scholarly journals Increased ubiquitination and reduced plasma membrane trafficking of placental amino acid transporter SNAT-2 in human IUGR

2015 ◽  
Vol 129 (12) ◽  
pp. 1131-1141 ◽  
Author(s):  
Yi-Yung Chen ◽  
Fredrick J. Rosario ◽  
Majida Abu Shehab ◽  
Theresa L. Powell ◽  
Madhulika B. Gupta ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Ubiquitin Ligase ◽  
Membrane Trafficking ◽  
Amino Acid Transporter ◽  
Acid Transport ◽  
Mechanistic Target Of Rapamycin ◽  
Neutral Amino Acid Transporter ◽  
Acid Transporter ◽  
Mtor Signalling

Inhibition of placental mechanistic target of rapamycin (mTOR) signalling, which activates NEDD4-2 (neural precursor cell expressed developmentally down-regulated protein 4-2) ubiquitin ligase leading to increased sodium-coupled neutral amino acid transporter 2 (SNAT-2) ubiquitination and removal from the syncytiotrophoblast plasma membrane may constitute a key mechanism underlying decreased placental amino acid transport in human IUGR.

scholarly journals Regulation of amino acid transporter trafficking by mTORC1 in primary human trophoblast cells is mediated by the ubiquitin ligase Nedd4-2

2016 ◽  
Vol 130 (7) ◽  
pp. 499-512 ◽  
Author(s):  
Fredrick J. Rosario ◽  
Kris Genelyn Dimasuay ◽  
Yoshikatsu Kanai ◽  
Theresa L. Powell ◽  
Thomas Jansson
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Ubiquitin Ligase ◽  
Membrane Trafficking ◽  
Molecular Mechanisms ◽  
Acid Transport ◽  
Trophoblast Cells ◽  
Human Trophoblast ◽  
Mtor Complex 1 ◽  
Human Trophoblast Cells

We demonstrate that mTOR complex 1 modulates amino acid transport in primary human trophoblast cells by regulating Nedd4-2 mediated ubiquitination and plasma membrane trafficking of specific transporter isoforms, which may constitute a molecular mechanisms underlying abnormal human fetal growth.

scholarly journals Amino acid sensing by enteroendocrine STC-1 cells: role of the Na+-coupled neutral amino acid transporter 2

2010 ◽  
Vol 298 (6) ◽  
pp. C1401-C1413 ◽  
Author(s):  
Steven H. Young ◽  
Osvaldo Rey ◽  
Catia Sternini ◽  
Enrique Rozengurt
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Transport ◽  
Isobutyric Acid ◽  
Amino Acid Transporter ◽  
Membrane Depolarization ◽  
Neutral Amino Acid ◽  
Acid Transport ◽  
Neutral Amino Acid Transporter ◽  
Acid Transporter

The results presented here show that STC-1 cells, a model of intestinal endocrine cells, respond to a broad range of amino acids, including l-proline, l-serine, l-alanine, l-methionine, l-glycine, l-histidine, and α-methyl-amino-isobutyric acid (MeAIB) with a rapid increase in the intracellular Ca2+ concentration ([Ca2+]i). We sought to identify the mechanism by which amino acids induce Ca2+ signaling in these cells. Several lines of evidence suggest that amino acid transport through the Na+-coupled neutral amino acid transporter 2 (SNAT2) is a major mechanism by which amino acids induced Ca2+ signaling in STC-1 cells: 1) the amino acid efficacy profile for inducing Ca2+ signaling in STC-1 cells closely matches the amino acid specificity of SNAT2; 2) amino acid-induced Ca2+ signaling in STC-1 cells was suppressed by removing Na+ from the medium; 3) the nonmetabolized synthetic substrate of amino acid transport MeAIB produced a marked increase in [Ca2+]i; 4) transfection of small interfering RNA targeting SNAT2 produced a marked decrease in Ca2+ signaling in response to l-proline in STC-1 cells; 5) amino acid-induced increase in [Ca2+]i was associated with membrane depolarization and mediated by Ca2+ influx, since it depended on extracellular Ca2+; 6) the increase in [Ca2+]i in response to l-proline, l-alanine, or MeAIB was abrogated by either nifedipine (1–10 μM) or nitrendipine (1 μM), which block L-type voltage-sensitive Ca2+ channels. We hypothesize that the inward current of Na+ associated with the function of SNAT2 leads to membrane depolarization and activation of voltage-sensitive Ca2+ channels that mediate Ca2+ influx, thereby leading to an increase in the [Ca2+]i in enteroendocrine STC-1 cells.

scholarly journals Ubiquitylation and endocytosis of the human LAT1/SLC7A5 amino acid transporter

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Céline Barthelemy ◽  
Bruno André
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Protein Kinase C ◽  
Amino Acid ◽  
Ubiquitin Ligase ◽  
Essential Amino Acids ◽  
Amino Acid Transporter ◽  
Kinase C ◽  
Acid Transporter ◽  
Mtorc1 Activation

AbstractThe human L-type amino acid transporter 1 (LAT1), also known as SLC7A5, catalyzes the transport of large neutral amino acids across the plasma membrane. As the main transporter of several essential amino acids, notably leucine, LAT1 plays an important role in mTORC1 activation. Furthermore, it is overexpressed in various types of cancer cells, where it contributes importantly to sustained growth. Despite the importance of LAT1 in normal and tumor cells, little is known about the mechanisms that might control its activity, for example by promoting its downregulation via endocytosis. Here we report that in HeLa cells, activation of protein kinase C by phorbol 12-myristate 13-acetate (PMA) triggers efficient endocytosis and degradation of LAT1. Under these conditions we found LAT1 downregulation to correlate with increased LAT1 ubiquitylation. This modification was considerably reduced in cells depleted of the Nedd4-2 ubiquitin ligase. By systematically mutagenizing the residues of the LAT1 cytosolic tails, we identified a group of three close lysines (K19, K25, K30) in the N-terminal tail that are important for PMA-induced ubiquitylation and downregulation. Our study thus unravels a mechanism of induced endocytosis of LAT1 elicited by Nedd4-2-mediated ubiquitylation of the transporter’s N-terminal tail.

scholarly journals Mutations of the basic amino acid transporter gene associated with cystinuria

1995 ◽  
Vol 310 (3) ◽  
pp. 951-955 ◽  
Author(s):  
K Miyamoto ◽  
K Katai ◽  
S Tatsumi ◽  
K Sone ◽  
H Segawa ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Transport ◽  
Basic Amino Acid ◽  
Amino Acid Transporter ◽  
Regulatory Subunit ◽  
Acid Transport ◽  
Transport Activity ◽  
Wild Type ◽  
Neutral Amino Acid Transporter ◽  
Acid Transporter

To investigate the function of a basic and neutral amino acid transporter-like protein (rBAT) which is a candidate gene for cystinuria, we analysed the rBAT gene in cystinuric patients. Patient 1 is a compound heterozygote with mutations in the rBAT gene causing a glutamine-to-lysine transition at amino acid 268, and a threonine-to-alanine transition at amino acid 341, who inherited these alleles from his mother (E268K) and father (T341A), respectively. Injection of T341A and E268K mutant cRNAs into oocytes decreased transport activity to 53.9% and 62.5% of control (L-cystine transport activity in oocytes injected with wild-type rBAT cRNA), respectively. Co-injection of E268K and T341A into oocytes strongly decreased amino acid transport activity to 28% of control. On the other hand, co-injection of wild-type and mutant rBAT did not decrease transport activity. Furthermore, immunological studies have demonstrated that the reduction of amino acid transport is not due to a decrease in the amount of rBAT protein expressed in oocyte membranes. These results indicate that mutations in the rBAT gene are crucial disease-causing lesions in cystinuria. In addition, co-injection experiments suggest that rBAT may function as a transport activator or regulatory subunit by homo- or hetero-multimer complex formation.

scholarly journals Identification and Characterization of Inhibitors of a Neutral Amino Acid Transporter, SLC6A19, Using Two Functional Cell-Based Assays

2018 ◽  
Vol 24 (2) ◽  
pp. 111-120 ◽  
Author(s):  
Sanjay J. Danthi ◽  
Beirong Liang ◽  
Oanh Smicker ◽  
Benjamin Coupland ◽  
Jill Gregory ◽  
...  
Keyword(s):  
Amino Acid ◽  
High Throughput Screening ◽  
Amino Acid Transporter ◽  
Neutral Amino Acid ◽  
Imaging Plate ◽  
Acid Uptake ◽  
Functional Assays ◽  
Neutral Amino Acid Transporter ◽  
Acid Transporter ◽  
Pharmacologically Active

SLC6A19 (B0AT1) is a neutral amino acid transporter, the loss of function of which results in Hartnup disease. SLC6A19 is also believed to have an important role in amino acid homeostasis, diabetes, and weight control. A small-molecule inhibitor of human SLC6A19 (hSLC6A19) was identified using two functional cell-based assays: a fluorescence imaging plate reader (FLIPR) membrane potential (FMP) assay and a stable isotope-labeled neutral amino acid uptake assay. A diverse collection of 3440 pharmacologically active compounds from the Microsource Spectrum and Tocriscreen collections were tested at 10 µM in both assays using MDCK cells stably expressing hSLC6A19 and its obligatory subunit, TMEM27. Compounds that inhibited SLC6A19 activity in both assays were further confirmed for activity and selectivity and characterized for potency in functional assays against hSLC6A19 and related transporters. A single compound, cinromide, was found to robustly, selectively, and reproducibly inhibit SLC6A19 in all functional assays. Structurally related analogs of cinromide were tested to demonstrate structure–activity relationship (SAR). The assays described here are suitable for carrying out high-throughput screening campaigns to identify modulators of SLC6A19.

scholarly journals B0AT1 amino acid transporter complexed with SARS-CoV-2 receptor ACE2 forms a heterodimer functional unit: in situ conformation using radiation inactivation analysis

Function ◽  
2021 ◽  
Author(s):  
Bruce R Stevens ◽  
J Clive Ellory ◽  
Robert L Preston
Keyword(s):  
Amino Acid ◽  
Membrane Trafficking ◽  
Functional Unit ◽  
Membrane Vesicles ◽  
High Energy ◽  
Amino Acid Transporter ◽  
Neutral Amino Acid ◽  
Transport Activity ◽  
Acid Transporter

Abstract The SARS-CoV-2 receptor, Angiotensin Converting Enzyme-2 (ACE2), is expressed at levels of greatest magnitude in the small intestine as compared to all other human tissues. Enterocyte ACE2 is co-expressed as the apical membrane trafficking partner obligatory for expression and activity of the B0AT1 sodium-dependent neutral amino acid transporter. These components are assembled as an [ACE2: B0AT1]2 dimer-of-heterodimers quaternary complex that putatively steers SARS-CoV-2 tropism in the gastrointestinal (GI) tract. GI clinical symptomology is reported in about half of COVID-19 patients, and can be accompanied by gut shedding of virion particles. We hypothesized that within this 4-mer structural complex, each [ACE2: B0AT1] heterodimer pair constitutes a physiological “functional unit.” This was confirmed experimentally by employing purified lyophilized enterocyte brush border membrane vesicles that were exposed to increasing doses of high-energy electron radiation from a 16 MeV linear accelerator. Based on established target theory, the results indicated the presence of Na+-dependent neutral amino acid influx transport activity functional unit with target size mw = 183.7 ± 16.8 kDa in situ in intact apical membranes. Each thermodynamically stabilized [ACE2: B0AT1] heterodimer functional unit manifests the transport activity within the whole ∼345 kDa [ACE2: B0AT1]2 dimer-of-heterodimers quaternary structural complex. The results are consistent with our prior molecular docking modeling and gut-lung axis approaches to understanding COVID-19. These findings advance the understanding of the physiology of B0AT1 interaction with ACE2 in the gut, and thereby potentially contribute to translational developments designed to treat or mitigate COVID-19 variant outbreaks and/or GI symptom persistence in long-haul Post-Acute Sequelae of SARS-CoV-2 (PASC).

scholarly journals Neutral amino acid transporter ASCT2 displays substrate-induced Na+ exchange and a substrate-gated anion conductance

2000 ◽  
Vol 346 (3) ◽  
pp. 705-710 ◽  
Author(s):  
Angelika BRÖER ◽  
Carsten WAGNER ◽  
Florian LANG ◽  
Stefan BRÖER
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Anion Channel ◽  
Amino Acid Transporter ◽  
Neutral Amino Acid ◽  
Xenopus Laevis Oocytes ◽  
Anion Conductance ◽  
Neutral Amino Acid Transporter ◽  
Inward Currents ◽  
Acid Transporter

The neutral amino acid transporter ASCT2 mediates electroneutral obligatory antiport but at the same time requires Na+ for its function. To elucidate the mechanism, ASCT2 was expressed in Xenopus laevis oocytes and transport was analysed by flux studies and two-electrode voltage clamp recordings. Flux studies with 22NaCl indicated that the uptake of one molecule of glutamine or alanine is accompanied by the uptake of four to seven Na+ ions. Similarly to the transport of amino acids, the Na+ uptake was mediated by an obligatory Na+ exchange mechanism that depended on the presence of amino acids but was not stoichiometrically coupled to the amino acid transport. Other cations could not replace Na+ in this transport mechanism. When NaCl was replaced by NaSCN in the transport buffer, the superfusion of oocytes with amino acid substrates resulted in large inward currents, indicating the presence of a substrate-gated anion channel in the ASCT2 transporter. The Km for glutamine derived from these experiments is in good agreement with the Km derived from flux studies; it varied between 40 and 90 μM at holding potentials of -60 and -20 mV respectively. The permeability of the substrate-gated anion conductance decreased in the order SCN- NO3- > I- > Cl- and also required the presence of Na+.

The SLC1 high-affinity glutamate and neutral amino acid transporter family

2013 ◽  
Vol 34 (2-3) ◽  
pp. 108-120 ◽  
Author(s):  
Yoshikatsu Kanai ◽  
Benjamin Clémençon ◽  
Alexandre Simonin ◽  
Michele Leuenberger ◽  
Martin Lochner ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Transporter ◽  
Neutral Amino Acid ◽  
High Affinity ◽  
Neutral Amino Acid Transporter ◽  
Transporter Family ◽  
Acid Transporter
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