Identification of a plasma membrane glutamine transporter from the rat hepatoma cell line H4-IIE-C3.
Glutamine is taken up into the rat hepatoma cell line H4-IIE-C3 by a Na+-dependent transport system which is specific for glutamine, alanine, serine, cysteine and asparagine and does not tolerate substitution of Na+ by Li+. Glutamine transport was relatively weakly inhibited by a 50-fold excess of leucine and was not inhibited by phenylalanine or N -methyl aminoisobutyrate. These general properties are characteristic of the recently identified ASCT/B0 family of transporters. Using a reverse transcriptase PCR-based homology cloning approach, we have characterized a cDNA for a novel member of this transporter family (H4-ASCT2) from H4-IIE-C3 cells. The cDNA encodes a 551-amino acid protein which exhibits similarities of between 75 and 85% with ASCT/B0 transporters previously cloned from other sources. When expressed in Xenopus oocytes, this transporter catalyses Na+-dependent glutamine uptake with characteristics very similar to those of glutamine uptake into the H4-IIE-C3 cells. This newly characterized transporter possesses a number of amino acid sequence differences from ASCT2 clones recently isolated from rat astroglial cells and from normal rat liver. In particular, the loop region between transmembrane helices 3 and 4 from H4-ASCT2 shares less than 60% sequence similarity with ASCT2 from rat liver; furthermore, there are some 25 single amino acid substitutions elsewhere in the H4-ASCT2 sequence compared with that from rat liver. Thus enhanced glutamine uptake in rat hepatoma cells is mediated by the expression of a novel ASCT/B0 transporter isoform rather than by increased expression of the ASCT2 mRNA found in normal rat liver.
Pollard
Matthew
M
Department of Biochemistry, School of Medical Sciences, University Walk, Bristol BS8 1TD, U.K.
Meredith
David
D
McGivan
John D
JD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000473:pathology; D002352:Carrier Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D018076:DNA, Complementary / Q000032:analysis; D008113:Liver Neoplasms / Q000473:pathology; D008969:Molecular Sequence Data; D051381:Rats; D017386:Sequence Homology, Amino Acid; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D018076:DNA, Complementary; C037853:glutamine transport proteins