Soluble c-Kit Proteins and Antireceptor Monoclonal Antibodies ...

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domains. To confine the ligand binding site to this portion of the receptor we generated a panel of murine .... tinct set of proteins that generate intracellular second mes- .... with methionine-free Dulbecco's modified Eagle's medium and grown.
Vol ,268, No.6,Issue of Februery 25. pp. 4399-4406,1993 Printed in U.S.A .

CHEMISTRY THEJOURNALOF BIOLOGICAL 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc

Soluble c-Kit Proteins and Antireceptor Monoclonal Antibodies Confine the Binding Site of the Stem Cell Factor* (Received for publication, September 11, 1992)

Janna M. Blechman$§, Sima Lev$, Maria Felice BrizzinII , Orit LeitnerS, Luigi Pegoraron, David Givol$, and Yosef Yarden$** From the $Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel and the flDipurtmento di Scienze Biomediche e Oncobgia Umanu, Universita di Torino, Torino 10126, Italy

The bindingof the stemcell factor (SCF) to the c-kit- promote formation of colonies from early hematopoietic proencoded receptor tyrosine kinase stimulatesa variety genitors (Zsebo et al., 1990a), its differential proliferative of biochemical responses that culminate in cellular pro- effect on normal mast cells, but not mast cells derived from liferation, migration, or survival. The extracellular W mutant mice (Nocka et al., 1990), and by its action onMCdomain of p145”” consists of five immunoglobulin-like 6 mast cells (Williams et al., 1990). This cytokine was respecdomains. To confine the ligand binding site to this tively named stem cell factor (SCF),’ c-kit ligand, and mast portion of the receptorwe generated a panel of murine cell growth factor. Besides its essential in vivo role in hemamonoclonal antibodies (mAbs) to the Kit protein and topoiesis, melanogenesis, and gametogenesis, in vitro SCF identified two mAbs that efficiently displaced recep- acts synergistically with other growth factors including erythtor-bound SCF and also inhibited proliferationof SCF- ropoietin, granulocyte-colony stimulating factor (G-CSF), dependent human megakaryocytes. To map the epi- granulocyte-macrophage colony stimulating factor (GM-CSF) topes of these mAbs we constructed and expressed soluble portions of the extracellular domain of Kit, and interleukins (ILs) 3 and 6 (reviewed in Besmer, 1991). which included either the twoamino-terminal Ig-like SCF exists both as a cell surface form and as a soluble form domains (denoted Kit 1-2), threeIg-like domains (Kit that corresponds to 164 amino-terminal residues of the long 1-2-3), or the entire extracellular portion (Kit-X). All protein. In addition, two alternatively spliced SCF mRNAs differ in the rate three recombinant proteins were recognized by the encode variant transmembrane proteins that ligand inhibitorymAbs, suggesting that theSCF bind- of processing into thesoluble form (Flanagan et al., 1991). All of the biological effects of SCF are initiated by its ing site resides in the amino-terminal half of the ectodomain. Consistent with this conclusion, all of the sol- binding to a cell surface receptor that is encoded by the c-kit uble proteins inhibited SCF binding to Kit-expressing proto-oncogene (Yarden et al., 1987;Qiu et al., 1988). The cells, and they also underwent specificcovalent cross- mouse kit gene is allelic with the white spotting ( W ) locus on linking to the radiolabeled ligand. However, whereas chromosome 5 (Chabot et al., 1988; Geissler et al., 1988).Early Kit 1-2-3and Kit-X displayed comparable ligand affin- observationsindicated that W mutants are phenotypically ities, deletion of the third Ig-like domain, in Kit 1-2, indistinguishable from 5’1 mutant mice and therefore predicted involved significant reduction in SCF binding. Hence, that these two loci interact on the same functional pathway the binding site of SCF probably includes Ig-like do- (Bennett, 1956; Russell, 1979). This prediction has been conmains l and 2, but structural determinants distal to firmed recently through the identification of the S1 and W this portionmay also participate in ligand recognition. gene products as a ligand (SCF) and its cognate receptor (Kit), respectively. High affinitybinding of noncovalently held dimers of SCF to~ 1 4 5protein ~ ’ ~ rapidly induces dimeriMice with mutations in the Steel ( S l )locus on chromosome zation of the receptor (Blume-Jensen et al., 1991; Lev et al., 10 exhibita complex phenotype that includes defects in 1992a). This stimulates the intrinsic cytoplasm-facing catamelanogenesis (whitehair coat), gametogenesis (sterility), lytic domain of Kit that functions as a tyrosine-specific proand hematopoiesis (macrocytic anemia) (Bennett, 1956; tein kinase. The next step in the signaling pathway includes McCulloh et al., 1964, 1965; Mintz and Russell, 1979; Silvers, tyrosine phosphorylation and physical association with a dis1979; Russell, 1979). S1 alleles contain deletions in agene that tinct set of proteins that generate intracellular second mesencodes a transmembrane protein which functions as a pre- sengers (Lev et al., 1991; Rottapel et al., 1991; Funasaka et cursor for a peptide growth factor (Zsebo et al., 1990b; Huang al., 1992). The interactions of the ligand-stimulated Kit with et al., 1990; Copeland et al., 1990). The soluble growth factor cellular substrates is mediated, at least in part, by the hydrowas identified independently on the basis of its ability to phillic kinase insert domain that lies within the catalytic core and strongly couples to proteins that contain src homology * This work was supported by National Institutes of Health Grant domains (Lev et al., 1992b; Rottapel et al., 1991). CA 51712, by the Citrina Foundation, an AIRC grant, and Consiglio The Kitprotein belongs, both structurallyand functionally, ~

~~

~~

Nazionale Ricerche Grant 92.02237.PF39. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Recipient of a postdoctoral fellowship from the Israel Cancer Research Fund. 11 Fellow of AIRC. ** Recipient of a research career development award from the Israel Cancer Research Fund. To whom correspondence should be addressed. Tel.: 972-8-343026;Fax: 972-8-344141.

’ The abbreviations used are: SCF, stem cell factor; CSF, colonystimulating factor; G-CSF, granulocyte CSF; GM-CSF, granulocytemacrophage CSF; IL, interleukin; PDGF, platelet-derived growth factor; mAb, monoclonal antibody; Kit-X, a recombinant extracellular domain of Kit/SCF receptor; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; bp, base pair(s); PAGE, polyacrylamide gel electrophoresis; EDAC, l-ethyl-3-(3-dimethylaminopropyl)carbodiimide; EGF, epidermal growth factor.

4399

4400

SCF Binding Site of Kit

to a subgroup of receptor tyrosine kinaseswhich includes the medium supplemented with 5% fetal calf serum and 5 ng/ml recombinanthuman IL-3.Growthfactordeprivation was achieved by receptors for the platelet-derived growth factors (PDGFs), colony-stimulatingfactor1,and a few receptors for still washing the cells twice in Iscove's modified Dulbecco's medium and culturingthem for 24 h in Iscove's modified Dulbecco's medium unknown ligands. These type 111 receptor tyrosine kinases supplemented with 5% fetal calf serum at a concentration of 2 X lo6/ (Yarden and Ullrich, 1988) are characterized by extracellular ml. Cells were preincubated for 50 min with different concentrations ligand binding portions that contain five immunoglobulin of the indicated antibody and seeded at 50,00O/well in 96-well plates ng/ml IL-3, or 30 ng/ml SCF. (1g)-like domains (Williams and Barclay, 1988; Hunkapiller with either 10ng/mlGM-CSF,10 was added (2 pCi/ and Hood, 1989).In addition, the ectodomains of these recep- Following a 24-h incubation,[rneth~l-~Hlthyrnidine 4 h. Alcohol/acid-precipitable radioactivity was well) for the last tors share many common short stretchesof amino acids that determined by liquid scintillation counting. are distributed throughout the whole length of the extracelMice-BALB/c and CD2/Fl mice were obtained from the Experilular portions (Yarden et al., 1987). This raises the question mental Animals Center of the Weizmann Institute of Science. of where along the extracellular structures the differential Buffered Solutions-HNTG buffer contained 20 mM HEPES (pH ligand binding specificities reside. One possibility is that a 7.5), 150 mM NaC1, 0.1% (w/v) Triton X-100, and 10% glycerol. Solubilization buffer contained 50 mM HEPES (pH 7.5), 150 mM specific Ig-like domain,or aspecific interdomainstretch, NaC1, 1% (w/v) Triton X-100, 10% glycerol, 2 mM EDTA and 1 mM functions as a ligand binding site. The Ig homology unit is phenylmethylsulfonyl fluoride. High wash contained 50 mM HEPES characterized by a primary sequence of70-100 residues in (pH 7.5), 500 mM NaCl, 0.2% (w/v) Triton X-100, 0.1% SDS, and 5 length, with an essentially invariant disulfide bridgespanning mM EDTA. Medium wash was like high wash except 150 mM NaCl. 40-60 amino acids. Severalother relatively conserved residues Low wash contained 10 mM Tris (pH 8.0), 0.1% (w/v) Triton X-100, are involved in establishing a tertiary structure referred to as and 5 mM EDTA. PBS contained 137 mM NaCl, 2.7 mM KC1,7.9 mM a n antibody fold (Amzel and Poljak, 1979). This structure is NaHP04 and1 mM KHZPO, (pH 7.2). Generation of Monoclonal Antibodies to Human Kit/SCF Recepshared by allmembers of the Ig supergene family,which tor-Kit-X was purified from the medium of transfected CHO cells includes molecules with immunological functions, and proby using lectin chromatography as we described previously (Lev et teins with no known immune function, such ascell adhesion al., 1992~). Ten micrograms of the protein was injected intraperitomolecules and receptors for lymphokines and growth factors neally into BALB/c mice (2 months old). Two additional injections (Williams and Barclay, 1988; Hunkapiller and Hood, 1989). were given at intervals of 2 weeks. The antisera were tested 10 days Kit, like other nonimmunological members of the family, after the third injection by using a direct binding assay to T-18 cells that overexpress c-kit. The spleens of mice that developed strong contains Ighomology units that correspond to the C2 type or immune responsewere selected, and theirsplenocytes were fused with the V type of Ig-like domains. NSO myeloma cells by using polyethylene glycol (Galfre et al., 1987). It is presently unclear how the compact andglobular struc- Hybridomas were selected for growth in hypoxanthine/aminopterin/ thymidine medium, and their supernatants were screened by using tures of the Ig homology units of receptor tyrosine kinases participate in the establishmentof ligand binding. However, an assay of antibody binding to T-18 cells. Positive wells of hybrisoluble recombinant portions of the PDGF receptor (Duan et domas were tested further by an immunoprecipitation assay using either radiolabeled Kit-X or T-18 cell extract followed by Western al., 1991) and Kit/SCF receptor(Lev et al., 1992c) fully retain blotting with a rabbit anti-Kit-X antibody. Hybridomas that scored high affinity ligand binding and alsoundergo ligand-induced positive were cloned twice by limiting dilut,ion. Large quantities of of the specific mAbs were produced by preparation of ascites fluid in CD2/ dimerization.Theseobservationsindicatedthatall structural determinants of ligand binding are confined to theF, mice. mAbs were purified from ascites fluid by affinity chromatogextracellular portionsof these receptors. To confine the ligand raphy on a Sepharose-protein A column that was equilibrated with binding cleft of Kit further we generated ligand-competitive 1.5 M glycine buffer (pH 8.9) containing 3 M NaC1. Elution of the antibodies from the column was done with 0.1 M citrate buffer (pH monoclonal antibodies (mAbs) and recombinantsoluble por- 5.0 for IgG, and pH 3.0 for other classes). tions of the ectodomain. By using these reagentswe obtained Construction and Expression of Mini-Kit-XProteins-To construct results thatprovide strong evidence that the SCF binding site expression vectors that direct the synthesis of portions of the extracellular domain of Kit we used the previously described human Kitis confined to the amino-terminal portion of Kit.

X cDNA cloned into the Bluescript (pBS; Stratagene, La Jolla, CA) plasmid (Lev et al., 1992~). The kit 1-2 expression vector, containing the 5' 719 bp of human c-kit cDNA was constructed as follows. An Materials-Radioactive materials were purchased from Amersham EcoRI-ClaI fragment was cut out from the pBS-kit-X plasmid, and the resulting ends were filled in with Klenow I enzyme and ligated Corp. Protein A coupled to Sepharose was from Pharmacia LKB Biotechnology Inc. or prepared in our laboratory. Recombinant hu- with T4 ligase. This resulted in juxtaposition of two in-frame stop man SCF and transforming growth factor-a were from Amgen (Thou- codons 3' next to the c-kitregion that encodes Ig-like domains 1 and sand Oaks, CA). Recombinant human IL-3 was a gift from Genetic 2. A BarnHI-XbaI fragment (704 bp) was cut out of the resulting Institute (Cambridge, MA), and recombinant human GM-CSF was a plasmid and inserted into the pLSV-DHFR mammalian expression gift from Sandoz (Basel, Switzerland). Molecular weight standards vector (Lev et al., 1992c) that was precut with XhoI and XbaI for gel electrophoresis were obtained from Bio-Rad. Calf serum was downstream of theinternal SV40 earlypromoter. The kit 1-2-3 from HyClone Laboratories (Logan, UT). Rabbitantibodies to mouse expression vector, containing 960 bp from the 5' end of human c-kit, Igs were from Jackson ImmunoResearch. Unless otherwise indicated, was similarly constructed from pBS-kit-X plasmid that was cleaved all other chemicals were from Sigma. Polyclonal antibodies to human with BarnHI and AsnI restriction endonucleases to yield a 960-bp fragment. This was ligated into a BamHI-SalI-cutshuttle vector Kit/SCF receptor were raised in rabbits that were injected with a recombinant Kit-X protein. The extracellular domain of the ~185"" (PATH2). After transformation of bacteria the resulting plasmid was receptor was purified from the medium of cells that were transfected cut with BamHI and ClaI, and the gel-purified fragment was ligated into pBS-kit-X vector from which the c-kit insert was removed by with the corresponding portion of the neu cDNA.' Cell Culture-CHO cells were cultured in F-12 medium containing using BarnHI and ClaI. The resulting plasmid contained the 5' 960 bp of c-kit and an in-frame stopcodon at its 3' end. A BamHI-XbaI 10% calf serum and antibodies and were maintained in a humidified incubator with 5% CO, in air at 37 "C. For transfection experiments fragment was then extracted from this plasmid and fused with a a CHO sublinethat lacks the dihydrofolate reductase (dhfr) gene was XhoI- and XbQI-CUtpLSV-DHFR vector. The mammalian expression were then separately COused (Urlaub and Chasin, 1980). Conditioned media were obtained vectorsencoding Kit1-2andKit1-2-3 by culturing the cells without serum. For binding experiments the transfected with a plasmid that confers neomycin resistance to dhfrmonolayers were grown on fibronectin(Boehringer Mannheim)- negative CHO cells (Urlauband Chasin, 1980). Stably expressing coated dishes (10 pg/ml fibronectin in PBS). M07e cells were rou- cells were selected by their ability to grow in gentimycin (0.8 mg/ml) tinely grown as a suspension culture in Iscove's modified Dulbecco's containing medium. Single-cell colonies were screened by biosynthetic labeling and immunoprecipitation of the corresponding [35s] methionine-labeled Kit protein. I. Stancovski, unpublished data. EXPERIMENTALPROCEDURES

'

SCF Binding Site of Kit

4401

Radiolabeling of Proteins-Affinity-purified rabbit antibodies to RESULTS mouse F(ab'):! were labeledwith "'1 by usingthechloramine-T Generation of Ligand-competitive Monoclonal Antibodies to method (Hunter and Greenwood, 1962). SCF was labeled by using IODO-GEN(Pierce Chemical Co.) as follows. IODO-GEN-coated the KitISCF Receptor-In an attempt to determine thecontubes (2pg/50 pl of chloroform) were incubated with SCF (8 pg) and tribution of specific regions of the extracellular domain of Kit Nal2'I (1 mCi) for 15 min a t 22 "C. The reaction mixture was then to the binding of SCF, we generated a panel of site-specific subjected to gel filtration on a Sephadex G-25 (fine) column (0.6 X 7 mouse mAbs to Kit by immunizing mice with a recombinant cm) thatwas presaturated with bovine serum albumin. whole ectodomain of human Kit (Kit-X; Lev et al., 1992~). Biosynthetic Labeling and Immunoprecipitation-Subconfluent monolayers of cells growing in six-well dishes (Costar) were washed The capacity of selected hybridoma clones to recognize spewith methionine-free Dulbecco's modified Eagle's medium and grown cifically human Kit was tested by immunoprecipitation of unlabeled Kit-X and Western blotting with rabbit polyclonal for 16 h in the same medium supplemented with 10% dialyzed calf serum and 50 pCi of [R'S]methionine/ml. The cell supernatants were antibodies (Fig. 1).The specificity of this blotting assay was spun for 10 min a t 4 "C in an Eppendorf centrifuge andsubjected to indicated by the lack of signal in immunoprecipitates of the immunoprecipitation. Protein A-conjugated Sepharose beads (3 mg/ extracellular portion of p185"" (denoted Neu-X), which was sample) were suspended in HNTG buffer and incubated for 30 min used as a control antigen. To confirm the specificity of the a t 22 "C with either a polyclonal rabbit antiserum to Kit-X (10 pl) or with rabbit anti-mouse F(ab')2 (5p g ) . The latter was followed, after mAbs to p145"', they were assayedfor bindingto c-kita brief wash, by incubation with mAbs (10 pg). The antibody-conju- transfected CHO cells (cell line T-18). All of the antibodies gated beads were washed three times with HNTG and incubated withthat immunoprecipitated Kit-X also recognized T-18cells but the cell supernatants for 2 h a t 4 "C. The immune complexes were did not react with the parental untransfected CHO cells (data washed twice with each of the high, medium, and low salt buffers. not shown). The results of the bindingof two mAbs, K44 and Then gel sample buffer was added to thewashed immunoprecipitates, and they were heated a t 95 "C (5 min) and resolved by SDS-PAGE K45, to c-kit-overexpressing cells are depicted in Fig. 2A. displayed saturable surface binding. and autoradiography. In the case of lZ'I-labeled samples or samples Evidently both antibodies To test the possibility that some of the mAbs that we that were not radioactivelylabeled, theimmunoprecipitates were generated are directed to the ligand recognition site of Kit, washed three times with HNTG. Western Blotting-Washed immunoprecipitates were mixed with the binding of l2'II-SCF to T-18 cells was determined in the SDS gel sample buffer and subjected to SDS-PAGE. The gel-sepa- presence or absence of each mAb. Fig. 2B depicts the results rated proteinswere transferred electrophoretically ontonitrocellulose that were obtained with mAbs K44 and K45. In contrast to filters. Filters were first saturated for 1 h at 22 "C with blocking solution (10% low fat milk in 20 mM Tris-HC1 (pH 7.6) and 17 mM the K45 antibody, which did not affect binding of lZ5I-SCF even a t high antibody concentrations, mAb K44 stoichiometNaC1). Antisera were then added inthesamesolution,andthe incubation was carried out for 1 h. For detection the filters were rically inhibited ligand binding at an approximately 1:l ratio washed three times (5 min each wash) with PBS, 0.05% Tween 20 of antibody to SCF. Only one other antibody, K57, similarly and reacted for 45 min at room temperature with horseradish perox- affected the binding of SCF (data not shown),implying that idase-conjugated protein A. The enzyme was removed by washing as both mAbs K44 and K57 are directed to the SCF binding site above. The filters were reacted for 1 min with a chemiluminescence or to a nearby determinant. To support possibility this further reagent (ECL, Amersham) and exposed to an autoradiography film we determined the effect of these mAbs on covalent crossfor 1-15 min. ' . the absence of mAbs two Binding Assays of Radiolabeled Ligands-The binding buffer con- linking of Iz5I-SCF to ~ 1 4 5 ~ ' In tained Dulbecco's modified Eagle's medium supplemented with 0.1% ligand-receptor complexes could be immunoprecipitated from bovine serum albumin and 20 mM HEPES-buffered at pH 7.5. The "-18 cells (Fig. 3A). These complexes correspond to receptor assays were carried out in 24-well dishes, and the monolayers were monomers and dimers (Lev et al., 1992a). However, incubation radiolabeled of the cells with "'1-SCF in thepresence of K44 or K57 mAb equilibrated in bindingbuffer 15 min prior to adding the ligand. For the determination of antibody binding the monolayers were incubated at 22 "C for 90 min with hybridoma supernatants or completely abolished both cross-linked receptor forms (Fig. with various concentrations of the purified mAbs. Following a brief 3A). In contrast, mAbs that did not inhibit SCF binding to wash with PBS, 0.1% bovine serum albumin, 1251-labeledrabbit anti- T-18 cells (e.g. K45 and K49) exertedonly limited effects on mouse F(ab')? (10' cpm/well)was added, and the incubation was cross-linking of '"I-SCF to the receptor. Similarly, mAbs K44 continued for an additional 90 min.Cell-bound radioactivity was and K57 did not immunoprecipitatepreformed covalent comdetermined by solubilization and harvest in 0.2 M NaOH and 0.1% plexes of '"I-SCF with ~ 1 4 5 ~(Fig. ' ' 3B), although they did SDS. For the determination of "'I-SCF binding cell monolayers were recognize theKit-Xprotein (Fig. 1B) and the full-length incubated a t 4 "C with 12'I-SCF for 2 h in the presence of various concentrations of unlabeled SCF. The amountof nonspecific binding was determined by performing the assay in the presence of a 100-fold Kit- X Neu-X excess of unlabeled SCF. At the endof the binding reaction thecells were washed, and their bound radioactivity was determined as described above. Ligand displacement analyses wereperformed in a similar way except that "'I-SCF (2 nM) was incubated with cellular monolayers for 4 h a t 4 "C in the presence of increasing concentrations of soluble Kit proteins. f 116 Covalent Cross-linking of Radiolabeled SCF-Affinity-purified recombinantproteins(Kit-X,Kit 1-2-3, Kit 1-2, and Neu-X) were incubated in PBS, 0.1% bovine serum albumin with "'I-SCF (100 ng/ml, lo5 cpm/ng) in the presence of variousconcentrations of 85 unlabeled SCF or transforming growth factor-n. The total reaction I6 Ab:AntiKit-X(rabbit) volume was 0.06 ml. After 4 h a t 22 "C the proteinswere subjected to covalent cross-linking by adding disuccinimidyl suberate toa 0.5 mM FIG. 1. Selection of hybridomas producing Kit-specific anfinal concentration, and the reaction wasallowed to continue for 40 tibodies. Supernatants of individual clones of hybridoma cells were min a t 22 "C. Gel sample buffer was then added, and the reaction was incubated with Kit-X protein (approximately200 ng) or with Neu-X terminated by boiling and SDS-PAGE. For cross-linking of SCF to protein (200 ng) as indicated. After gel electrophoresis and protein cultured cells the ligand was incubated with the cells for 2 h a t 4 "C, transfer toa nitrocellulose filter,the blotwas probed with apolyclonal washed, and the ligand-receptor complexes were covalently crossrabbit antibody to Kit-X followed by horseradish peroxidase-labeled linked by adding EDAC (15 mM) in PBS and further incubation for protein A. Chemiluminescence (ECL kit, Amersham) and autoradi40 min a t 22 "C. The cells were then lysed in solubilization buffer, ography (5-min exposure) were used to obtain the shown autoradiogram. IP, immunoprecipitated; IB, immunoblotted. and the Kitreceptor was immunoprecipitated as described above.

t

SCF Binding Site of Kit

4402

I P Ab! Anti-Klt-X ( r a b b i t )

[Ab] ; nM

[Ab]; nM FIG. 2. Antibody binding to Kit-overexpressing cells and competition with SCF. Panel A , confluent monolayers of T-18

FIG. 3. Effect of monoclonal antibodies on cross-linking of ''9-SCF to Kit and immunoprecipitation of the ligand-receptor complexes. Monolayers of T-18 cells (approximately lo6 cells) were incubated with 2 nM '*'I-SCF in the presence of the indicated B ) .Following mAbs a t 2 pg/ml (panel A ) or without antibodies (panel 2 h a t 4 "C the cells were washed, and the ligand-receptor complexes were covalently cross-linked by a 40-min incubation a t 22 "C with 15 mM EDAC. The complexes were then immunoprecipitated (ZP)with a polyclonal antibody to Kit-X (panelA ) or with the indicatedmAbs (panel B ) and analyzed by electrophoresis in 5% acrylamide gels followed by autoradiography (24h a t -70 "Cwith anintensifier screen). The locations of molecular mass marker proteins are indicated by bars, and the correspondingmasses are given in kDa. Note that both dimers and monomersof Kit were resolved.

TABLE I Effect of anti-Kit mAbs on DNA synthesis of M07e megakaryocyte cells M07e cells (50,00O/well) weredeprived of growth factors 24 h before starting the experiment.Antibodies were added to thecells 50 min priorto the additionof growth factors. The incorporationof [3H] thymidine into DNA was determined by a 4-h pulse that was given after 20 h of incubation a t 37 "C with antibodies andgrowth factors. The results given are averages of triplicate determinations in which the variation did not exceed 10%. The experiment was repeated twice. Treatment

cells, a CHO subline that overexpresses the human Kit protein (Lev Deprived cells et al., 1991), were incubated for90 min a t 22 "C withdifferent SCF (30 ng/ml) concentrations of mAb K44 (closed circles) or K45 (open squares). GM-CSF (10 ng/ml) After a brief washing step the monolayers were further incubated for K44 90 min a t 22 "C with radiolabeled rabbit Ig specific to mouse IgG. This was followed by extensive washingof the unbound radioactivity 10 and counting the cell-associated radioactivity in a y-counter. Panel 5d m 1 SCF (30 ng/ml) + K44 B, confluent monolayers of T-18 cells were incubated with lZ5I-SCF 0.5 pg/ml (2.5 ng/ml, lo5 cpm/ng) in the presence of different concentrations of mAb K44 (closed circles) or K45 (open squares).Following 4 h of 5d m 1 binding at 4 "Cthe monolayers were washed extensively withice-cold 10 d m 1 GM-CSF (10 ng/ml) K44 (10 pg/ml) PBS, and the cell-associated radioactivity was determined in a yK49 counter. Nonspecific binding of '251-SCFwas determined by performing the bindingreaction in the presenceof a 100-fold excess concen5 rg/ml tration of unlabeled SCF, and it was subtracted from the total amount 10 ccg/ml SCF (30 ng/ml) + K49 of bound radioactivity. The results shown are representative of two 0.5 pg/ml experiments. 5d m 1 10 u d m l

+

['HIThymidine incorporated cpm

1,463 57,966 59,632 1,069 1,196 3,536 2,622 2,977 68,587 1,552 2,773 50,715 39,383 28.667

receptor (data not shown) when was it not cross-linked to the ligand. On the basis of these experiments we concluded that mAbs K44 and K57 recognize determinants that are close to et ai., 1990). More recently it has been reported that M07e the SCF binding siteof ~ 1 4 5 ~unlike ", other antibodies that cells can proliferate also in thepresence of SCF (Hendrie et interact elsewhere on the receptor molecule. In separate ex- al., 1991). When tested on M07e cells, mAb K44 completely periments we addressed the possibility that K44 andK57 are inhibited SCF-induced incorporation of [3H]thymidine into directed against the same epitope. To this end, we radiolabeled macromolecules (Table I).However, K44 had noeffect on the K44 and analyzed its displacement from Kit-expressing cells induction of DNA synthesis by GM-CSF (Table I) or IL-3 by native SCF or K57 mAb. Interestingly, SCF efficiently (data not shown). As expected, mAb K49, which does not displaced surface-bound '251-K44, but K57 was ineffective a t affect SCF binding, exerted only a limited inhibitory effect concentrations that inhibited SCF bindingT-18 to cells (data on SCF-stimulated M07ecells. Construction and Expression of Two Deletion Mutants of not shown).We therefore concluded that the epitopes of mAbs K44 and K57 are nonoverlapping. the Extracellular Portion of Kit-In an attempt to localize the The capacity of mAb K44 to inhibit SCF binding to Kit epitopes of mAbs K44 and K57 that are apparently located was examined in uitro by using the M07e human megakar- close to the SCF binding cleft, we constructed two deletion yoblastic cell line. These cells display absolute dependence mutants of the Kit-X protein. The unique endonuclease refor survival and proliferation on GM-CSF and IL-3 (Avanzi striction sites EcoRI and AsnI of the human c-Kit cDNA

SCF Binding Site of Kit (Yarden et al., 1987) were used to introduce stop codons a t these sites, thereby terminatingtranslation a t eitherthe amino- or the carboxyl-terminal side of the third Ig domain of Kit (Fig. 4). The modified cDNAs were ligated separately into an eukaryotic expression vector that contained the dhfr gene as a selectable marker. These plasmids were used to transfect dhfr-deficient CHO cells (Urlaub andChasin, 1980), and drug-resistant colonies were grown individually. Clones that overexpressed the shorterdeletion mutant, containing Ig domains 1 and 2 (denoted Kit 1-2), or thelonger protein that contained also domain 3 (Kit 1-2-3), were selected by using an immunoprecipitation assay. A representative immunoprecipitation analysis of the selected clones is shown in Fig. 5. Monolayers of the selected cell lines and a control untransfected cell line were incubated for 16 h with [35S]methionine. The biosynthetically labeled Kit proteins that were secreted into the growth medium were then immunoprecipitated with a polyclonal rabbit antibodythat was raised against thewhole extracellular domain. As shown in Fig. 5, a 36-kDa protein was detectable in the medium of cells that were transfected with the kit 1-2 plasmid, whereas a 65-kDa protein was secreted by kit 1-2-3-transfected cells. These molecular masses are in agreement withthe expected sizes of the recombinant glycosylated proteins (Fig. 4 and Qiu et al., 1988). In addition to the indicated p36 and p65 proteins, two other bands of 120 and 180 kDa appeared in the immunoprecipitates. We attribute these molecules to nonspecific interactions with the polyclonal antiserum because they were precipitated also from untransfected cells (Fig. 5). Ligand Inhibitory mAbsRecognize the Recombinant Kit Proteins-In the next step we analyzed the ability of Kitspecific mAbs to recognize Kit 1-2-3 and Kit 1-2 proteins by using an immunoprecipitation assay. As shown in Fig. 6, Kit 1-2-3 wasrecognized by four mAbs, including the ligandcompetitive K44 and K57 antibodies. In addition, these two antibodies could immunoprecipitate the shorter mutant(Fig. 6, right panel), implying that theirepitopes are confined to Ig

Kit 1-2-3

Kit

TK2

4403

Kit-X

4' 4

J

I P Ab: Anti Kit-X(rabbit) FIG. 5. Expression of portions of the extracellular domain of Kit in CHO cells. cDNAs corresponding to thewhole extracellular domain of Kit (denoted Kit-X) or to portions of it (denoted Kit 1-2 and Kit 1-2-3;see Fig. 4) were cloned into thepLSV-DHFR eukaryotic expression vector and used to transfect cultured CHO cells. Clones of cells that overexpressed the transfected genes were selected and grown separately. Biosynthetically labeled supernatants from lo6cells were harvested after a 16-h incubation of the indicated cell lines with ["S]methionine. The Kitproteins were immunoprecipitated from the cleared supernatants by using a rabbit polyclonal antibody to Kit-X and visualized by SDS-PAGE and autoradiography (48 h at -70 "C with an intensifier screen). The locations of the Kit proteins are indicated by arrows. Also shown are thelocations of molecular mass marker proteins (in kDa). None refers to control untransfected CHO cells. Kit 1-2-3

I8 Ab: Anti Kit-X ( r a b b i t ) FIG. 6. Recognition of Kit 1-2 and Kit 1-2-3 by different monoclonal antibodies. Supernatants of CHO cell lines that overexpress Kit1-2-3 or Kit 1-2 (as indicated) were harvested and concentrated 20-fold in an ultrafiltration cell (Amicon). For control we used a CHO cell line that secretes the extracellular domain of the Neu protein (labeled Neu-X). Aliquots of 0.5 ml of each supernatant were then subjected to immunoprecipitation (ZP)with the indicated mAbs. The immunocomplexes were resolved by SDS-PAGE (8.5% acrylamide), transferred ontonitrocellulose, and immunoblotted (ZB) with a rabbit polyclonal antibody to Kit-X. Detection was performed by using chemiluminescence and autoradiography (left panel, 1 min; right panel, 15-min exposure of the x-ray film). An arrow marks the location of Kit 1-2 protein,and bars indicate the locations of molecular mass marker proteins.

domains 1and 2 and theintervening sequences. Interestingly, mAbs K27 and K69 did not react with Kit 1-2-3, suggesting that their recognition sites are distal to the first three Ig domains of Kit. Both K45 and K49mAbs,whichdo not interfere with ligand binding, recognized Kit 1-2-3 and also FIG. 4. Structural representation of the Kit protein and Kit 1-2 (Fig. 6 and data not shown). In conclusion, the portion recombinant forms of its extracellular domain. The various of the Kit protein which is included in the short deletion proteins are schematically presented, and the Ig-like domains are shown as loops. The proteins are drawn to scale, and thedesignations mutant Kit 1-2 contains thebinding sites of mAbs K44 and of their functional domains are indicated at the bottom. Also shown K57, and by extension also the SCFbinding site or part of it. are the locations of the unique sites of restriction endonucleases that Inhibition of Binding of Radiolabeled SCF to Wild-type Kit were used to construct the recombinant proteins. Numbers 1-5 indi- by Soluble Recombinant Proteins-Because of the soluble cate the respective Ig-like domains. The locations of all of the cysteine nature of the recombinant truncation mutants of Kit, simple residues of the extracellular domains are indicated by a circle and C. Tryptophan residues that belong to theconsensus structure of the Ig ligand binding assays were not feasible. In addition, immobihomology unit are shown by a circle and W. SP, signal peptide; TK, lization by using antibodies could interfere with the binding tyrosine kinase. assay. We therefore tested the ability of soluble Kit proteins

SCF Binding Site of Kit

4404

that were affinity purified over a mAb column (Fig. 7A) to inhibit bindingof SCF to Kit-overexpressingcells. This type of analysis has indicatedpreviously that Kit-X and wild-type Kit display comparable affinities to SCF (Lev et al., 1992~). The results presented in Fig. 7B further indicate that Kit 12-3 ispractically as potent as Kit-X in inhibitingbinding. SCF However, Kit 1-2, which lacksdomain3, was much less efficient than Kit 1-2-3 in displacing Kit-bound SCF (Fig. 7B). Since Kit 1-2 did not exceed 50% inhibition of SCF binding inseveral independentexperiments, we could not determine the relative ligand affinity of this mutant protein. However, we estimate that removal of Ig-like domain 3 involves 5-7-fold reduction in apparent ligand affinity.

Covalent Cross-linking of Radiolabeled SCF to Soluble Kit Proteins-To demonstrate directly the specific interaction between SCF andsoluble Kit proteins,we employed covalent cross-linking of the radiolabeled ligand. Immunoaffinity-purified Kit-X, Kit 1-2-3, or Kit 1-2 (or Neu-X as a negative control) were incubated with '251-SCF, and the cross-linking reagent disuccinimidyl suberate was added later to stabilize ligand-receptor complexes covalently. Gel electrophoresis that was performed afterward revealed radioactive bands of sizes that correspond tocomplexes of IZ5I-SCFwith the respective Kit protein (Fig. 8A). Thus, two bands that were identified previously as monomers and dimers of the extracellular domain of Kit (Lev et al., 19892~)were seen with Kit-X,whereas 85- and 55-kDa radioactive bands were observed with Kit 1-

A

Kit-X Kit

-1-2 Kit1.2-3

Mr(kd)

I72 57 K i t 1-2

i

K44A.P:

IB Ab:

rn-

- - 4- - -t Anti Kit-X (robbit)

CONCENlRAllON OF KTT P R O E I N ("MI

FIG. 7. Inhibition of binding of radiolabeled SCF to cells b y soluble Kit proteins. Panel A, purification of recombinant proteins. The indicated Kit proteins were harvested from the supernatantsof CHO cell lines that overexpress the respective portions of the ectodomain. Cleared cell supernatants were analyzed by immunoblotting ( I B ) either directly or after affinity purification on a column of the K44 mAbs (labeled K44 A.P.) as indicated. Panel B, inhibition of binding of lZ5I-SCF.Monolayers of CHO cells that overexpress human c-kit (T-18cells) were incubated at 4 "C with 2 nM"'1-SCF in the presence of the indicated concentrationsof purified Kit-X (squares), Kit 1-2-3 (open circles), or Kit 1-2 (closed circles). Following a 3-h incubation the monolayers were washed twice, and cell-bound radioactivity was determined ina y-counter. For determinationof nonspecific binding the experimentwas also performed in the presence of a 100-fold molar excess of unlabeled SCF, and thecell-bound radioac5% of total binding) was tivity under these conditions (less than subtracted from the signal obtained in the absence of unlabeled SCF. The results shown are representative of three experiments.

B

Kit

1-2

Mr (Kd 1

p;

Kif 1-2-3

57

FIG.8. Covalent cross-linking of "'I-SCF to portions of the extracellular domainof Kit. PanelA, affinity-purified Kit proteins or a recombinantectodomain of theNeuprotein(Neu-X) were incubated a t 4 "C for 4 h with 5 nM '2'I-SCF in 0.06 ml total volume. T o cross-link the radiolabeledliganddisuccinimidyl suberate was added to a 0.5 mM final concentration and the incubation continued for additional 40 min. Covalent complexes were then analyzed by SDS-PAGE (10% acrylamide gel) andautoradiography(20 h at -70 "C with an intensifier screen). Panel B, the indicatedKit-derived proteins (500 ng) were incubated at 4 "C for 4 h with "'I-SCF (5 nM) in a total volume of 0.06 ml. The reaction mixtures also contained unlabeled SCF (2 and 0.5 p ~ as, indicated), unlabeled transforming , no addition (-) as indicated growth factor-cu (TGF-a, 2.8 p ~ ) or above each lane. Covalent cross-linking was then induced by the addition of disuccinimidyl suberate andgel electrophoresis as in panel A. Autoradiography was for 24 h. Arrows indicate the locationsof the radiolabeled complexes of SCF and the corresponding Kitproteins. Note thata dimer of Kit-X was resolved, but no dimersof Kit 1-2-3 or Kit 1-2were detectable.

2-3 and Kit 1-2,respectively (Fig. 8A). The specificity of the covalent cross-linkingwas indicated by the absence of signal with the Neu-X protein(Fig. 8A). Additional experiments were performed to confirm the specificity of interaction of '251-SCF with the recombinant Kit proteins(Fig. 8B). These analysesshowed that unlabeled SCF (at0.5 or 2 nM), but not unlabeled transforming growth factor-0, reduced in a concentration-dependent manner the amount of radioactivity in the protein bands that correspond to Kit 1-2-3 and Kit1-2. Similar effects were observed when the cross-linking to Kit-X was examined (Fig. 8B). It was therefore concluded that the interactions between Iz5I-SCF and the recombinant proteins Kit 1-2-3 and Kit 1-2 are both ligand-specific and receptor-specific. DISCUSSION

Receptor tyrosine kinases that contain Ig-like domains in their ligand-binding portions now outnumber receptor tyrosinekinaseswithotherextracellularlandmarks(Hanks, 1991), and they include the PDGF receptor group, the receptors for the fibroblastgrowth factors as well as receptors for neurotrophic factors (reviewed in Ullrich and Schlessinger, 1990). Nevertheless, ligand binding to these receptors is less

SCF Binding Site of Kit

4405

which to test this model, and we currently employ chimeric understood than in the case of the EGF receptor and the insulin receptors. By using chimeras between chicken and Kit proteinsto address the mode of involvement of individual human EGF receptors (Lax et al., 1989) and direct peptide domains. The possibility that interdomain packing, rather than a et al., 1988; WU mapping of the binding sites of lZ5I-EGF (Lax et al., 1990) and EGF-competitive mAbs (Wu et al., 1989), it contiguous protein sequence, defines the ligand binding site was shown that the receptor’s portion that lies between the of Kit, is consistent with our observation that the SCFtwo cysteine-rich sequences specifies ligand binding. How- competitive mAbs (K44 and K57) recognize conformationshown). The aminoever, it appears that the amino-terminal portion of the EGF dependent epitopes of Kit(datanot receptor also participates in the formation of the ligand bind- terminal location of the binding site is also reminiscent of the ing cleft. Similarly, the binding of insulin and insulin-like finding that domain 2 of the aPDGF receptor confers specigrowth factor-1 to their related receptors appears to involve ficity to PDGF-AA (Heidaran et al., 1992). Interestingly, the both the amino-terminal portionsof the receptors (Wedekind first and thesecond Ig-like domains of another receptor with five such motifs, the intercellular cell adhesion molecule 1, et al., 1989; Kjeldsen et al., 1991) andtheircysteine-rich domains (Gustafson and Rutter, 1990; Yip et al., 1991). The contain the binding site for the integrin LFA-1 and also for a latter may function as a core binding cleft whereas ligand subgroup of human rhinoviruses (Staunton et al., 1990). Presumably, distal localization of the binding sites for soluble specificity is probably determined by adjacent sequences. Interestingly, studies of ligand interactions with receptor ligands ( i e . SCF and PDGF),integrin, or a bulky virus makes tyrosine kinases that contain Ig-like motifs also indicate that these cellular receptors accessible to interactions. In conclusion, our present studyconfined the ligand binding noncontiguous receptor segments may create the ligand binding sites of such receptors. In addition, determinantsof spec- site of Kit to its amino-terminal portion and implicated the ificity to different ligands may not coincide. For example, the involvement of noncontiguous protein sequences in SCF recamino-terminal three Ig-like domains of the PDGF receptor ognition. Further dissection of the amino-terminal half of the confer the ability to bind different PDGF isoforms (Heidaran ectodomain of Kit by internal deletions should make it poset al., 1990), but specificity of the a receptor to PDGF-AA sible to define more precisely the interaction between SCF resides in the second Ig-like domain and it is distinct from and Kit. Alternatively, crystallization of the recombinant Kit the PDGF-BB specificity determinant (Heidaranet al., 1992). proteins,and especially Kit 1-2-3, may provide molecular Likewise, the carboxyl-terminal half of the third Ig-like do- insights into the binding of SCF to itstarget cells. main of variants of the fibroblast growth factor receptors Acknowledgments-We thank Dr. Kris Zsebo (Amgen Center) for determines ligand specificity to thekeratinocyte growth factor or basic fibroblast growth factor, but binding of acidic fibro- recombinant human SCF, Magda David for help in the generation of blast growth factor is determined elsewhere in the receptor mAbs, and Miriam Fagan for typing this manuscript. (Werner et al., 1992; Miki et al., 1992; Yayon et al., 1992). REFERENCES By using SCF-competitive mAbs we localized the ligand Amzel, L. M., and Poljak, R. J. (1979)Annu. Reu. Biochem. 48,961-967 binding site to thetwo amino-terminal Ig-like domains of Kit Avanzi, G. C., Brizzi, M. F., Giannotti, J., Ciarletta, A., Yang, Y., Pegoraro. L., and Clark, S. C. (1990) J. Cell. Physiol. 146,458-464 (Fig. 3). However, a recombinant protein that contained only Bennett, D. (1956) J. Morphol. 9 8 , 199-234 the first two Ig-like domains (Kit 1-2) displayed reduced Besmer, P. (1991) Curr. Opin. Cell Biol. 3,939-946 affinity to SCF, as compared with the whole ectodomain of Blume-Jensen P., Claesson-Welsh L., Siegbahn, A. Zsebo, K. M., Westermark, B., and Heldin, C.-H. (1991) E d B O J. 10,412114128 Kit or a soluble protein encompassing the three amino-ter- Chabot, B., Stephenson,D. A., Chapman, V. M., Besmer, P., and Bernstein, A. (1988) Nature 335,8&89 minal Ig-like domains (Fig. 7B). It is therefore conceivable Copeland, N. G., Gilbert, D. J., Cho, B. C., Donovan, P. J., Jenkins, N. A,, that part of the high affinity binding site resides in the Coseman, D., Anderson, D., Lyman, S. D., and Williams, D. E. (1990) Cell 63, 175-183 receptor’s portion that is defined by the amino-terminal two Duan, D . 3 R., Pazin, M. J., Fretto, L. J., and Williams, L. T. (1991) J. Biol. Ig-like domains, but determinants which are included in the Chern. 266,413-418 third Ig loop are also involved in the formation of the ligand Flanagan, J. G., Chan, D., and Leder, P. (1991) Cell 64,1025-1035 Funasaka, Y., Boulton, T., Cobb, M., Yarden, Y., Fan, B.,L man, S. D., binding cleft. Covalent cross-linking experiments (Fig. 8) also Williams, D. E., Anderson, D. M., Zakut, R., Mishima, Y., an8Halaban. R. (1992) Mol. Biol. Cell 3 , 197-209 support our conclusion that the major portion of the SCF Galfre, G., Howe, S. C., Milstein, C., Butcher, G. W., and Howard, J. C. (1987) binding cleft is confined to the first and second Ig-like doNature 266,550-552 mains of Kit. However, this type of experiment cannotresolve Geissler, E. N., Ryan, M. A., and Housman, D. E. (1988) Cell 55,185-192 Gustafson, T. A., and Rutter, W. J. (1990)J. Biol. Chern. 265,18663-18667 differences in ligand binding affinities. Hanks, S. K. (1991) Curr. Opin. S t r u t . Biol. 1,369-383 An independent supportto theamino-terminal localization Heidaran, M. A., Pierce, J. H., Jensen, R. A., Matsui, T., and Aaronson, S. A. (1990) J. Biol. Chern. 265,18741-18744 of the SCF binding site was obtained recently in our labora- Heidaran, M. A., Yu, J.-C., Jensen, R. A., Pierce, J. H., and Aaronson, S. A. (1992) J. Biol. Chern. 267,2884-2887 tory by utilizing chimeric human-mouse Kit protein^.^ Since P. 0.. Miyazawa, K., Yang, Y. C., Langefeld, C. D., and Broxmeyer, murine Kit does not bind human SCF, it was possible to Hendrie, H. E. (1991) Exp. Hernatol. 19, 1031-1037 Huang, E., Nocka, K., Beier, D. R., Chu, T. Y., Buck, J. Lahm, H. W., Wellner, localize the binding site of human SCF through replacement D., Leder, P., and Besmer, P. 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