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Research Report

Characterization of Gpr101 expression and G-proteincoupling selectivity

Brian Batesa,⁎, Lynn Zhanga, Stan Nawoschikb, Sreekumar Kodangattila, Eugene Tsengb,David Kopscob, Angela Kramerb, Qin Shanb, Noël Taylora, Jeremy Johnsona, Ying Suna,Hui Min Chena, Maria Blatcherb, Janet E. Paulsena, Mark H. Pauschb

aWyeth Research, Biological Technologies, 87 Cambridge Park Drive, Cambridge, MA 02140, USAbWyeth Research, Neurosciences, Princeton, NJ 08543-8000, USA

A R T I C L E I N F O

⁎ Corresponding author. Fax: +1 617 65 8350.E-mail address: bbates@wyeth.com (B. Ba

0006-8993/$ – see front matter © 2006 Elsevidoi:10.1016/j.brainres.2006.02.123

A B S T R A C T

Article history:Accepted 26 February 2006Available online 27 April 2006

This report describes the identification and characterization of the murine orphan GPCR,Gpr101. Both human and murine genes were localized to chromosome X. Similar to itshuman ortholog, murine Gpr101 mRNA was detected predominantly in the brain withindiscrete nuclei. A knowledge-restricted hidden Markov model-based algorithm, capable ofaccurately predicting G-protein coupling selectivity, indicated that both human and murineGPR101 were likely coupled to Gs. This prediction was supported by the elevation of cyclicAMP levels and the activation of a cyclic AMP response element-luciferase reporter gene inHEK293 cells over-expressing human GPR101. Consistent with this, over-expression ofhuman GPR101 in a yeast-based system yielded an elevated, agonist-independent reportergene response in the presence of a yeast chimeric Gαs protein. These results indicate thatGPR101 participates in a potentially wide range of activities in the CNS via modulation ofcAMP levels.

© 2006 Elsevier B.V. All rights reserved.

Keywords:Orphan GPCRIn situ hybridizationYeastChimeric G proteinReporter gene assay

1. Introduction

The recent identification of numerous G-protein-coupledreceptors (GPCRs) in the mammalian genome has created alarge catalog of uncharacterized receptors (Vassilatis et al.,2003). This presents the significant challenge of identifyingcognate ligands for these numerous orphan GPCRs (oGPCRs).Investigators have used a diversity of approaches to identifyendogenous ligands for oGPCRs including bioinformaticmethods applied to oGPCR sequences, searching sequencedatabases for likely peptide ligands, testing panels ofbiologically active compounds and screening fractionatedtissue extracts in a process referred to as reverse pharma-

tes).

er B.V. All rights reserved

cology (Civelli et al., 2001; Lee et al., 2003; Wise et al., 2004). Inreverse pharmacology approaches, oGPCRs are firstexpressed in cell lines that permit efficient functionalexpression followed by detection of G-protein- and agonist-dependent activation by various methods. These methodstake advantage of well-characterized signal transductionpathways with robust downstream readouts, such as 35S-GTPγS binding, second messenger production (e.g. cAMP,inositol phosphates, intracellular Ca2+) and second messen-ger-dependent, G-protein-coupled reporter genes. Usingthese systems, oGPCR ligands have been identified byscreening known compound panels prioritized based on thesimilarity of oGPCRs to GPCRs with known ligands (Briscoe et

.

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al., 2003; Brown et al., 2003; Inbe et al., 2004; Itoh et al., 2003;Kotarsky et al., 2003; Liu et al., 2001; Morse et al., 2001;Nakamura et al., 2000; Nguyen et al., 2001; Nilsson et al.,2003; Oda et al., 2000; Zhu et al., 2001). Additionally, suchassays have been employed to screen biological extracts oftissues selected based on the expression pattern of theoGPCR (Hinuma et al., 1998; Kojima et al., 1999; Liu et al.,2003; Meunier et al., 1995; Reinscheid et al., 1995; Saito et al.,1999; Sakurai et al., 1998; Shimomura et al., 1999, 2002; Zhanget al., 2001).

The choice of a reverse pharmacology signal transductionassay can be tailored to the oGPCR if specific G-proteincoupling selectivity can be determined. Therefore, deciphe-ring coupling selectivity is an important step in understan-ding the biology of an oGPCR and in the development of cell-based assays for discovery of natural or surrogate ligands.

Fig. 1 – BestFit alignment of human (NM_054021) and murine Gdots indicate conserved substitutions, single dots semi-conserveby BestFit.

Because the coupling specificity of many oGPCRs has yet tobe experimentally determined, various computational stra-tegies have been developed (Cao et al., 2003; Moller et al.,2001; Sreekumar et al., 2004). These approaches have provento be quite accurate in defining the G-protein couplingselectivity of GPCRs with known ligands.

A recent report described the identification of the humanoGPCR, GPR101 (Lee et al., 2001). Alignment of this sequencewith all known GPCRs predicts that it is most similar toanother orphan receptor, GPR161 (also known as RE2), andfalls into a class of orphan GPCRs whose closest relativeswith known ligands are the melatonin receptors and theopsins (Vassilatis et al., 2003). However, the similaritybetween these receptors is low, making prediction of anendogenous ligand based on sequence similarity aloneunlikely. GPR101 is also weakly related to the histamine H2

PR101. Vertical lines indicate identical amino acids. Doubled. The two proteins are 70.75% identical as determined

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receptor. However, GPR101 lacks particular hallmarks ofbiogenic amine receptors, such as an aspartate in trans-membrane domain three, making this class of moleculesunlikely ligands.

The initial report of human GPR101 documented expres-sion in the central nervous system (CNS) but not the liver (Leeet al., 2001), making it likely that its endogenous ligand mightbe found in the CNS. Within the CNS, GPR101 transcript wasmost abundant in the hypothalamus, raising the possibilitythat it might play a role in modulating some of the diversefunctions of this brain area.

In order to characterize GPR101 in more detail, we isolatedits murine ortholog and determined its detailed expressionpattern in mouse brain. We also determined the signaltransduction pathways to which human and murine GPR101were coupled using a knowledge-restricted hidden Markovmodel (HMM) method and confirmed this finding by func-tional expression of human GPR101 in mammalian and yeastcells.

Fig. 2 – Murine and humanGPR101 are localized to a syntenic reg(green, arrows) detects human (A) GPR101 on Xq26–27 and murin(blue) to determine chromosomal identity. In illustrations in righrepresent location of FISH signals detected on each of ten chrom

2. Results

2.1. Identification of murine Gpr101

Southern analysis using human GPR101 (NM_054021) as aprobe against mouse genomic DNA identified a singlehybridizing band under both low and high stringencyconditions, suggesting a single murine ortholog (data notshown). Therefore, to isolate murine Gpr101, the full-lengthhuman gene was used as a probe to screen a mouse braincDNA library. Eight independent clones were isolated, all ofwhich encoded the same sequence or truncated versionsthereof. The largest cDNA isolated contained 5386 nucleo-tides and encoded a protein of 511 amino acids. It should benoted that the isolated cDNA differed from the predictedsequence (XM_141764). However, we have been unable toisolate the additional postulated upstream exons ofXM_141764 by RT-PCR (data not shown). Furthermore, the

ion of chromosome X. Fluorescent in situ hybridization signale (B) on XA5. The same spreads were also stained by DAPIt hand panels, dots next to schematized chromosomesosomal spreads analyzed.

Fig. 3 – Gpr101 is expressed predominately in brain.Northern blots showing Gpr101 expression in brain (A).Within brain, expression is highest in hypothalamus (B). Sizemarkers are indicated. Actin hybridization (A) and 28s rRNA(B) for their respective blots are shown. Abbreviations: OB,olfactory bulb; CTX, cerebral cortex, STR, striatum; HIP,hippocampus; HYP, hypothalamus; MB, midbrain; COLL,colliculus; CB; cerebellum; BS, brainstem; sk. musc., skeletalmuscle.

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predicted initiation codon of the isolated cDNA was preced-ed by a Kozak consensus sequence (ACC), and bioinformaticanalysis suggested no homology between mouse and humangenomic sequences upstream of this proposed initiationcodon (data not shown). Therefore, we believe the cDNAreported in this work encoded the full-length murine Gpr101gene. The amino acid sequence encoded by this cDNA was71% identical to its human counterpart (Fig. 1). BLASTsearching of the mouse genome database was unable toidentify a sequence of greater similarity to human GPR101.

To confirm the orthologous relationship of these genes,an analysis of synteny using chromosomal FISH wasconducted. Gpr101 mapped to mouse chromosome X, bandXA5, a region that is syntenic with the location of humanGPR101 (Fig. 2).

2.2. Expression of murine Gpr101

Northern blotting analysis detected a single Gpr101 transcriptof approximately 5.5 kb predominately in brain (Fig. 3). Themurine mRNA was detected in all areas of the brain analyzedwith strongest signals in the hypothalamus.

To localize expression in more detail, in situ hybridizationwas performed on mouse brain sections. Gpr101 transcriptwas detected in numerous areas and nuclei of the brain, withthe most prominent expression being highly restricted to afew nuclei (Table 1, Figs. 4, 5).

Within the olfactory bulb, hybridization was observed inoccasional, isolated neurons in the granular, glomerular andmitral cell layers (Fig. 4A).

In the septum, labeling was noted in all divisions ofthe lateral nucleus (Figs. 4B, D). The septum sends andreceives heavy innervation from the hippocampal forma-tion including afferents from the subiculum and entorhi-nal cortex. Within the hippocampus, we found prominentexpression of Gpr101 in the subiculum with relative levelsgreatest in its ventral aspect (Fig. 4C). Prominent labelingwas also noted in the source of the hippocampalperforant path, the entorhinal cortex (Fig. 4C), and inthe ventral-most portion of the pyramidal layer of thehippocampus beginning approximately at bregma −2.92(Fig. 4M).

In the forebrain basal ganglia, expression of Gpr101 waslimited to the nucleus accumbens. Within the accumbens,expressionwas strongest in the shell but weaker hybridizationwas occasionally seen in the core (Fig. 4B).

Extensive and strong hybridization was found broadlythrough the hypothalamus. In the anterior region, limitedlabeling was seen in the median preoptic nucleus (MnPO),anterior hypothalamic area and themedial preoptic area (Figs.4D, E). Stronger labeling was noted in the paraventricular andsupraoptic nuclei (PVN and SO, respectively) (Figs. 4E, G).Within the paraventricular nucleus, expression was mostprominent in the magnocellular divisions. Interestingly, weakGpr101 signal was also found in the subfornical organ (Fig. 4F),a circumventricular organ involved, alongwith theMnPO, PVNand SO in osmotic regulation and thirst. The strongesthypothalamic labeling was seen in the arcuate nucleus.Other areas of the hypothalamus in which we detectedGpr101 included the dorsomedial, medial mammillary and

perifornical nuclei and the lateral and posterior hypothalamicareas (Figs. 4H–K).

Murine Gpr101 was strongly expressed in several nuclei ofthe extended amygdala. In particular, heavy labeling wasseen in the amygdalohippocampal and amygdalopiriformtransition areas as well as in the central nuclei (Figs. 4K–N).Other positive amygdaloid nuclei included the anterioramygdaloid area, medial amygdaloid nuclei, amygdalostria-tal transition area and the bed nuclei of the stria terminalis(Fig. 4D).

In the thalamus, Gpr101 expression was rather limited,withweak labeling noted in the ventrolateral geniculate nucleiand the intergeniculate leaf (Fig. 4O).

Gpr101 transcript was detected in numerous nucleithroughout the midbrain and brain stem. In particular,hybridization was seen in limited neurons of the substantianigra pars compacta (SNC), substantia nigra pars reticulata(SNR) and ventral tegmental area (VTA) (Fig. 5A). The SNCand VTA are both dopaminergic nuclei, as evidenced by theirexpression of tyrosine hydroxylase (Fig. 5B). However, atpresent, it is not known if Gpr101 is expressed in thedopaminergic neurons found in these regions. Gpr101

Table 1 – Relative expression levels of Gpr101 in mousebrain

Olfactory bulbGlomerular layer +Granular layer +Mitral layer +

SeptumLateral septal nuc., intermed. +Lateral septal nuc., dorsal +Lateral septal nuc., ventral +

Basal gangliaAccumbens nuc. +

HypothalamusMedian preoptic nuc. +Medial preoptic area ++Paraventricular nuc. +++Supraoptic nuc. +++Anterior hypo. area +Dorsomedial hypo. nuc. ++Arcuate nuc. +++Lateral hypothalamic area +Perifornical nuc. +Posterior hypothalamic area +Medial mammillary nuc., lateral ++

Extended amygdalaAnterior amygdaloid area +Central amygdaloid nuc., medial ++Central amygdaloid nuc., capsular ++Medial amygdaloid nuclei +Amygdalostriatal transition area +Amygdalohippocampal area +++Amygdalopiriform transition area ++Bed nuclei of the ST +

HippocampusSubiculum ++Pyramidal layer ++Entorhinal cortex +

Midbrain and hindbrainNucleus of the pc +Magnocellular nucleus of the pc +Precommissural nucleus +Substantia nigra pars reticulata +Substantia nigra pars compacta +Ventral tegmental area +Deep mesencephalic nuc. +Nucleus of the brachium of the IC +Periaqueductal gray +Lateral periaqueductal gray +Ventrolateral periaqueductal gray +Dorsomedial periaqueductal gray +Dorsal raphe nuc. +Cuneiform nuc. +Lateral parabrachial nuc. +Laterodorsal tegmental nuc. +Dorsomedial tegmental area +Pontine reticular nuc., ventral +Pontine reticular nuc., caudal +Pontine reticular nuc., oral +Central gray of the pons ++Locus coeruleus +Prepositus nuc. +

Midbrain and hindbrainRaphe pallidus nuc. +Raphe magnus nuc. +Gigantocellular reticular nuc., alpha +Gigantocellular reticular nuc., ventral +Lateral paragigantocellular nuc. +Intermediate reticular nuc. +Parvicellular reticular nuc. +Lateral reticular nuc. +Medullary reticular nuc. +Nuc. of the solitary tract, medial +++Nuc. of the solitary tract, intermed. +

Circumventricular organsArea postrema +Subfornical organ +

ThalamusIntergeniculate leaf +Ventrolateral geniculate nuclei +

Abbreviations: nuc., nucleus; IC, inferior colliculus; pc, posteriorcommissure; ST, stria terminalis.

Table 1 (continued)

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expression was also seen in other important aminergic nucleiincluding the noradrenergic locus coeruleus (LC) (Fig. 5C) andthe serotoninergic dorsal raphe (DR), raphemagnus (RMg) andraphe pallidus (RPa) (Figs. 5E, G). These nuclei were alsodefinitively identified by their expression of aminergicmarker genes (tyrosine hydroxylase for the LC, Fig. 5D;tryptophan hydroxylase for the DR, RMg and RPa, Figs. 5F,H). As with the dopaminergic nuclei, at present, it is notknown if Gpr101 is co-expressed with these biogenic amines.

Gpr101 transcript was also detected in multiple areas inthe reticular formation and included the periaqueductalgray, central gray of the pons (Figs. 4P, R) and thepontine, gigantocellular, intermediate and medullary reti-cular nuclei (Figs. 4R, 5G). Very prominent expression wasalso found in the nucleus of the solitary tract withheaviest labeling in the medial portion (Fig. 4Q). Nospecific hybridization above background levels wasdetected in the cerebellum.

Outside the brain, expression of Gpr101 was noted in thespinal cord (Fig. 4T). Within the spinal cord, expression waslimited primarily to the superficial layers of the dorsal hornand surrounding the central canal in lamina X.

2.3. Prediction of G-protein coupling selectivity of GPR101

A knowledge-restricted hidden Markov model (HMM)-basedmethod, developed to predict GPCR G-protein couplingspecificity, was employed to predict the G-protein couplingselectivity of human and murine GPR101 (Sreekumar et al.,2004). The model predicted with high confidence that GPR101from both species belonged to the Gs class of GPCRs. E-valuesfor the match against Gs-specific HMMs are approximately 2orders of magnitude lower than that of the next best match(Fig. 6). In all 100 evaluations conducted, a Gs-specific HMMwas identified as the best match to the query sequences,further suggesting that GPR101 was not coupled to either Gαqor Gαi/o.

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2.4. Confirmation of Gs coupling by expression inmammalian and yeast cells

Expression plasmids encoding humanGPR101 or histamineH2receptor (H2R) were transiently transfected into HEK293 cellsthat stably maintained a CRE-Luc reporter gene activated byGs-coupled GPCRs. Under these conditions, H2R produced anelevated, agonist-independent luciferase response propor-tional to the amount of H2R expression plasmid transfected.The H2R-mediated luciferase response was further stimulatedby histamine or reversed by application of the inverse agonistcimetidine (Fig. 7A). Similarly, transfection of human GPR101produced an elevated, agonist-independent, luciferase res-ponse that was proportional to the amount of expressionplasmid transfected (Fig. 7B). Activation of the reporter gene inthis system is presumed to require an elevation in intracel-lular cAMP levels arising via Gs stimulation of adenyl cyclase.To confirm this, we directly measured cAMP levels aftertransfection of GPR101. Fig. 7C shows that intracellular cAMPlevels were increased in response to GPR101 transfection inamounts proportional to the amount of expression plasmidtransfected.

In an attempt to identify ligands that stimulate or inhibitGPR101, transfected cells were incubated with panels ofbiologically active compounds that included small molecules,peptides and bioactive lipids (data not shown). This approachfailed to identify selectively active compounds that furtherstimulated the GPR101-mediated luciferase response.

The predicted coupling of GPR101 to Gs was furtherexplored using a yeast-based GPCR expression system.Human GPR101 was introduced into yeast strains expressinga range of yeast Gα protein (Gpa1) chimeras each containingthe 5 C-terminal amino acids from mammalian Gα proteins.Expression of GPR101 in Gpa1-s5-containing cells elicited anelevated β-galactosidase response consistent with activationof the Gpa1-dependent MAP kinase signal transductionpathway by mass action (Fig. 8). No stimulation of β-galactosidase response over basal was detected whenhuman GPR101 was expressed in Gpa1-o5 or Gpa1-q5 cells.

Fig. 4 – In situ hybridization shows regional expression of Gpr10G–S, U–X) or sagittal (C, F) mouse brain sections or spinal cord (except in panel A where dark- and bright-field images are overdemonstrated in U–X where adjacent sections are hybridized wimages obtained using a 4× (C, D, H, J–N, P, R, and T) or 10× (A, BFilm autoradiograms of representative coronal sections are shoventricle; 7n, facial nerve; aca, anterior commissure anterior pahypothalamic area; AHi, amygdalohippocampal area; AP, area paqueduct (Sylvius); Arc, arcuate hypothalamic nucleus; BST, bedamygdaloid nucleus; CGPn, central gray of the pons; DM, dorsoperiaqueductal gray; Ent, entorhinal cortex; f, fornix; Gl, glomerolfactory bulb; ic, internal capsule; IGL, intergeniculate leaf; IRt, iLM, lateral mammillary nucleus; LSI, lateral septal nucleus intelateral ventricle; MePD, medial amygdaloid nucleus posterodorspart; Mi, mitral cell layer of the olfactory bulb; ML, medial mammmedial part; MPA, medial preoptic area; MPO, medial preoptic nparaventricular hypothalamic nucleus; PeF, perifornical nucleusnucleus caudal part; Pr, prepositus nucleus; Py, pyramidal cell lanucleus; SFO, subfornical organ; SO, supraoptic nucleus; SolM,geniculate nucleus; VLPAG, ventrolateral periaqueductal gray; V

Yeast cells expressing human GPR101 and Gpa1-s5 were usedto screen the same panels of biologically active compoundsused previously. Once again, no selectively active compoundswere identified (data not shown).

3. Discussion

In this report, we describe the cloning of the murineortholog of the human orphan GPCR, GPR101. The predictedamino acid sequences of the human and mouse proteins areapproximately 71% identical. Although relatively high, thisdegree of conservation prompted us to confirm the ortholo-gous relationship of these genes by independent means.Results of Southern blotting experiments (data not shown),bioinformatic database searches and chromosomal localiza-tion studies indicated that the mouse gene reported here is asingle copy, that there are no more highly related sequencesin the mouse genome and that the genes occupy syntenicchromosomal locations. Therefore, we are confident that thesequence reported here is the murine ortholog of humanGPR101.

Murine Gpr101 was expressed predominantly in the brainlike its human counterpart. Within the brain, expression wasseen in numerous limbic, autonomic and sensory areas. Thispatternof expression suggests thatGpr101and its ligandmightplay a neuromodulatory role in multiple, interrelatedpathways.

Expression in areas that receive and process internal andexternal sensory and humoral information, notably theolfactory bulb, subfornical organ, area postrema and nucleusof the solitary tract (NTS), puts Gpr101 in a position to affectthe response to numerous challenges to homeostasis andnovel stimuli. For example, the NTS is the entry point ofvisceral sensory information from the gustatory, gastrointes-tinal and cardiovascular systems. As such, Gpr101 couldmodulate NTS effects on satiety, body fluid homeostasis,gastric motility, blood pressure and heart rate (Blessing, 1997;Morton et al., 2005; Norgen, 1995).

1 in brain. In situ hybridization images of coronal (A, B, D, E,T) exposed to photographic emulsion. Images are dark fieldlaid to show details of the tissue. Probe specificity isith sense or anti-sense probes as indicated. Microscopic, E, F–G, I, O, Q, S, U–X) objective. Scale bars indicate 100 μm.wn in Y, Z and AA. Abbreviations 3V, 3rd ventricle; 4V, 4thrt; AcbSh, accumbens nucleus shell; AHA, anteriorostrema; APir, amygdalopiriform transition area; Aq,nucleus of the stria terminalis; CC, central canal; Ce, centralmedial hypothalamic nucleus; DMPAG, dorsomedialular layer of the olfactory bulb; GrO, granular layer of thentermediate reticular nucleus; LH, lateral hypothalamic area;rmediate part; LSV, lateral septal nucleus ventral part; LV,al part; MePV, medial amygdaloid nucleus posteroventralillary nucleus lateral part; MM, medial mammillary nucleusucleus; mt, mammillothalamic tract; opt, optic tract; Pa,; PH, posterior hypothalamic area; PnC, pontine reticularyer of the hippocampus; S, subiculum; SCh, suprachiasmaticnucleus of the solitary tract medial part; VLG, ventral lateralMH, ventromedial hypothalamic nucleus.

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Fig. 4 (continued).

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Perhaps the most prominent expression of Gpr101 wasfound in the hypothalamus. The hypothalamus is amajor regulator of the endocrine system, coordinatingresponses to internal and external sensory information aswell as information from limbic areas like the amygdala,septum and hippocampus. This broad array of activitiesimplicates Gpr101 in numerous hypothalamic roles suchas reproductive and ingestive behaviors as well as bloodpressure control and osmoregulation (Simerly, 1995). This

last function might be particularly likely given theexpression of Gpr101 in important osmoregulatory areasincluding the subfornical organ, median preoptic nucleus,area postrema, NTS, paraventricular nucleus and thesupraoptic nucleus (Denton et al., 1996; Oldfield andMcKinley, 1995).

The expression of Gpr101 in dopaminergic, serotonergicand noradrenergic nuclei is also intriguing. At present, it isnot known if the receptor is co-expressed with these

Fig. 5 – Gpr101 is expressed in dopaminergic, noradrenergic and serotonergic nuclei. In situ hybridization of Gpr101 (left) andimmunohistochemical (IHC) detection (right) of tyrosine hydroxylase (B, D) or tryptophan hydroxylase (F, H) in near-adjacentsections. Gpr101 expression is seen in dopaminergic nuclei (A; SNC, VTA), noradrenergic nuclei (C; LC) and serotonergic nuclei(E, G; DRVL, RPa, RMg). Microscopic images obtained using a 10× objective. Scale bars indicate 100 μm. Abbreviations: Aq,aqueduct (Sylvius); DRD, dorsal raphe nucleus dorsal part; DRV, dorsal raphe nucleus ventral part; DRVL, dorsal raphe nucleusventrolateral part; GiA, gigantocellular reticular nucleus alpha part; ml, medial lemniscus; LC, locus coeruleus; py, pyramidaltract; RMg, raphe magnus nucleus; ROb, raphe obscurus nucleus; RPa, raphe pallidus nucleus; SNC, substantia nigra compactpart; SNR, substantia nigra reticular part; VTA, ventral tegmental area.

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biogenic amines. However, its presence in these nucleisuggests that it could modulate some of the importanteffects, such as mood, motivational drive and attentionalstate, associated with them. Furthermore, numerous phar-macological agents modulate these aminergic systems toproduce desirable behavioral outcomes raising the possibi-lity that altering Gpr101 signaling might also have benefi-

cial effects in the areas of depression, anxiety or addiction(Sofuoglu and Kosten, 2005; Taylor et al., 2005).

Understanding the role of Gpr101 in the brain will begreatly facilitated by the identification of ligands that activateor inhibit its functions. However, given its limited similarity toknown receptors, identification of a natural ligand based onhomology alone is unlikely. Therefore, as a first step towards

Fig. 6 – G-protein coupling prediction. A radar plot ofE-values for matching the cytosolic residues of human andmouse GPR101 against G-protein coupling discriminativeknowledge-restricted HMMs is displayed. The radial distanceof a point from the center of the plot corresponds to theobserved E-values with each radial axis representing oneevaluation against the knowledge-restricted HMMs.

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designing an effective assay to identify biologically activeligands, wemade use of a knowledge-restricted HMMmodel topredict GPR101 G-protein coupling selectivity (Sreekumar etal., 2004). This methodwhich has been shown to have an errorrate of <1% predicted that both mouse and human GPR101were coupled to Gs.

Coupling of GPR101 to Gs was further suggested by theresults of reporter assays conducted in heterologous mam-malian- and yeast-based systems. In HEK293-CRE-Luc cells,dose-dependent expression of GPR101 produced a corres-ponding increase in luciferase activity. In this system,elevated luciferase levels are presumed to result from a risein intracellular cAMP mediated by Gs stimulation of adenylcyclase. This was confirmed by our direct observation of

Fig. 7 – GPR101 expression produced enhanced CRE-Lucresponse and increased intracellular cAMP levels. GPR101and histamine H2 receptors were transiently expressed inHEK293 CRE-Luc (A, B) or HEK293 cells (C) and assayed forinduced luciferase responses (A, B) or relative cAMP levels (C)as described in Experimental procedures. (A) Histamine H2receptor transfected (0.0125 μg expression plasmid per well;triplicate wells) cells were assayed in the absence of addedhistaminergic ligand (H2R) or with 10 μM histamine (Hist) orcimetidine (Cim). Empty vector (pcDNA) transfected cellswere assayed in the absence of added histaminergic ligand.Results displayed represent the mean of three independentexperiments. (B) HEK293 CRE-Luc cells transientlytransfectedwith GPR101 expression vector (open bars) or H2Rexpression vector (filled bars) in the indicated amounts wereassayed for agonist-independent-induced luciferase activity.(C) HEK293 cells transiently transfected with GPR101expression vector in the indicated amounts assayed forcAMP levels. Representative data from one of fourindependent experiments.

elevated intracellular cAMP levels after GPR101 transfection.Thus, these results are consistent with activation of a Gs-coupled signal transduction pathway by mass action due toincreasing amounts of GPR101 protein. In yeast, expression ofGPR101 in Gpa1-s5-containing cells elicited an elevated LacZreporter gene response indicative of mass action activation ofthe Gpa1-dependentMAP kinase signal transduction pathway.This result is also consistent with a GPR101 G-protein couplingpreference for Gαs and that the 5 C-terminal amino acids ofGαs play an important role in determining GPR101-G-proteincoupling selectivity. The yeast results agree with outcomesfrom the HMM and HEK293-CRE-Luc cell assays, both of whichimplicate Gαs as the preferred signaling partner of GPR101.

Screening panels of biologically active molecules using thedescribed reporter systems failed to identify GPR101 selec-tively active compounds. Although the assays used in thisstudy were validated for G-protein coupling and functionalresponse with a control Gs-coupled receptor (H2R), it remains

Fig. 8 – GPR101 expression in yeast cells expressingchimeric Gpa1-Gαs proteins. Yeast strains each expressingdistinct chimeric Gα proteins (Gpa1-s5, Gpa1-o5 andGpa1-q5) and containing either a GPR101 expression vector(pET189) or empty vector (p416GPD) were tested foragonist-independent β-galactosidase responses asdescribed in the Experimental procedures section.

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formally possible that they were not configured in a mannersuitable for detection of GPR101 agonists. In fact, the caveatthat such assays cannot be said to be truly suitable until anactive compound is identified is one of the substantialchallenges limiting the speedy identification of oGPCR ligands.Alternatively, our inability to identify active compounds maysimply reflect the incomplete representation of all naturaloGPCR ligands in the compound collections utilized in thisstudy. If so, then the most productive strategy for GPR101agonist identification would be to screen biological extracts.Therefore, the detailed expression data presented here shouldbe highly useful for selection of tissues likely to contain anatural agonist.

Taken together, our results suggest that Gpr101 is likely tomodulate numerous neurological pathways and have animpact on diverse behaviors. Furthermore, its strong expres-sion in the hypothalamus suggests that it might play amodulatory role in the neuroendocrine system. Future screen-ing efforts to identify surrogate or natural ligands will berequired in order to delineate the exact functions inwhich thisreceptor participates. Absent that, the creation of a geneticanimalmodelwould behighly useful to shed light onto the roleof this interesting receptor.

4. Experimental procedures

4.1. Mouse cDNA library screening

A cDNA librarywas constructed in pBS-KS (Stratagene, La Jolla,CA) frommousewhole brain polyA+ RNAusing the SuperscriptPlasmid System and the cDNA Synthesis and Cloning Kit(Invitrogen, Carlsbad, CA) following the manufacturer's proto-col. The plasmid library was mass-plated, and approximatelyone million colonies were transferred to nylon membranes.Membranes were probed with a 32P-random labeled, double-stranded cDNA probe which included the full-length open

reading frameof humanGPR101andhybridized for 20hat 60 °Cin a buffer containing 4× SSC, 5× Denhardt's, 1% SDS and100 μg/ml denatured salmon sperm DNA. Membranes werewashed twice in 2× SSC, 1% SDS at 60 °C for 20min and twice in0.1× SSC, 1% SDS at 60 °C for 30 min. Positive clones wereselected and their sequences determined.

4.2. Northern blot analysis

For Northern blot analysis, total RNAwas isolated frommousetissues using the RNeasy Midi kit (Qiagen, Valencia, CA) asspecified by the manufacturer. Ten micrograms of total RNAwas electrophoresed on formaldehyde/1.1% agarose gels andtransferred to Hybond-N+ nylon membranes (Amersham,Piscataway, NJ). In addition, a commercial Mouse MultipleTissue Northern (MTN) Blot (Clontech, Palto Alto, CA) wasprobed. Northern blots were hybridized in Quikhyb (Strata-gene, La Jolla, CA) for 2 h at 68 °C andwashed twicewith 2× SSCand 1% SDS at 65 °C for 20 min and twice with 0.1× SSC and 1%SDS at 65 °C for 30 min and exposed to X-ray film at −80 °C inthe presence of an intensifying screen. Random primed,double-stranded cDNA probes included the full-length openreading frame of murine Gpr101.

4.3. Chromosomal mapping

The chromosomal location of human and mouse GPR101 wasdetermined by fluorescent in situ hybridization (FISH) bytechnicians at SeeDNA Biotech Inc. (Windsor, Ontario,Canada) using standard procedures. Briefly, chromosomalspreads were made from isolated mouse lymphocytes andprobed with a biotinylated probe developed using the BioNickkit (Invitrogen, Carlsbad, CA). Hybridization and detection ofbound probe were carried out as described previously (Heng etal., 1992; Heng and Tsui, 1993). FISH signals and DAPI bandingimages were captured separately and superimposed todetermine chromosomal location.

4.4. In situ hybridization

All procedures involving animals were in strict accordancewith the National Institutes of Health Guidelines for the Care andUse of Laboratory Animals and approved by the InstitutionalAnimal Care and Use Committee. Male C57/Bl6 mice weredeeply anesthetized with Avertin and transcardially perfusedwith phosphate-buffered saline (PBS; pH 7.4) followed by 4%paraformaldehyde in PBS (pH 7.4). Tissues were removed andpost-fixed in 4% PFA for 4 to 8 h before transferring to 30%sucrose for cryoprotection. Tissues then were embedded inTissue Tek (Polysciences, PA, USA), and 10-μm-thick sectionswere cut on a cryostat. Sections were mounted onto SuperFrost/Plus slides and stored at −80 °C until use.

Linear DNA templates for in vitro synthesis of cRNA probeswere generated by polymerase chain reaction (PCR) from themurine Gpr101 cDNA using Gpr101-specific primers bearing T3or T7 RNA polymerase promoters. Sense or anti-sense probeswere transcribed using T3 or T7 RNA polymerases in thepresence of 35S-dUTP using the Riboprobe CombinationSystem (Promega, Madison, WI) following the manufacturer'sprotocol. Linear probe contained 53 nucleotides of coding

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sequence, the remainder comprised of 3′ untranslated mes-sage. In total, the probe encompassed nucleotides 1730–2528of the isolated cDNA. Gpr101-specific PCR primer sequencewas as follows: forward, 1730-5′-AGGGAGGTATTGAAGG-CAAG-3′-1749; reverse, 2528-5′-GGAGAAGGAGAGAAGAGA-GAG-3′-2508. Radioactive probes were purified by microSpinG-25 Columns (Amersham, Piscataway, NJ).

In situ hybridization was performed following standardprocedures. Briefly, sections were post-fixed in 4% PFA andacetylated with acetic anhydride. Hybridization was per-formed overnight at 55 °C with a probe concentration of50,000 cpm/μl in 50% formamide, 10 mM Tris–HCl, 200 μg/mltRNA, 1× Denhardt's solution, 10% dextran sulfate, 600 mMNaCl and 0.25% SDS. After hybridization, slides were treatedwith 40 μg/ml RNAseA for 30min at 37 °C thenwashed throughserial dilutions of SSC buffer. Two final high stringencywashesof 30 min each in 0.1× SSC at 65 °C were conducted.

Slides were exposed to film and then dipped in NTB2photographic emulsion (Kodak, NewHaven, CT). After 3 weeksof incubation at 4 °C, slides were developed for 2 min in D-19developer (Kodak), fixed for 5 min in Kodak fixer thencounterstained with cresyl violet, dehydrated and mounted.Slides hybridized with anti-sense probes were compared tothose with sense probe. Accumulation of silver grains abovesense background was qualitatively scored and assignedvalues of high (+++), medium (++) or low (+) relative expressionlevels. Determination of expressing brain regionswasmade bycomparing sections to the atlas of Paxinos and Franklin (2001)and was aided by acetycholinesterase and Nissl staining ofintervening sections.

4.5. G-protein coupling prediction

TMHMM 2.0 (Krogh et al., 2001) was used to predict theintracellular loops of human GPR101 and mouse Gpr101. Thepredicted cytosolic domains i1, i2, i3 and i4 were extracted andconcatenated as previously described (Sreekumar et al., 2004).The concatenated cytosolic residues were used as querysequence for ‘hmmpfam’ of the HMMER 2.2 package (Eddy,2001) to assess the match of a GPCR sequence against a set ofknowledge-restricted hidden Markov models (HMMs) thathave been shown to reliably predict GPCR/G-protein couplingspecificity (Sreekumar et al., 2004). Briefly, the set of knowl-edge-restricted HMMs consists of 100 different models foreach of the three classes of GPCRs viz. in Gi/o-, Gq/11- and Gs-specific, and hence the match between the query and themodels is assessed 100 times.

4.6. Construction of GPR101 expression vectors

In order to drive expression in tissue culture cells, the humanGPR101 protein-coding sequences were amplified using oligo-nucleotides that added BamHI and mammalian consensustranslational initiation sites to the 5′ end and an XhoI site tothe 3′ end. The fragments were cloned into correspondingsites in pCDNA3.1(+)neo (Invitrogen, Carlsbad, CA), producingplasmid pET120. The insert sequences were confirmed byautomated DNA sequencing. An expression plasmid encodinghuman H2R was constructed in similar fashion, yieldingpCDNA3.1-hH2R.

For expression in yeast, the human GPR101 protein-codingsequences were amplified using oligonucleotides that addBamHI and yeast consensus translational initiation sites to the5′ end and an XhoI site to the 3′ end. The fragment was clonedinto corresponding sites in the yeast expression vector,p416GPD (Mumberg et al., 1995), producing plasmid pET189.The sequences of the insert were confirmed by automatedDNA sequencing.

4.7. Mammalian-cell-based assay of GPR101 receptoractivity

HEK293-EBNA cells (Invitrogen, Carlsbad, CA) were co-trans-fected with the Mercury pCRE-Luc plasmid (Clontech, PaloAlto, CA) and pIRES-puro (Clontech, Palo Alto, CA) usingLipofectamine 2000 (Invitrogen, Carlsbad, CA) followed bydilution and clonal ring selection in puromycin-supplementedHEK293 EBNA medium containing 10% fetal bovine serum.Puromycin-selected HEK293 CRE-Luc cell clones were testedfor low-basal- and high-forskolin-induced CRE-Luc responses.Cell clones that produced the largest signal/basal responseupon forskolin stimulation were subsequently transientlytransfected with pCDNA3.1-human histamine H2 receptor(H2R) using Lipofectamine 2000 and tested for elevated basalCRE-Luc that was further stimulated by histamine. One stablecell line that produced reproducible elevated basal andhistamine induced CRE-Luc responses was designated HEK-CRE-Luc 2–48 and was selected for further analysis.

HEK-CRE-Luc 2–48 cells were transiently transfected in 24-well plates with human GPR101 or control H2R expressionplasmids by combining the receptor expression plasmid and acarrier plasmid (pCDNA3.1) with Lipofectamine 2000 andapplying the mixture to the cells for 4 h at 37 °C. The trans-fection complex mixture was then removed, fresh mediareplaced and cells cultured overnight. For H2R, appropriateagonist and antagonist ligands were added to the cellsfollowing overnight incubation and allowed to incubate for5 h. Induced luciferase activity was measured using theLuciferase Reporter Assay Kit (Clontech, Palo Alto, CA) accord-ing to the manufacturer's instructions. Briefly, cell lysis buffer(200 μl) was added to cell wells, aliquots removed (30 μl) andtransferred to a 96-well white opaque cell culture plate,luciferase substrates were added and light emission detectedusing a Dynex microtiter plate luminometer. Results wereplotted using GraphPad Prism.

Compounds contained in the LOPAC panels (Sigma-Aldrich, St. Louis, MO), Bioactive Lipid Library (BIOMOL,Plymouth Meeting, PA) and peptides (Bachem, Torrance, CA;Phoenix Pharmaceuticals, Belmont, CA) were tested foractivation of GPR101.

4.8. Yeast cell-based assay of GPR101 activity

The GPR101-yeast expression plasmid pET117 was introducedinto variants of MPY578fc cells (MATa ura3 his3 trp1 leu2 lys2ade2 far1::LYS2 fus1::FUS1-HIS3 sst2::SST2-G418R ste2::LEU2)(Pausch et al., 1998) using lithium acetate and selected foruracil prototrophy. In order to facilitate coupling of the GPR101receptor to G proteins,MPY578fc variant cells express chimericGpa1 proteins coupled to the mating signal transduction

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pathway. The chimeric constructs are expressed from theGPA1 locus andare composedofGpa1 sequences inwhich the 5C-terminal amino acids have been replaced with those ofmammalian Gα proteins (Erlenbach et al., 2001). Thus, thechimeric Gpa1 proteins containing the 5 C-terminal aminoacids from Gαs, Gαo and Gαq are referred to as Gpa1-s5, Gpa1-o5 and Gpa1-q5, respectively. A multicopy FUS1-LacZ reportergene plasmid, pMP283, was constructed by transferring a PstI–SalI fragment encoding the FUS1 promoter and FUS1-LacZfusion protein from pSL307 (McCaffrey et al., 1987) intocorresponding sites in pRS424 (Christianson et al., 1992). Theplasmidwas introduced intohumanGPR101-receptor-contain-ing cells and selected on media lacking trp and ura. Theresulting yeast strains were used for further analysis.

MPY578fc cells that express GPR101, chimeric Gα proteinand containing pMP283 were diluted in assay medium (SCD-ura-trp-his, pH 6.8, 25 mM PIPES, 1 mM 3-aminotriazole,0.16 mg/ml chlorophenyl red-β-D-galactopyranoside) anddispensed to the wells of 96-well microtiter dishes (5 × 105/ml, 200 μl/well). The plates were incubated with shaking(600 rpm) at 30 °C for 16 h after which absorbances at 570 nmwere measured using a Wallac Victor II plate reader. Assayswere conducted in quadruplicate, and results were plottedusing GraphPad Prism.

4.9. Measurement of cAMP levels

HEK293 cells were transiently transfected in 24-well plateswith human GPR101 using Lipofectamine 2000 and applyingthe mixture to the cells for 4 h at 37 °C. The transfectioncomplex mixture was then removed, fresh media replacedand cultured for 24 h. Induced cAMP activity was measuredusing the HitHunter Cyclic AMP XP CL kit (DiscoveRx, Fremont,CA) according to the manufacturer's instructions. Briefly, cellswere removed from their substrate and collected into Hanks'Balanced salt solution and spun down at low speed. The pelletwas resuspended in a solution containing anti-cAMP anti-body, and an aliquot was transferred to a 96-well microtiterplate. Detection reagent was added along with lysis solution,and cAMP activity was measured on a Victor-3 luminescentreader. Assays were conducted in triplicate, and at least fourindependent experiments were conducted.

Note added in proof

Themurine Gpr101 cDNA sequence described in this work hasbeen given the GenBank accession number DQ198079.

Acknowledgment

The authors wish to thank Roger Ford for help with thebioinformatic analysis.

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