The role of charged residues in the transmembrane helices of monocarboxylate transporter 1 and its ancillary protein basigin in determining plasma membrane expression and catalytic activity

Acute myeloid leukemia (AML) often presents as an oligoclonal disease whereby multiple genetically distinct subclones can co-exist within patients. Differences in signaling and drug sensitivity of such subclones complicates treatment and warrants tools to identify them and track disease progression. We previously identified over 50 AML-specific plasma membrane (PM) proteins and seven of these (CD82, CD97, FLT3, IL1RAP, TIM3, CD25 and CD123) were implemented in routine diagnostics in patients with AML (n=256) and MDS (n=33). We developed a pipeline termed CombiFlow in which expression data of multiple PM markers is merged, allowing a Principle Component-based analyses to identify distinctive marker expression profiles and to generate single cell tSNE landscapes to longitudinally track clonal evolution. Positivity for one or more of the markers after 2 courses of intensive chemotherapy predicted a shorter relapse-free survival supporting a role of these markers in measurable residual disease (MRD) detection. CombiFlow also allowed the tracking of clonal evolution in paired diagnosis and relapse samples (n=12). Extending the panel to 36 AML-specific markers further refined the CombiFlow pipeline. In conclusion, CombiFlow provides a valuable tool in the diagnosis, MRD detection, clonal tracking, and the understanding of clonal heterogeneity in AML.

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

Biochemical Journal ◽

10.1042/bj20100890 ◽

2010 ◽

Vol 431 (2) ◽

pp. 217-225 ◽

Cited By ~ 52

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.

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Bidirectional regulation of AQP2 trafficking and recycling: involvement of AQP2-S256 phosphorylation

AJP Renal Physiology ◽

10.1152/ajprenal.00291.2004 ◽

2005 ◽

Vol 288 (5) ◽

pp. F930-F938 ◽

Cited By ~ 104

Author(s):

Lene N. Nejsum ◽

Marina Zelenina ◽

Anita Aperia ◽

Jørgen Frøkiær ◽

Søren Nielsen

Keyword(s):

Plasma Membrane ◽

Rat Kidney ◽

Wild Type ◽

Membrane Expression ◽

Bidirectional Regulation ◽

Plasma Membrane Expression ◽

Intracellular Vesicles ◽

Membrane Treatment ◽

Microscopic Techniques

The present study examined the role of PKA and serine256 (S256) phosphorylation for AQP2 trafficking and recycling using cells transfected with wild-type AQP2 (AQP2-WT) or mutant AQP2 and high-resolution confocal microscopic techniques. In transiently transfected MDCK-C7 cells, stimulation with forskolin induced translocation of AQP2-WT to the plasma membrane. Treatment of AQP2-WT cells with the PKA inhibitor H-89 following forskolin stimulation resulted in internalization of AQP2-WT. Moreover, H-89 treatment of AQP2-S256D (mimicking constitutively phosphorylated AQP2 and hence localized to the plasma membrane) resulted in redistribution of AQP2-S256D to intracellular vesicles, even in the presence of forskolin. Both PGE2 and dopamine stimulation induced endocytosis of AQP2-WT and AQP2-S256D, respectively, in forskolin-stimulated cells. Consistent with this, dopamine in the presence of vasopressin stimulated endocytosis of AQP2 in slices of rat kidney inner medulla without substantial dephosphorylation. In conclusion, these results strongly suggest that 1) S256 phosphorylation is necessary but not sufficient for AQP2 plasma membrane expression, 2) active PKA is required for AQP2 plasma membrane expression, 3) PGE2 and dopamine induce internalization of AQP2 independently of AQP2 dephosphorylation, and 4) preceding activation of cAMP production is necessary for PGE2 and dopamine to cause AQP2 internalization.

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The actin cytoskeleton regulates exocytosis of all neutrophil granule subsets

AJP Cell Physiology ◽

10.1152/ajpcell.00384.2006 ◽

2007 ◽

Vol 292 (5) ◽

pp. C1690-C1700 ◽

Cited By ~ 76

Author(s):

Neelakshi R. Jog ◽

Madhavi J. Rane ◽

George Lominadze ◽

Gregory C. Luerman ◽

Richard A. Ward ◽

...

Keyword(s):

Plasma Membrane ◽

Actin Cytoskeleton ◽

Immunoblot Analysis ◽

Maximal Response ◽

Initial Increase ◽

Membrane Expression ◽

Plasma Membrane Expression ◽

Latrunculin A ◽

Specific Granules

A comprehensive analysis of the role of the actin cytoskeleton in exocytosis of the four different neutrophil granule subsets had not been performed previously. Immunoblot analysis showed that, compared with plasma membrane, there was less actin associated with secretory vesicles (SV, 75%), gelatinase granules (GG, 40%), specific granules (SG, 10%), and azurophil granules (AG, 5%). Exocytosis of SV, SG, and AG was measured as increased plasma membrane expression of CD35, CD66b, and CD63, respectively, with flow cytometry, and GG exocytosis was measured as gelatinase release with an ELISA. N-formylmethionyl-leucyl-phenylalanine (FMLP) stimulated exocytosis of SV, GG, and SG with an ED50of 15, 31, and 28 nM, respectively, with maximal response at 10−7M FMLP by 5 min, while no exocytosis of AG was detected. Disruption of the actin cytoskeleton by latrunculin A and cytochalasin D induced a decrease in FMLP-stimulated CD35 expression after an initial increase. Both drugs enhanced the rate and extent of FMLP-stimulated GG, SG, and AG exocytosis, while the EC50for FMLP was not altered. We conclude that the actin cytoskeleton controls access of neutrophil granules to the plasma membrane, thereby limiting the rate and extent of exocytosis of all granule subsets. Differential association of actin with the four granule subsets was not associated with graded exocytosis.

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

AJP Renal Physiology ◽

10.1152/ajprenal.00065.2019 ◽

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.

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Functional Role of Charged Residues in the Transmembrane Segments of the Yeast Plasma Membrane H+-ATPase

Journal of Biological Chemistry ◽

10.1074/jbc.m000546200 ◽

2000 ◽

Vol 275 (21) ◽

pp. 15709-15716 ◽

Cited By ~ 20

Author(s):

Valery V. Petrov ◽

Kristine P. Padmanabha ◽

Robert K. Nakamoto ◽

Kenneth E. Allen ◽

Carolyn W. Slayman

Keyword(s):

Plasma Membrane ◽

Functional Role ◽

Yeast Plasma Membrane ◽

Transmembrane Segments ◽

Charged Residues

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

AJP Cell Physiology ◽

10.1152/ajpcell.00454.2008 ◽

2009 ◽

Vol 296 (4) ◽

pp. C857-C867 ◽

Cited By ~ 20

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.

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

The AAPS Journal ◽

10.1208/s12248-017-0121-5 ◽

2017 ◽

Vol 19 (5) ◽

pp. 1377-1386 ◽

Cited By ~ 13

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

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Mechanism(s) of butyrate transport in Caco-2 cells: role of monocarboxylate transporter 1

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.2000.279.4.g775 ◽

2000 ◽

Vol 279 (4) ◽

pp. G775-G780 ◽

Cited By ~ 110

Author(s):

Christos Hadjiagapiou ◽

Larry Schmidt ◽

Pradeep K. Dudeja ◽

Thomas J. Layden ◽

Krishnamurthy Ramaswamy

Keyword(s):

Short Chain Fatty Acid ◽

Rnase Protection Assay ◽

Significant Inhibition ◽

Monocarboxylate Transporter ◽

Rnase Protection ◽

Monocarboxylate Transporter 1 ◽

Antisense Orientation ◽

Transporter Family ◽

Saturation Kinetics

The short-chain fatty acid butyrate was readily taken up by Caco-2 cells. Transport exhibited saturation kinetics, was enhanced by low extracellular pH, and was Na+independent. Butyrate uptake was unaffected by DIDS; however, α-cyano-4-hydroxycinnamate and the butyrate analogs propionate and l-lactate significantly inhibited uptake. These results suggest that butyrate transport by Caco-2 cells is mediated by a transporter belonging to the monocarboxylate transporter family. We identified five isoforms of this transporter, MCT1, MCT3, MCT4, MCT5, and MCT6, in Caco-2 cells by PCR, and MCT1 was found to be the most abundant isoform by RNase protection assay. Transient transfection of MCT1, in the antisense orientation, resulted in significant inhibition of butyrate uptake. The cells fully recovered from this inhibition by 5 days after transfection. In conclusion, our data showed that the MCT1 transporter may play a major role in the transport of butyrate into Caco-2 cells.

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