Na+/H+ exchanger isoform 6 (NHE6/SLC9A6) is involved in clathrin-dependent endocytosis of transferrin

2011 ◽  
Vol 301 (6) ◽  
pp. C1431-C1444 ◽  
Author(s):  
Lou Xinhan ◽  
Masafumi Matsushita ◽  
Manami Numaza ◽  
Akira Taguchi ◽  
Keiji Mitsui ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Ph Regulation ◽  
Microscopic Analysis ◽  
Cholera Toxin B Subunit ◽  
Wild Type ◽  
B Subunit ◽  
Epidermal Growth ◽  
Transferrin Uptake ◽  
In Cells

In mammalian cells, nine conserved isoforms of the Na+/H+ exchanger (NHE) are known to be important for pH regulation of the cytoplasm and organellar lumens. NHE1–5 are localized to the plasma membrane, whereas NHE6–9 are localized to distinct organelles. NHE6 is localized predominantly in endosomal compartments but is also found in the plasma membrane. To investigate the role of NHE6 in endocytosis, we established NHE6-knockdown HeLa cells and analyzed the effect of this knockdown on endocytotic events. The expression level of NHE6 in knockdown cells was decreased to ∼15% of the level seen in control cells. Uptake of transferrin was also decreased. No effect was found on the endocytosis of epidermal growth factor or on the cholera toxin B subunit. Moreover, in the NHE6-knockdown cells, transferrin uptake was found to be affected in the early stages of endocytosis. Microscopic analysis revealed that, at 2 min after the onset of endocytosis, colocalization of NHE6, clathrin, and transferrin was observed, which suggests that NHE6 was localized to endocytotic, clathrin-coated vesicles. In addition, in knockdown cells, transferrin-positive endosomes were acidified, but no effect was found on cytoplasmic pH. In cells overexpressing wild-type NHE6, increased transferrin uptake was observed, but no such increase was seen in cells overexpressing mutant NHE6 deficient in ion transport. The luminal pH in transferrin-positive endosomes was alkalized in cells overexpressing wild-type NHE6 but normal in cells overexpressing mutant NHE6. These observations suggest that NHE6 regulates clathrin-dependent endocytosis of transferrin via pH regulation.

scholarly journals Aluminum fluoride stimulates surface protrusions in cells overexpressing the ARF6 GTPase.

1996 ◽  
Vol 134 (4) ◽  
pp. 935-947 ◽  
Author(s):  
H Radhakrishna ◽  
R D Klausner ◽  
J G Donaldson
Keyword(s):  
Plasma Membrane ◽  
Aluminum Fluoride ◽  
Wild Type ◽  
Lipoxygenase Pathway ◽  
Transfected Cells ◽  
Defective Mutant ◽  
Transferrin Uptake ◽  
Adp Ribosylation Factor ◽  
Stimulation Of ◽  
In Cells

To study the effector function of the ADP- ribosylation factor (ARF) 6 GTP-binding protein, we transfected HeLa cells with wild-type, epitope-tagged ARF6. Previously shown to indirectly activate the ARF1 GTPase, aluminum fluoride (AIF) treatment of ARF6-transfected cells resulted in a redistribution of both ARF6 and actin to discrete sites on the plasma membrane, which became increasingly protrusive over time. The effects of AIF were reversible, specific to cells transfected with wild-type ARF6, and resembled the cellular protrusions observed in cells expressing the GTPase defective mutant of ARF6. Importantly, the protrusions observed in cells transfected with ARF6 were distinct from the enhanced stress fibers and membrane ruffles observed in cells transfected with RhoA and Rac1, respectively. In cells forming protrusions, there was an apparent stimulation of macropinocytosis and membrane recycling within the protrusive structures. In contrast, no block in transferrin uptake or alteration of the distribution of clathrin AP-2 complexes was detected in these cells. The AIF-induced, ARF6- dependent formation of protrusive structures was blocked by cytochalasin D and inhibitors of the lipoxygenase pathway. These observations support a novel role for the ARF6 GTPase in modeling the plasma membrane and underlying cytoskeleton.

8 FLOW CYTOMETRIC MONITORING OF CHOLERA TOXIN B SUBUNIT BINDING TO BOVINE SPERMATOZOA

2008 ◽  
Vol 20 (1) ◽  
pp. 84
Author(s):  
M. Boilard ◽  
M. Beaulieu ◽  
P. Blondin
Keyword(s):  
Flow Cytometry ◽  
Plasma Membrane ◽  
Lipid Rafts ◽  
Molecular Probes ◽  
Cholera Toxin B Subunit ◽  
Enhanced Fluorescence ◽  
Toxin B ◽  
B Subunit ◽  
Bovine Spermatozoa ◽  
High Fluorescence

In order to become able to fertilize, mammalian spermatozoa must undergo a series of biochemical modifications. This process called capacitation involves several changes of the content and the ultrastucture of the plasma membrane. Among these changes, loss of cholesterol from the plasma membrane is required. Lipid rafts are detergent-insoluble plasma membrane domains rich in cholesterol and sphingolipids. Some proteins are confined to lipid rafts while others are excluded. It has been hypothesized in the past that the loss of cholesterol could destabilize and relocate lipid rafts and would thus affect protein interactions in the plasma membrane, thereby leading to downstream events involved in the capacitation process. Thus, quantification of lipid rafts within the membrane of spermatozoa would become useful to monitor sperm functions and maturation level. The present study aimed to quantify lipid rafts in bovine spermatozoa using the Vibrant Lipid Raft detection kit from Molecular Probes (Invitrogen Canada, Inc., Burlingame, Ontario, Canada) and flow cytometry. The Vibrant kit uses the cholera toxin B subunit (CT-B) and claims to detect ganglioside Gm1 that sublocalizes within lipid rafts. Briefly, freshly ejaculated and frozen/thawed spermatozoa were washed once by centrifugation at 250g for five min in sp-Talp and were then re-suspended in sp-Talp containing 1 �g mL–1 CT-B. Then, cells were incubated at 4�C for 10 min, washed in chilled sp-Talp, incubated for 15 min in the presence of an anti-CT-B antibody coupled to the Alexa Fluor� 488 dye (Molecular Probes), and washed again to remove excess antibody. Spermatozoa were then analyzed with a BD LSR II flow cytometer (BD Biosciences, San Jose, CA, USA). Two populations showing different fluorescence levels were observed in all samples. Greater proportions of spermatozoa displayed the high fluorescence pattern in cryopreserved samples (37.9%) when compared to freshly ejaculated spermatozoa (8.2%) (P < 0.01). Also, when compared to freshly ejaculated spermatozoa, increased proportions of high fluorescence was detected following a 6-h incubation in sp-Talp containing bicarbonate and BSA. These results suggest that capacitation and cryopreservation both promote exposure of CT-B binding molecules in bovine spermatozoa. Microscopic observation of labeled cryopreserved spermatozoa did not yield the expected raft labeling patterns, but rather 5 different patterns of labeling. In the past, some of these patterns were recognized to be associated with capacitation and acrosome reaction. At this point, more work is needed to confirm which of the fluorescent patterns observed in microscopy corresponds to the enhanced fluorescence sperm population observed by flow cytometry and to directly associate this enhanced fluorescence to capacitation or the acrosome reaction. In conclusion, it appears that the Vibrant kit from Molecular Probes cannot be used to quantify lipid rafts by flow cytometry. Nevertheless, it might be an interesting tool to use in flow cytometry to monitor membrane changes associated with capacitation or cryo-damage.

scholarly journals Proline residues in transmembrane segment IV are critical for activity, expression and targeting of the Na+/H+ exchanger isoform 1

2004 ◽  
Vol 379 (1) ◽  
pp. 31-38 ◽  
Author(s):  
Emily R. SLEPKOV ◽  
Signy CHOW ◽  
M. Joanne LEMIEUX ◽  
Larry FLIEGEL
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Mammalian Cells ◽  
Integral Membrane Protein ◽  
Amide Hydrogen ◽  
Wild Type ◽  
Transmembrane Segment ◽  
Mutant Proteins ◽  
Transmembrane Segments ◽  
Induced Changes

NHE1 (Na+/H+ exchanger isoform 1) is a ubiquitously expressed integral membrane protein that regulates intracellular pH in mammalian cells. Proline residues within transmembrane segments have unusual properties, acting as helix breakers and increasing flexibility of membrane segments, since they lack an amide hydrogen. We examined the importance of three conserved proline residues in TM IV (transmembrane segment IV) of NHE1. Pro167 and Pro168 were mutated to Gly, Ala or Cys, and Pro178 was mutated to Ala. Pro168 and Pro178 mutant proteins were expressed at levels similar to wild-type NHE1 and were targeted to the plasma membrane. However, the mutants P167G (Pro167→Gly), P167A and P167C were expressed at lower levels compared with wild-type NHE1, and a significant portion of P167G and P167C were retained intracellularly, possibly indicating induced changes in the structure of TM IV. P167G, P167C, P168A and P168C mutations abolished NHE activity, and P167A and P168G mutations caused markedly decreased activity. In contrast, the activity of the P178A mutant was not significantly different from that of wild-type NHE1. The results indicate that both Pro167 and Pro168 in TM IV of NHE1 are required for normal NHE activity. In addition, mutation of Pro167 affects the expression and membrane targeting of the exchanger. Thus both Pro167 and Pro168 are strictly required for NHE function and may play critical roles in the structure of TM IV of the NHE.

scholarly journals The NR1 subunit of the N-methyl-d-aspartate receptor can be efficiently expressed alone in the cell surface of mammalian cells and is required for the transport of the NR2A subunit

2001 ◽  
Vol 356 (2) ◽  
pp. 539-547 ◽  
Author(s):  
Mónica GARCÍA-GALLO ◽  
Jaime RENART ◽  
Margarita DÍAZ-GUERRA
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Half Life ◽  
Mammalian Cells ◽  
Maturation Process ◽  
Aspartate Receptor ◽  
Heterologous System ◽  
The Stability ◽  
Nr2a Subunit ◽  
In Cells

We have used a heterologous system of expression of N-methyl-d-aspartate (NMDA) receptors based on the use of vaccinia virus to analyse the maturation, transport, assembly and differential expression of the NR1 and NR2A subunits of the receptors. We have demonstrated that the NR1 subunit is efficiently transported to the plasma membrane in cells expressing NR1 alone, similarly to cells producing NR1 and NR2A together. In contrast, NR2A requires NR1 expression to be located at the cell surface. The stability of both receptor subunits expressed alone is similar to that obtained in cells producing NR1 and NR2A. In pulse–chase experiments, the NR1 subunit displays a biphasic decay, with a fraction of the protein having a half-life of only 1h and the remaining presenting a turnover longer than 24h, similar to values obtained for the NR2A subunit. Our results also show a maturation process affecting the carbohydrate moiety in the NR1 subunit, such that immature NR1has a much shorter half-life than the mature form or the NR2A subunit. Finally, we show that only a fraction of mature NR1 interacts with NR2A to form multimeric functional complexes.

scholarly journals Shc Phosphotyrosine-Binding Domain Dominantly Interacts with Epidermal Growth Factor Receptors and Mediates Ras Activation in Intact Cells

1998 ◽  
Vol 12 (4) ◽  
pp. 536-543 ◽  
Author(s):  
Kazuhiko Sakaguchi ◽  
Yoshinori Okabayashi ◽  
Yoshiaki Kido ◽  
Sachiko Kimura ◽  
Yoko Matsumura ◽  
...  
Keyword(s):  
Growth Factor ◽  
Egf Receptor ◽  
Sh2 Domain ◽  
Wild Type ◽  
Intact Cells ◽  
Sh2 Domains ◽  
Epidermal Growth ◽  
Mutant Receptors ◽  
Ptb Domain ◽  
In Cells

Abstract The adaptor protein Shc contains a phosphotyrosine binding (PTB) domain and a Src homology 2 (SH2) domain, both of which are known to interact with phosphorylated tyrosines. We have shown previously that tyrosine 1148 of the activated epidermal growth factor (EGF) receptor is a major binding site for Shc while tyrosine 1173 is a secondary binding site in intact cells. In the present study, we investigated the interaction between the PTB and SH2 domains of Shc and the activated human EGF receptor. Mutant 52-kDa Shc with an arginine-to-lysine substitution at residue 175 in the PTB domain (Shc R175K) or 397 in the SH2 domain (Shc R397K) was coexpressed in Chinese hamster ovary cells overexpressing the wild-type or mutant EGF receptors that retained only one of the autophosphorylation sites at tyrosine 1148 (QM1148) or 1173 (QM1173). Shc R397K was coprecipitated with the QM1148 and QM1173 receptors, was tyrosine-phosphorylated, and associated with Grb2 and Sos. In contrast, coprecipitation of Shc R175K with the mutant receptors was barely detectable. In cells expressing the QM1173 receptor, Shc R175K was tyrosine-phosphorylated and associated with Grb2, while association of Sos was barely detectable. In cells expressing the QM1148 receptor, tyrosine phosphorylation of Shc R175K was markedly reduced. When both Shc R175K and 46-kDa Shc R397K were coexpressed with the mutant receptors, p46 Shc R397K was dominantly tyrosine-phosphorylated. In cells expressing the wild-type receptor, Shc R397K, but not Shc R175K, translocated to the membrane in an EGF-dependent manner. In addition, Ras activity stimulated by the immunoprecipitates of Shc R397K was significantly higher than that by the immunoprecipitates of Shc R175K. The present results indicate that tyrosine 1148 of the activated EGF receptor mainly interacts with the Shc PTB domain in intact cells. Tyrosine 1173 interacts with both the PTB and SH2 domains, although the interaction with the PTB domain is dominant. In addition, Shc bound to the activated EGF receptor via the PTB domain dominantly interacts with Grb2-Sos complex and plays a major role in the Ras-signaling pathway.

scholarly journals High-Resolution FRET Microscopy of Cholera Toxin B-Subunit and GPI-anchored Proteins in Cell Plasma Membranes

2000 ◽  
Vol 11 (5) ◽  
pp. 1645-1655 ◽  
Author(s):  
Anne K. Kenworthy ◽  
Nadezda Petranova ◽  
Michael Edidin
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Cholera Toxin ◽  
Lipid Rafts ◽  
Plasma Membranes ◽  
Membrane Lipid ◽  
Cholera Toxin B Subunit ◽  
Toxin B ◽  
Cholera Toxin B ◽  
B Subunit

“Lipid rafts” enriched in glycosphingolipids (GSL), GPI-anchored proteins, and cholesterol have been proposed as functional microdomains in cell membranes. However, evidence supporting their existence has been indirect and controversial. In the past year, two studies used fluorescence resonance energy transfer (FRET) microscopy to probe for the presence of lipid rafts; rafts here would be defined as membrane domains containing clustered GPI-anchored proteins at the cell surface. The results of these studies, each based on a single protein, gave conflicting views of rafts. To address the source of this discrepancy, we have now used FRET to study three different GPI-anchored proteins and a GSL endogenous to several different cell types. FRET was detected between molecules of the GSL GM1 labeled with cholera toxin B-subunit and between antibody-labeled GPI-anchored proteins, showing these raft markers are in submicrometer proximity in the plasma membrane. However, in most cases FRET correlated with the surface density of the lipid raft marker, a result inconsistent with significant clustering in microdomains. We conclude that in the plasma membrane, lipid rafts either exist only as transiently stabilized structures or, if stable, comprise at most a minor fraction of the cell surface.

scholarly journals Molecular and Functional Characterization of Caveolae in Mixed Cultures of Human NT-2 Neurons and Astrocytes

Neuroglia ◽  
2021 ◽  
Vol 2 (1) ◽  
pp. 68-88
Author(s):  
Jagdeep K. Sandhu ◽  
Maria Ribecco-Lutkiewicz ◽  
Abedelnasser Abulrob
Keyword(s):  
Plasma Membrane ◽  
Functional Characterization ◽  
Mixed Cultures ◽  
Cholera Toxin B Subunit ◽  
Mrna Levels ◽  
Caveolin 1 ◽  
B Subunit ◽  
A Cells ◽  
Human Neurons ◽  
Health And Disease

Caveolae are plasma membrane invaginations that are enriched in cholesterol-binding proteins called caveolins. The presence of caveolae and caveolins in mixed cultures of human neurons and glia has not been investigated. Here, we sought to determine the presence of caveolae and caveolins in human NTera-2 (NT2/D1) cells, differentiated with retinoic acid into neuron-like (NT2/N) and astrocyte-like (NT2/A) cells. We found that while caveolin-3 mRNA levels remained relatively constant, caveolin-1 and -2 levels were upregulated in NT2/A and downregulated in NT2/N. No caveolin-1 immunoreactivity was detected in NT2/N. Electron microscopy revealed numerous flask-shaped invaginations (~86–102 nm in diameter) in the plasma membrane of NT2/A and NT2/N cells, while only few were detected in NT2/D1 cells. Immunoelectron microscopy localized caveolin-1 gold particles in the flask-shaped structures on plasmalemma and cytoplasmic vesicles of NT2/A cells. Furthermore, NT2/A endocytosed Alexa 488 conjugated-cholera toxin B subunit (CTX-B) through a caveolae- and clathrin-dependent pathway, whereas NT2/N endocytosed CTX-B through a caveolae-independent pathway. We have established that while NT2/A expressed functional caveolae, the molecular identity of the plasma membrane invaginations in NT2/N is unknown. The expression of caveolin proteins was differentially regulated in these cells. Taken together, our findings support the usefulness of the human NT2 model system to study the role of caveolins in neuron–glia communication, and their involvement in brain health and disease.

scholarly journals Mutational Analysis of the Human Immunodeficiency Virus Type 1 Vpu Transmembrane Domain That Promotes the Enhanced Release of Virus-Like Particles from the Plasma Membrane of Mammalian Cells

1998 ◽  
Vol 72 (2) ◽  
pp. 1270-1279 ◽  
Author(s):  
Mousumi Paul ◽  
Suparna Mazumder ◽  
Nicholas Raja ◽  
M. Abdul Jabbar
Keyword(s):  
Amino Acids ◽  
Human Immunodeficiency Virus ◽  
Plasma Membrane ◽  
Human Immunodeficiency Virus Type ◽  
Hela Cells ◽  
Mammalian Cells ◽  
Transmembrane Domain ◽  
Wild Type ◽  
Immunodeficiency Virus

ABSTRACT Human immunodeficiency virus type 1 Vpu is a multifunctional phosphoprotein composed of the N-terminal transmembrane (VpuTM) and C-terminal cytoplasmic domains. Each of these domains regulates a distinct function of the protein; the transmembrane domain is critical in virus release, and phosphorylation of the cytoplasmic domain is necessary for CD4 proteolysis. We carried our experiments to identify amino acids in the VpuTM domain that are important in the process of virus-like particle (VLP) release from HeLa cells. VLPs are released from the plasma membrane of HeLa cells at constitutive levels, and Vpu expression enhanced the release of VLPs by a factor of 10 to 15. Deletion of two to five amino acids from both N- and C-terminal ends or the middle of the VpuTM domain generated mutant Vpu proteins that have lost the ability to enhance VLP release. These deletion mutants have not lost the ability to associate with the wild-type or mutant Vpu proteins and formed complexes with equal efficiency. They were also transported normally to the Golgi complex. Furthermore, a Vpu protein having the CD4 transmembrane and Vpu cytoplasmic domains was completely inactive, and Vpu proteins harboring hybrid Vpu-CD4 TM domains were also defective in the ability to enhance the release of VLPs. When tested for functional complementation in cotransfected cells, two inactive proteins were not able to reconstitute Vpu activity that enhances the release of Gag particles. Coexpression of functional CD4/Vpu hybrids or wild-type Vpu with inactive mutant CD4/Vpu proteins revealed that mutations in the VpuTM domain could dominantly interfere with Vpu activity in Gag release. Taken together, these results demonstrated that the structural integrity of the VpuTM domain is critical for Vpu activity in the release of VLPs from the plasma membrane of mammalian cells.

scholarly journals A dominant negative mutation suppresses the function of normal epidermal growth factor receptors by heterodimerization.

1991 ◽  
Vol 11 (3) ◽  
pp. 1454-1463 ◽  
Author(s):  
O Kashles ◽  
Y Yarden ◽  
R Fischer ◽  
A Ullrich ◽  
J Schlessinger
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Living Cells ◽  
Dominant Negative ◽  
Egf Receptors ◽  
Wild Type ◽  
Dominant Negative Mutation ◽  
Epidermal Growth ◽  
Mutant Receptors ◽  
In Cells

Recent studies provide evidence that defective receptors can function as a dominant negative mutation suppressing the action of wild-type receptors. This causes various diminished responses in cell culture and developmental disorders in murine embryogenesis. Here, we describe a model system and a potential mechanism underlying the dominant suppressing response caused by defective epidermal growth factor (EGF) receptors. We used cultured 3T3 cells coexpressing human wild-type receptors and an inactive deletion mutant lacking most of the cytoplasmic domain. When expressed alone, EGF was able to stimulate the dimerization of either wild-type or mutant receptors in living cells as revealed by chemical covalent cross-linking experiments. In response to EGF, heterodimers and homodimers of wild-type and mutant receptors were observed in cells coexpressing both receptor species. However, only homodimers of wild-type EGF receptors underwent EGF-induced tyrosine autophosphorylation in living cells. These results indicate that the integrity of both receptor moieties within receptor dimers is essential for kinase activation and autophosphorylation. Moreover, the presence of mutant receptors in cells expressing wild-type receptors diminished the number of high-affinity binding sites for EGF, reduced the rate of receptor endocytosis and degradation, and diminished biological signalling via EGF receptors. We propose that heterodimerization with defective EGF receptors functions as a dominant negative mutation suppressing the activation and response of normal receptors by formation of unproductive heterodimers.

BIOLOGICAL CHARACTERIZATION OF AMINO-TERMINAL EXON DELETIONS OF T-PA

1987 ◽  
Author(s):  
Glenn R Larsen ◽  
Kim Henson ◽  
Yitzak Blue
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Mammalian Cells ◽  
Specific Activity ◽  
Clot Lysis ◽  
Wild Type ◽  
Amino Terminal ◽  
Type Form ◽  
Epidermal Growth ◽  
Wild Type Form

The secreted form of t-pa is proposed to be a mosaic protein which contains 4 different domain elements based on amino acid homologies with fibronectin finger elements, epidermal growth factor, kringle structures, and the active site of serine proteases. Of the 12 exons which encode these domains only the finger and epidermal growth factor are encoded separately by single exons. To investigate whether a single exon can encode a functional element or domain within a protein, the following precise exon alterations were made by loop-out mutagenesis techniques which deleted either the fibronectin finger, epidermal growth factor, or combination finger/growth factor domain(s). These mutant proteins were expressed in mammalian cells and characterized with respect to affinity for fibrin, fibrinolytic and fibrinogenolytic potential. All mutants demonstrated significantly lower affinity for fibrin with respect to the wild-type protein. We estimate the KD of these mutants for fibrin to be at least 100-fold higher than the wild-type form which we determined to be approximately 0.3 uM. Each mutant retained characteristic activator stimulation by fibrin and were also shown to have the same approximate specific activity as the wild-type form. These mutants were further evaluated in citrated human plasma [125-I]-fibrin clot lysis assays over a range of activator concentrations and shown to behave similarly to wild-type t-pa at therapeutic thrombolytic concentrations. At some lower concentrations, however, reduced fibrinolysis was observed for the mutant forms relative to wild-type. All mutants were evaluated for their fibrinogenolytic potential and demonstrated no significant decrease in coagulable fibrinogen over a five hour period. This was in dramatic contrast to an equivalent activator concentration of urokinase which showed a precipitous decline in coaguable fibrinogen in the first hour.

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