Abstract
In renal epithelial cells amino acid deprivation induces an increase in L-Asp transport with a doubling of the Vmax and no change in Km (4.5 micronM) in a cycloheximide-sensitive process. The induction of sodium-depending L-aspartate transport was inhibited by single amino acids that are metabolized to produce glutamate but not by those that do not produce glutamate. The transaminase inhibitor aminooxyacetate in glutamine-free medium caused a decrease in cell glutamate content and an induction of glutamate transport. In complete medium aminooxyacetate neither decreased cell glutamate nor increased transport activity. These results are consistent with a triggering of induction of transport by low intracellular glutamate concentrations. High affinity glutamate transport in these cells is mediated by the excitatory amino acid carrier 1 (EAAC1) gene product. Western blotting using antibodies to the C-terminal region of EAAC1 showed that there is no increase in the amount of EAAC1 protein on prolonged incubation in amino acid-free medium. Conversely, the induction of high affinity glutamate transport by hyperosmotic shock was accompanied by an increase in EAAC1 protein. It is proposed that low glutamate levels lead to the induction of a putative protein that activates the EAAC1 transporter. A model illustrating such a mechanism is described.
Highlights
In mammalian cells intracellular glutamate concentrations are maintained at a high level by the presence of active transport systems for glutamate in the plasma membrane
System XAGϪ in NBL-1 cells has the same kinetic properties as glutamate transport induced in Xenopus oocytes by injection of excitatory amino acid carrier 1 (EAAC1) cRNA; since EAAC1 is known to be expressed in kidney while the other transporters are mainly restricted to brain, it is likely that EAAC1 encodes the NBL-1 cell glutamate transporter
We have shown that induction of System XAGϪ activity by hyperosmotic shock in NBL-1 cells is accompanied by a 3-fold increase in EAAC1-specific mRNA (14)
Summary
Materials—[U-14C]L-Aspartate, 35S-dATP, the Sequenase kit, and ECL reagents were from Amersham International (Amersham, UK). AOA, aminooxyacetic acid; EAAT, excitatory amino acid transporter; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; GRP78, 78-kDa glucoseregulated protein; GBP, glutamate-binding protein; Sulfo-SMCC, sulfosuccinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate. Transport Measurements—System XAGϪ activity was measured as the initial rate of sodium-dependent uptake of 50 M [U-14C]L-aspartate into NBL-1 cells over 5 min at 20 °C in the presence of 0.5 mM aminooxyacetic acid to inhibit aspartate metabolism (13). Cloning of the Hydrophilic Loop Region of EAAC1 and Preparation of an Anti-fusion Protein Antibody—The cDNA encoding the putative extracellular loop between helices 3 and 4 (amino acids 117–215) was first amplified from NBL-1 cell cDNA using nested PCR. A specific primer GCATGGCCTTTGCAGGG was used for sequencing across the pGEX2T multiple cloning site
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