Journal of Cell Science ee noigpoen htbt oiieyadngtvl euaeAT regulate negatively and positively both that proteins encoding genes o:10.1242/jcs.128280 doi: 5377–5390 126, Science Cell of Journal 2013 September 4 Accepted oet (AT rodents ( gene single fEF inligntok,weesohr,icuigTI,BXadCK,hv o enpeiul ikdt EGFR to linked between function previously proteins these been II that indicating not angiotensin EGF, by have with EGFR EGFR the CHKA, the of of and stimulation phosphorylation direct tyrosine BMX AT following attenuated activated occur TRIO, CHKA not the or including did BMX this others, but TRIO, stimulation, of whereas knockdown networks, Individual transactivation. signalling EGFR of meJ George J. Amee transactivation EGFR modulating II-mediated genes angiotensin identifies screen siRNA functional A Article Research ta. 00 iese l,20) n acr srvee yus by (AT reviewed as cancer, and 2010). 2009); 2008); al., al., et al., et (George Miners et (Li 2010; Zambidis dementia al., 2009; and et Zambidis, memory cognition, and including (Heringer-Walther Park neuromodulation pro- haematopoiesis 2009; 2010); 2001); al., Schiffrin, and al., et 2010; programming et al., cell Weiss et stem (Rompe 2007; responses al., Griendling, et inflammatory and Lu 2009; Mehta Horiuchi, and 2008; (Iwai angiogenesis and aneurysms (hypertrophy) dysfunction, growth endothelial dyslipidaemia; (fibrosis); cellular remodelling and include of development These subsequent also states. a and pathological can dysregulation influences for that targets processes (RAS), as modulatory serve and system principal homeostatic the renin–angiotensin of (AngII), selection the II of angiotensin balance, effector aldosterone fluid vasoconstriction, and in roles release, well-studied its from Apart Introduction words: Key (AT receptor 1 type angiotensin The Summary work this to equally ` contributed authors *These 8 7 6 5 4 3 2 1 oee,temlclrmcaim novdhv o e enrsle.T drs hs epromdafntoa iN cenof screen siRNA functional AT a robust performed a we demonstrate this, that address cells To epithelial resolved. mammary been human yet in not kinome have human involved the mechanisms molecular the however, AT at 06 et n redig 07.AtvtdAT Activated 2007). Griendling, and Mehta 2006; Catt, cancer. and disease cardiovascular functional in unbiased, remodelling of tissue power the for reveals important study mediators Our signalling crosstalk. new GPCR–EGFR in identify CHKA to for screens role genomic pervasive a indicating thrombin, ligand, oge Qian Hongwei osD Hannan D. Ross uhrfrcrepnec ( correspondence for Author eateto iceityadMlclrBooy oahUiest,Catn itra 80 Australia 3800, Australia Victoria, 3010, Clayton, Victoria, University, Parkville, Australia Monash Melbourne, 3010, Biology, of Victoria, Molecular University Parkville, and The Melbourne, Biochemistry Biology, of of Molecular University Department and The Australia Biochemistry Oncology, 3004, of of Victoria, Department Australia Department Australia Melbourne, 3010, MacCallum 3002, Institute, Victoria, Peter Victoria, Diabetes Australia Parkville, Sir Melbourne, and 3002, Melbourne, East Heart Victoria, of Cancer, IDI Melbourne, University MacCallum Baker East The Peter Centre, Pathology, Australia Genomics, Cancer of 4072, Functional MacCallum Department Queensland, for Peter Lucia, Centre Program, St. Victorian Control Queensland, The Growth of and University Signalling The Oncogenic Sciences, Biomedical of School 03 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2013. nI cspiaiyo h nitni ye1receptor 1 type angiotensin the on primarily acts AngII 1 1 –GRtasciain HAas eitdEF rnatvto nrsos oaohrGpoenculdrcpo (GPCR) receptor protein-coupled G another to response in transactivation EGFR mediated also CHKA transactivation. R–EGFR ) rti-ope eetr(PR noe ya by encoded (GPCR) receptor protein-coupled G a R), nitni,EF,Gpoenculdrcpo,sRA Transactivation siRNA, receptor, protein-coupled G EGFR, Angiotensin, 1A AGTR1 n AT and 1 5 n h GR ute netgto fTI n HArvae htteratvt slkl ob eurdfor required be to likely is activity their that revealed CHKA and TRIO of investigation Further EGFR. the and R rgr .Quaife-Ryan A. Gregory , 1,2,3,4 nhmn n w oooosioom in isoforms homologous two and humans in ) 1,2,3,6,7,8, [email protected] 1B d apr ta. 00 uyd and Hunyady 2000; al., et Gasparo (de ) roeW Purdue W. Brooke , * ) 1 )tasciae h pdra rwhfco eetr(GR omdaeclua growth, cellular mediate to (EGFR) receptor factor growth epidermal the transactivates R) 1 yi .Morgan A. Kylie , 1 ahy .Gould M. Cathryn , 1 Rs a)(eGsaoe l,20;Gilye l,21) h activated The 2010). al., et Guilluy 2000; AT al., et Gasparo (de Rac) opet h eeormrcGpoen G proteins G heterotrimeric the to couple n G and hshlps C phospholipase G48 ugsieo oefrGCsi eetrtyrosine receptor in GPCRs for role agonists a antagonist EGFR of with GPCR suggestive treated were which AG1478, cells thrombin, with when and inhibited be (LPA) stimulated could acid lysophosphatidic when 1, (ErbB2/HER2) endothelin oncoprotein phosphorylated neu the are and signalling (EGFR) tertiary receptor factor and secondary 2011). support Lefkowitz, and to (Shenoy which complexes scaffolds 2001), as al., also the and et endocytosis act and receptor (Qian mediate signalling, arrestins phosphorylation initial of terminate receptor Signal binding 2007). by and Griendling, recruitment mediated and is reactive Mehta 2000; 2006; termination of al., Catt, et N-terminal Gasparo generation and (de Jun channels Hunyady the ion various and pathway, and species MAPK extracellular oxygen JAK–STAT p38 the (MAPK; ERK1/2, kinase), kinases kinases, protein regulated mitogen-activated the 2 aln .Simpson J. Kaylene , abadclege is eotdta h pdra growth epidermal the that reported first colleagues and Daub 1 1 loculst oul n eetrtrsn kinases, tyrosine receptor and soluble to couples also R –GRtasciain aycniae r components are candidates Many transactivation. R–EGFR 3 ailW Thomas W. Daniel , s swl stemnmrcGpoen eg h,Rsand Ras Rho, (e.g. proteins G monomeric the as well as , 1 –GRtasciain eietfe ut of suite a identified We transactivation. R–EGFR b mdae acu oiiain,G mobilisation), calcium -mediated 3,4,6 3 an Handoko Yanny , atrG Thomas G. Walter , q/11 t stimulate (to 3 , i/o 1, * ,G , ` 5377 and 12/13 Journal of Cell Science hsise egnrtdarbs n rcal ua cellular human tractable and address robust AT To a lacking. of generated is model we detail issue, much and this defined poorly remains dniytepoen htaeivle nAT in involved are that proteins the identify model cardiomyocyte 2011). rat this al., a although et in mechanism (Smith 2003), the be Sadoshima, to appear and not does (Seta residue 319 tyrosine ee novdi h AT the novel in identify involved unbiasedly genes to screening siRNA functional perform inligmcieyadti scuilfrtedwsra AT downstream the for crucial is this and machinery signalling nodrt xmn h oeua ehnssunderlying mechanisms molecular the examine to AT order In model cellular AT stable a of of characterisation and Generation AT and the we that then, demonstrated Since have 1996). others al., et (Daub cross-talk signalling kinase Results direct or AT 1999), activated al., the with et EGFR (Eguchi the of activation interaction Pyk2 mediated ehnsshv endsrbdt xli AT explain to Various 2002). described al., et been Thomas 2005; Eguchi have al., 2002; et mechanisms Mifune al., 2001; et al., (Asakura et proliferation cellular and/or as such hypertrophy effects, functional and signalling growth mediated 5378 rnatvto nseii eltps lhuhti a been has this Although types. cell specific in transactivation h xc oeua ehns ikn h AT the linking Thus, mechanism biology. molecular underlying give exact the to of the failed picture has mechanistic approach broader this the not, than often more informative, iadsedn Mfn ta. 05 hs ta. 06,Ca 2006), al., et EGF Ohtsu 2005; al., metalloproteinase-mediated et (Mifune shedding ligand including transactivation, odt,ms tde aeuiie addt prahto approach candidate a utilised have studies most date, To 1 –GRtasciain ua ellrmdlwas model cellular human a transactivation, R–EGFR 1 –GRtransactivation R–EGFR ora fCl cec 2 (23) 126 Science Cell of Journal 1 –GRtasciain n sdtemdlto model the used and transactivation, R–EGFR 1 –GRtasciainprocess. transactivation R–EGFR 1 a hjc’EF family EGFR ‘hijack’ can R 1 -eitdEGFR R-mediated 1 oteEGFR the to R 1 ,truhits through R, 1 R–EGFR 1 2+ R- - nI ramn Fg C.Mxmlatvto fEF and EGFR of upon activation cells Maximal unstimulated 2C). the 2.5- (Fig. a above treatment and ERK1/2 2B) AngII (Fig. of cells activation unstimulated 2A). in (Fig. fold of level activation of pERK1/2) the 2-fold a above phosphorylation revealed (p42/44; EGFR data reproducible blot ERK1/2 western and of and Quantification robust (pY1068) the EGFR to led AngII ecie,ohrptwy oeitfrEK/ ciainfrom activation well ERK1/2 is for Ca prominent As exist (namely a stimulation. 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M na in nM) 1.7 au f2. nM, 24.8 of value 125 48n) hsdid this nM); 24.8 1 xrsinin expression R ]nI a not was I]AngII 1 n R 5 1 1 readily R )it a into R) 5 n 5 4 1 R- 1 2+ R Journal of Cell Science response amr pteilcl ie HMEC-HMLE-AT line, cell epithelial mammary eetasetdwt iNsta agtgnsivle nthe in involved genes target AT the that of those siRNAs specifically process, with transfected were HMEC-LST-AT aeilFg S3). Fig. material okokdw xrsino h a AT rat the of expression targeting down SMARTpools knock siGENOME to used We EGFR. the and AT the when confirmed xeiet aiae hssse ssial o eetn novel these detecting Together, for expected. suitable as as as AT well involved system protein, genes as this EGFR 3E,F) validated (Fig. total experiments ERK1/2 stimulation the and AngII reducing EGFR upon prevented of phosphorylation siRNAs knockdown phosphorylation ERK1/2 SMARTpool Similarly, and using 3C,D). 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Weinberg of obtained Robert HMEC- Institute (HMEC-LST, Professor were Massachusetts antigens from t Research, gift small Biomedical and a T (hTERT) were Large telomerase HMLE) SV40 human with with transformed immortalised and cells epithelial mammary Human culturing and lines Cell Methods and Materials hspoesadpri h sebyo h AT the of assembly the permit into insights and new process powerful offer this can approaches genome-wide h MCcl ie r ie nsplmnaymtra al S1. Table material supplementary in passaging given for are details lines Specific cell Australia). HMEC Cove, the Lane Coulter, (Beckman Counter utai) h Busrp KS-NHA-AT pBluescript The Australia). 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(BD supernatant Diva SE viral Vantage FACS the fresh 4 using (FACS) to Hill, of sorting cell exposed activated concentration to Castle grown were final hours, (Sigma-Aldrich, Cells 24 a Sequabrene cells. at target added and was Australia) Australia) South, Dandenong ˚ ncnlso,tepeetsuysree 2 iaegnsto genes kinase 720 screened study present the conclusion, In ih5 CO 5% with C olbrt the liberate to I 1 )plasmid. 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(http://www.ncbi.nlm.nih. sizes 110–160 amplicon (Primer3) predicted from with Blast junctions ranging 2000) Skaletsky, exon–exon Primer and over (Rozen NCBI designed gov/tools/primer-blast) using were possible) PCR quantitative (where real-time for PCR Primers quantitative real-time and Primers hswr a upre yteAsrla ainlHat and 472640, Health numbers [grant National grants Australian project the Council by Research supported Medical was work This Funding manuscript. the wrote and study this for funding stu obtained the data, discussed of designed framework screen, R.D.H conceptual and siRNA W.G.T the the manuscript; cell of the of execution reviewed testing and and screening; data design and discussed the generation siRNA mobilisation with the assisted calcium and with K.J.S assisted lines; and design H.Q knockdown and assay K.A.M siRNA assays; with with analyses; assisted assisted informatics G.A.Q-R Y.H the and performed C.M.G D.W.T and manuscript; the screening intracellula reviewed siRNA and the binding radioligand the analysed wrote and and experiments validation performed designed, A.J.G contributions Genomics Author Australasian Foundation. Fund Brockoff the the Investment Initiative, and (AMATA) Education Science Association Technologies Super Government’s the funding Development Australian by through Regional supported the Victorian Network and Phenomics from the Innovation Australian (ACRF), the Industry, (DIIRD), Foundation of Research Department Cancer lines. Australian cell HMEC Robert providing for Professor Dr USA) Technology, to and of Institute Thanks B constructs) for Australia). Institute MacCallum (plasmid (Whitehead Weinberg Melbourne, Peter Russell at Centre, (all Sarah Cancer manuscript) (proofreading Dr Hannan and Katherine Lesmana Darcy Analia Professor Associate Ms Phillip Jillian assistance), (technical Boast, Dr Szubert (FACS), Anna-Kristen Alison Milovak Ms Ms and Viki Ms microscopy), and (confocal Rossi Danne Ralph Mr to thanks Our Acknowledgements in outlined two-tailed are paired means screen the using are siRNA analysed graphically presented was data the described, Data for S1. Student’s performed Table material analyses supplementary statistical The 5.0d. analyses Prism Statistical GraphPad in drawn graphs and USA), WA, Redmond, ApidBoytm)weerltv uniiaino eeepeso was expression gene expression. GAPD of to normalised quantification was expression relative gene and where performed Biosystems) (Applied iePRSse o 0cce sn h eal ahn etnswt melt a with settings machine default the 0.7 using of cycles cDNA of 40 ramp volume for reaction curve Technologies), final System a Life PCR in primers Time Biosystems, reverse and forward (Applied of 20 nM Mix 300 and Master template Green SYBR db lutao S AoeSses a oe A S)t noprt the incorporate connecting interactions. Thicker to of screen. confidence increasing siRNA USA) represent the nodes) CA, from the (linking identified Jose, lines target San each in of redrawn Systems, level were (Adobe confidence Data option. CS5 interactions’ Illustrator confidence to ‘high Adobe submitted was the list using gene database screen the siRNA secondary The ‘interactome’. transactivation h itra etefrFntoa eoisi uddb the by funded is Genomics Functional for Centre Victorian The m a sae.Smlswr u nteApidBoytm tpn Real- StepOne Biosystems Applied the on run were Samples assayed. was l t 2 tsswti h rpPdPim50 rga.Uls otherwise Unless program. 5.0d Prism GraphPad the within -tests n trdat stored and O ˚ .Dt a nlsduigte70 D . QSoftware RQ 1.1 SDS 7000 the using analysed was Data C. 2 20 ˚ ro oue o eltm C nlss Fast analysis, PCR real-time For use. to prior C oeia eerh Massachusetts Research, iomedical acu oiiainasy and assays mobilisation calcium r yadeprmns nlsdand analysed experiments, and dy P aucit .. efre the performed B.W.P manuscript; , 0.05. 6 tnaderro h mean the of error standard 1 R–EGFR Journal of Cell Science eat . Serra-Page A., Debant, gci . epe,P . rn,G . oly .D n ngm,T. Inagami, and D. E. Motley, D., G. Frank, J., P. Dempsey, S., Eguchi, ePee . esree,V . ePte,C . aat .A n ulc,G R. G. Bullock, and A. L. Vakaet, R., T. C. Unger, Potter, De and L., V. W. Verstraeten, J. B., Paepe, Wright, De T., Inagami, J., K. Catt, M., Gasparo, de A. Ullrich, and C. Wallasch, U., F. Weiss, H., Daub, lnas . pro . ore,F,Feig .D,Zmni,D . Donaher, B., D. Zimonjic, D., M. Fleming, F., Koerner, L., Spirio, B., Elenbaas, Va A., Lymboussaki, N., Ekman, D., E. Motley, T., Yamakawa, K., Numaguchi, T., Inagami, H., Iwasaki, S., Eguchi, upeetr aeilaalbeoln at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.128280/-/DC1 online available material Supplementary 1022402]. number [grant to fellowship awarded research holds Foundation senior grant Courageous NHMRC also project Captain an the a R.D.H by and Australia (http://www.captaincourageousfoundation.com). R.D.H]; in funded and R.D.H, W.G.T. to 1024726 a,B,Km . i,Y,Go . u . ag . og . eae,J,Chen, J., Melamed, X., Kong, B., Wang, K., Xu, Z., Guo, Y., Xie, O., Kim, B., Dai, K. Ishidate, and H. Liao, C., Aoyama, Okigaki, S., E. Chiu, K., S. Logan, O., Kranenburg, L., J. M. S. Galisteo, Ram, J., and Andreev, J. P. Magalhaes, D., M. Abramoff, References gci . uauh,K,Iaai . asmt,T,Ymkw,T., Yamakawa, T., Matsumoto, H., Iwasaki, K., Numaguchi, S., Eguchi, oe,I,Mo,M,Tr,H,GiauGaosy . ay . zey . Weiss, B., Uziely, B., Maly, S., Grisaru-Granovsky, H., Turm, M., Maoz, I., Cohen, Trent, S., Telang, S., Arumugam, U., Goswami, A., Yalcin, L., A. Clem, F., B. Clem, J. Trejo, and L. G. Johnson, A., Russo, D., B. Cuevas, P., Arora, hn .W,Jnis . ioo . ann .D,Toa,W .adSmith, and G. W. Thomas, D., R. Hannan, L., Pipolo, A., Jenkins, W., H. Chan, E., Shanks, J., C. Kennedy, Y. D., Wrobel, X. T., Forster, Huang, M., L. and Selfors, A., T. 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