Biosynthesis of vascular endothelial growth factor-D involves proteolytic processing which generates non-covalent homodimers

Vascular endothelial growth factor-D (VEGF-D) binds and activates the endot helial cell tyrosine kinase receptors VEGF receptor-2 (VEGFR-2) and VEGF re ceptor-3 (VEGFR-3), is mitogenic for endothelial cells, and shares structur al homology and receptor specificity with VEGF-C, The primary translation p roduct of VEGF-D has long N- and C-terminal polypeptide extensions in addit ion to a central VEGF homology domain (VHD). The VHD of VEGF-D is sufficien t to bind and activate VEGFR-2 and VEGFR-3. Here we report that VEGF-D is p roteolytically processed to release the VHD. Studies in 293EBNA cells demon strated that VEGF-D undergoes Nand C-terminal cleavage events to produce nu merous secreted polypeptides including a fully processed form of M-r simila r to 21,000 consisting only of the VHD, which is predominantly a non-covale nt dimer. Biosensor analysis demonstrated that the VHD has similar to 290- and similar to 40-fold greater affinity for VEGFR-2 and VEGFR-3, respective ly, compared with unprocessed VEGF-D. In situ hybridization demonstrated th at embryonic lung is a major site of expression of the VEGF-D gene. Process ed forms of VEGF-D were detected in embryonic lung indicating that VEGF-D i s proteolytically processed in vivo.