Abstract
Cat-1 is a protein with a dual function, a high affinity, low capacity cationic amino acid transporter of the y+ system and the receptor for the ecotropic retrovirus. We have suggested that Cat-1 is required in the regenerating liver for the transport of cationic amino acids and polyamines in the late G1 phase, a process that is essential for liver cells to enter mitosis. In our earlier studies we had shown that the cat-1 gene is silent in the quiescent liver but is induced in response to hormones, insulin, and glucocorticoids, and partial hepatectomy. Here we demonstrate that cat-1 is a classic delayed early growth response gene in the regenerating liver, since induction of its expression is sensitive to cycloheximide, indicating that protein synthesis is required. The peak of accumulation of the cat-1 mRNA (9-fold) by 3 h was not associated with increased transcriptional activity of the cat-1 gene in the regenerating liver, indicating post-transcriptional regulation of expression of this gene. Induction of the cat-1 gene results in the accumulation of two mRNA species (7.9 and 3.4 kilobase pairs (kb)). Both mRNAs hybridize with the previously described rat cat-1/2.9-kb cDNA clone. However, the 3' end of a longer rat cat-1 cDNA (rat cat-1/6.5-kb) hybridizes only to the 7.9-kb mRNA transcript. Sequence analysis of this clone indicated that the two mRNA species result from the use of alternative polyadenylation signals. The 6. 5-kb clone contains a number of AT-rich mRNA destabilizing sequences which is reflected in the half-life of the cat-1 mRNAs (90 min for 7. 9-kb mRNA and 250 min for 3.4-kb mRNA). Treatment of rats with cycloheximide superinduces the level of the 7.9-kb cat-1 mRNA in the kidney, spleen, and brain, but not in the liver, suggesting that cell type-specific labile factors are involved in its regulation. We conclude that the need for protein synthesis for induction of the cat-1 mRNA, the short lived nature of the mRNAs, and the multiple sites for regulation of gene expression indicate a tight control of expression of the cat-1 gene within the regenerating liver and suggest that y+ cationic amino acid transport in liver cells is regulated at the molecular level.
Highlights
Cationic amino acids lysine, ornithine, and arginine are transported into cells through several transport systems that differ in their requirement for Naϩ [1,2]
Mammalian cationic amino acid transporters [3] are related to the yeast transporters for arginine, histidine, and choline [31], and this is supported by the recent findings of Kabat and associates [32] who demonstrated that the amino acids responsible for the function of the protein as a transporter are conserved through species
Evaluation of the Half-life (t1⁄2 ) of the cat-1 mRNAs—The sequence analysis of the cat-1/6.5 cDNA clone shows the presence of a stretch of 11 AT repeats and four copies of the ATTTA sequence motif (Fig. 3) within the 3Ј-untranslated region (3Ј-UTR)
Summary
Quiescent liver cells do not have an active high affinity yϩ transport system [1], transport of cationic amino acids into liver cells is induced in dividing primary hepatocytes and in transformed hepatoma cells [2]. This induction is accompanied by the accumulation of high levels of cat-1 mRNAs [2, 6, 24]. We report the molecular sites of regulation of expression of the cat-1 gene in rat tissues and hepatoma cells
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