scholarly journals Phospholipase D2 Localizes to the Plasma Membrane and Regulates Angiotensin II Receptor Endocytosis

2004 ◽  
Vol 15 (3) ◽  
pp. 1024-1030 ◽  
Author(s):  
Guangwei Du ◽  
Ping Huang ◽  
Bruce T. Liang ◽  
Michael A. Frohman
Keyword(s):  
Plasma Membrane ◽  
Angiotensin Ii ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Cell Types ◽  
Dominant Negative ◽  
Secretory Cells ◽  
Resting Cells ◽  
Angiotensin Ii Receptor ◽  
Multiple Cell

Phospholipase D (PLD) is a key facilitator of multiple types of membrane vesicle trafficking events. Two PLD isoforms, PLD1 and PLD2, exist in mammals. Initial studies based on overexpression studies suggested that in resting cells, human PLD1 localized primarily to the Golgi and perinuclear vesicles in multiple cell types. In contrast, overexpressed mouse PLD2 was observed to localize primarily to the plasma membrane, although internalization on membrane vesicles was observed subsequent to serum stimulation. A recent report has suggested that the assignment of PLD2 to the plasma membrane is in error, because the endogenous isoform in rat secretory cells was imaged and found to be present primarily in the Golgi apparatus. We have reexamined this issue by using a monoclonal antibody specific for mouse PLD2, and find, as reported initially using overexpression studies, that endogenous mouse PLD2 is detected most readily at the plasma membrane in multiple cell types. In addition, we report that mouse, rat, and human PLD2 when overexpressed all similarly localize to the plasma membrane in cell lines from all three species. Finally, studies conducted using overexpression of wild-type active or dominant-negative isoforms of PLD2 and RNA interference-mediated targeting of PLD2 suggest that PLD2 functions at the plasma membrane to facilitate endocytosis of the angiotensin II type 1 receptor.

Spatial Control of Epac2 Activity by cAMP and Ca2+-Mediated Activation of Ras in Pancreatic β Cells

2013 ◽  
Vol 6 (273) ◽  
pp. ra29-ra29 ◽  
Author(s):  
Olof Idevall-Hagren ◽  
Ida Jakobsson ◽  
Yunjian Xu ◽  
Anders Tengholm
Keyword(s):  
Plasma Membrane ◽  
Cell Types ◽  
Dominant Negative ◽  
Nucleotide Exchange ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Spatiotemporal Regulation ◽  
Nucleotide Exchange Factor ◽  
Multiple Cell ◽  
Guanosine Triphosphatase

The cAMP (adenosine 3′,5′-monophosphate)–activated guanine nucleotide exchange factor (GEF) Epac2 is an important mediator of cAMP-dependent processes in multiple cell types. We used real-time confocal and total internal reflection fluorescence microscopy to examine the spatiotemporal regulation of Epac2, which is a GEF for the guanosine triphosphatase (GTPase) Rap. We demonstrated that increases in the concentration of cAMP triggered the translocation of Epac2 from the cytoplasm to the plasma membrane in insulin-secreting β cells. Glucose-induced oscillations of the submembrane concentration of cAMP were associated with cyclic translocation of Epac2, and this translocation could be amplified by increases in the cytoplasmic Ca2+ concentration. Analyses of Epac2 mutants identified the high-affinity cAMP-binding and the Ras association domains as crucial for the translocation. Expression of a dominant-negative Ras mutant reduced Epac2 translocation, and Ca2+-dependent oscillations in Ras activity synchronized with Epac2 translocation in single β cells. The cyclic translocation of Epac2 was accompanied by oscillations of Rap GTPase activity at the plasma membrane, and expression of an inactive Rap1B mutant decreased insulin secretion. Thus, Epac2 localization is dynamically controlled by cAMP as well as by Ca2+-mediated activation of Ras. These results help to explain how oscillating signals can produce pulses of insulin release from pancreatic β cells.

The iodide channel of the thyroid: a plasma membrane vesicle study

1992 ◽  
Vol 263 (3) ◽  
pp. C590-C597 ◽  
Author(s):  
P. Golstein ◽  
M. Abramow ◽  
J. E. Dumont ◽  
R. Beauwens
Keyword(s):  
Plasma Membrane ◽  
Chloride Transport ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Anion Channel ◽  
Apical Plasma Membrane ◽  
Plasma Membrane Vesicle ◽  
Plasma Membrane Vesicles ◽  
Bovine Thyroid ◽  
Set Up

The uptake of radioactive iodide or chloride by plasma membrane vesicles of bovine thyroid was studied by a rapid filtration technique. A Na(+)-I- cotransport was demonstrated. When this Na(+)-I- cotransport is inactive (i.e., at 4 degrees C and in the absence of Na+), an uptake of iodide above chemical equilibrium could be induced, driven by the membrane potential. The latter was set up by allowing potassium to diffuse into the membrane vesicles in the presence of valinomycin and of an inward K+ gradient. This potential difference (positive inside) induced the uptake of iodide (or other anion present). The data support the existence of two anionic channels. The first one, observed at low near-physiological iodide concentration (micromolar range), which exhibits a high permeability and specificity for iodide (hence called the iodide channel), has a Km of 70 microM. The other one appears similar to the epithelial anion channel as described by Landry et al. (J. Gen. Physiol. 90: 779-798, 1987); it is still about fourfold more permeable to iodide than to chloride and presents a Km of 33 mM. Under physiological conditions the latter channel would mediate chloride transport, and the iodide channel, which is proposed to be restricted to the apical plasma membrane domain of the thyrocyte, transports iodide from the cytosol to the colloid space.

Internalisation of the type I angiotensin II receptor (ATI) and angiotensin II function in the rat adrenal zona glomerulosa cell.

1994 ◽  
Vol 141 (2) ◽  
pp. R5-R9 ◽  
Author(s):  
G. P. Vinson ◽  
M. M. Ho. ◽  
J.R. Puddefoot ◽  
R. Teja ◽  
S. Barker
Keyword(s):  
Monoclonal Antibody ◽  
Plasma Membrane ◽  
Angiotensin Ii ◽  
Zona Glomerulosa ◽  
Type I ◽  
Specific Monoclonal Antibody ◽  
Angiotensin Ii Receptor ◽  
Cellular Localisation ◽  
Glomerulosa Cells

ABSTRACT Little is known about the cellular localisation of the angiotensin II (AII) type 1 receptor (ATI) in the rat adrenal glomerulosa cell, but some studies have suggested that receptor internalisation and recycling may occur. Using a specific monoclonal antibody (6313/G2) to the first extracellular domain, we show here that most of the receptor is internalised in the unstimulated cell. When viable glomerulosa cells are incubated with 6313/G2, the receptor is transiently concentrated on the cell surface, and aldosterone output is stimulated. This stimulated output is enhanced by neither threshold nor maximal stimulatory concentrations of All amide, although the antibody does not inhibit All binding to the receptor. Conversely, the stimulatory actions of the antibody and those of ACTH are additive. The data suggest that recycling to the plasma membrane is constitutive, or regulated by unknown factors. Retention of the ATI receptor in the membrane is alone enough to allow sufficient G protein interaction to generate maximal stimulatory events.

scholarly journals Surface membrane vesicles from mononuclear cells stimulate erythroid stem cells to proliferate in culture

Blood ◽  
1982 ◽  
Vol 60 (3) ◽  
pp. 583-594 ◽  
Author(s):  
N Dainiak ◽  
CM Cohen
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Mononuclear Cells ◽  
Surface Membrane ◽  
Freeze Fracture ◽  
Membrane Vesicles ◽  
Cell Types ◽  
Target Cells ◽  
Freeze Fracture Electron Microscopy

Abstract In order to examine the contribution of cell surface materials to erythroid burst-promoting activity (BPA), we separated media conditioned by a variety of human cell types into pellets and supernatants by centrifugation. When added to serum-restricted cultures of nonadherent human marrow cells, pellets contained about half of the total stimulatory activity. Freeze-fracture electron microscopy of the pellets revealed the presence of unilamellar membrane vesicles ranging from 0.10 to 0.40 microM in diameter. The amount of BPA in culture increased with added vesicle concentration in a saturable fashion. Preparation of leukocyte conditioned medium (LCM) from 125I-wheat germ agglutinin labeled cells and studies comparing the glycoprotein composition of vesicles with that of leukocyte plasma membranes suggest that LCM-derived vesicles are of plasma membrane origin. Moreover, partially purified leukocyte plasma membrane preparations also contained BPA. While disruption of vesicles by freezing/thawing and hypotonic lysis did not alter BPA, heat, trypsin, or pronase treatment removed greater than 65% of BPA, implying that vesicle surface rather than intravesicular molecules express BPA. Results of BPA assays performed in two-layer clots indicated that proximity to target cells is required for vesicle BPA expression. We conclude that membrane vesicles spontaneously shed from cell surfaces may be important regulators of erythroid burst proliferation in vitro.

scholarly journals Analysis of the Antimalarial Drug Resistance Protein Pfcrt Expressed in Yeast

2002 ◽  
Vol 277 (51) ◽  
pp. 49767-49775 ◽  
Author(s):  
Hanbang Zhang ◽  
Ellen M. Howard ◽  
Paul D. Roepe
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Integral Membrane Protein ◽  
Chloroquine Resistance ◽  
Malarial Parasite ◽  
Plasma Membrane Vesicle ◽  
Wild Type ◽  
Stem Loop

Mutations in the novel membrane protein Pfcrt were recently found to be essential for chloroquine resistance (CQR) inPlasmodium falciparum, the parasite responsible for most lethal human malaria (Fidock, D. A., Nomura, T., Talley, A. K., Cooper, R. A., Dzekunov, S. M., Ferdig, M. T., Ursos, L. M., Sidhu, A. B., Naude, B., Deitsch, K. W., Su, X. Z., Wootton, J. C., Roepe, P. D., and Wellems, T. E. (2000)Mol. Cell6, 861–871). Pfcrt is localized to the digestive vacuolar membrane of the intraerythrocytic parasite and may function as a transporter. Study of this putative transport function would be greatly assisted by overexpression in yeast followed by characterization of membrane vesicles. Unfortunately, the very high AT content of malarial genes precludes efficient heterologous expression. Thus, we back-translated Pfcrt to design idealized genes with preferred yeast codons, no long poly(A) sequences, and minimal stem-loop structure. We synthesized a designed gene with a two-step PCR method, fused this to N- and C-terminal sequences to aid membrane insertion and purification, and now report efficient expression of wild type and mutant Pfcrt proteins in the plasma membrane ofSaccharomyces cerevisiaeandPichia pastorisyeast. To our knowledge, this is the first successful expression of a full-length malarial parasite integral membrane protein in yeast. Purified membranes and inside-out plasma membrane vesicle preparations were used to analyze wild typeversusCQR-conferring mutant Pfcrt function, which may include effects on H+transport (Dzekunov, S., Ursos, L. M. B., and Roepe, P. D. (2000)Mol. Biochem. Parasitol.110, 107–124), and to perfect a rapid purification of biotinylated Pfcrt. These data expand on the role of Pfcrt in conferring CQR and define a productive route for analysis of importantP. falciparumtransport proteins and membrane associated vaccine candidates.

scholarly journals Antigen-stimulated Activation of Phospholipase D1b by Rac1, ARF6, and PKCα in RBL-2H3 Cells

2002 ◽  
Vol 13 (4) ◽  
pp. 1252-1262 ◽  
Author(s):  
Dale J. Powner ◽  
Matthew N. Hodgkin ◽  
Michael J.O. Wakelam
Keyword(s):  
Plasma Membrane ◽  
Cell Types ◽  
Enzyme Activation ◽  
Dominant Negative ◽  
Ph Domain ◽  
Phosphatidylinositol 3 Kinase ◽  
Stimulation Of ◽  
Phosphatidylinositol 3

Phospholipase D (PLD) activity can be detected in response to many agonists in most cell types; however, the pathway from receptor occupation to enzyme activation remains unclear. In vitro PLD1b activity is phosphatidylinositol 4,5-bisphosphate dependent via an N-terminal PH domain and is stimulated by Rho, ARF, and PKC family proteins, combinations of which cooperatively increase this activity. Here we provide the first evidence for the in vivo regulation of PLD1b at the molecular level. Antigen stimulation of RBL-2H3 cells induces the colocalization of PLD1b with Rac1, ARF6, and PKCα at the plasma membrane in actin-rich structures, simultaneously with cooperatively increasing PLD activity. Activation is both specific and direct because dominant negative mutants of Rac1 and ARF6 inhibit stimulated PLD activity, and surface plasmon resonance reveals that the regulatory proteins bind directly and independently to PLD1b. This also indicates that PLD1b can concurrently interact with a member from each regulator family. Our results show that in contrast to PLD1b's translocation to the plasma membrane, PLD activation is phosphatidylinositol 3-kinase dependent. Therefore, because inactive, dominant negative GTPases do not activate PLD1b, we propose that activation results from phosphatidylinositol 3-kinase–dependent stimulation of Rac1, ARF6, and PKCα.

scholarly journals Association of β-Arrestin 1 with the Type 1A Angiotensin II Receptor Involves Phosphorylation of the Receptor Carboxyl Terminus and Correlates with Receptor Internalization

2001 ◽  
Vol 15 (10) ◽  
pp. 1706-1719
Author(s):  
Hongwei Qian ◽  
Luisa Pipolo ◽  
Walter G. Thomas
Keyword(s):  
Angiotensin Ii ◽  
Close Correlation ◽  
Dominant Negative ◽  
Receptor Internalization ◽  
Central Portion ◽  
Carboxyl Terminus ◽  
Angiotensin Ii Receptor ◽  
Receptor Phosphorylation ◽  
At1a Receptor ◽  
Mutant Receptors

Abstract Arrestins bind to phosphorylated G protein-coupled receptors and participate in receptor desensitization and endocytosis. Although arrestins traffic with activated type 1 (AT1A) angiotensin II (AngII) receptors, the contribution of arrestins to AT1A receptor internalization is controversial, and the physical association of arrestins with the AT1A receptor has not been established. In this study, by coimmunoprecipitating AT1A receptors and β-arrestin 1, we provide direct evidence for an association between arrestins and the AT1A receptor that was agonist- and time-dependent and contingent upon the level ofβ -arrestin 1 expression. Serial truncation of the receptor carboxyl terminus resulted in a graded loss of β-arrestin 1 association, which correlated with decreases in receptor phosphorylation. Truncation of the AT1A receptor to lysine325 prevented AngII-induced phosphorylation and β-arrestin 1 association as well as markedly inhibiting receptor internalization, indicating a close correlation between these receptor parameters. AngII-induced association was also dramatically reduced in a phosphorylation- and internalization-impaired receptor mutant in which four serine and threonine residues in the central portion of the AT1A receptor carboxyl terminus (Thr332, Ser335, Thr336, Ser338) were substituted with alanine. In contrast, substitutions in another serine/threonine-rich region (Ser346, Ser347, Ser348) and at three PKC phosphorylation sites (Ser331, Ser338, Ser348) had no effect on AngII-inducedβ -arrestin 1 association or receptor internalization. While AT1A receptor internalization could be inhibited by a dominant-negative β-arrestin 1 mutant (βarr1319–418), treatment with hyperosmotic sucrose to inhibit internalization did not abrogate the differences in arrestin association observed between the wild-type and mutant receptors, indicating that arrestin binding precedes, and is not dependent upon, receptor internalization. Interestingly, a substituted analog of AngII,[ Sar1Ile4Ile8]-AngII, which promotes robust phosphorylation of the receptor but does not activate receptor signaling, stimulated strong β-arrestin 1 association with the full-length AT1A receptor. These results identify the central portion of the AT1A receptor carboxyl terminus as the important determinant for β-arrestin 1 binding and internalization and indicate that AT1A receptor phosphorylation is crucial for β-arrestin docking.

scholarly journals Elevating CLIC4 in Multiple Cell Types Reveals a TGF-β Dependent Induction of a Dominant Negative Smad7 Splice Variant

PLoS ONE ◽  
2016 ◽  
Vol 11 (8) ◽  
pp. e0161410 ◽  
Author(s):  
Anjali Shukla ◽  
Yihan Yang ◽  
Sara Madanikia ◽  
Yan Ho ◽  
Mangmang Li ◽  
...  
Keyword(s):  
Splice Variant ◽  
Cell Types ◽  
Dominant Negative ◽  
Multiple Cell

The effect of angiotensin II on intracellular pH is mediated by AT1 receptor translocation

2008 ◽  
Vol 295 (1) ◽  
pp. C138-C145 ◽  
Author(s):  
Karina Thieme ◽  
Débora Mai N. Eguti ◽  
Margarida Mello-Aires ◽  
Maria Oliveira-Souza
Keyword(s):  
Plasma Membrane ◽  
Angiotensin Ii ◽  
Intracellular Ph ◽  
Recovery Rate ◽  
Intracellular Signaling ◽  
Mdck Cells ◽  
Ang Ii ◽  
Angiotensin Ii Receptor ◽  
Control Situation ◽  
Immunofluorescence Studies

The effect of ANG II on intracellular pH (pHi) recovery rate and AT1 receptor translocation was investigated in transfected MDCK cells. The pHi recovery rate was evaluated by fluorescence microscopy using the fluorescent probe BCECF-AM. The human angiotensin II receptor isoform 1 (hAT1) translocation was analyzed by immunofluorescence and confocal microscope. Our data show that transfected cells in control situation have a pHi recovery rate of 0.219 ± 0.017 pH U/min ( n = 11). This value was similar to nontransfected cells [0.211 ± 0.009 pH U/min ( n = 12)]. Both values were significantly increased with ANG II (10−9 M) but not with ANG II (10−6 M). Losartan (10−7 M) and dimethyl-BAPTA-AM (10−7 M) decreased significantly the stimulatory effect of ANG II (10−9 M) and induced an increase in Na+/H+ exchanger 1 (NHE-1) activity with ANG II (10−6 M). Immunofluorescence studies indicated that in control situation, the hAT1 receptor was predominantly expressed in cytosol. However, it was translocated to plasma membrane with ANG II (10−9 M) and internalized with ANG II (10−6 M). Losartan (10−7 M) induced hAT1 translocation to plasma membrane in all studied groups. Dimethyl-BAPTA-AM (10−7 M) did not change the effect of ANG II (10−9 M) on the hAT1 receptor distribution but induced its accumulation at plasma membrane in cells treated with ANG II (10−6 M). With ionomycin (10−6 M), the receptor was accumulated in cytosol. The results indicate that, in MDCK cells, the effect of ANG II on NHE-1 activity is associated with ligand binding to AT1 receptor and intracellular signaling events related to AT1 translocation.

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