RESUMEN
LAT1 (L-type amino acid transporter 1), one of the L-type amino acid transporters, transports the branched and aromatic amino acids. LAT1 requires the heavy chain of 4F2 antigen (4F2hc) for the functional expression as an amino acid transporter. The expression of this transporter is up-regulated in tumor cells and rapidly-growing cells to support their proliferation. Here, we studied the expression of LAT1 and 4F2hc in human cultured cells by real-time PCR and Western blot, and found that human brain astrocytomas, U343MGa, highly expressed LAT1 and 4F2hc mRNAs and proteins. The uptake of [14C]leucine by U343MGa cells is Na+-independent and inhibited by BCH (2-amino-2-norbornane carboxylic acid), and branched and aromatic amino acids, indicating that the LAT1 is expressed at the cell surface. Pulse chase labeling and surface labeling experiments of this cell line indicate that the protein synthesis of LAT1 and 4F2hc is slow, however, the heterodimeric complex assembled in the cells is very stable, and that the disulfide bond between the LAT1 and 4F2hc is not directly involved in the stability of the heterodimer.
Asunto(s)
Perfilación de la Expresión Génica , Transportador de Aminoácidos Neutros Grandes 1/genética , Aminoácidos/farmacología , Astrocitoma/genética , Astrocitoma/metabolismo , Astrocitoma/patología , Transporte Biológico/efectos de los fármacos , Western Blotting , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Radioisótopos de Carbono , Línea Celular , Línea Celular Tumoral , Electroforesis en Gel de Poliacrilamida , Cadena Pesada de la Proteína-1 Reguladora de Fusión/genética , Cadena Pesada de la Proteína-1 Reguladora de Fusión/metabolismo , Células HT29 , Células HeLa , Humanos , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Leucina/metabolismo , Leucina/farmacocinética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia Arriba/genéticaRESUMEN
2-Methyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine-3-acetonitrile (SCH 28080) is a reversible inhibitor specific for the gastric proton pump. The inhibition pattern is competitive with K(+). Here we studied the binding sites of this inhibitor on the putative three-dimensional structure of the gastric proton pump alpha-subunit that was constructed by homology modeling based on the structure of sarcoplasmic reticulum Ca(2+) pump. Alanine and serine mutants of Tyr(801) located in the fifth transmembrane segment of the gastric proton pump alpha-subunit retained the (86)Rb transport and K(+)-dependent ATPase (K(+)-ATPase) activities. These mutants showed 60-80-times lower sensitivity to SCH 28080 than the wild type in the (86)Rb transport activity. The K(+)-ATPase activities of these mutants were not completely inhibited by SCH 28080. The sensitivity to SCH 28080 was dependent on the bulkiness of the side chain at this position. Therefore, the side chain of Tyr(801) is important for the interaction with this inhibitor. In the three-dimensional structure of the E(2) form (conformation with high affinity for K(+)) of the gastric proton pump, Tyr(801) faces a cavity surrounded by the first, fourth, fifth, sixth, and eighth transmembrane segments and fifth/sixth, seventh/eighth, and ninth/tenth loops. SCH 28080 can dock in this cavity. However, SCH 28080 cannot dock in the same location in the E(1) form (conformation with high affinity for proton) of the gastric proton pump due to the drastic rearrangement of the transmembrane helices between the E(1) and E(2) forms. These results support the idea that this cavity is the binding pocket of SCH 28080.
