Abstract
BackgroundKnowledge about signaling pathways is typically compiled based on data gathered using different cell lines. This approach implicitly assumes that the cell line dependence is not important. However, different cell lines do not always respond to a particular stimulus in the same way, and lack of coherent data collected from closely related cellular systems can be detrimental to the efforts to understand the regulation of biological processes. To address this issue, we created a clone library of human mammary epithelial (HME) cells that expresses different levels of HER2 and HER3 receptors in combination with endogenous EGFR/HER1. Using our clone library, we have quantified the receptor activation patterns and systematically tested the validity of the existing hypotheses about the interaction patterns between HER1-3 receptors.ResultsOur study identified HER2 as the dominant dimerization partner for both EGFR and HER3. Contrary to earlier suggestions, we find that lateral interactions with HER2 do not lead to strong transactivation between EGFR and HER3, i.e., EGFR activation and HER3 activation are only weakly linked in HME cells. We also find that observed weak transactivation is uni-directional where stimulation of EGFR leads to HER3 activation whereas HER3 stimulation does not activate the EGFR. Repeating our experiments at lower cell confluency established that cell confluency is not a major factor in the observed interaction patterns. We have also quantified the dependence of the kinetics of Erk and Akt activation on different HER receptors. We found that HER3 signaling makes the strongest contribution to Akt activation and that, stimulation of either EGFR or HER3 leads to significant Erk activation.ConclusionOur study shows that clone cell libraries can be a powerful resource in systems biology research by making it possible to differentiate between various hypotheses in a consistent cellular background. Using our constructed clone library we profiled the cell signaling patterns to establish the role of HER2 in the crosstalk between EGFR and HER3 receptors in HME cells. Our results for HME cells show that the weak linkage between EGFR and HER3 pathways can lead to distinct downstream cellular signaling patterns in response to the ligands of these two receptors.
Highlights
Knowledge about signaling pathways is typically compiled based on data gathered using different cell lines
In order to understand the interactions between the HER molecules under physiological and pathological conditions, we need to study systems expressing different levels of these receptors, and need to do so in a common cellular background to ensure internal consistency of the generated datasets. This is the main objective of our study: We have developed a clone library of human mammary epithelial (HME) cell lines that express endogenous levels of EGFR and varying levels of HER2 and HER3 to experimentally examine the various hypothesized roles of HER2 in coupling EGFR and HER3 activation (Figure 1)
Expression of EGFR/HER2/HER3 in HME Cells HER receptor expression was examined in all cell lines using FACS
Summary
Knowledge about signaling pathways is typically compiled based on data gathered using different cell lines. Different cell lines do not always respond to a particular stimulus in the same way, and lack of coherent data collected from closely related cellular systems can be detrimental to the efforts to understand the regulation of biological processes To address this issue, we created a clone library of human mammary epithelial (HME) cells that expresses different levels of HER2 and HER3 receptors in combination with endogenous EGFR/HER1. HER2 receptors have no known ligand and HER3 lacks intrinsic tyrosine kinase activity [4,5], and upon ligand binding, they all undergo conformation changes that favor the formation or stabilization of receptor heteroand homo-dimers [6,7] This results in receptor phosphorylation, and the resultant formation of complexes with adaptor proteins to initiate signaling. This cascade of events leads to downstream signal transduction, and can trigger diverse biological responses [8,9,10]
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