ORA1, a Zebrafish Olfactory Receptor Ancestral to All Mammalian V1R Genes, Recognizes 4 ...

12 Pages·2014·2.26 MB·English

THEJOURNALOFBIOLOGICALCHEMISTRY VOL.289,NO.28,pp.19778–19788,July11,2014
©2014byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PublishedintheU.S.A.
ORA1, a Zebrafish Olfactory Receptor Ancestral to All
Mammalian V1R Genes, Recognizes 4-Hydroxyphenylacetic
Acid, a Putative Reproductive Pheromone
Receivedforpublication,April10,2014 Published,JBCPapersinPress,May15,2014,DOI10.1074/jbc.M114.573162
MaikBehrens‡,OliverFrank§1,HarshadraiRawel¶1,GauravAhuja(cid:2)1,ChristophPotting(cid:2),ThomasHofmann§,
WolfgangMeyerhof‡,andSigrunKorsching(cid:2)2
Fromthe‡DepartmentofMolecularGenetics,GermanInstituteofHumanNutritionPotsdam-Rehbruecke,Arthur-Scheunert-Allee
114-116,14558Nuthetal,the§ChairofFoodChemistryandMolecularSensoryScience,TechnischeUniversita¨tMu¨nchen,Lise-
Meitner-Strasse34,85354Freising,the¶InstituteofNutritionScience,UniversityofPotsdam,Arthur-Scheunert-Allee114-116,
14558Nuthetal,andthe(cid:2)InstituteofGenetics,UniversityatCologne,Zu¨lpicherStr.47A,50674Cologne,Germany
Background:Noligandsareknownforanyolfactoryreceptorofthev1r-relatedoragenefamily.
Results:ZebrafishORA1recognizeswithhighsensitivityandspecificity4-hydroxyphenylaceticacid.Thiscompoundelicits
ovipositionbehavior.
Conclusion:ORA1wasdeorphanizedwithaligandthatmaybeareproductivepheromone.
Significance:Pheromonereceptionconceivablymightbetheancestralfunctionoftheora/v1rfamily.
Theteleostv1r-relatedoragenesareasmall,highlyconserved andhighlyconservedolfactoryreceptorgenefamilyofjustsix
olfactory receptor gene family of only six genes, whose direct oragenes(6)constitutestheancestralrepertoire,fromwhich
orthologuescanbeidentifiedinlineagesasfarasthatofcarti- the large and dynamically evolving mammalian v1r families
laginous fish. However, no ligands for fish olfactory receptor originate. All mammalian v1r genes are monophyletic with a
classArelatedgenes(ORA)hadbeenuncoveredsofar.Herewe singlepairoforagenes,ora1andora2(6),whosedirectortho-
have deorphanized the ORA1 receptor using heterologous loguesarepresentalreadyincartilaginousfish(7).Inthelightof
expression and calcium imaging. We report that zebrafish suchdrasticdifferencesinevolutionarycharacteristicsforv1r
ORA1 recognizes with high specificity and sensitivity 4-hy- and v1r-related ora genes it would be interesting to compare
droxyphenylaceticacid.Thecarboxylgroupofthiscompoundis ORA ligands to those found for V1Rs. It is conceivable that
required in a particular distance from the aromatic ring, ligandsoftheslowlyevolvingoragenescouldbeclosertothose
whereasthehydroxylgroupinthepara-positionisnotessential, ofancestralv1rgenesthantheligandsofcontemporary,rapidly
butstronglyenhancesthebindingefficacy.Lowconcentrations evolvingv1rgenesthemselves.
of4-hydroxyphenylaceticacidelicitincreasesinovipositionfre- Thezebrafisholfactorysystemiswellcharacterized(Ref.8
quency in zebrafish mating pairs. This effect is abolished by andreferencestherein),andsowechosezebrafishoragenesfor
narisclosure.Wehypothesizethat4-hydroxyphenylaceticacid cloningandexpressioninamammaliancellline.Activationof
might function as a pheromone for reproductive behavior in thereceptorswasanalyzedbycalciumimaging,usingavariety
zebrafish.ORA1isancestraltomammalianV1Rs,anditsputa- ofplausibleassumptionsastopotentialligands.Althoughnone
tivefunctionaspheromonereceptorisreminiscentoftheroleof oftheseassumptionswereborneout,eventuallyoneleduson
severalmammalianV1Rsaspheromonereceptors. the track for a high affinity ligand of ORA1. We report the
structure-activity tuning of ORA1, and show that the most
effectiveligand,4-hydroxyphenylaceticacid,modulatesrepro-
Pheromonesplayessentialrolesinmanyintraspeciescom- ductivebehaviorofzebrafish.
munications,frommatingpreferencestocontrolofaggression
EXPERIMENTALPROCEDURES
and individual recognition. Chemical signaling also occurs
between species, e.g. for prey or predator detection. In mam- Cloning of Zebrafish ORA1 and ORA2 cDNA—ora1 and
malstwolargegenefamiliesarethoughttobemainlyrespon- ora2 are monoexonic genes, whose full-length coding
siblefordetectionofthesesignals,vomeronasalreceptorstype sequences wereamplifiedfromzebrafishgenomicDNA,using
1andtype2(V1RandV2R).3V2Rreceptorshavebeenshown thefollowingprimers:ORA1_FW5(cid:2)-TAGAATTCATGGACCT-
to recognize peptides (1, 2), whereas V1R ligands are found GTGTGTCACCA-3(cid:2), ORA1_RV 5(cid:2)-ATAGTTTAGCGGCCG-
amonglowmolecularweightmolecules,suchassteroids(Refs. CCGTTCTTGCCGCTGGAGTT-3(cid:2), ORA2_FW 5(cid:2)-CGGAAT-
3and4,seealsoRef.5).Wehaverecentlyshownthatasmall TCATGATTGCGGAGG-CTGTG-3(cid:2), ORA2_RV 5(cid:2)-ATA-
GTTTAGCGGCCG-CCGTGCATGGTCTCTGGCTG-3(cid:2).
Forward primers contain a 5(cid:2) EcoRI site and reverse primers
1Theseauthorscontributedequallytothisarticle. containaNotIsite.AfterPCRamplification,reactionproducts
2Towhomcorrespondenceshouldbeaddressed.Tel.:[phone];Fax: weredigestedwithEcoRIandNotI,andclonedintotheEcoRI
49-221-470-5172;E-mail:[email protected].
3The abbreviations used are: V1R and V2R, vomeronasal 1 receptor and and NotI sites of the modified vector pcDNA5FRT PM (9),
vomeronasal2receptor. therebyaddinganamino-terminalsst3epitopetofacilitateeffi-
This is an Open Access article under the CC BY license.
19778 JOURNALOFBIOLOGICALCHEMISTRY VOLUME289•NUMBER28•JULY11,2014
DeorphanizationofZebrafishOlfactoryReceptorORA1
cient cell surface localization and a carboxyl-terminal HSV OxidationofL-TyrosineandRelatedCompoundswithHydro-
(herpessimplexvirusglycoproteinD)tagtoenableimmunolo- genPeroxide—Todeterminewhetheroxidativeprocesseshave
gicaldetectionofthereceptors. resultedintheformationofagonisticcompoundsoriginating
Immunocytochemistry of ORA Constructs Transfected into from“aged”L-tyrosine,weincubatedcandidatesubstanceswith
HEK293Cells—ImmunocytochemicaldetectionofHEK293T hydrogenperoxidesolution.100mgoffreshlyorderedL-tyro-
cellsstablyexpressingtheGprotein-chimeraG(cid:2)16gust44was sineprovedtobeinactiveonORA1-transfectedcellsinfunc-
mainly done as described previously (10). Briefly, cells were tional calcium imaging experiments, as well as the same
seeded onto poly-D-lysine-coated glass coverslips in 24-well amount of L-dopamine and L-phenylalanine were mixed with
250(cid:3)lof30%hydrogenperoxidesolution(becauseoflimited
plates.Cellsweretransientlytransfectedwithconstructscod-
ingforORA1orORA2usingLipofectamine2000(Invitrogen), solubility600(cid:3)lwereusedforL-phenylalanine)andincubated
forseveralhoursatroomtemperature.Afterthis,thesamples
andincubatedfor24hat37°Cin5%CO .Next,thecellswere
2 were subjected to brief centrifugation, the supernatants were
washedtwicewithPBSandplacedfor30minonicetoblock
dilutedatleast10,000-foldandthentakenforsubsequentfunc-
endocytosis.Forcellsurfacelabelingbiotinylatedconcanavalin
tionalanalyses.TheremainingH O (atmost1mMor0.003%)
A(Sigma)wasappliedatadilutionof1:2,000(1honice).Repet- 2 2
hadnoeffectbyitself.
itivewashingwithice-coldPBSwasfollowedbymethanol/ac-
HPLC Purification of Aged L-Tyrosine—Both analytical and
tonefixation(1:1(v/v))for2min.FollowingwashingwithPBS
preparative RP-HPLC of the samples was performed on a
at room temperature, cells were incubated with 5% normal PRONTOSIL120-3-C18,SC150-ace-EPScolumn(150(cid:6)4.6
horse serum in PBS. Receptor proteins were detected with a mm, 3 (cid:3)m; Bischoff Analysentechnik und -geraete GmbH,
1:15,000 diluted anti-HSV antibody applied for 1 h atroom
Leonberg,Germany)usingaflowrateof0.8ml/min,UVdetec-
temperature in blocking reagent (5% normal horse serum in
tion at 280 nm, and a column temperature of 25°C with a
PBS). Excess antibodies were removed by washing with PBS,
JASCO(LaborundDatentechnikGmbH,Gross-Umstadt,Ger-
before1:2,000dilutedanti-mouseAlexa488and1:1,000diluted many)chromatographicsystem.Theseparationisbasedonthe
streptavidinAlexa633inPBS(cid:3)5%normalhorseserumwas hydrophobicinteractionsoftheanalyteswiththereversephase
appliedfor1hatroomtemperature.Finally,theglasscoverslips fillingofthecolumn.Adistilledwater(acidifiedwith2%acetic
werewashedthreetimeswithPBS,oncewithdeionizedH O, acid; v/v)/methanol gradient was applied under the following
2
and mounted with DAKO fluorescent mounting medium conditions: 0–20% methanol, 2 min; 20–35% methanol, 18
(DAKO).Imagesweretakenbyconfocallaserscanningmicros- min;35–68%methanol,2min;68%methanol,3min;0–68%
copy(LeicaTCSSP2).Fordeterminationofexpressionrates3 methanol,3min;0%methanol,12min(regeneration/equilibra-
representativeimagesperconstructweretakenandthenum- tion).Thetyrosinesamples(10mg/ml)weredissolvedindis-
berofgreen((cid:4)receptorexpressing)andred((cid:4)totalcellnum- tilledwater.Theconcentrationwasdecreasedincaseofana-
ber)cellswerecounted. lytical HPLC. The injection volume of the samples was
Functional Calcium Imaging Experiments—The functional 10–20(cid:3)l.Altogether10fractionswerecollected.Fraction2
calciumimagingexperimentswereperformedaccordingtoRef. was identified to contain pure tyrosine using an external
10. Briefly, HEK 293T cells stably expressing the G protein- standardandHPLC-MS(ShimadzuchromatographicLC-10
chimera G(cid:2)16gust44 were seeded onto 96-well plates coated system equipped with a mass spectrometer LC-MS 2010 EV,
with10(cid:3)g/mlofpoly-D-lysine.Thenextdaycellsweretran- Kyoto, Japan; MS conditions were as follows: Interface ESI,
CDL temperature/heating block (cid:4) 230°C, nebulizing gas
siently transfected with ORA1, ORA2, or human bitter taste
flow (cid:4) 1.5 ml/min; detector voltage (cid:4) 1.7 kV; interface volt-
receptorTAS2R16constructsusingLipofectamine2000(Invit-
age (cid:4) 4.5 V; CDL voltage (cid:4) 0. (cid:4) V; Q-array voltage, DC (cid:4)
rogen).After24hthecellswerewashedwithbufferC1(130mM 20–30V,R (cid:4)85–125V;scanmodus,eventtime(cid:4)0.8s,m/z(cid:4)
NaCl, 5 mM KCl, 10 mM Hepes, pH 7.4, 2 mM CaCl , 10 mM F
2 120–550)performedunderthesameseparationconditionsas
glucose), and loaded with the calcium-responsive dye Fluo-4
describedabove.Thecompoundinfraction6couldnotbeion-
AMinthepresenceof1mMprobenecide,aninhibitorofABC
izedundertheappliedMSconditions.Thetotalpeakareaofthe
transporter A1. To remove excessive fluorescence dye, cells
fractionswasusedtoestimatetheamountinfractions2and6
werewashedthreetimeswithbufferC1andtransferredintoa
usinganexternaltyrosinestandard.Therelativecomposition
fluorometricimagingplatereader(FLIPR,MolecularDevices)
wasdeterminedtobe67.6and5.2%,respectively.
formeasurement.TestsubstancesweredissolvedinC1buffer
Nuclear Magnetic Resonance (NMR) Spectroscopy—One-
and the changes in fluorescence after application of test sub- andtwo-dimensional1Hand13CNMRspectrawereacquired
stancesweremonitored.Forthecalculationofdose-response
ona500MHzAvanceIIIspectrometer(Bruker,Rheinstetten,
functionsdatafromatleasttwoindependentexperimentswere Germany),respectively.Dimethylsulfoxide-d MeOD(9:1,v/v)
6
obtained.Foreachexperimentthesignalsoftriplicatewellsfor wasusedassolventandchemicalshiftsarereportedinpartsper
eachconcentrationwereaveragedandthecorrespondingfluo- millionrelativetothesolventsignal.Homo-andheteronuclear
rescence changes of mock-transfected cells were subtracted. correlation experiments were carried out using the pulse
Graphs and calculations of EC concentrations were per- sequences taken from the Bruker software library. Data
50
formed using SigmaPlot. For the EC value determination processing was performed by using TopSpin (2.1; Bruker,
50
nonlinear regression analysis was performed using the equa- Rheinstetten, Germany) as well as Mestre-C (Mestrelab
tion:f(y)(cid:4)(a(cid:5)d)/1(cid:3)(x/EC )nh(cid:3)d. Research,ACorun˜a,Spain).
50
JULY11,2014•VOLUME289•NUMBER28 JOURNALOFBIOLOGICALCHEMISTRY 19779
DeorphanizationofZebrafishOlfactoryReceptorORA1
Behavioral Assays—Analysis of aversion or attraction was 1A).Thus,anessentialprerequisiteforfunctionalcharacteriza-
performedasdescribed(11).Individualadultzebrafish(Ab/Tu¨ tionisfulfilledforbothORA1andORA2.
strain,6–8monthsold)weretestedinanelongatedtank(10(cid:6) ORA1 but Not ORA2 Reacts to a Mixture of Amino Acids—
100cm,9litersfreshfilteredwater)after45minofhabituation. Previousstudieshavedemonstratedthataminoacidsandpher-
Fishmovementswerevideorecorded(30frames/second)from omonesrepresentpreferredolfactorystimuliforfish(21–23).
thesidefor5minbeforeandafterstimulusaddition(180(cid:3)lof Wehavetestedbothtypesofpotentialligands,usingaspositive
odorortankwaterascontrol)byanexperimenternotvisibleto controlcellsexpressingthebittertastereceptorTAS2R16stim-
thefish.Fishmovementswerevideorecordedfor5minbefore ulatedwiththebittercompoundD-((cid:5))-salicin(9).Noneofthe
andafterstimulusaddition(180(cid:3)lof1mM4-hydroxyphenyla- pheromonestested,amongthemthoseknowntoactivatesome
ceticacidorwater)andtrackedusingLoliTrackversion3auto- zebrafishglomeruli(21),couldactivateeitherORA1orORA2
mated motion tracker (12). Distance to the odor source and (Table1).
velocity were determined using Open Office (Apache). Mean Incontrast,themixtureofall20proteinogenicL-aminoacids
displacementwascalculatedasadifferenceofaveragelocation elicitedastrongcalciumsignalforORA1-transfectedcellsat1
andexpressedaspercentoftotaltanklength.Significancewas mM concentration per amino acid with a time course resem-
evaluatedbyStudent’sttest(two-sided,unpaired). blingthatofthepositivecontrol(Fig.1B).Stimulationoftrans-
Toexamineoviposition(egglaying),zebrafishwerekeptgen- fectedcellswithbufferalonehadnoeffect,andlikewisestimu-
der-separated for 1–2 weeks prior to the experiment. In the lationofmock-transfectedcells(emptyexpressionvector)with
evening preceding the experiment the breeding pairs were the full 20 amino acid mixture elicited no response (Fig. 1B).
gently transferred to breeding tanks (20 (cid:6) 10 cm, 600 ml of Thus the signal obtained for ORA1 appears to be a specific
water),withfemaleandmaleseparatedbyatranslucentdivider. receptor-mediatedresponsetotheaminoacidmixture.Ora2-
Thenextmorning,one-halfhourintothelightcyclethedivider transfectedcellswerenotactivatedbythemixture(Fig.1B).
was removed allowing the fish free movement. Pairs without ORA1 Activation Is Caused by a L-Tyrosine Contaminant—
eggs after the 90-min contact time were then supplemented Toidentifywhichofthe20L-aminoacidsareabletoactivate
with various concentrations of 4-hydroxyphenylacetic acid in ORA1,wenextseparatelyusedeachoftheaminoacidsatthe
10mMTris,pH7.4,orwithbufferalone.Thepairsweremon- same concentration (1 mM) (Fig. 1C). Only one amino acid,
itoredforthepresenceofeggs90minafterthestimulus.For tyrosine, mimicked the response elicited by the mixture of
generationoftransientlyanosmicfish,bothnostrilswereglued aminoacids,whereastheother19aminoacidshadnoeffect
with Histoacryl(cid:2) (Braun) and the fish were allowed a resting (Fig. 1C), suggesting that ORA1 activation in the previous
periodof24hbeforebeingtested.Anosmicfishshowednormal experimenthadbeensolelyduetothepresenceoftheL-ty-
motility.Significancewasestimatedby(cid:4)-squareanalysis. rosinereagent.
However, when we attempted to validate this result using
RESULTS
otherlotsoftyrosinewedidnotobserveanyactivationofORA1
ORA1andORA2AreEfficientlyExpressedandLocalizetothe (Fig.2A).ThisexcludesL-tyrosineasligandandsuggestsacon-
PlasmaMembrane—Wehavechosenzebrafishasthespecies taminantastheactivecompound.Becausethefirst,activelot
tosearchforORA1/ORA2agonists,becauseinthisspeciesthe hadagedforaprolongedperiodat(cid:3)4°Candindeedexhibited
expressionofallorafamilymembersinolfactorysensoryneu- anoff-color,wesuspectedcontaminationbysomedegradation
rons has been shown (6). For heterologous expression we product as the active ingredient, which presumably was only
selected a system that has been very efficient for functional present in trace amounts and therefore likely to be a high
expressionofbittertastereceptors(13),whicharetheclosest potencyagonist.
homologuesoftheora/v1rfamily(6).Inshort,thefull-length TheActiveCompoundIsGeneratedbyOxidationofTyrosine—
receptorsequenceisfusedtoanN-terminalsst3tagservingas We first examined whether the active ingredient might have
signal sequence (14) and a C-terminal hsv-epitope to enable formedbyoxidationoftyrosine,aprocessexpectedtohappen
detection (15). The constructs were transiently expressed in uponprolongedstorage.WereactedfreshL-tyrosinewith30%
HEK293TcellsstablytransfectedwiththebroadlyreactiveG hydrogenperoxideandtestedthereactionproductatdifferent
protein,G(cid:2)16gust44(16,17).Gprotein-coupledchemorecep- dilutions.Indeed,evenat1:10,000dilutionastrongsignalwas
tors are sometimes poorly transported to the plasma mem- generated,similartothesignalelicitedbytheagedtyrosinelot
braneinheterologoussystems(seeRefs.18–20),andwethere- (Fig. 2A), and in stark contrast to the complete inability of
foreanalyzedtheintracellulardistributionofORA1/ORA2by thefreshtyrosineitselftoactivateORA1(Fig.2A).Whenthe
immunocytochemicaldetection.Thereceptorswerevisualized closely related substances L-dopamine and L-phenylalanine
usinganantibodyagainsttheC-terminalhsv-epitopeandthe weresubjectedtothesameoxidationprocedure,noresponse
cellsurfacewasstainedbyconcanavalinA,whichservesbothas waselicitedeitherforthefresh(Table1)ortheoxidizedcom-
general cell marker and as label for plasma membrane (for pounds(Fig.2A).Furthermore,hydrogenperoxidebyitselfhad
detailssee“ExperimentalProcedures”). noeffect(Fig.2A).Theoutcomeofthisexperimentsuggested
BothORAreceptorsarerobustlyandreproduciblyexpressed. that the active substance in the aged L-tyrosine indeed origi-
Nearlytwo-thirdsofallcellsexpresseachreceptor(Fig.1A),anda natedfromoxidationofL-tyrosine.
largefractionoftheexpressedproteinappearstobelocalizedat Purification and Identification of the Active Compound by
theleveloftheplasmamembraneasseenbythesuperposition HPLCandLC-MS—Toidentifytheactivecompoundorcom-
ofthereceptorsignalswiththoseofthecellsurfacelabel(Fig. pounds, we first performed an analytical HPLC separation of
19780 JOURNALOFBIOLOGICALCHEMISTRY VOLUME289•NUMBER28•JULY11,2014
DeorphanizationofZebrafishOlfactoryReceptorORA1
FIGURE1.ActivationofORAreceptorsbyaminoacids.A,ORAreceptorslocalizetotheplasmamembrane.HEK293Tcellsweretransfectedasindicated,and
receptorproteinwasdetectedwithanantibodyagainsttheC-terminalHSVepitopeandAlexa488-coupledsecondaryantibody(green,upperrow).Cell
surfacesarevisualizedusingbiotinylatedconcanavalinAincombinationwithstreptavidin/Alexa633(redlabel,middlerow).Yellowcolorintheoverlay(bottom
row)indicatescloseproximityofreceptorswiththeplasmamembrane.Insetsshowgroupsofcellsmagnifiedbyafactorofthree.Notetheabsenceofgreen
signalsincellstransfectedwithemptyvector(thirdcolumn).Cellsexpressingtheoragenes(green)werecountedandexpressedaspercentageoftotalcells
(red)(cid:7)S.D.Scalebar,75(cid:3)m.B,ORA1butnotORA2isactivatedbyanaminoacidmixture.HEK293TcellsweretransientlytransfectedwithORA1,ORA2,
TAS2R16,oremptyvector,loadedwithFluo-4AM,andstimulatedwithamixtureofall20proteinogenicaminoacids(finalconcentration1mMeach).Changes
incalciumlevelswereanalyzedinafluorometricimagingplatereader.OnlyORA1-transfectedcellsshowaresponse,comparableinsizetothatofthepositive
control(TAS2R16and10mMoftheagonistD-((cid:5))-salicin(9)).Thus,ORA1appearstobespecificallyactivatedbyoneorseveralaminoacidspresentintheamino
acidmixture.C,ORA1-expressingcellsonlyrespondtotyrosine.HEK293TcellsweretransientlytransfectedwithORA1oremptyvector,loadedwiththe
calcium-sensitivedyefluo-4AM,andanalyzedinafluorometricimagingplatereaderbeforeandafterapplicationofsingleaminoacidsat1mMconcentration
each.Duplicatewells,oneofwhichisshownhere,weretestedforeachexperimentalcondition.Nochangeinfluorescencewasseenforuntransfectedcells
(middlecolumn)orcellstransfectedwithemptyvector(rightcolumn).TheORA1responsetotyrosineiscomparableinsizeandtimecoursetothatofthebitter
tastereceptorTAS2R16toastrongagonist(D-((cid:5))-salicin,10mM(9)).
agedtyrosineusingawater/methanolgradientforelutionfrom resulting in an elemental composition of C H O . LC-MS
8 8 3
areversedphaseC column(Fig.2B,fordetailssee“Experi- revealed an intense pseudo molecular ion [M-H](cid:5) with m/z
18
mental Procedures”). Ten major peaks were collected, dried 151.0andadditionalLC-MS/MSexperimentsshowedadaugh-
down,re-dissolvedin200(cid:3)lofH O,andtestedat1:6dilution terionm/z107.0,supportingthecleavageofamoleculeofCO ,
2 2
byfunctionalcalciumimagingofORA1-expressingcells(Fig. i.e.thepresenceofacarboxylicgroup.Analysisofthe1HNMR
2C).ORA1wasactivatedbyasinglepeakcontainedinfraction spectroscopicdatashowedatotalof3resonancesignals,twoof
6,andnosignalswereobservedinallotherfactions,consistent themaromatic(6.67and7.02ppm)andallthreesignalsresult-
withasinglecompoundunderlyingtheobservedactivationby ing from 2 protons each. The aromatic protons showed the
aged tyrosine. Fraction 6 amounted to 5.2% of the total peak typicalcouplingpatternofanAA(cid:2)XX(cid:2)spinsystemofapara-
area,i.e.itconstitutesaminorcomponentofagedtyrosine. substituted aromatic ring. The third signal at 3.37 ppm was
Toobtainenoughmaterialforstructuredeterminationthe shownbyDEPT-135aswellasheteronuclearsinglequantum
HPLCpurificationwasscaleduptoobtainabout10mgofpuri- coherenceexperimentstobederivedfromamethylenegroup.
fiedfraction6.TheexactmasswasdeterminedbyLC-TOF/MS The complete assignment of the structure was achieved by
JULY11,2014•VOLUME289•NUMBER28 JOURNALOFBIOLOGICALCHEMISTRY 19781
DeorphanizationofZebrafishOlfactoryReceptorORA1
TABLE1
BindingspectrumofORA1
19782 JOURNALOFBIOLOGICALCHEMISTRY VOLUME289•NUMBER28•JULY11,2014
DeorphanizationofZebrafishOlfactoryReceptorORA1
FIGURE 3. Concentration-dependent saturable activation of ORA1 by
4-hydroxyphenylacetic acid. HEK 293T cells transfected with ORA1 or
emptyvectorwerestimulatedwithdifferentconcentrationsof4-hydroxy-
phenylaceticacid,andcalciumresponseswereimaged.Foreachconcentra-
tionanaveragetraceofthreeidenticallytreatedwellsisshown(leftpanel).No
responseisseenforORA1-transfectedcellsstimulatedwithbufferandfor
emptyvector-transfectedcellsinallconditions(bottompanels).Notethat
ORA1-transfectedcellsareatleast10-foldmoresensitiveto4-hydroxy-
phenylaceticacidthantoagedtyrosine.Scalebar:yaxis,arbitraryunits;x
axis,2min.
suggestingthisacetylgroupasringsubstituent.Thesignalat
155.8ppmiswellinlinewithaquaternarycarbonsubstitutedin
the para-position with a phenolic system and showed the
expected correlations with protons H-C(2,2(cid:2)) and H-C(3,3(cid:2)),
seeFig.2Bfornomenclature.Takingallspectroscopicdatainto
consideration,thedegradationproductofL-tyrosinecouldbe
FIGURE2.ORA1isactivatedby4-hydroxyphenylaceticacid,acontami- unequivocally identified as 4-hydroxyphenylacetic acid (Fig.
nantofagedtyrosine.A,toprow,cellsweretransfectedwithORA1,and
challengedwith100(cid:3)Mofdifferentstimuliasindicated.Calciumtracesare 2B).Furthermore,spectroscopicdataofcommerciallyavailable
averagedfrom3wells.Onlyagedtyrosine(firstcolumn),i.e.thetyrosinelot 4-hydroxyphenylaceticacidshowedanexactmatchwiththose
usedinFig.1,andoxidizedfreshtyrosine(thirdcolumn)wereactive,butfresh
ofthefraction6compound.
tyrosineitself(secondcolumn)wasnot.Hydrogenperoxideitself,L-dopamine
(L-DOPA),andL-phenylalanine(L-Phe)hadnoeffect.Bottomrow,cellstrans- Validation of 4-Hydroxyphenylacetic Acid as ORA1 Ligand—
fectedwithemptyvectorshowednoresponsetoanyofthestimuli.Shown Calcium imaging of ORA1-transfected cells was performed
arecalciumtracesaveragedfrom2wells.Scaling:yaxis(cid:4)relativeunits,x
axis(cid:4)1min.B,asingleactivecompoundisdetectedinagedL-tyrosine.100 withdifferentconcentrationsofcommercial4-hydroxypheny-
(cid:3)gofagedtyrosinewasseparatedbyHPLCfractionationusingawater/meth- lacetic acid in the 3–300 (cid:3)M range. Responses were dose-de-
anolgradientwithUVdetectionat280nm.Tenfractionscorrespondingto pendentandsaturatedat30(cid:3)M,atleastanorderofmagnitude
peakabsorbancerates(labeled1–10)werecollectedatthefollowingtime
pointsduringtherun:1,3.4–5.6min;2,6.0–6.9min;3,7.5–8.2;4,8.2–9.2min; lowerthantheoriginalsource,agedtyrosine(Fig.3).Thisratio
5,9.2–10.2min;6,10.7–11.5min;7,13.7–14.9min;8,15.5–17.4;9,20.6–23 wasconsistentwiththeresultfromtheHPLCseparation,which
min;10,25–29.5min.yaxis,absorbanceunits(AU);xaxis,runtime(min).Inset
showedthat4-hydroxyphenylaceticacidwasacontaminantin
ofB,chemicalcharacterizationoffraction6showed4-hydroxyphenylacetic
acidasthemaincompound.Thecarbonatomsof4-hydroxyphenylaceticacid theoriginalsource,wellbelow10%.
arelabeledbyarbitrarynumberstoalloweasyassociationwiththefollowing Theabsenceofsignalsincellstransfectedwithemptyvector
analytical,massspectrometry,andNMRdatathatresultedinunambiguous
identification:UV-visible(0.1%aqueousHCOOH)/MeOH;6/4,v/v):(cid:5) (cid:4) (Fig.3)atteststothespecificityofthefunctionaldataforORA1-
228,276nm;LC/TOF-MS:C H O ;LC/MS(ESI(cid:5)):151.1(100;[M-H](cid:5));1HmNaxMR transfectedcells.Fromtheresultsofallthefunctionalexperi-
(65.6070[MmH,z2;Hd,imH-eCth(3y,l3s(cid:2)u)]l,fo7x.0id28e[-m8d6,/32MHe,OHD-C;(93/,13,(cid:2)v)]/;v1;3CCONSMY)R:(cid:6)(132.357M[sH,2zH;d,iHm-Ce(t5h)y]l, ments we concluded that 1) 4-hydroxyphenylacetic acid is a
sulfoxide-d /MeOD;9:1,v/v;DEPT-135,heteronuclearsinglequantumcoher- highpotencyagonistforthereceptorORA1,and2)thatrela-
ence,heter6onuclearmultiplebondcorrelation):(cid:6)40.3(CH ,C(5)),114.8(CH, tivelyminorstructuralchangesdestroytheagonisticproperties
C(3,3(cid:2))),125.6(C,C(1)),130.1(CH,C(2,2(cid:2))),155.8(C,C(4)),2173.2(C,C(6)).C,
suggestingapronouncedselectivityofthisreceptor.
eachofthecollectedfractionswasdried,redissolved,andusedtostimulate
ORA1-transfectedHEK293Tcells.Onlyfraction6resultedinfluorescence ActivationofORA1by4-Hydroxyphenylacetic-relatedCom-
changes.Lastcolumn,100(cid:3)MunpurifiedagedL-tyrosineservedaspositive pounds—Tobetterunderstandthemolecularfeaturesrequired
control((cid:3)con.).Cellstransfectedwithemptyvectorshowednoresponseto
for ORA1 agonists, we measured dose-response relationships
anyfraction.Scaling:yaxis,arbitraryunits;xaxis9min.
for a variety of compounds structurally related to 4-hydroxy-
means of heteronuclear multiple bond correlation optimized phenylaceticacid(Fig.4,Table1).Amongthisgroup,nobetter
for2,3J couplings.Asanexample,thecarbonylgroupreso- agonist than 4-hydroxyphenylacetic acid was found, which
C,H
natingat173.2ppmshowedacorrelationsignalwiththepro- remainsbyfarthebestagonistexhibitingthelowestthresh-
tonsofthemethylenegroup,indicatinga-CH COOHconfig- old concentration (approximately 0.1 (cid:3)M), the lowest EC
2 50
urationandthecarbonC(5)ofthelattershowedacross-peak concentration (1.9 (cid:7) 0.3 (cid:3)M S.E.), and the largest maximal
withthearomaticringprotonsH-C(2,2(cid:2))viaa3J coupling, signalamplitude((cid:8)F/F(cid:9)0.8).
C,H
JULY11,2014•VOLUME289•NUMBER28 JOURNALOFBIOLOGICALCHEMISTRY 19783
DeorphanizationofZebrafishOlfactoryReceptorORA1
FIGURE4.AgonistselectivityandefficacyofORA1.HEK293TG(cid:2)16gust44cellsweretransfectedwithORA1cDNAandstimulatedwithincreasingconcen-
trationsofcompoundsrelatedto4-hydroxyphenylaceticacidtomonitorthecorrespondingdose-responsefunctions.Resultingcalciumsignalsweremea-
suredbyfluorometricdetection.yaxis,(cid:8)F/F;xaxis,decadiclogarithmoftheconcentrationgiveninmicromolar.Structuralformulasofthesubstancesare
(cid:8)shFo/Fw.nSttroucthtueralelfatlotefrtahteioirnnsaamttehse.Ac,a4rb-hoyxdyrlogxroypuhpesnhyifltatcheeticdoacseid-raecstpivoantseescOuRrvAe1twoivtahrayninEgCd5e0gvraeleusetooft1h.e9r(cid:7)igh0t.3as(cid:3)wMealnladsaremdauxciemtahlesmiganxailmamalpsilgitnuadleaambpolviteud0.e8.
B,structuralalterationsatthepara-hydroxylgroupshiftthedose-responsecurvetotherightaswellasreducethemaximalsignalamplitude.Forcomparison,
thecalculateddose-responsecurvefortheoptimalagonist,4-hydroxyphenylaceticacid,isdepictedingray.C,10-foldreductioninaffinitybyapositive
inductiveormesomericeffectinpara-position.Forcomparison,thecalculateddose-responsecurvefortheoptimalagonist,4-hydroxyphenylaceticacid,is
depictedingray.
Of 55 compounds tested, only 11 4-hydroxyphenylacetic TheCarboxylGroupIsRequiredinaParticularDistanceto
acid-related compounds activated ORA1 and showed a dose- theAromaticRingStructure—Amidationormethylationofthe
dependentresponse(Fig.4,Table1).Theefficacyasestimated carboxylgroupincreasestheEC value100-fold,i.e.decreases
50
by the maximal signal amplitude of these compounds was the affinity by 2 orders of magnitude (4-hydroxyphenylacet-
clearly lower, maximally 60% of the value observed for 4-hy- amide and methyl-4-hydroxyphenylacetic acid, respectively,
droxyphenylaceticacid(Table1),suggestingthatallrepresent seeFig.4A,Table1).Slightlyincreasingthedistancefromthe
partialagonists.Thepotency,asestimatedbytheEC value, ringbyintercalatingamethylenegroup(3-(4-hydroxyphenyl)-
50
variedabout100-fold,withthatof4-hydroxyphenylaceticacid propionicacid)reducestheaffinitybythesameamount,butin
aboutanorderofmagnitudehigherthanthatofthenextbest additionimpairsthemaximalsignalamplitudemassively,down
agonists(Table1).Interestingly,thepotenciesdonotcorrelate to0.1(cid:8)F/F.Decreasingthedistanceofthecarboxylgroupfrom
wellwithmaximalsignalamplitudes(Table1),suggestingthat the ring abolishes the activity completely (4-hydroxybenzoic
potencyandefficacycanvaryindependentlyforthisreceptor. acid). Thus, the negative charge of the carboxyl group is
19784 JOURNALOFBIOLOGICALCHEMISTRY VOLUME289•NUMBER28•JULY11,2014
DeorphanizationofZebrafishOlfactoryReceptorORA1
requiredataparticulardistancefromtheringforefficientbind- zebrafishpairswith100(cid:3)M4-hydroxyphenylaceticacid(final
ing and in particular signal transduction. This hypothesis is concentration). We noted chasing behavior and in one case
supported by our observation that many related compounds, oviposition after contact with the odor in the case of female/
which were completely unable to activate ORA1 (Table 1), malepairsofzebrafish.Thissuggestedtousthat4-hydroxyphe-
showmodificationsinthispartofthemolecularstructure,e.g. nylaceticacidmightbeinvolvedinregulationofreproductive
theadditionofachargedgroup(4-hydroxy-L-phenylglycine). behavior.Itiswellknownthatseveralreproductivehormones
Thepara-HydroxyGroupIsNotAbsolutelyRequired—Omit- and their metabolites including prostaglandins and steroids
ting the para-hydroxy group (phenylacetic acid) results in andsofarunidentifiedcompoundsdodoubledutyasodorsthat
aboutanorderofmagnitudereducedpotencyandonly60%of signalthereproductivestateofthefemaletothemaleandvice
maximal signal amplitude (Fig. 4B, Table 1). However, many versa(seeRef.22).
variations of the para-substituent reduce the potency more Wetheninvestigatedapossibleeffectof4-hydroxyphenyla-
severely. For example, a negative mesomeric and inductive ceticacidonovipositionbehaviorbyexposingpairsoffemale
effect in the para-position by exchanging the hydroxyl group and male zebrafish to different concentrations of 4-hydroxy-
with an amino group (4-aminophenylacetic acid) leads to a phenylacetic acid. Because pairing of zebrafish by itself can
50-foldreductioninpotency(Fig.4B,Table1).Introducinga induceoviposition,wekeptthepairstogetherfor90minbefore
bulky(andcharged)substituentinthepara-positioneliminates stimulusorcontrolsolutionwasadded.Undertheexperimen-
theactivitycompletely(4-phenylenediaceticacid,Fig.4B). talconditionsused,thisresultedinovipositionduringthefirst
Apositiveinductiveormesomericeffectatthepara-position 90minin5%ofcases(n(cid:4)66).Asimilarfrequencyof8%(n(cid:4)
is only marginally better than omitting the para-substituent 25)wasobservedforcontrolpairsnotexposedtostimulusdur-
completely, as seen by the activities of 4-chlorophenylacetic ingthenext90min.Thisfrequencywasusedasaconservative
acid,4-toloylaceticacid,3,4-methylenedioxyphenylaceticacid, estimateofthebackgroundegg-layingfrequencyforthenext90
3,4-dihydroxyphenylacetic acid, and 4-methoxyphenylacetic min. We report that oviposition frequency increased several-
acid(Fig.4C,Table1).Interestingly,allfivecompoundspossess foldafteradditionof100(cid:3)M(finalconcentration)4-hydroxy-
undistinguishable EC values (Table 1), suggesting that the phenylaceticacid.Thisincreasewasblockedafterclosingthe
50
exactshapeandsizeofthepara-substituentisnotimportant. nostrils of the female with tissue glue (Fig. 5D). Both the
The latter two compounds exhibit reduced maximal signal increaseandtheblockweresignificant((cid:4)squaretest,n(cid:4)10,
amplitudescomparedwiththefirstthreecompounds(Fig.4B, p (cid:10) 0.001, and n (cid:4) 7, p (cid:10) 0.02, respectively). The induced
Table1).Thisuncouplingofaffinityandefficacywouldbecon- ovipositionpersistsat4-hydroxyphenylaceticacidconcentra-
sistent with the p-substituent boosting the efficacy of the tionsof30and10(cid:3)M(Fig.5D),althoughthenumberofeggs
ligand.Takentogether,thepara-hydroxylgroupisnotrequired laidappearedtodecreasewithdecreasingconcentration.The
perseforactivity,butenhancespotencyandefficacymassively. eggsappearedpartiallyimmatureandcontainedahigherthan
4-HydroxyphenylaceticAcidModulatesZebrafishReproduc- normalpercentageofopaque(dead)eggs.Wehypothesizethat
tiveBehavior—ThehighsensitivityofORA1for4-hydroxyphe- this could be due to the 4-hydroxyphenylacetic acid stimulus
nylaceticacidsuggeststhatthiscompoundmightbearelatively occurringoutofphysiologicalcontextinourexperimentalsit-
closefittotheendogenousligand,ifnotanendogenousligand uation. We conclude that 4-hydroxyphenylacetic acid at low
itself.Itspotencyisover10-foldbettercomparedwiththatofa concentrationscanelicitaphysiologicalresponseaspartofa
recentlyidentifiedligand-receptorpairthatsignalsaversionto reproductivebehaviorrepertoire.
decayingflesh(11).ThiswouldbeconsistentwithORA1serv-
DISCUSSION
ingaspheromonereceptor,whichgenerallyexhibithighersen-
sitivitythan“normal”olfactoryreceptors(cf.Refs.21and24). OurdeorphanizationofORA1constitutesthefirstinstance
Thus,weembarkedonasearchforinnatezebrafishbehavior ofaligandidentificationforanymemberoftheorafamilyof
elicitedby4-hydroxyphenylaceticacid. olfactor receptor genes. In fact, ORA1 is only the third deor-
First,weinvestigatedwhetherapointsourceof4-hydroxy- phanized fish olfactory receptor overall, with the other two
phenylaceticacidinastationarytankwouldelicitattractionor receptorsbelongingtootherfamilies(11,25).Thesmallfamily
avoidancebehavior(11).Fortheseexperiments180(cid:3)lof4-hy- ofjustsixoragenesisremarkableforitshighdegreeofconser-
droxyphenylaceticacidsolution(1mM)wasaddedtothetank vationanditsratherconstantfamilysize,bothdifferentfromall
byanexperimenterhiddenfromsightforthezebrafish.Using otherolfactoryreceptorgenefamiliesanalyzedsofar.Further-
the identical setup the same ligand concentration has been more,oragenesareunderstrongnegativeselectionandteleost
foundtoelicitmaximalaversionbehaviorfortheabovemen- ORA receptors possess direct orthologs even in cartilaginous
tionedligand/receptorpairwith10-foldloweraffinity(cf.Fig. fish(7),i.e.beforetheevolutionarydivergenceinteleostsand
5B). However, 4-hydroxyphenylacetic acid did not result in tetrapods.Thus,theorafamilyisancestraltothemammalian
detectable attraction or avoidance behavior (Fig. 5, A and B). andmoregenerallytetrapodv1rreceptorgenerepertoires.The
Furthermore,averagevelocity,ameasureofagitation,didnot v1rreceptorsformamonophyletictreewithtwoofthesixora
changeaftera4-hydroxyphenylaceticacidstimuluswasgiven, genes,ora1andora2.Therefore,fishora1andora2genesmay
and no incidents of freezing, a fear response, were observed remainclosertotheancestralv1rgenesthanthecontemporary
(Fig.5,AandC). v1r genes themselves, which show a very dynamic evolution,
Second,weconsideredapossiblefunctionof4-hydroxyphe- andexhibitmanygenebirthanddeathevents.Wehavethere-
nylacetic acid as a signal in social interactions, and tested foreattempteddeorphanizationofzebrafishORA1andORA2
JULY11,2014•VOLUME289•NUMBER28 JOURNALOFBIOLOGICALCHEMISTRY 19785
DeorphanizationofZebrafishOlfactoryReceptorORA1
FIGURE5.Zebrafishbehavioralresponsesto4-hydroxyphenylaceticacid.A,fishwereexposedtoapointsource(arrow)of4-hydroxyphenylaceticacid(1
mM),andtheirmovementswerevideotracked5minbeforeandafterstimulusaddition.Arepresentativetrackisshown,orange,prestimulus;brown,post-
stimulus.B,quantitativeevaluationoftracksshowsnoevidenceforattractionoraversionby4-hydroxyphenylaceticacid(pHPAA),n.s.,notsignificant.For
comparison,attractionbyfoodextract(food)andaversiontocadaverineisshown,rawdataforthesepanels,seeRef.11.C,nodifferenceinmotilitywasseen
afterexposureto4-hydroxyphenylaceticacid.Motilityismeasuredasaveragevelocityandexpressedaspercentoftheprestimulusvalue.D,oviposition
frequencyofzebrafishmatingpairsisincreasedafterexposureto4-hydroxyphenylaceticacid,finalconcentrationsasindicated.Bluelineindicatescontrol
value(leftgraybar).Allthreeconcentrationsof4-hydroxyphenylaceticacidareactive,***,p(cid:10)0.001.Narisclosure(rightgraybar)abolishestheincreasein
ovipositionevents,**,p(cid:10)0.02.E,schematicrepresentationoftheolfactory-mediatedresponseofzebrafishmatingpairsto4-hydroxyphenylaceticacid.
receptors, and have been successful for ORA1, for which we 4-Hydroxyphenylacetic acid is detected by ORA1 with at
identifiedandcharacterizedseveralagonists. leastanorderofmagnitudehighersensitivitythantypicalfood
WehaveshownORA1tobeahighlysensitiveandspecific odors,suchasaminoacids(EC valuesbetween10and100(cid:3)M
50
receptor for 4-hydroxyphenylacetic acid. An extensive search in vivo (24, 26, 27)) or the death-signaling odor cadaverine
ofstructurallyrelatedcompoundsyieldednocompoundwith (11), even though measurements in heterologous systems
similarorbetterpotencyandagonistefficacy.Albeitwecannot may exhibit lower sensitivities for odorants than are
excludetheexistenceofstructurallyunrelatedagonists,within observedinvivo(seee.g.Refs.28and29).Thishighsensitiv-
therangeofchemicalstructuresanalyzed,4-hydroxyphenyla- ityisconsistentwithapheromonalfunctionfor4-hydroxy-
ceticacidemergesastheoptimalactivatorforORA1.Thebest phenylacetic acid, because pheromones are expected to be
agonists among related compounds were about 10-fold less detectedatlowerconcentrationsthannormalodorants(21).
potentandexhibitedlessthantwo-thirdsoftheefficacycom- 4-Hydroxyphenylaceticacidisabiogeniccompound,which
paredwith4-hydroxyphenylaceticacid.Thislargedifferenceis occursinseveralmetabolicpathwaysincludingaminorcata-
consistentwiththehypothesisthat4-hydroxyphenylaceticacid bolic pathway for tyrosine (transamination, decarboxylation,
maybethephysiologicallyrelevantligandforORA1. and oxidation of the resulting aldehyde to the corresponding
19786 JOURNALOFBIOLOGICALCHEMISTRY VOLUME289•NUMBER28•JULY11,2014
DeorphanizationofZebrafishOlfactoryReceptorORA1
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JULY11,2014•VOLUME289•NUMBER28 JOURNALOFBIOLOGICALCHEMISTRY 19787