Prototypical Receptor Tyrosine Kinase Research Articles

EGF, TGF-α and Amphiregulin Differently Regulate Endometrium-Derived Mesenchymal Stromal/Stem Cells.

The prototypical receptor tyrosine kinase epidermal growth factor receptor (EGFR) is regulated by a set of its ligands, which determines the specificity of signaling and intracellular fate of the receptor. The EGFR signaling system is well characterized in immortalized cell lines such as HeLa derived from tumor tissues, but much less is known about EGFR function in untransformed multipotent stromal/stem cells (MSCs). We compared the effect of epidermal growth factor (EGF), transforming growth factor-α (TGF-α) and amphiregulin (AREG) on physiological responses in endometrial MSCs (enMSC) and HeLa cells. In addition, using Western blotting and confocal microscopy, we studied the internalization and degradation of EGFR stimulated by the three ligands in these cell lines. We demonstrated that unlike HeLa, EGF and TGF-α, but not AREG, stimulated enMSC proliferation and prevented decidual differentiation in an EGFR-dependent manner. In HeLa cells, EGF targeted EGFR for degradation, while TGF-α stimulated its recycling. Surprisingly, in enMSC, both ligands caused EGFR degradation. In both cell lines, AREG-EGFR internalization was not registered. In HeLa cells, EGFR was degraded within 2 h, restoring its level in 24 h, while in enMSC, degradation took more than 4-8 h, and the low EGFR level persisted for several days. This indicates that EGFR homeostasis in MSCs may differ significantly from that in immortalized cell lines.

EGFR Mutations in Head and Neck Squamous Cell Carcinoma.

EGFR is a prototypical receptor tyrosine kinase that is overexpressed in multiple cancers including head and neck squamous cell carcinoma (HNSCC). The standard of care for HNSCC remains largely unchanged despite decades of research. While EGFR blockade is an attractive target in HNSCC patients and anti-EGFR strategies including monoclonal antibodies and kinase inhibitors have shown some clinical benefit, efficacy is often due to the eventual development of resistance. In this review, we discuss how the acquisition of mutations in various domains of the EGFR gene not only alter drug binding dynamics giving rise to resistance, but also how mutations can impact radiation response and overall survival in HNSCC patients. A better understanding of the EGFR mutational landscape and its dynamic effects on treatment resistance hold the potential to better stratify patients for targeted therapies in order to maximize therapeutic benefits.

EGFR Membrane Dynamics and Organization Investigated by Camera-Based Multi-Parameter Fluorescence Imaging with High Spatiotemporal Resolution

The epidermal growth factor receptor is a prototypical receptor tyrosine kinase that has been investigated for four decades. However, the exact oligomerization state of the receptor in the cellular plasma membrane, its dynamics, and its relation to membrane domains and cytoskeletal structures is still under investigation. One of the hurdles is that the optimization of fluorescence methods to investigate structure, dynamics and oligomerization require different optimization strategies and thus their measurements have been performed in mutually exclusive ways. Here we overcome this problem by recording data in a total internal reflection microscope (TIRFM) at 1000 frames per second with optical resolution. We then use computational approaches to achieve super-resolution images (&lt100 nm) and extract quantitative parameters on fluorophore dynamics and oligomerization. For this purpose we combine imaging fluorescence correlation spectroscopy (imaging FCS) with super-resolution radial fluctuation (SRRF) analysis and number and brightness (N&B) analysis to extract spatial and temporal super-resolution data from the exact same sample simultaneously. Measurements were conducted on CHO cells that were co-transfected with a filamentous actin marker (LifeAct GFP) and mApple labeled epidermal growth factor receptor (EGFR-mApple). We achieve a spatial resolution of about 60 nm on actin filaments using SRRF, demonstrate diffusion with transient trapping for EGFR, and show that the majority of EGFR are dimerized in the absence of ligand. Interestingly, since the imaging FCS, N&B, and SRRF data were obtained simultaneously, we can show that the diffusion coefficient and the oligomerization state of EGFR are negatively correlated and that the diffusion coefficient shows no correlation with cytoskeletal actin structures.

Structure and Dynamics of the EGF Receptor as Revealed by Experiments and Simulations and Its Relevance to Non-Small Cell Lung Cancer.

The epidermal growth factor receptor (EGFR) is historically the prototypical receptor tyrosine kinase, being the first cloned and the first where the importance of ligand-induced dimer activation was ascertained. However, many years of structure determination has shown that EGFR is not completely understood. One challenge is that the many structure fragments stored at the PDB only provide a partial view because full-length proteins are flexible entities and dynamics play a key role in their functionality. Another challenge is the shortage of high-resolution data on functionally important higher-order complexes. Still, the interest in the structure/function relationships of EGFR remains unabated because of the crucial role played by oncogenic EGFR mutants in driving non-small cell lung cancer (NSCLC). Despite targeted therapies against EGFR setting a milestone in the treatment of this disease, ubiquitous drug resistance inevitably emerges after one year or so of treatment. The magnitude of the challenge has inspired novel strategies. Among these, the combination of multi-disciplinary experiments and molecular dynamic (MD) simulations have been pivotal in revealing the basic nature of EGFR monomers, dimers and multimers, and the structure-function relationships that underpin the mechanisms by which EGFR dysregulation contributes to the onset of NSCLC and resistance to treatment.

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

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.

The Dynamics and Localization of the Epidermal Growth Factor Receptor (EGFR) on Live Cell Plasma Membrane Studied by ITIR-FCS

The Epidermal Growth Factor Receptor (EGFR), a prototypical receptor tyrosine kinase, plays an important role in cellular processes such as proliferation, migration and apoptosis. EGFR is known to be strongly regulated by its localization and the state of activation on the plasma membrane. However, the spatial distribution of EGFR on the plasma membrane has not been resolved in detail.Here, we use Imaging Total Internal Reflection Fluorescence Correlation Spectroscopy (ITIR-FCS) to investigate EGFR behavior on live cells before and after stimulation. ITIR-FCS allows monitoring EGFR dynamics on cell membranes and in combination with the FCS diffusion laws provides information on the nanoscopic EGFR organization.Our results indicate that EGFR is partly trapped in cholesterol sensitive domains and also couples to the cytoskeleton. Stimulation by EGF activates EGFR in a dose dependent manner. At low-dose stimulation (10 ng/ml), the membrane diffusion and confinement of EGFR remain unchanged and EGFR internalization is cholesterol independent and EGFR recycles back to the membrane. At high-dose EGF treatment (100 ng/ml), EGFR endocytosis is cholesterol dependent and cholesterol-depletion impairs receptor internalization. In addition, cholesterol-depletion with concomitant EGF stimulation leads to stronger confinement of the receptor, independent of the EGF dose, as clusters accumulate on the membrane. Furthermore, the actin cytoskeleton promotes EGFR internalization after ligand stimulation. Disruption of the actin cytoskeleton inhibits EGFR endocytosis and induces clustering of EGFR on the cell membrane. This study demonstrates the capabilities of ITIR-FCS to follow receptor stimulation events on live cells. It demonstrates not only that EGFR activation is cholesterol and cytoskeleton dependent but also that the activation pathway is EGF dose dependent, thus providing new insights into receptor dynamics and organization on live cells.

Homogeneous time-resolved fluorescence-based assay to monitor extracellular signal-regulated kinase signaling in a high-throughput format.

The extracellular signal-regulated kinases (ERKs) are key components of multiple important cell signaling pathways regulating diverse biological responses. This signaling is characterized by phosphorylation cascades leading to ERK1/2 activation and promoted by various cell surface receptors including G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). We report the development of a new cell-based Phospho-ERK1/2 assay (designated Phospho-ERK), which is a sandwich proximity-based assay using the homogeneous time-resolved fluorescence technology. We have validated the assay on endogenously expressed ERK1/2 activated by the epidermal growth factor as a prototypical RTK, as well as various GPCRs belonging to different classes and coupling to different heterotrimeric G proteins. The assay was successfully miniaturized in 384-well plates using various cell lines endogenously, transiently, or stably expressing the different receptors. The validation was performed for agonists, antagonists, and inhibitors in dose–response as well as kinetic analysis, and the signaling and pharmacological properties of the different receptors were reproduced. Furthermore, the determination of a Z′-factor value of 0.7 indicates the potential of the Phospho-ERK assay for high-throughput screening of compounds that may modulate ERK1/2 signaling. Finally, our study is of great interest in the current context of investigating ERK1/2 signaling with respect to the emerging concepts of biased ligands, G protein-dependent/independent ERK1/2 activation, and functional transactivation between GPCRs and RTKs, illustrating the importance of considering the ERK1/2 pathway in cell signaling

The Insulin Receptor: Both a Prototypical and Atypical Receptor Tyrosine Kinase

Unlike prototypical receptor tyrosine kinases (RTKs), which are single-chain polypeptides, the insulin receptor (InsR) is a preformed, covalently linked tetramer with two extracellular α subunits and two membrane-spanning, tyrosine kinase-containing β subunits. A single molecule of insulin binds asymmetrically to the ectodomain, triggering a conformational change that is transmitted to the cytoplasmic kinase domains, which facilitates their trans-phosphorylation. As in prototypical RTKs, tyrosine phosphorylation in the juxtamembrane region of InsR creates recruitment sites for downstream signaling proteins (IRS [InsR substrate] proteins, Shc) containing a phosphotyrosine-binding (PTB) domain, and tyrosine phosphorylation in the kinase activation loop stimulates InsR's catalytic activity. For InsR, phosphorylation of the activation loop, which contains three tyrosine residues, also creates docking sites for adaptor proteins (Grb10/14, SH2B2) that possess specialized Src homology-2 (SH2) domains, which are dimeric and engage two phosphotyrosines in the activation loop.

Molecular Mechanisms that Regulate Epidermal Growth Factor Receptor Inactivation

The Epidermal Growth Factor Receptor (EGFR) is the prototypical receptor tyrosine kinase (RTK). These cell surface receptors are integral membrane proteins that bind ligands on their extracellular domain and relay that information to within the cell. The activated EGFR regulates diverse cell fates such as growth, proliferation, differentiation, migration, and apoptosis. These signaling properties are important for the appropriate development and maintenance of an organism. However, when inappropriately controlled, due to EGFR overexpression or hyperactivation, these signaling events are characteristic of many cancers. It remains unclear whether the uncontrolled EGFR activity leads to cell transformation or is a consequence of cell transformation. Regardless of the cause, increased EGFR activity serves both as a biomarker in the diagnosis of some cancers and is a molecular target for anti-cancer therapies. The promising results with current anti-EGFR therapies suggest that the receptor is a viable molecular target for a limited number of applications. However, to become an effective therapeutic target for other cancers that have elevated levels of EGFR activity, current approaches for inhibiting EGFR signaling will need to be refined. Here we describe the molecular mechanisms that regulate EGFR inactivation and discuss their potential as therapeutic targets for inhibiting EGFR signaling.

Phosphorylation of p68 RNA Helicase Plays a Role in Platelet-derived Growth Factor-induced Cell Proliferation by Up-regulating Cyclin D1 and c-Myc Expression

p68 RNA helicase is a protypical member of DEAD box family RNA helicase. The protein plays an important role in the cell developmental program and organ maturation. We demonstrated previously that, in response to growth factor platelet-derived growth factor (PDGF)-BB stimulation, p68 is phosphorylated at Tyr(593), and the phosphorylation of p68 promotes epithelial-mesenchymal transition via promoting beta-catenin nuclear translocation (Yang, L., Lin, C., and Liu, Z. R. (2006) Cell 127, 139-155). We show here that the tyrosine phosphorylation of p68 also mediates the effects of PDGF in stimulating cell proliferation. The phosphorylated p68 (referred to as phospho-p68) promotes cell proliferation by activating the transcription of cyclin D1 and c-Myc genes. We show that the ATPase/helicase activities of p68 are required for the activation of cyclin D1 transcription. The phospho-p68 participates in the complex assembled at the cyclin D1 and c-Myc promoters, which strongly suggests a direct role in transcriptional regulation. Furthermore, our data demonstrated that the phosphorylation of p68 at Tyr(593) plays a role in mediating the autocrine loop effects of PDGF, suggesting an important role for p68 phosphorylation in cell proliferation.