EXTRACELLULAR CLEAVAGE OF THE VASCULAR ENDOTHELIAL GROWTH-FACTOR 189-AMINO-ACID FORM BY UROKINASE IS REQUIRED FOR ITS MITOGENIC EFFECT

Alternative splicing of vascular endothelial growth factor (VEGF) mRNA results in three distinct molecular forms of 121 or 165 (V165) amino acids that are released in the conditioned medium of cultured cells an d one longer isoform of 189 amino acids (V189) that remains cell-assoc iated. V189 has been expressed in wild type CHO-K1 cells and in glycos aminoglycan deficient pgsA-745 Chinese hamster ovary (CHO) mutant cell s, It could be released from CHO-K1 cell membranes by heparin or a syn thetic peptide designed on the sequence encoded by exon 6 but was free ly released from CHO mutant cells, In both cases, the immunoreactive V 189 was mainly released as a 40-kDa cleaved form, provided that the se rine protease urokinase, but not plasmin, was active, Recombinant V189 was purified from insect cells infected with a recombinant baculoviru s as a nonmitogenic 50-kDa precursor that binds to the receptor Flt-1 but not to Flk-1. It could be matured by urokinase as a 38-kDa fragmen t able to bind to Flk-1 and to trigger cell proliferation, V165 and V1 89, however, could be cleaved by plasmin as 34-kDa fragments that exhi bit a decreased mitogenic activity, These findings indicate that the c arboxyl-terminal domain of V189 masks its binding do main to Flk-1.