Homotypic FADD interactions through a conserved RXDLL motif are required for death receptor-induced apoptosis

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1 (26), 1 1 & 26 Nture Pulishing Group All rights reserved /6 $3. Homotypi FADD intertions through onserved RXDLL motif re required for deth reeptor-indued poptosis JR Muppidi 1,2,3, AA Loito 1, M Rmswmy 1, JK Yng 4, L Wng 4,HWu 4 nd RM Siegel*,1 1 Immunoregultion Unit, Autoimmunity Brnh, NIAMS, NIH, Bethesd, MD 2892, USA 2 Deprtment of Phrmology, Toxiology nd Therpeutis, University of Knss Medil Center, Knss City, KS 6616, USA 3 HHMI-NIH, Reserh Sholors Progrm, Bethesd, MD 2814, USA 4 Deprtment of Biohemistry, Weill Medil College of Cornell University, New York, NY 121, USA * Corresponding uthor: RM Siegel, NIAMS, NIH, 9 Rokville pk, Bldg 1, Room 9N238, Bethesd, MD 2892, USA. Tel: þ ; Fx: þ ; E-mil: rsiegel@nih.gov Reeived 5.8.5; revised ; epted Edited y D Vux Astrt Deth reeptors in the TNF reeptor superfmily signl for poptosis vi the ordered reruitment of FADD nd spse-8 to deth-induing signling omplex (DISC). However, the nture of the protein protein intertions in the signling omplex is not well defined. Here we show tht FADD self-ssoites through onserved RXDLL motif in the deth effetor domin (DED). Despite exhiiting similr inding to oth Fs nd spse-8 nd preserved overll seondry struture, FADD RDXLL motif mutnts nnot reonstitute FsL- or TRAILindued poptosis nd fil to reruit spse-8 into the DISC of reonstituted FADD-defiient ells. Aolishing self-ssoition n trnsform FADD into dominnt-negtive mutnt tht interferes with Fs-indued poptosis nd formtion of mirosopilly visile reeptor oligomers. These findings suggest tht lterl intertions mong dpter moleules re required for deth reeptor poptosis signling nd implite self-ssoition into oligomeri ssemlies s key funtion of deth reeptor dpter proteins in inititing poptosis. dvne online pulition, 2 Jnury 26; doi:1.138/sj.dd Keywords: Fs; poptosis; FADD; oligomeriztion; TNF reeptor Arevitions: DISC, deth-induing signling omplex; DD, deth domin; DED, deth effetor domin; SPOTS, signling protein oligomeri trnsdution strutures; DEF, deth effetor filments Introdution The reeptors for FsL nd TRAIL diretly tivte poptosis pthwys ritil for lymphoyte homeostsis, self-tolerne nd tumor ell deth. Fs nd the TRAIL reeptors DR4 nd DR5 re TNFR superfmily memers ontining onserved deth domin (DD). Reeptor ligtion triggers reruitment of the DD in the dpter protein FADD to the reeptor through known intertion surfes in the two DDs In ddition to DD, FADD lso ontins n N-terminl deth effetor domin (DED) tht is struturlly similr to the DD. 4 The DED of FADD is known to reruit the prospse-8 (FLICE/MACH-1) to the deth-induing signling omplex (DISC) vi intertions with the tndem DEDs t the N-terminus of spse-8. 5 The omplex of the reeptor, FADD, nd spse-8 is referred to s the deth-induing signl omplex (DISC). 6,7 The DISC is thought to initite the spse sde nd poptosis y induing proximity etween spse-8 moleules. 8 1 More reent experiments hve shown tht in vitro, spse dimeriztion is neessry for spse tivtion nd preedes spse utoproessing. 11,12 However, the stoihiometry of FADD nd spse-8 in the DISC, nd how reeptor ligtion uses spse-8 tivtion is not ler. Reent dt hve highlighted the importne of reeptor oligomeriztion in triggering effiient Fs-indued poptosis. TNF-fmily reeptors n e pressoited prior to lignd inding in sumirosopi ssemlies, 13,14 nd we hve reently identified mirosopilly visile mirolusters of Fs termed signling protein oligomeri trnsdution strutures (SPOTS) tht n e seen within minutes of lignd inding prior to spse tivtion. 15 Reeptor pping nd internliztion follows SPOTS formtion, nd re lso importnt in Fs-indued poptosis. 16,17 FADD is required for formtion of SPOTS, suggesting tht lterl intertions etween FADD in reeptor-signling omplexes my e importnt in trnsduing n effiient deth signl. The FADD protein hs the potentil to e highly oligomeri. FADD ggregtes in vitro, 4 nd trnsfeted FADD in mmmlin ells forms lrge filmentous strutures termed deth effetor filments (DEF). 18 Site-direted mutgenesis experiments hve shown tht residues in region designted hydrophoi pth 1, entered round residue F25, re importnt for intertions etween FADD nd spse-8 (Figure 1). 4 In ddition, si surfe pth in the third -helix of the DED of FADD ws shown to e importnt in inding oth ellulr FLICE-like inhiitory protein (-FLIP) nd spse A region responsile for FADD self-ssoition hs not een determined. In mutgenesis studies of the DED of the poptosis inhiitor virl FLICE-like inhiitory protein (v-flip) MC159, hydrophoi pth 1 ws lso found to e importnt for intertions with FADD nd spse-8, ut nother domin ws lso found in the C-terminl portion of the DED tht ffeted its poptosis inhiitory funtion without loking intertions with FADD or spse-8. 2 This RXDLL motif is highly onserved mong proteins ontining DED, nd in proteins ontining tndem DED s this motif is more

2 2 FADD self-ssoition in poptosis signling d H73 EP L76 FADD- FADD- FADD DD- FADD DD- FADD DD- FADD DED- FADD DED- L75 H73 M1 F25(Y) R72.3% %.2% HP1 HP2 RXDLL motif D74 EP L76 F25(Y) FADD- FADD- 79.6% FADD- FADD DED- M1 L75 H73 R % 83.4% H73 D74 Figure 1 FADD self-ssoition is medited y its deth effetor domin (DED). () NMR struture of the DED of FADD. RXDLL motif (red), HP1 (green) nd HP2 (yellow) re shown. () sequene lignment of deth effetor domins showing onserved residues predited to e involved in hydrophoi pth 1 (green), hydrophoi pth 2 (yellow), nd the RXDLL motif (red). The sterisks (*) ove the FADD sequene indite FADD mutnts tht disrupted FADD selfssoition; 8: mutnts disrupting FADD ssoition with spse-8; : mutnts with no disernle phenotype. () Eletrostti potentil (EP) surfe of RXDLL motif of FADD. Red indites positive hrge nd lue, negtive hrge. (d) 293T ells were trnsfeted with onstruts enoding full-length wild-type FADD, FADD 1 79 (FADD DED) or FADD 8 29 (FADD DD) fused to either the yn fluoresent protein () or the yellow fluoresent protein () or lone. Cells were nlyzed y FACS for, nd fluoresene resonne energy trnsfer () etween nd. Cells were gted s indited on the sis of nd expression nd the mount of in gted ells is shown in the histogrm to the right. The perentge of -positive ells is shown in eh histogrm. Dt re representtive of t lest three independent experiments onserved in the first DED (Figure 1). The NMR struture of the DED of FADD 4 shows tht the R72, H73, nd D74 in this motif re exposed on the surfe, with L75 nd L76 diretly underneth (Figure 1). Here we show tht FADD self-ssoition is ritil for its dpter protein funtion in deth reeptor poptosis signling. We find tht FADD self-ssoition ours vi the DED, tht the FADD FADD interfe is not equivlent to the FADDspse-8 interfe nd tht FADD self-ssoition medited y the RXDLL motif is required for deth reeptor-medited poptoti signling nd spse-8 reruitment to the DISC. Results FADD self-ssoites through the DED We first investigted whether self-ssotion of FADD ours nd whih domin of FADD my e responsile for selfssoition. Fluoresene resonne energy trnsfer () llows protein protein intertions to e mesured in living ells. The use of flow ytometry to mesure etween yn- nd yellow fluoresent proteins ( nd ) hs llowed rpid quntittion of protein protein intertions in living ells in numer of settings. 13,14,21 23 When onstruts ontining full-length FADD fused to () nd (), were otrnsfeted, there ws high level of mong ells oexpressing oth fusion proteins when ompred to ells trnsfeted with unfused nd FADD-. ws ompletely rogted when the DED ws removed from one or oth of the onstruts (Figure 1d). Additionlly, full-length FADD nd FADD DED, ut not the FADD DD, o-immunopreipitted with full-length FADD (dt not shown). These dt show tht FADD self-ssoites in living ells nd tht self-ssoition is medited y the DED. A onserved short peptide motif in the DED medites FADD self-ssoition The NMR struture of the DED of FADD ontins two surfe hydrophoi pthes. 4 Hydrophoi pth 1 (HP1, Figure 1, green) medites inding to spse-8 nd onsists of the residues F25, L28, L26, L66, L7. A less onserved hydrophoi pth (HP2, Figure 1, yellow), fing opposite HP1, onsists of the residues M1, P3, L5, V6, L43, L5, P57 nd F82. Single nd multiple lnine muttions in HP2 mino ids did not disrupt FADD self-ssoition or funtion in Fs signling (dt not shown). These dt suggest tht FADD self-ssoition is not medited y HP2 nd tht HP2 does not prtiipte diretly in Fs poptosis signling. In ddition, the DED of FADD ontins the sequene RHDLL from positions 72 to 76 in the 6th lph helix of the DED tht is homologous to n RXDLL motif onserved etween mny DED-ontining proteins (Figure 1, red). In studies of this motif in the DED ontining ntipoptoti virl FLIP, MC159, it ws found tht the doule mutnts of the RHDLL to AHALL or RHDAA ut not point muttions in the motif were suffiient to redue ntipoptoti tivity. 2 Therefore, using site-direted mutgenesis, we ltered the RXDLL motif y mutting the leuines t positions 75 nd 76 to lnines (RHDAA) or, lterntively, the rginine t position

3 FADD self-ssoition in poptosis signling 3 72 nd the sprti id t position 74 to lnines (AHALL). 293T or FADD-defiient Jurkt ells (I2.1) were otrnsfeted with full-length wild-type (WT), RHDAA or AHALL mutnt FADD onstruts fused to or nd etween the two moleules ws nlyzed y FACS. The perentge of -positive ells ws sustntilly diminished in ells trnsfeted with RXDLL-mutnt FADD in oth 293T nd I2.1 FADD-defiient ells (Figure 2 nd ). There ws no resue of the diminished when AHALL- nd RHDAA- or AHALL- nd RHDAA- were otrnsfeted (dt not shown), inditing tht the two muttions in the RXDLL motif nnot omplement eh other. In ddition to the loss of shown etween the RXDLL mutnts, o-immunopreipittion of these mutnts with WT FADD ws impired (Figure 2). These FADD mutnts were lso unle to form DEF when overexpressed in COS-7 ells (Figure 2d), showing diffuse ytoplsmi stining with osionl ytoplsmi ggregtes. These dt show tht FADD self-ssoition is dependent on the RXDLL motif of the DED. The filure of these FADD mutnts to oligomerize in ells ould e due to generlized unfolding of the protein. If tht ws the se, other DED-medited intertions, suh s inding to spse-8, my e impired y muttions in the RXDLL motif. We therefore ssessed the ility of the RXDLL mutnts to intert with spse-8. WT FADD- or RXDLL mutnts were otrnsfeted with tlytilly intive spse-8 in 293T ells. As expeted, ws seen etween WT FADD nd spse-8. This intertion ws dependent on the DED of FADD, s the FADD DD lone filed to intert with spse-8 (Figure 3). Unlike the nerly omplete olition of FADD FADD intertions, the level of ws mintined etween spse-8 nd the AHALL FADD mutnt, nd ws onsistently inresed with the RHDAA mutnt (Figure 3). Co-immunopreipittion experiments onfirmed these results, with oth WT nd RXDLL mutnt FADD interting with HA-tgged spse-8 GFP in 293T ells (Figure 3). To onfirm tht the FADD DED hroring RXDLL motif muttions still folded normlly into the lph-helil DD struture, we ompred the irulr dihroism (CD) spetr of purified monomeri FADD DED with WT FADD, AHALL, AHDLL (R72A), nd RHDAA RXDLL motif sequenes. The CD spetr of ll of these RXDLL motif mutnts ompletely overlpped with tht of the wild-type protein, suggesting tht the glol folding of the FADD DED ws not pertured y the RXDLL motif muttions. This is onsistent with the retined ility of these mutnts to ind to spse-8 nd suggests tht the RXDLL motif is likely funtioning to medite FADD FADD intertions, nd is not required for proper folding of the FADD DED. FADD self-ssoition is ritil for deth reeptor-medited poptosis To test whether FADD self-ssoition is funtionlly importnt for poptosis signling indued y FsL nd TRAIL, we used the FADD-defiient Jurkt T-ell line, I2.1, whih is Cell numer FADD- FADD- 71.5% RHDAA- FADD-.2% AHALL- FADD- 293T FADD- RHDAA-.5% 16.4% FADD- AHALL- RHDAA- RHDAA- AHALL- AHALL- FADD-HA: GFP AHALL-GFP RHDAA-GFP IP:HA; IB: FADD Cell numer 4.9% 24.1% 14.1% FADD-HA IB: GFP IB: FADD 25 I2.1 (FADD ) d FADD- FADD- 55.3% DD- DD- RHDAA- RHDAA- AHALL- AHALL- Cell numer 1.5% 7.1% 6.3% RHDAA-GFP AHALL-GFP Figure 2 FADD self-ssoition is medited y the RXDLL motif. ( nd ) 293T ells () or FADD-defiient Jurkt I2.1 ells () were trnsfeted with onstruts enoding wild-type FADD or the RXDLL mutnts of FADD (RHDAA nd AHALL) fused to either or or lone. ws nlyzed s desried ove. () 293T ells were trnsfeted with onstruts enoding HA-FADD nd WT or RXDLL mutnt FADD fused to GFP. Cells were lysed nd immunopreipitted with nti-ha eds. Lystes nd immunopreiptes were run on gel nd immunolotted for oth HA nd GFP. (d) Cos7 ells were trnsfeted with onstruts enoding WT or RXDLL mutnt FADD fused to GFP nd imged y onfol mirosopy. All dt re representtive of t lest three independent experiments

4 4 FADD self-ssoition in poptosis signling Cspse-8- Cspse-8- FADD- Vetor DD-GFP RHDAA-GFP AHALL-GFP HA-Cspse-8 GFP:.9% Cspse-8- FADD DD- 31.1% Cspse-8- AHALL- HA-Csp8-GFP HA-Csp8-GFP IP:HA; IB: GFP Cell numer.3% 35.6% DD-GFP IB: GFP Cspse-8- FADD DED- 32.4% Cspse-8- RHDAA- 98.2% [θ] (1 3 deg m 3 dmol -1 ) WT(F25Y) RHDAA AHALL R72A Wvelength (nm) Figure 3 Cspse-8 inding nd overll protein folding re preserved in FADD RXDLL motif mutnts. () 293T ells were trnsfeted with onstruts enoding spse-8 nd WT, RXDLL mutnt, DD or DED FADD fused to or lone nd ws nlyzed s desried ove. () 293T ells were trnsfeted with HA-spse-8 GFP nd WT, RXDLL mutnt or DD FADD fused to GFP. Cells were lysed nd immunopreipitted with nti-ha eds. Lystes nd immunopreipittes were sujeted to SDS-PAGE nd immunolotted for GFP. All dt re representtive of t lest three independent experiments. () CD spetr of FADD F25Y nd the indited RXDLL motif mutnts. The verge wvelength dependene of molr elliptiity from five sns is shown, using 5-s integrtion time t 1.-nm wvelength inrements ompletely insensitive to poptosis indued y these deth reeptor lignds. 24 We trnsiently trnsfeted FADDdefiient ells with WT, RXDLL mutnt or DD only FADD onstruts fused to nd stimulted these ells with FsL, nti-fs or TRAIL to determine whether RXDLL mutnts ould reonstitute Fs or TRAIL reeptor-medited poptosis in these ells. WT FADD ws le to reonstitute FsL or TRAIL-medited poptosis in these ells, while the FADD DD ws not. Signifintly, the RXDLL mutnts RHDAA nd AHALL were lmost ompletely defiient in their ility to reonstitute poptosis signling in response to FsL or TRAIL (Figure 4 nd ). Following FsL stimultion, ells trnsfeted with these mutnt onstruts persisted in the nnexin-negtive gte while virtully ll ells with ny expression of wild-type FADD- eme nnexin positive (Figure 4). Although it hs een reported tht I2.1 expresses smller isoform of FADD reognized y ntiodies ginst the DED of FADD, 25 ntiodies ginst the FADD DD do not reognize ny speifi proteins in this line nd it is likely tht this isoform represents trunted form of FADD lking funtionl DD. Without DD, the trunted FADD would not e reruited to the DISC nd should not influene deth-reeptor-indued poptosis. In ddition, the RXDLL motif mutnts would likely not intert with the trunted FADD present in I2.1 ells sine these mutnts do not intert with WT FADD s shown in Figure 2. Interestingly, in spite of their inility to reonstitute deth reeptor-medited poptosis signling, trnsfetion of RXDLL mutnts resulted in levels of spontneous deth mong trnsfeted ells similr to WT FADD (upper right qudrnt of untreted ells nd legend, Figure 4). Spontneous poptosis indued y FADD hs een shown to e due to intertions with spse-8 medited y residues in the FADD DED hydrophoi pth 1, 4,18 nd is onsistent with the ility of the RXDLL mutnts to ind to spse Thus, the RXDLL motif tht is responsile for FADD FADD ssoition lso ppers neessry for FADD to funtion s deth reeptor dpter. To exmine the iohemil sis for the inility of RXDLL FADD mutnts to funtion, we studied the whether FADD RXDLL mutnts ould support ssemly of norml Fs-FADD-Cspse-8-signling omplex. Co-immunopreipittion experiments showed tht these FADD mutnts were le to intert normlly with Fs in 293T ells (Figure 4). We then reonstituted Jurkt I2.1 ells with WT FADD nd RXDLL FADD mutnts nd studied ssemly of the Fs DISC. In WT FADD trnsfetnts, oth FADD nd spse-8 were reruited to the DISC fter FsL stimultion. However, in I2.1 ells trnsfeted with either RHDAA or AHALL mutnt FADD, spse-8 ws sent from the DISC despite reltive preservtion of FADD reruitment (Figure 4d nd dt not shown). These dt suggest tht FADD self-ssoition is neessry for effiient

5 FADD self-ssoition in poptosis signling 5 Annexin V-APC FADD I2.1 (FADD ) FADD DD RHDAA AHALL F sl F sl Speifi ell deth (%) FADD- I2.1 (FADD ) FADD DD- RHDAA- nti-fs FsL TRAIL AHALL- HA-Fs- : FADD-FLAG FADD-FLAG HA-Fs- Vetor FADD-FLAG FADD-FLAG RHDAA-FLAG IP:HA; IB: FADD IB: FADD AHALL-FLAG d I2.1 (FADD ) FsL: Fs spse-8 FADD wtfadd RHDAA Lyste wtfadd RHDAA DISC 25 IP:HA; IB: Fs HA-Fs- 83 IB:Fs Figure 4 RXDLL mutnt FADD nnot reonstitute Fs-medited poptosis in FADD-defiient ells. ( nd ) The FADD-defiient Jurkt ell line, I2.1, ws trnsfeted with WT, RXDLL mutnt or DD FADD fused to. At 16 h fter trnsfetion, ells were treted with nti-fs or FsL or TRAIL for 5 h. Cell deth ws nlyzed y FACS with Annexin-APC nd PI. Cells were gted on PI-negtive ells to exlude deth due to trnsfetion nd GFP-positive ells were nlyzed for deth y Annexin-APC. Error rs indite S.E.M. of pooled dt from 2 3 independent experiments. Prior to stimultion viility (vg.7s.e.m.) of trnsfeted ells ws % for, % for FADD FL, % for FADD DD, % for RHDAA nd % for AHALL. () 293T ells were trnsfeted with HA-Fs- nd WT or RXDLL mutnt FADD. Cells were lysed nd immunopreipitted with nti-ha eds. Lystes nd immunopreipittes were run on gel nd immunolotted for Fs nd FADD. (d) I2.1 ells were trnsfeted with WT or RXDLL mutnt FADD. At 16 h fter trnsfetion, ells were stimulted with nti- Fs for 15 min. DISC formtion ws nlyzed y mesuring reruitment of trnsfeted FADD nd endogenous spse-8 to endogenous Fs. All dt re representtive of t lest three independent experiments reruitment nd tivtion of spse-8 to the DISC during Fs signling. To exmine whether muttions in the RXDLL motif ould trnsform FADD into dominnt-negtive inhiitor, we trnsfeted WT FADD nd the RXDLL mutnts into numer of different Fs-sensitive ell lines expressing endogenous FADD. Fs-expressing ells hve een divided into two tegories. In type I ell lines, the DISC is esily detetle nd Fs-indued ell deth nnot e loked y overexpression of ntipoptoti Bl-2 fmily moleules. In type II ell lines, the DISC is less esily deteted iohemilly nd Fs-indued ell deth n e loked y the overexpression of ntipoptoti Bl-2 moleules. 26 In type I ell lines, Fs is preferentilly lolized to lipid rft mirodomins, nd surfe reeptor lustering is more rpid nd prominent thn in type II ell lines. 15,27 We trnsfeted Jurkt, type II ell line, nd SKW6.4, type I ell line, with WT, RXDLL mutnt or the DD of FADD fused to nd ssessed the ility of trnsfeted ells to undergo poptosis in response to Fs stimultion. FADD DD- inhiited Fs-indued poptosis in Jurkt (type II) nd more prominently in SKW6.4 (type I) ells. FADD onstruts with muttions in the RXDLL motif inhiited Fsindued poptosis in SKW6.4 ut not in Jurkt ells (Figure 5 nd ). Sine ssemly nd tivtion of the DISC hs een ssoited with formtion of visile reeptor oligomers termed SPOTS 15 we exmined the suellulr loliztion of Fs in SKW 6.4 ells trnsfeted with RXDLL mutnt FADD nd found tht these mutnts interfered with the ility of Fs to form SPOTS nd reeptor ps fter reeptor rosslinking (Figure 5). These dt show tht non-self-ssoiting FADD mutnts n dominntly interfere with Fs-indued ell deth in type I ells, likely y interfering with SPOTS formtion medited y endogenous FADD. To define the limits of the RXDLL motif we reted individul lnine mutnts of R72, H73 nd D74 in the RXDLL motif s well s the djent R71 nd R77 residues. Like the more omplex RHDAA nd AHALL mutnts, the R72A nd D74A mutnts showed diminished inding to wild-type FADD nd did not fully reonstitute Fs-medited poptosis (Figure 6 nd ). It should lso e noted tht R72A, whih hd greter defet in its ssoition with WT FADD thn D74A, similrly hd greter defet in reonstitution of Fs-medited poptosis. In ontrst, R71A, H73A, nd R77A mutnts funtioned equivlently to wild-type FADD in these ssys (Figure 6 nd ). Although surfe-exposed residue, H73 is not onserved in the RXDLL motif nd is pprently not required for FADD funtion. Additionlly, the R72A FADD mutnt ut not R71A dominntly interfered with Fs-indued

6 6 FADD self-ssoition in poptosis signling Speifi ell deth (%) % Cells with Fs SPOTS or lusters SKW6.4 (Type I; FADD ) 1 1 1, nti-fs (ng/ml) SKW6.4 (Type I; FADD ) FADD- poptosis when trnsfeted into SKW 6.4 ells (Figure 6). These dt re in ontrst to report in whih FADD R71A ws found to e defiient in funtion nd inding to the Fs DD. 28 These results define R72, D74, L75 nd L76 s key AHALL- FADD- FADD DD- AHALL- RHDAA- Jurkt (Type II; FADD ) 1 FADD- 8 AHALL- RHDAA- 6 FADD DD- Speifi ell deth (%) , nti-fs (ng/ml) Figure 5 RXDLL mutnt FADD dominntly interferes with Fs signling nd reeptor lustering in type I ells. ( nd ) SKW 6.4 () or Jurkt () ells were trnsfeted with WT, RXDLL mutnt or DD FADD fused to. At 16 h fter trnsfetion, ells were treted with nti-fs for 5 h. Cell deth ws nlyzed s ove. Error rs indite S.E.M. of pooled dt from three independent experiments. In Jurkt ells, prior to stimultion viility (vg.7s.d.) of trnsfeted ells ws % for, % for FADD FL, % for FADD DD, % for RHDAA nd % for AHALL. In SKW 6.4 ells, prior to stimultion viility (vg.7s.d.) of trnsfeted ells ws % for, % for FADD FL, % for FADD DD, % for RHDAA nd % for AHALL. () SKW 6.4 ells were trnsfeted with the indited FADD- onstruts, stimulted with nti-fs for 1 h, fixed nd nlyzed for Fs loliztion y immmunofluoresene s desried in the Mterils nd Methods. Cells were visully sored for SPOTS nd reeptor ps nd the verge7s.e.m perent ells with SPOTS or reeptor ps re shown. All dt re representtive of t lest three independent experiments RHDAA- ell numer FADD- H73A- FADD- Speifi ell deth (%) FADD- I2.1 (FADD ) R71A - R72A- 7 SKW6.4 (FADD ) , nti-fs (ng/ml) Speifi ell deth (%) FADD- FADD- D74A- FADD- R71A- FADD- <.1% 74% 76.5% RHDAA- FADD- H73A- D74A - R72A- FADD- <.1% R77A- FADD- 78.4% 53.9%.2% 79.6% FsL R77A- FADD- R71A- FADD DD- R72A- Figure 6 Defiienies in FADD self-ssoition nd Fs signling re speifi for the RXDLL motif. () 293T ells were trnsfeted with onstruts enoding lone or FADD- nd WT, R71A, R72A, H73A, D74A, RHDAA or R77A FADD fused to nd ws nlyzed s desried ove. () FADDdefiient I2.1 ells were trnsfeted with WT, R71A, R72A H73A, D74A or R77A FADD fused to or lone. At 16 h fter trnsfetion, ells were treted with FsL for 5 h. Cell deth ws nlyzed s in Figure 4 nd re verges of triplite dt from 1 to 3 experiments7s.e.m. Prior to stimultion viility (vg.7s.d.) of trnsfeted ells ws % for, % for FADD FL, % for R71A, % for R72A, % for H73A, % for D74A nd % for R77A. () SKW 6.4 ells were trnsfeted with WT, R71A, R72A or DD FADD fused to or lone. At 16 h fter trnsfetion, ells were treted with nti-fs for 5 h. Cell deth ws nlyzed s ove. Error rs indite S.E.M. of pooled dt from 2 to 3 independent experiments. Prior to stimultion viility (vg.7s.d.) of trnsfeted ells ws % for, % for FADD FL, % for FADD DD, % for R71A nd % for R72A. All dt re representtive of t lest two independent experiments funtionl residues of the FADD FADD-inding motif nd indite tht nononserved residues inside nd outside the motif re not required. Densitometri nlysis of FADD expression in trnsiently trnsfeted Jurkt ells showed tht trnsfeted FADD

7 FADD self-ssoition in poptosis signling 7 FADD- 25 Speifi Cell Deth (%) FADD ws expressed etween three nd four-fold higher thn endogenous FADD (dt not shown). To determine whether the RXDLL motif is importnt for FADD expressed t more physiologil levels we generted stle ell lines of FADDdefiient I 2.1 Jurkt ells expressing lone, FADD- or D74A FADD-. The mount of FADD expressed y these ells ws found to e less thn or similr to tht of endogenous FADD expressed y Jurkt ells (Figure 7). At these levels, WT FADD still reonstituted sensitivity to FsLindued poptosis while line expressing slightly higher levels of D74A FADD mutnt did not (Figure 7). These dt indite tht ritil residues in the RXDLL motif re required for FADD funtion s n poptosis dpter t physiologil levels of FADD. Disussion I2.1 stle trnsfetnts FADD- Our results show tht self-ssoition medited y the RXDLL motif within the DED of FADD is ritil for deth reeptor D74A- IB: FADD IB: Atin I2.1 stle trnsfetnts 1 1 1, FsL (ng/ml) Jurkt 55kD 25kD 4kD FADD- D74A- Figure 7 Stle expression of RXDLL mutnt FADD in I2.1 ells fils to reonstitute Fs-medited poptosis. FADD-defiient I2.1 ells were stly trnsfeted with WT or D74A FADD fused to or lone, s desried in the Mterils nd Methods. () Immunolots of the indited trnsfetnts were proed with nti-fadd nd tin () The indited FADD stle trnsfetnts were treted overnight with FsL nd ell viility ws mesured y Annexin nd PI. Speifi ell deth ws nlyzed s ove. Initil viility of the ells ws 8871% (); 9171%(WT FADD); nd 8772% (FADD D74A). Dt re the verge of two independent experiments performed in triplite signling. We hve found tht the RXDLL motif is required for FADD self-ssoition, ut not ssoition with Fs or spse-8. An intt RXDLL motif is required to reonstitute deth reeptor-medited poptosis nd the DISC in FADDdefiient ells. Finlly, muttion of the RXDLL motif n onvert FADD into dominnt-negtive inhiitor of Fsindued poptosis. FADD muttions in the RXDLL motif pper to speifilly inhiit FADD FADD homotypi intertions rther thn upstrem intertions with Fs or downstrem intertions with spse-8. Beuse inding to Fs nd Cspse-8 nd the CD spetr of the purified DED re preserved, it is unlikely tht these muttions impir glol folding of the protein. Rther, speifi protein protein intertion surfe is likely disrupted. Our results implite R72 nd D74 s key surfe residues tht medite FADD self-ssoition, with L75 nd L76 forming the hydrophoi floor of the motif. In ord with its nononserved sttus, muttion of the H73 residue did not ffet FADD funtion. Interestingly, nlysis of the NMR struture of FADD shows tht R72 nd D74 ontriute to lternting surfe idi nd si pthes (Figure 1). It is likely tht the RXDLL motif inds to its ounterprt on nother DED, euse if nother moleulr surfe ws required, then RXDLL motif mutnts should e le to intert with the RXDLL motif in WT FADD through tht surfe. However, this ws not the se, s shown in Figure 2, RXDLL mutnts were s severely impired in inding WT FADD s themselves. FADD self-ssoition ppers to funtion y promoting the formtion of reeptor oligomers on the ell surfe fter reeptor ligtion. We previously reported tht these strutures, referred to s SPOTS, re ritil for full spse tivtion in the DISC. 15 We propose tht the RXDLL motif llows FADD to self-ssoite following its reruitment to the DISC. RXDLL-medited FADD self-ssoition my then drive the reruitment of spse-8 to the DISC y inresing the vidity of the omplex for spse-8. The RXDLL motif is neessry ut likely not suffiient for self-ssoition sine the motif is onserved in DED of the ntipoptoti MC159 vflip protein, whih does not self-ssoite or form deth-effetor filments, nd ts s dominnt-negtive inhiitor of FsLnd TRAIL-indued poptosis. 15,2 RXDLL mutnt FADD nd FADD DD re more potent dominnt-negtive inhiitors of Fs signling in type I ell lines suh s SKW 6.4 thn in type II ell lines suh s Jurkt. These differenes my rise euse reeptor oligomeriztion is more importnt for proximl signling in type I ell lines. We hve previously found tht in type I ut not type II ell lines, proportion of Fs lolizes to detergent-resistnt lipid rfts. Lipid rft loliztion redues the requirement for rosslinking of gonisti nti-fs ntiodies tht indued poptosis. 27 Disruption of lipid rfts in type I ells with ylodextrins inhiits lignd-indued SPOTS formtion nd reeptor pping (JRM nd RMS, unpulished oservtions). Thus in type II ells, the inresed Fs rosslinking required to produe n poptoti response my overome the dominnt-negtive effet of muttions tht inhiit FADD self-ssoition. The trnsformtion of FADD from n oligte dpter protein to dominnt-negtive inhiitor revels n importnt priniple in deth reeptor signling: tht in ddition to inding up nd downstrem signling moleules, dpter proteins

8 8 FADD self-ssoition in poptosis signling my need to self-ssoite to llow orret ssemly nd tivtion of spse-8 in the DISC. Wheres, oth RXDLL mutnts tht lk the ility to self-ssoite nd FADD mutnts lking the ility to ind spse-8, suh s F25G, fil to reonstitute Fs-indued poptosis, only RXDLL mutnt FADD n dominntly interfere with Fs-indued poptosis (JRM nd RMS, unpulished oservtions). These dt suggest tht loking the ility of FADD to self-ssoite nd form n rry of signling omplexes my e n effiient mehnism for loking deth-reeptor-indued poptosis. Indeed, sed on struturl studies it hs reently een proposed tht the ntipoptoti tivity of the DED-ontining virl FLIP moleule MC159 is dependent on its ility to inhiit FADD self-ssoition. 29 Reeptors in the TNF reeptor superfmily nd the toll-like reeptor/il-1 reeptor superfmily signl through the reruitment of modulr dpter proteins tht ssoite with intrellulr effetor moleules to trigger downstrem events. Rther thn eing liner sequene of vertil reruitment events, our results indite tht self-ssoition of dpter proteins n medite horizontl intertions tht mplify the initil lignd reeptor intertion signl nd trigger more effiient reruitment of ytoplsmi effetors. Signl mplifition through horizontl dpter protein intertions my produe inresed sensitivity to hnges in lignd onentrtion tht n fine tune ellulr responses. 3 This my e prtiulrly importnt for reeptors tht medite irreversile outomes suh s progrmmed ell deth. The memrneound signling omplexes promoted y FADD oligomeriztion pper to e ritil for reruitment nd proessing of spse-8. These omplexes my e similr to ytoplsmi high-moleulr weight omplexes tht hve een found to e ritil for tivtion of other spses suh s the poptosome for spse-9, the PIDDosome for spse-2 nd the inflmmsome for spse RXDLL-medited FADD self-ssoition my lso e n ttrtive trget for therpeuti intervention in poptosis signling pthwys tht depend on FADD nd other DED-ontining proteins. Mterils nd Methods Cells FADD-defiient Jurkt I2.1, Jurkt A3, SKW 6.4, Cos7 nd 293T ells were otined from ATCC. Jurkt nd SKW 6.4 ells were ultured in RPMI supplemented with fetl lf serum (1%), peniillin (5 mg/ml) nd streptomyin (5 mg/ml). Cos7 nd 293T ells were ultured in DMEM supplemented with fetl lf serum (1%), peniillin (5 mg/ml) nd streptomyin (5 mg/ml). All ells were grown in 5% CO 2 t 371C. Antiodies nd regents The Fs C2 ntiody ws otined from Snt Cruz Antiodies (Snt Cruz, CA, USA). The FADD ntiody ws otined from BD Trnsdution ls nd the spse-8 C15 ntiody ws otined from Dr. M Peter (University of Chigo, Chigo, IL, USA). Anti-GFP ws otined from Rohe. Anti-HA ntiody nd nti-ha ffinity mtrix ws otined from Covne. Plsmids nd mutgenesis FADD ws loned into pegfp-n1, pe-n1 nd pe-n1 using Pfu Turo (Strtgene) polymerse hin retion using primers flnking either end of FADD. FADD ws mutgenized y Quik Chnge sitedireted mutgenesis (Strtgene) using primers speifi for the desired muttions. Muttions were onfirmed y sequening. nlysis In ll, T ells per well were seeded in six-well dishes nd trnsfeted with expression vetors enoding fusion protein (5 ng) nd fusion protein (5 ng) using Fugene 6 trnsfetion regent (Rohe). Cells were hrvested 24 h fter trnsfetion, nd nlyzed on CyAn flow ytometer (Cytomtion). Exittion lsers nd filters for detetion hnnels were: 45 nm UV diode lser nd 45/ 5 nm ndpss filter for ; n 488 nm diode lser nd 546/1 nm ndpss filter for ; nd the 45 nm UV diode lser nd 546/1 nm ndpss filter for. All three signls were deteted from the sme individul ells y sequentilly illuminting them with the 488 nd 45 nm lsers. CD spetrosopy The F25Y solule vrint of the FADD AA 1 84 ws loned into the Pet28A vetor (Novgen) nd site-direted mutgenesis ws performed with the quikhnge method nd onfirmed y sequening in ll ses. Proteins were expressed in Esherihi oli strin BL21-CodonPlus(DE3)- RIPL (Strtgene), nd purified through Ni-ffinity hromtogrphy. Purified smples were deslted using HiPrep 26/1 Deslting olumn (Amershm Biosienes), nd djusted to onentrtion of.69 mg/ml. CD spetr were quired t 41C on n Aviv 62DS (Aviv Assoites) CD spetropolrimeter with 1 mm pth-length uvette. The wvelength dependene of molr elliptiity ws monitored s the verge of five sns, using 5-s integrtion time t 1.-nm wvelength inrements. Apoptosis indution ssys I2.1 (FADD defiient), A3 Jurkt or SKW 6.4 ell lines were trnsiently trnsfeted y eletroportion t 26 V, 725 O nd 125 mf s previously desried 2 using BTX ECM 61 eletroportor with onstruts s indited. At 16 h fter trnsfetion, ells were inuted with TRAIL (5 ng/ml; Alexis) or FsL (1 ng/ml; Alexis) plus nti-flag or ntihumn Fs APO1-3 (1 mg/ml; Kmiy Biomedil, Settle, WA, USA) plus nti-igg3 for 4 6 h. Cells were stined with Annexin V-APC (BD Phrmingen) nd propidium iodide (PI) ording to mnufturer s instrutions. Dt were olleted on FACS Cliur flow ytometer (Beton Dikinson) nd nlyzed using FlowJo (Treestr In.). Cells were gted on PI-negtive ells to exlude deth due to eletroportion, within the PI-negtive gte viility of GFP-positive ells ws ssessed on the sis of Annexin V. Speifi ell deth of trnsfeted ells ws determined using the formul (1 (% vile treted/% vile untreted)) 1. The S.E. in the men is shown for ell deth ssys run in triplite. Genertion of stly trnsfeted ell lines Construts enoding lone or wild-type or mutnt FADD fused to were linerized with AseI nd trnsfeted into the FADD-defiient Jurkt ell line, I 2.1, y eletroportion s desried ove. Cells were then ultured in medi ontining G418 (1 mg/ml; Strtgene, CA, USA) for

9 FADD self-ssoition in poptosis signling 9 2 weeks. Cells were then sorted sed on expression using MoFlo ell sorter (Dko Cytomtion, CA, USA) nd sorted -positive ells were ultured in medi ontining G418 (1 mg/ml) for nother 2 weeks to generte the stle ell lines. Immunopreipittion nd western nlysis In ll, T ells were seeded in six-well pltes nd trnsfeted with Fugene 6 with the indited onstruts. At 24 h fter trnsfetion, ells were hrvested nd lysed on ie in 1 ml lysis uffer (1% NP-4 (Sigm), 1% glyerol, Complete Protese Inhiitor (Rohe), 5 mm iodoeti id, 1 mm zvad (Envyme Systems Produts), 15 mm NCl, 15 mm EDTA, 1 mm Tris-HCl). Alterntively, I2.1 ells were trnsfeted y eletroportion with the indited onstruts. A totl of 15 trnsfetions for eh ondition were pooled. At 16 h fter trnsfetion, ells were stimulted for 15 min with reominnt CD8-FsL (2.5 mg/ml; Anell) nd lysed on ie in 1 ml lysis uffer. Lystes were immunopreipitted with the indited ntiodies nd Protein A grose eds (Rohe) or nti-ha ffinity mtrix eds (Covne) overnight t 41C. Immunopreipittes were wshed six times in lysis uffer. Lystes nd immunopreipittes were mixed with lithium dodeyl sulfte-ontining loding uffer (Invitrogen) nd DTT (Sigm) nd inuted t 81C for 2 min. Lystes nd immunopreipittes were run on 4 12% Bis-Tris gel (Bio-Rd) nd trnsferred to nitroellulose nd immunolotted s indited. Confol mirosopy Cos7 ells were grown on overslip hmers (L-Tek) nd trnsfeted s indited using Fugene 6 trnsfetion regent. At 16 h fter trnsfetion, ells were imged live using n Olympus onfol mirosope. Imges were nlyzed using Olympus nd Imris softwre. Quntittion of SPOTS formtion SKW 6.4 ells were trnsfeted y eletroportion with lone or WT or mutnt FADD-. At 16 h fter trnsfetion, ded ells were removed using Fioll density grdient medium (Amershm). Cells were stimulted with 1 mg/ml APO1-3 plus nti-igg3 or FsL plus nti-flag for 45 min nd dhered to poly-l lysine-oted overslips for 15 3 min. Cells were fixed on dry ie with ie-old 1% methnol for 7 min, followed y wshing nd stining of unstimulted ells with APO1-3 nd stining of ll ells with nti-mouse IgG-Alex-488 (Moleulr Proes) in PBS/.1% Tween-2/.1% BSA (IFA uffer). Quntittion of surfe reeptor lustering in positive trnsfeted ells ws performed s previously desried 15 y linded oserver nd t lest 5 ells were ounted for every ondition in n experiment. Cells with Fs SPOTS involving 425% of the plsm memrne or in memrne ps were ounted s positive. Aknowledgements We thnk B Ahvzi for protein modeling; NIAMS nd NIAID ore flow ytometry filities for tehnil ssistne; M Lenrdo, J Cohen, L Zheng nd D Kstner for ritilly reviewing the mnusript. JRM ws Howrd Hughes Medil Institute-Ntionl Institutes of Helth Reserh Sholr nd is supported y Grnt numer 5-T32-ES779 from the Ntionl Institute of Environmentl Helth Sienes (NIEHS), NIH. Referenes 1. Chinniyn AM, O Rourke K, Tewri M nd Dixit VM (1995) FADD, novel deth domin-ontining protein, interts with the deth domin of Fs nd initites poptosis. Cell 81: Mrtin DA, Zheng L, Siegel RM, Hung B, Fisher GH, Wng J, Jkson CE, Puk JM, Dle J, Strus SE, Peter ME, Krmmer PH, Fesik S nd Lenrdo MJ (1999) Defetive CD95/APO-1/Fs signl omplex formtion in the humn utoimmune lymphoprolifertive syndrome, type I. Pro. Ntl. Ad. Si. USA 96: Hung B, Eerstdt M, Olejnizk ET, Medows RP nd Fesik SW (1996) NMR struture nd mutgenesis of the Fs (APO-1/CD95) deth domin. Nture 384: Eerstdt M, Hung B, Chen Z, Medows RP, Ng SC, Zheng L, Lenrdo MJ nd Fesik SW (1998) NMR struture nd mutgenesis of the FADD (Mort1) deth-effetor domin. 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