Distinct Domains of CD98hc Regulate Integrins and Amino Acid Transport
2001; Elsevier BV; Volume: 276; Issue: 12 Linguagem: Inglês
10.1074/jbc.m011239200
ISSN1083-351X
AutoresCsilla A. Fenczik, Roy Zent, Melissa Dellos, David Calderwood, Joe Satriano, Carolyn Kelly, Mark H. Ginsberg,
Tópico(s)Metabolism and Genetic Disorders
ResumoCD98 is a cell surface heterodimer formed by the covalent linkage of CD98 heavy chain (CD98hc) with several different light chains to form amino acid transporters. CD98hc also binds specifically to the integrin β1A cytoplasmic domain and regulates integrin function. In this study, we examined the relationship between the ability of CD98hc to stimulate amino acid transport and to affect integrin function. By constructing chimeras with CD98hc and a type II transmembrane protein (CD69), we found that the cytoplasmic and transmembrane domains of CD98hc are required for its effects on integrin function, while the extracellular domain is required for stimulation of isoleucine transport. Consequently, the capacity to promote amino acid transport is not required for CD98hc's effect on integrin function. Furthermore, a mutant of CD98hc that lacks its integrin binding site can still promote increased isoleucine transport. Thus, these two functions of CD98hc are separable and require distinct domains of the protein. CD98 is a cell surface heterodimer formed by the covalent linkage of CD98 heavy chain (CD98hc) with several different light chains to form amino acid transporters. CD98hc also binds specifically to the integrin β1A cytoplasmic domain and regulates integrin function. In this study, we examined the relationship between the ability of CD98hc to stimulate amino acid transport and to affect integrin function. By constructing chimeras with CD98hc and a type II transmembrane protein (CD69), we found that the cytoplasmic and transmembrane domains of CD98hc are required for its effects on integrin function, while the extracellular domain is required for stimulation of isoleucine transport. Consequently, the capacity to promote amino acid transport is not required for CD98hc's effect on integrin function. Furthermore, a mutant of CD98hc that lacks its integrin binding site can still promote increased isoleucine transport. Thus, these two functions of CD98hc are separable and require distinct domains of the protein. complement dominant suppression Chinese hamster ovary 1,4-piperazinediethanesulfonic acid polyacrylamide gel electrophoresis activation index Hanks' buffered salt solution CD98hc is a widely distributed transmembrane protein that was originally discovered as a T-cell activation antigen (1Haynes B.F. Hemler M.E. Mann D.L. Eisenbarth G.S. Shelhamer J. Mostowski H.S. Thomas C.A. Strominger J.L. Fauci A.S. J. Immunol. 1981; 126: 1409-1414PubMed Google Scholar). CD98hc expression is tightly linked to cell proliferation, and antibodies against CD98hc can inhibit cell growth or induce apoptosis in specific cell types (2Yagita H. Masuko T. Hashimoto Y. Cancer Res. 1986; 46: 1478-1484PubMed Google Scholar, 3Warren A.P. Patel K. McConkey D.J. Palacios R. Blood. 1996; 87: 3676-3687Crossref PubMed Google Scholar). A compelling body of evidence implicates CD98hc in the transport of amino acids. CD98hc overexpression stimulates multiple amino acid transport systems including L, y+L, and xc− (4Verrey F. Meier C. Rossier G. Kuhn L.C. Pfluegers Arch. 2000; 440: 503-512Crossref PubMed Google Scholar). Furthermore, mutations in its closest paralogue, D2 (r-BAT), lead to a disorder of cysteine transport (5Calonge M.J. Gasparini P. Chillaron J. Chillon M. Gallucci M. Rousaud F. Zelante L. Testar X. Dallapiccola B. Di Silverio F. Nat. Genet. 1994; 6: 420-425Crossref PubMed Scopus (344) Google Scholar). Structurally, CD98 is a disulfide-bonded heterodimer of a common ∼80-kDa heavy chain (CD98hc) with one of several ∼40-kDa light chains. Because these light chains have multiple membrane-spanning domains, they resemble permeases and are believed to provide the amino acid transport activity of CD98 (6Mastroberardino L. Spindler B. Pfeiffer R. Skelly P.J. Loffing J. Shoemaker C.B. Verrey F. Nature. 1998; 395: 288-291Crossref PubMed Scopus (456) Google Scholar, 7Torrents D. Estevez R.A. Pineda M. Fernandez E. Lloberas J. Yun-Bo S. Zorzano A. Palacin M. J. Biol. Chem. 1998; 273: 32437-32445Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 8Kanai Y. Segawa H. Miyamoto K. Uchino H. Takeda E. Endou H. J. Biol. Chem. 1998; 273: 23629-23632Abstract Full Text Full Text PDF PubMed Scopus (871) Google Scholar). CD98hc may act to regulate the expression and cellular localization of the amino acid transporting activity of the light chain (6Mastroberardino L. Spindler B. Pfeiffer R. Skelly P.J. Loffing J. Shoemaker C.B. Verrey F. Nature. 1998; 395: 288-291Crossref PubMed Scopus (456) Google Scholar, 9Nakamura E. Sato M. Yang H. Miyagawa F. Harasaki M. Tomita K. Matsuoka S. Noma A. Iwai K. Minato N. J. Biol. Chem. 1999; 274: 3009-3016Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). Thus, this widely distributed membrane protein is strongly implicated in amino acid transport.There is also a growing literature implicating CD98hc in integrin function. Integrins are heterodimeric adhesion receptors expressed in almost every multicellular animal cell type (10Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8966) Google Scholar). Cells can rapidly modulate their integrins' affinity for extracellular ligands (activation), thereby regulating multiple integrin-dependent functions (11Hughes P.E. Pfaff M. Trends Cell Biol. 1998; 8: 359-364Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar). Integrin activation is inhibited by overexpression of isolated β1A integrin cytoplasmic domains (dominant suppression) (12Chen Y.-P. O'Toole T.E. Shipley T. Forsyth J. LaFlamme S.E. Yamada K.M. Shattil S.J. Ginsberg M.H. J. Biol. Chem. 1994; 269: 18307-18310Abstract Full Text PDF PubMed Google Scholar). CD98hc was identified as an integrin regulator in an expression-cloning scheme for proteins that can complement dominant suppression (CODS)1 (13Fenczik C.A. Sethi T. Ramos J.W. Hughes P.E. Ginsberg M.H. Nature. 1997; 390: 81-85Crossref PubMed Scopus (254) Google Scholar). In addition, CD98hc binds to integrin β1A cytoplasmic domains, and this interaction correlates with CODS (14Zent R. Fenczik C.A. Calderwood D.A. Liu S. Dellos M. Ginsberg M.H. J. Biol. Chem. 2000; 275: 5059-5064Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Furthermore, clustering CD98hc activates multiple integrin-dependent functions (13Fenczik C.A. Sethi T. Ramos J.W. Hughes P.E. Ginsberg M.H. Nature. 1997; 390: 81-85Crossref PubMed Scopus (254) Google Scholar,15Ohta H. Tsurudome M. Matsumura H. Koga Y. Morikawa S. Kawano M. Kusugawa S. Komada H. Nishio M. Ito Y. EMBO J. 1994; 13: 2044-2055Crossref PubMed Scopus (75) Google Scholar, 16Chandrasekaran S. Guo N.H. Rodrigues R.G. Kaiser J. Roberts D.D. J. Biol. Chem. 1999; 274: 11408-11416Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar) and mimics β1 integrin cosignaling in T-cells (17Warren A.P. Patel K. Miyamoto Y. Wygant J.N. Woodside D.G. McIntyre B.W. Immunology. 2000; 99: 62-68Crossref PubMed Scopus (27) Google Scholar). Thus, CD98hc physically and functionally interacts with integrin adhesion receptors. Indeed, clustering of CD98hc can stimulate several classes of integrins in multiple cell types (13Fenczik C.A. Sethi T. Ramos J.W. Hughes P.E. Ginsberg M.H. Nature. 1997; 390: 81-85Crossref PubMed Scopus (254) Google Scholar, 15Ohta H. Tsurudome M. Matsumura H. Koga Y. Morikawa S. Kawano M. Kusugawa S. Komada H. Nishio M. Ito Y. EMBO J. 1994; 13: 2044-2055Crossref PubMed Scopus (75) Google Scholar, 16Chandrasekaran S. Guo N.H. Rodrigues R.G. Kaiser J. Roberts D.D. J. Biol. Chem. 1999; 274: 11408-11416Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar).In the present study, we have assessed the relationship between the amino acid transport and integrin regulatory activities of CD98hc. We found that the CD98hc alone is necessary and sufficient for the interaction of CD98 with the β1A integrin tail and for CODS. By forming chimeras between CD98hc and another type II transmembrane protein, we found that the cytoplasmic and transmembrane domains of CD98hc are required for integrin interactions but not for stimulation of isoleucine transport. In contrast, the CD98hc extracellular domain was required for stimulation of amino acid transport. Thus, the amino acid transport activity and integrin interactions of CD98 are independent activities of the protein and are mediated by different domains of CD98hc.DISCUSSIONCD98hc combines with several different light chains to form a series of heterodimers that are involved in amino acid transport. CD98hc binds to integrin β1A cytoplasmic domains and blocks the capacity of β1A cytoplasmic domains to suppress integrin activation (CODS). We have compared the structural requirements of CD98hc for interaction with integrins with those involved in regulation of amino acid transport. Here we report that: 1) mutation of cysteines that disrupt CD98 heavy-light chain association and reduce amino acid transport do not disrupt its binding to β1A or its effect on integrin activation. 2) The cytoplasmic and transmembrane domains of CD98hc fused to another type II transmembrane protein are both necessary and sufficient for binding to the integrin β1A tail and for CODS. This chimera failed to stimulate amino acid transport. 3) Replacement of the cytoplasmic or transmembrane domains of CD98hc with those of CD69 blocked the capacity of CD98hc to bind to β1A and regulate integrin activation, but had minimal effects on the amino acid transport function of CD98. Thus, the amino acid transport function of CD98 is not required for its effects on integrin function, and amino acid transport can occur in the absence of CD98hc-integrin association.The formation of a covalent CD98 heterodimer is not required for its effects on integrin function. CD98hc has two extracellular cysteines Cys109 and Cys330. Cys109 is near the transmembrane domain of CD98hc and results in a disulfide bridge with a cysteine in an extracellular loop of the light chain between transmembrane domains 3 and 4 (25Pfeiffer R. Spindler B. Loffing J. Skelly P.J. Shoemaker C.B. Verrey F. FEBS Lett. 1998; 439: 157-162Crossref PubMed Scopus (90) Google Scholar). Mutation of Cys109 and Cys330 disrupted the covalent association with the light chain but did not impair interactions with or effects on integrins. While the covalent association was lost, it is possible that there was still a noncovalent interaction. Indeed, Pfeiffer et al. (25Pfeiffer R. Spindler B. Loffing J. Skelly P.J. Shoemaker C.B. Verrey F. FEBS Lett. 1998; 439: 157-162Crossref PubMed Scopus (90) Google Scholar) reported that the C109S mutant does still support the surface expression of the light chain. The C109S mutation still displays the same transport characteristics as the disulfide-bound heterodimers, albeit at a reduced rate. Moreover, we also found that overexpressed free heavy chains could also bind to the β1A tail. Furthermore, cotransfection of the hLAT1 light chain increased formation of heterodimers and amino acid transport but did not augment integrin interactions or effects. Consequently, our results indicate that the covalent association of CD98hc with a light chain is not required for its interaction with integrins or for the functional regulation of integrins.The cytoplasmic and transmembrane domains of CD98hc are both necessary and sufficient for binding to the integrin β1A tail and for effects on integrin function. When either of these domains was removed from CD98hc, integrin interactions were lost. Conversely, effects on integrins could be conveyed to CD69 by addition of these two domains. What is the role of the CD98hc transmembrane domain in binding to the β1A cytoplasmic tail? It is possible that the CD98hc transmembrane domain influences the conformation of the cytoplasmic domain to promote binding to integrin cytoplasmic domains. Alternatively, our integrin cytoplasmic domain model protein was based on that predicted from the sequence (ITB1_human) in the Swiss-Prot data base (Entry P05556). Glycosylation mapping studies have suggested that β1A (Lys752–Ile757) of the predicted "cytoplasmic" domain may reside in the membrane (27Armulik A. Nilsson I. von Heijne G. Johansson S. J. Biol. Chem. 1999; 274: 37030-37034Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Consequently, the CD98hc transmembrane domain may directly interact with a transmembrane portion of our model protein "tail." Furthermore, other integrin-binding proteins, such as cytohesin-1, Rack1, and skelemin also bind the membrane proximal region (28Liu S. Calderwood D.A. Ginsberg M.H. J. Cell Sci. 2000; 113: 3563-3571Crossref PubMed Google Scholar). Thus, the localization of this region in the membrane may specify preferential binding of integrin-associated proteins. Finally, the CD98hc solubilized from membranes could be associated with other proteins via the transmembrane domains. These "adapters" might contribute to the CD98hc-β1A tail interaction. In any case, our studies provide the first delineation of a specific functional role for the cytoplasmic and transmembrane domains of CD98hc, interaction with and regulation of β1A integrin function.Regulation of amino acid transport and integrins by CD98hc is a distinct and separable function of the polypeptide. Chimeras in which the cytoplasmic or transmembrane domains of CD98hc were replaced with those of CD69 lost the capacity to bind to β1A and regulate integrin activation. In contrast, these replacements had little effect on the amino acid transport function of CD98. Conversely, the exchange of the extracellular domain of CD98hc with that of CD69 resulted in a protein that was still capable of affecting integrin function but did not stimulate isoleucine transport. Thus, the amino acid transport activity of CD98hc is not required for its effect on integrin function.CD98hc functions as a chaperone to bring the associated light chains (LAT1, LAT2, y+LAT1, y+LAT2, xCT, and asc-1) to the plasma membrane (29Verrey F. Jack D.L. Paulsen I.T. Saier Jr., M.H. Pfeiffer R. J. Membr. Biol. 1999; 172: 181-192Crossref PubMed Scopus (143) Google Scholar, 30Palacin M. Bertran J. Zorzano A. Curr. Opin. Nephrol. Hypertens. 2000; 9: 547-553Crossref PubMed Scopus (35) Google Scholar). We found that the interaction of CD98hc with integrins and amino acid transporters are ascribable to distinct domains of the protein and are not mutually exclusive. Integrin-mediated adhesion often leads to the polarization of these receptors to the adherent cell surface. Consequently, the integrin-CD98hc interaction may serve to polarize the localization of CD98 and, consequently, amino acid transport. Conversely, CD98hc can influence multiple integrin-dependent functions, including virus-induced cell fusion, T-cell costimulation, and cell adhesion (13Fenczik C.A. Sethi T. Ramos J.W. Hughes P.E. Ginsberg M.H. Nature. 1997; 390: 81-85Crossref PubMed Scopus (254) Google Scholar,15Ohta H. Tsurudome M. Matsumura H. Koga Y. Morikawa S. Kawano M. Kusugawa S. Komada H. Nishio M. Ito Y. EMBO J. 1994; 13: 2044-2055Crossref PubMed Scopus (75) Google Scholar, 16Chandrasekaran S. Guo N.H. Rodrigues R.G. Kaiser J. Roberts D.D. J. Biol. Chem. 1999; 274: 11408-11416Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 17Warren A.P. Patel K. Miyamoto Y. Wygant J.N. Woodside D.G. McIntyre B.W. Immunology. 2000; 99: 62-68Crossref PubMed Scopus (27) Google Scholar). Thus, the CD98hc-integrin association can promote integrin-mediated cell adhesion that, in turn, could serve to localize the activities of CD98hc-linked amino acid transporters. CD98hc is a widely distributed transmembrane protein that was originally discovered as a T-cell activation antigen (1Haynes B.F. Hemler M.E. Mann D.L. Eisenbarth G.S. Shelhamer J. Mostowski H.S. Thomas C.A. Strominger J.L. Fauci A.S. J. Immunol. 1981; 126: 1409-1414PubMed Google Scholar). CD98hc expression is tightly linked to cell proliferation, and antibodies against CD98hc can inhibit cell growth or induce apoptosis in specific cell types (2Yagita H. Masuko T. Hashimoto Y. Cancer Res. 1986; 46: 1478-1484PubMed Google Scholar, 3Warren A.P. Patel K. McConkey D.J. Palacios R. Blood. 1996; 87: 3676-3687Crossref PubMed Google Scholar). A compelling body of evidence implicates CD98hc in the transport of amino acids. CD98hc overexpression stimulates multiple amino acid transport systems including L, y+L, and xc− (4Verrey F. Meier C. Rossier G. Kuhn L.C. Pfluegers Arch. 2000; 440: 503-512Crossref PubMed Google Scholar). Furthermore, mutations in its closest paralogue, D2 (r-BAT), lead to a disorder of cysteine transport (5Calonge M.J. Gasparini P. Chillaron J. Chillon M. Gallucci M. Rousaud F. Zelante L. Testar X. Dallapiccola B. Di Silverio F. Nat. Genet. 1994; 6: 420-425Crossref PubMed Scopus (344) Google Scholar). Structurally, CD98 is a disulfide-bonded heterodimer of a common ∼80-kDa heavy chain (CD98hc) with one of several ∼40-kDa light chains. Because these light chains have multiple membrane-spanning domains, they resemble permeases and are believed to provide the amino acid transport activity of CD98 (6Mastroberardino L. Spindler B. Pfeiffer R. Skelly P.J. Loffing J. Shoemaker C.B. Verrey F. Nature. 1998; 395: 288-291Crossref PubMed Scopus (456) Google Scholar, 7Torrents D. Estevez R.A. Pineda M. Fernandez E. Lloberas J. Yun-Bo S. Zorzano A. Palacin M. J. Biol. Chem. 1998; 273: 32437-32445Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 8Kanai Y. Segawa H. Miyamoto K. Uchino H. Takeda E. Endou H. J. Biol. Chem. 1998; 273: 23629-23632Abstract Full Text Full Text PDF PubMed Scopus (871) Google Scholar). CD98hc may act to regulate the expression and cellular localization of the amino acid transporting activity of the light chain (6Mastroberardino L. Spindler B. Pfeiffer R. Skelly P.J. Loffing J. Shoemaker C.B. Verrey F. Nature. 1998; 395: 288-291Crossref PubMed Scopus (456) Google Scholar, 9Nakamura E. Sato M. Yang H. Miyagawa F. Harasaki M. Tomita K. Matsuoka S. Noma A. Iwai K. Minato N. J. Biol. Chem. 1999; 274: 3009-3016Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). Thus, this widely distributed membrane protein is strongly implicated in amino acid transport. There is also a growing literature implicating CD98hc in integrin function. Integrins are heterodimeric adhesion receptors expressed in almost every multicellular animal cell type (10Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8966) Google Scholar). Cells can rapidly modulate their integrins' affinity for extracellular ligands (activation), thereby regulating multiple integrin-dependent functions (11Hughes P.E. Pfaff M. Trends Cell Biol. 1998; 8: 359-364Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar). Integrin activation is inhibited by overexpression of isolated β1A integrin cytoplasmic domains (dominant suppression) (12Chen Y.-P. O'Toole T.E. Shipley T. Forsyth J. LaFlamme S.E. Yamada K.M. Shattil S.J. Ginsberg M.H. J. Biol. Chem. 1994; 269: 18307-18310Abstract Full Text PDF PubMed Google Scholar). CD98hc was identified as an integrin regulator in an expression-cloning scheme for proteins that can complement dominant suppression (CODS)1 (13Fenczik C.A. Sethi T. Ramos J.W. Hughes P.E. Ginsberg M.H. Nature. 1997; 390: 81-85Crossref PubMed Scopus (254) Google Scholar). In addition, CD98hc binds to integrin β1A cytoplasmic domains, and this interaction correlates with CODS (14Zent R. Fenczik C.A. Calderwood D.A. Liu S. Dellos M. Ginsberg M.H. J. Biol. Chem. 2000; 275: 5059-5064Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Furthermore, clustering CD98hc activates multiple integrin-dependent functions (13Fenczik C.A. Sethi T. Ramos J.W. Hughes P.E. Ginsberg M.H. Nature. 1997; 390: 81-85Crossref PubMed Scopus (254) Google Scholar,15Ohta H. Tsurudome M. Matsumura H. Koga Y. Morikawa S. Kawano M. Kusugawa S. Komada H. Nishio M. Ito Y. EMBO J. 1994; 13: 2044-2055Crossref PubMed Scopus (75) Google Scholar, 16Chandrasekaran S. Guo N.H. Rodrigues R.G. Kaiser J. Roberts D.D. J. Biol. Chem. 1999; 274: 11408-11416Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar) and mimics β1 integrin cosignaling in T-cells (17Warren A.P. Patel K. Miyamoto Y. Wygant J.N. Woodside D.G. McIntyre B.W. Immunology. 2000; 99: 62-68Crossref PubMed Scopus (27) Google Scholar). Thus, CD98hc physically and functionally interacts with integrin adhesion receptors. Indeed, clustering of CD98hc can stimulate several classes of integrins in multiple cell types (13Fenczik C.A. Sethi T. Ramos J.W. Hughes P.E. Ginsberg M.H. Nature. 1997; 390: 81-85Crossref PubMed Scopus (254) Google Scholar, 15Ohta H. Tsurudome M. Matsumura H. Koga Y. Morikawa S. Kawano M. Kusugawa S. Komada H. Nishio M. Ito Y. EMBO J. 1994; 13: 2044-2055Crossref PubMed Scopus (75) Google Scholar, 16Chandrasekaran S. Guo N.H. Rodrigues R.G. Kaiser J. Roberts D.D. J. Biol. Chem. 1999; 274: 11408-11416Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). In the present study, we have assessed the relationship between the amino acid transport and integrin regulatory activities of CD98hc. We found that the CD98hc alone is necessary and sufficient for the interaction of CD98 with the β1A integrin tail and for CODS. By forming chimeras between CD98hc and another type II transmembrane protein, we found that the cytoplasmic and transmembrane domains of CD98hc are required for integrin interactions but not for stimulation of isoleucine transport. In contrast, the CD98hc extracellular domain was required for stimulation of amino acid transport. Thus, the amino acid transport activity and integrin interactions of CD98 are independent activities of the protein and are mediated by different domains of CD98hc. DISCUSSIONCD98hc combines with several different light chains to form a series of heterodimers that are involved in amino acid transport. CD98hc binds to integrin β1A cytoplasmic domains and blocks the capacity of β1A cytoplasmic domains to suppress integrin activation (CODS). We have compared the structural requirements of CD98hc for interaction with integrins with those involved in regulation of amino acid transport. Here we report that: 1) mutation of cysteines that disrupt CD98 heavy-light chain association and reduce amino acid transport do not disrupt its binding to β1A or its effect on integrin activation. 2) The cytoplasmic and transmembrane domains of CD98hc fused to another type II transmembrane protein are both necessary and sufficient for binding to the integrin β1A tail and for CODS. This chimera failed to stimulate amino acid transport. 3) Replacement of the cytoplasmic or transmembrane domains of CD98hc with those of CD69 blocked the capacity of CD98hc to bind to β1A and regulate integrin activation, but had minimal effects on the amino acid transport function of CD98. Thus, the amino acid transport function of CD98 is not required for its effects on integrin function, and amino acid transport can occur in the absence of CD98hc-integrin association.The formation of a covalent CD98 heterodimer is not required for its effects on integrin function. CD98hc has two extracellular cysteines Cys109 and Cys330. Cys109 is near the transmembrane domain of CD98hc and results in a disulfide bridge with a cysteine in an extracellular loop of the light chain between transmembrane domains 3 and 4 (25Pfeiffer R. Spindler B. Loffing J. Skelly P.J. Shoemaker C.B. Verrey F. FEBS Lett. 1998; 439: 157-162Crossref PubMed Scopus (90) Google Scholar). Mutation of Cys109 and Cys330 disrupted the covalent association with the light chain but did not impair interactions with or effects on integrins. While the covalent association was lost, it is possible that there was still a noncovalent interaction. Indeed, Pfeiffer et al. (25Pfeiffer R. Spindler B. Loffing J. Skelly P.J. Shoemaker C.B. Verrey F. FEBS Lett. 1998; 439: 157-162Crossref PubMed Scopus (90) Google Scholar) reported that the C109S mutant does still support the surface expression of the light chain. The C109S mutation still displays the same transport characteristics as the disulfide-bound heterodimers, albeit at a reduced rate. Moreover, we also found that overexpressed free heavy chains could also bind to the β1A tail. Furthermore, cotransfection of the hLAT1 light chain increased formation of heterodimers and amino acid transport but did not augment integrin interactions or effects. Consequently, our results indicate that the covalent association of CD98hc with a light chain is not required for its interaction with integrins or for the functional regulation of integrins.The cytoplasmic and transmembrane domains of CD98hc are both necessary and sufficient for binding to the integrin β1A tail and for effects on integrin function. When either of these domains was removed from CD98hc, integrin interactions were lost. Conversely, effects on integrins could be conveyed to CD69 by addition of these two domains. What is the role of the CD98hc transmembrane domain in binding to the β1A cytoplasmic tail? It is possible that the CD98hc transmembrane domain influences the conformation of the cytoplasmic domain to promote binding to integrin cytoplasmic domains. Alternatively, our integrin cytoplasmic domain model protein was based on that predicted from the sequence (ITB1_human) in the Swiss-Prot data base (Entry P05556). Glycosylation mapping studies have suggested that β1A (Lys752–Ile757) of the predicted "cytoplasmic" domain may reside in the membrane (27Armulik A. Nilsson I. von Heijne G. Johansson S. J. Biol. Chem. 1999; 274: 37030-37034Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Consequently, the CD98hc transmembrane domain may directly interact with a transmembrane portion of our model protein "tail." Furthermore, other integrin-binding proteins, such as cytohesin-1, Rack1, and skelemin also bind the membrane proximal region (28Liu S. Calderwood D.A. Ginsberg M.H. J. Cell Sci. 2000; 113: 3563-3571Crossref PubMed Google Scholar). Thus, the localization of this region in the membrane may specify preferential binding of integrin-associated proteins. Finally, the CD98hc solubilized from membranes could be associated with other proteins via the transmembrane domains. These "adapters" might contribute to the CD98hc-β1A tail interaction. In any case, our studies provide the first delineation of a specific functional role for the cytoplasmic and transmembrane domains of CD98hc, interaction with and regulation of β1A integrin function.Regulation of amino acid transport and integrins by CD98hc is a distinct and separable function of the polypeptide. Chimeras in which the cytoplasmic or transmembrane domains of CD98hc were replaced with those of CD69 lost the capacity to bind to β1A and regulate integrin activation. In contrast, these replacements had little effect on the amino acid transport function of CD98. Conversely, the exchange of the extracellular domain of CD98hc with that of CD69 resulted in a protein that was still capable of affecting integrin function but did not stimulate isoleucine transport. Thus, the amino acid transport activity of CD98hc is not required for its effect on integrin function.CD98hc functions as a chaperone to bring the associated light chains (LAT1, LAT2, y+LAT1, y+LAT2, xCT, and asc-1) to the plasma membrane (29Verrey F. Jack D.L. Paulsen I.T. Saier Jr., M.H. Pfeiffer R. J. Membr. Biol. 1999; 172: 181-192Crossref PubMed Scopus (143) Google Scholar, 30Palacin M. Bertran J. Zorzano A. Curr. Opin. Nephrol. Hypertens. 2000; 9: 547-553Crossref PubMed Scopus (35) Google Scholar). We found that the interaction of CD98hc with integrins and amino acid transporters are ascribable to distinct domains of the protein and are not mutually exclusive. Integrin-mediated adhesion often leads to the polarization of these receptors to the adherent cell surface. Consequently, the integrin-CD98hc interaction may serve to polarize the localization of CD98 and, consequently, amino acid transport. Conversely, CD98hc can influence multiple integrin-dependent functions, including virus-induced cell fusion, T-cell costimulation, and cell adhesion (13Fenczik C.A. Sethi T. Ramos J.W. Hughes P.E. Ginsberg M.H. Nature. 1997; 390: 81-85Crossref PubMed Scopus (254) Google Scholar,15Ohta H. Tsurudome M. Matsumura H. Koga Y. Morikawa S. Kawano M. Kusugawa S. Komada H. Nishio M. Ito Y. EMBO J. 1994; 13: 2044-2055Crossref PubMed Scopus (75) Google Scholar, 16Chandrasekaran S. Guo N.H. Rodrigues R.G. Kaiser J. Roberts D.D. J. Biol. Chem. 1999; 274: 11408-11416Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 17Warren A.P. Patel K. Miyamoto Y. Wygant J.N. Woodside D.G. McIntyre B.W. Immunology. 2000; 99: 62-68Crossref PubMed Scopus (27) Google Scholar). Thus, the CD98hc-integrin association can promote integrin-mediated cell adhesion that, in turn, could serve to localize the activities of CD98hc-linked amino acid transporters. CD98hc combines with several different light chains to form a series of heterodimers that are involved in amino acid transport. CD98hc binds to integrin β1A cytoplasmic domains and blocks the capacity of β1A cytoplasmic domains to suppress integrin activation (CODS). We have compared the structural requirements of CD98hc for interaction with integrins with those involved in regulation of amino acid transport. Here we report that: 1) mutation of cysteines that disrupt CD98 heavy-light chain association and reduce amino acid transport do not disrupt its binding to β1A or its effect on integrin activation. 2) The cytoplasmic and transmembrane domains of CD98hc fused to another type II transmembrane protein are both necessary and sufficient for binding to the integrin β1A tail and for CODS. This chimera failed to stimulate amino acid transport. 3) Replacement of the cytoplasmic or transmembrane domains of CD98hc with those of CD69 blocked the capacity of CD98hc to bind to β1A and regulate integrin activation, but had minimal effects on the amino acid transport function of CD98. Thus, the amino acid transport function of CD98 is not required for its effects on integrin function, and amino acid transport can occur in the absence of CD98hc-integrin association. The formation of a covalent CD98 heterodimer is not required for its effects on integrin function. CD98hc has two extracellular cysteines Cys109 and Cys330. Cys109 is near the transmembrane domain of CD98hc and results in a disulfide bridge with a cysteine in an extracellular loop of the light chain between transmembrane domains 3 and 4 (25Pfeiffer R. Spindler B. Loffing J. Skelly P.J. Shoemaker C.B. Verrey F. FEBS Lett. 1998; 439: 157-162Crossref PubMed Scopus (90) Google Scholar). Mutation of Cys109 and Cys330 disrupted the covalent association with the light chain but did not impair interactions with or effects on integrins. While the covalent association was lost, it is possible that there was still a noncovalent interaction. Indeed, Pfeiffer et al. (25Pfeiffer R. Spindler B. Loffing J. Skelly P.J. Shoemaker C.B. Verrey F. FEBS Lett. 1998; 439: 157-162Crossref PubMed Scopus (90) Google Scholar) reported that the C109S mutant does still support the surface expression of the light chain. The C109S mutation still displays the same transport characteristics as the disulfide-bound heterodimers, albeit at a reduced rate. Moreover, we also found that overexpressed free heavy chains could also bind to the β1A tail. Furthermore, cotransfection of the hLAT1 light chain increased formation of heterodimers and amino acid transport but did not augment integrin interactions or effects. Consequently, our results indicate that the covalent association of CD98hc with a light chain is not required for its interaction with integrins or for the functional regulation of integrins. The cytoplasmic and transmembrane domains of CD98hc are both necessary and sufficient for binding to the integrin β1A tail and for effects on integrin function. When either of these domains was removed from CD98hc, integrin interactions were lost. Conversely, effects on integrins could be conveyed to CD69 by addition of these two domains. What is the role of the CD98hc transmembrane domain in binding to the β1A cytoplasmic tail? It is possible that the CD98hc transmembrane domain influences the conformation of the cytoplasmic domain to promote binding to integrin cytoplasmic domains. Alternatively, our integrin cytoplasmic domain model protein was based on that predicted from the sequence (ITB1_human) in the Swiss-Prot data base (Entry P05556). Glycosylation mapping studies have suggested that β1A (Lys752–Ile757) of the predicted "cytoplasmic" domain may reside in the membrane (27Armulik A. Nilsson I. von Heijne G. Johansson S. J. Biol. Chem. 1999; 274: 37030-37034Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Consequently, the CD98hc transmembrane domain may directly interact with a transmembrane portion of our model protein "tail." Furthermore, other integrin-binding proteins, such as cytohesin-1, Rack1, and skelemin also bind the membrane proximal region (28Liu S. Calderwood D.A. Ginsberg M.H. J. Cell Sci. 2000; 113: 3563-3571Crossref PubMed Google Scholar). Thus, the localization of this region in the membrane may specify preferential binding of integrin-associated proteins. Finally, the CD98hc solubilized from membranes could be associated with other proteins via the transmembrane domains. These "adapters" might contribute to the CD98hc-β1A tail interaction. In any case, our studies provide the first delineation of a specific functional role for the cytoplasmic and transmembrane domains of CD98hc, interaction with and regulation of β1A integrin function. Regulation of amino acid transport and integrins by CD98hc is a distinct and separable function of the polypeptide. Chimeras in which the cytoplasmic or transmembrane domains of CD98hc were replaced with those of CD69 lost the capacity to bind to β1A and regulate integrin activation. In contrast, these replacements had little effect on the amino acid transport function of CD98. Conversely, the exchange of the extracellular domain of CD98hc with that of CD69 resulted in a protein that was still capable of affecting integrin function but did not stimulate isoleucine transport. Thus, the amino acid transport activity of CD98hc is not required for its effect on integrin function. CD98hc functions as a chaperone to bring the associated light chains (LAT1, LAT2, y+LAT1, y+LAT2, xCT, and asc-1) to the plasma membrane (29Verrey F. Jack D.L. Paulsen I.T. Saier Jr., M.H. Pfeiffer R. J. Membr. Biol. 1999; 172: 181-192Crossref PubMed Scopus (143) Google Scholar, 30Palacin M. Bertran J. Zorzano A. Curr. Opin. Nephrol. Hypertens. 2000; 9: 547-553Crossref PubMed Scopus (35) Google Scholar). We found that the interaction of CD98hc with integrins and amino acid transporters are ascribable to distinct domains of the protein and are not mutually exclusive. Integrin-mediated adhesion often leads to the polarization of these receptors to the adherent cell surface. Consequently, the integrin-CD98hc interaction may serve to polarize the localization of CD98 and, consequently, amino acid transport. Conversely, CD98hc can influence multiple integrin-dependent functions, including virus-induced cell fusion, T-cell costimulation, and cell adhesion (13Fenczik C.A. Sethi T. Ramos J.W. Hughes P.E. Ginsberg M.H. Nature. 1997; 390: 81-85Crossref PubMed Scopus (254) Google Scholar,15Ohta H. Tsurudome M. Matsumura H. Koga Y. Morikawa S. Kawano M. Kusugawa S. Komada H. Nishio M. Ito Y. EMBO J. 1994; 13: 2044-2055Crossref PubMed Scopus (75) Google Scholar, 16Chandrasekaran S. Guo N.H. Rodrigues R.G. Kaiser J. Roberts D.D. J. Biol. Chem. 1999; 274: 11408-11416Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 17Warren A.P. Patel K. Miyamoto Y. Wygant J.N. Woodside D.G. McIntyre B.W. Immunology. 2000; 99: 62-68Crossref PubMed Scopus (27) Google Scholar). Thus, the CD98hc-integrin association can promote integrin-mediated cell adhesion that, in turn, could serve to localize the activities of CD98hc-linked amino acid transporters. We thank our colleagues for their generosity in providing the reagents listed under "Experimental Procedures." We thank Drs. François Verrey and Manuel Palacin for reagents and for helpful discussions.
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