CD98 and Intracellular Adhesion Molecule I Regulate the Activity of Amino Acid Transporter LAT-2 in Polarized Intestinal Epithelia

We have previously shown that the heterodimer CD98/LAT-2 (LAT-2: amino acid transporter) is expressed in the basolateral membrane of intestinal epithelia and is associated with β1 integrin (Merlin, D., Sitaraman, S., Liu, X., Easterburn, K., Sun, J., Kucharzik, T., Lewis, B., and Madara, J. L. (2001) J. Biol. Chem. 276, 39282–39289). In the present study we examined the interaction of CD98/LAT2 with intracellular adhesion molecule I (ICAM-1) and the potential of such interaction on the activation of intracellular signal in Caco2-BBE cell monolayers. ICAM-1 was found to be expressed to the basolateral domain and to selectively coimmunoprecipitate with CD98/LAT-2 in Caco2-BBE monolayers. Using antibodies as ligands to CD98 and ICAM-1, we demonstrate that the basolateral cross-linking of CD98 and ICAM-1 differentially affects the intrinsic activity of the LAT-2 transporter. Whereas CD98 ligation decreases the Km and Vm of the LAT-2 transporter, ICAM-1 ligation increases Km and Vm of the amino acid transporter LAT-2. In addition, basolateral cross-linking of CD98 or ICAM-1 induces threonine phosphorylation of an ∼160-kDa supramolecular complex that is consistent with CD98/LAT-2-ICAM-1 complex. Together these findings demonstrate that (i) CD98/LAT-2 interacts with ICAM-1 in Caco2-BBE cell monolayers, and (ii) CD98 and ICAM-1 ligands generate intracellular signals that regulate the amino acids transporter (LAT-2) activity. Our data provide a novel mechanism by which events such as adhesion may be integrated by amino acid transport activity resulting from the direct interaction of cell surface molecules such as CD98 and ICAM-1. We have previously shown that the heterodimer CD98/LAT-2 (LAT-2: amino acid transporter) is expressed in the basolateral membrane of intestinal epithelia and is associated with β1 integrin (Merlin, D., Sitaraman, S., Liu, X., Easterburn, K., Sun, J., Kucharzik, T., Lewis, B., and Madara, J. L. (2001) J. Biol. Chem. 276, 39282–39289). In the present study we examined the interaction of CD98/LAT2 with intracellular adhesion molecule I (ICAM-1) and the potential of such interaction on the activation of intracellular signal in Caco2-BBE cell monolayers. ICAM-1 was found to be expressed to the basolateral domain and to selectively coimmunoprecipitate with CD98/LAT-2 in Caco2-BBE monolayers. Using antibodies as ligands to CD98 and ICAM-1, we demonstrate that the basolateral cross-linking of CD98 and ICAM-1 differentially affects the intrinsic activity of the LAT-2 transporter. Whereas CD98 ligation decreases the Km and Vm of the LAT-2 transporter, ICAM-1 ligation increases Km and Vm of the amino acid transporter LAT-2. In addition, basolateral cross-linking of CD98 or ICAM-1 induces threonine phosphorylation of an ∼160-kDa supramolecular complex that is consistent with CD98/LAT-2-ICAM-1 complex. Together these findings demonstrate that (i) CD98/LAT-2 interacts with ICAM-1 in Caco2-BBE cell monolayers, and (ii) CD98 and ICAM-1 ligands generate intracellular signals that regulate the amino acids transporter (LAT-2) activity. Our data provide a novel mechanism by which events such as adhesion may be integrated by amino acid transport activity resulting from the direct interaction of cell surface molecules such as CD98 and ICAM-1. Glycoprotein CD98 is a cell surface heterodimer formed by the covalent linkage of CD98 heavy chain with several different light chains to form amino acid transporters (1Verrey F. Meier C. Rossier G. Kuhn L.C. Pflug. Arch. 2000; 440: 503-512Google Scholar). Recently it has been demonstrated that the heterodimer CD98/LAT-2 is found only in tissues containing epithelial barriers (2Halestrap A.P. Price N.T. Biochem. J. 1999; 343: 281-299Google Scholar, 3Merlin D. Sitaraman S. Liu X. Eastburn K. Sun J. Kucharzik T. Lewis B. Madara J.L. J. Biol. Chem. 2001; 276: 39282-39289Google Scholar). The amino acid transporter LAT-2, a protein of 535 amino acids, is highly expressed in polarized epithelia (4Pineda M. Fernandez E. Torrents D. Estevez R. Lopez C. Camps M. Lloberas A. Palacin M. J. Biol. Chem. 1999; 274: 19738-19744Google Scholar, 5Rossier G. Meir C. Bauch C. Summa V. Sordat B. Verrey F. Kuhn C. J. Biol. Chem. 1999; 274: 34948-34954Google Scholar). In absorptive epithelia of the small intestine and in the small intestine-like cell line Caco2-BBE monolayers, the heterodimer CD98/LAT-2 is polarized to the basolateral side (3Merlin D. Sitaraman S. Liu X. Eastburn K. Sun J. Kucharzik T. Lewis B. Madara J.L. J. Biol. Chem. 2001; 276: 39282-39289Google Scholar). There is a growing literature implicating CD98 in integrin function (6Fenczik C. Sethi T. Ramos J.W. Hughes P.E. Ginsberg M.H. Nature. 1997; 390: 81-85Google Scholar, 7Zent R. Fenczik C.A. Calderwood. D.A. Liu S. Dellos M. Ginsberg M.H. J. Biol. Chem. 2000; 275: 5059-5064Google Scholar, 8Fenczik C.A. Zent R. Dellos M. Calderwood D.A. Satriano J. Kelly C. Ginsberg M.H. J. Biol. Chem. 2001; 276: 8746-8752Google Scholar). Recently we have demonstrated (3Merlin D. Sitaraman S. Liu X. Eastburn K. Sun J. Kucharzik T. Lewis B. Madara J.L. J. Biol. Chem. 2001; 276: 39282-39289Google Scholar) that β1 integrins, which are also polarized basolaterally in intestinal epithelial cells, associate with CD98/LAT-2. We found that CD98 not only influences β1 integrin distribution but also affects the cell shape and cytoskeletal order, features known to depend on β1 integrin function (14Mooseker M.S. Annu. Rev. Biol. 1985; 209: 209-241Google Scholar). Interestingly, CD98 interacts specifically with β1 integrins but not with the muscle-specific splice variant β1D or the leukocyte-specific β7 integrin (6Fenczik C. Sethi T. Ramos J.W. Hughes P.E. Ginsberg M.H. Nature. 1997; 390: 81-85Google Scholar). The basolateral location of CD98 suggests that this protein could be involved in some form of cell signaling where binding of ligand to the extracellular loop of CD98 results in an alteration in cellular function via the regulation of the amino acid transporter (LAT-2 in the intestine) and β1 integrin functions (9Rintoul R.C. Buttery R.C. Mackinnon A.C. Wong W.S. Mosher D. Haslett C. Sethi T. Mol. Biol. Cell. 2002; 13: 2841-2852Google Scholar). At present, possible ligands for CD98 remain to be determined, but it was reported that galectin-3, a 26-kDa β-galactosidase binding protein of the galectin family (10Dong S. Hughes R.C. FEBS Lett. 1996; 395: 165-169Google Scholar, 11Deves R. Boyd C.A.R. J. Membr. Biol. 1999; 173: 165-177Google Scholar), could bind to CD98 on T cells. It is possible that CD98 activation could regulate the activity of the amino acid transporter LAT-2. Because epithelial cells rest on the extracellular matrix (ECM), it is logical to expect specific interactions between basolateral “receptors” such as CD98 or ICAM-11 1The abbreviation used is: ICAM, intracellular adhesion molecule. 1The abbreviation used is: ICAM, intracellular adhesion molecule. and ECM. Indeed, upon binding to ECM ligands (outside), integrins deliver signals that control cell proliferation, gene induction, differentiation, and proliferation. The basolateral location of the heterodimer CD98/LAT-2 suggests that CD98 may interact with other adhesion molecules. Among adhesion proteins, the intercellular adhesion molecule ICAM-1 has been shown to be expressed in inflamed epithelial cells (12Kaiserlian D. Rigal D. Abello J. Revillard J.P. Eur. J. Immunol. 1991; 21: 2415-2421Google Scholar, 13Etienne-Manneville S. Manneville J.B. Adamson P. Wilbourn B. Greenwood J. Couraud P.O. J. Immunol. 2000; 165: 3375-3383Google Scholar). ICAM-1 is known to be the receptor to the heterodimer of CD11a, and CD18 (β2 integrin) is expressed in leukocytes. It is conceivable that in the intestinal epithelia, ICAM-1 may be part of a multicomponent web that includes CD98/LAT2 and integrin β1. The multicomponent web could orchestrate epithelial cell function such as LAT-2-mediated amino acid transport activity. We hypothesized that the amino acid transporter LAT-2 may be regulated by adhesion molecules such as ICAM-1 and CD98 in epithelial cells. In the present study we investigate (i) the expression of ICAM-1 in intestinal epithelial cell line Caco2-BBE, (ii) the possibility that ICAM-1 interacts with CD98/LAT-2, and (iii) the effects of cross-linking ICAM-1 and CD98 on LAT-2-mediated amino acid transport activity. Cell Culture—Caco2-BBE (14Mooseker M.S. Annu. Rev. Biol. 1985; 209: 209-241Google Scholar, 15Merlin D. Steel A. Gewirtz A.T. Si-Tahar M. Hediger M.A. Madara J.L J. Clin. Invest. 1998; 102: 2011-2018Google Scholar, 16Buyse M. Sitaraman S.V Liu X. Bado A. Merlin D. J. Biol. Chem. 2002; 277: 28182-28190Google Scholar) cells were grown as confluent monolayers in a 1:1 mixture of Dulbecco's Vogt-modified Eagle's media and Ham's F-12 medium supplemented with 15 mm HEPES buffer (pH 7.5), 14 mm NaHCO3, and 10% new-born calf serum. Monolayers were subcultured every 7 days by trypsinization with 0.1% trypsin and 0.9 mm EDTA in Ca2+/Mg2+-free phosphate-buffered saline. Cell surface biotinylation and cross-linking studies were carried out with confluent monolayers plated on collagen-coated permeable supports (area 0.3 cm2, pore size 0.4 μm) and examined 10 days post-plating. RT-PCR of ICAM-1 Expression—The expression of ICAM-1 in Caco2-BBE cells was determined using an RT-PCR method with oligonucleotide primers specific for ICAM-1. Total RNA was isolated from confluent Caco2-BBE cells cultured on plastic supports (area: 9.4 cm2) for 14 days with a Micro Fast Track™ kit (Invitrogen). The yield of RNA from each preparation was determined by ultraviolet spectrophotometry. 1 μg of total RNA was primed with oligo(dT) and reverse-transcribed with avian myeloblastosis virus-reverse transcriptase (cDNA cycle kit; Invitrogen). A dilution of the reverse transcription reaction was used as a template for amplification by PCR. After an initial denaturation at 94 °C for 5 min, PCR of the samples was carried out for 35 cycles under the following conditions: denaturation at 94 °C for 1 min, annealing at 55 °C for 2 min, and extension at 72 °C for 3 min. This was followed by a final extension step at 72 °C for 7 min. For detection of ICAM-1, the primers specific for ICAM-1 corresponded to nucleotide positions 53–70 (sense: 5′-TCGCTATGGCTCCCAGCA-3′) and 1662–1645 (antisense: 5′-ATAGGTTCAGGGAGGCG-3′) of the cDNA (GenBank™ GI:220-51567) were used that yield a product of 1,592 bp. PCR products were separated by electrophoresis on 1% agarose gels, visualized by ethidium bromide, and verified by DNA sequencing. Cell Surface Biotinylation—Filter-grown cells were rinsed twice with phosphate-buffered saline supplemented with 0.1 mm CaCl2 and 1 mm MgCl2. Basolateral or apical sides of the monolayers were incubated with freshly prepared sulfosuccinimidobiotin (s-NHS-biotine; Pierce) diluted in the same solution (0.5 mg/ml) for 30 min at room temperature. The reaction was quenched with 50 mm NH4Cl, and cells were lysed with a solution of 1% (wv) Triton X-100 in 20 mm Tris, pH 8.0, 50 mm NaCl, 5 mm EDTA, and 0.2% (w/v) bovine serum albumin supplemented with protease inhibitors. The protein solution was diluted with 1 ml of lysis buffer and then incubated with streptavidin-agarose (Pierce) for 24 h at 4 °C to bind biotinylated proteins. The protein solution was then boiled in sample buffer containing 2% SDS, 20% glycerol without β-mercaptoethanol at 100 °C for 5 min. Proteins were separated by SDS-PAGE and transferred overnight at 4 °C to nitrocellulose membranes. The blots were blocked for 1 h with 5% nonfat dry milk in blocking buffer. After washing with blocking buffer, the blots were incubated for 1 h at room temperature with a 1:1000 dilution of goat anti-CD98 (goat anti-CD98 from Santa Cruz Biotechnology), sheep anti-ICAM-1 (sheep anti-ICAM-1 from R&D Systems). They were further incubated for 30 min at room temperature with the appropriate horseradish peroxidase-conjugated antibody diluted 1:1000 and probed using ECL (Amersham Biosciences). Immunoprecipitation—Cells were washed with ice-cold phosphate-buffered saline and then lysed on ice in 1 ml of lysis buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1% Nonidet P40) containing 1 mg/ml aprotinin, 1 mm pepstatin, 2 mm serine proteases. The lysates were centrifuged at 10,000 × g for 15 min at 4 °C, and the resulting supernatants were subjected to immunoprecipitation and immunoblot analysis. For immunoprecipitation, the supernatants were incubated overnight at 4 °C with protein G-agarose suspension (50 μlof beads). The beads were pelleted by centrifugation at 12,000 × g for 20 s in a microfuge. Supernatants were transferred to fresh tubes, and the appropriate amount of specific antibody (1:1000 dilution of goat anti-CD98 (RDI), sheep anti-ICAM-1) was added and gently rocked for4hat 4 °C. Subsequently, 50 μl of protein G suspension was added to the mixture and incubated overnight at 4 °C. The complexes were collected by centrifugation at 12,000 × g for 20 s by microfuge. The beads were washed two times for 20 min with buffer 1 (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Nonidet P40), buffer 2 (50 mm Tris-HCl, pH 7.5, 500 mm NaCl, 1 mm EDTA, 0.1% Nonidet P40), and buffer 3 (10 mm Tris-HCl, pH 7.5, 0.1% Nonidet P40). 50 μl of gel loading buffer (1% (wv) Triton X-100 in 20 mm Tris, pH 8.0, 50 mm NaCl, 5 mm EDTA, 2% SDS, and 0.2% (w/v) bovine serum albumin supplemented with protease inhibitors 2% SDS) was added to the agarose pellet and boiled 5 min at 100 °C, subjected to SDS-PAGE, and transferred overnight at 4 °C to nitrocellulose membranes. The blots were blocked for 1 h with 5% nonfat dry milk in blocking buffer. After washing with blocking buffer, the blots were incubated for 1 h at room temperature with 1:1000 dilution of goat anti-CD98 (RDI), sheep anti-ICAM-1. They were further incubated for 30 min at room temperature with the appropriate horseradish peroxidase-conjugated antibody diluted 1:1000 and probed using ECL (Amersham Biosciences). Cross-linking CD98 and ICAM-1—Caco2-BBE cells were grown to confluence on permeable supports (area: 0.6 cm2; pore size: 0.4 μm). Before the experiment, Caco2-BBE monolayers were washed three times with buffer containing 100 mm NaCl, 2 mm KCl, 1 mm MgCl2, 1 mm CaCl2, and 10 mm HEPES (pH 7.4) at room temperature. Caco2-BBE monolayers were incubated apically with 200 μl of incubation medium and basolaterally with 500 μl of incubation medium containing 5, 10, or 20 μg/ml CD98 or ICAM-1 antibody with the appropriate secondary antibody (1:1000 dilution) or with only the secondary antibody (1:1000 dilution) for 1 h at 37 °C. Amino Acid Transport Uptake Assay—We investigated the effect of cross-linking CD98 or ICAM-1 on amino acid (leucine) transport across basolateral membranes of Caco2-BBE monolayers. Cells grown on filters were washed twice with a buffer containing 100 mm NaCl, 2 mm KCl, 1 mm MgCl2, 1 mm CaCl2, and 10 mm HEPES, pH 7.4. After CD98 or ICAM-1 cross-linking (see Caco2-BBE monolayers were transferred in in buffer containing at different 100 mm 2 mm KCl, 1 mm MgCl2, 1 mm CaCl2, 10 mm HEPES, pH to the basolateral and 100 mm 2 mm KCl, 1 mm MgCl2, 1 mm CaCl2, 10 mm HEPES, pH to the apical were for 2 min at 37 washing each in buffer solution at 4 °C the The of each was determined by Amino Acid Assay—We investigated the effect of cross-linking CD98 or ICAM-1 on amino acid (leucine) across basolateral membranes of Caco2-BBE monolayers. Cells grown on filters were washed twice with a buffer containing 100 mm NaCl, 2 mm KCl, 1 mm MgCl2, 1 mm CaCl2, and 10 mm HEPES, pH 7.4. After CD98 or ICAM-1 cross-linking (see Caco2-BBE monolayers were incubated basolaterally with 500 μl of incubation medium 100 mm 2 mm KCl, 1 mm MgCl2, 1 mm CaCl2, 10 mm HEPES, pH containing 2 and apically with μl of buffer medium for 1 h at 37 °C. At the of the incubation the monolayers were washed times on sides with incubation The containing the monolayers was to containing 500 μl of and μl of medium was added to the basolateral 500 μl of the incubation medium was from the basolateral 2 min at 37 °C. was by The of sample was determined by of ICAM-1 in Caco2-BBE of ICAM-1 was by RT-PCR in Caco2-BBE cells. were used that that Caco2-BBE expressed the The PCR product was from 1% agarose gel using a DNA kit and the to the of ICAM-1 ICAM-1 was at the protein by in Caco2-BBE cells Using the ICAM-1 Caco2-BBE cell lysates a to that was with the from the ICAM-1 ICAM-1 at and Basolateral of Caco2-BBE membrane of the ICAM-1 was in confluent Caco2-BBE monolayers. We examined the membrane expression of ICAM-1 by surface membrane cell surface membrane were by biotinylation of each membrane domain and using the ICAM-1 at that was expressed on apical and basolateral membranes in Caco2-BBE monolayers The ICAM-1 expression in the basolateral membrane of Caco2-BBE monolayers suggests that ICAM-1 could interact with other basolaterally expressed such as CD98 using the anti-CD98 at under that is expressed on the basolateral We have previously shown that this the covalent between CD98 and LAT-2 (14Mooseker M.S. Annu. Rev. Biol. 1985; 209: 209-241Google Scholar). ICAM-1 with CD98 investigate ICAM-1 with immunoprecipitation studies were Caco2-BBE cell lysates were subjected to immunoprecipitation for CD98 1 and or ICAM-1 2 and ICAM-1 and CD98 were by CD98 1 and or ICAM-1 3 and shown in 2 1 and and were found ICAM-1 and CD98 were by the CD98 The ∼160-kDa is to other and the the CD98 In addition, ICAM-1 and CD98 two at and by ICAM-1 antibody 3 and The ∼160-kDa is to be the of the between the CD98/LAT-2 heterodimer and ICAM-1 the CD98 or the ICAM-1 this probed by the the or the not The ICAM-1 the ICAM-1 the same by ICAM-1 antibody these results that ICAM-1 interacts with the CD98 and the CD98 heterodimer in Caco2-BBE cells. Cross-linking CD98 or ICAM-1 Uptake across Basolateral in Caco2-BBE light chain of CD98 has been demonstrated to function as an amino acid transporter. We examined cross-linking CD98 with the anti-CD98 or cross-linking with anti-ICAM-1 (see and effect on amino acid transport using an that cells. Uptake were in buffer at pH the for LAT-2-mediated amino acid CD98 or ICAM-1 ligation affects by the intrinsic activity of the amino acid transporter, the effect of basolateral CD98 or ICAM-1 ligation on the of was of the data that CD98 ligation the and the Km of the transporter In as shown in 4 ICAM-1 ligation the and the Km of the transporter Together these data demonstrate that CD98 and ICAM-1 ligation affects the intrinsic activity of the amino acid transporter in Caco2-BBE effects of cross-linking ICAM-1 on LAT-2-mediated transport across basolateral membrane in Caco2-BBE monolayers. The of for 2 min was determined in the solution at pH as under and The for transport was by cross-linking ICAM-1. The were to the 20 μg/ml of the ICAM-1 antibody with the appropriate secondary antibody (1:1000 dilution) or only the secondary antibody (1:1000 dilution) were added to the basolateral of Caco2-BBE monolayers for 1 h at 37 °C as under and data are the of three in Amino Acid across Basolateral of Caco2-BBE by CD98 and ICAM-1 of across basolateral membranes of the Caco2-BBE monolayers with was in the of extracellular in the extracellular medium (see and the extracellular medium not the not that is of the of LAT-2 amino acid transporter. 5, A and that cross-linking basolateral CD98 and cross-linking basolateral ICAM-1 in Caco2-BBE monolayers a of with the membranes of Caco2-BBE were incubated with only the appropriate secondary antibody (1:1000 The of to at 5 μg/ml CD98 or ICAM-1 antibody At 10 μg/ml CD98 antibody or ICAM-1 antibody the across basolateral membranes of Caco2-BBE monolayers was and and a at 20 μg/ml CD98 antibody and a at 20 μg/ml ICAM-1 antibody results demonstrate that CD98 and ICAM-1 may be for the LAT-2-mediated amino acid across basolateral membranes of Caco2-BBE monolayers. Basolateral CD98 and ICAM-1 of of a in Caco2-BBE CD98 and ICAM-1 in Caco2-BBE monolayers were °C as under and Caco2-BBE cell lysates were subjected to immunoprecipitation for CD98 or ICAM-1. ICAM-1 and CD98 were by The CD98 and ICAM-1 an ∼160-kDa that was by basolateral CD98 ligation control 2 and In addition, ICAM-1 the same ICAM-1 ligation ICAM-1 In CD98 not the ICAM-1 ligation ICAM-1 that ICAM-1 ligation a protein phosphorylation CD98 The protein at is to be CD98 or of the molecules to CD98 that at We have demonstrated that ICAM-1 is expressed on basolateral and apical membranes of Caco2-BBE monolayers. results are in with studies that the Caco2-BBE cell line the expression of ICAM-1 with other cell such as or (12Kaiserlian D. Rigal D. Abello J. Revillard J.P. Eur. J. Immunol. 1991; 21: 2415-2421Google Scholar). The expression of ICAM-1 in the basolateral of Caco2-BBE monolayers these cells an appropriate cellular for study of the interaction of ICAM-1 with other basolaterally expressed proteins. ICAM-1 is a cell adhesion molecule that an in matrix interactions and cellular interactions such as the A. A. T. K. J. 2002; Scholar). In the present we have demonstrated that ICAM-1 with the heterodimer This suggests that to a such as adhesion ICAM-1 not only as an receptor but also as a of supramolecular complexes at the membrane in epithelial cells. In addition, the of the heterodimer CD98/LAT-2 and ICAM-1 that may be cellular regulation by this supramolecular complex. The complex may via the amino acid transporter LAT-2, to regulate of cell For regulation of intracellular amino acid by LAT-2 transport activity may the which to phosphorylation of an intracellular In addition, it has been demonstrated that the intracellular amino acid several a of D.A. Biochem. J. 2000; Scholar). it has been shown that amino acid the activity of the signaling which to the activation of protein on protein K. K. C. J. J. Biol. Chem. 1998; Scholar, J. Biol. Chem. 1999; 274: Scholar). In the present we that cross-linking CD98 or ICAM-1, which ligands for these proteins, LAT-2-mediated transport activity. Interestingly, cross-linking CD98 and ICAM-1 differentially affects the LAT-2 transport activity. CD98 to LAT-2 is basolaterally expressed in intestinal epithelia and in Caco2-BBE (3Merlin D. Sitaraman S. Liu X. Eastburn K. Sun J. Kucharzik T. Lewis B. Madara J.L. J. Biol. Chem. 2001; 276: 39282-39289Google the resulting is the for a transporter for amino The extracellular domain of CD98 is for of LAT-2, and that extracellular domain to the membrane (1Verrey F. Meier C. Rossier G. Kuhn L.C. Pflug. Arch. 2000; 440: 503-512Google Scholar). The of regulation of amino acid transport by and and the possible between remain In the present study we that cross-linking CD98 affects the intrinsic activity of the LAT-2 transporter by the and the of LAT-2-mediated of In addition, we demonstrate that cross-linking CD98 the it be of to the transport of LAT-2 for the amino acids to this amino transporter an amino acid transport Interestingly, we demonstrate that cross-linking ICAM-1 decreases the and increases the of LAT-2-mediated cross-linking of ICAM-1 increases across the basolateral membranes of We that the transport activity are the of a direct or phosphorylation of the LAT-2 transporter. Cross-linking CD98 or ICAM-1 induces threonine phosphorylation of an ∼160-kDa supramolecular complex that is of Interestingly, LAT-2, and ICAM-1 have threonine phosphorylation cross-linking of CD98 or ICAM-1 may or of the complex of LAT-2, and ICAM-1. Cross-linking CD98 or ICAM-1 may phosphorylation of LAT-2 on threonine and transport activity of LAT-2, or phosphorylation of associated such as ICAM-1 or CD98 may LAT-2 transport activity. In the amino acid transporter LAT-2 is regulated by adhesion molecules such as ICAM-1 and CD98 in epithelial cells. CD98 and ICAM-1 may a in intracellular in amino acid transport activity resulting from CD98 and interaction may of events such as cell