Distinct Domains of CD98hc Regulate Integrins and Amino Acid Transport*

Csilla A. FenczikDagger§, Roy ZentDagger, Melissa Dellos, David A. Calderwood||, Joe Satriano**DaggerDagger, Carolyn Kelly**§§, and Mark H. Ginsberg¶¶

From the Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037 and the ** Department of Medicine, University of California San Diego and the Veterans Affairs Healthcare System (VASDHS), San Diego, California 92161

Received for publication, December 13, 2000


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta 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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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+L, and xc- (4). Furthermore, mutations in its closest paralogue, D2 (r-BAT), lead to a disorder of cysteine transport (5). Structurally, CD98 is a disulfide-bonded heterodimer of a common ~80-kDa heavy chain (CD98hc) with one of several ~40-kDa light chains. Because these light chains have multiple membrane-spanning domains, they resemble permeases and are believed to provide the amino acid transport activity of CD98 (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.

There is also a growing literature implicating CD98hc in integrin function. Integrins are heterodimeric adhesion receptors expressed in almost every multicellular animal cell type (10). Cells can rapidly modulate their integrins' affinity for extracellular ligands (activation), thereby regulating multiple integrin-dependent functions (11). Integrin activation is inhibited by overexpression of isolated beta 1A integrin cytoplasmic domains (dominant suppression) (12). CD98hc was identified as an integrin regulator in an expression-cloning scheme for proteins that can complement dominant suppression (CODS)1 (13). In addition, CD98hc binds to integrin beta 1A cytoplasmic domains, and this interaction correlates with CODS (14). Furthermore, clustering CD98hc activates multiple integrin-dependent functions (13, 15, 16) and mimics beta 1 integrin cosignaling in T-cells (17). Thus, CD98hc physically and functionally interacts with integrin adhesion receptors. Indeed, clustering of CD98hc can stimulate several classes of integrins in multiple cell types (13, 15, 16).

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 beta 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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Antibodies-- The hybridoma cell line 4F2(C13) (anti-CD98hc) was purchased from American Type Culture Collection (ATCC, Manassas, VA). The CD98hc antibody was purified from ascites produced in pristane-primed BALB/c mice by protein A affinity chromatography. Dr. S. Shattil (Scripps Research Institute) generously provided the activation-specific anti-alpha IIbbeta 3 monoclonal antibody, PAC1 (18). The anti-alpha IIbbeta 3-activating monoclonal antibody, anti-LIBS6, has been described previously (19). The anti-Tac antibody, 7G7B6, was obtained from the ATCC and was biotinylated with biotin-N-hydroxysuccinimide (Sigma) according to manufacturer's instructions. The alpha IIbbeta 3-specific peptide inhibitor, Ro43-5054(20) was a generous gift from B. Steiner (Hoffmann-La Roche, Basel, Switzerland). The mouse hybridoma cell line, 12CA5 (anti-HA), was purchased from ATCC. The anti-CD69 antibody (C1, a rabbit polyclonal antibody) was a generous gift from Drs. F. Diaz-Gonzalez and F. Sanchez-Madrid (University of Madrid, Spain).

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.

Tac-alpha 5 and Tac-beta 1A DNA in modified CMV-IL2R expression vectors (22) were generously provided by Drs. S. LaFlamme and K. Yamada (National Institutes of Health). Human 4F2 antigen (CD98hc) cDNA was provided by Dr. J. M. Leiden (University of Chicago, Chicago, IL) and was subcloned into pcDNA1 as an EcoRI fragment. Cless (C109S,C330S), C1 (C109S), and C2 (C330S) mutants of CD98hc were gifts from Dr. M. Palacin (Universitat de Barcelona). cDNA encoding hLAT1, a CD98 light chain, was a gift from Dr. F. Verrey (University of Zurich).

Construction of CD98 Chimeras-- HA-NH2 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. Delta 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. C69T98E98 contains amino acids 1-38 of CD69 (Swiss-Prot accession number Q07108) and amino acids 82-529 of CD98hc (Swiss-Prot accession number P08195). C98T69E98 contains amino acids amino acids 1-81 and 105-529 of CD98hc and amino acids 39-64 of CD69. C98T98E69 contains amino acids 1-104 of CD98hc and amino acids 65-199 of CD69. C98T69E69 contains amino acids 1-81 of CD98hc and amino acids 39-199 of CD69.

Cell Culture-- alpha beta Py cells, a CHO cell line expressing a constitutively active recombinant chimeric integrin containing the extracellular and transmembrane domains of human alpha IIbbeta 3 fused to the cytoplasmic domains of alpha 6Abeta 1 (alpha IIbalpha 6Abeta 3beta 1) (23), were maintained in Dulbecco's modified Eagle's medium (BioWhitaker) supplemented with 10% fetal calf serum (BioWhitaker), 1% nonessential amino acids (Life Technologies, Inc.), 1% glutamine (Sigma), 1% penicillin and streptomycin (Sigma), and 700 µg/ml G418 (Life Technologies, Inc.).

Cell Lysates-- 24 h after transfection with CD98hc or one of the chimeric cDNAs, alpha beta 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 Na3VO4, 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 µg/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 NH2-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), 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 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 µl 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 Ni2+ 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 alpha beta Py cells (cells positive for cotransfected Tac-alpha 5 or Tac-beta 1 as measured by 7G7B6 binding). Integrin activation was quantified as an activation index (AI) defined as (F - Fo)/(FLIBS6 - 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 saturating concentration of a competitive inhibitor (1 µM Ro43-5054), and FLIBS6 is the maximal median fluorescence intensity in the presence of the integrin activating antibody, anti-LIBS6 (2 µM). Percent reversal was calculated as (AI(beta 1A + CD98) - AIbeta 1A)/(AIalpha 5 - AIbeta 1A). AIbeta 1A is the activation index of cells transfected with Tac-beta 1A chimeras, AI(beta 1A + CD98) is the AI of cells cotransfected with CD98hc and Tac-beta 1A chimeras, and AIalpha 5 is the AI of cells transfected with Tac-alpha 5.

Amino Acid Transport-- 3H-Labeled Ile (77 Ci/mM) was purchased from Amersham Pharmacia Biotech. alpha beta 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+-free Hanks' buffered salt solution (HBSS) (136.6 mM ChCl, 5.4 mM KCl, 4.2 mM NaHCO3, 2.7 mM Na2HPO4, 1 mM CaCl2, 0.5 mM MgCl2, 0.44 mM KH2PO4, 0.41 mM MgSO4, pH 7.8), in which the sodium-containing salts were iso-osmotically replaced with choline, to remove extracellular Na+ and amino acids. Cells were equilibrated in warm choline-substituted HBSS for 10 min. The uptake of radiolabeled amino acids (2 µCi of [3H]Ile/ml) at 100 µmol/liter in 1 ml of choline-substituted HBSS was measured for 20 s at 37 °C. Uptake of [3H]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).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (Cys109) 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 (Cys109 and Cys330). 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 beta 1A cytoplasmic domain (Fig. 1B). Thus, covalent CD98 heterodimer formation is dispensable for the interaction of CD98hc with the integrin beta 1A cytoplasmic domain and for CODS.


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Fig. 1.   Stable formation of the CD98 heterodimer is not required for its effects on integrin function. A, effect of cysteine mutations on CODS. alpha beta Py cells were transfected with Tac-beta 1 and either wild-type CD98hc or CD98hc with serine substitutions at cysteines (Cys109, Cys330) involved in covalent heterodimer formation. Twenty-four 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-beta 1 suppression, which is calculated as 100 × (AITac-beta 1 + CD98 - AITac-beta 1)/(AITac-alpha 5 - AITac-beta 1). AI is the activation index of cells transfected with Tac-beta 1 alone or in combination with CD98hc or with Tac-alpha 5. Cless = CD98hc (C109S,C330S), C1 = CD98hc (C109S), C2 = CD98hc (C130S). B, CD98hc Cys109 and Cys130 are not required for binding to the integrin beta 1A cytoplasmic domain. alpha beta 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 beta 1A or alpha 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 ~80-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 (+DTT) of reducing agent. Biotinylated polypeptides were identified by streptavidin-peroxidase-generated chemiluminescence.

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 beta 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 beta 1A tail and for CODS.


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Fig. 2.   Effect of coexpression of the hLAT1 light chain on amino acid transport, heterodimer formation, and the binding of CD98hc to the beta 1A cytoplasmic domain. A, amino acid transport: alpha beta Py cells were transfected with cDNAs encoding CD98hc, hLAT1, CD98hc plus hLAT1 or vector DNA and assayed for the uptake of [3H]isoleucine after 24 h. Ile uptake was measured in a Na+-free solution, and the values are expressed as cpm/mg of protein. B, heterodimer formation: CHO cells were transfected with cDNA encoding CD98hc (4 µg) plus increasing amounts of hLAT1 light chain (0, 2, 6 µg). 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 beta 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 beta 1A or alpha 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.

The NH2 Terminus of CD98hc Is Intracellular, and the 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 NH2 terminus intracellular. To document the membrane topography of CD98hc, the NH2 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 NH2 or COOH terminus did not effect the overall surface expression of CD98hc as measured with an anti-CD98hc antibody (Fig. 3A). Furthermore, both the NH2- and COOH-terminal tags were present on the expressed protein (Fig. 3B). Consequently, we conclude that the NH2 terminus of CD98hc is intracellular, and the COOH terminus is extracellular.


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Fig. 3.   The NH2-terminal domain of CD98hc is cytoplasmic. CHO cells were transfected with CD98hc HA-tagged either at the NH2 terminus (HA-NH2) or at the COOH terminus (HA-COOH). A, twenty-four h after transfection cells were with stained with either an anti-HA antibody (top panels) or with anti-CD98hc antibody, 4F2 (bottom panels), and analyzed by flow cytometry. B, expression of HA tags: CHO cells transfected as in A were lysed and immunoprecipitated with an anti-CD98hc antibody (IP) or normal mouse IgG (IgG). The immunoprecipitates were fractionated by reduced SDS-PAGE. The immunoblots were stained with an anti-HA antibody and developed by peroxidase-mediated chemiluminescence.

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 beta 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 (C69T98E98) failed to complement dominant suppression (Fig. 4B) and failed to bind to the beta 1A cytoplasmic domain (Fig. 4C). Thus, the cytoplasmic domain of CD98hc is required for its capacity to interact with integrins.


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Fig. 4.   The cytoplasmic domain of CD98hc is required for integrin interactions. A, model of CD98hc/CD69 chimeras. B, complementation of dominant suppression (CODS): alpha beta Py cells were transfected with Tac-beta 1 and the CD98hc constructs 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-beta 1A suppression, as described in the legend to Fig. 1. C, binding to beta 1A tail: affinity chromatography with beta 1A or alpha IIb tails was performed using lysates of surface-biotinylated 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.


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Fig. 5.   The cytoplasmic and transmembrane domains of CD98hc are necessary and sufficient for binding to beta 1A cytoplasmic domains. CHO cells were transfected with each of the chimeric cDNAs depicted on the left-hand side of the figure. Twenty-four h later, surface proteins were labeled with Sulfo-Biotin N-hydroxysuccinimide, and the cells were lysed. Cell lysates were incubated with beads coated with model proteins containing beta 1A or alpha IIb cytoplasmic tails. Bound and eluted proteins that contain the extracellular domain of CD98hc (CD98hc, C98T69E98) were immunoprecipitated with anti-CD98hc antibody and fractionated by reduced SDS-PAGE, and the biotinylated polypeptides were detected by streptavidin-peroxidase chemiluminescence. Bound proteins that contain the extracellular domain of CD69 (CD69, C98T69E69, C98T98E69) were detected by immunoblot with the anti-CD69 antibody.

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 (C98T69E69) joined to the cytoplasmic domain of CD98hc failed to bind to the beta 1A cytoplasmic domain (Fig. 5). However, addition of the transmembrane domain of CD98hc (C98T98E69) 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 (C98T69E98). That construct also failed to bind to the beta 1A cytoplasmic domain (Fig. 5). Thus, binding of CD98hc to the beta 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 beta 1A cytoplasmic domain. To investigate whether the functional effects of CD98hc correlated with its binding to the beta 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 (C69T98E98; 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 (C98T69E98) 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 (C98T98E69) 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 beta 1A tail and for augmentation of integrin function.


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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: alpha beta Py cells were transfected with Tac-beta 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-beta 1 suppression, as described in the legend to Fig. 1. C, amino acid transport: alpha beta Py cells were transfected with cDNAs encoding the hLAT1 light chain and the CD98hc chimeras depicted in A. The uptake of [3H]isoleucine was measured 24 h after transfection as described under "Experimental Procedures." The uptake was measured in a Na+-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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

CD98hc combines with several different light chains to form a series of heterodimers that are involved in amino acid transport. CD98hc binds to integrin beta 1A cytoplasmic domains and blocks the capacity of beta 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 heavy-light chain association and reduce amino acid transport do not disrupt its binding to beta 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 beta 1A tail and for CODS. This chimera failed to stimulate amino acid transport. 3) Replacement of the cytoplasmic or transmembrane domains of CD98hc with those of CD69 blocked the capacity of CD98hc to bind to beta 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 Cys109 and Cys330. Cys109 is near the transmembrane domain of CD98hc and results in a disulfide bridge with a cysteine in an extracellular loop of the light chain between transmembrane domains 3 and 4 (25). Mutation of Cys109 and Cys330 disrupted the covalent association with the light chain but did not impair interactions with or effects on integrins. While the covalent association was lost, it is possible that there was still a noncovalent interaction. Indeed, Pfeiffer et al. (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 disulfide-bound heterodimers, albeit at a reduced rate. Moreover, we also found that overexpressed free heavy chains could also bind to the beta 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 beta 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 beta 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 beta 1A (Lys752-Ile757) 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-beta 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 beta 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 beta 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+LAT1, y+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, 15-17). 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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* This work was supported by Grants HL48728, HL59007, and AR27214 from the National Institutes of Health. This is publication number 13766-VB.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger These authors contributed equally to this work.

§ Supported by The United States Army Medical Research and Materiel Command under DMAD 17-97-1-7056.

Young Investigator Award of the National Kidney Foundation. Current address: Dept. of Medicine, Vanderbilt University, S-3223 Medical Center North, Nashville, TN 37232-2372.

|| Supported by grants from the Wellcome Trust and Susan G. Komen Breast Cancer Foundation.

Dagger Dagger Supported by National Institutes of Health Grant DK 28602.

§§ Supported by grants from the Veterans Affairs Medical Research Service.

¶¶ To whom correspondence should be addressed. Tel.: 858-784-7143; Fax: 858-784-7343; E-mail: ginsberg@scripps.edu.

Published, JBC Papers in Press, December 19, 2000, DOI 10.1074/jbc.M011239200

    ABBREVIATIONS

The abbreviations used are: CODS, complement dominant suppression; CHO, Chinese hamster ovary; Pipes, 1,4-piperazinediethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; AI, activation index; HBSS, Hanks' buffered salt solution.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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