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 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.

Structural and Functional Interactions of KCl Cotransport Proteins KCC1 and KCC3 in Sickle and Normal Erythrocyte Membranes

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2474-2474
Author(s):  
Mary Risinger ◽  
Jesse Rinehart ◽  
Scott Crable ◽  
Anna Ottlinger ◽  
Richard Winkelmann ◽  
...  
Keyword(s):  
Western Blot ◽  
Specific Antibody ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Erythrocyte Membranes ◽  
Western Blots ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
In Cells

Abstract The KCl cotransporter (KCC) mediates volume reduction in normal reticulocytes and exaggerated KCC activity in sickle red blood cells (SS RBC) (Joiner et al, Blood109:1728, 2007) contributes to pathological dehydration that potentiates sickling. Three separate genes (KCC1, KCC3, KCC4) are expressed in RBC (Crable et al, Exp. Hem.33:624, 2005). KCC1 and KCC3 proteins have been shown to interact in ex vivo expression systems (Simard et al, JBC282(25):18083, 2007), and co-expression of an N-terminal truncation of KCC1 reduces KCC activity mediated by full-length KCC1 or KCC3 (Casula et al. JBC276:41870, 2001), suggesting functional interaction. We show here via western blot analysis that SS RBC membranes contain more KCC1 protein (relative to KCC3) than AA RBC, independent of the reticulocytosis of sickle blood. Immunoprecipitation of solubilized SS RBC membranes with KCC3-specific antibody yielded a band at 125 kD on SDS PAGE which contained KCC1, as identified by western blotting with KCC1-specific antibody and by TOF mass spectroscopy. The effect of co-expression of KCC1 and KCC3 on KCC activity was assessed by measuring NEM-stimulated, Cl-dependent, (ouabain + bumetanide)-insensitive Rb uptake in HEK 293 cells. The Flip-In T-rex HEK 293 cell line (Invitrogen) containing a tetracycline-response promoter was transfected with a pcDNA5a plasmid containing KCC3a cDNA. Recombination of the plasmid with the integrated tet-promoter construct inserts the KCC3a gene under control of a tetracycline-responsive promoter. These cells were subsequently transduced with a retroviral vector (SF-91. Hildinger et at, Gene Ther. 5:1575, 1998) containing KCC1 cDNA linked to a GFP cassette. Control cells contained SF-91 vector lacking KCC1. Cells were selected for GFP expression and grown in the absence (un-induced, no KCC3a expression) or presence of tetracycline (induced, KCC3a expression). From this binary matrix, four types of cells were obtained: Cells with no additional KCC expression, representing endogenous KCC activity; cells with only KCC1 or KCC3a expression; cells with both KCC1 and KCC3a expression. Western blots indicated similar KCC1 expression in cells with KCC1 only and [KCC1 + KCC3] and similar KCC3 expression in cells with KCC3 only and [KCC1 + KCC3]. Thus, the expression of neither isoform was affected by the presence of the other. KCC activity in cells overexpressing KCC1 only was similar to endogenous activity in HEK 293 cells; i.e., transport activity of KCC1 alone was minimal. Cells overexpressing KCC3 only had a 5-fold increase in KCC activity over endogenous levels. When KCC1 was co-expressed with KCC3 in [KCC1 + KCC3] cells, an additional 50% increase in KCC activity was observed (p < 0.05 by paired t-test, N=4), despite similar levels of KCC3 expression by western blot analysis. This synergistic effect was dependent on the cytoplasmic N-terminus of KCC1, as it was not seen when the first 39 amino acids of KCC1 were removed. Interestingly, removal of the entire cytoplasmic N-terminal domain (117 aa) produced an inhibitory effect when co-expressed with KCC3a in HEK cells, as previously reported in Xenopus oocytes (Casula et al.). These data indicate that KCC1 and KCC3 interact structurally and functionally in RBC membranes, and provide another potential mechanism for regulation of KCC activity via multimeric associations between KCC isoforms. Thus, KCC activity could be modulated not only by transcriptional mechanisms and post-translational modification (phosphorylation), but also by altering the ratios of KCC isoforms or the kinetics of their association. We speculate that higher levels of KCC1 protein relative to KCC3 in SS RBC membranes could account for higher KCC activity in these cells relative to AA RBC.

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 BRET Analysis of GPCR Dimers in Neurons and Non-Neuronal Cells: Evidence for Inactive, Agonist, and Constitutive Conformations

2021 ◽  
Vol 22 (19) ◽  
pp. 10638
Author(s):  
Chayma El Khamlichi ◽  
Laetitia Cobret ◽  
Jean-Michel Arrang ◽  
Séverine Morisset-Lopez
Keyword(s):  
Cortical Neurons ◽  
G Protein Coupled Receptors ◽  
Constitutive Activity ◽  
Western Blots ◽  
Inverse Agonists ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Functional Consequences ◽  
G Protein Coupled

G-protein-coupled receptors (GPCRs) are dimeric proteins, but the functional consequences of the process are still debated. Active GPCR conformations are promoted either by agonists or constitutive activity. Inverse agonists decrease constitutive activity by promoting inactive conformations. The histamine H3 receptor (H3R) is the target of choice for the study of GPCRs because it displays high constitutive activity. Here, we study the dimerization of recombinant and brain H3R and explore the effects of H3R ligands of different intrinsic efficacy on dimerization. Co-immunoprecipitations and Western blots showed that H3R dimers co-exist with monomers in transfected HEK 293 cells and in rodent brains. Bioluminescence energy transfer (BRET) analysis confirmed the existence of spontaneous H3R dimers, not only in living HEK 293 cells but also in transfected cortical neurons. In both cells, agonists and constitutive activity of the H3R decreased BRET signals, whereas inverse agonists and GTPγS, which promote inactive conformations, increased BRET signals. These findings show the existence of spontaneous H3R dimers not only in heterologous systems but also in native tissues, which are able to adopt a number of allosteric conformations, from more inactive to more active states.

Gene expression profiles in HEK-293 cells with low or high store-operated calcium entry: can regulatory as well as regulated genes be identified?

2005 ◽  
Vol 21 (1) ◽  
pp. 14-33 ◽  
Author(s):  
Tatiana K. Zagranichnaya ◽  
Xiaoyan Wu ◽  
Arpad M. Danos ◽  
Mitchel L. Villereal
Keyword(s):  
Gene Expression ◽  
Transient Receptor Potential ◽  
Expression Profiles ◽  
Physiological Role ◽  
Gene Expression Profiles ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

Gene expression profiles were generated using cDNA microarray technology for clones of human embryonic kidney (HEK)-293 cells selected to have either high or low levels of store-operated Ca2+ entry (SOCE). For five high clones, three low clones, and control HEK-293 cells, duplicate Affymetrix U133A human gene arrays were run after extraction of total RNA from cells growing in the presence of serum. Of the ∼22,000 genes represented on the microarray, 58 genes had readings at least twofold higher, while 32 genes had readings at least twofold lower, in all five high SOCE clones compared with control HEK-293 cells. In the low SOCE clones, 92 genes had readings at least twofold higher, while 58 genes had readings at least twofold lower, than in HEK-293 cells. Microarray results were confirmed for 18 selected genes by real-time RT-PCR analysis; for six of those genes, predicted changes in the low SOCE clone were confirmed by an alternative method, monitoring mRNA levels in HEK-293 with SOCE decreased by expression of small interfering (si)RNA to canonical transient receptor potential protein-1. Genes regulated by SOCE are involved in signal transduction, transcription, apoptosis, metabolism, and membrane transport. These data provide insight into the physiological role of SOCE. In addition, a potential regulator of SOCE, insulin receptor substrate (IRS)-2, has been identified. A reduction of IRS-2 levels by siRNA methods in two high clones dramatically reduced SOCE, whereas overexpression of IRS-2 in a low SOCE clone elevated SOCE.

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 Calcium-sensing receptors control CYP27B1-luciferase expression. Transcriptional and post-transcriptional mechanisms

2021 ◽  
Author(s):  
Alice Huang ◽  
Lenah Binmahfouz ◽  
Dale P Hancock ◽  
Paul H Anderson ◽  
Donald T Ward ◽  
...  
Keyword(s):  
Fold Increase ◽  
Firefly Luciferase ◽  
Inhibitory Effect ◽  
Renilla Luciferase ◽  
Luciferase Expression ◽  
Mrna Levels ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Calcium Sensing ◽  
293 Cells

Abstract 25-hydroxyvitamin D 1α-hydroxylase (encoded by CYP27B1), which catalyses the synthesis of 1,25-dihydroxyvitamin D3, is subject to negative or positive modulation by extracellular Ca 2+ (Ca 2+o) depending on the tissue. However, the Ca 2+ sensors and underlying mechanisms are unidentified. We tested whether calcium-sensing receptors (CaSRs) mediate Ca 2+o-dependent control of 1α-hydroxylase using HEK-293 cells stably expressing the CaSR (HEK-CaSR cells). In HEK-CaSR cells, but not control HEK-293 cells, co-transfected with reporter genes for CYP27B1-Photinus pyralis (firefly) luciferase and control Renilla luciferase, an increase in Ca 2+o from 0.5 to 3.0 mM induced a 2-3 fold increase in firefly-luciferase activity as well as mRNA and protein levels. Surprisingly, firefly-luciferase was specifically suppressed at Ca 2+o ≥ 5.0 mM, demonstrating biphasic Ca 2+o control. Both phases were mediated by CaSRs as revealed by positive and negative modulators. However, Ca 2+o induced simple monotonic increases in firefly-luciferase and endogenous CYP27B1 mRNA levels, indicating that the inhibitory effect of high Ca 2+o was post-transcriptional. Studies with inhibitors and the CaSR C-terminal mutant T888A identified roles for PKC, phosphorylation of T888, and ERK1/2 in high Ca 2+o-dependent suppression of firefly-luciferase. Blockade of both PKC and ERK1/2 abolished Ca 2+o-stimulated firefly-luciferase, demonstrating that either PKC or ERK1/2 is sufficient to stimulate the CYP27B1 promoter. A key CCAAT box (–74 bp to –68 bp), which is regulated downstream of PKC and ERK1/2 was required for both basal transcription and Ca 2+o-mediated transcriptional upregulation. The CaSR mediates Ca 2+o-dependent transcriptional upregulation of 1α-hydroxylase and an additional CaSR-mediated mechanism is identified by which Ca 2+o can promote luciferase and possibly 1α-hydroxylase breakdown.

scholarly journals Activation of Vitamin D Receptor by the Wilms' Tumor GeneProduct Mediates Apoptosis of Renal Cells

2001 ◽  
Vol 12 (6) ◽  
pp. 1188-1196 ◽  
Author(s):  
KAY-DIETRICH WAGNER ◽  
NICOLE WAGNER ◽  
VIKAS P. SUKHATME ◽  
HOLGER SCHOLZ
Keyword(s):  
Vitamin D ◽  
Vitamin D Receptor ◽  
Kidney Development ◽  
Wilms Tumor ◽  
Kidney Cortex ◽  
Mrna Levels ◽  
Renal Cells ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

Abstract. The Wilms' tumor transcription factor WT1 is required for kidney development, but little is known about WT1 downstream signaling in renal cells. This study reported an approximately fivefold upregulation of vitamin D receptor (VDR) mRNA and protein in human embryonic kidney (HEK) 293 cells that stably expressed WT1 at a level comparable to the developing kidney in vivo. Co-transfection of HEK 293 cells with expression plasmids encoding four different WT1 splicing variants stimulated mouse vdr promoter activity more than fourfold. A 201-bp fragment was identified in the proximal vdr promoter that was required for transactivation by WT1. This critical sequence contained a predicted WT1 consensus site, which bound to recombinant WT1 protein. Temporal changes of vdr and wt1 mRNA levels in developing rat kidneys were correlated closely. The active metabolite 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) strongly inhibited the proliferation of wt1-transfected HEK 293 cells. Exposure to 1,25-(OH)2D3 caused apoptosis of cultured wt1 immunopositive cells from mouse embryonic kidney cortex. These findings suggest that transcriptional activation of the VDR by WT1 can mediate programmed death of renal embryonic cells in response to 1,25-(OH)2D3. The results provide the first evidence for a role of the vitamin D endocrine system in renal cell growth and differentiation during development.

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