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

2002 ◽  
Vol 157 (7) ◽  
pp. 1211-1222 ◽  
Author(s):  
László Hunyady ◽  
Albert J. Baukal ◽  
Zsuzsanna Gáborik ◽  
Jesus A. Olivares-Reyes ◽  
Márta Bor ◽  
...  
Keyword(s):  
Cell Surface ◽  
Kinase Inhibitors ◽  
Fluorescent Protein ◽  
Slow Component ◽  
Ang Ii ◽  
Hek 293 ◽  
Recycling Endosomes ◽  
293 Cells ◽  
Early Endosomes ◽  
Green Fluorescent

Agonist-induced endocytosis and processing of the G protein–coupled AT1 angiotensin II (Ang II) receptor (AT1R) was studied in HEK 293 cells expressing green fluorescent protein (GFP)– or hemagglutinin epitope–tagged forms of the receptor. After stimulation with Ang II, the receptor and its ligand colocalized with Rab5–GFP and Rab4–GFP in early endosomes, and subsequently with Rab11–GFP in pericentriolar recycling endosomes. Inhibition of phosphatidylinositol (PI) 3-kinase by wortmannin (WT) or LY294002 caused the formation of large endosomal vesicles of heterogeneous Rab composition, containing the ligand–receptor complex in their limiting membranes and in small associated vesicular structures. In contrast to Alexa®–transferrin, which was mainly found in small vesicles associated with the outside of large vesicles in WT-treated cells, rhodamine–Ang II was also segregated into small internal vesicles. In cells labeled with 125I-Ang II, WT treatment did not impair the rate of receptor endocytosis, but significantly reduced the initial phase of receptor recycling without affecting its slow component. Similarly, WT inhibited the early, but not the slow, component of the recovery of AT1R at the cell surface after termination of Ang II stimulation. These data indicate that internalized AT1 receptors are processed via vesicles that resemble multivesicular bodies, and recycle to the cell surface by a rapid PI 3-kinase–dependent recycling route, as well as by a slower pathway that is less sensitive to PI 3-kinase inhibitors.

scholarly journals Alteration of intracellular histamine H2 receptor cycling precedes antagonist-induced upregulation

2005 ◽  
Vol 289 (5) ◽  
pp. G880-G889 ◽  
Author(s):  
Satoshi Osawa ◽  
Masayoshi Kajimura ◽  
Seiji Yamamoto ◽  
Mutsuhiro Ikuma ◽  
Chihiro Mochizuki ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Inverse Agonist ◽  
Recycling Endosome ◽  
Histamine H2 Receptor ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Gfp Fluorescence ◽  
Term Administration ◽  
Green Fluorescent

Long-term administration of a histamine H2 receptor (H2R) antagonist (inverse agonist) induces upregulation of H2R in parietal cells, which may be relevant to the rebound hypersecretion of gastric acid that occurs after withdrawal of treatment. The mechanisms underlying this effect are unknown. We hypothesized that the H2R upregulation could be related to receptor trafficking and used H2R-green fluorescent protein (H2R-GFP) to test the hypothesis. Human H2R-GFP was generated and functionally expressed in HEK-293 cells. Binding of the H2R antagonist [3H]tiotidine was performed to quantify H2R expression, and H2R-GFP was imaged in living cells by confocal and evanescent wave microscopy. The binding affinity of [3H]tiotidine was not significantly different between H2R-GFP- and wild-type H2R-expressing HEK-293 cells, both of which had constitutive activity of adenylate cyclase. Visualization of H2R-GFP revealed that the agonist-induced H2R internalization and the antagonist-induced recycling of the internalized H2R from the recycling endosome within 2 h. Long exposure to the antagonist increased GFP fluorescence in the plasma membrane and also induced upregulation of H2R-GFP estimated by the binding assay, whereas long exposure to the agonist enhanced degradative trafficking of H2R-GFP. We examined whether the upregulation reflected an increase in receptor synthesis. Treatment with antagonist did not augment H2R mRNA, and subsequent inhibition of protein synthesis by cycloheximide had no effect on H2R upregulation. These findings suggested that upon exposure to an antagonist (inverse agonist), the equilibrium between receptor endocytosis and recycling is altered before H2R upregulation, probably via suppressing H2R degradation.

scholarly journals Distinct Subcellular Localization of a Group of Synaptobrevin-Like SNAREs in Paramecium tetraurelia and Effects of Silencing SNARE-Specific Chaperone NSF

2009 ◽  
Vol 9 (2) ◽  
pp. 288-305 ◽  
Author(s):  
Christina Schilde ◽  
Barbara Schönemann ◽  
Ivonne M. Sehring ◽  
Helmut Plattner
Keyword(s):  
Cell Surface ◽  
Membrane Trafficking ◽  
Fluorescent Protein ◽  
Close Association ◽  
Secretory Vesicle ◽  
Food Vacuole ◽  
Paramecium Tetraurelia ◽  
Content Type ◽  
Early Endosomes ◽  
Green Fluorescent

ABSTRACT We have identified new synaptobrevin-like SNAREs and localized the corresponding gene products with green fluorescent protein (GFP)-fusion constructs and specific antibodies at the light and electron microscope (EM) levels. These SNAREs, named Paramecium tetraurelia synaptobrevins 8 to 12 (PtSyb8 to PtSyb12), showed mostly very restricted, specific localization, as they were found predominantly on structures involved in endo- or phagocytosis. In summary, we found PtSyb8 and PtSyb9 associated with the nascent food vacuole, PtSyb10 near the cell surface, at the cytostome, and in close association with ciliary basal bodies, and PtSyb11 on early endosomes and on one side of the cytostome, while PtSyb12 was found in the cytosol. PtSyb4 and PtSyb5 (identified previously) were localized on small vesicles, PtSyb5 probably being engaged in trichocyst (dense core secretory vesicle) processing. PtSyb4 and PtSyb5 are related to each other and are the furthest deviating of all SNAREs identified so far. Because they show no similarity with any other R-SNAREs outside ciliates, they may represent a ciliate-specific adaptation. PtSyb10 forms small domains near ciliary bases, and silencing slows down cell rotation during depolarization-induced ciliary reversal. NSF silencing supports a function of cell surface SNAREs by revealing vesicles along the cell membrane at sites normally devoid of vesicles. The distinct distributions of these SNAREs emphasize the considerable differentiation of membrane trafficking, particularly along the endo-/phagocytic pathway, in this protozoan.

ANG II and calmodulin/CaMKII regulate surface expression and functional activity of NBCe1 via separate means

2007 ◽  
Vol 293 (1) ◽  
pp. F68-F77 ◽  
Author(s):  
Clint Perry ◽  
Hong Le ◽  
Irina I. Grichtchenko
Keyword(s):  
Fluorescent Protein ◽  
Surface Expression ◽  
Inhibitory Effect ◽  
Additive Effect ◽  
Xenopus Laevis Oocytes ◽  
Ang Ii ◽  
Early Endosomes ◽  
Green Fluorescent ◽  
Induced Changes

We recently reported that ANG II inhibits NBCe1 current and surface expression in Xenopus laevis oocytes (Perry C, Blaine J, Le H, and Grichtchenko II. Am J Physiol Renal Physiol 290: F417–F427, 2006). Here, we investigated mechanisms of ANG II-induced changes in NBCe1 surface expression. We showed that the PKC inhibitor GF109203X blocks and EGTA reduces surface cotransporter loss in ANG II-treated oocytes, suggesting roles for PKC and Ca2+. Using the endosomal marker FM 4-64 and enhanced green fluorescent protein (EGFP)-tagged NBCe1, we showed that ANG II stimulates endocytosis of NBCe1. To eliminate the possibility that ANG II inhibits NBCe1 recycling, we demonstrated that the recycling inhibitor monensin decreases surface expression, accumulates NBCe1-EGFP in endosomes, and inhibits NBCe1 current. Monensin and ANG II applied together produce greater inhibition of NBCe1 current than either did alone. This additive effect of monensin and ANG II suggests that ANG II stimulates internalization of NBCe1. We used the calmodulin (CaM) antagonist W13, which controls recycling by blocking the exit of the endocytosed cargo from early endosomes, to determine the role of CaM in NBCe1 trafficking. We demonstrated that W13 decreases surface expression of NBCe1, accumulates NBCe1-EGFP in endosomal-like formations, and inhibits NBCe1 current. W13 and ANG II applied together produce greater inhibition of NBCe1 current than either does alone, while W13 and monensin applied together do not. The additive effect of ANG II and W13 and lack of additive effect of monensin and W13 suggest that CaM is not involved in ANG II stimulation of internalization but controls recycling of endocytosed NBCe1. The CaM-activated enzyme CaM kinase II (CaMKII) applied with ANG II also gives an additive inhibitory effect, suggesting a role for CaMKII in NBCe1 recycling.

scholarly journals Dynamin Regulates Focal Exocytosis in Phagocytosing Macrophages

2003 ◽  
Vol 14 (5) ◽  
pp. 2016-2028 ◽  
Author(s):  
Anke Di ◽  
Deborah J. Nelson ◽  
Vytautas Bindokas ◽  
Mary E. Brown ◽  
Frances Libunao ◽  
...  
Keyword(s):  
Cell Surface ◽  
Actin Polymerization ◽  
Kinase Inhibitors ◽  
Fluorescent Protein ◽  
Glutathione S Transferase ◽  
Particle Uptake ◽  
Cytoplasmic Vesicles ◽  
Intracellular Ca ◽  
Particle Ingestion ◽  
Green Fluorescent

Phagocytosis in macrophages is thought to involve insertion of cytoplasmic vesicles at sites of membrane expansion before particle ingestion (“focal” exocytosis). Capacitance (Cm) measurements of cell surface area were biphasic, with an initial rise indicative of exocytosis followed by a fall upon phagocytosis. Unlike other types of regulated exocytosis, the Cm rise was insensitive to intracellular Ca2+, but was inhibited by guanosine 5′-O-(2-thio)diphosphate. Particle uptake, but not Cm rise, was affected by phosphatidylinositol 3-kinase inhibitors. Inhibition of actin polymerization eliminated the Cm rise, suggesting possible coordination between actin polymerization and focal exocytosis. Introduction of anti-pan-dynamin IgG blocked Cm changes, suggesting that dynamin controls focal exocytosis and thereby phagocytosis. Similarly, recombinant glutathione S-transferase•amphiphysin-SH3 domain, but not a mutated form that cannot bind to dynamin, inhibited both focal exocytosis and phagocytosis. Immunochemical analysis of endogenous dynamin distribution in macrophages revealed a substantial particulate pool, some of which localized to a presumptive endosomal compartment. Expression of enhanced green fluorescent protein•dynamin-2 showed a motile dynamin pool, a fraction of which migrated toward and within the phagosomal cup. These results suggest that dynamin is involved in the production and/or movement of vesicles from an intracellular organelle to the cell surface to support membrane expansion around the engulfed particle.

scholarly journals Wortmannin alters the intracellular trafficking of the bradykinin B2 receptor: role of phosphoinositide 3-kinase and Rab5

2003 ◽  
Vol 375 (1) ◽  
pp. 151-158 ◽  
Author(s):  
Steeve HOULE ◽  
François MARCEAU
Keyword(s):  
Fluorescent Protein ◽  
Dominant Negative ◽  
Bafilomycin A1 ◽  
Hek 293 ◽  
Agonist Stimulation ◽  
293 Cells ◽  
Dominant Negative Form ◽  
Phosphoinositide 3 Kinase ◽  
Green Fluorescent ◽  
Negative Form

Wortmannin reportedly induces the formation of enlarged cytoplasmic endosomes. Such vesicles were observed in a definite time window after wortmannin treatment (250 nM) in HEK-293 cells stably expressing a B2R (B2 receptor)–green fluorescent protein conjugate and other cell types. The alternative PI3K (phosphoinositide 3-kinase) inhibitor LY 294002 (100 μM) and a dominant-negative form of the enzyme (p85α ΔiSH2) induce a more modest vesicle enlargement. PI3K inhibition by drugs did not affect agonist-induced [3H]arachidonate release. The wortmannin-induced formation of giant endosomes also involves Rab5 activity, since a dominant-negative form of this GTPase (Rab5 S34N) partially inhibits the wortmannin effect and a constitutively active form of Rab5 (Rab5 Q79L) induces the formation of enlarged endosomes. Moreover, agonist stimulation targeted B2R–green fluorescent protein towards the periphery of the giant vesicles and led to partial receptor degradation only in wortmannin-treated cells. Receptor degradation was decreased by protease inhibitors and by bafilomycin A1, a drug that inhibits lysosome function. Accumulation of fluorescent material inside the enlarged endosomes was observed in cells treated with bafilomycin A1, wortmannin and an agonist. [3H]Bradykinin binding was decreased in HEK-293 cells treated with both wortmannin and the agonist, but not with either separately. Furthermore, a wortmannin-induced functional down-regulation of B2R was observed in rabbit jugular veins after repeated agonist stimulation (contractility assay). This is the first report of a G-protein-coupled receptor down-regulation induced by an alteration of its usual routing in the cell. These results suggest that both PI3K and Rab5 influence B2R intracellular trafficking.

scholarly journals Specific Retrieval of the Exocytic SNARE Snc1p from Early Yeast Endosomes

2000 ◽  
Vol 11 (1) ◽  
pp. 23-38 ◽  
Author(s):  
Michael J. Lewis ◽  
Benjamin J. Nichols ◽  
Cristina Prescianotto-Baschong ◽  
Howard Riezman ◽  
Hugh R. B. Pelham
Keyword(s):  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
Immunoelectron Microscopy ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Early Endosomes ◽  
Internal Structures ◽  
Green Fluorescent

Many endocytosed proteins in yeast travel to the vacuole, but some are recycled to the plasma membrane. We have investigated the recycling of chimeras containing green fluorescent protein (GFP) and the exocytic SNARE Snc1p. GFP-Snc1p moves from the cell surface to internal structures when Golgi function or exocytosis is blocked, suggesting continuous recycling via the Golgi. Internalization is mediated by a conserved cytoplasmic signal, whereas diversion from the vacuolar pathway requires sequences within and adjacent to the transmembrane domain. Delivery from the Golgi to the surface is also influenced by the transmembrane domain, but the requirements are much less specific. Recycling requires the syntaxins Tlg1p and Tlg2p but not Pep12p or proteins such as Vps4p and Vps5p that have been implicated in late endosome–Golgi traffic. Subtle changes to the recycling signal cause GFP-Snc1p to accumulate preferentially in punctate internal structures, although it continues to recycle to the surface. The internal GFP-Snc1p colocalizes with Tlg1p, and immunofluorescence and immunoelectron microscopy reveal structures that contain Tlg1p, Tlg2p, and Kex2p but lack Pep12p and Sec7p. We propose that these represent early endosomes in which sorting of Snc1p and late Golgi proteins occurs, and that transport can occur directly from them to the Golgi apparatus.

Effect of mibefradil on sodium and calcium currents

2005 ◽  
Vol 289 (2) ◽  
pp. G249-G253 ◽  
Author(s):  
Peter R. Strege ◽  
Cheryl E. Bernard ◽  
Yijun Ou ◽  
Simon J. Gibbons ◽  
Gianrico Farrugia
Keyword(s):  
Slow Wave ◽  
Interstitial Cells Of Cajal ◽  
Fluorescent Protein ◽  
Calcium Currents ◽  
Hek 293 ◽  
293 Cells ◽  
Ionic Conductances ◽  
Mouse Intestine ◽  
Green Fluorescent ◽  
Cells Of Cajal

Interstitial cells of Cajal (ICC) generate the electrical slow wave. The ionic conductances that contribute to the slow wave appear to vary among species. In humans, a tetrodotoxin-resistant Na+ current (NaV1.5) encoded by SCN5A contributes to the rising phase of the slow wave, whereas T-type Ca2+ currents have been reported from cultured mouse intestine ICC and also from canine colonic ICC. Mibefradil has a higher affinity for T-type over L-type Ca2+ channels, and the drug has been used in the gastrointestinal tract to identify T-type currents. However, the selectivity of mibefradil for T-type Ca2+ channels over ICC and smooth muscle Na+ channels has not been clearly demonstrated. The aim of this study was to determine the effect of mibefradil on T-type and L-type Ca2+ and Na+ currents. Whole cell currents were recorded from HEK-293 cells coexpressing green fluorescent protein with either the rat brain T-type Ca2+ channel α13.3b + β2, the human intestinal L-type Ca2+ channel subunits α1C + β2, or NaV1.5. Mibefradil significantly reduced expressed T-type Ca2+ current at concentrations ≥ 0.1 μM (IC50 = 0.29 μM), L-type Ca2+ current at > 1 μM (IC50 = 2.7 μM), and Na+ current at ≥ 0.3 μM (IC50 = 0.98 μM). In conclusion, mibefradil inhibits the human intestinal tetrodotoxin-resistant Na+ channel at submicromolar concentrations. Caution must be used in the interpretation of the effects of mibefradil when several ion channel classes are coexpressed.

Quantification of G-Protein Coupled Receptor Internalization Using G-Protein Coupled Receptor-Green Fluorescent Protein Conjugates with the ArrayScan™ High-Content Screening System

1999 ◽  
Vol 4 (2) ◽  
pp. 75-86 ◽  
Author(s):  
Bruce R. Conway ◽  
Lisa K. Minor ◽  
Jun Z. Xu ◽  
Joseph W. Gunnet ◽  
Robbin DeBiasio ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
G Protein ◽  
Fluorescent Protein ◽  
Receptor Internalization ◽  
Hek 293 ◽  
Whole Cells ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Green Fluorescent ◽  
G Protein Coupled

Many G-protein coupled receptors (GPCRs) undergo ligand-dependent homologous desensitization and internalization. Desensitization, defined as a decrease in the responsiveness to ligand, is accompanied by receptor aggregation on the cell surface and internalization via clathrin-coated pits to an intracellular endosomal compartment. In this study, we have taken advantage of the trafficking properties of GPCRs to develop a useful screening method for the identification of receptor mimetics. A series of studies were undertaken to evaluate the expression, functionality, and ligand-dependent trafficking of GPCR-green fluorescent protein (GFP) fusion conjugates stably transfected into HEK 293 cells. These GPCR-GFP expressing cells were then utilized in the validation of the ArrayScan™ (Cellomics™, Pittsburgh, PA), a microtiter plate imaging system that permits cellular and subcellular quantitation of fluorescence in whole cells. These studies demonstrated our ability to measure the internalization of a parathy-roid hormone (PTH) receptor-GFP conjugate after ligand treatment by spatially resolving internalized receptors. Internalization was time- and dose-dependent and appeared to be selective for PTH. Similar results were obtained for a β2-adrenergic receptor (β2 AR)-GFP conjugate stably expressed in HEK 293 cells. The internalized GFP-labeled receptors were visualized as numerous punctate "spots" within the cell interior. An algorithm has been developed that identifies and collects information about these spots, allowing quantification of the internalization process. Variables such as the receptor-GFP expression level, plating density, cell number per field, number of fields scanned per well, spot size, and spot intensity were evaluated during the development of this assay. The method represents a valuable tool to screen for receptor mimetics and antagonists of receptor internalization in whole cells rapidly.

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