Dynamic Analysis of the Epidermal Growth Factor (EGF) Receptor-ErbB2-ErbB3 Protein Network by Luciferase Fragment Complementation Imaging

Macdonald-Obermann, Jennifer L.; Adak, Sangeeta; Landgraf, Ralf; Piwnica‐Worms, David; Pike, Linda J.

doi:10.1074/jbc.m113.489534

Cited by 37 publications

(34 citation statements)

References 49 publications

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“…Cell-based approaches using indirect measurements, such as chemical crosslinking, nucleic acid aptamers, and bulk fluorescence complementation, provided evidence that HER3 oligomerizes before NRG binding, but upon binding the ligand it dissociates into monomeric receptors that are poised to heterodimerize with other HER receptors (43)(44)(45). More direct approaches, such as immunoelectron microscopy and quantum dot single-particle tracking analyses, have challenged this model and point to significant HER3 homodimerization and even clustering in response to NRG (30,46).…”

Section: Discussionmentioning

confidence: 99%

EGF and NRG induce phosphorylation of HER3/ERBB3 by EGFR using distinct oligomeric mechanisms

Lengerich

Agnew

Puchner

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Heteromeric interactions between the catalytically impaired human epidermal growth factor receptor (HER3/ERBB3) and its catalytically active homologs EGFR and HER2 are essential for their signaling. Different ligands can activate these receptor pairs but lead to divergent signaling outcomes through mechanisms that remain largely unknown. We used stochastic optical reconstruction microscopy (STORM) with pair-correlation analysis to show that EGF and neuregulin (NRG) can induce different extents of HER3 clustering that are dependent on the nature of the coexpressed HER receptor. We found that the presence of these clusters correlated with distinct patterns and mechanisms of receptor phosphorylation. NRG induction of HER3 phosphorylation depended on the formation of the asymmetric kinase dimer with EGFR in the absence of detectable higher-order oligomers. Upon EGF stimulation, HER3 paralleled previously observed EGFR behavior and formed large clusters within which HER3 was phosphorylated via a noncanonical mechanism. HER3 phosphorylation by HER2 in the presence of NRG proceeded through still another mechanism and involved the formation of clusters within which receptor phosphorylation depended on asymmetric kinase dimerization. Our results demonstrate that the higher-order organization of HER receptors is an essential feature of their ligand-induced behavior and plays an essential role in lateral cross-activation of the receptors. We also show that HER receptor ligands exert unique effects on signaling by modulating this behavior.HER/ERBB receptors | receptor tyrosine kinase signaling | receptor clustering | STORM | EGFR activation T he human epidermal growth factor receptors (HERs/ErbBs) are essential regulators of development and adult homeostasis (1). All four of them, EGF receptor (EGFR; HER1), HER2 (ErbB2), HER3 (ErbB3), and HER4 (ErbB4), are at the focus of therapeutic efforts in a variety of human diseases. Most of what we know about their activation mechanism has been revealed by the studies on EGFR, which showed that ligand binding induces EGFR dimerization through a series of structurally well-defined interactions between the extracellular and intracellular receptor domains (2). These interactions result in the formation of an asymmetric kinase dimer in which one kinase (termed the "activator kinase") is asymmetrically positioned to activate the second kinase (termed the "receiver kinase") allosterically (3). The receiver kinase then is poised to phosphorylate the receptor tails, resulting in the recruitment of downstream signaling molecules and signal propagation.One of the characteristic features of the HER receptor family is a significant degree of heteromeric interactions in response to ligand binding through which the receptors activate a variety of signaling pathways (1). These interactions are particularly important for signaling by the orphan receptor HER2 and the catalytically impaired HER3, which do not signal on their own under normal conditions. Although all HER receptors are assumed to form h...

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Section: Discussionmentioning

confidence: 99%

EGF and NRG induce phosphorylation of HER3/ERBB3 by EGFR using distinct oligomeric mechanisms

Lengerich

Agnew

Puchner

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…We have previously used luciferase fragment complementation assays to measure the formation of EGF receptor homodimers and EGFR/ErbB2 heterodimers (31,32). Therefore, we used this method to compare dimer formation induced by the four different ligands.…”

Section: Methodsmentioning

confidence: 99%

“…Therefore, we used this method to compare dimer formation induced by the four different ligands. Initially, we performed luciferase fragment complementation imaging using EGF receptors and ErbB2 that had been C-terminally truncated at the end of the transmembrane domain and thus lacked the entire intracellular domain (31,32). Therefore, in these truncated constructs, dimerization was mediated only by interactions between the extracellular and/or transmembrane domains of the receptor subunits.…”

Section: Methodsmentioning

confidence: 99%

Different Epidermal Growth Factor (EGF) Receptor Ligands Show Distinct Kinetics and Biased or Partial Agonism for Homodimer and Heterodimer Formation

Macdonald-Obermann

Pike

2014

Journal of Biological Chemistry

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105

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Background:The EGF receptor has multiple ligands that can induce different biological effects. Results: EGF and TGF␣, but not BTC or AREG, bias the EGF receptor toward heterodimer formation with ErbB2. AREG induces dimerization with different kinetics than the other ligands. Conclusion: EGFR ligands differentially induce receptor dimerization.Significance: This contributes to the biological differences elicited by different ligands.

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“…We have previously used a luciferase fragment complementation system to assess the ability of EGF to induce dimerization of the EGF receptor (22)(23)(24). In this study, we use our luciferase fragment complementation assay to visualize the recruitment of a variety of signaling proteins to the EGF receptor.…”

mentioning

confidence: 99%

Different Epidermal Growth Factor Receptor (EGFR) Agonists Produce Unique Signatures for the Recruitment of Downstream Signaling Proteins

Ronan¹,

Macdonald-Obermann

Huelsmann

et al. 2016

Journal of Biological Chemistry

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The EGF receptor can bind seven different agonist ligands.Although each agonist appears to stimulate the same suite of downstream signaling proteins, different agonists are capable of inducing distinct responses in the same cell. To determine the basis for these differences, we used luciferase fragment complementation imaging to monitor the recruitment of Cbl, CrkL, Gab1, Grb2, PI3K, p52 Shc, p66 Shc, and Shp2 to the EGF receptor when stimulated by the seven EGF receptor ligands. Recruitment of all eight proteins was rapid, dose-dependent, and inhibited by erlotinib and lapatinib, although to differing extents. Comparison of the time course of recruitment of the eight proteins in response to a fixed concentration of each growth factor revealed differences among the growth factors that could contribute to their differing biological effects. Principal component analysis of the resulting data set confirmed that the recruitment of these proteins differed between agonists and also between different doses of the same agonist. Ensemble clustering of the overall response to the different growth factors suggests that these EGF receptor ligands fall into two major groups as follows: (i) EGF, amphiregulin, and EPR; and (ii) betacellulin, TGF␣, and epigen. Heparin-binding EGF is distantly related to both clusters. Our data identify differences in network utilization by different EGF receptor agonists and highlight the need to characterize network interactions under conditions other than high dose EGF.

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Dynamic Analysis of the Epidermal Growth Factor (EGF) Receptor-ErbB2-ErbB3 Protein Network by Luciferase Fragment Complementation Imaging

Cited by 37 publications

References 49 publications

EGF and NRG induce phosphorylation of HER3/ERBB3 by EGFR using distinct oligomeric mechanisms

EGF and NRG induce phosphorylation of HER3/ERBB3 by EGFR using distinct oligomeric mechanisms

Different Epidermal Growth Factor (EGF) Receptor Ligands Show Distinct Kinetics and Biased or Partial Agonism for Homodimer and Heterodimer Formation

Different Epidermal Growth Factor Receptor (EGFR) Agonists Produce Unique Signatures for the Recruitment of Downstream Signaling Proteins

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