Abstract
Receptor tyrosine kinases (RTK)s are high-affinity cell surface receptors known to have a critical role in the development of many types of cancer. So far, approximately 20 different RTK classes have been identified. Among them the RTK class I (EGF receptor) and class I (Insulin receptor).We used an assay combining TIRF microscopy and micro-patterned surfaces, which can be applied for the detection of protein-protein interactions in and near the cell membrane in vivo.The first part of our work focuses on the Insulin-receptor (IR) and the Insulin-like growth factor 1 (IGF-1) receptor. Like other RTKs, the IR mediates its activity by causing the addition of phosphate groups to intracellular substrate proteins. Thus, cytosolic proteins named Insulin receptor substrates (IRS) are phosphorylated, which finally leads to an uptake of glucose by glucose transporters. We used the μ-patterning assay to analyze the interaction properties of different IRS-proteins with the IR and the IGF1-R. Our results indicate prominent differences in the interaction strength of IRS1 and IRS2 to the IR/IGF1-R, compared to the one of IRS3. FRAP-experiments proved different off-rates of IRS1 and IRS2. In the second part of the presented work we describe the interaction of the epidermal growth factor receptor (EGFR) with an important intracellular binding protein termed Grb2 using the same technique. Performed experiments confirm the strong interaction of these two molecules. Induction with EGF promotes the translocation of Grb2 into Clathrin Coated Pits (CCP)s only within EGFR-enriched membrane regions.Taken together presented results approve the power of the μ-patterning technique to study the interaction properties of plasma-membrane localized receptors. In the near future our established cellular systems will be used to study the effects of active pharmaceutical ingredients including plant metabolites on the interactions of membrane receptors.
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