Characterization of NMB, GRP and Their Receptors (BRS3, NMBR and GRPR) in Chickens

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c mo, l huang and others GRP, NMB and their receptors 59: 1 61–79 Research in chickens

Characterization of NMB, GRP and their receptors (BRS3, NMBR and GRPR) in chickens

Chunheng Mo*, Long Huang*, Lin Cui, Can Lv, Dongliang Lin, Liang Song, Guoqiang Zhu, Juan Li and Yajun Wang Correspondence should be addressed Key Laboratory of Bio-resources and Eco-environment of Ministry of Education, College of Life Sciences, to Y Wang or J Li Sichuan University, Chengdu, People’s Republic of China Email *(C Mo and L Huang contributed equally to this work) [email protected] or [email protected]

Abstract

The two structurally and functionally related peptides, gastrin-releasing peptide (GRP) Key Words and neuromedin B (NMB) play critical roles in many physiological/pathological processes ff chicken in mammals. However, the information regarding the expression and functionality ff NMB of avian NMB, GRP and their receptors is limited. Here, we characterized cNMB, cGRP ff GRP and their receptors (cNMBR, cGRPR and cBRS3) in chickens. Our results showed that: ff BRS3 (1) cNMBR and cGRPR expressed in CHO cells could be potently activated by cNMB and ff GRPR cGRP, respectively, as monitored by cell-based luciferase reporter assays, indicating that ff NMBR cNMBR and cGRPR are cNMB- and cGRP-specific receptors; strikingly, BRS3 of chickens ff pituitary hormone (/spotted gars), which is orthologous to mouse bombesin receptor subtype-3 (BRS3), could be potently activated by GRP and NMB, demonstrating that both peptides are the

Journal of Molecular Endocrinology endogenous ligands for chicken (/spotted gar) BRS3; (2) quantitative real-time PCR (qPCR) revealed that cGRPR is widely expressed in chicken tissues with abundant expression in the ovary, pancreas, proventriculus, spinal cord and brain, whereas cNMB, cNMBR and cBRS3 are mainly expressed in the brain and testes; (3) interestingly, qPCR, Western blot and immunostaining revealed that cGRP is predominantly expressed in the anterior pituitary and mainly localized to LH-cells, suggesting that cGRP is likely a novel pituitary hormone in chickens. In summary, our data help to uncover the roles of GRP, NMB and their receptors in birds, and provide the first persuasive evidence from an evolutionary prospective that in vertebrates, GRP and NMB are the endogenous ligands for BRS3, an Journal of Molecular orphan receptor that has puzzled endocrinologists for more than two decades. Endocrinology (2017) 59, 61–79

Introduction

Bombesin (BN) is a 14-amino acid peptide originally peptides encoded by separate genes, gastrin-releasing isolated from amphibian skin in 1971 (Erspamer et al. peptide (GRP) and neuromedin B (NMB), have also 1970, Anastasi et al. 1971), which has a potent stimulatory been identified in mammals (McDonald et al. 1979, action on preparations of intestinal, uterine and urine Minamino et al. 1983, Spindel et al. 1984, Krane et al. tract smooth muscle (Erspamer et al. 1972). Subsequently, 1988). Two active forms of GRP with an identical C

two structurally and functionally related bombesin-like terminus, GRP27 (27 amino acids) and GRP10 (10 amino

10.1530/JME-17-0020 Journal of Molecular Endocrinology BRS3 displaysanextremely lowaffinitytoGRP/NMB mammals ( (~58%) andNMBR(~48%), hasalsobeenidentifiedin (BRS3), whichshowshighsequenceidentitytoGRPR bombesin receptornamedsubtype-3 Jensen 1992 CNS andperipheraltissuesinmammals( and NMBRarereportedtobewidelyexpressedinthe with thebroadspectrumofGRP/NMBactions,GRPR signaling cascade( as calciummobilizationandactivationofMAPK/ERK receptors cantriggermultiplesignalingpathways,such 1995 and arefunctionallycoupledtoGqprotein( receptor familyofGprotein-coupled(GPCR) 1995 1990 and NMBreceptor(NMBR),respectively( their specificreceptors,namelyGRPreceptor(GRPR) the biologicalactionsofGRPandNMBaremediatedby Lach tissues ( and cellproliferationofbothnormalcancerous hormonesecretionintake, thermogenesis,pituitary physiological/pathological processes, such as food GRP, NMBisalsoinvolvedinthecontrolofmany Chen 2007 1988 normal tissuesandmanycancerous( contraction, GItractmotilityandcellproliferationin food intake,circadian rhythm, itching,smoothmuscle many physiological/pathologicalprocessesincluding with their wide distribution, GRP is reported to regulate Houben ( system, lungandanteriorpituitary gastrointestinal (GI)tract,urogenitalandreproductive ( system(CNS)andperipheraltissuesthe centralnervous ( have alsobeenidentifiedinporcine brainandspinalcord of NMB(designatedas after itsdiscovery, thetwoN-terminallyextendedforms from porcine spinalcord( 1984 cord, respectively( acids), have been isolated in porcine stomach and spinal Minamino Wada DOI: 10.1530/JME-17-0020 http://jme.endocrinology-journals.org Research In mammals,GRPandNMBarewidelyexpressedin t al et ). NMBisa10-aminoacidpeptideoriginallyisolated , , ). BothGRPRandNMBRbelongtobombesin , , Jensen et al Battey Merali Sano et al et al Rettori . 1995 . 1990 et al Gorbulev . 2008 , t al et et al t al et Jensen . 1993 et al t al et . 1985 , , . 2008 ). ApartfromGRPRandNMBR,athird . 1991 . 2004 Matusiak . 1999 McDonald Moody &Merali2004 Jensen , et al t al et Jensen ). . 1989 ). Uponligandstimulation,both , , , . 2008 . 1992 Wada Minamino McArthur Ohki-Hamazaki t al et t al et et al 30 , et al andNMB Ladenheim et al , , . 2008 c . 2005 Fathi

et al huang Jones ). Inconsistence ). Itisclearthat , ). Inaccordance ), includingthe Minamino 32 . 1983 Printed inGreatBritain . 2000 t al et , respectively) t al et . 1993 Spindel , . 2009 andothers t al et Wada Benya Benya Rozengurt ). Shortly , . 1994 . 2005 ). Since . 1992 ). Like Sun & t al et et al et al et al et al , . , . . . , . mammalian vertebrates,suchasinhibitionoffoodintake important rolesinmanyphysiologicalprocessesnon- are alsolinesofevidenceshowingthatGRP/NMBplay 2000 McDonald including birds,frogsandteleosts( receptors also existin non-mammalian vertebrates, 1997 Campbell secretion inchickensorturkeys( motility, crop-emptyingrate,pancreaticfluidandprotein 2015 administration ( in chickensandteleostsfollowingcentralorperipheral metabolic ratesandobesity( such asenergyhomeostasis,bodytemperature, CNS mayregulatemanyvitalphysiologicalprocesses, deficient micehassuggestedthatBRS3expressedinthe receptor. However, accumulatingevidencefromBRS3- question andBRS3hasbeenviewedasanorphan endogenous ligandofBRS3hasremainedanopen 1996 related peptides( ( hormone inanavianspecies (i.e.chicken). anew conceptthatGRPislikelyanovelpituitary forward perspective,andputs in vertebratesfromanevolutionary likely theendogenousligandsfororphanreceptorBRS3 first convincingevidencethatGRPandNMBaremost anterior pituitaries.Undoubtedly, ourstudy presentsthe we found that GRP is predominantly expressed in chicken effectively activated by GRP and NMB of BRS3.LikechickenBRS3,spottedgarBRS3couldbe activate BRS3, suggesting they are bothpotent ligands demonstrated that both GRP and NMB could potently NMBR andBRS3)forchickenGRP/NMB.Strikingly, we a result,weidentifiedthreefunctionalreceptors(GRPR, spatial expressionofGRP, NMBandtheirreceptors.As receptors forGRP/NMB;(2)systematicallyinvestigatethe to: (1)identifyandfunctionallycharacterizeallthe chicken asananimalmodel,ourpresentstudyaimed 2003 mammalian vertebratesincludingbirds( GRP andNMBhavenotbeenfullycharacterizedinnon- signaling property and expressionof the receptorsfor to theextensivestudiesinmammals,identity, Scanes &Pierzchala-Koziec2014 in chickens GRP, NMBandtheirreceptors K Published byBioscientifica Ltd. i

> As inmammals,bothGRPandNMBtheirputative > , ), andregulationofgastricacidsecretion,gallbladder , b , Xu &Volkoff 2009 , 1000 Mantey Ohki-Hamazaki Guan t al et et al nM) orothernaturallyoccurringbombesin- et al t al et . 1980 . 1991 Tachibana . 2010 Gorbulev . 1997 , , 1994 Nagalla , , t al et Downloaded fromBioscientifica.com at10/04/202101:03:26AM Yun Metzger , et al Ryan et al t al et . 2005 , et al . 2010 Tachibana t al et ). However, incontrast . 2015 Ohki-Hamazaki et al . 1992 Linari &1975 . 1992 ). Therefore,using Vaillant 59 a . 2010 . 1998 in vitro , ). Moreover, there b : 1 , , Iwabuchi Schroeter , Nagalla t al et Volkoff t al et ). ), thus,the . Moreover, . 2010 t al et . 1979 t al et t al et et al et al 62 via freeaccess b , . . , . , . . Research c mo, l huang and others GRP, NMB and their receptors 59:1 63 in chickens

Materials and methods Cloning the cDNAs of chicken GRPR, NMBR and BRS3

Chemicals, hormones, antibodies and primers According to the genomic sequences of cGRPR, cNMBR and cBRS3 (http://www.ensembl.org/Gallus_gallus), Chicken GRP (cGRP ), GRP (cGRP ) and NMB 27 27 10 10 gene-specific primers were designed to amplify the cDNA (cNMB) were synthesized using solid-phase Fmoc containing the open reading frame (ORF) of the receptors chemistry (GL Biochem, Shanghai, China). Goat anti- from the chicken brain with the use of high-fidelity Taq GRP polyclonal antibody was purchased from Santa Cruz DNA polymerase (TOYOBO). The amplified PCR products Biotechnology and monoclonal antibody for -actin was β were cloned into pTA2 vector and sequenced by ABI3100 obtained from Cell Signaling Technology. Anti-ACTH Genetic Analyzer (BGI). Finally, the cDNA sequence antibody was purchased from Abcam. Donkey anti-goat of each receptor was determined by sequencing three IgG (H + L) cross-adsorbed secondary antibody (DyLight independent clones. 488 conjugate) and anti-rabbit IgG (H + L) cross-adsorbed secondary antibody (Dylight 550 conjugate) were purchased from Thermo Fisher Scientific. The polyclonal Functional characterization of cNMBR, cGRPR and antibodies against recombinant full-length chicken GH, chicken/spotted gar/mouse BRS3 in CHO cells PRL and LHβ were prepared in our laboratory (Huang et al. Based on the cDNA sequences of chicken GRPR, NMBR 2014, Meng et al. 2014, Bu et al. 2016). All primers were and BRS3-like genes, gene-specific primers were used to synthesized by Beijing Genome Institute (BGI, China) amplify the ORF from adult chicken brain using high- and listed in Supplementary Table 1 (see section on fidelity Taq DNA polymerase (TOYOBO, Japan). The supplementary data given at the end of this article). amplified PCR products were cloned into the pcDNA3.1 (+) expression vector (Invitrogen) and sequenced. To Total RNA extraction compare the functional difference of chicken BRS3 with Adult chickens (1-year-old) of both sexes (Lohmann BRS3 from other vertebrate species, including mouse layer) were purchased from local commercial company. and spotted gar, we also amplified the complete coding Chickens were killed, and various tissues including regions of mouse and spotted gar BRS3 from brain tissues the different brain regions were isolated and collected and cloned them into the pcDNA3.1 (+) expression vector. for RNA extraction. Total RNA was prepared using According to our previously established methods, RNAzol (Molecular Research Center, Cincinnati, OH, the functionality and signaling property of each

Journal of Molecular Endocrinology USA) according to the manufacturer’s instructions. receptor was examined in Chinese hamster ovary (CHO) All experiments were performed according to the cells using pGL3-NFAT-RE-luciferase reporter system guidelines provided by the Animal Ethics Committee of (Wang et al. 2012), pGL4-SRE-luciferase reporter system Sichuan University. (Mo et al. 2015) and pGL3-CRE-luciferase reporter system (Wang et al. 2007a).

Reverse transcription and quantitative real-time PCR (qPCR) Western blot

Two micrograms of total RNA and 0.5 μg of To compare the protein level of cGRP in the anterior oligodeoxythymide were mixed in a total volume of 5 μL, pituitary, brain and proventriculus of adult chickens, incubated at 70°C for 10 min and cooled at 4°C for 2 min. Western blot analysis was performed. The respective tissue The first strand buffer, 0.5 mM each deoxynucleotide lysates were prepared for the detection of cGRP level using triphosphate and 100 U Moloney murine leukemia virus corresponding antibodies (1:300), as described in our (MMLV) reverse transcriptase (Takara) were then added recent study (Mo et al. 2015). In parallel, β-actin level was into the reaction mix in a total volume of 10 μL. Reverse examined in tissue lysates as a loading control. transcription (RT) was performed at 42°C for 90 min. To test whether cBRS3 activation can enhance ERK1/2 According to our previously established method (44/42 kDa) and CREB (43 kDa) phosphorylation, HEK293 (Cai et al. 2015), quantitative real-time PCR was conducted cells that transiently expressed cBRS3 were treated by

on the CFX96 Real-time PCR Detection System (Bio-Rad) cGRP27 (100 nM) for 10 min, and the phosphorylated to examine the mRNA levels of target genes in chicken ERK1/2 and CREB levels in cellular lysates were examined tissues. by Western blot, as described in our recent study

http://jme.endocrinology-journals.org © 2017 Society for Endocrinology Published by Bioscientifica Ltd. DOI: 10.1530/JME-17-0020 Printed in Great Britain Downloaded from Bioscientifica.com at 10/04/2021 01:03:26AM via free access Journal of Molecular Endocrinology Finally, cellswerecounterstainedwithDAPI thepituitary antibodies (1:300)for1 secondary washed and incubated with fluorochrome-conjugated well andincubatedat4°C overnight.Thecellswere in blockingbuffer(1:500or 1:1000),wereaddedtoeach and rabbitanti-cGH(oranti-cPRL/-cLH antibodies, goatanti-GRP After blocking,thetwoprimary room temperaturetominimizenon-specificadsorption. with theblockingbuffer(1%BSAinPBS)for30 Triton X-100for10 washed withPBS.Then,thecellsweretreatedby0.1% cellswerefixed in4%paraformaldehydeand pituitary previous study ( CO Cell-BIND 96-well plates (Corning) at37°C with 5% with 15%fetalbovineserum(Invitrogen)inaCorning chickens wereculturedinMedium199supplemented cells from adultfemale The dispersedanterior pituitary Double fluorescence staining control inparallel. at 4°Cwith100 cGRP sectionsincubatedwith in chickenpituitaries.Pituitary (1:300) wasusedtoprobethespatialdistributionofcGRP manufacturer’s instructions.Goatanti-GRPantibody kit (BosterBiologicalTechnology Ltd)accordingtothe using aStreptavidin–Biotin–PeroxidaseComplex(SABC) sections IHC stainingwasperformedinthepituitary paraformaldehyde, paraffinizedandslicedintosections. pituitaries fromadultfemalechickenswerefixedin4% cGRP (or cGH/ RNA were prepared for Western blot detection of cGRP by scalpels,andtheirrespectivetissuelysatestotal cephalic lobeandcaudalwereseparatedcarefully PBS toremoveanycontaminatingbloodcells.The collected fromadultfemalechickenswerewashedwith 2014 experiment, asdescribedinourrecentstudies( histochemical staining(IHC)wereusedinthis anterior pituitaries, qPCR, Western blot and immuno- To examinethespatialdistributionofGRPinchicken chicken anteriorpituitaries Detection ofGRPmRNAandprotein expression in examined andusedasaloadingcontrol. ( He DOI: 10.1530/JME-17-0020 http://jme.endocrinology-journals.org Research 2 t al et at a density of1 , mRNAexpression,respectively. ForIHC,anterior 27 -preabsorbed GRPantibody(incubatedovernight Mo 2016 . β -actin) proteinexpressionandqPCRassayof t al et , Meng ng/mL ofcGRP . 2015 Cui min, washedwithPBSandincubated t al et × et al 10 ). Inbrief,anteriorpituitaries 4 . 2014 . 2017

cells/well, as describedin our 27 ). After 4 ). c ) wasusedasanegative

huang β /-ACTH) diluted h of culture, the Printed inGreatBritain andothers Meng min at et al . our results. All experiments were repeated at least twice to validate evaluated withGraphPadPrism5(GraphPadSoftware). half-maximal effectiveconcentration(EC nonlinear regression models, and the corresponding wereconstructedusing test. Thedose-responsivecurves were analyzedbyone-wayANOVA followedbyDunnett’s control group(withoutpeptidetreatment).Thedata were expressed as relative fold increase compared to the expressing GRPR/NMBR/BRS3inpeptidetreatmentgroup to chosentissue.TheluciferaseactivitiesofCHOcells β The mRNAleveloftargetgenewasnormalizedbythat Data analysis ECLIPSE Ti). underafluorescencemicroscope(Nikon and observed mammalian vertebratesincluding westernpaintedturtles, this ( a previouslyreported‘bombesin receptorsubtype-3.5 Interestingly, wenotedthatchicken the chromosomallocationof and islocatedonchromosome4,whichdiffersfrom synteny analysis,wefoundthat uncertain whether and BRS3(48%). amino acidsequenceidentitieswithchickenGRPR(53%) bond formation( for Gproteincoupling,andtwocysteinesdisulfide transmembrane domains (TMD1–7), a DRY motif critical gars (74%).LikehumanNMBR,cNMBRcontainsseven Xenopus tropicalis acid sequenceidentitieswithNMBRofmice(72%), (accession no.: KU887757), which shows high amino is predictedtoencodeareceptorof388aminoacids brain ( the cDNAcontaininganORFof any non-mammalianvertebrates.Inthisstudy, wecloned lower vertebrates, Although in chickens Cloning ofNMBRcDNAandidentificationBRS3 Results in chickens GRP, NMBandtheirreceptors BRS3.5 -actin andthenexpressedasfolddifferencecompared Published byBioscientifica Ltd. As inchickens, Although BRS3.5 upeetr i. 1 Fig. Supplementary )’ ( NMBR Iwabuchi as BRS3 BRS3 ispredictedtoexistinchickensandother (82%),Niletilapia(68%)andspotted i. 1 Fig. NMBR inthisstudy. et al existsinmammals,itremains BRS3 BRS3 ). Moreover, cNMBRshowshigh . 2003 Downloaded fromBioscientifica.com at10/04/202101:03:26AM cDNAhasnotbeenreportedin existsinchickens.Using alsoexistsinothernon- ). Thecloned ), andthus,wedesignated NMBR NMBR BRS3 and BRS3 59 existsinchickens fromthechicken : 1 50 isidenticalto GRPR cNMBR ) valueswere ( i. 2 Fig. cDNA 64 via freeaccess ). Research c mo, l huang and others GRP, NMB and their receptors 59:1 65 in chickens

TMD1 Chicken NMBR--1------MEAELLSELTALS-FGGNGSVQLVPETTLLPPAWG-MAMFTVRCVIPSLYLLIITVGLLGNVTLMKIFISNSAMRS-74-- --Mouse NMBR--1------.PSKS..N.SVTTGANES...PEGW.RDF..ASD.TTTELVI...... IM.V....T...... -76-- Xenopus NMBR--1------MISGQTFANDSLIQ-.NVSSV.PSILPEVPV.HEQE-..A.II...... V..M...... I..V....T...... -75-- Tilapia NMBR--1------.DD.F..N.P.EA----LSPNSTEVPGPWSSADGAETVH.V....MT.V.I...V...... I..V....T...... -72-- --- Gar NMBR--1------MTME...NN.SFSG----.S.FP.NFT-DETV.DGRGTVD.II...... V.I...... I..V....T...... -72-- Chicken BRS3--1---MSQVYLHPSNQT.C.ATNGTELK.ILDNET.NEKWTEDS-FPGLEIL.T.YVT.AV..S...... AI.I.V.FKIKS.QT-79-- Chicken GRPR--1------.ASGECLL.DLETDNFILYNISANQSAN.SVLSDE-WFYPAFLYA..TI.GI..LI..I..I..I...CTVKS..N-75-- TMD2 TMD3 Chicken NMBR--75--VPNIFISSLAAGDLLLLVTCVPVDASRYFSEEWLFGEVGCKLIPAIQLTSVGVSVFTLTALSADRYKAIVKPMDIQTSSA-154- --Mouse NMBR--77--...... N...... L...... FD..M..K...... V...... R...N...M...G.-156- Xenopus NMBR--76--...... A...... F.F.F...IL...C...... N...... -155- Tilapia NMBR--73--...... F...... V...AA.....V...... N...... -152- ----Gar NMBR--73--...... I..F...FD..I..TMA.....V...... N...... -152- Chicken BRS3--80--...... T...F...... L...... T..IVDT.I..RI....LSF...... V...... R.....LEL...D.-159- Chicken GRPR--76--...L...... L...... LAD.....RI...... F...... R..E..A.H.-155- TMD4TMD5 chicken-NMBR--155-VLRTCAKAIAIWIISILLSVPEAVFSELAHIN---DTDNATFTACIPYPMTDDMHPKIHSVLIFLVYFLIPLAIISIYYY-231 --Mouse NMBR--157-L....V..MG..VV.V..A...... V.R.S---SL..SS...... Q..EL...... -233- Xenopus NMBR--156-..W..I..S...... A...... V...S---ES..VS...... QN.E...... Y....T...... -232- Tilapia NMBR--153-.FW..L..VS..LL.V..A....I..QVVSMQGHR.NT.V..VN.V...LSNQ...... M...... V...-232- ----Gar NMBR--153-..W..L...T.....V..A....I..QVV..K----DK.I.....V...LS.E...... IM..M...... -228- Chicken BRS3--160-L.K..C..GCV..V.MVFAI...... D.YSFSN--PEK.V..E..A...VSEKILQEV..LVC...FYIV...V..V..F-237- Chicken GRPR--156-LMKI.VR.AI...A.M..AI...... D.HPFHD--KGT.K..IS.A...HS.GL...... MAS..IFYI...SV..V...-233- TMD6 Chicken NMBR--232-HIAKSLIRSAHNIPGENSEHSKRQMETRKRLAKIVLVFVGFFAICWFPNHVLYMYRSFNYN-EIDSSIGHMVVTLVARVL-310- --Mouse NMBR--234-....T..K....L...YN..T.K...... C.IF...... I...... -...P.L...I...... -312- Xenopus NMBR--233-...RT..K...... Y...... C...... S-...Q.LR..II..I..I.-311- Tilapia NMBR--233-...RT..K...DM...V...T...... L..L...... H.H-QM.L.LA.L.I..L....-311- ----Gar NMBR--229-...RT..K...DM...F...... L..L...... R-.....VA.LII..L....-307- Chicken BRS3--238-L..RT.YK.TF.M.A.EHG.ARK.I.S...V..T...L.AL..F..L...I..L....T.HTSV.A.TF.LI..IFS.A.-317- Chicken GRPR--234-F...N.....Y...V.GNV.VRK.I.S.....RT.....CL..F..L.T.II.L...YH.S-.V.T.VL.FIASIC..I.-312- TMD7 Chicken NMBR--311-SFCNSCVNPFALYLLSESFRRHFNNQLRCRKKPQQERSASYLRNSSAIQTTSLKSNTRNTVTSVTQLNGHNLKQEMSS--388- --Mouse NMBR--313-..G...... S..C.GR.SY...GT...LS...VRM...... AK.M..NSVL....SM....AL--390- Xenopus NMBR--312-..S...... S..C.W..RH....T...L....V.M...... A..A..RTSL....S-R..V.L--388- Tilapia NMBR--312-..SS...... S....GRG.RP..Q....HST.H.RL..I.K..PTAAL-APAA..NPNR..VAL--388- ----Gar NMBR--308-...... SH.C..R.RYR..AT...QST...RM..I.K..PT.---T.L....S..R.V.L--382- Chicken BRS3--318-A.S...... W..K...Q..KK.VS.C.AKLCTKPP.APHSN.PPRAL.VTGS.HGSEI...L.TDYSITK.EE.V-396- Chicken GRPR--313-A.T...... K...KQ.....F.CRARLLI..Q.MA.STT--RM.....TNH-SLATFSLI..NHVCH.GYV--387-

Figure 1 Amino acid alignment of chicken NMBR (KU887757) with that of mice (NP_032729), Xenopus tropicalis (XP_012818691), Nile tilapia (XP_005452510) and spotted gars (XP_006643031), or with chicken BRS3 (NP_989737) and GRPR (NP_989738). The conserved ‘DRY motif’ is boxed and the seven transmembrane domains (TMD1–7) are shaded; the potential N-glycosylation site (NXT/S) is underlined; the pair of cysteine residues proposed for disulfide bond formation are boxed; dots indicate amino acids identical to chicken NMBR; dashes denote gaps in the alignment.

Xenopus tropicalis and spotted gars (Fig. 2). Interestingly, Receptor activation was then examined by pGL3-NFAT- Journal of Molecular Endocrinology BRS3 is likely lost in teleosts (e.g. Nile tilapia), as revealed RE-lcuiferase reporter, pGL4-SRE-luciferase reporter by synteny analysis. In addition, some of the paralogous and pGL3-CRE-luciferease reporter systems, which genes adjacent to BRS3/NMBR/GRPR (e.g. ADGRG2/4/6) could monitor receptor-activated calcium mobilization could be identified in these vertebrate species examined, (Wang et al. 2012), MAPK/ERK (Mo et al. 2015) and implicating that BRS3, GRPR and NMBR were likely adenylate cyclase (AC)/cAMP/PKA signaling pathways duplicated from a common ancestral gene by the 2 (Wang et al. 2007a), respectively. rounds of genome duplication (2R) occurred during Using the pGL3-NFAT-RE-luciferase reporter system, early vertebrate evolution (Fig. 2) (Yun et al. 2015). Using we demonstrated that cNMB could potently activate

RT-PCR, we cloned the cDNA of spotted gar BRS3 from cNMBR expressed in CHO cells (EC50: 8.2 nM). On the other

brain tissue (KX447669) (Supplementary Figs 2 and 3). hand, cGRP27 and cGRP10 are much less potent, indicating This finding further indicates the presence and expression that cNMBR is a NMB-specific receptor. By contrast,

of BRS3 in lower vertebrate species. cGRPR could be potently activated by cGRP27 and cGRP10

(with cGRP27 being slightly more potent than cGRP10), while cNMB is ineffective. These findings indicate that Functional characterization of chicken NMBR, BRS3 10 GRPR is a GRP-specific receptor. Strikingly, we found that and GRPR cBRS3 could be effectively activated by the three peptides

To compare the functional difference between the three (EC50 of cGRP27: 8.3 nM; EC50 of cGRP10: 9.7 nM; EC50 chicken bombesin receptors (cNMBR, cGRPR and cBRS3), of cNMB: 28.2 nM) (Fig. 3). This finding indicates that each receptor was transiently expressed in CHO cells cBRS3 can function as a receptor common for both cGRP

and treated by chicken GRP27, GRP10 and NMB10 (Fig. 3). and cNMB, and display a weak selectivity toward cGRP

10 /sGRP ). Furthermore,ourfindingsalsosuggestthatas Chicken Tu Chr 1 Chr 3 rtle 10 ); (B,C,D,EandF)effects ofcGRP

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andcGRP expressed inCHOcells,furthersupportingthenotion cGRP specific receptor( but notbycGRP, alsosupportingthatcNMBRisaNMB- could bepotentlyactivatedbycNMB(EC in chickens GRP, NMBandtheirreceptors Reptile NMBR GRP BRS3 Published byBioscientifica Ltd. R Gar LG7

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. offourreplicates( i. 4 Fig. ( duplication eventduringvertebrateevolution ( duplicated fromanancestral and mammals(e.g.mice),whichwerelikely (e.g. western paintedturtles),birds(e.g.chickens) lost), frogs(e.g. in spottedgars,teleosts(e.g.Niletilapia, showing theexistenceof chromosome unknown.(D)Schematicdiagram human genome.Chr, chromosome;ChrUn, were labeledbasedontheirgenesymbolsinthe syntenic genesidentifiedinthesespecies.Genes of interest,whiledottedlinesindicatethe BRS3 (WGD) event( round (3R)ofteleost-specificgenomeduplication are likelytobeparalogsgeneratedbythethird GRPR spotted garsandNiletilapia.Thetwocopiesof western paintedturtles,mice, syntenic regionsconservedbetweenchickens, (A), (A, BandC)Syntenyanalysisshowingthat Figure 2 Yun GRPR-L BRS3 islikelylost.Dashedlinesdenotethegenes et al ( 10 GRPRa ). Bycontrast,bothcGRP Downloaded fromBioscientifica.com at10/04/202101:03:26AM ) bythetworounds(2R)ofgenome ) couldpotentlyactivatecGRPR (B)and . 2015 and Meyer &Van dePeer2005 N ). Xenopus tropicalis =4). GRPR GRPRb 10 (C)arelocatedindistinct /mGRP ) identifiedinNiletilapia 59 BRS3 : 1 GRPR 10 , Xenopus tropicalis GRPR ) andspottedgar ), reptiles -like gene 50 5.8 nM), , and ), while BRS3 27 NMBR NMBR and 66 is via freeaccess

Research c mo, l huang and others GRP, NMB and their receptors 59:1 67 in chickens

Table 1 EC50 values of cGRP and cNMB in activating different that GRPR is a GRP-specific receptor. In contrast, cBRS3 signaling pathways in CHO cells expressing chicken (c-) GRPR/ could be effectively activated by all three peptides tested

NMBR/BRS3 or spotted gar (s-)/mouse (m-) BRS3. (EC50 of cGRP27: 7.5 nM; EC50 of cGRP10: 8.4 nM; EC50 of cNMB: 28.3 nM) and it shows a weak selectivity toward EC50 values (nM) GRP (Fig. 4) (Table 1). This finding substantiates the idea Peptide cGRPR cNMBR cBRS3 sBRS3 mBRS3 that cBRS3 can function as a receptor common for both Calcium signaling pathway a cGRP and cNMB. Meanwhile, our findings also suggest the cGRP27 3.4 >200 8.3 9.2 – a functional coupling of the three receptors to the MAPK/ cGRP10 15.7 >200 9.7 17.9 – cNMB – 8.2 28.2 44 – ERK signaling cascade. MAPK/ERK signaling pathway Using the pGL3-CRE-luciferease system, cGRPR and a cGRP27 6.1 ~100 7.5 4.6 – a cNMBR were also shown to be preferentially activated by cGRP10 16.9 >100 8.4 5.9 – cNMB >100a 5.8 28.3 30.8 – cGRP and cNMB, respectively, while cBRS3 was activated cAMP/PKA signaling pathway by both GRP and NMB with similar potencies (Fig. 5). This a cGRP27 66.8 >200 41.8 35.9 – a a finding suggests that all three receptors are also coupled cGRP10 >100 >200 71 42.2 – cNMB – 23.3 >200a 125.4 – to AC/cAMP/PKA pathway. However, we noted that much higher concentrations of cGRP/cNMB are required to ‘–’ means that the EC values could not be calculated based on the 50 activate the AC/cAMP/PKA signaling pathway than the experimental data. a other two pathways tested (Table 1). Indicates that EC50 value was roughly estimated based on the experimental data. To further verify the functional coupling of cGRPR, cNMBR and cBRS3 to the calcium mobilization, MAPK/ERK Journal of Molecular Endocrinology

Figure 4

(A, B, C, D and E) Effects of cGRP27, cGRP10 and cNMB10 on activating chicken (c-) GRPR (A), cNMBR (B), cBRS3 (C), spotted gar BRS3 (sBRS3) (D) and mouse BRS3 (mBRS3) (E) expressed in CHO cells, monitored by pGL4-SRE-luciferase reporter system; (F) CHO cells co-transfected with empty pcDNA3.1 (+) vector and pGL4-SRE-luciferase reporter construct were used as internal controls, and peptide treatment did not alter the luciferase activity of CHO cells at any concentration tested. Each data point represents mean ± s.e.m. of four replicates (N = 4).

huang 10 Printed inGreatBritain onactivatingchicken(c-)GRPR(A),cNMBR(B),cBRS3(C),spottedgarBRS3(sBRS3)(D)andmouse Fig. 6 Fig. 6 andothers , alldrugs ). kDa) ±

s . e . m . tested (10 be activatedbycGRPnor cNMBatanyconcentration reporter systems. In contrast, mouse BRS3 could neither potent thancNMB),asmonitoredbythethreeluciferase cGRP CHO cellscouldbeeffectivelyactivatedbycGRP(cGRP we found that like cBRS3, spotted gar BRS3 expressed in (a primitivefreshwaterfish).Usingthesameapproach, from other lower vertebrate species such as spotted gar BRS3 ledustotestwhetherGRP/NMBcouldactivate The highpotencyofGRPandNMBinactivatingchicken mouse BRS3 Functional characterizationofspottedgarand not shown). ofERK1/2andCREB phosphorylation cNMBR andcGRPRactivationcanalsoenhancethe and AC/cAMP/PKA/CREBpathways( offourreplicates( in chickens GRP, NMBandtheirreceptors Published byBioscientifica Ltd. 10 ) andcNMB(withcGRPbeingslightlymore −12 to10 N

= 4). (F)Western blotshowingthatcGRP −6 ) ( M) Figs 3 Downloaded fromBioscientifica.com at10/04/202101:03:26AM , 4 , 5 and 59 i. 5 Fig. Table 1 : 1 in vitro ). Similarly, ). 27 treatment (data 68 27 via freeaccess / Research c mo, l huang and others GRP, NMB and their receptors 59:1 69 in chickens

Figure 6

(A) Effects of 2-APB (100 µM) on cGRP27 (10 nM, 6 h)-induced luciferase activity of CHO cells expressing cGRPR, cNMBR or cBRS3, monitored by pGL3-NFAT-RE-luciferase reporter system; (B) effects of U73122 (U7, 20 µM) and

PD98059 (PD, 100 µM) on cGRP27 (10 nM, 6 h)-induced luciferase activity of CHO cells expressing cGRPR, cNMBR or cBRS3, monitored by pGL4-SRE-luciferase reporter system; (C) effects of MDL12330A (MDL, 20 µM) and H89 (10 µM) on

cGRP27 (100 nM, 6 h)-induced luciferase activity of CHO cells expressing cGRPR, cNMBR or cBRS3, monitored by pGL3-CRE-luciferase reporter system. Each drug was added 1 h before cGRP

Journal of Molecular Endocrinology treatment. In each graph, ‘T’ represents peptide treatment and ‘C’ represents control without peptide treatment. Each data point represents mean ± s.e.m. of four replicates (N = 4). *P < 0.01 vs control (in the absence of drug); #P < 0.01 vs peptide treatment (in the absence of drug).

Tissue expression of NMBR, GRPR, BRS3, GRP and NMB in the testes. In addition, cNMB is moderately expressed in in adult chickens the hypothalamus and ovary, and weakly in other tissues examined, except the kidneys, liver, lung and muscle. Using qPCR, we examined the mRNA expression of cGRPR is widely expressed in nearly all tissues cNMB, cNMBR, cGRP, cGRPR and cBRS3 in adult chicken examined except kidneys and fat. It is highly expressed in tissues, including the anterior pituitary, heart, kidneys, the ovary, many brain regions (hypothalamus, midbrain liver, lung, muscle, ovary, testes, spleen, pancreas, and hindbrain), spinal cord, proventriculus and pancreas, subcutaneous fat, skin, spinal cord, different brain and weakly expressed in other tissues examined. regions (telencephalon, midbrain, cerebellum, hindbrain Strikingly, unlike cGRPR, cGRP is predominantly and hypothalamus) and various parts of gastrointestinal and abundantly expressed in the anterior pituitary and tract (crop, proventriculus, gizzard, duodenum, jejunum, weakly expressed in most of the tissues examined. The ileum, cecum and colon). mRNA level of GRP in the anterior pituitary is, at least, As shown in Fig. 7, cNMBR is highly expressed in the a 100-fold higher than that in other tissues examined testes and weakly expressed in the duodenum, colon, including the proventriculus. Within the CNS, a relatively kidneys, spinal cord and various brain regions including high mRNA level of cGRP was detected in the spinal the hypothalamus. Like cNMBR, cNMB is highly expressed

huang Printed inGreatBritain Fig. 7 andothers ). Within cGRP cGRP is

anterior pituitary cGRP isexpressedincephalic andcaudallobeof anterior pituitary Characterization ofGRPexpression inchicken the spinalcord( including thehypothalamus,whileitisundetectable in in thetelencephalonandweaklyotherbrainregions skin andbrain.Within theCNS, in chickens GRP, NMBandtheirreceptors Published byBioscientifica Ltd. Fig. 7 (3 malesand3females). mean undetectable. Eachdatapointrepresentsthe ‘nd’ indicatesthatmRNAoftargetgeneis compared withthatofhypothalamus(Hp). β levels ofeachgenewerenormalizedtothat subcutaneous fat(Fat)andskin(Sk).ThemRNA ovary (Ov),testes(Te), spleen(Sp),pancreas(Pa), (Co), kidneys(Ki),liver(Li),lung(Lu),muscle(Mu), (Du), Jejunum(Je),ileum(IL),cecum(Ce),colon (Cp), proventriculus(Pr),gizzards(Gi),duodenum cord (Sc),anteriorpituitary(Pi),heart(He),crop (Cb), hindbrain(Hb),hypothalamus(Hp),spinal telencephalon (Tc), midbrain(Mb),cerebellum in adultchickentissues,includingthe cNMBR Quantitative real-timePCRassayof Figure 7 -actin ).

± andexpressedasthefolddifference The extremely abundant mRNA ,

s Downloaded fromBioscientifica.com at10/04/202101:03:26AM cGRP . e . m . of6adultchickens( , cGRPR cBRS3 and 59 cBRS3 ishighlyexpressed : 1 mRNAexpression N =6) cNMB

70 via freeaccess Research c mo, l huang and others GRP, NMB and their receptors 59:1 71 in chickens

expression of cGRP in chicken pituitaries led us to further cGRP-immunoreactive (cGRP-ir) cells were densely examine whether cGRP protein is highly expressed in the distributed in both lobes (Fig. 8). pituitary by Western blot. As shown in Fig. 8, a strong protein band corresponding to cGRP precursor (~13 kDa) cGRP is expressed mainly in LH-cells It is was detected in the anterior pituitary. In contrast, only documented that the chicken anterior pituitary a faint band was detected in the proventriculus, and no contains multiple hormone-producing cells, including visible band detected in the whole brain. somatotrophs (GH-cells) (Harvey et al. 1979), lactotrophs To probe the spatial distribution of cGRP within (PRL-cells) (Ohkubo et al. 2000, Liang et al. 2006), the anterior pituitary, qPCR, Western blot and corticotrophs (ACTH-cells) and gonadotrophs (e.g. immunohistochemical staining (IHC) were conducted. LH-cells) (Proudman et al. 1999, Scanes 2015). To qPCR and Western blot revealed that cGRP mRNA determine whether cGRP is produced by these hormone- and protein are abundantly expressed in caudal and producing cells, double immunofluorescence staining was cephalic lobes of chicken pituitaries. As a control, a performed in cultured dispersed pituitary cells. As shown protein band of 23 kDa corresponding to cGH was in Fig. 9, approximately 11.7% of cultured pituitary cells only detected in the caudal lobe (Fig. 8). In accordance are GRP-ir cells, and the majority of cGRP-ir cells (~70.6%) with this finding, IHC staining revealed that are LH-cells. Moreover, we also noted that most of the Journal of Molecular Endocrinology

Figure 8 (A) Amplification plot ofcGRP and β-actin using the same amount of adult chicken pituitary cDNA template; (B) qPCR assay of cGRP mRNA levels in the cephalic (Ce) and caudal (Ca) lobes of adult chicken anterior pituitaries. Each data point represents means ± s.e.m. of five female individuals N( = 5); (C) the cGRP band (13 kDa) was detected in pituitary lysate incubated with anti-GRP, whereas cGRP band was not detected in the pituitary lysate incubated with

anti-GRP pre-absorbed by 100 ng/mL cGRP27, validating the specificity of antibody in recognizing GRP expressed in chicken pituitary. (D) Western blot detection of cGRP band (13 kDa) in the whole brain (Br), anterior pituitary (Pi) and proventriculus (Pr) tissue lysates; (E) Western blot detection of cGRP (13 kDa) and cGH (23 kDa) in the cephalic (Ce) and caudal (Ca) lobes of adult chicken anterior pituitaries. (F, G and H) Immuno-histochemical staining of GRP protein in the anterior pituitary of adult female chickens. Histological examination was performed under bright-field illumination with a magnification at 40× (F) and 400× (H). GRP-ir cells were densely distributed in both cephalic (Ce) and cephalic (Ca) lobes of anterior pituitaries (F, H),

whereas no staining was observed in pituitary section incubated with anti-GRP pre-absorbed by cGRP27 (G).

s . e . m . of5replicates( N =5). c

huang Printed inGreatBritain andothers in chickens GRP, NMBandtheirreceptors Published byBioscientifica Ltd. Downloaded fromBioscientifica.com at10/04/202101:03:26AM 59 : 1 72 via freeaccess Research c mo, l huang and others GRP, NMB and their receptors 59:1 73 in chickens

LH-cells (~87.1%) are cGRP-positive. In contrast, cGRP cNMBR and cGRPR are functional receptors specific to signal was not detected in other hormone-producing cNMB and cGRP respectively cells, including GH-cells, PRL-cells and ACTH-cells. These In this study, an NMBR orthologous to human NMBR findings clearly indicate that cGRP is mainly expressed in was cloned in chickens. cNMBR shows a high degree of LH-cells. amino acid sequence identity (72–74%) with NMBR of humans and mice. Functional assays proved that cNMBR Discussion can be preferentially activated by cNMB. This finding is In this study, we characterized cNMBR, cBRS3, cGRPR, consistent with those observations in mammals, in which cGRP and cNMB in chickens. Functional assay demonstra NMBR can bind to NMB with higher affinity than to ted that cNMBR and cGRPR can be preferentially activated GRP and be preferentially activated by NMB (Wada et al. by cNMB and cGRP, respectively, while chicken (/spotted 1991, Benya et al. 1995). Our findings indicate that, as in gar) BRS3 can be activated by both cGRP and cNMB mammals (Jensen et al. 2008), cNMBR is a cNMB-specific effectively, indicating that GRP and NMB are the native receptor (Fig. 10).

ligand for BRS3 in chickens (/spotted gars). qPCR assay In parallel, we found that both cGRP27 and cGRP10 are

revealed that cGRPR is widely expressed in chicken tissues, 100-fold more potent than cNMB10 in activating cGRPR while cBRS3, cNMBR and cNMB are mainly expressed in expressed in CHO cells. This finding coincides with a the brain and testes. Furthermore, we found that cGRP is previous study, in which cGRPR expressed in CHO-K1 cells

predominantly expressed in the anterior pituitary. To our can bind to cGRP27 with a 1000-fold higher affinity than

knowledge, our study represents the first to present a new to porcine NMB10 and its activation causes the elevation

concept that cGRP is likely a novel pituitary hormone of intracellular calcium concentration upon 10-nM GRP27 in birds. Meanwhile, the discovery of the endogenous treatment (Iwabuchi et al. 2003). The findings from our ligands of BRS3 in chickens and spotted gars will facilitate present and previous studies indicate that, as in mammals our better understanding of the mysterious roles of BRS3 (Benya et al. 1995, Jensen et al. 2008), cGRPR is a functional play in vertebrates. receptor specific to cGRP.

A B Pituitary In chickens In mammals

Journal of Molecular Endocrinology GRP GRP NMB GRP NMB

Circulang GRPR BRS3 NMBR GRPR BRS3 NMBR GRP

Gq/Gs Gq/Gs Gq/Gs Gq Gq Gq GRP GRP

2+ 2+ GRPR GRPR GRPR GRPR MAPK/ERK [Ca ] cAMP/PKA MAPK/ERK [Ca ] GRP GRP ? ?

Proventriculus Pancreas Ovary Otherssues

Figure 10 (A) The bombesin receptors (NMBR, GRPR and BRS3) and their endogenous ligands identified in chickens and mammals. In chickens, GRPR and NMBR function as the receptors specific to GRP and NMB, respectively, whereas BRS3 can be potently activated by GRP and NMB and thus act as a receptor common for both peptides. In mammals, GRPR and NMBR function as the receptors specific to GRP and NMB, respectively, while BRS3 cannot be

potently activated by GRP/NMB in vitro (EC50 >>1000 nM) (Jensen et al. 2008). Interestingly, chicken GRPR, NMBR and BRS3 are likely coupled to Gq and Gs proteins, and their activation triggers calcium mobilization, MAPK/ERK and cAMP/PKA signaling pathways. In contrast, mammalian GRPR, NMBR and BRS3 are coupled to Gq protein, and their activation elevates calcium concentration and stimulates MAPK/ERK signaling pathway (Jensen et al. 2008). (B) The proposed actions of pituitary GRP (a novel pituitary hormone) on chicken peripheral tissues. Pituitary GRP, together with GRP from other tissues (e.g. proventriculus), may contribute to the circulating GRP pool (highlighted in blue) and thus play important roles in the proventriculus, pancreas, ovary and other peripheral tissues in an endocrine (/paracrine/autocrine) manner. The localization of cGRP in pituitary LH-cells (Fig. 9), together with abundant cGRPR expression in the ovary, strongly suggests that GRP–GRPR (marked by bold arrows) may play a critical role in the pituitary–ovary axis of chickens.

http://jme.endocrinology-journals.org © 2017 Society for Endocrinology Published by Bioscientifica Ltd. DOI: 10.1530/JME-17-0020 Printed in Great Britain Downloaded from Bioscientifica.com at 10/04/2021 01:03:26AM via free access Journal of Molecular Endocrinology cNMBR) ( cGRP/cNMB in activating their specific receptor (cGRPR/ cNMB, withhighpotencies comparabletothoseof chicken BRS3canbeactivated effectivelybycGRPand 2012 pathological rolesofBRS3signaling( has hinderedourunderstandingofthephysiological/ ligand(s) ofBRS3hasremainedanopenquestion,which ( any othernaturallyoccurringbombesin-relatedpeptides effectively activatedbyGRP/NMB(EC unlike NMBR/GRPR,humanormouseBRS3cannotbe Majumdar &Weber 2012 treat obesityanddiabetesinhumans( has beenviewedasapromisingtherapeutictargetto Guan insulin secretion in mice ( metabolic rates,obesity, bodyweightandpancreatic in theregulationoffoodintake,bodytemperature, There isgrowingevidencethatBRS3playscriticalroles spotted garBRS3 GRP andNMBare theendogenous ligandsforchicken/ levels uponligandstimulation( GRPR- orNMBR-transfectedcellsfailtoincreasecAMP finding contrastswiththemammalianstudy, inwhich signaling pathway for cGRPR/cNMBR. Our a secondary it impliesthatGs-AC/cAMP/PKApathwaymayrepresent concentrations ofcGRP/cNMBthantheotherpathways, activation ofAC/cAMP/PKApathwayrequireshigher may alsobecoupledtoGsprotein( cGRPR/cNMBR activation,suggestingthatbothreceptors cAMP/PKA signalingpathwayisalsolikelytriggeredby mobilization and MAPK/ERK signaling pathways, AC/ controlled byGq-PLC.Interestingly, apartfromcalcium noted thattheiractivationofMAPK/ERKpathwayislikely monitored bythecell-basedreporterassays.Moreover, we concentration and activates ERK signaling cascade, since their activation also increases intracellular calcium cGRPR andcNMBRarealsolikelycoupledtoGqprotein, 1995 signaling pathway( pathways, suchascalciummobilizationandERK subsequently triggersmultipledownstreamsignaling lead toIP3accumulationandDAGproduction,which GRPR arecoupledtoGqprotein,andtheiractivationcan Mantey DOI: 10.1530/JME-17-0020 http://jme.endocrinology-journals.org Research In thisstudy, wefoundthatunlikemouse BRS3, It isreportedthatinmammals,bothNMBRand , , Gonzalez Jensen t al et et al Table 1 . 2010 . 1997 t al et et al ). Thisfindingclearlyindicates thatboth , . 2008 Metzger , Fig. 10 . 2015 Ryan ). Asshowninourstudy, both , ). Gonzalez ) ( et al Ohki-Hamazaki t al et Benya . 1998 Benya . 2010 c

et al

huang > ), andthus,BRS3 . 1995 Guan . 2015 > i. 10 Fig. Printed inGreatBritain 1000 . 1995 andothers t al et , et al ). However, Lach nM), orby ). . 1997 ). Since . 2010 et al b . , , to GRP/NMB evolution, whichmayresultinthelossofitshighaffinity likely due to the dramatic structural change of BRS3 during inourpresentandpreviousstudies is treatment observed no responsiveness of mammalian BRS3 to GRP/NMB perspective. The little or BRS3 from an evolutionary likely tobetheputativenativeligand(s) for mammalian and spottedgarsstronglysuggeststhatGRPNMBare vertebrate species. act astheendogenousligandsforBRS3inotherlower gars. ThisfindingsuggeststhatGRPandNMBcanalso can beeffectivelyactivatedbyGRPandNMBinspotted PKA/CREB signalingpathways.Asinchickens,BRS3 activation alsostimulatesMAPK/ERKandAC/cAMP/ mammals ( can triggercalciummobilization,asdemonstratedin Furthermore, weprovedthatchicken BRS3 activation cGRP andcNMBarenativeligandsforchickenBRS3. [Phe Pharmacological studyshowed thatBB4canbindtofrog subtype (BB4)wasidentified in1995( mammalian vertebrates( that GRPandNMBarethenativeligandsofBRS3innon- activated byGRPandNMB,substantiatingourhypothesis like chickenBRS3,spottedgarBRS3canbepotently discrepancy betweenthetwostudiesisunclear. However, with anextremelylowaffinity( binds cGRP( study, inwhichcBRS3.5 (cBRS3) expressed in CHO-K1 cells and cNMBpotently. Ourfinding contrastswithaprevious demonstrated thatchickenBRS3canbeactivatedbycGRP BRS3.5 our synteny analysis clearly indicates that chicken BRS3.5, existsinchickens( combined receptor. GPCR(s) andmediateactionsofthecognateligandsits that likeGRPR,BRS3mayheterodimerizewithother 2011 opioid (morphine)-inducedscratchingbehavior( receptor (MOR1D)inmousespinalcordandmediatethe reported thatGRPRcanheterodimerizewithµ-opioid mammalian BRS3 to GRP/NMB protein(s) mayberequiredtoaidtheeffectivebindingof exclude thepossibilitythatotherunidentifiedaccessory of mammalianBRS3toGRP/NMB 1997 in chickens GRP, NMBandtheirreceptors Published byBioscientifica Ltd. The discovery ofnativeligandsBRS3inchickens The discovery In It isreportedthatanovelbombesinreceptor, named 13 ). Thisfindingpointsoutanotherpossibility , ]-bombesin withanaffinity ( Ryan isorthologoustomouse Bombina orientalis Ryan et al invitro K i . 1998

= et al 318 ( nM) orporcine NMB( . 1998 ). Despite the extremely low affinity Gorbulev Downloaded fromBioscientifica.com at10/04/202101:03:26AM Fig. 10 , thefourthbombesinreceptor Iwabuchi ). LikecGRPR/cNMBR,cBRS3 ). Iwabuchi in vivo et al BRS3 et al . 1992 in vitro Nagalla 59 . Recently, Liu K i M higher =0.2nM) . 2003 : 1 . Moreover, we et al , Mantey , wecannot et al . 2003 K ). However, i =678nM) Liu . 1995 ). The et al et al et al. 74 via freeaccess ). . .

Research c mo, l huang and others GRP, NMB and their receptors 59:1 75 in chickens

than to human GRP (Ki = 2.1 nM) and NMB (Ki = 30 nM). BRS3 signaling is less important and may be functionally In addition, [Phe13]-bombesin treatment causes calcium replaced by its family member(s), such as cGRPR, which is mobilization in Xenopus oocytes transiently expressing highly expressed in the hypothalamus (Fig. 7). BB4 (Nagalla et al. 1995). Interestingly, we noted that Bombina BB4 share a considerable amino acid sequence Tissue expression of NMB and NMBR in chickens identical to chicken/Xenopus tropicalis BRS3 (77%/84%) (Supplementary Fig. 4). Moreover, Bombina BB4 is expressed In this study, cNMB and cNMBR were detected to be exclusively in the forebrain and cortex (Nagalla et al. widely expressed in the CNS including the hypothalamus 1995), a tissue expression pattern nearly identical to (Fig. 7). This finding is consistent with those reports in chicken BRS3 (Fig. 7). The remarkable similarity in humans (Sano et al. 2004), pigs (Ma et al. 2016) and rats structure and tissue expression shared between Bombina (Wada et al. 1992), suggesting that cNMB may act as a BB4 and chicken BRS3 led us to hypothesize that Bombina neuropeptide to regulate some physiological processes, BB4 is orthologous to chicken BRS3, or a BRS3-like receptor such as food intake, as demonstrated in chickens and duplicated from BRS3 very recently, even though Bombina mammals (Jensen et al. 2008, Tachibana et al. 2010a,b). BB4 has evolved an unusual high affinity to frog [Phe13]- Among the peripheral tissues, the highest mRNA bombesin peptide (Nagalla et al. 1995), which only exists levels of cNMB and cNMBR were noted in the testes in some amphibian species (Nagalla et al. 1996). (Fig. 7). As in chickens, both NMB and NMBR are highly expressed in the testes of humans (Sano et al. 2004), pigs (Ma et al. 2016) and mice (Ohki-Hamazaki et al. 1997a). Tissue expression of BRS3 in chickens These findings strongly suggest that NMB may act as an In this study, cBRS3 is widely expressed in various autocrine/paracrine factor to regulate the functions of brain regions with the highest expression noted in testes in vertebrates. In addition, we noted that cNMB the telencephalon. Our finding is consistent with is expressed in the ovary and other peripheral tissues. previous reports, in which cBRS3.5 is highly expressed However, the low or no expression of cNMBR in these in the telencephalon by RT-PCR or in situ hybridization tissues questions an active role of cNMB in these tissues. (Iwabuchi et al. 2003, Ohki-Hamazaki et al. 2005). These In this study, cNMBR mRNA is undetectable in findings, together with the expression ofcGRP/cNMB the anterior pituitary. This finding contrasts with the detected in these regions (Fig. 7), strongly suggest that observation in rodents. In rats, both NMB and NMBR are cBRS3 can mediate the actions of cGRP/cNMB in chicken expressed in the anterior pituitary, and NMB can act as an

Journal of Molecular Endocrinology CNS. Apart from the CNS, cBRS3 and cNMB/cGRP are also autocrine/paracrine factor to inhibit thyroid-stimulating expressed in the testes, adipose tissue and skin, suggesting hormone (TSH) release (Rettori et al. 1989, Jones et al. a role of cBRS3 in mediating cGRP/cNMB actions in 1992). In mice, disruption of NMBR results in dysregulation these tissues. of the pituitary–thyroidal axis (Oliveira et al. 2006). The It is of particular interest to note that the tissue absence of NMBR expression in chicken anterior pituitary distribution of cBRS3 is similar, but non-identical, to that hints that unlike mammalian NMB (Rettori et al. 1989), in mammals (Ohki-Hamazaki et al. 2005). For instance, cNMB may not regulate pituitary functions, at least BRS3 is expressed in the brain and testes of chickens not directly. (Fig. 7), humans (Sano et al. 2004) and rodents (Fathi et al. 1993, Ohki-Hamazaki et al. 1997a); however, within the Tissue expression of cGRP and cGRPR: evidence for cGRP CNS, an obvious difference in BRS3 distribution has been being a novel potential pituitary hormone observed between chickens and mammals. BRS3 is mainly expressed in the hypothalamus of humans and rodents In this study, we found that cGRP mRNA is widely (Ohki-Hamazaki et al. 1997a, 2005, Sano et al. 2004), expressed in the spinal cord and various brain regions whereas BRS3 is highly expressed in chicken telencephalon, (Fig. 7). Similarly, cGRPR is highly or moderately expressed but not the hypothalamus (Fig. 7) (Ohki-Hamazaki et al. in the spinal cord and nearly all brain regions examined 2005). This remarkable difference hints that hypothalamic including the hypothalamus. Our findings partially BRS3 signaling plays a critical role in mammals such as coincide with an early study in chickens, in which the control of body weight and metabolic rate (Ohki- GRPR mRNA was reported to be widely expressed in all Hamazaki et al. 1997b), while in chicken hypothalamus, brain regions detected by RT-PCR (Iwabuchi et al. 2003).

http://jme.endocrinology-journals.org © 2017 Society for Endocrinology Published by Bioscientifica Ltd. DOI: 10.1530/JME-17-0020 Printed in Great Britain Downloaded from Bioscientifica.com at 10/04/2021 01:03:26AM via free access Journal of Molecular Endocrinology in both cephalic and caudal lobes. In accordance with both cGRPmRNAandprotein areabundantlyexpressed ( expressed intheanteriorpituitary gut ( in whichGRPismainlyproducedthebrainand/or Our finding contrasts with those in mammals and frogs, hormone. hypothesis thatcGRPislikelyanovelpituitary major source ofcGRPinchickens,butalsoraisethenew maybea not onlysuggestthattheanteriorpituitary same amountofcDNAtemplatewasused.Thesefindings value of by theiramplificationplotsshownin seems tobeevenhigherthanthatof assay showedthatthemRNAlevelof detected inanteriorpituitaries( with this finding, a strong cGRP protein band was expressed inchickenanteriorpituitaries.Inagreement is that chicken gonads. also supportinganautocrine/paracrineactionofGRPon cGRP on chickentissues( and thusexertitsendocrine/paracrine/autocrine actions circulation bytheseendocrine cellsundercertainstimuli These findingssuggestthatcGRPmaybesecretedintothe Yamanaka of proventriculus ( reported to be mainly expressed in the endocrine cells data coincidewithpreviousreports,inwhichGRPwas weakly expressedinotherpartsofGItract( mRNA ismoderatelyexpressedintheproventriculus,but several peripheraltissuesincludingtheGItract. 1994 or turkeys( secretion andpancreaticproteininchickens of GRPR)hasbeenshowntostimulateproventriculusacid inbirds,whichGRPorbombesin(anagonist observations tissues examined. This finding supports the previous proventriculus, pancreasandovary, andweaklyinother Sun cord, asdemonstratedinmammals( ( as inhibitionoffoodintakeinthehypothalamus a broadspectrumofGRPactionsinchickenCNS,such The wideexpressionof Tachibana DOI: 10.1530/JME-17-0020 http://jme.endocrinology-journals.org Research Using qPCR,IHCandWestern blot,weprovedthat ofourpresentstudy The moststrikingobservation Outside theCNS, et al ). Unlike Wada and cGRP . 2009 cGRP et al cGRPR et al t al et Linari &1975 mRNAisabundantlyandpredominantly ismuchlowerthanthatof ). . 1989 . 2010 cGRPR . 1990 were detected in the ovary andtestes, weredetectedintheovary Vaillant Fig. 10 a , , , , Scanes &Pierzchala-Koziec2014 cGRPR b cGRP Nagalla ), anditchsensationinthespinal cGRP ). Besidesproventriculus,both et al expressionisrestrictedto ishighlyexpressedinthe and . 1979 t al et c i. 8 Fig. ,

cGRP . 1992 β l Sun &Chen2007

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activity ( a coordinatedfashiontoregulatereproduction,GItract GRP fromothertissues(e.g.proventriculus),mayactin GRP,pancreas, itislikelythatpituitary togetherwith is highlyexpressedintheovary, proventriculusand circulating cGRPpool( may bereleasedandcontributetoalargeportionofthe hormone,which the notionthatcGRPisanovelpituitary cells(datanotshown). All these findings support pituitary stimulate GRPexpressionandsecretioninculturedchick that gonadotropin-releasinghormone1(GnRH1)can ( cells,andGRPismainlyexpressedinLH-cells pituitary elucidated that GRP-ir cells account for 11.7% of total is distributedthroughoutbothlobes( similar to that of gonadotrophs (e.g. LH-cells), which distributed inbothlobes( this finding,GRP-ircellswerealsoshowntobedensely including humans. the physiological/pathological roles ofBRS3inmammals, in non-mammalianspecies also promotesustore-think ofthenative ligandsforBRS3 two decades.Thediscovery an issuethathaspuzzledendocrinologistsformorethan ligands ofBRS3intworepresentativevertebratespecies, chickens, butalsoidentifiesGRPandNMBasthenative hormonein concept thatGRPislikelyanovelpituitary pituitary. Collectively, ourstudynotonlypresentsanew that cGRPispredominantlyexpressedintheanterior receptor forGRPandNMB.Moreover, wedemonstrated peptides. LikecBRS3,spottedgarBRS3canalsoactas a while cBRS3isafunctionalreceptorcommonforboth the receptorsspecifictocGRPandcNMB,respectively, assays revealedthatcGRPRandcNMBRcanfunctionas and BRS3 werecharacterized in this study. Functional been neglectedinvertebrates. ovarian physiology/pathology, atopicwhichhaslong novel facetofrolesplayedbybombesin-likepeptidesin Giles 2013 of age)ovariancancer( years and anextremelyhighincidence(30–35%at3.5 granulosa cellproliferation,differentiationandapoptosis, chicken ovary, adynamicorganshowingspectacular in chickens.OurfuturestudyonGRP–GRPRactions hints thatGRPmaybeanovel‘gonadotrophicfactor’ axisalso expression ofGRP–GRPRinthepituitary–ovary ( 1999 in chickens GRP, NMBandtheirreceptors i. 9 Fig. Campbell Published byBioscientifica Ltd. In summary, chickenGRP, NMB,GRPR,NMBR , Scanes 2015 ). Moreover, studydemonstrated ourpreliminary Campbell , Johnson &Lee2016 t al et . 1991 ). Doublefluorescencestainingfurther et al Fig. 10 . 1994 Downloaded fromBioscientifica.com at10/04/202101:03:26AM ). Meanwhile,theabundant Wang i. 8 Fig. ) andpancreaticfunctions ). ConsideringthatcGRPR et al ), willaidtodefinethe ), aspatialdistribution . 2007 59 : Proudman 1 b , Johnson & t al et 76 via freeaccess . Research c mo, l huang and others GRP, NMB and their receptors 59:1 77 in chickens

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Received in final form 15 April 2017 Accepted 3 May 2017