Direct Association of Interleukin-6 with a 130-kDa Component of the Interleukin-6 Receptor System*

Affinity cross-linking of membrane bound 12’I-inter- leukin-6 (IL-6) on several cell lines revealed a three-band pattern of IL-6-containing cross-linked com- plexes with molecular masses of 100, 120, and 150 kDa. To identify the membrane components that were associated with IL-6 in the three complexes, we employed the Denny- Jaffe reagent, a heterobifunctional, cleavable cross-linker that allows the transfer of ‘’‘1 from the ligand to its receptor. Samples cross-linked with Denny-Jaffe reagent were analyzed by two-di-mensional SDS-polyacrylamide gel electrophoresis in which the cross-linker was cleaved prior to the second dimension. This analysis revealed that IL-6 directly associates with a 130-kDa membrane protein thus al- lowing the formation of the 150-kDa complex. In addition, both the 100- and 120-kDa cross-linked com- plexes were shown to include an 80-kDa membrane glycoprotein associated with one and two IL-6 mole- cules, respectively. a

Interleukin 6 (IL-6)' is a cytokine that displays a broad range of biologic activities (reviewed in Refs. (1) and (2)) including the stimulation of immunoglobulin production by activated B cells (3), the support of myeloma (4, 5), plasmacytoma, and hybridoma proliferation (6,7), and the induction of acute phase responses in hepatocytes (8). IL-6 has also been shown to induce the differentiation of myeloid (9) and nerve cells (10) and has multiple colony-stimulating factor activity on hematopoietic progenitor cells (11).
IL-6 appears to mediate its effects by interacting with specific receptors on the cell surface. Binding studies have revealed the presence of both low affinity (12, 13) and high affinity (14, 15) binding sites for IL-6 on a variety of cells.
T h e cDNAs of two transmembrane glycoproteins that participate in the IL-6 receptor system, gp80 and gp130, with molecular masses of 80 and 130 kDa, have been cloned, sequenced, and expressed in eukaryotic cells (14,15). The gp80 receptor molecule directly binds IL-6 with low affinity (15) and has been shown to associate with gp130 in the presence of IL-6 (16). Antibodies to gp130 inhibit the formation of high affinity binding sites for IL-6 thus indicating that * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
' The abbreviations used are: IL-6, interleukin-6; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; gp, glycoprotein. 301-402-1608. gp130 participates in the formation of high affinity binding sites (14). Studies have also shown that the combination of IL-6 plus a soluble form of recombinant gp80, that lacks transmembrane and intracellular domains, can associate with gp130 to initiate IL-6 responses (16). This result implicates gp130 as a signal transducer in the generation of IL-6 responses (14,16) and indicates that the transmembrane and intracellular portions of gp80 are not necessary for signal transduction. Although gp130 participates in the formation of high affinity binding sites for IL-6 and in the generation of IL-6 responses, gp130 has thus far not been found to bind or directly interact with IL-6 itself. This has led to the hypothesis that gp130 is a nonligand binding member of the IL-6 receptor system and that only gp80 contributes to 16).
Although only gp80 has been documented to be an IL-6binding molecule, several studies incorporating cell surface affinity cross-linking with lZ5I-IL-6 have reported the formation of multiple IL-6-containing complexes ranging from 100 to 200 kDa (12,17,18), thus suggesting that the IL-6 receptor complex may consist of more than a single IL-6-binding protein. In this paper, we describe the characterization of the IL-6 receptor system on the human myeloma cell line, U266, using both homobifunctional and heterobifunctional affinity cross-linking techniques. Our results demonstrate that IL-6 associates directly with a 130-kDa membrane glycoprotein. In addition, our studies also reveal that two IL-6 molecules can be found complexed with an 80-kDa member of the receptor complex.

MATERIALS AND METHODS
Cell Culture-All cell culture was performed in complete medium, which consisted of RPMI 1640 (GIBCO) supplemented with 10% fetal calf serum (Whittaker Bioproducts, Walkersville, MD) and antibiotics, a t 37 "C in a humidified atmosphere of 7% C02. U266, a human myeloma cell line, and U937, a human histiocytic lymphoma cell line, were obtained from the American Type Culture Collection (Rockville, MD). H929, a human myeloma cell line, was generously provided by Dr. Adi Gazdar,National Cancer Institute,NIH,Bethesda,MD (19). Peripheral blood lymphocytes were prepared from the blood of normal donors using Ficoll fractionation and activated by culturing overnight in 10 pg/ml phytohemagglutinin. The IL-6dependent murine cell lines B9 and T1165 were grown in complete medium supplemented with 50 p~ 2-mercaptoethanol and recombinant IL-6.
Radiolabeling of Recombinant Human Interleukin-6-The biologic activity of the recombinant human IL-6 (R&D Systems, Minneapolis, MN) used in these studies was tested on two IL-6-dependent cell lines, B9 (20) and T1165 (6), and found to have 1 X lo9 hybridoma growth factor units/mg and 3 X lo7 plasmacytoma growth factor units/mg, respectively. The specific activity was essentially unchanged after iodination. IL-6 was labeled using the ['251]diiodo-Bolton-Hunter reagent (2200 Ci/mmol, Du Pont-New England Nuclear) according to the manufacturer's recommended procedure (21). Briefly, 2 pg of IL-6 in 15 pl of 33 mM sodium borate, 250 mM NaC1, a 150

Structural Characterization
of the II,-6 Reccptor p H 8.5, were romhined with 0.5 or 1.0 mCi of evaporated [""IJdiiodo-Holton-Hunter reagent for 4 h at 4 "C. The reaction was quenched with 5 pl of 1 M glycine for.15 min and then If;) gelatin was added as a carrier. Radiolabeled 11,-6 was separated from free Rolton-Hunter reagent by size exclusion chromatography on a Bio-Gel J'-6 (Hio-Rad) column previously blocked with 1"; bovine serum albumin and extensively washed with phosphate-buffered saline to remove unbound hovine serum alhumin. The recovery of labeled IL-6 was 76-XO%, and based on this value the specific activity of the '"'I-IL-6 was calculated to he 2-4 X 10" cpm/mmol. More than 99% of the radioactivity corresponded to a single hand of IL-6 when analyzed under both reducing and nonreducing SDS-PAGE and autoradiography. The hindahility of the '251-IIA; to LJ266 cells was determined to be 905 (22). Commercially prepared ""I-II,-G (Du Pont-New England Nuclear) was used in some experiments.
.SI)S-I"n(;l.:-Samples were analyzed by SDS-PAGE on 7% polyacrylamide slah gels (1.5 mm X 8 cm X 13 cm) in the presence of reducing or nonreducing conditions according to Laemmli ( 2 3 ) . After electrophoresis, gels were dried under vacuum prior to autoradiography.
Affinity Cross-/inf?ing of "'I-II,-fi to Its Receptor-Affinity crosslinking was performed using a modification of a previously published procedure (24). Brielly, cells were washed twice and resuspended a t 5 X 10' cells/ml in cold binding medium (RPMI 1640, 1% bovine serum albumin, 0.05% sodium azide). Binding was allowed to proceed for 1 h on ice with t.he indicated amount of "'I-1L-G. After binding, the cells were washed with cold RPMI 1640, 0.05% sodium azide to remove t.he unbound ""I-IL-6 and resuspended in 1 ml of phosphatehffered saline, 1 mM MgCI,, p H 8.3. Cross-linking was initiated by the addition of 300 pg/ml disuccinimidyl suberate, disuccinimidyl tartrate, o r dithiohis-(succinimidyl proprionate) (Pierce Chemical Co.), as indicated, and allowed to proceed at 4 "C for 15 min. The Cross-linking reaction was stopped by centrifugation and by immediate lysis of the cells with 50 mM Tris-HCI, 300 mM NaCI, 1% Nonidet 1'-40, 1 mM phenylmethylsulfonyl fluoride, 10 mM leupeptin, a n d 10 mM pepstatin, pH 7.5 (lysis buffer), for 30 min on ice. T h e lysate was cent.rifuged for 10 min a t 15,000 X R and the supernatant collected for immunoprecipitation (see helow) or for direct, analysis by SDS-PAGE under reducing or nonreducing conditions. ~mmunoprf~cipitntion-cLJ~.IL-6/~, a murine monoclonal anti-lL-6 antibody, and affinity-purified sheep anti-11,-6 polyclonal antibody have been descrihed elsewhere (25). Lysates obtained after affinity cross-linking with I2"1-II,-6 were incubated overnight at 4 "C with 2mt.i-11,-6 or control ant.ihodies and precipitated with protein G-Sepharose (Pharmacia LKB Biotechnology Inc.) for 4 h a t 4 "C. T h e precipitates were washed four times with lysis buffer and analyzed hy SDS-PAGE as indicated. In some experiments, antibodies conjugat,ed direct.ly to Sepharose were used with similar results. G/,vrosvlation Studirs-The contribution of glycosylation to the molecular mass of t.he cross-linked receptor complexes was assessed using a combination of: 1) inhibition of N-linked glycosylation and 2) subsequent cleavage of sialic acid and 0-linked glycosidic residues. Briefly, LJ266 cells, in the log phase of growth, were cultured for 18 h a t 6 X 10" cells/ml in complete medium containing 5 pg/ml tunicamycin. The cells were harvest,ed, cross-linked to ""I-11,-6, immunoprecipitated with sheep anti-IL-6, and immobilized on protein G-Sepharose beads as described above. To remove sialic acid residues, t h e immohilized immunoprecipitates were washed and resuspended in 0.1 M acet,ate huffer, pH 6.5, 10 mM CaC12 containing 0.3 unit/ml neuraminidase (Genzyme. Cambridge, MA) and incubated for 1 h a t 37 "C. 0-linked residues were then removed by resuspending the immohilized immunoprecipitates in the same buffer containing 10 mM I,-galactono-y-lactone plus 0.02 unit/mL 0-glycanase (Genzyme) and incubated overnight at. 37 "C. The samples were analyzed by

SDS-PAGE and autoradiography.
Affinity Cross-linhing with I1rnn.v-Jnffr Reagent-Recombinant IL-6 ( 1 pg) was laheled with the "'I-Denny-.Jaffe reagent (100 pCi, specific activity 2200 Ci/mmol, Du I'ont-New England Nuclear) following the procedure described above for the Bolton-Hunter reagent with the exception that the entire procedure was performed in the dark. The specific activit.y was calculated to be 1.5 X cpm/ mmol. For cross-linking experiments the cells were washed twice and resuspended at 5 X lO'cells/ml in cold binding medium (RPMI 1640, 1% bovine serum albumin. 800 pg/ml hacitracin). Binding was allowed to proceed in the dark for 1 h on ice with the indicated amount of 'Z'l-I~ennv-~Jaffe-II,-C,. After binding, the cells were washed in the dark with cold binding medium and resuspended in cold phosphatebuffered saline. Photocross-linking was accomplished by exposing samples for 15 min to 365 nm (long wave) LIV light. 'The cells were pelleted, resuspended in lysis buffer, and incubated for 30 min on ire. 'The lysate was centrifuged for 5 min at 15,000 X g, and the supernatant was immunoprecipitated with anti-11,-6 antihodies as indicated and analyzed hy one-and two-dimensional SI)S-I'A(;E. For two-dimensional analysis the immunoprecipitated lysate was applied to a 7% SDS-I'A(;E tube gel (first dimension) and elertrophoresed under reducing conditions. Cleavage of the azo linkage in the Denny-Jaffe cross-linker molecules was accomplished with three 15-min soakings of the first dimension tube gel in fresh 0.2 M sodium dit.hionite solution. The gel tube was then equilibrated with 50 m v Tris/HCl, pH 6.8, and 0.1% SDS prior to second dimension elertrnphoresis in a 7"X SDS-PAGE slab gel and autoradiography.

RESULTS
Previous studies have characterized only a single, 80-kDa, IL-6-binding membrane protein, termed q 8 0 (13,26). However, in several affinity cross-linking reports (12. 17, 18) and in our own preliminary studies, multiple IIA-containing cross-linked receptor complexes have been found. In order to characterize IL-&binding membrane proteins, we performed affinity cross-linking studies using "51-11,-6 and the homohifunctional cross-linking reagent disuccinimidvl suberate (12 A). In our studies, "'1-IL-6 was allowed to bind for 1 h a t 4 "C, and unbound ligand was removed prior to cross-linking.
More detailed studies were performed with the myeloma cell lines U266 and H929, both of which express a large number of IL-6 receptors (approximatelv 20,000/cell) (13, 16. 27). T h e loo-, 120-, and 150-kDa cross-linked complexes were obtained with either reducing or nonreducing conditions, indicating that interchain disulfide linkages were not responsi- 1*'1-11,-6 to n o r m a l a n d t r a n s f o r m e d h u m a n c e l l s . 1 X 1 0 ' cells were inruhatetl with 5 nsf ""I-IL f a t 4 "C for 60 min. After removal of unhountl ligand. the cells were cross-linked with :300 mg/ml disurrinimidyl suhrmte for 15 min at 4 "C and analyzed by reducing ST>S-I'A(;I' and autnratliography as described under "Materials and Met hods." Autoratliographies were exposed for different periods t o arhieve similar intensities: tJ266 and H929 lanes were exposed overnight, 11937 for :i (lays and peripheral hlood lymphocytes for 5 days. Me for the multiband pattern (Fig. 2 A ) . Immunoprecipitation with affinity-purified, anti-IL-6 antibodies also produced the same three labeled bands (Fig. 2R). The specificity of the binding was confirmed by competition with unlabeled IL-6 during the binding step. Fig. 2C, lane 2, shows that a 200-fold excess of unlabeled IL-6 completely inhibited the binding of '2'I-IL-6 to its receptor. The same affinity cross-linking patt,ern was also obtained using other homobifunctional crosslinkers including disuccinimidy! tartrate (6 A) and dithiobis-(succinimidyl propionate) (12 A) (data not shown). An identical pat,tern was obtained when binding proceeded for 1 h a t 37 "C (not shown), indicating the same associations were formed both at 4 and 37 "C. In addition, the 37 "C bands were less intense suggesting that binding at this temperature results in down-regulation of all three complexes.
Allowing for the 20-kDa mass of the IL-6 molecule, the format,ion of the three complexes suggested that as many as three IL-&binding proteins of different molecular masses existed (80, 100, and 130 kDa). We assessed the possibility that the three bands represented differently glycosylated forms of a single IL-6 receptor molecule cross-linked to I2"I-IL-6. Fig. 3 shows the result of deglycosylation and crosslinking experiments using a combination of 1) inhibition of N-linked glycosylation and 2) digestion of sialic acid and 0linked residues with glycosidases. Reductions in molecular weight occurred in all three cross-linked complexes. In addition, the continued presence of three distinct bands indicated that the multiple band pattern could not be explained by different levels of glycosylation on a single binding protein.
In order to further define the composition of the three complexes we employed an iodinated, heterobifunctional, cleavable cross-linker termed the '*'I-Denny-Jaffe reagent (28-30). With this procedure, IL-6 is first covalently labeled with the "'I-Denny-Jaffe cross-linking reagent. After receptor interaction, the bound '*'I-Denny-Jaffe-IL-6 is photocrosslinked to an adjacent protein. Upon cleavage of the crosslinker, the IL-6 is released and the '"1 remains covalently attached to the IL-6-binding protein. Due to the sequential order of this procedure, cross-linking occurs only between the labeled IL-6 molecule and a single associated protein and cannot occur between adjacent unlabeled proteins. When analyzed by SDS-PAGE, the photocross-linked U266 cells yielded the three radioact,ive complexes (100, 120, and 150 kDa) that are seen with conventional cross-linking (Fig 4A ). Cross-linked samples were then analyzed bv two-dimensional SDS-PAGE in which the Dennv-.Jaffe cross-linker was cleaved prior to electrophoresis in the second dimension. thus resulting in the release of the IL-6 molecule and the transfer of I2'I to the cross-linked protein. Fig. 4H shows that, after cleavage, two 80-kDa proteins ( a and b ) were released from positions on the diagonal that corresponded to the original 100-and 120-kDa complexes, whereas a separate l3O-kDa protein (c) was released from the original 150-kDa complex. Several conclusions can he drawn from these results. Hoth the 100and 120-kDa complexes include an 80-kDa rnernbrane protein. Furthermore, since no additional proteins other than IL-6 can be cross-linked into the complex, this finding also demonstrates that the 120-kDa complex consists of an 80-kDa membrane protein in association with two 20-kI)a molecules of IL-6. Equally important, these results also dernonstrate that IL-6 associates directly with a 180-kI)a rnernbrane protein.
The association of the 80-kDa molecule with two 11,-6 molecules to form the 120-kDa cross-linked complex was unexpected. We investigated the possibilitv that the 12O-kI)a complex was formed by the association of an 80-kDa receptor molecule with preexisting dimers of 11,-6. SIX-I'AGF: nntl autoradiography of '2"I-IL-6 yielded of onlv a single 'LO-kDa form, thus excluding the presence of covalent 11,-fi dimers. 'I'o test for noncovalent, dimers, we performed the photocrosslinking procedure, with 'Z"I-Denny-.Jaffe-lahelect 11,-6, in the absence of cells. Photocross-linking of noncovalent dimers of IL-6 would be expected to generate 40-kDa cross-linked complexes. Fig. 5 shows that onlv the 20-kDa monomeric form of IL-6 was generated using the Dennv-.Jaffe procedure, thus indicating that IL-6 dimers did not exist prior t o interaction with the 80-kDa membrane protein. This conclusion is also  After electrophoresis the Denny-.Jal'fe cross-linker was cleaved as descril)ed under "Materials and Methods," a n d t h e gel was snt),jectetl t o second dimension electrophoresis in a '7'';> SDS-l'A(;I.: slal) gel and autoradiography. supported hv gel filtration-HPLC analysis of radiolabled IL-6 in which only the monomeric form of IL-6 eluted from the column (not. shown). In addition, when cross-linking studies were performed using the HPLC-purified ""'I-IL-6, the same three complexes with M , values of 100, 120, and 150 were ohtained (data not. shown).

DlSClJSSlON
Although only a single 11,-6-hinding memhrane protein, gpp80, has thus far been documented (15). several affinity cross-linking studies have reported the format ion of multiple cross-linked complexes within the IL-6 receptor ssstem. Cross-linked bands of 110, 160. and 190 kDa have heen reported on the human Ivmphohlastoid line CESS (12), whereas loo-, 120-, and 200-kDa cross-linked complexes have been obtained using the human hepatoma, HepG2, and 3T:l fibrohlast,s transfected with the gp8OcDNA (15, 18). Although the gp80 IL-6 receptor was present in the latter studies, the exact composition of the individual cross-linked complexes has remained unknown. In this paper, we have structurally characterized the IL-6 receptor system on human cells using '2sII-IL-6 and affinity cross-linking techniques. Our studies revealed the formation of three IL-6-membrane protein crosslinked complexes, on both normal and transformed cells, with apparent molecular masses of 100, 120 and 150 kDa, respectively. In initial studies, we determined that the three-band pattern was not the product of interchain disulfide linkages within the complexes. We also established that differential glycosylation of proteins within the cross-linked complexes was not responsible for the three-band pattern. \Ve achieved identical results using homohifunctional crosslinkers ranging in length from 6 to 12 A. Considering that the distance between interacting hormone/receptor res,idues at the hinding interface of a related receptor is about 3 A ( 3 1 ), the ahility to bridge molecules with a 6-A cross-linker suggests that the associated proteins were in direct contact with the 11,-6 molecule. These results indicated that the association of II,-fi with its receptor may be more complex than previously dpscribed and prompted us to characterize the complexes in more detail.
T o identify the membrane components that were crosslinked to IL-6 in each of the three complexes, we employed an iodinated, photoactivatable, and cleavable cross-linker, the ""I-Denny-.Jaffe reagent, which, when coupled t o I I A , allows the t,ransfer of I2"I from the ligand to adjacent receptor components. Our analysis revealed several previously undocumented aspects of the I I A receptor complex. First, the 150-kDa complex includes IL-6 cross-linked to a I3O-kDa membrane glycoprotein. Equally important, our data reveal that both the 100-and 120-kDa complexes include IL-6 crosslinked to an 80-kDa membrane protein. Since all cross-linking events with the Denny-daffe reagent must occur hetween the ligand (IL-6) and only one other protein, this result also demonstrates that the 120-kDa complex contains two 11,-6 molecules cross-linked to a single 80-kDa glycoprotein.
In this st,udy we found that 1L-6 associates with two membrane glycoproteins whose molecular weights correspond to those of two transmembrane glycoproteins, gp80 and gp1:lO. that have previously heen shown to functinn in the 11,-6 receptor system. The cDNAs for both gp80 and gpl:lO have heen cloned, sequenced, and expressed in eukaryotic cells, and the mRNAs for both genes are expressed hv the IT266 myeloma cell line (14, 15). Forms of gp80 have been shown to directly bind IL-6 and to associate with cell surface gp140 in the presence of IL-6 (16). In addition, when expressed in 11,-%dependent cells, recombinant g p 1 : W can interact with I I A plus soluble forms of recombinant gp80 to generate a growth signal thus implicating gplpll0 as a signal transducer ( 1 4 ) .
Although gpl30 has been shown to participate in the generation of high affinity binding sites (It!), cells expressing only recombinant gp1.10 fail to bind 11,-fi (14). This ohservation has led to the h-ypothesis that gp1:lO does not hind or interact with IL-6 direct,ly but instead associates with m80 to stabilize the binding of IL-6 by gp80, thus converting gp80 a high affinity receptor (14,16). The above hypothesis has also been supported by the absence, in previous structural studies, of any data showing a direct association of gp130 with IL-6. Assuming the 130-kDa glycoprotein described in our study is in fact gp130, the finding presented here suggests another equally plausible hypothesis: specifically, that gp130 does bind IL-6, presumably together with gp80 in the high affinity complex. The concept that gp130 can act as a ligand-binding molecule is supported by recent studies demonstrating that recombinant-expressed gp130 alone directly binds oncostatin M with low affinity (32).
The formation of 100-and 120-kDa cross-linked complexes has also been observed by 18) who suggested that the 120-kDa complex may consist of gp80 plus two molecules of IL-6. In our experiments we have clearly demonstrated that the 100-and 120-kDa cross-linked complexes described here in fact contain one and two molecules of IL-6, respectively. We have also demonstrated that preexisting IL-6 dimers did not contribute to the formation of the 120-kDa complex. This suggests that the formation of the 120-kDa complex is a consequence of independent binding of the IL-6 molecules. Several models can be developed to explain the formation of the 100-and 120-kDa complexes. In one interpretation, the 120-kDa complex could be generated if gp80 possessed two binding sites for IL-6. We prefer an alternative model, also suggested by , in which two molecules of IL-6 would associate with two gp80 molecules to form a large tetrameric complex. Multiple crosslinking events within either of the possible models would generate both the 100-and 120-kDa cross-linked complexes.
T h e last model is supported by affinity cross-linking studies of  in which a 200-kDa IL-6-containing cross-linked complex was generated in 3T3 fibroblasts transfected with gp80 cDNA. Our experiments do not allow us to distinguish between these possibilities. Whatever form the IL-6/gp80 association takes, it is possible that structures consisting of two molecules of IL-6 and one or two molecules of gp80 could exist independently or may interact with gp130 t o engage the signal transduction pathway. Additional studies are needed to identify the mechanisms involved in this process.