This basic interaction between Grb2 and EGFR appeared without any additional EGFR activation and may be caused by the stimulating nature of the antibody coated surface and/or the presence of growth factor containing serum. bait-enriched region (purple arrows) remained stable for up to 50 min, whereas clusters in the bait-free area (yellow arrows) disappeared due to endocytotic events. Scale bar ?=? 3 m.(TIF) pone.0092151.s004.tif (346K) GUID:?6FD35A20-B939-41AD-96AA-7663A4B4EA3A Abstract The identification of the epidermal RCBTB1 growth factor receptor (EGFR) as an oncogene has led to the development of several anticancer therapeutics directed against this receptor tyrosine kinase. However, drug resistance and low efficacy remain a severe challenge, and have led to a demand for novel systems for an efficient identification and characterization of new substances. Here 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. It does not simply measure the phosphorylation of the receptor, but instead quantifies 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 Paris saponin VII to demonstrate an EGF dependent recruitment of Grb2 to the EGFR, which was significantly inhibited in the presence of clinically tested EGFR inhibitors, including small tyrosine kinase inhibitors and monoclonal antibodies targeting the EGF binding site. 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. Recruitment of the EGFR together with Grb2 to clathrin coated pits (CCPs) was found to be a key feature in our assay. Application of bleaching experiments enabled calculation of the Grb2 exchange rate, which significantly changed upon stimulation or the presence of EGFR activity inhibiting drugs. Introduction Receptor tyrosine kinases (RTKs) are a subclass of signaling receptors anchored at the cell surface and have intrinsic tyrosine kinase activity triggering signal transduction in response to ligand binding. RTKs are generally activated through ligand-induced oligomerization, typically dimerization, which leads to autophosphorylation of tyrosine residues in Paris saponin VII the kinase activation loop or the juxtamembrane region . These phosphotyrosine residues are important docking sites Paris saponin VII for a plethora of intracellular downstream signaling molecules and are typically bound by Src homology-2 (SH2) or phosphotyrosine-binding (PTB) domains . The epidermal growth factor receptor (EGFR) is a member of the ErbB family of receptors, a subclass of RTKs, and is expressed in all epidermal cells as well as stromal, glia and smooth muscle cells . EGFR signaling is one of the most important pathways that regulate growth, survival, proliferation and differentiation in mammalian cells . Thus, EGFR signaling is also critical for the development of many types of cancer. Mutations that lead to EGFR overexpression or overactivity have been associated with a number of cancers, including lung cancer, anal cancers and the glioblastoma multiforme , . Mutations involving the EGFR may lead to its constant activation, which results in uncontrolled cell division. Consequently, mutations of the EGFR have been identified in several types of cancer and it is the target of an expanding class of anticancer therapies C. The identification of EGFR as an oncogene has led to the development of anticancer therapeutics directed against the EGFR including AG1478, Gefitinib, Erlotinib, Lapatinib, Canertinib (small molecule kinase inhibitors) and Cetuximab (monoclonal antibody inhibitor) C. Resistance to these drugs has emerged as a major clinical problem limiting the efficacy of currently used inhibitors and their use in cancer patients . Thus, there is a demand for the optimization of existing, but also for the design of novel screening approaches to develop new inhibitors of RTKs. Current approaches are mainly based on the screening of purified.