A family of candidate taste receptors

26.Stoner,D.S.,Rinkevich,B.&Weissman,I.L.Heritable germ and somatic cell lineage competitions inchimeric colonial protochordates.Proc.Natl A96,9148±9153(1999).27.Turner,P.E.&Chao,L.Prisoner's dilemma in an RNA virus.Nature398,441±443(1999).28.Hodgkin,J.&Kaiser,D.Cell-to-cell stimulation of motility in nonmotile mutants of M.xanthus.Proc.Natl A74,2938±2942(1977).29.Wall,D.,Kolenbrander,P.E.&Kaiser,D.The Myxococcus xanthus pilQ(sglA)gene encodes a secretinhomolog required for type IV pilus biogenesis,social motility and development.J.Bacteriol.181,24±33(1999).30.Kroos,L.,Kuspa,A.&Kaiser,D.A global analysis of developmentally regulated genes in Myxococcusxanthus.Dev.Biol.117,252±266(1986).AcknowledgementsWe thank A.Garza,L.Shimkets,M.Singer and J.Strassman for helpful discussion, A.Garza for providing strain MS2021,and N.Hajela and J.Jiang for technical assistance. This research was supported by Michigan State University and an NSF grant to R.E.L. Correspondence and requests for materials should be addressed to G.J.V.(e-mail:velicerg@)..................................................................A family of candidate taste receptors in human and mouseHiroaki Matsunami*,Jean-Pierre Montmayeur*&Linda B.BuckHoward Hughes Medical Institute,Department of Neurobiology,Harvard Medical School,Boston,Massachusetts02115,USA*These authors contributed equally to this work .............................................................................................................................................. The gustatory system of mammals can sense four basic taste qualities,bitter,sweet,salty and sour,as well as umami,the taste of glutamate1±6.Previous studies suggested that the detection of bitter and sweet tastants by taste receptor cells in the mouth is likely to involve G-protein-coupled receptors2,7,8.Although two putative G-protein-coupled bitter/sweet taste receptors have been identi®ed9,the chemical diversity of bitter and sweet compounds leads one to expect that there is a larger number of different receptors8,10,11.Here we report the identi®cation of a family of candidate taste receptors(the TRBs)that are members of the G-protein-coupled receptor superfamily and that are speci®cally expressed by taste receptor cells.A cluster of genes encoding human TRBs is located adjacent to a Prp gene locus12,which in mouse is tightly linked to the SOA genetic locus that is involved in detecting the bitter compound sucrose octaacetate13±15.Another TRB gene is found on a human contig assigned to chromosome 5p15,the location of a genetic locus(PROP)that controls the detection of the bitter compound6-n-propyl-2-thiouracil in humans16,17.To search for taste receptors,we devised a strategy that was based on four ideas:®rst,taste receptors would be encoded by a family of related genes;second,some taste receptor genes would be found at genetic loci associated with the ability to taste speci®c compounds in mouse or human;third,taste receptors would be G-protein-coupled receptors(GPCRs)that have limited sequence similarity to other members of the GPCR superfamily;and last,taste receptor genes might be found by using the resources of the Human Genome Project to look for GPCR-encoding genes in genomic regions implicated in taste perception.We®rst asked whether there are genes encoding new GPCRs in the region of the human genome corresponding to the mouse SOA locus,which is tightly linked to a Prp gene13,ing the Jackson Laboratory Mouse Genome Informatics website (),we determined that the Prp gene and the SOA locus are located on mouse chromosome6 (63.6centimorgans(cM))and that the syntenic region in human is on chromosome12p13.We then determined whether any of the genes that mapped close to the mouse SOA locus had been cloned in human and were deposited in the National Center for Biotech-nology Information(NCBI)nr database();we used the sequences of those genes to search the NCBI Human Genome Sequence(HGS)database,focusing on human chromosome12.Among the genes we used for this search was a Prp gene,which we found on the chromosome-12contig NT_001856. By examining the contig map of chromosome12in the NCBI HGS database,we were able to identify contigs that¯anked NT_001856. This provided us with a focus set of contigs that might contain taste receptor genes.To®nd genes encoding GPCRs in this focus region,we®rst searched the human chromosome12database with large sets of GPCR protein sequences that we compiled from a GPCR database (/7tm).Although we identi®ed a few genes on human chromosome12that appeared to encode GPCRs,none was located in the focus set of contigs or their vicinity.We then searched the database with a member of the V1R family of candidate pheromone receptors(V1R5)18,19,because members of this family are not in the GPCR database and therefore had not been included in our GPCR sequence sets.We identi®ed two sequence stretches in contig NT_001856(which contains the Prp gene)encoding protein sequences distantly related to V1R5.When we retrieved the DNA sequence in and around one of these DNA regions and translated it, we determined that it contained an intronless gene encoding a putative receptor protein of311amino acids(hTRB2(for human taste receptor,family B,no.2)with weak homology to V1R5(Fig.1). We then asked whether hTRB2belongs to a family of related receptors,as we expected would be the case for taste ing hTRB2to search the chromosome12database,we identi®ed eight related genes,all in the NT_001856contig.Of the eight TRB genes, six encode receptors related to hTRB2(Fig.1)and two are ing these TRBs,we were unable to®nd any members of this family in either the NCBI nr or expressed sequence tag databases,consistent with the idea that these receptors might be expressed exclusively in taste tissue.However,we did identify a gene encoding a TRB family member(hTRB7,Fig.1)in a contig assigned to human chromosome5p15,the location of PROP,the genetic locus that governs the ability of humans to taste6-n-propyl-2-thiouracil,a bitter compound16.We also found a total of®ve TRB genes(one a pseudogene)on three chromosome-7contigs,two of which are assigned to7q31-32(data not shown).The candidate receptors encoded by the TRB genes on chromo-somes12and5(and7)share sequence motifs with one another, uniting them as members of the same receptor family.Although they have the seven-transmembrane domain structure characteristic of GPCRs,they are unrelated in sequence to both mGluR4,which detects glutamate20,21,and the candidate taste receptors TR1and TR2(ref.9).In addition,mGluR4,TR1and TR2have long extracellular amino-terminal domains that are proposed to bind ligand,whereas TRBs have very short N termini,suggesting that they use a different mode of ligand binding.Although TRBs are distantly related to V1Rs,TR1and TR2resemble V2Rs(refs22±24), candidate pheromone receptors that are expressed,with V1Rs,in the vomeronasal organ.The TRBs that we have identi®ed show high variability in protein sequence,suggesting that,like odorant receptors in the nose25,different family members may recognize chemicals with very different structures,such as chemically diverse bitter tastants.Are TRBs expressed in taste receptor cells,as they must be if they are truly taste receptors?To address this question,we turned to the mouse.We®rst asked whether we could isolate sequences encoding TRBs from either mouse genomic DNA or complementary DNA prepared from mouse taste tissue.We used polymerase chain reaction(PCR)with degenerate primers matching conserved sequences in TRBs to amplify related sequences,and then clonedand sequenced the PCR ing this approach,we obtained ®ve gene segments encoding TRBs from mouse genomic DNA (Fig.1),two of which appeared to be derived from pseudogenes (data not shown).We also obtained three TRB cDNAs from circumvallate and foliate taste papillae,two of which contained an uninterrupted open reading frame (Fig.1).These papillae are located on the tongue and contain taste buds where taste receptor cells are clustered 1,2.These cDNAs were nearly identical in sequence to three of the genes obtained from the genomic DNA.Given the error frequency of the polymerase used for PCR,these differences are unlikely to be due to PCR artifacts.Because the genomic DNA and cDNA were obtained from different mouse strains (DBA/2J and C57BL/6J,respectively),the nearly identical genes could either be closely related or allelic variants.The presence of a cluster of human genes encoding GPCRs adjacent to a Prp gene,which in mouse cosegregates with SOA,suggested that the TRBs may be taste receptors.Although the mouse and human TRBs are related,none of the mouse TRBs is similar enough to a human TRB identi®ed here to be considered an orthologue.Nonetheless,chromosome mapping of one mouse TRB gene (mTRB3)showed that it is located on mouse chromo-some 6between Prp/D6Mit13(63.6cM)and Kap/D6Mit111(63.7cM).This corresponds well with the SOA locus,which is tightly linked to the Prp gene 12.Reverse transcription±PCR (RT±PCR)with either degenerate TRB primers or primers speci®c for two mTRBs ampli®ed cDNAs of the correct size with RNA from mouse circumvallate and foliate taste papillae as well the tip of the tongue,which contains fungiform taste papillae,and the palate,which also has taste buds 1,2.Appropriatelysized PCR products were also obtained from testis,but not from a number of other tissues tested (Fig.2).This suggests that TRB genes may be expressed predominantly in taste tissue,as one might predict for genes encoding tastereceptors.Figure 1Candidate taste receptors encoded by human and mouse TRB genes.Putative receptor proteins encoded by TRB genes located on human chromosomes 12p13(hTRB1±6)and 5p15(hTRB7)or by TRB coding segments ampli®ed from mouse genomic DNA (mTRB1±3)or taste papillae cDNA (mTRB4±5)are aligned here with the putative pheromone receptor V1R5.Grey bars indicate amino acids found in 50%or more of the TRB proteins shown.Potential transmembrane regions are indicated by horizontal bars and roman numerals (I±VII).The TRBs share amino-acid sequence motifs,but are variable in sequence,consistent with an ability to recognize chemically diverse tastants.Palate Tip CV FOL Tongue OE VNO Brain Testis LiverabcdFigure 2TRB genes are expressed in taste papillae.RT±PCR was carried out with degenerate primers matching human TRBs (a )or primers speci®c for mTRB3(b ),mTRB2(c )or mouse b -actin (d ).The templates were cDNA prepared from RNA isolated from palate,tongue tip (the location of fungiform taste papillae)(Tip),circumvallate (CV)or foliate (FOL)taste papillae,tongue,olfactory epithelium (OE),vomeronasal organ (VNO),brain,testis or liver.PCR products were fractionated by size on agarose gels with 100-base pair (bp)ladder markers (far left and right lanes).The expected sizes of PCR products are indicated by arrows (a ,,780±800bp;b ,584bp;c ,556bp;d ,540bp).In situ hybridization experiments further showed that TRB genes are expressed in taste receptor cells (Fig.3).Consistent with the results of the RT±PCR experiments,labelled cRNA receptor probes hybridized to taste receptor cells in the taste buds of both circum-vallate and foliate papillae.Moreover,no hybridization was observed to other cells in the taste tissue sections or in sections through the vomeronasal organ,olfactory epithelium or brain.These results indicate that the receptor family that we have identi-®ed is probably selectively expressed in taste receptor cells.Notably,the TRB probes that we used labelled only a proportion of taste cells,while a probe matching the gene encoding gustducin,a G protein expressed in ,30±40%of taste receptor cells 26,27,labelled a greater number of taste receptor cells (Fig.3).A mix of TRB probes appeared to label a higher percentage of taste receptor cells than did one probe in the mix,suggesting that different taste receptor cells may express different TRBs (Fig.3).It may be that other mouse TRB genes not yet identi®ed are expressed either singly or in combinations in other taste receptor cells,and that this allows different taste receptor cells to recognize different tastants.In summary,we have identi®ed a family of receptors,the TRBs,that are speci®cally expressed in taste receptor cells in the mouth that detect gustatory stimuli.The TRBs are diverse in protein sequence,which is consistent with an ability to detect a variety of tastants with very different chemical structures.As only a propor-tion of the human genome sequence has been published,we expect that there will be many additional TRB genes beyond the 11that we identi®ed on chromosomes 12,5and 7.Given that previous studies have implicated G-protein signalling mechanisms in both bitter and sweet taste transduction,members of the TRB family might recog-nize either bitter or sweet compounds.However,the presence of at least one mouse TRB gene at the SOA locus,and the identi®cation of a cluster of TRB genes at the corresponding site in the human genome suggest that members of this family are likely to be involved in bitter taste sensation.MMethodsIdenti®cation of mouse genes and cDNAs encoding TRBsRNA was isolated from taste-bud-containing tissues dissected from C57BL/6J mice and then used to prepare cDNA 28.PCR reactions were carried out using these cDNAs or genomic DNA from DBA/2J mice (Jackson Laboratory)as described 22,25or using a `touch-down'PCR protocol 29.Two pairs of degenerate primers matching human TRBs were used:U1/U8or U2/U8(U1,C/TTIG/AG/CIAAT/CGGITTT/CATIGT/C/GIC/GTIGTIAA;U2,GGITTT/CATIGT/C/GIC/GTIGTIAAT/CT/GGIATGIGA;U8,TT/GA/GTTICCIAG/TIATIAGIAT/A/CIA/CAIGAA/GTG).PCR products were analysed on agarose gels and then gel-isolated and cloned into either pST Blue-1(Novagen)or pCR4-TOPO (Invitrogen).Analysis of mouse TRB gene expressionRNA was isolated from taste tissue and from other mouse tissues and used to prepare cDNA as above.The cDNAs were used in PCR reactions with the U1/U8degenerate primer pair (see above)or with primers speci®c for mTRB3(TGTGTGGTCACTACTCAC and GATTGCATGACAAGTGCC),mTRB2(GAAAGATCTCTGCAGTGGA and GCAAGCCTTTTATGTGGGC)or b -actin (Clontech).PCR was done as described 22except that the degenerate primers were annealed at 408C and the speci®c primers at 558C.For in situ hybridization experiments,digoxigenin cRNA probes were prepared from cloned mouse TRB gene or cDNA segments and hybridized to 16-m m tissue sections as described 22except that hybridization was done at 588C in a chamber saturated with 50%formamide and 5´sodium chloride/sodium citrate (SSC)22.Chromosome mapping of the mouse TRB3geneThe location of the mTRB3gene was determined using the T31-RH Mouse Radiation Hybrid mapping kit (Research Genetics).The data obtained were submitted to the Jackson Laboratory for determination of the chromosomal location of the gene.Received 13March;accepted 20March 2000.1.Roper,S.D.The cell biology of vertebrate taste receptors.Annu.Rev.Neurosci.12,329±353(1989).2.Lindemann,B.Taste reception.Physiol.Rev.76,718±766(1996).3.Lindemann,B.A taste for umami.Nature Neurosci.3,99±100(2000).4.Herness,M.S.&Gilbertson,T.A.Cellular mechanisms of taste transduction.Annu.Rev.Physiol.61,873±900(1999).5.Chaudhari,N.&Roper,S.D.Molecular and physiological evidence for 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buds.J.Neurosci.16,3817±3826(1996).21.Chaudhari,N.,Landin,A.M.&Roper,S.D.A metabotropic glutamate receptor variant functions as a taste receptor.Nature Neurosci.3,113±119(2000).22.Matsunami,H.,Buck,L.B.A multigene family encoding a diverse array of putative pheromone receptors in mammals.Cell 90,775±784(1997).23.Herrada,G.&Dulac,C.A novel family of putative pheromone receptors in mammals with a topographically organized and sexually dimorphic distribution.Cell 90,763±773(1997).24.Ryba,N.J.&Tirindelli,R.A new multigene family of putative pheromone receptors.Neuron 19,371±379(1997).25.Buck,L.&Axel,R.A novel multigene family may encode odorant receptors:a molecular basis for odor recognition.Cell 65,175±187(1991).26.Wong,G.T.,Ruiz-Avila,L.&Margolskee,R.F.Directing gene expression to gustducin-positive taste receptor cells.J.Neurosci.19,5802±5809(1999).27.McLaughlin,S.K.,McKinnon,P .J.&Margolskee,R.F.Gustducin is a taste-cell speci®c G protein closely related to the transducins.Nature 357,563±569(1992).28.Berghard,A.&Buck,L.B.Sensory transduction in vomeronasal neurons:evidence for G a o,G a i2,and adenyl cyclase II as major components of a pheromone signaling cascade.J.Neurosci.16,909±918(1996).Figure 3TRB genes are expressed in taste receptor cells in taste buds.Digoxigenin-labelled TRB cRNA probes or a gustducin cRNA probe were hybridized to sections through mouse circumvallate (a ±d ,i ,k )or foliate (e ±h ,j ,l )papillae.The probes used were prepared from sequences encoding mTRB2(a ,e ,l ),mTRB1(b ,f ,j ),three different TRBs (mTRB1,mTRB3,mTRB6)(c ,g ,k )or gustducin (d ,h ,l ).Scale bars,50m m (a ±h ;shown in h );10m m (i ±l ;shown in l ).29.Roux,K.H.in PCR Primer:A Laboratory Manual (eds Diffenbach,C.W.&Dveksler,G.S.)53±62(CSHL Press,Plainview,New York,1995).AcknowledgementsWe would like to thank E.Kurosawa-Pelletier and C.Gao for expert technical assistance and K.Fowler for help in preparing the manuscript.This work was supported by the Howard Hughes Medical Institute,grants from the National Institues of Health (to L.B.B.),and fellowship support from the Alice and Joseph Brook Fund (to J.-P.M.).Genbank accession numbers for mouse TRBs are AF24771±AF24775.Human TRB gene sequences are in the NCBI database in contigs NT_001856(chromosome 12),NT_000146(chromosome 5)and NT_001612,NT_000322and NT_002055(chromosome 7).Correspondence and requests for materials should be addressed to L.B.B.(e-mail:lbuck@)..................................................................High mobility of proteins in the mammalian cell nucleusRobert D.Phair *&Tom Misteli ²*BioInformatics Services,Rockville,Maryland 20854,USA²National Cancer Institute,NIH,Bethesda,Maryland 20892,USA..............................................................................................................................................The mammalian cell nucleus contains numerous sub-compart-ments,which have been implicated in essential processes such as transcription and splicing 1,2.The mechanisms by which nuclear compartments are formed and maintained are unclear.More fundamentally,it is not known how proteins move within the cell nucleus.We have measured the kinetic properties of proteinsin the nucleus of living cells using photobleaching techniques.Here we show that proteins involved in diverse nuclear processes move rapidly throughout the entire nucleus.Protein movement is independent of energy,which indicates that proteins may use a passive mechanism of movement.Proteins rapidly associate and dissociate with nuclear ing kinetic modelling,we determined residence times and steady-state ¯uxes of molecules in two main nuclear compartments.These data show that many nuclear proteins roam the cell nucleus in vivo and that nuclear compartments are the re¯ection of the steady-state association/dissociation of its `residents'with the nucleoplasmic space.Our observations have conceptual implications for under-standing nuclear architecture and how nuclear processes are organized in vivo .We measured the mobility of the nucleosomal binding protein HMG-17,the pre-mRNA splicing factor SF2/ASF and the rRNA processing protein ®brillarin in the nucleus of living cells.The three proteins are involved in three essential nuclear functions,transcrip-tion,pre-mRNA splicing and rRNA processing,respectively,and they represent three general types of distribution patterns that are observed for many nuclear proteins 1±3(Fig.1).HMG-17binds to nucleosomes and is thought to alter the higher order chromatin structure by modulating the access of transcriptional regulators to DNA 4.HMG-17is distributed evenly in foci throughout the nucleoplasm 5.SF2/ASF is an essential pre-mRNA splicing factor 6,7,which is concentrated in nuclear splicing factor compartments but is also present throughout the nucleoplasm 8.Fibrillarin associates with the U3small nucleolar RNA and is invovled in the processing of rRNA transcripts in the nucleolus 9,10.Fibrillarin is found almost exclusively in the nucleolus 11.We expressed these proteins as fusions with the auto¯uorescent green ¯uorescent protein (GFP)in HeLa or Baby Hamster Kidney cells.All three GFP-fusion proteinscolocal-Figure 1Colocalization of GFP-fusion constructs with endogenous proteins.Fusion proteins between GFP and SF2/ASF (a ),®brillarin (d )and HMG-17(g )were expressed in BHK cells.Endogenous SF2/ASF (b ),®brillarin (e )and HMG-17(h )were detected by indirect immuno¯uorescence using speci®c antibodies.The distribution pattern of theGFP-fusion proteins and the endogenous protein were identical and the labelling patterns were identical to that of the endogenous proteins in non-transfected cells (c ,f ,i ).Scale bar,10m m.。