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 VEGF(121) and VEGF(165) (121 and 165 residues long respectively) in fetal heart, lung, ovary, spleen, placenta and ovarian tumours. Unexpectedly, a 47 kDa species predominated in fetal intestine and muscle. The presumed initiation site in VEGF is an AUG codon (AUG(1039)), 1039 nt from its main transcriptional start site. AUG(1039) is preceded in the 5' untranslated region by an in-frame CUG at nt 499 (CUG(499)), which could produce the 47 kDa form with a 180-residue N-terminal extension. We therefore assessed whether CUG(499) functions as an initiator. CUG(499) initiation produced the 47 kDa VEGF(165) precursor, which was processed at two sites to yield VEGF and three N-terminal fragments. When CTG(499) was mutated to CGC, the precursor and N-terminal fragments were barely detectable. Although the precursor form was predominant in VEGF(165), both CUG(499) and AUG(1039) forms were found in VEGF(121). 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 CUG(499) and AUG(1039) initiation and processing of the inactive precursor and its products might be important in regulating angiogenesis.