THE CARBOXYL-TERMINAL DOMAIN-(111-165) OF VASCULAR ENDOTHELIAL GROWTH-FACTOR IS CRITICAL FOR ITS MITOGENIC POTENCY

Vascular endothelial growth factor (VEGF) is a potent and specific mit ogen for endothelial cells. VEGF is synthesized and secreted by many d ifferentiated cells in response to a variety of stimuli including hypo xia. VEGF is expressed in a variety of tissues as multiple homodimeric forms (121, 165, 189, and 206 amino acids/monomer) resulting from alt ernative RNA splicing. VEGF(121) is a soluble mitogen that does not bi nd heparin; the longer forms of VEGF bind heparin with progressively h igher affinity. The higher molecular weight forms of VEGF can be cleav ed by plasmin to release a diffusible form(s) of VEGF. We characterize d the proteolysis of VEGF by plasmin and other proteases. Thrombin, el astase, and collagenase did not cleave VEGF, whereas trypsin generated a series of smaller fragments. The isolated plasmin fragments of VEGF were compared with respect to heparin binding, interaction with solub le VEGF receptors, and ability to promote endothelial cell mitogenesis . Plasmin yields two fragments of VEGF as indicated by reverse phase h igh performance liquid chromatography and SDS-polyacrylamide gel elect rophoresis: an amino-terminal homodimeric protein containing receptor binding determinants and a carboxyl-terminal polypeptide which bound h eparin. Amino-terminal sequencing of the carboxyl-terminal peptide ide ntified the plasmin cleavage site as Arg(110)-Ala(111). A heterodimeri c form of VEGF(165/110), was isolated from partial plasmin digests of VEGF(165). The carboxyl-terminal polypeptide (111-165) displayed no af finity for soluble kinase domain region (KDR) or Fms-like tyrosine kin ase (FLT-1) receptors. The various isoforms of VEGF (165, 165/110, 110 , and 121) bound soluble kinase domain region receptor with similar af finity (approximately 30 pM). In contrast, soluble FLT-1 receptor diff erentiated VEGF isoforms (165, 165/110, 110, and 121) with apparent af finities of 10, 30, 120, and 200 pM, respectively. Endothelial cell mi togenic potencies of VEGF(110) and VEGF(121) were decreased more than 100-fold compared to that of VEGF(165). The present findings indicate that removal of the carboxyl-terminal domain, whether it is due to alt ernative splicing of mRNA or to proteolysis, is associated with a sign ificant loss in bioactivity.