MAGI-1 interacts with Slo1 channel proteins and suppresses Slo1 expression on the cell surface

Large conductance Ca2+-activated K+ (BKCa) channels encoded by the Slo1 gene (also known as KCNMA1) are physiologically important in a wide range of cell types and form complexes with a number of other proteins that affect their function. We performed a yeast two-hybrid screen to identify proteins that interact with BKCa channels using a bait construct derived from domains in the extreme COOH-terminus of Slo1. A protein known as membrane-associated guanylate kinase with inverted orientation protein-1 (MAGI-1) was identified in this screen. MAGI-1 is a scaffolding protein that allows formation of complexes between certain transmembrane proteins, actin-binding proteins, and other regulatory proteins. MAGI-1 is expressed in a number of tissues, including podocytes and the brain. The interaction between MAGI-1 and BKCa channels was confirmed by coimmunoprecipitation and glutathione S-transferase pull-down assays in differentiated cells of a podocyte cell line and in human embryonic kidneys (HEK)293T cells transiently coexpressing MAGI-1a and three different COOH-terminal Slo1 variants. Coexpression of MAGI-1 with Slo1 channels in HEK-293T cells results in a significant reduction in the surface expression of Slo1, as assessed by cell-surface biotinylation assays, confocal microscopy, and whole cell recordings. Partial knockdown of endogenous MAGI-1 expression by small interfering RNA (siRNA) in differentiated podocytes increased the surface expression of endogenous Slo1 as assessed by electrophysiology and cell-surface biotinylation assays, whereas overexpression of MAGI-1a reduced steady-state voltage-evoked outward current through podocyte BKCa channels. These data suggest that MAGI-1 plays a role in regulation of surface expression of BKCa channels in the kidney and possibly in other tissues.

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Nephrin binds to the COOH terminus of a large-conductance Ca2+-activated K+ channel isoform and regulates its expression on the cell surface

AJP Renal Physiology ◽

10.1152/ajprenal.00140.2008 ◽

2008 ◽

Vol 295 (1) ◽

pp. F235-F246 ◽

Cited By ~ 39

Author(s):

Eun Young Kim ◽

Kyoung-Jae Choi ◽

Stuart E. Dryer

Keyword(s):

Steady State ◽

Cell Surface ◽

Splice Variants ◽

Surface Expression ◽

K Channel ◽

Glutathione S Transferase ◽

Differentiated Cells ◽

Cell Surface Biotinylation ◽

Ion Channel Proteins ◽

Interfering Rna

We carried out a yeast two-hybrid screen to identify proteins that interact with large-conductance Ca2+-activated K+ (BKCa) channels encoded by the Slo1 gene. Nephrin, an essential adhesion and scaffolding molecule expressed in podocytes, emerged in this screen. The Slo1-nephrin interaction was confirmed by coimmunoprecipitation from the brain and kidney, from HEK-293T cells expressing both proteins, and by glutathione S-transferase pull-down assays. We detected nephrin binding to the Slo1VEDEC splice variant, which is typically retained in intracellular stores, and to the β4-subunit. However, we did not detect significant binding of nephrin to the Slo1QEERL or Slo1EMVYR splice variants. Coexpression of nephrin with Slo1VEDEC increased expression of functional BKCa channels on the surface of HEK-293T cells but did not affect steady-state surface expression of the other COOH-terminal Slo1 variants. Nephrin did not affect the kinetics or voltage dependence of channel activation in HEK-293T cells expressing Slo1. Stimulation of Slo1VEDEC surface expression in HEK-293T cells was also observed by coexpressing a small construct encoding only the distal COOH-terminal domains of nephrin that interact with Slo1. Reduction of endogenous nephrin expression by application of small interfering RNA to differentiated cells of an immortalized podocyte cell line markedly reduced the steady-state surface expression of Slo1 as assessed by electrophysiology and cell-surface biotinylation assays. Nephrin therefore plays a role in organizing the surface expression of ion channel proteins in podocytes and may play a role in outside-in signaling to allow podocytes to adapt to mechanical or neurohumoral stimuli originating in neighboring cells.

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The Florey Lecture, 1988 - From egg to embryo: the initiation of cell differentiation in Amphibia

Proceedings of the Royal Society of London Series B Biological Sciences ◽

10.1098/rspb.1989.0033 ◽

1989 ◽

Vol 237 (1286) ◽

pp. 11-25 ◽

Cited By ~ 3

Keyword(s):

Muscle Cell ◽

Gene Activation ◽

Cell Formation ◽

Gene Promoter ◽

Cell Types ◽

Specific Gene ◽

Embryonic Induction ◽

Differentiated Cells ◽

Wide Range ◽

Muscle Genes

Some of the principles by which different cell types first arise at the beginning of animal development are illustrated by muscle cell formation in Amphibia. If the nucleus of a differentiated muscle cell is transplanted to an enucleated egg, some of the resulting embryos develop into tadpoles with a wide range of normally differentiated cells. These experiments show that genes undergo major changes in activity as a response to components of egg cytoplasm. Two fundamental mechanisms account for the regional activation of genes in early embryos. One involves the effect of localized ‘determinants’ in egg cytoplasm, and the other concerns cell interactions or embryonic induction. Both these mechanisms seem to be responsible for muscle cell formation in amphibian development. The old problem of embryonic induction has recently become accessible to analysis at the molecular level, especially in the case of the mesoderm or muscle-forming induction. This has been greatly facilitated by using a sensitive and quantitative assay to detect the first transcripts of muscle genes a few hours after the start of induction. The role of early events and of interactions among like cells during response to induction is discussed. In analysing specific gene activation following induction, DNA injection into fertilized eggs has shown that a very small part of the cardiac actin gene promoter is sufficient to enable it to respond to induction. Although the experimental work summarized here has been done on amphibian embryos, which are more suitable than other embryos for embryological manipulation, the conclusions reached are believed to be generally applicable to the development of other organisms.

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Both Freshly Prepared and Frozen-Stored Amniotic Membrane Cells Express the Complement Inhibitor CD59

The Scientific World JOURNAL ◽

10.1100/2012/815615 ◽

2012 ◽

Vol 2012 ◽

pp. 1-5 ◽

Cited By ~ 5

Author(s):

Ágnes Füst ◽

Éva Pállinger ◽

Adrienn Stündl ◽

Eszter Kovács ◽

László Imre ◽

...

Keyword(s):

Cell Surface ◽

Amniotic Membrane ◽

Ocular Surface ◽

Cell Types ◽

Surface Expression ◽

Mesenchymal Cells ◽

Cell Surface Expression ◽

Complement Inhibitor ◽

Becton Dickinson ◽

Intracellular Expression

Amniotic membrane proved to be very effective tool in the treatment of a number of ocular surface diseases. The amniotic membrane, however, has to be stored before its transplantation onto the ocular surface followed by mandatory serologic control in order to exclude the transmission of certain viruses. Therefore it is most important to study if cryopreservation of the membrane affects cell surface expression of the molecules. We measured cell surface expression of CD59, a membrane-bound complement inhibitor on the cells of freshly prepared and cryopreserved amniotic membrane. Cells of amniotic membrane were separated mechanically. Epithelial and mesenchymal cells were identified by the intracellular expression of nanog and the cell surface ICAM1 positivity, respectively. Multicolor flow cytometric immunophenotyping was used for determination of the CD59 expression. CellQuest-Pro software program (Becton Dickinson) was used both for measurements and analysis. CD59-positive cells could be detected in all investigated samples and in all investigated cell types, although the expression level of CD59 differed. CD59 was expressed both on freshly prepared and frozen-stored samples. Higher level of CD59 was detected on ICAM1+ mesenchymal cells than on nanog+ epithelial cells. Our findings indicate that amniotic membranes maintain their complement inhibiting capacity after cryopreservation.

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Phosphorylation and internalization of gp130 occur after IL-6 activation of Jak2 kinase in hepatocytes.

Molecular Biology of the Cell ◽

10.1091/mbc.5.7.819 ◽

1994 ◽

Vol 5 (7) ◽

pp. 819-828 ◽

Cited By ~ 46

Author(s):

Y Wang ◽

G M Fuller

Keyword(s):

Protein Synthesis ◽

Cell Surface ◽

De Novo ◽

Cell Types ◽

Surface Expression ◽

Cell Surface Expression ◽

Protein Synthesis Inhibitors ◽

Different Cell Types ◽

Kinetics Of

Recent evidence has shown that members of the Jak kinase family are activated after IL-6 binds to its receptor complex, leading to a tyrosine phosphorylation of gp130, the IL-6 signal-transducing subunit. The different members of the IL-6 cytokine subfamily induce distinct patterns of Jak-Tyk phosphorylation in different cell types. Using monospecific antibodies to gp130, Jak2 kinase, and phosphotyrosine, we investigated the kinetics of IL-6 stimulation of members of this pathway in primary hepatocytes. Our findings show that Jak 2 is maximally activated within 2 min of exposure to IL-6, followed by gp130 phosphorylation that reaches its peak in another 2 min then declines to basal level by 60 min. In vitro phosphorylation experiments show that activated Jak 2 is able to phosphorylate both native gp130 and a fusion peptide containing its cytoplasmic domain, demonstrating gp130 is a direct substrate of Jak 2 kinase. Experiments designed to explore the cell surface expression of gp130 show that > or = 2 h are required to get a second round of phosphorylation after the addition of more cytokines. This finding suggests that activated gp130 is internalized from the cell surface after IL-6 stimulation. Additional experiments using protein synthesis inhibitors reveal that new protein synthesis is required to get a second cycle of gp130 phosphorylation indicating gp130 must be synthesized de novo and inserted into the membrane. These findings provide strong evidence that down regulation of the IL-6 signal in hepatocytes involves the internalization and cytosol degradation of gp130.

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Phosphatidylserine Is Not the Cell Surface Receptor for Vesicular Stomatitis Virus

Journal of Virology ◽

10.1128/jvi.78.20.10920-10926.2004 ◽

2004 ◽

Vol 78 (20) ◽

pp. 10920-10926 ◽

Cited By ~ 121

Author(s):

David A. Coil ◽

A. Dusty Miller

Keyword(s):

Cell Surface ◽

Vesicular Stomatitis Virus ◽

Virus Entry ◽

Annexin V ◽

Cell Types ◽

Membrane Lipid ◽

Cell Surface Receptor ◽

Wide Range ◽

Surface Receptor ◽

Vesicular Stomatitis

ABSTRACT The envelope protein from vesicular stomatitis virus (VSV) has become an important tool for gene transfer and gene therapy. It is widely used mainly because of its ability to mediate virus entry into all cell types tested to date. Consistent with the broad tropism of the virus, the receptor for VSV is thought to be a ubiquitous membrane lipid, phosphatidylserine (PS). However, the evidence for this hypothesis is indirect and incomplete. Here, we have examined the potential interaction of VSV and PS at the plasma membrane in more detail. Measurements of cell surface levels of PS show a wide range across cell types from different organisms. We demonstrate that there is no correlation between the cell surface PS levels and VSV infection or binding. We also demonstrate that an excess of annexin V, which binds specifically and tightly to PS, does not inhibit infection or binding by VSV. While the addition of PS to cells does allow increased virus entry, we show that this effect is not specific to the VSV envelope. We conclude that PS is not the cell surface receptor for VSV, although it may be involved in a postbinding step of virus entry.

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Actin-binding Protein α-Actinin-1 Interacts with the Metabotropic Glutamate Receptor Type 5b and Modulates the Cell Surface Expression and Function of the Receptor

Journal of Biological Chemistry ◽

10.1074/jbc.m608880200 ◽

2007 ◽

Vol 282 (16) ◽

pp. 12143-12153 ◽

Cited By ~ 27

Author(s):

Nuria Cabello ◽

Rosaria Remelli ◽

Laia Canela ◽

Ana Soriguera ◽

Josefa Mallol ◽

...

Keyword(s):

Cell Surface ◽

Glutamate Receptor ◽

Metabotropic Glutamate Receptor ◽

Surface Expression ◽

Receptor Type ◽

Actin Binding ◽

Cell Surface Expression ◽

Actin Binding Protein ◽

Metabotropic Glutamate ◽

And Function

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Neph1 regulates steady-state surface expression of Slo1 Ca2+-activated K+ channels: different effects in embryonic neurons and podocytes

AJP Cell Physiology ◽

10.1152/ajpcell.00354.2009 ◽

2009 ◽

Vol 297 (6) ◽

pp. C1379-C1388 ◽

Cited By ~ 19

Author(s):

Eun Young Kim ◽

Yu-Hsin Chiu ◽

Stuart E. Dryer

Keyword(s):

Steady State ◽

Cell Surface ◽

Splice Variants ◽

Surface Expression ◽

Immunoglobulin Superfamily ◽

Ciliary Ganglion ◽

Whole Cell ◽

Differentiated Cells ◽

Ciliary Ganglion Neurons ◽

Ganglion Neurons

Large-conductance Ca2+-activated K+ (BKCa) channels encoded by the Slo1 gene are often components of large multiprotein complexes in excitable and nonexcitable cells. Here we show that Slo1 proteins interact with Neph1, a member of the immunoglobulin superfamily expressed in slit diaphragm domains of podocytes and in vertebrate and invertebrate nervous systems. This interaction was established by reciprocal coimmunoprecipitation of endogenous proteins from differentiated cells of a podocyte cell line, from parasympathetic neurons of the embryonic chick ciliary ganglion, and from HEK293T cells heterologously expressing both proteins. Neph1 can interact with all three extreme COOH-terminal variants of Slo1 (Slo1VEDEC, Slo1QEERL, and Slo1EMVYR) as ascertained by glutathione S-transferase (GST) pull-down assays and by coimmunoprecipitation. Neph1 is partially colocalized in intracellular compartments with endogenous Slo1 in podocytes and ciliary ganglion neurons. Coexpression in HEK293T cells of Neph1 with any of the Slo1 extreme COOH-terminal splice variants suppresses their steady-state expression on the cell surface, as assessed by cell surface biotinylation assays, confocal microscopy, and whole cell recordings. Consistent with this, small interfering RNA (siRNA) knockdown of endogenous Neph1 in embryonic day 10 ciliary ganglion neurons causes an increase in steady-state surface expression of Slo1 and an increase in whole cell Ca2+-dependent K+ current. Surprisingly, a comparable Neph1 knockdown in podocytes causes a decrease in surface expression of Slo1 and a decrease in whole cell BKCa currents. In podocytes, Neph1 siRNA also caused a decrease in nephrin, even though the Neph1 siRNA had no sequence homology with nephrin. However, we could not detect nephrin in ciliary ganglion neurons.

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Phenotyping of Leukocytes and Leukocyte-Derived Extracellular Vesicles

Journal of Immunology Research ◽

10.1155/2016/6391264 ◽

2016 ◽

Vol 2016 ◽

pp. 1-12 ◽

Cited By ~ 12

Author(s):

Lotte Hatting Pugholm ◽

Rikke Bæk ◽

Evo Kristina Lindersson Søndergaard ◽

Anne Louise Schacht Revenfeld ◽

Malene Møller Jørgensen ◽

...

Keyword(s):

Immune System ◽

Cell Surface ◽

Extracellular Vesicles ◽

Cell Types ◽

Surface Expression ◽

Cell Surface Expression ◽

Parent Cell ◽

Phenotypic Differences ◽

Immunological Markers ◽

Cellular Expression

Extracellular vesicles (EVs) have a demonstrated involvement in modulating the immune system. It has been proposed that EVs could be used as biomarkers for detection of inflammatory and immunological disorders. Consequently, it is of great interest to investigate EVs in more detail with focus on immunological markers. In this study, five major leukocyte subpopulations and the corresponding leukocyte-derived EVs were phenotyped with focus on selected immunological lineage-specific markers and selected vesicle-related markers. The leukocyte-derived EVs displayed phenotypic differences in the 34 markers investigated. The majority of the lineage-specific markers used for identification of the parent cell types could not be detected on EVs released from monocultures of the associated cell types. In contrast, the vesicular presentation of CD9, CD63, and CD81 correlated to the cell surface expression of these markers, however, with few exceptions. Furthermore, the cellular expression of CD9, CD63, and CD81 varied between leukocytes present in whole blood and cultured leukocytes. In summary, these data demonstrate that the cellular and vesicular presentation of selected lineage-specific and vesicle-related markers may differ, supporting the accumulating observations that sorting of molecular cargo into EVs is tightly controlled.

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Vasopressin-inducible ubiquitin-specific protease 10 increases ENaC cell surface expression by deubiquitylating and stabilizing sorting nexin 3

AJP Renal Physiology ◽

10.1152/ajprenal.00001.2008 ◽

2008 ◽

Vol 295 (4) ◽

pp. F889-F900 ◽

Cited By ~ 48

Author(s):

Sheerazed Boulkroun ◽

Dorothée Ruffieux-Daidié ◽

Jean-Jacques Vitagliano ◽

Olivier Poirot ◽

Roch-Philippe Charles ◽

...

Keyword(s):

Cell Surface ◽

Surface Expression ◽

Cell Surface Expression ◽

Long Term Effects ◽

Hek 293 ◽

Fourfold Increase ◽

Sorting Nexin ◽

Cell Surface Biotinylation ◽

Specific Protease ◽

Ubiquitin Specific Protease

Adjustment of Na+ balance in extracellular fluids is achieved by regulated Na+ transport involving the amiloride-sensitive epithelial Na+ channel (ENaC) in the distal nephron. In this context, ENaC is controlled by a number of hormones, including vasopressin, which promotes rapid translocation of water and Na+ channels to the plasma membrane and long-term effects on transcription of vasopressin-induced and -reduced transcripts. We have identified a mRNA encoding the deubiquitylating enzyme ubiquitin-specific protease 10 (Usp10), whose expression is increased by vasopressin at both the mRNA and the protein level. Coexpression of Usp10 in ENaC-transfected HEK-293 cells causes a more than fivefold increase in amiloride-sensitive Na+ currents, as measured by whole cell patch clamping. This is accompanied by a three- to fourfold increase in surface expression of α- and γ-ENaC, as shown by cell surface biotinylation experiments. Although ENaC is well known to be regulated by its direct ubiquitylation, Usp10 does not affect the ubiquitylation level of ENaC, suggesting an indirect effect. A two-hybrid screen identified sorting nexin 3 (SNX3) as a novel substrate of Usp10. We show that it is a ubiquitylated protein that is degraded by the proteasome; interaction with Usp10 leads to its deubiquitylation and stabilization. When coexpressed with ENaC, SNX3 increases the channel's cell surface expression, similarly to Usp10. In mCCDcl1 cells, vasopressin increases SNX3 protein but not mRNA, supporting the idea that the vasopressin-induced Usp10 deubiquitylates and stabilizes endogenous SNX3 and consequently promotes cell surface expression of ENaC.

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