4F2 Antigen Research Articles

The heavy chain of 4F2 antigen promote prostate cancer progression via SKP-2

The heavy chain of 4F2 antigen promote prostate cancer progression via SKP-2

The heavy chain of 4F2 antigen promote prostate cancer progression via SKP-2

The 4F2 cell-surface antigen heavy chain (4F2hc) forms a heterodimeric complex with L-type amino acid transporter 1 (LAT1) and transports large neutral essential amino acids. However, in contrast to the traditional role of LAT1 in various cancers, the role of 4F2hc has largely remained unknown. The role of 4F2hc in prostate cancer was studied. Treatment of C4-2 cells with si4F2hc was found to suppress cellular growth, migratory and invasive abilities, with this effect occurring through the cell cycle, with a significant decrease in S phase and a significant increase in G0/G1 phase, suggesting cell cycle arrest. In addition, it was proven by RNA seq that the key to 4F2hc’s impact on cancer is SKP2. si4F2hc upregulates the protein expression of cyclin-dependent kinase inhibitors (P21cip1, P27kip1) through the downstream target SKP2. Furthermore, the expression of 4F2hc and LAT1 in prostate cancer cells suggests the importance of 4F2hc. Multivariate analysis showed that high 4F2hc expression was an independent prognostic factor for progression-free survival (HR 11.54, p = 0.0357). High 4F2hc was related to the clinical tumour stage (p = 0.0255) and Gleason score (p = 0.0035). Collectively, 4F2hc contributed significantly to prostate cancer (PC) progression. 4F2hc may be a novel marker and therapeutic target in PC.

A new monoclonal antibody, 4F2, specific for the oligodendroglial cell lineage, recognizes ATP‐dependent RNA helicase Ddx54: Possible association with myelin basic protein

Recent research in neural development has highlighted the importance of markers to discriminate phenotypic alterations of neural cells at various developmental stages. We isolated a new monoclonal antibody, 4F2, which was shown to be specific for an oligodendrocyte lineage. In primary cultures of oligodendroglial and mixed neural cells, the 4F2 antibody labeled a large proportion of Sox2(+) , Sox10(+) , A2B5(+) , NG2(+) , Olig2(+) , O4(+) , and myelin basic protein (MBP)(+) cells but did not label any GFAP(+) or NeuN(+) cells. In immunohistochemisty of rat embryos, the 4F2 antibody labeled a portion of neuroepithelial cells of the neural tube at embryonic day 9. The 4F2-positive cells were located initially in the ventricular zone as Musashi1(+) Tuj1(-) populations and distributed throughout the striatum; thereafter, they populated the whole brain and spinal cord. These cells showed ramified processes during embryonal development. The 4F2 antigen was associated with all four isoforms of MBP in coimmunoprecipitation experiments using brain homogenates or cell lysates of cultured oligodendrocytes. Immunoscreening of a brain cDNA library identified the antigen as DEAD (Asp-Glu-Ala-Asp) box polypeptide 54 (Ddx54), a member of the DEAD box family of RNA helicases involved in RNA metabolism, transcription, and translation. Cotransfection of the Ddx54 gene with MBP isoform genes increased the nuclear localization of the 21.5-kDa MBP isoform, which has been reported to function as a nuclear signal transduction molecule. These data indicate that Ddx54 might be not only a useful marker for investigating the ontogeny of oligodendrocytes but also an important factor in oligodendrocyte differentiation and myelination.

Loss of T cell CD98hc specifically ablates T cell clonal expansion and protects from autoimmunity. (102.16)

Abstract CD98hc (CD98 heavy chain, 4F2 antigen, Slc3a2) is a lymphocyte activation antigen with unclear function in T cells. Deletion of CD98hc in B cells leads to complete failure of B cell proliferation, plasma cell formation, and antibody secretion. Here we examined the role of T cell CD98 in protective and auto- immune responses by specifically deleting it in mouse T cells. Deletion of T cell CD98 prevented experimental autoimmune diabetes associated with reduced T cell clonal expansion. However, initial T cell homing to pancreatic islets was unimpaired. In contrast to B cells, CD98-null T cells showed only moderately impaired antigen-driven proliferation in vitro and nearly normal homeostatic proliferation. Furthermore, these cells were activated by antigen and produced cytokines (CD4) and efficiently killed targets (CD8). The integrin binding domain of CD98hc was necessary and sufficient for full clonal expansion, highlighting the role of adhesive signaling in T cell proliferation and autoimmune disease. When CD98-null T cells were pre-expanded in vitro, they adoptively transferred diabetes, establishing that impaired clonal expansion was responsible for protection from disease. The integrin binding domain of CD98hc is thus required for antigen-driven T cell clonal expansion in the pathogenesis of an autoimmune disease and may represent a useful therapeutic target.

Kaposi's Sarcoma-Associated Herpesvirus Forms a Multimolecular Complex of Integrins (αVβ5, αVβ3, and α3β1) and CD98-xCT during Infection of Human Dermal Microvascular Endothelial Cells, and CD98-xCT Is Essential for the Postentry Stage of Infection

Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with cell surface heparan sulfate (HS) and alpha3beta1 integrin during the early stages of infection of human dermal microvascular endothelial cells (HMVEC-d) and human foreskin fibroblasts (HFF), and these interactions are followed by virus entry overlapping with the induction of preexisting host cell signal pathways. KSHV also utilizes the amino acid transporter protein xCT for infection of adherent cells, and the xCT molecule is part of the cell surface heterodimeric membrane glycoprotein CD98 (4F2 antigen) complex known to interact with alpha3beta1 and alphaVbeta3 integrins. KSHV gB mediates adhesion of HMVEC-d, CV-1, and HT-1080 cells and HFF via its RGD sequence. Anti-alphaV and -beta1 integrin antibodies inhibited the cell adhesion mediated by KSHV-gB. Variable levels of neutralization of HMVEC-d and HFF infection were observed with antibodies against alphaVbeta3 and alphaVbeta5 integrins. Similarly, variable levels of inhibition of virus entry into adherent HMVEC-d, 293 and Vero cells, and HFF was observed by preincubating virus with soluble alpha3beta1, alphaVbeta3, and alphaVbeta5 integrins, and cumulative inhibition was observed with a combination of integrins. We were unable to infect HT1080 cells. Virus binding and DNA internalization studies suggest that alphaVbeta3 and alphaVbeta5 integrins also play roles in KSHV entry. We observed time-dependent temporal KSHV interactions with HMVEC-d integrins and CD98/xCT with three different patterns of association and dissociation. Integrin alphaVbeta5 interaction with CD98/xCT predominantly occurred by 1 min postinfection (p.i.) and dissociated at 10 min p.i., whereas alpha3beta1-CD98/xCT interaction was maximal at 10 min p.i. and dissociated at 30 min p.i., and alphaVbeta3-CD98/xCT interaction was maximal at 10 min p.i. and remained at the observed 30 min p.i. Fluorescence microscopy also showed a similar time-dependent interaction of alphaVbeta5-CD98. Confocal-microscopy studies confirmed the association of CD98/xCT with alpha3beta1 and KSHV. Preincubation of KSHV with soluble heparin and alpha3beta1 significantly inhibited this association, suggesting that the first contact with HS and integrin is an essential element in subsequent CD98-xCT interactions. Anti-CD98 and xCT antibodies did not block virus binding and entry and nuclear delivery of viral DNA; however, viral-gene expression was significantly inhibited, suggesting that CD98-xCT play roles in the post-entry stage of infection, possibly in mediating signal cascades essential for viral-gene expression. Together, these studies suggest that KSHV interacts with functionally related integrins (alphaVbeta3, alpha3beta1, and alphaVbeta5) and CD98/xCT molecules in a temporal fashion to form a multimolecular complex during the early stages of endothelial cell infection, probably mediating multiple roles in entry, signal transduction, and viral-gene expression.

Serum immunomodulatory factors in gastrointestinal diseases. A 30-50-kD serum fraction in Crohn's disease capable of modulating lymphocyte activation

We tested the hypothesis that serum factors present in Crohn's disease interfere with the process of lymphocyte activation. The mitogen-induced proliferation and the expression of early activation antigens by normal lymphocytes cultured in the presence of either Crohn's disease sera or sera from different controls were evaluated. The mitogen-induced proliferation was significantly impaired in the presence of Crohn's disease sera. These sera markedly inhibited the mitogen-induced interleukin-2 receptor (IL-2R) expression (48% inhibition), while the effect of sera on the expression of the transferrin receptor and the 4F2 antigen was much less pronounced. Diafiltration experiments showed that the inhibitory effect was confined to a 30-50-kD serum fraction. Such a serum property was not related to the patients' disease activity and disappeared after surgical removal of the affected bowel. The capability of inhibiting the mitogen-induced IL-2R expression was not restricted to Crohn's disease and was observed with sera from other inflammatory and neoplastic gastrointestinal disorders. This study indicates that a marked inhibition of the IL-2R is a mechanism underlying the immunosuppressive property of the serum in Crohn's disease and in other gastrointestinal conditions.

L-Type Amino Acid Transporter-1 Expressed in Human Astrocytomas, U343MGa

LAT1 (L-type amino acid transporter 1), one of the L-type amino acid transporters, transports the branched and aromatic amino acids. LAT1 requires the heavy chain of 4F2 antigen (4F2hc) for the functional expression as an amino acid transporter. The expression of this transporter is up-regulated in tumor cells and rapidly-growing cells to support their proliferation. Here, we studied the expression of LAT1 and 4F2hc in human cultured cells by real-time PCR and Western blot, and found that human brain astrocytomas, U343MGa, highly expressed LAT1 and 4F2hc mRNAs and proteins. The uptake of [14C]leucine by U343MGa cells is Na+-independent and inhibited by BCH (2-amino-2-norbornane carboxylic acid), and branched and aromatic amino acids, indicating that the LAT1 is expressed at the cell surface. Pulse chase labeling and surface labeling experiments of this cell line indicate that the protein synthesis of LAT1 and 4F2hc is slow, however, the heterodimeric complex assembled in the cells is very stable, and that the disulfide bond between the LAT1 and 4F2hc is not directly involved in the stability of the heterodimer.

Evaluation of CD98 Expression in Normal and Osteoarthritic Human Articular Chondrocytes.

Recent studies have provided evidence that integrins play roles in recognition of mechanical stimuli and its translation into a cellular response. Integrin signaling may be regulated by a number of mechanisms including accessory proteins such as CD98 (4F2 antigen). To determine CD98 expression by human articular chondrocytes and its involvement in human articular mechanotransduction. CD98 expression was assessed by immunostaining of cryostat sections of snap frozen articular cartilage and in cultured cells by western blotting. Chondrocytes enzymatically isolated from macroscopically normal and osteoarthritic (OA) articular cartilage were grown in short term, primary monolayer culture and used in a resting state or following mechanical stimulation at 0.33Hz. Human articular chondrocytes express CD98 and immunoreactivity revealed a similar heterogeneous pattern of CD98 in both normal and osteoarthritic (OA) human articular cartilage. No role of CD98 was detected by electrophysiological study. It appears that CD98 is expressed in a similar pattern in both normal and osteoarthritic (OA) cartilage. Although we detected no role for CD98 in chondrocyte mechanotransduction, it may be involved in other biological functions in chondrocyte intracellular signalling events.

C/ebpδ Null Mouse as a Model for the Double Knock-out of slc5a8 and slc5a12 in Kidney

slc5a8 and slc5a12 represent the high affinity and low affinity Na+/lactate co-transporters, respectively, in the kidney. Here we show that these transporters are expressed in the apical membrane of the proximal tubular cells in mouse kidney, indicating that these transporters are likely to mediate the first step in the renal reabsorption of lactate. Interestingly, the renal expression of both transporters is almost completely ablated in mice homozygous for the deletion of the transcription factor c/ebpdelta. This effect is tissue-specific since the expression of the transporters is not affected in non-renal tissues. The functional role of C/EBPdelta in the expression of SLC5A8 and SLC5A12 is demonstrable in HEK293 cells in reporter assays using gene-specific promoters. The ablation of the transporters in the kidney is accompanied by a marked increase in urinary excretion of lactate as well as a decrease in blood levels of lactate in c/ebpdelta-/- mice. These data provide evidence for an obligatory role for slc5a8 and slc5a12 in the renal absorption of lactate. In addition, we show that urinary excretion of urate is significantly elevated in c/ebpdelta-/- mice even though the expression of URAT1, the transporter responsible for the apical membrane uptake of urate in renal proximal tubule, is not altered. These data provide in vivo evidence for the functional coupling between lactate reabsorption and urate reabsorption in the kidney. Thus, the fortuitous double knock-out of slc5a8 and slc5a12 in kidney in c/ebpdelta-/- mice reveals the physiologic role of these transporters in the renal handling of lactate and urate.

Identification of LAT4, a Novel Amino Acid Transporter with System L Activity

System L amino acid transporters mediate the movement of bulky neutral amino acids across cell membranes. Until now three proteins that induce system L activity have been identified: LAT1, LAT2, and LAT3. The former two proteins belong to the solute carrier family 7 (SLC7), whereas the latter belongs to SLC43. In the present study we present a new cDNA, designated LAT4, which also mediates system L activity when expressed in Xenopus laevis oocytes. Human LAT4 exhibits 57% identity to human LAT3. Like LAT3, the amino acid transport activity induced by LAT4 is sodium-, chloride- and pH-independent, is not trans-stimulated, and shows two kinetic components. The low affinity component of LAT4 induced activity is sensitive to the sulfhydryl-specific reagent N-ethylmaleimide but not that with high affinity. Mutation in LAT4 of the SLC43 conserved serine 297 to alanine abolishes sensitivity to N-ethylmaleimide. LAT4 activity is detected at the basolateral membrane of PCT kidney cells. In situ hybridization experiments show that LAT4 mRNA is restricted to the epithelial cells of the distal tubule and the collecting duct in the kidney. In the intestine, LAT4 is mainly present in the cells of the crypt.

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

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. 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. These findings suggest that an increase in uptake of neutral amino acids induced by LysoPC results in enhancement of inflammatory responses of endothelial cells.

Multiple antigens are altered on T and B lymphocytes from peripheral blood and spleen of patients with Wiskott-Aldrich syndrome.

The gene for Wiskott-Aldrich syndrome (WAS) has been recently identified and cloned, but our knowledge of downstream events affected in WAS is limited to a few leucocyte cell surface molecules. To identify cell surface molecules whose abnormal expression could contribute to the functional impairment observed in WAS B and T lymphocytes, we studied the expression of a large panel of antigens on peripheral blood lymphoid cells (PBLC) and on isolated lymphocyte subpopulations from the spleen of WAS patients. WAS T lymphocytes from peripheral blood express increased levels of the activation antigens 4F2, CD49d, CD49e, CD53 and the activation/ memory marker CD45RO. In the spleen, however, WAS patients have more CD45RA CD4+ and CD8+ T lymphocytes than normal individuals, suggesting the selective accumulation of presumably naive cells in the WAS spleen. Interestingly, the naive phenotype of the lymphocytes that seem to accumulate in the WAS spleen is confirmed by the absence of increased expression of several activation antigens on their surface, and this correlated with their increased expression of CD43. These lymphocyte abnormalities were accompanied by an abnormal distribution of lymphocyte subsets within the spleen architecture, in particular by the lack of well developed germinal centres and T cell areas. We also found abnormal expression of CD43 and other sialylated proteins such as CDw75 and CD76, whose expression requires the action of specific sialyltransferases. This study shows that the combined impairment in cellular and humoral immunity observed in WAS is the result of multiple molecular abnormalities on the surface of WAS lymphocytes, that in turn might result in recirculation/migration anomalies.

Electrophile Response Element-mediated Induction of the Cystine/Glutamate Exchange Transporter Gene Expression

In mammalian cultured cells, the cystine/glutamate exchange transport mediated by system x(c)- is important to maintain intracellular GSH levels. System x(c)- consists of two protein components, xCT and the heavy chain of 4F2 antigen. The activity of system x(c)- is induced by various stimuli, including electrophilic agents like diethyl maleate. In the present study, we have investigated the mechanism of the transcriptional regulation of xCT mRNA by diethyl maleate. The xCT gene consisted of twelve exons and sequence analysis identified four electrophile response element (EpRE)-like sequences between -230 and -1 in the 5'-flanking region, designated EpRE-1 to EpRE-4. To identify sequences mediating the constitutive and induced expression of xCT, a series of 5'-deletion mutants created from the 5'-flanking region were cloned into a luciferase reproter vector and transfected into BHK21 cells. The 5'-deletion analysis revealed that the sequence between -116 and -82 is essential for the basal expression and the sequence between -226 and -116 containing EpRE-1 is essential in response to diethyl maleate. Mutational analysis demonstrated that EpRE-1 is critically involved in the response to diethyl maleate. Other stress agents like arsenite, cadmium, and hydroquinone seemed to induce system x(c)- activity via the same sequence. Furthermore, the experiments using the mouse embryonic fibroblasts derived from the Nrf2-deficient mice revealed that the induction of xCT gene by electrophilic agents is mediated by Nrf2. EpRE occurs in a broad spectrum of genes for the proteins that are involved in the defense against xenobiotics and regulates their expression. The present results have demonstrated that xCT is a novel member of this protein family.

Distribution of Cystine/Glutamate Exchange Transporter, System xc−, in the Mouse Brain

Mammalian cells express a transport system known as system x(c)-, which is an exchange agency specific for anionic forms of cystine and glutamate. System x(c)- activity is important to maintain both intracellular glutathione levels and the redox balance between cystine and cysteine in the extracellular milieu. We have shown that the cloned cDNAs encoding the transporter for system x(c)- consist of two components, xCT and the heavy chain of 4F2 antigen. In the present study, we have investigated the mRNA distribution for these components in the mouse brain by in situ hybridization. The xCT mRNA was expressed in the area postrema, subfornical organ, habenular nucleus, hypothalamic area, and ependymal cells of the lateral wall of the third ventricle in the adult mouse brain. A strong signal was also detected in the meninges in both adult and fetal mouse brains. The mRNA expression of the heavy chain of 4F2 antigen was detected in a more broad area, including all of the regions in which xCT mRNA was detected. These data are compatible with our biochemical evidence that xCT functions in combination with the heavy chain of 4F2 antigen to elicit system x(c)- activity. The expression of system x(c)- in meninges and some circumventricular organs may suggest that this transporter contributes to the maintenance of the redox state (i.e., cysteine/cystine ratio) in the CSF.

Heteromeric amino acid transporters: biochemistry, genetics, and physiology.

The heteromeric amino acid transporters (HATs) are composed of two polypeptides: a heavy subunit (HSHAT) and a light subunit (LSHAT) linked by a disulfide bridge. HSHATs are N-glycosylated type II membrane glycoproteins, whereas LSHATs are nonglycosylated polytopic membrane proteins. The HSHATs have been known since 1992, and the LSHATs have been described in the last three years. HATs represent several of the classic mammalian amino acid transport systems (e.g., L isoforms, y(+)L isoforms, asc, x(c)(-), and b(0,+)). Members of the HAT family are the molecular bases of inherited primary aminoacidurias cystinuria and lysinuric protein intolerance. In addition to the role in amino acid transport, one HSHAT [the heavy subunit of the cell-surface antigen 4F2 (also named CD98)] is involved in other cell functions that might be related to integrin activation. This review covers the biochemistry, human genetics, and cell physiology of HATs, including the multifunctional character of CD98.

Identification and characterisation of human xCT that co-expresses, with 4F2 heavy chain, the amino acid transport activity system xc-.

We have identified a new human complementary deoxyribonucleic acid (cDNA), for the xc- amino acid transporter (HGMW-approved name SLC7A11; also known as human xCT), that, when co-expressed with the heavy chain of surface antigen 4F2 (4F2hc, also termed CD98), but not with rBAT, (related to the bo,+ amino acid transporter), induces system xc- transport activity in Xenopus oocytes. Human xCT is the seventh human member of the family of amino acid transporters that are subunits of 4F2hc or rBAT and, inview of its amino acid sequence identity (89%) with mouse xCT, is most probably the human orthologue thereof. The amino acid transport activity induced by the co-expression of human 4F2hc and xCT in Xenopus oocytes was sodium independent and specific for L-cystine, L-glutamate and L-aspartate. This activity also functioned in an exchange mode (e.g. cystine/glutamate) with a substrate stoichiometry of 1:1. Expression of human xCT alone in oocytes did not induce amino acid transport activity and the expressed xCT protein was localised intracellularly. When human xCT was co-expressed with 4F2hc, the former localised to the oocyte plasma membrane. Tissue-expression studies showed that human SLC7A11 mRNA is expressed mainly in the brain, but also in pancreas and in cultured cell lines. The transport characteristics of human xCT and the distribution of its tissue expression strongly suggest that it corresponds to the human amino acid transporter system xc-.

Molecular and functional identification of large neutral amino acid transporters LAT1 and LAT2 and their pharmacological relevance at the blood-brain barrier.

We present here the evidence of molecular and functional expression of LAT1 and LAT2, subunits of the large neutral amino acid transporter system L, in cultured brain capillary endothelial cells of the rat. By means of the RT-PCR method, transcripts of LAT1, LAT2 and heavy chain of 4F2 antigen (4F2hc) were detected in rat primary cultured brain capillary endothelial cells (BCECs) and immortalized subline, RBEC1. The uptake properties of RBEC1, such as [3H]leucine and L-[3H]DOPA uptake, were similar to those of primary cultured BCECs. So, RBEC1 may retain almost native properties of the large neutral amino acid uptake activities. [3H]Leucine uptake by RBEC1 showed two saturable components and the Km values of the high- and low-affinity components were 8.92+/-3.18 and 119+/-45 microM, respectively. The Km value of the high-affinity component agreed well with that of LAT1 and the amino acid transport selectivity of RBEC1 was similar to that of LAT1. Therefore, it is suggested that LAT1 is important at the blood-brain barrier of rats. Additionally, the Km value of the low-affinity component was similar to that of LAT2. These observations indicate that LAT1 and LAT2 are involved as transporters for large neutral amino acids at the blood-brain barrier. Additionally, we concluded that RBEC1 is useful as an in-vitro model for evaluation of the pharmacological relevance of system L at the blood-brain barrier.

3-[123I]iodo-α-methyl-l-tyrosine transport and 4F2 antigen expression in human glioma cells

3[123I]iodo-α-methyl-L-tyrosine is a tracer of amino acid transport in brain tumors using single-photon emission-computed tomography and predominantly transported by amino acid transport system L. The 4F2 antigen has been identified to be linked to system L-like transport and is assumed to be a part of the transporter protein. We demonstrated that system L-mediated transport of IMT and 4F2 antigen expression are dependent on proliferation rate of human glioma cells and significantly correlated with each other.

Transport Properties of a System y+L Neutral and Basic Amino Acid Transporter: INSIGHTS INTO THE MECHANISMS OF SUBSTRATE RECOGNITION

The properties of system y(+)L-mediated transport were investigated on rat system y(+)L transporter, ry(+)LAT1, coexpressed with the heavy chain of cell surface antigen 4F2 in Xenopus oocytes. ry(+)LAT1-mediated transport of basic amino acids was Na(+)-independent, whereas that of neutral amino acids, although not completely, was dependent on Na(+), as is typical of system y(+)L-mediated transport. In the absence of Na(+), lowering of pH increased leucine transport, without affecting lysine transport. Therefore, it is proposed that H(+), besides Na(+) and Li(+), is capable of supporting neutral amino acid transport. Na(+) and H(+) augmented leucine transport by decreasing the apparent K(m) values, without affecting the V(max) values. We demonstrate that although ry(+)LAT1-mediated transport of [(14)C]l-leucine was accompanied by the cotransport of (22)Na(+), that of [(14)C]l-lysine was not. The Na(+) to leucine coupling ratio was determined to be 1:1 in the presence of high concentrations of Na(+). ry(+)LAT1-mediated leucine transport, but not lysine transport, induced intracellular acidification in Chinese hamster ovary cells coexpressing ry(+)LAT1 and 4F2 heavy chain in the absence of Na(+), but not in the presence of physiological concentrations of Na(+), indicating that cotransport of H(+) with leucine occurred in the absence of Na(+). Therefore, for the substrate recognition by ry(+)LAT1, the positive charge on basic amino acid side chains or that conferred by inorganic monovalent cations such as Na(+) and H(+), which are cotransported with neutral amino acids, is presumed to be required. We further demonstrate that ry(+)LAT1, due to its peculiar cation dependence, mediates a heteroexchange, wherein the influx of substrate amino acids is accompanied by the efflux of basic amino acids.

Glycoprotein-associated amino acid exchangers: broadening the range of transport specificity.

Members of the newly discovered glycoprotein-associated amino acid transporter family (gpaAT-family) share a similar primary structure with >40% identity, a predicted 12-transmembrane segment topology and the requirement for association with a glycoprotein (heavy chain) for functional surface expression. Five of the six identified gpaATs (light chains) associate with the surface antigen 4F2 heavy chain (4F2hc = CD98), a ubiquitous plasma membrane protein induced in cell proliferation, and which is also highly expressed at the basolateral surface of amino acid transporting epithelia. The differing tissue localizations of the 4F2hc-associated gpaATs appear to complement each other. As yet, a single gpaAT (b(0,+)AT) has been shown to associate with rBAT, a 4F2hc-related glycoprotein mainly localized in intestine and kidney luminal brush-border membranes. The transport characteristics of gpaATs have been shown, by expression in heterologous systems, to correspond to the previously described transport systems L, y+L, xc- and b(o,+). These (obligatory) exchangers of broad substrate specificity (with the exception of xCT) are expected to equilibrate the concentrations of their substrate amino acids across membranes. Thus, the driving force provided by a transmembrane gradient of one substrate amino acid, such as that generated by a parallel functioning unidirectional transporter, can be used by a gpaAT to fuel the secondary active vectorial transport of other exchangeable species. Vectorial transport of specific amino acids is also promoted by the intrinsic asymmetry of these exchangers. The fact that genetic defects of the epithelial gpaATs b(0,+)AT and y+LAT1 cause non-type I cystinuria and lysinuric protein intolerance, respectively, demonstrates that these gpaATs perform vectorial secondary and/or tertiary active transport of specific amino acids in vivo.