Kazushige Sakaguchi

Four of ten HLA-A2-restricted melanoma specific CTL that were derived from tumor-infiltrating lymphocytes (TIL) and administered to patients recognized the gp100 melanoma Ag and nine of ten recognized the MART-1 Ag. Adoptive transfer of the four gp100-reactive CTL, but not the other TIL, resulted in tumor regression when infused into autologous patients along with IL-2. Tumor regression was thus correlated with the recognition of gp100 by the administered T cells (p = 0.0048). To identify the epitopes recognized by these four gp100-reactive CTL, 169 peptides containing HLA-A2.1 binding motifs were synthesized and screened for their recognition by TIL using cytotoxicity and IFN-gamma release assays. Five gp100 epitopes (two for TIL620, three for TIL660, one for TIL1143, and two for TIL1200) were recognized by CTL derived from different patients. Five of eight HLA-A2 binding melanoma epitopes (five gp100, one MART-1/Melan-A, two tyrosinase) had intermediate binding affinity to HLA-A2.1. These gp100 epitopes may be responsible for mediating tumor rejection in vivo and thus may be useful for the development of immunotherapies for patients with melanoma.

Familial multiple endocrine neoplasia type 1 (MEN-1) is characterized by tumors of the parathyroids, endocrine pancreas, and anterior pituitary. Since the gene associated with MEN-1, located on chromosome 11 (11q13), may normally inhibit tumor proliferation, tumors could arise from inactivation of one or both of the alleles. However, parathyroid tumors in patients with MEN-1 have been considered to result from polyclonal hyperplasia. Using genetic probes, we tested parathyroid tumors for a monoclonal component, represented by a loss of alleles at any of eight loci along chromosome 11. Ten of 16 tumors from 14 patients with familial MEN-1 had losses of alleles from chromosome 11. Tumors with losses were larger than those without (1.6 vs. 0.2 g; P less than 0.002), suggesting that a monoclonal adenoma may develop after a phase of polyclonal hyperplasia. In 7 of 10 tumors, the subregion of loss was less than the full length of chromosome 11 but always included one copy of the MEN-1 locus. Of 34 sporadic adenomas from patients without MEN-1, 9 showed similar allelic losses in chromosome 11; in 7 the losses included the apparent MEN-1 locus. We conclude that many "hyperplastic" parathyroid tumors in familial MEN-1 are in fact monoclonal and may progress or even begin to develop by inactivation of the MEN-1 gene (at 11q13) in a precursor cell. Some sporadic adenomas have allelic losses on chromosome 11, which may also involve the MEN-1 gene.

MART-1 is an Ag expressed on melanomas and melanocytes, and is recognized by the majority of HLA-A2-restricted tumor-specific tumor-infiltrating lymphocytes (TIL) from melanoma patients. In the present study we have analyzed 10 potential 9-mer epitopes containing the HLA-A2.1 binding motifs for their ability to induce melanoma-specific T cell lines. Antimelanoma CTL could be generated only with MART-1(27-35) peptide, which has been previously shown to be recognized by a majority of HLA-A2-restricted TIL. Anti-MART-1(35-43)-specific CTL could also be induced, but these T cells did not recognize melanoma cells. MART-1(27-35)-specific CTL could be effectively generated from a total of 11 of 12 PBL and from 3 of 3 TIL derived from HLA-A2+ melanoma patients, as well as from 2 of 4 PBL from HLA-A2+ healthy donors by in vitro stimulation with autologous PBMC pulsed with the synthetic MART-1(27-35) peptide. These CTL lines specifically lysed and release cytokines (TNF-alpha, IFN-gamma, and GM-CSF) in response to T2 cells pulsed with MART-1(27-35), as well as to HLA-A2+ MART-1+ melanoma cells. CTL generated with MART-1(27-35) also lysed uncultured HLA-A2+ melanoma cells derived from tumor biopsies, indicating that this MART-1 epitope is likely to be expressed in association with HLA-A2 on the surface of tumor cells in vivo. CTL lines generated with MART-1(27-35) mediated 25- to 100-fold higher lytic activity than MART-1-reactive CTL grown from TIL in the presence of high dose IL-2. These results demonstrate that MART-1(27-35) peptide may represent an ideal candidate for Ag-specific immunotherapy in melanoma patients.

Hepatocyte growth factor/scatter factor (HGF/SF) is a heparin-binding polypeptide that stimulates cell proliferation, motility, and morphogenesis by activation of its receptor, the c-Met tyrosine kinase. HGF/SF consists of a series of structural units, including an amino-terminal segment with a hairpin loop, four kringle domains, and a serine protease-like region. In this study, we demonstrate that the amino-terminal (N) domain retains the heparin-binding properties of full-length HGF/SF. In contrast to a previous hypothesis, selected basic amino acid residues in the hairpin loop are not critical for heparin binding, although alanine substitution at a subset of these sites markedly reduced the biological activity of the HGF/SF isoform, HGF/NK1. Covalent cross-linking experiments performed with wild-type and heparan sulfate glycosaminoglycan (HSGAG)-deficient Chinese hamster ovary (CHO) cells revealed that Met-HGF/NK1 binding was strongly dependent on HSGAG. Addition of heparin to HSGAG-deficient CHO cells not only restored ligand binding, but also increased ligand-dependent Met tyrosine phosphorylation and c-fos expression. Moreover, our results showed that heparin stimulated ligand oligomerization through an interaction with the N domain. These findings establish the importance of the N domain for heparin-ligand and ligand-ligand interactions, and demonstrate a crucial role for HSGAG in receptor binding and signal transduction. Hepatocyte growth factor/scatter factor (HGF/SF) is a heparin-binding polypeptide that stimulates cell proliferation, motility, and morphogenesis by activation of its receptor, the c-Met tyrosine kinase. HGF/SF consists of a series of structural units, including an amino-terminal segment with a hairpin loop, four kringle domains, and a serine protease-like region. In this study, we demonstrate that the amino-terminal (N) domain retains the heparin-binding properties of full-length HGF/SF. In contrast to a previous hypothesis, selected basic amino acid residues in the hairpin loop are not critical for heparin binding, although alanine substitution at a subset of these sites markedly reduced the biological activity of the HGF/SF isoform, HGF/NK1. Covalent cross-linking experiments performed with wild-type and heparan sulfate glycosaminoglycan (HSGAG)-deficient Chinese hamster ovary (CHO) cells revealed that Met-HGF/NK1 binding was strongly dependent on HSGAG. Addition of heparin to HSGAG-deficient CHO cells not only restored ligand binding, but also increased ligand-dependent Met tyrosine phosphorylation and c-fos expression. Moreover, our results showed that heparin stimulated ligand oligomerization through an interaction with the N domain. These findings establish the importance of the N domain for heparin-ligand and ligand-ligand interactions, and demonstrate a crucial role for HSGAG in receptor binding and signal transduction. INTRODUCTIONDuring the past dozen years, several mitogens have been identified that possess the distinctive property of binding to heparin or closely related heparan sulfate glycosaminoglycan (HSGAG) 1The abbreviations used are: HSGAGheparan sulfate glycosaminoglycanHGF/SFhepatocyte growth factor/scatter factorHGF/NK1truncated HGF/SF isoform containing amino-terminal (N) domain and kringle 1HGF/NK2truncated HGF/SF isoform containing N domain and kringles 1 and 2K2second kringle domain in HGF/SFCHO-WTwild type Chinese hamster ovary (CHO) cell lineMDCKMadin-Darby canine kidney2A/NK1, 3A/NK1, and 5A/NK1alanine substitution mutants in HGF/NK1 with 2, 3, or 5 substitutions as described in the textPBSphosphate-buffered salinePAGEpolyacrylamide gel electrophoresisIGFinsulin-like growth factor (1Burgess W.H. Maciag T. Annu. Rev. Biochem. 1989; 58: 575-606Google Scholar, 2Ruoslahti E. Yamaguchi Y. Cell. 1991; 64: 867-869Google Scholar). Heparin-based affinity chromatography has been a convenient tool for purification of these factors, and several studies have shown that this binding phenomenon has functional relevance as well (2Ruoslahti E. Yamaguchi Y. Cell. 1991; 64: 867-869Google Scholar, 3Schreiber A.B. Kenney J. Kowalski W.J. Friesel R.T.M. Maciag T. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6138-6142Google Scholar, 4Rapraeger A.C. Krufka A. Olwin B.B. Science. 1991; 252: 1705-1708Google Scholar, 5Schlessinger J. Lax I. Lemmon M. Cell. 1995; 83: 357-360Google Scholar, 6Kan M. Wang F. Xu J. Crabb J.W. Hou J. McKeehan W.L. Science. 1993; 259: 1918-1921Google Scholar, 7Yayon A. Klagsbrun M. Esko J.D. Leder P. Ornitz D.M. Cell. 1991; 64: 841-848Google Scholar, 8Mach H. Volkin D.B. Burke C.J. Middaugh C.R. Linhardt R.J. Fromm J.R. Loganathan D. Mattsson L. Biochemistry. 1993; 32: 5480-5489Google Scholar, 9Ornitz D.M. Yayon A. Flanagan J.G. Svahn C.M. Levi E. Leder P. Mol. Cell. Biol. 1992; 12: 240-247Google Scholar, 10Spivak-Kroizman T. Lemmon M.A. Dikic I. Ladbury J.E. Pinchasi D. Huang J. Jaye M. Crumley G. Schlessinger J. Lax I. Cell. 1994; 79: 1015-1024Google Scholar). HSGAG-dependent stabilization (3Schreiber A.B. Kenney J. Kowalski W.J. Friesel R.T.M. Maciag T. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6138-6142Google Scholar) and/or localization of the growth factor on the cell surface may promote interaction with less abundant, higher affinity tyrosine kinase receptors involved in signal transduction (2Ruoslahti E. Yamaguchi Y. Cell. 1991; 64: 867-869Google Scholar, 4Rapraeger A.C. Krufka A. Olwin B.B. Science. 1991; 252: 1705-1708Google Scholar, 5Schlessinger J. Lax I. Lemmon M. Cell. 1995; 83: 357-360Google Scholar). Coupling of heparin or HSGAG either to the growth factor or its tyrosine kinase receptor may induce conformational changes in these molecules that augment signaling (3Schreiber A.B. Kenney J. Kowalski W.J. Friesel R.T.M. Maciag T. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6138-6142Google Scholar, 6Kan M. Wang F. Xu J. Crabb J.W. Hou J. McKeehan W.L. Science. 1993; 259: 1918-1921Google Scholar). Several laboratories have reported that heparin binding can provide a framework for ligand oligomerization, which may enhance signaling by stimulating dimerization of the tyrosine kinase receptor (5Schlessinger J. Lax I. Lemmon M. Cell. 1995; 83: 357-360Google Scholar, 8Mach H. Volkin D.B. Burke C.J. Middaugh C.R. Linhardt R.J. Fromm J.R. Loganathan D. Mattsson L. Biochemistry. 1993; 32: 5480-5489Google Scholar, 9Ornitz D.M. Yayon A. Flanagan J.G. Svahn C.M. Levi E. Leder P. Mol. Cell. Biol. 1992; 12: 240-247Google Scholar, 10Spivak-Kroizman T. Lemmon M.A. Dikic I. Ladbury J.E. Pinchasi D. Huang J. Jaye M. Crumley G. Schlessinger J. Lax I. Cell. 1994; 79: 1015-1024Google Scholar).Hepatocyte growth factor/scatter factor (HGF/SF) is a heparin-binding polypeptide that can function as a mitogen, motogen, or morphogen on a broad spectrum of cellular targets (11Rubin J.S. Bottaro D.P. Aaronson S.A. Biochim. Biophys. Acta. 1993; 1155: 357-371Google Scholar, 12Rosen E.M. Nigam S.K. Goldberg I.D. J. Cell Biol. 1994; 127: 1783-1787Google Scholar, 13Zarnegar R. Michalopoulos G.K. J. Cell Biol. 1995; 129: 1177-1180Google Scholar). HGF/SF is related to plasminogen and macrophage-stimulating protein, sharing with these molecules approximately 40-45% amino acid sequence identity and several structural motifs (14Nakamura T. Nishizawa T. Hagiya M. Seki T. Shimonishi M. Sugimura A. Tashiro K. Shimizu S. Nature. 1989; 342: 440-443Google Scholar, 15Yoshimura T. Yuhki N. Wang M.-H. Skeel A. Leonard E.J. J. Biol. Chem. 1993; 268: 15461-15468Google Scholar). HGF/SF is synthesized as an inactive monomer which undergoes internal proteolysis to yield a biologically active, disulfide-linked heterodimer (16Lokker N.A. Mark M.R. Luis E.A. Bennet G.L. Robbins K.A. Baker J.B. Godowski P.J. EMBO J. 1992; 11: 2503-2510Google Scholar, 17Naka D. Ishii T. Yoshiyama Y. Miyazawa K. Hara H. Hishida T. Kitamura N. J. Biol. Chem. 1992; 267: 20114-20119Google Scholar, 18Gak E. Taylor W.G. Chan A.M.-L. Rubin J.S. FEBS Lett. 1992; 311: 17-21Google Scholar). The heavy chain of the HGF/SF dimer (∼60 kDa) is derived from the amino terminus of the precursor and contains an amino-terminal segment followed by four kringle domains. A kringle consists of ∼80 amino acids and has a characteristic folding pattern defined by three internal disulfide bonds and additional conserved residues (19Patthy L. Trexler M. Vali Z. Banyai L. Varadi A. FEBS Lett. 1984; 171: 131-136Google Scholar). Two alternative transcripts have been identified that encode truncated variants of HGF/SF, terminating after either the first or second kringle domain (20Chan A.M.-L. Rubin J.S. Bottaro D.P. Hirshfield D.W. Chedid M. Aaronson S.A. Science. 1991; 254: 1382-1385Google Scholar, 21Miyazawa K. Kitamura A. Naka D. Kitamura N. Eur. J. Biochem. 1991; 197: 15-22Google Scholar, 22Hartmann G. Naldini L. Weidner K.M. Sachs M. Vigna E. Comoglio P.M. Birchmeier W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11574-11578Google Scholar, 23Cioce V. Csaky K.G. Chan A.M.-L. Bottaro D.P. Taylor W.G. Jensen R. Aaronson S.A. Rubin J.S. J. Biol. Chem. 1996; 271: 13110-13115Google Scholar). These smaller isoforms, designated HGF/NK1 and HGF/NK2, respectively, behave as antagonists or partial agonists, depending on the molecule and assay conditions (20Chan A.M.-L. Rubin J.S. Bottaro D.P. Hirshfield D.W. Chedid M. Aaronson S.A. Science. 1991; 254: 1382-1385Google Scholar, 22Hartmann G. Naldini L. Weidner K.M. Sachs M. Vigna E. Comoglio P.M. Birchmeier W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11574-11578Google Scholar, 23Cioce V. Csaky K.G. Chan A.M.-L. Bottaro D.P. Taylor W.G. Jensen R. Aaronson S.A. Rubin J.S. J. Biol. Chem. 1996; 271: 13110-13115Google Scholar, 24Lokker N.A. Godowski P.J. J. Biol. Chem. 1993; 268: 17145-17150Google Scholar, 25Schwall Godowski P.J. D.W. J. Cell Biol. 1996; Scholar). of these HGF/SF with affinity to the tyrosine kinase HGF/SF receptor, the Met (20Chan A.M.-L. Rubin J.S. Bottaro D.P. Hirshfield D.W. Chedid M. Aaronson S.A. Science. 1991; 254: 1382-1385Google Scholar, 22Hartmann G. Naldini L. Weidner K.M. Sachs M. Vigna E. Comoglio P.M. Birchmeier W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11574-11578Google Scholar, 23Cioce V. Csaky K.G. Chan A.M.-L. Bottaro D.P. Taylor W.G. Jensen R. Aaronson S.A. Rubin J.S. J. Biol. Chem. 1996; 271: 13110-13115Google Scholar, D.P. Rubin J.S. Chan A.M.-L. Aaronson S.A. Science. 1991; Scholar, L. Weidner K.M. Vigna E. G. A. G. R. Michalopoulos G.K. Birchmeier W. Comoglio P.M. EMBO J. 1991; with heparin-binding growth factors, the higher affinity cell surface binding sites for HGF/SF to heparan sulfate L. Weidner K.M. Vigna E. G. A. G. R. Michalopoulos G.K. Birchmeier W. Comoglio P.M. EMBO J. 1991; Scholar, R. L. Michalopoulos G. Biochem. Biophys. Scholar, N. S. Ishii T. M. S. H. H. Hishida T. Y. J. Biol. Chem. 1992; 267: Scholar, K. T. Naka D. Kitamura N. J. Biol. Chem. 1994; Scholar). on kringle and the hairpin loop in the amino-terminal (N) domain in heparin binding M. M. Y. Miyazawa K. Kitamura N. Biochemistry. 1992; Scholar, K. H. Hagiya M. Shimizu S. T. Y. T. J. Biol. Chem. 1994; although the importance of in this V. Csaky K.G. Chan A.M.-L. Bottaro D.P. Taylor W.G. Jensen R. Aaronson S.A. Rubin J.S. J. Biol. Chem. 1996; 271: 13110-13115Google Scholar). The of has been from a of cross-linking was of with that HSGAG for binding D. Ishii T. T. Hishida T. Hara H. Cell 1993; Scholar). of heparin to cells the biological to HGF/SF, although the on was either or depending on the assay R. Michalopoulos G. 1989; Scholar, E.M. Goldberg I.D. T. D.W. In Cell. Biol. 1989; Scholar, J.S. Chan A.M.-L. Bottaro D.P. W.H. Taylor W.G. A.C. Hirshfield D.W. J. T. P. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1991; Scholar, L. J. L. G.L. P. R.J. J. Biol. Chem. 1995; Scholar). that heparin stimulated dimerization of HGF/SF and HGF/NK1 in a the that a on the cell surface Met dimerization and activation Godowski P.J. D.W. J. Cell Biol. 1996; Scholar, L. J. L. G.L. P. R.J. J. Biol. Chem. 1995; Scholar) as for and (5Schlessinger J. Lax I. Lemmon M. Cell. 1995; 83: 357-360Google Scholar, 9Ornitz D.M. Yayon A. Flanagan J.G. Svahn C.M. Levi E. Leder P. Mol. Cell. Biol. 1992; 12: 240-247Google Scholar, 10Spivak-Kroizman T. Lemmon M.A. Dikic I. Ladbury J.E. Pinchasi D. Huang J. Jaye M. Crumley G. Schlessinger J. Lax I. Cell. 1994; 79: 1015-1024Google the study, we have the that heparin binding of HGF/SF is dependent on and basic amino acid residues in the hairpin loop of the N domain. also provide that HSGAG is for the binding of HGF/SF variants to Met and signal transduction. we demonstrate that heparin stimulates the oligomerization of HGF/SF in and cell and that the N domain has an role in this that the interaction of heparin-binding growth with heparan sulfate has a on biological (2Ruoslahti E. Yamaguchi Y. Cell. 1991; 64: 867-869Google Scholar, 3Schreiber A.B. Kenney J. Kowalski W.J. Friesel R.T.M. Maciag T. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6138-6142Google Scholar, 4Rapraeger A.C. Krufka A. Olwin B.B. Science. 1991; 252: 1705-1708Google Scholar, 5Schlessinger J. Lax I. Lemmon M. Cell. 1995; 83: 357-360Google Scholar, 7Yayon A. Klagsbrun M. Esko J.D. Leder P. Ornitz D.M. Cell. 1991; 64: 841-848Google Scholar, 8Mach H. Volkin D.B. Burke C.J. Middaugh C.R. Linhardt R.J. Fromm J.R. Loganathan D. Mattsson L. Biochemistry. 1993; 32: 5480-5489Google Scholar, 9Ornitz D.M. Yayon A. Flanagan J.G. Svahn C.M. Levi E. Leder P. Mol. Cell. Biol. 1992; 12: 240-247Google Scholar, 10Spivak-Kroizman T. Lemmon M.A. Dikic I. Ladbury J.E. Pinchasi D. Huang J. Jaye M. Crumley G. Schlessinger J. Lax I. Cell. 1994; 79: 1015-1024Google Scholar, 25Schwall Godowski P.J. D.W. J. Cell Biol. 1996; Scholar, R. Michalopoulos G. 1989; Scholar, E.M. Goldberg I.D. T. D.W. In Cell. Biol. 1989; Scholar, J.S. Chan A.M.-L. Bottaro D.P. W.H. Taylor W.G. A.C. Hirshfield D.W. J. T. P. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1991; Scholar, L. J. L. G.L. P. R.J. J. Biol. Chem. 1995; Scholar). In the of growth may strongly by binding to the on and in which of factor to cellular The was to the heparin-binding of HGF/SF and the role of heparin in growth factor binding and signal transduction. of the heparin-binding of HGF/SF to the N domain is a first in mutants with heparin in the importance of the interaction for growth factor activity and have in of HGF/SF mutants the N domain M. M. Y. Miyazawa K. Kitamura N. Biochemistry. 1992; Scholar) and the hairpin loop K. H. Hagiya M. Shimizu S. T. Y. T. J. Biol. Chem. 1994; Scholar) in heparin in studies of function not only from of the domain but also from folding of the that its or the also to heparin K. H. Hagiya M. Shimizu S. T. Y. T. J. Biol. Chem. 1994; a critical role for in this interaction that was with our that the N domain the binding properties of full-length HGF/SF, we demonstrate that the heparin-binding of HGF/SF in this the heparin-binding motifs of basic amino acid residues in a sequence 1989; Scholar, Rev. 1991; the role of sites in the hairpin of the N domain was The of alanine substitution at these to have on the interaction with that residues Two of basic residues the amino terminus of the N and can from from a truncated of HGF/SF, which to (14Nakamura T. Nishizawa T. Hagiya M. Seki T. Shimonishi M. Sugimura A. Tashiro K. Shimizu S. Nature. 1989; 342: 440-443Google Scholar, T. K. A. N. T. FEBS Lett. Scholar). Two and are of as well as residues that may to heparin binding 1989; Scholar, Rev. 1991; Scholar, P. E. Jaye M. W.H. J. Biol. Chem. 1995; our that HSGAG is for binding to in with in a Mark M.R. N.A. Luis E.A. Godowski P.J. J. Biol. Chem. 1992; 267: Scholar) affinity binding of HGF/SF to a containing the domain of Met and the heavy chain of heparin or HSGAG been to the These in several HSGAG may have with the protein, and been in the is the that the domain of heparin-binding growth tyrosine kinase receptor can heparin M. Wang F. Xu J. Crabb J.W. Hou J. McKeehan W.L. Science. 1993; 259: 1918-1921Google Scholar). is that of HGF/SF in the binding assay used by Mark M.R. N.A. Luis E.A. Godowski P.J. J. Biol. Chem. 1992; 267: Scholar) in of of the of HSGAG in affinity receptor binding (5Schlessinger J. Lax I. Lemmon M. Cell. 1995; 83: 357-360Google Scholar). of ligand from may not have been in a binding are the HSGAG for binding receptor dimerization as has been for (5Schlessinger J. Lax I. Lemmon M. Cell. 1995; 83: 357-360Google Scholar, 8Mach H. Volkin D.B. Burke C.J. Middaugh C.R. Linhardt R.J. Fromm J.R. Loganathan D. Mattsson L. Biochemistry. 1993; 32: 5480-5489Google Scholar, 9Ornitz D.M. Yayon A. Flanagan J.G. Svahn C.M. Levi E. Leder P. Mol. Cell. Biol. 1992; 12: 240-247Google Scholar, 10Spivak-Kroizman T. Lemmon M.A. Dikic I. Ladbury J.E. Pinchasi D. Huang J. Jaye M. Crumley G. Schlessinger J. Lax I. Cell. 1994; 79: 1015-1024Google Scholar) and HGF/SF Godowski P.J. D.W. J. Cell Biol. 1996; Scholar, L. J. L. G.L. P. R.J. J. Biol. Chem. 1995; this have been in the assay as findings that heparin or HSGAG was for binding and activation of receptor The of tyrosine phosphorylation and c-fos in cells by the of HGF/NK1 and heparin the biological relevance of binding in these to HGF/NK1 in to a of receptor binding that was the of by cross-linking binding either from of HSGAG on cells or of HSGAG. heparin at to a tyrosine phosphorylation not a this may a of the glycosaminoglycan to promote tyrosine as has been reported in cells the tyrosine kinase receptor G. M. EMBO J. 1995; results the importance of heparin not only for Met binding but also ligand reported for HGF/NK1 Godowski P.J. D.W. J. Cell Biol. 1996; cross-linking experiments performed in a that HGF/NK1 and the N domain have an to is markedly by the of the N a series of to in the of HGF/NK1 or N was with cells and cross-linking heparan sulfate or heparin was the experiments performed with cells and of the relevance of ligand oligomerization was the of heparin on ligand oligomerization with cell heparin increased dimer on CHO reduced oligomerization on and type CHO In the HGF/SF the heparin molecules that ligand-ligand less to the of heparin on oligomerization is dependent on the of HSGAG. ligand oligomerization is for signal the of heparin on the biological activity of HGF/SF with the and of on the cell INTRODUCTIONDuring the past dozen years, several mitogens have been identified that possess the distinctive property of binding to heparin or closely related heparan sulfate glycosaminoglycan (HSGAG) 1The abbreviations used are: HSGAGheparan sulfate glycosaminoglycanHGF/SFhepatocyte growth factor/scatter factorHGF/NK1truncated HGF/SF isoform containing amino-terminal (N) domain and kringle 1HGF/NK2truncated HGF/SF isoform containing N domain and kringles 1 and 2K2second kringle domain in HGF/SFCHO-WTwild type Chinese hamster ovary (CHO) cell lineMDCKMadin-Darby canine kidney2A/NK1, 3A/NK1, and 5A/NK1alanine substitution mutants in HGF/NK1 with 2, 3, or 5 substitutions as described in the textPBSphosphate-buffered salinePAGEpolyacrylamide gel electrophoresisIGFinsulin-like growth factor (1Burgess W.H. Maciag T. Annu. Rev. Biochem. 1989; 58: 575-606Google Scholar, 2Ruoslahti E. Yamaguchi Y. Cell. 1991; 64: 867-869Google Scholar). Heparin-based affinity chromatography has been a convenient tool for purification of these factors, and several studies have shown that this binding phenomenon has functional relevance as well (2Ruoslahti E. Yamaguchi Y. Cell. 1991; 64: 867-869Google Scholar, 3Schreiber A.B. Kenney J. Kowalski W.J. Friesel R.T.M. Maciag T. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6138-6142Google Scholar, 4Rapraeger A.C. Krufka A. Olwin B.B. Science. 1991; 252: 1705-1708Google Scholar, 5Schlessinger J. Lax I. Lemmon M. Cell. 1995; 83: 357-360Google Scholar, 6Kan M. Wang F. Xu J. Crabb J.W. Hou J. McKeehan W.L. Science. 1993; 259: 1918-1921Google Scholar, 7Yayon A. Klagsbrun M. Esko J.D. Leder P. Ornitz D.M. Cell. 1991; 64: 841-848Google Scholar, 8Mach H. Volkin D.B. Burke C.J. Middaugh C.R. Linhardt R.J. Fromm J.R. Loganathan D. Mattsson L. Biochemistry. 1993; 32: 5480-5489Google Scholar, 9Ornitz D.M. Yayon A. Flanagan J.G. Svahn C.M. Levi E. Leder P. Mol. Cell. Biol. 1992; 12: 240-247Google Scholar, 10Spivak-Kroizman T. Lemmon M.A. Dikic I. Ladbury J.E. Pinchasi D. Huang J. Jaye M. Crumley G. Schlessinger J. Lax I. Cell. 1994; 79: 1015-1024Google Scholar). HSGAG-dependent stabilization (3Schreiber A.B. Kenney J. Kowalski W.J. Friesel R.T.M. Maciag T. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6138-6142Google Scholar) and/or localization of the growth factor on the cell surface may promote interaction with less abundant, higher affinity tyrosine kinase receptors involved in signal transduction (2Ruoslahti E. Yamaguchi Y. Cell. 1991; 64: 867-869Google Scholar, 4Rapraeger A.C. Krufka A. Olwin B.B. Science. 1991; 252: 1705-1708Google Scholar, 5Schlessinger J. Lax I. Lemmon M. Cell. 1995; 83: 357-360Google Scholar). Coupling of heparin or HSGAG either to the growth factor or its tyrosine kinase receptor may induce conformational changes in these molecules that augment signaling (3Schreiber A.B. Kenney J. Kowalski W.J. Friesel R.T.M. Maciag T. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6138-6142Google Scholar, 6Kan M. Wang F. Xu J. Crabb J.W. Hou J. McKeehan W.L. Science. 1993; 259: 1918-1921Google Scholar). Several laboratories have reported that heparin binding can provide a framework for ligand oligomerization, which may enhance signaling by stimulating dimerization of the tyrosine kinase receptor (5Schlessinger J. Lax I. Lemmon M. Cell. 1995; 83: 357-360Google Scholar, 8Mach H. Volkin D.B. Burke C.J. Middaugh C.R. Linhardt R.J. Fromm J.R. Loganathan D. Mattsson L. Biochemistry. 1993; 32: 5480-5489Google Scholar, 9Ornitz D.M. Yayon A. Flanagan J.G. Svahn C.M. Levi E. Leder P. Mol. Cell. Biol. 1992; 12: 240-247Google Scholar, 10Spivak-Kroizman T. Lemmon M.A. Dikic I. Ladbury J.E. Pinchasi D. Huang J. Jaye M. Crumley G. Schlessinger J. Lax I. Cell. 1994; 79: 1015-1024Google Scholar).Hepatocyte growth factor/scatter factor (HGF/SF) is a heparin-binding polypeptide that can function as a mitogen, motogen, or morphogen on a broad spectrum of cellular targets (11Rubin J.S. Bottaro D.P. Aaronson S.A. Biochim. Biophys. Acta. 1993; 1155: 357-371Google Scholar, 12Rosen E.M. Nigam S.K. Goldberg I.D. J. Cell Biol. 1994; 127: 1783-1787Google Scholar, 13Zarnegar R. Michalopoulos G.K. J. Cell Biol. 1995; 129: 1177-1180Google Scholar). HGF/SF is related to plasminogen and macrophage-stimulating protein, sharing with these molecules approximately 40-45% amino acid sequence identity and several structural motifs (14Nakamura T. Nishizawa T. Hagiya M. Seki T. Shimonishi M. Sugimura A. Tashiro K. Shimizu S. Nature. 1989; 342: 440-443Google Scholar, 15Yoshimura T. Yuhki N. Wang M.-H. Skeel A. Leonard E.J. J. Biol. Chem. 1993; 268: 15461-15468Google Scholar). HGF/SF is synthesized as an inactive monomer which undergoes internal proteolysis to yield a biologically active, disulfide-linked heterodimer (16Lokker N.A. Mark M.R. Luis E.A. Bennet G.L. Robbins K.A. Baker J.B. Godowski P.J. EMBO J. 1992; 11: 2503-2510Google Scholar, 17Naka D. Ishii T. Yoshiyama Y. Miyazawa K. Hara H. Hishida T. Kitamura N. J. Biol. Chem. 1992; 267: 20114-20119Google Scholar, 18Gak E. Taylor W.G. Chan A.M.-L. Rubin J.S. FEBS Lett. 1992; 311: 17-21Google Scholar). The heavy chain of the HGF/SF dimer (∼60 kDa) is derived from the amino terminus of the precursor and contains an amino-terminal segment followed by four kringle domains. A kringle consists of ∼80 amino acids and has a characteristic folding pattern defined by three internal disulfide bonds and additional conserved residues (19Patthy L. Trexler M. Vali Z. Banyai L. Varadi A. FEBS Lett. 1984; 171: 131-136Google Scholar). Two alternative transcripts have been identified that encode truncated variants of HGF/SF, terminating after either the first or second kringle domain (20Chan A.M.-L. Rubin J.S. Bottaro D.P. Hirshfield D.W. Chedid M. Aaronson S.A. Science. 1991; 254: 1382-1385Google Scholar, 21Miyazawa K. Kitamura A. Naka D. Kitamura N. Eur. J. Biochem. 1991; 197: 15-22Google Scholar, 22Hartmann G. Naldini L. Weidner K.M. Sachs M. Vigna E. Comoglio P.M. Birchmeier W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11574-11578Google Scholar, 23Cioce V. Csaky K.G. Chan A.M.-L. Bottaro D.P. Taylor W.G. Jensen R. Aaronson S.A. Rubin J.S. J. Biol. Chem. 1996; 271: 13110-13115Google Scholar). These smaller isoforms, designated HGF/NK1 and HGF/NK2, respectively, behave as antagonists or partial agonists, depending on the molecule and assay conditions (20Chan A.M.-L. Rubin J.S. Bottaro D.P. Hirshfield D.W. Chedid M. Aaronson S.A. Science. 1991; 254: 1382-1385Google Scholar, 22Hartmann G. Naldini L. Weidner K.M. Sachs M. Vigna E. Comoglio P.M. Birchmeier W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11574-11578Google Scholar, 23Cioce V. Csaky K.G. Chan A.M.-L. Bottaro D.P. Taylor W.G. Jensen R. Aaronson S.A. Rubin J.S. J. Biol. Chem. 1996; 271: 13110-13115Google Scholar, 24Lokker N.A. Godowski P.J. J. Biol. Chem. 1993; 268: 17145-17150Google Scholar, 25Schwall Godowski P.J. D.W. J. Cell Biol. 1996; Scholar). of these HGF/SF with affinity to the tyrosine kinase HGF/SF receptor, the Met (20Chan A.M.-L. Rubin J.S. Bottaro D.P. Hirshfield D.W. Chedid M. Aaronson S.A. Science. 1991; 254: 1382-1385Google Scholar, 22Hartmann G. Naldini L. Weidner K.M. Sachs M. Vigna E. Comoglio P.M. Birchmeier W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11574-11578Google Scholar, 23Cioce V. Csaky K.G. Chan A.M.-L. Bottaro D.P. Taylor W.G. Jensen R. Aaronson S.A. Rubin J.S. J. Biol. Chem. 1996; 271: 13110-13115Google Scholar, D.P. Rubin J.S. Chan A.M.-L. Aaronson S.A. Science. 1991; Scholar, L. Weidner K.M. Vigna E. G. A. G. R. Michalopoulos G.K. Birchmeier W. Comoglio P.M. EMBO J. 1991; with heparin-binding growth factors, the higher affinity cell surface binding sites for HGF/SF to heparan sulfate L. Weidner K.M. Vigna E. G. A. G. R. Michalopoulos G.K. Birchmeier W. Comoglio P.M. EMBO J. 1991; Scholar, R. L. Michalopoulos G. Biochem. Biophys. Scholar, N. S. Ishii T. M. S. H. H. Hishida T. Y. J. Biol. Chem. 1992; 267: Scholar, K. T. Naka D. Kitamura N. J. Biol. Chem. 1994; Scholar). on kringle and the hairpin loop in the amino-terminal (N) domain in heparin binding M. M. Y. Miyazawa K. Kitamura N. Biochemistry. 1992; Scholar, K. H. Hagiya M. Shimizu S. T. Y. T. J. Biol. Chem. 1994; although the importance of in this V. Csaky K.G. Chan A.M.-L. Bottaro D.P. Taylor W.G. Jensen R. Aaronson S.A. Rubin J.S. J. Biol. Chem. 1996; 271: 13110-13115Google Scholar). The of has been from a of cross-linking was of with that HSGAG for binding D. Ishii T. T. Hishida T. Hara H. Cell 1993; Scholar). of heparin to cells the biological to HGF/SF, although the on was either or depending on the assay R. Michalopoulos G. 1989; Scholar, E.M. Goldberg I.D. T. D.W. In Cell. Biol. 1989; Scholar, J.S. Chan A.M.-L. Bottaro D.P. W.H. Taylor W.G. A.C. Hirshfield D.W. J. T. P. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1991; Scholar, L. J. L. G.L. P. R.J. J. Biol. Chem. 1995; Scholar). that heparin stimulated dimerization of HGF/SF and HGF/NK1 in a the that a on the cell surface Met dimerization and activation Godowski P.J. D.W. J. Cell Biol. 1996; Scholar, L. J. L. G.L. P. R.J. J. Biol. Chem. 1995; Scholar) as for and (5Schlessinger J. Lax I. Lemmon M. Cell. 1995; 83: 357-360Google Scholar, 9Ornitz D.M. Yayon A. Flanagan J.G. Svahn C.M. Levi E. Leder P. Mol. Cell. Biol. 1992; 12: 240-247Google Scholar, 10Spivak-Kroizman T. Lemmon M.A. Dikic I. Ladbury J.E. Pinchasi D. Huang J. Jaye M. Crumley G. Schlessinger J. Lax I. Cell. 1994; 79: 1015-1024Google the study, we have the that heparin binding of HGF/SF is dependent on and basic amino acid residues in the hairpin loop of the N domain. also provide that HSGAG is for the binding of HGF/SF variants to Met and signal transduction. we demonstrate that heparin stimulates the oligomerization of HGF/SF in and cell and that the N domain has an role in this