TYROSINE PHOSPHORYLATION AND EPIDERMAL GROWTH FACTOR-DEPENDENT REGULATION OF THE SODIUM-COUPLED AMINO-ACID TRANSPORTER B-0 IN THE HUMAN PLACENTAL CHORIOCARCINOMA CELL-LINE JAR

We have recently cloned an amino acid transporter from the human place ntal choriocarcinoma cell line JAR which, when functionally expressed in HeLa cells, induces an amino acid transport activity with character istics known to be associated with the amino acid transport system B-0 (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 B-0 (ATB (0)) has however not been previously described in these cells by funct ional studies, In the present investigation, we have obtained evidence for the existence of ATB(0) in JAR cells and delineated the functiona l characteristics of the transporter. The identifying characteristics include Nai-dependence and preference for neutral amino acids. In addi tion, we have used the JAR cells as a model system to investigate the regulatory aspects of ATB(0)., Treatment of the cells with the neuropr otective agent aurintricarboxylic acid (ATA) for 16 h lends to a signi ficant increase in ATB(0) activity. This increase is associated with e nhanced 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 incr eased tyrosine phosphorylation of two major proteins, 150 kDa and 140 kDa in size. The ISO 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 ATB(0) activity and on ATB(0 ) mRNA levels can be reproduced by EGF. Treatment of the cells with EG F for 24 h results in a significant increase in ATB(0) activity and th is effect is associated with an increase in the maximal velocity of th e transporter and with an increase in the steady state levels of the t ransporter mRNA. These data suggest that ATA influences ATB(0) activit y in JAR cells most likely by activating the EGF receptor through tyro sine phosphorylation. It is concluded that the human placental chorioc arcinoma cells functionally express the amino acid transport system B- 0 and that the expression of the system in these cells is stimulated b y EGF. (C) 1997 Elsevier Science B.V.