Vesiculin reveals the role of the <scp>IGF2</scp> C‐domain in receptor specificity within the insulin– <scp>IGF</scp> system

Insulin-like growth factor 2 (IGF2) engages multiple receptors of the insulin-hormone family, yet the structural determinants of its selectivity remain poorly defined. Vesiculin, a naturally occurring des[37-40]IGF2 isoform, represents a unique probe for dissecting these interactions. Here, we combine chemical synthesis, receptor-binding assays, phosphorylation studies, and molecular dynamics simulations to characterize vesiculin and compare it with wild-type IGF2. Vesiculin consistently displayed a reduced affinity for the majority of binding partners, with the most pronounced loss observed for IGF2R and its isolated domain 11 (D11). Computational analyses revealed that this reduction originates from the destabilization of the IGF2 C-domain, which in wild-type IGF2 forms cooperative contacts with IGF2R domains D6 and D8. Deletion of Arg37-Arg40 abolishes these interactions and enforces ineffective compensatory contacts. For the insulin receptor, the loss of these residues disrupts stabilizing electrostatic contacts with the α-CT and L2 domains. In contrast, IGF1R binding is only modestly affected, and vesiculin shows a markedly diminished affinity for IGFBP3, raising questions about its bioactivity. Together, these findings redefine the IGF2-IGF2R binding interface and highlight the IGF2 C-domain as a central determinant of receptor specificity across the insulin-IGF system, providing a framework for designing IGF2 analogs.