VEGF-A165, VEGF-A121, and VEGF-E-NZ2 interaction with VEGFR-2, HS, and NRP1. (A) Schematic outline of VEGF ligands used in this study with VEGF-A exon structure and bindings sites for VEGFR-2, HS, and NRP1 indicated. VEGF-A165 and VEGF-A121 were purchased or produced in-house by expressing canine cDNAs in Pichia pastoris. Similar results were obtained irrespective of source. VEGF-E-NZ2, VEGF-E-R126E (loss-of-function for NRP1 binding), and VEGF-A-NZ2 (gain-of-function for NRP1 binding) were also produced in Pichia pastoris (“Methods”). (B) PAE/VEGFR-2, NRP1 cells treated with 2 nM of VEGF-A165, VEGF-A121, or VEGF-E-NZ2 for 5 minutes were characterized by immunoprecipitation (IP)/immunoblotting (IB). Tyrosine phosphorylation of immunoprecipitated VEGFR-2 was detected by immunoblotting with antiphosphotyrosine mAb 4G10. Expression of VEGFR-2 and NRP1 was shown by immunoblotting on total cell lysates. The extent of VEGFR-2/NRP1 complex formation was assessed by immunoprecipitation of VEGFR-2 followed by immunoblotting for NRP1. The basal degree of complex formation in the absence of growth factors was set to 1. (C) PAE cells transfected with human VEGFR-2 and treated with 2 nM of the 3 VEGF ligands were analyzed for VEGFR-2 tyrosine phosphorylation as in panel B. Tyrosine phosphorylation of VEGFR-2 was induced by VEGF-A165 and VEGF-A121 but only inefficiently by VEGF-E-NZ2. Bottom panel shows equal VEGFR-2 loading. (D) [³⁵S]HS retention on nitrocellulose filter as a consequence of specific binding to VEGF-A165. [³⁵S]HS was not retained by incubation with VEGF-A121 or VEGF-E-NZ2.