Post-transcriptional regulation of the arginine transporter Cat-1 by amino acid availability.
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.
Aulak
K S
KS
Department of Nutrition, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.
Mishra
R
R
Zhou
L
L
Hyatt
S L
SL
de Jonge
W
W
Lamers
W
W
Snider
M
M
Hatzoglou
M
M
D020413:3' Untranslated Regions / Q000235:genetics; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D000906:Antibodies; D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000096:biosynthesis / Q000235:genetics*; D002460:Cell Line; D003470:Culture Media; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D004318:Doxycycline / Q000494:pharmacology; D005786:Gene Expression Regulation; D005910:Glioma; D007668:Kidney; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology; D010750:Phosphoproteins / Q000096:biosynthesis / Q000235:genetics*; D014176:Protein Biosynthesis / Q000187:drug effects; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D020413:3' Untranslated Regions; D000596:Amino Acids; D000906:Antibodies; D018797:Cell Cycle Proteins; D003470:Culture Media; C485648:Git1 protein, rat; D010446:Peptide Fragments; D010750:Phosphoproteins; D012333:RNA, Messenger; D003609:Dactinomycin; D003513:Cycloheximide; D004318:Doxycycline