Cloning of Trp1β isoform from rat brain: immunodetection and localization of the endogenous Trp1 protein

1999 ◽  
Vol 276 (4) ◽  
pp. C969-C979 ◽  
Author(s):  
Weiching Wang ◽  
Brian O’Connell ◽  
Raymond Dykeman ◽  
Takayuki Sakai ◽  
Christine Delporte ◽  
...  
Keyword(s):  
Amino Acids ◽  
Rat Brain ◽  
Protein A ◽  
Protein S ◽  
Polyclonal Antiserum ◽  
Cell Fractionation ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Mouse Tissues

The Trp gene product has been proposed as a candidate protein for the store-operated Ca2+channel, but the Trp protein(s) has not been identified in any nonexcitable cell. We report here the cloning of a rat brain Trp1β cDNA and detection and immunolocalization of the endogenous and expressed Trp1 protein. A 400-bp product, with >95% homology to mouse Trp1, was amplified from rat submandibular gland RNA. Rat-specific primers were used for cloning of a full-length rat brain Trp1β cDNA (rTrp1), encoding a protein of 759 amino acids. Northern blot analysis demonstrated the transcript in several rat and mouse tissues. The peptide (amino acids 523–536) was used to generate a polyclonal antiserum. The affinity-purified antibody 1) immunoprecipitated human Trp1 (hTrp1) from transfected HEK-293 cells, 2) reacted with a protein of ∼92 kDa, but not with hTrp3, in membranes of hTrp3-expressing HEK-293 cells, and 3) reacted with proteins of 92 and 56 kDa in human and rat brain membranes. Confocal microscopy and cell fractionation demonstrated that endogenous and expressed hTrp1 and expressed hTrp3 proteins were localized in the plasma membrane of HEK-293 cells, consistent with their proposed role in Ca2+ influx. The data demonstrate for the first time the presence of Trp1 protein in a nonexcitable cell.

scholarly journals Multidrug resistance-associated protein 9 (ABCC12) is present in mouse and boar sperm

2007 ◽  
Vol 406 (1) ◽  
pp. 31-40 ◽  
Author(s):  
Nobuhito Ono ◽  
Ingrid Van der Heijden ◽  
George L. Scheffer ◽  
Koen Van de Wetering ◽  
Elizabeth Van Deemter ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Kidney Cells ◽  
Cell Fractionation ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells ◽  
Hek 293 Cells ◽  
Drug Conjugates ◽  
293 Cells ◽  
Boar Sperm ◽  
Alternatively Spliced

The human and murine genes for MRP9 (multidrug resistance-associated protein 9; ABCC12) yield many alternatively spliced RNAs. Using a panel of monoclonal antibodies, we detected full-length Mrp9 only in testicular germ cells and mouse sperm; we obtained no evidence for the existence of the truncated 100 kDa MRP9 protein reported previously. In contrast with other MRPs, neither murine Mrp9 nor the human MRP9 produced in MRP9-transfected HEK-293 cells (human embryonic kidney cells) appears to contain N-linked carbohydrates. In mouse and boar sperm, Mrp9 localizes to the midpiece, a structure containing all sperm mitochondria. However, immunolocalization microscopy and cell fractionation studies with transfected HEK-293 cells and mouse testis show that MRP9/Mrp9 does not localize to mitochondria. In HEK-293 cells, it is predominantly localized in the endoplasmic reticulum. We have been unable to demonstrate transport by MRP9 of substrates transported by other MRPs, such as drug conjugates and other organic anions.

scholarly journals Optimal expression of cloned NMDAR1/NMDAR2A heteromeric glutamate receptors: a biochemical characterization

1993 ◽  
Vol 296 (3) ◽  
pp. 877-883 ◽  
Author(s):  
M Cik ◽  
P L Chazot ◽  
F A Stephenson
Keyword(s):  
Rat Brain ◽  
Biochemical Characterization ◽  
Radioligand Binding ◽  
Fold Increase ◽  
Binding Activity ◽  
Immunological Methods ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Adult Rat ◽  
293 Cells

The N-methyl-D-aspartate R1 (NMDAR1) and NMDAR2A subunits were expressed transiently either alone or in combination in human embryonic kidney (HEK) 293 cells. The biochemical and pharmacological properties of the cloned receptors were compared with those of adult rat brain NMDA receptors using both immunological methods with a newly developed anti-NMDAR2A-(1435-1445) antibody and [3H]MK801 radioligand binding activity. Anti-NMDAR2A-(1435-1445) antibodies recognized specifically four immunoreactive species with M(r)s of 180,000, 122,000, 97,000 and 54,000 in rat brain, but only a single band of M(r) 180,000 in HEK 293 cells singly transfected with plasmid pCISNMDAR2A. N-deglycosylation of HEK cell membranes yielded a 165,000-M(r) immunoreactive species, which is in agreement with the size predicted from the cDNA sequence for the mature NMDAR2A subunit. Co-expression of NMDAR1 and NMDAR2A subunits in HEK 293 cells resulted in cell death. Thus conditions were established for the optimum expression of heteromeric receptors in viable cells, including a requirement for DL-2-amino-5-phosphonopentanoic acid (AP5) in the culture medium post-transfection. Cells transfected with pCISNMDAR1 and pCISNMDAR2A combined yielded a 10-fold increase in the number of [3H]MK801 binding sites compared with single subunit expression. MK801 had similar affinity for the expressed receptors as for those found in adult rat and mouse brain. These results demonstrate that the NMDAR1 and NMDAR2A receptor subunits co-assemble to form a heteromeric complex with properties similar to those of the native receptors of adult mammalian forebrain. Furthermore, the conditions reported for maximal transient expression provide a basis for further structure-activity studies.

Potent and selective tools to investigate neuropeptide Y receptors in the central and peripheral nervous systems: BIBO3304 (Y1) and CGP71683A (Y5)

2000 ◽  
Vol 78 (2) ◽  
pp. 116-125 ◽  
Author(s):  
Yvan Dumont ◽  
Alain Cadieux ◽  
Henri Doods ◽  
Alain Fournier ◽  
Rémi Quirion
Keyword(s):  
Neuropeptide Y ◽  
Rat Brain ◽  
Binding Sites ◽  
Rat Colon ◽  
Receptor Subtypes ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
High Affinity ◽  
293 Cells ◽  
Receptor Cdna

We have evaluated 3 newly developed neuropeptide Y receptor antagonists in various in vitro binding and bioassays: BIBO3304 (Y1), T4[NPY33-36]4 (Y2), and CGP71683A (Y5). In rat brain homogenates, BIBO3304 competes for the same population of [125I][Leu31,Pro34] peptide YY (PYY) binding sites (75%) as BIBP3226, but with a 10 fold greater affinity (IC50 of 0.2 ± 0.04 nM for BIBO3304 vs. 2.4 ± 0.07 nM for BIBP3226),while CGP71683A has high affinity for 25% of specific [125I][Leu31,Pro34]PYY binding sites. Both BIBO3304 and CGP71683A (at 1.0 µM) were unable to compete for a significant proportion of specific [125I]PYY3-36/Y2 sites. The purported Y2 antagonist T4[NPY33-36]4 competed against [125I]PYY3-36 binding sites with an affinity of 750 nM. These results were confirmed in HEK 293 cells transfected with either the rat Y1, Y2, Y4, or Y5 receptor cDNA. BIBO3304, but not CGP71683A, competed with high affinity for [125I][Leu31,Pro34]PYY binding sites in HEK 293 cells transfected with the rat Y1 receptor cDNA, whereas the reverse profile was observed upon transfection with the rat Y5 receptor cDNA. Additionally, both molecules were inactive at Y2 and Y4 receptor subtypes expressed in HEK 293 cells. Receptor autoradiographic studies revealed the presence of [125I][Leu31,Pro34]PYY/BIBO3304-insensitive sites in the rat brain as reported previously for BIBP3226. Finally, the selective antagonistic properties of BIBO3304 were demonstrated in a Y1 bioassay (rabbit saphenous vein; pA2 value of 9.04) while being inactive in Y2 (rat vas deferens) and Y4 (rat colon) bioassays. These results confirm the high affinity and selectivity of BIBO3304 and CGP71683A for the Y1 and Y5 receptor subtypes, respectively, while the purported Y2 antagonist, T4[NPY33-36]4 possesses rather low affinity for this receptor.Key words: NPY receptor antagonist, receptor subtypes, bioassays, receptor binding assays, autoradiographic studies, receptor distribution.

scholarly journals Allosteric activation of the extracellular Ca2+-sensing receptor by L-amino acids enhances ERK1/2 phosphorylation

2007 ◽  
Vol 404 (1) ◽  
pp. 141-149 ◽  
Author(s):  
Heather J. Lee ◽  
Hee-Chang Mun ◽  
Narelle C. Lewis ◽  
Michael F. Crouch ◽  
Emma L. Culverston ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Hormone Secretion ◽  
Fine Tuning ◽  
Significant Enhancement ◽  
Acid Mixture ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
The Impact

The calcium-sensing receptor (CaR) mediates feedback control of Ca2+o (extracellular Ca2+) concentration. Although the mechanisms are not fully understood, the CaR couples to several important intracellular signalling enzymes, including PI-PLC (phosphoinositide-specific phospholipase C), leading to Ca2+i (intracellular Ca2+) mobilization, and ERK1/2 (extracellular-signal-regulated kinase 1/2). In addition to Ca2+o, the CaR is activated allosterically by several subclasses of L-amino acids, including the aromatics L-phenylalanine and L-tryptophan. These amino acids enhance the Ca2+o-sensitivity of Ca2+i mobilization in CaR-expressing HEK-293 (human embryonic kidney) cells and normal human parathyroid cells. Furthermore, on a background of a physiological fasting serum L-amino acid mixture, they induce a small, but physiologically significant, enhancement of Ca2+o-dependent suppression of PTH (parathyroid hormone) secretion. The impact of amino acids on CaR-stimulated ERK1/2, however, has not been determined. In the present study, we examined the effects of L-amino acids on Ca2+o-stimulated ERK1/2 phosphorylation as determined by Western blotting and a newly developed quantitative assay (SureFire). L-Amino acids induced a small, but significant, enhancement of Ca2+o-stimulated ERK1/2. In CaR-expressing HEK-293 cells, 10 mM L-phenylalanine lowered the EC50 for Ca2+o from approx. 2.3 to 2.0 mM in the Western blot assay and from 3.4 to 2.9 mM in the SureFire assay. The effect was stereoselective (L>D), and another aromatic amino acid, L-tryptophan, was also effective. The effects of amino acids were investigated further in HEK-293 cells that expressed the CaR mutant S169T. L-Phenylalanine normalized the EC50 for Ca2+o-stimulated Ca2+i mobilization from approx. 12 mM to 5.0 mM and ERK1/2 phosphorylation from approx. 4.6 mM to 2.6 mM. Taken together, the data indicate that L-phenylalanine and other amino acids enhance the Ca2+o-sensitivity of CaR-stimulated ERK1/2 phosphorylation; however, the effect is comparatively small and operates in the form of a fine-tuning mechanism.

scholarly journals MUPP1 complexes renal K+ channels to alter cell surface expression and whole cell currents

2009 ◽  
Vol 297 (1) ◽  
pp. F36-F45 ◽  
Author(s):  
Aleksandra Sindic ◽  
Chunfa Huang ◽  
An-Ping Chen ◽  
Yaxian Ding ◽  
William A. Miller-Little ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Surface ◽  
Pdz Domain ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Kidney Cortex ◽  
Whole Cell ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

We previously found that the Ca2+-sensing receptor (CaR) interacts with and inactivates the inwardly rectifying K+ channel Kir4.2 that is expressed in the kidney cortex and that has a COOH-terminal PDZ domain. To identify potential scaffolding proteins that could organize a macromolecular signaling complex involving the CaR and Kir4.2, we used yeast two-hybrid cloning with the COOH-terminal 125 amino acids (AA) of Kir4.2 as bait to screen a human kidney cDNA library. We identified two independent partial cDNAs corresponding to the COOH-terminal 900 AA of MUPP1, a protein containing 13 PDZ binding domains that is expressed in the kidney in tight junctions and lateral borders of epithelial cells. When expressed in human embryonic kidney (HEK)-293 cells, Kir4.2 coimmunoprecipitates reciprocally with MUPP1 but not with a Kir4.2 construct lacking the four COOH-terminal amino acids, Kir5.1, or the CaR. MUPP1 and Kir4.2 coimmunoprecipitate reciprocally from rat kidney cortex extracts. Coexpression of MUPP1 with Kir4.2 in HEK-293 cells leads to reduced cell surface expression of Kir4.2 as assessed by cell surface biotinylation. Coexpression of MUPP1 and Kir4.2 in Xenopus oocytes results in reduced whole cell currents compared with expression of Kir4.2 alone, whereas expression of Kir4.2ΔPDZ results in minimal currents and is not affected by coexpression with MUPP1. Immunofluorescence studies of oocytes demonstrate that MUPP1 reduces Kir4.2 membrane localization. These results indicate that Kir4.2 interacts selectively with MUPP1 to affect its cell surface expression. Thus MUPP1 and Kir4.2 may participate in a protein complex in the nephron that could regulate transport of K+ as well as other ions.

scholarly journals Glucagon receptor recycling: role of carboxyl terminus, β-arrestins, and cytoskeleton

2008 ◽  
Vol 295 (5) ◽  
pp. C1230-C1237 ◽  
Author(s):  
Lada Krilov ◽  
Amy Nguyen ◽  
Teruo Miyazaki ◽  
Cecilia G. Unson ◽  
Bernard Bouscarel
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Actin Filaments ◽  
Carboxyl Terminus ◽  
Glucagon Receptor ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Prolonged Duration ◽  
High Concentrations

Glucagon receptor (GR) activity and expression are altered in several diseases, including Type 2 diabetes. Previously, we investigated the mechanism of GR desensitization and internalization. The present study focused on the fate of internalized GR. Using both hamster hepatocytes and human embryonic kidney (HEK)-293 cells, we showed that internalized GR recycled to the plasma membrane within 30–60 min following stimulation of the cells with 100 nM glucagon. In HEK-293 cells and during recycling, GR colocalized with Rab4, Rab11, β-arrestin1, β-arrestin2, and actin filaments, in the cytosolic and/or perinuclear domains. Glucagon treatment triggered redistribution of actin filaments from the plasma membrane to the cytosol. GR coimmunoprecipitated with β-actin in both hepatocytes and HEK-293 cells. Downregulation of β-arrestin1 and β-arrestin2 or disruption of the cytoskeleton inhibited recycling, but not internalization of GR. Deletion of the GR carboxyl-terminal 70 amino acids abolished internalization of GR in response to glucagon while deletion of the last 40 amino acids only did not affect GR internalization and recycling. After exposure of the cells to either high concentrations or prolonged duration of glucagon, GR colocalized with lysosomes. GR degradation was inhibited by lysosomal, but not proteosomal, inhibitors. In conclusion, GR recycles through Rab4- and Rab11- positive vesicles. The actin cytoskeleton, β-arrestin1, β-arrestin2, and the receptor's carboxyl terminus are involved in recycling. Prolonged stimulation with glucagon targets GR for degradation in lysosomes. Therefore, the present study provides a better understanding of the GR recycling mechanism, which could become useful in the treatment of certain diseases, including diabetes.

scholarly journals Characteristics and requirements of basal autophagy in HEK 293 cells

Autophagy ◽  
2013 ◽  
Vol 9 (9) ◽  
pp. 1407-1417 ◽  
Author(s):  
Patience Musiwaro ◽  
Matthew Smith ◽  
Maria Manifava ◽  
Simon A. Walker ◽  
Nicholas T. Ktistakis
Keyword(s):  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

Halothane Inhibition of Recombinant Cardiac L-type Ca2+ Channels Expressed in HEK-293 Cells

2005 ◽  
Vol 103 (6) ◽  
pp. 1156-1166 ◽  
Author(s):  
Kevin J. Gingrich ◽  
Son Tran ◽  
Igor M. Nikonorov ◽  
Thomas J. Blanck
Keyword(s):  
Charge Transfer ◽  
Calcium Channels ◽  
Dependent Manner ◽  
Current Time ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Dependent Inactivation ◽  
Voltage Dependent

Background Volatile anesthetics depress cardiac contractility, which involves inhibition of cardiac L-type calcium channels. To explore the role of voltage-dependent inactivation, the authors analyzed halothane effects on recombinant cardiac L-type calcium channels (alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1), which differ by the alpha2/delta1 subunit and consequently voltage-dependent inactivation. Methods HEK-293 cells were transiently cotransfected with complementary DNAs encoding alpha1C tagged with green fluorescent protein and beta2a, with and without alpha2/delta1. Halothane effects on macroscopic barium currents were recorded using patch clamp methodology from cells expressing alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1 as identified by fluorescence microscopy. Results Halothane inhibited peak current (I(peak)) and enhanced apparent inactivation (reported by end pulse current amplitude of 300-ms depolarizations [I300]) in a concentration-dependent manner in both channel types. alpha2/delta1 coexpression shifted relations leftward as reported by the 50% inhibitory concentration of I(peak) and I300/I(peak)for alpha1Cbeta2a (1.8 and 14.5 mm, respectively) and alpha1Cbeta2aalpha2/delta1 (0.74 and 1.36 mm, respectively). Halothane reduced transmembrane charge transfer primarily through I(peak) depression and not by enhancement of macroscopic inactivation for both channels. Conclusions The results indicate that phenotypic features arising from alpha2/delta1 coexpression play a key role in halothane inhibition of cardiac L-type calcium channels. These features included marked effects on I(peak) inhibition, which is the principal determinant of charge transfer reductions. I(peak) depression arises primarily from transitions to nonactivatable states at resting membrane potentials. The findings point to the importance of halothane interactions with states present at resting membrane potential and discount the role of inactivation apparent in current time courses in determining transmembrane charge transfer.

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