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

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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Plasma membrane delivery of the gastric H,K-ATPase: the role of β-subunit glycosylation

AJP Cell Physiology ◽

10.1152/ajpcell.00068.2003 ◽

2003 ◽

Vol 285 (4) ◽

pp. C968-C976 ◽

Cited By ~ 17

Author(s):

O. Vagin ◽

S. Denevich ◽

G. Sachs

Keyword(s):

Plasma Membrane ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Glycosylation Site ◽

Β Subunit ◽

Wild Type ◽

Α Subunit ◽

Hek 293 ◽

Glycosylation Sites ◽

293 Cells

The factors determining trafficking of the gastric H,K-ATPase to the apical membrane remain elusive. To identify such determinants in the gastric H,K-ATPase, fusion proteins of yellow fluorescent protein (YFP) and the gastric H,K-ATPase β-subunit (YFP-β) and cyan fluorescent protein (CFP) and the gastric H,K-ATPase α-subunit (CFP-α) were expressed in HEK-293 cells. Then plasma membrane delivery of wild-type CFP-α, wild-type YFP-β, and YFP-β mutants lacking one or two of the seven β-subunit glycosylation sites was determined using confocal microscopy and surface biotinylation. Expression of the wild-type YFP-β resulted in the plasma membrane localization of the protein, whereas the expressed CFP-α was retained intracellularly. When coexpressed, both CFP-α and YFP-β were delivered to the plasma membrane. Removing each of the seven glycosylation sites, except the second one, from the extracellular loop of YFP-β prevented plasma membrane delivery of the protein. Only the mutant lacking the second glycosylation site (Asn103Gln) was localized both intracellularly and on the plasma membrane. A double mutant lacking the first (Asn99Gln) and the second (Asn103Gln) glycosylation sites displayed intracellular accumulation of the protein. Therefore, six of the seven glycosylation sites in the β-subunit are essential for the plasma membrane delivery of the β-subunit of the gastric H,K-ATPase, whereas the second glycosylation site (Asn103), which is not conserved among the β-subunits from different species, is not critical for plasma delivery of the protein.

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Palmitoylation is not required for trafficking of human anion exchanger 1 to the cell surface

Biochemical Journal ◽

10.1042/bj20030847 ◽

2004 ◽

Vol 378 (3) ◽

pp. 1015-1021 ◽

Cited By ~ 10

Author(s):

Joanne C. CHEUNG ◽

Reinhart A. F. REITHMEIER

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Palmitic Acid ◽

Anion Exchanger ◽

Cell Surface Expression ◽

Co2 Removal ◽

Anion Exchanger 1 ◽

Hek 293 ◽

Hek 293 Cells ◽

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

British Journal of Pharmacology ◽

10.1038/sj.bjp.0703563 ◽

2000 ◽

Vol 131 (2) ◽

pp. 370-374 ◽

Cited By ~ 13

Author(s):

Z Qi ◽

K Murase ◽

S Obata ◽

M Sokabe

Keyword(s):

Plasma Membrane ◽

Extracellular Atp ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells

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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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Protein kinase C-mediated Ca2+ entry in HEK 293 cells transiently expressing human TRPV4

British Journal of Pharmacology ◽

10.1038/sj.bjp.0705443 ◽

2003 ◽

Vol 140 (2) ◽

pp. 413-421 ◽

Cited By ~ 55

Author(s):

Feng Xu ◽

Eisaku Satoh ◽

Toshihiko Iijima

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Hek 293 ◽

Hek 293 Cells ◽

Kinase C ◽

293 Cells

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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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The current produced by the E779A mutant rat Na + /K + pump α1-subunit expressed in HEK 293 cells

Biochimica et Biophysica Acta (BBA) - Biomembranes ◽

10.1016/s0005-2736(00)00289-3 ◽

2000 ◽

Vol 1509 (1-2) ◽

pp. 155-166 ◽

Cited By ~ 4

Author(s):

Stefan Zillikens ◽

Günter Gisselmann ◽

Helfried Günther Glitsch

Keyword(s):

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

Α1 Subunit ◽

Mutant Rat

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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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Calcitonin Receptor-Mediated Growth Suppression of HEK-293 Cells Is Accompanied by Induction of p21WAF1/CIP1 and G2/M Arrest

Molecular Endocrinology ◽

10.1210/mend.13.10.0359 ◽

1999 ◽

Vol 13 (10) ◽

pp. 1738-1750 ◽

Cited By ~ 18

Author(s):

Andreas Evdokiou ◽

Liza-Jane Raggatt ◽

Gerald J. Atkins ◽

David M. Findlay

Keyword(s):

Cell Cycle ◽

Cell Growth ◽

Antisense Oligonucleotide ◽

Kinase Inhibitor ◽

Cyclin Dependent Kinase ◽

Hek 293 ◽

Hek 293 Cells ◽

Cyclin Dependent Kinase Inhibitor ◽

293 Cells ◽

Cdc2 Activity

Abstract We investigated the mechanisms by which calcitonin (CT) suppresses cellular proliferation, using HEK-293 cells stably transfected with either the rat C1a CT receptor (CTR) or the insert-negative form of the human CTR. CT treatment of clonal cell lines expressing either receptor type, but not untransfected HEK-293 cells, strongly suppressed cell growth in a concentration-dependent manner. The reduction in cell growth with CT treatment could not be attributed to cellular necrosis or apoptotic cell death, the latter assessed by both DNA fragmentation analysis and caspase 3 (CPP-32) assay. Growth inhibition was associated with an accumulation of cells in the G2 phase of the cell cycle. CT treatment of the human and rat CTR-expressing cell lines resulted in a rapid and sustained induction of mRNA encoding the cyclin-dependent kinase inhibitor, p21WAF1/CIP1, increased levels of which were maintained at least 48 h after initiation of treatment. Western blot analysis showed a rapid corresponding increase in p21WAF1/CIP1 protein, whereas protein levels of another member of the cyclin-dependent kinase inhibitor family, p27kip1, were unchanged. In parallel with the induction of p21, CT treatment reduced levels of p53 mRNA and protein. CT treatment resulted in a specific cell cycle block in G2, which was associated with inhibition of Cdc2/cyclin B kinase activity as measured by histone H1 phosphorylation. There was no evidence for p21 association with this complex despite the inhibition of Cdc2 activity. Evidence that p21 induction was causative of cell growth suppression was obtained from p21 antisense oligonucleotide experiments. Treatment with a p21 antisense oligonucleotide blocked induction of p21 expression and significantly reduced the CT-mediated growth inhibition. These observations suggest that p21 is required for the G2 arrest in response to CT, but argue against a direct role of p21 in the inhibition of Cdc2 activity. These studies suggest a novel regulation of cell cycle progression by CT and will provide a basis for detailed examination of the molecular mechanisms involved.

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