title: Analysis of insulin receptor substrate signaling dynamics on microstructured surfaces.
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.
authors: School of Engineering and Environmental Sciences, University of Applied Sciences Upper Austria, Wels, Austria.
authors: Peter
authors: P
authors: Lanzerstorfer
authors: Yosuke
authors: Y
authors: Yoneyama
authors: Fumihiko
authors: F
authors: Hakuno
authors: Ulrike
authors: U
authors: Müller
authors: Otmar
authors: O
authors: Höglinger
authors: Shin-Ichiro
authors: S
authors: Takahashi
authors: Julian
authors: J
authors: Weghuber
mesh_terms: D003287:Contrast Media / Q000737:chemistry; D036681:Fluorescence Recovery After Photobleaching; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism; D055504:Insulin Receptor Substrate Proteins / Q000378:metabolism*; D007333:Insulin Resistance; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D008853:Microscopy; D008856:Microscopy, Fluorescence; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017526:Receptor, IGF Type 1 / Q000378:metabolism; D011972:Receptor, Insulin / Q000378:metabolism*; D012680:Sensitivity and Specificity; D015398:Signal Transduction; D013499:Surface Properties
chemical_list: D003287:Contrast Media; D055504:Insulin Receptor Substrate Proteins; D049452:Green Fluorescent Proteins; D017526:Receptor, IGF Type 1; D011972:Receptor, Insulin; D051550:I-kappa B Kinase; D048031:JNK Mitogen-Activated Protein Kinases
keywords: IGF-I receptor; fluorescence recovery after photobleaching; insulin receptor; insulin receptor substrates; micropatterning