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.

There is a growing literature implicating CD98 in integrin function (6 -8). Recently we have demonstrated (3) that ␤ 1 integrins, which are also polarized basolaterally in intestinal epithelial cells, associate with CD98/LAT-2. We found that CD98 not only influences ␤ 1 integrin distribution but also affects the cell shape and cytoskeletal order, features known to depend on ␤ 1 integrin function (14). Interestingly, CD98 interacts specifically with ␤ 1 integrins but not with the musclespecific splice variant ␤ 1D or the leukocyte-specific ␤ 7 integrin (6). 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 ␤ 1 integrin functions (9). At present, possible ligands for CD98 remain to be determined, but it was reported that galectin-3, a 26-kDa ␤-galactosidase binding protein of the galectin family (10,11), 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 1 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 (12,13). ICAM-1 is known to be the receptor to the heterodimer of CD11a, and CD18 (␤ 2 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 ␤ 1 . 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.

MATERIALS AND METHODS
Cell Culture-Caco2-BBE (14 -16) 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 NaHCO 3 , and 10% new-born calf serum. Monolayers were subcultured every 7 days by trypsinization with 0.1% trypsin and 0.9 mM EDTA in Ca 2ϩ /Mg 2ϩ -free phosphate-buffered saline. Cell surface biotinylation and cross-linking studies were carried out with confluent * This work was supported by National Institutes of Health Grants DK-02831 (to D. M.) and DK-02802 (to S. S.) and a Senior Research Award from the Crohn's and Colitis Foundation of America (to D. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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 cm 2 ) for 14 days with a Micro Fast Track TM kit (Invitrogen). The yield of RNA from each preparation was determined by ultraviolet spectrophotometry. 1 g 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: 5Ј-TCGCTATGGCTCCCAGCA-3Ј) and 1662-1645 (antisense: 5Ј-ATAGGTTCAGGGAGGCG-3Ј) of the cDNA (GenBank TM 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 CaCl 2 and 1 mM MgCl 2 . 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 NH 4 Cl, 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 ␤-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 phosphatebuffered 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 ϫ 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 l of beads). The beads were pelleted by centrifugation at 12,000 ϫ 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 for 4 h at 4°C. Subsequently, 50 l of protein G suspension was added to the mixture and incubated overnight at 4°C. The complexes were collected by centrifugation at 12,000 ϫ 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 l 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).
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 MgCl 2 , 1 mM CaCl 2 , and 10 mM HEPES, pH 7.4. After CD98 or ICAM-1 cross-linking (see above), Caco2-BBE monolayers were transferred in new wells in Na ϩ -free buffer containing [ 14  Filter-grown Caco2-BBE monolayers were subjected to domain-specific biotinylation (Bs, basolateral domain; Ap, apical domain) for 30 min followed by Western blot analysis of total cell lysate from Caco2-BBE monolayers. Total cell protein was subjected to 10% SDS-PAGE followed by transfer to nitrocellulose membrane. The blot was immunostained with a sheep anti-ICAM-1 antibody. D, basolateral domain-specific plasma membrane delivery of CD98/LAT2 in Caco2-BBE monolayers. Filter-grown Caco2-BBE monolayers were subjected to domain-specific biotinylation (Bs, basolateral domain; Ap, apical domain) for 30 min followed by Western blot analysis of total cell lysate from Caco2-BBE monolayers. Total cell protein was subjected to 10% SDS-PAGE followed by transfer to nitrocellulose membrane. The blot was immunostained with a goat anti-CD98 antibody.
MgCl 2 , 1 mM CaCl 2 , 10 mM HEPES, pH 6.2, to the basolateral reservoir and 100 mM choline chloride, 2 mM KCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 10 mM HEPES, pH 7.2 to the apical reservoir. Uptakes were performed for 2 min at 37°C; washing each filter in buffer solution at 4°C stopped the reaction. The radioactivity of each filter was determined by liquid scintillation.
Amino Acid Efflux Assay-We investigated the effect of cross-linking CD98 or ICAM-1 on amino acid (leucine) efflux 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 MgCl 2 , 1 mM CaCl 2 , and 10 mM HEPES, pH 7.4. After CD98 or ICAM-1 cross-linking (see above), Caco2-BBE monolayers were incubated basolaterally with 500 l of incubation medium (Na ϩ free buffer: 100 mM choline chloride, 2 mM KCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 10 mM HEPES, pH 6.2) containing 2 M [ 14 C]leucine and apically with 250 l of unlabeled Na ϩ -free buffer medium for 1 h at 37°C. At the end of the incubation period, the monolayers were rapidly washed four times on both sides with unlabeled incubation medium. The filter containing the monolayers was moved to new wells containing 500 l of unlabeled medium, and 250 l of unlabeled medium was added to the upper reservoir. To measure basolateral leucine effluxes, 500 l of the incubation medium was taken from the basolateral reservoir after 2 min at 37°C. Radioactivity was measured by liquid scintillation counting. The radioactivity of sample was determined by liquid scintillation.

Expression of ICAM-1 in Caco2-BBE Monolayers-Expres-
sion of ICAM-1 mRNA was assessed by RT-PCR in Caco2-BBE cells. Primers were used that yielded ϳ1.6 kb product. Fig. 1A shows that Caco2-BBE expressed the 1.6-kb product. The PCR product was purified from 1% agarose gel using a DNA extraction kit (Qiagen Inc.), and the sequence showed 100% homology sequence to the published coding sequence of ICAM-1 (bases 53-1662). Additionally, ICAM-1 was detected at the protein level by Western blotting in Caco2-BBE cells (Fig. 1B). Using the anti-human ICAM-1 antibody, Caco2-BBE cell lysates displayed a single immunoreactive band corresponding to ϳ90 kDa (Fig. 1B, lane 1) that was comparable with the immunoreactive band from the recombinant ICAM-1 (Fig. 1B, lane 2).

ICAM-1 Is Expressed at Both Apical and Basolateral Membranes of Caco2-BBE Monolayers-
The membrane localization of the human ICAM-1 was assessed in confluent Caco2-BBE monolayers. We examined the plasma membrane expression of ICAM-1 by surface biotinylation. Plasma membrane domainspecific cell surface membrane glycoproteins were labeled by biotinylation of each plasma membrane domain (apical and basolateral). Western blot using the anti-human ICAM-1 displayed one immunoreactive band at ϳ90 kDa that was expressed on both apical and basolateral membranes in Caco2-BBE monolayers (Fig. 1C). The ICAM-1 expression in the basolateral membrane of Caco2-BBE monolayers suggests that ICAM-1 could interact with other basolaterally expressed proteins such as CD98 (Fig. 1D). As described previously, Western blot using the anti-CD98 displayed one immunoreactive band at ϳ130 kDa under non-reducing conditions (Fig. 1D) that is predominantly expressed on the basolateral membrane. We have previously shown that this 130 kDa represents the covalent association between CD98 and LAT-2 (14).

ICAM-1 Associates with CD98 -To investigate whether
ICAM-1 associates with CD98, immunoprecipitation studies were performed. Caco2-BBE cell lysates were subjected to immunoprecipitation for CD98 (Fig. 2, lanes 1 and 3) or ICAM-1 (lanes 2 and 4). ICAM-1 and CD98 immunoprecipitates were detected by either CD98 (lanes 1 and 2) or ICAM-1 (lanes 3 and 4) antibody. As shown in Fig. 2 (lanes 1 and 2) immunoreactive bands (ϳ160 and ϳ85 kDa) were found when ICAM-1 and CD98 immunoprecipitates were detected by the CD98 antibody. The ϳ160-kDa band is likely CD98/LAT2-complexed to other molecules, and the ϳ85 kDa represents the glycosylated CD98 monomer. In addition, ICAM-1 and CD98 immunoprecipitates displayed two immunoreactive bands at ϳ160 and ϳ90 kDa when detected by ICAM-1 antibody (Fig. 2, lanes 3  and 4). The ϳ160-kDa band is likely to be the result of the association between the CD98/LAT-2 heterodimer and ICAM-1 because the CD98 or the ICAM-1 immunoprecipitates displayed this particular band when probed by the anti-CD98, the anti-ICAM-1, or the anti-LAT-2 (data not shown) antibody. The ϳ90 kDa represents ICAM-1 because the recombinant ICAM-1 provided the same molecular band when detected by ICAM-1 antibody (Fig. 2, lane 5). Together, these results suggest that ICAM-1 interacts with the CD98 monomer and the CD98 heterodimer in Caco2-BBE cells.

Cross-linking CD98 or ICAM-1 Regulates Leucine Uptake across Basolateral Membranes in Caco2-BBE Monolayers-
The light chain of CD98 has been demonstrated to function as an L-type amino acid transporter. We examined whether crosslinking CD98 with the human anti-CD98 or cross-linking with anti-ICAM-1 (see "Materials and Methods") had any effect on amino acid transport using an assay that measures tritiated leucine uptake into cells. Uptake experiments were performed in Na ϩ -free buffer at pH 6.2, the optimal conditions for LAT-2-mediated amino acid uptake. To examine whether CD98 or ICAM-1 ligation affects leucine uptake by modifying the intrinsic activity of the amino acid transporter, the effect of basolateral CD98 or ICAM-1 ligation on the kinetics of [ 3 H]leucine uptake was studied. Kinetics analysis of the data (Fig. 3) indicated that CD98 ligation significantly decreased the V max FIG. 2. CD98/LAT-2 co-immunoprecipitate with ICAM-1. Caco2-BBE cell lysates were immunoprecipitated with anti-CD98 (lanes 1 and  3), anti ICAM-1 (lanes 2 and 4). Immunoprecipitates and 1 g of ICAM-1 recombinant protein (lane 5) were subjected to 10% SDSpolyacrylamide-gel electrophoresis followed by transfer to nitrocellulose membrane. The blot was immunostained with anti-CD98 (CD98) or anti-ICAM-1 (ICAM-1) antibody. Together these data demonstrate that CD98 and ICAM-1 ligation differently affects the intrinsic activity of the amino acid transporter in Caco2-BBE monolayers.
Basolateral CD98 and ICAM-1 Ligations Induce Same Pattern of Threonine Phosphorylation of a 160-kDa Protein in Caco2-BBE Monolayers-Basolateral CD98 and ICAM-1 ligations in Caco2-BBE monolayers were performed for 1 h at 37°C as described under "Materials and Methods." Caco2-BBE cell lysates were subjected to immunoprecipitation for CD98 or ICAM-1. ICAM-1 and CD98 immunoprecipitates were detected by anti-phosphothreonine antibody. The CD98 (Fig. 6, lane 1) and ICAM-1 (lane 5) immunoprecipitates display an ϳ160-kDa band that was induced by basolateral CD98 ligation (CD98 control ligation; Fig. 6, lanes 2 and 6). In addition, ICAM-1 (Fig.  6, lane 7) immunoprecipitate displays the same induced 160-kDa band after ICAM-1 ligation (control ICAM-1 ligation; Fig.  6, lane 8). In contrast, CD98 (Fig. 6, lane 3) immunoprecipitate did not display the 160-kDa band after ICAM-1 ligation (control ICAM-1 ligation; Fig. 6, lane 4), suggesting that ICAM-1 ligation induced a less potent protein phosphorylation than CD98 ligation. The detected phosphorylated protein at 160 kDa is likely to be CD98 or one of the molecules complexed to CD98 that immunoprecipitates at ϳ160 kDa. DISCUSSION We have demonstrated that ICAM-1 is constitutively expressed on basolateral and apical membranes of Caco2-BBE monolayers. These results are in agreement with studies showing that the well differentiated Caco2-BBE cell line shows the highest constitutive expression of ICAM-1 when compared with other less differentiated cell lines such as T84 or HT29 (12). The expression of ICAM-1 in the basolateral aspect of Caco2-BBE monolayers makes these cells an appropriate cellular model for study of the interaction of ICAM-1 with other basolaterally expressed proteins.
ICAM-1 is a cell adhesion molecule that plays an important role in cell-cell, cell-extracellular matrix interactions and cellular interactions such as the immune response (17). In the present study, we have demonstrated that ICAM-1 associates with the heterodimer CD98/LAT2. This result suggests that to accomplish a task such as adhesion ICAM-1 works not only as an individual receptor but also as a component of supramolecular complexes at the plasma membrane in epithelial cells. In addition, the association of the heterodimer CD98/LAT-2 and ICAM-1 suggest that there may be significant cellular regulation mediated by this supramolecular complex. The complex may induce signals, via the amino acid transporter LAT-2, to regulate multiple aspects of cell physiology. For example, regulation of intracellular amino acid availability mediated by LAT-2 transport activity may modulate the activity-signaling pathway, which leads to phosphorylation of an intracellular target protein. In addition, it has been demonstrated that the intracellular amino acid supply modulates several important FIG. 4. The effects of cross-linking ICAM-1 on leucine uptake LAT-2-mediated transport across basolateral membrane in Caco2-BBE monolayers. The concentration dependence of Lleucine uptake for 2 min was determined in the Na ϩ -free uptake solution at pH 6.20 as described under "Materials and Methods." The concentration dependence profile for leucine transport was shifted by cross-linking ICAM-1. The values were fitted to the Michaelis-Menten curve. 20 g/ml of the ICAM-1 antibody with the appropriate secondary antibody (1:1000 dilution) (ϩanti-ICAM-1) or only the secondary antibody (1:1000 dilution) (Ϫanti-ICAM-1) were added to the basolateral aspect of Caco2-BBE monolayers for 1 h at 37°C as described under "Materials and Methods." All data are the mean of three independent experiments performed in triplicate Ϯ S.D. regulatory translation factors through a variety of mechanisms (18). Furthermore, it has been shown that amino acid leucine availability regulates the activity of the signaling pathway, which leads to the activation of p70 S6 kinase (the 70-kDa protein kinase acting on ribosomal protein S6) (19,20). In the present study, we report that cross-linking CD98 or ICAM-1, which somehow mimics natural ligands for these proteins, modifies leucine LAT-2-mediated transport activity. Interestingly, cross-linking CD98 and ICAM-1 differentially affects the LAT-2 transport activity.
CD98 disulfide linked to LAT-2 is basolaterally expressed in intestinal epithelia and in Caco2-BBE (3); the resulting dimer is the minimal functional unit for a Na ϩ -independent trans-porter for zwitterionic amino acids. The extracellular domain of CD98 is responsible for recognition of LAT-2, and that extracellular domain ensures proper translocation to the plasma membrane (1). The mode of regulation of amino acid transport by both subunits (CD98 and LAT-2) and the possible interplay between them remain largely unstudied. In the present study we show that cross-linking CD98 affects the intrinsic activity of the LAT-2 transporter by increasing the affinity and reducing the capacity of LAT-2-mediated uptake of leucine. In addition, we demonstrate that cross-linking CD98 regulates the LAT-2dependent leucine efflux. However, it will be of interest to characterize the transport kinetics of LAT-2 for the export amino acids to know whether this amino transporter shows an asymmetric amino acid transport function. Interestingly, we demonstrate that cross-linking ICAM-1 decreases the affinity and increases the capacity of LAT-2-mediated leucine uptake. Furthermore, cross-linking of ICAM-1 increases leucine efflux across the basolateral membranes of Caco2-BBE.
We suggest that the transport activity changes are the result of a direct or indirect phosphorylation of the LAT-2 transporter. Cross-linking CD98 or ICAM-1 induces threonine phosphorylation of an ϳ160-kDa supramolecular complex that is likely composed of CD98/LAT-2-ICAM-1. Interestingly, CD98, LAT-2, and ICAM-1 proteins have predicted threonine phosphorylation sites; thus, cross-linking of CD98 or ICAM-1 may phosphorylate one or more proteins of the complex comprised of CD98, LAT-2, and ICAM-1. Cross-linking CD98 or ICAM-1 may induce phosphorylation of LAT-2 itself on threonine and change transport activity of LAT-2, or phosphorylation of associated proteins such as ICAM-1 or CD98 may influence LAT-2 transport activity.
In conclusion, 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 play a role in delivering intracellular signals. Changes in amino acid transport activity resulting from CD98 and ICAM interaction may ensure integration of events such as cell adhesion. 3 H]leucine for 60 min. Basolateral cross-linking CD98 at 5 g/ml, 10 g/ml, and 20 g/ml (A) or ICAM-1 at 5 g/ml, 10 g/ml, and 20 g/ml (B) in Caco2-BBE monolayers was performed for 1 h as described under "Materials and Methods." As a ligation control, secondary antibody against CD98 or ICAM-1 was incubated for 1 h (CTRL). The efflux of radioactivity was then measured for 2 min in Na ϩ -free solution at pH 6.20. Data correspond to the mean of three independent experiments performed in triplicate Ϯ S.D. p Ͻ 0.05 compared with control cells.