Physical and functional interactions between the serotonin transporter and the neutral amino acid transporter ASCT2

2016 ◽  
Vol 473 (13) ◽  
pp. 1953-1965 ◽  
Author(s):  
Pascal Seyer ◽  
Franck Vandermoere ◽  
Elisabeth Cassier ◽  
Joël Bockaert ◽  
Philippe Marin
Keyword(s):  
Plasma Membrane ◽  
Molecular Mechanisms ◽  
Physical Interaction ◽  
Maximal Velocity ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Concomitant Reduction ◽  
Solute Carrier ◽  
5 Ht Uptake

The activity of serotonergic systems depends on the reuptake of extracellular serotonin via its plasma membrane serotonin [5-HT (5-hydroxytryptamine)] transporter (SERT), a member of the Na+/Cl−-dependent solute carrier 6 family. SERT is finely regulated by multiple molecular mechanisms including its physical interaction with intracellular proteins. The majority of previously identified SERT partners that control its functional activity are soluble proteins, which bind to its intracellular domains. SERT also interacts with transmembrane proteins, but its association with other plasma membrane transporters remains to be established. Using a proteomics strategy, we show that SERT associates with ASCT2 (alanine–serine–cysteine–threonine 2), a member of the solute carrier 1 family co-expressed with SERT in serotonergic neurons and involved in the transport of small neutral amino acids across the plasma membrane. Co-expression of ASCT2 with SERT in HEK (human embryonic kidney)-293 cells affects glycosylation and cell-surface localization of SERT with a concomitant reduction in its 5-HT uptake activity. Conversely, depletion of cellular ASCT2 by RNAi enhances 5-HT uptake in both HEK-293 cells and primary cultured mesencephalon neurons. Mimicking the effect of ASCT2 down-regulation, treatment of HEK-293 cells and neurons with the ASCT2 inhibitor D-threonine also increases 5-HT uptake. Moreover, D-threonine does not enhance further the maximal velocity of 5-HT uptake in cells depleted of ASCT2. Collectively, these findings provide evidence for a complex assembly involving SERT and a member of another solute carrier family, which strongly influences the subcellular distribution of SERT and the reuptake of 5-HT.

scholarly journals Regulation of GPR54 Signaling by GRK2 and β-Arrestin

2009 ◽  
Vol 23 (12) ◽  
pp. 2060-2074 ◽  
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.

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

2004 ◽  
Vol 378 (3) ◽  
pp. 1015-1021 ◽  
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.

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

2007 ◽  
Vol 403 (1) ◽  
pp. 97-108 ◽  
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.

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

2000 ◽  
Vol 131 (2) ◽  
pp. 370-374 ◽  
Author(s):  
Z Qi ◽  
K Murase ◽  
S Obata ◽  
M Sokabe
Keyword(s):  
Plasma Membrane ◽  
Extracellular Atp ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

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

Endocrinology ◽  
2007 ◽  
Vol 148 (7) ◽  
pp. 3236-3245 ◽  
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.

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

2001 ◽  
Vol 281 (6) ◽  
pp. C1797-C1803 ◽  
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.

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

2007 ◽  
Vol 407 (3) ◽  
pp. 363-372 ◽  
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.

scholarly journals Attenuation of Oxidative Stress in HEK 293 Cells by the TCM Constituents Schisanhenol, Baicalein, Resveratrol or Crocetin and Two Defined Mixtures

2015 ◽  
Vol 18 (4) ◽  
pp. 661 ◽  
Author(s):  
John Richard Bend ◽  
Xue Yan (Iris) Xue Yan Xia ◽  
Daofeng Chen ◽  
Abudi Awaysheh ◽  
Andrea Lo ◽  
...  
Keyword(s):  
Real Time ◽  
Molecular Mechanisms ◽  
Nitrosative Stress ◽  
Fluorescent Protein ◽  
Antioxidant Properties ◽  
Oxidative And Nitrosative Stress ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

PURPOSE:  Our working hypothesis is that single bioactive phytochemicals with antioxidant properties that are important constituents of Traditional Chinese Medicine (TCM) and their defined mixtures have potential as chemoprotective agents for chronic conditions characterized by oxidative and nitrosative stress, including Alzheimer’s. Here we evaluate the ability of baicalein, crocetin, trans-resveratrol or schisanhenol and two defined mixtures of these TCM phytochemicals to attenuate the toxicity resulting from exposure to cell permeant t-butyl hydroperoxide (tBPH) in wild-type and bioengineered (to express choline acetyltransferase) HEK 293 cells. METHODS: Endpoints of tBHP-initiated oxidative and nitrosative stress in both types of HEK 293 cells and its attenuation by TCM constituents and mixtures included cytotoxicity (LDH release); depletion of intracellular glutathione (GSH); formation of S-glutathionylated proteins; oxidative changes to the disulfide proteome; and real-time changes in intracellular redox status. RESULTS: At low µM concentrations, each of the TCM constituents and mixtures effectively attenuated intracellular toxicity due to exposure of HEK 293 cells to 50 or 250 µM tBHP for 30 min to 3 h. Confocal microscopy of HEK 293 cells transfected with mutated green fluorescent protein (roGFP2) showed effective attenuation of tBHP oxidation by baicalein in real time. Three redox-regulated proteins prominent in the disulfide proteome of HEK 293 cells were identified by MALDI-TOF mass spectrometry. CONCLUSIONS: We conclude that single TCM chemicals and their simple mixtures have potential for use in adjunct chemoprotective therapy. Advantages of mixtures compared to single TCM constituents include the ability to combine compounds with varying molecular mechanisms of cytoprotection for enhanced biological activity; and to combine chemicals with complementary pharmacokinetic properties to increase half-life and prolong activity in vivo. This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page.

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