Differential PI 3-kinase dependence of early and late phases of recycling of the internalized AT1 angiotensin receptor

Hunyady, László; Báukal, Albert J.; Gáborik, Zsuzsanna; Olivares-Reyes, J. Alberto; Bor, Márta; Szaszák, Márta; Lodge, Robert; Catt, Kevin J.; Balla, Tamás

doi:10.1083/jcb.200111013

Cited by 165 publications

(168 citation statements)

References 52 publications

(61 reference statements)

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“…Expression vectors for GFP-Rab7, GFP-Rab5, and GFP-CD63 were previously described. 15,17 Cherry fluorescent protein (CherryFP)-conjugated Rab7 or its dominant negative guanosine diphosphate-locked (GDPlocked) mutant N125I were prepared by recloning the insert from the GFP-conjugated construction into the appropriate vector. GFP-LC3 labels autophagosomes in mammalian cells 18 ; the pEGFP-LC3 expression vector for this chimerical protein was a generous gift from Dr. T. Yoshimori (Osaka University, Japan).…”

Section: Microscopic Techniquesmentioning

confidence: 99%

“…15,16 Mitotic arrest without overt toxicity was also observed in vacuolar cells. [15][16][17][18] The present experiments aim to characterize the cell morphological and toxic responses to local anesthetics relative to the vacuolar-autophagic state. Novel questions addressed include the clinical relevance of drug concentrations that cause this response and its time frame, the relationship of the vacuolar response to cytotoxicity, the experimental dissociation of the three postulated steps of the vacuolar response ( Fig.…”

Section: Introductionmentioning

confidence: 99%

“…Two drugs were selected in relationship with the chemical classification of local anesthetics, i.e., the widely used amide, lidocaine, and the ester, procaine. A previously exploited primary cell model of nonneuronal origin, rabbit vascular smooth muscle cells, 15,17,19 was used to address the cellular effects of local anesthetics. These cells of mesenchymatous origin are related to pericytes known to be morphologically affected in vivo by local anesthetics, 11 are well adapted to morphological analysis (well spread, very adherent, transfectable), and may be used to represent the wide variety of nonneuronal cells affected in tissues by these drugs.…”

Section: Introductionmentioning

confidence: 99%

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Vacuolar ATPase-mediated sequestration of local anesthetics in swollen macroautophagosomes

Bawolak

Morissette

Marceau

2010

Can J Anesth/J Can Anesth

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Purpose Local anesthetics in their therapeutic concentration range cause a vacuolar cytopathology that has been observed in vivo and in various types of mammalian cells. We examined whether active concentration ranges of drugs and the kinetics of the vacuolar response are clinically relevant and whether this phenomenon is associated with cytotoxicity, autophagy, and cell stress signalling. Methods We compared procaine and lidocaine for morphological, functional, and signalling responses in a previously exploited non-neuronal system, primary smooth muscle cells. Several markers conjugated to fluorescent proteins allowed morphological and functional analysis of vacuolar cells. Signalling related to autophagy and cell stress was addressed (immunoblotting of cell lysates). Results Within 2-4 hr, lidocaine and procaine (C1 mM) induced massive cell vacuolization, a response abated by the V-ATPase inhibitor, bafilomycin A1, and activated macroautophagic signalling (LC3 II formation) but not other stress signalling (p38, ERK1/2, p53, no influence on serum-controlled Akt phosphorylation). Novel aspects of the morphological analysis include reduced LC3 labelling of the large vacuoles in cells treated with 3-methyl-adenine, inhibition of CD63 labelling of these vacuoles by co-expression of dominant negative Rab7, retention of secretory green fluorescent protein (GFP) possessing a signal sequence in vacuolar cells, and partial vacuole labelling with lysosomal-associated membrane protein 1 (LAMP1). Lidocaine (2.5-5 mM) was not overtly cytotoxic but arrested cell division over 48 hr. Conclusions V-ATPase-mediated sequestration of clinically relevant concentrations of local anesthetics sequentially involves vacuolization, macroautophagic signalling, and lysosome fusion to large vacuoles. Disruption of the secretory pathway and mitotic arrest were also observed over several hours without major cytotoxicity. RésuméObjectif A`concentration the´rapeutique, les anesthe´siques locaux causent une cytopathologie vacuolaire qui a e´teó bserve´e in vivo et dans divers types de cellules en culture. Nous avons examine´si les concentrations actives de me´dicaments et la cine´tique de cette re´ponse sont cliniquement pertinentes et si ce phe´nome`ne est associe´al a cytotoxicite´, l'autophagie et la signalisation de stress cellulaire.Méthode On a compare´les re´ponses morphologiques, fonctionnelles et signale´tiques a`la procaı¨ne et la lidocaı¨ne dans un syste`me non neuronal pre´alablement exploite´, le muscle lisse en culture primaire, afin d'e´clairer leurs effets toxicologiques a`des niveaux the´rapeutiques. Plusieurs marqueurs conjugue´s à des prote´ines fluorescentes ont permis des analyses morphologiques et fonctionnelles des cellules vacuolaires. La signalisation autophagique et celle relie´e au stress cellulaire ont e´te´e´value´es (immunobuvardage de lysats cellulaires). Résultats La lidocaı¨ne et la procaı¨ne (C1 mM) induisent en 2-4 hr une vacuolisation cellulaire massive, une

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Section: Microscopic Techniquesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Vacuolar ATPase-mediated sequestration of local anesthetics in swollen macroautophagosomes

Bawolak

Morissette

Marceau

2010

Can J Anesth/J Can Anesth

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“…The GFP and HA epitopes have been used to label G protein-coupled receptors, including α 2B -AR and AT1R, resulting in receptors with similar characteristics to the WT receptors [22][23][24]27] were mutated to alanines, were generated using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The sequence of each construct used in this study was verified by restriction mapping and nucleotide sequence analysis (Louisiana State University Health Sciences Center DNA Sequence Core).…”

Section: Plasmid Constructionsmentioning

confidence: 99%

Cell-surface targeting of α2-adrenergic receptors — Inhibition by a transport deficient mutant through dimerization

Zhou

Filipeanu

Duvernay

et al. 2006

Cellular Signalling

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We previously demonstrated that the α 2B -adrenergic receptor mutant, in which the F(x) 6 IL motif in the membrane-proximal carboxyl terminus were mutated to alanines (α 2B -ARm), is deficient in export from the endoplasmic reticulum (ER). In this report, we determined if α 2B -ARm could modulate transport from the ER to the cell surface and signaling of its wild-type counterpart. Transient expression of α 2B -ARm in HEK293T cells markedly inhibited cell-surface expression of wild-type α 2B -AR, as measured by radioligand binding. Subcellular localization demonstrated that α 2B -ARm trapped α 2B -AR in the ER. The α 2B -AR was shown to form homodimers and heterodimers with α 2B -ARm as measured by co-immunoprecipitation of the receptors tagged with green fluorescent protein and hemagglutinin epitopes. In addition to α 2B -AR, the transport of α 2A -AR and α 2C -AR to the cell surface was also inhibited by α 2B -ARm. Furthermore, transient expression of α 2B -ARm significantly reduced cell-surface expression of endogenous α 2 -AR in NG108-15 and HT29 cells. Consistent with its effect on α 2 -AR cell-surface expression, α 2B -ARm attenuated α 2A -AR-and α 2B -AR-mediated ERK1/2 activation. These data demonstrated that the ER-retained mutant α 2B -ARm conferred a dominant negative effect on the cell-surface expression of wild-type α 2 -AR, which is likely mediated through heterodimerization. These data indicate a crucial role of ER export in the regulation of cell-surface targeting and signaling of G protein-coupled receptors.

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“…Subsequent work with intact mammalian cells showed that pharmacologic inhibition of PI 3-kinase by the drugs LY294002 or wortmannin inhibits the intracellular trafficking of receptors, including the recycling of transferrin receptors [12][13][14][15] and the degradation of PDGF receptors [16,17]. A previous study also showed that wortmannin treatment reduces the recycling kinetics of the G protein-coupled angiotensin-1 receptor [18], indicating that postendocytic trafficking of GPCRs might be regulated by phosphoinositides. However, there is no information regarding the dependence of β 2 AR degradation or recycling on PtdIns3P production.…”

Section: Introductionmentioning

confidence: 99%

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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Differential PI 3-kinase dependence of early and late phases of recycling of the internalized AT1 angiotensin receptor

Cited by 165 publications

References 52 publications

Vacuolar ATPase-mediated sequestration of local anesthetics in swollen macroautophagosomes

Vacuolar ATPase-mediated sequestration of local anesthetics in swollen macroautophagosomes

Cell-surface targeting of α2-adrenergic receptors — Inhibition by a transport deficient mutant through dimerization

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

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