Despite a well-established role for the epidermal growth factor receptor (EGFR) in tumorigenesis, EGFR activities and endocytosis in tumors in vivo have not been studied. We labeled endogenous EGFR with GFP by genome-editing of human oral squamous cell carcinoma cells, which were used to examine EGFR-GFP behavior in mouse tumor xenografts in vivo. Intravital multiphoton imaging, confocal imaging of cryosections and biochemical analysis revealed that localization and trafficking patterns, as well as levels of phosphorylation and ubiquitylation of EGFR in tumors in vivo closely resemble patterns and levels observed in the same cells treated with 20–200 pM EGF in vitro. Consistent with the prediction of low ligand concentrations in tumors, EGFR endocytosis was kinase-dependent and blocked by inhibitors of clathrin-mediated internalization; and EGFR activity was insensitive to Cbl overexpression. Collectively, our data suggest that a small pool of active EGFRs is sufficient to drive tumorigenesis by signaling primarily through the Ras-MAPK pathway.
Signaling from epidermal growth factor receptor (EGFR) to extracellular-stimuli–regulated protein kinase 1/2 (ERK1/2) is proposed to be transduced not only from the cell surface but also from endosomes, although the role of endocytosis in this signaling pathway is controversial. Ras is the only membrane-anchored component in the EGFR–ERK signaling axis, and therefore, its location determines intracellular sites of downstream signaling. Hence, we labeled endogenous H-Ras (HRas) with mVenus fluorescent protein using gene editing in HeLa cells. mVenus-HRas was primarily located at the plasma membrane, and in small amounts in tubular recycling endosomes and associated vesicles. EGF stimulation resulted in fast but transient activation of mVenus-HRas. Although EGF:EGFR complexes were rapidly accumulated in endosomes together with the Grb2 adaptor, very little, if any, mVenus-HRas was detected in these endosomes. Interestingly, the activities of MEK1/2 and ERK1/2 remained high beyond the point of the physical separation of HRas from EGF:EGFR complexes and down-regulation of Ras activity. Paradoxically, this sustained MEK1/2 and ERK1/2 activation was dependent on the active EGFR kinase. Cell surface biotinylation and selective inactivation of surface EGFRs suggested that a small fraction of active EGFRs remaining in the plasma membrane is responsible for continuous signaling to MEK1/2 and ERK1/2. We propose that, under physiological conditions of cell stimulation, EGFR endocytosis serves to spatially separate EGFR–Grb2 complexes and Ras, thus terminating Ras-mediated signaling. However, sustained minimal activation of Ras by a small pool of active EGFRs in the plasma membrane is sufficient for extending MEK1/2 and ERK1/2 activities.
Pregnancy is a unique situation, in which placenta-derived small extracellular vesicles (sEVs) may communicate with maternal and foetal tissues. While relevant to homoeostatic and pathological functions, the mechanisms underlying sEV entry and cargo handling in target cells remain largely unknown. Using fluorescently or luminescently labelled sEVs, derived from primary human placental trophoblasts or from a placental cell line, we interrogated the endocytic pathways used by these sEVs to enter relevant target cells, including the neighbouring primary placental fibroblasts and human uterine microvascular endothelial cells. We found that trophoblastic sEVs can enter target cells, where they retain biological activity. Importantly, using a broad series of pharmacological inhibitors and siRNA-dependent silencing approaches, we showed that trophoblastic sEVs enter target cells using macropinocytosis and clathrin-mediated endocytosis pathways, but not caveolin-dependent endocytosis. Tracking their intracellular course, we localized the sEVs to early endosomes, late endosomes, and lysosomes. Finally, we used coimmunoprecipitation to demonstrate the association of the sEV microRNA (miRNA) with the P-body proteins AGO2 and GW182. Together, our data systematically detail endocytic pathways used by placental sEVs to enter relevant fibroblastic and endothelial target cells, and provide support for "endocytic escape" of sEV miRNA to P-bodies, a key site for cytoplasmic RNA regulation.
Endocytosis and postendocytic sorting of epidermal growth factor (EGF) receptor (EGFR) are the major regulators of EGFR signaling. EGFR endocytosis and ubiquitin-dependent lysosomal targeting are also considered to be the prototypic experimental system for studying the molecular mechanisms of stimulus-induced and constitutive endocytic trafficking. Therefore, elucidation of the mechanisms of EGFR endocytosis and its regulation of the signaling network is essential not only for better understanding of the EGFR biology but also for defining general regulatory principles in the endocytosis system. Comprehensive analysis of these mechanisms requires quantitative and physiologically relevant methodological approaches for measuring the rates of EGFR internalization, degradation, and recycling. Basic experimental protocols described in this chapter cover a combination of single-cell microscopy and biochemical methods that are used to follow EGF-induced endocytosis of EGFR in real time, measure the kinetic rate parameters of EGFR internalization and recycling, and analyze EGF-dependent ubiquitination and degradation of EGFR.
Concentrative nucleoside transporter 2 (CNT2) is a high-affinity adenosine transporter that may play physiological roles beyond nucleoside salvage. Previous reports relate CNT2 function to modulation of purinergic signaling and energy metabolism in intestinal and liver parenchymal cells (Duflot et al., 2004, Mol Cell Biol 24:2710-2719; Aymerich et al., 2006, J Cell Sci 119:1612-1621). In the present study, to further examine the link between CNT2 and energy metabolism, CNT2 protein partners were identified using the bacterial two-hybrid and GST pull-down approaches. The N-terminal segment of CNT2 was used as bait, since proteins lacking this domain display impaired plasma membrane insertion and intracellular retention. Glucose-regulated protein 58 (GRP58) was identified as a potential rCNT2 partner in pull-down experiments. Two-hybrid screening performed against a liver human cDNA library led to the identification of aldolase B as another hCNT2 partner. Aldolase B-RFP and endogenous GRP58 separately co-localized with CNT2 in HeLa cells transfected with YFPrCNT2. CNT2 interaction with GRP58 was validated using co-immunoprecipitation experiments. In HeLa cells, fluorescence resonance energy transfer (FRET) efficiency increased upon fructose addition, consistent with a transient interaction between aldolase B and the transporter. The physiological basis for in vivo interactions was derived from experiments in which GRP58 was inhibited or overexpressed and aldolase B activity stimulated towards glycolysis. GRP58 appeared to be a negative effector of CNT2 function, whereas aldolase B flux modulated CNT2 activity via a mechanism involving acquisition of higher affinity for its substrates. These findings support the theory that CNT2 plays roles other than salvage and establishes links with energy metabolism.
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