Analysis of insulin receptor substrate signaling dynamics on microstructured surfaces

Peter Lanzerstorfer, Yosuke Yoneyama, Fumihiko Hakuno, Ulrike Müller, Otmar Höglinger, Shin-Ichiro Takahashi, Julian Weghuber

Research output: Contribution to journalReview articlepeer-review

16 Citations (Scopus)

Abstract

Insulin receptor substrates (IRS) are phosphorylated by activated insulin/insulin-like growth factor I receptor tyrosine kinases, with this comprising an initial key event for downstream signaling and bioactivities. Despite the structural similarities, increasing evidence shows that IRS family proteins have nonredundant functions. Although the specificity of insulin/insulin-like growth factor signaling and biological responses partly reflects which IRS proteins are dominantly phosphorylated by the receptors, the precise properties of the respective IRS interaction with the receptors remain elusive. In the present study, we utilized a technique that combines micropatterned surfaces and total internal reflection fluorescence microscopy for the quantitative analysis of the interaction between IRS proteins and insulin/insulin-like growth factor in living cells. Our experimental set-up enabled the measurement of equilibrium associations and interaction dynamics of these molecules with high specificity. We revealed that several domains of IRS including pleckstrin homology and phosphotyrosine binding domains critically determine the turnover rate of the receptors. Furthermore, we found significant differences among IRS proteins in the strength and kinetic stability of the interaction with the receptors, suggesting that these interaction properties could account for the diverse functions of IRS. In addition, our analyses using fluorescent recovery after photobleaching revealed that kinases such as c-Jun N-terminal kinase and IκB kinase β, which phosphorylate serine/threonine residues of IRS and contribute to insulin resistance, altered the interaction kinetics of IRS with insulin receptor. Collectively, our experimental set-up is a valuable system for quantitifying the physiological interaction of IRS with the receptors in insulin/insulin-like growth factor signaling.

Original languageEnglish
Pages (from-to)987-1005
Number of pages19
JournalFEBS Journal
Volume282
Issue number6
DOIs
Publication statusPublished - Mar 2015

Keywords

  • IGF-I receptor
  • fluorescence recovery after photobleaching
  • insulin receptor
  • insulin receptor substrates
  • micropatterning
  • Phosphorylation
  • Plasmids/metabolism
  • Contrast Media/chemistry
  • Fluorescence Recovery After Photobleaching
  • Signal Transduction
  • Humans
  • JNK Mitogen-Activated Protein Kinases/metabolism
  • Insulin Resistance
  • I-kappa B Kinase/metabolism
  • Green Fluorescent Proteins/metabolism
  • Microscopy
  • Receptor, IGF Type 1/metabolism
  • Sensitivity and Specificity
  • Surface Properties
  • Protein Binding
  • HeLa Cells
  • Insulin Receptor Substrate Proteins/metabolism
  • Microscopy, Fluorescence
  • Receptor, Insulin/metabolism

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