Two amino acid residues determine the low substrate affinity of human cationic amino acid transporter-2A.
Mammalian cationic amino acid transporters (CAT) differ in their substrate affinity and sensitivity to trans-stimulation. The apparent Km values for cationic amino acids and the sensitivity to trans-stimulation of CAT-1, -2B, and -3 are characteristic of system y+. In contrast, CAT-2A exhibits a 10-fold lower substrate affinity and is largely independent of substrate at the trans-side of the membrane. CAT-2A and -2B demonstrate such divergent transport properties, even though their amino acid sequences differ only in a stretch of 42 amino acids. Here, we identify two amino acid residues within this 42-amino acid domain of the human CAT-2A protein that are responsible for the apparent low affinity of both the extracellular and intracellular substrate-binding sites. These residues are located in the fourth intracellular loop, suggesting that they are not part of the translocation pathway. Rather, they may be responsible for the low affinity conformation of the substrate-binding sites. The sensitivity to trans-stimulation is not determined by the same amino acid residues as the substrate affinity and must involve a more complex interaction between individual amino acid residues. In addition to the 42-amino acid domain, the adjacent transmembrane domain X seems to be involved in this function.
Habermeier
Alice
A
Department of Pharmacology, Johannes Gutenberg University, Obere Zahlbacher Strasse 67, 55101 Mainz, Germany.
Wolf
Sabine
S
Martiné
Ursula
U
Gräf
Petra
P
Closs
Ellen I
EI
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D001120:Arginine / Q000378:metabolism; D001665:Binding Sites; D001692:Biological Transport; D019921:Biotinylation; D015153:Blotting, Western; D027202:Cationic Amino Acid Transporter 2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins; D013329:Structure-Activity Relationship; D014162:Transfection; D014982:Xenopus laevis
D000596:Amino Acids; D027202:Cationic Amino Acid Transporter 2; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D001120:Arginine; D005982:Glutathione Transferase