Journal of Cell Science A93551,USA 94305-5317, CA h l usadpus(u ta. 05,n tde have studies no 2005), al., et (Luo pburs and burs for orthologs fly as knot- distinct proposed cystine the been R- 2012) vertebrate have al., four antagonists Although BMP et proteins. The containing pburs Lau 2011). and (de burs al., domain from et thrombospondin and Glinka repeats single furin-like 2011; a cysteine-rich, two al., al., contain et proteins et spondin with (Carmon Lau associate signaling contrast, de to R-spondin In to found 2011; mediate 2005). recently and leading were al., receptors 6 Wnt et pburs, and two 5 (Luo and LGR4, production of vertebrate cAMP burs DLGR2 consisting in ligands bursicon, increases 2004). knot-containing heterodimeric Hsueh, the cystine with and interacts (Avsian-Kretchmer multicellular in signaling metazoans extracellular in roles important 2005). play and al., et (Luo to morphogenesis ortholog exhibit and hardening The in cuticle mice 2004). 6 for and al., 5 LGR4-null and et LGR4, embryonic mammalian (Mazerbourg cells. is with lethality associated LGR4 progenitor perinatal retardation and growth and 2010), intrauterine adult including cells Clevers, tissues, diverse and stem of the cells for (Barker for proliferating marker are in a As cells expressed as C 2002). of stem role al., well-established subgroup et adult a (Hsu has in LGR5 INSL3 B, and with subgroup in those interact relaxin A for subgroups whereas subgroup receptors seven- in three hormones, LGRs of 2000). glycoprotein of al., group et G-protein-coupled consisting (Hsu conserved mammals evolutionarily receptors repeat-containing, an transmembrane are (leucine-rich receptors) LGRs Introduction BMP binding a of as capable pathways. ligand serves signaling rare into and Wnt a categorized is and receptor words: be norrin BMP Key Frizzled4 Thus, can for proteins. disease the important binding Norrie by are distinct with three mediated that patients the proteins signaling the in through receptors/binding Norrin, found signaling Wnt mammalian 6. three for mutations in activating and defects norrin 6, 5 to in of different and LGR4, according LGR5 antagonists capable that and subgroups by norrin indicated BMP also not between studies but several is interactions Mutagenesis LGR4, suggested receptors, is product, antagonist. by studies norrin 6 mediated gene that binding signaling showed LGR4, and disease Wnt we activate 5 stimulates sequences, Norrie only genome and LGR4, could available pburs norrin newly for and Although Using burs ligands pburs. cells. insect and cognate for burs ortholog to are vertebrate orthologous receptors proteins be a These to R-spondin postulated processes. been four physiological have diverse that vertebrates for indicated important a studies are DLGR2, that recent receptors to seven-transmembrane orthologous are 6 are and 5 LGR4, Mammalian Summary 10.1242/jcs.123471 doi: 2060–2068 126, ß Science Cell of Journal 2013 January 25 Accepted ( correspondence for *Author 2 1 Deng Cheng LGR4 the for ligand receptor a is norrin Multi-functional 2060 eateto ieSine n nttt fGnm cecs ainlYn-igUiest,Tie 1,Taiwan 112, Taipei University, Stanford, Yang-Ming Medicine, National of Sciences, School Genome University of Stanford Institute Gynecology, and and Sciences Obstetrics Life of of Department Department Biology, Cell Stem and Reproductive of Program 03 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2013. rtiswt ytn ntmtfaeuulyscee ycells by secreted usually are motif knot cystine a with Proteins orn G4 n inln,BPatgns,Lgn–eetrinteraction Ligand–receptor antagonist, BMP signaling, Wnt LGR4, Norrin, 1 rde Reddy Pradeep , [email protected] Drosophila Drosophila 1 unCheng Yuan , ) LR,i important is DLGR2, , eetratvtdb h uspushtrdmripratfrmrhgnss Although morphogenesis. for important heterodimer burs/pburs the by activated receptor 1 hn-e Luo Ching-Wei , iadfrFize4(z4 X ta. 04 n saBMP norrin a that Results as indicated and studies proteins. receptors/binding 2004) our distinct al., 2012), three in with al., et interacts LGR6, et (Xu a and (Xu as (Fzl4) norrin antagonist LGR5 Frizzled4 of roles not for known G-protein the ligand but with of Combined LGR4, cells. stimulation mammalian by Wnt the stimulates mediated norrin to burs/pburs, signaling contrast heterodimeric the In by burs invertebrate coupling genes. of ortholog pburs pburs an and be burs and to to norrin related genes vertebrate of found exudative evolution and the trace to familial able were in found prematurity 1992). of al., retinopathy et been (Berger and disease, have more Coates several vitroretinopathy, and mutations disease, Norrie with have norrin mutations patients norrin in 70 than identified norrin- More been 2002). in al., found et (Rehm also mice defects, were null Vascular function. blindness visual and of deafness loss severe sensori-neural a and retina the of hypovascularization by characterized with disorder X-linked an interact disease, to antagonists BMP receptors. of LGR4/5/6 vertebrate ability the demonstrated ecoeyrltdt netbr rpusgns(Avsian- and genes DAN-Ap) and pburs DAN DAN-Tc or and (gremlin-Tc, gremlin burs insects identified in We insect 2004). orthologs to Hsueh, to and found 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R&D from were 4 R-spondin and Wnt3A, BMP2 Recombinant Promega. and the 2 from pSV- purchased and were vector plasmid control TOPFLASH Super domain. transmembrane first 3 h sa o h rdcino laiepopaae(P–ornpoen and proteins (AP)–norrin phosphatase alkaline of production the for assay The assays Binding empty with transfected were (0.03 later plasmids hours receptor 24 or HEK293T and vector Briefly, plates, 2004). 24-well as into al., placed plasmid et were (Xu norrin cells modifications transfecting slight with by previously described performed as were assays 1994) TOPFLASH–luciferase Neighbor-Joining al., the assays et using Luciferase 2007) (Thompson al., replications. ClustalW et bootstrap (Tamura 1000 by and 4 method aligned MEGA with were implemented sequences Protein analysis Phylogenetic Pnri sdsrbdbfr X ta. 04.Clsgoigo coverslips on or growing hLGR6 hLGR5, Cells LGR4, 2004). 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LGR4 (Genhunter). using assay endogenous 2 activity assay, AP for concentration by binding The medium quantified PBS. was in serum-free diluted AP–norrin then of in and the concentrated hours was cultured Briefly, 24 AP–norrin at containing 2004). were and cells al., cells HEK293T et into later, transfected (Xu transiently described was plasmid previously AP-norrin were measurements affinity eimadteclsclue o nte 6husbfr uieaeassay. luciferase before hours serum-containing were the 16 cells another to added for the cultured then and was cells ng/ml) the medium and (20 serum-containing transfection, medium BMP4/2 after fresh hours. hours 24 to Six for plasmid. changed cultured norrin was the 0.02 medium of with doses the transfected different later, or 0.01 hours vector COS-7 24 and empty assay, kits and BRE–luciferase plasmid plates the reporter assay 24-well For BRE–luciferase into triplicate. luciferase placed in were times cells using three R- least or determined at Wnt3A performed without using were or normalized with and activities medium (Promega) serum-free Luciferase in hours spondin2. 16 another for treated ricesn mut fpamd noignri,geln rmi- n DAN and gremlin-2 gremlin, norrin, encoding Lipofectamine plasmids using of amounts increasing or uemxkt(i-a) xrsinlvl o G4wr omlzdt GAPDH to qPCRhLGR4-5 normalized kit were: Green were primers SYBR RT LGR4 q-PCR iTaq for The Sensiscript using levels levels. the Expression triplicate RNA (Bio-Rad). in using before kit a performed transcription Supermix water were using reverse DEPC-treated PCR isolated quantitative RNase-free, After were (Qiagen), with DNase. RNAs eluted with total and treatment The (Qiagen) control. kit negative extraction a as Dharmacon qPCRhLGR4-3 9 ;B,5 9 b -gguaagaaacuccuaauuauu-3 PBi:1w) h ihrslto ornsrcueiaewas image structure norrin resolution high The 1xwd). id: (PDB 9 CTAGTGAGTTTAATAGCACTAA. : TM 00(nirgn.A 4husatrtaseto,clswere cells transfection, after hours 24 At (Invitrogen). 2000 9 utasae eino ua G4wr aeby made were LGR4 human of region -untranslated K m d b ) pSV- g), auswr calculated. were values glcoiaeatvte.Aleprmnswere experiments All activities. -galactosidase 9 cabe S o-agtn olfrom Pool Non-Targeting (S) scrambled ; b m Gl(0.01 -Gal pSV- g 9 CTTTGTTTGCCATTTCCTA; : b Glpamd oehrwith together plasmid, -Gal 9 m -gaaaguaaacuguggucaauu- ) R56(0.01 LRP5/6 g), Dpn nyeadthe and enzyme I b -galactosidase m m )and/ g) )for g) m g Journal of Cell Science rblu castaneum Tribolium eou tropicalis Xenopus intestinalis Ciona n t uat o muoltigaayi PrzVlradHl,1997). Hill, and (Perez-Vilar analysis norrin ice. immunoblotting cell-matrix-bound on collect for to put mutants times plates its many culture and scraped were and plates added 200 minutes, were medium, 5 hydrochloride After residual guanidine removing M ml After 6 5 cells. containing with detached once washed remove were to plates culture PBS (ECM), matrix extracellular the to bound purpuratus Strongylocentrotus H70 eoewsigtie ie el eete nuae n2 MHepes 65 mM formaldehyde, 20 at in 3% incubated NaCl acetone, then were mM 60% seconds cells 150 30 HEPES, Fixed for 7.0), twice. mM fixed (pH washing (20 then before and Hank’s buffer 7.0) 7.0) in binding, pH pH BSA HEPES After Hepes, mg/ml hour. ice-cold mM (0.5 1 buffer 20 in binding with for the solution temperature with salt room times balanced three at washed AP–norrin were nM cells 50 with medium (3 Fzl4 etakD asCeesfrhmnLR n i lsis Dr plasmids; six LGR5; human and and LGR4 LGR4 mouse human encoding plasmids for for Clevers Liu QingYun Hans Dr thank We Acknowledgements ( its concentrated then and was medium norrin in Conditioned culture 50 with days. were 3 transfected cells for later, were medium day serum-free One plates (Invitrogen). 2000 six-well Lipofectamine using in mutants cultured and matrix cells extracellular HEK293T in mutants its medium and conditional norrin method. of assay analysis MTT of Expression 96 number Titer the Cell FBS, Promega 10% the using in determined culture was hours using cells 48 vector viable empty After or (Invitrogen). norrin of 2000 amounts Lipofectamine different with transfected were cells HEK293T assay MTT pellets, remove to centrifugation After hour. 1 for ice on 400 DNaseI] U/ml 30 and 2000 serum-free 500 Lipofectamine in in incubated lysed using then were and LGR4 cells hours 8 and/or transfection, for after norrin medium hours empty with encoding Thirty transfected were plasmids (Invitrogen). plate six-well or a in serum vector 10% in grown L-cells Mouse b respectively. Signaling), (Cell analysis antibody immunoblotting the myc another for or for used or medium was v5 plasmid Centricon serum-free with medium in DAN concentrated cultured or The were days. cells gremlin 2 later, gremlin, day One with vector. transfected empty DAN were and cells 2 gremlin HEK293T gremlin, secreted of Analysis XP_001635321, and Dm, LGR6-Hs; XP_425441,LGR5-Ga; DLGR2-Hm; LGR4-Mm; XP_002169896, DLGR2-Nv. NP_001017403, NP_766259, LGR4-Ct; DLGR2-Dm; LGR6-Mm; LGR4-Hs; XP_002123281, NP_001028581, DLGR2-Sp; BAD92980, LGR5-Mm; NP_476702, XP_782167, NP_034325, LGR5-Hs; LGR4-Gg; retrieved GenBank: XP_426162, also NP_003658, were NP_001103719; LGR6-Gg; receptors the XP_003642780, burs-Sp, LGR-B of NP_001107780; XP_973724; from Sequences NP_001103717. gremlin-Tc, pburs-Tc, pburs-Sp, NP_001107779; XP_972176; XP_001946298; burs-Tc, DAN-Tc, pburs-Ap, XP_001946341; NP_001158417; BAH71755; burs-Ap, DAN-Sk, NP_001006826; DAN-Ap, NP_650983; NP_001161561; DAN-Xt, burs-Dm, gremlin-Sk, NP_037504; NP_035955; NP_032701; NP_001083746; gremlin2-Mm, DAN-Mm, gremlin-Hs, gremlin-Xt, CAM45836; BAE22388; NP_000257; DAN-Hs, gremlin-Mm, norrin-Hs, AAH46632; gremlin2-Hs, NP_001154869; NP_035013; norrin-Xt, ABF06564; XP_002732831; norrin-Mm, gremlin-Nv, burs-like-Hm, pburs-Sk, XP_001961531; XP_002160711; NP_001256342; pburs-Dm, are: gremlin-like-Hm, XP_002162057; gremlin-Ce, proteins gremlin-Hm, XP_002156629; GenBank cystine-knot NP_001164702; The database. for GenBank gremlin-like-Sk, the numbers from retrieved accession were sequences protein The for receptors LGR-B analysis and phylogenetic proteins cystine-knot of Sequences hsht ahn ufr(. rsHl H94 eoesann troom at staining before 9.4) substrate. pH phosphatase alkaline Tris-HCl, BCIP/NBT M the with (0.1 overnight temperature buffer washing phosphate rsHl(H80,1m GA 1 N-dodecyl- EGTA, 0.1% NP-40, mM 1% 1 Roche), 8.0), (pH Tris-HCl niGPH(elSgaig antibodies. Signaling) (Cell anti- anti-GAPDH using immunoblotting before centrifugation by junctional adsorb to beads Ctnnsaiiainassay stabilization -Catenin 6 sn etio eoeimnbotn nlss o h eeto fnorrin of detection the For analysis. immunoblotting before Centricon using ) m ftespraatwr nuae o ora 4 at hour 1 for incubated were supernatant the of l m /el o 8hus rnfce el eete utrdi serum-free in cultured then were cells Transfected hours. 48 for g/well) Hm, ; Sk, : . rspiamelanogaster Drosophila acgosskowalevskii Saccoglossus yr magnipapillata Hydra b ctnn(ue l,20) uentnswr cleared were Supernatants 2004). al., et (Xu -catenin Hs, ; ˚ o 0 iue,wse nei alkaline in once washed minutes, 100 for C 6 b oosapiens Homo Dmloie(abohm,3m MgCl mM 3 (Calbiochem), -D-maltoside rtaeihbtrccti (EDTA-free; cocktail inhibitor protease m yi ufr[5 MNC,5 mM 50 NaCl, mM [150 buffer lysis l Nv, ; Ga, ; Ap, ; b ctnn(elSgaig and Signaling) (Cell -catenin Mm, ; eaotlavectensis Nematostella crhspo pisum Acyrthosiphon ˚ ihConcanavalin-A with C alsgallus Gallus u musculus Mus m fbuffer of l Sp, ; , Ct, ; Xt, ; Tc, ; 30– 2 ao . asbr,M,Mtu,T,Mhi . aaa . aao . Umezawa, R., Hatano, I., Kazama, Y., Mohri, T., Matsuo, M., Matsubara, S., Kato, s,S . aaaah,K,Nsi . uaa,J,Kd,M,Sewo,O .and D. O. Sherwood, M., Kudo, J., Kumagai, S., Nishi, K., Nakabayashi, Y., S. Hsu, s,S . uo . hn . aaaah,K,Bal,A,vndrSe,P . van J., P. Spek, der van A., Bhalla, K., Nakabayashi, T., Chen, M., Kudo, Y., S. Hsu, lna . od,C,Krc,N,Hag .L,Kznky,O,Igligr D., Ingelfinger, O., Kazanskaya, L., Y. Huang, N., Kirsch, C., Dolde, A., Glinka, u,C . ee,E . uo . wr . s,S . oegr .W and W. H. Honegger, Y., S. Hsu, J., Ewer, S., Sudo, M., E. Dewey, W., C. Luo, J. A. Hsueh, and M. K. R. B. Kobilka, Harland, Y., and Osuga, M., S. Kudo, M. Dionne, J., L. Brunet, D., Hsu, K., M. Khokha, J. M. Sternberg, and A. L. Kelley, e-hoo .adHuh .J. A. Hsueh, and A. I. S. Ben-Shlomo, Aaronson, H. and Clevers, A. and N. Gazit, Barker, A., Yaniv, G., Liu, A., Bafico, a,Y,Ktgw,K,Hrmtu . iuh,H,Ioe . hmd,M,Uchida, M., Shimada, T., Isobe, H., Kikuchi, Y., Hiramatsu, K., Kitagawa, Y., Gao, Kitagawa, and T. Oda, T., Hattori, C., Uchida, C. M., H. Shimada, K., Clevers, Kitagawa, Y., and Gao, B. Snel, B., W. Lau, de F. Fan, and K. Wood, P., Stecha, A., Paguio, Q. D., Liu, Garvin, and A. Z., Thomas, Cheng, Q., Lin, X., Do Gong, H., S., H. Ropers, K. P., Carmon, F. Cremers, J., T. Pol, de van A., Meindl, W., Berger, ao . or,Y,Mtu,T,Oaa . mzw,A,Ouaa .and R. Okuyama, A., Umezawa, E., Ogawa, T., Matsuo, Y., Mohri, S., Kato, vinKecmr .adHuh .J. A. Hsueh, and O. Avsian-Kretchmer, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.123471/-/DC1 online available material Supplementary after release [grant for Health PMC months. of in Deposited Institutes 12 A.H.]. National to the R21DK081898 by number supported was work This Funding paper. C.-W.L., and the data; Y.C., wrote analyzed A.J.W.H. P.R., A.J.W.H. and and C.D., C.D. C.D. research; research; performed designed C.-L.H., A.J.W.H. and C.D. contributions Author eLu . akr . o,T . o,B . i .S,Tuisn . uaa P., Kujala, H., Teunissen, S., V. Li, K., B. Koo, Y., T. Low, N., Barker, W., Lau, de n rJrm ahn o Pnri,hmnFl n LRP5/6 and Fzl4 human AP–norrin, for Nathans plasmids. Jeremy Dr and .adNsioi K. Nishimori, and A. 295 se,A J. A. Hsueh, otiigGpoenculdrcpos(G) dniiaino G6adLR and LGR7. LGR7 for and mechanism LGR6 of signaling identification the (LGR): J. receptors A. protein-coupled G Hsueh, containing and M. Duin, 1061. eetr eitn Wnt/ C. Niehrs, mediating and receptors M. C. Cruciat, M., Boutros, eeoiei ytn ntpoenta ciae rti-ope eetrLGR2. receptor protein-coupled USA G Sci. activates Acad. that Natl. Proc. protein knot cystine J. heterodimeric A. constitutive Hsueh, for loop. required intracellular are third receptor the hormone in luteinizing mutation 22478. human a the by of activation VI and V server. 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