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Anatomical Profiling of G Protein-Coupled Receptor Expression

Anatomical Profiling of G Protein-Coupled Receptor Expression
Anatomical Profiling of G Protein-Coupled Receptor Expression

Resource

Anatomical Pro?ling of

G Protein-Coupled Receptor Expression

Jean B.Regard,1,2,*Isaac T.Sato,1and Shaun R.Coughlin1,*

1Cardiovascular Research Institute,University of California,San Francisco,San Francisco,CA94158,USA

2Present address:National Institutes of Health,National Human Genome Research Institute,Building49,Room4C60,49Convent Dr., MSC4472,Bethesda,MD20892-4472,USA

*Correspondence:regardj@https://www.wendangku.net/doc/2a16346499.html,(J.B.R.),shaun.coughlin@https://www.wendangku.net/doc/2a16346499.html,(S.R.C.)

DOI10.1016/j.cell.2008.08.040

SUMMARY

G protein-coupled receptors(GPCRs)comprise the largest family of transmembrane signaling molecules and regulate a host of physiological and disease pro-cesses.To better understand the functions of GPCRs in vivo,we quanti?ed transcript levels of353nono-dorant GPCRs in41adult mouse tissues.Cluster analysis placed many GPCRs into anticipated ana-tomical and functional groups and predicted previ-ously unidenti?ed roles for less-studied receptors. From one such prediction,we showed that the Gpr91ligand succinate can regulate lipolysis in white adipose tissue,suggesting that signaling by this citric acid cycle intermediate may regulate energy homeo-stasis.We also showed that pairwise analysis of GPCR expression across tissues may help predict drug side effects.This resource will aid studies to un-derstand GPCR function in vivo and may assist in the identi?cation of therapeutic targets.

INTRODUCTION

Mammalian cells sense myriad signals in their environment via G protein-coupled receptors(GPCRs),the largest family of trans-membrane signaling molecules.GPCRs can be partitioned into two groups:odorant/sensory and nonodorant.Odorant/sensory receptors are restricted to specialized cells that detect external cues—odors,tastes,and pheromones—and regulate organis-mal behaviors such as feeding and mating.Nonodorant GPCRs are differentially expressed throughout the organism,respond to diverse endogenous ligands,and regulate a host of physiolog-ical processes including hematopoiesis,hemostasis,immune function,metabolism,neurotransmission,reproduction,cardiac function,and vascular tone.Accordingly,such receptors are the targets for about one-third of all approved drugs(Hopkins and Groom,2002;Muller,2000).Although in vivo roles have been de?ned for many of the approximately370nonodorant GPCRs in mice and humans,the expression and function of many such receptors are incompletely characterized,and a signi?cant fraction remain orphans(Fredriksson et al.,2003;Fre-driksson and Schioth,2005;Hill et al.,2002;Joost and Methner, 2002;Vassilatis et al.,2003).

To support studies of nonodorant GPCR function,we ana-lyzed the pattern of GPCR mRNA expression across tissues and the relative abundance for each of353nonodorant GPCRs in41tissues from adult mice.Hierarchical clustering analysis re-vealed groupings of tissues and receptors that predicted physi-ological functions for individual receptors and receptor clusters. We tested one such prediction by examining Gpr91,a receptor for the citric acid cycle intermediate succinate(He et al.,2004). Gpr91was grouped in the‘‘adipose cluster,’’but neither Gpr91 nor succinate was known to regulate adipocyte functions.We demonstrated that extracellular succinate can inhibit lipolysis in white adipose tissue in a manner consistent with its acting via adipocyte Gpr91.Overall,this data set provides a resource for those interested in?nding previously unidenti?ed roles for GPCRs with known ligands and hints regarding the functions of orphan GPCRs and the sources of their ligands.When com-pared with human expression data(SymAtlas,SAGEmap),these mouse data will aid the rational use of mice to model GPCR func-tion in human physiology and disease and may help point up new therapeutic targets and predict on-target side effects.In addi-tion,these quantitative data describing expression of a large number of related and relatively small genes across many tissues may support studies aimed at identifying cis-acting elements and transcription factors that dictate expression in particular tissues.

RESULTS

Tissue Pro?ling of GPCRs by qPCR

GPCRs are usually expressed at low levels.Indeed,nonodorant GPCRs comprise about1%of genes in the genome,but only 0.001%–0.01%of expressed sequence tags(ESTs)correspond to GPCRs(Fredriksson and Schioth,2005).Accordingly,we chose TaqMan-type quantitative real-time polymerase chain reaction(qPCR)for its high sensitivity,speci?city,and broad dy-namic range to measure GPCR mRNA expression.Primer/probe sets were validated as described in the Experimental Proce-dures;their sequences are provided in Table S3available online. Transcript levels for353GPCRs were pro?led in41adult tissues isolated from C57BL/6mice.Bar graphs indicating tran-script levels for each receptor in each tissue relative to internal controls(b-actin,cyclophilin,GAPDH,and ribosomal protein S9)are found in the Supplemental Data Set and at http://pdsp. https://www.wendangku.net/doc/2a16346499.html,/apGPCRe/.

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GPCR expression levels varied dramatically by tissue.Predict-ably,rhodopsin was the most abundantly expressed GPCR and among the most tissue speci?c,present at$350,000arbitrary units(a.u.)in eye but below25a.u.in other tissues(see below). Because rhodopsin presumably serves no function in extraocu-lar tissues,we adopted the convention that receptors with ex-pression values below25a.u.in a given tissue were‘‘absent.’’With this criterion,only25GPCRs were expressed above back-ground in all41tissues assayed(Table1);90were expressed in greater than half of tissues and238in less than half(Figure1A). Some ubiquitously expressed GPCRs were highly expressed in blood vessels[i.e.,Edg1(S1p1),F2r(Par1),Ednra,Ptger1], perhaps accounting for their presence in all tissues(Table1). Other ubiquitously expressed receptors,including Gpr56, Lec1(Lphn2),Lec2(Lphn1),Gpr107,Gpr108,Tm7sf1(Gpr137b), Tm7sf1l1(Gpr137),Tm7sf3,and Tpra40(Gpr175),were ex-pressed in?ve of?ve different nonvascular cell lines tested and may indeed be expressed by most cell types in vivo(Table1 and data not shown).

Save rhodopsin,nearly all GPCRs were expressed at levels below10,000a.u.To provide an overview of GPCR distribution, we designated receptors expressed at25–250,250–2500, and>2500a.u.as low,medium,and high expressors,respec-tively,and we grouped tissues by system(e.g.,central nervous system[CNS],endocrine,cardiovascular,pulmonary,metabolic, gastrointestinal,immune,reproductive,and cutaneous/barrier) (Figure1B).By these criteria,15or fewer receptors were ex-pressed at high level in any tissue group but CNS.

As expected,receptors that were highly expressed in a given tissue included receptors established to play an important role in that tissue(Table1).For example,light-detecting opsins were highly expressed in eye,and dopamine,gamma-aminobutyric acid(GABA),and glutamate receptors were highly expressed in CNS(Table1and Figure2A).In endocrine tissues,the extra-cellular calcium-sensing receptor(Casr),which regulates para-thyroid hormone secretion(Ho et al.,1995),was highly ex-pressed in parathyroid/thyroid;the growth hormone-releasing hormone receptor(Ghrhr),which regulates growth hormone se-cretion(Lin et al.,1993),was high in pituitary;and the glucagon-like peptide receptor1(Glp1r),which regulates insulin secretion (Scrocchi et al.,1996),was high in islets(Table1,Figure2B,and the Supplemental Data Set).The b3adrenergic receptor(Adrb3) and the niacin/ketone body receptor Hm74(Gpr109A)(Susulic et al.,1995;Tunaru et al.,2003),which regulate lipolysis,were among the most highly expressed receptors in adipose tissue (Table1and see below).Glucagon receptor(Gcgr),an important regulator of glucose homeostasis(Gelling et al.,2003),was highly expressed in liver,and the parathyroid hormone receptor (Pthr1),which regulates calcium and phosphate levels and modulates the activity of25-hydroxyvitamin D1-hydroxylase (Amizuka et al.,1997),was highly expressed in kidney(Table1 and Figure2C).In heart and blood vessels,angiotensin type1a re-ceptor(Agtr1),which regulates vascular tone(Ito et al.,1995;Su-gaya et al.,1995),thrombin receptor(F2r;Par1)and sphingosine-1-phosphate receptor(Edg1;S1p1),which play important roles in vascular development(Connolly et al.,1996;Liu et al.,2000),and the M2muscarinic acetylcholine receptor(Chrm2),which regu-lates heart rate(Gomeza et al.,1999)(Table1and Figure2D), were all highly https://www.wendangku.net/doc/2a16346499.html,r9,Cxcr4,and Il8rb(Cxcr2),which regulate leukocyte formation and function(Nagasawa et al., 1996;Shuster et al.,1995;Wurbel et al.,2001),were abundant in immune tissues(Table1).Taken together,these results pro-vided con?dence that high-level expression in speci?c tissues and tissue clusters correlates with physiological function and might predict roles for less-well-characterized receptors. Hierarchical Clustering of GPCR Expression

GPCR qPCR results were analyzed by unsupervised,hierarchical clustering to further explore potential relationships between re-ceptor expression and tissue function.The resulting

dendograms Table1.GPCRs Ubiquitously and Abundantly Expressed In Vivo

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for both the tissue and receptor axes showed functional clusters (Figure 3A for a thumbnail image and Figure S2for a full-size form).CNS tissues cerebellum,brainstem,hypothalamus,cere-bral cortex,hippocampus,striatum,olfactory bulb,retina,and whole eye clustered together,as did the immune/hematopoietic tissues spleen,thymus,and bone marrow.The steroidogenic or-gans adrenal gland and ovary clustered,but testes showed a very distinct pattern of GPCR expression.Liver,kidney,and gall blad-der formed a group,as did large intestine,small intestine,pan-creas,and stomach.Skin,esophagus,and tongue also formed a cluster,perhaps related to their common barrier function.Car-

diac atrium and ventricle,skeletal muscle,aorta,and urinary bladder formed a cluster,perhaps in part because of their sharing a relative abundance of muscle cells.Brown adipose tissue (BAT),white adipose tissue (WAT),isolated adipocytes,and vena cava formed an ‘‘adipose’’cluster.The presence of vena cava in this cluster likely represents the incomplete removal of surrounding fat from the samples.

A number of receptor axis clusters were easily recognized (Fig-ure 3).A portion of the ‘‘immune/hematopoietic’’cluster is shown in Figure 3B.Included are Ccr9,Cxcr6,Cxcr4,Ccr3,Ccxcr1(Xcr1),Blr1,Pgr16(Emr4),Gpr33,Ccr6,Gpr65(Tdag8),Cnr2,Edg6(S1p4),Gpr9(Cxcr3),Ccr7,Fksg79(Gpr174),G2a(Gpr132),P2Y10,Pgr27(Gpr114),Ebi2,Gpr18,and H963(Gpr171).Most of these receptors are known to be expressed in and/or to play a role in im-mune cells (Birkenbach et al.,1993;Forster et al.,1996,1999;Hum-bles et al.,2004;Karsak et al.,2007;Kim et al.,2001;Le et al.,2001;Malone et al.,2004;Nagasawa et al.,1996;Rao et al.,1999;Soto et al.,1998;Stacey et al.,2002;Varona et al.,2001;Wurbel et al.,2001),but no role for Fksg79(Gpr174),Pgr27(Gpr114),and H963(Gpr171)in this context has been described.

The pituitary cluster (Figure 3C)includes Bdkrb2,Gpr30(Gper),Ghrhr,Gnrhr,Drd3,Mc3r,Sstr5,Gpr2(Ccr10),and Hcrtr1(Ox1r);among these,Gpr2(Ccr10)has not been previously implicated in pituitary gland function (Brailoiu et al.,2007;Date et al.,2000;Kumar et al.,1997;Lin et al.,1993;Lorsignol et al.,1999;Tsut-sumi et al.,1992).

The CNS cluster was by far the largest.Our analysis (Figure 1B and Figure 3),as well as others (Vassilatis et al.,2003),suggests that more than 80%of all nonodorant GPCRs are expressed in CNS.In Figure 3D,we show a small portion of the CNS cluster that includes Gpr101,Hcrtr2(Ox2r),Oprk1,Gpr83,Ntsr1,Gpr45,Htr1a,Htr7,Oprm1,and Npy5r,all of which have been implicated in regulation of neuronal function (Bates et al.,2006;Filliol et al.,2000;Lovenberg et al.,1993;Marsh et al.,1998;Mazella et al.,1996;Popova et al.,2007;Simonin et al.,1995;Willie et al.,2003).Finally,Figure 3E shows the ‘‘eye/retina’’cluster,which contains Glp2r,Drd4,Grm6(Mglur6),Opn1mw,Opn1sw,Pgr5(Gpr152),Rho,Rrh,Vlgr1(Gpr98),Oa1(Gpr143),and Rgr.Save Glp2r and Pgr5(Gpr152),all are known to function in the eye (Chen et al.,2001;Chiu et al.,1994;Cohen et al.,1992;Dryja et al.,2005;Incerti et al.,2000;McGee et al.,2006;Nathans and Hogness,1983;Sun et al.,1997a,1997b ).

Taken together,the results outlined above reveal that tissues cluster into largely expected functional groups purely on the basis of their GPCR repertoires,and both expected and unique groups of receptors cluster by tissue function.These data identify sets of receptors involved in speci?c aspects of physiology and should prove useful in providing clues regarding in vivo roles for orphan GPCRs and new roles for receptors with known ligands.To test the latter prediction,we sought a possible role for a recently deorphanized receptor,Gpr91,found in the adipose cluster.Extracellular Succinate Inhibits Lipolysis,Likely via Gpr91

Thirteen GPCRs de?ned an adipose cluster (Figure 4A and cir-cled area in Figure 3A):Gpr64(He6),Hm74(Gpr109a),Adrb3,Gpr81,Pgr4(Gpr120),Gpr23(Lpa4),Tshr,Opn3,Oxtr,Sctr,Gpr91(Suncr1),Pthr1,and Ptger3(Ep3).Ligands for most

of

Figure 1.Distribution of Mouse GPCR mRNA Expression In Vivo

(A)The number of GPCRs expressed in tissues presented in pie chart form.Receptors expressed in all 41tissues assayed are ‘‘ubiquitous’’;receptors expressed in more than or less than half are ‘‘widespread’’or ‘‘restricted,’’respectively.A list of the ‘‘ubiquitous’’receptors is included in Table 1.

(B)GPCR expression by tissue systems.High,medium,low,and absent are de?ned in the Results .Tissue systems are de?ned as central nervous system (CNS:cerebellum,brainstem,hypothalamus,cerebral cortex,hippocampus,striatum,olfactory bulb,olfactory epithelium,retina,whole eye),endocrine (pituitary gland,islets of Langerhans,adrenal gland,thyroid/parathyroid),car-diovascular (aorta,vena cava,heart atrium,heart ventricle),pulmonary (lung,trachea),metabolic (brown adipose tissue,white adipose tissue,isolated ad-ipocytes,liver,skeletal muscle,kidney),gastroenteric (pancreas,gall bladder,large intestine,small intestine,stomach,urinary bladder),reproductive (ovary,testes,uterus),barrier (tongue,esophagus,skin),and immune (spleen,thy-mus,bone marrow).A list of the highly expressed GPCRs in individual tissue systems is included in Table 1.

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these receptors are known to affect adipocyte function (Butcher and Carlson,1970;Gotoh et al.,2007;Moskowitz and Fain,1969;Rodbell,1964;Sinha et al.,1976;Susulic et al.,1995;Tunaru

et al.,2003;Valet et al.,1998),but,to our knowledge,no role for Gpr64(He6),Gpr81,Opn3,or Gpr91(Suncr1)in adipose tissue has been reported.He et al.demonstrated that the citric acid

cycle

Figure 2.qRT-PCR Tissue Distribution Yields Predicted Patterns of Expression

Representative GPCRs that were highly expressed in distinct tissue systems were selected from Figure 1B and Table 1to illustrate tissue speci?city,range of expression levels,and reproducibility.Examples are shown for the following:CNS:metabotropic glutamate receptor 1[Grm1(Mglur1)]and rhodopsin [Rho](A);endocrine system:growth hormone releasing hormone receptor [Ghrhr]and the extracellular calcium-sensing receptor [Casr](B);metabolic tissues:parathyroid hormone receptor 1[Pthr1]and glucagon receptor [Gcgr](C);and cardiovascular system:M2muscarinic receptor [Chrm2]and the sphingosine-1-phosphate receptor 1[Edg1(S1p1)](D).Values are plotted as the mean ±SEM;n =2–5.

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intermediate succinate can activate Gpr91and that Gpr91,pre-sumably in kidney,mediates elevation of plasma renin levels and blood pressure in response to exogenous succinate (He et al.,2004).Like Adrb3and Hm74(Gpr109a),Gpr91(Suncr1)mRNA was most highly expressed in white adipose tissue (WAT)and was abundant in puri?ed adipocytes (Figure 4B).Gpr91is at least partially G i coupled (He et al.,2004),and G i -coupled GPCRs are known to inhibit lipolysis in adipose tissues (Moreno et al.,1983).Accordingly,we examined the effect of succinate on isoprotere-nol-induced lipolysis in isolated WAT (Isoproterenol has relatively low af?nity for Adrb3and most likely acts via Adrb2in this context).Succinate inhibited lipolysis in a dose-dependent manner with an apparent IC 50of 44m M (Figure 4C),a concentration similar to the EC 50for succinate activation of Gpr91heterologously ex-pressed in 293cells (He et al.,2004).Inhibition of lipolysis by succinate was ablated by pertussis toxin pretreatment.3T3-L1adipocyte-like cells,which do not express Gpr91mRNA

(J.B.R.

Figure 3.Unsupervised Hierarchical Clus-tering of GPCR Expression across Tissues

(A)Transformed qRT-PCR data for the 353GPCRs assayed in the 41tissues was evaluated by unsu-pervised hierarchical clustering with average linkage with Cluster 3.0and visualized with Java TreeView (see the Experimental Procedures ).A thumbnail image is shown here;a full-size version is shown in Figure S2.Multiple clusters and subclusters were seen,and ?ve were chosen for further analysis.The ?fth cluster (blue circle to-ward the bottom)is discussed in Figure 4.

(B)A portion of the ‘‘immune/hematopoietic’’clus-ter is shown;note the abundance of chemokine receptors.

(C)The ‘‘pituitary’’cluster contains many well-documented regulators of pituitary function,including Ghrhr and Gnrhr.

(D)A small portion of the ‘‘CNS’’cluster,by far the largest.This portion contains receptors for impor-tant neurotransmitters including serotonin,neuro-peptide Y,orexin and opiates.

(E)The ‘‘eye/retinal’’cluster contains light-sensing opsins as well as other receptors known to regu-late vision.

and S.R.C.,unpublished data)and were not sensitive to the antilipolytic effects of succinate,became succinate sensi-tive when transfected with a mammalian expression vector for GPR91(Figure 4E).These data strongly suggest that succi-nate inhibits lipolysis in WAT via a G i -coupled GPCR,presumably Gpr91.

Pairwise Analysis of GPCR

Coexpression May Help Identify Multiple Roles for Individual GPCRs To explore whether coexpression of GPCRs might provide additional clues to function,we compared the expression pattern of each GPCR to that of every

other to generate the map shown in Figure 5.Positive and neg-ative correlation of expression patterns are indicated by yellow and blue colors,respectively (Figure 5A and Figure S3).Such correlation maps are a powerful tool for organizing and analyzing gene-gene and protein-protein interactions on a global scale (Collins et al.,2007a,2007b;Krogan et al.,2006;Schuldiner et al.,2005;Segre et al.,2005).

Not surprisingly,tissue-speci?c receptor clusters similar to those seen in Figure 3were,for the most part,recapitulated as distinct blocks along the diagonal.Examples are shown in Fig-ures 5B,5C,and 5D which largely reproduce the immune/hema-topoietic,eye/retina,and adipose clusters of Figures 3B,3E,and 4A,respectively.Off-diagonal blocks drew attention to possible roles for receptors in other contexts.By pointing out possible roles for receptors outside of their main physiological cluster,such analysis may be useful in understanding and predicting on-target drug side effects (see the Discussion ).

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Figure4.Gpr91(Sucnr1)and Its Ligand,Succinate,can Inhibit Lipolysis in Adipocytes

(A)The‘‘adipose’’cluster(blue circle,lower part Figure3A)contained numerous regulators of adipocyte function.

(B)Adrb3,Hm74(Gpr109a)and Gpr91(Sucnr1)were expressed at similarly high levels in white adipose tissues(WAT)and isolated adipocytes.

(C)Isolated WAT was treated with isoproterenol(20nM)to stimulate lipolysis,as measured by glycerol release at3hr.Succinate added concurrently with isoproterenol inhibited lipolysis in a concentration-dependent manner with an apparent IC50of approximately44m M.

(D)Isolated WAT was pretreated either with KRH/BSA or KRH/BSA containing pertussis toxin(PTX;100ng/mL)for3hr prior to exposure to the indicated con-ditions.Isoproterenol(20nM)-stimulated glycerol release was inhibited by the addition of80m M succinate.Pretreatment of WAT with PTX abrogated this effect, suggesting succinate’s inhibition of lipolysis occurred in a G i/o-dependent manner,consistent with Gpr91activation.

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DISCUSSION

We have quantitated mRNA levels for the nonodorant G protein-coupled receptors encoded in the mouse genome in 41tissues and provide this data set as a resource for predicting roles for incompletely characterized GPCRs,exploring tissue-speci?c gene expression,and other purposes.The fact that tissues that comprise classical physiological systems (cardiovascular,gas-trointestinal,etc.)were clustered together simply on the basis of their GPCR repertoires speaks to the key roles that GPCRs play in homeostatic regulation.

Our anatomic expression pro?ling yielded a large amount of in-formation consistent with known physiology,and high-level ex-pression of a GPCR in a particular tissue cluster or speci?c tissue correlated well with its physiological role.Although this result is not surprising,it does provide con?dence that roles for orphan receptors or GPCRs not known to play a role in a particular phys-iological process might be predicted by presence in a given clus-ter.Our demonstration that Gpr91expression pointed to a role for extracellular succinate in regulating lipolysis in adipocytes validates this notion and is also of intrinsic interest.

The concentration of succinate in plasma has been reported at 5–125m M,a range that surrounds the EC 50for Gpr91activation (He et al.,2004)and the IC 50for inhibition of lipolysis in WAT (Fig-ure 4).Succinate concentrations increase during exercise and metabolic acidosis and,in rodents,in hyperglycemic metabolic states (Forni et al.,2005;Hochachka and Dressendorfer,1976;Krebs,1950;Kushnir et al.,2001;Nordmann and Nordmann,1961;Sadagopan et al.,2007).Thus,excursions in the levels of extracellular succinate do occur and might regulate adipocyte function in vivo.Adipocyte function was not investigated in mice lacking Gpr91,which are grossly healthy (He et al.,2004).Overall,a physiological role for succinate in regulating adipocyte metabolism is plausible,but when and how such a system might be important and/or redundant with other systems that govern adipocyte function remains unknown.

Forty-nine of the 353GPCRs pro?led were expressed in only one or two of the 41tissues examined (see Table S1).Such con-?ned expression might point out targets of pharmaceutical inter-est.For example,testes showed a GPCR expression pattern very distinct from that of other tissues.Gpr150,Gpr66,Gpr15,Mtnr1a,and Pgr23were almost perfectly speci?c to testes.Whether such receptors play a role in spermatogenesis or other testicular functions and their potential utility as targets for drugs aimed at controlling fertility is unknown.

A comparison of each receptor’s expression pattern with that of every other (Figure 5)provided a means of pointing out possible roles for a given receptor outside its main physiological cluster.

(E)Differentiated 3T3-L1cells,which do not express Gpr91mRNA,were transfected with mammalian expression vectors containing b 2adrenergic receptor (Adrb2)and control (green ?uorescent protein;GFP)or Adrb2and GPR91.Succinate (100m M)inhibited isoproterenol (10nM)-induced lipolysis in cells cotrans-fected with GPR91but not GFP.The data shown are mean ±SD,n =3;p =0.025,unpaired student’s t test.This experiment was done three times with similar

results.

Figure 5.Hierarchical-Cluster Analysis of Pairwise GPCR Expression Reveals Addi-tional Levels of Interaction

(A)Pearson correlation r coef?cients were calcu-lated for interactions between each GPCR with all others on the basis of tissue expression pat-terns.The resulting data set was further analyzed with Cluster 3.0with complete linkage and visual-ized with TreeView.A thumbnail image is shown here,and a full-size image is available in Fig-ure S3.Receptors with similar distributions are shown in yellow;distinct distributions are shown in blue.The x and y axes are mirror images of one another.The diagonal represents each receptor interacting with itself (perfect similarity in distribution).

(B–D)Clustering of receptors by similarity of expression reveals an immune/hematopoietic grouping very similar to that in Figure 3B (B),an eye/retinal cluster similar to that in Figure 3E (C),and an adipose cluster similar to that in Figure 4A (D).However,analysis of GPCR inter-action clusters off the diagonal suggested recep-tor functions outside of their most obvious phys-iological roles.This might aid in understanding and prediction of on-target drug side effects (see the Discussion and Figure S1).

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Hm74(Gpr109a),the ketone body receptor that is activated ther-apeutically by niacin to treat dyslipidemias(Soga et al.,2003;Tu-naru et al.,2003;Wise et al.,2003),provides an interesting exam-ple.By traditional clustering analysis,Hm74(Gpr109a)is placed in the‘‘adipose’’cluster(Figure4A),and activation of adipocyte Hm74likely mediates the antilipolytic actions of niacin(Tunaru et al.,2003).It was recently shown that the skin-?ushing side ef-fect of niacin(Carlson,2005)is mediated by Hm74expression by bone-marrow-derived epidermal Langerhans cells that release of vasodilatory prostanoids(Benyo et al.,2006;Benyo et al.,2005). By quantitative pro?ling across tissues,Hm74was noted to be expressed relatively highly in skin and other‘‘barrier-cluster’’tis-sues as well as adipose(Figure4B).By expression correlation analysis,Hm74was not found with the adipose cluster on the diagonal but instead clustered with receptors with more wide-spread expression(Figures S1and S3).A search of off-diagonal interactions revealed that Hm74interacts not only with the adi-pose cluster but also with receptors in both immune and barrier clusters(Figure S1and S3).Thus,analysis of GPCR expression data from these different perspectives may generate hypotheses regarding on-target side effects of drugs.

GPCR genes are usually relatively small,often intronless,and range from closely to distantly related.These features,plus the availability of quantitative expression data across multiple tis-sues for hundreds of related genes that show clusters based on shared tissue-speci?c patterns,may provide a resource for those interested in identifying the combinations of cis-acting elements that specify gene expression in a given cell type. Some GPCRs are thought to function as heterodimers,and cluster analysis for coexpression might point to potential recep-tor pairs.For example,Gabbr1is currently thought to function as an obligate heterodimer with Gpr51(Gabbr2)(Jones et al.,1998; Kaupmann et al.,1998;Kuner et al.,1999;White et al.,1998).Our data(Supplemental Data Set)and those of others(Calver et al., 2000)demonstrate similar expression patterns for these recep-tors in CNS and divergent expression in the periphery.These re-sults are consistent with Gabbr1and Gabbr2heterodimer forma-tion in the CNS but raise the possibility that in the periphery, expression of one or the other partner is regulated,that these re-ceptors may use other partners,and/or that heterodimerization may not be required in all settings(Cheng et al.,2007). Several caveats should be stated regarding interpreting our expression data.(1)Relative mRNA levels,of course,will not always re?ect relative protein expression levels or the relative importance of a particular receptor in a particular tissue.Indeed, some important receptors such as adrenergic receptors were expressed at relatively low levels.(2)Tissues are comprised of multiple cell types,and receptor expression can be restricted to a minority cell type.In the extreme,receptor expression in a minority population can be missed by whole-tissue analysis. For example,neither our qPCR data(Supplemental Data Set) nor those of others(Liberles and Buck,2006)detected trace amine associated receptor1(Taar1)expression in brain,but a Taar1Lac-Z knockin mouse revealed Taar1expression in dis-crete neuronal populations and Taar1-dependent regulation of dopaminergic activity(Lindemann et al.,2008).(3)The presence of a receptor within a particular anatomical cluster does not exclude important functions in tissues outside that cluster.For example,the D2dopamine receptor(Drd2)is recognized as the major dopamine receptor subtype in pituitary(Kelly et al., 1997;Saiardi et al.,1997),but Drd2was in the CNS cluster, whereas the D3dopamine receptor(Drd3)was in the pituitary cluster(Figure3C).The cluster analysis presented here used the Pearson correlation,which normalizes expression levels (see the Experimental Procedures)to focus on gaining informa-tion from the pattern of gene expression at the cost of ignoring absolute levels of receptor expression,and the Drd cluster re-sults are presumably a result of the fact that Drd2is expressed at moderate levels in numerous CNS structures whereas Drd3 is expressed at very low levels in only a few structures(Supple-mental Data Set).However,our quantitative expression data (Supplemental Data Set)reveal that Drd2is expressed approxi-mately100-fold higher than Drd3in pituitary,consistent with the dominant role of Drd2in pituitary function.Thus,this resource is best utilized when the data are analyzed from several perspec-tives and should be viewed as a means of generating hypotheses to be tested experimentally.Raw expression data for the353 GPCRs in the41organ samples are available at http://pdsp. https://www.wendangku.net/doc/2a16346499.html,/apGPCRe/,for those who wish to perform their own analyses.

Lastly,expression of individual GPCRs in speci?c tissues can be different in human and mouse.When used together with GPCR expression patterns in human(SymAtlas,SAGEmap), our data set should facilitate rational use of mouse to model the roles of GPCRs in human physiology and disease. EXPERIMENTAL PROCEDURES

qRT-PCR

Ten-week-old C56BL/6mice(Jackson Labs)were housed in University of California,San Francisco(UCSF)animal facilities for2weeks prior to organ harvest.Mice were anesthetized with ketamine/xylazine and transcardially-perfused with saline for the removal of https://www.wendangku.net/doc/2a16346499.html,ans were dissected,rapidly frozen in liquid nitrogen,and stored atà80 C until the time of RNA isolation. Corresponding organs from two male and two female mice were pooled to yield a single tissue RNA sample(i.e.,for each separate replicate,organs of four mice were pooled),with the exception of sex-speci?c organs,which were pooled from two mice.Pancreas was removed and stored for24hr in RNAlater at4 C prior to storage atà80 C.Islets of Langerhans were isolated from20male C57BL/6males by the UCSF islet isolation core.Primary adipo-cytes were isolated from sex organ fat pads as described(Rodbell,1964).Tis-sues were?rst homogenized,and crude RNA was extracted with Trizol(Invitro-gen).RNA was further puri?ed with RNeasy columns(QIAGEN)with on-column DNase I digestion.RNA samples were treated with DNase I a second time and concentrated with the Zymo Research DNA-free RNA kit.First-strand synthe-sis was performed with the iScript kit(Biorad).qPCR assays were performed in a384-well format with an ABI7900HT,Platinum qPCR mix(Invitrogen)and 10m l reactions.Cycle threshold(Ct)values were collected at0.2for all sam-ples;Ct values for individual GPCRs were compared to Ct values for four inter-nal controls(b-actin,cyclophilin,GAPDH,and ribosomal protein S9)for all tis-sues.Taqman primer/probes have been described(Regard et al.,2007),and sequences are available in Table S3.There were22mouse GPCRs for which we were unable to generate primer/probe sets that produced a signal above background in any tissue.A lack of data should be viewed as‘‘no data’’for these receptors,which are listed in Table S2.PCR ef?ciencies were calculated as described(Peirson et al.,2003).2(CtGPCRàCtcontrol)multiplied by215was used for graphical representation of qPCR data.Repeated qPCR analysis of a given tissue RNA sample,i.e.,technical replicates,yielded variations of less than10%.Two to?ve independently prepared RNA samples from sepa-rate tissue isolations were analyzed for assessment of in vivo expression

568Cell135,561–571,October31,2008a2008Elsevier Inc.

variability,which was also generally less than10%.Data presented in the Supplemental Data Set and elsewhere represent the mean±SEM(n=2–5).

Lipolysis

C57BL6mice were euthanized by cervical dislocation.Sex organ fat pads were dissected and?nely minced and washed four times with Krebs-Ringer HEPES buffer containing4%fatty-acid-free bovine serum albumin(BSA),5mM glu-cose,and0.1mM ascorbic acid(KRH-BSA).Twenty-?ve to30milligrams of WAT mincate was placed in the upper well of transwells in a24-well plate and placed in a tissue culture incubator at37 C with the speci?ed cocktail for the indicated amount of time.KRH-BSA samples were taken from the wells at the speci?ed time for quanti?cation of glycerol release and were divided by the weight of the mincate.Pertussis toxin(Calbiochem)pretreatment was per-formed for4hr in a tissue culture incubator with mild shaking.3T3-L1cells (ATCC)were cultured and differentiated via standard protocols.Fully differen-tiated3T3-L1cells were transfected(Amaxa,Nucleofection)with mammalian expression vectors containing b2-adrenergic receptor(Adrb2—generously provided by Dr.J.Silvio Gutkind)and GPR91(Missouri S&T)and allowed to recover for48–72hr prior to lipolysis assays.Free glycerol was quanti?ed with the Free Glycerol reagent(Sigma).IC50for succinate inhibition of isopro-terenol-induced lipolysis was calculated with Prism4.

Hierarchical Clustering

All analysis of the data was performed with Python,Scipy,and Excel.For pair-wise correlation in Figure5,Pearson correlation r values were computed between the expression vectors of every pair of genes in the data set.Trans-formed data were hierarchically clustered with Cluster3.0(Eisen et al.,1998) and visualized with Java TreeView(Saldanha,2004).

SUPPLEMENTAL DATA

Supplemental Data include three?gures,three tables,and one data set and can be found with this article online at https://www.wendangku.net/doc/2a16346499.html,/supplemental/ S0092-8674(08)01129-X.

ACKNOWLEDGMENTS

We thank the Coughlin lab for valuable discussions,Ivo Cornelissen,Gerard Honig,and Grant Li,for technical assistance in isolating mouse tissues,and Stuart Peirson for sharing qPCR ef?ciency transformations.We also thank Dale Webster and Joseph Derisi for help with informatics,Silvio Gutkind for providing the Adrb2construct,and Bryan Roth for critical reading of the man-uscript.This work was supported by the National Institutes of Health(S.R.C.) and Sandler Family Foundation(J.B.R.).

Received:January29,2008

Revised:June27,2008

Accepted:August28,2008

Published:October30,2008

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