Mast cell surfaceome characterization reveals CD98 heavy chain is critical for optimal cell function.

BACKGROUND: Mast cells are involved in many distinct pathologic conditions, suggesting that they recognize and respond to various stimuli and thus require a rich repertoire of cell surface proteins. However, mast cell surface proteomes have not been comprehensively characterized. OBJECTIVE: We aimed to further characterize the mast cell surface proteome to obtain a better understanding of how mast cells function in health and disease. METHODS: We enriched for glycosylated surface proteins expressed in mouse bone marrow-derived cultured mast cells (BMCMCs) and identified them using mass spectrometry analysis. The presence of novel surface proteins in mast cells was validated by real-time quantitative PCR and flow cytometry analysis in BMCMCs and peritoneal mast cells (PMCs). We developed a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing approach to disrupt genes of interest in BMCMCs. RESULTS: The glycoprotein enrichment approach resulted in the identification of 1270 proteins in BMCMCs, 378 of which were localized to the plasma membrane. The most common protein classes among plasma membrane proteins were small GTPases, receptors, and transporters. One such cell surface protein was CD98 heavy chain (CD98hc), encoded by the Slc3a2 gene. Slc3a2 gene disruption resulted in a significant reduction in CD98hc expression, adhesion, and proliferation. CONCLUSIONS: Glycoprotein enrichment coupled with mass spectrometry can be used to identify novel surface molecules in mast cells. Moreover, CD98hc plays an important role in mast cell function.

CD98hc (SLC3A2) sustains amino acid and nucleotide availability for cell cycle progression.

CD98 heavy chain (CD98hc) forms heteromeric amino acid (AA) transporters by interacting with different light chains. Cancer cells overexpress CD98hc-transporters in order to meet their increased nutritional and antioxidant demands, since they provide branched-chain AA (BCAA) and aromatic AA (AAA) availability while protecting cells from oxidative stress. Here we show that BCAA and AAA shortage phenocopies the inhibition of mTORC1 signalling, protein synthesis and cell proliferation caused by CD98hc ablation. Furthermore, our data indicate that CD98hc sustains glucose uptake and glycolysis, and, as a consequence, the pentose phosphate pathway (PPP). Thus, loss of CD98hc triggers a dramatic reduction in the nucleotide pool, which leads to replicative stress in these cells, as evidenced by the enhanced DNA Damage Response (DDR), S-phase delay and diminished rate of mitosis, all recovered by nucleoside supplementation. In addition, proper BCAA and AAA availability sustains the expression of the enzyme ribonucleotide reductase. In this regard, BCAA and AAA shortage results in decreased content of deoxynucleotides that triggers replicative stress, also recovered by nucleoside supplementation. On the basis of our findings, we conclude that CD98hc plays a central role in AA and glucose cellular nutrition, redox homeostasis and nucleotide availability, all key for cell proliferation.

SLC3A2/CD98hc, autophagy and tumor radioresistance: a link confirmed.

SLC3A2/CD98hc (solute carrier family 3 member 2) and its light chain subunits constitute the heterodimeric transmembrane complexes that mediate amino acid transport and regulate MTOR and macroautophagy/autophagy. Despite the proven tumorigenic role of SLC3A2 in a number of cancers including head and neck squamous cell carcinomas (HNSCC), the link between SLC3A2, autophagy regulation and tumor radioresistance remained elusive. In a recently published study we demonstrated that low levels of SLC3A2 and SLC7A5/LAT1 protein expression significantly correlate with good clinical prognosis in locally advanced HNSCC treated with primary radiochemotherapy. The SLC3A2-deficient HNSCC cells show a higher radiosensitivity and increased autophagy levels. We found that autophagy activation is a tumor survival strategy to overcome nutrient stress by lack of SLC3A2 and to withstand radiation-mediated cell damage. Inhibition of the autophagy activation in SLC3A2 knockout HNSCC cells by knockdown of ATG5 expression or treatment with bafilomycin A1 results in radiosensitivity. Consequently, the expression levels of ATG5 correlates with overall survival in HNSCC patients, and autophagy inhibition in combination with SLC3A2-targeted therapy can be a promising strategy for HNSCC radiosensitization. Abbreviations: CD98hc: CD98 heavy chain CSC cancer stem cells; EAA: essential amino acids; GSH: glutathione; MTOR: mammalian target of rapamycin; HNSCC: head and neck squamous cell carcinoma; RCTx: primary radiochemotherapy; PORT-C: postoperative radiochemotherapy; ROS: reactive oxygen species; SLC3A2: solute carrier family 3 member 2; TCA cycle: tricarboxylic acid cycle.

Negative regulation of amino acid signaling by MAPK-regulated 4F2hc/Girdin complex.

Amino acid signaling mediated by the activation of mechanistic target of rapamycin complex 1 (mTORC1) is fundamental to cell growth and metabolism. However, how cells negatively regulate amino acid signaling remains largely unknown. Here, we show that interaction between 4F2 heavy chain (4F2hc), a subunit of multiple amino acid transporters, and the multifunctional hub protein girders of actin filaments (Girdin) down-regulates mTORC1 activity. 4F2hc interacts with Girdin in mitogen-activated protein kinase (MAPK)- and amino acid signaling-dependent manners to translocate to the lysosome. The resultant decrease in cell surface 4F2hc leads to lowered cytoplasmic glutamine (Gln) and leucine (Leu) content, which down-regulates amino acid signaling. Consistently, Girdin depletion augments amino acid-induced mTORC1 activation and inhibits amino acid deprivation-induced autophagy. These findings uncovered the mechanism underlying negative regulation of amino acid signaling, which may play a role in tightly regulated cell growth and metabolism.

CD98hc (SLC3A2) drives integrin-dependent renal cancer cell behavior.

BACKGROUND: Overexpression of CD98hc (SLC3A2) occurs in a variety of cancers and is suspected to contribute to tumor growth. CD98, a heterodimeric transmembrane protein, physically associates with certain integrin ß subunit cytoplasmic domains via its heavy chain, CD98hc. CD98hc regulates adhesion-induced intracellular signal transduction via integrins, thereby, affecting cell proliferation and clonal expansion. Disruption of CD98hc led to embryonic lethality in mice (E 3.5 and E 9.5) and CD98hc -/- embryonic stem cell transplantation failed to form teratomas, while CD98hc over-expression in somatic cells resulted in anchorage-independent growth. However, it is unclear whether interference with CD98hc expression tumor cell behavior. METHODS: Renal cell cancer cell lines have been used to determine the effect of CD98hc expression on cancer cell behavior using cell adhesion, cell trans-migration and cell spreading assays. Flow cytometric analysis was performed to study the rate of apoptosis after detachment or serum starvation. shRNA-lentiviral constructs were used to stably knockdown or reconstitute full length or mutated CD98hc. The role of CD98 as a promotor of tumorigenesis was evaluated using an in in vivo tumor transplantation animal model. Immunohistochemical analysis was performed to analyze cell proliferation and CD98 expression in tumors. RESULTS: This report shows that CD98hc silencing in clear cell renal cancer cells reverts certain characteristics of tumorigenesis, including cell spreading, migration, proliferation and survival in vitro, and tumor growth in vivo. Acquisition of tumorigenic characteristics in clear cell renal cancer cells occurred through the integrin binding domain of CD98hc. A CD98hc/integrin interaction was required for adhesion-induced sustained FAK phosphorylation and activation of the major downstream signaling pathways PI3k/Akt and MEK/ERK, while overexpression of a constitutive active form of FAK rescued the CD98hc deficiency. CONCLUSIONS: In this study we demonstrate that loss of CD98hc blocks tumorigenic potential in renal cell cancer.

Manipulation of CD98 resolves type 1 diabetes in nonobese diabetic mice.

The interplay of CD4(+) and CD8(+) T cells targeting autoantigens is responsible for the progression of a number of autoimmune diseases, including type 1 diabetes mellitus (T1D). Understanding the molecular mechanisms that regulate T cell activation is crucial for designing effective therapies for autoimmune diseases. We probed a panel of Abs with T cell-modulating activity and identified a mAb specific for the H chain of CD98 (CD98hc) that was able to suppress T cell proliferation. The anti-CD98hc mAb also inhibited Ag-specific proliferation and the acquisition of effector function by CD4(+) and CD8(+) T cells in vitro and in vivo. Injection of the anti-CD98hc mAb completely prevented the onset of cyclophosphamide-induced diabetes in NOD mice. Treatment of diabetic NOD mice with anti-CD98hc reversed the diabetic state to normal levels, coincident with decreased proliferation of CD4(+) T cells. Furthermore, treatment of diabetic NOD mice with CD98hc small interfering RNA resolved T1D. These data indicate that strategies targeting CD98hc might have clinical application for treating T1D and other T cell-mediated autoimmune diseases.

CD98hc (SLC3A2) is a key regulator of keratinocyte adhesion.

BACKGROUND: Adhesion of keratinocytes is crucial for maintaining the integrity of the skin, as demonstrated by the number of dermatological disorders of genetic origin that are associated with a defect of basal keratinocyte adhesion. Integrins are the main component of the molecular networks involved in this phenomenon, but there are many others. In a recent description of proteins associated to caveolae at the plasma membrane of human basal epidermal cells, we demonstrated that CD98hc is localized with ß1 integrin. OBJECTIVES: We investigated the CD98hc proteins interactions and the role of CD98hc in keratinocyte adhesion. METHODS: CD98hc protein interaction was identified following co-immunoprecipitation and proteomic analysis using LTQ-FT mass spectrometer. Extinction of CD98hc gene expression using specific short hairpin RNA or over-expression of CD98hc lacking the ß1 integrin binding site was used to evaluate the role of this protein in keratinocyte fate. RESULTS: We show that CD98hc forms molecular complexes with ß1 and ß4 integrins in primary human keratinocytes and, using immunofluorescence, that these complexes are localized at the plasma membrane, in keeping with a role in adhesion. We confirmed that this protein is a key player of keratinocyte adhesion because in absence of interaction between CD98hc and integrins, ß1 integrin failed to translocate from the cytoplasm to the plasma membrane and keratinocytes expressed epidermal differentiation markers. CONCLUSIONS: All these data strongly suggested that CD98hc is involved in integrin trafficking and by consequence, in keratinocyte adhesion and differentiation.

Arginine transport in human erythroid cells: discrimination of CAT1 and 4F2hc/y+LAT2 roles.

Since arginine metabolites, such as nitric oxide and polyamines, influence the expression of genes involved in erythroid differentiation, the transport of the cationic amino acid may play an important role in erythroid cells. However, available data only concern the presence in these cells of CAT1 transporter (system y(+)), while no information exists on the role of the heterodimeric transporters of system y(+)L (4F2hc/y(+)LAT1 and 4F2hc/y(+)LAT2) which operates transmembrane arginine fluxes cis-inhibited by neutral amino acids in the presence of sodium. Using erythroleukemia K562 cells and normal erythroid precursors, we demonstrate here that arginine transport in human erythroid cells is due to the additive contributions of a leucine-sensitive and leucine-insensitive component. In both cell types, leucine inhibition of arginine influx is much less evident in the absence of sodium, a hallmark of system y(+)L. In K562 cells, N-ethylmaleimide, a known inhibitor of CAT transporters (system y(+)), suppresses only a fraction of arginine influx corresponding to leucine-insensitive uptake. Moreover, in Xenopus oocytes coexpressing 4F2hc and y(+)LAT2, leucine exerts a marked inhibition of arginine transport, partially dependent on sodium, while no inhibition is seen in oocytes expressing CAT1. Lastly, silencing of SLC7A6, the gene for y(+)LAT2, lowers arginine transport and doubles the intracellular content of the cationic amino acid in K562 cells. We conclude that arginine transport in human erythroid cells is due to both system y(+) (CAT1 transporter) and system y(+)L (4F2hc/y(+)LAT2 isoform), which mainly contribute, respectively, to the influx and to the efflux of the cationic amino acid.

Drosophila expresses a CD98 transporter with an evolutionarily conserved structure and amino acid-transport properties.

Mammalian CD98 heterodimeric amino acid transporters consist of a promiscuous single-pass transmembrane glycoprotein, CD98hc (CD98 heavy chain), and one of six multipass transmembrane proteins or 'light chains'. The heterodimeric complexes of CD98hc and the light chains LAT1 (L-type amino acid transporter 1) or LAT2 specifically promote sodium-independent System L exchange of neutral amino acids, including leucine. CD98hc is also implicated in other processes, including cell fusion, cell adhesion and activation of TOR (target of rapamycin) signalling. Surprisingly, recent reports suggested that insects lack a membrane-bound CD98hc, but in the present study we show that Drosophila CG2791 encodes a functional CD98hc orthologue with conservation in intracellular, transmembrane and extracellular domains. We demonstrate by RNA-interference knockdown in Drosophila Schneider cells that CG2791 and two Drosophila homologues of the mammalian CD98 light chains, Mnd (Minidiscs) and JhI-21, are required for normal levels of System L transport. Furthermore, we show that System L activity is increased by methoprene, an analogue of the developmentally regulated endocrine hormone juvenile hormone, an effect that is potentially mediated by elevated Mnd expression. Co-expression of CG2791 and JhI-21, but not CG2791 and Mnd, in Xenopus oocytes mediates System L transport. Finally, mapping of conserved sequences on to the recently determined crystal structure of the human CD98hc extracellular domain highlights two conserved exposed hydrophobic patches at either end of the domain that are potential protein-protein-interaction surfaces. Therefore our results not only show that there is functional conservation of CD98hc System L transporters in flies, but also provide new insights into the structure, functions and regulation of heterodimeric amino acid transporters.

Inhibition of system L (LAT1/CD98hc) reduces the growth of cultured human breast cancer cells.

It has been suggested that system L (LAT1/CD98hc) is up-regulated in cancer cells, including breast tumour cells, and is therefore a promising molecular target to inhibit or limit tumour cell growth. In view of this, we have examined the effect of BCH and other inhibitors of system L on the growth of MCF-7, ZR-75-1 and MDA-MB-231 cells. Treating cells with BCH markedly inhibited the metabolism of WST-1 in a dose-dependent fashion. Similarly, melphalan and D-leucine inhibited the growth of cultured breast cancer cells whereas MeAIB, an inhibitor of system A, was without effect. The effects of BCH and melphalan on cell growth were non-additive suggesting that both compounds were acting at a single locus. The results indicate that system L is required to maintain MCF-7, ZR-75-1 and MDA-MB-231 cell growth and support the notion that LAT1/CD98hc may be a suitable target to inhibit breast cancer progression.

CD98hc (SLC3A2) interaction with the integrin beta subunit cytoplasmic domain mediates adhesive signaling.

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 (Thr(755)-Thr(802)) 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.

Apical and basolateral 4F2hc and the amino acid exchange of L-DOPA in renal LLC-PK1 cells.

The present study aimed to examine the presence and define the role of 4F2hc, a glycoprotein associated with the LAT2 amino acid transporter, in L-DOPA handling by LLC-PK1 cells. For this purpose we have measured the activity of the apical and basolateral inward and outward transport of [14C] L-DOPA in cell monolayers and examined the influence of 4F2hc antisense oligonucleotides on [14C] L-DOPA handling. The basal-to-apical transepithelial flux of [14C] L-DOPA progressively increased with incubation time and was similar to the apical-to-basal transepithelial flux. The spontaneous and the L-DOPA-stimulated apical fractional outflow of [14C] L-DOPA were identical to that through the basal cell side. The L-DOPA-induced fractional outflow of [14C] L-DOPA through the apical or basal cell side was accompanied by marked decreases in intracellular levels of [14C] L-DOPA. In cells treated with an antisense oligonucleotide complementary to 4F2hc mRNA for 72 h, [14C] L-DOPA inward transport and 4F2hc expression were markedly reduced. Treatment with the 4F2hc antisense oligonucleotide markedly decreased the spontaneous fractional outflow of [14C] L-DOPA through the apical or the basal cell side. It is likely that the Na+-independent and pH-sensitive uptake of L-DOPA include the hetero amino acid exchanger LAT2/4F2hc, which facilitates the trans-stimulation of L-DOPA and its outward transfer at both the apical and basal cell sides.

CD98hc (SLC3A2) mediates integrin signaling.

Integrins regulate cellular behaviors through signaling pathways, including Rho GTPases and kinases. CD98 heterodimers, comprised of a heavy chain (CD98hc, SLC3A2) and one of several light chains, interact with integrins through CD98hc. CD98hc overexpression leads to anchorage-independent cell growth and tumorigenesis in 3T3 fibroblasts and activates certain integrin-regulated signaling pathways. To establish the biological function of CD98hc, we disrupted the gene and analyzed CD98hc-null cells. Here we report that CD98hc contributes to integrin-dependent cell spreading, cell migration, and protection from apoptosis. Furthermore, CD98hc is required for efficient adhesion-induced activation of Akt and Rac GTPase, major contributors to the integrin-dependent signals involved in cell survival and cell migration. CD98 promotes amino acid transport through its light chains; however, a CD98hc mutant that interacts with beta1 integrins, but not CD98 light chains, restored integrin-dependent signaling and protection from apoptosis. beta1 integrins are involved in the pathogenesis of certain cancers. CD98hc deletion markedly impaired the ability of embryonic stem cells to form teratocarcinomas in mice; teratocarcinoma formation was reconstituted by reexpression of CD98hc or of the mutant that interacts exclusively with integrins. Thus, CD98hc is an integrin-associated protein that mediates integrin-dependent signals, which promote tumorigenesis.

CD98hc (SLC3A2) interaction with beta 1 integrins is required for transformation.

CD98hc (SLC3A2) constitutively and specifically associates with beta(1) integrins and is highly expressed on the surface of human tumor cells irrespective of the tissue of origin. We have found here that expression of CD98hc promotes both anchorage- and serum-independent growth. This oncogenic activity is dependent on beta(1) integrin-mediated phosphoinositol 3-hydroxykinase stimulation and the level of surface expression of CD98hc. Using chimeras of CD98hc and the type II membrane protein CD69, we show that the transmembrane domain of CD98hc is necessary and sufficient for integrin association in cells. Furthermore, CD98hc/beta(1) integrin association is required for focal adhesion kinase-dependent phosphoinositol 3-hydroxykinase activation and cellular transformation. Amino acids 82-87 in the putative cytoplasmic/transmembrane region appear to be critical for the oncogenic potential of CD98hc and provide a novel mechanism for tumor promotion by integrins. These results explain how high expression of CD98hc in human cancers contributes to transformation; furthermore, the transmembrane association of CD98hc and beta(1) integrins may provide a new target for cancer therapy.

Lysophosphatidylcholine enhances cytokine production of endothelial cells via induction of L-type amino acid transporter 1 and cell surface antigen 4F2.

OBJECTIVE: A diverse range of lipid oxidation products detected in oxidized low-density lipoprotein (oxLDL) and atherosclerotic lesions are capable of eliciting biological responses in vascular cells. We performed DNA microarray experiments to explore novel responses of human umbilical vein endothelial cells (HUVECs) to oxLDL and its components. METHODS AND RESULTS: cDNA microarray analysis showed that oxLDL, lysophosphatidylcholine (LysoPC), 4-hydroxy-2-nonenal, and oxysterols altered gene expression specifically, but some genes were commonly induced in HUVECs. Solute carrier family 3 member 2 and family 7 member 5, encoding the heavy chain of the cell surface antigen 4F2 (4F2hc) and the L-type amino acid transporter 1 (LAT1), respectively, were induced by oxLDL and many oxidation products. LAT1 requires 4F2hc to form a heterodimeric functional complex to transport neutral amino acids into the cell. LysoPC increased membrane protein levels of LAT1 confirmed by Western blot analysis and also uptake of L-[(14)C]leucine, which was inhibited by a competitive inhibitor for LAT1. The release of interleukin 6 (IL-6) and IL-8 was increased in LysoPC-treated cells and was attenuated by the LAT1 inhibitor. CONCLUSIONS: These findings suggest that an increase in uptake of neutral amino acids induced by LysoPC results in enhancement of inflammatory responses of endothelial cells.

The ancillary proteins of HATs: SLC3 family of amino acid transporters.

The heteromeric amino acid transporters (HATs) are composed of a light and a heavy subunit linked by a disulfide bridge. The heavy subunits are the SLC3 members (rBAT and 4F2hc), whereas the light subunits are members of the SLC7 family of amino acid transporters. SLC3 proteins are type II membrane glycoproteins (i.e., one single transmembrane domain and the C-terminus located outside the cell) with a bulky extracellular domain that shows homology with alpha-glucosidases. rBAT heterodimerizes with b(0,+)AT (SLC7A9) constituting the amino acid transport b(0,+), the main system responsible for the apical reabsorption of cystine in kidney. The defect in this system causes cystinuria, the most common primary inherited aminoaciduria. 4F2hc subserves various amino acid transport systems by dimerization with different SLC7 proteins. The main role of SLC3 proteins is to help routing of the holotransporter to the plasma membrane. A working model for the biogenesis of HATs based on recent data on the rBAT/b(0,+)AT heterodimeric complex is presented. 4F2hc is a multifunctional protein, and in addition to its role in amino acid transport, it may be involved in other cellular functions. Studies on two SLC7 members (Asc-2 and AGT1) demonstrate heterodimerization with unknown heavy subunits.

Expression of L-type amino acid transporter 1 (LAT1) and 4F2 heavy chain (4F2hc) in liver tumor lesions of rat models.

BACKGROUND AND OBJECTIVES: It has been said that amino acid transporters play an important role in supplying nutrition to cells and for cell proliferation. In this study, we examined whether LAT1 and 4F2hc are closely related to tumor growth. METHODS: Rat colon cancer cells (RCN-9) were injected into the spleen of 12 male rats (inbred F344/DuCrj). In each rat, liver samples including tumor lesions were immunostained with anti-LAT1 and anti-4F2hc antibodies. The staining area of LAT1 and 4F2hc tumor lesions was calculated by computer analysis. RESULTS: Sixty-eight tumor nodules were observed in 12 livers. Out of the 68 tumor nodules, 36 nodules (52.9%) indicated a positive staining of LAT1 and 32 (47.1%) had a negative staining of LAT1. However, the LAT1 expression was scarcely detected in non-tumor areas. In terms of the 4F2hc expression, there were 56 nodules (82.4%) with 4F2hc positive and 12 (17.6) with 4F2hc negative. In addition, the expression of 4F2hc in non-tumor areas was almost the same as the expression of 4F2hc in tumor lesions. The average tumor size of the group with LAT1 positive and 4F2hc positive (n = 31) was 0.845 +/- 0.232 mm(2), which was significantly larger than that of the group with LAT1 negative and 4F2hc negative group (n = 7) (0.090 +/- 0.028 mm(2)) or the group with LAT1 positive and 4F2hc negative (n = 5) (0.097 +/- 0.025 mm(2)), respectively (P = 0.0017, P = 0.007). CONCLUSION: LAT1 was related to tumor growth. We think that LAT1 can possibly enhance its ability to promote tumor growth in cooperation with 4F2hc.