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The 4F2hc surface antigen is necessary for expression of system L-like neutral amino acid-transport activity in C6-BU-1 rat glioma cells: evidence from expression studies in Xenopus laevis oocytes.

ABSTRACT: Mammalian cells possess a variety of amino acid-transport systems with overlapping substrate specificity. System L is one of the major amino acid-transport systems in all non-epithelial cells. Its molecular structure is not known. To clone the neutral amino acid-transporter system L, we followed an expression cloning strategy using Xenopus laevis oocytes. A cDNA library derived from C6-BU-1 rat glioma cells was used as a source, because high expression of system L activity could be demonstrated with polyadenylated RNA isolated from these cells, when injected into Xenopus laevis oocytes [Bröer, Bröer and Hamprecht (1994) Biochim. Biophys. Acta 1192, 95-100]. A single clone (ILAT) was identified, the sense cRNA of which, on injection into Xenopus laevis oocytes, stimulated sodium-independent isoleucine transport by about 100-fold. Further characterization revealed that transport of cationic amino acids was also stimulated. Sequencing of the cDNA showed that the identified clone is the heavy chain of the rat 4F2 surface antigen, a marker of tumour cells and activated lymphocytes. Uptake of neutral and cationic amino acids was not stimulated by the presence of Na+ ions. Antisense cRNA transcribed from this clone or antisense oligonucleotides, when co-injected with polyadenylated RNA from C6-BU-1 rat glioma cells, completely suppressed system L-like isoleucine-transport activity. We conclude that ILAT is necessary for expression of system L-like amino acid-transport activity by polyadenylated RNA from C6-BU-1 rat glioma cells.

SUBMITTER: Broer S

PROVIDER: S-EPMC1136194 | biostudies-other | 1995 Dec

REPOSITORIES: biostudies-other

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Project description:BackgroundA new type of influenza virus, known as type D, has recently been identified in cattle and pigs. Influenza D virus infection in cattle is typically asymptomatic; however, its infection in swine can result in clinical disease. Swine can also be infected with all other types of influenza viruses, namely A, B, and C. Consequently, swine can serve as a "mixing vessel" for highly pathogenic influenza viruses, including those with zoonotic potential. Currently, the only antiviral drug available targets influenza M2 protein ion channel is not completely effective. Thus, it is necessary to develop an M2 ion channel blocker capable of suppressing the induction of resistance to the genetic shift. To provide a basis for developing novel ion channel-blocking compounds, we investigated the properties of influenza D virus M2 protein (DM2) as a drug target.ResultsTo test the ion channel activity of DM2, the DNA corresponding to DM2 with cMyc-tag conjugated to its carboxyl end was cloned into the shuttle vector pNCB1. The mRNA of the DM2-cMyc gene was synthesized and injected into Xenopus oocytes. The translation products of DM2-cMyc mRNA were confirmed by immunofluorescence and mass spectrometry analyses. The DM2-cMyc mRNA-injected oocytes were subjected to the two-electrode voltage-clamp (TEVC) method, and the induced inward current was observed. The midpoint (Vmid) values in Boltzmann modeling for oocytes injected with DM2-cMyc RNA or a buffer were -152 and -200 mV, respectively. Assuming the same expression level in the Xenopus oocytes, DM2 without tag and influenza C virus M2 protein (CM2) were subjected to the TEVC method. DM2 exhibited ion channel activity under the condition that CM2 ion channel activity was reproduced. The gating voltages represented by Vmid for CM2 and DM2 were -141 and -146 mV, respectively. The reversal potentials observed in ND96 for CM2 and DM2 were -21 and -22 mV, respectively. Compared with intact DM2, DM2 variants with mutation in the YxxxK motif, namely Y72A and K76A DM2, showed lower Vmid values while showing no change in reversal potential.ConclusionThe M2 protein from newly isolated influenza D virus showed ion channel activity similar to that of CM2. The gating voltage was shown to be affected by the YxxxK motif and by the hydrophobicity and bulkiness of the carboxyl end of the molecule.

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The 4F2hc surface antigen is necessary for expression of system L-like neutral amino acid-transport activity in C6-BU-1 rat glioma cells: evidence from expression studies in Xenopus laevis oocytes.

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