Tyrosine phosphorylation of the orphan receptor ESDN/DCBLD2 serves as a scaffold for the signaling adaptor CrkL

•Functional proteomics identifies ESDN as a novel CrkL-SH2 binding protein.•Fyn and other tyrosine kinases induce the binding of ESDN to both the CrkL and Fyn SH2 domains.•Mass spectrometry-based identification of regulated tyrosine phosphorylation sites on ESDN. A quantitative proteomics screen to identify substrates of the Src family of tyrosine kinases (SFKs) whose phosphorylation promotes CrkL-SH2 binding identified the known Crk-associated substrate (Cas) of Src as well as the orphan receptor endothelial and smooth muscle cell-derived neuropilin-like protein (ESDN). Mutagenesis analysis of ESDN’s seven intracellular tyrosines in YxxP motifs found several contribute to the binding of ESDN to the SH2 domains of both CrkCT10 regulator of kinase Crk-Like (CrkL) and a representative SFK Fyn. Quantitative mass spectrometry showed that at least three of these (Y565, Y621 and Y750), as well as non-YxxP Y715, are reversibly phosphorylated. SFK activity was shown to be sufficient, but not required for the interaction between ESDN and the CrkL-SH2 domain. Finally, antibody-mediated ESDN clustering induces ESDN tyrosine phosphorylation and CrkL-SH2 binding. CrkL-SH2physically interacts with ESDN and CAS by pull down (View interaction) CrkL-SH2physically interacts with ESDN by pull down (View Interaction: 1, 2) Fyn-SH2physically interacts with ESDN by pull down (View interaction)