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Insulin Receptors and Downstream Substrates Associate with Membrane Microdomains after Treatment with Insulin or Chromium(III) Picolinate

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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.

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Acknowledgments

This study was supported by the NSF (CHE0628260, MCB1024668) and by the American Heart Association (AHA0650081Z).

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Authors and Affiliations

  1. Department of Biomedical Sciences, Colorado State University, Fort Collins, CO, 80523, USA

    Abeer Al-Qatati, Amber L. Wolf-Ringwall & Deborah A. Roess

  2. Cell and Molecular Biology Program, Colorado State University, Fort Collins, CO, 80523, USA

    Peter W. Winter, Debbie C. Crans, Deborah A. Roess & B. George Barisas

  3. Department of Chemistry, Colorado State University, Campus Mail 1872, Fort Collins, CO, 80523, USA

    Pabitra B. Chatterjee, Alan K. Van Orden, Debbie C. Crans & B. George Barisas

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  1. Abeer Al-Qatati
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  2. Peter W. Winter
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  4. Pabitra B. Chatterjee
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Correspondence to B. George Barisas.

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Abeer Al-Qatati and Peter W. Winter were contributed equally to this study.

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Al-Qatati, A., Winter, P.W., Wolf-Ringwall, A.L. et al. Insulin Receptors and Downstream Substrates Associate with Membrane Microdomains after Treatment with Insulin or Chromium(III) Picolinate. Cell Biochem Biophys 62, 441–450 (2012). https://doi.org/10.1007/s12013-011-9326-x

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