Distinct Domains of CD98hc Regulate Integrins and Amino Acid Transport*

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.

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 ␤ 1A 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.
CD98hc is a widely distributed transmembrane protein that was originally discovered as a T-cell activation antigen (1). CD98hc expression is tightly linked to cell proliferation, and antibodies against CD98hc can inhibit cell growth or induce apoptosis in specific cell types (2,3). A compelling body of evidence implicates CD98hc in the transport of amino acids. CD98hc overexpression stimulates multiple amino acid transport systems including L, y ϩ L, and x c Ϫ (4). Furthermore, mutations in its closest paralogue, D2 (r-BAT), lead to a disorder of cysteine transport (5). Structurally, CD98 is a disulfidebonded heterodimer of a common ϳ80-kDa heavy chain (CD98hc) with one of several ϳ40-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 (6 -8). CD98hc may act to regulate the expression and cellular localization of the amino acid transporting activity of the light chain (6,9). Thus, this widely distributed membrane protein is strongly implicated in amino acid transport.
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 ␤ 1A 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.
DNA Constructs and Recombinant Proteins-cDNAs encoding the expressed integrin cytoplasmic domains joined to four heptad repeats were cloned into the modified pET-15 vector as described previously (21). Recombinant expression in BL21 (DE3) pLysS cells (Novagen) and purification of the recombinant model proteins were performed in accordance with the manufacturer's 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 they varied by less than 4 daltons from those predicted by the desired sequence.
Construction of CD98 Chimeras-HA-NH 2 was constructed by polymerase chain reaction with primers that included a nine-amino acid influenza hemagglutinin (HA)-tag followed by a three-Gly linker, which was placed directly after the initiator Met. The primers used to create HA-COOH added the HA-tag, preceded by a three-Gly linker to the COOH-terminal portion of CD98hc. All of the CD98hc chimeras were made by overlap polymerase chain reaction. ⌬CD98 deletes amino acids 2-77 (all numbering uses the amino acid sequence reported in entry 4F2_human (entry P08195) of the Swiss-Prot data base as of November 2000), which removes the entire cytoplasmic domain of CD98hc, maintaining the initiator methionine as well as the presumptive stop transfer sequence Val-Arg-Thr-Arg. C 69 T 98 E 98 contains amino acids 1-38 of CD69 (Swiss-Prot accession number Q07108) and amino acids 82-529 of CD98hc (Swiss-Prot accession number P08195). C 98 T 69 E 98 contains amino acids amino acids 1-81 and 105-529 of CD98hc and amino acids 39 -64 of CD69. C 98 T 98 E 69 contains amino acids 1-104 of CD98hc and amino acids 65-199 of CD69. C 98 T 69 E 69 contains amino acids 1-81 of CD98hc and amino acids 39 -199 of CD69.
Cell Lysates-24 h after transfection with CD98hc or one of the chimeric cDNAs, ␣␤Py cells were washed twice in phosphate-buffered saline and surface-biotinylated using Sulfo-Biotin N-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 Na 3 VO 4 , 50 mM NaF, 40 mM sodium pyrophosphate, 10 mM Pipes, 50 mM NaCl, 150 mM sucrose, pH 6.8) containing 1% Triton X-100, 0.5% sodium deoxycholate, 1 mM EDTA, and protease inhibitors (aprotinin, 5 g/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride) and clarified by centrifugation.
Affinity Chromatography Experiments-Recombinant proteins were expressed in BL21 (DE3) pLysS cells (Novagen) and bound to His-bind resin (Novagen) through their NH 2 -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% NaN 3 . Beads were added to cell lysates diluted in buffer A (0.05% Triton X-100, 3 mM MgCl 2 , and protease inhibitors), incubated overnight at 4°C, and then washed five times with buffer A. 100 l of SDS-sample buffer was added to the beads, and the mixture was heated at 100°C for 5 min. After centrifugation at 10,000 rpm for 30 min in a microcentrifuge (5417C Eppendorf), the supernatant was fractionated by SDS-PAGE and analyzed by immunoblotting. Chimeras with the extracellular domain of CD69 were analyzed in the absence of dithiothreitol, because the anti-CD69 antibody employed only detects the nonreduced form of CD69. In some experiments, proteins were eluted off the beads with 100 l of elution buffer (1 M imidazole, 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 anti-CD98hc antibody prebound to protein A-Sepharose beads (Amersham Pharmacia Biotech). The following day, the beads were washed three times with the immunoprecipitation buffer and heated in sample buffer for SDS-PAGE containing 1 mM dithiothreitol. Samples were separated on 4 -20% SDS-polyacrylamide gels (Novex) and transferred to nitrocellulose membranes. Membranes were blocked with 5% nonfat milk powder in Tris-buffered saline 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 Ni 2ϩ beads with recombinant proteins were verified by Coomassie Blue staining of SDS-PAGE profiles of SDS-eluted proteins.
Flow Cytometry-Analytical two-color flow cytometry was performed as described previously (24). PAC1 binding was assessed in a subset of transiently transfected ␣␤Py cells (cells positive for cotransfected Tac-␣ 5 or Tac-␤ 1 as measured by 7G7B6 binding). Integrin activation was quantified as an activation index (AI) defined as ( . AI ␤1A is the activation index of cells transfected with Tac-␤ 1A chimeras, AI (␤1A ϩ CD98) is the AI of cells cotransfected with CD98hc and Tac-␤ 1A chimeras, and AI ␣5 is the AI of cells transfected with Tac-␣ 5 .
Amino Acid Transport-3 H-Labeled Ile (77 Ci/mM) was purchased from Amersham Pharmacia Biotech. ␣␤Py cells were transfected with cDNAs encoding CD98hc or one of the chimeric cDNAs in the presence or absence of cDNA encoding hLAT1 using LipofectAMINE 2000 (Life Sciences Technologies). Transport studies were performed on cells that were transfected with 80 -90% efficiency, as judged by staining for CD98 in flow cytometry. Before the transport assays, cells were rinsed with warm Na ϩ -free Hanks' buffered salt solution (HBSS) (136.6 mM ChCl, 5.4 mM KCl, 4.2 mM NaHCO 3 , 2.7 mM Na 2 HPO 4 , 1 mM CaCl 2 , 0.5 mM MgCl 2 , 0.44 mM KH 2 PO 4 , 0.41 mM MgSO 4 , pH 7.8), in which the sodium-containing salts were iso-osmotically replaced with choline, to remove extracellular Na ϩ and amino acids. Cells were equilibrated in warm choline-substituted HBSS for 10 min. The uptake of radiolabeled amino acids (2 Ci of [ 3 H]Ile/ml) at 100 mol/liter in 1 ml of cholinesubstituted HBSS was measured for 20 s at 37°C. Uptake of [ 3 H]Ile was linearly dependent on incubation time up to at least 3 min. Uptake was terminated by washing the cells rapidly four times with 1 ml/well of ice-cold HBSS. The cells were lysed overnight with 1 ml 2 M NaOH. A 0.95-ml aliquot from each well was mixed with scintillation fluid, and radioactivity was quantified in a Beckman LS 6000SC liquid scintillation counter. The remaining 0.1 ml was analyzed for protein content using the BCA protein assay reagent (Pierce).

CD98 Heavy Light Chain Association Is Not Required for the
Interaction of CD98 with Integrins-CD98 is a heterodimer formed by a common heavy chain (CD98hc) disulfide-bonded to one of a number of light chains that mediate amino acid transport. CD98hc has two extracellular cysteines, one of which (Cys 109 ) is involved in covalent association of the heavy and light chains (25). To examine the role of CD98 heavy-light chain association on its interactions with integrins, we first investigated the effect of mutation of these cysteines (Cys 109 and Cys 330 ). The C109S (C1) mutation alone, or in combination with the C330S (Cless) mutation, is reported to reduce the amino acid transport activity of CD98 (7,25,26). However, the capacity of CD98hc to complement dominant suppression was not impaired by mutation of both or either cysteines (Fig. 1A). We confirmed that the mutant lacking cysteines (Cless) failed to form stable disulfide-bonded heterodimers with CD98 light chains (Fig. 1C) and found that it, like wild-type CD98hc, bound to the integrin ␤ 1A cytoplasmic domain (Fig. 1B). Thus, covalent CD98 heterodimer formation is dispensable for the interaction of CD98hc with the integrin ␤ 1A cytoplasmic domain and for CODS.
As an alternative approach to evaluate the role of the CD98 heavy-light chain association, we examined the effect of increased expression of a CD98 light chain (hLAT1) on integrin interactions. When CHO cells were transfected with CD98hc, there was a substantial quantity of free heavy chain (Fig. 2B). Transfection of increasing quantities of a light chain, hLAT1, resulted in an increasing proportion of CD98 heterodimers (Fig. 2B). As expected, formation of increased CD98 heterodimers led to a marked increase in amino acid transport ( Fig. 2A). However, increased heterodimer formation did not detectably alter the ability of CD98hc to bind to ␤ 1A cytoplasmic tails (Fig. 2C). Thus, while the formation of CD98 heterodimers is important for the stimulation of amino acid transport, CD98hc alone is sufficient for binding to the integrin ␤ 1A tail and for CODS.
The NH 2 Terminus of CD98hc Is Intracellular, and the FIG. 1. Stable formation of the CD98 heterodimer is not required for its effects on integrin function. A, effect of cysteine mutations on CODS. ␣␤Py cells were transfected with Tac-␤ 1 and either wild-type CD98hc or CD98hc with serine substitutions at cysteines (Cys 109 , Cys 330 ) involved in covalent heterodimer formation. Twentyfour h after transfection, cells were collected and the Tac-positive subset of cells was analyzed for the ability to bind to the PAC1 antibody. Data are expressed as percentage reversal of Tac-␤ 1 suppression, which is calculated as 100 ϫ (AI Tac-␤1 ϩ CD98 Ϫ AI Tac-␤1 )/(AI Tac-␣5 Ϫ AI Tac-␤1 ). AI is the activation index of cells transfected with Tac-␤ 1 alone or in combination with CD98hc or with Tac-␣ 5 . Cless ϭ CD98hc (C109S,C330S), C1 ϭ CD98hc (C109S), C2 ϭ CD98hc (C130S). B, CD98hc Cys 109 and Cys 130 are not required for binding to the integrin ␤ 1A cytoplasmic domain. ␣␤Py cells were transfected with either CD98hc, the Cless mutant, or vector DNA. After 24 h surface proteins were labeled with Sulfo-Biotin N-hydroxysuccinimide, cell lysates were incubated with beads coated with model proteins containing ␤ 1A or ␣ IIb cytoplasmic tails. The bound proteins were eluted and immunoprecipitated with anti-CD98hc and fractionated by reduced SDS-PAGE. Biotinylated CD98hc was identified by streptavidin-peroxidase-generated chemiluminescence staining of an ϳ80-kDa polypeptide. In addition, the starting cell lysate was immunoprecipitated with either anti-CD98hc antibodies (Lysate) or with a control IgG (IgG). Proteins were fractionated by reduced SDS-PAGE, and biotinylated proteins were detected by streptavidin-peroxidase-generated chemiluminescence. C, mutation of CD98hc cysteines 109 and 130 disrupts covalent heterodimer formation. Biotinylated lysates of surface labeled cells transfected with either CD98hc or Cless CD98hc were immunoprecipitated with anti-CD98hc antibodies as described in B. The lysates were fractionated by SDS-PAGE in the absence or presence (ϩDTT) of reducing agent. Biotinylated polypeptides were identified by streptavidin-peroxidase-generated chemiluminescence.

FIG. 2.
Effect of coexpression of the hLAT1 light chain on amino acid transport, heterodimer formation, and the binding of CD98hc to the ␤ 1A cytoplasmic domain. A, amino acid transport: ␣␤Py cells were transfected with cDNAs encoding CD98hc, hLAT1, CD98hc plus hLAT1 or vector DNA and assayed for the uptake of [ 3 H]isoleucine after 24 h. Ile uptake was measured in a Na ϩ -free solution, and the values are expressed as cpm/mg of protein. B, heterodimer formation: CHO cells were transfected with cDNA encoding CD98hc (4 g) plus increasing amounts of hLAT1 light chain (0, 2, 6 g). Cell surface proteins were biotinylated, and CD98 was immunoprecipitated from cell lysates with an anti-CD98hc antibody. Heterodimers were visualized by running nonreduced SDS-PAGE as described in the legend to Fig. 1C. C, binding to the ␤ 1A cytoplasmic domain: CHO cells were transfected with cDNA encoding CD98hc and increasing amounts of hLAT1. Twenty-four h later cells were surface-biotinylated and lysed. Lysates were mixed with beads coated with ␤ 1A or ␣ IIb tails. Beads were washed, bound, and eluted proteins were immunoprecipitated with anti-CD98hc antibody and fractionated by reduced SDS-PAGE. The biotinylated polypeptides were detected by streptavidin-peroxidase chemiluminescence.
COOH Terminus Is Extracellular-In the foregoing experiments, we found that the interactions of CD98 with integrins could be ascribed to its heavy chain. Consequently, we wished to analyze the structural determinants in the heavy chain responsible for these interactions. CD98hc is predicted to be a type II transmembrane protein, with the COOH terminus extracellular and the NH 2 terminus intracellular. To document the membrane topography of CD98hc, the NH 2 and COOH terminus of CD98hc were separately tagged using a HA-tag. When cells were transfected with the COOH-terminal-tagged cDNA, the epitope tag was readily detected on the cell surface as indicated by a rightward shift in the fluorescence intensity (Fig. 3A). In contrast, the amino-terminal tag was not detected on the cell surface. The presence of the epitope tag on the NH 2 or COOH terminus did not effect the overall surface expression of CD98hc as measured with an anti-CD98hc antibody (Fig.  3A). Furthermore, both the NH 2 -and COOH-terminal tags were present on the expressed protein (Fig. 3B). Consequently, we conclude that the NH 2 terminus of CD98hc is intracellular, and the COOH terminus is extracellular.
The Cytoplasmic and Transmembrane Domains of CD98hc Are Required for Its Interaction with Integrins-Having confirmed the membrane topography of CD98hc, we wished to examine the role of its cytoplasmic and extracellular domains in the integrin interaction. We first deleted the cytoplasmic domain of CD98hc (Fig. 4A). This abolished its ability to complement dominant suppression (Fig. 4B), although it did not block surface expression (data not shown, but see Fig. 5). Deletion of the CD98hc cytoplasmic domain completely abolished its ability to bind to the ␤ 1A cytoplasmic domain (Fig. 4C). As an alternative approach, we exchanged the cytoplasmic domain of CD98hc with another type II transmembrane protein (CD69).
That chimera (C 69 T 98 E 98 ) failed to complement dominant suppression (Fig. 4B) and failed to bind to the ␤ 1A cytoplasmic domain (Fig. 4C). Thus, the cytoplasmic domain of CD98hc is required for its capacity to interact with integrins.
To assess whether the CD98hc cytoplasmic domain was sufficient for this interaction, additional chimeric exchanges were made. A construct containing the extracellular and transmembrane domains of CD69 (C 98 T 69 E 69 ) joined to the cytoplasmic domain of CD98hc failed to bind to the ␤ 1A cytoplasmic domain (Fig. 5). However, addition of the transmembrane domain of CD98hc (C 98 T 98 E 69 ) resulted in binding that was comparable with that observed with full-length CD98hc. To test whether the transmembrane domain of CD98hc was required for binding to integrin tails, a construct was made in which only the transmembrane domain was replaced with that of CD69 and the extracellular and cytoplasmic domains were retained (C 98 T 69 E 98 ). That construct also failed to bind to the ␤ 1A cytoplasmic domain (Fig. 5). Thus, binding of CD98hc to the ␤ 1A cytoplasmic domain requires both its cytoplasmic and transmembrane domains.
Amino Acid Transport Activity and CODS Require Structurally Distinct Regions of CD98hc-The foregoing studies established that both the cytoplasmic and transmembrane domains of CD98hc were required for its capacity to bind to the ␤ 1A cytoplasmic domain. To investigate whether the functional effects of CD98hc correlated with its binding to the ␤ 1A cytoplasmic domain, each of these chimeras (Fig. 6A) was analyzed for its capacity to mediate CODS and to promote Ile transport. In these experiments, the expression of each chimera was verified by flow cytometry, and equivalent expression was observed for each one (data not shown). As previously noted, the substitution of the cytoplasmic domain of CD98hc with that of CD69 abolished CODS (C 69 T 98 E 98 ; Fig. 6B). However, this chimera stimulated Ile transport to comparable levels with wild-type CD98hc (Fig. 6C). Similarly, substitution of the transmembrane domain of CD98hc (C 98 T 69 E 98 ) markedly suppressed effects on integrin function ( Fig. 6B) but had little effect on the ability to stimulate Ile transport (Fig. 6C). Thus, the amino acid transport activity of CD98hc is not sufficient for CODS. Conversely, the substitution of the extracellular domain of CD98hc with that of CD69 (C 98 T 98 E 69 ) preserved effects on integrin function (Fig. 6B) but abolished amino acid transport activity (Fig. 6C). Thus, the extracellular domain of CD98hc is necessary (in the context of another type II transmembrane protein) for its ability to stimulate isoleucine transport. Conversely, the transmembrane and cytoplasmic domains of CD98hc are necessary and sufficient for binding to the ␤ 1A tail and for augmentation of integrin function.

DISCUSSION
CD98hc combines with several different light chains to form a series of heterodimers that are involved in amino acid transport. CD98hc binds to integrin ␤ 1A cytoplasmic domains and blocks the capacity of ␤ 1A 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 heavylight chain association and reduce amino acid transport do not disrupt its binding to ␤ 1A 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 ␤1A tail and for CODS. This chimera failed to stimulate amino acid transport.  Fig. 1. C, binding to ␤ 1A tail: affinity chromatography with ␤ 1A or ␣ IIb tails was performed using lysates of surfacebiotinylated CHO cells that had been transfected with the constructs described in A. Bound proteins that contain the extracellular domain of CD98hc (panels a, b, and c) were eluted from the beads, immunoprecipitated with anti-CD98hc antibody, fractionated by SDS-PAGE, and the biotinylated polypeptides were detected by streptavidin-peroxidase chemiluminescence. Bound proteins that contain the extracellular domain of CD69 (panel d) were fractionated by SDS-PAGE, and CD69 was detected by immunoblot with anti-CD69 antibodies. 3) Replacement of the cytoplasmic or transmembrane domains of CD98hc with those of CD69 blocked the capacity of CD98hc to bind to ␤ 1A 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 Cys 109 and Cys 330 . Cys 109 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 (25). Mutation of Cys 109 and Cys 330 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. (25) 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 disulfidebound heterodimers, albeit at a reduced rate. Moreover, we also found that overexpressed free heavy chains could also bind to the ␤ 1A 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 ␤ 1A 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 ␤ 1A 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 ␤ 1A (Lys 752 -Ile 757 ) of the predicted "cytoplasmic" domain may reside in the membrane (27). 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 (28). 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-␤ 1A 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 ␤ 1A 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 ␤ 1A 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 ϩ LAT1, y ϩ LAT2, xCT, and asc-1) to the plasma membrane (29,30). 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 (13,(15)(16)(17). 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.
Acknowledgments-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.
FIG. 6. The cytoplasmic and transmembrane domains of CD98hc are necessary and sufficient for its effect on integrin function, but not amino acid transport. A, model of CD98hc/CD69 chimeras. B, CODS: ␣␤Py cells were transfected with Tac-␤ 1 and the CD98hc chimeras depicted in A. Twenty-four h after transfection, cells were collected, and the Tac-positive subset of cells were analyzed for the ability to bind to the PAC1 antibody. Data are expressed as percentage reversal of Tac-␤ 1 suppression, as described in the legend to Fig. 1. C, amino acid transport: ␣␤Py cells were transfected with cDNAs encoding the hLAT1 light chain and the CD98hc chimeras depicted in A. The uptake of [ 3 H]isoleucine was measured 24 h after transfection as described under "Experimental Procedures." The uptake was measured in a Na ϩ -free solution, and the values are expressed as cpm/mg protein, where the base-line uptake in cells transfected with hLAT1 alone has been subtracted.