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J Biol Chem, Vol. 274, Issue 43, 30424-30432, October 22, 1999
From the The regulation of the high affinity cationic
amino acid transporter (Cat-1) by amino acid availability has been
studied. In C6 glioma and NRK kidney cells, cat-1 mRNA
levels increased 3.8-18-fold following 2 h of amino acid
starvation. The transcription rate of the cat-1 gene
remained unchanged during amino acid starvation, suggesting a
post-transcriptional mechanism of regulation. This mechanism was
investigated by expressing a cat-1 mRNA from a
tetracycline-regulated promoter. The cat-1 mRNA
contained 1.9 kilobase pairs (kb) of coding sequence, 4.5 kb of
3'-untranslated region, and 80 base pairs of 5'-untranslated region.
The full-length (7.9 kb) mRNA increased 5-fold in amino
acid-depleted cells. However, a 3.4-kb species that results from the
usage of an alternative polyadenylation site was not induced,
suggesting that the cat-1 mRNA was stabilized by
cis-acting RNA sequences within the 3'-UTR. Transcription
and protein synthesis were required for the increase in full-length cat-1 mRNA level. Because omission of amino acids from
the cell culture medium leads to a substantial decrease in protein
synthesis, the translation of the increased cat-1 mRNA
was assessed in amino acid-depleted cells. Western blot analysis
demonstrated that cat-1 mRNA and protein levels changed
in parallel. The increase in protein level was significantly lower than
the increase in mRNA level, supporting the conclusion that
cat-1 mRNA is inefficiently translated when the supply
of amino acids is limited, relative to amino acid-fed cells. Finally,
y+-mediated transport of arginine in amino acid-fed and
-starved cells paralleled Cat-1 protein levels. We conclude that the
cat-1 gene is subject to adaptive regulation by amino acid
availability. Amino acid depletion initiates molecular events that lead
to increased cat-1 mRNA stability. This causes an
increase in Cat-1 protein, and y+ transport once amino
acids become available.
Post-transcriptional Regulation of the Arginine Transporter
Cat-1 by Amino Acid Availability
,,,,
Department of Nutrition and
¶ Department of Biochemistry, Case Western Reserve University
School of Medicine, Cleveland, Ohio 44106 and the
§ Laboratory of Anatomy and Embryology, University of
Amsterdam, Amsterdam, The Netherlands
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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