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Originally published In Press as doi:10.1074/jbc.M702877200 on June 27, 2007

J. Biol. Chem., Vol. 282, Issue 33, 24477-24484, August 17, 2007
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CD98hc (SLC3A2) Interaction with the Integrin beta Subunit Cytoplasmic Domain Mediates Adhesive Signaling*

Gerald W. Prager, Chloé C. Féral, Chungho Kim, Jaewon Han, and Mark H. Ginsberg1

From the Department of Medicine, University of California San Diego, La Jolla, California 92093

Received for publication, April 4, 2007 , and in revised form, June 20, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In mammals, beta1 integrin adhesion receptors generate signals that mediate cell spreading, migration, proliferation, and survival. CD98, a heterodimeric transmembrane protein, physically associates with certain integrin beta subunit cytoplasmic domains (tails) via its heavy chain, CD98hc (SLC3A2), and loss of CD98hc impairs integrin signaling. Here we have used the lack of CD98hc interaction with the Drosophila integrin betaPS tail for a homology scanning analysis that implicated the C-terminal 8 residues of beta3 (Thr755-Thr802) in CD98hc binding. We then identified point mutations in the beta3 C terminus (T755K and T758M) that abolish CD98hc association and a double mutation in the corresponding residues in the betaPS tail (K839T,M842T), which resulted in gain of CD98hc interaction. Furthermore, the loss of function beta3(T755K) mutation or the gain of function beta3/betaPS(K839T,M842T) led to a loss or gain of integrin-mediated cell spreading, respectively. Thus, we have identified critical integrin residues required for CD98hc interaction and in doing so have shown that CD98c interaction with the integrin beta tail is required for its ability to mediate integrin signaling. These studies also provide new insights into how CD98hc may cooperate with other cytoplasmic domain binding proteins to modulate integrin functions and into the evolution of integrin signaling.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Integrins mediate cell-cell and cell-matrix adhesion to support the development and functioning of multicellular organisms (1-4). In addition to mediating adhesion, these receptors are bona fide signaling receptors that regulate cytoskeletal organization, gene expression, and cell proliferation, differentiation, and survival. These effects are achieved via signaling pathways including Rho GTPases and kinases (1, 4-7); the actions of these signaling pathways result in the cell spreading that follows adhesion to integrin ligands. Integrins are composed of non-covalently associated {alpha} and beta subunits, the N terminus of each subunit is extracellular, and a single transmembrane domain separates the ~700-1200-residue extracellular domain from a usually short (5-65 residue) cytoplasmic domain or tail (8). The cytoplasmic domains of integrins play an essential role in integrin signaling by interacting with cytoplasmic proteins (9). In addition, a genetic complementation strategy identified an interaction between a transmembrane protein, CD98, and the integrin beta1A cytoplasmic domain (10), suggesting that this interaction might also participate in integrin signaling.

CD98 heterodimers, comprised of a heavy chain, CD98hc (SLC3A2, 4F2hc), and one of several light chains (11) interact with integrins via the cytoplasmic domain of CD98hc (10, 12), a type II transmembrane protein. CD98hc cross-linking or over-expression activates (13-15) and CD98hc deletion inhibits (16) certain integrin-regulated signaling pathways. The CD98hc intracellular domain is necessary and sufficient for interaction with integrins and promotion of integrin signaling (12, 16), whereas the CD98hc extracellular domain interacts with the light chains to promote amino acid transport (12). These data suggest that the CD98hc-integrin interaction is required for its capacity to mediate integrin signaling. Here, we have tested this hypothesis by fine mapping of the integrin residues required for binding to CD98hc. Authentic CD98hc orthologues are not evident in invertebrates (17), and we report that an invertebrate beta integrin cytoplasmic domain, Drosophila betaPS, fails to bind mammalian CD98hc. We use this species difference and a "homology scanning" strategy to identify two residues in the integrinbeta tail that control the binding specificity for CD98hc but have no effect on talin binding. Using loss of function and gain of function mutants at these residues, we find that CD98hc binding to integrin beta tails is required for efficient adhesion-dependent cell spreading. Thus, we show that the interaction of CD98hc with the integrin beta subunit tail mediates adhesive signaling.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies—The hybridoma cell line 4F2(C13) (anti-CD98) was purchased from American Type Culture Collection. The CD98 antibody was purified from ascites produced in pristine-primed BALB/c mice by protein A affinity chromatography. Mouse CD98hc-specific antibody (M20) and anti-FAK2 antibody (C20) were purchased from Santa Cruz Biotechnology and talin antibody (clone 8d4) was from Sigma. An antibody specific for phosphorylated FAK Tyr576 was obtained from BIOSOURCE. Monoclonal antibody against human {alpha}IIbbeta3 (D-57) has been described previously (18).

Cell Culture—Mouse embryonic fibroblasts (MEF) were derived from CD98hc conditional homozygote embryos as described previously.3 Briefly, exon 1, encoding the transmembrane domain of CD98hc, and exon 2 were targeted and flanked with lox P sites. Two homologous ES cell recombinant clones were injected into host blastocysts. Germ line transmission was obtained, and heterozygote animals were bred to obtained homozygotes CD98hcfl/fl mice. The CD98hc-/- MEFs were generated by infecting CD98hcfl/fl MEFs with Adeno-CRE encoding CRE recombinase.3 MEFs were cultured in complete Dulbecco's modified Eagle's medium with high glucose (Invitrogen), supplemented with 10% fetal bovine serum (HyClone), 0.1 mM nonessential amino acids (Invitrogen), 2 mM L-glutamine (Invitrogen), 1% penicillin/streptomycin (Sigma), 1% HEPES (Invitrogen), 0.2% mercaptoethanol. Chinese hamster ovary cell line (CHO) expressing the polyoma large T antigen were transiently transfected with CD98hc, {alpha}IIbbeta3, or its mutant using Nucleofector II device (AMAXA Biosystems). Similar expression levels were checked by flow cytometry as described below.

Flow Cytometry—Analytical two color flow cytometry was performed as described previously (19). Expression levels of transiently transfected CHO cells were assessed by biotinylated D-57 binding and 488 Alexa Flour-labeled streptavidin (Molecular Probes). CD98hc expression levels in MEF cells were measured by anti-mouse CD98hc (clone H202-141, Pharmingen) binding. Goat fluorescein isothiocyanate-conjugated anti-rat IgG was obtained from BioSource International and was used as secondary antibody for anti-CD98hc detection.

DNA Constructs and Recombinant Proteins—cDNA encoding integrin cytoplasmic domains joined to four heptad repeats were cloned into the modified pET-15 vector as described previously (20) and were biotinylated in vivo as described (21). The repeats form parallel coiled-coil dimers so that the tails are dimerized and parallel, thus, mimicking clustered integrins. CD98hc also binds to a heterodimeric construct formed with {alpha}IIb and beta3 tails that mimic the cytoplasmic face of a single integrin (22). Point mutations in beta3 tails were performed utilizing the QuikChange kit (Stratagene) or, for the swap mutations between beta3 and betaPS cytoplasmic tails, with megaprimer polymerase chain reaction techniques. Recombinant expression in BL21(DE3) cells (Novagen) and biotinylation of the avidin tag as well as purification of the recombinant products were done in accordance with the manufacturer's instruction (Novagen), with an additional purification step on a reverse phase C18 high performance liquid chromatography (HPLC) column (Vydac). Polypeptide masses were confirmed by matrix-assisted laser desorption time of flight mass spectrometry and varied by less than 0.1% from those predicted by the desired sequence. Synthetic peptides used in the competition assays were synthesized on an ABI 430 peptide synthesizer and were 95% homogeneous as judged by a reverse phase C18 HPLC column (Vydac).

Construction of beta3 Mutants or Chimera—Point mutations in beta3 or betaPS were performed utilizing the QuikChange kit (Stratagene). To generate beta3/betaPS chimera, whereby the cytoplasmic domain of beta3 was replaced by betaPS, a SphI restriction enzyme site was created via a silent mutation at nucleotide 1899 (C to A) of beta3 cDNA sequence using site-directed mutagenesis. beta3betaPS and beta3betaPS(K839T,M842T) chimeras were generated by ligation of PCR-generated beta3 transmembrane domain and betaPS cytoplasmic domain fusion sequences into the wild type beta3 expressing vector, CDHYG3A.SphI (23).

Cell Lysates—MEF cells were washed 3 times in phosphate-buffered saline before they were lysed in buffer A (1 mM Na3VO4, 50 mM NaF, 40 mM sodium pyrophosphate, 10 mM Pipes, 50 mM NaCl, 150 mM sucrose, 1% Triton X-100, 0.5% deoxycholate, and protease inhibitors (Roche Applied Science), pH 6.8) for 30 min at 4 °C. The cell lysate was then centrifuged at 14,000 rpm before supernatant was used for affinity chromatography.

Integrin Tail Affinity Chromatography (HPLC)—HPLC was performed as described (20). Briefly, 1.2 mg of each recombinant integrin cytoplasmic domain dissolved in 1 ml of 20 mM Pipes, 50 mM NaCl, pH 6.8 (PN buffer), plus 0.2 ml of 100 mM sodium acetate was bound to 150 µl of immobilized NeutravidinTM beads (Pierce), 50% slurry at 4 °C overnight. Neutravidin beads were then washed 3 times in PN Buffer and 2 times in buffer A (1 mM Na3VO4, 50 mM NaF, 40 mM sodium pyrophosphate, 10 mM Pipes, 50 mM NaCl, 150 mM sucrose, 0.5% Triton X-100, pH 6.8). Equal loading of Immobilized Neutravidin beads with recombinant proteins was verified by Coomassie Blue staining of SDS-PAGE profiles of SDS eluted proteins. Beads were added to cell lysates diluted in buffer A and protease inhibitors and incubated overnight at 4 °C and then washed 5 times with buffer A. 75 µl of 2x reducing SDS-sample buffer was added to the beads, and the mixture was heated 95 °C for 10 min. After 14,000 rpm centrifugation in a tabletop microcentrifuge (Beckman, GS15R), the supernatant was fractionated by SDS-PAGE and analyzed by Western blotting.

Cell Spreading Assays—Assays of cell spreading on fibrinogen or fibronectin were performed as described previously (24). Briefly, 24-well plates were coated with 10 µg/ml fibrinogen or fibronectin in a coating buffer (150 mM NaCl, 50 mM Na2HPO4, pH 8.0, at 4 °C overnight) and blocked with 1% heat-denatured bovine serum albumin for 60 min at 37 °C. Cells were detached, washed twice with Dulbecco's modified Eagle's medium plus 1 mg/ml bovine serum albumin, and resuspended in the same medium at 5 x 105 cells/ml. The cells (5 x 104 cells/well) were permitted to attach at 37 °C and incubated for the times indicated. Unattached cells were washed away with phosphate-buffered saline, attached cells were fixed with 3.7% formaldehyde for 5 min at room temperature, washed twice with PBS, and stained with 0.5% crystal violet in 25% methanol for 30 min at room temperature, washed with distilled water 3 times, and observed with bright field microscopy. Photographic images were acquired with FAST1394 CCD. Cells that exhibited flattening and the presence of lamellipodia under microscope examination were scored as spreading cells. Cell area was assessed by Image J 1.32 software (National Institutes of Health).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CD98hc Is Required for {alpha}IIbbeta3 Integrin-mediated Cell Spreading—Previous studies with CD98hc-deficient embryonic stem cells suggested that CD98hc is important in beta1 integrin signaling (16). As a further test of that idea, we used conditional null CD98fl/fl MEF and examined the effect of in vitro deletion of CD98hc by adenovirus-mediated infection of Cre recombinase (Fig. 1A). As expected, deletion of CD98hc blocked {alpha}5beta1 integrin-mediated cell spreading on fibronectin, a consequence of beta1 integrin signaling (Fig. 1, B and C). Having confirmed that acute deletion of CD98hc in MEFs had similar effects to those observed in ES cells, we next sought to establish a system to enable mapping of CD98hc binding sequences in integrin beta cytoplasmic domains. Previous studies showed that CD98hc-expressing and CD98hc-deficient MEFs express no beta3 integrins.4 Because beta3 but not beta1 integrins mediate cell attachment to fibrinogen in most cell types, the platelet integrin can be used as a model to analyze structural requirements for integrin signaling in these cells (25). We expressed recombinant integrin {alpha}IIbbeta3 in these MEFs (Fig. 1D) and {alpha}IIbbeta3-transfected cells, but not untransfected cells, adhered to fibrinogen (not shown). Because, CD98hc interacts with the beta3 cytoplasmic domain (22) and integrin {alpha}IIbbeta3,4 we next examined the role of CD98hc in {alpha}IIbbeta3-mediated cell spreading. The CD98hc null MEFs attached to fibrinogen but failed to spread; in contrast, the floxed CD98hc parental cells spread extensively (Fig. 1, E and F). Thus, we have established a system in which cell spreading is mediated by a recombinant integrin ({alpha}IIbbeta3) and is CD98hc-dependent.


Figure 1
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  		FIGURE 1.
Spreading of MEF expressingbeta3 integrin depends on the presence of CD98hc. A, the effect of in vitro deletion of CD98hc by adenovirus-mediated infection of Cre recombinase in conditional null CD98hcfl/fl MEFs was examined by FACS analysis using a Rat anti-mouse CD98hc antibody as a primary and a fluorescein isothiocyanate-labeled anti-rat antibody as a secondary antibody. B, cell spreading assays. Twenty min after cell adhesion to 10 µg ml-1 fibronectin, the surface area of MEFs was measured using NIH Image J. Spreading of CD98hc-/- MEFs was reduced as compared with CD98hcfl/fl MEFs. Scale bar, 20 µm. C, time course of cell spreading. CD98hc fl/fl or CD98hc-/- MEFs were plated on 10 µg/ml fibronectin, and cell area was measured at the indicated times. D, FACS analysis of expression levels of {alpha}IIbbeta3 integrins. 48 h after CD98-expressing or CD98hc-deficient MEFs were transiently transfected with cDNAs encoding integrin{alpha}IIb and beta3 and stained for {alpha}IIbbeta3 expression using D57 antibody to generate the indicated dot plots. The histograms exhibit the expression of integrin {alpha}IIbbeta3 in the gated integrin-positive and propidium iodide (PI)-negative populations. E, CD98hcfl/fl or CD98hc-/- MEFs transiently expressing {alpha}IIbbeta3 were plated on fibrinogen and allowed to adhere for 20 min. Twenty min after adhesion to 10 µgml-1 fibrinogen, the surface area of the MEFs was measured using Image J. wt, wild type. F, time course of cell spreading on fibrinogen. CD98hcfl/fl or CD98hc-/- cells were adhered to fibrinogen as described in E, and spreading was measured at the indicated times after attachment. *, p < 0.005.

 


Figure 2
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  		FIGURE 2.
CD98hc binds to the beta3 cytoplasmic tails but not to betaPS cytoplasmic tail. A, alignment of the amino acid sequences of human beta3 and D. melanogaster betaPS integrin cytoplasmic tails. B, affinity chromatography. Immobilized Neutravidin beads were loaded with beta3(HisAvi-B3), a beta3 with randomized amino acid sequence(HisAvi-B3rand) or betaPS (HisAviBPS) tail proteins. Bound proteins from CD98hc+/+ or CD98hc-deficient MEF cell lysates were separated on 4-20% SDS-PAGE under reducing conditions, transferred to a nitrocellulose membrane, and immunoblotted either with antibodies specific for CD98hc or talin. Lysates represents 10% of the starting cell lysate from each affinity purification experiment. Loading of each tail protein was assessed by Coomassie Blue staining (Loading). Depicted is a representative result from five independent experiments.

 
Mapping Integrin Sequences Required for CD98hc Binding—Having established an assay for CD98hc-dependent integrin signaling in a cell in which we could manipulate integrin sequences, we next sought a CD98hc non-binding integrin for a homology scanning analysis similar to that used to map the filamin binding sequence in integrin beta tails (26). We first compared the beta3 tail sequence to that of beta7 and beta1D, two beta tails that fail to bind CD98hc (22), and noted substantial divergence from beta3 sequence. We also examined betaPS from Drosophila melanogaster because the cytoplasmic tail of betaPS is 68% identical with the human beta3 cytoplasmic domain (Fig. 2A) and because Drosophila were reported to lack an authentic CD98hc orthologue (17). Because the complete genome of D. melanogaster is now available, we performed a BLAST search with the human CD98hc sequence and identified maltase H precursor (P07190 [GenBank] ), which is similar to a region of the CD98hc extracellular domain but does not appear to have a transmembrane domain and, thus, does not appear to be a true orthologue. Similarly, both UniProtKB/TrEMBL and Homologene databases list only vertebrate CD98hc orthologues. Consistent with a lack of a CD98hc orthologue in Drosophila, the betaPS cytoplasmic domain failed to bind human CD98hc (Fig. 2B). In contrast, mammalian talin, a critical integrin cytoplasmic domain-binding protein in all species so far examined (27, 28), bound as strongly to the betaPS tail as it did to the beta3 tail, confirming the functionality of the betaPS cytoplasmic domain. Thus, CD98hc binds to beta3 but not to the similar betaPS cytoplasmic domain.


Figure 3
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  		FIGURE 3.
CD98hc binding involves the C terminus of beta3. A, amino acid sequences of C-terminal swapmutations of beta3 with the betaPS cytoplasmic domain. B, affinity chromatography using lysate of MEFs was performed as described in Fig. 2, and bound CD98hc was detected by immunoblotting. Equivalent loading of the recombinant beta tails was verified by Coomassie Blue staining. CD98hc binds to beta3 and to betaPS chimeras containing the C-terminal eight amino acids of beta3(betaPS (839-846 beta3)). C, MEF lysates were added to immobilized Neutravidin beads loaded with beta3-cytoplasmic tail proteins in the absence or presence of beta3 tail synthetic peptides. Bound CD98hc was detected by SDS-PAGE separation and immunoblotting. Bound CD98hc was quantified by densitometry, and these values were used to calculate percentage inhibition = 100 x (I0 -I)/I0, where I0 = intensity in the absence of a competitor, and I = intensity in the presence of competitor.

 
We next studied the effect of exchanges between beta3 and betaPS on CD98hc binding. CD98hc binds to beta1A but not to its splice variant, beta1D (22). The main differences between these two integrins are in the C-terminal amino acids. Therefore, we hypothesized that the beta sequences important for CD98hc binding are located within the C-terminal region of the cytoplasmic domain of beta3. Swapping the C-terminal eight amino acids between beta3 and betaPS switched CD98hc binding phenotypes (Fig. 3A). betaPS tail chimeras containing the last eight amino acids of beta3 (betaPS (839-846 beta3)) bound CD98hc to a similar extent as full-length beta3. Conversely, a beta3 tail containing the C-terminal eight amino acids of betaPS (beta3 (755-762 betaPS)) lost its capacity to bind CD98hc (Fig. 3B). Thus, these exchanges indicate that residues 755-762 of the beta3 tail are critical for high affinity CD98hc binding.

We next employed a competitive inhibition approach to test the importance of the C terminus of the integrin beta subunit in CD98hc binding. Synthetic peptides that spanned either the C-terminal or the N-terminal region of the beta3 cytoplasmic domain were used to compete for CD98hc binding to the full-length beta3 tail. The C-terminal peptide (beta3 (748-762)) competed for CD98hc binding with a calculated IC50 of ~300 µM (Fig. 3C). In sharp contrast, the N-terminal peptide, beta3 (721-738), had no effect at concentrations up to 10 mM. The difference in activity of these two peptides is not likely to be due to charge effects since they have similar calculated (ExPASy ProtParam) isoelectric points (beta3 (748-762) pI = 8.59; beta3 (721-738) pI = 8.50). Thus, these data together with the data obtained from the homology exchange mutants suggest that the C-terminal region of the beta3 cytoplasmic domain binds to CD98hc.

Amino Acid Residues Essential for CD98hc Interaction—To assess the role of individual amino acid residues within the beta3 cytoplasmic domain that are important for CD98hc binding, we generated point-mutated beta-tail proteins. Because swapping the C-terminal eight amino acids between beta3 and betaPS led to a reversal in CD98hc binding, we studied differences in the sequence of the C-terminal end. Truncation of the three C-terminal amino acids of beta1A does not influence CD98hc binding, whereas deleting the last seven residues abolishes it (22), suggesting that beta3 Arg760-Thr762 is dispensable for CD98hc binding. Consistent with this hypothesis, CD98hc bound to a three-residue truncation of beta3 (beta3(760X)) (Fig. 4A). A comparison of the C-terminal sequences of CD98hc binding integrins (beta3 or beta1A) with non-CD98hc binding beta integrins (beta1D or betaPS) (Fig. 4B) suggested that Thr755 and Thr758 might be important in CD98hc binding and led us to examine the effect of substituting betaPS residues into these positions in beta3. Mutation of Thr755 to Lys755 or Thr758 to Met758 in beta3 each reduced CD98hc binding to near background levels. In contrast, talin binding to the mutated cytoplasmic tails was not reduced (Fig. 4A), confirming their functionality. Thus, Thr755 and Thr758 are important for CD98hc binding to the beta3 cytoplasmic domain.

The foregoing experiments showed that substitution of beta3 Thr755 to Lys755 resulted in a loss of CD98hc binding to the beta3 tail. This residue is a Val in beta1A, an integrin that interacts with CD98hc, suggesting that a Thr is not absolutely required in this position for CD98 binding. Furthermore, these data suggest a Lys is not permitted in this position for retention of CD98 binding; indeed, a Lys substitution for Val796 in beta1A, which corresponds to the Thr755 position in beta3, led to a decrease in CD98hc binding with no effect on talin binding (Fig. 4B).

Two Threonine Substitutions in betaPS Results in CD98hc Binding—The experiments described above identify two amino acids within the C-terminal region of beta3 involved in CD98hc binding (Thr755 and Thr758). The corresponding residues in betaPS are a Lys839 and Met842. Changing both of these residues in betaPS cytoplasmic domain to Thr resulted in a dramatic gain of CD98hc binding function in betaPS (K839T,M842T) (Fig. 5A). Consistent with the loss of CD98hc when either Thr755 or Thr758 in integrin beta3 tail was mutated (see Fig. 4), single Thr substitutions at either at Lys839 or at Met842 resulted in no increase of CD98hc binding (Fig. 5A). Thus, introducing Thr at position 839 and 842 into betaPS resulted in a gain of CD98hc binding function.


Figure 4
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  		FIGURE 4.
Point mutations in the beta3 and beta1A cytoplasmic tails that inhibit CD98hc association. A, beta3(T755K) or beta3(T758K) mutations disrupt CD98hc interaction. Affinity chromatography was performed as described in Fig. 2. Lysate represents 10% of the starting lysate. B, a single amino acid mutation in beta1A (V796K), which corresponds to beta3 Thr755, disrupts CD98hc interaction with beta1A cytoplasmic tail. As reported previously (22), CD98hc interacts with beta1A but not with beta1D. Talin binding to beta tails is not disrupted by any of these mutations. Loading of each tail was assessed by Coomassie Blue staining (Loading).

 


Figure 5
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  		FIGURE 5.
betaPS(K839T,M842T) gains the capacity to interact with CD98hc. A, amino acid sequences of integrin betaPS tail proteins with the single or double threonine substitutions indicated. B, CD98hc binding to integrin betaPS mutations. Immobilized integrin cytoplasmic tails were incubated with lysates of MEFs, and bound proteins were fractionated by SDS-PAGE. Binding of CD98hc was analyzed by immunoblotting. Equivalent loading of the beta tail protein was verified by Coomassie Blue staining of the eluted proteins (Loading). C, the intensity of bands on CD98hc blots were quantified by densitometric analysis and normalized to the content of CD98hc in 10% of the cell lysate (defined as 1). Results are expressed as the mean ± S.D. of triplicate determinations.

 
Integrin-CD98hc Interaction Mediates Adhesive Signaling—Having defined conservative substitutions that selectively alter CD98hc-integrin interactions, we tested the potential biological role of CD98hc-integrin interaction. CHO cells expressing either wild type {alpha}IIbbeta3 integrin or a mutant in which Thr755 had been replaced with lysine (T755K) were studied. Similar expression levels of recombinant proteins were confirmed by FACS analysis (Fig. 6A). Cells expressing beta3(T755K) exhibited markedly reduced spreading on fibrinogen (Fig. 6B). This difference was not due to generalized defects in cell spreading because both cell lines spread to a similar extent on fibronectin, a ligand for endogenous hamster integrin {alpha}5beta1 (Fig. 6B). Thus, a point mutation that disrupts CD98hc binding to beta3 resulted in impaired cell spreading.

Conversely, we asked whether a gain of CD98hc binding function mutant of betaPS (K839T,M842T), as described above, would enhance cell spreading. We created chimeras in which the extracellular and transmembrane domains of beta3 were joined to the cytoplasmic domain of betaPS and examined the spreading of cells expressing this mutant on the beta3-specific ligand, fibrinogen. Wild type beta3 and the two chimeras, beta3betaPS and beta3betaPS(K839T,M842T), were expressed at similar levels when they were co-transfected with a cDNA encoding integrin {alpha}IIb (Fig. 6C). The cells expressing {alpha}IIbbeta3betaPS exhibited markedly reduced spreading compare with those expressing {alpha}IIbbeta3 (Fig. 6D). Strikingly, the cells expressing {alpha}IIbbeta3betaPS(K839T,M842T) spread to a much greater extent than those expressing {alpha}IIbbeta3betaPS (Fig. 6D). Thus, the CD98hc-integrin interaction mediates the signaling events required for integrin-dependent cell spreading.


Figure 6
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  		FIGURE 6.
CD98hc interaction with the beta tail regulates integrin-mediated cell spreading. A, FACS analysis of {alpha}IIbbeta3 or {alpha}IIbbeta3(T755K) integrin expression. CHO cells were transfected with the indicated integrin and analyzed 48 h after transfection as described in Fig. 1. Both integrins were expressed at similar levels (fluorescence intensity for {alpha}IIbbeta3, 35.1 arbitrary units; for {alpha}IIbbeta3(T755K), 32.2 arbitrary units). B, cell spreading assays. CHO cells expressing either wild type or mutated {alpha}IIbbeta3(T755K) were allowed to spread on fibrinogen-(left panel) or a recombinant fragment of fibronectin containing the cell binding domain (right panel)-coated coverslips. Quantification of cell area was performed after 60 min and is expressed as percentage of average cell area measured in wild type {alpha}IIbbeta3-expressing CHO cells. The data represent the mean ± S.E. of four independent experiments. *, p < 0.001. C, FACS analysis of expression levels of {alpha}IIbbeta3, {alpha}IIbbeta3betaPS chimera, or {alpha}IIbbeta3betaPS (K839T,M842T) mutants in CHO cells 48 h after transfection. All constructs were expressed at a comparable levels (fluorescence intensity for {alpha}IIbbeta3, 791 arbitrary units; for {alpha}IIbbeta3betaPS chimera, 838 arbitrary units; for {alpha}IIbbeta3betaPS (K839T,M842T) mutants, 803 arbitrary units). D, cell area measurements of CHO cells expressing {alpha}IIbbeta3,{alpha}IIbbeta3betaPS chimera, or{alpha}IIbbeta3betaPS (K839T,M842T) mutants. CHO cells expressing recombinant integrins were allowed to spread on fibrinogen-coated coverslips. Quantification was done as described in B. Cell spreading on fibrinogen of CHO cells expressing {alpha}IIbbeta3betaPS chimera was markedly reduced when compared with CHO cells expressing {alpha}IIbbeta3 integrins. CHO cells expressing {alpha}IIbbeta3betaPS (K839T,M842T) mutants spread to a greater extent than CHO cells expressing wild type {alpha}IIbbeta3betaPS chimeras. *, p < 0.001.

 


Figure 7
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  		FIGURE 7.
Sequence comparison of integrin beta cytoplasmic domains. Shown is an alignment of the sequences of the cytoplasmic tails of human beta3 (3), chicken beta3 (gallus) Xenopus laevis beta3 (Xenopus), Zebrafish beta3 (Danio), human beta1A (1a), human beta1D (1d), D. melanogaster betaPS (PS), Pseudoplusia includens: beta1 (pseudplusia), Biomphalaria glabrata: beta (biomphalaria), Strongylocentrotus purpuratus: betaL (strongylocentrotus), C. elegans betaPAT3 (elegans). Note at the dotted line boxed amino acid residue is a conserved Thr in beta3 and a Val (or Ile) in vertebrate beta1A. As shown here, a Lys residue at this position is incompatible with association with CD98hc. A Lys residue is present in beta1D and in betaPS, which do not interact with CD98hc, and is present in all of the invertebrate integrins (solid line box). The asterisks indicate nucleotides in that column are identical in all sequences in the alignment. The colon indicated that conserved substitutions have been observed. The periods indicate that semi-conserved substitutions are observed.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our results implicate the C-terminal eight residues of the integrin beta3 tail in CD98hc interaction and pinpoint Thr755 and Thr758 as being of particular significance. This site is distinct from both the phosphotyrosine binding-like and membrane proximal binding sites for talin that are responsible for integrin affinity modulation (29-31). Because CD98hc does not appear to interact with integrin tail sites required for affinity modulation, these data explain why deletion of CD98hc fails to alter integrin affinity (16). Furthermore, the Src family kinases require the C-terminal 3 residues of beta3 for binding (21), whereas CD98hc does not, suggesting that the CD98hc and Src binding sites may be distinct. In contrast, the filamin binding site in beta7 (32) spans residues analogous to the CD98hc site in beta1A, suggesting that filamin might inhibit CD98hc binding to integrin beta tails, thus partially explaining the capacity of filamin to block cell spreading and cell migration when it binds to integrin beta1A (26). In sum, we have identified critical residues required for CD98hc binding and in doing so have provide new insights into how this protein may cooperate with other cytoplasmic domain binding proteins to modulate integrin function.

The identification of residues critical for CD98hc binding also suggests new directions for analysis of the regulation of the CD98hc-integrin interaction. This region of beta3 is flexible and unstructured in aqueous solution (33-35); however, when the beta3 tail interacts with DPC micelles, this region has a strong helical propensity, and the two critical Thr residues would come to lie along the same face of a helix and be brought close to the plasma membrane (36). Vinogradova et al. (36) propose that integrin activation and tail separation may modulate this membrane interaction and could, thus, regulate the CD98hc integrin interaction by changing the conformation of beta3-(755-759). The membrane interaction could change the relationship of beta3-(755-759) to the cytoplasmic membrane-proximal region of CD98hc, a site implicated in binding to integrins (14). Furthermore, Thr755 and Thr758 have the potential to be phosphorylated; however, to date biochemical studies in platelets have demonstrated phosphorylation of Thr751 and Thr753 but not the two Thr residues critical for CD98hc binding (37). Platelets express little CD98hc,4 so future studies will be required to determine whether these residues can be phosphorylated in other CD98hc-expressing cells. Thus, our results raise the possibilities that integrin cytoplasmic domain separation or phosphorylation can regulate the integrin interaction with CD98hc.

Our finding that the interaction of CD98hc with the integrin beta tail is required for efficient integrin signaling explains some of the biological consequences of alternative splicing of the beta1 integrin cytoplasmic domain. beta1D is a splice variant that is expressed primarily in differentiated striated muscle, and beta1D is less efficient than beta1A in mediating cell spreading (38) and ERK mitogen-activated protein kinase-mediated cell proliferation (39). Each of these events involves CD98hc-dependent integrin signaling (16). The residue corresponding to beta3 Thr755 is Lys in beta1D, and making this single substitution in beta1A blocked CD98hc binding. Thus, our data explain the failure of beta1D to bind to CD98hc (22) and the relative inability of beta1D to engage the signaling pathways required for cell motility and proliferation. Furthermore, enforced replacement of beta1A by beta1D results in embryonic lethality with the plethora of developmental defects, in part caused by impaired migration of neuroepithelial cells (40); similarly, deletion of CD98hc results in early embryonic lethality (41). Thus, our studies provide a biochemical explanation for some of the biological consequences of switching from the integrin beta1A to beta1D splice variant.

Our studies also suggest that the CD98hc-integrin beta tail interaction is a vertebrate specialization that has expanded the functional capacity of integrins. Authentic CD98hc first appears in the most primitive vertebrates (17), and motifs required to bind CD98hc seem well preserved in vertebrates. For example a TNITY motif is found in the beta3 tails of amphibians and fish (Fig. 7). Furthermore, as shown here, beta1A contains a Val at the locus corresponding to beta3 Thr755. beta1A capacity to bind CD98hc requires the absence of a Lys at this position, a feature also conserved in amphibians and fish. In contrast, in invertebrates such Drosophila and Strongylocentrotus, a Lys residue occurs at this position (Fig. 7), and we show here that the presence of this Lys residue contributes to the failure of Drosophila betaPS to bind to CD98hc. CD98hc promotes the integrin-dependent phosphorylation of pp125FAK (16), a central player in mammalian integrin signaling that mediates cell migration and survival (42). In striking contrast to mammals, deletion of the pp125FAK orthologue in Drosophila does not lead to lethality or to obvious defects in integrin function (43). Thus, we propose that CD98hc-mediated integrin signaling pathways are a vertebrate specialization that enable the efficient adhesion-mediated activation of pp125FAK and other key enzymes (16) required for integrin-dependent cell migration and cell survival.


   FOOTNOTES
 
* This work was supported in part by Austrian Science Foundation Fellowship J2511-B11 (to G. W. P.), National Institutes of Health Grants HL078784, AR27214, and HL31950, and by Cell Migration Consortium Grant U54 GM064346. 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. Back

1 To whom correspondence should be addressed: University of California, Leichtag Bldg., Rm. 181, 9500 Gilman Dr., Dept. 0726, La Jolla, CA 92093-0726. Tel.: 858-822-6432; Fax: 858-822-6458; E-mail: mhginsberg{at}ucsd.edu.

2 The abbreviations used are: FAK, focal adhesion kinase; HPLC, high performance liquid chromatography; MEF, mouse embryonic fibroblast; CHO, Chinese hamster ovary; Pipes, 1,4-piperazinediethanesulfonic acid; FACS, fluorescence-activated cell sorter. Back

3 Feral, C. C., Zijlstra, A., Tkachenko, E., Prager, G., Gardel, M. L., Slepak, M., and Ginsberg, M. H. (2007) J. Cell. Biol., in press. Back

4 G. W. Prager, C. C. Féral, C. Kim, J. Han, and M. H. Ginsberg, unpublished results. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Schwartz, M. A., Schaller, M. D., and Ginsberg, M. H. (1995) Annu. Rev. Cell Dev. Biol. 11, 549-599[CrossRef][Medline] [Order article via Infotrieve]
  2. Humphries, M. J., and Newham, P. (1998) Trends Cell Biol. 8, 78-83[Medline] [Order article via Infotrieve]
  3. Geiger, B., Bershadsky, A., Pankov, R., and Yamada, K. M. (2001) Nat. Rev. Mol. Cell Biol. 2, 793-805[CrossRef][Medline] [Order article via Infotrieve]
  4. Hynes, R. O. (2002) Cell 110, 673-687[CrossRef][Medline] [Order article via Infotrieve]
  5. Hannigan, G., Troussard, A. A., and Dedhar, S. (2005) Nat. Rev. Cancer 5, 51-63[CrossRef][Medline] [Order article via Infotrieve]
  6. Guo, W., and Giancotti, F. G. (2004) Nat. Rev. Mol. Cell Biol. 5, 816-826[CrossRef][Medline] [Order article via Infotrieve]
  7. Giancotti, F. G., and Ruoslahti, E. (1999) Science 285, 1028-1032[Abstract/Free Full Text]
  8. Hynes, R. O. (1992) Cell 69, 11-25[CrossRef][Medline] [Order article via Infotrieve]
  9. Liu, S., Calderwood, D. A., and Ginsberg, M. H. (2000) J. Cell Sci. 113, 3563-3571[Abstract]
  10. Fenczik, C. A., Sethi, T., Ramos, J. W., Hughes, P. E., and Ginsberg, M. H. (1997) Nature 370, 81-85
  11. Verrey, F., Closs, E. I., Wagner, C. A., Palacin, M., Endou, H., and Kanai, Y. (2004) Pfluegers Arch. Eur. J. Physiol. 447, 532-542[CrossRef][Medline] [Order article via Infotrieve]
  12. Fenczik, C. A., Zent, R., Dellos, M., Calderwood, D. A., Satriano, J., Kelly, C., and Ginsberg, M. H. (2001) J. Biol. Chem. 276, 8746-8752[Abstract/Free Full Text]
  13. Rintoul, R. C., Buttery, R. C., Mackinnon, A. C., Wong, W. S., Mosher, D., Haslett, C., and Sethi, T. (2002) Mol. Biol. Cell 13, 2841-2852[Abstract/Free Full Text]
  14. Henderson, N. C., Collis, E. A., Mackinnon, A. C., Simpson, K. J., Haslett, C., Zent, R., Ginsberg, M., and Sethi, T. (2004) J. Biol. Chem. 279, 54731-54741[Abstract/Free Full Text]
  15. Cai, S., Bulus, N., Fonseca-Siesser, P. M., Chen, D., Hanks, S. K., Pozzi, A., and Zent, R. (2005) J. Cell Sci. 118, 889-899[Abstract/Free Full Text]
  16. Feral, C. C., Nishiya, N., Fenczik, C. A., Stuhlmann, H., Slepak, M., and Ginsberg, M. H. (2004) Proc. Natl. Acad. Sci. U. S. A. 102, 355-360[CrossRef][Medline] [Order article via Infotrieve]
  17. Uinuk-Ool, T., Mayer, W. E., Sato, A., Dongak, R., Cooper, M. D., and Klein, J. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 14356-14361[Abstract/Free Full Text]
  18. Loftus, J. C., O'Toole, T. E., Plow, E. F., Glass, A., Frelinger, A. L., III, and Ginsberg, M. H. (1990) Science 249, 915-918[Abstract/Free Full Text]
  19. O'Toole, T. E., Katagiri, Y., Faull, R. J., Peter, K., Tamura, R. N., Quaranta, V., Loftus, J. C., Shattil, S. J., and Ginsberg, M. H. (1994) J. Cell Biol. 124, 1047-1059[Abstract/Free Full Text]
  20. Pfaff, M., Liu, S., Erle, D. J., and Ginsberg, M. H. (1998) J. Biol. Chem. 273, 6104-6109[Abstract/Free Full Text]
  21. Arias-Salgado, E. G., Lizano, S., Shattil, S. J., and Ginsberg, M. H. (2005) J. Biol. Chem. 280, 29699-29707[Abstract/Free Full Text]
  22. Zent, R., Fenczik, C. A., Calderwood, D. A., Liu, S., Dellos, M., and Ginsberg, M. H. (2000) J. Biol. Chem. 275, 5059-5064[Abstract/Free Full Text]
  23. Partridge, A. W., Liu, S., Kim, S., Bowie, J. U., and Ginsberg, M. H. (2005) J. Biol. Chem. 280, 7294-7300[Abstract/Free Full Text]
  24. Liu, S., Thomas, S. M., Woodside, D. G., Rose, D. M., Kiosses, W. B., Pfaff, M., and Ginsberg, M. H. (1999) Nature 402, 676-681[CrossRef][Medline] [Order article via Infotrieve]
  25. Ylanne, J., Chen, Y.-P., O'Toole, T. E., Loftus, J. C., Takada, Y., and Ginsberg, M. H. (1993) J. Cell Biol. 122, 223-233[Abstract/Free Full Text]
  26. Calderwood, D. A., Huttenlocher, A., Kiosses, W. B., Schwartz, M. A., and Ginsberg, M. H. (2001) Nat. Cell Biol. 12, 1060-1068
  27. Brown, N. H., Gregory, S. L., Rickoll, W. L., Fessler, L. I., Prout, M., White, R. A., and Fristrom, J. W. (2002) Dev. Cell 3, 569-579[CrossRef][Medline] [Order article via Infotrieve]
  28. Tadokoro, S., Shattil, S. J., Eto, K., Tai, V., Liddington, R. C., de Pereda, J. M., Ginsberg, M. H., and Calderwood, D. A. (2003) Science 302, 103-106[Abstract/Free Full Text]
  29. Calderwood, D. A., Yan, B., de Pereda, J. M., Garcia-Alvarez, B., Fujioka, Y., Liddington, R. C., and Ginsberg, M. H. (2002) J. Biol. Chem. 277, 21749-21758[Abstract/Free Full Text]
  30. Garcia-Alvarez, B., de Pereda, J. M., Calderwood, D. A., Critchley, D. R., Ulmer, T. S., Campbell, I. D., Ginsberg, M. H., and Liddington, R. C. (2003) Mol. Cell 11, 49-58[CrossRef][Medline] [Order article via Infotrieve]
  31. Wegener, K. L., Partridge, A., Han, J., Pickford, A. R., Liddington, R. C., Ginsberg, M. H., and Campbell, I. D. (2007) Cell 128, 171-182[CrossRef][Medline] [Order article via Infotrieve]
  32. Kiema, T., Lad, Y., Jiang, P., Oxley, C. L., Baldassarre, M., Wegener, K. L., Campbell, I. D., Ylanne, J., and Calderwood, D. A. (2006) Mol. Cell 21, 337-347[CrossRef][Medline] [Order article via Infotrieve]
  33. Vinogradova, O., Velyvis, A., Velyviene, A., Hu, B., Haas, T., Plow, E., and Qin, J. (2002) Cell 110, 587-597[CrossRef][Medline] [Order article via Infotrieve]
  34. Ulmer, T. S., Yaspan, B., Ginsberg, M. H., and Campbell, I. D. (2001) Biochemistry 40, 7498-7508[Medline] [Order article via Infotrieve]
  35. Weljie, A. M., Hwang, P. M., and Vogel, H. J. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 5878-5883[Abstract/Free Full Text]
  36. Vinogradova, O., Vaynberg, J., Kong, X., Haas, T. A., Plow, E. F., and Qin, J. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 4094-4099[Abstract/Free Full Text]
  37. Lerea, K. M., Cordero, K. P., Sakariassen, K. S., Kirk, R. I., and Fried, V. A. (1999) J. Biol. Chem. 274, 1914-1919[Abstract/Free Full Text]
  38. Belkin, A. M., Retta, S. F., Pletjushkina, O. Y., Balzac, F., Silengo, L., Fassler, R., Koteliansky, V. E., Burridge, K., and Tarone, G. (1997) J. Cell Biol. 139, 1583-1595[Abstract/Free Full Text]
  39. Belkin, A. M., and Retta, S. F. (1998) J. Biol. Chem. 273, 15234-15240[Abstract/Free Full Text]
  40. Baudoin, C., Goumans, M. J., Mummery, C., and Sonnenberg, A. (1998) Genes Dev. 12, 1202-1216[Abstract/Free Full Text]
  41. Tsumura, H., Suzuki, N., Saito, H., Kawano, M., Otake, S., Kozuka, Y., Komada, H., Tsurudome, M., and Ito, Y. (2003) Biochem. Biophys. Res. Commun. 308, 847-851[CrossRef][Medline] [Order article via Infotrieve]
  42. Cohen, L. A., and Guan, J. L. (2005) Curr. Cancer Drug Targets 5, 629-643[CrossRef][Medline] [Order article via Infotrieve]
  43. Grabbe, C., Zervas, C. G., Hunter, T., Brown, N. H., and Palmer, R. H. (2004) Development 131, 5795-5805[Abstract/Free Full Text]

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