PI3P signaling regulates receptor sorting but not transport in the endosomal pathway

Petiot, Anne; Fauré, Julien; Stenmark, Harald; Grüenberg, Jean

doi:10.1083/jcb.200303018

Cited by 132 publications

(143 citation statements)

References 35 publications

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“…5). This finding is consistent with previous studies on the effects of PI 3-kinase inhibitors on the degradation of other signaling receptors, such as the PDGF receptor [16] and the EGF receptor [25,26] There are at least three PI 3-kinases in mammalian cells, and the most commonly used PI 3-kinase inhibitors act on all three [27]. Evidence from other studies implicates the type III PI 3-kinase hVps34 in controlling traffic to lysosomes [28,29].…”

Section: Discussionsupporting

confidence: 91%

Inhibitors of phosphoinositide 3-kinase cause defects in the postendocytic sorting of β2-adrenergic receptors

Awwad

Iyer

Rosenfeld

et al. 2007

Experimental Cell Research

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Phosphatidylinositol 3-kinase inhibitors have been shown to affect the endocytosis or subsequent intracellular sorting in various receptor systems. Agonist-activated β 2 -adrenergic receptors undergo desensitization by mechanisms that include the phosphorylation, endocytosis and degradation of receptors. Following endocytosis, most internalized receptors are sorted to the cell surface, but some proportion is sorted to lysosomes for degradation. It is not known what governs the ratio of receptors that recycle versus receptors that undergo degradation. To determine if phosphatidylinositol 3-kinases regulate β 2 -adrenergic receptor trafficking, HEK293 cells stably expressing these receptors were treated with the phosphatidylinositol 3-kinase inhibitors LY294002 or wortmannin. We then studied agonist-induced receptor endocytosis and postendocytic sorting, including recycling and degradation of the internalized receptors. Both inhibitors amplified the internalization of receptors after exposure to the β-agonist isoproterenol, which was attributable to the sorting of a significant fraction of receptors to an intracellular compartment from which receptor recycling did not occur. The initial rate of β 2 -adrenergic receptor endocytosis and the default rate of receptor recycling were not significantly altered. During prolonged exposure to agonist, LY294002 slowed the degradation rate of β 2 -adrenergic receptors and caused the accumulation of receptors within rab7-positive vesicles. These results suggest that phosphatidylinositol 3-kinase inhibitors (1) cause a misrouting of β 2 -adrenergic receptors into vesicles that are neither able to efficiently recycle to the surface nor sort to lysosomes, and (2) delays the movement of receptors from late endosomes to lysosomes.

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Section: Discussionsupporting

confidence: 91%

Inhibitors of phosphoinositide 3-kinase cause defects in the postendocytic sorting of β2-adrenergic receptors

Awwad

Iyer

Rosenfeld

et al. 2007

Experimental Cell Research

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“…Thus, expression of the 2xFYVE-probe mimicked the endosomal accumulation of PI(3)P-binding effector proteins seen upon loss of MTM1. This blocks accessibility of PI(3)P for PI(3)P-dependent motor proteins and PI(3)P metabolizing enzymes (Petiot, Faure et al 2003). To allow dissociation of the FYVE-probe from endosomes, a single FYVE domain under a weak promoter (Hayakawa, Hayes et al 2004;Stuffers, Malerod et al 2010) or knockin of the GFP-tagged FYVE domain using CRISPR/ Cas9 technology within a genomic safe harbour locus (Doudna and Charpentier 2014) should be used.…”

Section: Pi Conversion In Endosomal Exocytosis Parallels Switching Ofmentioning

confidence: 99%

A phosphoinositide conversion mechanism for exit from endosomes

Ketel¹,

Krauß²,

Nicot³

et al. 2016

Nature

165

177

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I owe special thanks to Marnix Wieffer and Michael Krauß, who spent a lot of time helping me to get started in the lab, to overcome numerous experimental problem, I was not sure how to deal with, and frequently joined in discussions about my project with excellent advice. I am really grateful for your experimental support, Michael, and the time you invested in the project during our paper revision.Further, I would like to thank Jocelyn Laporte and his group, especially Anne-Sophie Nicot, at the IGBMC in Strasbourg for their support and a very fruitful collaboration, and Carsten Schultz and his group at the EMBL in Heidelberg for their support and constant supply with PIP/AMs. I owe special thanks to Dmytro Puchkov for the ultrastructural analysis and Eberhard Krause for mass spectrometry done within this project. I also need to acknowledge Markus Wenk and Federico Torta at the Singapore Lipidomics Incubator for joining the project at a very late stage, when we urgently needed help from lipidomics specialists. It was a pity that experiments did not work out as planned.I would further like to express my gratitude to two very skilled technicians in the AG Haucke lab: Silke Zillmann and Delia Löwe. Without your help it would have been impossible to achieve so much within the limited amount of time I had during the paper revision. by VPS34-IN1 treatment................................................... 52 2.3.11 Fluorescence microscopy................................................................................. 53 2.3.12 Flow cytometry............................................................................................ III SummaryPhosphoinositides (PIs) are a minor class of short-lived phospholipids that serve as crucial signposts of membrane identity. Thereby, PIs full fill important functions in cell signaling and membrane transport. PI 4-phosphates such as phosphatitylinositol-4-phosphate (PI(4)P) and phosphatitylinositol-4,5-bisphosphate (PI(4,5)P 2 ) are enriched at the plasma membrane (PM), on secretory organelles and lysosomes, while PI 3-phosphates, i.e.phosphatitylinositol-3-phosphate (PI(3)P), are a hallmark of the endosomal system.Directional transport between these compartments, thus, requires regulated PI conversion.However, PI conversion in exit from PI(3)P-enriched endosomes en route to the PI(4)P-and PI(4,5)P 2 -positive PM in endosomal recycling remained unknown.Here, we report that cargo exit from endosomes requires removal of PI(3)P by the PI(3)P 3-phosphatase myotubularin 1 (MTM1), and concomitant PI(4)P synthesis by PI 4-kinase type II α (PI4K2α). Loss of MTM1 causes endosomal accumulation of PI(3)P and PI(3)P effector proteins, i.e. sorting nexins, Kif16b-mediated outward traffic of PI(3)P containing endosomes and sub-plasmalemmal accumulation of exocytosis-deficient endosomes. As PI4K2α associates with MTM1 and thereby facilitates membrane recruitment of MTM1, these phenotypic changes are mimicked by loss of PI4K2α. The conversion of PI(3)P-to-PI(4)P is paralleled by a switch i...

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“…We used a BHK cell line expressing human EGF receptor tagged with GFP under the control of a tetracycline promoter (Petiot et al, 2003; Supplemental Figure S3, A and B; the antibody we used only recognized the human receptor), because fractionation of endosomes is optimal in these cells with our protocol. After incubation on ice with anti-EGF receptor antibody followed by a Cy5-coupled secondary antibody, cells were challenged with 200 ng/ml Texas Red-human EGF and further incubated for 50 min at 37°C.…”

Section: Molecular Biology Of the Cell 4946mentioning

confidence: 99%

“…In some experiments, we used BHK cells expressing the human EGF receptor tagged with the GFP under the control of a tetracycline promoter (Petiot et al, 2003). Cells were mock-treated or treated with siRNAs against Alix, Tsg101, or both for 3 d. EGF receptor expression was induced with 1 g/ml doxycycline 24 h before the experiment.…”

Section: Manipulations Of Cells In Vivomentioning

confidence: 99%

“…Baby hamster kidney cells (BHK-21; Gruenberg et al, 1989) and BHK-21 cells expressing human green fluorescent protein (GFP)-tagged EGF receptor were cultured as described (Petiot et al, 2003). Reagents were obtained from the following sources: protein assay reagent and horseradish peroxidase (HRP)-conjugated secondary antibodies from Bio-Rad Laboratories (Hercules, CA); HPTS (8-hydroxypyrene-1,3,6-trisulfonic acid), DAPI, and Texas Red-coupled EGF from Molecular Probes (Eugene, OR); DPX (p-xylene-bis-pyridinium bromide) from Chemie Brunschwig (Basel, Switzerland); U18666A (3-␤-[2-(diethylamino)ethoxy]androst-5-en-17-one) from Biomol Research Laboratories (Plymouth Meeting, PA); Lipofectamine 2000, Oligofectamine, TRIzol, SuperscriptT-MRT, and TOPO vector from Invitrogen (Basel, Switzerland); oligonucleotides and small interfering RNA (siRNA) from Qiagen (Germantown, MD); doxycycline from Clontech (Saint-Germain-en-Laye, France); glutathione Sepharose 4B from Amersham Biosciences (Little Chalfont, United Kingdom); anti-Tsg101 mAb (clone 4A10) from Genetex (San Antonio, TX); anti-EGF receptor and anti-annexin A2 monoclonal antibodies (BD Biosciences, San Diego, CA); anti-GFP mAb (Roche Diagnostics, Basel, Switzerland); anti-Vps4 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA); fluororescently labeled secondary antibodies from Jackson ImmunoResearch Laboratories (West Grove, PA).…”

Section: Cells Reagents and Antibodiesmentioning

confidence: 99%

See 1 more Smart Citation

In Vitro Budding of Intralumenal Vesicles into Late Endosomes Is Regulated by Alix and Tsg101

Falguières

Luyet

Bissig

et al. 2008

MBoC

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Endosomes along the degradation pathway leading to lysosomes accumulate membranes in their lumen and thus exhibit a characteristic multivesicular appearance. These lumenal membranes typically incorporate down-regulated EGF receptor destined for degradation, but the mechanisms that control their formation remain poorly characterized. Here, we describe a novel quantitative biochemical assay that reconstitutes the formation of lumenal vesicles within late endosomes in vitro. Vesicle budding into the endosome lumen was time-, temperature-, pH-, and energy-dependent and required cytosolic factors and endosome membrane components. Our light and electron microscopy analysis showed that the compartment supporting the budding process was accessible to endocytosed bulk tracers and EGF receptor. We also found that the EGF receptor became protected against trypsin in our assay, indicating that it was sorted into the intraendosomal vesicles that were formed in vitro. Our data show that the formation of intralumenal vesicles is ESCRT-dependent, because the process was inhibited by the K173Q dominant negative mutant of hVps4. Moreover, we find that the ESCRT-I subunit Tsg101 and its partner Alix control intralumenal vesicle formation, by acting as positive and negative regulators, respectively. We conclude that budding of the limiting membrane toward the late endosome lumen, which leads to the formation of intraendosomal vesicles, is controlled by the positive and negative functions of Tsg101 and Alix, respectively. INTRODUCTIONIn eukaryotic cells, molecules of the plasma membrane, ligands and solutes are internalized into early endosomes, from where they can be recycled back to the plasma membrane, transported to the trans-Golgi network, or targeted to late endosomes and lysosomes (Gruenberg, 2001;Maxfield and McGraw, 2004;Mayor and Pagano, 2007). The latter pathway is followed in particular by ubiquitinated signaling receptors that need to be down-regulated. These receptors are incorporated into invaginations of the early endosomal membrane that form within their lumen (Hurley and Emr, 2006), thus giving rise to nascent multivesicular bodies (MVBs) or endosomal carrier vesicles (ECVs; Gruenberg and Stenmark, 2004;Piper and Katzmann, 2007), which function as intermediates between early and late endosomes. Eventually, intralumenal vesicles are delivered to lysosomes, where they are degraded together with their receptor cargo.Major progress has been made in our understanding of the molecular mechanisms that control the sorting of ubiquitinated receptors, in particular the epidermal growth factor (EGF) receptor (Hurley and Emr, 2006;Slagsvold et al., 2006;Piper and Katzmann, 2007;Williams and Urbe, 2007). These receptors interact via the ubiquitin moiety with hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), which in turn binds clathrin and phosphatidyl-inositol-3-phosphate (PtdIns3P), thereby concentrating the receptor in early endosomal membrane domains (Raiborg et al., 2001;Urbe et al., 2003;Raiborg et al., ...

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PI3P signaling regulates receptor sorting but not transport in the endosomal pathway

Cited by 132 publications

References 35 publications

Inhibitors of phosphoinositide 3-kinase cause defects in the postendocytic sorting of β2-adrenergic receptors

Inhibitors of phosphoinositide 3-kinase cause defects in the postendocytic sorting of β2-adrenergic receptors

A phosphoinositide conversion mechanism for exit from endosomes

In Vitro Budding of Intralumenal Vesicles into Late Endosomes Is Regulated by Alix and Tsg101

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