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Michael Lübbert - One of the best experts on this subject based on the ideXlab platform.
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the aml1 eto target gene LAT2 interferes with differentiation of normal hematopoietic precursor cells
Leukemia Research, 2014Co-Authors: Aitomi Essig, Jesus Duqueafonso, Heike L Pahl, Sven Schwemmers, Michael LübbertAbstract:The adaptor protein linker activator of T-cells 2 (LAT2) is a known AML1/ETO target gene whose function during normal hematopoiesis is unknown. We addressed the role of LAT2 during erythroid and myeloid differentiation of normal human CD34+ hematopoietic cells. LAT2 is expressed at low levels in CD34+ cells and upregulated during cytokine-induced myeloid and erythroid differentiation. Forced LAT2 expression leads to a delay of erythroid and myeloid differentiation keeping CD34+ cells in a more immature state, whereas LAT2 knockdown accelerates differentiation. It is tempting to speculate that by affecting the differentiation capacity of normal hematopoietic progenitors, LAT2 may contribute to the pathogenesis of AML.
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The AML1/ETO target gene LAT2 interferes with differentiation of normal hematopoietic precursor cells
Leukemia research, 2013Co-Authors: Aitomi Essig, Jesus Duque-afonso, Heike L Pahl, Sven Schwemmers, Michael LübbertAbstract:The adaptor protein linker activator of T-cells 2 (LAT2) is a known AML1/ETO target gene whose function during normal hematopoiesis is unknown. We addressed the role of LAT2 during erythroid and myeloid differentiation of normal human CD34+ hematopoietic cells. LAT2 is expressed at low levels in CD34+ cells and upregulated during cytokine-induced myeloid and erythroid differentiation. Forced LAT2 expression leads to a delay of erythroid and myeloid differentiation keeping CD34+ cells in a more immature state, whereas LAT2 knockdown accelerates differentiation. It is tempting to speculate that by affecting the differentiation capacity of normal hematopoietic progenitors, LAT2 may contribute to the pathogenesis of AML.
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The HDAC class I-specific inhibitor entinostat (MS-275) effectively relieves epigenetic silencing of the LAT2 gene mediated by AML1/ETO
Oncogene, 2011Co-Authors: Jesus Duque-afonso, A Yalcin, Mohammad Abdelkarim, Olaf Heidenreich, T. Berg, Michael LübbertAbstract:The chromosomal translocation (8;21) fuses the hematopoietic transcription factor AML1 ( RUNX1 ) with ETO ( RUNX1T1, MTG8 ), resulting in the leukemia-specific chimeric protein AML1/ETO. This fusion protein has been implicated in epigenetic silencing, recruiting histone deacetylases (HDACs) and DNA methyltransferases to target promoters. Previously, we have identified a novel in vivo AML1/ETO target gene, LAT2 (NTAL/LAB/WBSCR5), which is involved in FcɛR I, c-Kit, B-cell and T-cell receptor signalling. We have now addressed the molecular mechanisms of AML1/ETO-mediated LAT2 repression. In Kasumi-1 cells, where AML1/ETO bound to the LAT2 gene, small interfering RNA (siRNA)-mediated AML1/ETO depletion caused upregulation of LAT2 , suggesting a possible direct mechanism of repression. Expression of AML1/ETO was associated with a decrease in acetylation of histones H3, H3K9 and H4, and an increase in H3K9 and H3K27 trimethylation. The class I-specific HDAC inhibitors entinostat (MS-275) and mocetinostat (MGCD0103) induced LAT2 expression specifically in AML1/ETO-expressing cells, resulting in induction of several activating histone marks on the LAT2 gene, including trimethylation of histone H3K4. The combination of entinostat and decitabine increased acetylation of histones H3 and H4, as well as LAT2 mRNA expression, in an at least additive fashion. In conclusion, several repressive histone modifications mark the LAT2 gene in the presence of AML1/ETO, and LAT2 gene derepression is achieved by pharmacological inhibition of HDACs.
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regulation of the adaptor molecule LAT2 an in vivo target gene of aml1 eto runx1 runx1t1 during myeloid differentiation
British Journal of Haematology, 2011Co-Authors: Jesus Duqueafonso, Leticia Solari, Aitomi Essig, Heike L Pahl, Tobias Berg, Michael LübbertAbstract:SummaryThe leukaemia-specific fusion oncoprotein RUNX1/RUNX1T1 (AML1/ETO), resulting from the chromosomal translocation (8;21) in acutemyeloid leukaemia (AML), imposes a striking genotype–phenotyperelationship upon this distinct subtype of AML, which is mediated bymultiple, co-ordinate downstream effects induced by this chimerictranscription factor. We previously identified the LAT2 gene, encoding theadaptor molecule LAT2 (NTAL, LAB), which is phosphorylated by KIT andhas a role in mast cell and B-cell activation, as a target of the repressor activityof RUNX1/RUNX1T1. These results were confirmed and extended bydemonstrating downregulation of the LAT2 protein in response toconditional RUNX1/RUNX1T1 expression, and its absence in primaryAML with the t(8;21). In contrast, in a cohort of 43 AML patients, higherlevels of LAT2 were associated with myelomonocytic features. Differentiationof HL-60 and NB4 cells towards granulocytes by all trans-retinoic acid(ATRA) resulted in downregulation of LAT2; conversely, it was upregulatedduring phorbol ester-induced monocytic differentiation of HL-60 cells.Forced expression of LAT2 in Kasumi-1 cells resulted in a striking block ofATRA- and phorbol ester-induced differentiation, implicating disturbancesof the graded expression of this adaptor molecule in the maturation block ofmyeloid leukaemia cells.Keywords: chromosomal translocations, acute myeloid leukaemia, transcrip-tion factor, myeloid differentiation, adaptor molecule.
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Regulation of the adaptor molecule LAT2, an in vivo target gene of AML1/ETO (RUNX1/RUNX1T1), during myeloid differentiation
British Journal of Haematology, 2011Co-Authors: Jesus Duque-afonso, Leticia Solari, Aitomi Essig, Heike L Pahl, Tobias Berg, Michael LübbertAbstract:SummaryThe leukaemia-specific fusion oncoprotein RUNX1/RUNX1T1 (AML1/ETO), resulting from the chromosomal translocation (8;21) in acutemyeloid leukaemia (AML), imposes a striking genotype–phenotyperelationship upon this distinct subtype of AML, which is mediated bymultiple, co-ordinate downstream effects induced by this chimerictranscription factor. We previously identified the LAT2 gene, encoding theadaptor molecule LAT2 (NTAL, LAB), which is phosphorylated by KIT andhas a role in mast cell and B-cell activation, as a target of the repressor activityof RUNX1/RUNX1T1. These results were confirmed and extended bydemonstrating downregulation of the LAT2 protein in response toconditional RUNX1/RUNX1T1 expression, and its absence in primaryAML with the t(8;21). In contrast, in a cohort of 43 AML patients, higherlevels of LAT2 were associated with myelomonocytic features. Differentiationof HL-60 and NB4 cells towards granulocytes by all trans-retinoic acid(ATRA) resulted in downregulation of LAT2; conversely, it was upregulatedduring phorbol ester-induced monocytic differentiation of HL-60 cells.Forced expression of LAT2 in Kasumi-1 cells resulted in a striking block ofATRA- and phorbol ester-induced differentiation, implicating disturbancesof the graded expression of this adaptor molecule in the maturation block ofmyeloid leukaemia cells.Keywords: chromosomal translocations, acute myeloid leukaemia, transcrip-tion factor, myeloid differentiation, adaptor molecule.
Gerd Krause - One of the best experts on this subject based on the ideXlab platform.
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Molecular Mechanisms of Thyroid Hormone Transport by l-Type Amino Acid Transporter.
Experimental and clinical endocrinology & diabetes : official journal German Society of Endocrinology [and] German Diabetes Association, 2019Co-Authors: Gerd Krause, Katrin M. HinzAbstract:Thyroid hormones (TH) pass through the plasma membrane into the target cells via transporter proteins. Thyroid hormone transporters that have been identified until now belong to two different solute carrier (SLC) subfamilies i) the major facilitator superfamily (MFS) and ii) the amino acid polyamine-organocation (APC) superfamily. Both are comprised by 12 transmembrane helices, however with different structural topology. The TH transporter MCT8, MCT10 and OATP1C1 are members of the MSF. The l-type amino acid transporters (LATs) are transporting neutral amino acids across the membrane. Two LAT subtypes, LAT1 and LAT2 are members of the APC superfamily, need the escort protein 4F2hc and facilitate uptake but no efflux of TH-subtypes. Homology models of LAT2 that are based on crystal structures of APC transporters guided mutagenesis, revealed molecular structure-function determinants for recognition and transition for import and export of TH-subtypes. The recently solved cryo-EM structure of LAT1 confirmed the structural input. Two other LAT subtypes, LAT3 and LAT4 are members of the MFS. From previous observed negative effect of LAT3 and LAT4 on 3,3’-T2 uptake by LAT1 and LAT2 it was indirectly concluded that LAT3 might export 3,3’-T2. There are still open questions that need to be addressed in order to fully understand the molecular recognition pattern and traversing mechanism of import and export of particular TH-subtypes by LAT1 and LAT2. Moreover, clarification is needed whether LAT3 and LAT4 are exporting TH. Recent new data could not verify the initial hypothesis of TH export by LAT3. Therefore, further investigations are necessary to explain the negative effect of LAT3 on the TH import by LAT2.
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Thyroid hormone transport across L-type amino acid transporters: What can molecular modelling tell us?
Molecular and cellular endocrinology, 2017Co-Authors: Gerd Krause, Katrin M. HinzAbstract:Abstract Thyroid hormones (THs) and their derivatives require transmembrane transporters (TTs) to mediate their translocation across the cell membrane. Among these TTs, the L-type amino acid transporters (LAT) not only transport amino acids (AAs) but also certain THs and their derivatives. This review summarizes available knowledge concerning structure function patterns of the TH transport by LAT1 and LAT2. For example, LAT2 imports 3,3′-T 2 and T 3 , but not rT 3 and T 4 . In contrast to amino acids, THs are not at all exported by LAT2. Homology modelling of LAT1 and LAT2 is based on available crystal structures from the same superfamily the amino acid/polyamine/organocation transporter (APC). Molecular model guided mutagenesis has been used to predict substrate interaction sites. A common recognition feature for amino acid- and TH-derivatives has been suggested in an interior cavity of LAT1 and LAT2. Therein additional distinct molecular determinants that are responsible for the bidirectional AA transport but allowing only unidirectional import of particular THs have been confirmed for LAT2 by mutagenesis. Characterized substrate features that are needed for TH translocation and distinct LAT2 properties will be highlighted to understand the molecular import and export mechanisms of this transporter in more detail.
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Transport of Iodothyronines by Human L-Type Amino Acid Transporters
Endocrinology, 2015Co-Authors: Chantal Zevenbergen, Marcel E. Meima, Elaine C. Lima De Souza, Robin P. Peeters, Anita Kinne, Gerd Krause, W. Edward Visser, Theo J. VisserAbstract:Thyroid hormone (TH) transporters facilitate cellular TH influx and efflux, which is paramount for normal physiology. The L-type amino acid transporters LAT1 and LAT2 are known to facilitate TH transport. However, the role of LAT3, LAT4, and LAT5 is still unclear. Therefore, the aim of this study was to further characterize TH transport by LAT1 and LAT2 and to explore possible TH transport by LAT3, LAT4, and LAT5. FLAG-LAT1-5 constructs were transiently expressed in COS1 cells. LAT1 and LAT2 were cotransfected with the CD98 heavy chain. Cellular transport was measured using 10 nM (125)I-labeled T4, T3, rT3, 3,3'-T2, and 10 μM [(125)I]3'-iodotyrosine (MIT) as substrates. Intracellular metabolism of these substrates was determined in cells cotransfected with either of the LATs with type 1 or type 3 deiodinase. LAT1 facilitated cellular uptake of all substrates and LAT2 showed a net uptake of T3, 3,3'-T2, and MIT. Expression of LAT3 or LAT4 did not affect transport of T4 and T3 but resulted in the decreased cellular accumulation of 3,3'-T2 and MIT. LAT5 did not facilitate the transport of any substrate. Cotransfection with LAT3 or LAT4 strongly diminished the cellular accumulation of 3,3'-T2 and MIT by LAT1 and LAT2. These data were confirmed by metabolism studies. LAT1 and LAT2 show distinct preferences for the uptake of the different iodocompounds, whereas LAT3 and LAT4 specifically facilitate the 3,3'-T2 and MIT efflux. Together our findings suggest that different sets of transporters with specific influx or efflux capacities may cooperate to regulate the cellular thyroid state.
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Involvement of the L-Type Amino Acid Transporter LAT2 in the Transport of 3,3′-Diiodothyronine across the Plasma Membrane
European Thyroid Journal, 2015Co-Authors: Anita Kinne, Katrin M. Hinz, Josef Köhrle, Eva K. Wirth, Melanie Wittner, Ralf Schülein, Gerd KrauseAbstract:Thyroid hormones are transported across cell membranes by transmembrane transporter proteins, for example by members of the monocarboxylate transporter (MCT) and the L-type amino acid transporter (LAT) families. LATs consist of a light chain (e.g. LAT2) and a heavy chain (CD98), which is essential for their cell surface expression and functionality. The specificity of LAT2 for thyroid hormones and their metabolites and its role in their transport was not fully clear. This fact motivated us to establish a cell system to elucidate the uptake of thyroid hormones and their metabolites by mouse LAT2. The coinjection of cRNA coding for LAT2 and CD98 into Xenopus laevis oocytes resulted in a markedly increased level of 3,3′-diiodo-L-thyronine (3,3′-T2) and to some extent also enhanced T3 transport. To gain insight into properties of thyroid hormones and their metabolites transported by LAT2, we inhibited 3,3′-T2 uptake by various iodothyronine derivatives. T1 and T2 derivatives as well as 2-aminobicyclo-[2,2,1]-heptane-2-carboxylic acid strongly competed with 3,3′-T2 uptake. In addition, we performed T2 uptake measurements with the thyroid hormone-specific transporter MCT8. For both LAT2 and MCT8, Km values in a low micromolar range were calculated. We demonstrated that oocytes are a suitable system for thyroid hormone transport studies mediated by LAT2. Our data indicates that LAT2 compared to other thyroid hormone transporters prefers 3,3′-T2 as the substrate. Thus, LAT2 might contribute to the availability of thyroid hormone by importing and/or exporting 3,3′-T2, which is generated either by T3 inactivation or by rapid deiodinase 1-mediated rT3 degradation.
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Involvement of the L-Type Amino Acid Transporter LAT2 in the Transport of 3,3 ' -Diiodothyronine across the Plasma Membrane
European thyroid journal, 2015Co-Authors: Anita Kinne, Katrin M. Hinz, Josef Köhrle, Eva K. Wirth, Melanie Wittner, Ralf Schülein, Gerd KrauseAbstract:Thyroid hormones are transported across cell membranes by transmembrane transporter proteins, for example by members of the monocarboxylate transporter (MCT) and the L-type amino acid transporter (LAT) families. LATs consist of a light chain (e.g. LAT2) and a heavy chain (CD98), which is essential for their cell surface expression and functionality. The specificity of LAT2 for thyroid hormones and their metabolites and its role in their transport was not fully clear. This fact motivated us to establish a cell system to elucidate the uptake of thyroid hormones and their metabolites by mouse LAT2. The coinjection of cRNA coding for LAT2 and CD98 into Xenopus laevis oocytes resulted in a markedly increased level of 3,3'-diiodo-L-thyronine (3,3'-T2) and to some extent also enhanced T3 transport. To gain insight into properties of thyroid hormones and their metabolites transported by LAT2, we inhibited 3,3'-T2 uptake by various iodothyronine derivatives. T1 and T2 derivatives as well as 2-aminobicyclo-[2, 2,1]-heptane-2-carboxylic acid strongly competed with 3,3'-T2 uptake. In addition, we performed T2 uptake measurements with the thyroid hormone-specific transporter MCT8. For both LAT2 and MCT8, Km values in a low micromolar range were calculated. We demonstrated that oocytes are a suitable system for thyroid hormone transport studies mediated by LAT2. Our data indicates that LAT2 compared to other thyroid hormone transporters prefers 3,3'-T2 as the substrate. Thus, LAT2 might contribute to the availability of thyroid hormone by importing and/or exporting 3,3'-T2, which is generated either by T3 inactivation or by rapid deiodinase 1-mediated rT3 degradation.
Hitoshi Endou - One of the best experts on this subject based on the ideXlab platform.
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High expression of L-type amino acid transporter 1 as a prognostic marker in bile duct adenocarcinomas
Cancer medicine, 2014Co-Authors: Nobuyuki Yanagisawa, Hitoshi Endou, Norihiro Nakada, Kiyomi Hana, Masaaki Ichinoe, Isao Okayasu, Wasaburo Koizumi, Yoshiki MurakumoAbstract:Oncocytic L-type amino acid transporter (LAT) 1 may be a prognostic indicator and target of new molecular therapeutic agents against malignancies. To investigate whether LAT1 expression influence the outcomes of patients with bile duct cancer, the expression of LAT1, LAT2, CD98, and Ki-67 was investigated immunohistochemically in 134 surgically resected bile duct adenocarcinomas, including 84 distal extrahepatic bile duct adenocarcinomas, 21 hilar cholangiocarcinomas, 15 intrahepatic cholangiocarcinomas, and 14 ampullary adenocarcinomas. LAT1 expression was weakly correlated with CD98 expression and Ki-67 labeling index (LI). Kaplan–Meier analysis showed a significant difference in prognosis between patients with bile duct adenocarcinomas having LAT1-high and -low scores, whereas LAT2 and CD98 expression and Ki-67 LI were not predictive of poor prognosis. Prognosis tended to be worse in patients having tumors with LAT1-high/LAT2-low than LAT1-low/LAT2-high scores (P = 0.0686). Multivariable analyses revealed that LAT1 expression, surgical margin, pT stage were independent prognostic factors. In conclusion, aberrant overexpression of LAT1 in bile duct adenocarcinoma predicts poor prognosis, suggesting that LAT1 may be a potential target of anticancer therapy.
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Unique and selective expression of L-amino acid transporter 1 in human tissue as well as being an aspect of oncofetal protein.
Histology and histopathology, 2013Co-Authors: Norihiro Nakada, Hitoshi Endou, Tetuo Mikami, Kiyomi Hana, Masaaki Ichinoe, Nobuyuki Yanagisawa, Tsutomu Yoshida, Isao OkayasuAbstract:Dysregulated expression of L-type amino acid transporter 1 (LAT1), which transports large neutral amino acids, is a characteristic of various human cancers and possibly offers a molecular target for chemotherapy. LAT2, in contrast, shows lower expression in neoplasms. LAT1 is presumed to be a biomarker of many cancers, suggesting a kind of oncoprotein. However, no precise analysis of LAT1 and LAT2 expression has been performed in systemic normal tissues. To see characteristics of LAT1 and LAT2, immunohisto-chemical expression of LAT1 and LAT2 was assessed and compared in normal human systemic organs and tissues from 3 adults, 3 children and 3 fetuses in the present study. Cardiac muscles, hepatocytes, thymic epithelial cells and primitive neuroectodermal cells in fetus were positive with LAT1, whereas no expression was found in the respective adult tissues, indicating an aspect of oncofetal protein. In adult tissues, LAT1 was found to be expressed proximal to proliferative zones in gastrointestinal mucosa by double immunostaining of LAT1 and Ki-67. Testicular Sertoli cells, ovarian follicular cells, and pancreatic islet cells showed strong expression. Although the systemic capillary endothelium did not express LAT1, but did express LAT2, capillaries corresponding to the blood-brain, blood-follicle, and blood-retinal barriers demonstrated strong LAT1 immunoreactions. In conclusion, LAT1 was expressed in gonad tissues and several kinds of cells having special functions, as well as being discovered to be an aspect of oncofetal protein. In addition, ubiquitous LAT2 expression was confirmed immunohistochemically in systemic tissues, indicating constitutional function
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Transport of 3-Fluoro-l-α-Methyl-Tyrosine by Tumor-Upregulated L-Type Amino Acid Transporter 1: A Cause of the Tumor Uptake in PET
Journal of nuclear medicine : official publication Society of Nuclear Medicine, 2012Co-Authors: Pattama Wiriyasermkul, Shushi Nagamori, Ryuichi Ohgaki, Hidekazu Tanaka, Hideyuki Tominaga, Noboru Oriuchi, Kyoichi Kaira, Hidekazu Nakao, Takeru Kitashoji, Hitoshi EndouAbstract:L-3- 18 F-a-methyl tyrosine ( 18 F-FAMT) has been developed as a PET radiotracer for tumor imaging. Clinical studies have demonstrated the usefulness of 18 F-FAMT PET for the prediction of prognosis and the differentiation of malignant tumors and benign lesions. 18 F-FAMT exhibits higher cancer specificity in peripheral organs than other amino acid PET tracers and 18F-FDG. The accumulation of 18F-FAMT is strongly correlated with the expression of L-type amino acid transporter 1 (LAT1), an isoform of system L highly upregulated in cancers. In this study, we examined the interaction of 3-fluoro-L-a-methyltyrosine (FAMT) with amino acid transporters to assess the mechanisms of 18 F-FAMT uptake in PET. Methods: We applied in vitro assays using established mammalian cell lines stably expressing LAT1 or a non–cancer-type system L isoform LAT2. The inhibitory effect on L- 14 C-leucine uptake and the induction effect on efflux of preloaded L- 14 C-leucine were examined for FAMT and other amino acid tracers. FAMT transport was compared among cell lines with varied LAT1 expression level. Results: FAMT prominently inhibited LAT1-mediated L-14C-leucine uptake in a competitive manner but had less of an effect on LAT2. In the efflux experiments, FAMT induced the efflux of preloaded L-14C-leucine through LAT1, indicating that FAMT is transported by LAT1 and not by LAT2. Among amino acid–related compounds examined in this study, including those used for PET tracers, the compounds with an a-methyl group such as FAMT, 2-fluoro-L-a-methyl-tyrosine, 3-iodo-L-a-methyl-tyrosine, and L-a-methyl-tyrosine were well transported by LAT1 but not by LAT2. However, L-methionine, L-tyrosine, 3-fluoro-L-tyrosine, 2-fluoro-L-tyrosine, and O-(2fluoroethyl)-L-tyrosine were transported by both LAT1 and LAT2, suggesting that the a-methyl moiety is responsible for the LAT1 selectivity of FAMT. FAMT transport rate and LAT1 protein level were well correlated, supporting the importance of LAT1 for the cellular uptake of FAMT. Conclusion: Distinct from other amino acid PET tracers, because of its a-methyl moiety, FAMT is selective to LAT1 and not transported by LAT2. This property of FAMT is proposed to contribute to highly tumorspecific accumulation of 18 F-FAMT in PET.
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role of amino acid transporter LAT2 in the activation of mtorc1 pathway and the pathogenesis of crescentic glomerulonephritis
Laboratory Investigation, 2011Co-Authors: Ryota Kurayama, Yoshimichi Sai, Noriko Ito, Yukino Nishibori, Daisuke Fukuhara, Yoshihiro Akimoto, Eiji Higashihara, Yasuhito Ishigaki, Kenichi Miyamoto, Hitoshi EndouAbstract:Molecular mechanisms and signaling pathways leading to cellular proliferation and lesion formation in the crescentic glomerulonephritis (CGN) remain elusive. In the present study we have explored a potential role of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway and amino acid transporter (LAT) in the pathogenesis of CGN. Immunohistochemistry and western blot analysis of glomeruli isolated from a rat model of CGN revealed that activation of mTORC1 preceded crescent formation in glomerular parietal epithelial cells (PECs) and podocytes. Daily treatment of rats with the mTOR inhibitor everolimus just after induction of CGN was not beneficial and instead led to increased cellular necrosis of PECs. However, daily treatment starting 7 days after the onset of CGN was beneficial and maintained intact glomeruli. Out of three forms of L-type neutral amino acid transporters (LAT1–LAT3) studied here, only LAT2 was found to be upregulated in the PECs and podocytes in advance of the crescent formation as well as in the crescent lesion itself. Cell culture study revealed that plasma membrane expression of LAT2 markedly stimulated mTORC1 signaling pathway, which was significantly abrogated by coexistence of LAT inhibitor. Finally, LAT inhibitor significantly abrogated development of crescent formation of CGN on day 7. Our data suggest that LAT2 may have a pivotal role in the pathogenesis of CGN by activating the mTORC1 pathway in the glomerular epithelial cells.
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Establishment and characterization of mammalian cell lines stably expressing human L-type amino acid transporters.
Journal of pharmacological sciences, 2008Co-Authors: Emiko Morimoto, Yoshikatsu Kanai, Kyung Kim, Arthit Chairoungdua, Hye Won Choi, Michael F. Wempe, Naohiko Anzai, Hitoshi EndouAbstract:System L (SL), a basolateral amino acid transporter, transports large neutral amino acids (LNAAs) in a Na+-independent manner. Previously, we identified two isoforms of transporters: L-type amino acid transporter 1 (LAT1) and 2 (LAT2) and revealed their distinct substrate selectivity and transport properties. In this study, to establish more stable human LAT1 (hLAT1) and LAT2 (hLAT2) in vitro assay systems, we established mouse cell lines stably expressing hLAT1 (S2-LAT1) and hLAT2 (S2-LAT2). Real-time quantitative RT-PCR analysis revealed that S2-LAT1 and S2-LAT2 cells express hLAT1 and hLAT2 mRNAs at 20 – 1000-fold higher levels than those of endogenous mouse Lat1 and LAT2. S2-LAT1 and S2-LAT2 mediated [14C]L-leucine transport properties were measured and corresponded to results observed via Xenopus oocytes. Using these cells, the data demonstrate that hLAT1 and hLAT2 exhibit different characters in the acceptance of α-methyl amino acids and amino acid–related compounds with bulky side chains such as thyroid hormones and melphalan. S2-LAT1 and S2-LAT2 cells are expected to facilitate hLAT1 and hLAT2 substrate recognition research and contribute to drug development by providing an efficient assay system to screen for chemical compounds that interact with hLAT1 and hLAT2.
Katrin M. Hinz - One of the best experts on this subject based on the ideXlab platform.
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Molecular Mechanisms of Thyroid Hormone Transport by l-Type Amino Acid Transporter.
Experimental and clinical endocrinology & diabetes : official journal German Society of Endocrinology [and] German Diabetes Association, 2019Co-Authors: Gerd Krause, Katrin M. HinzAbstract:Thyroid hormones (TH) pass through the plasma membrane into the target cells via transporter proteins. Thyroid hormone transporters that have been identified until now belong to two different solute carrier (SLC) subfamilies i) the major facilitator superfamily (MFS) and ii) the amino acid polyamine-organocation (APC) superfamily. Both are comprised by 12 transmembrane helices, however with different structural topology. The TH transporter MCT8, MCT10 and OATP1C1 are members of the MSF. The l-type amino acid transporters (LATs) are transporting neutral amino acids across the membrane. Two LAT subtypes, LAT1 and LAT2 are members of the APC superfamily, need the escort protein 4F2hc and facilitate uptake but no efflux of TH-subtypes. Homology models of LAT2 that are based on crystal structures of APC transporters guided mutagenesis, revealed molecular structure-function determinants for recognition and transition for import and export of TH-subtypes. The recently solved cryo-EM structure of LAT1 confirmed the structural input. Two other LAT subtypes, LAT3 and LAT4 are members of the MFS. From previous observed negative effect of LAT3 and LAT4 on 3,3’-T2 uptake by LAT1 and LAT2 it was indirectly concluded that LAT3 might export 3,3’-T2. There are still open questions that need to be addressed in order to fully understand the molecular recognition pattern and traversing mechanism of import and export of particular TH-subtypes by LAT1 and LAT2. Moreover, clarification is needed whether LAT3 and LAT4 are exporting TH. Recent new data could not verify the initial hypothesis of TH export by LAT3. Therefore, further investigations are necessary to explain the negative effect of LAT3 on the TH import by LAT2.
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Thyroid hormone transport across L-type amino acid transporters: What can molecular modelling tell us?
Molecular and cellular endocrinology, 2017Co-Authors: Gerd Krause, Katrin M. HinzAbstract:Abstract Thyroid hormones (THs) and their derivatives require transmembrane transporters (TTs) to mediate their translocation across the cell membrane. Among these TTs, the L-type amino acid transporters (LAT) not only transport amino acids (AAs) but also certain THs and their derivatives. This review summarizes available knowledge concerning structure function patterns of the TH transport by LAT1 and LAT2. For example, LAT2 imports 3,3′-T 2 and T 3 , but not rT 3 and T 4 . In contrast to amino acids, THs are not at all exported by LAT2. Homology modelling of LAT1 and LAT2 is based on available crystal structures from the same superfamily the amino acid/polyamine/organocation transporter (APC). Molecular model guided mutagenesis has been used to predict substrate interaction sites. A common recognition feature for amino acid- and TH-derivatives has been suggested in an interior cavity of LAT1 and LAT2. Therein additional distinct molecular determinants that are responsible for the bidirectional AA transport but allowing only unidirectional import of particular THs have been confirmed for LAT2 by mutagenesis. Characterized substrate features that are needed for TH translocation and distinct LAT2 properties will be highlighted to understand the molecular import and export mechanisms of this transporter in more detail.
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Involvement of the L-Type Amino Acid Transporter LAT2 in the Transport of 3,3′-Diiodothyronine across the Plasma Membrane
European Thyroid Journal, 2015Co-Authors: Anita Kinne, Katrin M. Hinz, Josef Köhrle, Eva K. Wirth, Melanie Wittner, Ralf Schülein, Gerd KrauseAbstract:Thyroid hormones are transported across cell membranes by transmembrane transporter proteins, for example by members of the monocarboxylate transporter (MCT) and the L-type amino acid transporter (LAT) families. LATs consist of a light chain (e.g. LAT2) and a heavy chain (CD98), which is essential for their cell surface expression and functionality. The specificity of LAT2 for thyroid hormones and their metabolites and its role in their transport was not fully clear. This fact motivated us to establish a cell system to elucidate the uptake of thyroid hormones and their metabolites by mouse LAT2. The coinjection of cRNA coding for LAT2 and CD98 into Xenopus laevis oocytes resulted in a markedly increased level of 3,3′-diiodo-L-thyronine (3,3′-T2) and to some extent also enhanced T3 transport. To gain insight into properties of thyroid hormones and their metabolites transported by LAT2, we inhibited 3,3′-T2 uptake by various iodothyronine derivatives. T1 and T2 derivatives as well as 2-aminobicyclo-[2,2,1]-heptane-2-carboxylic acid strongly competed with 3,3′-T2 uptake. In addition, we performed T2 uptake measurements with the thyroid hormone-specific transporter MCT8. For both LAT2 and MCT8, Km values in a low micromolar range were calculated. We demonstrated that oocytes are a suitable system for thyroid hormone transport studies mediated by LAT2. Our data indicates that LAT2 compared to other thyroid hormone transporters prefers 3,3′-T2 as the substrate. Thus, LAT2 might contribute to the availability of thyroid hormone by importing and/or exporting 3,3′-T2, which is generated either by T3 inactivation or by rapid deiodinase 1-mediated rT3 degradation.
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Involvement of the L-Type Amino Acid Transporter LAT2 in the Transport of 3,3 ' -Diiodothyronine across the Plasma Membrane
European thyroid journal, 2015Co-Authors: Anita Kinne, Katrin M. Hinz, Josef Köhrle, Eva K. Wirth, Melanie Wittner, Ralf Schülein, Gerd KrauseAbstract:Thyroid hormones are transported across cell membranes by transmembrane transporter proteins, for example by members of the monocarboxylate transporter (MCT) and the L-type amino acid transporter (LAT) families. LATs consist of a light chain (e.g. LAT2) and a heavy chain (CD98), which is essential for their cell surface expression and functionality. The specificity of LAT2 for thyroid hormones and their metabolites and its role in their transport was not fully clear. This fact motivated us to establish a cell system to elucidate the uptake of thyroid hormones and their metabolites by mouse LAT2. The coinjection of cRNA coding for LAT2 and CD98 into Xenopus laevis oocytes resulted in a markedly increased level of 3,3'-diiodo-L-thyronine (3,3'-T2) and to some extent also enhanced T3 transport. To gain insight into properties of thyroid hormones and their metabolites transported by LAT2, we inhibited 3,3'-T2 uptake by various iodothyronine derivatives. T1 and T2 derivatives as well as 2-aminobicyclo-[2, 2,1]-heptane-2-carboxylic acid strongly competed with 3,3'-T2 uptake. In addition, we performed T2 uptake measurements with the thyroid hormone-specific transporter MCT8. For both LAT2 and MCT8, Km values in a low micromolar range were calculated. We demonstrated that oocytes are a suitable system for thyroid hormone transport studies mediated by LAT2. Our data indicates that LAT2 compared to other thyroid hormone transporters prefers 3,3'-T2 as the substrate. Thus, LAT2 might contribute to the availability of thyroid hormone by importing and/or exporting 3,3'-T2, which is generated either by T3 inactivation or by rapid deiodinase 1-mediated rT3 degradation.
François Verrey - One of the best experts on this subject based on the ideXlab platform.
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Differential expression of system L amino acid transporter subtypes in rat placenta and yolk sac
Placenta, 2020Co-Authors: Wejdan H. Owaydhah, Nick Ashton, François Verrey, Jocelyn D. GlazierAbstract:Abstract Introduction Amino acid transport across the placenta is crucial for fetal growth. In rodent models, the visceral yolk sac (referred to as yolk sac hereafter) is also likely to contribute to fetal amino acid provision. System L amino acid transporters mediate the transport of essential amino acids. System L activity is mediated by light chains LAT1 (Slc7a5) and LAT2 (Slc7a8) which form functional complexes by heterodimeric linkage to CD98 (Slc3a2). LAT4 (Slc43a2) is monomeric, possessing overlapping amino acid substrate specificity with LAT1 and LAT2. Methods This study investigates the expression of these LAT subtypes in fetus-matched rat placenta and yolk sac. Results Slc7a5, Slc7a8 and Slc43a2 transcripts were expressed in placenta and yolk sac with similar expression patterns between sexes. LAT1 expression was significantly higher in placenta than yolk sac. Conversely, LAT2 and LAT4 expression was significantly higher in yolk sac than placenta; CD98 expression was comparable. LAT1, LAT2, LAT4 and CD98 were distributed to rat placental labyrinth zone (LZ) and junctional zone (JZ). LAT1 and LAT4 demonstrated higher expression in LZ, whilst LAT2 was more intensely distributed to JZ. LAT1, LAT2, LAT4 and CD98 were expressed in yolk sac, with punctate LAT1 staining to endodermal cell cytoplasm, contrasting with the intense LAT2, LAT4 and CD98 endodermal cell basolateral distribution, accounting for greater LAT2 and LAT4 expression in yolk sac compared to placenta. Conclusion LAT1, LAT2 and LAT4 are expressed in rat placenta and yolk sac implicating a combined role for these LAT subtypes in supporting fetal growth and development.
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Cooperation of Antiporter LAT2/CD98hc with Uniporter TAT1 for Renal Reabsorption of Neutral Amino Acids.
Journal of the American Society of Nephrology : JASN, 2018Co-Authors: Clara Vilches, Antonio Zorzano, Emilia Boiadjieva-knöpfel, Susanna Bodoy, Simone M. R. Camargo, Miguel López De Heredia, Esther Prat, Aida Ormazabal, Rafael Artuch, François VerreyAbstract:Background Reabsorption of amino acids (AAs) across the renal proximal tubule is crucial for intracellular and whole organism AA homeostasis. Although the luminal transport step is well understood, with several diseases caused by dysregulation of this process, the basolateral transport step is not understood. In humans, only cationic aminoaciduria due to malfunction of the basolateral transporter y+LAT1/CD98hc (SLC7A7/SLC3A2), which mediates the export of cationic AAs, has been described. Thus, the physiologic roles of basolateral transporters of neutral AAs, such as the antiporter LAT2/CD98hc (SLC7A8/SLC3A2), a heterodimer that exports most neutral AAs, and the uniporter TAT1 (SLC16A10), which exports only aromatic AAs, remain unclear. Functional cooperation between TAT1 and LAT2/CD98hc has been suggested by in vitro studies but has not been evaluated in vivoMethods To study the functional relationship of TAT1 and LAT2/CD98hc in vivo, we generated a double-knockout mouse model lacking TAT1 and LAT2, the catalytic subunit of LAT2/CD98hc (dKO LAT2-TAT1 mice).Results Compared with mice lacking only TAT1 or LAT2, dKO LAT2-TAT1 mice lost larger amounts of aromatic and other neutral AAs in their urine due to a tubular reabsorption defect. Notably, dKO mice also displayed decreased tubular reabsorption of cationic AAs and increased expression of y+LAT1/CD98hc.Conclusions The LAT2/CD98hc and TAT1 transporters functionally cooperate in vivo, and y+LAT1/CD98hc may compensate for the loss of LAT2/CD98hc and TAT1, functioning as a neutral AA exporter at the expense of some urinary loss of cationic AAs. Cooperative and compensatory mechanisms of AA transporters may explain the lack of basolateral neutral aminoacidurias in humans.
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Recycling of aromatic amino acids via TAT1 allows efflux of neutral amino acids via LAT2-4F2hc exchanger
Pflugers Archiv : European journal of physiology, 2007Co-Authors: Tamara Ramadan, Simone M. R. Camargo, Brigitte Herzog, Mauro Bordin, Klaas M. Pos, François VerreyAbstract:The rate of amino acid efflux from individual cells needs to be adapted to cellular demands and plays a central role for the control of extracellular amino acid homeostasis. A particular example of such an outward amino acid transport is the basolateral efflux from transporting epithelial cells located in the small intestine and kidney proximal tubule. Because LAT2-4F2hc (Slc7a8-Slc3a2), the best known basolateral neutral amino acid transporter of these epithelial cells, functions as an obligatory exchanger, we tested whether TAT1 (Slc16a10), the aromatic amino-acid facilitated diffusion transporter, might allow amino acid efflux via this exchanger by recycling its influx substrates. In this study, we show by immunofluorescence that TAT1 and LAT2 indeed colocalize in the early kidney proximal tubule. Using the Xenopus laevis oocytes expression system, we show that L-glutamine is released from oocytes into an amino-acid-free medium only when both transporters are coexpressed. High-performance liquid chromatography analysis reveals that several other neutral amino acids are released as well. The transport function of both TAT1 and LAT2-4F2hc is necessary for this efflux, as coexpression of functionally inactive but surface-expressed mutants is ineffective. Based on negative results of coimmunoprecipitation and crosslinking experiments, the physical interaction of these transporters does not appear to be required. Furthermore, replacement of TAT1 or LAT2-4F2hc by the facilitated diffusion transporter LAT4 or the obligatory exchanger LAT1, respectively, supports similar functional cooperation. Taken together, the results suggest that the aromatic amino acid diffusion pathway TAT1 can control neutral amino acid efflux via neighboring exchanger LAT2-4F2hc, by recycling its aromatic influx substrates.
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activation of system l heterodimeric amino acid exchangers by intracellular substrates
The EMBO Journal, 2002Co-Authors: Christian Meier, Zorica Ristic, Stefan Klauser, François VerreyAbstract:System L-type transport of large neutral amino acids is mediated by ubiquitous LAT1-4F2hc and epithelial LAT2-4F2hc. These heterodimers are thought to function as obligatory exchangers, but only influx properties have been studied in some detail up until now. Here we measured their intracellular substrate selectivity, affinity and exchange stoichiometry using the Xenopus oocyte expression system. Quantification of amino acid influx and efflux by HPLC demonstrated an obligatory amino acid exchange with 1:1 stoichiometry. Strong, differential trans-stimulations of amino acid influx by injected amino acids showed that the intracellular substrate availability limits the transport rate and that the efflux selectivity range resembles that of influx. Compared with high extracellular apparent affinities, LAT1- and LAT2-4F2hc displayed much lower intracellular apparent affinities (apparent Km in the millimolar range). Thus, the two system L amino acid transporters that are implicated in cell growth (LAT1-4F2hc) and transcellular transport (LAT2-4F2hc) are obligatory exchangers with relatively symmetrical substrate selectivities but strongly asymmetrical substrate affinities such that the intracellular amino acid concentration controls their activity.
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LAT2, a new basolateral 4F2hc/CD98-associated amino acid transporter of kidney and intestine.
The Journal of biological chemistry, 1999Co-Authors: Grégoire Rossier, François Verrey, Christian Meier, Christian Bauch, Vanessa Summa, Bernard Sordat, Lukas C. KühnAbstract:Glycoprotein-associated amino acid transporters (gpaAT) are permease-related proteins that require heterodimerization to express their function. So far, four vertebrate gpaATs have been shown to associate with 4F2hc/CD98 for functional expression, whereas one gpaAT specifically associates with rBAT. In this study, we characterized a novel gpaAT, LAT2, for which mouse and human cDNAs were identified by expressed sequence tag data base searches. The encoded ortholog proteins are 531 and 535 amino acids long and 92% identical. They share 52 and 48% residues with the gpaATs LAT1 and y(+)LAT1, respectively. When mouse LAT2 and human 4F2hc cRNAs were co-injected into Xenopus oocytes, disulfide-linked heterodimers were formed, and an L-type amino acid uptake was induced, which differed slightly from that produced by LAT1-4F2hc: the apparent affinity for L-phenylalanine was higher, and L-alanine was transported at physiological concentrations. In the presence of an external amino acid substrate, LAT2-4F2hc also mediated amino acid efflux. LAT2 mRNA is expressed mainly in kidney and intestine, whereas LAT1 mRNA is expressed widely. Immunofluorescence experiments showed colocalization of 4F2hc and LAT2 at the basolateral membrane of kidney proximal tubules and small intestine epithelia. In conclusion, LAT2 forms with LAT1 a subfamily of L-type gpaATs. We propose that LAT1 is involved in cellular amino acid uptake, whereas LAT2 plays a role in epithelial amino acid (re)absorption.
