New vascular endothelial growth factor isoform generated by internal ribosome entry site-driven CUG translation initiation.
We recently demonstrated that the very long 5'-untranslated region (5'-UTR) of the vascular endothelial growth factor (VEGF) mRNA contains two independent internal ribosome entry sites (IRES A and B). In the human sequence, four potential CUG translation initiation codons are located in between these IRES and are in frame with the classical AUG start codon. By in vitro translation and COS-7 cell transfections, we demonstrate that a high mol wt VEGF isoform [called large VEGF (L-VEGF)] is generated by an alternative translation initiation process, which occurs at the first of these CUG codons. Using a bicistronic strategy, we show that the upstream IRES B controls the translation initiation of L-VEGF. This isoform is 206 amino acids longer than the classical AUG-initiated form. With a specific antibody raised against this NH2 extension, we show that the L-VEGF is present in different mouse tissues or in transfected COS-7 cells. We also demonstrate that L-VEGF is cleaved into two fragments: a 23-kDa NH2-specific fragment and a fragment with an apparent size similar to that of the classical AUG-initiated form. This cleavage requires the integrity of a hydrophobic sequence located in the central part of the L-VEGF molecule. This sequence actually plays the role of signal peptide in the classical AUG-initiated form. The AUG-initiated form and the COOH cleavage product of the L-VEGF are both secreted. In contrast, the large isoform and its NH2 fragment present an intracellular localization. These data unravel a further level of complexity in the regulation of VEGF expression.
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Institut National de la Santé et la Recherche Médicale Unité 397, Endocrinologie et Communication Cellulaire, Centre Hospitalier Universitaire Rangueil, 31403 Toulouse cedex 04, France.
Bornes
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Bresson
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Créancier
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Prats
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D020121:5' Untranslated Regions / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002522:Chlorocebus aethiops; D018387:Codon, Initiator; D016228:Endothelial Growth Factors / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008222:Lymphokines / Q000235:genetics / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D020033:Protein Isoforms; D012270:Ribosomes / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D020121:5' Untranslated Regions; D018387:Codon, Initiator; D016228:Endothelial Growth Factors; D008222:Lymphokines; D020033:Protein Isoforms; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors