Extracellular ATP-dependent activation of plasma membrane Ca2+
 pump in HEK-293 cells

AE1 (anion exchanger 1) is a glycoprotein found in the plasma membrane of erythrocytes, where it mediates the electroneutral exchange of chloride and bicarbonate, a process important in CO2 removal from tissues. It had been previously shown that human AE1 purified from erythrocytes is covalently modified at Cys-843 in the membrane domain with palmitic acid. In this study, the role of Cys-843 in human AE1 trafficking was investigated by expressing various AE1 and Cys-843Ala (C843A) mutant constructs in transiently transfected HEK-293 cells. The AE1 C843A mutant was expressed to a similar level to AE1. The rate of N-glycan conversion from high-mannose into complex form in a glycosylation mutant (N555) of AE1 C843A, and thus the rate of trafficking from the endoplasmic reticulum to the Golgi, were comparable with that of AE1 (N555). Like AE1, AE1 C843A could be biotinylated at the cell surface, indicating that a cysteine residue at position 843 is not required for cell-surface expression of the protein. The turnover rate of AE1 C843A was not significantly different from AE1. While other proteins could be palmitoylated, labelling of transiently transfected HEK-293 cells or COS7 cells with [3H]palmitic acid failed to produce any detectable AE1 palmitoylation. These results suggest that AE1 is not palmitoylated in HEK-293 or COS7 cells and can traffic to the plasma membrane.

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Extracellular ATP dissociates nonmuscle myosin from P2X7complex: this dissociation regulates P2X7pore formation

AJP Cell Physiology ◽

10.1152/ajpcell.00079.2009 ◽

2009 ◽

Vol 297 (2) ◽

pp. C430-C439 ◽

Cited By ~ 54

Author(s):

Ben J. Gu ◽

Catherine Rathsam ◽

Leanne Stokes ◽

Andrew B. McGeachie ◽

James S. Wiley

Keyword(s):

Specific Inhibitor ◽

Life Time ◽

Cell Types ◽

Interferon Γ ◽

Extracellular Atp ◽

Nonmuscle Myosin ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

Myosin Va

The P2X7receptor is a ligand-gated cation channel that is highly expressed on monocyte-macrophages and that mediates the pro-inflammatory effects of extracellular ATP. Dilation of the P2X7channel and massive K+efflux follows initial channel opening, but the mechanism of secondary pore formation is unclear. The proteins associated with P2X7were isolated by using anti-P2X7monoclonal antibody-coated Dynabeads from both interferon-γ plus LPS-stimulated monocytic THP-1 cells and P2X7-transfected HEK-293 cells. Two nonmuscle myosins, NMMHC-IIA and myosin Va, were found to associate with P2X7in THP-1 cells and HEK-293 cells, respectively. Activation of the P2X7receptor by ATP caused dissociation of P2X7from nonmuscle myosin in both cell types. The interaction of P2X7and NMMHC-IIA molecules was confirmed by fluorescent life time measurements and fluorescent resonance of energy transfer-based time-resolved flow cytometry assay. Reducing the expression of NMMHC-IIA or myosin Va by small interfering RNA or short hairpin RNA led to a significant increase of P2X7pore function without any increase in surface expression or ion channel function of P2X7receptors. S- l-blebbistatin, a specific inhibitor of NMMHC-IIA ATPase, inhibited both ATP-induced ethidium uptake and ATP-induced dissociation of P2X7-NMMHC-IIA complex. In both cell types nonmuscle myosin closely interacts with P2X7and is dissociated from the complex by extracellular ATP. Dissociation of this anchoring protein may be required for the transition of P2X7channel to a pore.

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Critical roles for p22phox in the structural maturation and subcellular targeting of Nox3

Biochemical Journal ◽

10.1042/bj20060819 ◽

2007 ◽

Vol 403 (1) ◽

pp. 97-108 ◽

Cited By ~ 53

Author(s):

Yoko Nakano ◽

Botond Banfi ◽

Algirdas J. Jesaitis ◽

Mary C. Dinauer ◽

Lee-Ann H. Allen ◽

...

Keyword(s):

Plasma Membrane ◽

Subcellular Targeting ◽

Hek 293 ◽

Human Embryonic Kidney Cells ◽

Regulatory Subunits ◽

Hek 293 Cells ◽

293 Cells ◽

A Subunit ◽

And Function

Otoconia are small biominerals in the inner ear that are indispensable for the normal perception of gravity and motion. Normal otoconia biogenesis requires Nox3, a Nox (NADPH oxidase) highly expressed in the vestibular system. In HEK-293 cells (human embryonic kidney cells) transfected with the Nox regulatory subunits NoxO1 (Nox organizer 1) and NoxA1 (Nox activator 1), functional murine Nox3 was expressed in the plasma membrane and exhibited a haem spectrum identical with that of Nox2, the electron transferase of the phagocyte Nox. In vitro Nox3 cDNA expressed an ∼50 kDa primary translation product that underwent N-linked glycosylation in the presence of canine microsomes. RNAi (RNA interference)-mediated reduction of endogenous p22phox, a subunit essential for stabilization of Nox2 in phagocytes, decreased Nox3 activity in reconstituted HEK-293 cells. p22phox co-precipitated not only with Nox3 and NoxO1 from transfectants expressing all three proteins, but also with NoxO1 in the absence of Nox3, indicating that p22phox physically associated with both Nox3 and with NoxO1. The plasma membrane localization of Nox3 but not of NoxO1 required p22phox. Moreover, the glycosylation and maturation of Nox3 required p22phox expression, suggesting that p22phox was required for the proper biosynthesis and function of Nox3. Taken together, these studies demonstrate critical roles for p22phox at several distinct points in the maturation and assembly of a functionally competent Nox3 in the plasma membrane.

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Activation of the Novel Estrogen Receptor G Protein-Coupled Receptor 30 (GPR30) at the Plasma Membrane

Endocrinology ◽

10.1210/en.2006-1605 ◽

2007 ◽

Vol 148 (7) ◽

pp. 3236-3245 ◽

Cited By ~ 290

Author(s):

E. Filardo ◽

J. Quinn ◽

Y. Pang ◽

C. Graeber ◽

S. Shaw ◽

...

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

G Protein ◽

Intracellular Camp ◽

Calcium Stores ◽

G Protein Coupled Receptor ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

G Protein Coupled

G protein-coupled receptor 30 (GPR30), a seven-transmembrane receptor (7TMR), is associated with rapid estrogen-dependent, G protein signaling and specific estrogen binding. At present, the subcellular site of GPR30 action is unclear. Previous studies using antibodies and fluorochrome-labeled estradiol (E2) have failed to detect GPR30 on the cell surface, suggesting that GPR30 may function uniquely among 7TMRs as an intracellular receptor. Here, we show that detectable expression of GPR30 on the surface of transfected HEK-293 cells can be selected by fluorescence-activated cell sorting. Expression of GPR30 on the cell surface was confirmed by confocal microscopy using the lectin concanavalin A as a plasma membrane marker. Stimulation of GPR30-expressing HEK-293 cells with 17β-E2 caused sequestration of GPR30 from the cell surface and resulted in its codistribution with clathrin and mobilization of intracellular calcium stores. Evidence that GPR30 signals from the cell surface was obtained from experiments demonstrating that the cell-impermeable E2-protein conjugates E2-BSA and E2-horseradish peroxidase promote GPR30-dependent elevation of intracellular cAMP concentrations. Subcellular fractionation studies further support the plasma membrane as a site of GPR30 action with specific [3H]17β-E2 binding and G protein activation associated with plasma membrane but not microsomal, or other fractions, prepared from HEK-293 or SKBR3 breast cancer cells. These results suggest that GPR30, like other 7TMRs, functions as a plasma membrane receptor.

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Insulin increases plasma membrane content and reduces phosphorylation of Na+-K+ pump α1-subunit in HEK-293 cells

AJP Cell Physiology ◽

10.1152/ajpcell.2001.281.6.c1797 ◽

2001 ◽

Vol 281 (6) ◽

pp. C1797-C1803 ◽

Cited By ~ 23

Author(s):

Gary Sweeney ◽

Wenyan Niu ◽

Victor A. Canfield ◽

Robert Levenson ◽

Amira Klip

Keyword(s):

Plasma Membrane ◽

Kinase Inhibitor ◽

Serine Phosphorylation ◽

Intact Cells ◽

Α Subunit ◽

Hek 293 ◽

Hek 293 Cells ◽

Kinase C ◽

293 Cells ◽

Α1 Subunit

Insulin stimulates K+ uptake and Na+ efflux via the Na+-K+ pump in kidney, skeletal muscle, and brain. The mechanism of insulin action in these tissues differs, in part, because of differences in the isoform complement of the catalytic α-subunit of the Na+-K+ pump. To analyze specifically the effect of insulin on the α1-isoform of the pump, we have studied human embryonic kidney (HEK)-293 cells stably transfected with the rat Na+-K+ pump α1-isoform tagged on its first exofacial loop with a hemagglutinin (HA) epitope. The plasma membrane content of α1-subunits was quantitated by binding a specific HA antibody to intact cells. Insulin rapidly increased the number of α1-subunits at the cell surface. This gain was sensitive to the phosphatidylinositol (PI) 3-kinase inhibitor wortmannin and to the protein kinase C (PKC) inhibitor bisindolylmaleimide. Furthermore, the insulin-stimulated gain in surface α-subunits correlated with an increase in the binding of an antibody that recognizes only the nonphosphorylated form of α1 (at serine-18). These results suggest that insulin regulates the Na+-K+ pump in HEK-293 cells, at least in part, by decreasing serine phosphorylation and increasing plasma membrane content of α1-subunits via a signaling pathway involving PI 3-kinase and PKC.

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Regulation of GPR54 Signaling by GRK2 and β-Arrestin

Molecular Endocrinology ◽

10.1210/me.2009-0013 ◽

2009 ◽

Vol 23 (12) ◽

pp. 2060-2074 ◽

Cited By ~ 60

Author(s):

Macarena Pampillo ◽

Natasha Camuso ◽

Jay E. Taylor ◽

Jacob M. Szereszewski ◽

Maryse R. Ahow ◽

...

Keyword(s):

Plasma Membrane ◽

Cell Line ◽

Molecular Mechanisms ◽

Control Cell ◽

Cytoplasmic Tail ◽

Intracellular Loop ◽

Membrane Expression ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells

Abstract Kisspeptin and its receptor, GPR54, are major regulators of the hypothalamic-pituitary-gonadal axis as well as regulators of human placentation and tumor metastases. GPR54 is a Gq/11-coupled G protein-coupled receptor (GPCR), and activation by kisspeptin stimulates phosphatidy linositol 4, 5-biphosphate hydrolysis, Ca2+ mobilization, arachidonic acid release, and ERK1/2 MAPK phosphorylation. Physiological evidence suggests that GPR54 undergoes agonist-dependent desensitization, but underlying molecular mechanisms are unknown. Furthermore, very little has been reported on the early events that regulate GPR54 signaling. The lack of information in these important areas led to this study. Here we report for the first time on the role of GPCR serine/threonine kinase (GRK)2 and β-arrestin in regulating GPR54 signaling in human embryonic kidney (HEK) 293 cells, a model cell system for studying the molecular regulation of GPCRs, and genetically modified MDA MB-231 cells, an invasive breast cancer cell line expressing about 75% less β-arrestin-2 than the control cell line. Our study reveals that in HEK 293 cells, GPR54 is expressed both at the plasma membrane and intracellularly and also that plasma membrane expression is regulated by cytoplasmic tail sequences. We also demonstrate that GPR54 exhibits constitutive activity, internalization, and association with GRK2 and β- arrestins-1 and 2 through sequences in the second intracellular loop and cytoplasmic tail of the receptor. We also show that GRK2 stimulates the desensitization of GPR54 in HEK 293 cells and that β-arrestin-2 mediates GPR54 activation of ERK1/2 in MDA-MB-231 cells. The significance of these findings in developing molecular-based therapies for treating certain endocrine-related disorders is discussed.

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Heterodimerization, trafficking and membrane topology of the two proteins, Ostα and Ostβ, that constitute the organic solute and steroid transporter

Biochemical Journal ◽

10.1042/bj20070716 ◽

2007 ◽

Vol 407 (3) ◽

pp. 363-372 ◽

Cited By ~ 45

Author(s):

Na Li ◽

Zhifeng Cui ◽

Fang Fang ◽

Jin Young Lee ◽

Nazzareno Ballatori

Keyword(s):

Plasma Membrane ◽

Membrane Topology ◽

Bimolecular Fluorescence Complementation ◽

Mrna Levels ◽

Western Blots ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

Pngase F ◽

Organic Solute

Co-immunoprecipitation studies using mouse ileal proteins and transfected HEK-293 (human embryonic kidney-293) cells revealed that the two proteins, Ostα and Ostβ, which generate the organic-solute transporter are able to immunoprecipitate each other, indicating a heteromeric complex. Mouse ileal Ostα protein appeared on Western blots largely as bands of 40 and 80 kDa, the latter band consistent with an Ostα homodimer, and both of these bands were sensitive to digestion by the glycosidase PNGase F (peptide:N-glycosidase F). Ostβ appeared as bands of 17 and 19 kDa, and these bands were not sensitive to PNGase F. Both the 40 and 80 kDa forms of Ostα, and only the 19 kDa form of Ostβ, were detected among the immunoprecipitated proteins, indicating that the interaction between Ostα and Ostβ is associated with specific post-translational processing. Additional evidence for homodimerization of Ostα and for a direct interaction between Ostα and Ostβ was provided by BiFC (bimolecular fluorescence complementation) analysis of HEK-293 cells transfected with Ostα and Ostβ tagged with yellow-fluorescent-protein fragments. BiFC analysis and surface immunolabelling of transfected HEK-293 cells also indicated that the C-termini of both Ostα and Ostβ are facing the intracellular space. The interaction between Ostα and Ostβ was required not only for delivery of the proteins to the plasma membrane, but it increased their stability, as noted in transfected HEK-293 cells and in tissues from Ostα-deficient (Ostα−/−) mice. In Ostα−/− mice, Ostβ mRNA levels were maintained, yet Ostβ protein was not detectable, indicating that Ostβ protein is not stable in the absence of Ostα. Overall, these findings identify the membrane topology of Ostα and Ostβ, demonstrate that these proteins are present as heterodimers and/or heteromultimers, and indicate that the interaction between Ostα and Ostβ increases the stability of the proteins and is required for delivery of the heteromeric complex to the plasma membrane.

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pH of TGN and recycling endosomes of H+/K+-ATPase-transfected HEK-293 cells: implications for pH regulation in the secretory pathway

AJP Cell Physiology ◽

10.1152/ajpcell.00008.2003 ◽

2003 ◽

Vol 285 (1) ◽

pp. C205-C214 ◽

Cited By ~ 36

Author(s):

Terry E. Machen ◽

Mary Jae Leigh ◽

Carmen Taylor ◽

Tohru Kimura ◽

Shinji Asano ◽

...

Keyword(s):

Plasma Membrane ◽

Secretory Pathway ◽

Ph Regulation ◽

Buffer System ◽

Β Cells ◽

Hek 293 ◽

Recycling Endosomes ◽

Hek 293 Cells ◽

293 Cells ◽

Golgi Network

The influences of the gastric H+/K+ pump on organelle pH during trafficking to and from the plasma membrane were investigated using HEK-293 cells stably expressing the α- and β-subunits of human H+/K+-ATPase (H+/K+-α,β cells). The pH values of trans-Golgi network (pHTGN) and recycling endosomes (pHRE) were measured by transfecting H+/K+-α,β cells with the pH-sensitive GFP pHluorin fused to targeting sequences of either TGN38 or synaptobrevin, respectively. Immunofluorescence showed that H+/K+-ATPase was present in the plasma membrane, TGN, and RE. The pHTGN was similar in both H+/K+-α,β cells (pHTGN 6.36) and vector-transfected (“mock”) cells (pHTGN 6.34); pHRE was also similar in H+/K+-α,β (pHRE 6.40) and mock cells (pHRE 6.37). SCH28080 (inhibits H+/K+-ATPase) caused TGN to alkalinize by 0.12 pH units; subsequent addition of bafilomycin (inhibits H+ v-ATPase) caused TGN to alkalinize from pH 6.4 up to a new steady-state pHTGN of 7.0–7.5, close to pHcytosol. Similar results were observed in RE. Thus H+/K+-ATPases that trafficked to the plasma membrane were active but had small effects to acidify the TGN and RE compared with H+ v-ATPase. Mathematical modeling predicted a large number of H+ v-ATPases (8,000) active in the TGN to balance a large, passive H+ leak (with PH ∼10–3 cm/s) via unidentified pathways out of the TGN. We propose that in the presence of this effective, though inefficient, buffer system in the Golgi and TGN, H+/K+-ATPases (estimated to be ∼4,000 active in the TGN) and other transporters have little effect on luminal pH as they traffic to the plasma membrane.

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Transport activity of chimaeric AE2-AE3 chloride/bicarbonate anion exchange proteins

Biochemical Journal ◽

10.1042/bj20030007 ◽

2003 ◽

Vol 371 (3) ◽

pp. 687-696 ◽

Cited By ~ 21

Author(s):

Jocelyne FUJINAGA ◽

Frederick B. LOISELLE ◽

Joseph R. CASEY

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Cytoplasmic Domain ◽

Anion Transport ◽

Transport Activity ◽

Surface Processing ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

Anion Transport Activity

Chloride/bicarbonate anion exchangers (AEs), found in the plasma membrane of most mammalian cells, are involved in pH regulation and bicarbonate metabolism. Although AE2 and AE3 are highly similar in sequence, AE2-transport activity was 10-fold higher than AE3 (41 versus 4 mM · min−1 respectively), when expressed by transient transfection of HEK-293 cells. AE2–AE3 chimaeras were constructed to define the region responsible for differences in transport activity. The level of AE2 expression was approx. 30% higher than that of AE3. Processing to the cell surface, studied by chemical labelling and confocal microscopy, showed that AE2 is processed to the cell surface approx. 8-fold more efficiently than AE3. The efficiency of cell-surface processing was dependent on the cytoplasmic domain, since the AE2 domain conferred efficient processing upon the AE3 membrane domain, with a predominant role for amino acids 322–677 of AE2. AE2 that was expressed in HEK-293 cells was glycosylated, but little of AE3 was. However, AE2 expressed in the presence of the glycosylation inhibitor, tunicamycin, was not glycosylated, yet retained 85 ± 8% of anion-transport activity. Therefore glycosylation has little, if any, role in the cell-surface processing or activity of AE2 or AE3. We conclude that the low anion-transport activity of AE3 in HEK-293 cells is due to low level processing to the plasma membrane, possibly owing to protein interactions with the AE3 cytoplasmic domain.

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GTPase of the immune-associated nucleotide-binding protein 5 (GIMAP5) regulates calcium influx in T-lymphocytes by promoting mitochondrial calcium accumulation

Biochemical Journal ◽

10.1042/bj20120516 ◽

2012 ◽

Vol 449 (2) ◽

pp. 353-364 ◽

Cited By ~ 14

Author(s):

Xi Lin Chen ◽

Daniel Serrano ◽

Marian Mayhue ◽

Hans-Joachim Wieden ◽

Jana Stankova ◽

...

Keyword(s):

T Cells ◽

Plasma Membrane ◽

T Lymphocytes ◽

Actin Cytoskeleton ◽

Survival Function ◽

Nucleotide Binding ◽

Membrane Channels ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells

Mature T-lymphocytes undergo spontaneous apoptosis in the biobreeding diabetes-prone strain of rats due to the loss of the functional GIMAP5 (GTPase of the immune-associated nucleotide-binding protein 5) protein. The mechanisms underlying the pro-survival function of GIMAP5 in T-cells have not yet been elucidated. We have previously shown that GIMAP5 deficiency in T-cells impairs Ca2+ entry via plasma membrane channels following exposure to thapsigargin or stimulation of the T-cell antigen receptor. In the present study we report that this reduced Ca2+ influx in GIMAP5-deficient T-cells is associated with the inability of their mitochondria to sequester Ca2+ following capacitative entry, which is required for sustained Ca2+ influx via the plasma membrane channels. Consistent with a role for GIMAP5 in regulating mitochondrial Ca2+, overexpression of GIMAP5 in HEK (human embryonic kidney)-293 cells resulted in increased Ca2+ accumulation within the mitochondria. Disruption of microtubules, but not the actin cytoskeleton, abrogated mitochondrial Ca2+ sequestration in primary rat T-cells, whereas both microtubules and actin cytoskeleton were needed for the GIMAP5-mediated increase in mitochondrial Ca2+ in HEK-293 cells. Moreover, GIMAP5 showed partial colocalization with tubulin in HEK-293 cells. On the basis of these findings, we propose that the pro-survival function of GIMAP5 in T-lymphocytes may be linked to its requirement to facilitate microtubule-dependent mitochondrial buffering of Ca2+ following capacitative entry.

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