scholarly journals Co-expression of metabotropic glutamate receptor type 1α with Homer-1a/Vesl-1S increases the cell surface expression of the receptor

1999 ◽  
Vol 341 (3) ◽  
pp. 795-803 ◽  
Author(s):  
Francisco CIRUELA ◽  
Mikhail M. SOLOVIEV ◽  
R. A. Jeffrey McILHINNEY
Keyword(s):  
Cell Surface ◽  
Metabotropic Glutamate Receptor ◽  
Inositol Phosphates ◽  
Immunofluorescent Staining ◽  
Cell Surface Expression ◽  
Immunochemical Analysis ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Metabotropic Glutamate ◽  
293 Cells

Homer-1a is a 30 kDa protein that forms part of a family of conserved Homer-related proteins that interact with the C-termini of the metabotropic glutamate receptors mGluR1α and mGluR5a. Analysis of HEK-293 cells by PCR showed that they contained mRNA coding for members of the Homer family with the predominant form being Homer-1b, which is consistent with the immunochemical analysis of these cells. Homer-1a could not be detected by immunochemical analysis. To examine the function of Homer-1a, HEK-293 cells were transfected with cDNA encoding mGluR1α or Homer-1a or co-transfected with both cDNAs. When cells were co-transfected with the cDNAs for both proteins, immunofluorescent staining and biotinylation of cell surface molecules revealed a significant increase in the amount of receptor present at the cell surface in contrast to cells transfected with mGluR1α cDNA alone. This finding was consistent with a concomitant increase in the production of inositol phosphates after treatment of the doubly transfected cells with agonist. Intracellular immunostaining for both proteins revealed that they were co-localized and underwent a redistribution into a large vesicular compartment when they were co-expressed.

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 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 Trafficking defects of the Southeast Asian ovalocytosis deletion mutant of anion exchanger 1 membrane proteins

2005 ◽  
Vol 392 (3) ◽  
pp. 425-434 ◽  
Author(s):  
Joanne C. Cheung ◽  
Emmanuelle Cordat ◽  
Reinhart A. F. Reithmeier
Keyword(s):  
Cell Surface ◽  
Anion Exchanger ◽  
Mdck Cells ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Intercalated Cells ◽  
Anion Exchanger 1 ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

Human AE1 (anion exchanger 1) is a membrane glycoprotein found in erythrocytes and as a truncated form (kAE1) in the BLM (basolateral membrane) of α-intercalated cells of the distal nephron, where they carry out electroneutral chloride/bicarbonate exchange. SAO (Southeast Asian ovalocytosis) is a dominant inherited haematological condition arising from deletion of Ala400–Ala408 in AE1, resulting in a misfolded and transport-inactive protein present in the ovalocyte membrane. Heterozygotes with SAO are able to acidify their urine, without symptoms of dRTA (distal renal tubular acidosis) that can be associated with mutations in kAE1. We examined the effect of the SAO deletion on stability and trafficking of AE1 and kAE1 in transfected HEK-293 (human embryonic kidney) cells and kAE1 in MDCK (Madin–Darby canine kidney) epithelial cells. In HEK-293 cells, expression levels and stabilities of SAO proteins were significantly reduced, and no mutant protein was detected at the cell surface. The intracellular retention of AE1 SAO in transfected HEK-293 cells suggests that erythroid-specific factors lacking in HEK-293 cells may be required for cell-surface expression. Although misfolded, SAO proteins could form heterodimers with the normal proteins, as well as homodimers. In MDCK cells, kAE1 was localized to the cell surface or the BLM after polarization, while kAE1 SAO was retained intracellularly. When kAE1 SAO was co-expressed with kAE1 in MDCK cells, kAE1 SAO was largely retained intracellularly; however, it also co-localized with kAE1 at the cell surface. We propose that, in the kidney of heterozygous SAO patients, dimers of kAE1 and heterodimers of kAE1 SAO and kAE1 traffic to the BLM of α-intercalated cells, while homodimers of kAE1 SAO are retained in the endoplasmic reticulum and are rapidly degraded. This results in sufficient cell-surface expression of kAE1 to maintain adequate bicarbonate reabsorption and proton secretion without dRTA.

scholarly journals Cross-Signaling between the Metabotropic Glutamate Receptor 2 and the Serotonin 2A Receptor in HEK-293 Cells

2015 ◽  
Vol 108 (2) ◽  
pp. 416a
Author(s):  
Lia Baki ◽  
Jason Younkin ◽  
Jose Miguel Eltit ◽  
Miguel Fribourg ◽  
Amr Ellaithy ◽  
...  
Keyword(s):  
Glutamate Receptor ◽  
Metabotropic Glutamate Receptor ◽  
Hek 293 ◽  
Serotonin 2A Receptor ◽  
Hek 293 Cells ◽  
Metabotropic Glutamate ◽  
293 Cells ◽  
Serotonin 2A

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.

Essential role for Ca2+in regulation of IL-1β secretion by P2X7nucleotide receptor in monocytes, macrophages, and HEK-293 cells

2003 ◽  
Vol 285 (2) ◽  
pp. C286-C299 ◽  
Author(s):  
Lalitha Gudipaty ◽  
Jonathan Munetz ◽  
Philip A. Verhoef ◽  
George R. Dubyak
Keyword(s):  
Essential Role ◽  
Signal Sequence ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Nucleotide Receptors ◽  
Hek 293 ◽  
External Stimulation ◽  
Hek 293 Cells ◽  
293 Cells

Interleukin (IL)-1β is a proinflammatory cytokine that elicits the majority of its biological activity extracellularly, but the lack of a secretory signal sequence prevents its export via classic secretory pathways. Efficient externalization of IL-1β in macrophages and monocytes can occur via stimulation of P2X7nucleotide receptors with extracellular ATP. However, the exact mechanisms by which the activation of these nonselective cation channels facilitates secretion of IL-1β remain unclear. Here we demonstrate a pivotal role for a sustained increase in cytosolic Ca2+to potentiate secretion of IL-1β via the P2X7receptors. Using HEK-293 cells engineered to coexpress P2X7receptors with mature IL-1β (mIL-1β), we show that activation of P2X7receptors results in a rapid secretion of mIL-1β by a process(es) that is dependent on influx of extracellular Ca2+and a sustained rise in cytosolic Ca2+. Moreover, reduction in extracellular Ca2+attenuates ∼90% of P2X7receptor-mediated IL-1β secretion but has no effect on enzymatic processing of precursor IL-1β (proIL-1β) to mIL-1β by caspase-1. Similar experiments with THP-1 human monocytes and Bac1.2F5 murine macrophages confirm the unique role of Ca2+in P2X7receptor-mediated secretion of IL-1β. In addition, we report that cell surface expression of P2X7receptors in the absence of external stimulation also results in enhanced release of IL-1β and that this can be repressed by inhibitors of P2X7receptors. We clarify an essential role for Ca2+in ATP-induced IL-1β secretion and indicate an additional role of P2X7receptors as enhancers of the secretory apparatus by which IL-1β is released.

scholarly journals Protein Kinase C Activation Regulates Human Serotonin Transporters in HEK-293 Cells via Altered Cell Surface Expression

1997 ◽  
Vol 17 (1) ◽  
pp. 45-57 ◽  
Author(s):  
Yan Qian ◽  
Aurelio Galli ◽  
Sammanda Ramamoorthy ◽  
Stefania Risso ◽  
Louis J. DeFelice ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cell Surface ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Hek 293 ◽  
Kinase C ◽  
293 Cells ◽  
Altered Cell ◽  
Protein Kinase C Activation
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