Bröer A, etal., Biochem J. 2001 May 1;355(Pt 3):725-31. doi: 10.1042/bj3550725.
Heterodimeric amino acid transporters are comprised of a type-II membrane protein named the heavy chain (4F2hc or rBAT) that may associate with a number of different polytopic membrane proteins, called light chains. It is thought that the heavy chain is mainly involved in the trafficking of the comp
lex to the plasma membrane, whereas the transport process itself is catalysed by the light chain. The 4F2 heavy chain (4F2hc) associates with at least six different light chains to induce distinct amino acid-transport activites. To test if the light chains are specifically recognized and to identify domains involved in the recognition of light chains, C-terminally truncated mutants of 4F2hc were constructed and co-expressed with the light chains LAT1, LAT2 and y(+)LAT2. The truncated isoform T1, comprised of only 133 amino acids that form the cytosolic N-terminus and the transmembrane helix, displayed only a slight reduction in its ability to promote LAT1 expression at the membrane surface compared with the 529 amino acid wild-type 4F2hc protein. Co-expression of increasingly larger 4F2hc mutants caused a delayed translocation of LAT1. In contrast to the weak effects of 4F2hc truncations on LAT1 expression, surface expression of LAT2 and y(+)LAT2 was almost completely lost with all truncated heavy chains. Co-expression of LAT1 together with the other light chains did not result in displacement of LAT2 and y(+)LAT2. The results suggest that extracellular domains of the heavy chain are responsible mainly for recognition of light chains other than LAT1 and that the extracellular domain ensures proper translocation to the plasma membrane.
Zielinska M, etal., J Neurochem. 2015 Dec;135(6):1272-81. doi: 10.1111/jnc.13387. Epub 2015 Nov 6.
One of the aspects of ammonia toxicity to brain cells is increased production of nitric oxide (NO) by NO synthases (NOSs). Previously we showed that ammonia increases arginine (Arg) uptake in cultured rat cortical astrocytes specifically via y(+)L amino acid transport system, by activation of its
member, a heteromeric y(+)LAT2 transporter. Here, we tested the hypothesis that up-regulation of y(+)LAT2 underlies ammonia-dependent increase of NO production via inducible NOS (iNOS) induction, and protein nitration. Treatment of rat cortical astrocytes for 48 with 5 mM ammonium chloride ('ammonia') (i) increased the y(+)L-mediated Arg uptake, (ii) raised the expression of iNOS and endothelial NOS (eNOS), (iii) stimulated NO production, as manifested by increased nitrite+nitrate (Griess) and/or nitrite alone (chemiluminescence), and consequently, (iv) evoked nitration of tyrosine residues of proteins in astrocytes. Except for the increase of eNOS, all the above described effects of ammonia were abrogated by pre-treatment of astrocytes with either siRNA silencing of the Slc7a6 gene coding for y(+)LAT2 protein, or antibody to y(+)LAT2, indicating their strict coupling to y(+)LAT2 activity. Moreover, induction of y(+)LAT2 expression by ammonia was sensitive to Nf-kappaB inhibitor, BAY 11-7085, linking y(+)LAT2 upregulation to the Nf-kappaB activation in this experimental setting as reported earlier and here confirmed. Importantly, ammonia did not affect y(+)LAT2 expression nor y(+)L-mediated Arg uptake activity in the cultured cerebellar neurons, suggesting astroglia-specificity of the above described mechanism. The described coupling of up-regulation of y(+)LAT2 transporter with iNOS in ammonia-exposed astrocytes may be considered as a mechanism to ensure NO supply for protein nitration. Ammonia (NH4(+)) increases the expression and activity of the L-arginine (Arg) transporter (Arg/neutral amino acids [NAA] exchanger) y(+)LAT2 in cultured rat cortical astrocytes by a mechanism involving activation (nuclear translocation) of the transcription factor nuclear factor-Nuclear factor-kappaB (Nf-kappaB-p65). Up-regulation of y(+)LAT2 transporter is coupled with increased inducible nitric oxide synthase (iNOS) expression, which leads to increase nitric oxide (NO) synthesis and protein nitration.
The transmembrane adaptor protein (TRAP), NTAL, is phosphorylated in mast cells following FcvarepsilonRI aggregation whereby it cooperates with LAT to induce degranulation. The Kit ligand, stem cell factor (SCF), enhances antigen-induced degranulation and this also appears to be NTAL-dependent. Howe
ver, Kit and FcvarepsilonRI appear to utilize different mechanisms to induce NTAL phosphorylation. Thus, we examined whether the responsible kinases selectively phosphorylated distinct tyrosines in NTAL and explored the implications for downstream signaling. Whereas FcvarepsilonRI required Lyn and Syk for NTAL phosphorylation, Kit appeared to directly phosphorylate NTAL. Furthermore, co-transfection studies with NTAL constructs revealed that Lyn, Syk, and Kit phosphorylate different tyrosines in NTAL. The tyrosines principally phosphorylated by Syk were recognized as Grb2-binding sites, whereas Lyn and Kit phosphorylated other tyrosines, both inside and outside of these motifs. Pull down studies revealed that PLCgamma1 associated with the two terminal Syk-phosphorylated Grb2-binding sites, which would help to explain the observed decrease in antigen-induced calcium signal and degranulation in NTAL-knock down-human mast cells. The observations reported herein support the conclusion that NTAL may be differentially utilized by specific receptors for relaying alternative signals and this suggests a flexibility in the function of TRAPs not previously appreciated.
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 aci
d 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.
Zielińska M, etal., Neurochem Int. 2012 Sep;61(4):531-5. doi: 10.1016/j.neuint.2012.02.021. Epub 2012 Feb 27.
Increased l-Arg (Arg) uptake to astrocytes and neurons is thought to contribute to enhanced nitric oxide (NO) synthesis and oxidative/nitrosative stress associated with hyperammonemia (HA). Recently we had shown that HA increases the expression in the brain of y(+)LAT2
, an isoform of the y(+)L heteromeric transporter which promotes [(3)H]Arg efflux form brain cells in the presence of l-glutamine (Gln) (Zielińska et al., 2011). In this study, we demonstrate that a significant proportion of [(3)H]Arg uptake to cultured cortical astrocytes is likewise mediated by system y(+)L, in addition to the uptake showing characteristics of systems y(+), B(0+) and b(0+). However, stimulation of [(3)H]Arg uptake by treatment with 5mM ammonium chloride ("ammonia") for 48 h could be solely ascribed to the y(+)L-mediated component of the uptake. Ammonia treatment increased the expression of the brain specific y(+)L isoform, y(+)LAT2, both at the mRNA and protein level, and silencing of the Slc7a6 gene coding for y(+)LAT2 protein specifically reduced the ammonia-induced [(3)H]Arg uptake. This study suggests an important role of y(+)LAT2 in the modulation of NO synthesis in the ammonia-exposed astrocytes.
