Signaling from endosomes: Location makes a difference

Sadowski, Lukasz; Pilecka, Iwona; Miączyńska, Marta

doi:10.1016/j.yexcr.2008.09.021

Cited by 169 publications

(160 citation statements)

References 93 publications

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“…Although active signaling from within endosomes has been reported for a selected group of membrane receptors (reviewed in refs. [23][24][25], here we directly demonstrate that internalized CD3ζ is signaling competent. The important, albeit nontrivial, questions that remain to be answered are whether distinct TcR/CD3-dependent signaling pathways can be traced specifically to the plasma-membrane and intracellular pools of CD3ζ and how these pathways depend on TcR/CD3 trafficking within the cell.…”

Section: Discussionsupporting

confidence: 55%

Imaging T-cell receptor activation reveals accumulation of tyrosine-phosphorylated CD3ζ in the endosomal compartment

Yudushkin

Vale²

2010

Proc. Natl. Acad. Sci. U.S.A.

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Phosphorylation of the T-cell receptor complex (TcR/CD3) mediates the survival and antigen-induced activation of T cells. TcR/CD3 phosphorylation is usually monitored using phospho-specific antibodies, which precludes dynamic measurements. Here, we have developed genetically encoded, live-cell reporters that enable simultaneous monitoring of the phosphorylation state and intracellular trafficking of CD3ζ, the major signal-transducing subunit of the TcR/CD3. We show that these reporters provide accurate readouts of TcR/CD3 phosphorylation and are sensitive to the local balance of kinase and phosphatase activities acting upon TcR/CD3. Using these reporters, we demonstrate that, in addition to the expected activation-dependent phosphorylation at the plasma membrane, tyrosine-phosphorylated CD3ζ accumulates on endosomal vesicles distinct from lysosomes. These results suggest that an intracellular pool of phosphorylated CD3ζ may help to sustain TcR/ CD3 signaling after the receptor internalization. Recent studies using high-resolution imaging have demonstrated that signaling via TcR/CD3 displays complex spatial organization. TcR/CD3 molecules are preclustered at the plasma membrane and, upon ligation, nucleate downstream signaling components seconds after T-cell activation (4, 5). TcR/CD3 is constitutively internalized and recycled to the plasma membrane in naïve and activated T cells (reviewed in ref. 6), but the function of this turnover as well as the phosphorylation state of internalized receptor remain unknown.Currently, phosphorylation of TcR/CD3 in cells and in vitro is monitored using phospho-specific antibodies, and there are no methods that enable dynamic monitoring of the spatial organization of CD3 phosphorylation in live cells. Here, we have constructed genetically encoded fluorescent reporters compatible with imaging of live and fixed cells and demonstrate that they accurately monitor the dynamics and intracellular organization of CD3ζ phosphorylation in Jurkat T cells. Using the reporters, we observed that in addition to the expected activationdependent phosphorylation at the plasma membrane, tyrosinephosphorylated CD3ζ accumulated in the perinuclear endosomal vesicles. Our results demonstrate that endosomal CD3ζ remains signaling-competent and suggest the possibility that internalized CD3ζ pool may help to sustain long-term signaling in T cells. Results Design and Characterization of the CD3ζ Phosphorylation Reporters.To generate a Förster's resonant energy transfer (FRET)-based monomolecular reporter for phosphorylation of the key CD3 signal-transducing subunit ζ, we fused the C terminus of CD3ζ to a pair of green and red fluorescent proteins, eGFP and mCherry, linked by a flexible spacer and followed by the tandem SH2 domains of human ZAP-70 (residues 1-259; tSH2 ). We reasoned that intramolecular binding of the SH2 domains to tyrosine-phosphorylated ITAMs of CD3ζ (7) would result in conformational rearrangement of the adjacent fluorescent proteins and change in the FRET efficiency between the dono...

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Section: Discussionsupporting

confidence: 55%

Imaging T-cell receptor activation reveals accumulation of tyrosine-phosphorylated CD3ζ in the endosomal compartment

Yudushkin

Vale²

2010

Proc. Natl. Acad. Sci. U.S.A.

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“…Besides activation of MAPKs (49), plasma membrane NCL has been shown to increase cytoplasmic Ca 2ϩ levels (52), or activate PI3K (53). On the other hand, accumulating evidence suggests that signaling events related to cell migration require ligand/receptor endocytosis (54). VEGF and VEGF receptor 2 internalization are required for endothelial recovery in a wound healing assay (55), whereas low platelet-derived growth factor (PDGF) concentrations induce cell migration by promoting clathrin-mediated endocytosis of PDGF receptor (56).…”

Section: Discussionmentioning

confidence: 99%

Interplay between αvβ3 Integrin and Nucleolin Regulates Human Endothelial and Glioma Cell Migration

Κουτσιούμπα

Polytarchou

Courty

et al. 2013

Journal of Biological Chemistry

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Background: Cell surface nucleolin (NCL) is a promising target for development of anticancer agents. Results: A novel pathway that includes ␣ ␤ 3 integrin and leads to cell surface NCL localization and cell migration has been identified. Conclusion: ␣ ␤ 3 can be used as a biomarker for the use of NCL antagonists. Significance: This pathway is active in endothelial and glioma cells, as well as in human glioblastomas.

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“…These results indicate that functional intracellular angiotensin II receptors are located at endo-lysosomal level. Endosomes have previously been implicated as important sites for receptor-initiated signaling, as they contain a wide range of signaling molecules in their membranes and are able to recruit scaffolding proteins and signaling mediators (40,43,46). Apparently this is an intriguing observation because in endolysosomal system the NH 2 -terminal of receptors (binding site) is located in the lumenum and COOH-terminal is located in the cytoplasmic side.…”

Section: Discussionmentioning

confidence: 99%

Intracellular angiotensin II activates rat myometrium

Deliu

Tica

Motoc

et al. 2011

American Journal of Physiology-Cell Physiology

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Deliu E, Tica AA, Motoc D, Brailoiu GC, Brailoiu E. Intracellular angiotensin II activates rat myometrium. Am J Physiol Cell Physiol 301: C559 -C565, 2011. First published May 22, 2011; doi:10.1152/ajpcell.00123.2011.-Angiotensin II is a modulator of myometrial activity; both AT1 and AT2 receptors are expressed in myometrium. Since in other tissues angiotensin II has been reported to activate intracellular receptors, we assessed the effects of intracellular administration of angiotensin II via microinjection on myometrium, using calcium imaging. Intracellular injection of angiotensin II increased cytosolic Ca 2ϩ concentration ([Ca 2ϩ ]i) in myometrial cells in a dose-dependent manner. The effect was abolished by the AT 1 receptor antagonist losartan but not by the AT2 receptor antagonist PD-123319. Disruption of the endo-lysosomal system, but not that of Golgi apparatus, prevented the angiotensin II-induced increase in [Ca 2ϩ ]i. Blockade of AT1 receptor internalization had no effect, whereas blockade of microautophagy abolished the increase in [Ca 2ϩ ]i produced by intracellular injection of angiotensin II; this indicates that microautophagy is a critical step in transporting the peptide into the endo-lysosomes lumenum. The response to angiotensin II was slightly reduced in Ca 2ϩ -free saline, indicating a major involvement of Ca 2ϩ release from internal stores. Blockade of inositol 1,4,5-trisphosphate (IP 3) receptors with heparin and xestospongin C or inhibition of phospholipase C (PLC) with U-73122 abolished the response to angiotensin II, supporting the involvement of PLC-IP 3 pathway. Angiotensin II-induced increase in [Ca 2ϩ ]i was slightly reduced by antagonism of ryanodine receptors. Taken together, our results indicate for the first time that in myometrial cells, intracellular angiotensin II activates AT 1-like receptors on lysosomes and activates PLC-IP 3-dependent Ca 2ϩ release from endoplasmic reticulum; the response is further augmented by a Ca 2ϩ -induced Ca 2ϩ release mechanism via ryanodine receptors activation. calcium imaging; cytosolic calcium concentration; endoplasmic reticulum; microinjection ANGIOTENSIN II is an eight amino acid peptide that exerts its actions by activation of G protein-coupled receptors, namely AT 1 and AT 2 receptors. Increasing evidence suggests that angiotensin II acts not only as an endocrine/paracrine factor, but also as an autacoid or intracrine peptide, with the ability to signal from within the cell, without stimulating plasma membrane receptors (21, 38). We previously reported that intracellular administration of angiotensin II via liposomes increases contractility of rat aorta via activation of intracellular receptors (7). Similarly, microinjection of angiotensin II increases cytosolic Ca 2ϩ concentration ([Ca 2ϩ ] i ) in vascular smooth muscle cells (13, 16) or kidney proximal tubule cells (49). The intracellular angiotensin II can result either via uptake from the interstitium or by intracellular synthesis. In the latter case, intracellular renin conver...

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Signaling from endosomes: Location makes a difference

Cited by 169 publications

References 93 publications

Imaging T-cell receptor activation reveals accumulation of tyrosine-phosphorylated CD3ζ in the endosomal compartment

Imaging T-cell receptor activation reveals accumulation of tyrosine-phosphorylated CD3ζ in the endosomal compartment

Interplay between αvβ3 Integrin and Nucleolin Regulates Human Endothelial and Glioma Cell Migration

Intracellular angiotensin II activates rat myometrium

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