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Two distinct and independent sites on IL‐6 trigger gp 130 dimer formation and signalling.

Two distinct and independent sites on IL‐6 trigger gp 130 dimer formation and signalling. The EMBO Journal vol.14 no.9 pp.1942-1951, 1995 Two distinct and independent sites on IL-6 trigger gpl3O dimer formation and signalling IL-6Ra (also referred to as the and Giacomo Rita ax subunit) gpl 30 (also Paonessa1, Graziani, referred to as the B subunit). IL-6Ra specifically binds Anna De Serio, Rocco Savino, IL-6 at a low relatively affinity (10-9 M; Yamasaki et al., Armin Laura Ciapponi, Lahm, 1988). The IL-6-IL-6Rax complex associates with gpl3O Anna Laura Salvati, Carlo Toniatti and at high affinity (10-11 M) thus promoting signal transduc- Gennaro Ciliberto tion inside the cell (Hibi et al., 1990). Both Istituto di Ricerche di Biologia Molecolare IL-6Rax and gpl30 belong to the haematopoietin (IRBM), P.Angeletti, Via Pontina Km 30 600, 1-00040 Pomezia, Rome, Italy receptor superfamily that includes, among others, the growth hormone (GH) receptor (GHbp) (Cosman, 1993). author 'Corresponding These receptors all share sequence and structural similarities in a 220 amino acid region called the cytokine The helical cytokine interleukin 6 and its (IL) specific binding domain (CBD), located in the extracellular subunit IL-6Ra form a 1:1 portion binding complex which, by of the molecule all the corres- promoting homodimerization of the signalling subunit (Bazan, 1990b). Moreover, ponding cytokines are predicted to fold as a bundle of gpl3O on the surface of intra- target cells, triggers four a-helices cellular We (Bazan, 1990a, 1991). responses. expressed differently tagged From and structural similarities with the GH forms of and used them in sequence gpl3O solution-phase binding receptor whose structure has been solved X- to show that the soluble extracellular domains complex, by assays Vos et we built a of dimerization in the absence of ray crystallography (de al., 1992), gpl3O undergo 3-D model of the membranes. In vitro reactions were computer-assisted IL-6-IL-6Ra-gp 130 receptor assembly interaction et Lahm et In also performed in the of two sets of IL-6 (Savino al., 1994a,b; al., 1995). presence the was variants amino acid substitutions in two analogy with GH, IL-6 receptor complex envisaged carrying as a heterotrimer in which a molecule distinct areas of the surface single IL-6 bridges cytokine (site 2, comprising In the residues in the A and C and site in the IL-6Rax and the gpl3O CBDs. this complex, exposed helices, 3, with is formed the terminal of the CD The IL-6 contact surface IL-6Rax by residues part loop). binding affinity D AB to IL-6Ra of these variants is normal but their bio- in the putative helix and loop (site 1), whereas its here in A logical activity is poor or absent. We demonstrate binding site to gpl30 constitutes exposed residues the that both the site 2 and site 3 IL-6 variants and C helices (site 2; Savino et al., complexed 1994a,b). Furthermore, with bind a molecule but are residues in the E2 strand and AB2 of the CBDs of IL-6Rax single gpl3O loop unable to dimerize whereas the combined site 2/3 gpl30 and IL-6Rax form a third interface et it, (Yawata al., with The variants lose the to interact 1993; Lahm et al., 1995). The of the model ability gpl3O. reliability in binding properties of these variants vitro, and the was tested site-directed of both IL-6 and by mutagenesis monoclonal result of using a neutralizing antibody IL-6Rax; variants amino acid substitutions of bearing lead to the conclusion that directed against site 3, residues to be at or postulated receptor-cytokine is formed direct at two gpl3O dimer through binding interfaces confirmed the receptor-receptor phenotypes and oriented sites on IL-6. independent differently predicted from the model et (Savino al., 1994a,b). further reveal that Immunoprecipitation experiments This heterotrimeric model does not, however, explain the assembled is of fully receptor complex composed additional A event in the findings. key IL-6 signalling two IL-6, two IL-6Ra and two molecules. We gpl3O cascade is the dimerization of the 30 chain gpl (Murakami propose here a model representing the IL-6 receptor et that activates associated al., 1993) (i) tyrosine kinases complex as hexameric, which might be common to of the Jak and the family (ii) phosphorylation of a set other helical cytokines. of intracellular the substrates, including intracytoplasmic Key words: gp13O dimer/IL-6 receptor antagonist/IL-6 domain of and gpl30 transcription factors of the STAT receptor complex/IL-6Ra et et family (Stahl al., 1994; Zhong al., 1994). These observations have led to the conclusion that a second gpl30 molecule in the participates active IL-6 receptor complex. Introduction Additional information on the complexity of the IL-6 Interleukin (IL) 6 is a multifunctional cytokine that acts receptor complex has emerged from the recently developed on different target cells to induce a variety of biological IL-6 receptor antagonists. Different strategies have been responses (Van Snick, 1990). Transmembrane glyco- adopted to design these IL-6 variants, aimed at generating that form proteins the IL-6 receptor complex have been molecules that can still effectively bind IL-6Ra but can identified and cloned (Yamasaki et al., 1988; Hibi et al., carry amino acid substitutions that impair the activation 1990). Two membrane proteins are both necessary and of gpl3O by the IL-6-IL-6Rcx complex. Interestingly, sufficient to constitute a functional receptor complex: two strategies developed independently produced IL-6 1942 assembly IL-6 receptor complex antagonists with the expected properties by mutating two A C. B C.t different portions of the IL-6 four-helix bundle. The first is a patch of four exposed residues in the A and portion e M C helices, which we called site 2 (Savino et al., 1994a,b) because its location is similar to site 2 in GH. The second is formed by residues located at the beginning of the D helix and AB loop [referred to as site II by Brakenhoff sgpl3O -72 kDa _ et al. (1994) and Ehlers et al. (1994)], which we call site 3. The location of site 3 does not fit into the GH paradigm. To investigate further the unique properties of the IL-6 sIL-6Rt_ v receptor complex, we developed a very sensitive assay =~40 kDa ^ 4 ~~~ to analyse the biochemical steps of receptor assembly. Differentially tagged versions of soluble gpl30 and IL-6Rax receptors are expressed in insect cells and are reactions with wild-type and used in immunoprecipitation IL-6Rx molecules. Our results indicate variant IL-6 and anti-FLAG anti-myc that the IL-6 -IL-6Rx complex binds two gp 130 molecules in insect cells. (A) Western Fig. 1. Production of sgpl3O and sIL-6Ra binding sites. Furthermore, at two distinct and independent Five cells infected with blotting of supernatants (50 of High gl) that two IL-6, two IL-6Rx and two we provide evidence sgpl30-FLAG, sIL-6Ramyc and recombinant baculovirus expressing are present in the final complex, and gp130 molecules 1 monoclonal antibodies. M, 30-myc and stained with their specific sgp 35S metabolically labelled on their topological location in this putative molecular weight marker. (B) In vitro give details in insect cells. M, sgpl3O-FLAG and sIL-6Ra (15 expressed is presented which reconciles this 'hexamer'. A model gl) molecular weight marker. that of the trimeric GH receptor complex structure with that a similar assembly process might also and suggests sIL-6Ra were estimated to be 1- for other helical cytokines. forms of sgpl 30 and of take place 5-7 mgIl for sIL-6Rcx after affinity 2 mg/l for sgp 130 and unpublished observations). purification (G.Paonessa, Results the gpl 30 dimerization assay is The strategy adopted for 2A. In a typical experiment, unlabelled An in vitro assay for gpl30 homodimerization depicted in Figure was immobilized on protein A-Sepharose Homodimerization of gpl 30 has been shown to take place sgpl30-myc with the anti-myc monoclonal anti- on the surface of cells exposed to IL-6 (Murakami et al., beads coated specific immunoprecipitations were per- To test whether gpl30 requires the transmembrane body. After washing, 1993). of various combinations of IL-6, and intracytoplasmic regions to dimerize or, on the con- formed in the presence is an intrinsic property of the extracellular domain, IL-6Rax (labelled or unlabelled) and [35S]gpl30-FLAG. trary, latter is not recognized by the anti-myc we decided to set up an in vitro dimerization assay. Because the its immunoprecipitation can only be To do this, we expressed gpl30 in its soluble form monoclonal antibody, that dimers had been formed. truncated at the predicted junction between explained by assuming gpl30 (sgp 130), this experiment are shown in Figure 2B, extracellular and transmembrane domains, and modified The results of two different epitope tags: the efficient in vitro formation of gpl30 dimers at the C-terminus so as to carry where (Evan et al., 1985) and was confirmed by the immunoprecipitation of labelled sgpl3O-myc carrying a myc tag with a FLAG tag (Hopp et al., 1988). This by anti-myc antibodies. As expected, dimers sgpl 30-FLAG gpl 30-FLAG sgpl30 molecules recognizable were only formed in the presence of both IL-6 and IL- makes the different antibodies. Likewise, two 6Ra. IL-6Rax was immunoprecipitated only in by specific monoclonal Likewise, forms of IL-6Ra were generated, one the of IL-6 (Figure 2B). recombinant soluble presence residue 322 of the extracellular domain, the truncated at to the same C-terminal Dimerization of gp 130 is triggered by two other with a myc tag attached sites on IL-6 encoded their natural leader peptide independent binding residue. All cDNAs have been which act as cDNAs were inserted in the Recently, IL-6 variants generated sequence. The modified et al., 1994; Savino et al., vector under the baculovirus polyhedrin receptor antagonists (Brakenhoff recombination amino acid substitutions were Recombinant were transfected in Sf9 1994a,b). In these variants, promoter. plasmids distinct areas of the IL-6 surface, as and the recombinant viruses expressing the various introduced in two cells the 3-D model shown in Figure 3. These were and amplified. Recombinant recep- predicted by receptors purified can be subdivided into two classes: (i) by infecting insect cells in serum-free mutant proteins tors were produced 2 mutations in helix A and C, and medium. were assayed by Western blot for site variants, bearing Supematants at the of bearing mutations beginning recombinant production (Figure IA). Individual (ii) site 3 variants, protein of two distant sites on IL-6 with helix D. The identification were metabolically labelled [35S]methionine receptors in transduction, and low compared with similar roles promoting signal at high specific activity background evidence that the subunit 1 Receptors expressed in with the signalling with other proteins (Figure B). together that these a molecular weight of -40 kDa for sIL- dimerization, immediately suggest insect cells show undergoes sites for the two 130 gp kDa for that these molecules sites are independent binding 6Rax and 72 gp 130, suggesting subunits. to modifications but to a are subjected post-translation we decided to evaluate their test this than when in mammalian cells To hypothesis lesser extent expressed 30 and dimerize it in vitro. to bind baculovirus The levels of the two ability sgpl et al., 1994). expression (Sporeno 1943 G.Paonessa et al. 7 :t 1? 441 II 11 t' "1411 l { i Z i f , ^ 0l -'_ suI) J)iflercnt conlhina<tionr -. ,I .-.oR. " N .41 ._- 1) s.!li tilt - II..h RK H.- low Illctithation sh S1.)S IPAGCLF." is Fig. 2. sgpl30 is able to dimerize in vitro in the presence of IL-6-sIL-6Rax. (A) Experimental strategy. sgpl3O-myc attached to protein and incubated with different combinations of and The A-Sepharose beads via anti-myc 9E10 monoclonal antibody IL-6, sIL-6RaX sgpl30-FLAG. use of a 35S-labelled form of a chain. After incubation and participation of a specific molecule in the complex is detected by the specific receptor is with buffer and to PAGE. The has been washing of the Sepharose beads, the bound material eluted SDS loading subjected (B) experiment shown in Cold attached on beads was incubated with the performed according to the experimental strategy (A). sgpl30-myc protein A-Sepharose of each lane in the amounts: 3 500 cold and 50 molecules indicated on top following tg IL-6, supernatant containing sIL-6Roa supernatant Plt gl Asterisks indicate 35S-labelled material. containing 35S-labelled receptors. Rx, slL-6Rax; 130, sgpl3O-FLAG. the site 2 variants we selected the four amino From acid substitution Y31 D/G35F/S 118RNV 121 also called NH2 D, which acts as a full IL-6 on a DFRD, antagonist variety of human cell lines et For site the (Savino al., 1994b). 3, variant was described which also has low Q159E/T162P and acts as a on some biological activity partial antagonist but not all human cell lines et To (Brakenhoff al., 1994). avoid residues which affect introducing proline might or we two new variants folding stability, generated (WI57R/Dl60R and T162D) which map in the same area and have similar to The properties Q159E/T162P. in this are properties of all the IL-6 variants used study in vitro to summarized in Table I, which reports binding measured ELISA et sIL-6Rcx as by (Savino al., 1994a) on two different human cell lines and biological activity with different of to human IL-6. Both degrees sensitivity site 2 and site variants have normal to 3 binding affinity as well as a IL-6Rax, strongly impaired biological activity. To measure to beads coated binding gpl30, Sepharose with anti-myc monoclonal antibody were incubated with the myc-tagged preparation of sIL-6Rx and then used to co-immunoprecipitate labelled sgp 130-FLAG in the presence of the wild-type or mutant IL-6 molecule. All three variants (DFRD, W157R/D160R and T162D) with an -2- immunoprecipitated sgp130 efficiency only to 4-fold lower than wild-type IL-6 (Figure 4A). This result differs from that reported previously with a different site 2 variant 31D/35F using Chinese hamster ovary Fig. 3. Location of putative receptor binding sites on the 3-D model of human IL-6. Schematic RIBBONS (Carson, 1987) representation of (CHO)-derived sgp130, where a stronger defect in gp130 the IL-6 model (Savino et al., 1994a) showing the three sites involved binding was observed (Savino et al., 1994a). This discrep- in receptor interaction: site 1 (blue), the site of interaction with ancy has also been observed consistently with the site 2 IL-6Rx around on Arg179 helix D (also including parts of the AB and site 3 variants used in this study (results not shown). Savino et site 2 loop; al., 1993); (red), residues from helix A and C, to be the site of We believe this is due to a significant difference in receptor postulated interaction with one gp130 molecule and required for signalling (Savino et al., 1994a,b); site 3 (green), centred concentration, whereby the use of baculovirus sgp 130 around the N-terminal end of helix D, important for signalling and reveals residual binding of these variants that cannot be also originally identified as responsible for binding (Brakenhoff gpl30 detected under the limiting conditions imposed by the use et al., 1994). For site 3, only the IL-6 residues mutagenized in this of the CHO-derived sgpl30. study are shown. 1944 IL-6 receptor complex assembly Table I. Receptor binding and biological activity of the IL-6 variants described in the text IL-6 variants sIL-6Rc binding Biological activity (% of wild-type) (% of wild-type) XG-1 (myeloma cells) HepG2 (hepatoma cells) wt IL-6 100 100 DFRD ± 97 15 <0.01 <0.01 157R/160R ± ± 220 + 51 6 4 2.9 0.2 162D 104 18 8 3 1.3 0.3 DFRD/157R/160R ± 160 40 <0.01 <0.01 DFRD/162D 45 ± 7 <0.01 <0.01 Binding to the sIL-6Rx was determined as described (Savino et al., 1994a). The IL-6 biological measured activity on the XG-1 myeloma cell line is the stimulation of cell replication (Savino et al., 1994b). The IL-6 biological activity measured on the cell line is the of HepG2 hepatoma activation the transfected IL-6-inducible C-reactive protein gene promoter (Savino et al., 1994b). The biological activity of each variant (expressed as percentage of wild-type IL-6 activity) on both cell lines was determined as the ratio between the concentration of IL-6 and the wild-type concentration of variant IL-6 necessary to give 50% of wild-type IL-6 maximal stimulation. 157R 160R 162DI l)FRD DFRD 157R160R 162D DFRD wtIL-6 no .*-sgpt30 157R'16(0H.t 162D DFRD DFRD 157R 160R 162D wt IL-6 DFRD no I -iiIIf. .Zz. Lfi.~.LI LfIZz,,I.L-6 M 4- sgpl30 Fig. 4. gpl 30 binding and dimerization of wild-type and IL-6 variants. (A) Binding of sgpl30 to wild-type and variants of IL-6 complexed with sIL-6Rax. sIL-6Ramyc was immobilized on protein A-Sepharose beads via anti-myc 9E10 monoclonal antibody and incubated with 35S-labelled sgpl30-FLAG (50 in 1 supematant) the presence of increasing concentrations (10 gg, ,ug, 100 ng and 10 ng) of wild-type or variant IL-6, as gl indicated on of the top lanes. After incubation and washing, the bound material was subjected to PAGE. M, molecular weight marker. (B) Dimerization of sgpl30 in the presence of wild-type and variants of IL-6. sgpl3O-myc was immobilized on protein A-Sepharose beads via anti- myc 9E10 monoclonal antibody and incubated with 35S-labelled sgpl30-FLAG (50 ,tl supernatant) in the presence of cold sIL-6Ra (500 ,l supernatant) and 1 increasing concentrations (10 and 100 ng) of wild-type or variants of IL-6, as indicated. After incubation and washing, the gg, gg bound material was subjected to PAGE. M, molecular weight marker. When the dimerization assay was performed using the mutation involving four amino acid residues is to likely protocol described in the previous all abolish the section, the IL-6 completely interaction with the subunit gp130 variants showed a selective and dramatic decrease (Figure binding at site 2, while the mutations W1 leaky 57R/ in 4B), precise parallel with their impairment of D160R and T162D do not bioactivity fully impair the interaction of In (Table I). fact, DFRD, which does not residual the at possess respective gpl30 subunits binding site 3. bioactivity and is a full antagonist (Savino et the two areas of the IL-6 surface al., 1994b), Apparently, affected does not dimerize whereas gpl3O at all, variants W157R/ by these mutations behave as independent gpl30 binding Dl 60R T 1 and 62D show a residual activity of -3 and 1% sites, because both site 2 and site 3 variants are able to in cells and are respectively HepG2 reduced in 130 bind one gpl30 molecule To this gp efficiently. analyse dimerization to -6 and 4% From these results two more variants simultaneous mutations respectively. further, bearing we can conclude that both site 2 and site 3 while at sites 2 and 3 were variants, generated (DFRD/W157R/D160R still the to recruit in the and These variants maintain normal IL- maintaining capability gpl30 DFRD/T162D). presence of IL-6Ra, cannot form 130 dimers. This 6Ra as are gp binding (Table I) and, expected, biologically What for finding explains the lack of has inactive. When tested with biological activity. sgpl30 binding, although DFRD variants to be taken into is that the only slight both were almost com- account, however, differences, 1945 et aL G.Paonessa wt IL-6 'l:I I I:,sI:Iii 1.i:3 Ir '10 - !N O I;0 1 'r '1 _. a- am ,lb v - - -W *GM mode A + B Fig. 5. Immunoprecipitation with mAb 16 of complexes formed with Site 3 mutant Site 2 mutant wild-type or variant IL-6. Wild-type or variant IL-6 (as indicated on of the lanes) was immobilized on protein A-Sepharose beads top incubated with the receptor molecules in the using mAb 16 and amounts: 500 of containing cold sIL-6Raz and following gl supematant 50 of 5S-labelled receptors. Rax, sIL-6Rax; ,ul supernatant containing 130, sgpl3O-FLAG; M, molecular weight marker. Asterisks indicate 35S-labelled material. After incubation and washing, the bound material was subjected to PAGE. mode A pletely unable to bind sgpl30 (Figure 4A). The residual binding can be explained if we assume that residual still occurs at site 3 as discussed above. As mode B binding these variants did not dimerize gpl30 expected, of A and B modes of interaction of Fig. 6. Schematic representation (Figure 4B). the IL-6Rax-IL-6 with molecules. complex gpl30 The 'two sites' hypothesis was tested independent further the anti-IL-6 monoclonal antibody using specific variant shows no residual to 30. Most mAb 16 et al., 1990). mAb 16 has been DFRD binding sgpl (Brakenhoff both sites are unavailable for this interaction, shown to inhibit the biological activity of probably, previously cell surface 2 because of the mutation and site 3 because of the IL-6 without with its binding site interfering led to the that hindrance due to the bound molecule. et This hypothesis steric antibody (Brakenhoff al., 1992). with but In conclusion, by using a variety of tools it was possible mAb 16 impairs the interaction of IL-6 gp130 modes of interaction of mAb 16 was to two not with IL-6Rcx. The epitope recognized by distinguish independent in between and and 30 with the IL-6- IL-6Roa complex (outlined Figure localized within the region Q154 T162, gpl oriented in a similar for the of site 3 In mode A one chain is this information was used development 6). gpl3O chain in the GH et fashion to the second GHbp receptor IL-6 receptor antagonists (Brakenhoff al., 1992, 1994). in mode B the second we that if IL-6 complex (de Vos et al., 1992); In the light of these findings, speculated on and is oriented sites for site 2 gp 130 chain binds site 3 IL-6 differently presents two independent binding gpl30, of mAb 16 could block site to one side of the IL-6-IL-6Ra complex (Figure 6). and site 3, the binding only IL-6 both modes 2 free to interact with another gp 130 In the presence of wild-type operate 3, leaving site and lead to homodimerization. molecule. simultaneously gpl30 we immunoprecipitations with Therefore performed in E2 of the mAb 16 or mutant IL-6, and tested their A variant the strand cytokine binding using wild-type labelled IL-6Ra of is in 130 to bind and immunoprecipitate domain IL-6Rla selectively impaired gp ability that of the site 3 dimerization and sgpl30. Initial results showed the mAb 16 Previous with human IL-6Ra mutations, W157R/D160R destroys epitope mutagenesis experiments still be the some information about the composition of the whereas T162D can recognized by antibody provided interface (Yawata et al., 1993). Out of a (data not shown). IL-6Rx-gpl3O limited subset of As shown in Figure 5, all the variants tested bind sIL- set of receptor variants, only a large in was able to in with the observation that amino acid substitutions the IL-6Ra CBD 6Rax efficiently, agreement to while the mAb 16 does not interfere with this interaction. Moreover, selectively reduce binding gpl30 leaving still to interact efficiently intact. The of these mutations mAb 16 allowed wild-type IL-6 affinity for IL-6 majority its co-immunoprecipitation. Not in the E2 strand and AB2 loop of subdomain 2 of with sgpl30, promoting map this interaction took place only in the presence the CBD, which is also the location of the binding surprisingly, receptor of IL-6Ra, thus excluding the possibility that the neutraliz- interface between the two GH receptor chains in the ing effect of this monoclonal antibody is due to a complete GH-(GHbp)2 complex (de Vos et al., 1992; Yawata et al., the IL-6-IL-6Ra inhibition of gpl30 interaction with 1993). Based on these findings we produced an IL-6Ra the effect of mAb 16 is due variant two substitutions in the E2 strand (H280S/ complex. Hence, biological carrying to a selective inhibition of gpl130 dimerization. Indeed, 1 which was called Mutl. The construction of Mut 1 D28 V) while the site 3-type T162D variant is still able to bind and the characterization of its biochemical properties will in the presence of mAb 16, the site 2-type be described elsewhere (A.L.Salvati, A.Lahm, G.Paonessa, sgpl3O 1946 IL-6 receptor complex assembly :\ -II t:g-t e~~~~~~Ii -f;it to that obtained when the used is complex formed by wild-type IL-6Rca and a combined IL-6 site 2/3 variant -------- (Figure 4B). The IL-6 receptor complex is a hexamer which contains two IL-6, two IL-6Ra and two gpl30 molecules Structural comparisons and functional studies had sug- that gested previously the assembly of the IL-6 receptor 13 IbFt ¢ Ii w ]-ii V complex requires the formation of an IL-6-IL-6Ra- gpl 30 heterotrimer which is structurally and topologically I.-t;v M II similar to the one assembled by GH-(GHbp)2 (Savino et al., 1994a,b; Lahm et al., 1995). The experiments here presented might indicate, however, that the functional complex is instead a tetramer, differing from the trimer an additional by binding of a second gpl30 subunit with a 'novel' orientation. Alternatively, and most probably, it is that the functional possible complex has a different structure in which two trimeric complexes of the GH- type cluster close to each other to form a higher order Fig. 7. The sIL-6Rca variant Mutl has lost the ability to dimerize symmetrical structure. To gain further insight into the gpl30. (A) Dimerization of sgpl30 in the presence of sIL-6Ra variant stoichiometry of the IL-6 receptor complex, we designed Mutl. sgpl3O-myc was immobilized on protein A-Sepharose beads immunoprecipitation experiments to test if in vitro com- via anti-myc 9E10 monoclonal antibody and incubated with 50 of Al plexes contain more than one IL-6Ra and/or IL-6 supernatant containing 35S-labelled sgpl3O-FLAG and wild-type or variant sIL-6Ra (Mutl; 500 p1 supernatant) in the molecule. absence and presence of 1 and 10 ,ug of wild-type IL-6, as indicated. After To this end, Sepharose beads were coated with un- incubation and washing, the bound material was subjected to PAGE. labelled myc-tagged sIL-6Ra (via anti-myc monoclonal M, molecular weight marker. (B) Binding of wild-type or variant antibody) and immunoprecipitations were performed in sIL-6Rax (Mutl) in the presence of wild-type and variants of IL-6. the presence of various combinations of IL-6, sgpl30-myc was immobilized on protein A-Sepharose sgpl30 beads via anti- myc 9E10 monoclonal antibody and incubated with sgpl30-FLAG (labelled or unlabelled) and [35S]IL-6Rx. The results (500 supernatant) and 50 ,l of supematant containing 35S-labelled gi (Figure 8A) demonstrate that for IL-6Ra a second wild-type or 1 variant sIL-6Ra (Mutl) in the presence of and 10 ,ug of molecule is also recruited in the receptor complex in the wild-type and variants of IL-6, as indicated. After incubation and presence of the other two components, namely IL-6 washing, the bound material was subjected to PAGE. M, molecular weight and marker. sgp 130. to assess Finally, whether a second IL-6 molecule is also associated with the complex we used an IL-6 variant G.Ciliberto and C.Toniatti, manuscript in preparation). which bears the myc tag at its N-terminus (called hereafter Mutl is able to bind IL-6 with an affinity similar to that IL-6myc). This molecule, produced in Escherichia coli of wild-type IL-6Ra; therefore, the Mut I -wtIL-6 complex and purified to is as homogeneity, biologically active as was tested for binding and its ability to promote dimeriz- wild-type IL-6 (data not shown) and has the advantage of ation of The gp130. results are shown in Figure 7A. being specifically recognized the by anti-myc monoclonal Interestingly, Mut I in combination with wild-type IL-6 still antibody. The usual immunoprecipitation strategy was interacts with gp 130 but displays a selective impairment in followed using cold IL-6myc as the 'first' IL-6 molecule, gpl30 dimerization: this behaviour mirrors that of site 2 in vitro 35S-labelled wild-type IL-6 as the 'second' IL-6 and site 3 IL-6 variants. molecule and various combinations of receptors. As shown According to the GH-(GHbp)2-based trimeric model in Figure 8B, IL-6 also undergoes in vitro dimerization of the IL-6-IL-6Ra-gpl30 interaction, the site defined but only in the presence of both IL-6Ra and 30. gpl In by Mutl in IL-6Rax is projected in the same orientation conclusion, these experiments show that the incubation of (and towards the same gpl30 as site 2 on IL-6 with the extracellular molecule) IL-6. domains of IL-6Ra and gp 130 This implies that gpl30 cannot bind in mode A causes the formation (Figure of complexes in which not only two 6) to the Mutl - wtIL-6 complex, and that the residual gp 130 but also two subunits, IL-6 molecules and two binding is due to gpl30 interactions in the B mode. As IL-6Ra are subunits, present. discussed before, this second gp 130 appeared to be recruited in the with complex the contribution of site 3 in Discussion IL-6. To test this hypothesis we performed immuno- precipitation stimulation of cells experiments combining Mutl with a repre- IL-6 target causes covalent dimeriza- sentative site 2 or site 3 variant. The results are shown in tion of the signal-transducing chain and these gp 130, Figure 7B. In agreement with our the incuba- dimers have been found to be associated predictions, constantly with tion of Mutl with DFRD, which is a site 2 IL-6 functional et variant, receptor complexes (Davis al., 1993; still results in In dimerization binding to the Murakami et 130 has gp130. contrast, although al., 1993). Although gp site 3 T162D variant has normal for on cell membrane and therefore with Muti been observed the binding affinity not formed and show here that this (data shown), the complex Mutl T162D whole receptor molecule, we event by has lost residual to 130: this result is similar is driven the extracellular domains of both the any binding gp by ca 1947 G.Paonessa et aL precipitation reactions distinguished two distinct areas on IL-6 - IL-6 - IL-6 - IL-6 (sites 2 and 3), each of which is responsible for the -- - *Rct *Ra *Ra *Rci independent binding of two gpl30 chains with different - - 130 130 *130 '130 orientations. This provides a molecular explanation for how the cytokine recruits two gpl30 molecules, causes sgp130_- their dimerization and promotes signalling. An important issue is what are the driving forces that bring about this process. Our study provides some important clues. Both site 2 and site 3 on IL-6 are unable to establish stable interactions with gpl30 in the absence sIL-6Ra-t of IL-6Rax. Although we cannot exclude the possibility that these sites on IL-6 are 'activated' by conformational changes upon interaction with IL-6Rcx, it may be that portions of the IL-6Ra surface are annexed, thus extending the interaction surfaces between gpl30 and the IL-6-IL- 6Rx complex. We favour the latter explanation because this is the mechanism in operating receptor assembly of + + + + + 35S-ILL-6 GH, where it has been shown that when this hormone - Rct - - REx binds to its receptor, no major structural rearrangements - 130 - 130 - occur et (Ultsch al., 1994). Experiments using combina- in 2 tions of IL-6 variants site or site 3 and the receptor variant Mutl our choice: site 2 in strengthen IL-6 plus residues 280/281 in IL-6Rx are towards the projected same 130 molecule and form a gp together single combined whereas the other site is of site, composed a combination IL6 -_ w of site 3 and a still unidentified of IL-6Ra. In each part combined site the alteration of either the IL-6 or the IL- causes loss of to the 6Rax component binding facing gpl30 chain. Immunoprecipitation experiments demonstrated that the 8. Dimerization of IL-6Ra and IL-6. Dimerization of IL-6Ra. Fig. (A) receptor complex assembled in vitro contains not only was immobilized on beads via sIL-6Ramyc protein A-Sepharose two gpl30 chains but also two IL-6 and two IL-6Rx 9E10 monoclonal and incubated with the molecules anti-myc antibody indicated on the of each lane in the amounts: 3 molecules. We therefore propose that the active receptor top following IL-6, gg 500 cold and 50 ,ul supematant containing sgpl30-FLAG gl hexamer with an complex is made of a IL-6-IL-6Ra- 35S-labelled supernatant containing receptors. Rax, sIL-6Rax; 130, gpl3O stoichiometry of 2:2:2. Based on previous 3-D Asterisks indicate 35S-labelled material. After sgpl3O-FLAG. in modelling predictions and on the data presented this incubation and the bound material was to PAGE. washing, subjected paper, we present a schematic model for this Dimerization of IL-6. was immobilized on topological (B) IL-6myc protein and A-Sepharose beads via 9E10 monoclonal anti-myc antibody new structure that can be called the 'hexameric IL-6 of each lane in the incubated with the molecules indicated on the top The in receptor complex'. model is shown Figure 9 both tl amounts: 50 35S-labelled 500 following ,l IL-6, supernatant from a side view and two Two projected along planes. containing cold or sIL-6Ra. sgpl3O-FLAG Ra, sIL-6Rax; 130, distinct have indeed to be defined in the hexamer: regions marker. After incubation and sgpl3O-FLAG; M, molecular weight the bound material was to PAGE. the in which the is and the washing, subjected top part cytokine located, bottom formed the second subdomains of the part by and f3 subunits because we CBDs of both and in which direct contacts (IL-6Ra) (gpl30) receptor IL-6Rax gpl30 observe efficient dimerization in the absence of both the are established the chains. by receptor transmembrane and We believe that Although it is possible to consider the hexamer as cytoplasmic regions. these dimers faithfully reproduce what occurs on the cell formed the of two GH-like trimers by clustering juxtaposed surface, because their formation depends on in inverted orientations, we think that this is highly completely the presence of IL-6-IL-6Rax complexes and they are not reductive because we believe that the hexamer is 'held' formed when IL-6 variants with normal binding together by several interaction surfaces, some of which affinity for but low or no have been identified and others of IL-6Rx, biological activity, are used in already which can only place of IL-6. be additional contacts could in wild-type hypothesized. Many fact The design of IL-6 receptor to be used for be at both for direct antagonists envisaged levels, example interaction the treatment of IL-6 disregulation was approached with between the two IL-6 molecules. It is to note interesting the idea that such molecules should possess normal (or that affinity cross-linking experiments on IL-6-responsive improved) IL-6Ra binding but affinity impaired interaction cells with iodinated IL-6 revealed the presence of IL-6 with gpl3O (Brakenhoff et al., 1994; Savino et al., either alone or dimers, associated with an IL-6Roc molecule 1994a,b). Interestingly, our results revealed that IL-6 (Rose-John et al., 1991; D'Alessandro et al., 1993; Stoyan like DFRD receptor antagonists (Savino et al., 1994b) et al., 1993). However, none of our site 2 and site 3 IL-6 function an unusual through mechanism: they are able variants map in the putative cytokine-cytokine contact to recruit a single gp 130 chain but fail to induce its surface because they do not show selective impairment homodimerization, thus lacking signalling capacity. of IL-6 dimerization in immunoprecipitation reactions Furthermore, their use in solution binding/immuno- (G.Paonessa and R.Graziani, unpublished results). From 1948 IL-6 receptor complex assembly Top Fig. 9. Model of the hexameric IL-6 receptor complex. Shown are a side view of the IL-6 hexameric receptor complex (left) and two cross-sections through the complex (right): one that includes the bound cytokine (top), and a second (bottom) formed by the second subdomains of the CBDs of At this level, two asterisks in the IL-6Ra both IL-6Ro and gp130 where only interactions between the receptor molecules are postulated to occur. the in the IL-6Ra variant Mut1. For IL-6, numbers identify the sites indicate hypothetical location of the amino acids H280 and D281 mutagenized of interaction with (sites 1-3). The colour code is blue for green for IL-6RCa and pink for IL-6. For the the various receptor components gpl30, receptors, only the part corresponding to their CBDs is shown. the it could also be expected that gp 130 has at IL- 12 to also as natural model, paration). Interestingly, (referred least two sites of interaction with IL-6 and two with killer cell stimulatory factor), which is by itself always IL-6Ra, and targeted mutagenesis of the extracellular found as a disulfide-linked heterodimer between a 35 kDa region of gpl 30 should be very useful in supporting this. related to IL-6 et al., 1992) and a polypeptide (Merberg Lastly, homodimerization contact surfaces between two 40 kDa related to IL-6Roa (Gearing and polypeptide gp 130 and/or two IL-6Rx could be located at the bottom could also form a 2+2 receptor complex Cosman, 1991), level formed by the second subdomains of the CBDs. with its receptor subunit IL-12R which is strongly homo- While this manuscript was being prepared it was logous to gpl3O (Chua et al., 1994). Because IL-6 binds reported that the incubation of purified IL-6-IL-6Rax first to a receptor chain that does not have any direct role complexes with soluble gp130 produced in CHO cells in cell signalling, it is tempting to speculate that the active gave rise to complexes whose molecular weight is com- cytokine is indeed the IL-6-IL-6Rcx complex; this is also patible only with the formation of hexamers with a 2:2:2 supported by the evidence that soluble forms of IL-6Rax et stoichiometry (Ward et al., 1994). Therefore, identical act as potentiators of cytokine activity (Taga al., 1989) conclusions have been obtained using a different approach. and are present at high levels in the circulation (Honda thus be a common The use of wild-type molecules only did not, however, et al., 1992). Hexamer formation could information about the relationship which evolved to ensure the formation provide any topological biological strategy of the in of sites and various molecules the assembled receptor complex high-affinity binding firmly trap cytokines et and/or factors into active (Ward al., 1994). growth configurations. The hexamer is a novel type of receptor complex first discovered for IL-6. We propose, based on sequence Materials and methods homology data, that hexamers could be a common feature and of the of receptor complexes assembled by other cytokines whose Tagging expression soluble receptors cDNA for soluble forms of at the C- Mutant coding gpl3O tagged are to be specific ligand binding receptors predicted with et or FLAG et terminus myc (Evan al., 1985) (Hopp al., 1988) similar to IL-6Rcx. structurally and functionally Among were double-strand at epitopes generated by inserting oligonucleotides factor et these are ciliary neurotrophic (CNTF) (Davis al., for amino acids 605- the EcoRI site of 130 located around the codons gp and also IL- 11 et in the case of 1991) probably (Hilton al., 1994). 606 The were myc, E(605)- (E-F). resulting C-termini, in the case called and, a FEEQKLISEEDL-Stop (hereafter sgpl3O-myc) Indeed, immunoprecipitations performed using strategy called of sgpl30-FLAG). FLAG, E(605)-FDYKDDDDK-Stop (hereafter similar to that described in this for IL-6 paper strongly for soluble forms of with and without Mutant cDNA coding IL-6Rax the CNTF-CNTF-R that the dimerization of suggest at the C-terminus were PCR suitable myc tag epitope generated by using takes in solution called sIL- complex place (A.De Serio, R.Graziani, In the case of oligonucleotides. untagged sIL-6Ra (hereafter 322. In the case of in a codon was inserted after G.Ciliberto and manuscript pre- 6Ra), stop proline R.Laufer, G.Paonessa, 1949 G.Paonessa et al. sIL-6Rax tagged with myc, myc epitope was inserted after proline (1990) Structure-function analysis of human IL-6. J. Immunol., 145, 322 followed by a stop codon; the resulting C-terminus was P(322)- 561-568. GGEQKLISEEDL-Stop (hereafter called sIL-6Ramyc). The cDNAs of Brakenhoff,J.P.J., de Hon,F.D., Fontaine,V., Hart,M., de Groot,E.R., the soluble receptor variants sgpl3O-myc, sgpl3O-FLAG, sIL-6Rax and Content,J. and Aarden,L.A. (1992) Two different sites on the IL-6 sIL-6Raxmyc were cloned into the BamHI site of the baculovirus molecule are in involved biological activity. In Revel,M. (ed.), IL-6: expression vector pBluBac III (Invitrogen). The generation of and Physiopathology Clinical Potential. Serono Symposium recombinant baculovirus expressing variants of gpl3O and IL-6Ra was Publication, Raven Press, New York, Vol. 88, pp. 33-41. performed with MAXBAC kit (Invitrogen) according to the instructions Brakenhoff,J.P.J., de Hon,F.D., Fontaine,V., Boekel,E.T., Schooltink,H., of the manufacturer. The expression of cold receptors was accomplished Rose-John,S., Heinrich,P.C., Content,J. and Aarden,L.A. (1994) by infecting High Five cells (Invitrogen) with a high titre stock of Development of a human interleukin-6 receptor antagonist. J. Biol. recombinant virus at a m.o.i. of 5-10. After 2 h the inoculum was 86-93. Chem., 269, removed and the cells were incubated at 27°C in SF-900 II medium M. Ribbon models of macromolecules. Mol. Carson, (1987) J. Graphics, (Gibco) until all cells detached from the plate (48-72 h). Medium was 5, 103-106. collected and centrifuged, and the supernatant was assayed by Western et al. of a human IL-12 Chua,A.O. (1994) Expression cloning receptor blot for recombinant protein production. A new member of the with component. cytokine receptor superfamily strong homology to gpl3O. J. Immunol., 153, 128-136. The Cosman,D. (1993) hematopoietin receptor superfamily. Cytokine, 5, of recombinant labelling receptors 135S]Methionine 95-106. Recombinant soluble were labelled to receptors metabolically according D'Alessandro,F., Colamonici,O.R. and Nordan,R.P. (1993) Direct the following protocol. Semi-confluent High Five cells in a 6 cm plate association of interleukin-6 with a 130-kDa component of the were infected (m.o.i. of 5-10) with high titre recombinant baculovirus. interleukin-6 receptor system. J. Biol. Chem., 268, 2149-2153. After 36-48 h, medium was replaced with 2 ml of methionine-free Davis,S., Aldrich,T.H., Valenzuela,D.M., Wong,V., Furth,M.E., Squinto, Grace's insect medium and incubated at for 1 h. The medium was 27°C S.P. and Yancopoulos,G.D. (1991) The receptor for ciliary neurotrophic then replaced with 1.5 ml of methionine-free Grace's insect medium factor. Science, 253, 59-63. 50 of act. >1000 containing TRAN[35S]LABEL (sp. Ci/mmol, gt Davis,S., Aldrich,T.H., Pan,L., Taga,T., Kishimoto,T., Ip,N.Y. Stahl,N., 10 mCi/ml; ICN Biomedical Inc.) and incubated for a further 2 h. and Yancopoulos,G.D. (1993) LIFR, and gpl30 as heterodimerizing Medium was then collected, cell debris centrifuged and the supernatant signal transducers of the tripartite CNTF receptor. Science, 260, used for the experiments. 1805-1808. de Vos,A.M., Ultsch,M. and Kossiakoff,A.A. (1992) Human growth Immunoprecipitations hormone and extracellular domain of its receptor: crystal structure of Immunoprecipitation experiments were typically performed according the complex. Science, 255, 306-312. to the 500 baculovirus following protocols. supernatant expressing gl Ehlers,M., Grotzinger,J., deHon,F.D., Mullberg,J., Brakenhoff,J.P.J., myc-tagged receptors and 5 wild-type IL-6, variant IL-6 or IL-6myc gg and Identification of two novel Liu,J., Wollmer,A. Rose-John,S. (1994) were incubated with 4 anti-myc 9E 10 monoclonal antibody (Evan et al., gu of human IL-6 for and regions responsible receptor binding signal 1985) or 1 ,ug monoclonal antibody IL-6- 16 (CLB, The Netherlands) plus transduction. J. Immunol., 153, 1744-1753. 40 50% in PBS of to hereafter slurry protein A-Sepharose (referred gl and Isolation of Evan,G.I., Lewis,G.K., Ramsay,G. Bishop,J.M. (1985) as at After three times with PAS; Pharmacia) overnight 4°C. washing monoclonal antibodies for human specific proto-oncogene c-myc PBSTB (I X PBS, 0.05% Tween 20, 0.2% Brij 96), the Sepharose beads Mol. Cell. 3610-3616. product. Biol., 5, were incubated with other receptor(s) and/or cytokine (for further details and of the subunit of Gearing,D.P. Cosman,D. (1991) Homology p40 see legends to the respective figures) for at least 12 h at 4°C. The beads Natural Killer Cell Factor with the extracellular Stimulatory (NKSF) were then washed three times with PBSTB, resuspended in SDS loading domain of the interleukin-6 9-10. receptor. Cell, 66, heated for 5 min at and to SDS-PAGE. buffer, 95°C subjected and Hibi,M., Murakami,M., Saito,M., Hirano,T., Taga,T. Kishimoto,T. (1990) Molecular cloning and expression of an IL-6 signal transducer, Generation and of IL-6 variants expression gpl3O. Cell, 63, 1149-1157. Generation and of IL-6 variants were as expression performed exactly et al. of a murine IL-l1 Hilton,D.J. (1994) Cloning receptor tX-chain; 1 amino acid described previously (Savino et al., 994b). For the numbering requirement for gpl 30 for high affinity binding and signal transduction. with at the of IL-6 see Savino et al. (1994a). IL-6 tagged myc epitope EMBO J., 13, 4765-4775. N-terminus (IL-6myc) was generated by PCR using suitable oligonucleo- Honda,M., Yamamoto,S., Cheng,M., Suzuki,H., Yasukawa,K., Saito,T., The tides; it was cloned in pT7.7 vector (Studier et al., 1990). resulting and Human soluble IL-6 Osugi,Y., Tokunaga,T. Kishimoto,T. (1992) sequence of the N-terminus is the following: Met-EQKLISEEDL-wtIL-6, receptor: its detection and enhanced release by HIV infection. J. is valine in where the first amino acid of IL-6 represented by position Immunol., 148, 2175-2180. 2. 35S-labelling of wild-type IL-6 was performed with rabbit reticulocyte Hopp,T.P., Prickett,K.S., Price,V.L., Libby,R.T., March,C.J., Cerretti,D.P., to the instructions. lysate (Promega) according supplier's and short marker Urdal,D.L. Conlon,P.J. (1988) polypeptide useful for recombinant identification and sequence protein purification. In and 1204-1210. vitro receptor binding assays bioassays BiolTechnology, 6, vitro and on and et al. The molecular of human IL-6 In receptor binding assays bioassays HepG2 XG-1 Lahm,A. (1995) design receptor lines were as described Ann. NY Acad. 762. cell performed exactly previously (Savino antagonists. Sci., et and al., 1994b). Merberg,D.M., Wolf,S.F. Clark,S.C. (1992) Sequence similarity between NKSF and the IL-6/G-CSF Immunol. family. Today, 13, 77-78. Nakagawa,T., Yasukawa,K., Murakami,M., Hibi,M., Nakagawa,N., Acknowledgements Yamanishi,K., Taga,T. and Kishimoto,T. (1993) IL-6-induced We thank Anna Maurizio Laufer and Tramontano, Sollazzo, Ralph Janet homodimerization of gpl30 and associated activation of a tyrosine Clench for this and Yves and critically reading manuscript Cully Manuela kinase. Science, 260, 1808-1810. Emili for artwork. Rose-John,S., Hipp,E., Lenz,D., Legres,L.G., Korr,H., Hirano,T., and Structural and functional Kishimoto,T. Heinrich,P.C. (1991) studies on the human interleukin-6 Biol. receptor. J. Chem., 266, 3841-3846. References Savino,R., Lahm,A., Giorgio,M., Cabibbo,A., Tramontano,A. and Ciliberto,G. (1993) Saturation mutagenesis of the human interleukin Bazan,J.F. (1990a) Hemapoietic receptors and helical cytokines. 6 site: receptor-binding Implications for its three-dimensional structure. Immunol. Today, 11, 350-354. Proc. Natl Acad. Sci. USA, 90, 4067-4071. Bazan,J.F. (1990b) Structural design and molecular evolution of a Savino,R., Lahm,A., Ciapponi,L., Sporeno,E., Altamura,S., Salvati,A.L., cytokine receptor superfamily. Proc. Natl Acad. Sci. USA, 87, 6934- Paonessa,G., Toniatti,C. and Ciliberto,G. (1994a) Generation of interleukin-6 receptor antagonists by molecular-modelling guided Bazan,J.F. (1991) Neuropoietic cytokines in the hematopoietic fold. mutagenesis of residues important for activation. EMBO 1., 13, gpl30 197-208. 1357-1367. Neuron, 7, de Di and Brakenhoff,J.P.J., Hart,M., Groot,E.R., Aarden,L.A. Padova,F. Savino,R., Lahm,A., Demartis,A., Ciapponi,L., Cabibbo,A., Toniatti,C., 1950 IL-6 receptor complex assembly and Delmastro.P.. Altamura,S. Ciliberto.G. (1994b) Rational design of a receptor of human interleukin-6. super-antagonist EMBO J., 13, 5863-5870. Sporeno.E.. Paonessa.G.. Salvati.A.L.. Graziani.R.. Delmastro.P., Ciliberto.G. and Toniatti,C. (1994) Oncostatin M binds directly to gp 130 and behaves as interleukin-6 antagonist on a cell line expressing gp130 but lacking functional oncostatin M receptors. J. Biol. Chem., 10991-10995. 269, et al. Association and Stahl,N. (1994) activation of Jak-Tyk kinases by CNTF-LIF-OSM-IL-6 beta receptor components. Sc ienice. 263. 92-95. StoyanT.. Van Michaelis,U., Schooltink.H., Dam.M.. Rudolph.R., Heinrich,P.C. and (1993) Recombinant soluble Rose-John,S. interleukin-6 receptor: expression in Escherichia c o/i. renaturation and purification. Elur J. Biochem.. 216, 239-245. Studier.F.W., Rosemberg.A.H.. and Dunn,J.J. Dubendorff,J.W. (1990) Use of T7 RNA polymerase to direct expression of cloned genes. Methods Encviznol., 185, 60-89. Taga,T.. Hibi,M., Hirata.Y., Yamasaki,K., Matsuda.T., Yasukawa,K._ Hirano.T. and Kishimoto.T. (1989) Interleukin-6 triggers the association of its receptor with a possible signal transducer, gpl30. Cell, 58. 573-581. Ultsch.M.H., Somers,W., Kossiakoff.A.A. and de (1994) The Vos,A.M. crystal structure of affinity-maturated human Growth Hormone at 2 resolution. J. Mol. Biol., 236, 286-299. Van Snick,J. (1990) Interleukin-6: an overview. Aon)lu. Rev. Immuidnol., 8. 253-278. Howlett.G.J.. Ward,L.D., Discolo,G., Yasukawa,K.. Hammacher.A.. Moritz.R. and Simpson.R.J. (1994) High affinity interleukin-6 receptor is a hexameric complex consisting of two molecules each of interleukin-6 and gp- 130. interleukin-6, receptor, J. Biol. Clietz., 269. 23286-23289. Yamasaki.K.. Yawata.H., Taga,T.. Hirata,Y., Kawanishi.Y.. Seed.B.. Hirano.T. and TaniguchiT., Kishimoto,T. (1988) Cloning and expression of the human interleukin-6 receptor. (BSF-2/IFNP2) Science, 241, 825-828. Yawata,H., Yasukawa,K., Shunji,N., Murakami,M.. Yamasaki,K., Hibi,M., Taga,T. and (1993) Structure-function Kishimoto,T. analysis of human IL-6 receptor: dissociation of amino acid residues required for IL-6 binding and for IL-6 signal transduction 130. through gp EMBO J.. 1705-1712. Wen.Z. and Zhong.Z., Darnell,J.E.,Jr (1994) Stat3: a STAT family member activated by tyrosine phosphorylation in response to epidermal factor growth and interleukin-6. Science. 95-98. 264, Received oni JanLuarv 20, 1995 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals

Two distinct and independent sites on IL‐6 trigger gp 130 dimer formation and signalling.

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Springer Journals
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Copyright © European Molecular Biology Organization 1995
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0261-4189
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1460-2075
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10.1002/j.1460-2075.1995.tb07186.x
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Abstract

The EMBO Journal vol.14 no.9 pp.1942-1951, 1995 Two distinct and independent sites on IL-6 trigger gpl3O dimer formation and signalling IL-6Ra (also referred to as the and Giacomo Rita ax subunit) gpl 30 (also Paonessa1, Graziani, referred to as the B subunit). IL-6Ra specifically binds Anna De Serio, Rocco Savino, IL-6 at a low relatively affinity (10-9 M; Yamasaki et al., Armin Laura Ciapponi, Lahm, 1988). The IL-6-IL-6Rax complex associates with gpl3O Anna Laura Salvati, Carlo Toniatti and at high affinity (10-11 M) thus promoting signal transduc- Gennaro Ciliberto tion inside the cell (Hibi et al., 1990). Both Istituto di Ricerche di Biologia Molecolare IL-6Rax and gpl30 belong to the haematopoietin (IRBM), P.Angeletti, Via Pontina Km 30 600, 1-00040 Pomezia, Rome, Italy receptor superfamily that includes, among others, the growth hormone (GH) receptor (GHbp) (Cosman, 1993). author 'Corresponding These receptors all share sequence and structural similarities in a 220 amino acid region called the cytokine The helical cytokine interleukin 6 and its (IL) specific binding domain (CBD), located in the extracellular subunit IL-6Ra form a 1:1 portion binding complex which, by of the molecule all the corres- promoting homodimerization of the signalling subunit (Bazan, 1990b). Moreover, ponding cytokines are predicted to fold as a bundle of gpl3O on the surface of intra- target cells, triggers four a-helices cellular We (Bazan, 1990a, 1991). responses. expressed differently tagged From and structural similarities with the GH forms of and used them in sequence gpl3O solution-phase binding receptor whose structure has been solved X- to show that the soluble extracellular domains complex, by assays Vos et we built a of dimerization in the absence of ray crystallography (de al., 1992), gpl3O undergo 3-D model of the membranes. In vitro reactions were computer-assisted IL-6-IL-6Ra-gp 130 receptor assembly interaction et Lahm et In also performed in the of two sets of IL-6 (Savino al., 1994a,b; al., 1995). presence the was variants amino acid substitutions in two analogy with GH, IL-6 receptor complex envisaged carrying as a heterotrimer in which a molecule distinct areas of the surface single IL-6 bridges cytokine (site 2, comprising In the residues in the A and C and site in the IL-6Rax and the gpl3O CBDs. this complex, exposed helices, 3, with is formed the terminal of the CD The IL-6 contact surface IL-6Rax by residues part loop). binding affinity D AB to IL-6Ra of these variants is normal but their bio- in the putative helix and loop (site 1), whereas its here in A logical activity is poor or absent. We demonstrate binding site to gpl30 constitutes exposed residues the that both the site 2 and site 3 IL-6 variants and C helices (site 2; Savino et al., complexed 1994a,b). Furthermore, with bind a molecule but are residues in the E2 strand and AB2 of the CBDs of IL-6Rax single gpl3O loop unable to dimerize whereas the combined site 2/3 gpl30 and IL-6Rax form a third interface et it, (Yawata al., with The variants lose the to interact 1993; Lahm et al., 1995). The of the model ability gpl3O. reliability in binding properties of these variants vitro, and the was tested site-directed of both IL-6 and by mutagenesis monoclonal result of using a neutralizing antibody IL-6Rax; variants amino acid substitutions of bearing lead to the conclusion that directed against site 3, residues to be at or postulated receptor-cytokine is formed direct at two gpl3O dimer through binding interfaces confirmed the receptor-receptor phenotypes and oriented sites on IL-6. independent differently predicted from the model et (Savino al., 1994a,b). further reveal that Immunoprecipitation experiments This heterotrimeric model does not, however, explain the assembled is of fully receptor complex composed additional A event in the findings. key IL-6 signalling two IL-6, two IL-6Ra and two molecules. We gpl3O cascade is the dimerization of the 30 chain gpl (Murakami propose here a model representing the IL-6 receptor et that activates associated al., 1993) (i) tyrosine kinases complex as hexameric, which might be common to of the Jak and the family (ii) phosphorylation of a set other helical cytokines. of intracellular the substrates, including intracytoplasmic Key words: gp13O dimer/IL-6 receptor antagonist/IL-6 domain of and gpl30 transcription factors of the STAT receptor complex/IL-6Ra et et family (Stahl al., 1994; Zhong al., 1994). These observations have led to the conclusion that a second gpl30 molecule in the participates active IL-6 receptor complex. Introduction Additional information on the complexity of the IL-6 Interleukin (IL) 6 is a multifunctional cytokine that acts receptor complex has emerged from the recently developed on different target cells to induce a variety of biological IL-6 receptor antagonists. Different strategies have been responses (Van Snick, 1990). Transmembrane glyco- adopted to design these IL-6 variants, aimed at generating that form proteins the IL-6 receptor complex have been molecules that can still effectively bind IL-6Ra but can identified and cloned (Yamasaki et al., 1988; Hibi et al., carry amino acid substitutions that impair the activation 1990). Two membrane proteins are both necessary and of gpl3O by the IL-6-IL-6Rcx complex. Interestingly, sufficient to constitute a functional receptor complex: two strategies developed independently produced IL-6 1942 assembly IL-6 receptor complex antagonists with the expected properties by mutating two A C. B C.t different portions of the IL-6 four-helix bundle. The first is a patch of four exposed residues in the A and portion e M C helices, which we called site 2 (Savino et al., 1994a,b) because its location is similar to site 2 in GH. The second is formed by residues located at the beginning of the D helix and AB loop [referred to as site II by Brakenhoff sgpl3O -72 kDa _ et al. (1994) and Ehlers et al. (1994)], which we call site 3. The location of site 3 does not fit into the GH paradigm. To investigate further the unique properties of the IL-6 sIL-6Rt_ v receptor complex, we developed a very sensitive assay =~40 kDa ^ 4 ~~~ to analyse the biochemical steps of receptor assembly. Differentially tagged versions of soluble gpl30 and IL-6Rax receptors are expressed in insect cells and are reactions with wild-type and used in immunoprecipitation IL-6Rx molecules. Our results indicate variant IL-6 and anti-FLAG anti-myc that the IL-6 -IL-6Rx complex binds two gp 130 molecules in insect cells. (A) Western Fig. 1. Production of sgpl3O and sIL-6Ra binding sites. Furthermore, at two distinct and independent Five cells infected with blotting of supernatants (50 of High gl) that two IL-6, two IL-6Rx and two we provide evidence sgpl30-FLAG, sIL-6Ramyc and recombinant baculovirus expressing are present in the final complex, and gp130 molecules 1 monoclonal antibodies. M, 30-myc and stained with their specific sgp 35S metabolically labelled on their topological location in this putative molecular weight marker. (B) In vitro give details in insect cells. M, sgpl3O-FLAG and sIL-6Ra (15 expressed is presented which reconciles this 'hexamer'. A model gl) molecular weight marker. that of the trimeric GH receptor complex structure with that a similar assembly process might also and suggests sIL-6Ra were estimated to be 1- for other helical cytokines. forms of sgpl 30 and of take place 5-7 mgIl for sIL-6Rcx after affinity 2 mg/l for sgp 130 and unpublished observations). purification (G.Paonessa, Results the gpl 30 dimerization assay is The strategy adopted for 2A. In a typical experiment, unlabelled An in vitro assay for gpl30 homodimerization depicted in Figure was immobilized on protein A-Sepharose Homodimerization of gpl 30 has been shown to take place sgpl30-myc with the anti-myc monoclonal anti- on the surface of cells exposed to IL-6 (Murakami et al., beads coated specific immunoprecipitations were per- To test whether gpl30 requires the transmembrane body. After washing, 1993). of various combinations of IL-6, and intracytoplasmic regions to dimerize or, on the con- formed in the presence is an intrinsic property of the extracellular domain, IL-6Rax (labelled or unlabelled) and [35S]gpl30-FLAG. trary, latter is not recognized by the anti-myc we decided to set up an in vitro dimerization assay. Because the its immunoprecipitation can only be To do this, we expressed gpl30 in its soluble form monoclonal antibody, that dimers had been formed. truncated at the predicted junction between explained by assuming gpl30 (sgp 130), this experiment are shown in Figure 2B, extracellular and transmembrane domains, and modified The results of two different epitope tags: the efficient in vitro formation of gpl30 dimers at the C-terminus so as to carry where (Evan et al., 1985) and was confirmed by the immunoprecipitation of labelled sgpl3O-myc carrying a myc tag with a FLAG tag (Hopp et al., 1988). This by anti-myc antibodies. As expected, dimers sgpl 30-FLAG gpl 30-FLAG sgpl30 molecules recognizable were only formed in the presence of both IL-6 and IL- makes the different antibodies. Likewise, two 6Ra. IL-6Rax was immunoprecipitated only in by specific monoclonal Likewise, forms of IL-6Ra were generated, one the of IL-6 (Figure 2B). recombinant soluble presence residue 322 of the extracellular domain, the truncated at to the same C-terminal Dimerization of gp 130 is triggered by two other with a myc tag attached sites on IL-6 encoded their natural leader peptide independent binding residue. All cDNAs have been which act as cDNAs were inserted in the Recently, IL-6 variants generated sequence. The modified et al., 1994; Savino et al., vector under the baculovirus polyhedrin receptor antagonists (Brakenhoff recombination amino acid substitutions were Recombinant were transfected in Sf9 1994a,b). In these variants, promoter. plasmids distinct areas of the IL-6 surface, as and the recombinant viruses expressing the various introduced in two cells the 3-D model shown in Figure 3. These were and amplified. Recombinant recep- predicted by receptors purified can be subdivided into two classes: (i) by infecting insect cells in serum-free mutant proteins tors were produced 2 mutations in helix A and C, and medium. were assayed by Western blot for site variants, bearing Supematants at the of bearing mutations beginning recombinant production (Figure IA). Individual (ii) site 3 variants, protein of two distant sites on IL-6 with helix D. The identification were metabolically labelled [35S]methionine receptors in transduction, and low compared with similar roles promoting signal at high specific activity background evidence that the subunit 1 Receptors expressed in with the signalling with other proteins (Figure B). together that these a molecular weight of -40 kDa for sIL- dimerization, immediately suggest insect cells show undergoes sites for the two 130 gp kDa for that these molecules sites are independent binding 6Rax and 72 gp 130, suggesting subunits. to modifications but to a are subjected post-translation we decided to evaluate their test this than when in mammalian cells To hypothesis lesser extent expressed 30 and dimerize it in vitro. to bind baculovirus The levels of the two ability sgpl et al., 1994). expression (Sporeno 1943 G.Paonessa et al. 7 :t 1? 441 II 11 t' "1411 l { i Z i f , ^ 0l -'_ suI) J)iflercnt conlhina<tionr -. ,I .-.oR. " N .41 ._- 1) s.!li tilt - II..h RK H.- low Illctithation sh S1.)S IPAGCLF." is Fig. 2. sgpl30 is able to dimerize in vitro in the presence of IL-6-sIL-6Rax. (A) Experimental strategy. sgpl3O-myc attached to protein and incubated with different combinations of and The A-Sepharose beads via anti-myc 9E10 monoclonal antibody IL-6, sIL-6RaX sgpl30-FLAG. use of a 35S-labelled form of a chain. After incubation and participation of a specific molecule in the complex is detected by the specific receptor is with buffer and to PAGE. The has been washing of the Sepharose beads, the bound material eluted SDS loading subjected (B) experiment shown in Cold attached on beads was incubated with the performed according to the experimental strategy (A). sgpl30-myc protein A-Sepharose of each lane in the amounts: 3 500 cold and 50 molecules indicated on top following tg IL-6, supernatant containing sIL-6Roa supernatant Plt gl Asterisks indicate 35S-labelled material. containing 35S-labelled receptors. Rx, slL-6Rax; 130, sgpl3O-FLAG. the site 2 variants we selected the four amino From acid substitution Y31 D/G35F/S 118RNV 121 also called NH2 D, which acts as a full IL-6 on a DFRD, antagonist variety of human cell lines et For site the (Savino al., 1994b). 3, variant was described which also has low Q159E/T162P and acts as a on some biological activity partial antagonist but not all human cell lines et To (Brakenhoff al., 1994). avoid residues which affect introducing proline might or we two new variants folding stability, generated (WI57R/Dl60R and T162D) which map in the same area and have similar to The properties Q159E/T162P. in this are properties of all the IL-6 variants used study in vitro to summarized in Table I, which reports binding measured ELISA et sIL-6Rcx as by (Savino al., 1994a) on two different human cell lines and biological activity with different of to human IL-6. Both degrees sensitivity site 2 and site variants have normal to 3 binding affinity as well as a IL-6Rax, strongly impaired biological activity. To measure to beads coated binding gpl30, Sepharose with anti-myc monoclonal antibody were incubated with the myc-tagged preparation of sIL-6Rx and then used to co-immunoprecipitate labelled sgp 130-FLAG in the presence of the wild-type or mutant IL-6 molecule. All three variants (DFRD, W157R/D160R and T162D) with an -2- immunoprecipitated sgp130 efficiency only to 4-fold lower than wild-type IL-6 (Figure 4A). This result differs from that reported previously with a different site 2 variant 31D/35F using Chinese hamster ovary Fig. 3. Location of putative receptor binding sites on the 3-D model of human IL-6. Schematic RIBBONS (Carson, 1987) representation of (CHO)-derived sgp130, where a stronger defect in gp130 the IL-6 model (Savino et al., 1994a) showing the three sites involved binding was observed (Savino et al., 1994a). This discrep- in receptor interaction: site 1 (blue), the site of interaction with ancy has also been observed consistently with the site 2 IL-6Rx around on Arg179 helix D (also including parts of the AB and site 3 variants used in this study (results not shown). Savino et site 2 loop; al., 1993); (red), residues from helix A and C, to be the site of We believe this is due to a significant difference in receptor postulated interaction with one gp130 molecule and required for signalling (Savino et al., 1994a,b); site 3 (green), centred concentration, whereby the use of baculovirus sgp 130 around the N-terminal end of helix D, important for signalling and reveals residual binding of these variants that cannot be also originally identified as responsible for binding (Brakenhoff gpl30 detected under the limiting conditions imposed by the use et al., 1994). For site 3, only the IL-6 residues mutagenized in this of the CHO-derived sgpl30. study are shown. 1944 IL-6 receptor complex assembly Table I. Receptor binding and biological activity of the IL-6 variants described in the text IL-6 variants sIL-6Rc binding Biological activity (% of wild-type) (% of wild-type) XG-1 (myeloma cells) HepG2 (hepatoma cells) wt IL-6 100 100 DFRD ± 97 15 <0.01 <0.01 157R/160R ± ± 220 + 51 6 4 2.9 0.2 162D 104 18 8 3 1.3 0.3 DFRD/157R/160R ± 160 40 <0.01 <0.01 DFRD/162D 45 ± 7 <0.01 <0.01 Binding to the sIL-6Rx was determined as described (Savino et al., 1994a). The IL-6 biological measured activity on the XG-1 myeloma cell line is the stimulation of cell replication (Savino et al., 1994b). The IL-6 biological activity measured on the cell line is the of HepG2 hepatoma activation the transfected IL-6-inducible C-reactive protein gene promoter (Savino et al., 1994b). The biological activity of each variant (expressed as percentage of wild-type IL-6 activity) on both cell lines was determined as the ratio between the concentration of IL-6 and the wild-type concentration of variant IL-6 necessary to give 50% of wild-type IL-6 maximal stimulation. 157R 160R 162DI l)FRD DFRD 157R160R 162D DFRD wtIL-6 no .*-sgpt30 157R'16(0H.t 162D DFRD DFRD 157R 160R 162D wt IL-6 DFRD no I -iiIIf. .Zz. Lfi.~.LI LfIZz,,I.L-6 M 4- sgpl30 Fig. 4. gpl 30 binding and dimerization of wild-type and IL-6 variants. (A) Binding of sgpl30 to wild-type and variants of IL-6 complexed with sIL-6Rax. sIL-6Ramyc was immobilized on protein A-Sepharose beads via anti-myc 9E10 monoclonal antibody and incubated with 35S-labelled sgpl30-FLAG (50 in 1 supematant) the presence of increasing concentrations (10 gg, ,ug, 100 ng and 10 ng) of wild-type or variant IL-6, as gl indicated on of the top lanes. After incubation and washing, the bound material was subjected to PAGE. M, molecular weight marker. (B) Dimerization of sgpl30 in the presence of wild-type and variants of IL-6. sgpl3O-myc was immobilized on protein A-Sepharose beads via anti- myc 9E10 monoclonal antibody and incubated with 35S-labelled sgpl30-FLAG (50 ,tl supernatant) in the presence of cold sIL-6Ra (500 ,l supernatant) and 1 increasing concentrations (10 and 100 ng) of wild-type or variants of IL-6, as indicated. After incubation and washing, the gg, gg bound material was subjected to PAGE. M, molecular weight marker. When the dimerization assay was performed using the mutation involving four amino acid residues is to likely protocol described in the previous all abolish the section, the IL-6 completely interaction with the subunit gp130 variants showed a selective and dramatic decrease (Figure binding at site 2, while the mutations W1 leaky 57R/ in 4B), precise parallel with their impairment of D160R and T162D do not bioactivity fully impair the interaction of In (Table I). fact, DFRD, which does not residual the at possess respective gpl30 subunits binding site 3. bioactivity and is a full antagonist (Savino et the two areas of the IL-6 surface al., 1994b), Apparently, affected does not dimerize whereas gpl3O at all, variants W157R/ by these mutations behave as independent gpl30 binding Dl 60R T 1 and 62D show a residual activity of -3 and 1% sites, because both site 2 and site 3 variants are able to in cells and are respectively HepG2 reduced in 130 bind one gpl30 molecule To this gp efficiently. analyse dimerization to -6 and 4% From these results two more variants simultaneous mutations respectively. further, bearing we can conclude that both site 2 and site 3 while at sites 2 and 3 were variants, generated (DFRD/W157R/D160R still the to recruit in the and These variants maintain normal IL- maintaining capability gpl30 DFRD/T162D). presence of IL-6Ra, cannot form 130 dimers. This 6Ra as are gp binding (Table I) and, expected, biologically What for finding explains the lack of has inactive. When tested with biological activity. sgpl30 binding, although DFRD variants to be taken into is that the only slight both were almost com- account, however, differences, 1945 et aL G.Paonessa wt IL-6 'l:I I I:,sI:Iii 1.i:3 Ir '10 - !N O I;0 1 'r '1 _. a- am ,lb v - - -W *GM mode A + B Fig. 5. Immunoprecipitation with mAb 16 of complexes formed with Site 3 mutant Site 2 mutant wild-type or variant IL-6. Wild-type or variant IL-6 (as indicated on of the lanes) was immobilized on protein A-Sepharose beads top incubated with the receptor molecules in the using mAb 16 and amounts: 500 of containing cold sIL-6Raz and following gl supematant 50 of 5S-labelled receptors. Rax, sIL-6Rax; ,ul supernatant containing 130, sgpl3O-FLAG; M, molecular weight marker. Asterisks indicate 35S-labelled material. After incubation and washing, the bound material was subjected to PAGE. mode A pletely unable to bind sgpl30 (Figure 4A). The residual binding can be explained if we assume that residual still occurs at site 3 as discussed above. As mode B binding these variants did not dimerize gpl30 expected, of A and B modes of interaction of Fig. 6. Schematic representation (Figure 4B). the IL-6Rax-IL-6 with molecules. complex gpl30 The 'two sites' hypothesis was tested independent further the anti-IL-6 monoclonal antibody using specific variant shows no residual to 30. Most mAb 16 et al., 1990). mAb 16 has been DFRD binding sgpl (Brakenhoff both sites are unavailable for this interaction, shown to inhibit the biological activity of probably, previously cell surface 2 because of the mutation and site 3 because of the IL-6 without with its binding site interfering led to the that hindrance due to the bound molecule. et This hypothesis steric antibody (Brakenhoff al., 1992). with but In conclusion, by using a variety of tools it was possible mAb 16 impairs the interaction of IL-6 gp130 modes of interaction of mAb 16 was to two not with IL-6Rcx. The epitope recognized by distinguish independent in between and and 30 with the IL-6- IL-6Roa complex (outlined Figure localized within the region Q154 T162, gpl oriented in a similar for the of site 3 In mode A one chain is this information was used development 6). gpl3O chain in the GH et fashion to the second GHbp receptor IL-6 receptor antagonists (Brakenhoff al., 1992, 1994). in mode B the second we that if IL-6 complex (de Vos et al., 1992); In the light of these findings, speculated on and is oriented sites for site 2 gp 130 chain binds site 3 IL-6 differently presents two independent binding gpl30, of mAb 16 could block site to one side of the IL-6-IL-6Ra complex (Figure 6). and site 3, the binding only IL-6 both modes 2 free to interact with another gp 130 In the presence of wild-type operate 3, leaving site and lead to homodimerization. molecule. simultaneously gpl30 we immunoprecipitations with Therefore performed in E2 of the mAb 16 or mutant IL-6, and tested their A variant the strand cytokine binding using wild-type labelled IL-6Ra of is in 130 to bind and immunoprecipitate domain IL-6Rla selectively impaired gp ability that of the site 3 dimerization and sgpl30. Initial results showed the mAb 16 Previous with human IL-6Ra mutations, W157R/D160R destroys epitope mutagenesis experiments still be the some information about the composition of the whereas T162D can recognized by antibody provided interface (Yawata et al., 1993). Out of a (data not shown). IL-6Rx-gpl3O limited subset of As shown in Figure 5, all the variants tested bind sIL- set of receptor variants, only a large in was able to in with the observation that amino acid substitutions the IL-6Ra CBD 6Rax efficiently, agreement to while the mAb 16 does not interfere with this interaction. Moreover, selectively reduce binding gpl30 leaving still to interact efficiently intact. The of these mutations mAb 16 allowed wild-type IL-6 affinity for IL-6 majority its co-immunoprecipitation. Not in the E2 strand and AB2 loop of subdomain 2 of with sgpl30, promoting map this interaction took place only in the presence the CBD, which is also the location of the binding surprisingly, receptor of IL-6Ra, thus excluding the possibility that the neutraliz- interface between the two GH receptor chains in the ing effect of this monoclonal antibody is due to a complete GH-(GHbp)2 complex (de Vos et al., 1992; Yawata et al., the IL-6-IL-6Ra inhibition of gpl30 interaction with 1993). Based on these findings we produced an IL-6Ra the effect of mAb 16 is due variant two substitutions in the E2 strand (H280S/ complex. Hence, biological carrying to a selective inhibition of gpl130 dimerization. Indeed, 1 which was called Mutl. The construction of Mut 1 D28 V) while the site 3-type T162D variant is still able to bind and the characterization of its biochemical properties will in the presence of mAb 16, the site 2-type be described elsewhere (A.L.Salvati, A.Lahm, G.Paonessa, sgpl3O 1946 IL-6 receptor complex assembly :\ -II t:g-t e~~~~~~Ii -f;it to that obtained when the used is complex formed by wild-type IL-6Rca and a combined IL-6 site 2/3 variant -------- (Figure 4B). The IL-6 receptor complex is a hexamer which contains two IL-6, two IL-6Ra and two gpl30 molecules Structural comparisons and functional studies had sug- that gested previously the assembly of the IL-6 receptor 13 IbFt ¢ Ii w ]-ii V complex requires the formation of an IL-6-IL-6Ra- gpl 30 heterotrimer which is structurally and topologically I.-t;v M II similar to the one assembled by GH-(GHbp)2 (Savino et al., 1994a,b; Lahm et al., 1995). The experiments here presented might indicate, however, that the functional complex is instead a tetramer, differing from the trimer an additional by binding of a second gpl30 subunit with a 'novel' orientation. Alternatively, and most probably, it is that the functional possible complex has a different structure in which two trimeric complexes of the GH- type cluster close to each other to form a higher order Fig. 7. The sIL-6Rca variant Mutl has lost the ability to dimerize symmetrical structure. To gain further insight into the gpl30. (A) Dimerization of sgpl30 in the presence of sIL-6Ra variant stoichiometry of the IL-6 receptor complex, we designed Mutl. sgpl3O-myc was immobilized on protein A-Sepharose beads immunoprecipitation experiments to test if in vitro com- via anti-myc 9E10 monoclonal antibody and incubated with 50 of Al plexes contain more than one IL-6Ra and/or IL-6 supernatant containing 35S-labelled sgpl3O-FLAG and wild-type or variant sIL-6Ra (Mutl; 500 p1 supernatant) in the molecule. absence and presence of 1 and 10 ,ug of wild-type IL-6, as indicated. After To this end, Sepharose beads were coated with un- incubation and washing, the bound material was subjected to PAGE. labelled myc-tagged sIL-6Ra (via anti-myc monoclonal M, molecular weight marker. (B) Binding of wild-type or variant antibody) and immunoprecipitations were performed in sIL-6Rax (Mutl) in the presence of wild-type and variants of IL-6. the presence of various combinations of IL-6, sgpl30-myc was immobilized on protein A-Sepharose sgpl30 beads via anti- myc 9E10 monoclonal antibody and incubated with sgpl30-FLAG (labelled or unlabelled) and [35S]IL-6Rx. The results (500 supernatant) and 50 ,l of supematant containing 35S-labelled gi (Figure 8A) demonstrate that for IL-6Ra a second wild-type or 1 variant sIL-6Ra (Mutl) in the presence of and 10 ,ug of molecule is also recruited in the receptor complex in the wild-type and variants of IL-6, as indicated. After incubation and presence of the other two components, namely IL-6 washing, the bound material was subjected to PAGE. M, molecular weight and marker. sgp 130. to assess Finally, whether a second IL-6 molecule is also associated with the complex we used an IL-6 variant G.Ciliberto and C.Toniatti, manuscript in preparation). which bears the myc tag at its N-terminus (called hereafter Mutl is able to bind IL-6 with an affinity similar to that IL-6myc). This molecule, produced in Escherichia coli of wild-type IL-6Ra; therefore, the Mut I -wtIL-6 complex and purified to is as homogeneity, biologically active as was tested for binding and its ability to promote dimeriz- wild-type IL-6 (data not shown) and has the advantage of ation of The gp130. results are shown in Figure 7A. being specifically recognized the by anti-myc monoclonal Interestingly, Mut I in combination with wild-type IL-6 still antibody. The usual immunoprecipitation strategy was interacts with gp 130 but displays a selective impairment in followed using cold IL-6myc as the 'first' IL-6 molecule, gpl30 dimerization: this behaviour mirrors that of site 2 in vitro 35S-labelled wild-type IL-6 as the 'second' IL-6 and site 3 IL-6 variants. molecule and various combinations of receptors. As shown According to the GH-(GHbp)2-based trimeric model in Figure 8B, IL-6 also undergoes in vitro dimerization of the IL-6-IL-6Ra-gpl30 interaction, the site defined but only in the presence of both IL-6Ra and 30. gpl In by Mutl in IL-6Rax is projected in the same orientation conclusion, these experiments show that the incubation of (and towards the same gpl30 as site 2 on IL-6 with the extracellular molecule) IL-6. domains of IL-6Ra and gp 130 This implies that gpl30 cannot bind in mode A causes the formation (Figure of complexes in which not only two 6) to the Mutl - wtIL-6 complex, and that the residual gp 130 but also two subunits, IL-6 molecules and two binding is due to gpl30 interactions in the B mode. As IL-6Ra are subunits, present. discussed before, this second gp 130 appeared to be recruited in the with complex the contribution of site 3 in Discussion IL-6. To test this hypothesis we performed immuno- precipitation stimulation of cells experiments combining Mutl with a repre- IL-6 target causes covalent dimeriza- sentative site 2 or site 3 variant. The results are shown in tion of the signal-transducing chain and these gp 130, Figure 7B. In agreement with our the incuba- dimers have been found to be associated predictions, constantly with tion of Mutl with DFRD, which is a site 2 IL-6 functional et variant, receptor complexes (Davis al., 1993; still results in In dimerization binding to the Murakami et 130 has gp130. contrast, although al., 1993). Although gp site 3 T162D variant has normal for on cell membrane and therefore with Muti been observed the binding affinity not formed and show here that this (data shown), the complex Mutl T162D whole receptor molecule, we event by has lost residual to 130: this result is similar is driven the extracellular domains of both the any binding gp by ca 1947 G.Paonessa et aL precipitation reactions distinguished two distinct areas on IL-6 - IL-6 - IL-6 - IL-6 (sites 2 and 3), each of which is responsible for the -- - *Rct *Ra *Ra *Rci independent binding of two gpl30 chains with different - - 130 130 *130 '130 orientations. This provides a molecular explanation for how the cytokine recruits two gpl30 molecules, causes sgp130_- their dimerization and promotes signalling. An important issue is what are the driving forces that bring about this process. Our study provides some important clues. Both site 2 and site 3 on IL-6 are unable to establish stable interactions with gpl30 in the absence sIL-6Ra-t of IL-6Rax. Although we cannot exclude the possibility that these sites on IL-6 are 'activated' by conformational changes upon interaction with IL-6Rcx, it may be that portions of the IL-6Ra surface are annexed, thus extending the interaction surfaces between gpl30 and the IL-6-IL- 6Rx complex. We favour the latter explanation because this is the mechanism in operating receptor assembly of + + + + + 35S-ILL-6 GH, where it has been shown that when this hormone - Rct - - REx binds to its receptor, no major structural rearrangements - 130 - 130 - occur et (Ultsch al., 1994). Experiments using combina- in 2 tions of IL-6 variants site or site 3 and the receptor variant Mutl our choice: site 2 in strengthen IL-6 plus residues 280/281 in IL-6Rx are towards the projected same 130 molecule and form a gp together single combined whereas the other site is of site, composed a combination IL6 -_ w of site 3 and a still unidentified of IL-6Ra. In each part combined site the alteration of either the IL-6 or the IL- causes loss of to the 6Rax component binding facing gpl30 chain. Immunoprecipitation experiments demonstrated that the 8. Dimerization of IL-6Ra and IL-6. Dimerization of IL-6Ra. Fig. (A) receptor complex assembled in vitro contains not only was immobilized on beads via sIL-6Ramyc protein A-Sepharose two gpl30 chains but also two IL-6 and two IL-6Rx 9E10 monoclonal and incubated with the molecules anti-myc antibody indicated on the of each lane in the amounts: 3 molecules. We therefore propose that the active receptor top following IL-6, gg 500 cold and 50 ,ul supematant containing sgpl30-FLAG gl hexamer with an complex is made of a IL-6-IL-6Ra- 35S-labelled supernatant containing receptors. Rax, sIL-6Rax; 130, gpl3O stoichiometry of 2:2:2. Based on previous 3-D Asterisks indicate 35S-labelled material. After sgpl3O-FLAG. in modelling predictions and on the data presented this incubation and the bound material was to PAGE. washing, subjected paper, we present a schematic model for this Dimerization of IL-6. was immobilized on topological (B) IL-6myc protein and A-Sepharose beads via 9E10 monoclonal anti-myc antibody new structure that can be called the 'hexameric IL-6 of each lane in the incubated with the molecules indicated on the top The in receptor complex'. model is shown Figure 9 both tl amounts: 50 35S-labelled 500 following ,l IL-6, supernatant from a side view and two Two projected along planes. containing cold or sIL-6Ra. sgpl3O-FLAG Ra, sIL-6Rax; 130, distinct have indeed to be defined in the hexamer: regions marker. After incubation and sgpl3O-FLAG; M, molecular weight the bound material was to PAGE. the in which the is and the washing, subjected top part cytokine located, bottom formed the second subdomains of the part by and f3 subunits because we CBDs of both and in which direct contacts (IL-6Ra) (gpl30) receptor IL-6Rax gpl30 observe efficient dimerization in the absence of both the are established the chains. by receptor transmembrane and We believe that Although it is possible to consider the hexamer as cytoplasmic regions. these dimers faithfully reproduce what occurs on the cell formed the of two GH-like trimers by clustering juxtaposed surface, because their formation depends on in inverted orientations, we think that this is highly completely the presence of IL-6-IL-6Rax complexes and they are not reductive because we believe that the hexamer is 'held' formed when IL-6 variants with normal binding together by several interaction surfaces, some of which affinity for but low or no have been identified and others of IL-6Rx, biological activity, are used in already which can only place of IL-6. be additional contacts could in wild-type hypothesized. Many fact The design of IL-6 receptor to be used for be at both for direct antagonists envisaged levels, example interaction the treatment of IL-6 disregulation was approached with between the two IL-6 molecules. It is to note interesting the idea that such molecules should possess normal (or that affinity cross-linking experiments on IL-6-responsive improved) IL-6Ra binding but affinity impaired interaction cells with iodinated IL-6 revealed the presence of IL-6 with gpl3O (Brakenhoff et al., 1994; Savino et al., either alone or dimers, associated with an IL-6Roc molecule 1994a,b). Interestingly, our results revealed that IL-6 (Rose-John et al., 1991; D'Alessandro et al., 1993; Stoyan like DFRD receptor antagonists (Savino et al., 1994b) et al., 1993). However, none of our site 2 and site 3 IL-6 function an unusual through mechanism: they are able variants map in the putative cytokine-cytokine contact to recruit a single gp 130 chain but fail to induce its surface because they do not show selective impairment homodimerization, thus lacking signalling capacity. of IL-6 dimerization in immunoprecipitation reactions Furthermore, their use in solution binding/immuno- (G.Paonessa and R.Graziani, unpublished results). From 1948 IL-6 receptor complex assembly Top Fig. 9. Model of the hexameric IL-6 receptor complex. Shown are a side view of the IL-6 hexameric receptor complex (left) and two cross-sections through the complex (right): one that includes the bound cytokine (top), and a second (bottom) formed by the second subdomains of the CBDs of At this level, two asterisks in the IL-6Ra both IL-6Ro and gp130 where only interactions between the receptor molecules are postulated to occur. the in the IL-6Ra variant Mut1. For IL-6, numbers identify the sites indicate hypothetical location of the amino acids H280 and D281 mutagenized of interaction with (sites 1-3). The colour code is blue for green for IL-6RCa and pink for IL-6. For the the various receptor components gpl30, receptors, only the part corresponding to their CBDs is shown. the it could also be expected that gp 130 has at IL- 12 to also as natural model, paration). Interestingly, (referred least two sites of interaction with IL-6 and two with killer cell stimulatory factor), which is by itself always IL-6Ra, and targeted mutagenesis of the extracellular found as a disulfide-linked heterodimer between a 35 kDa region of gpl 30 should be very useful in supporting this. related to IL-6 et al., 1992) and a polypeptide (Merberg Lastly, homodimerization contact surfaces between two 40 kDa related to IL-6Roa (Gearing and polypeptide gp 130 and/or two IL-6Rx could be located at the bottom could also form a 2+2 receptor complex Cosman, 1991), level formed by the second subdomains of the CBDs. with its receptor subunit IL-12R which is strongly homo- While this manuscript was being prepared it was logous to gpl3O (Chua et al., 1994). Because IL-6 binds reported that the incubation of purified IL-6-IL-6Rax first to a receptor chain that does not have any direct role complexes with soluble gp130 produced in CHO cells in cell signalling, it is tempting to speculate that the active gave rise to complexes whose molecular weight is com- cytokine is indeed the IL-6-IL-6Rcx complex; this is also patible only with the formation of hexamers with a 2:2:2 supported by the evidence that soluble forms of IL-6Rax et stoichiometry (Ward et al., 1994). Therefore, identical act as potentiators of cytokine activity (Taga al., 1989) conclusions have been obtained using a different approach. and are present at high levels in the circulation (Honda thus be a common The use of wild-type molecules only did not, however, et al., 1992). Hexamer formation could information about the relationship which evolved to ensure the formation provide any topological biological strategy of the in of sites and various molecules the assembled receptor complex high-affinity binding firmly trap cytokines et and/or factors into active (Ward al., 1994). growth configurations. The hexamer is a novel type of receptor complex first discovered for IL-6. We propose, based on sequence Materials and methods homology data, that hexamers could be a common feature and of the of receptor complexes assembled by other cytokines whose Tagging expression soluble receptors cDNA for soluble forms of at the C- Mutant coding gpl3O tagged are to be specific ligand binding receptors predicted with et or FLAG et terminus myc (Evan al., 1985) (Hopp al., 1988) similar to IL-6Rcx. structurally and functionally Among were double-strand at epitopes generated by inserting oligonucleotides factor et these are ciliary neurotrophic (CNTF) (Davis al., for amino acids 605- the EcoRI site of 130 located around the codons gp and also IL- 11 et in the case of 1991) probably (Hilton al., 1994). 606 The were myc, E(605)- (E-F). resulting C-termini, in the case called and, a FEEQKLISEEDL-Stop (hereafter sgpl3O-myc) Indeed, immunoprecipitations performed using strategy called of sgpl30-FLAG). FLAG, E(605)-FDYKDDDDK-Stop (hereafter similar to that described in this for IL-6 paper strongly for soluble forms of with and without Mutant cDNA coding IL-6Rax the CNTF-CNTF-R that the dimerization of suggest at the C-terminus were PCR suitable myc tag epitope generated by using takes in solution called sIL- complex place (A.De Serio, R.Graziani, In the case of oligonucleotides. untagged sIL-6Ra (hereafter 322. In the case of in a codon was inserted after G.Ciliberto and manuscript pre- 6Ra), stop proline R.Laufer, G.Paonessa, 1949 G.Paonessa et al. sIL-6Rax tagged with myc, myc epitope was inserted after proline (1990) Structure-function analysis of human IL-6. J. Immunol., 145, 322 followed by a stop codon; the resulting C-terminus was P(322)- 561-568. GGEQKLISEEDL-Stop (hereafter called sIL-6Ramyc). The cDNAs of Brakenhoff,J.P.J., de Hon,F.D., Fontaine,V., Hart,M., de Groot,E.R., the soluble receptor variants sgpl3O-myc, sgpl3O-FLAG, sIL-6Rax and Content,J. and Aarden,L.A. (1992) Two different sites on the IL-6 sIL-6Raxmyc were cloned into the BamHI site of the baculovirus molecule are in involved biological activity. In Revel,M. (ed.), IL-6: expression vector pBluBac III (Invitrogen). The generation of and Physiopathology Clinical Potential. Serono Symposium recombinant baculovirus expressing variants of gpl3O and IL-6Ra was Publication, Raven Press, New York, Vol. 88, pp. 33-41. performed with MAXBAC kit (Invitrogen) according to the instructions Brakenhoff,J.P.J., de Hon,F.D., Fontaine,V., Boekel,E.T., Schooltink,H., of the manufacturer. The expression of cold receptors was accomplished Rose-John,S., Heinrich,P.C., Content,J. and Aarden,L.A. (1994) by infecting High Five cells (Invitrogen) with a high titre stock of Development of a human interleukin-6 receptor antagonist. J. Biol. recombinant virus at a m.o.i. of 5-10. After 2 h the inoculum was 86-93. Chem., 269, removed and the cells were incubated at 27°C in SF-900 II medium M. Ribbon models of macromolecules. Mol. Carson, (1987) J. Graphics, (Gibco) until all cells detached from the plate (48-72 h). Medium was 5, 103-106. collected and centrifuged, and the supernatant was assayed by Western et al. of a human IL-12 Chua,A.O. (1994) Expression cloning receptor blot for recombinant protein production. A new member of the with component. cytokine receptor superfamily strong homology to gpl3O. J. Immunol., 153, 128-136. The Cosman,D. (1993) hematopoietin receptor superfamily. Cytokine, 5, of recombinant labelling receptors 135S]Methionine 95-106. Recombinant soluble were labelled to receptors metabolically according D'Alessandro,F., Colamonici,O.R. and Nordan,R.P. (1993) Direct the following protocol. Semi-confluent High Five cells in a 6 cm plate association of interleukin-6 with a 130-kDa component of the were infected (m.o.i. of 5-10) with high titre recombinant baculovirus. interleukin-6 receptor system. J. Biol. Chem., 268, 2149-2153. After 36-48 h, medium was replaced with 2 ml of methionine-free Davis,S., Aldrich,T.H., Valenzuela,D.M., Wong,V., Furth,M.E., Squinto, Grace's insect medium and incubated at for 1 h. The medium was 27°C S.P. and Yancopoulos,G.D. (1991) The receptor for ciliary neurotrophic then replaced with 1.5 ml of methionine-free Grace's insect medium factor. Science, 253, 59-63. 50 of act. >1000 containing TRAN[35S]LABEL (sp. Ci/mmol, gt Davis,S., Aldrich,T.H., Pan,L., Taga,T., Kishimoto,T., Ip,N.Y. Stahl,N., 10 mCi/ml; ICN Biomedical Inc.) and incubated for a further 2 h. and Yancopoulos,G.D. (1993) LIFR, and gpl30 as heterodimerizing Medium was then collected, cell debris centrifuged and the supernatant signal transducers of the tripartite CNTF receptor. Science, 260, used for the experiments. 1805-1808. de Vos,A.M., Ultsch,M. and Kossiakoff,A.A. (1992) Human growth Immunoprecipitations hormone and extracellular domain of its receptor: crystal structure of Immunoprecipitation experiments were typically performed according the complex. Science, 255, 306-312. to the 500 baculovirus following protocols. supernatant expressing gl Ehlers,M., Grotzinger,J., deHon,F.D., Mullberg,J., Brakenhoff,J.P.J., myc-tagged receptors and 5 wild-type IL-6, variant IL-6 or IL-6myc gg and Identification of two novel Liu,J., Wollmer,A. Rose-John,S. (1994) were incubated with 4 anti-myc 9E 10 monoclonal antibody (Evan et al., gu of human IL-6 for and regions responsible receptor binding signal 1985) or 1 ,ug monoclonal antibody IL-6- 16 (CLB, The Netherlands) plus transduction. J. Immunol., 153, 1744-1753. 40 50% in PBS of to hereafter slurry protein A-Sepharose (referred gl and Isolation of Evan,G.I., Lewis,G.K., Ramsay,G. Bishop,J.M. (1985) as at After three times with PAS; Pharmacia) overnight 4°C. washing monoclonal antibodies for human specific proto-oncogene c-myc PBSTB (I X PBS, 0.05% Tween 20, 0.2% Brij 96), the Sepharose beads Mol. Cell. 3610-3616. product. Biol., 5, were incubated with other receptor(s) and/or cytokine (for further details and of the subunit of Gearing,D.P. Cosman,D. (1991) Homology p40 see legends to the respective figures) for at least 12 h at 4°C. The beads Natural Killer Cell Factor with the extracellular Stimulatory (NKSF) were then washed three times with PBSTB, resuspended in SDS loading domain of the interleukin-6 9-10. receptor. Cell, 66, heated for 5 min at and to SDS-PAGE. buffer, 95°C subjected and Hibi,M., Murakami,M., Saito,M., Hirano,T., Taga,T. Kishimoto,T. (1990) Molecular cloning and expression of an IL-6 signal transducer, Generation and of IL-6 variants expression gpl3O. Cell, 63, 1149-1157. Generation and of IL-6 variants were as expression performed exactly et al. of a murine IL-l1 Hilton,D.J. (1994) Cloning receptor tX-chain; 1 amino acid described previously (Savino et al., 994b). For the numbering requirement for gpl 30 for high affinity binding and signal transduction. with at the of IL-6 see Savino et al. (1994a). IL-6 tagged myc epitope EMBO J., 13, 4765-4775. N-terminus (IL-6myc) was generated by PCR using suitable oligonucleo- Honda,M., Yamamoto,S., Cheng,M., Suzuki,H., Yasukawa,K., Saito,T., The tides; it was cloned in pT7.7 vector (Studier et al., 1990). resulting and Human soluble IL-6 Osugi,Y., Tokunaga,T. Kishimoto,T. (1992) sequence of the N-terminus is the following: Met-EQKLISEEDL-wtIL-6, receptor: its detection and enhanced release by HIV infection. J. is valine in where the first amino acid of IL-6 represented by position Immunol., 148, 2175-2180. 2. 35S-labelling of wild-type IL-6 was performed with rabbit reticulocyte Hopp,T.P., Prickett,K.S., Price,V.L., Libby,R.T., March,C.J., Cerretti,D.P., to the instructions. lysate (Promega) according supplier's and short marker Urdal,D.L. Conlon,P.J. (1988) polypeptide useful for recombinant identification and sequence protein purification. In and 1204-1210. vitro receptor binding assays bioassays BiolTechnology, 6, vitro and on and et al. The molecular of human IL-6 In receptor binding assays bioassays HepG2 XG-1 Lahm,A. (1995) design receptor lines were as described Ann. NY Acad. 762. cell performed exactly previously (Savino antagonists. Sci., et and al., 1994b). Merberg,D.M., Wolf,S.F. Clark,S.C. (1992) Sequence similarity between NKSF and the IL-6/G-CSF Immunol. family. Today, 13, 77-78. Nakagawa,T., Yasukawa,K., Murakami,M., Hibi,M., Nakagawa,N., Acknowledgements Yamanishi,K., Taga,T. and Kishimoto,T. (1993) IL-6-induced We thank Anna Maurizio Laufer and Tramontano, Sollazzo, Ralph Janet homodimerization of gpl30 and associated activation of a tyrosine Clench for this and Yves and critically reading manuscript Cully Manuela kinase. Science, 260, 1808-1810. Emili for artwork. Rose-John,S., Hipp,E., Lenz,D., Legres,L.G., Korr,H., Hirano,T., and Structural and functional Kishimoto,T. Heinrich,P.C. (1991) studies on the human interleukin-6 Biol. receptor. J. Chem., 266, 3841-3846. References Savino,R., Lahm,A., Giorgio,M., Cabibbo,A., Tramontano,A. and Ciliberto,G. (1993) Saturation mutagenesis of the human interleukin Bazan,J.F. (1990a) Hemapoietic receptors and helical cytokines. 6 site: receptor-binding Implications for its three-dimensional structure. Immunol. Today, 11, 350-354. Proc. Natl Acad. Sci. USA, 90, 4067-4071. Bazan,J.F. 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Ultsch.M.H., Somers,W., Kossiakoff.A.A. and de (1994) The Vos,A.M. crystal structure of affinity-maturated human Growth Hormone at 2 resolution. J. Mol. Biol., 236, 286-299. Van Snick,J. (1990) Interleukin-6: an overview. Aon)lu. Rev. Immuidnol., 8. 253-278. Howlett.G.J.. Ward,L.D., Discolo,G., Yasukawa,K.. Hammacher.A.. Moritz.R. and Simpson.R.J. (1994) High affinity interleukin-6 receptor is a hexameric complex consisting of two molecules each of interleukin-6 and gp- 130. interleukin-6, receptor, J. Biol. Clietz., 269. 23286-23289. Yamasaki.K.. Yawata.H., Taga,T.. Hirata,Y., Kawanishi.Y.. Seed.B.. Hirano.T. and TaniguchiT., Kishimoto,T. (1988) Cloning and expression of the human interleukin-6 receptor. (BSF-2/IFNP2) Science, 241, 825-828. Yawata,H., Yasukawa,K., Shunji,N., Murakami,M.. Yamasaki,K., Hibi,M., Taga,T. and (1993) Structure-function Kishimoto,T. analysis of human IL-6 receptor: dissociation of amino acid residues required for IL-6 binding and for IL-6 signal transduction 130. through gp EMBO J.. 1705-1712. Wen.Z. and Zhong.Z., Darnell,J.E.,Jr (1994) Stat3: a STAT family member activated by tyrosine phosphorylation in response to epidermal factor growth and interleukin-6. Science. 95-98. 264, Received oni JanLuarv 20, 1995

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Published: May 1, 1995

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