We have examined the association of insulin receptors (IR) and downstream signaling molecules with membrane microdomains in rat basophilic leukemia (RBL-2H3) cells following treatment with insulin or tris(2-pyridinecarbxylato)chromium(III) (Cr(pic) 3). Single-particle tracking demonstrated that individual IR on these cells exhibited reduced lateral diffusion and increased confinement within 100 nm-scale membrane compartments after treatment with either 200 nM insulin or 10 μM Cr(pic) 3. These treatments also increased the association of native IR, phosphorylated insulin receptor substrate 1 and phosphorylated AKT with detergent-resistant membrane microdomains of characteristically high buoyancy. Confocal fluorescence microscopic imaging of Di-4-ANEPPDHQ labeled RBL-2H3 cells also showed that plasma membrane lipid order decreased following treatment with Cr(pic) 3 but was not altered by insulin treatment. Fluorescence correlation spectroscopy demonstrated that Cr(pic) 3 did not affect IR cell-surface density or compete with insulin for available binding sites. Finally, Fourier transform infrared spectroscopy indicated that Cr(pic) 3 likely associates with the lipid interface in reverse-micelle model membranes. Taken together, these results suggest that activation of IR signaling in a cellular model system by both insulin and Cr(pic) 3 involves retention of IR in specialized nanometer-scale membrane microdomains but that the insulin-like effects of Cr(pic) 3 are due to changes in membrane lipid order rather than to direct interactions with IR.
- Fourier transform infrared spectroscopy
- Lipid order
- Membrane microdomain
- Reverse micelle
- Single-particle tracking