Sungwon Choi, Mike Lee, Amy L. Shiu, Sek Jin Yo and Gregory W. Aponte

16 Pages·2006·0.75 MB·English

Sungwon Choi, Mike Lee, Amy L. Shiu, Sek Jin Yo and Gregory W. Aponte
Am J Physiol Gastrointest Liver Physiol 292:98-112, 2007. First published Aug 24, 2006;
doi:10.1152/ajpgi.00295.2006
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GPR93 activation by protein hydrolysate induces CCK transcription and secretion in
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AJP - Gastrointestinal and Liver Physiology publishes original articles pertaining to all aspects of research involving normal or
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Society. ISSN: 0193-1857, ESSN: 1522-1547. Visit our website at http://www.the-aps.org/.
AmJPhysiolGastrointestLiverPhysiol292:G98–G112,2007.
FirstpublishedAugust24,2006;doi:10.1152/ajpgi.00295.2006.
Identification of a protein hydrolysate responsive G protein-coupled receptor
in enterocytes
Sungwon Choi, Mike Lee, Amy L. Shiu, Sek Jin Yo, and Gregory W. Aponte
DepartmentofNutritionalSciencesandToxicology,UniversityofCaliforniaatBerkeley,Berkeley,California
Submitted5July2006;acceptedinfinalform21August2006
Choi S, Lee M, Shiu AL, Yo SJ, Aponte GW. Identification of a tively(15,19,33,52,65).Signalingeventsinitiatedbyluminal
proteinhydrolysateresponsiveGprotein-coupledreceptorinenterocytes. protein hydrolysate have been a focus of studies for defining
Am J Physiol Gastrointest Liver Physiol 292: G98–G112, 2007. First themechanismsleadingtothereleaseofCCK(48,49).Inthe
publishedAugust24,2006;doi:10.1152/ajpgi.00295.2006.—Gprotein- enteroendocrine STC-1 cells, protein hydrolysate activates
coupledreceptors(GPCRs)havethepotentialtoplayaroleasmolecular
ERK1/2,CaMKpathways,aswellasthePKApathways(19).
sensorsresponsivetoluminaldietarycontents.Althoughsucharolefor
Several studies have demonstrated that the uptake of protein
GPCRshasbeenimplicatedintheintestinalresponsetoproteinhydro-
hydrolysate in the intestine is through the proton-coupled
lysate, no GPCR directly involved in this process has been previously
oligopeptide transporter PepT1 (1, 13, 14). Oligopeptides up-
identified. In the present study, for the first time, we identified GPR93
take through this transporter is linked to events that subse-
expressioninenterocytesanddemonstrateditsactivationinthesecellsby
protein hydrolysate with EC of 10.6 mg/ml as determined by the quently lead to an induced transcription of CCK (19) and the
inductionofintracellularfree5C0a2(cid:1).Inenterocytes,GPR93wassyner- releaseofCCK(14).Therearenumerousexamplesofnutrient- Do
gisticallyactivatedbyproteinhydrolysateincombinationwithanagonist, sensing events in the lumen that may be mediated by trans- w
n
oleoyl-L-(cid:2)-lysophosphatidicacid(LPA),whichactivatedthereceptorin porters or receptors. The indirect involvement of G protein- lo
theseenterocyteswithEC50of7.9nM.TheincreasedintracellularCa2(cid:1) coupled receptors (GPCRs) in these sensing events in which ade
byGPR93activationwasobservedwithouttheadditionofapromiscuous the release of their ligands is directly induced by luminal d
G(cid:2)proteinandwaspertussistoxinsensitive,whichsuggestsG(cid:2)q-and nutrients is well characterized. For example, free fatty acids fro
G(cid:2)-mediatedpathways.ActivatedGPR93alsoinducedpertussistoxin- m
sensiitive ERK1/2 phosphorylation. Both nuclear factor of activated T dacirteivcatltyesinndeuucroeptehpetidreeleYas(eNoPfYp)erpetciedpetoYrsY,o(r3)p,rowtehiinchhyindrotulyrn- ajp
cellsand12-O-tetradecanoylphorbol13-acetateresponsiveelementsre- g
porteractivitieswereinducedbyproteinhydrolysateincellsexogenously sate induces the release of CCK (12, 42, 48), which in turn i.p
expressingGPR93.Thepeptidomimeticcefaclorbyitselfdidnotactivate activates the receptor CCK1R (63). The role of GPCRs as hy
s
GPR93 but potentiated the protein hydrolysate response and further sensorsthatcandirectlyrespondtochangesinluminalcontents io
amplified the synergistic enhancement of GPR93 activation by protein is not well defined. The characteristics of their seven trans- log
hydrolysate and LPA. These data suggest that, physiologically, the membrane configuration not only allow for a given GPCR to y.o
compositionofstimulimightdetermineGPR93activityoritssensitivity recognizeawiderangeofmolecularstructuresbutalsoactivate rg
towardagivenactivatorandsuggestanewmechanismoftheregulation multiple pathways depending on the conditions of the stimuli. o
n
ofmucosalcellproliferationanddifferentiationandhormonalsecretion SomeGPCRshavebeenreportedtobedirectlyactivatedby F
e
bydietaryproductsinthelumen. basicL-aminoacidssuchastheCa2(cid:1)-sensingreceptorCaR(9); bru
extracellular signal-regulated protein kinase 1/2; intestine; GPR92; the GPRC6A, which has wide tissue distribution outside the a
GPR93;lysophosphatidicacid intestine(61);andtheheterodimerictastereceptorT1R1/T1R3 ry 5
(47).TheidentificationofG(cid:2)gustducinandtransducin,which , 2
associate with taste receptors, in the gastrointestinal (GI) mu- 0
0
INTESTINAL MUCOSAL HOMEOSTASIS necessitates the coordination cosaaswellasthepresenceofT2RintheGItractsuggeststhis 8
of physiological and chemical factors that regulate functions family of GPCRs can act as sensors that may respond to
from cellular renewal and differentiation along the crypt-to- luminal contents such amino acids and toxins (27, 66).
villus axis to the regulation of hormone secretion, immune Using the enterocyte-like hybrid Berkeley Rat Intestine
response,andnutrientassimilationalongtheproximal-to-distal Epithelial 380 (hBRIE 380i) cells, which do not express
intestine. This coordination is partly facilitated by cellular PepT1, we explored the possibility that signaling cascades in
factors that initiate intracellular signals in response to the the enterocytes initiated by protein hydrolysate could be di-
luminal content. The importance of the presence of luminal rectly mediated by the activation of a GPCR. In the present
dietary nutrients is well exemplified in patients undergoing study, we identified for the first time a GPCR, GPR93, in the
total parenteral nutrition (TPN). Chronic TPN resulting in a rat enterocytes that is directly responsive to protein hydroly-
dramatic mucosal remodeling can lead to a compromised sate.GPR93activationbyproteinhydrolysatemobilizedintra-
absorptive capacity and intestinal immune function and the cellular Ca2(cid:1) concentration ([Ca2(cid:1)]) and activated ERK1/2
i
development of intestinal and liver diseases. throughbothG(cid:2) -andG(cid:2)-mediatedpathways.Proteinhydro-
q i
Studies investigating nutrient-induced signaling and gene lysate and oleoyl-L-(cid:2)-lysophosphatidic acid (LPA) synergisti-
regulation have mostly centered on the effects of fatty acids callyactivatedGPR93.OurdatasuggestthatGPR93couldbe
andglucoseontheexpressionofproteinsinvolvedintransport partly responsible for luminal protein hydrolysate-induced
and metabolism in adipocytes and pancreatic (cid:3)-cells, respec- ERK1/2 activation and for the subsequent effects of this
Addressforreprintrequestsandothercorrespondence:G.W.Aponte,Univ. Thecostsofpublicationofthisarticleweredefrayedinpartbythepayment
ofCalifornia,Dept.ofNutritionalSciencesandToxicology,119MorganHall, ofpagecharges.Thearticlemustthereforebeherebymarked“advertisement”
Berkeley,CA94720-3104(e-mail:[email protected]). inaccordancewith18U.S.C.Section1734solelytoindicatethisfact.
G98 0193-1857/07$8.00Copyright©2007theAmericanPhysiologicalSociety http://www.ajpgi.org
PROTEINHYDROLYSATERESPONSIVEGPCR G99
activation, such as alterations in cell proliferation and differ- G AG
entiation. CA GC
MAMTaEtReIrAiaLlSs.AANlDl rMeaEgTeHntOs,DiSncluding meat protein hydrolysate GGTTCAGCTGGGTACCGGTTCAGCT
(aAcc(cCIwia2dacp2sceletn4ahlociie,tllya3dCtlelloinphlhlssn7r2irvyeetinie°o4obcl1eszu,Clsof,nhiee’0asesafsfxe,t%ec2inouiw)npduuo5dtmrshrlhnnt)ee5ebteayi.eo1ulr.%nosmeepeid0rEsvnxseTeiis0stlsxiCpnfihatanhtIepegi(cid:4)ceiihmOeedrsneroadegi2rdeencnmdsi-/hnoieadmmdD9tocBeutnvlpi5awefuenltLdtaRat%irinltrenyosssPorbsIytestednEcAseriaarawyencuisgdn,wpa(rtcetm3eie3dC.uwtnofre8o5FfeddH’wrhpe0mn(-soyeBmrihOifuer,cteyrermrmCphmnoe)cwceanextmSieebnolwnerapcltdy;efstrliteyeeoi.frsseaaHuer.l(cid:5)rpsrsliecmmupyesmuwtT8aesucisued0eeohlerwsdc(odlan%cnedIelunnhite-M.dtuslcdearitch,fsTeohu)sDfooicB,enaorphnrneMedr1aRrregmlflaol0lWdIGc;sfutah0EshireteiswPueoBIeBinhnUnnrpRsmteR3Riecvtapz/r9es8ymIimiIelerntS3Ee0E(drnopaelmiCiogcnrdl33gpskaoehbmau88eettenralnhebnnni0o0artrdndess-)----iti (cid:8)(cid:8)ReversePrimer(5to3) CGTCGGGCCTCGCCAGTGTCCAGAAGACCGGAGGTGAAGTTGAAACCACATTTTGC CCGTCCGTAGGTTAATTTTCTGTTGGAGCACTGTAGCTGAACCTGAAAGACAATTCCTG(cid:8)ACATTATGCTTCCTTCATCTGTGGAGACCCAGAGGGCTGAATCTTGGGGGCCGTTGTCCCTTACAGTTCATGGCCACTACCAAAAGTAGAGAGCTTCAGATTTTTTCATTGTCATTCATACTCGTCTTAAAGAGTAGAAAGCCTACACTACAGTTAGCTAGCTTAGACCAGATTGTACTCCTTCAGGGTTCCCCGTGTCACAACAGGTTGATCTCGTCCATAGCTAGCTCAGAAGAGGCCACAGTCCTTCAG (cid:3)(cid:3)pe1;AR,-adrenergicreceptor.22 Downloa
ing), coated with rat-tail collagen type I (2). For transfection, GG ACCTGCCCGC bty de
cceellllss/mwle),reantdrypinscinuibzaetde,drwesituhsptehnedpedlasimnidIMDDNMA i(n7.a5 v(cid:6)olu1m0e6 C ptorsu d from
between 0.2 and 0.7 ml at room temperature for 5 min. The AG ece a
total amount of DNA in each transfection was 8 (cid:4)g/106 of TGCCG Yr jpg
PachqpGrGiciraipnfinneedeemBuoruu(cid:2)4sctielldorPmlemliRlae1pda1sesssrslp58eltbitI,dateii0,nautrEiwfieorsuon(n.ssGrm4netI(nreeits(3onmnd(-B(cid:2)rli(8Imevewcdgao(cid:3)nqti0tsliurAohr,mtiot2sti[ecsetr-heACNnEhooicRgboenlenegRaQcrrPictnags2asal2hsueuY)dl)t(cid:1)ttinevs,,epvrwen)P(cid:4),r)]e.udGdlewrw.ifeatsgcetcutPrDttmOteEiitecDeaeensirRmexoNdbnoDeeNapp1sinlebTtAmtyNpteAnar0A.ioaelrlno30AmmeeTEirbeT.sdtz,.cxpslehQ2esieaeTtiPpladuep5ceadotlrr2hi.ihdblo1incoeelloaYekntElio.osdnyytptoiVoswee5nlnEpmseiefa,alrlsnenoeeyacsstPefatqgdrgctsuhorfn2r1ruuahayaoonefemdYcteyf)salpco(ttnrl.eNip9Ide/taooe9tc1iM,rrnorirocwP6e(n0eeaadcenSs0YDsecg6nsthpaeitloG:dor1oMetolnh(cid:4)fvoa3cnRnerfcBPmteFra6-tndae)rr12mRw,oBtg,roch0YAilofIeDpaue(cid:3)aai%oEnfa1gEssos-n2Nr0tfeiork-Qa8ncto3a)aAnwe2rBa0gfat8dgru0irwf0ecC0roGercrpearceioiantlhSeler(cid:4)enPeeesm.o.dGcsdeoRPcgdsevTTrwtte/Cio9uagilremrohhndloaeu3ciaRrsneeeces---tltt,, RT-PCRanalysesandcDNAcloning (cid:8)(cid:8)ForwardPrimer(5to3) CTGCCTGGGCGTGTGGGCTCTCATCCTGCAGGAACCACTGGCTGGACTGGGCTAAAG GACCACATCTGAATACCAAGGCGACCGCCTAGTCTCAAGAGTGGTGAACCCCTGAGATATCCACCATGGGCAGCAATAGCACTTTGCCAATTCTTCAGCCAACACCTCTTCCAGGCGCTCAACATCACCGCGGAGCAGTTCGATCCACCATGAACTCAACTCTGTTCTCCGCTGAGAGCGTCTGGGCACTGAAAGCCCACCATGACTCTGGAGTCCATCATGGCGTGCTGCCCCACCATGGCCCGGTCCCTGACTTGGGGCTGCTGTCCCACCATGGGGTGCTGCCTGAGCGAGGAGGCCAAG indesigningtheprimersforP2Y.NPY1R,neuropeptide10 on February 5, 2008 i.physiology.org
wasligatedintothemultiplecloningsiteofpCI-neoexpression for GCTAAG TGTGTCCTGATGATGGCGACCTGGCGCAGT used
vector (Promega), and each G protein cDNA was ligated into d so
the multiple cloning site of pcDNA3.0 (Invitrogen). G(cid:2)q use sal
wfftvcGhurDeai(cid:2)esctmNih(cid:7)tooe6eAnrqnt)h(ih5,i(ewmtaCwsnyalesracosntse(noh1udtpcae0soncec,gnhcdo3rse)de5t.daeor,sunTn3gch6artrfleee)e.mduPemGonCobrPtvReyAflResuEcldt9ioeegQ3urnmaestttisp(ainc5nlgpgae3ggrtno,PeetttdhC5efpe8iRoanr)trGo),tta(thPieEh.teieRentG.hC9,Fec3(OCoPGBoaOn)Pa2psHm(cid:1)Rteernx--9uHstp3ceeIrrt-rneiesEmoasisGtidnsietniiiFonvuooPngesff ucleotideprimers AccessionNumber XM_575667NM_057121 BC098703XM_228501XM_228500XM_575667NM_198199ZI1504BC086538NM_031036NM_053542 ence(NW_048043)wa
n u
A9asaotmn9frEd-uhiQbnculatoimesgcedDaaatcnfNeuirddcsaAiyginmntm(goAtiocteooYhnpcvrt6hcoee0Domrn4lnNaec0dpo0Arcpdi0a3ioi)nn.xs0gwgii.dgistTtanhyhsPaeneltAthNhsseueerHqebtfiisu2ucrp-sentotnoeitn3crlimsVegbe)oiIanI(ensIJaleu0el(scmi43nlr8eee3c2onplu3tatliad)md(,ciTeniewnRgdpaoErtswih)amciectaohien2dnrd5ass- OligoTable1. Gene RT-PCRanalysisGPR93PepT1cDNAcloningP2Y5P2Y9P2Y10GPR93GPR103NPYIR(cid:3)AR2(cid:2)Gq(cid:2)G15(cid:2)G(cid:7)6qi5my Ratgenomicseq
AJP-GastrointestLiverPhysiol•VOL292•JANUARY2007•www.ajpgi.org
G100 PROTEINHYDROLYSATERESPONSIVEGPCR
nuclear factor of activated T cells (NFAT) luciferase reporter from laser-captured villus and crypt regions (15 patches
constructs are pBV-luc (a generous gift from Dr. Bert Vo- from the crypt and villus area, separately, were pooled) was
gelstein,TheJohns HopkinsKimmelCancerCenter)with 9(cid:6) isolatedusingRNeasyMicroKit(Qiagen).Semiquantitative
TPA response elements (TGACTAA) or 8(cid:6) NFAT response RT-PCR was then performed. The purity of harvested LMD
elements (GGAGGAAAAACTGTTTCATACAGAAGGCGT), sampleswasconfirmedbycomparingtheexpressionlevelof
respectively,insertedinitsmultiplecloningsite.Allconstructs villin and I-FABP, differentiation markers of intestinal ep-
wereverifiedbyDNAsequencing(DNASequencingFacility, ithelial cells.
University of California at Berkeley). Localization of GPR93 in CHO cells. CHO cells were
TissuepreparationandRNAisolationforGPR93expression transfectedwiththeGPR93-EGFPfusionconstructbyelectro-
profile. Overnight fasted male Sprague-Dawley rats (14 wk poration(4(cid:4)gplasmidDNA/106cells).After24hofrecovery
old) were used as tissues sources (n (cid:9) 4). The protocol for incubation in IMDM-10% BCS under normal culture condi-
animalusewasreviewedandapprovedbytheAnimalCareand tions, cells were trypsinized, resuspended in phenol red-free
UseCommitteeoftheUniversityofCaliforniaatBerkeley.GI IMDM-10% BCS, plated on six-well slides coated with colla-
tissue samples were prepared as follows: intestines were ex- gen type I at a density of 104/well, and incubated for 1 h in
tracted and cut into segments (each (cid:5)5 cm long). Residual humidifiedpetridishes.Slideswerethentransferredintofresh
luminal contents were removed by running ice-cold PBS media and further incubated for 16 h under normal culture
through the intestinal segments. The mucosal layer of the conditions. The localization of EGFP-tagged GPR93 was vi-
intestine was obtained by gentle scraping of the exposed sualizedbyusingLaserScanningConfocalMicroscopy(Zeiss
luminal surface. The muscle layer was obtained after further 510 UV/Vis Meta system).
scraping to remove the residual mucosal layer. The stomach AEQ-based [Ca2(cid:1)]i mobilization assay. [Ca2(cid:1)]i mobiliza- Dow
mucosa was prepared in a similar fashion. The purity of the tion assay was performed as previously described (54) with n
mucosa was verified by the relative expression of villin and slight modifications. Briefly, mtAEQ expression vector was lo
a
intestinal fatty acid binding proteins (I-FABP), differentiation coelectroporated(2(cid:4)g/106cells)withotherplasmidconstructs de
d
maNrkoenr-sGoIf tiinstseusetinsaalmenptleersocwyeterse, aosbtdaeinteerdmibnyedrbemyoRvTin-PgCtRh.e arescoinvdeircafoterd20inhthine IfiMguDrMes,-1a0n%d BthCeS.ceFllosr wCeHrOe aclelollws,ecdeltlos fro
m
organs, followed by rinsing with ice-cold PBS twice and weredislodgedwith5mMEDTA-PBSandloadedwith5(cid:4)M a
mincingwithsurgicalscissors.Immediately,thetissuesamples coelenterazine-h (Promega)-300 (cid:4)M glutathione in IMDM jp
g
wereimmersedinice-coldTRIzol(Invitrogen),homogenized, (2 (cid:6) 106 cells/ml) at 37°C for 2 h with gentle rolling. For i.p
h
andfrozeninliquidnitrogen.Alltissuepreparationstepswere hBRIE 380i cells, cells were trypsinized and gently rolled for y
s
doneonice.RNAwasisolatedfromtheTRIzoltissuehomog- 1 h in IMDM-10% BCS, followed by loading in HBSS (In- io
lo
enate according to the manufacturer’s protocol. vitrogen) for 1 h under the same condition as used for CHO g
Semiquantitative RT-PCR. Reverse transcription was as de- cells. A 100-(cid:4)l aliquot (5 (cid:6) 104 cells in HBSS) was then y.o
scribed previously (40). PCR was carried out using Taq DNA assayed in a luminometer equipped with an injector (Turner rg
o
polymerase (New England Biolab). GPR93 specific primers BioSystem).Thestimuluswasinjectedintothecellsuspension n
amplified a cDNA fragment of 249 bp. PCR parameters for in a 100-(cid:4)l aliquot at a 2(cid:6) final concentration in PBS. A F
e
GPR93 were as follows: 20 s at 94°C, 15 s at 55°C, and 30 s 100-(cid:4)l aliquot of lysis buffer (300 mM CaCl2 and 300 (cid:4)M bru
at 72°C for 32 cycles. The primers and PCR condition for digitonin) was injected 40 s later to react with the remaining a
ry
villin, I-FABP, and 18S RNA were as previously described AEQ. Luminescence [as relative light units (RLU)] was re- 5
(40).AmplifiedcDNAfragmentswereanalyzedbyagarosegel cordedcontinuously.FractionalRLUisincreasedRLUdueto , 2
0
electrophoresis followed by densitometry. The specificity of a stimulus normalized to the total RLU, i.e., the integrated 0
8
the PCR products was confirmed by DNA sequencing. RLUvaluefor30safterinjectionofthestimulusplusthatfor
Laser microscopy dissection. Rat duodenum tissue sections 20 s after the addition of the lysis buffer. All reagents tested
were prepared for cryostat as previously described (24). were dissolved in PBS (pH 7.4).
Briefly, the duodenum section was removed from overnight TodeterminewhetherprotonscouldactivateGPR93,stimuli
fastedmaleSprague-Dawleyrats(12wkold),andtheresidual in the form of the buffer at varying pH were used. Phosphate
luminal contents were washed out by running ice-cold PBS buffer (PB; 10 mM Na HPO and 2 mM KH PO ) was pH
2 4 2 4
throughtheduodenumsegment.Theduodenumwasfurthercut adjustedwithHClorNaOHto6.5(acidic),7.4(neutral),or8.5
into 2-mm sections in the horizontal direction after short (basic), and NaCl was added to bring the osmolality to 300
fixation in 70% ethanol in PBS (pH 7.4). The tissue sections osmol/kgH O, as determined by microosmometer (Precision
2
werebrieflyrinsedwithice-coldPBSandimmersedinice-cold Systems). To test whether osmotic pressure could stimulate
30% (wt/vol) sucrose in PBS overnight at 4°C. The sucrose- GPR93, modified PB (PB at 100 osmol/kgH O, adjusted with
2
equilibrated sections were embedded into optimum cutting NaCl, pH. 7.4), 2% glycerol in modified PB (211 osmol/
temperature compound (TissueTeK), frozen on dry ice, cryo- kgH O), and 4% glycerol in modified PB (553 osmol/kgH O)
2 2
sectioned at 10 (cid:4)m thickness, and then stored at (cid:10)80°C. were used as stimuli.
For the laser microscopy dissection (LMD) procedure, 10 BSA digestion. Fatty acid-free BSA (Roche) was dissolved
(cid:4)m cryosections were mounted on slides, fixed with 70% in PBS (pH 7.4) at a concentration of 10 mg/ml (wt/vol).
ethanol for 30 s, and stained with eosin, followed by a 5-s DigestedBSAwaspreparedbyincubatingaBSAsolution(10
dehydration step in each of 70%, 95%, and 100% ethanol. mg/ml)withproteinaseK(5(cid:4)genzyme/mgBSA,Invitrogen)
Afterbriefairdrying,thesectionswerelasermicrodissected at37°Cfor20hwithgentleagitation.Thesolutionwasheated
using a Leica AS LMD system with the following setting: at 80°C for 15 min to inactivate proteinase K and then centri-
aperture, 6–10; intensity, 45; and speed, 2–5. Total RNA fuged at 16,000 g at 4°C for 5 min.
AJP-GastrointestLiverPhysiol•VOL292•JANUARY2007•www.ajpgi.org
PROTEINHYDROLYSATERESPONSIVEGPCR G101
Luciferase reporter assay. Two micrograms of the reporter
construct/106 of either CHO or hBRIE 380i cells were elec-
troporatedwithotherconstructsasindicatedinthefigures,and
cells were seeded into 12-well plates at 5 (cid:6) 105 cells/well in
IMDM-10% BCS. For the NFAT reporter study, cells were
allowed to recover in IMDM-10% BCS for 20 h after trans-
fection.Onthedayofeachexperiment,cellswerefirstwashed
three times with PBS, serum starved for 2 h, and then treated
with either 10 (cid:4)M LPA or 50 mg/ml peptone in serum-free
IMDMfor6h.FortheTREreporterstudy,cellswereallowed
to recover in IMDM-10% BCS for 36 h. On the day of each Fig.1. InductionofERK1/2phosphorylationbypeptoneinhybridBerkeley
RatIntestineEpithelial380(hBRIE380i)cells.ThehBRIE380icellswere
experiment, cells were washed three times with PBS and
laiddownin60-mmtissueculturedishesatthedensityof2(cid:6)105/dish.After
treated with either 10 (cid:4)M LPA or 50 mg/ml peptone in
a24-hincubationat37°CinIscove’smodifiedDulbecco’smedium(IMDM)-
serum-freeIMDMfor12h.Fattyacid-freeBSA,ataconcen- 10%bovinecalfserum(BCS),cellswereserumstarvedinIMDM-0.1%BCS
tration of 0.1% (wt/vol), was added as a carrier in the treat- for12h,followedbya1-hincubationinHBSS.Thecellsweretreatedwith10
or20mg/mlofpeptonefor4min.ThelevelsofERK1/2phosphorylationwere
ments.Fortymicrolitersofpassivelysisbuffer(Promega)were
determinedbyWesternblotanalysisasdescribedinMATERIALSANDMETHODS.
added to each well after the treatments. The luciferase activi- A representative image was shown. The intensity of the phosphorylated
ties of the samples were determined according to the manu- ERK1/2(pERK1/2)bandswasnormalizedtototalERK1,andtheresultwas
facturer’sprotocolusingaluminometerandnormalizedtothe expressedasfoldchangewiththevaluewithoutpeptonearbitrarilysetto1. D
o
totalproteinconcentration,determinedbytheBio-Radprotein w
n
assay (Bio-Rad). lo
Inhibitors treatment. For [Ca2(cid:1)] mobilization assay, cells RESULTS ad
i e
wbeefroereintchuebaastesdayw. Uith-783012n2g,/morliptseritnuascstiisvteoxaninal(oPgTUX-)7f3o3r4234, aht nalOduiretoabryjescitgivnealwsainsttootshteudinytethsteinGalPeCpRitshethliaatltcrealnlssd.uWceeecxhtoesre- d fro
m
10 or 20 (cid:4)M, was mixed with the stimulus without preincu- protein hydrolysate (peptone), a mixture of enzymatically de- a
bation. Nifedipine (10 (cid:4)M) and thapsigargin (20 nM) were rived peptide fragments (mostly between 120 and 1,200 Da) jp
g
added to the cells 5 min and 30 min, respectively, before a and free amino acids, as a representative dietary component i.p
h
stimulus was added. For Western blot analysis, cells were since peptone mimics dietary proteins digest in the luminal y
s
preincubatedwith100ng/mlPTXfor20h.Thepreincubation chyme (12). Protein hydrolysate stimulates mucosal brush io
with 100 nM wortmannin, 5 (cid:4)M U-73122 or U-73343, or 50 border ERK1/2 (6), but a GPCR that mediates this event has log
(cid:4)M PD-98059, was for 30 min. yettobeidentified.ThesignificanceofERK1/2activityinthe y.o
Westernimmunoblottinganalysis.Todeterminetheeffectof intestinal mucosa has been illustrated by the prominent sub- rg
o
protein hydrolysate on ERK1/2 activation, hBRIE 380i cells cellularlocalizationofERK1/2inthebrushborderandbythe n
were transfected with 6 (cid:4)g GPR93 cDNA/106 cells and laid responsivenessofERK1/2signalingtochangesintheluminal Fe
down in 60-mm dishes at a density of 2(cid:6) 105 per dish. After content in response to feeding (6). bru
a 24-h incubation in IMDM-10% BCS, cells were serum We determined that peptone also stimulated the phosphor- ary
starved in IMDM-0.1% BCS for 12 h, followed by a 1-h ylation of ERK1/2 in enterocyte-derived hBRIE 380i cells 5
incubation in HBSS. The cells were treated with 20 mg/ml of (Fig.1).TheoligopeptidetransporterPepT1,areportedmedi- , 2
0
peptone or 100 nM LPA for 4 min, unless otherwise noted in ator of peptone responses in intestinal cells (14, 44), is not 08
expressedinhBRIE380icellsasdeterminedbyRT-PCR(data
thefigures.Afterthetreatment,cellswereimmediatelyplaced
onice,rinsedtwicewithice-coldPBS,andscrapedwith40(cid:4)l notshown).Wetookadvantageofthisfacttosearchforother
of 2(cid:6) Laemmli sample buffer per 60-mm dish. Western blot mediators of peptone response using hBRIE 380i cells.
We focused on orphan GPCRs because the activation of
analysis on polyvinylidene difluoride membrane was carried
many GPCRs often leads to increased ERK1/2 (28, 57), and
out as previously described (24). Protein concentration was
GPCRs have a wide variety of activators: from large glyco-
determined by the Bio-Rad protein assay. The amount of
proteinhormones,neurotransmitters,andmetabolitestoexter-
proteinperlanewas0.2(cid:4)gforERK1detectionand10(cid:4)gfor
nalstimuli,suchaslightandodorantmolecules.Besidesbeing
phosphorylated ERK1/2 (pERK1/2). The primary antibody
activated by their high affinity ligands, GPCRs can also be
againstERK1(SantaCruzBiotechnology)wasusedat1:3,000
stimulated by activators with different molecular characteris-
and against pERK1/2 (Cell Signaling) at 1:2,000. The horse- tics; for example, the Ca2(cid:1)-sensing GPCR can also be acti-
radish peroxidase-conjugated secondary antibody was used at
vatedbyL-aminoacids(9),andtheproton-sensingGPCRcan
1:10,000.
also be activated by lysolipids (59).
Sequence analysis. Identity score between different GPCRs The GenBank and Ensembl genomic DNA databases were
was determined by the ClustalW alignment method. searched for putative GPCRs with non-olfactory-like se-
Statistical analysis. Data are expressed as means (cid:11) SD. quences.RNAsfromsmallintestinalepithelialcellsisolatedby
Statisticaldifferencebetweenmultiplegroupswasdetermined dispersion (2) and from hBRIE 380i cells were used to screen
by one-way ANOVA with Tukey’s post hoc test performed candidateGPCRsbyRT-PCR(unpublisheddata).Theselected
usingSPSSversion11.SignificancewasacceptedatP(cid:12)0.05. candidate GPCRs were tested for their responsiveness to pep-
Dose-response curves were generated using the curve-fitting tone in a [Ca2(cid:1)] mobilization assay by cotransfecting the
i
software GraphPad Prism version 4. promiscuous G(cid:2) (unpublished data).
15
AJP-GastrointestLiverPhysiol•VOL292•JANUARY2007•www.ajpgi.org
G102 PROTEINHYDROLYSATERESPONSIVEGPCR
We determined GPR93 was a candidate GPCR that was yield an observable [Ca2(cid:1)] peak. In contrast, upon its activa-
i
responsive to peptone. O’Dowd and coworkers (39) reported tion, GPR103 yielded an increased [Ca2(cid:1)] similar to that of
i
its expression in several mouse tissues. GPR93 is a family A GPR93withouttheadditionofapromiscuousG(cid:2)protein.This
GPCR that belongs to a group of purinoreceptor-like GPCRs suggests that a G(cid:2) -mediated pathway is downstream of
q
(29),i.e.,P2Y ,P2Y ,andP2Y .Theaimofthisstudywasto GPR93 activation. A [Ca2(cid:1)] flux due to a GPCR activation
5 9 10 i
characterizeGPR93activationbypeptoneinhBRIE380icells, can be caused by G(cid:2) or G(cid:3)(cid:13) dissociated from G(cid:2). PTX (80
q i
focusing on the collective net [Ca2(cid:1)] flux and ERK1/2 phos- ng/ml),aspecificinhibitorofG(cid:2),reducedpeptonestimulation
i i
phorylation. by50%andLPAstimulationby60%(Fig.2D),suggestingthat
PeptoneandLPAinduce[Ca2(cid:1)] inCHOcellsoverexpress- GPR93 activation partially leads to a pathway involving G(cid:2).
i i
ing GPR93. The first step of the characterization of GPR93 GPCRs often exert intracellular signaling events by coupling
activation was performed in CHO cells using the mtAEQ- with more than one kind of G proteins; as an example, LPA
based [Ca2(cid:1)] mobilization assay. The proper localization of receptor1,2,and3cancouplewithbothG(cid:2) andG(cid:2) (8).The
i i q
the transfected GPR93 to the cell surface plasmalemma was possibleinvolvementofotherG(cid:2)proteinsinGPR93-mediated
confirmedbyfluorescenceconfocalmicroscopyusingGPR93- [Ca2(cid:1)] increase in our system remains to be determined. A
i
EGFPfusion(Fig.2A,inset).Althoughintheinitialscreening possible downstream effector of G(cid:2)- or G(cid:2) -coupled recep-
i q
process G(cid:2) was cotransfected with GPR93, cells transiently tors mediating the increase of [Ca2(cid:1)] is PLC-(cid:3). A treatment
15 i
transfected only with GPR93 also showed a significant, tran- with PLC-(cid:3) inhibitor U-73122, but not its inactive analog
sient[Ca2(cid:1)] fluxinresponsetopeptone.Therewasno[Ca2(cid:1)] U-73343, almost abolished peptone induction and inhibited
i i
intheemptyvectortransfectantinresponsetopeptone,withor LPAstimulationbymorethan50%(Fig.2E).PLC-(cid:3)liberates
without a promiscuous G(cid:2) (data not shown). Thus all experi- inositoltriphosphate,whichactivates theCa2(cid:1)channelonthe Do
ments were carried out with GPR93-transfected cells without endoplasmic reticulum membrane resulting in a [Ca2(cid:1)]i flux. wn
cotransfecting a promiscuous G(cid:2) (G(cid:2) or G(cid:2) ). Nifedipine (10 (cid:4)M), an inhibitor of plasma membrane L-type lo
6qi5myr 15 a
We tested the possibility that peptone might activate other voltage-gated Ca2(cid:1) channel, did not affect the [Ca2(cid:1)]i flux de
d
GprPesCsRinsgnGoPnRsp1e0c3ifi,cNaPllYy.1RPe(pctootnrea-ntsrefeacteteddCwHitOh Gce(cid:2)lls ove)r,eoxr- isnhdouwcne)d.Tbhyappesipgtaorngeino(r20LPnAM)s,taimnuinlahtiibointorofofGsParRc9o3(en(ddaot)aplnaos-t fro
6qi5myr m
(cid:3)2AR (cotransfected with G(cid:2)15) did not show an increase in mic reticulum Ca2(cid:1) ATPase, completely eliminated the in- a
[Ca2(cid:1)]. P2Y , P2Y , and P2Y , with or without cotransfec- crease in [Ca2(cid:1)], suggesting that GPR93 activation induces jp
i 5 9 10 i g
tion with G(cid:2)15, were also not stimulated by peptone (data not Ca2(cid:1) release from intracellular stores (data not shown). i.ph
shown). The possibility of GPR93 activation by compounds We repeated the characterization of GPR93 activation in y
s
otherthanpeptone,mostofwhichcouldbeinthediet,wasalso hBRIE 380i cells. In hBRIE 380i cells transiently transfected io
esixmpliolarrede.xteSnotytopproetpetionneh.yTdhroelycsoamteposutinmdsultahtaetddiGdPnRo9t3indinucea cwoittrhanGsPfeRc9ti3n,gpeaptpornoemoisrcLuoPuAsinGd(cid:2)ucpedroatei[nC.a2T(cid:1)h]ei fliunxcrewaistehoiunt logy.o
[Ca2(cid:1)] are listed in Table 2. The possibility of the activation [Ca2(cid:1)] levels was concentration dependent (Fig. 3A). The rg
i i o
opfloGrePdR. p9H3 bfryompro6t.o5nt/opH8.5o,rahsywpeol/lhayspeorstmonoilcailtiytywfarosmal1so00exto- 3EAC)5.0PfTorXpreepdtounceedwtahse1p0e.p6tomnge/smtilmaunldati7o.n9 nbMy 6f8o%r LaPnAd (LFPigA. n F
e
553 osmol/kgH2O, did not induce [Ca2(cid:1)]i in GPR93-overex- stimulation by 50% (Fig. 3B). U-73122 decreased peptone bru
pressing CHO cells. stimulationby83%,whereasU-73343hadnoeffects(Fig.3C). a
ry
AnothercompoundthatwastestedwasLPA.ExternalLPA The effect of U-73122 was dose responsive (data not shown). 5
can be present in the diet as it is or as a product of PLA2 Similarly, U-73122, but not U-73343, inhibited LPA stimula- , 2
0
digestioninthelumenoftheintestine.P2Y9,whichhasa30% tionby52%(Fig.3C).Thesedataareconsistentwithwhatwas 08
amino acid identity with GPR93, was recently reported to be observed in CHO cells.
activated by LPA (37). We treated GPR93-transfected cells GPR93 activation leads to induced NFAT- and TRE-lucif-
with 5 (cid:4)M LPA and observed that [Ca2(cid:1)] was significantly erase reporter activities. We tested whether the increase in
i
induced, but there was no [Ca2(cid:1)] release observed in the [Ca2(cid:1)] levels due to GPR93 activation could lead to down-
i i
empty vector transfectant (data not shown). Therefore, LPA stream gene responses by the luciferase reporter assay. We
was included as a stimulus to further characterize GPR93 examinedtheluciferaseactivityofreporterconstructscontain-
activation. ing response elements to NFAT and TPA (TRE) as indicators
Peptone and LPA activation of GPR93 in CHO and hBRIE forincreased[Ca2(cid:1)].GPR93activationbypeptoneorLPAin
i
380i cells is PTX sensitive and mediated by PLC-(cid:1). GPR93- CHO cells induced luciferase activity from a construct con-
mediated[Ca2(cid:1)] fluxinresponsetopeptoneorLPAwasdose taining NFAT responsive elements (Fig. 4A). The NFAT lu-
i
responsive (Fig. 2, A and C). The EC was 4 mg/ml for ciferasereportercouldnotbeusedinhBRIE380icellsbecause
50
peptoneand3.6nMforLPA(Fig.2C).Forcomparison,known eventhepositivecontrol(TPAplusionomycin)didnotinduce
GPCRs were activated by their agonists, i.e., NPY1R (a G(cid:2)- the luciferase activity, which might be due to the lack of
i
GPCR)with/withoutchimericG(cid:2) (achimericGprotein expression of the transcription factor (unpublished data).
6qi5myr
toenhancesignalsfromG(cid:2)-coupledreceptor)byNPY,(cid:3) AR Therefore, we used a luciferase reporter construct containing
i 2
(a G(cid:2)-GPCR) with/without G(cid:2) (to enhance signals from TRE. The activation of GPR93 by peptone or LPA induced
s 15
G(cid:2)-coupled receptor) by isoproterenol, and GPR103 [a G(cid:2) - luciferase expression (Fig. 4B). Interestingly, the effect of
s q
GPCR (31)] by RFamide P518 (Fig. 2B). Cotransfecting peptoneonNFAT-drivenreporterwasweakerthanthatofLPA
G(cid:2) was needed to observe a significant increase in in CHO cells, but it was LPA that had a weaker effect on the
6qi5myr
[Ca2(cid:1)] flux elicited through NPY1R. This was similar to TREluciferasereporterinhBRIE380icells.PeptoneandLPA
i
(cid:3) AR, for which a cotransfection with G(cid:2) was required to also induced luciferase expression from the TRE-luciferase
2 15
AJP-GastrointestLiverPhysiol•VOL292•JANUARY2007•www.ajpgi.org
PROTEINHYDROLYSATERESPONSIVEGPCR G103
Fig. 2. Dose-dependent intracellular Ca2(cid:1)
concentration([Ca2(cid:1)])increasemediatedby
i
G(cid:2) andPLC-(cid:3)inresponsetoGPR93stim-
i
ulationbypeptoneandoleoyl-L-(cid:2)-lysophos-
phatidic acid (LPA) in Chinese hamster
ovary (CHO) cells. A: the localization of
enhanced green fluorescent protein-tagged
GPR93onthecellsurfaceplasmalemmawas
visualized by laser scanning confocal mi-
croscopy (inset; scale bar (cid:9) 10 (cid:4)m). CHO
cellstransfectedwithmitochondria-targeted
aequorin(mtAEQ)andGPR93cDNAwere
loadedwith5(cid:4)Mcoelenterazine-hfor2h,
and[Ca2(cid:1)] wasassayedinthepresenceof
i
peptone or LPA at the indicated concentra-
tionsasdescribedinMATERIALSANDMETH-
ODS. A representative plot of [Ca2(cid:1)]i in-
crease vs. time was shown (n (cid:9) 3). Frac-
tionalrelativelightunits(RLUs)istheRLU
atatimepointovertotalRLUasdescribedin
MATERIALS AND METHODS. The arrow indi- Do
cates the time when peptone or LPA was w
introduced. B: for comparison, [Ca2(cid:1)]i of nlo
CHO cells cotransfected with the cDNA of a
d
GPR103, neuropeptide Y (NPY) receptor e
sGu(cid:2)b(cid:7)ty6pqie5my1r(a(cNhPimYe1rRic),GoprroteNinPYto1Renhawnicthe d fro
Ca2(cid:1)signalfromG(cid:2)-coupledreceptor),and m
(cid:3) -adrenergicreceptoir((cid:3) AR)or(cid:3) ARwith a
G2(cid:2)15 (to enhance Ca2(cid:1)2signal fr2om G(cid:2)s- jpg
coupledreceptor)inresponsetotheirrespec- i.p
tive ligands as indicated was assayed. A hy
representativeplotof[Ca2(cid:1)]iprofileforeach sio
of the G protein-coupled receptors vs. time lo
was shown. C: CHO cells transfected with g
y
mtAEQcDNAandtheemptyvector(Œ)or .o
GPR93cDNA(})werestimulatedwithvar- rg
ious doses of peptone or LPA. Integrated o
n
RLU represents the integrated fractional F
RLUvaluesfora30-speriodaftertheintro- e
b
ductionofpeptoneorLPA.Datapointsare ru
means (cid:11) SD (n (cid:9) 3). D: [Ca2(cid:1)] of CHO a
cells expressing GPR93 treatediwith 20 ry 5
masgsa/myeldofafpteerptaon2e4-ohr 1p0reinnMcuboaftioLnPAinwthaes , 20
presence or absence of 80 ng/ml pertussis 0
8
toxin (PTX). Fold change is the ratio of
integratedRLUinthepresenceofstimulito
that in the absence of stimuli (substituted
withPBSasacontrol),whichwasarbitrarily
designatedas1.Barsaremeans(cid:11)SD(n(cid:9)
3).aP(cid:12)0.05,relativetobar2.bP(cid:12)0.05,
relative to bar 3. E: [Ca2(cid:1)] of CHO cells
i
expressingGPR93treatedwith20mg/mlof
peptoneor10nMofLPAwasassayedinthe
presenceorabsenceof20(cid:4)MofU-73122or
U-73343. Bars are means (cid:11) SD (n (cid:9) 3).
aP (cid:12) 0.05, relative to bar 4. bP (cid:12) 0.05,
relativetobar5.Whitebarsrepresentcon-
trols, black bars represent peptone treat-
ments, and gray bars represent LPA treat-
ments.
reporter in GPR93-transfected CHO cells with LPA having a GPR93activationmediatesenhancedERK1/2phosphoryla-
strongereffect(datanotshown).Thedifferenceinpeptoneand tion in hBRIE 380i cells that is sensitive to PTX, mediated by
LPA effects on the reporter gene suggests that activation of PLC-(cid:1), and dependent on MAPKK. Cells transfected with
GPR93 by peptone and LPA is distinct and cell type specific. GPR93 exhibited enhanced ERK1/2 activation in response to
AJP-GastrointestLiverPhysiol•VOL292•JANUARY2007•www.ajpgi.org
G104 PROTEINHYDROLYSATERESPONSIVEGPCR
Table 2. The list of compounds that did not activate GPR93 Peptone and LPA synergistically activate GPR93. Whether
peptone and LPA could act in concert to activate GPR93 was
Category CompoundsTested tested by treating GPR93-transfected hBRIE 380i cells with
Aminoacids Casaminoacids varyingdosesofpeptoneandLPAsimultaneouslyina[Ca2(cid:1)]i
Eachofthe20aminoacids mobilization assay (Fig. 6A). Peptone or LPA individually
Di_tripeptides Glycyl-glycine activated GPR93. However, [Ca2(cid:1)] increase was more than
i
(cid:13)-Glutamyl-cysteinyl-glycine additivewhenbothwerecombinedandusedasastimulus(Fig.
Monosaccharides Arabinose 6A, bar 4 vs. 2 and 3; and bar 7 vs. 5 and 6). Fatty acid-free
Glucose
BSA at 0.1% and peptone were added together in this partic-
Xylose
ular experiment because LPA solutions used in all our exper-
Fructose
Galactose iments contained 0.1% fatty acid-free BSA as a carrier. We
Disaccharides (cid:2)-Lactose observed that GPR93 did not significantly respond to BSA
(cid:3)-Lactose stimulationupto100mg/ml(datanotshown);however,BSA
Maltose reduced [Ca2(cid:1)] flux due to peptone (data not shown) or LPA
Melibiose i
stimulation (Fig. 7A, bar 6 vs. 5; and bar 8 vs. 7). Therefore,
Sucrose
Trehalose [Ca2(cid:1)]iinductionbypeptoneinFig.6wastoalessextentthan
Sugaralcohol Sorbitol what was shown in Fig. 3C.
Shortchainfattyacids Aceticacid GPR93couldbeactivatedbypeptoneandLPAasshownin
Butyricacid Fig. 3. We tested whether endogenous GPR93 activation in
Long-chainsaturatedfattyacids PLraoupriiocnaiccidacid hBRIE 380i cells could also be determined by [Ca2(cid:1)]i mobi- Dow
lization assay using hBRIE 380i cells stably expressing n
Myristicacid mtAEQ. At the cell stage before confluency, peptone or LPA lo
Palmiticacid a
Long-chainunsaturatedfattyacid Oleicacid didnotincrease[Ca2(cid:1)]i(datanotshown).However,whenthe de
d
Bileacids CDheooxliyccahcoildicacid ceenlclys,wpeerpetoanlleowanedd tLoPbAecionmdueceddiff[eCrean2(cid:1)tia]ite(dFiagt.86-Bd)p.oInstdcuocntflioun- from
Taurocholicacid by peptone was much weaker than LPA compared with what a
Nucleotides CMP wasobservedinproliferatingcellsinthetransienttransfection jp
g
ADP system. This might be due to increased expression of other i.p
CDP h
endogenous LPA receptors in our cell culture system that, y
ATP s
UTP unlike GPR93, are not peptone responsive. The induction of io
Sugarnucleotides UDP-glucose [Ca2(cid:1)]i was synergistically enhanced when peptone and logy
UDP-galactose LPA were combined (Fig. 6B, bar 4 vs. 2 and 3; and bar 6 .o
UDP-glucuronicacid vs. 2 and 5). rg
UDP-N-acetylglucosamine o
We used two approaches to verify that the peptide compo- n
nentsinpeptonewereindeedresponsibleforGPR93activation F
e
and for synergistic effects of peptone plus LPA on GPR93 bru
activation. We tested the effect of proteinase K-digested fatty a
peptone or LPA compared with cells transfected with the acid-free BSA on the [Ca2(cid:1)]i flux in the GPR93-overexpress- ry 5
empty vector. The level of pERK1/2 was induced rapidly in inghBRIE380icells.IncontrasttoundigestedBSA,whichdid , 2
responsetopeptoneorLPA,reachingamaximumat4minand not induce a change in [Ca2(cid:1)]i, digested BSA (50 mg/ml) 008
then gradually decreasing (data not shown). Peptone at a dose showedasignificantincreaseoverbasal(Fig.7A,bar4vs.1).
between 20 and 50 mg/ml maximally induced ERK1/2 phos- Thechangein[Ca2(cid:1)] waslessthanthatobservedwithpeptone
i
phorylation in GPR93-overexpressing cells, whereas specific atasimilardose.Thisislikelyduetothefactthatthepeptide
LPA stimulation was only observed at 100 nM (data not componentsinpeptonearederivedfrommorethanonekindof
shown). protein. Digested BSA synergistically induced [Ca2(cid:1)] release
i
TodeterminethecorrelationbetweenGPR93activationand when it was combined with LPA (Fig. 7A, bar 9 vs. 2 and 5;
theincreaseinpERK,PTXandU-73122wereused.PTX(100 and bar 10 vs. 3 and 5). We also utilized cefaclor, a peptido-
ng/ml)almostabolishedpeptone(Fig.5A)aswellasLPA(Fig. mimetic substrate of the oligopeptide transporter PepT1 (7),
5A) induction. Likewise, U-73122 decreased pERK induction whichdidnotinduce[Ca2(cid:1)] releaseinhBRIE380icells(Fig.
i
bypeptone(Fig.5B)andLPA(Fig.5B)tothebasallevels.The 7B, bar 5; and tested up to 5 mM; data not shown). Cefaclor
treatment with phosphatidylinositol 3-kinase inhibitor wort- synergisticallyenhancedGPR93activationbypeptoneorLPA
mannin (100 nM) did not change the effect of peptone on (Fig. 7B, bar 6 vs. 2 and 5; and bar 7 vs. 3 and 5). Enhanced
pERK(datanotshown).Theseresultssuggestaninvolvement activation of GPR93 by a combination of peptone and LPA
ofG(cid:3)(cid:13)subunitsfromG(cid:2) andPLC-(cid:3)activationintheinduc- wasalsofurtheramplifiedinthepresenceofcefaclor(Fig.7B,
i
tion of pERK. This does not exclude the possibility of other bar 8 vs. 4).
pathways, which were not explored in this study, contributing TheimplicationofthisresultisthatLPAandpeptonemight
toERK1/2activation(includingthosethatareG(cid:2) mediated). act on different sites of the GPR93 molecule. Although at the
q
The observed induction of pERK due to GPR93 activation doses of peptone and LPA that were used, the effects were
requiresMAPKK(MEK1/2),sincePD-98059(MEK1/2inhib- synergisticwhethertheGPR93activationbysaturateddosesof
itor) at 50 (cid:4)M abolished induced pERK by peptone (Fig. 5C) peptonecouldbeamplifiedbyLPAandviceversacouldnotbe
and LPA (Fig. 5C). determined due to the nature of the assay. The physiological
AJP-GastrointestLiverPhysiol•VOL292•JANUARY2007•www.ajpgi.org
PROTEINHYDROLYSATERESPONSIVEGPCR G105
Fig. 3. G(cid:2)- and PLC-(cid:3)-mediated, dose-re-
i
sponsive peptone and LPA induction of
[Ca2(cid:1)] in GPR93-transfected hBRIE 380i
i
cells. A: dose responsiveness of [Ca2(cid:1)] in-
i
crease due to GPR93 activation by peptone
(left) or LPA (right) was determined as de-
scribedinFig.2C.Datapointsaremeans(cid:11)
SD(n(cid:9)3).B:theeffectof80ng/mlPTXon
[Ca2(cid:1)] when cells were treated with 20
i
mg/ml peptone or 30 nM LPA was deter-
mined.Barsaremeans(cid:11)SD(n(cid:9)3).aP(cid:12)
0.05,relativetobar2.bP(cid:12)0.05,relativeto
bar3.C:[Ca2(cid:1)] responseduetoatreatment
i D
with20mg/mlpeptoneor30nMLPAwas o
assayedinthepresenceorabsenceof20(cid:4)M w
U-73122orU-73343.Barsaremeans(cid:11)SD nlo
(n(cid:9)3).aP(cid:12)0.05,relativetobar4.bP(cid:12) a
d
0.05,relativetobar5.Whitebarsrepresent e
d
cmoenntrtso,lsa,nbdlagckraybabrsarrseprreepsreenstenptepLtoPnAe ttrreeaatt-- fro
m
ments.
a
jp
g
i.p
h
y
s
io
lo
g
y
.o
rg
o
n
F
e
b
ru
a
ry
5
, 2
0
0
Fig.4. InductionofnuclearfactorofactivatedT 8

cells (NFAT)- and TPA response element
(TRE)-luciferase reporter activities by GPR93
activation.A:CHOcellsweretransfectedwith2
(cid:4)gNFAT-luciferasereporterplasmidDNAplus
6(cid:4)gofeithertheemptyvectororGPR93cDNA
construct.Twentyhoursaftertransfection,cells
wereserumstarvedfor2h,followedbyeither50
mg/mlpeptoneor10(cid:4)MLPAtreatmentin50%
IMDMfor6h.Barsaremeans(cid:11)SD(n(cid:9)3).
FoldchangerepresentsthechangesinRLUper
microgram total protein of the cells stimulated
withpeptoneorLPAnormalizedtothecontrol
value (of cells treated with PBS), which was
arbitrarilydesignatedas1.aP(cid:12)0.05,relativeto
bar4.B:hBRIE380icellsweretransfectedwith
2(cid:4)gTRE-luciferasereporterplasmidDNAplus
6(cid:4)gofeithertheemptyvectororGPR93cDNA
construct. Thirty-six hours after transfection,
cellsweretreatedwitheither50mg/mlpeptone
or 10 (cid:4)M LPA in IMDM-0.1% fatty acid-free
BSA for 12 h. Bars are means (cid:11) SD (n (cid:9) 3).
Fold change is as described in A. aP (cid:12) 0.05,
relativetobar4.
AJP-GastrointestLiverPhysiol•VOL292•JANUARY2007•www.ajpgi.org
G106 PROTEINHYDROLYSATERESPONSIVEGPCR
Fig. 5. G(cid:2)- and PLC-(cid:3)-mediated and
i
MAPKK (MEK1/2)-dependent increase of
ERK1/2 phosphorylation due to GPR93 acti-
vationbypeptoneorLPAinhBRIE380icells.
A: hBRIE 380i cells were transfected with
either 6 (cid:4)g of the empty vector or GPR93
expression construct/106 cells. The levels of
ERK1/2 phosphorylation in response to 20
mg/ml peptone or 100 nM LPA were deter-
minedbyWesternblotanalysisasdescribedin
MATERIALSANDMETHODS.Arepresentativeim-
agewasshown.TheintensityofthepERK1/2
bands was normalized to that of total ERK1,
and the result was expressed as fold change
D
withthevaluewithoutpeptoneorLPAtreat-
o
mentarbitrarilysetto1.Theeffectsofa20-h w
n
preincubationwith100ng/mlPTX,a30-min lo
preincubation with either 5 (cid:4)M U-73122 or a
U-73343 (B) and 50 (cid:4)M PD-98059 (C) on de
d
iancdtiuvcaetdionERwKa1s/2deptheormspihnoerdy.laTtihoen bceyllsGPwRe9r3e fro
m
treatedwith20mg/mlpeptoneor100nMLPA
for4min.Datawerepresentedasdescribed ajp
inA. g
i.p
h
y
s
io
lo
g
y
.o
rg
o
n
F
e
b
ru
a
ry
significance of the synergistic effect of peptone plus LPA on myenteric plexus or vagal nerves where it could also serve as 5
GPR93activationisthatthesensitivityofendogenousGPR93 a signal transducer of luminal activators. The level of GPR93 , 2
0
toward its activators is likely modulated depending on the in the mucosal layer was three times higher than that in the 0
8
compositionofthedietarycomponentsthatareincontactwith muscle layer of the respective section; the most abundant was
enterocytesinsitu.TheeffectsofdigestedBSAorcefacloron in the duodenal mucosa with a slight progressive decrease
GPR93activationillustrateamodulationofGPCRactivitiesby toward the colon. GPR93 in the stomach mucosa was at a
compounds that by themselves result in lower or no activity lower level than in the intestinal mucosa. We further charac-
and suggest that various components of peptone might act in terized the pattern of GPR93 expression in the intestinal
concert, leading to the observed net activation of GPR93. mucosa by LMD in both the area of cell proliferation (crypt)
GPR93isexpressedthemostinthesmallintestinalmucosa and the area of cells that are nonproliferative and terminally
andinpostconfluenthBRIE380icells.GPR93mRNAlevelsin differentiated (villus) (Fig. 8B). The growth stage of the laser
rattissuesweredeterminedbysemiquantitativeRT-PCR(Fig. microdissectedcellsfromtissuewasconfirmedbytheexpres-
8A). GPR93 is highly expressed in the intestine. The level of sion of villin (an early differentiation marker) and I-FABP (a
GPR93 in the hypothalamic region of the brain was (cid:5)10% of late differentiation marker). GPR93 mRNA level was the
thatintheduodenummucosa.TheGPR93wasalsodetectedin highest in the villar tip in an area corresponding to the brush
kidney, spleen, and heart. Like other GPCRs found in the border, which is composed mostly of enterocytes (Fig. 8C).
intestine, GPR93 was found in both neuronal as well as This in situ distribution corresponded with the pattern of
nonneuronal tissues. The significance of GPR93 expression in expression in the hBRIE 380i cell line. GPR93 expression
muscle tissues will be more readily elucidated when the char- patterninhBRIE380icellswasdeterminedbyRT-PCRwhen
acterization of endogenous ligands or activators is resolved. the cells were at the state of near/early confluency and post-
GiventhatthedistributionofGPR93intheintestinemightbe confluency(Fig. 8D). Villinexpressionwasusedtoverifythe
similar to that of other GPCRs found in the intestine and that stateofdifferentiation.Consistentwiththeresultoftheinvivo
they are present in both neuronal and nonneuronal tissues, it tissue distribution, GPR93 transcript was detected at near
alsoremainstobedeterminedwhetherGPR93ispresentinthe confluency (similar to crypt cells), and its level continued to
AJP-GastrointestLiverPhysiol•VOL292•JANUARY2007•www.ajpgi.org