Investigating extracellular <i>in situ</i> EGFR structure and conformational changes using FRET microscopy

Roberts, Selene K.; Tynan, Christopher J.; Winn, Martyn; Martin-Fernandez, Marisa L.

doi:10.1042/bst20110632

Cited by 8 publications

(4 citation statements)

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“…The fact that the linker region is flexible allows other orientations of EGFR, such that inactive, unliganded dimers might form as proposed by Jura et al , or asymmetric dimers might form as seen with the invertebrate Drosophila EGFR . The latter viewpoint has been championed by the group of Martin‐Fernandez , which has focused on biophysical measurements to elucidate the structure of the “high‐affinity” EGFR implicated by Scatchard EGF‐binding plots showing upward curvature (negative cooperativity) at very low‐EGF concentrations. This group has presented evidence from FRET/FLIM measurements with a labeled EGF ligand (<1 nM) as donor and a membrane carbocyanine dye as acceptor for the existence of a small cohort (10–15%) of “high‐affinity” dimers, which would be asymmetric and lie on (parallel to) the cell membrane.…”

Section: Discussionmentioning

confidence: 99%

Dynamic conformational transitions of the EGF receptor in living mammalian cells determined by FRET and fluorescence lifetime imaging microscopy

Ziomkiewicz

Loman²,

Klement³

et al. 2013

Cytometry Pt A

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We have revealed a reorientation of ectodomain I of the epidermal growth factor receptor (EGFR; ErbB1; Her1) in living CHO cells expressing the receptor, upon binding of the native ligand EGF. The state of the unliganded, nonactivated EGFR was compared to that exhibited after ligand addition in the presence of a kinase inhibitor that prevents endocytosis but does not interfere with binding or the ensuing conformational rearrangements. To perform these experiments, we constructed a transgene EGFR with an acyl carrier protein sequence between the signal peptide and the EGFR mature protein sequence. This protein, which behaves similarly to wild-type EGFR with respect to EGF binding, activation, and internalization, can be labeled at a specific serine in the acyl carrier tag with a fluorophore incorporated into a 4 0 -phosphopantetheine (P-pant) conjugate transferred enzymatically from the corresponding CoA derivative. By measuring F€ orster resonance energy transfer between a molecule of Atto390 covalently attached to EGFR in this manner and a novel lipid probe NR12S distributed exclusively in the outer leaflet of the plasma membrane, we determined the apparent relative separation of ectodomain I from the membrane under nonactivating and activating conditions. The data indicate that the unliganded domain I of the EGFR receptor is situated much closer to the membrane before EGF addition, supporting the model of a selfinhibited configuration of the inactive receptor in quiescent cells. V C 2013 International Society for Advancement of CytometryKey terms lifetime imaging; time-correlated single-photon counting; epidermal growth factor receptor; tethered configuration; EGF; FRET The X-ray crystallographic structures of the extracellular domain of the epidermal growth factor receptor (EGFR) have been determined in both the liganded (1,2) and unliganded (3) states. In the structures with bound EGF or TGFa, the ligand is associated with an extended region comprising domains I and III, and a homodimeric structure is generated by interaction of b-hairpins protruding from the two domains II of the constituent monomers, confirming an earlier model of Lemmon et al. (4). This extended structure was also seen in the extracellular domain of the ErbB2 member of the ErbB family (5). ErbB2 lacks a known ligand but features a potent kinase domain requiring activation by its preferred heterodimerization partners EGFR and ErbB3. In contrast, the unliganded extracellular domain of EGFR crystallized in a selfinhibitory structure in which the domain II dimerization arm is completely suppressed by intramolecular interactions with domain IV (3). This structure has been presumed to be the form assumed by the inactive native receptor in the plasma membrane. A number of in vivo studies featuring mutated EGFR (6), antibody binding (7), and co-crystal structures of mAbs with native EGFR (8,9) have confirmed the Iwona Ziomkiewicz and Anastasia Loman contributed equally to the work.

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Section: Discussionmentioning

confidence: 99%

Dynamic conformational transitions of the EGF receptor in living mammalian cells determined by FRET and fluorescence lifetime imaging microscopy

Ziomkiewicz

Loman²,

Klement³

et al. 2013

Cytometry Pt A

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“…The EGFR gene is located on chromosome 7p11.2 and contains 28 exons. The protein it encodes has an extracellular domain, a transmembrane domain, and a cytoplasmic domain ( 7 ). The cytoplasmic domain (also called the tyrosine kinase domain) is responsible for the phosphorylation of its downstream targets and self-regulation.…”

Section: Introductionmentioning

confidence: 99%

Multiple mutations in the EGFR gene in lung cancer: a systematic review

Castañeda-González¹,

Chaves²,

Parra‐Medina³

2022

Transl Lung Cancer Res

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Background: Lung cancer is a public health problem worldwide. Currently, identifying genetic mutations in the epidermal growth factor receptor (EGFR) has brought significant changes in diagnosing and managing patients with lung cancer. The presence of multiple mutations, defined as the presence of more than one EGFR mutation, has been reported in a few studies. Therefore, we carried out this systematic review to describe the most common multiple mutations in the EGFR gene.Methods: We conduct a systematic review of descriptive studies, cohorts, and clinical trials published in Scopus, PubMed, Scielo, and Virtual Health Library literature. The inclusion criteria for the systematic review were descriptive studies, cohorts, and clinical trials with the presence of multiple mutations in the EGFR gene. It was followed the Preferred Reporting Items for Systematic Meta-Analyses (PRISMA) guidelines.Results: In the systematic review, 41 articles were included. Four hundred and forty-six cases with multiple mutations in the EGFR gene were found (0.95% of the patients included in the studies). The most prevalent dual mutations observed were T790M + L858R and deletions in exon 19 + T790M. Triple mutations were found in 9 cases (2.017%). According to reports, the presence of T790M mutation in the multiple mutations has been associated with poor clinical outcomes.Discussion: The presence of multiple mutations in the EGFR gene is rare. It is of great importance to consider the T790M mutation since it generates resistance to pharmacological management and has worse outcomes. The most important limitation was that clinical information data and follow-up could not be collected in a large percentage of patients. Therefore, future work should be focused on clinical characteristics, follow-up and repercussions in the treatment of patients with multiple mutations.

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“…This suggests that the FN2 domain could lie on the cell membrane “sideways on,” possibly presenting an extended interaction surface to the headgroups of the lipid molecules via one of its β sheets. Significantly, both computational ( Arkhipov et al., 2013 , Arkhipov et al., 2014 , Franco-Gonzalez et al., 2014 , Kaszuba et al., 2015 , Kästner et al., 2009 ) and experimental ( Roberts et al., 2012 ) studies suggest that other RTKs may interact with lipid bilayers via their ectodomains.…”

Section: Introductionmentioning

confidence: 99%

Structures of the EphA2 Receptor at the Membrane: Role of Lipid Interactions

et al. 2016

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SummaryEphs are transmembrane receptors that mediate cell-cell signaling. The N-terminal ectodomain binds ligands and enables receptor clustering, which activates the intracellular kinase. Relatively little is known about the function of the membrane-proximal fibronectin domain 2 (FN2) of the ectodomain. Multiscale molecular dynamics simulations reveal that FN2 interacts with lipid bilayers via a site comprising K441, R443, R465, Q462, S464, S491, W467, F490, and P459–461. FN2 preferentially binds anionic lipids, a preference that is reduced in the mutant K441E + R443E. We confirm these results by measuring the binding of wild-type and mutant FN2 domains to lipid vesicles. In simulations of the complete EphA2 ectodomain plus the transmembrane region, we show that FN2 anchors the otherwise flexible ectodomain at the surface of the bilayer. Altogether, our data suggest that FN2 serves a dual function of interacting with anionic lipids and constraining the structure of the EphA2 ectodomain to adopt membrane-proximal configurations.

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Investigating extracellular in situ EGFR structure and conformational changes using FRET microscopy

Cited by 8 publications

References 50 publications

Dynamic conformational transitions of the EGF receptor in living mammalian cells determined by FRET and fluorescence lifetime imaging microscopy

Dynamic conformational transitions of the EGF receptor in living mammalian cells determined by FRET and fluorescence lifetime imaging microscopy

Multiple mutations in the EGFR gene in lung cancer: a systematic review

Structures of the EphA2 Receptor at the Membrane: Role of Lipid Interactions

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