drosophila orphan receptor q9vhw1_dro branch out along with muscarinic acetylcholine and histamine receptors in this tree and might bind to acetylcholine or histamines for its activation. q9vaa2_dro is closely related to that of adenosine receptors. super conserved receptors expressed in brain from vertebrate species form a separate branch and might represent potential novel subfamily of gpcrs binding to undiscovered endogenous biogenic amine ligands. high-affinity lysophosphatidic acid receptor homologs o43898_hum and gpr63_hum form a distinct branch. similarly, orphan receptors gp21_hum and gp51_hum, gpr62_hum and q8tdv4_hum, q8ndv2_hum and q8ngv3_hum and q9vmi4_dro form a distinct branch, suggesting only distant relationship with other members of the cluster. receptors of biogenic amines, few serotonergic receptors and many orphan receptors are represented in different branches in cluster drosophila dopamine 2-like receptor, q8is45_dro, groups well with the human counterparts suggesting that their evolution extends much before drosophila. interestingly, dop2_dro is grouped with the adrenaline receptors instead with dopaminergic receptors and shows similar sequence identity with vertebrate alpha 1-, and beta-adrenergic, and d1-like, d2-like dopaminergic and serotonergic receptors. this drosophila receptor has been discussed as a novel structural class of dopamine receptors. drosophila octopamine receptor isoforms in mushroom bodies branch out with human alpha adrenergic _hum) receptors since they share high sequence identity in tm regions with alpha adrenergic receptors. q9ve32_dro branches out from human alpha adrenergic receptors and may have adrenaline as its ligand for activation. orphan striatum-specific g protein-coupled receptor, though grouped with biogenic amine receptors, may represent a novel subtype of gpcr due to the lack of conservation of key functional residues. orphan receptors, q9w3v5_dro and q8tdv5_hum, q96p66_hum and q8n6u8_hum, q9vhp6_dro and q9vbg4_dro form their own branch sharing distant relationship with other receptors in this tree and might represent potential novel subfamilies of biogenic amine gpcrs. class b receptors class b receptors are represented by two clusters consisting of classical hormone receptors and drosophila methuselah like proteins. the ligands for receptors of cluster are structurally related polypeptide hormones of 27– amino-acid residues, secretin, calcitonin, corticotropin-releasing factor, urocortins, growth-hormone-releasing hormone, vasoactive intestinal peptide, glucagon, glucagon-like peptides and glucose-dependent insulinotropic polypeptide. drosophila orphan receptors, q9v716_dro and q9v6c7_dro are closely related to the human receptor for corticotropin releasing factor receptor which binds to urocortins. q9v6n4_dro, q9vyh9_dro and q9nef7_dro are related to calcitonin and calcitonin gene-related peptide type receptors. three small accessory proteins, called receptor activity-modifying proteins, interact with these calcitonin receptors and can generate six pharmacologically distinct receptors. if this phenomenon of ramp-enabled receptor diversity exists in other receptors, then it will further complicate the ligand-receptor interactions of gpcrs, assuming they still bind to structurally similar ligands. human orphan receptor, q8nhb4_hum, is very closely related to ptrr_hum receptor binding to parathyroid hormone and parathyroid hormone-related protein. methuselah receptors and its paralogs of drosophila solely represent cluster the drosophila mutant methuselah was identified from a screen for single gene mutations that extended average lifespan of an organism and also increased resistance to several forms of stress, including starvation, heat, and oxidative damage. there are no obvious homologues of these receptors within human or c. elegans genomes. drosophila receptors, q8inm0_dro, q8ipd0_dro and q95nu7_dro, are closely related to previously identified mth members and may be new paralogs of these receptors. cell adhesion receptors large number of gpcrs with long extracellular n-termini, containing gpcr proteolytic site domain, are represented in cluster. several of these receptors also have one or many functional domains such as epidermal growth factor, leucine rich repeat, hormone-binding domain and immunoglobulin domains. these form several distantly related branches. except cd97_hum, all the receptors in this cluster are orphans with no known ligands. there are only four drosophila sequences representing these receptors. class c receptors receptors of class c are divided mainly into four clusters: metabotropic glutamate receptors, γ-aminobutryic acid receptors, calcium-sensing receptors and retinoic acid-inducible g-protein-coupled receptors. cluster consists of human and drosophila mgrs. human mgrs are sub-grouped into three different branches: first contains mgr1_hum and mgr5_hum and second contains mgr2_hum and mgr3_hum. the third branch, including mgr4_hum, 6– and drosophila mgrs represent a separate subgroup. drosophila orphan receptor q9v4u4_dro is closely related to mgr_dro and might bind to glutamate for its activation. calcium-sensing receptor forms cluster along with a set of orphan receptors. these orphan receptors either may have ligands and/or function similar to that of casr_hum or they may act as pheromone/olfactory receptors. phylogenetic tree of most members of family gpcrs across different genomes, caenorhabditis elegans, drosophila melanogaster, japanese pufferfish, goldfish, human, mouse, rat and salmon ) have shown casr_hum forms a separate branch part of pheromone/olfactory cluster of class c gpcrs. to note that olfactory and gustatory/taste receptors are not considered in this work. cluster consists of retinoic acid-inducible g-protein-coupled receptors. raigs have short extracellular amino-terminal domains as opposed to the other receptors currently assigned to class c. boss_dro also has short atd and branch out very early with the members of raigs and may represent new single member subfamily of class c receptors. the gabab receptors are present in cluster it is represented by four sub-branches, of which three are gababr1-3_hum type receptors and fourth sub-branch of drosophila orphan receptors related to that of gaba receptors. gabab is exclusively present in drosophila as separate branch whose function is not yet known. previous results have only been able to functionally characterize d-gababr and r when the two subtypes are co-expressed either in xenopus laevis oocytes or mammalian cell lines, whilst d-gababr was inactive in any combination. this suggests d-gababr requires a counterpart other than d-gababr and r to form a functional heterodimer. thus the current clustering approach suggests that q9vka4_dro or q9vr40_dro may interact with d-gababr and form a functional heterodimer. frizzled/smoothened receptors cluster comprises receptors with a long n-terminus and conserved cysteine rich domains which are likely to participate in wnt ligand binding. these receptors control the specification of cell fate, cell adhesion, migration, polarity and proliferation. this cluster is represented by ten human and four drosophila frizzled receptors together with smoothened receptors. the topology of the phylogenetic tree shows one smoothened and four frizzled branches. frz1_dro is closely related to human fzd3_hum and fzd6_hum. frz2_dro is related to fzd5_hum and fzd8_hum, whereas frz3_hum and frz4_hum form separate branches distantly related to other receptors. unassociated gpcrs thirty one gpcr sequences could not be included in any cluster with appreciable bootstrap values or blastp similarity. this can either be viewed as members of single member clusters with certain atypical parts of their sequences that could be a result of chimeric origin of the receptors or due to evolutionary pressure not shared by their closest phylogenetic neighbors. we have therefore placed these receptors separately as unassociated gpcrs, although these receptors clearly do not belong to the same group. most of the unassociated receptors remain as orphan receptors.  the phylogenetic analyses performed using human and drosophila gpcrs suggest that the sequences can be divided into clusters and reveals unexpected level of similarity between human and drosophila gpcrs. clusters group drosophila and human gpcrs together suggesting high evolutionary conservation across species for gpcr sequences. there are clusters, four of nucleotide-lipid receptors three clusters of peptide receptors and two clusters of chemokine and one cluster of glutamate receptors that do not contain any representation from drosophila gpcrs in our current dataset of sequences considered. perhaps the immune-related receptors, such as the chemokine ones, are not either recognized yet or not present in lower organisms such as drosophila. if there is a clear absence of such classes of receptors, this might also suggest that immune defense is regulated by proteins other than gpcrs in drosophila. interestingly, there is one cluster of secretin drosophila receptors where there is no human representation. these proteins are involved in aging in drosophila. furthermore, in this analysis, we also notice that out of the clusters that co-cluster human and drosophila gpcrs, drosophila gpcrs remain isolated sub-clusters in of them leaving behind only nine clusters that allow easy inter-mixing of the two sets of sequences. this includes clusters each of peptide and biogenic amine receptors and one cluster each of class b, c and frizzled receptors. the current clustering analysis provides ligand class association to drosophila and human orphan receptors could be associated with probable ligand classes using co-clustering principles as earlier observed within human gpcr sequences alone. further, similar cellular localizations have been suggested for drosophila orphan receptors that belong to the opsin family. gpcrs with similar extracellular domain architecture also co-cluster suggesting this similarity is encoded even within the gpcr domain. further this analysis also suggests dimerizing partner for d-gababr that might form a functional heterodimer. we have determined the relationship of the receptors within subgroups of the large gpcr superfamily by means of a cross-genome phylogenetic clustering approach. these studies also revealed a higher-level phylogenetic organization in which clusters with common ligand structure or chemistry, or a shared function, are evident across genomes. we hope that this approach proves valuable for identifying the natural ligands of drosophila and human orphan receptors. 
cross-genome phylogenetic analyses were performed to identify eight major groups of gpcrs dividing them into clusters of human and drosophila proteins and reveal unexpected levels of evolutionary conservation across human and drosophila gpcrs. similarly, a group of drosophila gpcrs, associated in aging, is not present in humans. a higher level of phylogenetic organization was revealed in which clusters with common domain architecture or cellular localization or ligand structure or chemistry or a shared function are evident across human and drosophila genomes. such analyses will prove valuable for identifying the natural ligands of drosophila and human orphan receptors that can lead to a better understanding of physiological and pathological roles of these receptors. phylogenetic clustering methods were used to elucidate the chemical nature of receptor ligands, which led to the identification of natural ligands for many orphan receptors. we have clustered human and drosophila receptors with known ligands and orphans through cross genome phylogenetic analysis and hypothesized higher relationship of co-clustered members that would ease ligand identification, as related receptors share ligands with similar structure or class. a large number of putative gpcrs are'orphans' with no identified natural ligands. our analysis suggests ligand class association to unknown drosophila receptors and unknown human gpcrs. the first step in understanding the function of orphan gpcrs is to identify their ligands. we also observe that members of human chemokine receptors, involved in immune response, and most of nucleotide-lipid receptors do not have counterparts in drosophila.
 the cell-membrane g-protein coupled receptors are one of the largest known superfamilies and are the main focus of intense pharmaceutical research due to their key role in cell physiology and disease. a large number of putative gpcrs are'orphans' with no identified natural ligands. the first step in understanding the function of orphan gpcrs is to identify their ligands. phylogenetic clustering methods were used to elucidate the chemical nature of receptor ligands, which led to the identification of natural ligands for many orphan receptors. we have clustered human and drosophila receptors with known ligands and orphans through cross genome phylogenetic analysis and hypothesized higher relationship of co-clustered members that would ease ligand identification, as related receptors share ligands with similar structure or class.  cross-genome phylogenetic analyses were performed to identify eight major groups of gpcrs dividing them into clusters of human and drosophila proteins and reveal unexpected levels of evolutionary conservation across human and drosophila gpcrs. we also observe that members of human chemokine receptors, involved in immune response, and most of nucleotide-lipid receptors do not have counterparts in drosophila. similarly, a group of drosophila gpcrs, associated in aging, is not present in humans.  our analysis suggests ligand class association to unknown drosophila receptors and unknown human gpcrs. a higher level of phylogenetic organization was revealed in which clusters with common domain architecture or cellular localization or ligand structure or chemistry or a shared function are evident across human and drosophila genomes. such analyses will prove valuable for identifying the natural ligands of drosophila and human orphan receptors that can lead to a better understanding of physiological and pathological roles of these receptors. 