Abstract
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.
Original language | English (US) |
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Pages (from-to) | 441-450 |
Number of pages | 10 |
Journal | Cell biochemistry and biophysics |
Volume | 62 |
Issue number | 3 |
DOIs | |
State | Published - Apr 2012 |
Externally published | Yes |
Bibliographical note
Funding Information:Acknowledgments This study was supported by the NSF (CHE0628260, MCB1024668) and by the American Heart Association (AHA0650081Z).
Keywords
- Chromium
- Fourier transform infrared spectroscopy
- Insulin
- Lipid order
- Membrane microdomain
- Picolinate
- Reverse micelle
- Single-particle tracking