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Analysis of insulin receptor substrate signaling dynamics on microstructured surfaces
Author(s) -
Lanzerstorfer Peter,
Yoneyama Yosuke,
Hakuno Fumihiko,
Müller Ulrike,
Höglinger Otmar,
Takahashi ShinIchiro,
Weghuber Julian
Publication year - 2015
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.13213
Subject(s) - insulin receptor , insulin receptor substrate , grb10 , receptor , microbiology and biotechnology , biology , phosphotyrosine binding domain , fluorescence recovery after photobleaching , biochemistry , insulin , phosphorylation , signal transduction , irs1 , irs2 , tyrosine kinase , receptor tyrosine kinase , insulin like growth factor , cell signaling , insulin resistance , growth factor , sh2 domain , endocrinology , membrane
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.