Quantification and Kinetic Analysis of Grb2-EGFR Interaction on Micro-Patterned Surfaces for the Characterization of EGFR-Modulating Substances

<div><p>The identification of the epidermal 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 (μ-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 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.</p></div

Sensitivity of EGFR-Grb2 interaction on μ-biochips.

<p>(A) HeLa cells expressing EGFR-GFP or Grb2-YFP, respectively, grown on anti-EGFR antibody coated μ-biochips at indicated time points after EGF (170 nM) addition. (B) Temporal resolution of the change in contrast induced by EGF (n = 40 cells). (C) Time course of the corresponding F+ and F- signals of EGFR and Grb2, respectively (n = 40 cells). (D) EGF concentration dependent increase of Grb2 contrast (n = 40). Fluorescent contrast was normalized to the value prior to EGF stimulation. Error bars are based on the standard error of the mean. Scale bar  = 5 μm.</p

Inducibility of EGFR signaling on μ-structured surfaces.

<p>(A) Representative HeLa cell expressing Grb2-YFP grown on an anti-EGFR antibody coated μ-biochip. Induction by EGF (170 nM) for 15 minutes significantly increases the basic fluorescent contrast. (B) Quantification of EGFR-Grb2 interaction by contrast evaluation. Addition of EGF leads to an increase in the Grb2 contrast from ∼0.33 to ∼0.55. Error bars are based on the standard error of the mean (n > 300 cells). Scale bar  =  5 μm.</p

Inhibition of EGFR-Grb2 interaction by the small tyrosine kinase inhibitor AG1478.

<p>(A) Representative HeLa cell pretreated with 1 μM AG1478 for 4 hours expressing Grb2-YFP grown on an anti-EGFR antibody coated μ-biochip at the indicated time points after EGF addition (170 nM). (B) Time course of the Grb2 contrast change upon EGF stimulation in control and AG1478 pretreated (1, 10 μM) cells (n = 50). Pretreatment with a tyrosine kinase inhibitor leads to a decreased contrast compared to untreated cells. Fluorescent contrast was normalized to the value prior to EGF stimulation. (C) Dose-response relationship of the EGFR-Grb2 interaction in cells pretreated with indicated AG1478 concentrations (n = 40). (D) Temporal resolution of Grb2 contrast at different AG1478 concentrations. Cells were incubated with AG1478 and the decrease in contrast was observed for 90 min. Contrast was normalized to the value prior AG1478 addition (n =  40). Error bars are based on the standard error of the mean.</p

Inhibition of Grb2 recruitment to the EGFR by EGFR-activity modulating drugs.

<p>Grb2 contrast increase upon EGF stimulation (170 nM, 15 min) in cells pre-incubated with the indicated EGFR antagonists for 4 hours in comparison to untreated cells. Error bars are based on the standard error of the mean (n = 40 cells).</p

Basic principle of the μ-patterning screening assay.

<p>(A) Schematic diagram of EGFR-Grb2 interaction in living cells grown on a functionalized μ-biochip. Cells attach within 2–3 hours after seeding on the μstructured glass surface and endogenous EGFR is captured by anti-EGFR antibodies. Specific interaction of fluorescently labeled Grb2 with the EGFR is characterized by enhanced co-patterning in anti-EGFR antibody enriched regions. (B) Proof of principle. HeLa cells expressing EGFR-GFP are grown on an anti-EGFR antibody coated μ-biochip. Accurate alignment of anti-EGFR antibodies into unblocked spots leads to a strong enrichment of the bait protein (EGFR) into μ-patterns (upper row). Co-localization of the fluorescently labeled prey protein (Grb2-YFP) in anti-EGFR antibody positive regions indicates specific protein-protein interactions (lower row). Scale bars  =  5 μm.</p

Accumulation of EGFR and Grb2 in clathrin-coated pits (CCPs).

<p>Representative TIRF microscopy images of HeLa cells transfected with clathrin light chain-GFP (CliC-GFP), EGFR-GFP or Grb2-YFP, respectively, co-stained by Transferrin-Alexa647 as a marker for CCPs. Cells were stimulated with EGF for 15 min with simultaneous addition of Tfr-647 (25 μg/ml). Fluorescence intensity profiles are shown on the right for the indicated line scans. Scale bars  = 5 μm.</p

Stabilization of CCPs on the μ-patterned surface.

<p>(A) Representative FRAP images of a single pattern in HeLa cells expressing Grb2-YFP after EGF stimulation. Preformed CCPs remain stable and are not endocytosed as indicated by recovery of Grb2 at the same position (black arrows). (B) Mean fluorescence recovery curves of bleached cluster (red circle)/non-cluster (green circle) regions within a single pattern of untreated/AG1478-treated (1 μM for 4 hours) cells. Vertical dashed lines represent slow half-times of the different samples. Error bars are based on the standard error of the mean (n = 40 for cluster/non-cluster regions of 10 different cells). Scale bar  =  1 μm. (C) Cartoon indicating the formation and stabilization of CCPs on anti-EGFR antibody coated μ-biochips.</p

Kinetic FRAP parameters. Results are given as mean ± SEM.

<p>40 cluster/non-cluster regions of 10 different cells were analyzed.</p