Ligand binding potential of recombinant alternative splice variants
Among the 60 ASV cloned, we selected 10 for initial functional testing (Table 2). The selected ASV (corresponding to ASV derived from VEGFR1, VEGFR2, VEGFR3, Tie1, Met, Kit, CSF1R, PDGFRβ, FGFR1, and RAGE) represent diverse members of gene families, possess known functional domains such as ligand binding domains, and encode novel amino acids compared with previously reported splice variant sequences.
Table 2  Alternative splice variants selected for functional testing  Ten alternative splice variants were selected for functional testing. aLengths of the alternative splice variant open-reading frames (ORF) and lengths of the wildtype receptor extracellular domains (ECD) are indicated by the numbers of amino acids. bNovel C-terminal amino acids of each alternative splice variant are shown. *Stop codon.  Efficient expression and secretion of the selected 10 recombinant ASV (VEGFR1, VEGFR2, VEGFR3, Tie1, Met, Kit, CSF1R, PDGFRβ, FGFR1, and RAGE) from HEK293 cells was confirmed by western blot analysis of the cell culture supernatants, using anti-Myc antibody to detected the Myc-tagged ASV (Figure 3a). Furthermore, we observed ligand binding by ASV proteins derived from VEGFR1, VEGFR2, PDGFRβ, Met, and CSF1R – which bound to VEGF-A, VEGF-C, PDGF, hepatocyte growth factor, and CSF, respectively (Figure 3b). For evaluation of Tie1-751, purified recombinant protein was used for binding to Ang-1, and a dissociation constant (Kd) of approximately 89nM was measured (Figure 3b).
Figure 3  Expression and ligand binding of recombinant alternative splice variants. (a) HEK293 cells were transiently transfected with the indicated cDNA constructs. Conditioned media of HEK293 cells were collected after 48 hours, separated on SDS-PAGE gels and probed with an anti-Myc antibody to detect the Myc-tagged alternative splice variants (ASV). Molecular weights (kDa) are indicated. (b) For VEGFR1-541, VEGFR2-712, PDGFRβ-336, Met-877 and CSF1R-306, conditioned media from untransfected (Control, dashed lines) or ASV-transfected (Specific, solid lines) HEK293 cells were applied to plates precoated with the receptor-specific ligands. Unbound ASV were detected using antibodies against the extracellular domains of the receptors. Purified Tie1-751(6His) was used for Ang-1 binding, as above. Kd, dissociation constant. (c) Solution binding of VEGF-D to VEGFR3-765-Myc. Binding was carried out by combining VEGF-D with conditioned medium from either VEGFR3-765-Myc-expressing cells (lanes 1 to 3) or untransfected cells (lane 4). Subsequent immunoprecipitation was performed using anti-VEGF-D antibody and detected using anti-Myc antibody. To confirm the specificity of interaction between VEGF-D and VEGFR3-765-Myc, binding was performed in the presence of fivefold molar excess of either recombinant human VEGFR3/Fc chimera (lane 2) or soluble recombinant human VEGFR1/Fc chimera (lane 3). Molecular weights (kDa) are indicated. CM, Conditioned medium; IP, Immunprecipitation; WB, Western blot.   Not all receptor–ligand interactions could be detected by plate-based binding, which may be a consequence of steric issues associated with binding receptor or ligand to the surface of the plate. Binding of VEGF-D to VEGFR3-765, for example, was demonstrated only when the assay was performed in solution (Figure 3c). Specificity of VEGF-D binding to VEGFR3-765 was confirmed using a soluble VEGFR3/Fc chimera, which was able to compete with VEGFR3-765 binding to VEGF-D – unlike a soluble VEGFR1/Fc chimera (Figure 3c).