S-acylation regulates Kv1.5 channel surface expression

2007 ◽  
Vol 293 (1) ◽  
pp. C152-C161 ◽  
Author(s):  
Lian Zhang ◽  
Karyn Foster ◽  
Qiuju Li ◽  
Jeffrey R. Martens
Keyword(s):  
Cell Surface ◽  
Time Course ◽  
Electrical Response ◽  
Proteasome Inhibitors ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Channel Protein ◽  
Kv Channels ◽  
Channel Expression ◽  
Intracellular Compartments

The number of ion channels expressed on the cell surface shapes the complex electrical response of excitable cells. An imbalance in the ratio of inward and outward conducting channels is unfavorable and often detrimental. For example, over- or underexpression of voltage-gated K+ (Kv) channels can be cytotoxic and in some cases lead to disease. In this study, we demonstrated a novel role for S-acylation in Kv1.5 cell surface expression. In transfected fibroblasts, biochemical evidence showed that Kv1.5 is posttranslationally modified on both the NH2 and COOH termini via hydroxylamine-sensitive thioester bonds. Pharmacological inhibition of S-acylation, but not myristoylation, significantly decreased Kv1.5 expression and resulted in accumulation of channel protein in intracellular compartments and targeting for degradation. Channel protein degradation was rescued by treatment with proteasome inhibitors. Time course experiments revealed that S-acylation occurred in the biosynthetic pathway of nascent channel protein and showed that newly synthesized Kv1.5 protein, but not protein expressed on the cell surface, is sensitive to inhibitors of thioacylation. Sensitivity to inhibitors of S-acylation was governed by COOH-terminal, but not NH2-terminal, cysteines. Surprisingly, although intracellular cysteines were required for S-acylation, mutation of these residues resulted in an increase in Kv1.5 cell surface channel expression, suggesting that screening of free cysteines by fatty acylation is an important regulatory step in the quality control pathway. Together, these results show that S-acylation can regulate steady-state expression of Kv1.5.

scholarly journals Roles of the N- and C-termini of GLUT4 in endocytosis

2002 ◽  
Vol 115 (1) ◽  
pp. 131-140 ◽  
Author(s):  
Hadi Al-Hasani ◽  
Raghu K. Kunamneni ◽  
Kevin Dawson ◽  
Cynthia S. Hinck ◽  
Dirk Müller-Wieland ◽  
...  
Keyword(s):  
Cell Surface ◽  
Glucose Transporter ◽  
Surface Expression ◽  
Dominant Negative ◽  
Cell Surface Expression ◽  
Target Cells ◽  
Dileucine Motif ◽  
Intracellular Compartments ◽  
Targeting Signal

In insulin target cells, the predominantly expressed glucose transporter isoform GLUT4 recycles between distinct intracellular compartments and the plasma membrane. To characterize putative targeting signals within GLUT4 in a physiologically relevant cell type, we have analyzed the trafficking of hemagglutinin (HA)-epitope-tagged GLUT4 mutants in transiently transfected primary rat adipose cells. Mutation of the C-terminal dileucine motif (LL489/90) did not affect the cell-surface expression of HA-GLUT4. However, mutation of the N-terminal phenylalanine-based targeting sequence (F5) resulted in substantial increases, whereas deletion of 37 or 28 of the 44 C-terminal residues led to substantial decreases in cell-surface HA-GLUT4 in both the basal and insulin-stimulated states. Studies with wortmannin and coexpression of a dominant-negative dynamin GTPase mutant indicate that these effects appear to be primarily due to decreases and increases, respectively, in the rate of endocytosis. Yeast two-hybrid analyses revealed that the N-terminal phenylalanine-based targeting signal in GLUT4 constitutes a binding site for medium chain adaptins μ1, μ2, and μ3A, implicating a role of this motif in the targeting of GLUT4 to clathrin-coated vesicles.

scholarly journals Tissue factor trafficking in fibroblasts: involvement of protease-activated receptor–mediated cell signaling

Blood ◽  
2007 ◽  
Vol 110 (1) ◽  
pp. 161-170 ◽  
Author(s):  
Samir K. Mandal ◽  
Usha R. Pendurthi ◽  
L. Vijaya Mohan Rao
Keyword(s):  
Cell Surface ◽  
Cell Signaling ◽  
Tissue Factor ◽  
Neutralizing Antibodies ◽  
Factor Viia ◽  
Surface Expression ◽  
Coagulation Cascade ◽  
Cell Surface Expression ◽  
Clotting Factor ◽  
Intracellular Compartments

Tissue factor (TF) is the cellular receptor for clotting factor VIIa (FVIIa), and the formation of TF-FVIIa complexes on cell surfaces triggers the activation of the coagulation cascade and the cell signaling. Our recent studies have shown that a majority of TF resides in various intracellular compartments, predominantly in the Golgi, and that FVIIa binding to cell surface TF induces TF endocytosis and mobilizes the Golgi TF pool to translocate it to the cell surface. This present study is aimed to elucidate the mechanisms involved in TF endocytosis and its mobilization from the Golgi. Activation of protease-activated receptor 1 (PAR1) and PAR2 by specific peptide agonists and proteases, independent of FVIIa, mobilized TF from the Golgi store and increased the cell surface expression of TF. Blocking PAR2 activation, but not PAR1, with neutralizing antibodies fully attenuated the FVIIa-induced TF mobilization. Consistent with these data, silencing the PAR2 receptor, and not PAR1, abrogated the FVIIa-mediated TF mobilization. In contrast to their effect on TF mobilization, PAR1 and PAR2 activation, in the absence of FVIIa, had no effect on TF endocytosis. However, PAR2 activation is found to be critical for the FVIIa-induced TF endocytosis. Overall the data herein provide novel insights into the role of PARs in regulating cell surface TF expression.

scholarly journals Stimulation of Na+ transport by AVP is independent of PKA phosphorylation of the Na-K-ATPase in collecting duct principal cells

2005 ◽  
Vol 289 (5) ◽  
pp. F1031-F1039 ◽  
Author(s):  
David Mordasini ◽  
Mauro Bustamante ◽  
Martine Rousselot ◽  
Pierre-Yves Martin ◽  
Udo Hasler ◽  
...  
Keyword(s):  
Cell Surface ◽  
Time Course ◽  
Collecting Duct ◽  
Pump Current ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Intact Cells ◽  
Principal Cells ◽  
Na Pump ◽  
Stimulation Of

Arginine-vasopressin (AVP) stimulates Na+ transport and Na-K-ATPase activity via cAMP-dependent PKA activation in the renal cortical collecting duct (CCD). We investigated the role of the Na-K-ATPase in the AVP-induced stimulation of transepithelial Na+ transport using the mpkCCDc14 cell model of mammalian collecting duct principal cells. AVP (10−9 M) stimulated both the amiloride-sensitive transepithelial Na+ transport measured in intact cells and the maximal Na pump current measured by the ouabain-sensitive short-circuit current in apically permeabilized cells. These effects were associated with increased Na-K-ATPase cell surface expression, measured by Western blotting after streptavidin precipitation of biotinylated cell surface proteins. The effects of AVP on Na pump current and Na-K-ATPase cell surface expression were dependent on PKA activity but independent of increased apical Na+ entry. Time course experiments revealed that in response to AVP, the cell surface expression of both endogenous Na-K-ATPase and hybrid Na pumps containing a c- myc-tagged wild-type human α1-subunit increased transiently. Na-K-ATPase cell surface expression was maximal after 30 min and then declined toward baseline after 60 min. Immunoprecipitation experiments showed that PKA activation did not alter total phosphorylation levels of the endogenous Na-K-ATPase α-subunit. In addition, mutation of the PKA phosphorylation site (S943A or S943D) did not alter the time course of increased cell surface expression of c- myc-tagged Na-K-ATPase in response to AVP or to dibutyryl-cAMP. Therefore, stimulation of Na-K-ATPase cell surface expression by AVP is dependent on PKA but does not rely on α1-subunit phosphorylation on serine 943 in the collecting duct principal cells.

scholarly journals Different Steady State Subcellular Distributions of the Three Splice Variants of Lysosome-associated Membrane Protein LAMP-2 Are Determined Largely by the COOH-terminal Amino Acid Residue

1997 ◽  
Vol 137 (5) ◽  
pp. 1161-1169 ◽  
Author(s):  
Nancy R. Gough ◽  
Douglas M. Fambrough
Keyword(s):  
Cell Surface ◽  
Splice Variants ◽  
Single Gene ◽  
Surface Expression ◽  
Site Directed Mutagenesis ◽  
Cell Surface Expression ◽  
Cytosolic Domain ◽  
Cytosolic Domains ◽  
Intracellular Compartments ◽  
Targeting Signals

The extensively glycosylated lysosome-associated membrane proteins (LAMP)-2a, b, and c are derived from a single gene by alternative splicing that produces proteins with differences in the transmembrane and cytosolic domains. The lysosomal targeting signals reside in the cytosolic domain of these proteins. LAMPs are not restricted to lysosomes but can also be found in endosomes and at the cell surface. We investigated the subcellular distribution of chimeras comprised of the lumenal domain of avian LAMP-1 and the alternatively spliced domains of avian LAMP-2. Chimeras with the LAMP-2c cytosolic domain showed predominantly lysosomal distribution, while higher levels of chimeras with the LAMP-2a or b cytosolic domain were present at the cell surface. The increase in cell surface expression was due to differences in the recognition of the targeting signals and not saturation of intracellular trafficking machinery. Site-directed mutagenesis defined the COOH-terminal residue of the cytosolic tail as critical in governing the distributions of LAMP-2a, b, and c between intracellular compartments and the cell surface.

scholarly journals PSD-95 and SAP97 Exhibit Distinct Mechanisms for Regulating K+ Channel Surface Expression and Clustering

2000 ◽  
Vol 148 (1) ◽  
pp. 147-157 ◽  
Author(s):  
Amanda M. Tiffany ◽  
Louis N. Manganas ◽  
Eunjoon Kim ◽  
Yi-Ping Hsueh ◽  
Morgan Sheng ◽  
...  
Keyword(s):  
Cell Surface ◽  
Ion Channel ◽  
Membrane Vesicles ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
K Channel ◽  
Neuronal Membrane ◽  
K Channels ◽  
Kv Channels ◽  
Abundance And Distribution

Mechanisms of ion channel clustering by cytoplasmic membrane-associated guanylate kinases such as postsynaptic density 95 (PSD-95) and synapse-associated protein 97 (SAP97) are poorly understood. Here, we investigated the interaction of PSD-95 and SAP97 with voltage-gated or Kv K+ channels. Using Kv channels with different surface expression properties, we found that clustering by PSD-95 depended on channel cell surface expression. Moreover, PSD-95–induced clusters of Kv1 K+ channels were present on the cell surface. This was most dramatically demonstrated for Kv1.2 K+ channels, where surface expression and clustering by PSD-95 were coincidentally promoted by coexpression with cytoplasmic Kvβ subunits. Consistent with a mechanism of plasma membrane channel–PSD-95 binding, coexpression with PSD-95 did not affect the intrinsic surface expression characteristics of the different Kv channels. In contrast, the interaction of Kv1 channels with SAP97 was independent of Kv1 surface expression, occurred intracellularly, and prevented further biosynthetic trafficking of Kv1 channels. As such, SAP97 binding caused an intracellular accumulation of each Kv1 channel tested, through the accretion of SAP97 channel clusters in large (3–5 μm) ER-derived intracellular membrane vesicles. Together, these data show that ion channel clustering by PSD-95 and SAP97 occurs by distinct mechanisms, and suggests that these channel-clustering proteins may play diverse roles in regulating the abundance and distribution of channels at synapses and other neuronal membrane specializations.

Time course of recovery of endothelial cell surface thrombin receptor (PAR-1) expression

1999 ◽  
Vol 276 (1) ◽  
pp. C38-C45 ◽  
Author(s):  
Chad A. Ellis ◽  
Chinnaswamy Tiruppathi ◽  
Raudel Sandoval ◽  
Walter D. Niles ◽  
Asrar B. Malik
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Surface ◽  
Electrical Resistance ◽  
Time Course ◽  
De Novo ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Thrombin Receptor ◽  
Endothelial Cell Surface

We studied dynamics of cell surface expression of proteolytically activated thrombin receptor (PAR-1) in human pulmonary artery endothelial cells (HPAEC). PAR-1 activation was measured by changes in cytosolic calcium concentration ([Ca2+]i) and HPAEC retraction response (determined by real-time transendothelial monolayer electrical resistance). [Ca2+]iincrease in response to thrombin was abolished by preexposure to 25 nM thrombin for >60 min, indicating PAR-1 desensitization, but preexposure to 25 nM thrombin for only 30 min or to 10 nM thrombin for up to 2 h did not desensitize PAR-1. Exposure to 10 or 25 nM thrombin decreased monolayer electrical resistance 40–60%. Cells preexposed to 10 nM thrombin, but not those preexposed to 25 nM thrombin, remained responsive to thrombin 3 h later. Loss of cell retractility was coupled to decreased cell surface PAR-1 expression as determined by immunofluorescence. Cell surface PAR-1 disappeared upon short-term (30 min) thrombin exposure but reappeared within 90 min after incubation in thrombin-free medium. Exposure to 25 nM thrombin for >60 min prevented rapid cycloheximide-insensitive PAR-1 reappearance. Cycloheximide-sensitive recovery of cell surface PAR-1 expression required 18 h. Therefore, both duration and concentration of thrombin exposure regulate the time course of recovery of HPAEC surface PAR-1 expression. The results support the hypothesis that initial recovery of PAR-1 surface expression in endothelial cells results from a rapidly mobilizable PAR-1 pool, whereas delayed recovery results from de novo PAR-1 synthesis. We conclude that thrombin itself regulates endothelial cell surface PAR-1 expression and that decreased surface expression interferes with thrombin-induced endothelial cell activation responses.

Persistent HIV Transcription Induces Sialyl-Lewis-X Glyco-Antigen Cell-Surface Expression

2020 ◽  
Author(s):  
Florent Colomb ◽  
Leila B. Giron ◽  
Leticia Kuri Cervantes ◽  
Tongcui Ma ◽  
Samson Adeniji ◽  
...  
Keyword(s):  
Cell Surface ◽  
Surface Expression ◽  
Sialyl Lewis X ◽  
Cell Surface Expression ◽  
Lewis X ◽  
Hiv Transcription ◽  
Sialyl Lewis

Influence of β-D-mannuronic acid, as a new member of non-steroidal anti-inflammatory drugs family, on expression pattern of chemokines and their receptors in rheumatoid arthritis

2019 ◽  
Vol 16 ◽  
Author(s):  
Mona Aslani ◽  
Arman Ahmadzadeh ◽  
Zahra Aghazadeh ◽  
Majid Zaki-Dizaji ◽  
Laleh Sharifi ◽  
...  
Keyword(s):  
Cell Surface ◽  
Mrna Expression ◽  
Surface Expression ◽  
Phase Iii ◽  
Cell Surface Expression ◽  
Optimum Dose ◽  
High Dose ◽  
Mannuronic Acid ◽  
Anti Inflammatory ◽  
High Doses

Background: : Based on the encouraging results of phase III clinical trial of β-D-mannuronic acid (M2000) (as a new anti-inflammatory drug) in patients with RA, in this study, we aimed to evaluate the effects of this drug on the expression of chemokines and their receptors in PBMCs of RA patients. Methods:: PBMCs of RA patients and healthy controls were separated and the patients' cells were treated with low, moderate and high doses (5, 25 and 50 μg/mL) of M2000 and optimum dose (1 μg/mL) of diclofenac, as a control in RPMI-1640 medium. Real-time PCR was used for evaluating the mRNA expression of CXCR3, CXCR4, CCR2, CCR5 and CCL2/MCP-1. Cell surface expression of CCR2 was investigated using flow cytometry. Results:: CCR5 mRNA expression reduced significantly, after treatment of the patients' cells with all three doses of M2000 and optimum dose of diclofenac. CXCR3 mRNA expression down-regulated significantly followed by treatment of these cells with moderate and high doses of M2000 and optimum dose of diclofenac. CXCR4 mRNA expression declined significantly after treatment of these cells with moderate and high doses of M2000. CCL2 mRNA expression significantly reduced only followed by treatment of these cells with high dose of M2000, whereas, mRNA and cell surface expressions of CCR2 diminished significantly followed by treatment of these cells with high dose of M2000 and optimum dose of diclofenac. Conclusion:: According to our results, M2000 through the down-regulation of chemokines and their receptors may restrict the infiltration of immune cells into the synovium.

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