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 indepen dent internal ribosome entry sites (IRES A and B). In the human sequence, f our potential CUG translation initiation codons are located in between thes e IRES and are in frame with the classical AUG start codon. By in vitro tra nslation and COS-7 cell transfections, we demonstrate that a high mol wt VE GF isoform [called large VEGF (L-VEGF)] is generated by an alternative tran slation 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 t han the classical AUG-initiated form. With a specific antibody raised again st this NH, extension, we show that the L-VEGF is present in different mous e tissues or in transfected COS-7 cells. We also demonstrate that L-VEGF is cleaved into two fragments: a 23-kDa NH,-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 t he central part of the L-VEGF molecule. This sequence actually plays the ro le 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 con trast, the large isoform and its NH, fragment present an intracellular loca lization. These data unravel a further level of complexity in the regulatio n of VEGF expression.