Dimerization Drives EGFR Endocytosis Through Two Sets of Compatible

inWn,Xne hnadZiin Wang* Zhixiang and Chen Xinmei Wang, Qian of sets codes two endocytic through compatible endocytosis EGFR drives Dimerization ARTICLE RESEARCH ß eevd2 uy21;Acpe 9Dcme 2014 December 19 Accepted 2014; July 23 Received ([email protected]) correspondence for *Author 2H7, T6G AB Edmonton, Alberta, Group, of Research University Transduction Dentistry, Canada. Signal and Medicine and of Genetics Faculty Medical of Department The 250-amino-acid 229-residue a by a followed core contains kinase tyrosine protein domain conserved cytoplasmic intracellular single 622- a is glycosylated domain 23-residue transmembrane heavily The binding dimerization. ligand receptor The for and responsible is domains. domain extracellular Her4/ErbB4 three amino-acid and of 1989) glycoprotein Yamamoto al., membrane composed 170-kDa a family et is 1986; EGFR 1993). (Kraus receptor al., et al., Her3/ErbB3 (Plowman ErbB 1986), the et al., of (Bargmann et members Sliwkowski, Her2/ErbB2/neu three and and include other epithelial Yarden of The 1999; those 2001). (Wells, including widely lineages types, is and cell mesenchymal activity many kinase in superfamily tyrosine the expressed intrinsic of with member prototypal receptors the of is known ErbB1, also and (EGFR), HER1 receptor as (EGF) factor growth epidermal The INTRODUCTION spatially be Dual codes to Dimerization, endocytic Endocytosis, receptor, have EGF WORDS: KEY molecule, one receptor codes, endocytic each coordinated. compatible of in sets contained two the of the endocytosis dimer, the codes mediate receptor to endocytic that found of we sets Finally, required. compatible are two that indicating PDGFR competent, and EGFR found of both We EGFR–PDGFR although sets endocytosis. two receptor an for that that required indicate are results codes These endocytic endocytosis. EGFR in mutant impaired endocytosis-deficient EGFR of the heterodimer the and ErbB3 Moreover, deficient. heterodimers and endocytosis EGFR–ErbB3 also ErbB2 are or that EGFR–ErbB2 endocytic of Interestingly, lack show the codes. to We owing deficient endocytosis mutants. are homodimers this their tested and various we of hetero-dimers receptors Here, or homo- proximity. specific generating close receptor by hypothesis in by drives in monomer, contained driven one receptor dimerization is codes, each endocytic the endocytosis of sets EGFR two how bringing that dimerization, clear propose not We (EGF) dimerization. internalization. is factor EGFR by it growth controlled epidermal However, is that endocytosis (EGFR) previously receptor shown have We ABSTRACT 05 ulse yTeCmayo ilgssLd|Junlo elSine(05 2,9590doi:10.1242/jcs.160374 935–950 128, (2015) Science Cell of Journal | Ltd Biologists of Company The by Published 2015. a hlcltasebaeppie h 542-residue The peptide. transmembrane -helical b eeoie sedctssdeficient, endocytosis is heterodimer b ooiesaeendocytosis- are homodimers D 0511 salso is 1005–1017 vnsidcdb G-idn ieyrglt EGFR–EGFR regulate likely EGF-binding by induced 2008; events Burgess, 2006; Yarden, and and 2009). development MacDonald, the (Citri and in cells Hynes role tumor key a of play growth to shown additional been receptor normal this has numerous of family members many in of activity aberrant involved for the responses, is cellular growth essential and Although is processes 2006). morphogenic signaling Yarden, EGFR and factor-induced Citri and signal 2001; Yarden survival, 1999; complex Sliwkowski, (Wells, proliferation, differentiation a and cell EGFR migration initiates modulates adhesion, The kinases that residues. cascade tyrosine transduction tyrosine receptor regulatory of family with tail C-terminal emnlsqec faioais10–07addluiemotif dileucine and EGFR 1005–1017 C- L EGFR acids by the amino and of controlled activation, sequence terminal is kinase EGFR internalization than rather EGFR dimerization, We that autophosphorylation. shown and activity have kinase tyrosine intrinsic of activation dimerization, receptor include events These endocytosis. xmn h noyoi frcpo ooiesand homodimers receptor of endocytosis The the mechanism. examine underlying difference. the this for is account homodimerization might what by heterodimerization induced or clear changes not conformational different still al., is but et internalization, it Shankaran EGFR rather improve 1999; homodimerization endocytosis might receptor heterodimerization al., that than in et indicate data Worthylake impaired These 1999; 2006). al., was et al., heterodimer However, (Wang both EGF 1994;). et al., EGFR–ErbB2 et form Goldman following Qian the 1994; 1987; can Cantley, ErbB2 and Schlessinger, EGFR Carraway 1990; and with that of (Yarden heterodimers shown capable stimulation that, been are and suggest results homodimers has homodimers These ErbB2 It 1997). Gilboa homodimers, endocytosis. al., 1985; et EGFR al., strictly Klapper et like is 1995; (Drebin al., of endocytosis bivalency et ErbB2 capable antibody mAb-induced on be dependent that to antibodies al., monoclonal and reported et certain to (mAbs), been with Muthuswamy binding Cbl 1996; has upon al., of internalization ErbB2 et binding However, (Levkowitz poor 1999). recycling contribute The might the heterodimer 1998). to EGFR–ErbB2 the the al., or ErbB3 that et al., ErbB2, et indicated Lenferink Waterman 1996; been al., et 1998; also (Pinkas-Kramarski back surface has recycled cell often the It to are al., ErbB3 1999). and et EGF-responsive ErbB2 al., Baulida endocytosis-inefficient et using 1993; Wang Carpenter, by and 1996; endocytosis (Sorkin in receptors chimeric impaired are family known internalization. not receptor is the it However, drives 2007). dimerization al., the et how Wang 2005; activity al., kinase et receptor (Wang of independent endocytosis, EGFR driven 1010 h noyoi fEF siiitdb idn oEF The EGF. to binding by initiated is EGFR of endocytosis The nti td,w xli otolbesse ospecifically to system controllable a exploit we study, this In receptor ErbB the of members other the that shown been has It L 1011 ucina noyi oe omdaedimerization- mediate to codes endocytic as function 935

Journal of Cell Science n h R uinrcpo a ptp-agdwt Ato HA with epitope-tagged was antibodies. with receptor Myc, detection with fusion specific epitope-tagged allow FRB also the were of receptors and All fusion S1). FKBP Fig. material the (supplementary receptor receptor FKBP-fused FRB-fused AP21967, the and heterodimerizer between the heterodimerization induce with can stimulation homodimerizer these and fused the FKBP, receptors of with C- between with homodimerization transfection the stimulation induce to can cells, Following AP20187, FRB 293T receptors. or 2006) into FKBP these chimeras al., fusing of by a et chimeras constructed terminus we Zhan receptor study, of this 1999; In series S1). al., Fig. 1, material et protein (supplementary associated (Muthuswamy FKBP12-rapamycin-binding FKBP-12-rapamycin respectively the from and domain FKBP (FRB) with two heterodimerize fused or proteins proteins (FKBP) system, two protein homodimerize FK506-binding this with specifically fused to In used be kits. can chemicals dimerization receptor Heterodimerization niaeta w noyi oe r eurdfrreceptor for required are codes results endocytic These EGFR. two in endocytosis-deficient codes that endocytic the indicate of the presence the and Moreover, despite EGFR deficient. EGFR mutant endocytosis between in EGFR–ErbB3 also codes or heterodimer endocytic are EGFR–ErbB2 of Interestingly, lack heterodimers the are C-termini. homodimers to their ErbB3 owing and deficient ErbB2 endocytosis that show We heterodimers. ARTICLE RESEARCH eentedctsdfloigA217adto o ,1 and 936 15 5, for ErbB3–FKBP surface addition and or AP20187 ErbB2–FKBP following EGF cell both endocytosed min, EGFR– of not 30 were addition whereas a to following 5 2B,C, for Fig. internalized following AP20187 strongly in of was by shown endocytosis FKBP As ErbB3–FKBP, the homodimers signal. examined biotinylation and receptor then We EGFR–FKBP these 2A). (Fig. of respectively by homodimerization EGFR– encoded (HRG, min, the 30 heregulin homodimers. and formed for all EGF ErbB3–FKBP stimulation in and shown AP20187 ErbB2–FKBP As FKBP, assay. following cross-linking 2A, a Fig. by the determined of receptor was dimerization on receptors The AP20187 endocytosis. of and activation effect or dimerization, the ErbB2–FKBP examined both EGFR–FKBP, and with with ErbB3–FKBP, cells We FKBP 293T 1A). fused transfected (Fig. (ErbB3–FKBP) then we ErbB3 al., ErbB3, and the (ErbB2–FKBP) or drives et ErbB2 ErbB2 also AP20187 (Wang homodimerization of whether EGFR–FKBP EGF, internalization determine of to To the internalization 2005). similar to led and that which EGFR–FKBP, activation of shown homodimerization the previously induced have homodimers receptor We family ErbB of Endocytosis ARGENT ARGENT the receptor and of utilized hetero-dimerization internalization we Homodimerization and the heterodimers, Regulated homo- study or specifically controllable homodimers to a system develop To and homo- hetero-dimerization controlled and chimeras Receptor RESULTS coordinated. spatially be each to receptor required. in have the contained molecule, one of are codes, receptor endocytosis endocytic the codes compatible two mediate the endocytic to dimer, EGFR that compatible show both we PDGFR two Finally, although and that EGFR indicates deficient, the PDGFR endocytosis that and is show further heterodimer We endocytosis. b D ooiesaeedctsscmeet which competent, endocytosis are homodimers 0511 sas mardi endocytosis in impaired also is 1005–1017 NRG1 lostimulated also ) TM Regulated TM b eiinymgtb u oalc fedctccdsi h C- the in this codes endocytic and receptors. of the lack deficient, of a domains endocytosis to regulatory terminal due are be might homodimers deficiency ErbB2 that ErbB3 indicate EGFR– data with these and transfected phosphorylation together, Taken cells the 2D,E). 293T (Fig. induced in FKBP EGFR AP20187 of and shown endocytosis have EGF and we both control, a that As lack 2D). its here to (Fig. owing activity kinase HRG with intrinsic ligand of physiological stimulation its upon or 2003;). phosphorylated AP20187 al., either not et was Garrett ErbB3 readily 2002; However, exposed (Schlessinger, is constitutively domain with its ErbB2 to dimerization consistent owing overexpressed phosphorylation is that for the which available 1996; induced showing AP20187 2D), al., reports (Fig. that previous et showed ErbB2 also Baulida of We internalization phosphorylation 1993; 1999). are al., have Carpenter, et homodimers which Wang and ErbB3 reports (Sorkin or and previous deficient ErbB2 ErbB2 with either that consistent these of shown with is endocytosis of incubation the which endocytosis 2D,E, stimulate ErbB3, Fig. not the in did examine shown AP20187 As further homodimers. to receptor immunofluorescence conducted Indirect of endocytosis 2B,C). was the (Fig. stimulate not either did ErbB3–FKBP, it homodimerization 2A), induced (Fig. had ErbB3–FKBP HRG of whereas Moreover, min. 30 itnlto inl(i.3,) hc niae htEGFR– that deficient. indicates endocytosis also which are 3E,F), heterodimers (Fig. ErbB3 surface either cell of signal the following endocytosis shown biotinylation the as strong 3D). ErbB3-FKBP stimulate or (Fig. a not EGFR–FRB ErbB3–FKBP did stimulated EGFR–ErbB2 AP21967 and AP21967 However, EGFR–FRB and between that HRG association both indicated ErbB3–FKBP, ErbB2–FKBP results or deficient. endocytosis homodimers of are our heterodimers EGFR–FRB formation the the the homodimers, without of induces heterodimers, formation the specifically (EGFR–FRB)–(ErbB2–FKBP) stimulate 3B,C). AP21967 (Fig. the not ErbB2–FKBP that or did EGFR–FRB Given either AP21967 of signal, endocytosis cell the biotinylation following co- by shown and surface cells as EGFR–FRB However, and between 3A). 293T association (Fig. EGF ErbB2–FKBP in strong both a ErbB2–FKBP, that induced and AP21967 showed EGFR–FRB We with transfected immunoblot. was by by heterodimers followed receptor analysis experiments of co-immunoprecipitation formation by ErbB2– The determined EGF, and AP21967. with stimulated EGFR–FRB or then HRG were with cells The cells ErbB3–FKBP. EGFR/ErbB3 or 293T 1A) FKBP transfected (Fig. and (EGFR–FRB) then EGFR To EGFR–ErbB2 with and hypothesis. FRB of our fused the we test endocytosis heterodimers, lack and to the EGFR–ErbB2 ErbB3 models good of examine and as heterodimers ErbB2 serve the as EGFR–ErbB3 both EGFR Thus, homodimers that codes. ErbB3 the endocytic suggests and contrast, By ErbB2 above of 2007). of al., shown endocytosis endocytosis et Wang impaired 2005; the the the al., in et mediate contained (Wang codes homodimer shown C-terminus endocytic have the We that EGFR internalization. previously are dimer, and EGFR the endocytic above mediating of of molecule for sets receptor each two required in that contained is one underlying internalization codes, the this that propose for We EGFR mechanism 2005). by al., controlled et is (Wang internalization dimerization EGFR that shown have We EGFR–ErbB3 and heterodimers EGFR–ErbB2 of endocytosis Impaired iial,fr23 el otasetdwt GRFBand EGFR–FRB with co-transfected cells 293T for Similarly, ora fCl cec 21)18 3–5 doi:10.1242/jcs.160374 935–950 128, (2015) Science Cell of Journal

Journal of Cell Science EERHARTICLE RESEARCH h noyoi eiinyo GREb2o EGFR–ErbB3 caused or C-terminus EGFR–ErbB2 ErbB3 of and heterodimers. deficiency ErbB2 the endocytosis of endocyticthe for effect of inhibitory codes sets alternatively, the or endocytosis, two endocytic receptor mediate within to that required necessary are suggest codes molecule results the EGFR are these contains internalization, the heterodimers heterodimer that EGFR–ErbB3 the Given confirmed and deficient. results EGFR–ErbB2 endocytosis Our both ErbB3–FKBP. that and EGFR– ErbB2–FKBP of endocytosis the stimulate FRB, not did shown AP21967 3G,H, As immunofluorescence. Fig. in indirect by examined further was h noyoi fEF–rB n GREb3heterodimers EGFR–ErbB3 and EGFR–ErbB2 of endocytosis The h eue GRedctssi epnet G sas an also is EGF to response in homodimer. EGFR endocytosis internalized of EGFR formation reduced the efficiently to The 1994). due is observed al., is the endocytosis et that homodimer likely EGFR Soltoff is EGFR it 1994; dimerization, EGF-induced al., the following ErbB2 et that well and Qian EGFR 1990; Given co-expressing al., is EGFR cells et both heterodimer in (Goldman It of and preferred formation is heterodimers, formation the 3B,C,G,H). EGFR–ErbB2 (Fig. induce and can stimulated homodimers EGF not internalization but that did ErbB2–FKBP, established EGFR EGF of of internalization some addition the the stimulate ErbB2–FKBP, and EGFR–FRB ti neetn ont htfr23 el co-expressing cells 293T for that note to interesting is It ora fCl cec 21)18 3–5 doi:10.1242/jcs.160374 935–950 128, (2015) Science Cell of Journal eetrwseioetge ihMc TM, Myc. domain. with transmembrane tagged epitope fusion was FRB receptor the also and were HA receptors with fusion epitope-tagged FKBP the research. of this All in used and constructed mutants and receptors of various representation Schematic 1. Fig. 937

Journal of Cell Science EERHARTICLE RESEARCH tmlt n noyoi fEF n rB Fg 3D–H). (Fig. ErbB3 not 938 and did both EGFR HRG of expressing of endocytosis cells addition in any the 293T impaired stimulate ErbB3–FKBP, for is and contrast, heterodimer EGFR–FRB By EGFR–ErbB2 the internalization. that indication 2. Fig. e etpg o legend. for page next See u o GR hsrsl scnitn ihtefnig htboth that impaired endocytosis. findings are in the heterodimers with EGFR–ErbB3 and consistent homodimers is ErbB3 result ErbB3, this of EGFR, homodimerization not the but stimulates only HRG that Given ora fCl cec 21)18 3–5 doi:10.1242/jcs.160374 935–950 128, (2015) Science Cell of Journal

Journal of Cell Science itnlto inla ecie nteMtrasadMtos oto,total Control, Methods. and surface Materials cell the the in following described by as examined signal was biotinylation The receptors biotinylation. ErbB surface of cell of endocytosis by Endocytosis homodimerization (B) following protein antibody. receptors the anti-Myc ErbB and with lysed were immunoblotted dimer; cells were the with Dim, samples then crosslinked heregulin; and HRG, were (DSS), proteins free; suberate serum The disuccinimidyl SF, ng/ Dimerization. indicated. (50 (A) as HRG monomer. or or Mon, nM), min (100 30 AP20187 for ng/ml), ml) (50 EGF ErbB3– with or treated receptors. ErbB2–FKBP were EGFR–FKBP, family FKBP with ErbB transfected of transiently endocytosis cells and Dimerization 2. Fig. ARTICLE RESEARCH eius10–07adted-ecn oi L motif acid di-leucine amino the especially and EGFR, EGFR C- 1005–1017 of the mutant of that residues domain shown EGFR previously regulatory endocytosis an have terminus We and the codes. mediate EGFR endocytic to checked lacking wild-type required between are we dimer heterodimers EGFR internalization, the sets in its two endocytosis-inhibitory whether codes of examine endocytic further presence To of ErbB3. the and code or ErbB2 endocytic in ErbB3, an signals of and lack the ErbB2 either in to due EGFR–ErbB3 be and might EGFR–ErbB2 heterodimers the of endocytosis impaired The endocytosis the dimers for EGFR required of are codes endocytic of sets Two 4 at MESNA mM 100 with incubation without receptors biotinylated Fg B.Te,w xrse EGFR expressed we fused We Then, 2007). 1B). al., (Fig. et (Wang endocytosis EGFR EGFR for essential he ifrn xeiet.* experiments. different three inl u nylcsteedctccdsi t -emns our C-terminus, its in codes endocytic the lacks only but signal, and EGFR EGFR–FRB the of that with heterodimer the EGFR treated that the indicate further when or results EGFR–FRB either However, of EGFR internalization 3L,M). detectable (Fig. no but EGFR AP21967, reduced, eliminated greatly of was internalization EGFR–FRB not of the endocytosis observe The stimulation, FKBP. not EGF upon did results, we biotinylation our with Consistent (EGFR detectable the no that endocytosis. indicate in EGFR EGFR impaired results the AP21967, and These EGFR–FRB or 3J,K). homodimers. of (Fig. heterodimer with EGFR–FRB EGFR–FRB observed was either some FKBP of treated of of internalization formation internalization when the might the which to However, 3J,K), EGF (Fig. due observe eliminated by not be upon but not reduced, shown signal, greatly the was As did biotinylation induced 3I). EGFR (Fig. surface AP21967 we receptors cell and stimulation, two the EGF these following both of that co-immunoprecipitation association The showed AP21967. or the assays EGF stimulated and either cells with 293T cells in EGFR–FRB described above the n ehd.Ec au stemean the is value Each Methods. and bars: Scale Methods. and Materials 20 the indirect in double described by as analyzed immunofluorescence and were (green) red) (pY; ErbB3–FKBP phosphotyrosine or ErbB2–FKBP EGFR–FKBP, The of following immunofluorescence. localization mean receptors indirect the ErbB by is of determined value Endocytosis homodimerization Each (D) B. experiments. from three dimerizers. data least or the ligands of with Quantification incubation the (C) on without proteins and sulfo-NHS-SS-biotin-labeled surface the from cell biotin remove to min 10 efrhraaye h noyoi fte(EGFR–FRB)– the of endocytosis the analyzed further We m .()Qatfcto ftedt rmDa ecie nteMaterials the in described as D from data the of Quantification (E) m. D D D D 0511 ihFB (EGFR FKBP with 1005–1017 0511–KPwsosre Fg LM.These 3L,M). (Fig. observed was 1005–1017–FKBP EGFR–FRB of internalization The 1005–1017–FKBP. 0511)dmrb nietimmunofluorescence. indirect by dimer 1005–1017) D 0511 uati mardi noyoi.Given endocytosis. in impaired is mutant 1005–1017 D 0511 uatde o oti ninhibitory an contain not does mutant 1005–1017 P , .1we oprdwt SF. with compared when 0.01 6 ...o tlategtclsfrom cells eight least at of s.e.m. D 0511–KPand 1005–1017–FKBP D 0511 uatis mutant 1005–1017 D 1005–1017–FKBP) 1010 D D 1005–1017– 1005–1017– 6 L ...o at of s.e.m. 1011 ˚ for C 293T are , EGFR of endocytosis the block EGFR not the did with AG1478 EGFR Treatment inhibitor 5A,B). (Fig. kinase internalized strongly etdwehrti uatEGFR mutant this whether tested xrse ohEF–R n EGFR and EGFR–FRB both expressed fedctssadol ifri noyi oe,orresults our codes, endocytic in differ only and endocytosis of n P08 ramn o –0mn EGFR EGF min, both 5–30 upon for signal, treatment biotinylation AP20187 surface and by cell shown the As following homodimerization. following endocytosis undergo ev sago oe ots hsnto.W ue PDGFR fused We notion. PDGFR this and test (PDGFR PDGFR to EGFR FKBP model of good with a heterodimer as a serve of Thus, PDGFR homodimerization stimulate 2010). that to sufficient shown and receptor endocytic have EGFR different, factor but growth of We mediate platelet-derived functional, of sets codes. sets to created two two we endocytic containing this, order any dimer test receptor To in are endocytosis. a receptor words, for compatible sufficient other codes or need In codes identical endocytic endocytosis. of be sets two the to whether examined next We induce to internalization failed EGFR codes endocytic non-compatible Two n hntetdteclswt P16.A hw nFg 5E, Fig. in EGFR shown As EGFR–FRB AP21967. and between heterodimerization with the cells stimulated the AP21967 treated then and ybt yfloigtecl ufc itnlto signal 5H,I). biotinylation (Fig. surface immunofluorescence cell EGFR indirect and EGFR–FRB the by that Given and following 5F,G) by (Fig. both by EGFR min, 15 AP20187 for and EGF both treatment upon that immunofluorescence indirect eeoie fEF–R n EGFR and EGFR–FRB of heterodimer homodimers. EGFR the for that codes indicate endocytic by an results activity kinase These its 5C,D). PDGFR (Fig. of inhibition treatment without AG1478 or with internalized eut niaeta w eso noyi oe r eurdto required are codes endocytic of internalization. sets EGFR mediate two that indicate results noyi oe aal fmdaigteedctsso EGFR of endocytosis PDGFR the mediating and of EGFR capable both immunofluorescence codes As endocytic of and 4D,E). analysis 4B,C) EGFR–PDGFR (Fig. both (Fig. the microscopy by biotinylation shown that surface as with found cell internalized, cells not also the was We of heterodimer 4A). treatment (Fig. the following AP20187 PDGFR heterodimer and a formed EGFR–FRB that co-immunoprecipitation noyi oe fEF n PDGFR and EGFR of codes endocytic PDGFR and aaae l,21) ecntutdaFB–uinEF and EGFR FKBP–fusion PDGFR a constructed the we of PDGFR 2010), al., 952–965 et residues Pahara PDGFR hydrophobic for acid crucial the is that amino terminus Given of receptor. we PDGFR EGFR–FKBP region the dimers, the with receptor in code code of endocytic EGFR endocytosis the the replaced mediating for required mediate dimers. to receptor insufficient of are endocytosis the codes endocytic non-compatible two 07rsdehdohbcrgo ihte9295residue 952–965 the with PDGFR region of hydrophobic fragment residue 1017 o,w eeal oaayeteedctsso the of endocytosis the analyze to able were we Now, ofrhrts hte w optbeedctccdsare codes endocytic compatible two whether test further To PDGFR PDGFR b b b FB n GRFBi 9Tcls esoe by showed We cells. 293T in EGFR–FRB and –FKBP ora fCl cec 21)18 3–5 doi:10.1242/jcs.160374 935–950 128, (2015) Science Cell of Journal noyi oe(eius9295 a lofnto as function also can 952–965) (residues code endocytic wpmtn ihterpaeeto h GR1004– EGFR the of replacement the with mutant swap b FB eeoie a o nenlzd sshown as internalized, not was heterodimer –FKBP by showed we Similarly, 5A,B). (Fig. –FKBP PDGFR ooies epciey u aasgetta the that suggest data our respectively, homodimers, FB,hwvr h GRFBand EGFR–FRB the however, –FKBP, b b (EGFR FB)(i.1)adepesdboth expressed and 1C) (Fig. –FKBP) b PDGFR nenlzto Mr ta. 1991; al., et (Mori internalization PDGFR PDGFR b FB)(i.1) ethen We 1C). (Fig. –FKBP) PDGFR noyoi Phr tal., et (Pahara endocytosis PDGFR b b FB a strongly was –FKBP FB r ohcapable both are –FKBP (PDGFR r o optbeand compatible not are PDGFR FB a beto able was –FKBP FBi 9Tcells 293T in –FKB PDGFR FB.W co- We –FKBP. b snecessary is ) FB was –FKBP b b endocytic b b –FKBP contain would b 939 C- b b

Journal of Cell Science EERHARTICLE RESEARCH 940 3. Fig. e etpg o legend. for page next See ora fCl cec 21)18 3–5 doi:10.1242/jcs.160374 935–950 128, (2015) Science Cell of Journal

Journal of Cell Science obeidrc muoloecne cl a:20 bar: Scale immunofluorescence. indirect double EGFR and (red) hw nFg F hsmtn eetrwsdmrzdupon dimerized was receptor mutant this 6F, Fig. in shown hshrltdfloigA217tetet o ee of level low A treatment. ErbB2 of AP20187 internalization following phosphorylated uatrcpo ErbB2 receptor create to mutant 1005–1017 sequence acid a with amino C-terminus C- code ErbB2 the endocytic entire in EGFR the other signals the replaced inhibitory of we are ErbB2, lack there of ErbB2. that terminus the in possibility sequence either inhibitory the to an exclude can of To due presence 1005–1017 the The be or homodimer. might codes residues restoration endocytic ErbB2 full the of of of fragment lack internalization the EGFR restore the partially endocytic the within and that 6B,C) suggests codes (Fig. which signal immunofluorescence, biotinylation indirect surface by cell the following both inl oto sa ecie ntelgn o i.2.()Qatfcto of Quantification (C) 2B. Fig. for legend the biotinylation in described surface EGFR– as cell is the the Control of following signal. Endocytosis by (B) examined Methods. heterodimer and as ErbB2 (IP) Materials co-immunoprecipitation the by in (dimerization) examined described was association free. ErbB2 The serum and SF, ErbB2. EGFR indicated. and between as EGFR or or of ng/ml) min Heterodimerization (50 30 (A) HRG for ng/ml), nM) (50 EGF (500 with AP21967 treated were ErbB2– ErbB3–FKBP and or EGFR–FRB FKBP with co-transfected transiently cells receptors 293T family receptors. ErbB of mutants. endocytosis and and Heterodimerization 3. Fig. ARTICLE RESEARCH u otelc fedctccds eisre GRresidues an EGFR generate inserted to we ErbB2 (ErbB2 of mutant codes, fusion region ErbB2–FKBP endocytic corresponding of the be into might lack 1005–1017 ErbB2 the of to deficiency due internalization the that manner Considering the dimeric drive a to in required internalization are signals internalization Multiple the endocytosis. not the of is stimulate activation phosphorylation to kinase sufficient the that indicates stimulated which 5E), AP21967 (Fig. the heterodimer interesting that is It note dimer. receptor to the of internalization unable the are mediate codes to endocytic non-compatible of sets two that suggest mean the * is experiments. value different Each L. from data the is value Each J. from data the of Quantification (K) mean 2B. Fig. for legend and EGFR of heterodimer EGFR of Endocytosis (J) immunoprecipitation. EGFR for and and nM) EGFR–FRB FRB (500 of AP21967 Heterodimerization or (I) ng/ml) indicated. EGFR (50 as EGF or with min treated 30 then were EGFR cells and The EGFR–FRB with co-transfected transiently EGFR and EGFR–FRB * mean experiments. the indirect is double value by Each examined 20 was bar: (green) Scale ErbB3 immunofluorescence. or (red) EGFR (green) of ErbB2 endocytosis and The as immunofluorescence. heterodimers the indirect EGFR–ErbB3 by of and determined Quantification mean EGFR–ErbB2 (F) the of 2B. is Endocytosis Fig. value (G) for Each legend E. the from signal. in data biotinylation described surface as cell is the Control EGFR–ErbB3 following the by of examined was Endocytosis heterodimer and ErbB3 (E) EGFR and co-immunoprecipitation. of EGFR by Heterodimerization between examined (D) (dimerization) error. association standard The the ErbB3. is bar mean error the the is and value Each B. from data the n xmndteitraiaino ErbB2 of internalization the examined and hw nFg A hnepesdi 9Tclsb transient by cells 293T in expressed when ErbB2 6A, transfection, Fig. in shown 6 D D ...o tlattreeprmns L noyoi fEGFR–FRB of Endocytosis (L) experiments. three least at of s.e.m. 0511 ycl ufc itnlto.Cnrli sdsrbdi the in described as is Control biotinylation. surface cell by 1005–1017 EGFR– between (dimerization) association The 1005–1017-FKBP. P AH eeoieiainadedctsso rBfamily ErbB of endocytosis and Heterodimerization (A–H) , D D 0511-KPwseaie yco- by examined was 1005–1017-FKBP 0511–KP(re)htrdmr a xmndby examined was heterodimers (green) 1005–1017–FKBP .1we oprdwt F IM noyoi fthe of Endocytosis (I–M) SF. with compared when 0.01 EGFR1005–1017 P D 6 , 0511–KPhtrdmr.23 el were cells 293T heterodimers. 1005–1017–FKBP ...o tlategtclsfo he different three from cells eight least at of s.e.m. .1we oprdwt SF. with compared when 0.01 EGFR1005–1017 D C EGFR1005–1017 m 6 FB a ooieie and homodimerized was –FKBP 6 .()Qatfcto fdt rmG. from data of Quantification (H) m. ...o tlat8clsfo three from cells 8 least at of s.e.m. ...o tlattreexperiments. three least at of s.e.m. 6 EGFR1005–1017 ...o tlattreexperiments three least at of s.e.m. FB a loosre by observed also was –FKBP EGFR1005–1017 FB Fg D.As 1D). (Fig. –FKBP m .()Qatfcto of Quantification (M) m. D FB)(i.1D), (Fig. -FKBP) 1005–1017–FKBP. FB.As –FKBP. ocet yrdrcpo ErbB2 receptor hybrid C-terminus a EGFR the create with C-terminus to of we ErbB2 internalization possibility, entire this the the confirm replaced drive To 1005– dimerization. efficiently after sequence to receptors acid required ErbB amino also both the are that beyond 1017 suggest signals by data endocytic by These following other and 6I,J). shown 6G,H) (Fig. (Fig. endocytosis immunofluorescence signal indirect biotinylation as surface in cell this the impaired following homodimerization, Moreover, completely phosphorylated. AP20187-induced not was but mutant treatment, AP20187 oin eeaie h noyoi fte(EGFR–FRB)– the of endocytosis signals the this endocytic confirm examined identical (ErbB2 To of endocytosis. we by receptor pair for notion, a sufficient both be of not presence and by might indicate the 7B,C) results (Fig. shown even These signal that 7D,E). as biotinylation (Fig. immunofluorescence surface heterodimer, deficient, indirect cell This the endocytosis following treatment. is AP21967 however, upon phosphorylated hshrlto fErbB2 of phosphorylation ErbB2 eiin,dsiebt h GRFBhmdmradthe and indirect homodimer EGFR–FRB and the 7G,H) both the (Fig. endocytosis ErbB2 is despite following heterodimer signal by deficient, this AP21967 7I,J), both biotinylation (Fig. upon by immunofluorescence surface shown phosphorylated as cell were Interestingly, treatment. and heterodimerized GRCtriu s33(rm6497 o GRad360 of and EGFR start for the 644–957) acids and (from amino domain 313 total transmembrane is The of C-terminus ErbB2. EGFR end in the 1035 between residue after terminus t noyoi.Teadto fA217idcdthe induced AP20187 of addition examined and ErbB2 The cells of 293T homodimerization endocytosis. into receptor its hybrid this transfected rmtelc fedctccdsi t C-terminus. its results in ErbB2 codes endocytic C-terminus, of of deficiency lack EGFR endocytosis the results from the These the pattern. that residues dimeric confirmed within a also including in signals we internalization codes, EGFR other mediate Therefore, endocytic and 6N,O) multiple 1005–1017 (Fig. that immunofluorescence concluded indirect by GRCtriu nldsteaioai eunefo 5 to ErbB2 957 the constructed from 1, we sequence Fig. When acid in amino 1186. shown the includes As C-terminus required. of EGFR is sets this two codes the for containing endocytic of coordination compatible explanation spatial both possible that is One and endocytosis impaired codes. 6K–O), endocytic (Fig. identical dimerization the the following the examined the We mediate receptors. (EGFR–FRB)–(ErbB2 to ErbB codes of endocytic the compatible internalization of needed of are sets internalization conditions two additional whether for determined we spatially internalization Next, be dimer to mediate need to codes coordinated endocytic compatible of sets Two hw nFg A hnc-xrse n23 el,EGFR–FRB cells, ErbB2 293T in co-expressed As when and molecule. 7A, each Fig. (EGFR in in code codes shown one endocytic 1005–1017), identical sequence of acid amino pair a (EGFR–FRB)–(ErbB2 contains heterodimer The heterodimers. ihA207fr53 i togysiuae h noyoi of endocytosis the stimulated ErbB2 strongly min treatment 5–30 signal, for biotinylation AP21087 surface with cell the following by shown oepesdi 9Tcls GRFBadErbB2 when and 7F, EGFR–FRB Fig. cells, in 293T shown EGFR in As entire molecule. co-expressed each (the in codes set one endocytic C-terminus), EGFR complete and identical D EGFR-C EGFR-C EGFR-C C ora fCl cec 21)18 3–5 doi:10.1242/jcs.160374 935–950 128, (2015) Science Cell of Journal EGFR1005–1017 EGFR1005–1017 FB Fg LM.Smlrrslswr obtained were results Similar 6L,M). (Fig. –FKBP FB ooie en outyendocytosed robustly being homodimer –FKBP FB)htrdmrta otistost of sets two contains that heterodimer –FKBP) FB Fg F,ti a olwdby followed was this 6F), (Fig. –FKBP FB eeoieie n were and heterodimerized –FKBP EGFR-C EGFR-C FB Fg K.Mroe,as Moreover, 6K). (Fig. –FKBP FB Fg K,btunlike but 6K), (Fig. –FKBP EGFR-C EGFR-C FB Fg D.We 1D). (Fig. –FKBP eptteEF C- EGFR the put we , EGFR1005–1017 EGFR1005–1017 EGFR-C –FKBP) –FKBP) –FKBP 941

Journal of Cell Science nomto,w eiv httelc fedctssfrthe for endocytosis of lack this on the ErbB2 Based and that EGFR EGFR. of in heterodimer believe than we membrane plasma information, the from away htcnan h noyi oe ihnErbB2 within codes endocytic the contains that 9Tclstgte ihwl yeEF–R,the EGFR–FRB, type wild with (EGFR–FRB)–(EGFR together heterodimer in of co-expressed internalization cells when when However, with that, EGFR 8A–D). 293T as (Fig. treatments mutant AP20187 showed EGFR and this wild-type EGF We both of cells, upon sets internalized compromised. 293T was two in the is between expressed codes coordination endocytic identical the of endocytic of spatially, sets wild-type domain but two with transmembrane codes, contains heterodimerizes heterodimer the mutant the this to EGFR, when closer Thus, codes endocytic spatially EGFR. its shifted but EGFR, are EGFR wild-type mutant as domain This intracellular of domain. end transmembrane its the (EGFR internalization C-terminal to at the closer acids shift to codes amino EGFR of 15 domain kinase deleting the by domain intracellular coordination. EERHARTICLE RESEARCH 942 ErbB2 for 675–1035) (from PDGFR and EGFR–FRB the of Endocytosis 4. Fig. tlategtclsfo he ifrn experiments. different three from 20 mean cells bar: the Scale eight PDGFR is immunofluorescence. least and value indirect EGF–FRB at double Each of by B. Heterodimerization examined from (A) heterodimers data free. (green) serum the SF, of indicated. Quantification as (C) or min 30 EGFR PDGFR for and and nM) EGFR–FRB (500 between AP21967 (dimerization) with association treated then were cells The w eso dnia cmail)edctccdsadperhaps and codes the endocytic of (compatible) coordination identical identical spatial of of the sets sets and to two sufficient, endocytosis, two not EGFR but that necessary, mediate are suggest codes endocytic results AP21967 (compatible) by These stimulation 8E–H). upon (Fig. reduced significantly was FKBP) ofrhrts hspsiiiy esotndteEGFR the shortened we possibility, this test further To D 4–5–KP Fg E otisa lotidentical almost an contains 1E) (Fig. 943–957–FKBP) b FB eeoieswseaie yfloigtecl ufc itnlto inl oto sa ecie nteFg Blegend. 2B Fig. the in described as is Control signal. biotinylation surface cell the following by examined was heterodimers -FKBP EGFR-C EGFR-C hs h GRC-terminus EGFR the Thus, . sdet mrprspatial improper to due is b FB heterodimers. –FKBP D EGFR-C 943–957–FKBP D 0511–KPwseaie yc-muorcptto.()Edctsso EGF-FRB of Endocytosis (B) co-immunoprecipitation. by examined was 1005–1017–FKBP 6 D ...o tlattreeprmns D noyoi fEFFB(e)adPDGFR and (red) EGF–FRB of Endocytosis (D) experiments. three least at of s.e.m. 943–957– sfurther is 9Tclswr rninl otasetdwt G–R n PDGFR and EGF–FRB with co-transfected transiently were cells 293T oan(hne l,19;Cnil ta. 91 hoe al., et Ohno C-terminus 1991; al., EGFR et the Canfield in 1990; al., codes et by endocytic (Chen the domain hypothesis on our dependent or homodimers receptor of homodimers of receptor test endocytosis heterodimers. and the various the specifically studying of can specifically permitting kinase we by formation system, heterodimers, receptor Second, the dimerization induce internalization. artificial induce two this and and using offers phosphorylation dimerization, EGF- system the activation, mimic EGFR This can system induced dimerization S1. this First, Table including advantages. kinases, material PDGFR and tyrosine supplementary and to receptor wild-type kit ErbB3 other dimerization ErbB2, heterodimerize and receptor EGFRs or a we mutant using Here, homodimerize by internalization. close specifically into hypothesis EGFR receptor, this drives each in tested then to codes of which was set sets study proximity, one two this brings codes, dimerization of endocytic that EGFR. propose of goal we of results, the endocytosis our on 2005), the Based regulates al., dimerization et how Wang determine (Wang 2007; activation al., kinase EGFR et by than driven rather is dimerization endocytosis receptor EGFR that demonstration our Following DISCUSSION te odtosaerqie omdaeteedctsso the of endocytosis the mediate to dimers. required receptor are conditions other thsbe hw htEF nenlzto slargely is internalization EGFR that shown been has It m .()Qatfcto fdt rmD ahvlei h mean the is value Each D. from data of Quantification (E) m. ora fCl cec 21)18 3–5 doi:10.1242/jcs.160374 935–950 128, (2015) Science Cell of Journal b h eut r umrzdin summarized are results The . b FB.The –FKBP. 6 b ...of s.e.m. b –FKBP. –FKBP

Journal of Cell Science EERHARTICLE RESEARCH i.5 noyoi fteEGFR the of Endocytosis 5. Fig. G uniiaino h aafo .Ec au stemean the is value Each F. from data the of Quantification (G) EGFR EGFR of Endocytosis (A) B uniiaino h aafo .Ec au stemean the is value Each A. from data the of Quantification (B) EGFR G 5 gm)o P08 10n)wt rwtotA17 o 0mno o h niae ie E h soito dmrzto)btenEF–R and EGFR–FRB between (dimerization) association The (E) time. indicated the for or min 30 for AG1478 without or with nM) (100 EGFR AP20187 or ng/ml) (50 EGF xeiet.* 20 experiments. bar: Scale immunofluorescence. indirect gen eeoieswseaie ydul nietimnfursec.Saebr 20 bar: Scale immunofluorescence. indirect double by examined was heterodimers (green) fa es ih el rmtredfeetexperiments. different three from cells eight least at of PDGFR PDGFR PDGFR FB eeoieswseaie yfloigtecl ufc itnlto inl oto sa ecie ntelgn o i.2B. Fig. for legend the in described as and is EGF–FRB Control of signal. Endocytosis biotinylation (F) surface (IP). cell co-immunoprecipitation the by following examined by were examined receptors was the heterodimers of –FKBP phosphorylation the and free. –FKBP serum SF, indicated. as or min 30 for AG1478 without or with nM) (100 AP20187 or ng/ml) (50 EGF with treated then were cells The –FKBP. P , .1we oprdwt F EI 9Tclswr otasetdwt GRFBadEGFR and EGFR–FRB with co-transfected were cells 293T (E–I) SF. with compared when 0.01 PDGFR FB a xmndb olwn h elsraeboiyainsga.Cnrli sdsrbdi h eedfrFg 2B. Fig. for legend the in described as is Control signal. biotinylation surface cell the following by examined was –FKBP PDGFR FB ooie n t eeoie ihEGFR–FRB. with heterodimer its and homodimer –FKBP m .()Qatfcto fdt rmC ahvlei h mean the is value Each C. from data of Quantification (D) m. 6 6 ...o tlattreeprmns H noyoi fEFFB(e)adEGFR and (red) EGF–FRB of Endocytosis (H) experiments. three least at of s.e.m. ...o tlattreeprmns C noyoi fEGFR of Endocytosis (C) experiments. three least at of s.e.m. m .()Qatfcto fdt rmH ahvlei h mean the is value Each H. from data of Quantification (I) m. ora fCl cec 21)18 3–5 doi:10.1242/jcs.160374 935–950 128, (2015) Science Cell of Journal AD 9Tclswr rninl rnfce with transfected transiently were cells 293T (A–D) 6 ...o tlategtclsfo he different three from cells eight least at of s.e.m. PDGFR FB.Teclswr hntetdwith treated then were cells The –FKBP. PDGFR FB a xmndby examined was -FKBP PDGFR 6 –FKBP s.e.m. 943

Journal of Cell Science a lobe eotdta h mardedctsso rB or ErbB2 of endocytosis impaired the It that 2007). reported al., been et (Wang also dimers has EGFR of endocytosis the mediating EERHARTICLE RESEARCH 944 the especially 1005–1017, L sequence EGFR motif the acid dileucine that shown amino have we C-terminal Recently, 1996). Mellman, 1995; 6. Fig. e etpg o legend. for page next See 1010 L 1011 sa motn noyi oein code endocytic important an is , u bv yohsssae,w xmndwehrtost of the of sets endocytosis two the mediating whether for examined required test are we To codes 1999). stated, endocytic al., et hypothesis Wang above 1999; al., our Baulida et 1993; Muthuswamy in 1996; Carpenter, codes al., and endocytic et (Sorkin of lack domains the intracellular to their due likely is homodimers ErbB3 ora fCl cec 21)18 3–5 doi:10.1242/jcs.160374 935–950 128, (2015) Science Cell of Journal

Journal of Cell Science Fg AD.Hwvr h eeoie fti uatwt wild- with mutant [(EGFR–FRB)–(EGFR this EGFR treatment of type heterodimer AP20187 the and However, 5A–D). EGF (Fig. both upon efficiently endocytosed i.6 noyoi ftehmdmr fvrosEb2mtnsfused mutants ErbB2 ErbB2 various of FKBP. homodimers the with of Endocytosis 6. Fig. ARTICLE RESEARCH mn cdcd 0411 ihtecrepnigPDGFR corresponding (EGFR the 952–965 code with acid and 1004–1017 amino EGFR endocytic code an the 2005; acid constructed al., of also amino et we endocytosis Wang Second, the 2007). 2010; PDGFR al., mediating al., et et endocytic of Wang the (Pahara containing capable homodimers First, molecule are receptor receptor evidence. that each following PDGFR codes despite and the 4), EGFR (Fig. by of This supported compatible. heterodimer be must is codes 3) endocytic finding (Fig. of sets to two endocytosis required the dimers, are in codes dimers. receptor endocytic of impaired of endocytosis the sets is mediate fact two The heterodimer that endocytosis. its demonstrates for this essential is that that possesses EGFR code endocytic EGFR–FRB and one codes heterodimer, endocytic this complete Within endocytic of examined heterodimer. pair next (EGFR–FRB)–(EGFR We the a endocytosis. of that endocytosis receptor endocytic the deficient the suggest for contain data required our only is endocytosis codes receptor, heterodimers one these are in that codes Given heterodimers 2). (Fig. EGFR–ErbB3 heterodimers EGFR–ErbB2 that and showed first We dimers. receptor C uniiaino h aafo .Ec au stemean the ErbB2 is of value Endocytosis 2B. Each (D) Fig. B. experiments. for from three legend data least the the in of described Quantification as (C) is Control signal. biotinylation and Dimerization (A) free. serum SF, indicated. as ErbB2 or of min phosphorylation 30 for nM) (100 niae.()Dmrzto n h hshrlto fErbB2 of phosphorylation the and Dimerization (K) indicated. httehmdmro hssa uatEGFR showed mutant We swap 2010). this al., et of Pahara homodimer 2007; al., the et that Wang 2005; al., et eiin Fg EI ept h atta h nydifference only the that fact the despite 5E–I) (Fig. deficient olwdb muoltig G noyoi fErbB2 of Endocytosis of (G) phosphorylation immunoblotting. and by Dimerization followed (F) indicated. as or ErbB2 min 30 for nM) (100 C xeiet.(–)23 el eetasetytasetdwt ErbB2 with transfected transiently were cells 293T (K–O) mean experiments. the is value Each 20 bar: Scale immunofluorescence. xmndb nietimnfursec.Saebr 20 bar: Scale immunofluorescence. indirect by examined M uniiaino h aafo .Ec au stemean the ErbB2 is of value Endocytosis Each (N) 2B. L. experiments. Fig. from three for data legend least the the in of described Quantification as (M) is Control signal. biotinylation surface rslnigeprmnsfloe yimnbotn.()Edctssof Endocytosis (B) immunoblotting. ErbB2 by followed experiments crosslinking eeeaie ycosikn xeiet olwdb immunoblotting. ErbB2 by of followed Endocytosis experiments (L) crosslinking by examined were F FJ 9Tclswr rninl rnfce with transfected transiently ErbB2 were cells * 293T experiments. (F–J) different SF. three from cells eight 20 bar: Scale immunofluorescence. indirect by examined ih el rmtredfeeteprmns * experiments. mean SF. the different with is three value from Each cells N. eight from data of Quantification (O) sa ecie ntelgn o i.2.()Qatfcto ftedt from data the ErbB2 of Quantification of mean (H) Endocytosis the 2B. (I) is Fig. value for Each legend the G. 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KPis FKBP –FKBP EGFR- – b hwdhr htteEGFR–ErbB2 following the internalized we that Interestingly, is here 1999). al., showed domain et (Wang intracellular domain dimerization extracellular EGF-induced EGFR ErbB2 the the of and composed other receptor chimeric and endocytosis, 6). are EGFR C-terminus (Fig. in required mediate 1005–1017 also they region of to that upstream here codes sufficient found endocytic we not 2007), al., are et (Wang endocytosis EGFR n h L the and PDGFR with compared 952–965). 1005–1017 acids acids amino endocytic amino the is (EGFR heterodimer the code within receptors two the between oana h idtp GR u t -emns(which C-terminus its but EGFR, wild-type intracellular the (EGFR identical as EGFR the almost mutant domain contains a that constructed test the 957–FKBP–HA) further we of To required. possibility, coordination is codes spatial this endocytic for the compatible explanation of that the sets possible is two of one endocytosis domain Thus, impaired transmembrane EGFR. this the within from than away receptor further be might acrteais(prdne l,20;Fida ta. 05.It other 2005). with al., et combination Friedman of in 2002; al., used et formation been (Spiridon dimerization therapies has cancer a ErbB2, the Pertuzumab, reducing for in Recently, inhibitor helpful promote signaling. be would receptor which ErbB therefore of kinase-inactive both homodimers, and ErbB3 its and homodimers monoclonal inhibit EGFR EGFR a internalization-efficient can ErbB3, including that receptors, arm possible ErbB and dimerization other be ErbB2 with might heterodimerization the It against therapy. antibody useful be cancer would that insights for key that yield mechanisms might the endocytosis exploring regulate that suggests EGFR model internalization- Our receptors. internalized. the only be to with EGF, fail interact and codes protein(s) to cannot EGFR– regulating endocytic or heterodimers response EGFR–ErbB2 arranged ErbB3 but endocytosis, in properly undergo homodimers and a Thus, interact endocytic only be identical thus, simultaneously. downstream either and, two sites might binding The identical with endocytosis, two monomer. have EGFR or endocytic dimer each for identical EGFR, of required in of sets protein(s) two dimerization one of the exposure induces codes, the the EGFR in describe results to the which to EGF model, model this of In ‘dimerization’ endocytosis. binding EGFR a EGF-induced propose of regulation we S1, Table crucial a also endocytosis. is in receptors the between EGFR factor dimerized coordination of spatial in The codes the endocytic that 2010). implicated the indicate Schlessinger, study been and this of (Bae has results dimers receptor coordination molecules of spatial receptor activation and The codes spatial two required. endocytic The are between compatible conditions endocytosis. of other sets EGFR perhaps two mediate necessary, the the are to results of codes coordination sufficient, These endocytic that identical not 8). of (Fig. but showed sets two reduced that We significantly suggest (EGFR–FRB)–(EGFR the was EGFR. heterodimer to 957–FKBP) the closer of of spatially internalization shifted domain is codes) transmembrane endocytic the contains noyoi eiin Fg FJ.A hw nFg ,teEGFR ErbB2 the within 1, codes still Fig. endocytic was the in codes, contains shown that endocytic As C-terminus intact 7F–J). and (Fig. deficient identical endocytosis of pair a contains ehv hw rvosyta h eeoie fEF n a and EGFR of heterodimer the that previously shown have We lhuhw aesonpeiul htrsde 1005–1017 residues that previously shown have we Although noyoi sa motn a odwrglt elsurface cell downregulate to way important an is Endocytosis material supplementary in summarized as data these on Based ora fCl cec 21)18 3–5 doi:10.1242/jcs.160374 935–950 128, (2015) Science Cell of Journal 1010 L 1011 oi ihnti euneaeesnilfor essential are sequence this within motif EGFR-C eeoie,which heterodimer, D D EGFR-C 943– 943– 945 b

Journal of Cell Science au stemean the is value EERHARTICLE RESEARCH 946 FKBP. with fused mutants ErbB2 various and ErbB2 EGFR–FRB and between EGF–FRB heterodimers with the transfected of Endocytosis 7. Fig. fEFFBadErbB2 and EGF–FRB of A eeoieiaino G–R n ErbB2 and EGF–FRB of Heterodimerization (A) i.2.()Qatfcto ftedt rmG ahvlei h mean the is value Each G. from data the of ErbB2 Quantification (H) 2B. Fig. GRFBadErbB2 and EGFR–FRB ih el rmtredfeeteprmns FJ 9Tclswr rninl otasetdwt G–R n ErbB2 and EGF–FRB with co-transfected transiently were cells 293T (F–J) 20 experiments. different bar: three Scale from immunofluorescence. cells indirect eight by examined were heterodimers ihA297(0 M o 0mno sidctd F eeoieiaino G–R n ErbB2 and EGF–FRB of Heterodimerization (F) indicated. as or min 30 for nM) (500 AP21967 with h hshrlto fEF–R eeeaie yc-muorcptto I)adimnbotn.()Edctsso G–R n ErbB2 and EGF–FRB of Endocytosis (B) immunoblotting. and (IP) co-immunoprecipitation by examined were EGFR–FRB of phosphorylation the KPhtrdmr a xmndb olwn h elsraeboiyainsga.Cnrli sdsrbdi h eedfrFg B C Quantificat (C) 2B. Fig. for legend the in described as is mean Control the signal. is biotinylation value surface Each cell the B. following from by data examined was heterodimers FKBP EGFR-C FB gen eeoieswr xmndb obeidrc muoloecne cl a:20 bar: Scale immunofluorescence. indirect double by examined were heterodimers (green) –FKBP 6 ...o tlategtclsfo he ifrn experiments. different three from cells eight least at of s.e.m. EGFR-C EGFR-C FB n h hshrlto fEF–R eeeaie yc-muorcptto n muoltig G Endocytosis (G) immunoblotting. and co-immunoprecipitation by examined were EGFR–FRB of phosphorylation the and –FKBP FB eeoieswseaie yfloigtecl ufc itnlto inl oto sa ecie ntelgn for legend the in described as is Control signal. biotinylation surface cell the following by examined was heterodimers –FKBP 6 EGFR1005–1017 ...o tlattreeprmns D noyoi fEFFB(e)adErbB2 and (red) EGF–FRB of Endocytosis (D) experiments. three least at of s.e.m. EGFR1005–1017 FB.Teclswr hntetdwt P16 50n)fr3 i ra niae.S,srmfree. serum SF, indicated. as or min 30 for nM) (500 AP21967 with treated then were cells The –FKBP. FB.Teascain(ieiain ewe GRFBadErbB2 and EGFR–FRB between (dimerization) association The -FKBP. 6 ...o tlattreeprmns I noyoi fEFFB(e)and (red) EGF–FRB of Endocytosis (I) experiments. three least at of s.e.m. m .()Qatfcto fdt rmD ahvlei h mean the is value Each D. from data of Quantification (E) m. ora fCl cec 21)18 3–5 doi:10.1242/jcs.160374 935–950 128, (2015) Science Cell of Journal EGFR-C FB.Teascain(ieiain between (dimerization) association The –FKBP. m .()Qatfcto fdt rmI Each I. from data of Quantification (J) m. AE 9Tclswr rninl co- transiently were cells 293T (A–E) EGFR-C FB.Teclswr hntreated then were cells The –FKBP. EGFR1005–1017 EGFR1005–1017 FB (green) –FKBP 6 ...o tleast at of s.e.m. EGFR1005–1017 FB and –FKBP o fthe of ion –

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Journal of Cell Science eetr r loapial oteercpo yoiekinases. tyrosine receptor these to ErbB 2002; applicable for findings also Yarden, our are that and possible receptors dimerized (Oved is it are binding Thus, 2002). ligand receptor, Schlessinger, after factor internalized growth and ErbB2 hepatocyte and the receptor of tumour mammary vaccines inhibiting peptide in effective that growth are reported loop been dimerization also has ARTICLE RESEARCH 948 fusion FKBP pC4-R make the to or Pharmaceuticals) proteins, (Ariad vector and pC4-Fv1E the ErbB2 subcloned into then EGFR, respectively, pcDNA3.1/Myc-His(-)A/ErbB3, full-length and pcDNA3.1/Myc-His(-)A/ ErbB2, pEYFP-N1/EGFR, ErbB3, vector from amplified and our pRSET-B-mCherry were ErbB3 ErbB2 the in was EGFR, into the FRB-fused constructed inserted using gene Tsien) Roger and from EGFR (previously (gift PCR wild-type by pEYFP-N1/EGFR construct To laboratory) the 1. from Fig. EGFR, in amplified shown are wild-type research this mCherry-tagged in used constructs the All treated construction fusion Plasmid then AP21967 37 and FRB at heterodimerizer h min and or 24 30 for nM) FKBP Following for nM) (100 (500 serum-starved or AP20187 techniques. were homodimerizer receptors with cells cloning fusion 293T molecular FKBP receptors, some we and of using Briefly, ErbB3, the transfection ErbB2, to manufacturer. receptors FRB EGFR, or like the FKBP mutant receptors fusing of different by of chimeras instructions receptor C-terminus of the series 2006). a al., to constructed et Zhan according 2006; out Clackson, Neff 2002; 1999; al., al., et et Wilde Welm Stockwell 1999; 2001; 1998; al., Blau, al., et and et Muthuswamy (Keenan 1998; respectively Schreiber, proteins FRB and the two specifically and heterodimerize FKBP chemicals or with the the FKBP fused heterodimers, system, with this fused study Inc. proteins In homodimerize or Pharmaceuticals, used. specifically were ARIAD MA) from homodimers (Cambridge, to kit’ Heterodimerization us receptor Regulated hetero- and allowed homo- of ‘ARGENT receptor that regulated internalization system and controllable dimerization a develop hetero-dimerization To and homo- receptor Regulated were CRL-11268) ATCC 37 cells, kidney at embryonic grown (human cells transfection 293T and treatment culture, Cell pC4-R and pC4-Fv1E pcDNA3.1/Myc-His(-)A include ON), research this Burlington, in used (Invitrogen, plasmids The chemicals and Antibodies METHODS AND MATERIALS otiig1%FS eiiln n tetmcn(0 /l n were and CO U/ml) 5% (100 a streptomycin in and maintained penicillin, FBS, 10% containing tews pcfe,alo h hmcl n egnswr bandfrom obtained MO). were Louis, reagents and (St. Unless Cruz chemicals Sigma MA). the Santa (Boston, of all Products from specified, Research otherwise obtained Oncogene (Ab-1) from were EGFR obtained against (Y11) were antibodies ErbB2 monoclonal HA Mouse EGFR(1005), and CA. of against against Biotechnology, (C17), antibodies University antibodies ErbB3 PDGFR polyclonal (C18), monoclonal Tsien, rabbit (9E10), and Mouse Roger Myc (pY99), CA). from (9G6), Diego, ErbB2 gift San (a CA), California View, pRSET-B-mCherry (Mountain pEYFP-N1 and MA), Cambridge, Pharmaceuticals, rHG(0n/l o 0mno sindicated. as or ng/ml) min (50 30 EGF for AP21967, ng/ml) nM (50 500 cells AP20187, HRG induce nM or transfected 100 specifically the with treated To heterodimerization, were or phosphate. homodimerization calcium receptor with transfection transient euae eetrhm-adhtr-ieiainasy eecarried were assays hetero-dimerization and homo- receptor Regulated l ftercposadmtnswr xrse n23 el by cells 293T in expressed were mutants and receptors the of All ayrcpos nldn DG,nregot factor growth nerve PDFGR, including receptors, Many nvivo in TM ˚ nDlec’ oiidEgesmdu (DMEM) medium Eagle’s modified Dulbecco’s in C euae ooieiainkt n ‘ARGENT and kit’ Homodimerization Regulated Alne l,2007). al., et (Allen H 2 etr(ra hraetcl)t aeFRB make to Pharmaceuticals) (Ariad vector E atmosphere. ˚ C. 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Journal of Cell Science nlzdadrslsaemean are results and analyzed sn Student’s using ooieiainktadARGENT and kit Homodimerization eetr ntescin h ifrnei h loecneintensity fluorescence the in ( difference areas polygon small The and polygon section. large the between the in receptors Na ARTICLE RESEARCH le,S . art,J . aae .V,Jns .L,Pilp,G,Fri G., Forni, G., Phillips, L., A. Jones, V., S. Rawale, T., J. Garrett, D., S. Allen, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.160374/-/DC1 online available material Supplementary material Supplementary Health of Z.W.]. Institutes to Canadian the RES0006924 from number grants [grant by part, Research in supported, was work This Funding paper. the the of version of final draft the first wrote Z.W. the experiments. wrote some performed and X.C. experiments paper. most performed and designed Q.W. contributions Author interests. financial or competing no declare authors The interests Competing ARGENT the providing for Inc. 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V V L S nalthe all in nalthe all in z 20) etd acnsof vaccines Peptide (2007). ai eeaqie and acquired were -axis V Z Z L - - 2 sections sections 4 li B/Mon PBS/CM in Cl V S z ersnsthe represents ) scino the of -section TM | Regulated 100 % ald,J,Kas .H,Aiad,M,D ir,P .adCretr G. Carpenter, and P. P. Fiore, Di M., Alimandi, H., M. Kraus, J., Baulida, A. R. Weinberg, and C. M. Hung, I., C. Bargmann, ru,M . sig . ii . oec,N .adArno,S A. S. Aaronson, and C. N. Popescu, T., Miki, W., Issing, H., M. Kraus, and Y. Yarden, R., Pinkas-Kramarski, E., Hurwitz, N., Vaisman, N., L. Klapper, arwy .L,IIadCnly .C. L. Cantley, and III L., K. Carraway, W. A. Burgess, J. Schlessinger, and H. J. Bae, ye,N .adMcoad G. MacDonald, and E. N. Hynes, Y. Yarden, and E. Peles, B., R. Levy, R., Goldman, ela,I. Mellman, Y. Yarden, and M. Sela, A., Freywald, E., Tzahar, N., L. Klapper, G., Levkowitz, J., M. Vugt, van L., M. Poll, de van R., Pinkas-Kramarski, E., A. Lenferink, Rivera, E., M. Pavone, T., C. Rollins, L., N. Courage, R., D. Yaeger, T., Keenan, art,T . cen .M,Lu . lea,T . dm,T . Lovrecz, E., T. Adams, C., T. Elleman, M., Lou, M., N. McKern, P., S., T. S. Garrett, Bacus, S., Lavi, L., Lyass, B., Schechter, A., Rinon, M., L. I. M. Friedman, Greene, and A. R. Weinberg, F., D. Stern, C., V. Link, A., J. Drebin, T. Clackson, Y. Yarden, and A. Citri, S. M. Brown, and L. J. Goldstein, J., W. Chen, S. Kornfeld, and W. Gregory, D., R. Ye, F., K. Johnson, M., W. Canfield, iba . e-ey . adn .adHns .I. Y. Henis, and Y. Yarden, R., Ben-Levy, L., Gilboa, uhsay .K,Gla,M n rge .S. J. Brugge, and M. Gilman, K., S. Muthuswamy, H. C. Heldin, and L. Claesson-Welsh, S., Mori, aaa . h,H,Ce,X n ag Z. Wang, and X. Chen, H., Shi, J., Pahara, Y. Yarden, and S. Oved, A. C. Blau, and T. Neff, ho . twr,J,Fune,M . osat . he . iaae S., Miyatake, I., Rhee, H., Bosshart, C., M. Fournier, J., Stewart, H., Ohno, ciain ftenuocgn yapitmtto leigtetransmembrane the altering mutation point p185. a of by domain oncogene neu the of activations E2bok rstl ihgot atrreceptors. factor growth with crosstalk blocks HER2 M. Sela, l rBrcposohrta h pdra rwhfco eetrare receptor factor growth epidermal the than other impaired. receptors endocytosis ErbB All tyrosine receptor of autophosphorylation or activation tumor kinases. mammary in inhibiting arrangements in effective are vivo. loop in growth dimerization HER-2/neu the h rB1adeb- eetr nhmnbes acnm el:amechanism a cells: carcinoma transregulation. breast human receptor in for receptors erbB-2 and erbB-1 the 19) opigo h -b rtocgn rdc oErbB-1/EGF-receptor proteins. to ErbB product other protooncogene to c-Cbl not the but of Coupling heterodimers. (1996). receptor and J. to E. superiority Zoelen, signaling J. van confers EMBO M., kinases Sela, tyrosine H., ErbB Waterman, E., Y. Tzahar, Yarden, N., L. Klapper, human of subset a in overexpression tumors. ERBB/epidermal for the mammary evidence of family: member receptor third factor a growth ERBB3, of characterization and Isolation of dimerization regulated the for ligands al. FKBP12 et T. bivalent Clackson, F., of J. proteins. Amara, activity T., Guo, and W., Yang, M., V. cancer. in yoieknssb obntoso As mlctosfrcancer for implications mAbs: of combinations the Y. by immunotherapy. of Yarden, kinases reversion and tyrosine M. and Sela, product protein antibodies. monoclonal oncogene by phenotype an transformed of Down-modulation Biol. Cell Mol. Rev. of Nat. internalization pit-mediated receptor. coated lipoprotein for density required low is the tails, cytoplasmic signaling. in growth found cation- in heterodimerization bovine receptor for the to role a of receptor erbB4: tail. internalization cytoplasmic factor-II the rapid of growth 24-29 for acids 6-phosphate/insulin-like amino signal mannose the independent of Localization targets. therapeutic yolsi yoieadtrsn iaeatvt nteitrcin fNeu of interactions the in activity pits. kinase coated tyrosine active with receptors and an tyrosine reveals cytoplasmic ectodomain receptors. al. ErbB2 ErbB et W. other truncated A. with a Burgess, interact 505. C., to of E. poised Nice, structure conformation, N., R. crystal Jorissen, The M., Kofler, O., G. yrpoi eini h abxltriu ftepaee-eie rwhfactor endocytosis. growth ligand-mediated 266 for platelet-derived important the is of which terminus beta-receptor carboxyl the in region hydrophobic 12 hmclyidcddimerization. induced chemically eetredctsstruhaCtria yrpoi oi hrdb EGF by shared motif hydrophobic C-terminal a receptor. through endocytosis receptor cells. by signaling differential for evidence heterodimers. provides and receptors homo- ErbB of dimerization at,T,Glusr . ichue,T n oiaio .S. J. proteins. Bonifacino, clathrin-associated and with T. signals sorting Science Kirchhausen, tyrosine-based A., of Interaction Gallusser, T., Saito, 97 575-625. , 2535-2540. , 21158-21164. , Nature 19) ucaso uo-niioymncoa nioist ErbB-2/ to antibodies monoclonal tumor-inhibitory of subclass A (1997). 269 o.Cells Mol. iog e.Chem. Med. Bioorg. x.Cl Res. 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