CONICET | Buscador de Institutos y Recursos Humanos

Insulin signaling comprises a complex cascade of events playing a key role in the regulation of glucose metabolism and in cellular growth. Impaired response to insulin is the hallmark of diabetes while upregulated insulin activity occurs in many cancers. Insulin receptor (IR) is a tetrameric receptor tyrosine kinase. Two splice variants exist in mammalian cells: IR-A lacking exon 11, considered to be involved in mitogenesis, and the full length IR-B responsible for the activation of the metabolic cascade. Although considerable biochemical data exist on insulin binding and downstream signal transduction, little is known about the differential dynamics of these isoforms and its possible effects on insulin signaling. Increased signaling from internalized receptors in endosomes has been correlated with mitogenicity, suggesting that inside the endosomes IR bound with the ligand could be activated and could still phosphorylate downstream effectors. To gain insight into the dynamics of the activation and internalization of the IR isoforms by microscopy we combined 2 powerful labeling techniques: streptavidin-QDs conjugated with biotinylated ligands; and visible fluorescent proteins (VFPs). We generated IR fused to eGFP, CFP and YFP without affecting functionality. Using confocal and programmable array microscopy (PAM) in live cells, we demonstrated that biotin amido caproyl bovine insulin is able to bind IR and IR-VFPs (A and B) in living cells, activating it to trigger insulin signaling and to induce internalization. A cell-by-cell study revealed a higher rate of internalization of IR-A compared to IR-B. These differences in internalization showed a correlation with a higher and sustained activation in response to insulin of IR-A. Differential IR isoform activation also correlates with distinctive ERK activation profiles suggesting a mechanism for the divergent regulation of gene expression in response to insulin.