Dataset Information

A new temperature-dependent strategy to modulate the epidermal growth factor receptor.

ABSTRACT: The dynamic manipulation of kinases remains a major obstacle to unraveling cell-signaling networks responsible for the activation of biological systems. For example, epidermal growth factor (EGF) stimulates the epidermal growth factor receptor (EGFR/ErbB1); however, EGF also recruits other kinases (HER2/ErbB2) involved with various signaling pathways. To better study EGFR we report a new strategy to selectively activate receptor tyrosine kinases fused to elastin-like polypeptides (ELPs), which can be visualized inside mammalian cells using fixed and live-cell fluorescence microscopy. ELPs are high molecular weight polypeptides that phase separate abruptly upon heating. When an EGFR-ELP fusion is heated, it clusters, initiates receptor internalization, phosphorylates, initiates downstream kinase signaling, and undergoes retrograde transport towards the cell body. Unlike other strategies to block EGFR (small molecule inhibitors, RNAi, or transcriptional regulators), EGFR-ELP clustering can be specifically switched on or off within minutes. Live-cell imaging suggests that EGFR-ELPs assemble in most cells with only a 3?°C increase in temperature. This strategy was found reversible and able to dynamically control the downstream phosphorylation/activation of the ERK1/2 pathway. For the first time, this strategy enables the rational engineering of specific temperature-sensitive receptors that may have broad applications in the study and manipulation of biological processes.

SUBMITTER: Li Z

PROVIDER: S-EPMC6338443 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

A new temperature-dependent strategy to modulate the epidermal growth factor receptor.

Li Zhe Z Tyrpak David R DR Park Mincheol M Okamoto Curtis T CT MacKay J Andrew JA

Biomaterials 20180811

The dynamic manipulation of kinases remains a major obstacle to unraveling cell-signaling networks responsible for the activation of biological systems. For example, epidermal growth factor (EGF) stimulates the epidermal growth factor receptor (EGFR/ErbB1); however, EGF also recruits other kinases (HER2/ErbB2) involved with various signaling pathways. To better study EGFR we report a new strategy to selectively activate receptor tyrosine kinases fused to elastin-like polypeptides (ELPs), which c ...[more]

PMID: 30196151

Similar Datasets

Project description:AimsEpidermal growth factor receptor (EGFR) is essential to the development of multiple tissues and organs and is a target of cancer therapeutics. Due to the embryonic lethality of global EGFR deletion and conflicting reports of cardiac-overexpressed EGFR mutants, its specific impact on the adult heart, normally or in response to chronic stress, has not been established. Using complimentary genetic strategies to modulate cardiomyocyte-specific EGFR expression, we aim to define its role in the regulation of cardiac function and remodelling.Methods and resultsA floxed EGFR mouse model with α-myosin heavy chain-Cre-mediated cardiomyocyte-specific EGFR downregulation (CM-EGFR-KD mice) developed contractile dysfunction by 9 weeks of age, marked by impaired diastolic relaxation, as monitored via echocardiographic, haemodynamic, and isolated cardiomyocyte contractility analyses. This contractile defect was maintained over time without overt cardiac remodelling until 10 months of age, after which the mice ultimately developed severe heart failure and reduced lifespan. Acute downregulation of EGFR in adult floxed EGFR mice with adeno-associated virus 9 (AAV9)-encoded Cre with a cardiac troponin T promoter (AAV9-cTnT-Cre) recapitulated the CM-EGFR-KD phenotype, while AAV9-cTnT-EGFR treatment of adult CM-EGFR-KD mice rescued the phenotype. Notably, chronic administration of the β-adrenergic receptor agonist isoproterenol effectively and reversibly compensated for the contractile dysfunction in the absence of cardiomyocyte hypertrophy in CM-EGFR-KD mice. Mechanistically, EGFR downregulation reduced the expression of protein phosphatase 2A regulatory subunit Ppp2r3a/PR72, which was associated with decreased phosphorylation of phospholamban and Ca2+ clearance, and whose re-expression via AAV9-cTnT-PR72 rescued the CM-EGFR-KD phenotype.ConclusionsAltogether, our study highlights a previously unrecognized role for EGFR in maintaining contractile homeostasis under physiologic conditions in the adult heart via regulation of PR72 expression.

Project description:Oesophageal squamous cell carcinomas and oesophageal adenocarcinomas show distinct patterns of ErbB expression and dimers. The functional effects of specific ErbB homodimers or heterodimers on oesophageal (cancer) cell behaviour, particularly invasion during early carcinogenesis, remain unknown. Here, a new cellular model system for controlled activation of epidermal growth factor receptor (EGFR) or human epidermal growth factor receptor 2 (HER2) and EGFR-HER2 or HER2-human epidermal growth factor receptor 3 (HER3) homodimers and heterodimers was studied in non-neoplastic squamous oesophageal epithelial Het-1A cells. EGFR, HER2 and HER3 intracellular domains (ICDs) were fused to dimerization domains (DmrA/DmrA and DmrC), and transduced into Het-1A cells lacking ErbB expression. Dimerization of EGFR, HER2 or EGFR-HER2 and HER2-HER3 ICDs was induced by synthetic ligands (A/A or A/C dimerizers). This was accompanied by phosphorylation of the respective EGFR, HER2 and HER3 ICDs and activation of distinct downstream signalling pathways, such as phospholipase C?1, Akt, STAT and Src family kinases. Phenotypically, ErbB dimers caused cell rounding and non-apoptotic blebbing, specifically in EGFR-HER2 and HER2-HER3 heterodimer cells. In a Transwell assay, cell migration velocity was elevated in HER2 dimer cells as compared with empty vector cells. In addition, HER2 dimer cells showed in increased cell invasion, reaching significance for induced HER2-HER3 heterodimers (P?=?0.015). Importantly, in three-dimensional organotypic cultures, empty vector cells grew as a superficial cell layer, resembling oesophageal squamous epithelium. In contrast, induced HER2 homodimer cells were highly invasive into the matrix and formed cell clusters. This was associated with partial loss of cytokeratin 7 (when HER2 homodimers were modelled) and p63 (when EGFR-HER2 heterodimers were modelled), which suggests a change or loss of squamous cell differentiation. Controlled activation of specific EGFR, HER2 and HER3 homodimers and heterodimers caused oesophageal squamous epithelial cell migration and/or invasion, especially in a three-dimensional microenvironment, thereby functionally identifying ErbB homodimers and heterodimers as important drivers of oesophageal carcinogenesis. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Dataset Information

A new temperature-dependent strategy to modulate the epidermal growth factor receptor.

Publications

A new temperature-dependent strategy to modulate the epidermal growth factor receptor.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets