Ligand-resolved in vivo phosphoproteomics of the adult heart maps ErbB signaling by EGF and NRG1β

ErbB receptor tyrosine kinases orchestrate phosphorylation-based signaling in response to extracellular ligands and are key drivers in cancer biology. Although ErbB-targeted therapies have transformed cancer care, some agents cause cardiac adverse events. Yet, the acute phosphorylation programs engaged by ErbB ligands in the adult heart remain incompletely defined. Here, we applied in vivo quantitative phosphoproteomics with dual enrichment using TiO₂ and anti-phosphotyrosine antibodies to map acute cardiac phosphorylation responses to epidermal growth factor (EGF; EGFR/ErbB1) and neuregulin-1β (NRG1β; ErbB3/ErbB4) in adult mouse hearts. EGF triggered robust receptor tyrosine kinase signaling, convergence with insulin-associated nodes independent of insulin receptor activation, and phosphorylation of calcium-handling proteins including phospholamban, SERCA, NCX1, and CaV1.2, implicating CaMK2δ. NRG1β elicited a coordinated ErbB-dependent response featuring activation of Akt, MAPK, and stress kinases, with engagement of sarcomere and metabolic modules. Comparative analysis identified shared core signaling alongside ligand-specific differences in kinase and transcription factor phosphorylation, with EGF displaying broader network breadth. These data provide a phosphorylation-centric framework for ligand-resolved ErbB signaling in the heart and offer mechanistic insight into how ErbB-targeted therapies may influence cardiac function.