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- W2019928409 abstract "Receptor tyrosine kinases (RTKs) are cell membrane receptors with intrinsic tyrosine kinase activity that trigger signal transduction. RTKs are known key regulators of essential cellular processes and have a critical role in the development of many types of cancer. Here we introduce an assay using micro-patterned surfaces in combination with TIRF microscopy for the analysis of EGF-receptor (EGFR) and insulin/insulin-like growth factor-receptor (IR/IGFR) downstream signaling. In addition, we applied the technique to validate the efficacy of medically relevant RTK modulators. We used micropatterning to force bait-EGFR/IR/IGFR molecules into microscopic domains on the surface of living cells, while monitoring co-recruitment of a fluorescent intracellular prey molecule (Grb2/IRS), respectively. First, micropatterning of HeLa cells expressing the prey Grb2 enabled us to quantify the EGFR-Grb2 interaction, and we could show that Grb2 was recruited to the EGFR in a phosphorylation dependent manner. Pretreatment with pharmacologically active ingredients used for the treatment of human cancers significantly reduced the inducibility of the signaling system. Based on these results we could set up a dose-response relationship in a live cell context. Second, we performed fluorescence recovery after photobleaching (FRAP) experiments with RKT class II receptors (IR and IGFR) and different cytosolic insulin receptor substrate (IRS) proteins. Our results indicate prominent differences in the interaction strength of IRS1 and IRS2 to the IR/IGF1-R, compared to the one of IRS3. Taken together the results approve the power of the micropatterning technique to study the interaction properties of plasma-membrane localized receptors. This method enables a fast analysis of the samples with high sensitivity in a live cell context. We could quantify the molecular effects of different RTK modulators and we will use the system in the near future to study further active pharmaceutical ingredients." @default.
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- W2019928409 date "2014-01-01" @default.
- W2019928409 modified "2023-10-01" @default.
- W2019928409 title "A Live Cell Tirf-Microscopy Based Screening Assay for Fast Analysis of Receptor Tyrosine Kinase Modulators" @default.
- W2019928409 doi "https://doi.org/10.1016/j.bpj.2013.11.3972" @default.
- W2019928409 hasPublicationYear "2014" @default.
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