Native Serotonin 5-HT2C Receptors Are Expressed as Homodimers on the Apical Surface of ...

14 Pages·2015·1.63 MB·English

1521-0111/87/4/660–673$25.00 http://dx.doi.org/10.1124/mol.114.096636
MOLECULARPHARMACOLOGY MolPharmacol87:660–673,April2015
Copyrightª2015byTheAmericanSocietyforPharmacologyandExperimentalTherapeutics
Native Serotonin 5-HT Receptors Are Expressed as
2C
Homodimers on the Apical Surface of Choroid Plexus
Epithelial Cells
Katharine Herrick-Davis, Ellinor Grinde, Tara Lindsley, Milt Teitler, Filippo Mancia,
Ann Cowan, and Joseph E. Mazurkiewicz
CenterforNeuropharmacology & Neuroscience,Albany Medical College,Albany, NewYork (K.H.-D.,E.G., T.L., M.T.,J.E.M.);
DepartmentofPhysiologyandCellularBiophysics,ColumbiaUniversity,NewYork,NewYork(F.M.);andCenterforCellAnalysis
andModeling, Universityof Connecticut HealthCenter, Farmington, Connecticut (A.C.)
D
ReceivedOctober31,2014;acceptedJanuary21,2015 ow
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ABSTRACT e
d
Gprotein–coupledreceptors(GPCRs)areaprominentclassof molecular brightness analysis. FCS with PCH revealed molec- fro
plasmamembraneproteinsthatregulatephysiologicresponses ularbrightnessvaluesfornative5-HT receptorsequivalentto m
2C m
to a wide variety of stimuli and therapeutic agents. Although the molecular brightness of a homodimer. 5-HT receptors
2C o
GPCRoligomerizationhasbeenstudiedextensivelyinrecombi- displayed a diffusion coefficient of 5 (cid:1) 1029 cm2/s and were lp
h
nant cells, it remains uncertain whether native receptors expressedat32receptors/mm2ontheapicalsurfaceofchoroid a
rm
expressed in their natural cellular environment are monomers, plexusepithelialcells.Thefunctionalsignificanceandsignaling
.a
dimers,oroligomers.Thegoalofthisstudywastodeterminethe capabilities of the homodimer were investigated in human s
p
monomer/oligomer status of a native GPCR endogenously embryonickidney293cellsusingagoniststhatbindinawash- etjo
erexcperpestosresdininchiotsronidatpuleraxlusceelpluitlahreleianlvcierollnsmweenret.eNvaaltuivaeted5-uHsTin2gC rWeshiesrteaanst magaonnniestrbtoinodninegotor boontehpprroottoommeerrsreosuflttheedhinomGopdriomteeirn. urn
a
fluorescence correlation spectroscopy (FCS) with photon activation, maximal stimulation required occupancy of both ls
counting histogram (PCH). An anti–5-HT2C fragment antigen protomers.Thisstudyisthefirsttodemonstratethehomodimeric .org
binding protein was used to label native 5-HT receptors. structure of 5-HT receptors endogenously expressed in their a
A known monomeric receptor (CD-86) served as2aC control for native cellular en2vCironment, and identifies the homodimer as t A
S
decoding the oligomer status of native 5-HT receptors by afunctionalsignalingunit. P
2C E
T
J
o
u
Introduction formationhasbeenreportedtoregulateallaspectsofGPCR rna
ls
G protein–coupled receptors (GPCRs) represent oneof the function including synthesis, ligand binding, G protein on
coupling, and trafficking (reviewed in Herrick-Davis, 2013; J
largestfamiliesofcellmembranesignalingproteins.Theyare a
Milligan, 2013). Even so, the presence and functional n
u
presentonthesurfaceofmostcellsandregulatethephysiologic relevance of GPCR dimerization in vivo is widely debated ary
functionsofallmajororgansystemsinthehumanbody.GPCRs (reviewed in Ferré et al., 2014). Concerns have been raised 2
8
modulate physiologic responses to light, odorants, hormones, about the functional relevance of GPCR dimerization, as , 2
neurotransmitters, and therapeutic agents. Although physio- monomeric receptors have been reported to activate 01
9
logicprocessesregulatedbyGPCRactivationandblockadehave G proteins in reconstituted systems (Bayburt et al., 2007;
been studied for decades, it is still uncertain whether the Whortonetal.,2007),andthecrystalstructureoftheagonist-
functional signaling unit is a monomer, dimer, or higher- occupiedb -adrenergicreceptorrevealedamonomericreceptor
2
orderoligomer. incomplexwithasingleGprotein(Rasmussenetal.,2011).On
Currently, there are hundreds of reports in the published theotherhand,structuralstudiesofnativerhodopsinreceptors
literaturedescribingthedimericoroligomericnatureofGPCRs haverevealedtheirdimericorganizationinrodoutersegments
expressed in recombinant cell systems. Dimer/oligomer (Liang et al., 2003) and their association with G transducin
(Jastrzebskaetal.,2013b).Additionally,leukotriene,dopamine,
FundingforthisworkwasprovidedbyagrantfromtheNationalInstitutes andserotoninreceptorshavebeensolubilizedashomodimersin
ofHealth National Institute ofMental Health [R21-MH086796] awarded to
complexwithasingleGprotein(BanèresandParello,2003;Han
K.H.-D.
dx.doi.org/10.1124/mol.114.096636. etal.,2009;Pellissieretal.,2011).
ABBREVIATIONS: Cab,cabergoline;CPSM,countspersecondpermolecule;2D,two-dimensional;3D,three-dimensional;DMEM,Dulbecco’s
minimalessentialmedium;ER,endoplasmicreticulum;Erg,ergotamine;FBS,fetalbovineserum;Fab,fragmentantigenbinding;FCS,fluorescence
correlationspectroscopy;GFP,greenfluorescentprotein;GPCR,Gprotein–coupledreceptor;HEK293,humanembryonickidney293;IP,inositol
phosphate; mAb, monoclonal antibody; PBS, phosphate-buffered saline; PCH, photon counting histogram; TIRF, total internal reflection
fluorescence.
660
FCSAnalysisofNativeGPCR 661
Materials and Methods
ThepresenceofGPCRdimersoroligomersinintactnative
tissues and in vivo is inferred from studies using indirect
CellCultureandTransfection. HEK293cells(AmericanType
biophysical methods, immunofluorescence in fixed tissue CultureCollection,Manassas,VA)wereculturedinDulbecco’sminimal
sections, and functional studies in transgenic animals. For
essential medium (DMEM; Cellgro, Manassas, VA) with 10% fetal
example,ligand-basedfluorescenceresonanceenergytransfer bovineserum(FBS;HyClone,Logan,UT)inahumidifiedincubatorat
studies identified oxytocin homodimers in rat mammary 37°C, 5% CO . For the confocal microscopy and FCS experiments,
2
gland tissue (Albizu et al., 2010), and heterodimers of HEK293cellswere plated insix-wellplates fitted with 25-mm poly-
D1andD2dopaminereceptorsinstriatalneuronsareinferred D-lysine–coatedglasscoverslips (Fisher Scientific, Pittsburgh, PA) at
from antibody-based fluorescence resonance energy transfer adensityof4(cid:1)105cellspercoverslipandtransfectedwith200ngof
infixedbrainslices(Hasbietal.,2011).Supportingevidence the indicated plasmid DNA using lipofectamine reagent (Invitrogen,
Carlsbad,CA)for5hours.Followingtransfection,cellswereculturedin
isprovidedbystudiesusingheterodimer-selectiveantibodies
minimal essential medium (without phenol red) with 10% charcoal-
(Berg et al., 2012) and transgenic mice expressing mutant
strippedserum(Gibco/LifeTechnologies,GrandIsland,NY)for20hours
receptors that restore (Rivero-Müller et al., 2010) or inhibit
at37°C,5%CO.PriortoFCSanalysis,coverslipswerewashedtwicein
2
(González et al., 2012) normal receptor function. Pharmaco-
HEPES-bufferedKrebs-Ringersolution.
logical approaches have used heterodimer-selective agonists
Plasmid. cDNAs encoding the 5-HT receptor and the VSV
2A
(Waldhoeretal.,2005;Fujitaetal.,2014)orantagoniststhat isoformofthe5-HT receptorwereclonedintopEGFP-N1,pECFP-
2C
bindinapseudo-irreversiblemannertooneprotomerwithin N1,andpmCherry-N1vectors(TakaraClontech,MountainView,CA)
D
the homodimer (Smith et al., 2011). However, studies using tocreatechimericreceptorswithfluorescenttagsontheC-terminusof o
w
direct biophysical methods capable of monitoring protein each receptor. CD-86/GFP and CD-86/GFP-GFP were generously n
interactionsinlivingtissuesareneededtosubstantiatethese providedbyG.Milligan(UniversityofGlasgow,Glasgow,UK).Site- loa
findings. directedmutagenesis(Stratagene,LaJolla,CA)wasusedtocreatean de
d
Although recent studies have used direct methods with Ael2im06inKamteuptoatteionntiainlaagllgrgergeaentiofnlu(oZraeschceanrtiapsreotteainl.,(2G0F0P2)).constructsto fro
near single-molecule sensitivity, such as total internal re- m
flectionfluorescence(Calebiroetal.,2013;KasaiandKusumi, GFP-Tagged,Anti–5-HT2CFragmentAntigenBindingProtein mo
2014; Teichmann et al., 2014) and fluorescence correlation (2C-Fab-GFP). Amonoclonalantibodyrecognizinganexternaldomain lp
spectroscopy(FCS)(Briddonetal.,2008;Malengoetal.,2008; 5of-HthTe 5r-eHcTep2CtorrsecienpttroarnwsfaecstepdreHvEioKus2l9y3dceevlleslo(Mpeadncainadetshalo.w,2n00t7o).laTbheel harm
GangulyandChattopadhyay,2010;Herrick-Davisetal.,2012, cDNA2Csequencesencodingtheheavyandlightchainsofthemonoclonal .as
2u0si1n3g;rCecoormridbeinnanettcaell.l,l2in0e1s4.)T,htehepsuerpsotusedioefsthweeprerespeenrftosrtmudedy ainnttoi–m5-aHmTm2Calfiraangmexepnrtesasniotnigevnecbtoinrsdi(nAgsspurroteetinal.(,F2a0b0)7w)earnedspulbacslmoniedds petjo
u
was to determine the monomer/oligomer status of a native containing clone 1C4 (Mancia et al., 2007) were used in the present rn
a
GPCRendogenouslyexpressedinitsnaturalcellularenviron- study. The light-chain cDNA sequence was cloned into an expression ls
ment.Theserotonin5-HT2Creceptorwasselectedbecauseitis vector upstream of an internal ribosomal entry site followed by red .org
highly expressed in choroid plexus epithelial cells, an estab- fluorescentproteinasaselectionmarker(Assuretal.,2007).Forthe a
lished primary cell culture for studying native 5-HT presentstudy,theheavy-chaincDNAsequencewasmodifiedasfollows: t A
2C a His6 tag was added in place of the stop codon, and the cDNA was S
receptors (Esterle and Sanders-Bush, 1992), and monoclonal P
ligated into the pEGFP-N1 expression vector (Clontech) for direct E
antibodies recognizing the native conformation of the 5-HT T
2C labeling of the heavy-chain C-terminus with GFP. Site-directed J
receptor have already been developed (Mancia et al., 2007). o
mutagenesis (Stratagene) was used to create an A206K mutation in u
Previously, we demonstrated the application of FCS with theGFPtagtoeliminatepotentialaggregation(Zachariasetal.,2002). rna
photon countinghistogram(PCH), a sensitive method for the HEK293cellsweretransfectedbycalciumphosphateprecipitationwith ls o
direct quantification of photons emitted from individual a mixture of the light- and heavy-chain plasmids. A stable cell line n
J
fluorescent molecules, for determining the oligomeric status expressingbothlightandheavychainswasidentifiedbyfluorescence an
of GPCR expressed in recombinant cell lines (Herrick-Davis microscopy and Western blot. Stably transfected cells were plated at ua
et al., 2012, 2013). In FCS, fluorescence-tagged proteins that adensityof5(cid:1)106cells/100-mmdishandcultured(37°C,5%CO2)for ry 2
associate with one another or are part of a larger protein 1puwriefeiekdifnroDmMtEheMcuwlittuhre10m%edlioawonIgnGicFkBelSre(HsinyCcololunme).n2sC(T-Fhaebrm-GoFFPiswhaesr 8, 2
complex will codiffuse with one another. Autocorrelation 0
analysisrevealsthediffusioncharacteristicsoffluorescence- Scientific,Waltham,MA)accordingtothemanufacturer’sprotocol. 19
Following column purification, samples were dialyzed in HEPES-
tagged proteins in the sample and provides an estimate of
bufferedKrebs-Ringer(pH7.4),andconcentratedto0.1mMinspin
the number of fluorescent proteins codiffusing together as
columns(30,000nominalmolecularweightcut-off;EMDMillipore,
acomplexwithintheplasmamembrane. Billerica,MA).
In the present study, a monoclonal anti–5-HT2C fragment WesternBlot. Intactchoroidplexustissueisolatedfromthethird
antigen binding protein was used as a probe for labeling and lateral ventricles of an adult female Sprague-Dawley rat was
native5-HT receptorsinchoroidplexusepithelialcells.FCS placedinamicrocentrifugetubecontaining50mlofcoldRIPAbuffer
2C
and PCH were used to monitor the diffusion characteristics withproteaseinhibitorcocktail(Sigma-Aldrich,St.Louis,MO).The
and oligomer status of 5-HT receptors expressed in their tubewasplacedonicefor15minutes,thensonicatedinanice-cold
2C
nativecellularenvironment.Thefunctionalsignificancewas water bath for 15 minutes, and placed on ice again for 15 minutes
prior to centrifugation at 30,000g for 30 minutes at 4°C. Ten
investigatedinhumanembryonickidney293(HEK293)cells
microliters of the supernatant was diluted 1:1 in nonreducing
using ergots that bind in a wash-resistant manner to one
Laemmlisamplebuffer.Purified2C-Fab-GFPandprestainedprotein
protomer(cabergoline)orbothprotomers(ergotamine)ofthe
standards(Bio-Rad,Hercules,CA)werealsodilutedinnonreducing
homodimer. These studies provide the first pharmacological
Laemmli sample buffer. The samples were heated at 70°C for
and functional evidence for the presence of 5-HT2C receptor 15minutesandrunona10%Tris-HClBio-RadReadyGelat95V
homodimers and highlight the signaling capabilities of the for70minutes.Gelproteinsweretransferredtonitrocellulose(Hybond
individualprotomers. ECL; Amersham/GEHealthcare, Pittsburgh, PA) andprobedwith
662 Herrick-Davisetal.
2C-Fab-GFPovernightat4°Cin1%milk/1%bovineserumalbumin trichloroaceticacidwasaddedtoeachwell.Theplatewasincubated
blockingsolution.FollowingincubationwithGFP(B-2)–horseradish onicefor20minutestoreleasesurface-boundligandfromthecells
peroxidase(1:3000;SantaCruz,Dallas,TX),proteinswerevisual- withoutcelllysis.Followingincubation,450mlofthetrichloroacetic
izedusingenhancedchemiluminescence(Amersham). acidsolutionwasremovedfromeachwell,mixedwith5mlofEcoscint
ChoroidPlexusEpithelialCellCultureandImmunostaining. cocktail, and counted in a Beckman liquid scintillation counter
Primarychoroidplexusepithelialcellswerepreparedasdescribedby (BeckmanCoulter,Pasadena,CA).
Esterle and Sanders-Bush (1992). In brief, choroid plexuses were Fluorescence Correlation Spectroscopy. For FCS measure-
dissected from the third and lateral ventricles of fetuses of timed- ments,cellswerewashedtwicewithHEPES-bufferedKrebs-Ringer
pregnant Sprague-Dawley rats at19days of gestation.The choroids (withoutglucose),andthecoverslipwasplacedinanAttofluorCell
wereplacedindigestbuffercontainingphosphate-bufferedsaline,pH Chamber (Molecular Probes/Life Technologies, Grand Island, NY)
7.4,with0.33mg/mlpronase(Sigma-Aldrich)and0.25mg/mlDNase1 with 1 ml of HEPES-buffered minimal essential medium (without
(Sigma-Aldrich)for25minutesat37°C,washedtwice,thendissociated phenolred).FCSmeasurementsweremadeusingaZeissLSM-780
bytriturationinDMEM/F12(1:1)(Cellgro)with0.13mg/mlDNase1. confocalmicroscope(Zen2010software;CarlZeiss,Jena,Germany)
Thesupernatantcontainingdissociatedepithelialcellswascentrifuged equippedwitha32elementgalliumarsenidephosphidelineararray
at1100rpmfor3minutes.Epithelialcellswereresuspendedinculture detector,as previouslydescribed (Herrick-Davis andMazurkiewicz,
media containing DMEM/F12 (1:1) with 10% charcoal-stripped FBS 2013).One-photonexcitationwithacontinuous-waveargonionlaser
(Gibco), 1% N2 supplement (Invitrogen), 10 ng/ml epidermal growth wasperformedusinga40(cid:1)(numericalaperture1.2)C-apochromat
factor(Invitrogen),and1%PenStrep(Invitrogen),thentransferredtoa water immersion objective to create an observation volume on the
60-mm dish and incubated (37°C, 5% CO2) for 2 hours to allow order of 10215 liters. FCS measurements were made on the upper D
fibroblasts to adhere to the dish. The culture medium containing plasmamembraneoftransfectedHEK293cellsandtheapicalsurface ow
unattached epithelial cells was removed from the dish, and the of primary choroid plexus epithelial cells labeled with the 2C-Fab- nlo
epithelialcellswereplatedon25-mmglasscoverslips(FisherScientific) GFPantibody.Positioningoftheplasmamembraneinthecenterof a
d
coatedwithlaminin(Gibco).After3weeksinculture,primarychoroid theobservationvolumewasachievedbymonitoringthephotoncounts ed
epithelial cells were labeled with the monoclonal anti–5-HT2C–Fab- per molecule in real time while simultaneously focusing upward fro
GFP antibody (2C-Fab-GFP, diluted 1:3 in HEPES-buffered Krebs- throughthecellcytosoland thenplasmamembranetoidentifythe m
Ringer)for40minutesat23°CimmediatelypriortoFCSrecording.For focal plane corresponding to the maximal photon counts per mole- m
o
costaining with antitransthyretin (Bioss, Woburn, MA), 3-week-old cule.FCSmeasurementswererecordedat23°Cfor100seconds,as lp
cultures of choroid epithelial cells were fixed in phosphate-buffered 10consecutive10-secondintervals.GFPwasexcitedbythe488-nm ha
3.7%paraformaldehyde(10minutesatroomtemperature),permeabi- laser set at 0.1% to minimize photobleaching. The time-dependent rm
lized with 0.1% triton, blocked with 4% donkey serum, and stained fluctuations in fluorescence intensity were recorded on the gallium .as
p
overnight at 4°C with 2C-Fab-GFP (diluted 1:10) and rabbit anti- arsenidephosphidedetectorforfluorescenceemissionintherangeof e
transthyretin(diluted1:100)inHEPES-bufferedKrebs-Ringer,pH7.4, 520–625nm.Apinholeofoneairyunitwasused.FCSrecordingswere tjo
u
with 1% donkey serum. The rabbit antitransthyretin was visualized analyzed by a digital temporal correlator (using nonlinear least- rn
withanAlexa488–conjugateddonkeyanti-rabbitIgG(diluted1:1000; squares minimization, Zeiss Aim 4.2 software) to calculate the als
InvIintroosgietno)l.PhosphateAssay. HEK293cellswereseededat2(cid:1)105 aduectaocyoirnrefllautoiroenscfeunnccetifolnucGtu(ta)t,iownhiinchtenrespitryesaesnitnsetqh.e1t:ime-dependent a.org
cells/well, in 24-well plates, in DMEM with 10% FBS. Cells were t A
tVrSaVnsifseocftoerdmwoitfhth1e005-HngT2oCfrpelcaespmtoidrucosnintgailnipinogfeccDtaNmAineenrceoadgienngttfhoer GðtÞ5 ÆdFðtÞÆF× dðtFÞðæ2t1tÞæ (1) SPET
5hoursat37°C.Followingtransfection,cellswereculturedinDMEM J
o
winiotshit1o0l-%freFe,BsSerfuorm2-f4reheoDurMs,EtMhenwiwthas0h.5edmaCnidofla[3bHel]emdyoovineornsiitgohl.tAint fwluhcetrueatGio(tn)inistetnhseity,(tdiFm)eatasvoemraegtei.meopfotihnet(tc)haanndgeatiantfilmueorienstceernvcael urna
thestartoftheexperiment,cellswerewashedtwicewithphosphate- later(t1t),dividedbythesquareoftheaveragefluorescenceintensity. ls o
bufferedsaline(PBS)andwerepretreatedwithserum-freeDMEMin Autocorrelation analyses were performed using the Zeiss Aim 4.2 n J
the absence or presence of drug for 60 minutes (or as indicated), softwarepackagewithanautocorrelationbintimeof0.2microseconds. an
u
followed by drug washout. The washout period consisted of four FCSdatawerefittoatwo-dimensional(2D)model(forlateraldiffusion a
washes with PBS over a 30-minute period: one wash immediately withintheplasmamembrane)withtwocomponentsasineq.2: ry 2
fmtooleltdohwieaiicnnhgcautnhbgeaetesonrad.tF1oof0l-dlmorwuinginupgtredeitrnrutegeartwvmaaelssnhwto,uhftoe,lrlteohiwneeitndhoebsyciettolhllsrpewheeosrsuepbrhesaettuqeru(neIePndt) GðtÞ511AN21(cid:1)F1(cid:3)11t=tD1(cid:4)211F2(cid:3)11t=tD2(cid:4)21(cid:5)(cid:6) (2) 8, 2019
assay was initiated by adding lithium chloride to the assay media,
withorwithoutdrugsasindicated,foranadditional60minutes.Total whereNisthenumberofmoleculesintheobservationvolume.F1,F2,
[3H]IPproductionwasmeasuredbyanionexchangechromatography andtD1,tD2representtherespectivefractionsanddiffusiontimesof
as previously described (Berridgeet al., 1983). Data were analyzed thetwocomponents.Apre-exponentialtermisincludedtoaccountfor
usingGraphPadPrismsoftware(GraphPadSoftware,LaJolla,CA). photophysicalproperties(e.g.,blinking)ofthefluorescentprobe,
Whole-Cell Radioligand Binding Assay. HEK293 cells were n (cid:7) (cid:8) o
seededat 4 (cid:1) 105 cells/well, in 12-well plates, in DMEM with 10% A511 Tbe2ttb ð12TbÞ21 ;
FBS. Cells were transfected with 200 ng plasmid containing cDNA
encodingtheVSVisoformofthe5-HT receptorinserum-freemedia whereT andt representtheblinkingfractionandrelaxationtime,
2C b b
using lipofectamine reagent for 5 hours at 37°C. Following trans- respectively.ItshouldbenotedthatindividualGFPmoleculesarenot
fection,cellswereculturedinDMEMwith10%FBSfor24hours,then always fluorescent. They can exhibit blinking, exist in a prolonged
washed and incubated overnight in serum-free DMEM. Cells were darkstate,orbeimmatureandnonfluorescent(UlbrichandIsacoff,
pretreatedwiththeindicateddrugsfor60minutesfollowedbyfour 2007).
washeswithPBSovera30-minuteperiod.Washedcellswerelabeled Theautocorrelationcurvedepictsthefluorescenceintensityfluctua-
with[3H]mesulergine(2.5nM)in0.5mlofserum-freeDMEM,inthe tionsasafunctionofparticlenumberanddiffusiontime.Theaverage
absenceandpresenceof1mM5-HTtodefinespecificbindingtocell dwelltimeofthefluorescentspecieswithintheobservationvolume(t )
D
surface5-HT receptors,for20minutesat37°C.Cellswerewashed is calculated from the midpoint of the autocorrelation curve. The
2C
withPBStoremoveunboundradioligand,and0.5mlofice-cold3% diffusion coefficient (D) for lateral diffusion of fluorescence-tagged
FCSAnalysisofNativeGPCR 663
receptorswithintheplasmamembraneiscalculatedasineq.3,where brightness of GFP-tagged 5-HT receptors. A known monomeric
2C
v istheradiusoftheobservationvolumeinthehorizontaldimension: plasmamembranereceptorwithasingleC-terminalGFPtag(CD-86/
o
GFP) and with a tandem GFP tag (CD-86/GFP-GFP) was used to
v2 determine the molecular brightness of a single GFP and two GFP
D5 o (3).
4t tags.AllGFPconstructscontainedanA206Kmutationtoeliminate
D
potential self-aggregation of GFP (Zacharias et al., 2002). To
The radius of the observation volume (vo) was determined determine the contribution of background autofluorescence from
experimentallybymeasuringthewidthatthehalf-maximumheight cytoplasmicproteins,adilutesolutionofpurifiedmonomericGFPwas
of a Gaussian distribution fitted to the image of subresolution evaluated.Themolecularbrightnessof2C-Fab-GFPinsolution[8795
fluorescent beads (0.1 mm FluoSpheres; Invitrogen), as previously counts per second per molecule (CPSM)] was similar to GFP from
described (Cole et al., 2011). In our experimental setup, vo was pEGFP plasmid expressed in the cytosol of HEK293 cells (9075
determined to be 0.30 mm. Thus, the surface area of the plasma CPSM),indicatingthatbackgroundautofluorescencefromcytoplas-
membraneintheobservationvolumeisapproximatedbypvo2tobe mic proteins was minimal (approximately 3%) in our experimental
0.28mm2. setup.
The amplitude of the autocorrelation function G(0) (equal to the
y-intercept) is inversely related to the number of molecules in the
Results
observationvolume(N )asineq.4:
PSF
1 Creation of a Monoclonal, Monovalent Anti–5-HT
N 5  × g (4) 2C D
PSF Gð0Þ21 Fragment Antigen Binding Protein with GFP Tag o
w
wheregisthepointspreadfunction(PSF)whichdescribestheshape w(2eCre-Fgaenbe-GraFtePd)b.yAimntmi–u5n-HizTin2gCmmicoenwociltohnaaldeatnetrigbeondti-seoslu(mbiAlizbesd) nloa
oftheobservationvolume.Thenumericalvalueofgdiffersdepending d
and purified form of the rat 5-HT receptor (Mancia et al., e
o0n.3t5hefomroadetlhsreelee-cdteimdefonrsiaonnaallys(i3sDa)ndGiasu0s.s5iafnorm2DodFeClSusaendalyinsisPaCnHd 2007). The mAb clone 1C4, rec2oCgnizing an extracellular d fro
epitope and demonstrating selectivity for rat over human m
analysis. m
Theaveragefluorescenceintensityoraveragephotoncountrate(k) 5-HT2C receptors (Mancia et al., 2007), was selected for the o
recorded for a given sample is determined by the number of present study. The rat and human 5-HT2C receptor protein lph
fluorescent molecules (NPSF) and their molecular brightness («), as sequences differ by only one or two amino acids in the arm
describedineq.5: extracellular loop regions, whereas the N-terminal domains .a
s
differby11aminoacids(Saltzmanetal.,1991).Native5-HT p
k5NPSF׫ (5). receptorsexpressedinmammaliancellsareglycosylatedattw2Co etjo
u
Thus,dividingthecountrate(k)bythenumberofmolecules(NPSF) sites: N39 in the N-terminus and N204/205 in the second rna
providesanestimateofthemolecularbrightness(«)ofthesample. extracellular loop (Backstrom et al., 1995). Since the mAb ls
Photon Counting Histogram. Fluorescence fluctuation data recognizesthenativeconformationofthereceptor,andbinding .org
recorded during a FCS experiment can be used to generate photon of the mAb does not interfere with deglycosylation (Mancia a
counting histograms, which provide quantitative information about etal.,2007),theseobservationsimplicatetheN-terminusasthe t A
S
thenumberoffluorescentmoleculesandthenumberofphotoncounts likelyepitopeforbindingofthemAbtothe5-HT receptor. P
2C E
permolecule(Chenetal.,1999).Tenmeasurementsweremadeonthe Figure 1A illustrates the architecture of the anti–5-HT T
upperplasmamembraneofeachcellbymonitoringthephotoncount Fab.Theheavy-andlight-chainvariableregionsoftheant2i–C Jo
ratefor100seconds,as10consecutive10-secondobservationperiods. 5-HT Fabarelinkedtotheconstantregionsofantilysozyme urn
Although the laser intensity was set to 0.1% to minimize photo- 2C a
D1.3 from pASK84 (Skerra, 1994). The antilysozyme D1.3 ls
bleaching, photo-bleaching was apparent during the first10-second o
observationperiod.Therefore,theaveragemolecularbrightnessfrom allowsproperassemblyoftheFabviadisulfidelinkageofthe n J
the second through 10th observation periods was calculated and constant regions following expression in mammalian cells. an
rfleupoorretsecdenacsethinetemnoslietycutlraarcberitghhattnesshsowfoerdthlaartgceelslp.iSkeegsmoerntdsrioftfsthine sTuhbeclloinghedt ianntdo mheaamvmy aclhiaaninsexopfretshseionanvtei–ct5o-rHsTa2sCilFluasbtrwateerde uary 2
falnuaolryessicse.nTcoeginenteenrsaitteya(dhuisettoogrcaemll,meoavcehm1e0n-ste)cwoenrdeoebxscelruvdaetdiofnropmertihode iinntFerign.a1lBri.bToshoemliaglhetn-cthrayinsitceDNanAdsreeqdueflnucoereisscfeonlltowpreodtebiynaans 8, 20
wasbrokendownintoonemillionintervalsorbins(PCHbintime5 aselectionmarker(Assuretal.,2007).Theheavy-chaincDNA 19
10 microseconds ). Histograms were constructed using the PCH
sequencewassubclonedintopEGFP,upstreamandinframe
module in the Zeiss Aim 4.2 software in which the number of
withtheGFPsequencefordirectlabelingoftheheavychain
10-microsecondbinswasplottedonthey-axisandphotoncountson
with a GFP tag. Following coexpression of the light- and
thex-axis.Theresultinghistogramdepictsthenumberofbinsthat
registered1,2,3photoncountsetc.duringone10-secondobservation heavy-chainplasmidsinaHEK293stablecellline,theanti–
period. Since a constant intensity light source produces a photon 5-HT2CFabwithGFPtag(2C-Fab-GFP)wasisolatedfromthe
count distribution that follows Poisson statistics, as fluorescent culture media (structure shown in Fig. 1C). GFP was used
molecules enter and diffuse through the nonhomogenously illumi- insteadofchemicallabelingtoensureanexact1:1stoichiom-
natedobservationvolume,thefluctuationsinfluorescenceintensity etry of GFP tag per Fab, essential for molecular brightness
resultinabroadeningofthePoissondistribution.Thissuper-Poisson analysis.AlthoughitisnotedthatindividualGFPmolecules
characteristicisobservedinthetailofthePCHcurve.PCHdatawere
arenotalwaysfluorescent,asGFPexhibitsblinkinganddark
fittoaone-componentmodelinwhichconcentrationandmolecular
states (Ulbrich and Isacoff, 2007), the use of appropriately
brightnesswereallowedtobefree(andthefirst-ordercorrectionwas
matchedmonomericanddimericGFPcontrolsamplesinthe
fixedatzero) todeterminetheaveragemolecularbrightnessofthe
sample.Residualsofthecurvefitandreducedx2analyseswereused presentstudyallowsaccuratedeterminationofGPCRoligomer
todeterminethegoodnessoffit(Mülleretal.,2000). statusbymolecularbrightnessanalysis.
Controls for Molecular Brightness Analysis. Both cytosolic Characterization of 2C-Fab-GFP. Analysis of purified
and plasma membrane controls were used to decode the molecular 2C-Fab-GFPbyWesternblotrevealedasinglebandmigrating
664 Herrick-Davisetal.
SeparatepoolsofHEK293cellswereindependentlytransfected
withplasmidscontainingcDNAencodingeithertherat5-HT
2C
receptorwithaC-terminalmCherrytagorthe5-HT receptor
2A
with a C-terminal cyan fluorescent protein tag. Twenty-four
hoursaftertransfection,theseparatepoolsofHEK293cellswere
mixedtogetherandplatedonthesamecoverslip.Thelivecells
were immunostained with purified 2C-Fab-GFP and examined
byconfocalmicroscopy(Fig.3A).The2C-Fab-GFPimmunostain-
ingcolocalizedwithplasmamembranemCherry-tagged5-HT
2C
receptors but was absent in untransfected cells and cells
expressingcyanfluorescentprotein–tagged5-HT receptors.
2A
Ablastsearchoftheratgenomewasconductedtoidentify
potentialoff-targetlabelingsitesforthe2C-Fab-GFPprobein
choroid epithelial cells. When the results of the search were
limitedtoproteinsexpressedonthesurfaceofepithelialcells,
the protein with the highest homology to the N-terminal
domain of the 5-HT receptor was aminopeptidase M. This
2C D
protein displayed 43% sequence homology to residues 31–46 o
w
oinfgthree5si-dHuTe2sCoNf-taemrmininopaelpdtoimdaasien.MHow(6e5v8e–r6,7t3h)eacorrerelsopcaontedd- nloa
d
within an intracellular region of the protein and thus would e
d
Fig.1. DesignoftheGFP-taggedanti–5-HT2Cfragmentantigenbinding nlaobteblenaactcievsesib5l-eHtTo intereracecpttworitsho2nC-Fthaeb-GsFurPfawcehenofusinedtatcot from
pvarortiaebinle(2rCeg-Fioanbs-G(VFHP)a.(nAd)TVhL)eoFfaabimscoonmocploonseadloafntthibeohdeyavrayi-saenddalgigahint-scthtahine epithelial cells. It is2Cimportant to note that the anti–5-HT2C mo
5-HT2Creceptorandtheconstantregions(CH1andCL)ofantilysozymeD1.3 monoclonalFabdemonstratesspeciesselectivityforratover lph
ferxopmrespsAioSnKv8e4ct.o(rBw)itThhaenliingthetr-nchalarinibocsDomNAalesnetqruyesnictee(IwRaEsSc)lfoonlleodweidntboyaann human5-HT2Creceptors(Manciaetal.,2007),eventhoughthe arm
red fluorescent protein (RFP) selection marker. The heavy-chain cDNA ratandhumanproteinsequencesshare78%homologywithin .a
s
sequence was cloned into an expression vector upstream and in frame theN-terminaldomainofthe5-HT receptor(Saltzmanetal., p
2C e
withthecDNAsequenceforGFP,fordirectlabelingoftheheavychainwith 1991). These results indicate that the 2C-Fab-GFP probe is tjo
aGFPtag.(C)2C-Fab-GFPwaspurifiedfromtheculturemediaofastable u
HEK293celllinecoexpressingtheheavy-andlight-chainplasmids.CMV, highlyspecificforlabelingnative5-HT2Creceptorsonthesurface rn
a
cytomegalovirus. of choroid plexus epithelial cells, with minimal to no off-target ls
labeling. .org
CharacterizationofPrimaryChoroidPlexusEpithelial a
nearthe100kDastandardmolecularmassmarker(Fig.2A). Cell Cultures. Primary epithelial cell cultures expressing t A
S
These results are consistent with the predicted migration native5-HT receptorswerepreparedfromratchoroidplexus P
2C E
patternofthelightchain/heavychain/GFPcomplex,inwhich tissue.Ofthe14different5-HTreceptorsubtypesidentifiedto T
J
each component contributes approximately 30 kDa to the date,the5-HT receptoristheonlyonethatisexpressedinthe o
2C u
overallsizeofthecomplex.Totestthespecificityofthe2C-Fab- choroidplexus.Choroidplexusepithelialcellsaretheonlycells rn
a
GFPprobeforlabeling5-HT receptors,atotalproteinextract inthe brain that expresstransthyretin,a thyroxin and retinol ls
2C o
waspreparedfromfreshlyisolated,intactchoroidplexustissue carrierproteinthatisalsofoundinthebloodandliver(Dickson n
J
andanalyzedbyWesternblot(Fig.2B).Westernblotanalysis a
n
u
bofettwheee5n-H45T2aCnrdec6e5ptkoDrai,srperperdeiscetnedtintgo ismhomwatbuarnedasnmdimgraattuinrge ary 2
formsofthereceptor(Backstrometal.,1995).The5-HT2C 8, 2
receptorhasbeenshowntoformdetergent-sensitivehomodimers 0
1
(Herrick-Davis et al., 2004); thus, under mild denaturing 9
conditions, fainter bands representing homodimers should
appear between 90 and 130 kDa. Since several GPCRs have
beensolubilizedashomodimersinapentamericassemblywith
theircognateGprotein(BanèresandParello,2003;Hanetal.,
2009; Pellissier et al., 2011; Jastrzebska et al., 2013b), an
additionalbandrepresentingthe5-HT receptorhomodimer
2C
incomplexwithaheterotrimericGproteinwouldbeexpected
atapproximately200kDa.Thepredominantbandsidentified
withthe2C-Fab-GFPprobeontheWesternblotshowninFig.
2Bfallwithinthesepredictedsizeranges. Fig.2. Westernblotsof2C-Fab-GFP.(A)Lane1:molecularmassmarkers
(kilodaltons); lane 2: 2C-Fab-GFP purified from the culture media of
Next, the specificity of the 2C-Fab-GFP probe for labeling
a stable HEK293 cell line coexpressing the heavy- and light-chain
thenativeconformationofthe5-HT2Creceptorexpressedon plasmids. The light chain, heavy chain, and GFP are approximately
theplasmamembraneoflivingcellswasexamined.The5-HT 30kDaeach.(B)Lane1:molecularmassmarkers(kilodaltons);lane2:
2A
receptoristheproteinmostcloselyrelatedtothe5-HT receptor totalproteinextractfromchoroidplexustissuelabeledwith2C-Fab-GFP.
2C Bands within the predicted ranges for immature to mature 5-HT
in the genome. Therefore, the 5-HT2A receptor was used as receptors (45–60 kDa), homodimers (90–120 kDa), and homodimers 2inC
acontroltodemonstratethespecificityofthe2C-Fab-GFPprobe. complexwithGprotein(170–200kDa)arepresent.
FCSAnalysisofNativeGPCR 665
surface of the epithelial cells (Hartig et al., 1990). This
observation was confirmed in our cultures using confocal
microscopy.The2C-Fab-GFPimmunostainingwaspresenton
the apical surface and lateral plasma membrane but not on
the basolateral surface of the cultured epithelial cells (Fig.
3C). The functionality of the 5-HT receptors and the
2C
specificityofthe2C-Fab-GFPprobeareclearlydemonstrated
by the pronounced redistribution of fluorescence from the
plasmamembranetointracellularvesiclesfollowingexposure
to5-HT,asshowninFig.3D.
The epithelial cells were large and polygonal. The apical
surfaceareawasdeterminedusingthepolygonalmeasuring
tool in the Zeiss Aim 4.2 software. The mean and standard
errorof themeanfrom10differentfieldsofcells(52cellsin
total)wasdeterminedtobe32636387mm2.Thisvaluewas
used to calculate the level of native 5-HT receptors
2C
endogenouslyexpressedontheapicalsurfaceoftheepithelial
D
cells,asdescribedlater. o
w
FCS and PCH Analysis of the 2C-Fab-GFP Probe. n
FCSisperformedusingahighnumericalapertureobjectiveto loa
d
focusalaserbeamintoasmalldiffraction-limitedspot,creating e
d
adetectionorobservationvolumeontheorderof0.5(cid:1)10215 fro
liters(reviewedinElson,2011;Herrick-DavisandMazurkiewicz, m
m
2013). As fluorescent molecules pass through the observation
o
volume, they are excited by a laser and give off bursts of lp
h
photons,whicharerecordedinrealtimebyasensitivephoton arm
countingdetector.FCSrecordingsweremadeusingapurified .a
s
solutionof2C-Fab-GFPandontheupperplasmamembraneof p
e
HEK293 cells expressing fluorescence-tagged 5-HT2C recep- tjou
tors.Theresultingfluctuationsinfluorescence,producedbythe rn
a
fluorescent molecules entering and leaving the observation ls
volume,areshowninthefluorescenceintensitytracesinFig. .org
Fig.3. 2C-Fab-GFPspecificity.(A)HEK293cellsweredividedintotwo 4A.Autocorrelationanalysisofthefluorescenceintensitytrace a
groupsandwereseparatelytransfectedwithplasmidcontainingmCherry- is performed using a nonlinear least-squares fitting routine t A
tagged5-HT (5-HT /mCherry,red)orcyanfluorescentprotein(CFP)– S
2C 2C whichgraphicallyrepresentstheautocorrelationfunctionG(t) P
tagged5-HT2A(5-HT2A/CFP,blue).Followingtransfection,thecellswere E
mixedandplatedonasinglecoverslipandstainedlivewith2C-Fab-GFP ontheordinateanddiffusiontimeontheabscissa.Theaverage T
(green)for30minutesatroomtemperature.Whitescalebar=10mm.The dwelltimeofthefluorescentmoleculeswithintheobservation Jo
mergedimage(rightpanel)shows2C-Fab-GFPplasmamembranelabeling volumeiscalculatedfromthemidpointoftheautocorrelation urn
oceflclselelsxperxepsrseinssginthge5m-HosTt2Ccl/omsCelhyerrerlyat(esdhopwronteaisnyineltlohwe)geannodmneo,tlhaebe5l-iHngT2oAf decaycurve. als o
reencdeopgteonrou(nsolyoevxeprlraepsseodf ginreecnhoraonidd pblleuxeu).s(Bep)itNhealtiiavlec5el-lHsTl2aCberleecdepwtoitrhs theAulatboceolerdre5la-HtioTncurrevceesptfoorrsthaere2sCh-oFwabn-GinFFPigin. 4sBol.uAtiuontoaconrd- n Ja
2C-Fab-GFP (green) are shown in the left panel. The same field ofcells 2C nu
cpoasnteali.nTedhewmitheragendainmtiabgoedyistoshtorwannstinhytrheetinrig(rhetdp)aisneslh.oWwhnitienstchaelembiadrdl=e rae3laDtiomnoadnelalfyosrisBorfowthneia2nC-dFifafub-sGioFnP. Tsohleutbiiopnhawsaics baeusttocfoitrrbey- ary 2
5th0emampi.c(aCl)suOrpfaticcea,llasteecrtaiolnpsla(0sm.5ammmemthbicrka)nfer,oamndabZa-ssotalactker(a6.l0remgmio)nsshoofwceinllgs lation curves for the fluorescence-tagged 5-HT2C receptors 8, 2
werebestfitbya2Dmodelforthelateraldiffusionofproteins 0
costainedwith2C-Fab-GFPandtransthyretin.Therelativepositionwithin 1
the Z-stack is noted in the lower-left corner of each image. (D) Choroid within the plasma membrane, with a very fast component 9
plexusepithelialcellslabeledwiththe2C-Fab-GFPprobeinthepresence relatedtothephoto-physicalpropertiesofGFP(ontheorder
of1mM5-HT.Theleftpanelshowsanentirefieldofcells(whitescalebar=
of 300 microseconds) and a slower component representing
50mm).Themiddlepanelshowsasinglecellathighermagnification(white
scale bar = 50 mm). The right panel shows the differential interference the translational diffusion of receptors within the plasma
contrast(DIC)overlayofthecellinthemiddlepanel.Thewhitearrowspoint membrane(ontheorderof40–50milliseconds).Autocorrela-
to the lateral plasma membrane where there is little fluorescence, in tion analysis revealed diffusion coefficients on the order of
contrasttocellsculturedintheabsenceof5-HT(Fig.3C,middlepanel).
0.5–0.6mm2/sforthefluorescence-tagged5-HT receptorsin
2C
HEK293cells(Table1).
et al., 1985). Thus, transthyretin is commonly used as an Theamplitude (y-intercept)of theautocorrelationcurveis
intracellularbiomarkertoidentifyepithelialcellsisolatedfrom inversely related to the number of fluorescent molecules
intactchoroidplexustissue. presentintheobservationvolume.Forillustrationpurposes,
The primary epithelial cell cultures displayed the charac- autocorrelationcurvesfromsampleswithsimilarnumbersof
teristic cobblestone pattern and stained positive for 5-HT fluorescent molecules or fluorescent complexes are shown in
2C
receptorsandtransthyretin(Fig.3B).Astheculturesmature Fig. 4B.Note that,although theamplitude of the autocorre-
overaperiodof2weeks,theepithelialcellsbecomepolarized lationcurveisthesameforallthreesamples,thecorrespond-
(Villalobos et al., 1997). Radioligand binding studies have ing fluorescence intensity traces (Fig. 4A) show average
reported that 5-HT receptors are expressed on the apical photoncountratesofapproximately65kHzfor2C-Fab-GFP
2C
666 Herrick-Davisetal.
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8
Fig.4. FCSrecordingsfromasolutionof2C-Fab-GFP,theplasmamembraneoftransfectedHEK293cellsexpressingthe5-HT2Creceptorlabeledwith , 2
2(AC)-FFalubo-GreFsPce,nacnedinthteenpsliatsymtraamceesmfobrraonneeo1f0t-rsaencosfnedctoebdsHerEvaKt2io9n3pceelrlisoedx.p(Bre)sAsiuntgocaocrhreimlaetiroinc5a-nHaTly2CsisreocfepthtoerfwluiotrheascCen-tceerminitneanlsGitFyPtrtaacges(5.-THhTe2rCe/GdFliPn)e. 019
representstheautocorrelationoftheobservedfluorescencesignal,andthegreenlinerepresentsthefittoatwo-componentmodel:afastcomponent(on
theorderof300microseconds)relatedtothephoto-physicalpropertiesofGFP,andaslowercomponent(ontheorderof40milliseconds)representingthe
translationaldiffusionoffluorescence-taggedreceptorsintheplasmamembrane.Dividingtheaveragephotoncountrate(kilohertz)determinedfrom
thefluorescenceintensitytrace(A)bythenumberoffluorescentmoleculesdeterminedfromtheamplitudeoftheautocorrelationcurve(B)predictsthe
averagemolecularbrightnessofthesample.(C)PhotoncountinghistogramsofthecorrespondingFCSrecordings.Togeneratethehistograms,each
10-secondfluorescenceintensitytrace(A)wasbrokendownintoonemillion10-microsecondintervalsorbins(PCHbintime=10microseconds).The
numberofbinsisplottedonthey-axisandphotoncountsonthex-axis.(D)ResidualsofthePCHcurvefittoaone-componentmodel.Theresidualsofthe
curvefitarelessthan2standarddeviationsandarerandomlydistributedaboutzero,indicatingagoodfittotheselectedmodel.
and 135 kHz for 2C-Fab-GFP–labeled 5-HT receptors and receptorsinchoroidepithelialcells(Fig.5B),indicatingthat
2C
5-HT receptors with a C-terminal GFP tag (5-HT /GFP). the transfected HEK293 cells were expressing receptors
2C 2C
These results indicate that the fluorescence-tagged 5-HT withinthenativephysiologicrange.
2C
receptors produce approximately twice as many photon Fluorescence fluctuation data recorded during an FCS
counts and thus are approximately twice as bright as the experiment can be used to generate a PCH, which provides
2C-Fab-GFPinsolution.Theamplitudeoftheautocorrelation quantitative information about the number of fluorescent mole-
curve for the 2C-Fab-GFP–labeled receptors expressed in cules and the number of photon counts produced by individual
HEK293 cells (Fig. 4B) was similar to that of native 5-HT fluorescentmolecules(Chenetal.,1999).Thenumberofphoton
2C
FCSAnalysisofNativeGPCR 667
TABLE1
Diffusion(FCS)andmolecularbrightness(PCH)offluorescenttagsinsolutionorinthecytoplasm,andfluorescence-taggedreceptorsontheplasma
membrane
HEK293cellsexpressingaknownmonomericreceptor(CD-86)wereusedasacontroltodeterminethemolecularbrightnessofamonomer(CD-86/GFP).Themolecular
brightnessofadimerwasdeterminedusingatandemGFPtag(CD-86/GFP-GFP).Diffusionreportedinmillisecondsrepresentstheaveragedwelltimeofthereceptorinthe
observationvolume.Diffusioncoefficients(micrometerssquaredpersecond)werecalculatedusinga2Dmodelforthelateraldiffusionofreceptorswithintheplasma
membrane.PCHmolecularbrightnessvaluesarereportedasCPSM.Reducedx2valuesarereportedforthePCHdatafittoaone-componentmodelforasingle,homogeneous
populationofmonomersorhomodimers.Datarepresentthemean6S.E.M.forthenumberofcellsexamined(N).
FluorescentTagsandLabeledReceptors FCSDiffusion PCHBrightness Reduced N
ms mm2/s CPSM x2
2C-Fab-GFP(S) —a — 87956145 1.1460.03 35
mGFPinHEK293cells(C) — — 90756287 1.0760.02 25
CD-86/GFPinHEK293cells(PM) 37.062.0 0.6160.03 10,3336364 1.0960.07 12
CD-86/GFP-GFPinHEK293cells(PM) 37.462.2 0.6060.04 19,6916530 1.3360.10 10
5-HT /GFPinHEK293cells(PM) 40.461.9 0.5660.03 18,3306674 1.1960.07 15
2C
2C-Fab-GFP–labeled5-HT receptorsinHEK293cells(PM) 48.762.2 0.4660.02 18,7836384 1.5160.12 20
2C
2C-Fab-GFP–labeled5-HT receptorsinchoroidplexusepithelial 42.661.8 0.5360.02 18,4666361 1.5560.22 10
2C
cells(PM)
Cytoplasm,C;plasmamembrane,PM;solution,S.
aNotcalculated. D
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w
n
counts per molecule provides an estimate of the molecular aboutzerowithsmallvariationsinamplitude(asinFig.4D) loa
d
brightness. Since the molecular brightness is proportional to andreducedx2valuesclosetounity(asinTable1),thedata e
d
thenumberoffluorescentmoleculestravelingtogetherwithin areagoodfitfortheselectedmodel(Mülleretal.,2000).The fro
a protein complex, the oligomer size of a fluorescence-tagged residuals of the curve fit indicate that the PCH data are m
m
proteincanbedeterminedbycomparingmolecularbrightness adequatelydescribedbyaone-componentmodelforhomodimers,
o
valueswithestablishedmonomericcontrolscontainingasingle withoutmonomersortetramers,indicatingthatthe2C-Fab- lp
h
fluorescenttagorwithtwofluorescenttags(Chenetal.,1999). GFP–labeled5-HT2CreceptorsexpressedinHEK293cellsare arm
Thus,aGPCRhomodimerwithtwofluorescenttagswouldbe homodimers. .a
s
twiceasbrightasamonomer,atetramerwithfourfluorescent FCSandPCHAnalysisofNative5-HT Receptorsin p
2C e
tagswouldbe4timesasbright,andsoforth. ChoroidPlexusEpithelialCells. Native5-HT2Creceptors tjou
Photoncounting histogramsgenerated fromthe FCS data expressed in primary choroid plexus epithelial cells were rn
a
arepresentedinFig.4C.Togenerateahistogram,each10-second labeledwith2C-Fab-GFP.Theapicalplasmamembranewas ls
fluorescenceintensitytrace(showninFig.4A)wasbrokendown positioned within the laser-illuminated observation volume .org
intoonemillion10-microsecondintervalsorbins(PCHbintime byfocusingupwardfromthemiddleofthecelltothetop,while a
510microseconds).Histogramswereconstructedinwhichthe simultaneously monitoring the photon counts per molecule. t A
S
number of 10-microsecond bins was plotted on the y-axis and Optimalpositioningoftheplasmamembranewithinthecenter P
E
photon counts on the x-axis. The shape of the histogram is of the observation volume is critical for accurate molecular T
J
a function of the number of fluorescent molecules and their brightness analysis because the observation volume is not o
u
molecular brightness. For the 2C-Fab-GFP–labeled receptors illuminated homogeneously and the detected photon counts rn
a
and5-HT /GFP,thehistogramsshowanaverageof1.1photon decreaseasfluorescentmoleculestravelawayfromthecenter ls
2C o
counts per 10-microsecond bin time, which is equivalent to oftheobservationvolume.Asthefreelydiffusing,fluorescence- n
J
110,000 counts per second. Dividing by the average number of taggedreceptorspassedthroughtheobservationvolume,they a
n
u
molecules,calculatedfromtheamplitudeoftheautocorrelation wereexcitedbythelaser,andthefluctuationsinfluorescence a
curvesinFig.4B(usingeq.4witha3DPCHmodelwhereN56), intensitywererecorded(Fig.5A).Similarto5-HT2Creceptors ry 2
yieldsanaveragemolecularbrightnessof18,333CPSMforthe transiently expressed in HEK293 cells, the autocorrelation 8, 2
fluorescence-tagged receptors. The PCH for the 2C-Fab-GFP curvefornative5-HT receptorsinprimaryepithelialcellswas 0
2C 1
solutionregisteredhalfthenumberofphotoncountsasthePCH bestfitbya2Dmodelwithtwocomponents,afastcomponent 9
forthefluorescence-taggedreceptors,indicatingthatthesolution related to the photo-physical properties of the GFP tag and
washalfasbright.Molecularbrightnessvaluesforthe2C-Fab- aslowercomponentrepresentingthetranslationaldiffusionof
GFPsolutionandformonomericGFPexpressedinthecytosol receptors withinthe plasmamembrane. Autocorrelationanal-
of HEK293 cells were on the order of 9000 CPSM (Table 1), ysisrevealeddiffusioncoefficientsontheorderof0.5mm2/sfor
roughlyhalfthebrightnessofthefluorescence-tagged5-HT native5-HT receptorsinchoroidplexusepithelialcells.
2C 2C
receptors. TheamplitudeoftheFCSautocorrelationcurvefornative
The PCHs shown in Fig. 4C were generated by fitting the 5-HT receptorsinprimaryepithelialcellspresentedinFig.
2C
data to a one-component model for a single population of 5Bindicatesanaverageofninefluorescentmoleculeswithin
fluorescent species, monomers for the 2C-Fab-GFP solution, the observation volume (calculated using eq. 4 with a 2D
and homodimers for the fluorescence-tagged receptors. The model). A plasma membrane observation area of 0.28 mm2
residualsofthePCHcurvefit(Fig.4D)areanindicatorofhow (calculated from the radius of the observation volume as
well the data fit the selected model. When the residuals are describedinMaterialsandMethods)yieldsanapproximate
systematicwithlargevariationsinamplitude(greaterthan2 expression level of 32 receptors/mm2 of plasma membrane.
standarddeviations)andreducedx2valuesgreaterthan3.0, An average apical surface area of 3263 mm2 predicts an
theselectedmodelisapoorfit(Mülleretal.,2000).However, expression level on the order of 105 5-HT receptors per
2C
whenthe residuals of thecurve fitare randomly distributed choroidepithelialcell.
668 Herrick-Davisetal.
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Fig.5. FCSrecordingsfromtheplasmamembraneofchoroidplexusepithelialcellslabeledwith2C-Fab-GFP,andtheplasmamembraneoftransfected 8
HEK293cellsexpressingmonomericCD-86withoneGFPtag(CD-86–GFP)ortwoGFPtags(CD-86–GFP-GFP).(A)Fluorescenceintensitytracesfor , 2
0
one10-secondobservationperiod.(B)Autocorrelationanalysisofthefluorescenceintensitytraces.Theredlinerepresentstheautocorrelationofthe 1
observedfluorescencesignal,andthegreenlinerepresentsthefittoatwo-componentmodel:afastcomponent(ontheorderof300microseconds)related 9
tothephoto-physicalpropertiesofGFP,andaslowercomponent(ontheorderof40milliseconds)representingthetranslationaldiffusionoffluorescence-
taggedreceptorsintheplasmamembrane.Dividingtheaveragephotoncountrate(kilohertz)determinedfromthefluorescenceintensitytrace(A)bythe
numberoffluorescentmoleculesdetermined fromtheamplitude oftheautocorrelationcurve(B)predictstheaveragemolecularbrightnessofthe
sample.(C)PhotoncountinghistogramsofthecorrespondingFCSrecordings.Togeneratethehistograms,each10-secondfluorescenceintensitytrace
(A)wasbrokendownintoonemillion10-microsecondintervalsorbins(PCHbintime=10microseconds).Thenumberofbinsisplottedonthey-axisand
photoncountsonthex-axis.(D)ResidualsofthePCHcurvefittoaone-componentmodel.Theresidualsofthecurvefitarelessthan2standard
deviationsandarerandomlydistributedaboutzero,indicatingagoodfittotheselectedmodel.
PCH analysis of native 5-HT receptors labeled with trace for CD-86–GFP displayed a photon count rate that was
2C
2C-Fab-GFP(Fig.5C)producedhistogramswithsimilarnum- approximately half that of CD-86–GFP-GFP (Fig. 5A), even
bersofphotoncountsandmolecularbrightnessvaluesasob- thoughtheamplitudeoftheautocorrelationcurvewassimilar
servedfor5-HT receptorsexpressedinHEK293cells(Table1). forbothsamples(Fig.5B).TheseresultspredictthattheCD-86
2C
Aknownmonomericreceptor,CD-86,wasusedasacontrolfor receptorwithasingleGFPtagishalfasbrightasCD-86with
decodingthemonomer/oligomerstatusofnative5-HT receptors twoGFPtags.
2C
inchoroidepithelialcells.FCSandPCHanalysesforCD-86with Thefluorescenceintensitytracesfornative5-HT receptors
2C
aC-terminalGFPtag(CD-86–GFP)andtandemGFPtag(CD- labeled with 2C-Fab-GFP and CD-86–GFP-GFP displayed
86–GFP-GFP)areshowninFig.5.Thefluorescenceintensity similarphotoncountrates(Fig.5A),andthecorresponding
FCSAnalysisofNativeGPCR 669
histograms (Fig. 5C) predict similar molecular brightness
values.PCHanalysisproducedamolecularbrightnessvalue
formonomericCD-86–GFP(10,333CPSM)thatwassimilar
tothemonomeric2C-Fab-GFPinsolution(8795CPSM),but
was approximately half the brightness of CD-86–GFP-GFP
(19,691 CPSM) and native 5-HT receptors labeled with
2C
2C-Fab-GFP(18,466CPSM),asreportedinTable1.Whenfit
to a one-component model for homodimers, without mono-
mers or tetramers, the residuals of the PCH (fit deviation
showninFig.5D)werelessthan2standarddeviations,were
randomly distributed around zero, and yielded reduced x2
valuesclosetounity(Table1),indicatingthatthePCHdataare
adequately described by the selected model. These results
demonstrate the homodimericnature ofnative 5-HT recep-
2C
torsinchoroidplexusepithelialcells.
Fig. 6. Representative dose-response curves for stimulation of IP
SignalingPropertiesof5-HT2CReceptorHomodimers. measured in HEK293 cells expressing 5-HT2C receptors. IP production
Teitler and colleagues used a pharmacological approach for wasmeasuredinthepresenceofincreasingconcentrationsofeachdrugas
D
identifyingandinvestigatingthesignalingpropertiesofGPCR indicated. Basal IP levels determined in cells treated with serum-free o
culturemediaweresubtractedout.Dataareexpressedasthepercentage w
hbionmdoidnimaewrsa(sShm-reitshisettanalt.,,p20se1u1d).oI-nirtrheveeirrssitbuldeym,aanntnaegrontoistosntehaort owfitmha5x-iHmTa,ltIhPepinrtordiuncsticioenffaiccahciieevsaobfleEwrgitahndeaCchabdwruegr.eI0n.9c0omanpdar0i.s8o5n, nloa
d
bothprotomerswithinthehomodimerwereidentifiedandused respectively. e
d
toprovidethefirstpharmacologicalevidenceforthepresenceof fro
5-HT homodimers(Smithetal.,2011).Werationalizedthatif m
7 m
agonists with similar binding characteristics were available, it fourtimesovera30-minuteperiod(drugwashout) toremove
o
would be possible to investigate the signaling properties of drug that binds in a competitive, reversible manner. At the lp
h
individualprotomerswithinthehomodimer.Suchstudieswould startofthe IP assay, the cells were only treated withserum- arm
havethedistinctadvantageofallowingtheinvestigationofthe free culture media containing lithium chloride (to prevent IP .a
s
signalingpropertiesofwild-typeGPCRinsteadofrequiringthe breakdown). No drugs were added during the IP assay. As p
e
use of mutant receptors with altered binding properties or expected, pretreatment with 5-HT followed by drug washout tjo
u
signalingdefectivemutantreceptors,asusedinpreviousstudies resulted in minimal stimulation of IP production (Fig. 7B), rn
a
ofthisnature. indicatingthat5-HTbindsinacompetitive,reversiblemanner ls
Ergotderivativesareknownagonistswithmoderatetohigh to5-HT2Creceptors.Incontrast, approximately half-maximal .org
2a0ff0in2i;tKynfoigrh5t-HetTa2lA.,,22B0,20C4r).eRceepcteonrtsly(,Ntehwemcrayns-tTaalnstcrruedctiuertesalo.f, IwPithproCdaubctiaonndresmubasineqeudefnotllowwaisnhgouat,60a-nmdinnuetearp-mreatxreimatamleInPt at A
S
the5-HT and5-HT receptorsweresolvedinthepresence productionwasobservedfollowingErgpretreatmentanddrug P
1B 2B E
of ergotamine (Wacker et al., 2013), indicating ergotamine washout(Fig.7B). T
J
forms a relatively stable complex with the receptor. These Whole-cell radioligand binding studies were performed in o
u
observations led us to investigate and subsequently identify parallel with the IP signaling experiments to monitor drug rn
a
ergotderivativesthatbindinawash-resistantmannertoone interactions with cell surface 5-HT receptors. Intact cells ls
2C o
or both protomers of the 5-HT receptor homodimer. The expressing5-HT receptorswereincubatedwithasaturating n
ergotswereusedtoinvestigatet2hCesignalingpropertiesofthe concentrationof2[3CH]mesulergineintheabsenceandpresence Ja
n
u
homodimer,asdescribedlater. of1mM5-HTtodefinespecificbindingtocellsurfacereceptors a
Dose-responsecurvesforthestimulationofIPproductionin (Fig. 7C). 5-HT pretreatment followed by drug washout ry 2
HEK293 cells expressing wild-type 5-HT2C receptors are resulted in minimal inhibition of [3H]mesulergine binding, 8, 2
shown in Fig. 6. Ergotamine (Erg), 5-HT, and cabergoline demonstrating that 5-HT binds in a competitive, reversible 0
1
(Cab)stimulatedIPproductioninadose-dependentmanner, manner to cell surface 5-HT receptors. However, pretreat- 9
2C
withEC valuesof1.3,2.3,and527nM,respectively,similar ment and washout of Cab and Erg inhibited specific radio-
50
to previously published results (Newman-Tancredi et al., ligand binding by 45 and 81%, respectively (Fig. 7C). The
2002; Knight et al., 2004). The data are expressed as the inhibition of radioligand binding following drug washout
percentageofmaximalIPproductionachievedwiththatdrug. indicates that Cab and Erg bind in a wash-resistant manner
TheefficaciesofErgandCabwithrespectto5-HTwere0.90 with slow dissociation kinetics or in a pseudo-irreversible
and0.85,respectively. mannertocellsurface5-HT receptors.Themagnitudeofthe
2C
Next,thesignalingandbindingpropertiesbeforeandafter observed effect was identical to the half-maximal and near-
drug washout were investigated (Fig. 7). For these experi- maximal residual signaling properties displayed by Cab and
ments,eachdrugwastestedatasingleconcentration20-fold Erg,respectively,intheIPassay.
over the EC value of that drug. In the initial experiment Thetimecourseforestablishingthewash-resistantsignaling
50
shown in Fig. 7A, the drugs were added with the lithium ofErgandCabwasinvestigated(Fig.8).Cellswerepretreated
chloride at the beginning of the IP assay, and IP production with drug for various times, ranging from 5 to 60 minutes,
was measured. As expected, similar maximal levels of IP followed by drug washout and subsequent IP assay in the
production were achieved with each drug. In a second set of absence of drug. Establishment of the Cab wash-resistant IP
experimentsshowninFig.7,BandC,thecellswerepretreated signaling occurred gradually over the 60-minute test period,
withdrugsfor60minutestoensureequilibration,thenwashed reachingmaximallevelswithin30minutesofdrugtreatment.