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

2005 ◽  
Vol 288 (5) ◽  
pp. F930-F938 ◽  
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.

scholarly journals CombiFlow: Combinatorial AML-specific plasma membrane expression profiles allow longitudinal tracking of clones

2021 ◽  
Author(s):  
Roos Houtsma ◽  
Nisha K. van der Meer ◽  
Kees Meijer ◽  
Linde Morsink ◽  
Shanna M. Hogeling ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Residual Disease ◽  
Expression Profiles ◽  
Clonal Evolution ◽  
Membrane Expression ◽  
Routine Diagnostics ◽  
Free Survival ◽  
Longitudinal Tracking ◽  
Plasma Membrane Expression

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.

scholarly journals Palmitoylation of the Rous Sarcoma Virus Transmembrane Glycoprotein Is Required for Protein Stability and Virus Infectivity

2001 ◽  
Vol 75 (23) ◽  
pp. 11544-11554 ◽  
Author(s):  
Christina Ochsenbauer-Jambor ◽  
David C. Miller ◽  
Charles R. Roberts ◽  
Sung S. Rhee ◽  
Eric Hunter
Keyword(s):  
Plasma Membrane ◽  
Rous Sarcoma Virus ◽  
Double Mutant ◽  
Surface Expression ◽  
Virus Infectivity ◽  
Wild Type ◽  
Membrane Expression ◽  
Rous Sarcoma ◽  
Plasma Membrane Expression ◽  
Sarcoma Virus

ABSTRACT The Rous sarcoma virus (RSV) transmembrane (TM) glycoprotein is modified by the addition of palmitic acid. To identify whether conserved cysteines within the hydrophobic anchor region are the site(s) of palmitoylation, and to determine the role of acylation in glycoprotein function, cysteines at residues 164 and 167 of the TM protein were mutated to glycine (C164G, C167G, and C164G/C167G). In CV-1 cells, palmitate was added to env gene products containing single mutations but was absent in the double-mutant Env. Although mutant Pr95 Env precursors were synthesized with wild-type kinetics, the phenotypes of the mutants differed markedly. Env-C164G had properties similar to those of the wild type, while Env-C167G was degraded faster, and Env containing the double mutant C164G/C167G was very rapidly degraded. Degradation occurred after transient plasma membrane expression. The decrease in steady-state surface expression and increased rate of internalization into endosomes and lysosomes paralleled the decrease in palmitoylation observed for the mutants. The phenotypes of mutant viruses were assessed in avian cells in the context of the pATV8R proviral genome. Virus containing the C164G mutation replicated with wild-type kinetics but exhibited reduced peak reverse transcriptase levels. In contrast, viruses containing either the C167G or the C164G/C167G mutation were poorly infectious or noninfectious, respectively. These phenotypes correlated with different degrees of glycoprotein incorporation into virions. Infectious revertants of the double mutant demonstrated the importance of cysteine-167 for efficient plasma membrane expression and Env incorporation. The observation that both cysteines within the membrane-spanning domain are accessible for acylation has implications for the topology of this region, and a model is proposed.

scholarly journals Mutations in sixth transmembrane domain of AQP2 inhibit its translocation induced by vasopression

2000 ◽  
Vol 278 (3) ◽  
pp. F395-F405 ◽  
Author(s):  
Yumi Yamashita ◽  
Keiji Hirai ◽  
Yoshifumi Katayama ◽  
Kiyohide Fushimi ◽  
Sei Sasaki ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Immunofluorescence Microscopy ◽  
Transmembrane Domain ◽  
Signal Sequence ◽  
Collecting Duct ◽  
Water Channel ◽  
Wild Type ◽  
Membrane Expression ◽  
Plasma Membrane Expression ◽  
Golgi Network

Vasopression-induced phosphorylation of serine 256 of the aquaporin-2 (AQP2) water channel triggers translocation of the protein from cystolic reservoir vesicles to the apical membrane of collecting duct principal cells. Dileucine motifs are located in the sixth transmembrane domain (6TM) of AQP2 and are known as the signal sequence for internalization, sorting from the trans-Golgi network to endosomes/lysosomes, and basolateral sorting. In this study, involvement of 6TM in vasopressin-induced translocation of the protein was investigated. A series of mutations in 6TM of AQP2 was introduced to rat cDNA and expressed in LLC-PK1 cells. Immunofluorescence microscopy indicated that the mutant AQP2 proteins were retained in the cytoplasm after vasopressin stimulation, which actually promoted the plasma membrane expression of wild-type protein. Immunoelectron microscopy showed that the mutant AQP2 proteins reached the endosomes but did not reach the plasma membrane. These results demonstrate that 6TM has essential domains for vasopressin-induced translocation from endosomes to the plasma membrane.

The actin cytoskeleton regulates exocytosis of all neutrophil granule subsets

2007 ◽  
Vol 292 (5) ◽  
pp. C1690-C1700 ◽  
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.

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 Dopamine desynchronizes the retinal clock through a melanopsin-dependent regulation of acetylcholine retinal waves during development

2021 ◽  
Author(s):  
Chaimaa Kinane ◽  
Hugo Calligaro ◽  
Antonin Jandot ◽  
Christine Coutanson ◽  
Nasser Haddjeri ◽  
...  
Keyword(s):  
Ganglion Cells ◽  
Critical Role ◽  
Wild Type ◽  
Retinal Waves ◽  
Dopaminergic Cells ◽  
Bidirectional Regulation ◽  
Chemical Synapses ◽  
External Time ◽  
Da Release

AbstractDopamine (DA) plays a critical role in retinal physiology, including resetting of the retinal circadian clock that in turn regulates DA release. DA acts on major classes of retinal cells by reconfiguring electrical and chemical synapses. Although a bidirectional regulation between intrinsically photosensitive melanopsin ganglion cells (ipRGCs) and dopaminergic cells has been demonstrated during development and adulthood, DA involvement in the ontogeny of the retinal clock is still unknown.Using wild-typePer2Lucand melanopsin knockout (Opn4-/-::Per2Luc) mice at different postnatal stages, we found that the retina can generate self-sustained circadian rhythms from postnatal day 5 that emerge in the absence of external time cues in both genotypes. Intriguingly, DA lengthens the endogenous period only in wild-type retinas, suggesting that this desynchronizing effect requires melanopsin. Furthermore, blockade of cholinergic retinal waves in wild-type retinas induces a shortening of the period, similarly toOpn4-/-::Per2Lucexplants. Altogether, these data suggest that DA desynchronizes the retinal clock through a melanopsin-dependent regulation of acetylcholine retinal waves, thus offering a new role of melanopsin in setting the period of the retinal clock during development.

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