Ligand-induced EGF Receptor Oligomerization Is Kinase-dependent and Enhances Internalization

Hofman, Erik; Bader, Arjen N.; Voortman, Jarno; Heuvel, D.J. van den; Sigismund, Sara; Verkleij, Arie J.; Gerritsen, Hans C.; Henegouwen, Paul M.P. van Bergen en

doi:10.1074/jbc.m110.164731

Cited by 97 publications

(119 citation statements)

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“…Direct predimerization, at least among ErbB3 molecules, has been reported previously (11,14). Predimerization has also been detected for ErbB1 (32). Therefore, we assumed that the two-step association sites were predimers of HRG receptors in the kinetic models.…”

Section: Discussionmentioning

confidence: 99%

Dynamically varying interactions between heregulin and ErbB proteins detected by single-molecule analysis in living cells

Hiroshima

Saeki

Okada‐Hatakeyama

et al. 2012

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

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Heregulin (HRG) belongs to the family of EGFs and activates the receptor proteins ErbB3 and ErbB4 in a variety of cell types to regulate cell fate. The interactions between HRG and ErbB3/B4 are important to the pathological mechanisms underlying schizophrenia and some cancers. Here, we observed the reaction kinetics between fluorescently labeled single HRG molecules and ErbB3/B4 on the surfaces of MCF-7 human breast cancer cells. The equilibrium association and the dissociation from equilibrium were also measured using single-molecule imaging techniques. The unitary association processes mirrored the EGF and ErbB1 interactions in HeLa cells [Teramura Y, et al. (2006) , suggesting that the predimerization of the receptors, followed by intermediate formation (between the first and second ligand-binding events to a receptor dimer), accelerated the formation of doubly liganded signaling dimers of the receptor molecules. However, the dissociation analysis suggested that the first HRG dissociation from the doubly liganded dimer was rapid, but the second dissociation from the singly liganded dimer was slow. The dissociation rate constant from the liganded monomer was intermediate. The dynamic changes in the association and dissociation kinetics in relation to the dimerization of ErbB displayed negative cooperativity, which resulted in apparent low-and high-affinity sites of HRG association on the cell surface.ligand-receptor interaction | cell signaling | epidermal growth factors T he ErbB family includes four member proteins, which localize to the plasma membrane of various types of cells and mediate signal transduction involved in cell differentiation and proliferation (1, 2). Among the ErbB family members, ErbB3 and ErbB4 are receptors for several extracellular protein ligands, including heregulin (HRG, also called neuregulin). ErbB3 is involved in the development of the heart and nervous system, and ErbB4 plays a role in lactation (3, 4). The dysregulation of ErbB3 and B4 (referred to hereafter as "HRG receptors") is often related to malignancy in human cancers (2). The long-term exposure of MCF-7 cells, a cultured cell line derived from a human breast carcinoma, to HRG induces the production of differentiation markers, such as milk proteins or lipid droplets, whereas similar exposure to epidermal growth factor induces proliferation (5).ErbB molecules must dimerize to transduce signals across the plasma membrane (3,4,6). Single ErbB molecules associate with single ligand molecules, such as HRG, and two liganded receptor molecules form a doubly liganded dimer on the plasma membrane (7). Dimerization is essential for the activation of ErbB molecules. This activation involves the phosphorylation of multiple sites along the cytoplasmic tail domain, which is catalyzed by the cytoplasmic kinase domain of the dimerization partner. In addition to homodimerization, some combinations of heterodimerization between members of the ErbB family have been described (1, 2). Several forms of unliganded and liganded homoand heterodi...

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Section: Discussionmentioning

confidence: 99%

Dynamically varying interactions between heregulin and ErbB proteins detected by single-molecule analysis in living cells

Hiroshima

Saeki

Okada‐Hatakeyama

et al. 2012

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

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“…This model has been supported by crystal structures of the extracellular domain of EGFR (7,8) as well as by reports of EGFR dimerization after stimulation with EGF. However, numerous studies have also reported the presence of EGFR dimers or even larger oligomers in the membranes of resting cells (9)(10)(11)(12)(13). The presence of preformed EGFR dimers and oligomers indicates that regulation of EGFR signaling is more complex than implied by the model outlined above (Fig.…”

Section: Introductionmentioning

confidence: 93%

The Epidermal Growth Factor Receptor Forms Location-Dependent Complexes in Resting Cells

Yavas

Macháň

Wohland

2016

Biophysical Journal

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The epidermal growth factor receptor (EGFR) is a prototypical receptor tyrosine kinase involved in cell growth and proliferation and associated with various cancers. It is commonly assumed that after activation by binding of epidermal growth factor to the extracellular domain it dimerizes, followed by autophosphorylation of tyrosine residues at the intracellular domain. However, its oligomerization state before activation is controversial. In the absence of ligands, EGFR has been found in various, inconsistent amounts of monomeric, inactive dimeric, and oligomeric forms. In addition, evidence suggests that the active conformation is not a simple dimer but contains higher oligomers. As experiments in the past have been conducted at different conditions, we investigate here the influence of cell lines (HEK293, COS-7, and CHO-K1), temperature (room temperature and 37 C), and membrane localization on the quantitation of preformed dimers using SW-FCCS, DC-FCCS, quasi PIE-FCCS, and imaging FCCS. While measurement modality, temperature, and localization on upper or lower membranes have only a limited influence on the dimerization amount observed, the cell line and location to periphery versus center of the cell can change dimerization results significantly. The observed dimerization amount is strongly dependent on the expression level of endogenous EGFR in a cell line and shows a strong cell-to-cell variability even within the same cell line. In addition, using imaging FCCS, we find that dimers have a tendency to be found at the periphery of cells compared to central positions.

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“…Quantitation of EGF receptor expression in each line by 125 I-EGF saturation binding indicated that the number of cell surface EGF receptors expressed in each line is within Ϯ20% of the average level of receptor expression (data not shown). Cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% FetalPlex, 100 g/ml G418, 100 g/ml hygromycin, and 100 g/ml Zeocin and maintained in an incubator at 37°C in 5% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

“…In the absence of ligand, the EGF receptor is thought to exist as a monomer, although inactive "pre-dimers" are known to form (1)(2)(3)(4)(5). Upon binding an agonist ligand, the EGF receptor dimerizes leading to the activation of its tyrosine kinase and the phosphorylation of tyrosine residues in the C-terminal tail of the receptor (6 -8).…”

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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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Ligand-induced EGF Receptor Oligomerization Is Kinase-dependent and Enhances Internalization

Cited by 97 publications

References 30 publications

Dynamically varying interactions between heregulin and ErbB proteins detected by single-molecule analysis in living cells

Dynamically varying interactions between heregulin and ErbB proteins detected by single-molecule analysis in living cells

The Epidermal Growth Factor Receptor Forms Location-Dependent Complexes in Resting Cells

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

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