Expression Of LAT1 Research Articles

Expression of LAT1 predicts progression of transitional cell carcinoma of the upper urinary tract

LAT1, a neutral amino acid transporter, requires covalent association with the heavy chain of 4F2 cell surface antigen (4F2hc) for its functional form. We investigated the importance of LAT1 and 4F2hc expressions to progression in transitional cell carcinoma of the upper urinary tract. In 124 cases, we examined their expressions using immunohistochemistry and in situ hybridization, and also their relationships to the expression of proliferating cell nuclear antigen (PCNA), clinicopathologic parameters, and clinical outcome. Positive expressions of LAT1 (protein and mRNA), and 4F2hc (protein) were recognized in 79.8%, 89.5%, and 87.9% of samples, respectively. In tumor cells, LAT1 protein was detected as a nodular pattern or membranous pattern (plasma membrane or diffuse cytoplasm plus plasma membrane), while LAT1 mRNA was detected as nodular or diffuse (diffuse cytoplasm) patterns and 4F2hc protein was detected as a diffuse pattern. A correlation with stage and both overall and disease-free survival rates in the univariate analysis, but not with PCNA index, was shown for: LAT1 protein expression, a membranous pattern of LAT1 protein, a diffuse pattern of LAT1 mRNA, a cooperative expression of LAT1 protein (membranous pattern) and 4F2hc protein, and a cooperative expression of LAT1 mRNA (diffuse pattern) and 4F2hc protein. In conclusion, the detection of LAT1 protein would appear to be of value in informing the progression of TCC-UUT.

Expression and function of LAT1, a neutral amino acid exchanger, in renal porcine epithelial cell line LLC‐PK1

The present study examined the expression of LAT1 and the functional characteristics of the inward and outward [14C] l-leucine transporter in the renal porcine epithelial cell line LLC-PK1. LLC-PK1 cells were cultured in polycarbonate filters and accumulation and transepithelial flux of the substrate monitored with [14C] l-leucine. LAT1 transcripts were examined by RT-PCR. LAT1 protein was detected by immunoblotting. The accumulation of [14C] l-leucine in the cell and the [14C] l-leucine transepithelial flux were four- and twofold, respectively, when the substrate was added from the basal cell side, suggesting that the basolateral membrane is endowed with a high density of transport units, when compared with the apical membrane. Increases in the transepithelial flux of [14C] l-leucine by unlabelled l-leucine were also more pronounced when unlabelled l-leucine was added from the basolateral membrane. In the absence of Na+, unlabelled l-leucine increased the basal and apical fractional outflow of [14C] l-leucine, this being similar at pH 7.4 and pH 6.2. RT-PCR and immunoblotting detected LAT1 transcript and protein, respectively. LLC-PK1 cells are endowed with the LAT1 transcript and protein and transport l-leucine through the Na+-independent and pH-insensitive LAT1 transporter. The density of transporter units in LLC-PK1 cells may be higher at the basolateral membranes, although be also present in the apical membranes.

Expression of LAT1 and LAT2 amino acid transporters in human and rat intestinal epithelial cells

The present study evaluated the presence of LAT1 and LAT2 amino acid transporters in human Caco-2 cells and rat IEC-6 cells along the mucosa of the rat digestive tract. The LAT1 cDNA was amplified by PCR using two sets of primers (one specific for rat LAT1 and another simultaneously specific for human, rat and mice). The LAT2 cDNA was amplified by PCR using one set of primers simultaneously specific for human, rat and mice LAT2. The presence of LAT1 and LAT2 protein was examined by means of immunoblotting using an antibody raised against the rat LAT1 and mouse LAT2. Caco-2 and IEC-6 cells, as well as the rat intestinal mucosa, are endowed with both LAT1 and LAT2 transporter transcripts and protein. LAT1 protein is most abundant in IEC-6 cells, which is in agreement with functional data previously reported. The findings in the rat intestinal mucosa indicate that LAT1 protein is most abundant in the colon and its abundance markedly decreases at the level of jejunum and ileum, which contrast with relative homogeneous presence of LAT2 across the digestive tract. In conclusion, Caco-2 and IEC-6 cells, as well as the rat intestinal mucosa, are endowed with both LAT1 and LAT2 amino acid transporter transcripts and protein.

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.

Organ-Specific Overexpression of Renal LAT2 and Enhanced Tubular l -DOPA Uptake Precede the Onset of Hypertension

Spontaneously hypertensive rats (SHR) might have increased renal production of dopamine. L-3,4-Dihydroxyphenylalanine (L-DOPA) uptake in renal epithelial cells is promoted through the type 2 L-type amino acid transporter (LAT2), and this might rate-limit the synthesis of renal dopamine. The present study evaluated L-DOPA uptake in isolated renal proximal tubules of SHR and normotensive controls (Wistar-Kyoto rats [WKY]). Expression of LAT1 and LAT2 in the renal cortex and intestinal mucosa was also evaluated. Tubular uptake of L-DOPA in WKY and SHR was a saturable process, being greater in the latter than the former at both 4 and 12 weeks of age. cDNA fragments (LAT1, 688 bp; LAT2, 729 bp) labeled with 32P were used as probes for Northern blot analysis. Expression of LAT2 in SHR kidneys was higher than in WKY kidneys. This increase was more marked at 4 than at 12 weeks of age. Intestinal LAT2 expression, however, was identical in SHR and WKY at both 4 and 12 week of age. By Northern blot analysis, the LAT1 transcript was not identified in either the kidney or intestine. Kidney total RNA was then reverse-transcribed and amplified by polymerase chain reaction with specific primers for LAT1. The presence of a fragment of the expected size for LAT1 led to the conclusion that LAT1 mRNA is a rare message in kidney. We conclude that overexpression of LAT2 in the SHR kidney might contribute to the enhanced L-DOPA uptake, which is organ specific and precedes the onset of hypertension.

Functional and molecular characteristics of system l in human breast cancer cells

The functional and molecular properties of system l in human mammary cancer cells (MDA-MB-231 and MCF-7) have been examined. All transport experiments were conducted under Na +-free conditions. α-Aminoisobutyric acid (AIB) uptake by MDA-MB-231 and MCF-7 cells was almost abolished by BCH (2-amino-2-norbornane-carboxylic acid). AIB uptake by MDA-MB-231 cells was also inhibited by l-alanine (83.6%), l-lysine (75.6%) but not by l-proline. Similarly, l-lysine and l-alanine, respectively, reduced AIB influx into MCF-7 cells by 45.3% and 63.7%. The K m of AIB uptake into MDA-MB-231 and MCF-7 cells was, respectively, 1.6 and 8.8 mM, whereas the V max was, respectively, 9.7 and 110.0 nmol/mg protein/10 min. AIB efflux from MDA-MB-231 and MCF-7 cells was trans-stimulated by BCH, l-glutamine, l-alanine, l-leucine, l-lysine and AIB (all at 2 mM). In contrast, l-glutamate, l-proline, l-arginine and MeAIB had no effect. The interaction between l-lysine and AIB efflux was one of low affinity. The fractional release of AIB from MDA-MB-231 cells was trans-accelerated by d-leucine and d-tryptophan but not by d-alanine. MDA-MB-231 and MCF-7 cells expressed LAT1 and CD98 mRNA. MCF-7 cells also expressed LAT2 mRNA. The results suggest that AIB transport in mammary cancer cells under Na +-free conditions is predominantly via system l which acts as an exchange mechanism. The differences in the kinetics of AIB transport between MDA-MB-231 and MCF-7 cells may be due to the differential expression of LAT2.

Association of 4F2hc with light chains LAT1, LAT2 or y+LAT2 requires different domains.

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 complex 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.

Increased expression of an amino acid transporter LAT1 in healing of acetic acid-induced chronic gastric ulcer in rats

Increased expression of an amino acid transporter LAT1 in healing of acetic acid-induced chronic gastric ulcer in rats

Increased expression of an amino acid transporter LAT1 in healing of acetic acid-induced chronic gastric ulcer in rats

Increased expression of an amino acid transporter LAT1 in healing of acetic acid-induced chronic gastric ulcer in rats