FIGURE 2.

Interaction of VEGFR-2 and VEGFR-3 with VEGF-DΔNΔC variants. A, representation of structure for part of the N-terminal α-helix (⁹³FYDIETLKVIDEEWQ¹⁰⁷) in human mature VEGF-D with the mAb 286 binding site shown in red. B, analysis of binding of VEGF-DΔNΔC variants to VEGFR-2 (left) and VEGFR-3 (right) by ELISA (see “Experimental Procedures”). y axes show binding of variant proteins compared with VEGF-DΔNΔC (the latter defined as 100%), and x axes define the mutated amino acid in each variant. The same amount of each VEGF-DΔNΔC variant was used. VEGF-D, VEGF-DΔNΔC. Assays were conducted three times. Columns, mean; error bars, S.D. C, bioassays for binding and cross-linking of the extracellular domains of VEGFR-2 (left) and VEGFR-3 (right) by VEGF-DΔNΔC variants. The same amount of each VEGF-DΔNΔC variant was used in each assay. Results are expressed as a percentage of fluorescence units generated by VEGF-DΔNΔC variants relative to VEGF-DΔNΔC (y axes). x axes define the mutated amino acid in each variant. Assays were conducted five times. Columns, mean; error bars, S.D. D, receptor phosphorylation induced by selected VEGF-DΔNΔC variants. Adult LECs were stimulated with matched quantities of VEGF-DΔNΔC or its variants or left unstimulated (No GF). Lysates were immunoprecipitated (IP) with an antibody against VEGFR-2 (left) or VEGFR-3 (right) and analyzed by reducing SDS-PAGE and Western blotting with an antibody against phosphotyrosine (pY) to assess activation of receptors (top blot in each pair) or with an antibody against VEGFR-2 (bottom blot in each pair on the left) or VEGFR-3 (right bottom blot) to confirm the presence of each receptor. VEGFR-2 migrated predominantly at ∼230 kDa, whereas VEGFR-3 migrated as three bands, a ∼125-kDa cleaved form and two uncleaved forms of ∼175 and ∼195 kDa that differed in degree of glycosylation. Sizes of molecular mass markers (in kDa) are shown to the left of the panels. Dotted lines indicate where irrelevant tracks have been excised from images.