Tyrosine kinase receptors (RTKs) are transmembrane proteins which are activated following binding with peptide growth factors or hormones and play key roles in processes such as cellular growth, metabolism, motility and differentiation. Recent evidence suggests that these receptors are involved in the pathogenesis and progression of a variety of cancers. In addition, dysfunction in IR and IGF-IR signaling is strongly linked to obesity and type 2 diabetes mellitus. Due to the fact that these receptors have major influences on human health, they were intensively investigated and became major drug targets for the above mentioned diseases. Aim of this thesis was to set up a novel and workable live cell analysis platform based on micropatterned surfaces for studying the influence of tyrosine kinase receptor signaling modulating substances on the one hand, and on the other hand a screening platform for the identification and characterization of insulin mimetic substances. The respective chapters were published in different scientific journals.
(1) Quantification and Kinetic Analysis of Grb2-EGFR Interaction on Micro-Patterned Surfaces for the Characterization of EGFR-Modulating Substances. PLOS ONE (2014).
In this chapter we report on a technique which combines micro-patterned surfaces and total internal reflection fluorescence (TIRF) microscopy (-patterning assay) for the quantitative analysis of EGFR activity.
Here we quantified the interaction of the key signal transmitting protein Grb2 (growth factor receptor-bound protein 2) with the EGFR in a live cell context. It was possible to demonstrate an EGF dependent recruitment of Grb2 to the EGFR, which was significantly inhibited in the presence of clinically tested EGFR inhibitors. Importantly, in addition to its potential use as a screening tool, our experimental setup offers the possibility to provide insight into the molecular mechanisms of bait-prey interaction. Application of bleaching experiments enabled calculation of the Grb2 exchange rate, which significantly changed upon stimulation or the presence of EGFR activity inhibiting drugs.
(2) Analysis of insulin receptor substrate (IRS) signaling dynamics on micro- structured surfaces. FEBS Journal (2015).
Here we provide additional insight into IR/IGF-IR downstream signaling via insulin receptor substrate (IRS) proteins. Interaction of IR/IGF-IR with IRS proteins is an initial key event for downstream signaling and bioactivities. Despite the structural similarities, increasing evidences show that IRS family proteins have non-redundant functions. Although the specificity of insulin/IGF signaling and biological responses in part reflects which IRS proteins are dominantly phosphorylated by the receptors, the precise properties of respective IRS interaction with the receptors remain elusive. Again we used the -patterning assay for the quantitative analysis of the interaction between IRS proteins and IR/IGF-IR in living cells. Our experimental setup 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 IRS functions. In addition, our analyses using fluorescent recovery after photobleaching revealed that kinases such as c-Jun N-terminal kinase and IkB kinase beta, which phosphorylate serine/threonine residues of IRS and contribute to insulin resistance, altered the interaction kinetics of IRS with insulin receptor. (3) Identification of Novel Insulin Mimetic Drugs by Quantitative Total Internal Reflection Fluorescence (TIRF) Microscopy (2014).
Finally, we used quantitative TIRFM to investigate the glucose transporter 4 (GLUT4) translocation modulatory properties of selected phytochemicals (chapter 5). For this purpose, TIRFM was applied to quantify GLUT4 translocation in highly insulin-sensitive CHO-K1 cells expressing a GLUT4-myc-GFP fusion protein. Using our approach, we demonstrated the influence of selected phytamines on GLUT4 translocation and identified novel potential insulin mimetics. An increase in the TIRF signal was found to correlate with an elevated glucose uptake.
Variations in the expression level of the human insulin receptor (hInsR) showed that the insulin mimetics identified stimulate GLUT4 translocation by a mechanism that is independent of the presence of the hInsR. Taken together, the results indicate that TIRF microscopy is an excellent tool for the quantification of GLUT4 translocation and for identifying insulin mimetic drugs.