Preformed Oligomeric Epidermal Growth Factor Receptors Undergo an Ectodomain Structure Change during Signaling

Martin-Fernandez, Marisa L.; Clarke, David T.; Tobin, Mark J.; Jones, Samantha V.; Jones, Gareth R.

doi:10.1016/s0006-3495(02)75585-9

Cited by 109 publications

(95 citation statements)

References 60 publications

(113 reference statements)

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“…promote lateral signal propagation (Martin-Fernandez et al, 2002;Yu et al, 2002;Bader et al, 2009). Our observation that only a subset of BRI1-SERK3 heterooligomers is present after ligand depletion is comparable to findings for EGFR in mammalian cells, as demonstrated by Bader et al (2009).…”

Section: Discussionsupporting

confidence: 79%

Visualization of BRI1 and BAK1(SERK3) Membrane Receptor Heterooligomers during Brassinosteroid Signaling

Bücherl¹,

Esse²,

Kruis³

et al. 2013

Plant Physiology

110

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The leucine-rich repeat receptor-like kinase BRASSINOSTEROID-INSENSITIVE1 (BRI1) is the main ligand-perceiving receptor for brassinosteroids (BRs) in Arabidopsis (Arabidopsis thaliana). Binding of BRs to the ectodomain of plasma membrane (PM)-located BRI1 receptors initiates an intracellular signal transduction cascade that influences various aspects of plant growth and development. Even though the major components of BR signaling have been revealed and the PM was identified as the main site of BRI1 signaling activity, the very first steps of signal transmission are still elusive. Recently, it was shown that the initiation of BR signal transduction requires the interaction of BRI1 with its SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) coreceptors. In addition, the resolved structure of the BRI1 ectodomain suggested that BRI1-ASSOCIATED KINASE1 [BAK1](SERK3) may constitute a component of the ligand-perceiving receptor complex. Therefore, we investigated the spatial correlation between BRI1 and BAK1(SERK3) in the natural habitat of both leucine-rich repeat receptor-like kinases using comparative colocalization analysis and fluorescence lifetime imaging microscopy. We show that activation of BR signaling by exogenous ligand application resulted in both elevated colocalization between BRI1 and BAK1(SERK3) and an about 50% increase of receptor heterooligomerization in the PM of live Arabidopsis root epidermal cells. However, large populations of BRI1 and BAK1(SERK3) colocalized independently of BRs. Moreover, we could visualize that approximately 7% of the BRI1 PM pool constitutively heterooligomerizes with BAK1(SERK3) in live root cells. We propose that only small populations of PMlocated BRI1 and BAK1(SERK3) receptors participate in active BR signaling and that the initiation of downstream signal transduction involves preassembled BRI1-BAK1(SERK3) heterooligomers.

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Section: Discussionsupporting

confidence: 79%

Visualization of BRI1 and BAK1(SERK3) Membrane Receptor Heterooligomers during Brassinosteroid Signaling

Bücherl¹,

Esse²,

Kruis³

et al. 2013

Plant Physiology

110

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“…Although several lines of evidence (3,4), support this view, both recent and earlier evidence (16)(17)(18)(20)(21)(22) have suggested that two aspects of the classical model need to be reconsidered: (i) inactive receptors are not necessarily monomeric, such that (ii) higher-order clusters of inactive and/or activated receptors may exist. We chose the novel technique of N&B analysis (23) to investigate these issues.…”

Section: Discussionmentioning

confidence: 99%

“…The findings suggest the existence of preformed (i.e., ligand-independent, ErbB1 receptor dimers) (16,17) or larger aggregates (18) in addition to the classical dimers formed upon ligand-induced activation of receptor monomers. However, the different techniques, cell types, and conditions of the experiments have resulted in conflicting and contradictory results.…”

mentioning

confidence: 92%

“…However, the different techniques, cell types, and conditions of the experiments have resulted in conflicting and contradictory results. Several studies using fixed cells and/or low temperature incubations based on hetero-or homo-FRET have indicated that <10% to as much as 50% of ErbB1 may exist as preformed dimers or oligomers (17,19,20). Inhibition of the tyrosine kinase activity of the receptor led to disaggregation of the transient dimers and clusters to monomers (18), suggesting that some measurements may have been made on nonstarved cells with preactivated receptors.…”

mentioning

confidence: 99%

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Distribution of resting and ligand-bound ErbB1 and ErbB2 receptor tyrosine kinases in living cells using number and brightness analysis

Nagy

Claus

Jovin

et al. 2010

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

164

159

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Ligand-driven dimerizations of ErbB receptor subunits fulfill a fundamental role in their activation. We have used the number and brightness analysis technique to investigate the existence of preformed ligand-independent dimers and clusters and to characterize the initial steps in the activation of ErbB1 and ErbB2. In cells expressing 50,000-200,000 receptors, ErbB1 was monomeric in the absence of ligand stimulation, whereas in CHO cells with receptor levels >500,000 as much as 30% of ErbB1 was present as preformed dimers. EGF induced the formation of ErbB1 dimers as well as larger clusters (up to pentamers) that colocalized with clathrin-coated pits. The distribution of unstimulated ErbB2 in cells expressing 3·10 5 − 10 6 receptors was fundamentally different, in that this receptor was present in preformed homoassociated aggregates containing 5-10 molecules. These constitutive ErbB2 homoclusters colocalized with caveolae, increased in size at subphysiological temperatures, but decreased in size upon EGF stimulation. We conclude that these ErbB2 clusters are promoted primarily by membrane-mediated interactions and are dispersed upon ligand stimulation.EGFR | epidermal growth factor | ErbB proteins | receptor clusters | signal transduction E rbB proteins (ErbB1-4, HER1-4) constitute the best characterized family of receptor tyrosine kinases (1). Biochemical analysis has demonstrated that ErbB1, the prototypical member of the family (also known as the epidermal growth factor receptor, EGFR or HER1) undergoes ligand-induced homodimerization as a key step in its activation (2). More recent crystallographic studies reveal that ligand binding induces a transition from a closed conformation of ErbB1 to an extended configuration with the capacity to dimerize via intermolecular interactions mediated by domain II (3, 4). The ultrastructural data also confirm that dimerization of ErbB1 plays a fundamental role in activating the kinase domain by a mechanism resembling that of cyclin dependent kinases (5, 6). The coreceptor ErbB2 has no known ligand but upon transactivation expresses the most potent kinase activity of the ErbB family, thereby increasing the efficiency of signaling mediated by ErbB2-containing heterodimers (7). ErbB2 constitutively adopts an extended conformation potentiating the formation of heterodimers (8, 9) that can be inhibited by pertuzumab, a monoclonal antibody sterically blocking the heterodimerization arm of ErbB2 (10). Although the extracellular domain of ErbB2 has failed to form crystallographic homodimers, molecular biological and fluorescence resonance energy transfer (FRET) experiments have shown that full-length ErbB2 exists in dimers or higher-order aggregates in the plasma membrane (11,12). The implication is that the transmembrane, juxtamembrane and other intracellular domains (5, 13, 14) act in conjunction with the membrane environment (15) to mediate the dimerization and, thereby, functional states of ErbB proteins.Many investigators have sought to determine the distribution of ErbB1 in ...

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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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Preformed Oligomeric Epidermal Growth Factor Receptors Undergo an Ectodomain Structure Change during Signaling

Cited by 109 publications

References 60 publications

Visualization of BRI1 and BAK1(SERK3) Membrane Receptor Heterooligomers during Brassinosteroid Signaling

Visualization of BRI1 and BAK1(SERK3) Membrane Receptor Heterooligomers during Brassinosteroid Signaling

Distribution of resting and ligand-bound ErbB1 and ErbB2 receptor tyrosine kinases in living cells using number and brightness analysis

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

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