Three fluorescent protein voltage sensors exhibit low plasma membrane expression in mammalian cells

2007 ◽  
Vol 161 (1) ◽  
pp. 32-38 ◽  
Author(s):  
B.J. Baker ◽  
H. Lee ◽  
V.A. Pieribone ◽  
L.B. Cohen ◽  
E.Y. Isacoff ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Membrane Expression ◽  
Voltage Sensors ◽  
Plasma Membrane Expression ◽  
Expression In Mammalian Cells

scholarly journals The inhibition of monocarboxylate transporter 2 (MCT2) by AR-C155858 is modulated by the associated ancillary protein

2010 ◽  
Vol 431 (2) ◽  
pp. 217-225 ◽  
Author(s):  
Matthew J. Ovens ◽  
Christine Manoharan ◽  
Marieangela C. Wilson ◽  
Clarey M. Murray ◽  
Andrew P. Halestrap
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Monocarboxylate Transporter ◽  
Xenopus Laevis Oocytes ◽  
Transport Activity ◽  
Membrane Expression ◽  
Binding Partner ◽  
Reverse Transcription Pcr ◽  
C Terminus ◽  
Plasma Membrane Expression

In mammalian cells, MCTs (monocarboxylate transporters) require association with an ancillary protein to enable plasma membrane expression of the active transporter. Basigin is the preferred binding partner for MCT1, MCT3 and MCT4, and embigin for MCT2. In rat and rabbit erythrocytes, MCT1 is associated with embigin and basigin respectively, but its sensitivity to inhibition by AR-C155858 was found to be identical. Using RT (reverse transcription)–PCR, we have shown that Xenopus laevis oocytes contain endogenous basigin, but not embigin. Co-expression of exogenous embigin was without effect on either the expression of MCT1 or its inhibition by AR-C155858. In contrast, expression of active MCT2 at the plasma membrane of oocytes was significantly enhanced by co-expression of exogenous embigin. This additional transport activity was insensitive to inhibition by AR-C155858 unlike that by MCT2 expressed with endogenous basigin that was potently inhibited by AR-C155858. Chimaeras and C-terminal truncations of MCT1 and MCT2 were also expressed in oocytes in the presence and absence of exogenous embigin. L-Lactate Km values for these constructs were determined and revealed that the TM (transmembrane) domains of an MCT, most probably TM7–TM12, but not the C-terminus, are the major determinants of L-lactate affinity, whereas the associated ancillary protein has little or no effect. Inhibitor titrations of lactate transport by these constructs indicated that embigin modulates MCT2 sensitivity to AR-C155858 through interactions with both the intracellular C-terminus and TMs 3 and 6 of MCT2. The C-terminus of MCT2 was found to be essential for its expression with endogenous basigin.

The adaptor protein 14-3-3 binds to the calcium-sensing receptor and attenuates receptor-mediated Rho kinase signalling

2012 ◽  
Vol 441 (3) ◽  
pp. 995-1007 ◽  
Author(s):  
Ajanthy Arulpragasam ◽  
Aaron L. Magno ◽  
Evan Ingley ◽  
Suzanne J. Brown ◽  
Arthur D. Conigrave ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Rho Kinase ◽  
Adaptor Protein ◽  
Proximal Region ◽  
Calcium Sensing Receptor ◽  
Membrane Expression ◽  
Hek 293 ◽  
Calcium Sensing ◽  
Plasma Membrane Expression

A yeast two-hybrid screen performed to identify binding partners of the CaR (calcium-sensing receptor) intracellular tail identified the adaptor protein 14-3-3θ as a novel binding partner that bound to the proximal membrane region important for CaR expression and signalling. The 14-3-3θ protein directly interacted with the CaR tail in pull-down studies and FLAG-tagged CaR co-immunoprecipitated with EGFP (enhanced green fluorescent protein)-tagged 14-3-3θ when co-expressed in HEK (human embryonic kidney)-293 or COS-1 cells. The interaction between the CaR and 14-3-3θ did not require a putative binding site in the membrane-proximal region of the CaR tail and was independent of PKC (protein kinase C) phosphorylation. Confocal microscopy demonstrated co-localization of the CaR and EGFP–14-3-3θ in the ER (endoplasmic reticulum) of HEK-293 cells that stably expressed the CaR (HEK-293/CaR cells), but 14-3-3θ overexpression had no effect on membrane expression of the CaR. Overexpression of 14-3-3θ in HEK-293/CaR cells attenuated CaR-mediated Rho signalling, but had no effect on ERK (extracellular-signal-regulated kinase) 1/2 signalling. Another isoform identified from the library, 14-3-3ζ, exhibited similar behaviour to that of 14-3-3θ with respect to CaR tail binding, cellular co-localization and impact on receptor-mediated signalling. However, unlike 14-3-3θ, this isoform, when overexpressed, significantly reduced CaR plasma membrane expression. Results indicate that 14-3-3 proteins mediate CaR-dependent Rho signalling and may modulate the plasma membrane expression of the CaR.

scholarly journals Claudin-19 mediates the effects of NO on the paracellular pathway in thick ascending limbs

2019 ◽  
Vol 317 (2) ◽  
pp. F411-F418
Author(s):  
Casandra M. Monzon ◽  
Jeffrey L. Garvin
Keyword(s):  
Plasma Membrane ◽  
Control Period ◽  
Paracellular Pathway ◽  
Membrane Expression ◽  
No Donor ◽  
Charge Selectivity ◽  
Plasma Membrane Expression ◽  
Cgmp Signaling ◽  
Blocking Antibodies ◽  
Dilution Potential

Claudins are a family of tight junction proteins that provide size and charge selectivity to solutes traversing the paracellular space. Thick ascending limbs (TALs) express numerous claudins, including claudin-19. Nitric oxide (NO), via cGMP, reduces dilution potentials in perfused TALs, a measure of paracellular permeability, but the role of claudin-19 is unknown. We hypothesized that claudin-19 mediates the effects of NO/cGMP on the paracellular pathway in TALs via increases in plasma membrane expression of this protein. We measured the effect of the NO donor spermine NONOate (SPM) on dilution potentials with and without blocking antibodies and plasma membrane expression of claudin-19. During the control period, the dilution potential was −18.2 ± 1.8 mV. After treatment with 200 μmol/l SPM, it was −14.7 ± 2.0 mV ( P < 0.04). In the presence of claudin-19 antibody, the dilution potential was −12.7 ± 2.1 mV. After SPM, it was −12.9 ± 2.4 mV, not significantly different. Claudin-19 antibody alone had no effect on dilution potentials. In the presence of Tamm-Horsfall protein antibody, SPM reduced the dilution potential from −9.7 ± 1.0 to −6.3 ± 1.1 mV ( P < 0.006). Dibutyryl-cGMP (500 µmol/l) reduced the dilution potential from −19.6 ± 2.6 to −17.2 ± 2.3 mV ( P < 0.002). Dibutyryl-cGMP increased expression of claudin-19 in the plasma membrane from 29.9 ± 3.8% to 65.9 ± 10.1% of total ( P < 0.011) but did not change total expression. We conclude that claudin-19 mediates the effects of the NO/cGMP signaling cascade on the paracellular pathway.

scholarly journals Counterregulation of clathrin-mediated endocytosis by the actin and microtubular cytoskeleton in human neutrophils

2009 ◽  
Vol 296 (4) ◽  
pp. C857-C867 ◽  
Author(s):  
Silvia M. Uriarte ◽  
Neelakshi R. Jog ◽  
Gregory C. Luerman ◽  
Samrath Bhimani ◽  
Richard A. Ward ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Actin Cytoskeleton ◽  
Human Neutrophils ◽  
Functional Responses ◽  
Membrane Expression ◽  
Granule Exocytosis ◽  
Plasma Membrane Expression ◽  
Microtubular Cytoskeleton ◽  
Latrunculin A ◽  
Azurophil Granule

We have recently reported that disruption of the actin cytoskeleton enhanced N-formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated granule exocytosis in human neutrophils but decreased plasma membrane expression of complement receptor 1 (CR1), a marker of secretory vesicles. The present study was initiated to determine if reduced CR1 expression was due to fMLP-stimulated endocytosis, to determine the mechanism of this endocytosis, and to examine its impact on neutrophil functional responses. Stimulation of neutrophils with fMLP or ionomycin in the presence of latrunculin A resulted in the uptake of Alexa fluor 488-labeled albumin and transferrin and reduced plasma membrane expression of CR1. These effects were prevented by preincubation of the cells with sucrose, chlorpromazine, or monodansylcadaverine (MDC), inhibitors of clathrin-mediated endocytosis. Sucrose, chlorpromazine, and MDC also significantly inhibited fMLP- and ionomycin-stimulated specific and azurophil granule exocytosis. Disruption of microtubules with nocodazole inhibited endocytosis and azurophil granule exocytosis stimulated by fMLP in the presence of latrunculin A. Pharmacological inhibition of phosphatidylinositol 3-kinase, ERK1/2, and PKC significantly reduced fMLP-stimulated transferrin uptake in the presence of latrunculin A. Blockade of clathrin-mediated endocytosis had no significant effect on fMLP-stimulated phosphorylation of ERK1/2 in neutrophils pretreated with latrunculin A. From these data, we conclude that the actin cytoskeleton functions to limit microtubule-dependent, clathrin-mediated endocytosis in stimulated human neutrophils. The limitation of clathrin-mediated endocytosis by actin regulates the extent of both specific and azurophilic granule exocytosis.

Optimization and Application of a Biotinylation Method for Quantification of Plasma Membrane Expression of Transporters in Cells

2017 ◽  
Vol 19 (5) ◽  
pp. 1377-1386 ◽  
Author(s):  
Vineet Kumar ◽  
Tot Bui Nguyen ◽  
Beáta Tóth ◽  
Viktoria Juhasz ◽  
Jashvant D. Unadkat
Keyword(s):  
Plasma Membrane ◽  
Membrane Expression ◽  
Plasma Membrane Expression ◽  
In Cells

scholarly journals Low Plasma Membrane Expression of the Miltefosine Transport Complex Renders Leishmania braziliensis Refractory to the Drug

2009 ◽  
Vol 53 (4) ◽  
pp. 1305-1313 ◽  
Author(s):  
María P. Sánchez-Cañete ◽  
Luís Carvalho ◽  
F. Javier Pérez-Victoria ◽  
Francisco Gamarro ◽  
Santiago Castanys
Keyword(s):  
Plasma Membrane ◽  
Cutaneous Leishmaniasis ◽  
Oral Drug ◽  
Leishmania Braziliensis ◽  
Β Subunit ◽  
Membrane Expression ◽  
Plasma Membrane Expression ◽  
Highly Sensitive ◽  
P Type ◽  
Transport Complex

ABSTRACT Miltefosine (hexadecylphosphocholine, MLF) is the first oral drug with recognized efficacy against both visceral and cutaneous leishmaniasis. However, some clinical studies have suggested that MLF shows significantly less efficiency against the cutaneous leishmaniasis caused by Leishmania braziliensis. In this work, we have determined the cellular and molecular basis for the natural MLF resistance observed in L. braziliensis. Four independent L. braziliensis clinical isolates showed a marked decrease in MLF sensitivity that was due to their inability to internalize the drug. MLF internalization in the highly sensitive L. donovani species requires at least two proteins in the plasma membrane, LdMT, a P-type ATPase involved in phospholipid translocation, and its β subunit, LdRos3. Strikingly, L. braziliensis parasites showed highly reduced levels of this MLF translocation machinery at the plasma membrane, mainly because of the low expression levels of the β subunit, LbRos3. Overexpression of LbRos3 induces increased MLF sensitivity not only in L. braziliensis promastigotes but also in intracellular amastigotes. These results further highlight the importance of the MLF translocation machinery in determining MLF potency and point toward the development of protocols to routinely monitor MLF susceptibility in geographic areas where L. braziliensis might be prevalent.

scholarly journals Reduced Plasma Membrane Expression of Dysferlin Mutants Is Attributed to Accelerated Endocytosis via a Syntaxin-4-associated Pathway

2010 ◽  
Vol 285 (37) ◽  
pp. 28529-28539 ◽  
Author(s):  
Frances J. Evesson ◽  
Rachel A. Peat ◽  
Angela Lek ◽  
Fabienne Brilot ◽  
Harriet P. Lo ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Expression ◽  
Plasma Membrane Expression ◽  
Syntaxin 4
Sign in / Sign up

Export Citation Format

Share Document