scholarly journals The PDZ-interacting domain of TRPC4 controls its localization and surface expression in HEK293 cells

2002 ◽  
Vol 115 (17) ◽  
pp. 3497-3508
Author(s):  
Laurence Mery ◽  
Bettina Strauß ◽  
Jean F. Dufour ◽  
Karl H. Krause ◽  
Markus Hoth
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Surface Expression ◽  
Hek293 Cells ◽  
Cell Surface Expression ◽  
Hek 293 ◽  
Protein Insertion ◽  
Cell Surface Biotinylation ◽  
Surface Localization ◽  
Interacting Domain

Mammalian homologs of the Drosophila TRP protein have been shown to form cation-permeable channels in the plasma membrane but very little is known about the mechanisms that control their cell surface localization. Recently it has been demonstrated that the last three C-terminal amino acids(TRL) of TRPC4 comprise a PDZ-interacting domain that binds to the scaffold protein EBP50 [ezrin/moesin/radixin-binding phosphoprotein 50]. In this report, we have examined the influence of the TRL motif on the subcellular distribution of TRPC4 in human embryonic kidney (HEK) 293 cells. We have also analyzed the consequences of the interaction between EBP50 and the membrane-cytoskeletal adaptors of the ezrin/radixin/moesin (ERM) family for the cell surface expression of TRPC4. Using immunofluorescence microscopy, we found that the mutant lacking the TRL motif accumulated into cell outgrowths and exhibited a punctate distribution pattern whereas the wild-type channel was evenly distributed on the cell surface. Deletion of the PDZ-interacting domain also decreased the expression of TRPC4 in the plasma membrane by 2.4-fold, as assessed by cell surface biotinylation experiments. Finally, in a large percentage of cells co-expressing TRPC4 and an EBP50 mutant lacking the ERM-binding site, TRPC4 was not present in the plasma membrane but co-localized with the truncated scaffold in a perinuclear compartment (most probably representing the Golgi apparatus) and in vesicles associated with actin filaments. Our data demonstrate that the PDZ-interacting domain of TRPC4 controls its localization and surface expression in transfected HEK293 cells. They also point to a yet unexplored role of the EBP50-ERM complex in the regulation of protein insertion into the plasma membrane.

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 Inhibition of endocytosis causes phosphorylation (S256)-independent plasma membrane accumulation of AQP2

2004 ◽  
Vol 286 (2) ◽  
pp. F233-F243 ◽  
Author(s):  
Hua Lu ◽  
Tian-Xiao Sun ◽  
Richard Bouley ◽  
Karen Blackburn ◽  
Margaret McLaughlin ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Collecting Duct ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Wild Type ◽  
Independent Manner ◽  
Cell Surface Biotinylation ◽  
Extensive Cell ◽  
Medullary Collecting Duct

Inhibition of clathrin-mediated endocytosis by expression of a GTPase-deficient dynamin mutant (dynamin-2/K44A) for 16 h results in an accumulation of plasma membrane aquaporin-2 (AQP2) in epithelial cells stably transfected with wild-type AQP2. We now show a similar effect of K44A dynamin in LLC-PK1 cells transfected with an S256 phosphorylation-deficient AQP2 mutant, AQP2(S256A), and in AQP2-transfected inner medullary collecting duct (IMCD) cells. More acute blockade of endocytosis in these cells with the cholesterol-depleting agent methyl-β-cyclodextrin (mβCD; 10 mM) resulted in a rapid and extensive cell-surface accumulation of both wild-type AQP2 and AQP2 (S256A) within 15 min after treatment. This effect was similar to that induced by treatment of the cells with vasopressin. Blockade of endocytosis by mβCD was confirmed using quantitative analysis of FITC-dextran uptake and AQP2 membrane insertion was verified by cell-surface biotinylation. These data indicate that AQP2 recycles constitutively and rapidly between intracellular stores and the cell surface in LLC-PK1 and IMCD cells. The constitutive trafficking process is not dependent on phosphorylation of the serine-256 residue of AQP2, which is, however, an essential step for regulated vasopressin/cAMP-mediated translocation of AQP2. Our data show that rapid and extensive plasma membrane accumulation of AQP2 can occur in a vasopressin receptor (V2R)- and phosphorylation-independent manner, pointing to a potential means of bypassing the mutated V2R in X-linked nephrogenic diabetes insipidus to achieve cell surface expression of AQP2.

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.

Effect of aging on CD11b and CD69 surface expression by vesicular insertion in human polymorphonuclear leucocytes

1999 ◽  
Vol 97 (3) ◽  
pp. 323-329 ◽  
Author(s):  
J. M. NOBLE ◽  
G. A. FORD ◽  
T. H. THOMAS
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Surface Expression ◽  
Cell Surface Receptor ◽  
Cell Surface Expression ◽  
Elderly Subjects ◽  
Polymorphonuclear Leucocytes ◽  
Cd11b Expression ◽  
Cd69 Expression ◽  
Vesicle Exocytosis

The exocytosis of intracellular vesicles is an important function of the plasma membrane, which is responsible for hormone secretion, cell surface expression of antigens, ion transporters and receptors, and intracellular and intercellular signalling. Human aging is associated with many physiological and cellular changes, many of which are due to alterations in plasma membrane functioning. Alterations in vesicle externalization with age could account for many of these changes. We investigated whether alterations in vesicle exocytosis occur with increasing age by flow-cytometric determination of CD11b and CD69 expression on the surface of human polymorphonuclear leucocytes (PMN) stimulated with phorbol myristate acetate (PMA), a tumour promoter which binds to and activates protein kinase C (PKC) directly, or with formyl-Met-Leu-Phe (fMLP), which activates PKC indirectly via interactions with a cell surface receptor and G-protein, and subsequent inositol phosphate hydrolysis. Following stimulation with PMA, a decrease in the proportion of PMN expressing CD69 at high levels was observed in elderly compared with young subjects (young, 55.3%; elderly, 43.9%; P = 0.01). No aging-related differences in the proportion of PMN expressing CD11b (young, 73.7%; elderly, 68.4%; P = 0.15), or in the number of molecules of CD69 or CD11b expressed per cell, were observed. Stimulation with fMLP or low PMA concentrations resulted in full CD11b expression but minimal CD69 expression in both young and elderly subjects. Cells which expressed CD69 had no CD11b expression, while those cells expressing CD11b had minimal CD69 expression. Thus the PMA-induced expression of CD11b and CD69 in human PMN represents two separate processes, only one of which is affected in aging. CD11b expression appears to require a lesser degree of PKC stimulation compared with that required for CD69 expression. The age-associated reduction in PMA-stimulated CD69 expression may occur either at or distal to PKC activation. Such a decrease may contribute to the age-associated impairments in PMN function that contribute, in turn, to immunosenescence.

scholarly journals Serine 129 phosphorylation of membrane-associated α-synuclein modulates dopamine transporter function in a G protein–coupled receptor kinase–dependent manner

2013 ◽  
Vol 24 (11) ◽  
pp. 1649-1660 ◽  
Author(s):  
Susumu Hara ◽  
Shigeki Arawaka ◽  
Hiroyasu Sato ◽  
Youhei Machiya ◽  
Can Cui ◽  
...  
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
Dopamine Transporter ◽  
Surface Expression ◽  
Hek293 Cells ◽  
Cell Surface Expression ◽  
Receptor Kinase ◽  
Wild Type ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled

Most α-synuclein (α-syn) deposited in Lewy bodies, the pathological hallmark of Parkinson disease (PD), is phosphorylated at Ser-129. However, the physiological and pathological roles of this modification are unclear. Here we investigate the effects of Ser-129 phosphorylation on dopamine (DA) uptake in dopaminergic SH-SY5Y cells expressing α-syn. Subcellular fractionation of small interfering RNA (siRNA)–treated cells shows that G protein–coupled receptor kinase 3 (GRK3), GRK5, GRK6, and casein kinase 2 (CK2) contribute to Ser-129 phosphorylation of membrane-associated α-syn, whereas cytosolic α-syn is phosphorylated exclusively by CK2. Expression of wild-type α-syn increases DA uptake, and this effect is diminished by introducing the S129A mutation into α-syn. However, wild-type and S129A α-syn equally increase the cell surface expression of dopamine transporter (DAT) in SH-SY5Y cells and nonneuronal HEK293 cells. In addition, siRNA-mediated knockdown of GRK5 or GRK6 significantly attenuates DA uptake without altering DAT cell surface expression, whereas knockdown of CK2 has no effect on uptake. Taken together, our results demonstrate that membrane-associated α-syn enhances DA uptake capacity of DAT by GRKs-mediated Ser-129 phosphorylation, suggesting that α-syn modulates intracellular DA levels with no functional redundancy in Ser-129 phosphorylation between GRKs and CK2.

scholarly journals GLUT8 Subcellular Localization and Absence of Translocation to the Plasma Membrane in PC12 Cells and Hippocampal Neurons

Endocrinology ◽  
2005 ◽  
Vol 146 (11) ◽  
pp. 4727-4736 ◽  
Author(s):  
Mathieu Widmer ◽  
Marc Uldry ◽  
Bernard Thorens
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Cell Surface ◽  
Pc12 Cells ◽  
Hippocampal Neurons ◽  
Osmotic Shock ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Intracellular Compartment ◽  
Early Endosomes

GLUT8 is a high-affinity glucose transporter present mostly in testes and a subset of brain neurons. At the cellular level, it is found in a poorly defined intracellular compartment in which it is retained by an N-terminal dileucine motif. Here we assessed GLUT8 colocalization with markers for different cellular compartments and searched for signals, which could trigger its cell surface expression. We showed that when expressed in PC12 cells, GLUT8 was located in a perinuclear compartment in which it showed partial colocalization with markers for the endoplasmic reticulum but not with markers for the trans-Golgi network, early endosomes, lysosomes, and synaptic-like vesicles. To evaluate its presence at the plasma membrane, we generated a recombinant adenovirus for the expression of GLUT8 containing an extracellular myc epitope. Cell surface expression was evaluated by immunofluorescence microscopy of transduced PC12 cells or primary hippocampal neurons exposed to different stimuli. Those included substances inducing depolarization, activation of protein kinase A and C, activation or inhibition of tyrosine kinase-linked signaling pathways, glucose deprivation, AMP-activated protein kinase stimulation, and osmotic shock. None of these stimuli-induced GLUT8 cell surface translocation. Furthermore, when GLUT8myc was cotransduced with a dominant-negative form of dynamin or GLUT8myc-expressing PC-12 cells or neurons were incubated with an anti-myc antibody, no evidence for constitutive recycling of the transporter through the cell surface could be obtained. Thus, in cells normally expressing it, GLUT8 was associated with a specific intracellular compartment in which it may play an as-yet-uncharacterized role.

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