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J Biol Chem, Vol. 275, Issue 7, 5059-5064, February 18, 2000

Class- and Splice Variant-specific Association of CD98 with Integrin  Cytoplasmic Domains^*

Roy Zent^§, Csilla A. Fenczik^§¶, David A. Calderwood, Shouchun Liu^**, Melissa Dellos, and Mark H. Ginsberg

From the Department of Vascular Biology, Scripps Research Institute, La Jolla, California 92037

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CD98 is a type II transmembrane protein involved in neutral and basic amino acid transport and in cell fusion events. CD98 was implicated in the function of integrin adhesion receptors by its capacity to reverse suppression of integrin activation by isolated integrin _1A domains. Here we report that CD98 associates with integrin  cytoplasmic domains with a unique integrin class and splice variant specificity. In particular, CD98 interacted with the ubiquitous _1A but not the muscle-specific splice variant, _1D, or leukocyte-specific ₇ cytoplasmic domains. The ability of CD98 to associate with integrin cytoplasmic domains correlated with its capacity to reverse suppression of integrin activation. The association of CD98 with integrin _1A cytoplasmic domains may regulate the function and localization of these membrane proteins.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The development and function of multicellular animals requires integrin adhesion receptors (1). Integrin-dependent cell adhesion is regulated, in part, by ligand binding affinity ("activation") changes controlled by cellular signaling cascades (1-3). Regulation of integrin affinity is important in cell migration (4-6), extracellular matrix assembly (7), and morphogenesis (8). Integrin activation is energy-dependent and is mediated by cell type specific signals operating through integrin cytoplasmic domains (9).

Complementation of dominant suppression (CODS)¹ is an expression cloning scheme used to identify proteins that modulate integrin affinity (10). CODS depends on the ability of an isolated integrin _1A cytoplasmic domain, in the form of a chimera with the  subunit of the interleukin-2 receptor, to block integrin activation (dominant suppression). Proteins involved in integrin activation are isolated by their ability to complement dominant suppression. CD98, a type II transmembrane protein first discovered as a T-cell activation antigen (11), was identified utilizing CODS. CD98, although widely expressed on proliferating cells, is generally down-regulated in quiescent cells (12). CD98 forms disulfide-bonded heterodimers with several light chains that strongly resemble permeases (13-20). CD98 regulates the transport of neutral and positively charge amino acids through these light chains (14, 15, 17, 18). Thus, CODS has identified an unexpected connection between cell adhesion and certain amino acid transporters.

The mechanism by which CD98 influences integrin function is not yet clear. CODS was predicated on the idea that it would identify integrin  cytoplasmic domain binding proteins (10). Many  cytoplasmic domains manifest overall sequence similarity (1, 2); however, the cytoskeletal protein, talin, binds to the muscle-specific splice variant, _1D, more tightly than to _1A. In addition, the leukocyte-specific ₇ cytoplasmic domain binds to filamin more tightly than to _1A (21). We have now examined interactions between CD98 and recombinant parallel-dimerized integrin _1A, _1D, and ₇ cytoplasmic domains by affinity chromatography (21). Here we report that CD98 interacts with the _1A but not _1D or ₇ integrin cytoplasmic domains. Furthermore, the CD98 interaction is insensitive to  cytoplasmic domain mutations that abolish the binding of talin and filamin. The capacity of CD98 to complement dominant suppression correlates with its capacity to bind to the suppressive  cytoplasmic domains. The interaction of the integrin _1A cytoplasmic domain with CD98 may thus serve to regulate the localization and the function of these membrane proteins.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Antibodies-- The hybridoma cell line 4F2(C13) (anti-CD98) was purchased from American Type Culture Collection (ATCC). The CD98 antibody was purified from ascites produced in pristane-primed BALB/c mice by protein A affinity chromatography. Filamin antibody (monoclonal antibody 1680) was purchased from Chemicon and talin antibody (clone 8d4) from Sigma. Dr. S. Shattil (Scripps Research Institute) generously provided the activation-specific anti-_IIb3 monoclonal antibody, PAC1 (22). The anti-_IIb3 activating monoclonal antibody, anti-LIBS6, has been described previously (23). The anti-Tac antibody, 7G7B6, was obtained from the American Tissue Culture Collection (Rockville, MD) and was biotinylated with biotin-N-hydroxysuccinimide (Sigma) according to manufacturer's instructions. The _IIb3-specific peptide inhibitor, Ro43-5054 (24), was a generous gift from B. Steiner (Hoffmann-La Roche, Basel, Switzerland).

DNA Constructs and Recombinant Proteins-- cDNA encoding the expressed integrin cytoplasmic domains joined to 4 heptad repeats (Fig. 1) were cloned into the modified pET-15 vector as described previously (21). Point mutations in _1D and ₇ (Fig. 1) were performed utilizing the Quickchange kit (Stratagene). Recombinant expression in BL21 (DE3)pLysS cells (Novagen) and purification of the recombinant products were made in accordance with the manufacturers 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 varied by less than 4 daltons from those predicted by the desired sequence.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Amino acid sequences of integrin  cytoplasmic domains. Depicted is an alignment of the integrin cytoplasmic domains used in this study. The underlined tyrosine (Y) was mutated to an alanine (A) to form the YA mutants. All integrin sequences with the exception of 7 correspond to those human sequences published in the Swiss-Protein data base as of May 15, 1999. In 7, the amino-terminal Arg was changed to Lys in order to introduce a HindIII restriction site.

Tac-5 and Tac-_1A DNA in modified CMV-IL2R expression vectors (25) were generously provided by Drs. S. LaFlamme and K. Yamada (National Institutes of Health, Bethesda, MD). Inserts encoding Tac-_1D, Tac-₇, Tac-_1A(Y788A), and Tac-_1A(801X) were subcloned into the modified CMV-IL2R expression vector as HindIII-XhoI fragments.

Cell Culture-- py cells, a Chinese hamster ovary cell line expressing the polyoma large T antigen and a constitutively active recombinant chimeric integrin, _IIb6A31 (26), were maintained in Dulbecco's modified Eagle's medium (BioWhitaker); supplemented with 10% fetal calf serum (BioWhitaker), 1% non-essential amino acids (Life Technologies, Inc.), 1% glutamine (Sigma), 1% penicillin and streptomycin (Sigma), and 700 µg/ml G418 (Life Technologies, Inc.). Human Jurkat T cell lines were obtained from ATCC and maintained in RPMI1680 (BioWhitaker) supplemented with 10% fetal calf serum, 1% nonessential amino acids, 1% glutamine, and 1% penicillin and streptomycin. The filamin-1-deficient human melanoma cell line M2 and a reconstituted line A7 (27) (kindly donated by T. P Stossel) were cultured in Eagle's medium (BioWhitaker), supplemented with 10% fetal calf serum, 1% nonessential amino acids, 1% glutamine, and 1% penicillin and streptomycin.

Cell Lysates-- Jurkat 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). Platelet lysates were prepared as described previously (21).

Subcellular fractionation of Jurkat cells was performed after surface biotinylation. The cells were washed three times in Hepes-saline (200 mM Hepes, 12 mM CaCl₂·2H₂O, 16 mM MgSO₄, pH 7.3-7.4), suspended in 20 mM Hepes, and homogenized with a Dounce homogenizer. An equal quantity of buffer B (20 mM Hepes, 0.5 M sucrose, 10 mM MgCl₂, 0.1 M KCl, 2 mM CaCl₂·H₂O with protease inhibitors) was added to the homogenate, and the mixture was centrifuged at 500 × g at 4 °C for 15 min. The supernatant was collected and centrifuged at 100 000 × g for 30 min in a Beckman model L7-65 centrifuge. The cytoplasmic fraction (supernatant) was removed and the membrane fraction (pellet) washed in a 1:1 mixture of 20 mM Hepes and buffer B. The membrane fraction was resuspended in buffer A, 1 mM EDTA, and protease inhibitors and centrifuged at 30,000 × g for 20 min.

Affinity Chromatography Experiments-- Recombinant proteins were expressed in BL21(DE3)pLysS cells (Novagen) and bound to His-bind resin (Novagen) through their N-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) and 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 10,000 rpm centrifugation in a microcentrifuge, the supernatant was fractionated by SDS-PAGE and analyzed by Western blotting. 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 4F2 antibody pre-bound to protein A-Sepharose beads (Amersham Pharmacia Biotech). The following day, the beads were washed three times with the immunoprecipitation buffer and heated in reducing sample buffer for SDS-PAGE under reducing conditions. Samples were separated on 4-20% SDS-polyacrylamide gels (Novex) and transferred to nitrocellulose membranes. Membranes were blocked with Tris-buffered saline, 5% nonfat milk powder 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 (9). PAC1 binding was assessed in a subset of transiently transfected py cells (cells positive for co-transfected Tac-5 as measured by 7G7B6 binding). Integrin activation was quantified as an activation index (AI) defined as (F F_o)/(F_LIBS6  F_o), in which F is the median fluorescence intensity of PAC1 binding, F_o is the median fluorescence intensity of PAC1 binding in the presence of competitive inhibitor (Ro43-5054, 1 µM), and F_LIBS6 is the maximal median fluorescence intensity of PAC1 binding in the presence of the integrin activating antibody anti-LIBS6 (2 µM). Percentage of reversal is calculated as (AI_{(x + CD98)}  AI_x)/(AI₅  AI_x). AI_x is the activation index of cells transfected with Tac-_x chimeras, AI_{(x + CD98)} is the AI of cells co-transfected with CD98 and Tac _x chimeras, and AI₅ is the AI of cells transfected with Tac-5. The x of _x can have values of 1A, 1D, and 7 for the Tac-_1A, Tac-_1D, and Tac-₇ chimeras, respectively.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CD98 Binds to the _1A Integrin Cytoplasmic Domain-- CD98 can block reduced integrin affinity caused by overexpression of free _1A cytoplasmic domains, suggesting a physical interaction between _1A and CD98 (10). To assess this potential interaction, we examined the binding of solubilized membrane proteins to the _1A cytoplasmic domain. For affinity matrices, we used model proteins in which the integrin cytoplasmic domain was joined to four heptad repeats (21). The repeats form parallel coiled-coil dimers so that the tails are dimerized and parallel. When a Jurkat cell lysate was exposed to such an affinity matrix, a cell surface polypeptide of 88 kDa bound to the _1A but not to the _IIb tail (Fig. 2A). This polypeptide was immunoprecipitated by the anti-CD98 antibody, 4F2 (Fig. 2B). Based on its mass and reactivity with anti-CD98 antibody, the _1A tail binding polypeptide was identified as CD98.

View larger version (42K): [in this window] [in a new window]   Fig. 2.   1A cytoplasmic domains bind CD98. Jurkat human T cells were surface-labeled with Sulfo-Biotin N-hydroxysuccinimide, and the cells were lysed in buffer A (see "Experimental Procedures"). Panel A depicts a reduced SDS-PAGE analysis of the biotinylated proteins that bound to Ni2+ beads, coated with model proteins containing 1A (1A) or IIb (IIb) cytoplasmic tails. Adjacent lanes show the surface proteins present in the lysate (lysate) or the ones that bound to uncoated Ni2+ beads (0). In panel B, the biotinylated surface proteins that bound to the 1A (1A) or IIb (IIb) tails or uncoated beads (0) were immunoprecipitated with CD98 antibody (IP) or a control IgG (IgG). The immunoprecipitates were fractionated by reduced SDS-PAGE, and biotinylated proteins were detected by streptavidin-peroxidase-generated chemiluminescence.

To assess the specificity of CD98 binding to  integrin tails, affinity chromatography was performed with _1D, ₃, and ₇ cytoplasmic domains. CD98 did not bind to ₇ and binding to _1D was weak and variable (Fig. 3A). In contrast, talin and filamin (Fig. 3A) bound strongly to _1D and ₇ tails, respectively, as reported (21). CD98 also bound to ₃, and binding was not altered by the presence of the _IIb cytoplasmic domain (Fig. 3B). Thus, CD98 binding to integrin tails is integrin class- and splice-variant-specific.

View larger version (36K): [in this window] [in a new window]   Fig. 3.   CD98 binds to the 1A and 3 cytoplasmic domain rather than that of 1D or 7. A, in the upper panel, surface-biotinylated Jurkat cell lysates were allowed to bind to model proteins containing the 1A, 7, 1D, or IIb integrin tails. The bound fractions were immunoprecipitated with CD98 antibody and analyzed by SDS-PAGE, as described under "Experimental Procedures." In the lower two panels, human platelet lysates were incubated with the same tail constructs, and bound proteins were fractionated by reduced SDS-PAGE and immunoblotted with antibodies to talin or to filamin. The loading of each tail was verified by Coomassie Blue staining of the model proteins eluted from the beads and fractionated by SDS-PAGE (data not shown). B, the surface-labeled Jurkat T cell lysate used in panel A was allowed to bind to model proteins containing a heterodimer of the IIb and 3 tails, or to model proteins containing only the individual tails. Bound fractions were immunoprecipitated with CD98 antibody and analyzed by SDS-PAGE, as described under "Experimental Procedures."

Differential CD98 Binding to  Integrin Tails Is Independent of Filamin and Talin Binding-- CD98 binds well to the _1A integrin cytoplasmic domain but not to those of _1D or ₇. The binding assays were performed using talin- and filamin-1-containing cell extracts. Thus, these CD98 binding differences could be due to competition for CD98 binding by filamin-1 or talin, which bind preferentially to ₇ or _1D, respectively (21). To test this possibility, we used filamin-1-deficient human melanoma cells (M2) and reconstituted cells (A7) (27) to examine the role of filamin-1 in CD98 binding. CD98 bound to the _1A tail, but not ₇, when lysates of M2 cells were used (Fig. 4A), showing that filamin-1 is not required for CD98 binding to _1A. CD98 binding to ₇ was not observed in the filamin-1 null (M2) cells. Consequently, competition with filamin-1 does not account for the failure of ₇ to bind CD98.

View larger version (37K): [in this window] [in a new window]   Fig. 4.   Differences in CD98 binding to 1A and 7 cytoplasmic domains are independent of the presence of filamin. Affinity chromatography was performed using surface-biotinylated M2 (F) or A7 (F+) cell lysates and various cytoplasmic tails (IIb, 1A, 7). Bound proteins were immunoprecipitated with anti-CD98 antibody and fractionated by reduced SDS-PAGE, and the biotinylated polypeptides were detected by streptavidin-peroxidase chemiluminescence (panel A, CD98). Lysates of A7 and M2 cells were incubated with the indicated integrin cytoplasmic tails, and bound proteins were fractionated by SDS-PAGE and immunoblotted with anti-filamin monoclonal antibody 1680 (panel A, filamin). In panel B, surface-biotinylated Jurkat cell lysates were incubated with 1A and 7 tails and their corresponding YA (1YA, 7YA) mutants. CD98 and filamin binding was assessed as described in panel A. Loading of integrin tails was equal as verified by Coomassie Blue staining (data not shown).

To examine the role of talin, we used cell membrane preparations with a greatly reduced talin content (Fig. 5A). CD98 extracted from these membranes bound _1A but not _1D cytoplasmic domains (Fig. 5B). Thus, talin does not prevent CD98 binding to _1D, nor is it required for CD98 binding to _1A.

View larger version (39K): [in this window] [in a new window]   Fig. 5.   Differing CD98 binding to 1A and 1D cytoplasmic domains is independent of talin. Jurkat cells were surface-labeled with biotin, lysed in buffer A, and fractionated into membrane and cytosolic fractions. Whole cell lysate (Total), membrane (Membrane), and cytosolic fractions (Cytsosol) were fractionated by SDS-PAGE and immunoblotted with an anti-talin antibody (panel A). The membrane fraction and whole cell lysate were incubated with IIb, 1A, or 1D integrin tails and bound CD98 was detected by immunoprecipitation as described under "Experimental Procedures" (panel B). In panel C, lysates of Jurkat cells (upper) and platelets (lower) were analyzed for binding of CD98 and talin to 1A and 1D tails, and their corresponding YA (1YA, 1DYA) mutants as described in Fig. 4. Loading of integrin tails was equal as verified by Coomassie Blue staining (data not shown).

The Y788A mutation of _1A (Fig. 1) disrupts filamin (Fig. 4B) and talin (Fig. 5C) binding (21). Similar Tyr to Ala mutations in ₇ and _1D tails, corresponding to the Y788A mutation in _1A (Fig. 1), also disrupted filamin (Fig. 4B) and talin (Fig. 5C) binding. CD98 binding to  integrin tails was not affected by Tyr to Ala mutations (Figs. 4B and 5C). The Tyr to Ala mutation introduced into _1D or ₇ did not increase CD98 binding, nor was CD98 binding reduced in the _1A(Y788A) mutant. These results confirm that talin or filamin competition does not account for the lack of CD98 binding to _1D and ₇ and that talin or filamin binding is not required for CD98 binding to the _1A cytoplasmic domain.

CD98 Binding to Integrin Cytoplasmic Domains Correlates with Complementation of Dominant Suppression-- Overexpression of isolated integrin _1A cytoplasmic domains, in the form of a Tac-_1A chimera, results in suppression of integrin activation. Dominant suppression is reversed by overexpression of CD98 (10). Tac-_1A, Tac-_1D, and Tac-₇ induced dominant suppression of integrin activation (Fig. 6A). As noted above (Fig. 3), CD98 bound poorly to _1D and ₇ tails, showing that CD98 binding is not required for dominant suppression. However, CD98 was much less effective at reversing the suppression induced by Tac-_1D and Tac-₇ (Fig. 6B). Thus the capacity of CD98 to rescue suppression correlates with its binding to the suppressive  cytoplasmic domain.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   A,  tails induce varying amounts of integrin suppression. py cells were transfected with Tac-1 (0.5 µg), Tac-1D (1.0 µg), Tac-7 (3.0 µg), or Tac-5 (1.0 µg). After 24 h, cells were collected and analyzed for PAC1 binding to the Tac-positive subset of cells. The activation index was calculated for cells expressing different amounts of each Tac chimera. Activation index is defined as 100(Fo- FR)/(FLIBS6  FR), where Fo is the median fluorescence intensity of PAC1 binding; FR is the background fluorescence intensity of PAC1 binding in the presence of a competitive inhibitor (1 µM Ro43-5054), and FLIBS6 is the maximal fluorescence intensity in the presence of 2 µM anti-LIBS6, an activating monoclonal antibody. The mean ± S.D. of at least five independent experiments for each Tac chimera is shown. B, CD98 binding to  tails correlates with its ability to reverse dominant suppression. py cells were transfected with each of the Tac chimeras in the presence or absence of 4 µg of cDNA encoding full-length CD98. 24 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, which is calculated as (AIx + CD98  AIx)/(AI5  AIx). AI is the activation index, AIx is the AI of cells transfected with Tac  chimeras, AIx + CD98 the AI of cells transfected with CD98 and Tac x chimeras, and AI5 is the AI of cells transfected with the Tac-5. The x of x can have values of 1A, 1D, and 7 for the Tac-1A, Tac-1D, and Tac-7 chimeras, respectively. The expression of the Tac-1A and Tac-1D chimeras were similar (mean fluorescence intensity = 340 ± 20 and 370 ± 50 units, respectively), while Tac-7 was better expressed (mean fluorescence intensity = 530 ± 90 units). In the absence of CD98, Tac-7 (55 ± 6% suppression) inhibited activation less than Tac-1A or Tac-1D (77 ± 4% and 82 ± 7% suppression, respectively).

CD98 Binding Is Not Sufficient to Induce Dominant Suppression-- As noted above, _1A tails suppress integrin activation and bind CD98. To assess whether CD98 binding alone is sufficient to induce dominant suppression, we first examined CD98 binding to a series of _1A truncation mutants (Fig. 1). CD98 binding was lost when the C-terminal seven residues were deleted (_1AC797X)) but not when the last three amino acids were eliminated (_1A(801X)) (Fig. 7A). Despite maintaining its capacity to bind to CD98, the Tac-_1A(801X) mutant was a poor suppressor of integrin activation (Fig. 7B), and this was not due to a quantitative reduction in the association of CD98 with _1A(801X) (Fig. 7C). Furthermore, the _1A(Y788A) mutant, which also bound CD98 (Figs. 4 and 5), failed to suppress integrin activation (Fig. 7B). Consequently, integrin  cytoplasmic domain binding to CD98 is not sufficient to induce dominant suppression.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   A, binding of CD98 to truncated 1A cytoplasmic domains. Truncation mutants of 1A cytoplasmic tails were made as described under "Experimental Procedures." Lysates of surface-biotinylated Jurkat cells were incubated overnight with affinity matrices containing 1A, 1A(779X), 1A(783X), 1A(791X), 1A(797X), or 1A(801X) integrin tails, and the bound fractions were immunoprecipitated with CD98 antibody and analyzed by SDS-PAGE (panel A). Biotinylated polypeptides were detected by streptavidin-peroxidase chemiluminescence (CD98). Loading of the affinity matrix with each tail was verified by Coomassie Blue staining of model proteins eluted from the resin and fractionated by SDS-PAGE (Coomassie Blue). B, CD98 binding is not sufficient to induce dominant suppression. py cells were transfected with Tac-1 (0.5 µg), Tac-1A(801X) (1.0 µg), Tac-1A(Y788A) (1.0 µg), or Tac-5 (1.0 µg). After 24 h, cells were detached and analyzed for PAC1 binding to the Tac-positive subset of cells by flow cytometry as described under "Experimental Procedures." The activation index was calculated for cells expressing different amounts of each Tac chimera as described in Fig. 6. Note that the 1A(801X) and 1A(Y788A) tails induced little suppression, even though they bound CD98. C, similar association of 1A(801X) and 1A tails with CD98. Lysates of surface-biotinylated Jurkat cells were incubated overnight with affinity matrices containing the indicated quantities of 1A or 1A(801X) integrin tails, and the bound fractions were immunoprecipitated with CD98 antibody and fractionated by SDS-PAGE. Biotinylated polypeptides were detected by streptavidin-peroxidase-dependent chemiluminescence and quantified by scanning densitometry. Data are reported as percentage of binding relative to the maximal binding at 50 µg of 1A model protein.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CD98 is implicated in several cellular functions, including amino acid transport, cell fusion events, and integrin activation (12). We previously found that CD98 reverses dominant suppression of integrin function (10). We now report that: 1) CD98 associates with the _1A integrin cytoplasmic domain; 2) CD98 interacts differentially with  cytoplasmic tails in a class- and splice variant-specific manner, which is independent of the capacity of the tails to bind the cytoskeletal proteins talin and filamin; 3) CD98's capacity to associate with integrin tails correlates with its ability to overcome dominant suppression of integrin activation; 4) CD98 association with integrin tails is neither necessary nor sufficient for dominant suppression of integrin activation. Thus, the association of CD98 with integrin cytoplasmic domains may regulate the function and localization of these membrane proteins.

CD98 physically associates with _1A integrin cytoplasmic domains. This association was observed utilizing model protein mimics of dimerized integrin cytoplasmic tails, and it may account for the physical association of certain ₁ integrins with CD98.² The specificity of the interaction was confirmed by the lack of binding to mimics containing cytoplasmic domains from _IIb or several other  subunits. CD98 was added to the tails in the presence of other cellular proteins, so it remains possible that an intermediary protein is required for this interaction. However, CD98 was the only surface protein observed binding to the _1A tail (Fig. 2). Moreover, we observed CD98 binding in the absence of two known integrin binding proteins, talin and filamin (Figs. 3 and 4). CD98 failed to bind to _1D and ₇ cytoplasmic domains, even though these tails bind many of the same polypeptides as _1A (21). Thus, we conclude that CD98 associates with the _1A tail and that the interaction is potentially direct.

CD98 binds to integrin  cytoplasmic domains with unique splice variant and class specificity. CD98 bound well to the _1A tail and the ₃ tail. Binding to the _1D and ₇ tails was negligible. The specificity of CD98 binding differs markedly from the specificity of talin and filamin binding, since talin binds preferentially to the _1D tail and filamin to the ₇ tail (21). Moreover, the binding of both cytoskeletal proteins is sensitive to the Tyr substitution with Ala in the first "NPXY" (21) in _1A and, as shown here, in ₇ and _1D. Strikingly, CD98 binding was insensitive to this mutation. Finally, although the last three residues of _1A were dispensable, the last seven residues were required for binding. Thus, the features of the  tail defined here for CD98 binding identifies a novel structural specificity for integrin  tail function.

CD98 binding to  tails correlates with its capacity to complement dominant suppression. CD98 was implicated in integrin activation by its capacity to reverse the suppression of integrin activation caused by an isolated _1A cytoplasmic domain (10). In the present work, we found that CD98 binds to the _1A cytoplasmic domain, but fails to bind well to the ₇ or _1D cytoplasmic domain. Strikingly, CD98 failed to complement dominant suppression initiated by either ₇ or _1D cytoplasmic domains. Consequently, the mechanism of CODS appears to involve CD98 binding to the suppressive  tail. Furthermore, cross-linking of CD98 stimulates integrin ₃₁-dependent adhesion in small cell lung cancer cells (10) and in certain breast cancer cell lines (28) and ₁ integrin-dependent cell fusion events (29-36). Thus, our finding that CD98-₁ cytoplasmic domain interactions correlate with effects on integrin function is relevant to integrin-dependent events involved in mulinucleate giant cell formation, virally induced cell fusion, and regulation of cell adhesion.

The physical interaction of CD98 with integrin cytoplasmic domains may be involved in modulating amino acid transport regulation. CD98 is known to regulate y+L and L type amino acid transport (14, 15, 17, 18). This regulation is probably due to disulfide-bonded heterodimer formation with a variety of light chains, that resemble permease amino acid transporters (13-20). In fact, mutations in one of these light chains (15) are a likely cause of lysinuric protein intolerance (37). CD98 may function to regulate both the expression and localization of its light chains (18). In certain cells CD98 has a basolateral localization (38). _1A integrins also manifest basolateral polarization in many cells (39, 40), probably due to interactions with underlying matrix components (41) or recruitment to lateral cell contacts (42). It is noteworthy that ₇ integrins are primarily involved in lymphocyte homing and _1D integrins primarily form mechanical linkages in striated and cardiac muscle (43, 44). Thus, the failure of these cytoplasmic domains to bind to CD98 correlates well with their lack of a role in establishing polarity in epithelial or mesenchymal cells. Consequently, the physical association of CD98 with _1A integrin cytoplasmic domains may participate in the polarization and regulation of amino acid transporters and to modulate the function of certain integrins.

	ACKNOWLEDGEMENTS

We thank our colleagues for their generosity in providing the reagents listed under "Experimental Procedures." We thank Drs. Thomas Stossel and Sandy Shattil for critical reviews of the manuscript.

	FOOTNOTES

^* This work was supported in part by Grants HL48728 and AR27214 from the National Institutes of Health. This is publication 12537-VB from the Scripps Research Institute.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.

Fellow of the National Kidney Foundation.

^§ These authors contributed equally to this work.

^¶ Supported by United States Army Medical Research and Material Command Grant DAMD 17-97-1-7056.

Wellcome Trust International Prize Traveling Fellow.

^** Supported by a National Service Research Award IF32HL 09922-01.

To whom correspondence should be addressed: Dept. of Vascular Biology, Scripps Research Inst., 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-784-7143; Fax: 619-784-7343; E-mail: ginsberg@ scripps.edu.

² M. Hemler, personal communication.

	ABBREVIATIONS

The abbreviations used are: CODS, complementation of dominant suppression; PAGE, polyacrylamide gel electrophoresis; Pipes, 1,4-piperazinediethanesulfonic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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