Evolution of Brain-Expressed Biogenic Amine Receptors into Olfactory Trace Amine-Associated Receptors

1.2 MB·English

by  Lingna Guo, Wenxuan Dai, Zhengrong Xu, Qiaoyi Liang, Eliot T Miller, Shengju Li, Xia Gao, Maude W Baldwin, Renjie Chai, Qian Li

Evolution of Brain-Expressed Biogenic Amine Receptors into
Olfactory Trace Amine-Associated Receptors
Lingna Guo,†,1,2,3,4 Wenxuan Dai,†,1,2 Zhengrong Xu,†,1,2,5,6 Qiaoyi Liang,7 Eliot T. Miller,8 Shengju Li,1,2
Xia Gao,5,6 Maude W. Baldwin ,7 Renjie Chai,*,3,4,6 and Qian Li *,1,2,9
1CenterforBrainScience,ShanghaiChildren’sMedicalCenter,SchoolofMedicine,ShanghaiJiaoTongUniversity,Shanghai,China
2Department of Anatomy and Physiology, Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education,
ShanghaiJiaoTongUniversitySchoolofMedicine,Shanghai,China
3State Key Laboratory of Bioelectronics, Department of Otolaryngology Head and Neck Surgery, Zhongda Hospital, School of Life
SciencesandTechnology,JiangsuProvinceHigh-TechKeyLaboratoryforBio-MedicalResearch,SoutheastUniversity,Nanjing,China
4Co-InnovationCenterofNeuroregeneration,NantongUniversity,Nantong,China
5DepartmentofOtolaryngologyHeadandNeckSurgery,AffiliatedDrumTowerHospitalofNanjingUniversityMedicalSchool,Jiangsu
ProvincialKeyMedicalDiscipline(Laboratory),Nanjing,China D
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6ResearchInstituteofOtolaryngology,Nanjing,China n
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7MaxPlanckInstituteforOrnithology,EvolutionofSensorySystemsResearchGroup,Seewiesen,Germany ad
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8MacaulayLibrary,CornellLabofOrnithology,Ithaca,NY,USA d fro
9ShanghaiResearchCenterforBrainScienceandBrain-InspiredIntelligence,Shanghai,China m
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†Theseauthorscontributedequallytothiswork. ttp
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*Correspondingauthors:E-mails:[email protected];[email protected]. ://a
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Associateeditor:JulianEchave ad
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Abstract .o
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The family of trace amine-associated receptors (TAARs) is distantly related to G protein-coupled biogenic aminergic o
m
receptors. TAARs are found in the brain as well as in the olfactory epithelium where they detect biogenic amines. /m
b
However,thefunctionalrelationshipofreceptorsfromdistinctTAARsubfamiliesandindifferentspeciesisstilluncer- e
/a
tain.Here,weperformathoroughphylogeneticanalysisof702TAAR-like(TARL)andTAARsequencesfrom48species. rtic
WeshowthatacladeofTarlgeneshasgreatlyexpandedinlampreys,whereastheotherTarlcladeconsistsofonlyoneor le
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two orthologs in jawed vertebrates and is lost in amniotes. We also identify two small clades of Taar genes in sharks 9/3
relatedtotheremainingTaargenesinbonyvertebrates,whicharedividedintofourmajorclades.Wefurtheridentify /m
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ligands for 61 orphan TARLs and TAARs from sea lamprey, shark, ray-finned fishes, and mammals, as well as novel ac
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ligandsfortwo5-hydroxytryptaminereceptor4orthologs,aserotoninreceptorsubtypecloselyrelatedtoTAARs.Our 0
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results reveal a pattern of functional convergence and segregation: TARLs from sea lamprey and bony vertebrate A/65
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olfactoryTAARsunderwentindependentexpansionstofunctionaschemosensoryreceptors,whereasTARLsfromjawed 3
t5
vertebratesretainancestralresponseprofilesandmayhavesimilarfunctionstoTAAR1inthebrain.Overall,ourdata ic06 b
provideacomprehensiveunderstandingoftheevolutionandligandrecognitionprofilesofTAARsandTARLs. ly
e g
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Keywords:traceamine-associatedreceptor,olfactoryreceptor,GPCR,receptorevolution,receptordeorphanization, e
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site-directedmutagenesis,homologymodeling. t o
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Introduction ril 2
traceamines,andalloftheotherTAARshighlyexpressedin 0
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Trace amine-associated receptors (TAARs) form a distinct themainolfactoryepithelium(MOE)recognizingexogenous
subfamily of G protein-coupled receptors (GPCRs) that are biogenic amines (Liberles and Buck 2006; Xu and Li 2020;
specialized to detect both endogenous and exogenous bio- Dewan2021).SeveralTAARsarealsodistributedthroughout
genicamines.TAARswereinitiallydiscoveredasreceptorsfor thebodyandarefoundinothertissuessuchaskidney,heart,
a number of trace amines that are structurally similar to and testes, albeit with lower expression levels (Gainetdinov
monoamine neurotransmitters (e.g., serotonin, dopamine, et al. 2018). Nonolfactory TAAR1 negatively regulates excit-
and histamine) but present in trace concentrations in the abilityandmonoamineneurotransmittertransmissionofdo-
brain(Borowskyetal.2001;Bunzowetal.2001).Thesubse- paminergic and serotonergic neurons. Psychostimulant
quent studies revealed a functional dichotomy of TAARs, abuse-related behaviors are reduced by TAAR1 activation
withTAAR1expressedinthebrainandsensingendogenous and enhanced by TAAR1 knockout in mice (Bradaia et al.
(cid:2)TheAuthor(s)2022.PublishedbyOxfordUniversityPressonbehalfoftheSocietyforMolecularBiologyandEvolution.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(https://creativecommons.
org/licenses/by/4.0/),whichpermitsunrestrictedreuse,distribution,andreproductioninanymedium,providedtheoriginalworkis Open Access
properlycited.
Mol.Biol.Evol.39(3):msac006 doi:10.1093/molbev/msac006 AdvanceAccesspublicationJanuary11,2022 1
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Guoetal. . doi:10.1093/molbev/msac006
2009; Revel et al. 2011; Achat-Mendes et al. 2012; Liu et al. Asp3.32 and Asp5.42, and recognize diamines (which have
2020). Olfactory TAARs, on the other hand, are crucial for twoaminogroups)(Lietal.2015).However,withtheexcep-
perceptionofamines,anecologicallyimportantclassofodor- tion of receptors from a few model species, most of the
ants. Amines are produced by amino acid decarboxylation TAARs in different species remain orphan receptors, which
duringdecompositionofproteins,andareoftenenrichedin restrict the functional analyses of this specific family of
animal body fluids. Therefore, they have been proposed to receptors.
mediateintra-andinterspecificcommunicationthroughthe Here,wesoughttoinvestigatetheevolutionaryhistoryand
TAARolfactorysubsystem.Forinstance,trimethylamineisa functional responses of TAARs, TARLs, and HTR4s. In total,
species-andsex-specificurineodorthatactivatesTAAR5to weretrieved702TAARandTARLsequencesfrom48verte-
attractmiceandrepelrats(Lietal.2013;Saraivaetal.2016). brate species and constructed a phylogenetic tree. Among
TheTAAR4ligand2-phenylethylamineisenrichedinpreda- severalaminergicreceptorsselectedasoutgroups,HTR4sare
tor urine and elicits innate avoidance behaviors in rodents theclosestrelativetoTAARsandTARLs.TARLsaregrouped
(Ferrero et al. 2011; Dewan et al. 2013). Cadaverine can be intotwosubfamilies.Onesubfamilyexpandsgreatlyinlamp-
D
produced from decaying animal carcasses and activates reys,whereastheotheroneexhibitsverylittleduplicationin o
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TAAR13c, triggering aversive behavior in zebrafish (Hussain jawedvertebratesandappearstobelostinamniotes.Wealso n
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et al. 2013). Thus, TAARs play important roles in both psy- foundthatTAARscanbeclusteredintofourdistinctclades— a
d
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chostimulantaddictionandsocialbehaviors. Ia, Ib, II, and III. To further analyze their functional relation- d
SinceTaargeneswerefirstclonedin2001,theyhavebeen ships, we next identified novel ligands for 61 TAARs and fro
m
identifiedinseveralvertebrategenomes(Hussainetal.2009; TARLs as well as two HTR4s. Surprisingly, HTR4s, TARLs h
Gao et al. 2017; Eyun 2019; Sharma et al. 2019; Dieris et al. from jawed fishes and nonolfactory TAAR1 have similar li- ttp
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2021).ThenumbersofintactTaargenesvaryacrossspecies, gandresponseprofiles,whereassomelampreyTARLsrecog- ://a
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ranging fromzeroindolphins,sixinhumans, 15 inmiceto nize ligands that are also recognized by olfactory TAARs, ad
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112 in zebrafish. TAARs are distantly related to classic bio- albeit with a distinct structural basis. Consistent with the m
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genic amine receptors, including dopamine and serotonin distinct functional profile of the two TARL subfamilies, we .o
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receptors.Phylogeneticstudieshaveindicatedthatthe5-hy- found that TARLs in jawed fishes such as zebrafish are p
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droxytryptamine receptor 4 (HTR4), a serotonin receptor expressedinthebrain,whereaslampreyTARLsareexpressed om
subtype, is more closely related to the TAAR subfamily intheMOE.Insum,ourcomprehensiveanalysisoftheevo- /m
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than other biogenic amine receptors (Hashiguchi and lutionandfunctionofTAARs/TARLsuncoverstheevolution- e/a
Nishida 2007; Hussain et al. 2009; Li and Liberles 2016; arytransitionsfrombrain-expressedbiogenicaminereceptors rtic
Dieris et al. 2021). However, there has been controversy toolfactoryaminedetectors.Ourstudyprovidesevidencefor le/3
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over the exact timing of the origin of Taar genes. Initial functionaldistinctionandconvergencebetweenTAARsand /3
reportssuggestedthattheTaargenefamilyemergedinjaw- TARLs. /m
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lessvertebratessuchassealamprey(HashiguchiandNishida c
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2007; Libants et al. 2009). Other studies suggested a later Results 06/6
origin in early jawed fish (Hussain et al. 2009; Eyun et al. 5
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2016); because so-called sea lamprey TAARs form a mono- EvolutionaryHistoryofHTR4s,TARLs,andTAARs 35
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phyletic cladeandlacktheclassic TAARmotifinthetrans- To explore the evolutionary relationships of HTR4s, TARLs, 6
b
membrane a-helix VII, they were named Taar-like (Tarl) andTAARs,weretrievedathoroughcollectionofsequences y g
genes. The earliest-branching Taar genes with the intact from different species. These species included two jawless ue
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TAAR motif are found in cartilaginous fishes, the earliest- fishes (hagfish and sea lamprey), three cartilaginous fishes t o
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branchingextantjawedvertebrates,includingelephantshark, (ghostshark,bambooshark,andwhaleshark),seventeleost 0
5
catshark, white shark, and whale shark (Hussain et al. 2009; fishes (medaka, catfish, goldfish, pufferfish, stickleback, fugu, A
p
Marra et al. 2019; Sharma et al. 2019). Recently, a compre- andzebrafish),oneholostfish(spottedgar),onelobe-finned ril 2
hensive phylogenetic analysis of Tarl and Taar genes places fish(coelacanth),oneamphibian(clawedfrog),sevenreptiles, 0
2
2
lampreyTarlsassistertootherTarlgenes(calledtheTaarV and26mammalianspecies.Intotal,17sequencesofHTR4s,
subfamilyinpreviousstudies)(HashiguchiandNishida2007; and702homologoussequencesofTARLsorTAARsfrom48
Eyun et al. 2016), which together are sister to the classical specieswereobtained(supplementarytableS1anddataS1,
Taars(Dierisetal.2021). Supplementary Material online). In addition, we selected 39
Apart from the Tarl clade, classical TAAR receptors are classicalbiogenicaminereceptorsasoutgroups,consistingof
classified into three major clades (called class I, II, and III by seven a adrenergic receptors, three b adrenergic receptors,
Hussain et al. 2009, or called clades I, II, and III by Li et al. four dopamine receptors, nine muscarinic acetylcholine
[2015]). ThetetrapodTAARsaregroupedincladesIandII, receptors, seven histamine receptors, and nine serotonin
whereascladeIIIisteleost-specific.Interestingly,almostallof receptorsfromwhaleshark,zebrafish,coelacanth,andmouse.
the clade I and II TAARs possess the canonical amine- Thephylogeneticanalysesofthereceptorsequenceswere
recognition motif (Asp3.32; Ballesteros–Weinstein indexing performed using maximal likelihood (ML) in IQ-TREE (sup-
for GPCRs). This motif is lost in the vast majority of clade plementary fig. S1A and B, Supplementary Material online)
IIITAARs,whichevolvethenoncanonicalamine-recognition andnodalsupportwasassessedbothby5,000ultrafast(UF)
motif, Asp5.42. Furthermore, a few TAARs contain both bootstrapreplicates(Hoangetal.2018)aswell100standard
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Brain-ExpressedBiogenicAmineReceptorsintoOlfactoryTraceAmine-AssociatedReceptors . doi:10.1093/molbev/
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bootstrapreplicates;similartreetopologiesandsupportval- and the four clades of TAARs (supplementary fig. S2A,
ues were obtained from multiple independent runs. SupplementaryMaterialonline).Inagreementwiththepre-
Consistent with previous results, we recover monophyletic viousstudy,theTAARmotifiswellconservedinallofthefour
clades of HTR4s, lamprey TARLs, and TAARs with maximal clades ofTAARs. InTARLLandTARLs, weidentifiedsimilar
support (fig. 1A and supplementary fig. S1A and B, butdistinctfingerprintmotifs.TARLLshaveaconsensusmo-
SupplementaryMaterial online). Likein lamprey TARLs, the tif of SxxNPxLxxxxNxxF, whereas TARLs have a consensus
classicalTAARmotifisalsomissinginTARLsfromthisclade motifofNSxxNPxLYxxxxxSF(fig.1B).Wealsoidentifiedsev-
(fig. 1B). To differentiate between those TARLs from the eral conserved amino acids of TARLs and TARLLs that are
jawed vertebrates and the lamprey TARLs, we renamed the absent in TAARs. Most of those are in transmembrane or
lampreyTARLsasTARLLs(thelastLindicatinglamprey).The extracellular regions, suggesting a role in ligand recognition,
TARLLsshowalargeexpansion,consistingof33membersin distinctfromthatseeninTAARs(fig.1C).
sea lamprey that can be further subdivided into four subfa- Wefurtheranalyzedthepresenceofpseudogenesinsev-
milies, TARLL1-4 (supplementary fig. S1A, Supplementary eral species. We did not find any TAAR/TARL/TARLL pseu-
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Materialonline).Bycontrast,thereappearedtobegenerally dogenes in hagfish, confirming that TARLLs specifically o
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onlyoneTARLinmostofjawedvertebrates(TARL1incar- emerge in lamprey. The percentages of pseudogenes differ n
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tilaginous fishes, ray-finned fishes, coelacanth, and amphib- across species, varying from 6% in mouse to 44% in whale a
d
e
ians)andtwoTARLsinsomespecies(TARL1andTARL2in shark (supplementary fig. S2B, Supplementary Material d
whale shark and coelacanth, supplementary fig. S1A, online). fro
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Supplementary Material online). We did not find any h
TARLsinamniotes(supplementarytableS1,Supplementary Cell-BasedHigh-ThroughputScreeningto ttp
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Materialonline).TARLLsandTARLsthereforedisplaydistinct DeorphanizeReceptors ://a
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diversificationpatternsinjawlessandjawedvertebrates,sug- Next, we conducted high-throughput screening to further ad
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gestingdistinctfunctionalroles. explore the functional properties of these aminergic recep- m
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Next, we investigated the evolutionary dynamics of tors. We cloned 248 TAARs from 11 mammals (human, .o
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TAARs. TAARs form a large monophyletic clade, including mouse,rat,guineapig,hamster,cat,horse,rabbit,sheep,rhe- p
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receptorsfromspeciesrangingfromjawedfishes,amphibians, sus, and wild boar), two reptiles (alligator and chicken), six om
reptiles,andmammals.TheTAARscanbefurthersegregated teleosts(zebrafish,catfish,fugu,medaka,salmon,andpuffer- /m
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intofourmajormonophyleticgroupsorclades(Ia,Ib,II,and fish),onelobed-finnedfish(coelacanth),oneholostfish(spot- e/a
III), largely corresponding to previously defined groups tedgar),andonecartilaginousfish(bambooshark)(fig.2A). rtic
(Hussainetal.2009;Lietal.2015).Apreviousstudyinvesti- WealsoclonedeightTARLsfromcoelacanth,zebrafish,cat- le/3
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gatedaround300TAARsequencesandsegregatedtheminto fish,fugu,medaka,spottedgar,shark,and28TARLLsfromsea /3
threecladesdesignatedclassI,II,andIII(Hussainetal.2009). lamprey (supplementary table S2, Supplementary Material /m
s
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Inourstudy,withanexpandedsetofspeciesanalyzed,classI online).Inaddition,weclonedtwoHTR4sfrommouseand c
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TAARs were separated into two clades, which we named zebrafish.Wethenchallengedalloftheclonedreceptorswith 06
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clades Ia and Ib. Furthermore, Hussain et al. subdivided aligandlibrarycomprised97aminesat100mM.Intotal,we 5
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TAARs into 28 different subfamilies (TAAR1–28). Here, we successfully identified novel ligands for 50 TAARs, eight 35
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found several TAARs that could not be included into the TARLs, three TARLLs, and two HTR4s. The deorphaned 6
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existing 28 subfamilies, so we applied new subfamily names receptors are spread across the major receptor clades y g
to those TAARs (subfamilies are distinguished if they form (fig. 2B and supplementary table S3, Supplementary ue
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separategroupsinsidetheclades).ForcladeIIIteleostTAARs, Materialonline).Forthosereceptorsshowinghighresponses, t o
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we additionally named three subfamilies (TAAR29, 30, and we subsequently performed dose–response curves. Most of 0
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31).ForotherTAARs,wenamedthesubfamiliesbythemain theligandsactivatethereceptorswithhalfmaximaleffective A
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species in which they occur, including TAARS1–2 (S for concentrations(EC50)of1–100mM,andafewligandsshow ril 2
shark), TAARC1–3 (C for coelacanth), TAARSG1–6 (SG for extremely sensitive activation with an EC of 20 pM (sup- 0
50 2
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spottedgar),TAARR1(Rforreptile),andTAARM1–2(Mfor plementarytableS4,SupplementaryMaterialonline).
metatherian)(fig.1A).Interestingly,TAARsfromcartilaginous Tobetterunderstandtheligandresponseprofiles,weclas-
fishes display a distinct phylogenetic distribution. sifiedthe97aminesintoeightchemicalclustersaccordingto
Cartilaginous fish TAARs form two subfamilies, TAARS1 their chemical features, including predefined atom symbols,
and TAARS2. TAARS1 is sister to the remaining clade Ia bond types, atom environment properties, and atom prop-
TAARs, whereas TAARS2 is sister to all subsequent TAARs, erties(supplementaryfig.S3,SupplementaryMaterialonline).
including clades Ib, II, and III TAARs (fig. 1A). This phyloge- Thelargestthreeclustersconsistofagroupmainlycomposed
netic pattern suggests that the divergence of clade Ia and ofprimaryorsecondaryamines(cluster2),anothergroupis
other TAARs (clades Ib, II, and III) occurred prior to the di- primarilycomposedofamineswitharomaticrings(cluster3),
vergenceofcartilaginousfishandbonyfish. and cluster 5 is mainly composed of tertiary amines. The
Thecharacteristicfingerprintmotif(NSxxNPxxYxxxYxWF, otherfiveclustersincludecluster1(taurine),cluster4(creat-
wherexrepresentsnonconservedaminoacids)inthetrans- inine),cluster6(isopropylamine),cluster7(aniline)andclus-
membranea-helixVIIwasidentifiedforTAARs(Hussainetal. ter8(indoleanditsderivatives).Itisworthnotingthatmost
2009).WeanalyzedtheconsensusmotifsinTARLLs,TARLs, biogenic amine neurotransmitters are included in chemical
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A
Clade Ia
27
10
11
TARLL
SG1 12
1
TAARS2 Clade Ib
TARL
HTR4 4
C1
TAARS1 C4
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Outgroup 5 loa
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Tetrapod 20 SG3 m
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Lobe-finned fish 29 .o
18 17 13 up
Ray-finned Holost fish 16 .co
Teleost fish 32 m
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Cartilaginous fish b
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Jawless fish /a
Outgroup 2625 SG4 rtic
SG5 14 le
SG6 /3
22 9
30233128 Clade III /3/m
24 s
a
0.4 c
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0
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B C /6
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TARLL TARL 0
Clade III LFCVWIFLAVTILFMGVFNHCQYLFMCAINSCSCGSATLFMVINPLMVIVIYGALFMTILFYRPKSWF 350
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TAAR CCCllaaladddeee II baII WWWLLLFFFGCVIIGGGAVAATTLYYYWLLLFFFSYYINNNSSSALMSAAATTTLLFFFMCCCVINNNPPPLLLFFMVIIIVIIIHYYYGSGAVAALLFFFMFFFMSHYYYLHPPPRNSSTRRCWWWFFF Y FP PH SHP IT by guest on 0
TARLL WLFMVIGTHYLFMVINSSMGATFAVINPLYVILFYVIMGAVLGSATLFINSLRKMPQSLFY 5 Ap
TARL WLGYLSVTINSGSATLMINPLFILYGALFVLFYVHNRKQPSFY ril 2
HTR4 WLGYINSGLNPFLYAFLNRAF R 02
AV MAFI LI Y KST TARLLTARL 2
Outgroup WLFCVILCGYWLCVAINSSLCGSATLFVIN7.P50LFMAVILCVIYPCATLRFCTILFCSNHMSERKEDNSTLFY 90% > ccoonnsseerrvvaattiioonn >> 8900%%
FIG.1.PhylogeneticanalysisshowingtheevolutionaryhistoryofHTR4,TAARsandTAAR-likereceptors(TARLandTARLL).(A)RadiallayoutofML
phylogenetictreeofHTR4,TAARs,TARLs,andTARLLs.About758aminoacidsequencesfrom48specieswereincludedforphylogeneticinference.
Outgroupsincludesevenaadrenergicreceptors,threebadrenergicreceptors,fourdopaminereceptors,ninemuscarinicacetylcholinereceptors,
sevenhistaminereceptors,andnineserotoninreceptorsfromwhaleshark,zebrafish,coelacanth,andmouse.Differentgroupsofvertebratespecies
arecolor-coded(scalebar¼0.4substitutionspersite).Accordingtoourupdatednomenclature,cladeIaTAARsaremainlycomposedofTAARC1,
TAARSG1,TAAR1fromjawedvertebrates,andteleost-specificTAAR10,11,21,27subfamilies.CladeIbTAARscontainTAARC4,tetrapod-specific
TAAR2–4,andteleost-specificTAAR12 subfamilies.CladeII TAARs arecomposedofTAARC2,TAARR1,TAARM1–2,andmammal-specific
TAAR5–9subfamilies.CladeIIITAARscontainTAARC3,TAARSG2–6,andteleost-specificTAAR13–20,22–26,28–31subfamilies.(B)Sequence
logoplotsoftransmembranesegmentVIIofthefourmajorTAARclades(cladesIa,Ib,II,andIII),aswellasofTARLL,TARL,HTR4,andoutgroup
receptors.ThebrownaminoacidsrepresenttheTAARfingerprintmotif.ThegreenandorangeaminoacidsrepresentthemotifsforTARLLand
TARL,respectively.Themostconservedprolineat7.50siteintheBallesteros–Weinsteinindexingsystemisalsolabeled.(C)Conservedsitesin
TARLLandTARLsubfamiliesshowninthetransmembranesegmentsandextracellularloopsareshown.
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FIG.2.Summaryofdeorphanedreceptors.(A)Theevolutionaryrelationshipof47ofthe48species(exceptbambooshark)whosereceptorswere
included in this study were depicted. The presence of receptor genes was marked by the filled circles, which are color-coded according to
phylogeneticgroupasinfigure1A.TheemergenceandlossofTARLL,TARL,andTAARcladesareindicatedbycoloredstarsandcross.Namesof
speciesinredtextindicateaspecieswhosereceptorswereclonedandtestedinthisstudy.TheasteriskssignifythatligandsforTAARsofthose
specieswerenotstudiedbefore.(B)Thenumbersofreceptorsclonedanddeorphanedweresummarized,anddividedintofourgroups:receptors
newlydeorphanedinthisstudy(red),receptorswithnovelligandsidentifiedinthisstudy(lightgreen),receptorsdeorphanedinpreviousstudies
(lightblue),andreceptorsclonedandtestedbutnotdeorphaned(gray).
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cluster 3, and most of the other known TAAR ligands are is clustered into PAM group 2. Since Dieris et al. (2021)
included in clusters 2 and 5. The 52 identified ligands are showed a different topology on the clades of TARLL, TARL,
mainlyincluster2,3,5,6,7,and8.Allofthemoleculesare andTAARwithhighersupportvalues,wealsoexaminedthe
positivelychargedinphysiologicalconditions,exceptformol- effectofthedifferenceintopologiesbyanalyzingresponsesof
eculesincluster7andcluster8,whichareneutralinphysi- areducedsetofreceptorspresentinbothphylogenies.The
ologicalconditions. clustering results from the topology comparison are similar,
and are consistent with the results from the full data set
FunctionalRelationshipsofHTR4s,TARLLs,TARLs, (supplementaryfig.S5A–D,SupplementaryMaterialonline).
andTAARs
Next, we combined our datawith known ligands ofTAARs TAARsShowDistinctLigand-BindingFeatures
frommodelorganisms,suchashuman,mouse,rat,andzebra- WeidentifiednovelligandsforoneTAARS1(TAARS1b),five
fish(XuandLi2020).Comparisonofligandresponsesshowed cladeIa,tencladeIb,24cladeII,andtencladeIIITAARsfrom
that in general, receptors in different clades have distinct 18differentspecies(supplementarytableS3,Supplementary
D
ligand profiles, responding to chemicals in distinct clusters, Material online), providing an opportunity to understand o
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whereas receptors within the same clades tend to detect TAAR-ligand interaction in depth. We previously reported n
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ligands from the same chemical clusters (fig. 3A). Further, that clade III TAARs evolved the noncanonical Asp5.42 to a
d
e
TAAR and TARL orthologs from different species generally detect amine ligands, whereas other TAARs utilize the con- d
detectverysimilarligands,suggestingconservationofrecep- served Asp3.32 to detect amine ligands (Li et al. 2015). We fro
m
torfunctionacrossspecies(fig.3A).However,wedidobserve observedthesamepatterninthecurrentstudy(supplemen- h
broaderligandprofilesforreceptororthologsinsomespecies, tary fig. S6A, Supplementary Material online). Further, we ttps
althoughwecouldnotruleoutthepossibilitythatreceptor found that Asp3.32 (but not Asp5.42) is well conserved in ://a
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orthologs may not function equally well in vitro. It is also sequences of HTR4, TARLL, and TARL, implying that HTR4, ad
e
worth noting that TAAR-expressing olfactory sensory neu- TARLL,andTARLusetheconservedAsp3.32todetectamines. m
ic
rons generally recognize the same ligands identified by Interestingly, a few clade II mammalian TAARs, including .o
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in vitro assays, yet with enhanced sensitivity and increased TAAR6 and TAAR8, have Asp5.43 instead of Asp5.42. This p
.c
numberofligands,likelyduetooptimizedsignalingcompo- results from the loss of an amino acid at 5.44 in the fifth om
nentsandchaperonsinneuronsinvivo(Pacificoetal.2012; transmembranesegmentsofthosereceptors(supplementary /m
b
Zhangetal.2013). fig.S6A,SupplementaryMaterialonline).Inspiteofthisfact, e/a
To better illustrate the functional relevance of HTR4s, simulationsdosuggestthatthoseTAARsmayrecognizedia- rtic
TARLLs,TARLs,andTAARs,weperformedphylogeneticprin- mines according to computational simulations (Izquierdo le/3
9
cipal component analysis (phyloPCA) (Revell and Collar et al. 2018). However, our results clearly showed that /3
2009)onallofthedeorphanedreceptorsbasedontheligand Asp5.43-containing TAAR6 and TAAR8 family members, in- /m
s
a
profilesandtheMLphylogenetictree.Inourprimarydataset, cludingTAAR6frommouse,rat,cat,TAAR8afromrat,wild c
0
since the top six principal components (PCs) together only boar, and TAAR8b from rabbit, can only be activated by 06
/6
explain 63.9% of the variance (supplementary fig. S4A–C, monoamines other than diamines (supplementary fig. S6B 5
0
Supplementary Material online), we further applied dimen- and table S3, Supplementary Material online). We suspect 35
0
sionality reduction using Uniform Manifold Approximation thatAsp5.43mightbedirectedawayfromthepotentialbind- 6
b
and Projection (UMAP) on all phyloPCA scores for better ing pocket, and thus may not be involved in amine y g
visualization (fig. 3B). To quantitatively classify receptors by recognition. ue
s
their phyloPCA scores on all PC axes, we used a clustering Our previous study also proposed a two-step model for t o
n
algorithm(PartitionAroundMedoids,PAM)afteridentifying Asp5.42 acquisition, suggesting that an ancestral TAAR of 0
5
the optimal number of groups (Maechler et al. 2019; cladeIIITAARsacquiredAsp5.42,gainingdiaminesensitivity, A
p
Kassambara and Mundt 2020). We found that all of these andsubsequentlylostAsp3.32(Lietal.2015).Interestingly,we ril 2
receptors were assigned into two groups by PAM (supple- foundthatbothAsp3.32andAsp5.42arepresentinthecarti- 0
2
2
mentary fig. S4D, Supplementary Material online), overlap- laginous fish TAARS2 subfamily (supplementary fig. S6A,
ping with the UMAP visualization (fig. 3B). We noted that Supplementary Material online). The facts that TAARS2 al-
almostallofthereceptorsfromcladeIaTAARs(exceptzebra- readyhasbothAsp3.32andAsp5.42togetherwiththeobser-
fishTAAR10bandmedakaTAAR21e),TARLs,andHTR4sare vation that some clade II mammalian TAARs contain both
clusteredinPAMgroup2,suggestingthattheysharesimilar Asp3.32andAsp5.43suggesttwoalternativescenarios.Inone
ligand response profiles. Some clade Ib TAARs (TAAR4 and scenario, thesetwo residuesmay havearisen independently
TAAR12)andacladeIIITAAR(catfishTAAR13g)alsoappear and convergently multiple times, in shark TAARS2, and in
intheUMAPplotclosetoPAMgroup2,whichmightbedue some clade II and clade III TAARs. Alternatively, after the
totheirresponsestosomeligandsincluster3(amineswith divergence of clade Ia, the ancestor of the subsequent
aromatic rings). However, most clades Ib, II, and III TAARs TAARs may have evolved both Asp3.32 and Asp5.42. Asp5.42
clusterinPAMgroup1togetherwithsharkTAARS1bbothin mayhavethenbeenlostincladeIbTAARsbutretainedinthe
thePAM clustering andthe UMAP plot. For thefour deor- commonancestorofcladeIIandIIITAARs,followedbysub-
phanedsealampreyTARLLs,three(TARLL2a,TARLL3h,and sequentlossofAsp5.42insomecladeIITAARsorshiftingto
TARLL4a)areclusteredintoPAMgroup1,andonlyTARLL1b Asp5.43 in other clade II TAARs (as a result of a deletion,
6
Brain-ExpressedBiogenicAmineReceptorsintoOlfactoryTraceAmine-AssociatedReceptors . doi:10.1093/molbev/
MBE
msac006
A HTR4 ZebrMafoisuhs eH HTTRR4c4
Sea lamprey TARLL1b
TARLL SSSeeeaaa lllaaammmppprrreeeyyy TTTAAARRRLLLLLL234aha ligAamndo ucnluts otef rs
Shark TARL2 4
ZeMberadfaiskha TTAARRLL11 3
TARL CaFtufigshu TTAARRLL11 2
Spotted gar TARL1 1
Coelacanth TARL1
Coelacanth TARL2
TAARS1 CoelacSahnatrhk TTAAAARRCS11bb Amount of
Zebrafish TAAR1 ligands
Spotted gar TAAR1b
Mouse TAAR1 1
Clade Ia ZebraHfuismhRa aTntA TTAAARAA1RR01a1 24
Zebrafish TAAR10b 8
MMeeddaakkaa TTAAAARR2211ef 16
Fugu TAAR21e
Fugu TAAR27
Mouse TAAR2
Mouse TAAR3
Guinea pig TAAR3
Hamster TAAR3
Horse TAAR3
Mouse TAAR4
Clade Ib GuHinaemas pteigr TTAAAARR44
Cat TAAR4
Horse TAAR4
Zebrafish TAAR12h
Zebrafish TAAR12i
ZebCraatffiisshh TTAAAARR1122bj D
MouRCsaaett TTTAAAAAARRR555 ow
Hamster TAAR5 n
WildH boorsaer TTAAAARR55 lo
Sheep TAAR5 a
Human TAAR5 d
Mouse TAAR6 e
Clade II MMMMoooouuuRRusssRCaaseeeaatte ttTTTTT TTTAAAAAAAAAAAAAAAARRRRRRRR77777766abbdef d from h
RRaatt TTAAAARR77dh ttp
GuHWinaSeilmdah s ebpteeoigpar rTTT TAAAAAAAARRRR777b7ac s://a
MMRaoobuuRRbssaaeiettt TTTTTAAAAAAAAAARRRRR88888baacc cade
Wild boar TAAR8a m
MouRsaet TTAAAARR99 ic
HaRmasbtbeirt TTAAAARR99 .o
Cat TAAR9 u
Zebrafish TAAR13a p
ZZeebbrraaffiisshh TTAAAARR1133dc .c
Zebrafish TAAR13e o
Catfish TAAR13c m
Clade III CCCaaatttfffiisisshhh TTTAAAAAARRR111333hfg /m
Zebrafish TAAR14d b
SpoZZteteebbdrr aagffaiissrh hT TATAAAAARRRS11G662cf e/a
12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152 Lig2and c3luste5r rtic
6 7 8 le
/3
9
B (i.e., Butylamine) (i.e., Phenylethylamine) (i.e., Triethylamine) (i.e., Indole) /3/m
10 sa
c
0
0
6
0 /6
5
P2 Clade Ia 0
UMA−10 CCClllaaadddeee IIIbIII 3506
HTTTAAATRARRRLL4LS1 by gu
−20 e
s
−4 −2 0UMAP12 4 6 t o
Figure 3 n
0
5
A
FIG.3.LigandresponseprofileofHTR4s,TARLs,TARLLs,andTAARs.(A)Overviewoftheligandresponseprofileofallofthedeorphanedreceptors. p
Theredsquaresrepresentpositiveligand–receptorinteractionandtheyellowsquaresdenotenoresponses.Thehorizontallinesshowbordersof ril 2
differentclades.Thetestedcompoundswereclassifiedintoeightchemicalclusters(supplementaryfig.S4,SupplementaryMaterialonline)(only 02
2
sixclustersofcompoundswerefoundtobeligandsofanyreceptor);differentclustersareseparatedbytheverticallines.Themoleculestructures
underneathpanel(A)arechosenasrepresentativesofeachchemicalcluster.Thenamesofthoseligandsareshownbelow:1)butylamine,2)
hexylamine,3)isoamylamine,4)isobutylamine,5)cadaverine,6)pyrrolidine,7)putrescine,8)2-methylbutylamine,9)3-methylthiopropylamine,
10)agmatine,11)cyclohexylamine,12)cyclopentylamine,13)piperidine,14)spermidine,15)5-amino-1-pentanol,16)spermine,17)amylamine,
18)octylamine,19)heptylamine,20)propylamine,21)benzylamine,22)tryptamine,23)phenylethylamine,24)histamine,25)tyramine,26)
octopamine,27)3-methoxytyramine,28)5-methoxytryptamine,29)4-methoxyphenethylamine,30)5-methoxy-N,N-dimethyltryptamine,31)
gramine,32)phenylethanolamine,33)dopamine,34)serotonin,35)N,N-dimethylcyclohexylamine,36)1-methylpyrrolidine,37)1-methylpiper-
idine,38)trimethylamine,39)N,N-dimethylphenethylamine,40)N,N-dimethylbenzylamine,41)N,N-dimethylisopropylamine,42)N,N,N0,N0-
tetramethyl-1,4-butanediamine,43)N,N,N0,N0-Tetramethyl-1,3-propanediamine,44)triethylamine,45)isopropylamine,46)aniline,47)indole,48)
1-methylindole,49)3-methylindole,50)5-methylindole,51)6-methylindole,and52)7-methylindole.Thetuningbreadthofeachreceptoriscolor-
codedontheright.(B)TheUMAPreductionandvisualizationofphyloPCAresultsofthedeorphanedreceptorsisshown.Theclusterinthelower
rightquadrantisoutlinedwithadashedrectangleandhighlightedintheinset.Withinthiscluster,dotswithblackoutlinesindicatethereceptors
(HTR4,TARL,cladeIaTAAR,andaTARLL)whichbelongtogroup2bythePAMclusteringmethod;dotsarecoloredaccordingtophylogenetic
clade.Notethatthereceptornamesofthisgroup(PAMgroup2)arecoloredinblackin(A);thenamesofPAMgroup1receptorsclosetoPAM
group2areitalicizedin(A);allothernamesofPAMgroup1receptorsareingray.
7
MBE
Guoetal. . doi:10.1093/molbev/msac006
supplementaryfig.S6A,SupplementaryMaterialonline),and ofTARLLstoaminesareveryspecificastheyarenotrespon-
lossofAsp3.32inthemajorityofcladeIIITAARs(supplemen- sive to other chemicals including aldehydes, ketones, acids,
taryfig.S6C,SupplementaryMaterialonline). esters,andalcohols(fig.4A).Theligandprofileincludeslong
In addition, it was previously reported that clade Ib alkylchainamines,suchashexylamineandheptylamine,and
TAAR2-4 mainly recognize primary and secondary amines, amineswhosenitrogenisconnectedtoasecondarycarbon,
but clade II TAAR5-9 mainly recognize tertiary amines suchasisopropylamineandcyclohexylamine(fig.4Bandsup-
(Ferrero et al. 2012). Here, we found that clade II TAARs plementary fig. S9B, Supplementary Material online). This
can also recognize a relatively large number of cluster 2 suggests that the binding pocket of TARLL4a is flexible in
ligandswithprimaryandsecondaryamines.Thisisconsistent itsvolumetofitthesizesofavarietyofligands.
with previous studies showing that TAAR3 and TAAR4 are Interestingly, TARLL3h recognizes tetramethyl-1,4-
more broadly tuned in vivo, and could recognize tertiary butanediamine and N,N,N0,N0-tetramethyl-1,3-propanedi-
amines as well (Zhang et al. 2013; Dewan et al. 2018). We amine, diamines with two amino groups (fig. 4C).
also found that clade Ib TAARs respond predominantly to RecognitionofdiaminesinTAARsinvolvestwoasparticacids,
cluster 3 ligands (amines with aromatic rings), and clade II Asp3.32 and Asp5.42 (Li et al. 2015; Sharma et al. 2016). Do
TAARs respond predominantly to cluster 5 ligands (tertiary However,althoughAsp3.32isretainedinTARLL3h,Asp5.42is wn
lo
amines) (supplementary fig. S7A and B, Supplementary not found, implying a distinct diamine recognition mecha- a
d
e
Materialonline). nism in TARLL3h. To investigate this further, we performed d
Anotherinterestingfindingisthefunctionalconvergence homology modeling and ligand docking in TARLL3h. In ad- fro
m
betweensharkTAARS1andTAAR5.SharkTAARS1appears ditiontoAsp3.32thatformsasaltbridgewithN,N,N0,N0-tet- h
closely related to clade Ia TAARs according to the phyloge- ramethyl-1,4-butanediamine or N,N,N0,N0-tetramethyl-1,3- ttp
s
netictree(fig.1A).However,theligandprofileofTAARS1is propanediamine, Tyr3.33 binds to the other amino group ://a
c
moresimilartoTAAR5incladeIIthantoTAARsincladeIa, through pi–cation interactions in TARLL3h (fig. 4D). ad
which mainly recognizes cluster 3 molecules (amines with Notably, the Tyr3.33 site is one of the well-conserved amino em
ic
aromaticrings). BamboosharkTAARS1bcanrecognizeone acidsintheTARLLclade(fig.1C).Toverifythedockingresults, .o
u
ligand from cluster 2 (primary or secondary amines), one we performed site-directed mutagenesis in TARLL3h. p
.c
ligand from cluster 3 (amines with aromatic rings), and five Mutation of Asp3.32 or Tyr3.33 to alanine (D3.32A or om
ligandsfromcluster5(tertiaryamines),whicharetheligands Y3.33A)completelyeliminatedthereceptoractivity,whereas /m
b
of TAAR5 as well (fig. 3A and B; supplementary fig. S8A, mutation of Tyr3.33 to phenylalanine (Y3.33F) retained pi– e/a
Supplementary Material online). We used the R package cation interactions and the receptor activity yet resulted in rtic
l1ou (Khabbazian et al. 2016) to search across the phyloge- slightly reduced affinity (EC50 three times higher than wild le/3
9
netic treefor well-supported shifts to newoptimainligand type receptor) (supplementary fig. S9C, Supplementary /3
response profiles (supplementary fig. S8B, Supplementary Materialonline). Theseresults suggest thepotential rolesof /m
s
Material online). A notable shift was indeed discovered in Asp3.32andTyr3.33ofTARLL3hindiaminerecognition. ac
0
shark TAARS1b following the divergence from clade Ia Moreover,TARLLsseemtoextendligandprofilesbeyond 06
/6
TAARS(supplementaryfig.S8B,SupplementaryMaterialon- the profiles seen in TAARs. TARLL1b can be activated by 5
0
line).ThisfunctionalconvergenceofTAARS1andTAAR5is indole and its derivatives, which are not ligands for any 35
0
intriguing:asTAAR5ligandsarepotentallomonesinmam- TAARs (fig. 4E and supplementary fig. S9D and E, 6
b
mals (Li et al. 2013), the ecological relevance of TAARS1b Supplementary Material online). Indole and its derivatives y g
ligandsforcartilaginousfisheswillbeinterestingtoexplore. areneutralinphysiologicalconditions,andarefoundtoac- ue
s
tivate members of odorant receptor family (Pfister et al. t o
n
IndependentDevelopmentandFunctional 2020). To probe the structural basis of indole recognition, 0
5
ConvergenceofTARLLandTAAR we performed homology modeling and ligand docking in A
p
Nstuexdti,ewsheaevxeasmugingeedstethdethTaAtRTLALRcLlaLdsearienemxporreessdeedtainil.tPhreeMvioOuEs PThAeR5L.3L81ibs t(hfieg.cr4itFic).alTshitee tdoocsktainbgilizeresinudltoslesuthggroeustgehdpti–hapti ril 20
2
2
andmediateolfactoryfunctioninthesealamprey(Scottetal. interactions with the aromatic rings of indole. This site is
2019).However,detailedanalysesofthosereceptorsarestill also important for recognition of indole derivatives such as
lacking. Here, we successfully deorphaned three lamprey 1-methylindole(fig.4F).
TARLLs(TARLL1b,3h,4a)outof28clonedandfunctionally
testedreceptors,andfurtherinvestigatedthestructuralbasis TARLandTAAR1PreservetheLigandProfilesofHTR4
in TARLLs for ligand recognition (supplementary table S3, andIndependentlyGainNewFunctions
Supplementary Material online). Expression patterns of the AsunderscoredbytheUMAPplot(fig.3B),TARL,HTR4,and
threerelatedTarllgenesdeterminedbyinsituhybridization TAAR1appeartohavesimilarligandprofilesandarecluster
intheMOEofsealampreyalsosupportsthehypothesisthat together (PAM group 2). Most of their ligands belong to
lampreyTARLLsmainlyfunctionasolfactoryreceptors(sup- cluster 3 ligands (amines with aromatic rings), which are
plementary fig. S9A, Supplementary Material online). mainly neurotransmitters and their derivatives (fig. 3A). In
TARLL4a has a broad ligand profile of amines with eight addition,theanalysisofexpressionlevelsinapanelofzebra-
ligands from cluster 2 (primary or secondary amines) and fish tissues demonstrates that these receptors are mainly
one ligand from cluster 6 (isopropylamine). The responses expressed in the brain, instead of in the MOE where other
8
Brain-ExpressedBiogenicAmineReceptorsintoOlfactoryTraceAmine-AssociatedReceptors . doi:10.1093/molbev/
MBE
msac006
A B
20 Sea lamprey TARLL4a
y Sea lamprey TARLL4a
vit
cti15 20 20
ReceptorA150 or Activity1105 H2N 1105 H2N
pt
0 ce5 5
No DliegcPNaylho nOenadcltnadyynll e alahalycddeeteIHhalseydopdtebeahultydydreeahlyddbeUeehnDynidzdheaeycldadCrnyeojachllayoBsdheemenoxznaoenpo2h-nenen3o3,o-n4nh-aeehn2peot-xncaaeonnuoenmdieaornaenMounsβNec-olononanOenocotniace DnaeoicciHca denApoticica dnaVociiacPd nrilaloicipciH doeaTnxciihiacd onagolicIiyc sdcaoolcbiicud tyarAVcililacld ylBe atuicpticAyhl ld epafGnchoieyralrd -atmaneBacrtyuletpe tiyalan tcylbee tuatatcyrEeettylhN altyelra eycplt yiartsuIHeovsebaoptuttbeyylor raantcylee Otacatctyeelt a(at-c)e-et(ma+te)e-ntmhe1o-lntb1hu-tolpaen1n-otlhaenEptotlhaParnnooleptah2inP-oehlptrehoinpoyllaent5o-hlamnIeton6l-hdyomlli7eent-dhmyolleitenhdylToilrnye3d-ptoAlmaeemtmiyhlBnyaluettmy5hiHli-neaoaxepmyrmilionanpeomy-il1na-epmienPnteutarCneaolsdciavneerine Re0 0 0P.3hen1yleth3yla1m0ine3 (0µM10)03000 0 1 B3utyla1m0in3e0 (µ1M00)3001000
Aldehyde Ketone Acid Ester Alcohol Amine
C D
Sea lamprey TARLL3h TARLL3h TARLL3h
8
y N,N,N’,N’-tetramethyl-1,4-butanediamine N,N,N’,N’-tetramethyl-1,3-propanediamine
or Activit 46 N N Dow
Recept 2 Y3.33 nload
e
0 d
N,N0,N',N1'-tet3ram1e0thy3l-01,41-b0u0ta3n0e0d1ia0m00ine (µM) Y3.33 D3.32 fro
m
4 h
Activity 3 N N D3.32 ttps://a
ptor 2 cad
ece 1 em
R ic
0 .o
0 1 3 10 30 1003001000 u
N,N,N',N'-tetramethyl-1,3-propanediamine (µM) p
.c
o
E F m
Sea lamprey TARLL1b /m
TARLL1b TARLL1b
ceptor Activity1122305050 HN indoleF5.38 1-methylindolFe5.38 be/article/39/3
Re 5 /m
s
0 a
00.10.3 1 3 10 100 1000 c0
Indole (µM) 06
6 /65
0
y N 3
ctivit 4 506
or A D3.32 by
pt 2 D3.32 g
ece ue
R s
00 0.10.31 3 10 100 1000 t on
1-methylindole (µM) 0
5
FIG.4.StructuralbasisforligandrecognitionofTARLLs.(A)ResponsesofsealampreyTARLL4atodifferentgroupsofchemicalsincludingaldehyde, Ap
ketone,acid,ester,alcohol,andamines(pinkcolor).(B)Dose-dependentresponsesofTARLL4atophenylethylamineandbutylamine.(C)Dose- ril 2
dependentresponsesofTARLL3htoN,N,N0,N0-tetramethyl-1,4-butanediamineandN,N,N0,N0-tetramethyl-1,3-propanediamine.(D)Homology 02
2
modelingandliganddockingforTARLL3hbindingtotwoligands,N,N,N0,N0-tetramethyl-1,4-butanediamine(left)andN,N,N0,N0-tetramethyl-1,3-
propanediamine(right).BothAsp3.32andTyr3.33werefoundtointeractwithligands.(E)Dose-dependentresponsesofTARLL1btoindoleand1-
methylindole.(F)HomologymodelingandliganddockingforTARLL3hbindingtoindoleand1-methylindole.Phe5.38wasfoundtoplayarolein
ringrecognitionofindolethroughpi–piinteractions.
TAARs and TARLLs are expressed (Liberles and Buck 2006; cluster 2(primary or secondary amines), 11moleculesfrom
Scottetal.2019)(fig.5A).Theexpressionpatternandligand chemical cluster 3 (amines with aromatic rings), and two
profileofTARLssuggestapossiblefunctionasaneurotrans- molecules from chemical cluster 5 (tertiary amines). We
mitter receptor in the brain rather than as peripheral then compared the responses of HTR4 from mouse and
chemosensors. zebrafish,TARL1fromzebrafish,catfish,fugu,medaka,spot-
Tobettercomparetheirligandprofiles,weselectedthe16 ted gar, and coelacanth, TARL2 from coelacanth and shark,
ligandsthatactivateHTR4,TARL,andTAAR1inourscreen- TAAR1frommouseandzebrafishtothe16ligandsat10mM
ing assay (supplementary table S3, Supplementary Material (fig.5B).Ingeneral,allreceptorsshowedhighactivationlevels
online). This set included three molecules from chemical tothefourHTR4ligands,suggestingfunctionalconservation
9
MBE
Guoetal. . doi:10.1093/molbev/msac006
A
es 300 Htr4 2000 Tarl 250 Taar1 100 Taar10a 15000 Gapdh
NAcopiRNA 200 11050000 125000 6800 10000
ofcD10ng 100 500 15000 4200 5000
erer 0 0 0 0 0
Numbp Brain MOEHeartLiverKidney Brain MOEHeartLiverKidney Brain MOE HeartLiverKidney Brain MOEHeartLiverKidney Brain MOE HeartLiverKidney
B C
HTR4 TARL1 TARL2 TAAR1 20
2 Hexylamine
2 Octylamine 10~20 Fugu TARL1 Receptor
2 Heptylamine HTR4
5~10 TAAR1
3 Tryptamine TARL1
Spotted gar TARL1 TARL2
3 Phenylethylamine 2~5 Coelacanth TARL1 D
33 T3-ymraemthinoexytyramine 0~2 %)0.5 Medaka TARL1 5−methox5y−−mSNee,rtNho−otodxnyimitnreypthtyalmtryinpetamine Lig235and cluster ownloa
3 5-methoxytryptamine 0 9.5 Zebrafish TARL1 de
33 45--mmeetthhooxxyyp-Nh,eNn-edtihmyelathmyilntreyptamine PC2 (1 Zebrafish HTR4c d from
3 Gramine 0.0 Coelacanth TAARC1b h
3 Phenylethanolamine N,N−dimethylphenethylamine ttp
3 Dopamine CaPt4fih−sehmn eyTtlAehtoRhxLyy1lapmheinneethylamine Shark TAZReLb2rafish TACAoRe1lacanth TARL2 s://a
3 Serotonin Mouse HTR4 ca
5 N,N-dimethylphenethylamine Mouse TAAR1 3−methoxytyramine de
m
5 N,N-dimethylbenzylamine −1 0 1 ic
MoZuesber ZaeHfibTsrhRa f4iHsCTh aRtTf4iAsRh LFT1uMAgeSRudp LaoTt1ktAaeC RdoT Leg1AlaaRrC cLoaT1enlAtahRc LaCT1noAtelhRS aLhTc1aAarknR tLhT 2AMTRoAZLuAse2Reb rCTa1fiAbsAh RT1AAR1 PC1 (70.9%) .oup.com/m
b
D TAAR1 only HMuomusaen E Clade Ib Clade II e/article
TARL &TAAR1 Chicken /39
TARL only Alligator Clade Ia TAA /3/m
Frog RS2 sa
Coelacanth c
0
0
Spotted gar TAAR1 6
/6
Goldfish TARLL 50
Zebrafish TAARS1 3
5
0
Catfish 6
Medaka TARL by
Fugu HTR4 gu
e
Pufferfish st o
Whale shark Clade III n
Ghostshark Outgroup 05
A
Sea lamprey p
Hagfish Brain-functioning aminergic receptors ril 2
HTR4 TARL1 TARL2 TAAR1 022
Olfactory-functioning aminergic receptors
FIG.5. LigandprofilesandexpressionpatternsofHTR4,TARL,andTAAR1.(A)qPCRanalysisofdifferentreceptorsinzebrafishtissues.Gapdh
servesasacontrol.(B)ResponseofHTR4,TARL,TAARC1,andTAAR1to16ligandsat10lM.Theheatmapisbasedontheratiooffluorescence
intensityofligand–receptorinteractionandnoligandcontrolsintheluciferaseassay.TAARC1isalsoincludedbecauseitresidesincladeIacloseto
TAAR1andshowsasimilarligandprofiletoHTR4,TARL,andTAAR.(C)PhyloPCAanalysisontheresponsesofHTR4,TARL,TAARC1,andTAAR1
tothese16ligands.Inthisanalysis,PC1explains70.9%ofthevarianceandPC2explains19.5%ofthevariance.Dotscoloredbyreceptorclades
representtheligandresponsesofreceptorsonthefirsttwoPCaxes.Ligandeigenvectorsarerepresentedbylinesandcoloredaccordingtothe
ligandclusters.Aligandnameisshownatthetipofthelineifitseigenvectorhasahighercontribution(comparedwithotherligands)toeitherPC1
orPC2.RedandbluedashedcirclesindicatetwogroupsofreceptorsseparatedbyPC1andPC2.(D)ThepresenceofHTR4,TARL1,TARL2,and
TAAR1 in several representative species in vertebrates is plotted. (E) A model for the evolution and functional diversification of aminergic
receptors.Thecladeslabeledingreen(includingTARLL,TAARS1,TAARS2,thenon-TAAR1partofcladeIa,cladeIb,cladeII,andcladeIIITAARs)
playimportantrolesinolfaction.Thecladeslabeledinred,includingHTR4,TARL,andTAAR1arelikelytodetectamineneurotransmittersand
traceaminesinthebrain.
10