Cell surface CD147 protein promotes production of matrix metalloproteinases and hyaluronan, associates with monocarboxylate transporters and integrins, and is involved in reproductive, neural, inflammatory, and tumor functions. Here we combined covalent cross-linking, mass spectrometric protein identification, and co-immunoprecipitation to show selective CD147 association with three major types of transporters (CD98 heavy chain (CD98hc)-L-type amino acid transporter, ASCT2, and monocarboxylate transporters) as well as a regulator of cell proliferation (epithelial cell adhesion molecule). In the assembly of these multicomponent complexes, CD147 and CD98hc play a central organizing role. RNA interference knock-down experiments established a strong connection between CD147 and CD98hc expression and a strong positive association of CD147 (and CD98hc) with cell proliferation. As the CD147-CD98hc complex and proliferation diminished, AMP-activated protein kinase (a cellular "fuel gauge") became activated, indicating a disturbance of cellular energy metabolism. Our data point to a CD147-CD98 cell surface supercomplex that plays a critical role in energy metabolism, likely by coordinating transport of lactate and amino acids. Furthermore we showed how covalent cross-linking, together with mass spectrometry, can be used to identify closely associated transmembrane proteins. This approach should also be applicable to many other types of transmembrane proteins besides those associated with CD98hc and CD147. (+info)
Amino acids as modulators of endothelium-derived nitric oxide.
(18/54)
To examine the mechanisms whereby amino acids modulate nitric oxide (NO) production and blood flow in the renal vasculature, chemiluminescence techniques were used to quantify NO in the renal venous effluent of the isolated, perfused rat kidney as different amino acids were added to the perfusate. The addition of 10(-4) or 10(-3) M cationic amino acids (l-ornithine, l-lysine, or l-homoarginine) or neutral amino acids (l-glutamine, l-leucine, or l-serine) to the perfusate decreased NO and increased renal vascular resistance. Perfusion with anionic amino acids (l-glutamate or l-aspartate) had no effect on either parameter. The effects of the cationic and neutral amino acids were reversed with 10(-3) M l-arginine and prevented by deendothelialization or NO synthase inhibition. The effects of the neutral amino acids but not the cationic amino acids were dependent on extracellular sodium. Cationic and neutral amino acids also decreased calcimycin-induced NO, as assessed by DAF-FM-T fluorescence, in cultured EA.hy926 endothelial cells. Inhibition of system y(+) or y(+)L by siRNA for the cationic amino acid transporter 1 or the CD98/4F2 heavy chain diminished the NO-depleting effects of these amino acids. Finally, transport studies in cultured cells demonstrated that cationic or neutral amino acids in the extracellular space stimulate efflux of l-arginine out of the cell. Thus the present experiments demonstrate that cationic and neutral amino acids can modulate NO production in endothelial cells by altering cellular l-arginine transport through y(+) and y(+)L transport mechanisms. (+info)
Expression of the cystine-glutamate exchanger (xc-) in retinal ganglion cells and regulation by nitric oxide and oxidative stress.
(19/54)
The cystine-glutamate exchanger, system x(c)(-), mediates the Na(+)-independent exchange of cystine into cells, coupled to the efflux of intracellular glutamate. System x(c)(-) plays a critical role in glutathione homeostasis. Early studies of brain suggested that system x(c)(-) was present primarily in astrocytes but not neurons. More recent work indicates that certain brain neurons have an active system x(c)(-). In the retina, system x(c)(-) has been demonstrated in Muller and retinal pigment epithelial cells. We have recently suggested that two protein components of system x(c)(-), xCT and 4F2hc, are present in ganglion cells of the intact retina. Here, we have used (1) molecular and immunohistochemical assays to determine whether system x(c)(-) is present in primary ganglion cells isolated from neonatal mouse retinas and (2) functional assays to determine whether its activity is regulated by oxidative stress in a retinal ganglion cell line (RGC-5). Primary mouse ganglion cells and RGC-5 cells express xCT and 4F2hc. RGC-5 cells take up [(3)H]glutamate in the absence of Na(+), and this uptake is blocked by known substrates of system x(c)(-) (glutamate, cysteine, cystine, quisqualic acid). Treatment of RGC-5 cells with NO and reactive oxygen species donors leads to increased activity of system x(c)(-) associated with an increase in the maximal velocity of the transporter with no significant change in the substrate affinity. This is the first report of system x(c)(-) in primary retinal ganglion cells and RGC-5 cells. Oxidative stress upregulates this transport system in RGC-5 cells, and the process is associated with an increase in xCT mRNA and protein but no change in 4F2hc mRNA or protein. (+info)
Possible activation of murine T lymphocyte through CD98 is independent of interleukin 2/interleukin 2 receptor system.
(20/54)
CD98 is a widely expressed cell surface heterodimeric protein of 125 kDa. Its expression is upregulated during lymphocyte activation induced by mitogen, superantigen, conventional antigen, and a combination of phorbol myristate acetate (PMA) and ionomycin. However, the role of CD98 in the immune system is not so well understood. The role of CD98 in murine T lymphocyte proliferation was investigated, especially in correlation with the interleukin 2 (IL-2)/interleukin 2 receptor (IL-2R) system. Monoclonal antibody (mAb) directed against murine CD98 heavy chain (mCD98HC) suppressed the proliferation of lymphocytes stimulated with concanavalin A (Con A). Anti-mCD98HC mAb did not suppress the expression of IL-2Ralpha. Anti-IL-2Ralpha mAb, which suppressed DNA synthesis, did not inhibit the expression of CD98HC. Murine IL-2 (recombinant), which induced considerable DNA synthesis by lymphocytes stimulated with a sub-optimal dose of Con A, did not induce CD98HC expression in lymphocytes. In addition, anti-mCD98HC mAb did not inhibit the production of IL-2 by lymphocyte stimulated with Con A. Taken together with these findings, it was speculated that the CD98 system is independent of the IL-2/IL-2R system in murine T lymphocyte activation. (+info)
The structural and functional units of heteromeric amino acid transporters. The heavy subunit rBAT dictates oligomerization of the heteromeric amino acid transporters.
(21/54)
Heteromeric amino acid transporters are composed of a catalytic light subunit and a heavy subunit linked by a disulfide bridge. We analyzed the structural and functional units of systems b0,+ and xC-, formed by the heterodimers b0,+ AT-rBAT and xCT-4F2hc, respectively. Blue Native gel electrophoresis, cross-linking, and fluorescence resonance energy transfer in vivo indicate that system b0,+ is a heterotetramer [b0,+ AT-rBAT]2, whereas xCT-4F2hc seems not to stably or efficiently oligomerize. However, substitution of the heavy subunit 4F2hc for rBAT was sufficient to form a heterotetrameric [xCT-rBAT]2 structure. The functional expression of concatamers of two light subunits (which differ only in their sensitivity to inactivation by a sulfhydryl reagent) suggests that a single heterodimer is the functional unit of systems b0,+ and xC-. (+info)
Expression of amino acid transporter LAT1 and 4F2hc in the healing process after the implantation of a tooth ash and plaster of Paris mixture.
(22/54)
BACKGROUND: In order to elucidate the expression pattern of L-type amino acid transporter 1 (LAT1) in the bone formation process, the expressions of LAT1 and its subunit 4F2 heavy chain (4F2hc) were investigated in the healing process after the implantation of a tooth ash-plaster of Paris mixture in rats with calvarial osseous defect. MATERIALS AND METHODS: Circular calvarial defects (8 mm in diameter) were made midparietally. The rats were divided into 2 groups, 1 control group and 1 experimental group. In the control group, the defect was only covered with a soft tissue flap (control group); in the experimental group, it was filled with a mixture of tooth ash and plaster of Paris (2:1 by weight; mixture group). The rats were sacrificed at 1, 2, 4 and 8 weeks after operation and RT-PCR and immunohistochemical analyses were performed. RESULTS: In the RT-PCR analysis, the expressions of the LAT1 and 4F2hc mRNAs were slightly stronger in the experimental group than in the control group. In the immunohistochemical analysis, at 1 week after operation, the LAT1 protein and its subunit 4F2hc protein were mainly expressed in the osteoblasts, osteocytes and interstitial tissues of the area around the defect and the inner part of newly forming bone in both groups. The expressions of LAT1 and 4F2hc proteins were decreased at 2 and 4 weeks after operation. The LAT1 and 4F2hc proteins were scarcely expressed at 8 weeks after operation in both groups. The expressions of LAT1 and 4F2hc proteins were slightly stronger in the mixture group than in the control group. CONCLUSION: These results suggest that the LAT1 and its subunit 4F2hc are highly expressed in the early stage of new bone formation and may have an important role in providing cells with neutral amino acids, including several essential amino acids, at that stage. (+info)
Functional characterization of two S-nitroso-L-cysteine transporters, which mediate movement of NO equivalents into vascular cells.
(23/54)
System L amino acid transporters have been shown to be responsible for cellular uptake of S-nitroso-L-cysteine (l-CSNO). In this study, we examined the characteristics of L-CSNO uptake in Xenopus laevis oocytes expressing system L transporters and found that uptake increased only when both 4F2 heavy chain (4F2HC) and either L-type amino acid transporter 1 (LAT1) or LAT2 light chain were coexpressed. The K(m) for transport was 57 +/- 8 microM for 4F2HC-LAT1 and 520 +/- 52 microM for 4F2HC-LAT2. Vascular endothelial and smooth muscle cells were shown to express transcripts for 4F2HC and for both LAT1 and LAT2. Transport of L-CSNO into red blood cells, endothelial cells, and smooth muscle cells was inhibited by 2-aminobicyclo(2.2.1)heptane-2-carboxylic acid (BCH) and by large neutral amino acids demonstrating functional system L transporters in each cell type. Uptake of L-CSNO led to accumulation of cellular S-nitrosothiols and inhibition of both growth factor-induced ERK phosphorylation and TNF-alpha-mediated IkappaB degradation. Similar effects were seen when cells were incubated simultaneously with S-nitrosoalbumin and L-cysteine but not with d-cysteine or with S-nitrosoalbumin alone. In each case, nitrosylation of proteins and cellular responses were blocked by BCH. Together, these data suggest that transmembrane movement of nitric oxide (NO) equivalents from the plasma albumin NO reservoir is mediated by cysteine, which serves as a carrier. The mechanism requires transnitrosylation from S-nitrosoalbumin to free cysteine and activity of system L transporters, thereby providing a unique pathway for cellular responses to S-nitrosoalbumin. (+info)
Differential effect of cross-linking the CD98 heavy chain on fusion and amino acid transport in the human placental trophoblast (BeWo) cell line.
(24/54)
CD98 (otherwise known as 4F2) is an integral membrane protein with multiple functions including amino acid transport, integrin activation, cell fusion and cell activation. The molecular mechanisms coordinating these multiple functions remain unclear. We have studied CD98 heavy chain (hc) function in a human placental trophoblast cell line (BeWo). We show that cross-linking of CD98hc by incubation of cells in the presence of functional monoclonal antibodies causes cellular re-distribution of the protein from the cytoplasm to the plasma membrane as measured by flow cytometry, western blotting and quantitative immuno-electron microscopy. The latter technique also indicated that CD98hc is trafficked between cell surface and cytoplasmic pools in vesicles. Increased cell surface CD98 correlates with increased cellular fusion in BeWo cells. In addition, we show reduced LAT 1 surface expression and neutral amino acid transport in the presence of the CD98 mabs. The results thus suggest that the function of CD98 in cell fusion is distinct from its role in cellular nutrient delivery. (+info)