Ž .Molecular Brain Research 71 1999 96–103www.elsevier.comrlocaterbres

Research report

Two related G protein-coupled receptors: The distribution of GPR7 in ratbrain and the absence of GPR8 in rodents

Dennis K. Lee a, Tuan Nguyen c, Calvin A. Porter d, Regina Cheng c, Susan R. George a,b,c,Brian F. O’Dowd a,c,)

a Department of Pharmacology, UniÕersity of Toronto, Medical Sciences Building, 8 Taddle Creek Rd. Rm. 4353, Toronto, Ontario, Canada, M5S 1A8b Department of Medicine, UniÕersity of Toronto, Medical Sciences Building, Toronto, Ontario, Canada, M5S 1A8

c Centre for Addiction and Mental Health, 33 Russell Street, Toronto, Ontario, Canada, M5S 2S1d Department of Biological Sciences, Texas Tech UniÕersity, Lubbock, TX 79409 USA

Accepted 4 May 1999

Abstract

Ž .GPR7 and GPR8, orphan G protein-coupled receptor GPCR genes, expressed in the brain and periphery share highest sequenceidentity to each other and significant similarity with opioid and somatostatin receptors. To further our knowledge of GPR7’s physiologicalfunction, we performed in situ hybridization analyses of rat brain to reveal specific patterns of expression in the brain. GPR7 mRNA wasfound to be discretely localized in areas of the amygdala, hippocampus, hypothalamus and cortex. We previously reported that GPR7 was

w xhighly conserved in both human and rodent orthologs while GPR8 was not found in the rodent 9 . We speculated that GPR8 originatedafter the divergence of the human and rodent. Using primers designed from human GPR8, we isolated lemur GPR8 and subsequentlyaligned human, monkey, and lemur GPR8 orthologs to design primers recognizing highly conserved regions of GPR8. Using theseprimers, orthologs of GPR7 and GPR8 were isolated by the PCR from rabbit, tree shrew, and flying lemur, as well as GPR7 in the rat.Subsequent analysis of the clones obtained demonstrated that both GPR7 and GPR8 sequences were highly conserved amongst thespecies studied, but a rodent GPR8 was not isolated. The absence of a GPR8 gene in the rodent suggests that GPR8 originated from geneduplication of GPR7 after the rodent line diverged from the rabbit, tree shrew, flying lemur, lemur, monkey and human lines. In addition,the taxonomic distribution of GPR8 is consistent with molecular studies grouping rabbits with primates, tree shrews and flying lemursrather than with rodents. q 1999 Elsevier Science B.V. All rights reserved.

Keywords: G protein-coupled receptor; mRNA distribution; Ortholog

1. Introduction

Neuropeptides are involved in numerous central ner-vous system processes such as reward, behaviour, andlearning, and their actions are mediated mainly throughspecific cell membrane receptors. Many ofm these neu-ropeptide receptors are members of the G protein-coupled

Ž .receptor GPCR superfamily, which share a commonŽ .structure of seven transmembrane TM domains and many

conserved amino acid residues. In our search for novelGPCRs we reported the discovery of two human GPCR-

) Corresponding author. Department of Pharmacology, University ofToronto, Medical Sciences Building, 8 Taddle Creek Rd. Rm. 4353,Toronto, Ontario, Canada, M5S 1A8. fax: q1-416-971-2868; E-mail:brian.odowd@utoronto.ca

encoding genes, GPR7 and GPR8. Sharing a 70% nu-cleotide and a 64% amino acid identity with each otherand significant identities to opioid and somatostatin recep-tor genes, we predicted that GPR7 and GPR8 bind thesame endogenous peptide ligand, possibly opioid or so-matostatin-like. However, only the GPR7 receptor wasfound to bind a non-selective opioid ligand with low

w xaffinity, while neither bound somatostatin 9 .The rat and mouse orthologs of GPR7 were previously

w xcloned 9 and the rat ortholog used as a probe for themapping of mRNA expression in rat brain described in thisreport. GPCR gene sequences are generally conserved

w xamong human, rat and mouse species 7 to the degree thatthe isolation of a novel human GPCR gene is usually

Žfollowed by the prompt cloning of a rodent ortholog and.vice versa by PCR screening. However, we were unable

0169-328Xr99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.Ž .PII: S0169-328X 99 00171-0

( )D.K. Lee et al.rMolecular Brain Research 71 1999 96–103 97

to isolate a rodent ortholog of GPR8 by PCR with GPR8-specific primers despite repeated attempts with different

w xprimer pairs and annealing temperatures 9 .In order to study the evolution of GPR7 and GPR8, we

screened genomic DNA of rodent species and speciesrepresenting phylogenetic intermediates between humanand rodents with the PCR, using degenerate oligonucleo-tides. We now report the mRNA distribution of GPR7 inthe rat brain; the isolation of GPR8 orthologs in lemur,flying lemur, tree shrew and rabbit; and GPR7 orthologs inflying lemur, tree shrew and rabbit.

2. Materials and methods

2.1. Cloning of GPR7 and GPR8 orthologs

ŽPCR analysis was carried out using lemur Propithecus. Ž .Õerreauxi genomic DNA 1 mg with primers designed

Ž .from the sequence encoding transmembrane region TM 2Ž X X. Ž X5 -CTGAACCTGGCCGTCGCCGAC-3 and TM7 5 -

X.GTAGGACATACTGATGAC-3 of human GPR8. Theconditions for the PCR were 30 cycles of 40 s at 958C, 40s at 458C and 1 min at 728C followed by a 5 min extension

Ž .at 728C. Flying lemur Cynocephalus Õariegatus , treeŽ . Ž .shrew Tupaia glis , fruit bat Dobsonia moluccensis ,Ž . Ž .rabbit SylÕalagus floridana , mouse Mus musculus and

Ž . Ž .rat Rattus norÕegicus genomic DNA 1 mg was ampli-fied with degenerate primers designed from the TM3

Ž Xsequences 5 -TACTTTCTbGCCGTGATGAGCGTG-X .GACGdTA-3 ; bsA, C or G, dsA or C and the TM6Ž Xsequences 5 -GACeGAGGCCAGGTGGAAGGGCGTC-

X .CAGCA-3 ; esA or G of human, monkey and lemurGPR8. The conditions for the PCR were 30 cycles of 40 sat 958C, 40 s at 488C and 1 min extension at 728Cfollowed by a 5 min extension at 728C. PCR products werephenolrchloroform extracted, precipitated with ethanol,phosphorylated with T4 polynucleotide kinase and blunt-ended with Klenow enzyme. These products were thenelectrophoresed on a 0.5% low-melt agarose gel and DNAin the expected size ranges were cut from the gel. TheDNA was then subcloned into the EcoRV site of thepBluescript vector, and transformed into bacteria. Colonieswere selected, plasmid DNA purified and subjected tosequence analysis.

2.2. In situ hybridization of rat brain sections

Preparation of rat brain sections and in situ hybridiza-tion procedures were carried out as previously describedw x14 . Briefly, brains were removed from male Sprague–

Ž .Dawley rats Jackson laboratories within 30 s of decapita-tion, frozen, sectioned at 14 mm thickness using a micro-tome cryostat, thaw-mounted onto microscope slides andstored at y708C. A fragment encoding the rat ortholog ofGPR7 from TM3 to TM7 was labeled by random priming

w35 x Ž .using S dCTP NEN, Dupont . The rat brain slices wereincubated for 2 h in prehybridization solution, hybridized

Ž 6 .with the labelled probe 10 dpmrslice for 16 h, andwashed in conditions of increasing temperature and de-creasing salt concentrations. The hybridized sections weredehydrated in a graded alcohol series and exposed to X-ray

Ž .film Dupont MRF-34 for 4–6 weeks at y708C anddeveloped. For use as controls, adjacent sections werehybridized following treatment with RNase, to confirm thespecificity of hybridization.

3. Results

3.1. Isolation of GPR7 and GPR8 orthologs

GPR8-specific primer pairs designed from conservedTM domains were unable to amplify sequences encodingGPR8 from rodent genomic DNA. This suggested eitherthat the rodent GPR8 ortholog identity to human GPR8was low enough to preclude its amplification or the ab-sence of such an ortholog. Human GPR8 is most closelyrelated to GPR7, which has rodent orthologs. Since arodent ortholog for GPR8 appeared to be absent, wespeculated that GPR7 was replicated after its divergencefrom the rodent evolutionary lines, diverging over time tobecome GPR7 and GPR8.

We carried out a systematic search for GPR8 in mam-mals to isolate orthologs and a representative of its earliestprogenitor. PCR amplification of lemur genomic DNA,with primers designed from human GPR8 TM2 and TM7regions, resulted in the cloning of a DNA fragment approx-imately 600 bp in length. This fragment was subcloned andsequenced to reveal a nucleotide sequence identity of 93%and a predicted protein sequence identity of 91% to humanGPR8, confirming the isolation of a lemur GPR8 ortholog.Previously, we had isolated GPR8 in the African green

Table 1Ž .GPR7 and GPR8 ortholog identities. Ortholog identities for A GPR7

Ž .and B GPR8. Percent amino acid sequence identities are shown betweeneach species, percent nucleotide sequence identities are in parentheses.Humshuman, FL s flying lemur, TSs tree shrew, Rabs rabbit andMussmouse

FL TS Rab Mus

( )A GPR7 Ortholog IdentitiesŽ . Ž . Ž . Ž .Hum 90 90 93 91 93 88 92 87

Ž . Ž . Ž .FL 90 89 88 87 88 82Ž . Ž .TS 92 89 92 85

Ž .Rab 96 86

( )B GPR8 Ortholog IdentitiesFL TS Rab

Ž . Ž . Ž .Hum 89 88 82 86 85 83Ž . Ž .FL 83 86 88 83

Ž .TS 82 82

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Fig. 1. Amino acid alignment of GPR8 orthologs from various species. GPR8shuman, MonGPR8smonkey, ProGPR8s lemur, FLGPR8s flying lemur, TSGPR8s tree shrew, and RabGPR8s rabbit.Shaded and boxed amino acids are divergent from the human GPR8 sequence. Transmembrane regions are shown and amino acids are numbered at the right. The sequences reported in this paper have beendeposited in the Genbank database under accession numbers AF042362 through AF042369.

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Fig. 2. Amino acid alignment of GPR7 orthologs from various species. GPR7shuman, FLGPR7s flying lemur, TSGPR7s tree shrew, RabGPR7s rabbit and MusGPR7smouse. Shaded and boxed aminoacids are divergent from the human GPR7 sequence. Transmembrane regions are shown and amino acids are numbered at the right.

( )D.K. Lee et al.rMolecular Brain Research 71 1999 96–103100

Fig. 3. GPR7 mRNA distributions patterns in rat brain by in situ hybridization. In situ hybridization of coronal rat brain sections presented rostrocaudally,w x Ž . Ž . Ž . Ž .showing in distance from bregma according to the stereotactic coordinates by Paxinos and Watson 10 . A q1.7 mm, B q1.2 mm, C y1.3 mm, D

Ž . Ž .y1.8 mm, E y3.3 mm, and F y4.3 mm. Abbreviations are as follows: TTs taenia tecta, ICjs islands of Calleja, ICjMsmajor island of Calleja,POsprimary olfactory cortex, Ensendopyriform nucleus, OTsolfactory tubercle, SOssupraoptic nucleus, SCNssuprachiasmatic nucleus, DGsdentate gyrus, CAs fields of Ammon’s horn, PVNsparaventricular nucleus of hypothalamus, DMHsdorsomedial nucleus of hypothalamus, VMHsventromedial nucleus of hypothalamus, Arcsarcuate nucleus, PVTsperiventricular nucleus of thalamus, MeAsmedial amygdaloid nucleus, PmCAsposteromedial amygdaloid nucleus.

( )D.K. Lee et al.rMolecular Brain Research 71 1999 96–103 101

w xmonkey 9 . Sequences encoding human, monkey and thelemur GPR8 were aligned in order to design degenerateprimers based on sequences encoding TM3 and TM6.Using these primers, PCR amplification of flying lemur,tree shrew, fruit bat, rabbit, mouse, and rat genomic DNAamplified fragments approximately 400 bp in length. Thesefragments were subcloned and sequenced. Sequence analy-sis revealed GPR7 and GPR8 orthologs in the flyinglemur, tree shrew and rabbit, each with high identity to

Ž .human GPR7 and GPR8, respectively Table 1 . A GPR8ortholog was found in neither the rat or mouse, and neitherGPR7 or GPR8 orthologs were found in the fruit bat. Thedegenerate primers amplified fragments encoding or-thologs to other human genes including GPR1, GPR7,GPR9, and somatostatin SSTR3 in the rat as well asGPR9, GPR12, and SSTR3 in the bat.

The sequences for the receptors encoded by humanGPR8 and its orthologs from monkey, lemur, flying lemur,

Ž .tree shrew, and rabbit were aligned Fig. 1 as were thereceptors encoded by human GPR7 and its orthologs from

Ž .flying lemur, tree shrew, rabbit and mouse Fig. 2 . Theprotein sequences of GPR7 and GPR8 orthologs werehighly conserved with no observed shifts in reading frameand no amino acid deletions or insertions.

3.2. GPR7 mRNA distribution in the rat brain

GPR7 mRNA was expressed in discrete areas of ratŽ .brain Fig. 3 . Dense signals were detected in the taenia

tecta and islands of Calleja, including the major island.There was scattered mRNA in the olfactory tubercle andthe primary olfactory cortex, with little signal detected inthe remainder of the cerebral cortex. In hypothalamus,there was discrete and very dense expression of GPR7mRNA in the suprachiasmatic nucleus and in the parvocel-lular division of the paraventricular nucleus. Dense expres-sion of mRNA was also present in the supraoptic, dorso-medial and ventromedial nuclei of the hypothalamus. Inthe hippocampus, moderate expression was seen in thedentate gyrus and in the CA fields of Ammon’s horn.Some of the amygdaloid nuclei also expressed GPR7mRNA, with particular abundance in the medial amyg-daloid nucleus.

4. Discussion

4.1. Phylogenetic analysis of GPR7 and GPR8 orthologs

GPR7 and GPR8 orthologs were isolated from speciesof the closely related taxonomic superorders Archonta and

Ž . ŽGlires Fig. 4 . The human, monkey Cercopithecus. Ž . Žaethiops and lemur P. Õerreauxi species of the Primate

.order belong to the superorder Archonta, which alsoŽ .encompasses tree shrews T. glis, of the order Scandentia ,

Ž .flying lemurs C. Õariegatus, of the order Dermoptera and

Fig. 4. Phylogenetic tree showing hypothesized relationships among fivemammalian orders. The ellipse represents the hypothesized origin of theGPR8 gene. The relationships among tree shrews, flying lemur, primates,and lagomorphs are unresolved. The traditional taxa Archonta and GliresŽ .not supported by this study are indicated.

Žthe bare-backed fruit bat D. moluccensis, of the order. w x ŽChiroptera 11 . The mouse M. musculus, of the order

. Ž .Rodentia , rat R. norÕegicus, of the order Rodentia andŽ .rabbit S. floridana, of the order Lagomorpha are grouped

w xtogether in the superorder Glires 2 . However, there ismolecular evidence for a Primate, Scandentia, and Der-moptera grouping with Lagomorpha with the exclusion of

w xChiroptera 1,2,4,8 . The human, flying lemur, tree shrew,and rabbit GPR8 ortholog sequences display high identities

Ž .of greater than 80% to each other Table 1 , supportingthis grouping.

Previously, we stated that GPR7 and GPR8 likely sharea common progenitor gene, based on the high level ofidentity between the two genes and the common intronless

w xgene structure they share 9 . We compared GPR8 orthologidentities between human, flying lemur, tree shrew andrabbit sequences representing the Primate, Dermoptera,

Ž .Scandentia and Lagomorpha orders, respectively Fig. 4 .While our GPR8 ortholog amino acid identities ranged

Ž .from 82% to 89% 82% to 88% for nucleotide identitiesthe GPR7 ortholog amino acid identities seem to be greater,

Žranging from 88% to 96% 82% to 91% for nucleotide.identities despite covering a greater observed phyloge-

netic range than the GPR8 orthologs by the inclusion of aŽ .mouse ortholog representing the order Rodentia Table 1 .

The similar nucleotide identity ranges suggests that bothGPR7 and GPR8 undergo similar mutation rates in thesemammals. However, the difference in amino acid identityranges suggests that GPR7 is under somewhat greatersequence conservation than GPR8. Sharing a commonprogenitor and a nucleotide identity of 70%, we can specu-late that GPR7 and GPR8 diverged relatively recently, latein mammalian evolution. Stronger evolutionary conserva-tion pressure on GPR7 and the presence of orthologs inboth rat and mouse suggest that GPR7 underwent a dupli-cation event after diverging from the rodent to give rise toGPR8. The high identities existing between our isolatedGPR8 orthologs would predict rodent GPR8 orthologs of

( )D.K. Lee et al.rMolecular Brain Research 71 1999 96–103102

high identity as well, based on the grouping of superordersGlires and Archonta. However, since no such rodent or-tholog appears to exist, GPR8 may have originated as areplicate of GPR7 after the divergence of the rodent fromthe evolutionary lines leading to human, monkey, lemur,flying lemur, tree shrew and rabbit. A comparable exampleof GPCR duplication and genetic drift has been reportedfor the angiotensin II type 1 receptor. Through sequencingof cDNA and genomic DNA and evolutionary analyses,

w xYoshida et al. 13 revealed that the mouse and rat possessŽtwo subtypes of the angiotensin II type 1 receptor AII

.types 1A and 1B while the bovine and human possessonly a single gene.

4.2. GPR7 mRNA distribution in rat brain

To understand the possible neurophysiological roles ofGPR7, we examined the distribution of GPR7 mRNAs inthe rat brain. In general, GPR7 showed moderate to denseexpression in the factory cortex, hippocampus and hypo-thalamus. We previously reported GPR7 expression in

w xmouse hippocampus, hypothalamus, and midbrain 9 . Bycomparison, GPR7 rat brain expression patterns appear tobe more widespread than in the mouse. Aside from theventral tegmental area of the midbrain, GPR7 rat brainexpression overlaps and exceeds the expression patternseen in the mouse. Expression patterns were similar be-tween rat and mouse in the hippocampus and amygdala. Inthe rat hypothalamus, expression was seen in the paraven-tricular, dorsomedial and supraoptic nuclei, in addition tothe suprachiasmatic, ventromedial and arcuate nuclei asseen in the mouse. Finally, dense signals were present inthe rat taenia tecta and islands of Calleja with moderate,scattered expression in the primary olfactory cortex andolfactory tubercle, areas not perceived to express GPR7mRNA in the mouse.

With the exception of GPR8, GPR7 had the highestprotein sequence identities with the opioid, somatostatinand nociceptin receptors at approximately 37%, as re-

w xvealed by a BLAST search 3 . Furthermore, a comparisonof the GPR7 expression pattern with the opioid, somato-statin and nociceptin receptors revealed overlap in regionsof the rodent brain including the hypothalamus, hippocam-pus and the amygdala and in the case of the opioid

w xreceptors, the olfactory cortex 5,6,12 . A strong contrastwas seen between the wider tissue distribution of theopioid, somatostatin, and nociceptin receptor mRNAs vs.the restricted and discrete distribution of GPR7. For exam-ple, in comparison with the opioid receptors, GPR7 ex-pression in rat brain corresponded best with that of thekappa opioid receptor, specifically in the primary olfactorycortex, olfactory tubercle, islands of Calleja, dentate gyrus,CA fields of Ammon’s horn, and the medial amygdaloid,suprachiasmatic, supraoptic, paraventricular, arcuate, ven-

w xtromedial and dorsomedial nuclei 5 . However, GPR7expression was absent in other regions reported to express

significant levels of the kappa receptor, such as the medialhabenula, substantia nigra, caudate putamen and various

w xareas and nuclei of the cortex and thalamus 5 . Overall,GPR7 rat brain expression was seen to overlap with butnot closely match the expression patterns of opioid, so-matostatin or nociceptin receptors. However, localizationin regions of the limbic, hippocampal and hypothalamicregions suggest GPR7 may have a role in memory, learn-ing, olfaction and modulation of neuroendocrine effects.

Acknowledgements

This research was supported by grants from the Na-tional Institute on Drug Abuse, the Medical ResearchCouncil of Canada, the Smokeless Tobacco ResearchCouncil and by a PMACrMRC grant from Merck-FrosstResearch Laboratories. We would like to thank Mai Nguyenfor her excellent technical assistance. Tree shrew, flyinglemur, fruit bat and rabbit genomic DNA and tissue sam-ples were provided by the frozen tissue collection of TheMuseum, Texas Tech University and by Dr. Morris Good-man of Wayne State University School of Medicine.

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