u igZo* onYugN,RsaNer n ra Seed* Brian and Niedra Rasma Na, Soon-Young Zhou*, Ling Guo cells in with recycling mice and from receptor-mediated in Deficits ARTICLE RESEARCH ß 3916 2014 April 15 Accepted 2013; May 15 Received [email protected]) ([email protected]; correspondence for USA. *Authors 02114, MA Boston, General growth Street, Massachusetts Cambridge Biology, lipoprotein, 185 Integrative Hospital, and density Computational for Center low , as cargo of such endocytosis receptor-mediated ligands, supports date, to process Schmid, and 2010). noteworthy Pucadyil Kirchhausen, show 1999; and Harrison McMahon, mechanisms 2009; and assembly (Marsh their to , yeast similarity that from vesicle-coat conserved and are that COPII, human and shown COPI , have including the studies to structure to Crystallographic determination endosomes. and endosomes to vesicles endocytosis membrane caveolar from clathrin-coated (5) through transport (TGN) plasma network retrograde trans-Golgi from (3) (4) Golgi, been endosomes, to endocytosis ER have (COPI), from I transport clathrin-mediated to transport anterograde Golgi mediated coat of from COPII by (2) transport mediated routes retrograde (ER) (1) reticulum five endoplasmic – versa, studied well to vice especially environment of from and exchange flows interior vesicular the intracellular the and broker Among extracellular compartments. to between nutrients secretory pathways and regulated information transport highly endocytic evolved and have organisms Eukaryotic INTRODUCTION Clathrin, , Endocytosis lethality, Embryonic receptors. deficits GPR107, surface WORDS: of KEY subset selective limited a highly with indirectly be or membrane The directly might plasma in the GPR107 compartments. to observed that receptors endocytic and and of return from VPS35 studies the protein for Colocalization responsible clathrin with toxins. the associates GPR107 and to that (LRP1) suggest resistance analyses protein-1 proteomic and receptor-related (LDL) cargo lipoprotein of uptake low-density decreased LRP2). internalization, transferrin as reduced known exhibit cells also is receptor endocytic (megalin cubilin–megalin complex the in of implicated representation in G-protein-coupled the reduction in multiple decrease a of a by and abundance family characterized transcript is cubilin of the that deletion phenotype of that lethal initially embryonic shows was member report that a This protein receptors. be membrane integral to III predicted type a is GPR107 ABSTRACT 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,31–97doi:10.1242/jcs.135269 3916–3927 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. ltrnmdae eiua rnpr,tems ihystudied highly most the transport, vesicular Clathrin-mediated Gpr107 nl el niaeta P17interacts GPR107 that indicate cells -null Gpr107 Gpr107 Gpr107 ou disruption nl fibroblast -null ed oan to leads ftasernrcpo TF1 lokona FC,LRP1 TFRC), as LRP2). known as also known (TRFC1; and (also receptor megalin transferrin of and cubilin genes multiple Gpr107 by that pathway of mediated receptor expression endocytic is in multi- post-coitus. the defect days in a 12.5 participating and exhibit 11.5 embryos between Null lethality of embryonic ablation in Genetic characterized. and generated aebe dniidadtersrcue eemnd nldn a including determined, vesicle structures clathrin-coated their the and of identified up Many been make 1999). have that McMahon, molecules and ancillary (Harrison Marsh the vesicles 2010; coat Kirchhausen, to triskelions together structures and The which cage-like into triskelions). (CLC), assembled (called are chains trimmers key heavy three-legged 25-kDa 190-kDa a form of understood, and composed Clathrin, (CHC) is poorly 2009). pits, chains coated still Schmid, of and element complex, structural membrane (Pucadyil a process plasma plasma the undergo sorting from of detach and invaginate, deformation that membrane pits by coated cellular into cell entrapped domains the for on are factors receptors ligand-bound surface essential pathway, this and In proliferation. complexes factors, erto.GR0 ooaie ihcahi n E1 and EEA1, and VPS35. and clathrin clathrin fibronectin with with interacts and colocalizes physically uptake GPR107 cargo secretion. LRP1-mediated internalization, in by paralog encoded protein closest the the with sequence by acid encoded protein mammals, The birds, conserved 2007). mammals, yeast and (Edgar, among rice highly cress, the nematodes, including dipterans, arthropods, throughout kingdoms, fish, is insects, found animal be and receptor GPR107 can plant G-protein-coupled counterparts 2007). and the is phylogenetically (Edgar, of that extracellular domain members superfamily transmembrane large seven of a C-terminal characteristic a peptide, and an signal bear domain to predicted hydrophobic was in protein N-terminal encoded expression The for 2007). enriched (Edgar, cDNAs lung among identified Human 1999; report. initially this was of McMahon, subject the is endocytosis and mediated (Marsh and of receptors 2009). 1 Schmid, recognition and on through Pucadyil epsin of sorting motifs number cargo AP180/CALM, a mediate specific that as including I, well endophilin as proteins, AP1–AP4, proteins, accessory adaptor of family is 4o hc eeecsdtgte iha2k upstream 2-kb a with together excised were the exons, which 18 of contains 14 first murine homologous The (BAC)-mediated method. recombination artificial bacterial a The of Generation RESULTS nti work, this In receptor- of regulation the in GPR107 of participation The Gpr107 nl mroi irbat xii eue expression reduced exhibit fibroblasts embryonic -null Gpr108 ou nebyncse el a irpe yusing by disrupted was cells stem embryonic in locus a Gpr107 ,wt orsodn eet ntransferrin in defects corresponding with 5, Gpr107 Egr 07,sae 9 dniyb amino by identity 49% shares 2007), (Edgar, 2 / 2 mice ooyosnl iehv been have mice null homozygous Gpr107 Gpr107 nmice. in GPR107 results

Journal of Cell Science n a eee ytasetepeso fR ners in into heterozygous integrase injected The mice. R4 were chimeric of clones produce The to expression Selected blastocysts 1A. transient clones. mouse Fig. by targeted in sites deleted correctly attP indicated and attB was probes integrase R4 and by the flanked was using cassette neomycin 1B) multiplex (Fig. al., by in et identified (Langerak 2005) illustrated (MLPA) were as amplification clones probe cassette, ligation-dependent targeted neomycin Correctly a 1A. with Fig. replaced and region ARTICLE RESEARCH i.1. Fig. fro pups 164 matings, these From for heterozygous PCR Mice genomic of shown). of not Effect (data progeny using MLPA using homozygous identified confirmed and and were 1C) (Fig. animals animals chimeric heterozygous of progeny n 2 3adP sdfrPRgntpn n 5adP o h 4dlto etaeas hw.Tepie ufxsLadRdnt etadright, and left denote R and L suffixes MT1 as primer well The as shown. cells, P1 also wild-type primers in are The events test indicated. The target deletion are different MT2. R4 C2 for in the C2 and shown C1, for C1 as neo, neo, P6 Using removed, 3m, 3m, and 5m, cells. were 5m, P5 3wt, stem elements 3wt, 5wt, and del embryonic resistance 5wt, of were genotyping products the targeted analysis 13 PCR fragment the cells, to MLPA for 1 generate MT1 Representative used exons to in (B) sites, MLPA P4 respectively. integrase attP for and and R4 used P3 attB of S5) integrase P2, Table expression R4 and material by After supplementary flanked line. in tk-2A-neo cell marker (shown stem selection probes the embryonic bearing MT1 BAC the targeting a yielding Using exons. 17 contains locus (G). ic fyl a r mle hntoeo eeoyosltemts lhuhte aenra opooy (F,G) morphology. normal have they although littermates, heterozygous of those than smaller are sac yolk of piece h ee on nwl-yeebys E Smaller (E) embryos. wild-type in found level the ubr f5,3 n e eeietfe ssnl oyo rae yaayi falebyncse elcoe xiiigrssac oG1.I MT2 In G418. to wild- resistance of exhibiting PCR clones by (D) co cell obtained markers. the genotypes stem M, and mouse embryonic indicated. Individual cells (right). all (C) are wild-type sac of integrase. mice in yolk R4 analysis (KO) those by and by knockout excision with (left) greater following homozygous compared embryos or observed and cells copy not (Het) MT1 was single heterozygous signal targeted as MLPA (WT), correctly identified neo in the were cells, half neo stem one and embryonic approximately 3m by 5m, diminished of be numbers to observed were intensities erse yE25 No E12.5. by regressed Gpr107 Gpr107 Gpr107 2 / 2 nl mro oevaiiyaon E10.5–11.5. around viability lose embryos -null mro eeietfe srgesd(ersne ytergteby)i min(,)truhPRaayi sn ml ic fyl sac. yolk of piece small a using analysis PCR through (F,G) amnion in embryo) right the by (represented regressed as identified were embryos nciaino mroi development embryonic on inactivation Gpr107 Gpr107 2 / 2 werematedtogenerateF2offspring. Gpr107 mro eeietfe rmnra mro rpeetdb h eteby)i min()o ihu minadyl sac yolk and amnion without or (F) amnion in embryo) left the by (represented embryos normal from identified were embryos Hp1 7lteswr bandand obtained were litters 27 m as nw as known (also RAwsasn rmnl mro Epei)adyl as( rfx n rsn nhtrzgtsat heterozygotes in present and prefix) (Y sacs yolk and prefix) (E embryos null from absent was mRNA Gpr107 2 / 2 Cbx5 mro tE11.5. at embryos C)and (C1) ) A ceai iga ftecntuto of construction the of diagram Schematic (A) Itgb3 Gpr107 C)a nenlcnrl onraieitniis h w n w signal 3wt and 5wt the intensities, normalize to controls internal as (C2) oa N a rprd n ees rncito n quantitative and post-coitus. transcription days reverse 10.5 and at prepared, sac was yolk RNA and Total embryo whole of isolation for size. reduced ab smalle exhibited organ or number the tissue small conspicuous homozygous of a analysis but the wild-type 10.5, However, segregation and of day heterozygous at Most Mendelian from embryos indistinguishable S2). normal were Table embryos with material consistent (supplementary isolated, embryos homozygous 32 were and heterozygous 53 wild-type, 27 10.5, day indistin physically were eoye.O h us 0 eehtrzgu for heterozygous were 106 pups, the Of genotyped. uainidcsa mroi ehlphenotype. lethal embryonic an induces mutation homozygous No S1). Gpr107 Table material (supplementary wild-type were rgatfml iefo eeoyosmtnswr dissected were matings heterozygous from mice female Pregnant 2 / 2 mro htwr dniidtruhPRaayi yuigasmall a using by analysis PCR through identified were that embryos nl uswr dniid niaigta h targeted the that indicating identified, were pups -null ora fCl cec 21)17 9632 doi:10.1242/jcs.135269 3916–3927 127, (2014) Science Cell of Journal Gpr107 RAaudnei T e n knockout and Het WT, in abundance mRNA usal rmterwl-yeltemts At littermates. wild-type their from guishable Gpr107 omlt pr rmtesize. the from apart normality Gpr107 koku ie h murine The mice. -knockout mro i o dniyany identify not did embryos r 2 / 2 mro aesubstantially have embryos Gpr107 Gpr107 +/ , Gpr107 n MT2 and 2 n 58 and 0 of 50% py 3917 mice eted, type

Journal of Cell Science rcinbtnti h yolsi rcino eosiue el;hwvr hr a o-pcfcbn nctpamcfato.C,cytoplasmi CE, fraction. cytoplasmic in band non-specific a was there 25 however, cells; bars: reconstituted Scale of extraction. fraction cytoplasmic membrane the in not but fraction o nkoku el.Wsenbotn eetdteepce ullnt P17( GPR107 reconstituted full-length in expected immunoblotting the and immunofluorescence detected using blotting by Western detected was cells. expression knockout E10.5 GPR107 in (B,C) from cells. not derived (KO) were knockout cells and (Rc) (MEF) reconstituted fibroblast embryonic Mouse Flag-tagged cells. hTERT. knockout and reconstituted produce as(i.1) tdy1.,1 mle rmrel deformed markedly or smaller 18 11.5, yolk day and At embryos nonviable wild-type 1D). in (Fig. found sacs that the half of no approximately abundance contained showed transcript embryos tissues heterozygous yolk-sac and mRNA, and embryo analyze homozygous to performed were RT-PCR ARTICLE RESEARCH 3918 of Generation 2. Fig. pooled of sequencing quantitative by whole analysis expression mRNA E10.5 vn ewe 15ad1. aspost-coitus. days 12.5 and 11.5 between event and that implants indicate small for findings two from homozygous several isolated However, were be S2). could tissues Table embryonic material (supplementary gestations xrsinwsls from lost was protein Cubilin expression shown). not immunofluorescence (data by as well S1), proteins as Fig. material (supplementary of sacs matrix yolk and abundance embryos multiple some in reduced as (RT)-PCR real-time quantitative The by validated well was transcripts S3). cubilin as complex Table 2002), material receptor of (supplementary Birn, proteins endocytic and null interacting multi-ligand the (Christensen and in cubilin–megalin ligands genes receptors, the under-expressed encode of that cluster cells a identified (E)10.5 aeilTbeS) xmlsaesoni i.1.A a 12.5, day At 1E. Fig. (supplementary in shown embryos are live homozygous examples no S2); as Table identified material were and noyi eetrcomplex multi-ligand receptor cubilin–megalin endocytic the with associated genes Gpr107 Gpr107 Gpr107 Gpr107 nl mro xii erae xrsinof expression decreased exhibit embryos -null 2 2 / / 2 2 2 Gpr107 mro ol eietfe rmattlo 40 of total a from identified be could embryos / 2 Gpr107 and Gpr107 mro eeietfe mn 4fetuses 64 among identified were embryos Gpr107 xrsinvco repyFa etr eeitoue into introduced were vectors Flag empty or vector expression Gpr107 Gpr107 nl el and cells -null nl mro ufracatastrophic a suffer embryos -null +/+ m m. 2 irpin(i.1,) These 1F,G). (Fig. disruption / 2 Gpr107 mro tebyncday embryonic at embryos mro u o rmthe from not but embryos Gpr107 eeepeso.The expression. gene rcntttdclsfrom cells -reconstituted Gpr107 Gpr107 eunea h emns(i.2) oto elln that line cell control A 2A). (Fig. terminus C lacked the reintroduced at the sequence a which a lines, in of fibroblast transduction retroviral isolated independently Gpr107 from between arising effects variation confounding potential avoid To transduction. hog nuainwt lx-4-ojgtdtasernfor transferrin Alexa-546-conjugated with incubation through an with 2B,C). blotting (Fig. and GPR107 2A). against immunofluorescence (Fig. antibody using insert by the confirmed lacking vector ta. 99 ihradHwe 06 oyaiadGflt 2007). Gofflot, Willnow and Kozyraki 1999; 2006; al., Howie, and et Fisher 1999; (Kozyraki al., embryogenesis et crucial normal play in that S3), roles Table (Shh), hedgehog material (supplementary sonic retinoids as 2001; and such morphogens, al., and et lipids Nykjaer of transport 2001; al., et Kozyraki Asse 1999; are al., nutrients et that other as (Kozyraki and S3), folate of such transport maternal–fetal Table the proteins, in material involved complex-interacting (supplementary (Amn) as well as and transferrin transthyretin, M, Apo endocytic E, multi-ligand Apo ApoA1, the including complex, receptor of ligands several encoding of transcripts Ablation sac. yolk visceral os mroi irbat(E)clue eeiiitdfrom initiated were cultures E10.5 (MEF) fibroblast embryonic Mouse fibroblasts in embryo defects pathway transport Transferrin , rnfri nenlzto a etdi h w ellines cell two the in tested was internalization Transferrin 6ka n rnae P17pout( product GPR107 truncated a and kDa) 66 ´ a ta. 05 oyaiadGflt 07,a ela the as well as 2007), Gofflot, and Kozyraki 2005; al., et mat nl mroi irbatcells. fibroblast embryonic -null Gpr107 Gpr107 ora fCl cec 21)17 9632 doi:10.1242/jcs.135269 3916–3927 127, (2014) Science Cell of Journal rcntttdMFcl iewscetdb using by created was line cell MEF -reconstituted Gpr107 2 nl E el yrtoia rndcint create to transduction retroviral by cells MEF -null koku)wscntutdb rndcino the of transduction by constructed was (knockout) / 2 mro n motlzdb sn hTERT using by immortalized and embryos Gpr107 Gpr107 Gpr107 nl mro n motlzdwith immortalized and embryos -null Gpr107 A iga fpoesto process of Diagram (A) DAepeso cassette expression cDNA a agdwt Flag a with tagged was eue h xrsinof expression the reduced Gpr107 , 0ka ntemembrane the in kDa) 40 Gpr107 xrsinwas expression xrcin ME, extraction; c nl mouse -null el but cells

Journal of Cell Science oa n ufc RC rti D eedcesdi ulMFcls 1 eaieysann el;M,pstvl tiigcls cl as 25 bars: Scale cells. staining positively M2, cells; staining negatively M1, cells. MEF cells. null knockout pink, in cells; decreased reconstituted were blue, control; (D) isotype protein Green, TRFC1 analysis. cytometry surface flow and using total by measured was level protein TRFC1 ute uniidb esrn h aeo nenlzto of internalization of was rate transport the Transferrin 3B). measuring (Fig. by cells quantified knockout cells further reconstituted in in not h and 2 but transferrin, within diminished reconstituted unlabeled signals with transferrin until When Alexa-546-conjugated the chased appear with subsequently 3A). labeled not were (Fig. been transferrin did had cells that but knockout cells cells knockout in reconstituted apparent min in was 20 transferrin min 2.5 Endocytosed time. within of periods different ARTICLE RESEARCH mgsrcre t25 ,1,2 n 0mnaeson le uli e,Aea56cnuae rnfri.()Atr1hpleo Alexa-546-conjuga assess of were pulse cells 1-h (KO) After knockout (B) and ( transferrin. (Rc) 1 Alexa-546-conjugated reconstituted receptor red, in Transferrin nuclei; (red) (C) Blue, abundances chases. shown. Transferrin 2-h are transferrin. and min unlabeled 1- 30 with 0, and chased 20 were 10, cells 5, transferrin, 2.5, in attenuated at is recorded recycling Images and internalization Transferrin 3. Fig. using recycling receptor transferrin of extent the 125 and transferrin hooun rbflmcnA,aet htices lysosomal increase that agents A1, bafilomycin or 3C,D). chloroquine substantially (Fig. cells were reconstituted in abundance than cells protein knockout surface in lower and protein mRNA, TFRC1 total However, knockout shown). not between (data abundance cells reconstituted TFRC2 and in difference any did reveal Immunoblotting not cells. into transport Fig. iron transferrin-mediated material (supplementary cells reconstituted S2C,D). knockout to to bound than transferrin cells less cells knockout and cells, by a reconstituted cells. internalized by knockout showed than was than transferrin cells recycling less receptor reconstituted Substantially transferrin min, of Fig. 50 rate material higher after (supplementary However, receptor cells S2A,B). transferrin reconstituted and than internalization recycling transferrin of rate higher eosiue n ncotclswr rae ihmonensin, with treated were cells knockout and Reconstituted support to known are TFRC2, and TFRC1 receptors, Two -aee rnfri.Koku el eefudt xii a exhibit to found were cells Knockout transferrin. I-labeled Trfc1 RAwsmaue yuigrvretasrpinqatttv TPR Means RT-PCR. quantitative transcription reverse using by measured was mRNA ) Gpr107 nl cells -null E cells MEF n yooorpcdei h w eltpssoe that showed 4C). (Fig. types cells cell reconstituted in in two than colocalized knockout the highly than more in were in fluorescence cells lysosomal dye TRFC1 and of knockout TRFC1 lysosomotropic colocalization a in the greater of and a receptor Comparison liberates cells. internalized compartments reconstituted acidic changes. of of pH other modest the fraction of or more elevation that with view the the with albeit consistent general and are TRFC1, data the knockout These surface followed both TRFC1 of in Total pattern TRFC1 4B). (Fig. surface cells produced in reconstituted particular in increase in than A1 substantial cells Bafilomycin a reconstituted 4B). treatment, (Fig. in after cells higher even total knockout was however, density cells; and TRFC1 reconstituted surface knockout TRFC1 in treated surface in Surface than amount cytometry. cells higher and a flow by increased using mechanisms, proportionately measured different were TRFC1 through pH hlxtxni neooi sltdfo marine from isolated exotoxin an is toxin Cholix E10.5 in disrupted is endocytosis LRP1-mediated n a enrpre ob noyoe,lk xtxnA, 2008). exotoxin al., 0.5 like with et incubated endocytosed, (Jørgensen were that be process cells to LRP1-mediated Knockout reported an been through has and to related structurally is that estv otetxnadde ihn2–8h(i.5) After 5A). (Fig. h were 24–48 cells within reconstituted died and whereas toxin effects, the its to sensitive to resistant were A ieoreo rnfri paei eosiue n ncotcells. knockout and reconstituted in uptake transferrin of Timecourse (A) . ora fCl cec 21)17 9632 doi:10.1242/jcs.135269 3916–3927 127, (2014) Science Cell of Journal suooa aeruginosa Pseudomonas m Trfc1 /lcoi toxin cholix g/ml 6 ..aeshown. are s.d. Vibrio RA() and (C), mRNA Gpr107 xtxnA exotoxin dfollowing ed m m. strains ted 3919 -null

Journal of Cell Science EERHARTICLE RESEARCH 3920 labeling. concentrated. and medium collected isotope unlabeled, was in or supernatant deuterated was the incubated stable were and that respectively, were proteins lysine differential and cells arginine secreted containing knockout using and of Reconstituted by analysis performed proteomic Quantitative E10.5 in (supplementary Integrin- cells reconstituted in acidic in S4A). prominently that Fig. increased material more than that LRP1 cells surface agents knockout increased that pH N- showed compartment types Flow biotin detection. analysis cell water-soluble blot a cytometry both by using followed in was ester labeling LRP1 hydroxysulfosuccinimide same vectorial Surface the S3). by Fig. assessed was material supplementary LRP1 of 5C,D; ratio and (Fig. mature the in but to decreased cells, RT-PCR significantly reconstituted precursor with were compared protein, cells Quantitative LRP1 knockout surface than that and LDL 5B). total showed (Fig. less studies internalize conjugated to immunoblotting cells with found Alexa- incubation were of reconstituted of cells internalization functional h knockout of 1 LDL, to rate LRP1 After diverse the LDL. attributable An of of 488-conjugated analysis 2001). was by internalization established Strickland, that the which and for deficit domain, (Herz cytoplasmic responsible ligands and are trans-membrane that together chain light a 85-kDa ligand-binding associated contains of non-covalently clusters a four and domains containing chain 5A). heavy (Fig. cells 515-kDa was knockout of that that formazan was of of cells third density reconstituted one a approximately (MTS) in yielding reduction transport tetrazolium impaired, electron a substantially toxin, that the showed with assay treatment of h 72 R1 ut-ag eetrmglnhmlg opie a comprises homolog, megalin receptor multi-cargo a LRP1, a Gpr107 -eitdfboetn1sceini compromised is secretion fibronectin-1 5-mediated nl E cells MEF -null Gpr107 Lrp1 RA swl as well as mRNA, nciainwas inactivation oprdwt hto ulcls(i.6,) h bnac of abundance The cells 6B,C). (Fig. integrin reconstituted cells in encoding null higher mRNA of fourfold that with to fibronectin compared that three- of showed was Digestion peptides analysis expression constituent cells. quantitative showed and knockout analysis fibronectin-1, not spectrometry gel but mass from 6A) using and identified (Fig. was by cells protein fractionated reconstituted expressed were highly A mixtures electrophoresis. protein secreted The n aulrpoensrig3 VS5 eetetpputative the top validate To the pathways. were these from (VPS35) GPR107 of 35 partners sorting binding (CLTC) protein to Clathrin vacuolar clathrin- TGN S4). and Table the in over- material from (supplementary involved transport prominent endosomes retrograde proteins a and from endocytosis showed mediated peptides proteins of co-precipitated representation liberated the was peptides the alone from cells, of MEF analysis tag spectrometry knockout Mass and the respectively. reconstituted of or lysates GPR107 from performed tagged VPS35 and of chain heavy Immunoprecipitation clathrin with interacts GPR107 xrsino ncotclst rae ereta that than degree Fig. greater material a (supplementary cells to S4B). reconstituted cells with knockout observed that on the integrin agents the that expression to enhanced cells showed pH knockout compartment also related acidic analysis in elevated cytometric be receptor Flow 6E). could fibronectin (Fig. of secretion that expression suggesting fibronectin-1 decreased cells, reconstituted reduced than significantly the cells were knockout abundance) in integrin lower (total for density integrin found membrane the both was that showed change a analyses cytometry significant Flow no 6D). (Fig. whereas and cells RT-PCR, null quantitative using receptor by the analyzed fibronectin-1 was the respectively, comprise which elsraedniyadtecmie nrclua n plasma and intracellular combined the and density surface cell 5 Itga5 ora fCl cec 21)17 9632 doi:10.1242/jcs.135269 3916–3927 127, (2014) Science Cell of Journal RAlvlwsfudt erdcdi the in reduced be to found was level mRNA a 5( Itga5 RC on TRFC1 surface of proportion a greater exposes pH vesicular agents elevating to Exposure 4. Fig. AI le cl as 25 bars: Scale blue. DAPI, cells. knockout in and (Red) reconstituted LAMP1 and (green) TRFC1 of staining Immunofluorescence (C) control. line the cell to untreated relative changes fold represent the bars the above numbers The cells. for knockout shown and is reconstituted staining IgG control and staining antibody TRFC1 between ratio (MFI) intensity Mean fluorescence (B) black). (Baf, A1 bafilomycin and green) (Chl, red), chloroquine (Mon, monensin include used agents The blue). (UT, cells untreated with compared pH elevate vesicular that agents with treatment following cells and (KO) (Rc) knockout reconstituted on gray) (IgG, staining antibody control matched isotype- to relative analysis by cytometry measured were TRFC1 total cells. reconstituted n integrin and ) Gpr107 a nl el than cells -null and A ufc and Surface (A) b a 1( b surface 5 m Gpr107 m. chains, Itgb1 Itgb1 ), -

Journal of Cell Science h ci inlitniyt omlz h aa bnac aisdrvdfo nlsso h w ellnsaesonblwteimage. the below shown are lines cell two the of analysis imagin from fluorescence derived infrared ratios ratiometric by Abundance quantified data. was a the blot The lysate normalize LRP1. cell to against Total antibody intensity adsorption. an bead signal with streptavidin blotted actin by and the gels labeling asse SDS-PAGE sulfo-NHS-SS-biotin 4–12% was following in (green) separated isolated LDL were were 25 bound proteins and bars: medium Surface Scale fresh cells. h. with reconstituted 5 fed were and cells 2 knockout 1, and 0, reconstituted for LDL, Alexa-488-conjugated incubation of after pulse 1-h a After exocytosis. EERHARTICLE RESEARCH h ans--hsht eetr(lokona IGF2R), as known endosome and (also early RAB9 markers receptor the endosome mannose-6-phosphate late with the perinuclear the seen less whereas slightly a EEA1, was but marker fraction similar, in co-staining A discrete 7D). structures concordant (Fig. a observed with vesicular was localization GPR107 green clathrin-associated of with of colocalization high A fused GPR107. of reconstituted against of antibody degree GPR107 an analysis with similar staining expressed following by cells or that (GFP) protein microscopy cells fluorescent confocal of using by analysis studied was localization GPR107 structures vesicular and endosomal clathrin-containing of early fraction a similar with colocalizes yielding GPR107 VPS35, and Flag-tagged 7C). CLTC in (Fig. analysis which against results blot performed to in subjected was cells using and immunoprecipitated experiment MEF these was knockout similar GPR107 between and association A reconstituted an 7B). (Fig. blotting demonstrated by proteins also followed VPS35 GPR107 tagged for and an CLTC using against specific proteins reaction antibody co-immunoprecipitation VPS35 A reciprocal and A VPS35. immunoblot CLTC 7A). and with or (Fig. detected 293ETN by CLTC readily line was against tagged followed interaction cell antibodies VPS35, using immunoprecipitation human analysis or the to in CLTC subjected expressed and was GPR107 GPR107 between interaction Means 5. Fig. 2hwe vlae yuiga T sa.Means assay. MTS an using by evaluated when h 72 0.5 to m 6 Gpr107 /lcoi oi u ncot(O el eerssat hlxtxnsosgetrcttxct to cytotoxicity greater shows toxin Cholix resistant. were cells (KO) knockout but toxin cholix g/ml ..aeshown, are s.d. nl el r eitn oLP-eedn oi yooiiyaditraieLLpoorly. LDL internalize and cytotoxicity toxin LRP1-dependent to resistant are cells -null n 5 .()LP bnac ntecl ufc n nitaellrcmatet slwrin lower is compartments intracellular in and surface cell the on abundance LRP1 (D) 3. m .(C) m. 6 ..aeshown, are s.d. Lrp1 RAi eue in reduced is mRNA n 5 ,*** 3, P 5 n h yooemre yorce hwdvr limited very showed LysoTracker marker 7D). (Fig. lysosome colocalization the and eitdgn agtn strategy. targeting gene mediated the study, co- this In be could DISCUSSION m3 8C). (Fig. analyses microscopy m1or cells, the 293ETN transfected with consistent in Neither expression The poor 8C). exhibited protein (Fig. shown). m3 m2 mutant unlike not CLTC, with immunoprecipitated conspicuously (data was cells transfected dim the the of in intensity protein fluorescence fusion The vesicles. m3–GFP of subset for a signals provides to restriction domain cytoplasm, C-terminal the deleted throughout the was located that vesicles m3 suggesting but on type, dispersed Mutants wild be 8B). to to distribution (Fig. similar seen cells a retained HeLa m2 in and characterized m1 was GFP mutants the to of distribution fused intracellular The 8A). sequence substitute (Fig. a m2 mutant by in replaced N- was 3 The Loop respectively. domain. m3, m1 and mutants C-terminal give to short truncated were domains C-terminal a and terminal and loops intracellular domain, N-terminal extracellular three predicted in a (shown has constructed GPR107 were 8A). m3, Fig. and m2 m1, mutants, with interaction three mediate CLTC, GPR107 on domains which map to for order In important are clathrin GPR107 with of interaction domains C-terminal and N- .0.(B) 0.004. Gpr107 ora fCl cec 21)17 9632 doi:10.1242/jcs.135269 3916–3927 127, (2014) Science Cell of Journal koku el oprdwt hto eosiue cells. reconstituted of that with compared cells -knockout Gpr107 nl E el xii eet nLLlaigand loading LDL in defects exhibit cells MEF -null Gpr107 Gpr107 (A) Gpr107 rcntttdclsta ncotclswithin cells knockout than cells -reconstituted ou a irpe yuigaBAC- a using by disrupted was locus Gpr107 rcntttd(c el eesensitive were cells (Rc) -reconstituted Gpr107 nl K)clscmae with compared cells (KO) -null bainhdprofound had ablation dsraeproteins surface nd using g 3921 ssed

Journal of Cell Science EERHARTICLE RESEARCH bevdebyncltaiy mroi eet nmatrix phenotype 3922 organismic in the in defects implicated the be Embryonic also to might lethality. contributed assembly additionally embryonic 2002), have placental–maternal al., observed homozygote might et stabilizes in factors (Iwaki development mechanical levels embryonic fibrinogen reduced during fetal attachment at Because jeopardize found to cubilin embryos. were expected subtypes of fibrinogen collagen be encoding the incapacitation and transcripts could addition, to partial In function maternal or development. the megalin loss from the and Because transport circulation, 2007). 2002; nutrient fetal Gofflot, Verroust, and mediates and Kozyraki 2011; cubilin 2006; Christensen Howie, 2001; al., al., 2002; and et et Fisher Birn, Nykjaer (Kaseda 2001; soluble and al., ligands et Christensen water Kozyraki heterogeneous 1999; proteins al., of poorly et mineral-binding Kozyraki variety proteins, proteins, a binding vitamins and lipid-binding their of and transportation D3, the hormones and visceral and in kidney the intestine, participate B12 sac, in and yolk roles gland embryonic their thyroid the exert of cells megalin epithelial and cubilin receptors the of start the gene for E10.5, Cubilin period at lethality. critical diminished embryonic in prominently observed was the defects expression for account selective or contribute to to identified abundance appeared Transcript which sequencing trafficking, post-coitus. cubilin–megalin-related days viability through of 12.5 profiling loss and a 11.5 in between resulting embryogenesis, for consequences b labeled were cells knockout and reconstituted from and proteins cells The reconstituted cells. from null isolated from isolated peptides those fibronectin-1 than Characteristic abundant (B) ( more medium. threefold culture are (Rc) spectrometry reconstituted mass with in compared reduced medium is culture secretion Fibronectin-1 6. Fig. rncit ssmlrbtentetocl ye (ITG types cell two integrin the between receptor similar fibronectin-1 is of transcripts Abundance (D) cells. knockout el.Gen stp oto;bu,rcntttdcls ik ncotcls 1 eaieysann el;M,pstvl tiigcells. staining positively M2, cells; staining negatively M1, cells. knockout pink, cells; reconstituted blue, control; isotype Green, cells. 13 C, rvosdt aesonta h ut-iadendocytic multi-ligand the that shown have data Previous 15 )adlgtsal stps( isotopes stable light and N) Gpr107 ooyoesurvival. homozygote 12 C, 14 ) epciey n itnuse ymlclrms.()TemN ee ffboetn1( fibronectin-1 of level mRNA The (C) mass. molecular by distinguished and respectively, N), Gpr107 b ) E ohsraeadttlintegrin total and surface Both (E) 1). koku E cells. MEF -knockout a (ITG 5 a )tasrpsi erae nkoku el,weesaudneo integrin of abundance whereas cells, knockout in decreased is transcripts 5) A n ao pce idctdb e ro)wsrdcdi ncot(KO) knockout in reduced was arrow) red by (indicated species major One (A) ulclswsosre htcudb xlie ytedecreased the on by explained receptors be surface could of that density observed integrin was clathrin-coated cells and null undergo LRP1 TRFC1, that Transport by a represented receptors embryos. recycling, membrane mouse vesicular null plasma from in of cells found derived fibroblast were embryonic been defects from had established were that lines cell null Goossens 2009). 2007; al., Nishiyama, et and (Arai remodeling integrin and fibronectin deposition the of subunit expression in diminished defect, receptor to synthesis related fibronectin be might The in turn, cells. fibronectin-1 fibroblasts of mouse expression diminished null the of light in ltrndpnetedctssi h al tg fvesicle of stage by early affected the be in to endocytosis appeared clathrin-dependent receptors of gene. set the EEA1-associated of that limited or disruption observation the a clathrin-associated with consistent of only finding is The GPR107 subset contains observed. a vesicles was only of lysosome Little late the subset that with EEA1. or associated a marker were markers as that endosome endosome structures well vesicular early to as localization the clathrin, a containing containing with vesicles vesicles colocalizes GPR107 of that subset indicated microscopy confocal .Asbtnildces ncroitraiainby internalization cargo in decrease substantial A 5. obte nesadtefnto of function the understand better To utpeaatrpoen aebe eie htfacilitate that defined been have proteins adaptor Multiple and VPS35, and clathrin with co-immunoprecipitated GPR107 a ora fCl cec 21)17 9632 doi:10.1242/jcs.135269 3916–3927 127, (2014) Science Cell of Journal r eue nkoku el oprdwt hto reconstituted of that with compared cells knockout in reduced are 5 Gpr107 a ,wihpriiae nmatrix in participates which 5, nl el htivle subset a involved that cells -null Gpr107 Gpr107 nl cells. -null eosiue and reconstituted , Fn1 sdcesdin decreased is ) dniidby identified Gpr107 Gpr107 heavy y b 1 - -

Journal of Cell Science igetp fcrowr lofudt eifune by influenced be to found also were cargo of type single a a undergo might that functions receptors transport multi-ligand support. the These they to large related cargo. pathway for sorting their specialized abundance and transcript receptors presence compartments. reduced its different on showed depend are into that that sorting receptors steps of for processing subset endocytic the that perform for role signaling important to the eukaryotic a cell to have attributed the might fundamental been GPR107 instructs not date, a highly to has receptor function support phylogenetically KDEL a to such is Although appears GPR107 process. and receptor, KDEL conserved Also cell. nearly the the is within 2001), and like structures al., vesicular organization in et transmembrane expressed Majoul ER seven exclusively 1991; a the Pelham, has 1990; like GPR107 motif Pelham, However, from KDEL and the topology. (Lewis recognizes differ because which transmembrane (KDELR1), organization proteins receptor retention a structural adapter lack fundamental The they their 2007). in Trejo, GPR107 and (Wolfe EERHARTICLE RESEARCH omto n htrcgieseii ois uha PYor NPxY as such motifs, specific recognize Yxx that and formation vector empty Flag exper or co-immunoprecipitation VPS35-Flag the CLTC-Flag, in cells. used 293ETN examin lysates in were cell co-immunoprecipitation immunoprecipitates the reciprocal The of by beads. 10% CLTC anti-Flag represents with using Input interaction Gpr107 (IP) VPS35. GPR107 immunoprecipitated or of were CLTC Verification lysates Flag, (B) cell recognizing and antibodies cells, using 293ETN immunoblotting VPS35. into and transfected CLTC was with vector interacts GPR107 7. Fig. tiigiaehglgt h rasoni h ne mg.Rdarw nAadCidct eetdpoenbns cl as 25 bars: Scale bands. protein detected Clathr indicate the C in and box A white in The arrows load (green). against Red GPR107 sample antibodies GPR1 expressing image. high with against cells inset a stained antibody HeLa the using an were in as in fraction cells visualized well shown membrane also MEF as area the were red), Reconstituted R, (red) the in vesicles. (M-6-P LysoTracker highlights detected EEA1 receptor and n image be mannose-6-phosphate and RAB9 is marker staining could marker clathrin band endosome but endosome of late GPR107 late lysate the fraction The The bead or cell a cells. EEA1 (green). anti-Flag total with marker MEF after the endosome colocalizes (KO) Flag in early GPR107 knockout the against concentration (D) clathrin, and antibody low 2). (Rc) an to Fig. reconstituted by owing in in detected sample (shown experiment be input Co-immunoprecipitation can the CLTC-Flag (C) in antibody. enrichment. visible anti-GPR107 before or not anti-Flag but using enrichment immunoblotting by examined were oercposta aebe rdtoal osdrdt ferry to considered traditionally been have that receptors Some W nrcpos rdigcroldnrcposadclathrin and receptors cargo-laden bridging receptors, on etrwr otasetdit 9ENclsa niae,adcl yae eeimnpeiiae sn niFa ed.Teimmunoprecipitate The beads. anti-Flag using immunoprecipitated were lysates cell and indicated, as cells 293ETN into co-transfected were vector Gpr107 A oimnpeiiaineprmn n23T cells. 293ETN in experiment Co-immunoprecipitation (A) nl embryos -null el a identified has cells 2001; Pfeffer, proteins. a clathrin from 2007; these receptors and containing retrieves GPR107 from compartment al., GPR107 that indicate between might et receptors VPS35 association and an (Wassmer The transmembrane plays 2005). TGN mammals, recycling Seaman, to in in SNX6 endosomes and role SNX5 important SNX2, SNX1 possibly VPS35, identified The VPS29, and VPS26, GPR107. Two were includes with which VPS35, be density. complex, co-immunoprecipitated retromer protein surface also that retromer proteins receptor might among and of TGN CLTC reduction to proteins, the endosome in from implicated clathrin-mediated transport membrane, plasma retrograde the integrin to proteins transporting whereas GPR107, Gpr107 of absence unaffected. the was integrin the expression but in Similarly, process unaffected. reduced GPR107-mediated the a was where on 2 latter type dependent and 1 was type former receptors transferrin of behaviors the of ablation eety owr eei ceni ali mroi stem embryonic haploid in screen genetic forward a Recently, in receptors affected of density surface decreased the Although ora fCl cec 21)17 9632 doi:10.1242/jcs.135269 3916–3927 127, (2014) Science Cell of Journal nl irbat scnitn ihalso npathways in lesion a with consistent is fibroblasts -null Gpr107 tiigdsrmnto a on between found was discrimination striking A . Gpr107 Gpr107 sacuildtriato ricin of determinant crucial a as Fa xrsinvco rFa empty Flag or vector expression -Flag m a m. xrsinwas expression 5 dby ed iment. and 3923 b ot 07 in 1 s

Journal of Cell Science osqec ftercrodlvr ehnss hthighly GPR107. initiate that of can mechanisms, receptors client delivery that the cargo with receptors their overlap as of possibly The subset, consequence distinct ricin. a a constitute and might cholix transport as toxin retrograde such ER, shiga the to toxin, transport retrograde undergo that toxins without pathway finding mutated The excluded. be be cannot that cannot the that viability is However, receptor cellular 2011). specific that compromising al., a et view of (Elling a existence identified screen proteins ricin-resistance receptor, membrane the plasma a proteinaceous in of paucity is specific the by ricin no supported the that Because has supposed 2011). commonly toxin been al., has isolated it et lectin, independently galactose-binding (Elling among clones mutations ricin-resistant identified frequently EERHARTICLE RESEARCH 3924 pEAK15. Gpr107 from constructions Alexa-488- Plasmid obtained Massachusetts were of the transferrin, MitoTracker committee by and Technologies. Alexa-546-conjugated Life LysoTracker approved usage LDL, protocols and Hospital. conjugated under care General conducted animal were institutional studies Mouse reagents and Animals METHODS AND MATERIALS in Lesions 2011). al., et (Elling sensitivity eeas netdit h xrsinvco pCMV-3 vector expression the into inserted also pCMV-3 were vector expression the into te IadFa aswr netdit h Zm ia vector. viral pZome the into inserted were tags Flag and II Strep Gpr107 DAwtotteFa a a netdit xrsinvector expression into inserted was tag Flag the without cDNA Gpr107 Gpr107 baincnespoeto uprsteve htthe that view the supports protection confers ablation , Vps35 usre ih aepriua motnefor importance particular have might subserves and Cltc 6 DA ihFa a eeinserted were tag Flag with cDNAs Flag. Gpr107 Gpr107 n t he mutants three its and 6 HA. eetemost the were Gpr107 with anand n aigwsiiitdt eeaehmzgu mutants. homozygous generate to initiated was mating lines. and stem maintained, the embryonic transmitted which engineered of two the Gpr107 produced, from were animals derived chimeric male been Seven had 8–12 PCR that with blastocysts cells C57BL/6 S5). injecting MLPA. by the by generated Table of were affirmed mice Removal Chimeric was material events. sequences deletion of coding supplementary the phosphotransferase pair confirm neomycin in a to integrase sequenced with shown R4 were analysis products P6; PCR an using (P5, with by primers tested transfected were ganciclovir were resultant cells tested. The (GCV)-resistant cassette. 5H02, 96 TK-neo the of delete and out to (supplementary plasmid identified expression 5G06 probes were lines, clones MLPA positive Two multiplex Six S5). with Table material MLPA (200 G418 using to resistant screened were in that Clones a (Taconic). RP23-361I14 at cells linearized stem was clone BAC targeting PI- BAC The single 2003). Seed, and and (Yang vector elsewhere targeting coli small Escherichia recombination homologous a using by constructed between was BAC targeting The of Generation anti-integrin- ab28320); L2420); (Sigma-Aldrich; (Abcam; anti-LRP1-heavy-chain sc- anti-LRP1-light-chain Biotechnology, ab83810); anti-TFRC2 ab633335); Cruz (Abcam; (Abcam; Santa anti-TFRC1 ab2731); chain; Abcam, Biotechnology; 271178; (heavy Cruz (Santa anti-clathrin commercially anti-cubilin sc-23644); for used: sources were following antibodies rabbits available The immunizing conjugates. by KLH-peptide produced with were antibodies peptide Anti-GPR107 Antibodies niEA SgaAdih 75) niFa 2(Sigma-Aldrich; M2 anti-M-6-P-receptor ab2792); anti-Flag (Abcam; E7659); ab10099); anti-PDI F1804). (Sigma-Aldrich; (ab2810); (Abcam; anti-RAB9 anti-EEA1 ab2733); anti-VPS35 (Abcam; ab25076); ora fCl cec 21)17 9632 doi:10.1242/jcs.135269 3916–3927 127, (2014) Science Cell of Journal uainit h etgnrto.Htrzgu iewere mice Heterozygous generation. next the into mutation Sce ieadeetooae nomueW/2S embryonic W4/129S6 mouse into electroporated and site I eetdpoenbands. protein indicate detected arrows Red immunoprecipitation. IP, (3 VPS35. control three Flag-tagged or with GPR107 mutants (WT) by wild-type cells HA-tagged domain. 293ETN co-expressing N-terminal in the performed of to was deletion binding Co-immunoprecipitation by but affected domain, only C-terminal is deletion or upon VPS35 N-terminal lost is the CLTC either to of Binding alters (C) domain cells. GFP. C-terminal in with GPR107 localization fusion the by of visualized Deletion was (B) mutants GPR107 of three Localization the blue). and (dark domain deletion C-terminal a the has in m3 and loop) with loop (green the loop) GGGSGGGSGGGSEGG of (red replacement EASATDGKAAINLAK a sequence the has 3 m2 retains box), but (green domain peptide has N-terminal signal m1 the tag. in (HA) deletion hemagglutinin a a with to constructed binds were that GPR107 of domain CLTC. the Mapping 8. Fig. Gpr107 olwn eea rcdrsta aebe described been have that procedures general following , A he uat fGR0:m,m n m3, and m2 m1, GPR107: of mutants Three (A) nl mice -null 6 lgvco) LCor CLTC vector), Flag a m (Abcam; 5 /l were g/ml)

Journal of Cell Science n utrdi soesmdfe ubcosmdu ID)with (IMDM) acids medium amino 10 non-essential Dulbecco’s and glutamine, modified serum, fetuses calf Iscove’s iron-supplemented 10.5-day-old 10% in from derived cultured were and fibroblasts embryonic Primary cells MEF GPR107 reconstituted of Derivation ARTICLE RESEARCH etoso oesiswr nuae vrih t4 at Tissue overnight serum. incubated blocking goat were using 1% coverslips by with or blocked (BSA) supplemented sections albumin was Biosciences) serum fluorescence bovine (BD Nonspecific 5% solution permeabilized with min. and PBS 10 were min in Cells 15 for X-100 min. for 4 Triton Biosciences) for 0.3% (BD plate. PBS with in buffer 12-well SDS fixation 0.5% a in in paraformaldehyde fixed permeabilized 2% in and in fixed min coverslips were 15 coverslips onto for on seeded cells or were sections Tissue in cells cells in of 10,000 microscopy tissues For culture, frozen compound. sectioning (OCT) temperature by cutting prepared optimal were sections tissue Embryonic Immunofluorescence for uterus. specialized maternal those from dissected except sacs yolk primers or from embryos (Qiagen) All from RNeasy or an cells using purified MEF and extracted was copy RNA RT-PCR Total the quantitative transcript and transcripts, estimated preparation the RNA an of to length converted the then was and number. proportional reads number of counted. are order of counts copy then the read number on transcript the was is and the mRNA cell 2005) each al., to each et in kept. (Carter transcripts matched was copies of match 500,000 that number best total the reads using the only Assuming of database read, each mRNA number Genome For the UCSC The BLAST. to or the Bowtie matched from either were downloaded Sequences (http://hgdownload.cse.ucsc.edu/downloads. was html#mouse). mRNAs Sequencing database manufacturer’s mouse Center. (Illumina). The Medical browser of the University kit Tufts list (Qiagen). of to core sequencing A Genomics the columns mRNA according Trizol in performed an was using prepared RNeasy using embryos was using instructions whole library E10.5 purified sequencing from and extracted (Invitrogen) was RNA Total analysis data and RNA-Seq niae nioisfr2ha omtmeaue olwdby 40 were Images equipped combination. a followed lasers. microscope helium-neon using the confocal temperature, nm AOBS acquired with 543/594 SP5 room and stained Imaging Leica argon-krypton, were a with at antibodies. on Cells secondary performed h min. 2 was fluorescence-labeled 30 with for for incubation serum) BSA antibodies 3% goat 0.3% containing indicated with v/v (PBS permeabilized solution 5% were blocking and cells in The prepared X-100 PBS. Triton with times five rinsed ie o 0mnwt %(/)prfradhd nPSa room at PBS at in methanol paraformaldehyde (w/v) 100% 4% with with or min temperature 10 were for slides fixed with before microscope fluorescent 33258 a using Hoechst by a63 visualized with a were Cells Invitrogen). using counterstained mounted. 200; visualized was in was (1 DNA IgG Staining anti-rabbit cubilin. Alexa-546-conjugated secondary against antibody polyclonal rwt mt lgvco,rsetvl,in respectively, vector, Flag empty with or of expression stable lines, Biological cell (Applied and MEF virus cells) subsequent hTERT Two with transduction Materials). by generated were ntilct naBoRdi5isrmn.Pie eune r itdin listed are sequences S6. Primer Table instrument. material iQ5 Bio-Rad supplementary a on triplicate Transcriptase in Reverse II iQ SuperScript and with (Invitrogen) quantitative prepared and were transcription mixes Reverse RT-PCR S5. Table material in supplementary detailed are and (http://pga.mgh.harvard.edu/primerbank/) PrimerBank E el rw ngascvrlp eewse ihPSand PBS with washed were coverslips glass on grown cells MEF 6 i-meso objective. oil-immersion m /lgnaii.hTERT-immortalized gentamicin. g/ml Gpr107 Gpr107 TM ncot(etrcnrl,wr salse through established were control), (vector knockout Gpr107 6 SYBR nl 1. E el n salsmn of establishment and cells MEF E10.5 -null r63 or DAfsdwt lgtgcdn sequences Flag-tag-coding with fused cDNA H re uemx(i-a)adperformed and (Bio-Rad) Supermix Green 6 betv n h prpit filter appropriate the and objective 2 Gpr107 Gpr107 20 Gpr107 ˚ Cfor1h.Cellswere Gpr107 nl E cells. MEF -null eeslce from selected were c(reconstituted Rc ˚ iharabbit a with C nl E cells MEF -null eosiue n ncotcls(3 cells knockout internalization and transferrin Reconstituted of kinetics of Measurement confocal using by observed and times different transferrin microscopy. at for fixed medium fresh were with Cells fed chase. was Biosciences). transferrin fresh culture with washing the After For (BD medium, h. serum-free immunofluorescence. 1 buffer for transferrin for to exposed fixation were above cells recycling, in described fixed as and immunostaining using visualized were by localized was cells protein transferrin GPR107 differentSubsequently the at PBS For and with washing times by transferrin. stopped was reaction medium human serum-free the internalization, fresh with Alexa-546-conjugated replaced was medium containing The h. 2 serum-free for with with incubated medium and plates times were three cells medium 12-well the serum-free day, with into next washed The overnight. seeded medium in were incubated and coverslips cells knockout cells MEF and in Reconstituted recycling and internalization Transferrin eosiue n ncotclswr rae ih2 with treated were cells pH vesicular knockout elevate that and agents with Reconstituted cells MEF of Treatment were wells 37 competition at h and 1 for the triplicate above, of described the as day transferrin, the in radioactive with On cells loaded as assay. experiment, the internalization the before the experiment, day for one previously plates 6-well described into recycling seeded receptor were transferrin Cells of kinetics of Measurement eyln a eie ytertobtenterdociiyof radioactivity the medium in between (transferrin the receptor transferrin ratio total determine transferrin). Transferrin the and internalized to and medium transferrin. by the tubes in defined internalized transferrin to for was the transferred tube recycling the and of to transferred solubilized internalized radioactivity and of wash were a recycling cells as allow cells transferred to the indicated was to for min) medium the 60 tube efflux for a the 40, incubated points, into time 20, and plate different medium The 10, At efflux min. transferrin. 5, 1 of (2, of ml course 1 times the medium with over fed 100 efflux times 7.2), was pre-warmed three (pH HEPES transferrin) with mM human washed 20 ml BSA, then 0.2% with were medium cells (serum-free The min. 2 ufr(5 MNC,1m CaCl mM 1 NaCl, mM (150 buffer o1m eu-remdu ih02 S otiig2 containing BSA 0.2% with medium serum-free ml 1 to naee rnfri a de otecmeiinwl.Clswere Cells well. competition the to 37 at added of excess incubated was 500-fold a and transferrin wells triplicate unlabeled to transferrin) human ng/ml 1500 ypotn h ai fitra rnfri osraetasernrtoa a as ratio transferrin surface to time. analyzed transferrin of was internal function of internalization ratio transferrin the the of plotting the by kinetics of subtracting radioactivity The average by wells. the from calculated triplicate well was competition the transferrin in radioactivity surface for and solubilized intracellular and times six o h taysaesraetasernmaueet h lt was plate the measurement, transferrin to surface 4 transferred at and steady-state incubated NaOH the N 0.1 For with min with for washed 5 solubilized suitable were for then cells and tubes acid) the and twice acetic Finally, M buffer buffer transferrin. 0.5 surface PBS PBS NaCl, remove M pre-chilled to (0.5 ice with buffer on washed 2.5 pH ice, with on incubated placed min), 60 h el eewse ihsrmfe MMadicbtdi serum- 37 in at incubated h 1 and experiment, for the IMDM BSA 0.2% of serum-free with prepared, day with medium the free were washed On were condition competition. cells each for the for well additional wells one Triplicate plus for experiment. allow of to experiment day the before day one ltswr eoe tvrostmsadwse ihpre-warmed with washed and times various at (37 removed were Plates aee ua rnfri 50ng/ml (500 transferrin human labeled SgaAdih r5 or (Sigma-Aldrich) 25 (M5273), loecneatvtdcl otn analysis. sorting cell activated fluorescence ˚ C)serum-freemediumandthenincubatedwithpre-warmedpH4.6 ora fCl cec 21)17 9632 doi:10.1242/jcs.135269 3916–3927 127, (2014) Science Cell of Journal m ˚ o nta f37 of instead h 2 for C hooun C68,1 MbflmcnA (B1793) A1 bafilomycin nM 10 (C6628), chloroquine M c ˚ ,rmvda h niae iepit 2 ,1,30, 10, 5, (2, points time indicated the at removed C, cutn.Aohr1m fefu eimwsadded was medium efflux of ml 1 Another -counting. c cutn.Ec ieon a rae individually. treated was timepoint Each -counting. m /lbeednA(ilgn)fr1 olwdby followed h 16 for (Biolegend) A brefeldin g/ml c cutn.Teseii aiatvt of radioactivity specific The -counting. 2 6 0m oimaeaebfe)for buffer) acetate sodium mM 20 , ˚ n hnwse ihPSbuffer PBS with washed then and C 10 125 5 eepae na6wl plate 6-well a on plated were ) -rnfri PriEmr and (PerkinElmer) I-transferrin ˚ .Teclswr hnexposed then were cells The C. , 09%cnlec nthe on confluence 80–90% c cutn.The -counting. m monensin M m g/ml 3925 125 m ˚ C. g/ I-

Journal of Cell Science oesisadicbtdoengt h etdy h el eeserum- were cells the with day, plates next 12-well The onto overnight. seeded incubated and were coverslips cells knockout and Reconstituted assay internalization LDL (3 seeded were cells knockout cells and MEF Reconstituted of treatment toxin Cholix ARTICLE RESEARCH 3926 was (SILAC) and reconstituted culture ( of heavy cell containing numbers medium in equal in growing cells acids knockout and amino seeding by by performed labeling isotope culture Stable cell in acids quantification amino protein by labeling isotope Stable were cells MEF knockout 4 at and overnight amounts reconstituted streptavidin–agarose Equal from with mg/ml) instructions. (1 incubated protein manufacturer’s cellular sulfo-NHS-SS-biotin the of to and according (Pierce), sulfo-NHS-LC-biotin membrane-impermeable the using reagents biotinylated were proteins surface Cell analysis protein Surface CaCl mM digitonin, 2 (0.02% NaCl, buffer mM 150 HE 7.5, in protease pH were cells HEPES, a fractions mM containing lysing 50 membrane NP-40) sequentially 1% and by 8, 8, Cytoplasmic pH extracted pH Tris-HCl, (Roche). EDTA, mM cocktail mM (50 inhibitor 5 buffer obtained lysis NaCl, were standard mM cells in 150 MEF cells or the 293ETN incubating transfected by from lysates cell Total analysis Immunoblot PBS- (1 with treatment Cells by detached were EDTA. cells knockout serum-free and LDL- Reconstituted with Alexa-488-conjugated chased cytometry Flow and with h incubated 1 times. various for and for medium medium h serum-free 2 containing for starved alnBooia asSetoer aiiyo avr University. Harvard the by of at Facility analyzed Spectrometry (LC-MS/MS) and Mass The spectrometry Biological excised Blue. mass Taplin were Brilliant tandem Coomassie chromatography bands 4– liquid with a protein in stained expressed Amicon separated and differentially cutoff were gel mixtures weight SDS-PAGE protein molecular 12% secreted 10-kDa The system. a filtration through concentrated and ( aemd n rtaeihbtr)adlssbfe 1 MTi,p 7.4, pH Tris, mM (10 buffer lysis and inhibitors) protease and maleimide eebokdwt dse lcigbfe L-O Biosciences), and (LI-COR TFRC2 against or monoclonal buffer LRP1 mouse against blocking antibodies a polyclonal Odyssey rabbit with Membranes with incubated (Millipore). membranes 10% blocked SDS-PAGE. PVDF to X-100, were to subjected Triton transferred and method 1% were Bradford Proteins Protein EGTA, the inhibitors). using mM protease quantified and 1 was deoxycholate EDTA, 0.5% SDS, mM 0.1% 1 glycerol, NaCl, mM 100 R1a efre ynraiigteseii rb adintensity of band quantification probe specific and of the using Biosciences) that normalizing to performed by (LI-COR an was performed software sizing using LRP1was infrared Band scanned 3.0 with (1 System. were Odyssey IgG Imaging membranes incubation anti-mouse Infrared Biosciences), or After Odyssey anti-rabbit LI-COR 680 Tween-20. 15,000, IRDye in and 0.2% 800 IRDye with secondary buffer TBS nioisaantTF1adintegrin and TRFC1 against antibodies ie.Tepoen eeeue ihSSsml ufrfrSDS-PAGE for buffer sample SDS analysis. with immunoblot eluted and were proteins The times. elpaeadtetdwt 0.5 with treated and plate well xrsin fewrs h el eewse ihPStieand FlowJo. or twice software Pro PBS Quest with Cell Data Biosciences). with washed BD analyzed (FACSCalibur, were were cytometry flow were using cells by surface or analyzed the and internal expression both Afterwards, visualize surface to expression. antibodies cell above the then visualize and with Biosciences) incubated to (BD solution h permeabilization 1 using permeabilized for antibody control T sa Poea a sdt esr elvaiiyacrigt the to according viability cell measure instructions. to manufacturer’s used was (Promega) assay MTS 12 C, 14 )(ire,rsetvl.Tredycluemdawr collected were media culture Three-day respectively. (Pierce), N) b -actin. 6 10 5 eete nuae ihFITC-conjugated with incubated then were ) b atna 4 at -actin m /lcoi oi o 8had7 .An h. 72 and h 48 for toxin cholix g/ml ˚ vrih n ahdwith washed and overnight C a 13 ra isotope-matched an or 5 C, 6 10 15 ˚ )adlgtisotopes light and N) 4 n ahdthree washed and C nowlso 96- a of wells into ) 2 0m N-ethyl mM 10 , uhrcontributions Author interests. competing no declare authors The interests Competing affiliated (all proteomics with MA). help Hospital, for for General Wang Cao Massachusetts Huajun Zhifang with cells, and stem analysis embryonic of data microinjection sequence for Lu Naifang thank We Acknowledgements groups. between differences Student’s analyses Statistical beads 4 anti-Flag at with h incubated 4 for were (Sigma-Aldrich) described They as analysis. prepared immunoblotting were cells for knockout and reconstituted from Lysates co-immunoprecipitation and Proteomics wk,T,Snoa-opr .J,Pia . oaah,T,Pols .A and A. V. Ploplis, T., Kobayashi, M., Paiva, J., M. Sandoval-Cooper, T., Iwaki, J. A. Edgar, J. P. Verroust, and H. I. E. Birn, Christensen, and E., I. C. E. Carmack, B., Christensen, D. Dudekula, V., VanBuren, A., A. Sharov, G., M. Carter, Asse T. Nishiyama, and Y. K. Arai, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.135269/-/DC1 online available material Supplementary material Supplementary Hospital. General Massachusetts by supported was work This Funding article. the prepared experiments, the and designed prepared data Seed and Brian interpreted experiments. data performed interpreted Niedra Na Rasma data Soon-Young article. interpreted article. experiments, the executed prepared and and designed conceived, Zhou Ling Guo fe ahn,tebudpoen eeeue nSSsml loading to sample followed SDS was proteins. interacting in above candidate described eluted identify procedure were immunoblotting proteins The bound buffer. incubated the beads. were A/G washing, protein cells on After adsorbed 293ETN then transfected and antibodies or corresponding the cells with MEF from and lysates sections the several cell assay, co-immunoprecipitation in For analysis. sliced spectrometry was mass for peptide. gel sent stained The Flag and Blue. gel using Brilliant SDS-PAGE by Coomassie 4–12% a with eluted in separated were were proteins proteins eluted binding The the times, three salt ez .adSrcln,D K. D. Strickland, and J. Herz, T. Kirchhausen, and C. S. Harrison, J. L. Peelman, and H. Favoreel, A., Zeveren, Van A., Soom, Van K., Goossens, E. S. Howie, and E. C. Fisher, P., S. Demers, R., O’Malley, G., Wirnsberger, J., Taubenschmid, U., Elling, uigebyncdvlpeti h mouse. the in development 1034. embryonic during J. F. Castellino, development. during and function tubule receptors. endocytic microarray. S. oligonucleotide M. whole-genome the mouse Ko, during and C. nutrients Nelson, of internalization embryo. the rodent in the 1086. cubilin of of development peri-implantation role and of Expression Louvet-Valle half P., second Verroust, the extracellular placental during of placenta regulation the mouse expression. in matrix the involved factors in possible pregnancy: RNAs messenger matrix ns n nlsso nern scniaeF1rcposi bovine in receptor. signaling receptors FN1 candidate coats. as vesicle of novel two embryos. analysis including preimplantation variants, splice and (FN1) 1 ones, fibronectin of Quantification (2009). cells. stem embryonic Schnuetgen, similar mouse D., al. Schramek, having G., et Schmauss, animals, F. I., A. and Shukalyuk, Q., plants Vanhaelen, both in receptors. coupled found G-protein proteins to topology of family LUSTR development. mt . io,S,Gflt . islNtck,P,Ile,F,Cha F., Illien, P., Linsel-Nitschke, F., Gofflot, S., Vinot, E., ´mat, ora fCl cec 21)17 9632 doi:10.1242/jcs.135269 3916–3927 127, (2014) Science Cell of Journal 21) owr n ees eeistruhdrvto fhaploid of derivation through genetics reverse and Forward (2011). t ts a sdt eemn h ttsia infcneof significance statistical the determine to used was -test 20) ua P17admrn p18aemmeso the of members are Gpr108 murine and GPR107 Human (2007). e.Biol. Dev. Nature 20) irngnsaiie lcna-aenlattachment placental-maternal stabilizes Fibrinogen (2002). il Reprod. Biol. .Ci.Invest. Clin. J. a.Rv o.Cl Biol. Cell Mol. Rev. Nat. 466 296 1048-1049. , M e.Biol. Dev. BMC e . inne,F n oyai R. Kozyraki, and F. Rinninger, S., ´e, 20) rncitcp ubretmto sn a using estimation number copy Transcript (2005). 279-297. , 20) h oeo eai LP2G30 during (LRP-2/Gp330) megalin of role The (2006). ˚ elSe Cell Stem Cell .Atrwsigwt ellssbfe with buffer lysis cell with washing After C. 20) eeomna hne nextracellular in changes Developmental (2007). 20) R:amliucinlsaegrand scavenger multifunctional a LRP: (2001). 77 20) eai n uii:multifunctional cubilin: and Megalin (2002). 108 923-933. , 20) eai n uii,rl nproximal in role cubilin, and Megalin (2002). 21) tutrlbooy osrainin Conservation biology: Structural (2010). 779-784. , eit.Nephrol. Pediatr. N Seq. DNA 9 ,1. 9 3 563-574. , 256-266. , m .Pathol. J. Am. eoeBiol. Genome 18 il Reprod. Biol. 235-241. , 17 993-999. , 6 R61. , 160 72 tlt F., ˆtelet, 1021- , 1079- , (2005).

Journal of Cell Science ei,M .adPla,H R. H. Pelham, and J. M. Lewis, aol . tab . el .W,Ddn .adSo and R. Duden, W., S. Hell, M., Straub, I., Majoul, H. Jacobs, and P. J. Schouten, O., A. Nygren, P., Langerak, Gburek, A., Dautry-Varsat, C., Jacobsen, J., P. S., Verroust, J., Cui, Fyfe, R., C., Kozyraki, Jacobsen, C., Gerdes, M., Kristiansen, J., Fyfe, R., Kozyraki, F. A. Gofflot, and Saito, R. Kozyraki, and H. Sato, M., Hosojima, R., Kaseda, øgne,R,Pry .E,Fedos,R . ibr .S,Brlt,D H. D. Bartlett, S., M. Kimber, J., R. Fieldhouse, E., A. Purdy, R., Jørgensen, ARTICLE RESEARCH ag euae neatosbtenpoen novdi OIvscetraffic: vesicle FRET. COPI using in cells involved living proteins in between measurements interactions regulates cargo recombination non-homologous receptor. and MLPA. oligonucleotide homologous three using of events detection apical the quantitative for epithelia. pathway polarized K. major in S. a 12491-12496. transferrin Moestrup, is of and I. endocytosis uptake E. cubilin-mediated Christensen, dependent E., T. Willnow, J., A-I apolipoprotein high-affinity lipoprotein. a high-density of is al. endocytosis et facilitating cubilin, R. receptor receptor, Krahe, A., B12 Chapelle, factor-vitamin la de intrinsic M., Aminoff, I., E. Christensen, amnionless. and megalin cubilin, of roles D(3). vitamin of the in cubilin irocholerae. R. Vibrio A. Merrill, and Nature .Bo.Chem. Biol. J. 348 20) hlxtxn oe D-ioyaigfco from factor ADP-ribosylating novel a toxin, Cholix (2008). 162-163. , 20) utlgn noyoi n ogntldefects: congenital and endocytosis Multiligand (2007). 19) ua ooou fteyatHDEL yeast the of homologue human A (1990). 283 10671-10678. , ur hr.Des. Pharm. Curr. hr pe.Dial. Apher. Ther. e.Cell Dev. uli cd Res. Acids Nucleic rc al cd c.USA Sci. Acad. Natl. Proc. 21) oeo eai and megalin of Role (2011). ig .D. H. ling, ¨ 1 139-153. , a.Med. Nat. 5Spl 1 Suppl. 15 13 20) ai and Rapid (2005). 20) Megalin- (2001). 33 3038-3046. , 20) KDEL- (2001). e188. , 19) The (1999). 5 656-661. , 14-17. , 98 , as,M n cao,H T. H. McMahon, and M. Marsh, ag .adSe,B. Seed, and Y. Yang, J. Trejo, and L. B. Wolfe, J. Herz, and A. Nykjaer, E., T. Willnow, emn .N. M. Seaman, L. S. Schmid, and J. T. Pucadyil, R. S. Pfeffer, R. H. Pelham, S., M. Nielsen, C., Jacobsen, R., J. Leheste, R., Kozyraki, C., J. Fyfe, A., Nykjaer, ase,T,Atr . un,M . aly . re,C .adCle,P J. P. Cullen, and J. C. Traer, J., Oakley, V., M. Bujny, N., Attar, T., Wassmer, el ihitc atra riiilchromosomes. artificial bacterial intact with cells endocytosis. receptor coupled proteins. ancient for cieGPssi otdvsceformation. vesicle 10.1126/science.1171004. doi: coated 1217-1220. in GTPases active R109-111. complex. Golgi and al. 25(OH) et hormone K. steroid S. the D(3). Moestrup, of vitamin metabolism A., abnormal Chapelle, causes la dysfunction de Cubilin M., Aminoff, J., P. Verroust, 285 20) oso-ucinsre eel N5adSX spotential as SNX6 and SNX5 reveals retromer. mammalian screen the of loss-of-function components A (2007). 68-75. 215-220. , ora fCl cec 21)17 9632 doi:10.1242/jcs.135269 3916–3927 127, (2014) Science Cell of Journal 20) ebaetasot ermrt h rescue. the to retromer transport: Membrane (2001). 19) eyln fpoen ewe h nolsi reticulum endoplasmic the between proteins of Recycling (1991). rc al cd c.USA Sci. Acad. Natl. Proc. 20) eyl orrcposwt retromer. with receptors your Recycle (2005). 20) ieseii eetreigi os mroi stem embryonic mouse in targeting gene Site-specific (2003). ur pn elBiol. Cell Opin. Curr. a.Cl Biol. Cell Nat. 20) ltrndpnetmcaim fGprotein- G of mechanisms Clathrin-dependent (2007). 19) h tutrleao endocytosis. of era structural The (1999). Traffic 20) osre ucin fmembrane of functions Conserved (2009). 1 E157-E162. , 8 19) iorti eetr:nwroles new receptors: Lipoprotein (1999). 462-470. , 98 .Cl Sci. Cell J. 3 13895-13900. , 585-591. , cec NwYr,N.Y.) York, (New Science a.Biotechnol. Nat. 120 45-54. , rnsCl Biol. Cell Trends 21 ur Biol. Curr. 447-451. , Science (2001). 3927 325 15 11 , , ,

Journal of Cell Science