COVALENT DIMERIZATION OF VASCULAR-PERMEABILITY FACTOR VASCULAR ENDOTHELIAL GROWTH-FACTOR IS ESSENTIAL FOR ITS BIOLOGICAL-ACTIVITY - EVIDENCE FROM CYS TO SER MUTATIONS

Vascular permeability factor, or vascular endothelial growth factor (V PF/VEGF) is an important factor in the regulation of vascular growth a nd vascular permeability. VPF is a secreted, dimeric protein and has 8 cysteine residues conserved with platelet-derived growth factor (PDGF ). To study the role of some of these cysteine residues in maintaining the structure and function of VPF, we replaced the codons for the sec ond, third, fourth, and fifth cysteine by serine codons, and expressed the mutant proteins in a mammalian expression system. Cysteine residu es 2 and 4 in VPF were found to be directly involved in anti parallel interchain disulfide bonds, as in PDGF. VPF mutants lacking one of the se cysteines were severely impaired in their S-linked dimerization, wh ile upon coexpression of both mutants the ability to form dimers was r estored. The VPF mutants lacking cysteine residue 2 or 4 also competed poorly for receptor binding of labeled VPF and had low biological act ivity, but these defects were also complemented by coexpressing the tw o mutants, indicating that for efficient receptor binding and activati on VPF needs to be a covalent dimer, unlike PDGP-BB. Furthermore, cyst eine residue 5 was found to be essential for VPF dimerization and acti vity, while the mutant lacking cysteine residue 3 was only mildly affe cted in its ability to dimerize and had partial biological activity.