Activation of Insulin Receptor Signaling Pathway Using Ligand Microbeads in Mammalian Cells and Isolation of Receptor-Bead Complex

The study of cell surface receptors and their associated signaling pathways on the plasma membrane are vital in understanding cellular responses. The insulin receptor is a tyrosine kinase that is activated in response to insulin microbeads presented to three mammalian cell lines: CHO, NIH-3T3 and COS-7. Phosphatidylinositol 3-kinase (PI3K) signaling, a major insulin receptor signaling pathway, phosphorylates the 3-position hydroxyl group of the inositol ring of phosphatidylinositol-4,5-biphosphate, resulting in phosphatidylinositol-3,4,5-triphosphate (PIP3) that acts by recruiting specific pleckstrin homology (PH) domain containing proteins to cell membranes. Akt (protein kinase B) containing PH domain gets activated, binds to PIP3 and localizes to the site of receptor activation. The PH domain of Akt was fused to green fluorescent protein (GFP) to create a biosensor for phosphatidylinositol. Confocal microscopy confirmed Akt-PH recruitment to the cell membrane. Immunofluorescence staining (IF) and western blots confirmed insulin receptor and phosphotyrosine activity in cells. The activated insulin receptor complex was captured and isolated by insulin coated microbeads on the surface of cells.