Variant Exons v6 and v7 Together Expand the Repertoire of Glycosaminoglycans Bound by CD44
1997; Elsevier BV; Volume: 272; Issue: 50 Linguagem: Inglês
10.1074/jbc.272.50.31837
ISSN1083-351X
AutoresJonathan P. Sleeman, Kazuhiro Kondo, Jürgen Moll, Helmut Ponta, Peter Herrlich,
Tópico(s)Cell Adhesion Molecules Research
ResumoIsoforms of the glycoprotein CD44 are cell surface receptors for the glycosaminoglycan hyaluronate. They have been implicated in many biological processes, but their function in these is poorly understood and cannot be explained solely by hyaluronate binding. In the present work we examine the ligand binding properties of alternatively spliced CD44 variant isoforms which are functionally involved in the immune system, embryonic development, and tumor behavior. We show that these isoforms bind directly to the purified glycosaminoglycans chondroitin sulfate, heparin, and heparin sulfate, in addition to being able to bind to hyaluronate. Binding to this extended repertoire of glycosaminoglycans by CD44 depends on the inclusion of peptide sequences encoded by the alternatively spliced exons v6 and v7, and occurs both when the CD44 is solubilized from the plasma membrane and when it is expressed on intact cells. A single point mutation in the most N-terminal hyaluronate binding motif of CD44 ablates both hyaluronate and chondroitin sulfate binding, suggesting that glycosaminoglycans are bound through a common motif, and that only one of the hyaluronate binding motifs is responsible for the majority of glycosaminoglycan binding by CD44 on the cell surface. Taken together, these observations indicate that alternative splicing regulates the ligand binding specificity of CD44 and suggest that structural changes in the CD44 protein have a profound effect on the range of ligands to which this molecule can bind with potentially wide-ranging functional consequences. Isoforms of the glycoprotein CD44 are cell surface receptors for the glycosaminoglycan hyaluronate. They have been implicated in many biological processes, but their function in these is poorly understood and cannot be explained solely by hyaluronate binding. In the present work we examine the ligand binding properties of alternatively spliced CD44 variant isoforms which are functionally involved in the immune system, embryonic development, and tumor behavior. We show that these isoforms bind directly to the purified glycosaminoglycans chondroitin sulfate, heparin, and heparin sulfate, in addition to being able to bind to hyaluronate. Binding to this extended repertoire of glycosaminoglycans by CD44 depends on the inclusion of peptide sequences encoded by the alternatively spliced exons v6 and v7, and occurs both when the CD44 is solubilized from the plasma membrane and when it is expressed on intact cells. A single point mutation in the most N-terminal hyaluronate binding motif of CD44 ablates both hyaluronate and chondroitin sulfate binding, suggesting that glycosaminoglycans are bound through a common motif, and that only one of the hyaluronate binding motifs is responsible for the majority of glycosaminoglycan binding by CD44 on the cell surface. Taken together, these observations indicate that alternative splicing regulates the ligand binding specificity of CD44 and suggest that structural changes in the CD44 protein have a profound effect on the range of ligands to which this molecule can bind with potentially wide-ranging functional consequences. Alternative splicing and/or post-translational modification generate multiple CD44 isoforms (reviewed in Ref. 1Sherman L. Sleeman J. Herrlich P. Ponta H. Curr. Opin. Cell Biol. 1994; 6: 726-733Crossref PubMed Scopus (380) Google Scholar). CD44 isoforms have been implicated in a wide variety of adhesion-dependent cellular processes, such as T-cell signaling and activation, lymphocyte recirculation, cell-cell and cell-matrix interactions, and cell migration and metastasis (2Herrlich P. Zöller M. Pals S.T. Ponta H. Immunol. Today. 1993; 14: 395-399Abstract Full Text PDF PubMed Scopus (270) Google Scholar, 3Lesley J. Hyman R. Kincade P.W. Adv. Immunol. 1993; 54: 271-335Crossref PubMed Scopus (1026) Google Scholar, 4Knudson C.B. Knudson W. FASEB J. 1993; 7: 1233-1241Crossref PubMed Scopus (599) Google Scholar). The ligand binding activities of CD44 which mediate these adhesive processes, and functional differences between the different isoforms are both poorly understood.The majority of CD44 isoform diversity is generated by the incorporation of amino acid stretches encoded by 10 alternatively spliced exons into a membrane proximal position of the extracellular portion. Transcripts in which these variant exons are spliced out encode the most common and widely expressed 85-kDa isoform (CD44s). The expression of CD44 isoforms containing sequences encoded by the variant exons (CD44v), however, is tightly regulated. Constitutive expression of these isoforms is restricted to a limited selection of epithelia and leukocytes, while transient and regulated isoform expression is observed during several physiological processes (5Arch R. Wirth K. Hofmann M. Ponta H. Matzku S. Herrlich P. Zöller M. Science. 1992; 257: 682-685Crossref PubMed Scopus (400) Google Scholar, 6Wainwright D. Sherman L. Sleeman J. Ponta H. Herrlich P. Ann. N. Y. Acad. Sci. 1996; 758: 345-349Crossref Scopus (12) Google Scholar). The expression of CD44v isoforms is up-regulated in many tumors during tumor progression (7Ponta H. Sleeman J. Dall P. Moll J. Sherman L. Herrlich P. Invasion & Metastasis. 1995; 14: 82-86Google Scholar).It is well documented that CD44 isoforms bind to the extracellular matrix glycosaminoglycan (GAG) 1The abbreviations used are: GAG, glycosaminoglycan; CPC, cetylpyridinium chloride; CS, chondroitin sulfate; CS-A, -B, and -C, chondroitin sulfate type A, B, and C (chondroitin-4-sulfate, dermatan sulfate and chondroitin-6-sulfate, respectively); HA, hyaluronate; HS, heparin sulfate; KS, keratan sulfate; H, heparin; FCS, fetal calf serum; PCR, polymerase chain reaction; PBS, phosphate-buffered saline.1The abbreviations used are: GAG, glycosaminoglycan; CPC, cetylpyridinium chloride; CS, chondroitin sulfate; CS-A, -B, and -C, chondroitin sulfate type A, B, and C (chondroitin-4-sulfate, dermatan sulfate and chondroitin-6-sulfate, respectively); HA, hyaluronate; HS, heparin sulfate; KS, keratan sulfate; H, heparin; FCS, fetal calf serum; PCR, polymerase chain reaction; PBS, phosphate-buffered saline. hyaluronate (HA), and HA binding motifs in the extracellular portion of the protein have been identified (e.g. Refs. 3Lesley J. Hyman R. Kincade P.W. Adv. Immunol. 1993; 54: 271-335Crossref PubMed Scopus (1026) Google Scholar, 8Yang B. Yang B.L. Savani R.C. Turley E.A. EMBO J. 1994; 13: 286-296Crossref PubMed Scopus (335) Google Scholar, and 9Sleeman J.P. Arming S. Moll J.F. Hekele A. Rudy W. Sherman L.S. Kreil G. Ponta H. Herrlich P. Cancer Res. 1996; 56: 3134-3141PubMed Google Scholar). However, other ligand binding activities must exist. For example, CD44s-mediated lymphocyte binding to mucosal high endothelial venules is independent of HA binding (10Culty M. Miyake K. Kincade P.W. Silorski E. Butcher E.C. Underhill C. J. Cell Biol. 1990; 111: 2765-2774Crossref PubMed Scopus (314) Google Scholar). Moreover, an antibody specific for CD44 variant isoforms containing an exon v6-encoded epitope blocks tumor growth, lymphocyte activation, and limb bud outgrowth, but does not block CD44-mediated HA binding (5Arch R. Wirth K. Hofmann M. Ponta H. Matzku S. Herrlich P. Zöller M. Science. 1992; 257: 682-685Crossref PubMed Scopus (400) Google Scholar, 6Wainwright D. Sherman L. Sleeman J. Ponta H. Herrlich P. Ann. N. Y. Acad. Sci. 1996; 758: 345-349Crossref Scopus (12) Google Scholar, 9Sleeman J.P. Arming S. Moll J.F. Hekele A. Rudy W. Sherman L.S. Kreil G. Ponta H. Herrlich P. Cancer Res. 1996; 56: 3134-3141PubMed Google Scholar). Although no specific ligands have been identified for CD44v isoforms, a plethora of ligands have been ascribed to CD44s, including mucosal addressin (11Picker L.J. Nakache M. Butcher E.C. J. Cell Biol. 1989; 109: 927-937Crossref PubMed Scopus (254) Google Scholar), collagen type I (12Faasen A. Schrager J. Klein D. Oegema T. Couchman J. McCarthy J. J. Cell Biol. 1992; 116: 521-531Crossref PubMed Scopus (236) Google Scholar), fibronectin (13Jalkanen S. Jalkanen M. J. Cell Biol. 1992; 116: 817-825Crossref PubMed Scopus (448) Google Scholar), MIP-1β (14Tanaka Y. Adams D.H. Hubscher S. Hirano H. Siebenlist U. Shaw S. Nature. 1993; 361: 79-82Crossref PubMed Scopus (843) Google Scholar), the chondroitin-sulfated form of invariant chain (15Naujokas M.F. Morin M. Anderson M.S. Peterson M. Miller J. Cell. 1993; 74: 257-268Abstract Full Text PDF PubMed Scopus (201) Google Scholar), serglycin (16Toyama-Sorimachi N. Sorimachi H. Tobita Y. Kitamura F. Yagita H. Suzuki K. Miyasaka M. J. Biol. Chem. 1995; 270: 7437-7444Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar), and osteopontin (17Weber G. Ashkar S. Glimcher M. Cantor H. Science. 1996; 271: 509-512Crossref PubMed Scopus (806) Google Scholar). While certain of these interactions are mediated by binding of the ligand to sugar moieties on the CD44s protein, the nature of the interaction with the other ligands is unclear. At least for serglycin, binding by CD44s is dependent on the presence of chondroitin sulfate (CS) modifications on the serglycin protein (16Toyama-Sorimachi N. Sorimachi H. Tobita Y. Kitamura F. Yagita H. Suzuki K. Miyasaka M. J. Biol. Chem. 1995; 270: 7437-7444Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). In this case, however, the CD44s protein could not bind directly to CS, a finding also reported by others (18Miyake K. Underhill C.B. Lesley J. Kincade P.W. J. Exp. Med. 1990; 172: 69-75Crossref PubMed Scopus (557) Google Scholar). Work with purified CD44s proteins, on the other hand, suggests that CD44s has a weak affinity for CS (19Underhill C.B. Chi-Rosso G. Toole B.P. J. Biol. Chem. 1983; 258: 8086-8091Abstract Full Text PDF PubMed Google Scholar, 20Aruffo A. Stamenkovic I. Melnick M. Underhill C.B. Seed B. Cell. 1990; 61: 1303-1313Abstract Full Text PDF PubMed Scopus (2135) Google Scholar, 21Peach R.J. Hollenbaugh D. Stamenkovic I. Aruffo A. J. Cell Biol. 1993; 122: 257-264Crossref PubMed Scopus (321) Google Scholar), a conclusion supported by CD44 transfection experiments in a B cell lymphoma line (22Sy M.S. Guo Y.-J. Stamenkovic I. J. Exp. Med. 1991; 174: 859-866Crossref PubMed Scopus (302) Google Scholar).The role of CD44 splice variants in certain aggressive tumors arising from cells which constitutively express only CD44s and their restricted expression compared with CD44s in normal tissues indicate that these isoforms possess molecular properties in addition to those exhibited by CD44s. The variant portion of CD44v proteins may either mediate binding to new ligands, or modulate the function of domains expressed on all CD44 proteins, such as the HA-binding domain. Here we show that CD44 variants containing sequences encoded by exons v6 and v7 bind directly and avidly to multiple GAGs, both as soluble proteins and when expressed on the cell surface. Binding requires the N-terminal HA binding motif, suggesting that variant exon expression extends the spectrum of GAG binding via this motif. These findings suggest that alternative splicing of CD44 serves to regulate and define the range of ligands to which the protein can bind.DISCUSSIONThe elucidation of ligands bound by CD44 is of paramount importance if the biological role of this molecule is to be understood. Furthermore, differences in ligand binding properties between CD44s and CD44v isoforms are likely to be pivotal for their differential function in physiological and pathological processes. The data in this paper reveal new GAG binding properties of CD44, which moreover are only present in variant isoforms containing both exons v6 and v7. Like HA binding, these GAG binding properties require the most N-terminal HA binding motif.The focus of this paper is to document the existence and structural requirements of CD44 binding to a wide range of GAGs. These GAG binding properties potentially extend the ability of CD44 to bind to many proteinaceous ligands, for while HA exists as a free carbohydrate within the extracellular matrix, other GAGs are always found as carbohydrate modifications on proteins. Many biological signaling molecules, cell surface molecules, and components of the extracellular matrix have been reported to be modified by CS, H, or HS (reviewed in Ref. 34Ruoslahti E. J. Biol. Chem. 1989; 264: 13369-13372Abstract Full Text PDF PubMed Google Scholar). The extended GAG binding properties of CD44 isoforms bearing both exon v6- and v7-encoded sequences may permit these isoforms to interact with such molecules. An attractive model would be one in which the variant portion of CD44 not only permits interaction with GAG modifications on these molecules, but also itself interacts with the protein portion of the molecule to confer avidity and specificity on the binding interaction. We have tested the ability of CD44v4-v7 to bind to the CS-modified forms of the cytokine macrophage colony-stimulating factor (35Price L.K. Choi H.U. Rosenberg L. Stanley E.R. J. Biol. Chem. 1992; 267: 2190-2199Abstract Full Text PDF PubMed Google Scholar), but were unable to detect binding (data not shown). This would be compatible with the notion that two discrete binding activities on CD44 may be required, each of which is insufficient to allow a fruitful CD44-ligand interaction. Such a model could suggest a mechanism by which the anti-CD44v6 antibody 1.1ASML blocks lymphocyte activation (5Arch R. Wirth K. Hofmann M. Ponta H. Matzku S. Herrlich P. Zöller M. Science. 1992; 257: 682-685Crossref PubMed Scopus (400) Google Scholar), limb bud outgrowth (6Wainwright D. Sherman L. Sleeman J. Ponta H. Herrlich P. Ann. N. Y. Acad. Sci. 1996; 758: 345-349Crossref Scopus (12) Google Scholar), and metastatic tumor growth (9Sleeman J.P. Arming S. Moll J.F. Hekele A. Rudy W. Sherman L.S. Kreil G. Ponta H. Herrlich P. Cancer Res. 1996; 56: 3134-3141PubMed Google Scholar, 36Seiter S. Arch R. Komitowski D. Hofmann M. Ponta H. Herrlich P. Matzku S. Zöller M. J. Exp. Med. 1993; 177: 443-455Crossref PubMed Scopus (326) Google Scholar) while not inhibiting HA binding (9Sleeman J.P. Arming S. Moll J.F. Hekele A. Rudy W. Sherman L.S. Kreil G. Ponta H. Herrlich P. Cancer Res. 1996; 56: 3134-3141PubMed Google Scholar), or other CD44-GAG interactions (data not shown). Additionally, the GAG binding properties of CD44v6- and v7-containing isoforms may also increase their affinity for the CS-modified ligands ascribed to CD44s, a possibility which we are exploring.CD44 interactions with GAG-bearing proteins may have several functional consequences. First, they may promote the binding of the CD44 to other cell surface or extracellular matrix components, resulting in enhanced cell-cell or cell-matrix interactions. Second, the interaction may trigger signal transduction via the CD44 molecule. Signal transduction activity through CD44 expressed on T lymphocytes has been reported (37Taher T.E.I. Smit L. Griffioen A.W. Schilder-Tol E.J.M. Borst J. Pals S.T. J. Biol. Chem. 1996; 271: 2863-2867Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Third, the CD44 may bind to biological signaling molecules and present them to their receptors, permitting signal transduction through these receptors by facilitating high affinity interactions. In this case, CD44 would act analogously to cell surface proteoglycans such as the syndecan family. These sequester cytokines and growth factors such as members of the fibroblast growth factor family, which bind to GAG modifications on the cell surface proteoglycan and are subsequently presented to their receptor (for a recent review, see Ref. 38Salmivirta S. Jalkanen M. Experientia. 1995; 51: 863-872Crossref PubMed Scopus (81) Google Scholar). Indeed, it has already been shown that GAG-modified CD44 isoforms can present growth factors in this way (39Bennett K.L. Jackson D.G. Simon J.C. Tanczos E. Peach R. Modrell B. Stamenkovic I. Plowman G. Aruffo A. J. Cell Biol. 1995; 128: 687-698Crossref PubMed Scopus (359) Google Scholar). However, if a CD44 isoform were to act as a growth factor presentation molecule by means of its GAG binding properties, it would instead bind to GAG modifications on the growth factor and then present the factor to its receptor.It has been suggested that the chondroitin sulfate-modified invariant chain functions as an accessory molecule during T cell responses through its interaction with CD44 (15Naujokas M.F. Morin M. Anderson M.S. Peterson M. Miller J. Cell. 1993; 74: 257-268Abstract Full Text PDF PubMed Scopus (201) Google Scholar). In this regard, it is interesting to note that CD44 variants are transiently up-regulated during lymphocyte activation, and that this up-regulation is necessary for an immune response to develop (5Arch R. Wirth K. Hofmann M. Ponta H. Matzku S. Herrlich P. Zöller M. Science. 1992; 257: 682-685Crossref PubMed Scopus (400) Google Scholar, 40Koopman G. Heider K.-H. Horst E. Adolf G.R. van den Berg F. Ponta H. Herrlich P. Pals S.T. J. Exp. Med. 1993; 177: 897-904Crossref PubMed Scopus (318) Google Scholar). Clearly, one possibility would be that CD44 isoforms bearing exon v6- and v7-encoded peptides may confer on activated T cells the ability to interact more avidly with the chondroitin sulfate-modified invariant chain on cellular targets during the development of the immune response due to their enhanced GAG binding properties.The mere expression of CD44 on the cell surface has been amply documented to be insufficient to confer constitutive HA binding properties on cells (3Lesley J. Hyman R. Kincade P.W. Adv. Immunol. 1993; 54: 271-335Crossref PubMed Scopus (1026) Google Scholar). There is mounting evidence to suggest that the binding to HA by CD44 is regulated at multiple levels, and to date these data point toward a crucial role for the structural conformation of the protein in this regulation. Thus, although the extracellular portion of CD44 possesses two functional HA binding motifs (8Yang B. Yang B.L. Savani R.C. Turley E.A. EMBO J. 1994; 13: 286-296Crossref PubMed Scopus (335) Google Scholar), we show here that the most N-terminal motif provides the vast majority of GAG binding activity by CD44 on the cell surface. This is in agreement with fusion protein studies (21Peach R.J. Hollenbaugh D. Stamenkovic I. Aruffo A. J. Cell Biol. 1993; 122: 257-264Crossref PubMed Scopus (321) Google Scholar), and suggests that the tertiary structure of CD44 masks the HA binding activity of the second, membrane proximal HA binding motif. Furthermore, we demonstrate that incorporation of the peptide sequence encoded by exon v7 into CD44 in some instances abrogates HA binding. This has also been observed for other exons (41Bennett K. Modrell B. Greenfield B. Bartolazzi A. Stamenkovic I. Peach R. Jackson D. Spring F. Aruffo A. J. cell Biol. 1995; 131: 1623-1633Crossref PubMed Scopus (145) Google Scholar), and further suggests that alterations in the tertiary structure of the protein regulate HA binding. In this regard, it is interesting to note that glycosylation can have a positive or negative effect on HA binding (33Sleeman J. Rudy W. Hofmann M. Moll J. Herrlich P. Ponta H. J. Cell Biol. 1996; 135: 1139-1150Crossref PubMed Scopus (120) Google Scholar, 41Bennett K. Modrell B. Greenfield B. Bartolazzi A. Stamenkovic I. Peach R. Jackson D. Spring F. Aruffo A. J. cell Biol. 1995; 131: 1623-1633Crossref PubMed Scopus (145) Google Scholar, 42Katoh S. Zheng Z. Oritani K. Shimozato T. Kincade P. J. Exp. Med. 1995; 182: 419-429Crossref PubMed Scopus (230) Google Scholar, 43Lesley J. English N. Perschl A. Gregoroff J. Hyman R. J. Exp. Med. 1995; 182: 431-437Crossref PubMed Scopus (176) Google Scholar, 44Bartolazzi A. Nocks A. Aruffo A. Spring F. Stamenkovic I. J. Cell Biol. 1996; 132: 1199-1208Crossref PubMed Scopus (156) Google Scholar). Here we demonstrate a novel twist in this structural story, namely that the incorporation of the amino acid sequences encoded by variant exons 6 and 7 into the CD44 protein permits the N-terminal HA binding motif to bind to several different types of GAG in addition to HA.There is obviously specificity in the extension of CD44 GAG binding properties mediated by v6- and v7-mediated structural changes, as we detected no or only low KS binding by CD44 in these experiments. On the basis of the data presented here, it is not possible to determine whether the exon v6- and v7-encoded epitopes only indirectly alter the structure of the protein to expose latent GAG binding properties, or whether they actively collaborate with the HA binding motif to extend the types of GAG bound by CD44. The weak CS binding capacity of CD44s suggests that the former possibility is more likely. Very long exposures of the Western blots in Figs. 3 and 7 revealed a weak interaction of CD44s and CD44v6 with GAGs other than HA (data not shown). Clearly, the incorporation of variant exon-encoded sequences dramatically up-regulates the GAG binding capacity of CD44, but other structural parameters may also regulate this on a cell line-specific basis.A number of CD44 isoforms have been described which are GAG-modified (38Salmivirta S. Jalkanen M. Experientia. 1995; 51: 863-872Crossref PubMed Scopus (81) Google Scholar, 45Brown T.A. Bouchard T. St. John T. Wayner E. Carter W.G. J. Cell Biol. 1991; 113: 207-221Crossref PubMed Scopus (300) Google Scholar). The ability of isoforms of CD44 containing sequences encoded by exons v6 and v7 to bind to CS, heparin, and HS raises the possibility that these isoforms may be able to bind to other, GAG-modified CD44 proteins. This cannot, however, be the explanation for the CD44 multimerization we have previously reported (33Sleeman J. Rudy W. Hofmann M. Moll J. Herrlich P. Ponta H. J. Cell Biol. 1996; 135: 1139-1150Crossref PubMed Scopus (120) Google Scholar), as the isoforms involved are not GAG-modified (data not shown). Furthermore, it is possible that other variant exon combinations not analyzed in this present study may also be able to promote GAG binding by CD44. In this regard, it is interesting that Droll et al. (46Droll A. Dougherty S.T. Chiu R.K. Dirks J.F. McBride W.H. Cooper D.L. Dougherty G.J. J. Biol. Chem. 1995; 270: 11567-11573Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) reported adhesive interactions between alternatively spliced CD44 isoforms.In conclusion, our results show that changes in CD44 splicing alter the specificity of ligand binding by the CD44 protein. The implications of this are wide ranging as CD44 variant isoforms have been shown to play a functional role in many aspects of immunology, embryology, and tumor biology. Further understanding of the ligand binding activities of CD44 will reveal how CD44 functions in these varied processes. Alternative splicing and/or post-translational modification generate multiple CD44 isoforms (reviewed in Ref. 1Sherman L. Sleeman J. Herrlich P. Ponta H. Curr. Opin. Cell Biol. 1994; 6: 726-733Crossref PubMed Scopus (380) Google Scholar). CD44 isoforms have been implicated in a wide variety of adhesion-dependent cellular processes, such as T-cell signaling and activation, lymphocyte recirculation, cell-cell and cell-matrix interactions, and cell migration and metastasis (2Herrlich P. Zöller M. Pals S.T. Ponta H. Immunol. Today. 1993; 14: 395-399Abstract Full Text PDF PubMed Scopus (270) Google Scholar, 3Lesley J. Hyman R. Kincade P.W. Adv. Immunol. 1993; 54: 271-335Crossref PubMed Scopus (1026) Google Scholar, 4Knudson C.B. Knudson W. FASEB J. 1993; 7: 1233-1241Crossref PubMed Scopus (599) Google Scholar). The ligand binding activities of CD44 which mediate these adhesive processes, and functional differences between the different isoforms are both poorly understood. The majority of CD44 isoform diversity is generated by the incorporation of amino acid stretches encoded by 10 alternatively spliced exons into a membrane proximal position of the extracellular portion. Transcripts in which these variant exons are spliced out encode the most common and widely expressed 85-kDa isoform (CD44s). The expression of CD44 isoforms containing sequences encoded by the variant exons (CD44v), however, is tightly regulated. Constitutive expression of these isoforms is restricted to a limited selection of epithelia and leukocytes, while transient and regulated isoform expression is observed during several physiological processes (5Arch R. Wirth K. Hofmann M. Ponta H. Matzku S. Herrlich P. Zöller M. Science. 1992; 257: 682-685Crossref PubMed Scopus (400) Google Scholar, 6Wainwright D. Sherman L. Sleeman J. Ponta H. Herrlich P. Ann. N. Y. Acad. Sci. 1996; 758: 345-349Crossref Scopus (12) Google Scholar). The expression of CD44v isoforms is up-regulated in many tumors during tumor progression (7Ponta H. Sleeman J. Dall P. Moll J. Sherman L. Herrlich P. Invasion & Metastasis. 1995; 14: 82-86Google Scholar). It is well documented that CD44 isoforms bind to the extracellular matrix glycosaminoglycan (GAG) 1The abbreviations used are: GAG, glycosaminoglycan; CPC, cetylpyridinium chloride; CS, chondroitin sulfate; CS-A, -B, and -C, chondroitin sulfate type A, B, and C (chondroitin-4-sulfate, dermatan sulfate and chondroitin-6-sulfate, respectively); HA, hyaluronate; HS, heparin sulfate; KS, keratan sulfate; H, heparin; FCS, fetal calf serum; PCR, polymerase chain reaction; PBS, phosphate-buffered saline.1The abbreviations used are: GAG, glycosaminoglycan; CPC, cetylpyridinium chloride; CS, chondroitin sulfate; CS-A, -B, and -C, chondroitin sulfate type A, B, and C (chondroitin-4-sulfate, dermatan sulfate and chondroitin-6-sulfate, respectively); HA, hyaluronate; HS, heparin sulfate; KS, keratan sulfate; H, heparin; FCS, fetal calf serum; PCR, polymerase chain reaction; PBS, phosphate-buffered saline. hyaluronate (HA), and HA binding motifs in the extracellular portion of the protein have been identified (e.g. Refs. 3Lesley J. Hyman R. Kincade P.W. Adv. Immunol. 1993; 54: 271-335Crossref PubMed Scopus (1026) Google Scholar, 8Yang B. Yang B.L. Savani R.C. Turley E.A. EMBO J. 1994; 13: 286-296Crossref PubMed Scopus (335) Google Scholar, and 9Sleeman J.P. Arming S. Moll J.F. Hekele A. Rudy W. Sherman L.S. Kreil G. Ponta H. Herrlich P. Cancer Res. 1996; 56: 3134-3141PubMed Google Scholar). However, other ligand binding activities must exist. For example, CD44s-mediated lymphocyte binding to mucosal high endothelial venules is independent of HA binding (10Culty M. Miyake K. Kincade P.W. Silorski E. Butcher E.C. Underhill C. J. Cell Biol. 1990; 111: 2765-2774Crossref PubMed Scopus (314) Google Scholar). Moreover, an antibody specific for CD44 variant isoforms containing an exon v6-encoded epitope blocks tumor growth, lymphocyte activation, and limb bud outgrowth, but does not block CD44-mediated HA binding (5Arch R. Wirth K. Hofmann M. Ponta H. Matzku S. Herrlich P. Zöller M. Science. 1992; 257: 682-685Crossref PubMed Scopus (400) Google Scholar, 6Wainwright D. Sherman L. Sleeman J. Ponta H. Herrlich P. Ann. N. Y. Acad. Sci. 1996; 758: 345-349Crossref Scopus (12) Google Scholar, 9Sleeman J.P. Arming S. Moll J.F. Hekele A. Rudy W. Sherman L.S. Kreil G. Ponta H. Herrlich P. Cancer Res. 1996; 56: 3134-3141PubMed Google Scholar). Although no specific ligands have been identified for CD44v isoforms, a plethora of ligands have been ascribed to CD44s, including mucosal addressin (11Picker L.J. Nakache M. Butcher E.C. J. Cell Biol. 1989; 109: 927-937Crossref PubMed Scopus (254) Google Scholar), collagen type I (12Faasen A. Schrager J. Klein D. Oegema T. Couchman J. McCarthy J. J. Cell Biol. 1992; 116: 521-531Crossref PubMed Scopus (236) Google Scholar), fibronectin (13Jalkanen S. Jalkanen M. J. Cell Biol. 1992; 116: 817-825Crossref PubMed Scopus (448) Google Scholar), MIP-1β (14Tanaka Y. Adams D.H. Hubscher S. Hirano H. Siebenlist U. Shaw S. Nature. 1993; 361: 79-82Crossref PubMed Scopus (843) Google Scholar), the chondroitin-sulfated form of invariant chain (15Naujokas M.F. Morin M. Anderson M.S. Peterson M. Miller J. Cell. 1993; 74: 257-268Abstract Full Text PDF PubMed Scopus (201) Google Scholar), serglycin (16Toyama-Sorimachi N. Sorimachi H. Tobita Y. Kitamura F. Yagita H. Suzuki K. Miyasaka M. J. Biol. Chem. 1995; 270: 7437-7444Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar), and osteopontin (17Weber G. Ashkar S. Glimcher M. Cantor H. Science. 1996; 271: 509-512Crossref PubMed Scopus (806) Google Scholar). While certain of these interactions are mediated by binding of the ligand to sugar moieties on the CD44s protein, the nature of the interaction with the other ligands is unclear. At least for serglycin, binding by CD44s is dependent on the presence of chondroitin sulfate (CS) modifications on the serglycin protein (16Toyama-Sorimachi N. Sorimachi H. Tobita Y. Kitamura F. Yagita H. Suzuki K. Miyasaka M. J. Biol. Chem. 1995; 270: 7437-7444Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). In this case, however, the CD44s protein could not bind directly to CS, a finding also reported by others (18Miyake K. Underhill C.B. Lesley J. Kincade P.W. J. Exp. Med. 1990; 172: 69-75Crossref PubMed Scopus (557) Google Scholar). Work with purified CD44s proteins, on the other hand, suggests that CD44s has a weak affinity for CS (19Underhill C.B. Chi-Rosso G. Toole B.P. J. Biol. Chem. 1983; 258: 8086-8091Abstract Full Text PDF PubMed Google Scholar, 20Aruffo A. Stamenkovic I. Melnick M. Underhill C.B. Seed B. Cell. 1990; 61: 1303-1313Abstract Full Text PDF PubMed Scopus (2135) Google Scholar, 21Peach R.J. Hollenbaugh D. Stamenkovic I. Aruffo A. J. Cell Biol. 1993; 122: 257-264Crossref PubMed Scopus (321) Google Scholar), a conclusion supported by CD44 transfection experiments in a B cell lymphoma line (22Sy M.S. Guo Y.-J. Stamenkovic I. J. Exp. Med. 1991; 174: 859-866Crossref PubMed Scopus (302) Google Scholar). The role of CD44 spl
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