Post-transcriptional regulation of the arginine transporter Cat-1 by amino acid availability

J Biol Chem. 1999 Oct 22;274(43):30424-32. doi: 10.1074/jbc.274.43.30424.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 3' Untranslated Regions / genetics
  • Amino Acids / metabolism*
  • Animals
  • Antibodies
  • Base Sequence
  • Cell Cycle Proteins / biosynthesis
  • Cell Cycle Proteins / genetics*
  • Cell Line
  • Culture Media
  • Cycloheximide / pharmacology
  • Dactinomycin / pharmacology
  • Doxycycline / pharmacology
  • Gene Expression Regulation*
  • Glioma
  • Kidney
  • Kinetics
  • Molecular Sequence Data
  • Peptide Fragments / immunology
  • Phosphoproteins / biosynthesis
  • Phosphoproteins / genetics*
  • Protein Biosynthesis / drug effects
  • RNA Processing, Post-Transcriptional
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Rats
  • Transcription, Genetic
  • Tumor Cells, Cultured

Substances

  • 3' Untranslated Regions
  • Amino Acids
  • Antibodies
  • Cell Cycle Proteins
  • Culture Media
  • Git1 protein, rat
  • Peptide Fragments
  • Phosphoproteins
  • RNA, Messenger
  • Dactinomycin
  • Cycloheximide
  • Doxycycline