Cbl and Cbl-b independently regulate EGFR through distinct receptor interaction modes.

Highly homologous E3 ubiquitin ligases, Cbl and Cbl-b, mediate ubiquitination of EGF receptor (EGFR), leading to its endocytosis and lysosomal degradation. Cbl and Cbl-b, are thought to function in a redundant manner by binding directly to phosphorylated Y1045 (pY1045) of EGFR and indirectly via the Grb2 adaptor. Unexpectedly, we found that inducible expression of Cbl or Cbl-b mutants lacking the E3 ligase activity but fully capable of EGFR binding does not significantly affect EGFR ubiquitination and endocytosis in human oral squamous cell carcinoma (HSC3) cells which endogenously express Cbl-b at a relatively high level. Each endogenous Cbl species remained associated with ligand-activated EGFR in the presence of an overexpressed counterpart species or its mutant, although Cbl-b overexpression partially decreased Cbl association with EGFR. Binding to pY1045 was the preferential mode for Cbl-b:EGFR interaction, whereas Cbl relied mainly on the Grb2-dependent mechanism. Overexpression of the E3-dead mutant of Cbl-b slowed down EGF-induced degradation of active EGFR, while this mutant and a similar mutant of Cbl did not significantly affect MAPK/ERK1/2 activity. EGF-guided chemotaxis migration of HSC3 cells was diminished by overexpression of the E3-dead Cbl-b mutant but was not significantly affected by the E3-dead Cbl mutant. By contrast, the inhibitory effect of the same Cbl mutant on the migration of OSC-19 cells expressing low Cbl-b levels was substantially stronger than that of the Cbl-b mutant. Altogether, our data demonstrate that Cbl and Cbl-b may operate independently through different modes of EGFR binding to jointly control receptor ubiquitination, endocytic trafficking, and signaling.

Internalization of trophoblastic small extracellular vesicles and detection of their miRNA cargo in P-bodies.

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.

EGF receptor signaling, phosphorylation, ubiquitylation and endocytosis in tumors in vivo.

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.

Endocytosis separates EGF receptors from endogenous fluorescently labeled HRas and diminishes receptor signaling to MAP kinases in endosomes.

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.

Methods to study endocytic trafficking of the EGF receptor.

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.

Fluorescent nucleoside derivatives as a tool for the detection of concentrative nucleoside transporter activity using confocal microscopy and flow cytometry.

The abundance and function of transporter proteins at the plasma membrane are likely to be crucial in drug responsiveness. Functional detection of human concentrative nucleoside transporters (hCNTs) is of interest for predicting drug sensitivity because of their ability to transport most nucleoside-derived drugs. In the present study, two fluorescent nucleoside analogues, uridine-furan and etheno-cytidine, were evaluated as tools to study in vivo nucleoside transporter-related functions. These two molecules showed high affinity interactions with hCNT1 and hCNT3 and were shown to be substrates of both transporters. Both fluorescence microscopy and flow cytometry experiments showed that uridine-furan uptake was better suited for distinguishing cells that express hCNT1 or hCNT3. These data highlight the usefulness of fluorescent nucleoside derivatives, as long as they fulfill the requirements of confocal microscopy and flow cytometry, for in vivo analysis of hCNT-related function.

rCNT2 extracellular cysteines, Cys(615) and Cys(649), are important for maturation and sorting to the plasma membrane.

rCNT2 is a purine-preferring concentrative nucleoside transporter implicated in the regulation of extracellular adenosine levels and purinergic signaling. This study addressed the analysis of the CNT2 C-terminus tail as a domain likely to be implicated in transporter sorting. The topological mapping of this segment revealed that Cys(615) and Cys(649) are important residues for the proper trafficking of CNT2 to the plasma membrane. The inhibition of protein disulfide isomerase (PDI) and ER glycosidase I and II impaired rCNT2 trafficking to the cell surface, similarly to Cys(615) and Cys(649) mutants. The present work suggests these two cysteine residues are relevant for the proper sorting of the transporter and its functional performance.

Ribavirin uptake into human hepatocyte HHL5 cells is enhanced by interferon-α via up-regulation of the human concentrative nucleoside transporter (hCNT2).

Ribavirin is a broad spectrum antiviral that increases the response rate in chronic hepatitis C patients when administered in combination with IFNα. Ribavirin is a purine nucleoside derivative, transported into hepatocytes by nucleoside transporters. hCNT2 is the best candidate to mediate ribavirin uptake into hepatocytes due to its high-affinity for purines and its capacity to concentrate its substrates intracellularly. The aim of this study was to determine whether hCNT2 function is under IFNα modulation. IFNα treatment of the nontransformed human hepatocyte-derived cell line HHL5 induced a rapid and transient increase in hCNT2 activity after cytokine addition. hCNT2 activity up-regulation was associated with increased ribavirin accumulation into cells. This increase was consistent with the translocation of hCNT2-containing vesicles to the plasma membrane via a mechanism requiring ERK 1/2 and ROCK activation and cytoskeleton integrity. Longer treatments with IFNα induced transcriptional activation of the hCNT2-encoding gene (SLC28A2), resulting in a sustained increase in hCNT2-related activity. These observations are proof of concept for at least one of the putative mechanisms underlying the synergistic responses induced by combination therapy with IFNα and ribavirin.

Role of the transporter regulator protein (RS1) in the modulation of concentrative nucleoside transporters (CNTs) in epithelia.

SLC28 genes encode three plasma membrane transporter proteins, human concentrative nucleoside transporter (CNT)1, CNT2, and CNT3, all of which are implicated in the uptake of natural nucleosides and a variety of nucleoside analogs used in the chemotherapy of cancer and viral and inflammatory diseases. Mechanisms determining their trafficking toward the plasma membrane are not well known, although this might eventually become a target for therapeutic intervention. The transporter regulator RS1, which was initially identified as a short-term, post-transcriptional regulator of the high-affinity, Na(+)-coupled, glucose transporter sodium-dependent glucose cotransporter 1, was evaluated in this study as a candidate for coordinate regulation of membrane insertion of human CNT-type proteins. With a combination of studies with mammalian cells, Xenopus laevis oocytes, and RS1-null mice, evidence that RS1 down-regulates the localization and activity at the plasma membrane of the three members of this protein family (CNT1, CNT2, and CNT3) is provided, which indicates the biochemical basis for coordinate regulation of nucleoside uptake ability in epithelia and probably in other RS1-expressing cell types.

New role of the human equilibrative nucleoside transporter 1 (hENT1) in epithelial-to-mesenchymal transition in renal tubular cells.

Epithelial-to-mesenchymal transition (EMT) is an important pro-fibrotic event in which tubular epithelial cells are transformed into myofibroblasts. Nucleoside transporters (NT) are regulated by many factors and processes, some of which are involved in fibrosis, such as cytokines, inflammation, and proliferation. Equilibrative nucleoside transporter 1 (ENT1) has been proved to be the most widely expressed adenosine transporter. In that sense, ENT1 may be a key player in cell damage signaling. Here we analyze the role of human ENT1 (hENT1) in the EMT process in proximal tubular cells. Addition of the main inducer of EMT, the transforming growth factor-ß1, to HK-2 cells increased hENT1 mRNA and protein level expression. ENT1-mediated adenosine uptake was also enhanced. When cells were incubated with dipyridamole to evaluate the potential contribution of ENT1 to EMT by blocking its transport activity, EMT was induced. Moreover, the knock down of hENT1 with siRNA induced EMT and collagen production in HK-2 cells. Kidneys isolated from ENT1 knockout mice showed higher levels of interstitial collagen and α-SMA positive cells than wild-type mice. Our results point to a new potential role of hENT1 as a modulator of EMT in proximal tubular cells. In this sense, hENT1 could be involved in renal protection processes, and the loss or reduced expression of hENT1 would lead to an increased vulnerability of cells to the onset and/or progression of renal fibrosis.

Structural determinants for rCNT2 sorting to the plasma membrane of polarized and non-polarized cells.

rCNT2 (rat concentrative nucleoside transporter 2) (Slc28a2) is a purine-preferring concentrative nucleoside transporter. It is expressed in both non-polarized and polarized cells, where it is localized in the brush border membrane. Since no information about the domains implicated in the plasma membrane sorting of rCNT2 is available, the present study aimed to identify structural and functional requirements for rCNT2 trafficking. The comprehensive topological mapping of the intracellular N-terminal tail revealed two main features: (i) a glutamate-enriched region (NPGLELME) between residues 21 and 28 that seems to be implicated in the stabilization of rCNT2 in the cell surface, since mutagenesis of these conserved glutamates resulted in enhanced endocytosis; and (ii) mutation of a potential protein kinase CK2 domain that led to a loss of brush border-specific sorting. Although the shortest proteins assayed (rCNT2-74AA, -48AA and -37AA) accumulated intracellularly and lost their brush border membrane preference, they were still functional. A deeper analysis of CK2 implication in CNT2 trafficking, using a CK2-specific inhibitor [DMAT (2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole)] and other complementary mutations mimicking the negative charge provided by phosphorylation (S46D and S46E), demonstrated an effect of this kinase on rCNT2 activity. In summary, the N-terminal tail of rCNT2 contains dual sorting signals. An acidic region is responsible for its proper stabilization at the plasma membrane, whereas the putative CK2 domain (Ser(46)) is implicated in the apical sorting of the transporter.

Link between high-affinity adenosine concentrative nucleoside transporter-2 (CNT2) and energy metabolism in intestinal and liver parenchymal cells.

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.