Class- and Splice Variant-specific Association of CD98 with Integrin β Cytoplasmic Domains
2000; Elsevier BV; Volume: 275; Issue: 7 Linguagem: Inglês
10.1074/jbc.275.7.5059
ISSN1083-351X
AutoresRoy Zent, Csilla A. Fenczik, David Calderwood, Shouchun Liu, Melissa Dellos, Mark H. Ginsberg,
Tópico(s)Cell Adhesion Molecules Research
ResumoCD98 is a type II transmembrane protein involved in neutral and basic amino acid transport and in cell fusion events. CD98 was implicated in the function of integrin adhesion receptors by its capacity to reverse suppression of integrin activation by isolated integrin β1A domains. Here we report that CD98 associates with integrin β cytoplasmic domains with a unique integrin class and splice variant specificity. In particular, CD98 interacted with the ubiquitous β1A but not the muscle-specific splice variant, β1D, or leukocyte-specific β7 cytoplasmic domains. The ability of CD98 to associate with integrin cytoplasmic domains correlated with its capacity to reverse suppression of integrin activation. The association of CD98 with integrin β1A cytoplasmic domains may regulate the function and localization of these membrane proteins. CD98 is a type II transmembrane protein involved in neutral and basic amino acid transport and in cell fusion events. CD98 was implicated in the function of integrin adhesion receptors by its capacity to reverse suppression of integrin activation by isolated integrin β1A domains. Here we report that CD98 associates with integrin β cytoplasmic domains with a unique integrin class and splice variant specificity. In particular, CD98 interacted with the ubiquitous β1A but not the muscle-specific splice variant, β1D, or leukocyte-specific β7 cytoplasmic domains. The ability of CD98 to associate with integrin cytoplasmic domains correlated with its capacity to reverse suppression of integrin activation. The association of CD98 with integrin β1A cytoplasmic domains may regulate the function and localization of these membrane proteins. complementation of dominant suppression polyacrylamide gel electrophoresis 1,4-piperazinediethanesulfonic acid The development and function of multicellular animals requires integrin adhesion receptors (1.Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9026) Google Scholar). Integrin-dependent cell adhesion is regulated, in part, by ligand binding affinity ("activation") changes controlled by cellular signaling cascades (1.Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9026) Google Scholar, 2.Schwartz M.A. Schaller M.D. Ginsberg M.H. Annu. Rev. Cell Dev. Biol. 1995; 11: 549-599Crossref PubMed Scopus (1474) Google Scholar, 3.Hughes P.E. Renshaw M.W. Pfaff M. Forsyth J. Keivens V.M. Schwartz M.A. Ginsberg M.H. Cell. 1997; 88: 521-530Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar). Regulation of integrin affinity is important in cell migration (4.Huttenlocher A. Ginsberg M.H. Horwitz A.F. J. Cell Biol. 1996; 134: 1551-1562Crossref PubMed Scopus (314) Google Scholar, 5.Huttenlocher A. Palecek S.P. Lu Q. Zhang W. Mellgren R.L. Lauffenburger D.A. Ginsberg M.H. Horwitz A.F. J. Biol. 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Complementation of dominant suppression (CODS)1 is an expression cloning scheme used to identify proteins that modulate integrin affinity (10.Fenczik C.A. Sethi T. Ramos J.W. Hughes P.E. Ginsberg M.H. Nature. 1997; 370: 81-85Crossref Scopus (260) Google Scholar). CODS depends on the ability of an isolated integrin β1A cytoplasmic domain, in the form of a chimera with the α subunit of the interleukin-2 receptor, to block integrin activation (dominant suppression). Proteins involved in integrin activation are isolated by their ability to complement dominant suppression. CD98, a type II transmembrane protein first discovered as a T-cell activation antigen (11.Haynes 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), was identified utilizing CODS. CD98, although widely expressed on proliferating cells, is generally down-regulated in quiescent cells (12.Diaz Jr., L.A. Fox D.A. J. Biol. Reg. Homeostat. Agents. 1998; 12: 25-32PubMed Google Scholar). CD98 forms disulfide-bonded heterodimers with several light chains that strongly resemble permeases (13.Mannion B.A. Kolesnikova T.V. Lin S.W. Wang S. Thompson N.L. Hemler M.E. J. Biol. Chem. 1998; 273: 33127-33129Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 14.Kanai 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 (901) Google Scholar, 15.Torrents D. Estevez R. Pineda M. Fernandez E. Lloberas J. Shi Y.-B. Zorzano A. Palacin M. J. Biol. Chem. 1998; 273: 32437-32445Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 16.Estevez R. Camps M. Rojas A.M. Tesrar X. Deves R. Hediger M.A. Zorzano A. Palacin M. FASEB. 1998; 12: 1319-1329Crossref PubMed Scopus (72) Google Scholar, 17.Mastroberardino L. Spindler B. Pfeiffer R. Loffing J. Skelley P.J. Shoemaker C.B. Verrey F. Nature. 1998; 395: 288-291Crossref PubMed Scopus (470) Google Scholar, 18.Pfeiffer R. Rossier G. Spindler B. Meier C. Kuhn L. Verrey F. EMBO J. 1999; 18: 49-57Crossref PubMed Scopus (239) Google Scholar, 19.Pfeiffer R. Spindler B. Loffing J. Skelley P.J. Shoemaker C.B. Verrey F. FEBS Lett. 1998; 439: 157-162Crossref PubMed Scopus (90) Google Scholar, 20.Tsurudome M. Ito M. Takebayashi S. Okumura K. Nishio M. Kawano M. Kusawaga S. Komada S. Ito Y. J. Immunol. 1999; 162: 2462-2466PubMed Google Scholar). CD98 regulates the transport of neutral and positively charge amino acids through these light chains (14.Kanai 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 (901) Google Scholar, 15.Torrents D. Estevez R. Pineda M. Fernandez E. Lloberas J. Shi Y.-B. Zorzano A. Palacin M. J. Biol. Chem. 1998; 273: 32437-32445Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 17.Mastroberardino L. Spindler B. Pfeiffer R. Loffing J. Skelley P.J. Shoemaker C.B. Verrey F. Nature. 1998; 395: 288-291Crossref PubMed Scopus (470) Google Scholar, 18.Pfeiffer R. Rossier G. Spindler B. Meier C. Kuhn L. Verrey F. EMBO J. 1999; 18: 49-57Crossref PubMed Scopus (239) Google Scholar). Thus, CODS has identified an unexpected connection between cell adhesion and certain amino acid transporters. The mechanism by which CD98 influences integrin function is not yet clear. CODS was predicated on the idea that it would identify integrin β cytoplasmic domain binding proteins (10.Fenczik C.A. Sethi T. Ramos J.W. Hughes P.E. Ginsberg M.H. Nature. 1997; 370: 81-85Crossref Scopus (260) Google Scholar). Many β cytoplasmic domains manifest overall sequence similarity (1.Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9026) Google Scholar, 2.Schwartz M.A. Schaller M.D. Ginsberg M.H. Annu. Rev. Cell Dev. Biol. 1995; 11: 549-599Crossref PubMed Scopus (1474) Google Scholar); however, the cytoskeletal protein, talin, binds to the muscle-specific splice variant, β1D, more tightly than to β1A. In addition, the leukocyte-specific β7 cytoplasmic domain binds to filamin more tightly than to β1A (21.Pfaff M. Liu S. Erle D.J. Ginsberg M.H. J. Biol. Chem. 1998; 273: 6104-6109Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). We have now examined interactions between CD98 and recombinant parallel-dimerized integrin β1A, β1D, and β7 cytoplasmic domains by affinity chromatography (21.Pfaff M. Liu S. Erle D.J. Ginsberg M.H. J. Biol. Chem. 1998; 273: 6104-6109Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). Here we report that CD98 interacts with the β1A but not β1D or β7 integrin cytoplasmic domains. Furthermore, the CD98 interaction is insensitive to β cytoplasmic domain mutations that abolish the binding of talin and filamin. The capacity of CD98 to complement dominant suppression correlates with its capacity to bind to the suppressive β cytoplasmic domains. The interaction of the integrin β1A cytoplasmic domain with CD98 may thus serve to regulate the localization and the function of these membrane proteins. The hybridoma cell line 4F2(C13) (anti-CD98) was purchased from American Type Culture Collection (ATCC). The CD98 antibody was purified from ascites produced in pristane-primed BALB/c mice by protein A affinity chromatography. Filamin antibody (monoclonal antibody 1680) was purchased from Chemicon and talin antibody (clone 8d4) from Sigma. Dr. S. Shattil (Scripps Research Institute) generously provided the activation-specific anti-αIIbβ3 monoclonal antibody, PAC1 (22.Shattil S.J. Hoxie J.A. Cunningham M. Brass L.F. J. Biol. Chem. 1985; 260: 11107-11114Abstract Full Text PDF PubMed Google Scholar). The anti-αIIbβ3 activating monoclonal antibody, anti-LIBS6, has been described previously (23.Frelinger III, A.L. Du X. Plow E.F. Ginsberg M.H. J. Biol. Chem. 1991; 266: 17106-17111Abstract Full Text PDF PubMed Google Scholar). The anti-Tac antibody, 7G7B6, was obtained from the American Tissue Culture Collection (Rockville, MD) and was biotinylated with biotin-N-hydroxysuccinimide (Sigma) according to manufacturer's instructions. The αIIbβ3-specific peptide inhibitor, Ro43-5054 (24.Alig L. Edenhofer A. Hadvary P. Hurzeler M. Knopp D. Muller M. Steiner B. Trzeciak A. Weller T. J. Med. Chem. 1992; 35: 4393-4407Crossref PubMed Scopus (202) Google Scholar), was a generous gift from B. Steiner (Hoffmann-La Roche, Basel, Switzerland). cDNA encoding the expressed integrin cytoplasmic domains joined to 4 heptad repeats (Fig. 1) were cloned into the modified pET-15 vector as described previously (21.Pfaff M. Liu S. Erle D.J. Ginsberg M.H. J. Biol. Chem. 1998; 273: 6104-6109Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). Point mutations in β1D and β7 (Fig. 1) were performed utilizing the Quickchange kit (Stratagene). Recombinant expression in BL21 (DE3)pLysS cells (Novagen) and purification of the recombinant products were made in accordance with the manufacturers instructions (Novagen), with an additional final purification step on a reverse phase C18 high performance liquid chromatography column (Vydac). Polypeptide masses were confirmed by electrospray ionization mass spectrometry on an API-III quadrupole spectrometer (Sciex, Toronto, Ontario, Canada) and varied by less than 4 daltons from those predicted by the desired sequence. Tac-α5 and Tac-β1A DNA in modified CMV-IL2R expression vectors (25.LaFlamme S.E. Thomas L.A. Yamada S.S. Yamada K.M. J. Cell Biol. 1994; 126: 1287-1298Crossref PubMed Scopus (207) Google Scholar) were generously provided by Drs. S. LaFlamme and K. Yamada (National Institutes of Health, Bethesda, MD). Inserts encoding Tac-β1D, Tac-β7, Tac-β1A(Y788A), and Tac-β1A(801X) were subcloned into the modified CMV-IL2R expression vector asHindIII-XhoI fragments. αβpy cells, a Chinese hamster ovary cell line expressing the polyoma large T antigen and a constitutively active recombinant chimeric integrin, αIIbα6Aβ3β1(26.Baker E.K. Tozer E.C. Pfaff M. Shattil S.J. Loftus J.C. Ginsberg M.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1973-1978Crossref PubMed Scopus (67) Google Scholar), were maintained in Dulbecco's modified Eagle's medium (BioWhitaker); supplemented with 10% fetal calf serum (BioWhitaker), 1% non-essential amino acids (Life Technologies, Inc.), 1% glutamine (Sigma), 1% penicillin and streptomycin (Sigma), and 700 μg/ml G418 (Life Technologies, Inc.). Human Jurkat T cell lines were obtained from ATCC and maintained in RPMI1680 (BioWhitaker) supplemented with 10% fetal calf serum, 1% nonessential amino acids, 1% glutamine, and 1% penicillin and streptomycin. The filamin-1-deficient human melanoma cell line M2 and a reconstituted line A7 (27.Cunningham C.C. Gorlin J.B. Kwiatkowski D.J. Hartwig J.H. Janmey P.A. Byers H.R. Stossel T.P. Science. 1992; 255: 325-327Crossref PubMed Scopus (498) Google Scholar) (kindly donated by T. P Stossel) were cultured in Eagle's medium (BioWhitaker), supplemented with 10% fetal calf serum, 1% nonessential amino acids, 1% glutamine, and 1% penicillin and streptomycin. Jurkat cells were washed twice in phosphate-buffered saline and surface-biotinylated using Sulfo-BiotinN-hydroxysuccinimide in phosphate-buffered saline according to the manufacturer's instructions (Pierce). They were then washed twice with Tris-buffered saline and lysed by sonication on ice in buffer A (1 mm Na3VO4, 50 mm NaF, 40 mm sodium pyrophosphate, 10 mm Pipes, 50 mm NaCl, 150 mmsucrose, pH 6.8) containing 1% Triton X-100, 0.5% sodium deoxycholate, 1 mm EDTA, and protease inhibitors (aprotinin, 5 μg/ml leupeptin, and 1 mmphenylmethylsulfonyl fluoride). Platelet lysates were prepared as described previously (21.Pfaff M. Liu S. Erle D.J. Ginsberg M.H. J. Biol. Chem. 1998; 273: 6104-6109Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). Subcellular fractionation of Jurkat cells was performed after surface biotinylation. The cells were washed three times in Hepes-saline (200 mm Hepes, 12 mmCaCl2·2H2O, 16 mmMgSO4, pH 7.3–7.4), suspended in 20 mm Hepes, and homogenized with a Dounce homogenizer. An equal quantity of buffer B (20 mm Hepes, 0.5 m sucrose, 10 mm MgCl2, 0.1 m KCl, 2 mm CaCl2·H2O with protease inhibitors) was added to the homogenate, and the mixture was centrifuged at 500 × g at 4 °C for 15 min. The supernatant was collected and centrifuged at 100 000 ×g for 30 min in a Beckman model L7-65 centrifuge. The cytoplasmic fraction (supernatant) was removed and the membrane fraction (pellet) washed in a 1:1 mixture of 20 mm Hepes and buffer B. The membrane fraction was resuspended in buffer A, 1 mm EDTA, and protease inhibitors and centrifuged at 30,000 × g for 20 min. Recombinant proteins were expressed in BL21(DE3)pLysS cells (Novagen) and bound to His-bind resin (Novagen) through their N-terminal His tag in a ratio of 1 ml of beads/liter of culture. Coated beads were washed with PN (20 mm Pipes, 50 mm NaCl, pH 6.8) and stored at 4 °C in an equal volume of PN containing 0.1% NaN3. Beads were added to cell lysates diluted in buffer A, (0.05% Triton X-100, 3 mm MgCl2, and protease inhibitors) and incubated overnight at 4 °C and then washed five times with buffer A. 100 μl of SDS-sample buffer was added to the beads and the mixture was heated at 100 °C for 5 min. After 10,000 rpm centrifugation in a microcentrifuge, the supernatant was fractionated by SDS-PAGE and analyzed by Western blotting. In some experiments, proteins were eluted off the beads with 100 μl of elution buffer (1 mimidazole, 500 mm NaCl, 20 mm Tris-HCl, pH 7.9) and 1 ml of immunoprecipitation buffer (20 mm Tris-HCl, 150 mm NaCl, 10 mm benzamidine HCl, 1% Triton X-100, 0.05% Tween 20, and protease inhibitors) was then added. The eluted proteins were immunoprecipitated overnight at 4 °C with an 4F2 antibody pre-bound to protein A-Sepharose beads (Amersham Pharmacia Biotech). The following day, the beads were washed three times with the immunoprecipitation buffer and heated in reducing sample buffer for SDS-PAGE under reducing conditions. Samples were separated on 4–20% SDS-polyacrylamide gels (Novex) and transferred to nitrocellulose membranes. Membranes were blocked with Tris-buffered saline, 5% nonfat milk powder and stained with streptavidin-peroxidase or with specific antibodies and appropriate peroxidase conjugates. Bound peroxidase was detected with an enhanced chemiluminescence kit (Amersham Pharmacia Biotech). Equal loading of Ni2+ beads with recombinant proteins were verified by Coomassie Blue staining of SDS-PAGE profiles of SDS eluted proteins. Analytical two-color flow cytometry was performed as described previously (9.O'Toole T.E. Katagiri Y. Faull R.J. Peter K. Tamura R.N. Quaranta V. Loftus J.C. Shattil S.J. Ginsberg M.H. J. Cell Biol. 1994; 124: 1047-1059Crossref PubMed Scopus (581) Google Scholar). PAC1 binding was assessed in a subset of transiently transfected αβpy cells (cells positive for co-transfected Tac-α5 as measured by 7G7B6 binding). Integrin activation was quantified as an activation index (AI) defined as (F−Fo)/(F LIBS6 −Fo), in which F is the median fluorescence intensity of PAC1 binding, Fo is the median fluorescence intensity of PAC1 binding in the presence of competitive inhibitor (Ro43-5054, 1 μm), andF LIBS6 is the maximal median fluorescence intensity of PAC1 binding in the presence of the integrin activating antibody anti-LIBS6 (2 μm). Percentage of reversal is calculated as (AI (βx + CD98) −AI βx)/(AI α5− AI βx).AI βx is the activation index of cells transfected with Tac-βx chimeras,AI (βx + CD98) is the AIof cells co-transfected with CD98 and Tac βxchimeras, and AI α5 is the AI of cells transfected with Tac-α5. The x of βx can have values of 1A, 1D, and 7 for the Tac-β1A, Tac-β1D, and Tac-β7chimeras, respectively. CD98 can block reduced integrin affinity caused by overexpression of free β1A cytoplasmic domains, suggesting a physical interaction between β1A and CD98 (10.Fenczik C.A. Sethi T. Ramos J.W. Hughes P.E. Ginsberg M.H. Nature. 1997; 370: 81-85Crossref Scopus (260) Google Scholar). To assess this potential interaction, we examined the binding of solubilized membrane proteins to the β1A cytoplasmic domain. For affinity matrices, we used model proteins in which the integrin cytoplasmic domain was joined to four heptad repeats (21.Pfaff M. Liu S. Erle D.J. Ginsberg M.H. J. Biol. Chem. 1998; 273: 6104-6109Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). The repeats form parallel coiled-coil dimers so that the tails are dimerized and parallel. When a Jurkat cell lysate was exposed to such an affinity matrix, a cell surface polypeptide of 88 kDa bound to the β1A but not to the αIIb tail (Fig.2 A). This polypeptide was immunoprecipitated by the anti-CD98 antibody, 4F2 (Fig. 2 B). Based on its mass and reactivity with anti-CD98 antibody, the β1A tail binding polypeptide was identified as CD98. To assess the specificity of CD98 binding to β integrin tails, affinity chromatography was performed with β1D, β3, and β7 cytoplasmic domains. CD98 did not bind to β7 and binding to β1D was weak and variable (Fig. 3 A). In contrast, talin and filamin (Fig. 3 A) bound strongly to β1D and β7 tails, respectively, as reported (21.Pfaff M. Liu S. Erle D.J. Ginsberg M.H. J. Biol. Chem. 1998; 273: 6104-6109Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). CD98 also bound to β3, and binding was not altered by the presence of the αIIb cytoplasmic domain (Fig.3 B). Thus, CD98 binding to integrin tails is integrin class- and splice-variant-specific. CD98 binds well to the β1A integrin cytoplasmic domain but not to those of β1D or β7. The binding assays were performed using talin- and filamin-1-containing cell extracts. Thus, these CD98 binding differences could be due to competition for CD98 binding by filamin-1 or talin, which bind preferentially to β7 or β1D, respectively (21.Pfaff M. Liu S. Erle D.J. Ginsberg M.H. J. Biol. Chem. 1998; 273: 6104-6109Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). To test this possibility, we used filamin-1-deficient human melanoma cells (M2) and reconstituted cells (A7) (27.Cunningham C.C. Gorlin J.B. Kwiatkowski D.J. Hartwig J.H. Janmey P.A. Byers H.R. Stossel T.P. Science. 1992; 255: 325-327Crossref PubMed Scopus (498) Google Scholar) to examine the role of filamin-1 in CD98 binding. CD98 bound to the β1A tail, but not β7, when lysates of M2 cells were used (Fig.4 A), showing that filamin-1 is not required for CD98 binding to β1A. CD98 binding to β7 was not observed in the filamin-1 null (M2) cells. Consequently, competition with filamin-1 does not account for the failure of β7 to bind CD98. To examine the role of talin, we used cell membrane preparations with a greatly reduced talin content (Fig.5 A). CD98 extracted from these membranes bound β1A but not β1D cytoplasmic domains (Fig. 5 B). Thus, talin does not prevent CD98 binding to β1D, nor is it required for CD98 binding to β1A. The Y788A mutation of β1A (Fig. 1) disrupts filamin (Fig.4 B) and talin (Fig. 5 C) binding (21.Pfaff M. Liu S. Erle D.J. Ginsberg M.H. J. Biol. Chem. 1998; 273: 6104-6109Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). Similar Tyr to Ala mutations in β7 and β1D tails, corresponding to the Y788A mutation in β1A (Fig. 1), also disrupted filamin (Fig. 4 B) and talin (Fig. 5 C) binding. CD98 binding to β integrin tails was not affected by Tyr to Ala mutations (Figs. 4 B and 5 C). The Tyr to Ala mutation introduced into β1D or β7 did not increase CD98 binding, nor was CD98 binding reduced in the β1A(Y788A) mutant. These results confirm that talin or filamin competition does not account for the lack of CD98 binding to β1D and β7 and that talin or filamin binding is not required for CD98 binding to the β1Acytoplasmic domain. Overexpression of isolated integrin β1A cytoplasmic domains, in the form of a Tac-β1A chimera, results in suppression of integrin activation. Dominant suppression is reversed by overexpression of CD98 (10.Fenczik C.A. Sethi T. Ramos J.W. Hughes P.E. Ginsberg M.H. Nature. 1997; 370: 81-85Crossref Scopus (260) Google Scholar). Tac-β1A, Tac-β1D, and Tac-β7 induced dominant suppression of integrin activation (Fig. 6 A). As noted above (Fig. 3), CD98 bound poorly to β1D and β7 tails, showing that CD98 binding is not required for dominant suppression. However, CD98 was much less effective at reversing the suppression induced by Tac-β1D and Tac-β7 (Fig. 6 B). Thus the capacity of CD98 to rescue suppression correlates with its binding to the suppressive β cytoplasmic domain. As noted above, β1A tails suppress integrin activation and bind CD98. To assess whether CD98 binding alone is sufficient to induce dominant suppression, we first examined CD98 binding to a series of β1A truncation mutants (Fig. 1). CD98 binding was lost when the C-terminal seven residues were deleted (β1AC797X)) but not when the last three amino acids were eliminated (β1A(801X)) (Fig.7 A). Despite maintaining its capacity to bind to CD98, the Tac-β1A(801X) mutant was a poor suppressor of integrin activation (Fig. 7 B), and this was not due to a quantitative reduction in the association of CD98 with β1A(801X) (Fig. 7 C). Furthermore, the β1A(Y788A) mutant, which also bound CD98 (Figs. 4 and 5), failed to suppress integrin activation (Fig. 7 B). Consequently, integrin β cytoplasmic domain binding to CD98 is not sufficient to induce dominant suppression. CD98 is implicated in several cellular functions, including amino acid transport, cell fusion events, and integrin activation (12.Diaz Jr., L.A. Fox D.A. J. Biol. Reg. Homeostat. Agents. 1998; 12: 25-32PubMed Google Scholar). We previously found that CD98 reverses dominant suppression of integrin function (10.Fenczik C.A. Sethi T. Ramos J.W. Hughes P.E. Ginsberg M.H. Nature. 1997; 370: 81-85Crossref Scopus (260) Google Scholar). We now report that: 1) CD98 associates with the β1A integrin cytoplasmic domain; 2) CD98 interacts differentially with β cytoplasmic tails in a class- and splice variant-specific manner, which is independent of the capacity of the tails to bind the cytoskeletal proteins talin and filamin; 3) CD98's capacity to associate with integrin tails correlates with its ability to overcome dominant suppression of integrin activation; 4) CD98 association with integrin tails is neither necessary nor sufficient for dominant suppression of integrin activation. Thus, the association of CD98 with integrin cytoplasmic domains may regulate the function and localization of these membrane proteins. CD98 physically associates with β1A integrin cytoplasmic domains. This association was observed utilizing model protein mimics of dimerized integrin cytoplasmic tails, and it may account for the physical association of certain β1 integrins with CD98. 2M. Hemler, personal communication. The specificity of the interaction was confirmed by the lack of binding to mimics containing cytoplasmic domains from αIIb or several other β subunits. CD98 was added to the tails in the presence of other cellular proteins, so it remains possible that an intermediary protein is required for this interaction. However, CD98 was the only surface protein observed binding to the β1A tail (Fig. 2). Moreover, we observed CD98 binding in the absence of two known integrin binding proteins, talin and filamin (Figs. 3 and 4). CD98 failed to bind to β1D and β7 cytoplasmic domains, even though these tails bind many of the same polypeptides as β1A (21.Pfaff M. Liu S. Erle D.J. Ginsberg M.H. J. Biol. Chem. 1998; 273: 6104-6109Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). Thus, we conclude that CD98 associates with the β1A tail and that the interaction is potentially direct. CD98 binds to integrin β cytoplasmic domains with unique splice variant and class specificity. CD98 bound well to the β1Atail and the β3 tail. Binding to the β1Dand β7 tails was negligible. The specificity of CD98 binding differs markedly from the specificity of talin and filamin binding, since talin binds preferentially to the β1D tail and filamin to the β7 tail (21.Pfaff M. Liu S. Erle D.J. Ginsberg M.H. J. Biol. Chem. 1998; 273: 6104-6109Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). Moreover, the binding of both cytoskeletal proteins is sensitive to the Tyr substitution with Ala in the first "NPXY" (21.Pfaff M. Liu S. Erle D.J. Ginsberg M.H. J. Biol. Chem. 1998; 273: 6104-6109Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar) in β1A and, as shown here, in β7 and β1D. Strikingly, CD98 binding was insensitive to this mutation. Finally, although the last three residues of β1A were dispensable, the last seven residues were required for binding. Thus, the features of the β tail defined here for CD98 binding identifies a novel structural specificity for integrin β tail function. CD98 binding to β tails correlates with its capacity to complement dominant suppression. CD98 was implicated in integrin activation by its capacity to reverse the suppression of integrin activation caused by an isolated β1A cytoplasmic domain (10.Fenczik C.A. Sethi T. Ramos J.W. Hughes P.E. Ginsberg M.H. Nature. 1997; 370: 81-85Crossref Scopus (260) Google Scholar). In the present work, we found that CD98 binds to the β1A cytoplasmic domain, but fails to bind well to the β7 or β1D cytoplasmic domain. Strikingly, CD98 failed to complement dominant suppression initiated by either β7 or β1D cytoplasmic domains. Consequently, the mechanism of CODS appears to involve CD98 binding to the suppressive β tail. Furthermore, cross-linking of CD98 stimulates integrin α3β1-dependent adhesion in small cell lung cancer cells (10.Fenczik C.A. Sethi T. Ramos J.W. 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Pineda M. Feliubadalo L. Esteves R.A. de Cid R. Sanjurjo P. Zorzano A. Nunes V. Huoponen K. Reinikainen A. Simell O. Savontaus M.L. Aula P. Palacin M. Nat. Genet. 1999; 21: 293-296Crossref PubMed Scopus (240) Google Scholar). CD98 may function to regulate both the expression and localization of its light chains (18.Pfeiffer R. Rossier G. Spindler B. Meier C. Kuhn L. Verrey F. EMBO J. 1999; 18: 49-57Crossref PubMed Scopus (239) Google Scholar). In certain cells CD98 has a basolateral localization (38.Nakamura E. Sato M. Yang H. Miyagawa F. Harasaki M. Tomita K. Matsuoka S. Noma A. Iwai K. Minato M. J. Biol. Chem. 1999; 274: 3009-3016Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). β1A integrins also manifest basolateral polarization in many cells (39.Simon E.E. Liu C.H. Das M. Nigam S. Broekelmann T.J. McDonald J.A. Am. J. Physiol. 1994; 267: F612-F623PubMed Google Scholar, 40.Zambruno G. Marchisio P.C. Marconi A. Vaschieri C. Melchiori A. Giannetti A. De Luca M. 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Thus, the failure of these cytoplasmic domains to bind to CD98 correlates well with their lack of a role in establishing polarity in epithelial or mesenchymal cells. Consequently, the physical association of CD98 with β1Aintegrin cytoplasmic domains may participate in the polarization and regulation of amino acid transporters and to modulate the function of certain integrins. We thank our colleagues for their generosity in providing the reagents listed under "Experimental Procedures." We thank Drs. Thomas Stossel and Sandy Shattil for critical reviews of the manuscript.
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