Tyrosine phosphorylation-and epidermal growth factor-dependent regulation of the sodium-coupled amino acid transporter B0 in the human placental choriocarcinoma cell line JAR

摘要

We have recently cloned an amino acid transporter from the human placental choriocarcinoma cell line JAR which, when functionally expressed in HeLa cells, induces an amino acid transport activity with characteristics known to be associated with the amino acid transport system B0 (R. Kekuda, P.D. Prasad, Y.J. Fei, V. Torres-Zamorano, S. Sinha, T.L. Yang-Feng, F.H. Leibach, and V. Ganapathy, J. Biol. Chem. 271, 18657–18661, 1996). The presence of the amino acid transport system B0 (ATB0) has however not been previously described in these cells by functional studies. In the present investigation, we have obtained evidence for the existence of ATB0 in JAR cells and delineated the functional characteristics of the transporter. The identifying characteristics include Na+-dependence and preference for neutral amino acids. In addition, we have used the JAR cells as a model system to investigate the regulatory aspects of ATB0. Treatment of the cells with the neuroprotective agent aurintricarboxylic acid (ATA) for 16 h leads to a significant increase in ATB0 activity. This increase is associated with enhanced maximal velocity of the transporter and with increased steady state levels of the transporter mRNA. The effect of ATA is blocked by the tyrosine kinase inhibitor genistein. ATA treatment results in increased tyrosine phosphorylation of two major proteins, 180 kDa and 140 kDa in size. The 180 kDa protein is likely to be the epidermal growth factor (EGF) receptor because exposure of the cells to EGF also leads to enhanced tyrosine phosphorylation of a protein of similar molecular size. Furthermore, the effects of ATA on ATB0 activity and on ATB0 mRNA levels can be reproduced by EGF. Treatment of the cells with EGF for 24 h results in a significant increase in ATB0 activity and this effect is associated with an increase in the maximal velocity of the transporter and with an increase in the steady state levels of the transporter mRNA. These data suggest that ATA influences ATB0 activity in JAR cells most likely by activating the EGF receptor through tyrosine phosphorylation. It is concluded that the human placental choriocarcinoma cells functionally express the amino acid transport system B0 and that the expression of the system in these cells is stimulated by EGF.