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

2008 ◽  
Vol 295 (1) ◽  
pp. F235-F246 ◽  
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.

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

2009 ◽  
Vol 297 (6) ◽  
pp. C1379-C1388 ◽  
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.

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

2009 ◽  
Vol 297 (1) ◽  
pp. C55-C65 ◽  
Author(s):  
Lon D. Ridgway ◽  
Eun Young Kim ◽  
Stuart E. Dryer
Keyword(s):  
Cell Surface ◽  
Outward Current ◽  
Cell Types ◽  
Surface Expression ◽  
Scaffolding Protein ◽  
Actin Binding ◽  
Differentiated Cells ◽  
Cell Surface Biotinylation ◽  
Bait Construct ◽  
Wide Range

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.

The NITY motif of the beta-chain cytoplasmic domain is involved in stimulated internalization of the beta3 integrin A isoform

2001 ◽  
Vol 114 (6) ◽  
pp. 1101-1113
Author(s):  
M. Gawaz ◽  
F. Besta ◽  
J. Ylanne ◽  
T. Knorr ◽  
H. Dierks ◽  
...  
Keyword(s):  
Steady State ◽  
Cell Surface ◽  
Molecular Mechanisms ◽  
Chinese Hamster Ovary Cells ◽  
Surface Expression ◽  
Cytoplasmic Domain ◽  
Cell Surface Expression ◽  
Beta Chain ◽  
Single Chain ◽  
Beta3 Integrin

Beta3 integrin adhesion molecules play important roles in wound repair and the regulation of vascular development and three beta3 integrin isoforms (beta3-A, -B, -C) have been described so far. Surface expression of beta3 integrins is dynamically regulated through internalization of beta3 integrins, however, the molecular mechanisms are understood incompletely. To evaluate the role of the cytoplasmic domain of beta3 integrins for internalization, we have generated single chain chimeras with variant and mutated forms of beta3 cytoplasmic domains. Upon transient transfection into chinese hamster ovary cells, it was found that the beta3-A chimera had strongly reduced cell surface expression compared with the corresponding beta3-B, or beta3-C fusion proteins, or the tail-less constructs, whereas steady state levels of all chimeras were near identical. Studies employing cytoplasmic domain mutants showed that the NITY motif at beta3-A 756–759 is critical for plasma membrane expression of beta3-A. Furthermore, delivery of beta3-A to the cell surface was specifically modulated by the cytoplasmic protein beta3-endonexin, a previously described intracellular protein. Coexpression of the native, long form of beta3-endonexin, which does not interact with the beta3 tail, acted as a dominant negative inhibitor of beta3-A-internalization and enhanced steady-state surface expression of the beta3-A-chimera. Furthermore, anti-beta3 antibody-induced internalization of the native beta3 integrin (alpha(IIb)beta3 was dramatically reduced for the Tyr(759)-Ala substitution mutant (alpha(IIb)beta3) (Y759A) and expression of the long isoform of beta3-endonexin substantially decreased the internalization of wild-type alpha(IIb)beta3. Thus, the NITY motif of the beta-chain cytoplasmic domain is involved in stimulated internalization of the beta3 integrin A isoform and beta3-endonexin appears to couple the beta3-A isoform to a specific receptor-recycling pathway.

scholarly journals Differential Expression and Functional Characterization of Luteinizing Hormone Receptor Splice Variants in Human Luteal Cells: Implications for Luteolysis

Endocrinology ◽  
2009 ◽  
Vol 150 (6) ◽  
pp. 2873-2881 ◽  
Author(s):  
Rachel E. Dickinson ◽  
Alan J. Stewart ◽  
Michelle Myers ◽  
Robert P. Millar ◽  
W. Colin Duncan
Keyword(s):  
Cell Surface ◽  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Splice Variants ◽  
Primary Cultures ◽  
Surface Expression ◽  
Full Length ◽  
Luteinizing Hormone Receptor ◽  
Cell Surface Expression ◽  
Truncated Form

The human LH receptor (LHR) plays a key role in luteal function and the establishment of pregnancy through its interaction with the gonadotropins LH and human chorionic gonadotropin. We previously identified four splice variants of the LHR in human luteinized granulosa cells (LGCs) and corpora lutea (CL). Real-time quantitative PCR revealed that expression of the full-length LHR (LHRa) and the most truncated form (LHRd) changed significantly in CL harvested at different stages of the ovarian cycle (P < 0.01, ANOVA). LHRa expression was reduced in the late luteal CL (P < 0.05). Conversely, an increase in LHRd expression was observed in the late luteal CL (P < 0.01). Chronic manipulation of human chorionic gonadotropin in LGC primary cultures supported the in vivo findings. LHRd encodes a protein lacking the transmembrane and carboxyl terminal domains. COS-7 cells expressing LHRd were unable to produce cAMP in response to LH stimulation. COS-7 cells coexpressing LHRd and LHRa also failed to generate cAMP in response to LH, suggesting that this truncated form has a negative effect on the signaling of LHRa. Immunofluorescence staining of LGC and COS-7 cells implied that there is a reduction in cell surface expression of LHRa when LHRd is present. Overall, these results imply expression of LHR splice variants is regulated in the human CL. Furthermore, during functional luteolysis a truncated variant could modulate the cell surface expression and activity of full-length LHR.

Ku80 autoantigen as a cellular coreceptor for human parvovirus B19 infection

Blood ◽  
2005 ◽  
Vol 106 (10) ◽  
pp. 3449-3456 ◽  
Author(s):  
Yasuhiko Munakata ◽  
Takako Saito-Ito ◽  
Keiko Kumura-Ishii ◽  
Jie Huang ◽  
Takao Kodera ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Surface ◽  
Cell Lines ◽  
Bone Marrow Cells ◽  
Parvovirus B19 ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Human Parvovirus B19 ◽  
Erythroid Cells ◽  
Interfering Rna

AbstractHuman parvovirus B19 (B19) infects human erythroid cells expressing P antigen. However, some cell lines that were positive for P antigen failed to bind B19, whereas some cell lines had an ability to bind B19 despite undetectable expression of P antigen. We here demonstrate that B19 specifically binds with Ku80 autoantigen on the cell surface. Furthermore, transfection of HeLa cells with the gene of Ku80 enabled the binding of B19 and allowed its entry into cells. Moreover, reduction of cell-surface expression of Ku80 in KU812Ep6 cells, which was a high-sensitive cell line for B19 infection, by short interfering RNA for Ku80 resulted in the marked inhibition of B19 binding in KU812Ep6 cells. Although Ku80 originally has been described as a nuclear protein, human bone marrow erythroid cells with glycophorin A or CD36, B cells with CD20, or T cells with CD3 were all positive for cell-surface expression of Ku80. B19 infection of KU812Ep6 cells and bone marrow cells was inhibited in the presence of anti-Ku80 antibody. Our data suggest that Ku80 functions as a novel coreceptor for B19 infection, and this finding may provide an explanation for the pathologic immunity associated with B19 infection.

scholarly journals C-terminal region regulates the functional expression of human noradrenaline transporter splice variants

2006 ◽  
Vol 401 (1) ◽  
pp. 185-195 ◽  
Author(s):  
Chiharu Sogawa ◽  
Kei Kumagai ◽  
Norio Sogawa ◽  
Katsuya Morita ◽  
Toshihiro Dohi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Surface ◽  
Splice Variants ◽  
Functional Expression ◽  
Surface Expression ◽  
Terminal Region ◽  
Site Directed Mutagenesis ◽  
Cell Surface Expression ◽  
C Terminus ◽  
And Function

The NET [noradrenaline (norepinephrine) transporter], an Na+/Cl−-dependent neurotransmitter transporter, has several isoforms produced by alternative splicing in the C-terminal region, each differing in expression and function. We characterized the two major isoforms of human NET, hNET1, which has seven C-terminal amino acids encoded by exon 15, and hNET2, which has 18 amino acids encoded by exon 16, by site-directed mutagenesis in combination with NE (noradrenaline) uptake assays and cell surface biotinylation. Mutants lacking one third or more of the 24 amino acids encoded by exon 14 exhibited neither cell surface expression nor NE uptake activity, with the exception of the mutant lacking the last eight amino acids of hNET2, whose expression and uptake resembled that of the WT (wild-type). A triple alanine replacement of a candidate motif (ENE) in this region mimicked the influences of the truncation. Deletion of either the last three or another four amino acids of the C-terminus encoded by exon 15 in hNET1 reduced the cell surface expression and NE uptake, whereas deletion of all seven residues reduced the transport activity but did not affect the cell surface expression. Replacement of RRR, an endoplasmic reticulum retention motif, by alanine residues in the C-terminus of hNET2 resulted in a similar expression and function compared with the WT, while partly recovering the effects of the mutation of ENE. These findings suggest that in addition to the function of the C-terminus, the common proximal region encoded by exon 14 regulates the functional expression of splice variants, such as hNET1 and hNET2.

scholarly journals FAP-1 Association with Fas (Apo-1) Inhibits Fas Expression on the Cell Surface

2003 ◽  
Vol 23 (10) ◽  
pp. 3623-3635 ◽  
Author(s):  
Vladimir N. Ivanov ◽  
Pablo Lopez Bergami ◽  
Gabriel Maulit ◽  
Taka-Aki Sato ◽  
David Sassoon ◽  
...  
Keyword(s):  
Cell Surface ◽  
Fas Ligand ◽  
Surface Expression ◽  
Dominant Negative ◽  
Intracellular Pool ◽  
C Terminus ◽  
Interfering Rna ◽  
Insight Into ◽  
Advanced Tumors

ABSTRACT As revealed by intracellular pools of nonactive Fas (Apo-1), export of Fas to the cell surface is often impaired in human tumors, thereby inactivating Fas ligand-mediated apoptosis. Here, we demonstrate that association with Fas-associated phosphatase 1 (FAP-1) attenuates Fas export to the cell surface. Forced expression of FAP-1 reduces cell surface Fas levels and increases the intracellular pool of Fas within the cytoskeleton network. Conversely, expression of dominant-negative forms of FAP-1, or inhibition of FAP-1 expression by short interfering RNA, efficiently up-regulates surface expression of Fas. Inhibition of Fas surface expression by FAP-1 depends on its association with the C terminus of Fas. Mutation within amino acid 275 results in decreased association with FAP-1 and greater export of Fas to the cell surface in melanomas, normal fibroblasts, or Fas null cells. Identifying the role of FAP-1 in binding to, and consequently inhibition of, Fas export to the cell surface provides novel insight into the mechanism underlying the regulation of Fas trafficking, which is commonly impaired in advanced tumors with FAP-1 overexpression.

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

2008 ◽  
Vol 295 (4) ◽  
pp. F889-F900 ◽  
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.

scholarly journals Internalization of UT-A1 urea transporter is dynamin dependent and mediated by both caveolae- and clathrin-coated pit pathways

2010 ◽  
Vol 299 (6) ◽  
pp. F1389-F1395 ◽  
Author(s):  
Haidong Huang ◽  
Xiuyan Feng ◽  
Jieqiu Zhuang ◽  
Otto Fröhlich ◽  
Janet D. Klein ◽  
...  
Keyword(s):  
Cell Surface ◽  
Lipid Raft ◽  
Surface Expression ◽  
Membrane Lipid ◽  
Hek293 Cells ◽  
Cell Surface Expression ◽  
Protein Accumulation ◽  
Intracellular Loop ◽  
Cell Plasma ◽  
Cell Surface Biotinylation

Dynamin is a large GTPase involved in several distinct modes of cell endocytosis. In this study, we examined the possible role of dynamin in UT-A1 internalization. The direct relationship of UT-A1 and dynamin was identified by coimmunoprecipitation. UT-A1 has cytosolic NH2 and COOH termini and a large intracellular loop. Dynamin specifically binds to the intracellular loop of UT-A1, but not the NH2 and COOH termini. In cell surface biotinylation experiments, coexpression of dynamin and UT-A1 in HEK293 cells resulted in a decrease of UT-A1 cell surface expression. Conversely, cells expressing dynamin mutant K44A, which is deficient in GTP binding, showed an increased accumulation of UT-A1 protein on the cell surface. Cell plasma membrane lipid raft fractionation experiments revealed that blocking endocytosis with dynamin K44A causes UT-A1 protein accumulation in both the lipid raft and nonlipid raft pools, suggesting that both caveolae- and clathrin-mediated mechanisms may be involved in the internalization of UT-A1. This was further supported by 1) small interfering RNA to knock down either caveolin-1 or μ2 reduced UT-A1 internalization in HEK293 cells and 2) inhibition of either the caveolae pathway by methyl-β-cyclodextrin or the clathrin pathway by concanavalin A caused UT-A1 cell membrane accumulation. Functionally, overexpression of dynamin, caveolin, or μ2 decreased UT-A1 urea transport activity and decreased UT-A1 cell surface expression. We conclude that UT-A1 endocytosis is dynamin-dependent and mediated by both caveolae- and clathrin-coated pit pathways.

scholarly journals Mutational analysis of histidine residues in the rabbit Na+/dicarboxylate co-transporter NaDC-1

1998 ◽  
Vol 331 (1) ◽  
pp. 257-264 ◽  
Author(s):  
Ana M. PAJOR ◽  
Ning SUN ◽  
Heidi G. VALMONTE
Keyword(s):  
Cell Surface ◽  
Mutational Analysis ◽  
Surface Expression ◽  
Site Directed Mutagenesis ◽  
Cell Surface Expression ◽  
Transport Activity ◽  
Membrane Expression ◽  
Histidine Residues ◽  
Cell Surface Biotinylation

Succinate transport by the rabbit Na+/dicarboxylate co-transporter, NaDC-1, expressed in Xenopusoocytes was inhibited by the histidyl-selective reagent diethyl pyrocarbonate (DEPC). Therefore the role of histidine residues in the function of NaDC-1 was examined by site-directed mutagenesis. All 11 histidine residues in NaDC-1 were converted to alanine, but only mutant H106A exhibited a decrease in succinate transport. Additional mutations of NaDC-1 at position 106 showed that aspartic acid and asparagine, but not arginine, can substitute for histidine. Examination of succinate and citrate kinetics of H106A revealed a decrease in Vmax with no change in Km. Cell surface biotinylation experiments showed that the transport activity of all four mutants at position 106 was correlated with the amount of cell surface expression, suggesting a role of His-106 in membrane expression rather than function. Two of the histidine mutants, H153A and H569A, exhibited insensitivity to inhibition by DEPC, indicating that these residues are involved in binding DEPC. Neither of these residues is required for transport activity; thus DEPC probably inhibits NaDC-1 function by hindrance of the mobility of the carrier. We conclude that histidine residues are not critical for transport function in NaDC-1, although His-106 might be involved in determining protein expression or stability in the membrane.

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