A precursor form of vascular endothelial growth factor arises by initiation from an upstream in-frame CUG codon

Vascular endothelial growth factor (VEGF) is a mitogen in physiological and pathological angiogenesis. Understanding the expression of different VEGF isoforms might be important for distinguishing angiogenesis in tissue development, vascular remodelling and tumour formation. We examined its expression and noted the presence of the isoforms VEGF121 and VEGF165 (121 and 165 residues long respectively) in fetal heart, lung, ovary, spleen, placenta and ovarian tumours. Unexpectedly, a 47kDa species predominated in fetal intestine and muscle. The presumed initiation site in VEGF is an AUG codon (AUG1039), 1039nt from its main transcriptional start site. AUG1039 is preceded in the 5′ untranslated region by an in-frame CUG at nt 499 (CUG499), which could produce the 47kDa form with a 180-residue N-terminal extension. We therefore assessed whether CUG499 functions as an initiator. CUG499 initiation produced the 47kDa VEGF165 precursor, which was processed at two sites to yield VEGF and three N-terminal fragments. When CTG499 was mutated to CGC, the precursor and N-terminal fragments were barely detectable. Although the precursor form was predominant in VEGF165, both CUG499 and AUG1039 forms were found in VEGF121. VEGF precursor induced neither the proliferation of human umbilical vein endothelial cells nor the expression of angiopoietin 2, which can be induced by, and act with, VEGF to induce tumour angiogenesis. The precursor also adheres to the extracellular matrix (ECM), suggesting that it might be a storage form for generating active VEGF in the cell or ECM. Alternate CUG499 and AUG1039 initiation and processing of the inactive precursor and its products might be important in regulating angiogenesis.