scholarly journals Allosteric activation of preformed EGF receptor dimers by binding of a single ligand

2021 ◽  
Author(s):  
Endang Purba ◽  
Ei-ichiro Saita ◽  
Reetesh Akhouri ◽  
Lars-Göran Öfverstedt ◽  
Gunnar Wilken ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Single Molecule ◽  
Molecular Mechanisms ◽  
Egf Receptor ◽  
Electron Tomography ◽  
Growth Factor Receptor ◽  
Full Length ◽  
Cancer Therapies ◽  
Receptor Dimer

Abstract Aberrant activation of the epidermal growth factor receptor (EGFR) by mutations has been implicated in a variety of human cancers. Elucidation of the structure of the full-length receptor is essential to understand the molecular mechanisms underlying its activation. Unlike previously anticipated, here, we report that purified full-length EGFR adopts a homodimeric form in vitro before and after activation. Cryo-electron tomography analysis of the purified receptor also showed that the extracellular domains of the receptor dimer, which are conformationally flexible before activation, are stabilised by ligand binding. Consistently, optical single-molecule observation also demonstrated that binding of only one ligand activates the receptor dimer on the cell surface. Based on these results, we propose an allosteric model for the activation of EGFR dimers by ligand binding. Our results demonstrate how oncogenic mutations spontaneously activate the receptor and shed light on the development of novel cancer therapies.

scholarly journals Allosteric activation of preformed EGF receptor dimers by a single ligand binding event

2021 ◽  
Author(s):  
Endang R. Purba ◽  
Ei-ichiro Saita ◽  
Reetesh R. Akhouri ◽  
Lars-Göran Öfverstedt ◽  
Gunnar Wilken ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Single Molecule ◽  
Molecular Mechanisms ◽  
Egf Receptor ◽  
Active Form ◽  
Growth Factor Receptor ◽  
Full Length ◽  
Receptor Dimer

Abstract Aberrant activation of the epidermal growth factor receptor (EGFR) by mutations has been implicated in a variety of human cancers. Elucidation of the structure of the full-length receptor is essential to understand the molecular mechanisms underlying its activation. Unlike previously anticipated, here, we report that purified full-length EGFR adopts a homodimeric form in vitro before and after ligand binding. Cryo-electron tomography analysis of the purified receptor also showed that the extracellular domains of the receptor dimer, which are conformationally flexible before activation, are stabilized by ligand binding. This conformational flexibility stabilization most likely accompanies rotation of the entire extracellular domain and the transmembrane a-helix, resulting in dissociation of the intracellular kinase dimer and, thus, rearranging it into an active form. Consistently, mutations of amino acid residues at the interface of the inactive, symmetric kinase dimer spontaneously activate the receptor in vivo. Optical single-molecule observation also demonstrated that binding of only one ligand activates the receptor dimer on the cell surface. Based on these results, we propose an allosteric model for the activation of EGFR dimers by ligand binding. Our results demonstrate how oncogenic mutations spontaneously activate the receptor and shed light on the development of novel cancer therapies.

Met -/- kidneys express epithelial cells that chemotax and form tubules in response to EGF receptor ligands

1997 ◽  
Vol 272 (2) ◽  
pp. F222-F228
Author(s):  
C. Kjelsberg ◽  
H. Sakurai ◽  
K. Spokes ◽  
C. Birchmeier ◽  
I. Drummond ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epithelial Cell ◽  
Egf Receptor ◽  
Transforming Growth Factor ◽  
Growth Factor Receptor ◽  
Receptor Ligands ◽  
Tubule Formation ◽  
Human Papillomavirus 16 ◽  
Immortalized Cells

The growth factor/receptor combination of hepatocyte growth factor (HGF)/c-met has been postulated to be critical for mesenchymal-to-epithelial conversion and tubule formation in the developing kidney. We therefore isolated and immortalized cells from embryonic kidneys of met -/- transgenic mice to determine whether these cells were epithelial and able to chemotax and form tubules in vitro. The cells were immortalized with retrovirus expressing human papillomavirus 16 (HPV 16) E6/E7 genes. Two rapidly dividing clones were isolated and found to express the epithelial cell markers cytokeratin, zonula occludens-1, and E-cadherin but not to express the fibroblast marker vimentin. The met -/- cells were able to chemotax in response to epidermal growth factor and transforming growth factor-alpha (TGF-alpha) and form tubules in vitro in response to TGF-alpha but not HGF. These experiments suggest that the HGF/c-met axis is not essential for epithelial cell development in the embryonic kidney and demonstrate that other growth factors are capable of supporting early tubulogenesis.

scholarly journals Infrared nanospectroscopy reveals the molecular interaction fingerprint of an aggregation inhibitor with single Aβ42 oligomers

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Francesco Simone Ruggeri ◽  
Johnny Habchi ◽  
Sean Chia ◽  
Robert I. Horne ◽  
Michele Vendruscolo ◽  
...  
Keyword(s):  
Small Molecules ◽  
Single Molecule ◽  
Protein Misfolding ◽  
Molecular Mechanisms ◽  
Chemical Sensitivity ◽  
Incomplete Knowledge ◽  
Parkinson’S Diseases ◽  
Aggregation Inhibitor ◽  
Misfolding Diseases

AbstractSignificant efforts have been devoted in the last twenty years to developing compounds that can interfere with the aggregation pathways of proteins related to misfolding disorders, including Alzheimer’s and Parkinson’s diseases. However, no disease-modifying drug has become available for clinical use to date for these conditions. One of the main reasons for this failure is the incomplete knowledge of the molecular mechanisms underlying the process by which small molecules interact with protein aggregates and interfere with their aggregation pathways. Here, we leverage the single molecule morphological and chemical sensitivity of infrared nanospectroscopy to provide the first direct measurement of the structure and interaction between single Aβ42 oligomeric and fibrillar species and an aggregation inhibitor, bexarotene, which is able to prevent Aβ42 aggregation in vitro and reverses its neurotoxicity in cell and animal models of Alzheimer’s disease. Our results demonstrate that the carboxyl group of this compound interacts with Aβ42 aggregates through a single hydrogen bond. These results establish infrared nanospectroscopy as a powerful tool in structure-based drug discovery for protein misfolding diseases.

scholarly journals Transmembrane-truncated αvβ3 integrin retains high affinity for ligand binding: evidence for an ‘inside-out’ suppressor?

1998 ◽  
Vol 330 (2) ◽  
pp. 861-869 ◽  
Author(s):  
J. Raj MEHTA ◽  
Beate DIEFENBACH ◽  
Alex BROWN ◽  
Eilish CULLEN ◽  
Alfred JONCZYK ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Wound Repair ◽  
Molecular Mechanisms ◽  
Full Length ◽  
Αvβ3 Integrin ◽  
Cytoplasmic Domains ◽  
Bivalent Cation ◽  
High Affinity ◽  
Cellular Environment

The molecular mechanisms of αvβ3 integrin affinity regulation have important biological implications in tumour development, wound repair and angiogenesis. We expressed, purified and characterized recombinant forms of human αvβ3 (r-αvβ3) and compared the activation state of these with αvβ3 in its cellular environment. The ligand specificity and selectivity of recombinant full-length and double transmembrane truncations of r-αvβ3 cloned in BacPAK6 vectors and expressed in Sf9 and High Five insect cells were compared with those of native placental αvβ3 and the receptor in situ on the cell surface. r-αvβ3 integrins were purified by affinity chromatography from detergent extracts of cells (full-length), and from the culture medium of cells expressing double-truncated r-αvβ3. r-αvβ3 had the same epitopes, ligand-binding specificities, bivalent cation requirements and susceptibility to RGD-containing peptides as native αvβ3. On M21-L4 melanoma cells, αvβ3 mediated binding to vitronectin, but not to fibrinogen unless activated with Mn2+. Non-activated αIIbβ3 integrin as control in M21-L-IIb cells had the opposite profile, mediating binding to fibrinogen, but not to vitronectin unless activated with Mn2+. Thus these receptors had moderate to low ligand affinity. In marked contrast, purified αvβ3 receptors, with or without transmembrane and cytoplasmic domains, were constitutively of high affinity and able to bind strongly to vitronectin, fibronectin and fibrinogen under physiological conditions. Our data suggest that, in contrast with the positive regulation of αIIbβ3 in situ, intracellular controls lower the affinity of αvβ3, and the cytoplasmic domains may act as a target for negative regulators of αvβ3 activity.

Involvement of Shc in insulin- and epidermal growth factor-induced activation of p21ras

1994 ◽  
Vol 14 (3) ◽  
pp. 1575-1581
Author(s):  
G J Pronk ◽  
A M de Vries-Smits ◽  
L Buday ◽  
J Downward ◽  
J A Maassen ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Egf Receptor ◽  
Growth Factor Receptor ◽  
Nucleotide Exchange ◽  
Similar Time ◽  
Epidermal Growth

Shc proteins are phosphorylated on tyrosine residues and associate with growth factor receptor-bound protein 2 (Grb2) upon treatment of cells with epidermal growth factor (EGF) or insulin. We have studied the role of Shc in insulin- and EGF-induced activation of p21ras in NIH 3T3 cells overexpressing human insulin receptors (A14 cells). A14 cells are equally responsive to insulin and EGF with respect to activation of p21ras. Analysis of Shc immunoprecipitates revealed that (i) both insulin and EGF treatment resulted in Shc tyrosine phosphorylation and (ii) Shc antibodies coimmunoprecipitated both Grb2 and mSOS after insulin and EGF treatment. The induction of tyrosine phosphorylation of Shc and the presence of Grb2 and mSOS in Shc immunoprecipitates followed similar time courses, with somewhat higher levels after EGF treatment. In mSOS immunoprecipitates, Shc could be detected as well. Furthermore, Shc immune complexes contained guanine nucleotide exchange activity toward p21ras in vitro. From these results, we conclude that after insulin and EGF treatment, Shc associates with both Grb2 and mSOS and therefore may mediate, at least in part, insulin- and EGF-induced activation of p21ras. In addition, we investigated whether the Grb2-mSOS complex associates with the insulin receptor or with insulin receptor substrate 1 (IRS1). Although we observed association of Grb2 with IRS1, we did not detect complex formation between mSOS and IRS1 in experiments in which the association of mSOS with Shc was readily detectable. Furthermore, whereas EGF treatment resulted in the association of mSOS with the EGF receptor, insulin treatment did not result in the association of mSOS with the insulin receptor. These results indicate that the association of Grb2-nSOS with Shc may be an important event in insulin-induced, mSOS-mediated activation of p21ras.

scholarly journals Effects of Pathological Mutations on the Prion-Like Polymerisation of MyD88

2018 ◽  
Author(s):  
Ailís O’Carroll ◽  
Brieuc Chauvin ◽  
James Brown ◽  
Ava Meagher ◽  
Joanne Coyle ◽  
...  
Keyword(s):  
Single Molecule ◽  
Self Assembly ◽  
Point Mutations ◽  
Adaptor Protein ◽  
Full Length ◽  
Binary Response ◽  
Death Domain ◽  
Tir Domain ◽  
The Impact

AbstractA novel concept has emerged whereby the higher-order self-assembly of proteins provides a simple and robust mechanism for signal amplification. This appears to be a universal signalling mechanism within the innate immune system, where the recognition of pathogens or danger-associated molecular patterns need to trigger a strong, binary response within cells. Previously, multiple structural studies have been limited to single domains, expressed and assembled at high protein concentrations. We therefore set out to develop new in vitro strategies to characterise the behaviour of full-length proteins at physiological levels. In this study we focus on the adaptor protein MyD88, which contains two domains with different self-assembly properties: a TIR domain that can polymerise similarly to the TIR domain of Mal, and a Death Domain that has been shown to oligomerise with helical symmetry in the Myddosome complex. To visualize the behaviour of full-length MyD88 without purification steps, we use single-molecule fluorescence coupled to eukaryotic cell-free protein expression. These experiments demonstrate that at low protein concentration, only full-length MyD88 forms prion-like polymers. We also demonstrate that the metastability of MyD88 polymerisation creates the perfect binary response required in innate signalling: the system is silenced at normal concentrations but upstream signalling creates a “seed” that triggers polymerisation and amplification of the response. These findings pushed us to re-interpret the role of polymerisation in MyD88-related diseases and we studied the impact of disease-associated point mutations L93P, R196C and L252P/L265P at the molecular level. We discovered that all mutations completely block the ability of MyD88 to polymerise. We also confirm that L252P, a gain-of-function mutation, allows the MyD88 mutant to form extremely stable oligomers, even when expressed at low nanomolar concentrations. Thus, our results are consistent with and greatly add to the findings on the Myddosomes digital ‘all-or-none’ responses and the behaviour of the oncogenic mutation of MyD88.

scholarly journals Kinesins relocalize the chromosomal passenger complex to the midzone for spindle disassembly

2018 ◽  
Vol 217 (5) ◽  
pp. 1687-1700 ◽  
Author(s):  
Itziar Ibarlucea-Benitez ◽  
Luke S. Ferro ◽  
David G. Drubin ◽  
Georjana Barnes
Keyword(s):  
Single Molecule ◽  
Molecular Mechanisms ◽  
Chromosomal Passenger Complex ◽  
Late Anaphase ◽  
Chromosomal Passenger ◽  
Spindle Disassembly ◽  
Spindle Midzone ◽  
Chromosome Separation

Mitotic spindle disassembly after chromosome separation is as important as spindle assembly, yet the molecular mechanisms for spindle disassembly are unclear. In this study, we investigated how the chromosomal passenger complex (CPC), which contains the Aurora B kinase Ipl1, swiftly concentrates at the spindle midzone in late anaphase, and we researched the role of this dramatic relocalization during spindle disassembly. We showed that the kinesins Kip1 and Kip3 are essential for CPC relocalization. In cells lacking Kip1 and Kip3, spindle disassembly is severely delayed until after contraction of the cytokinetic ring. Purified Kip1 and Kip3 interact directly with the CPC and recruit it to microtubules in vitro, and single-molecule experiments showed that the CPC diffuses dynamically on microtubules but that diffusion stops when the CPC encounters a Kip1 molecule. We propose that Kip1 and Kip3 trap the CPC at the spindle midzone in late anaphase to ensure timely spindle disassembly.

scholarly journals Domain deletion in the extracellular portion of the EGF-receptor reduces ligand binding and impairs cell surface expression.

1990 ◽  
Vol 1 (2) ◽  
pp. 173-188 ◽  
Author(s):  
I Lax ◽  
F Bellot ◽  
A M Honegger ◽  
A Schmidt ◽  
A Ullrich ◽  
...  
Keyword(s):  
Cell Surface ◽  
Ligand Binding ◽  
Protein Tyrosine Kinase ◽  
Egf Receptor ◽  
Surface Display ◽  
Growth Factor Receptor ◽  
Cell Surface Expression ◽  
Receptor Interaction ◽  
Amino Terminal ◽  
Mutant Receptors

Cultured NIH-3T3 cells were transfected with cDNA constructs encoding human epidermal growth factor-receptor (EGF-R)* and two deletion mutants in the extracellular portion of the receptor molecule. One mutant is devoid of 124 amino-terminal amino acids, and the other lacks 76 residues. Mutant receptors were not delivered to the cell surface unless the transfected cells contained also endogenous EGF-Rs, suggesting that receptor interaction complements the mutation and allows surface display of mutant receptors. Immunoprecipitation experiments revealed an association between mutant and endogenous EGF-Rs when both proteins were expressed in the same cell. Hence, receptor-oligomers may exist in the plane of the membrane even in the absence of ligand binding, and oligomerization may play a role in normal trafficking of EGF-Rs to the cell surface. Mutant receptors retained partial ligand binding activity as 125I-labeled EGF was covalently cross-linked to both mutant receptors, and EGF stimulated, albeit weakly, their protein tyrosine kinase activity. Both mutant EGF-Rs bind EGF with a 10-fold lower affinity than that of the solubilized wild type EGF-R. These results provide further evidence that the region flanked by the two cysteine-rich domains plays a crucial role in defining ligand-binding specificity of EGF-R.

scholarly journals Epidermal growth factor receptor downregulation in cultured bovine cumulus cells: reconstitution of calcium signaling and stimulated membrane permeabilization

Reproduction ◽  
2005 ◽  
Vol 130 (4) ◽  
pp. 517-528 ◽  
Author(s):  
Zhong Zhao ◽  
Damien Garbett ◽  
Julia L Hill ◽  
David J Gross
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Cumulus Cell ◽  
Growth Factor Receptor ◽  
Cumulus Cells ◽  
Membrane Permeabilization ◽  
Egf Receptors ◽  
Epidermal Growth

Cumulus cell–oocyte complexes (COCs), culturedin vitro, are competent for maturation and fertilization. Inclusion of epidermal growth factor (EGF) in the COC culture medium enhancesin vitromaturation and subsequent embryonic development. It has been shown that isolated COCs exposed to EGF respond with a prolonged and pulsatile release of Ca2+into the extra-cellular medium and that cumulus cells (CCs) of complexes exhibit both a slow rise in intracellular [Ca2+] ([Ca2+]i) and plasma membrane permeabilization in response to EGF. These unusual signaling responses were examined in isolated, cultured bovine CCs. Few individual CCs showed [Ca2+]iincreases; the lack of response was found to be due to decrease of expression of endogenous EGF receptors after dissociation. CCs transfected with a human EGF receptor–GFP fusion protein showed robust, prolonged, EGF-stimulated [Ca2+]ielevations characteristic of CC responses in intact COCs. Many CCs that responded to EGF stimulation with a [Ca2+]irise also released entrapped fura-2 dye at the peak of the [Ca2+]iresponse, suggesting that CC permeabilization and death follows activation of the EGF receptor. The [Ca2+]ielevation due to EGF stimulation and subsequent membrane permeabilization was shown to be mediated by the inositol triphosphate signaling pathway.

scholarly journals How IGF-1 activates its receptor

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Jennifer M Kavran ◽  
Jacqueline M McCabe ◽  
Patrick O Byrne ◽  
Mary Katherine Connacher ◽  
Zhihong Wang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Growth Factor ◽  
Ligand Binding ◽  
Insulin Receptor ◽  
Conformational Change ◽  
Growth Factor Receptor ◽  
Full Length ◽  
Type I ◽  
Insulin Like Growth Factor ◽  
Growth And Survival

The type I insulin-like growth factor receptor (IGF1R) is involved in growth and survival of normal and neoplastic cells. A ligand-dependent conformational change is thought to regulate IGF1R activity, but the nature of this change is unclear. We point out an underappreciated dimer in the crystal structure of the related Insulin Receptor (IR) with Insulin bound that allows direct comparison with unliganded IR and suggests a mechanism by which ligand regulates IR/IGF1R activity. We test this mechanism in a series of biochemical and biophysical assays and find the IGF1R ectodomain maintains an autoinhibited state in which the TMs are held apart. Ligand binding releases this constraint, allowing TM association and unleashing an intrinsic propensity of the intracellular regions to autophosphorylate. Enzymatic studies of full-length and kinase-containing fragments show phosphorylated IGF1R is fully active independent of ligand and the extracellular-TM regions. The key step triggered by ligand binding is thus autophosphorylation.

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