Abstract 323: ABCA1 Expression Promotes ApoAI Acidification on the Plasma Membrane via Recruitment of Vacuolar ATPase

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Shuhui Wang ◽  
Gregory Brubaker ◽  
Jonathan D Smith
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Vacuolar Atpase ◽  
Atpase Inhibitor ◽  
Ph Indicator ◽  
Cell Surface Biotinylation ◽  
Abca1 Expression ◽  
Helical Hairpin ◽  
Inhibitor Dose ◽  
Cell Surface Level

Objective: We have shown that ABCA1 mediates unfolding of apoAI N-terminal helical hairpin on the cell surface. Others have shown that apoAI can solubilize liposomes made with egg PC:PS (8:2) only at acidic pH, where apoAI has unfolded to expose more hydrophobic surfaces. We postulated that ABCA1 may mediate the local acidification of the plasma membrane to promote apoAI unfolding and lipidation. Methods and Results: We found that apoAI unfolding in guanidine was facilitated at pH 5.0 vs. pH 7.5 (EC50 = 0.75 M and 0.94 M guanidine, respectively). We confirmed that apoAI solubilization of and binding to POPC:PS liposomes was only effective at pH < 5.5. We then designed a fluorescent ratiometric apoAI pH indicator labeling apoAI with FITC and Alexa647, and validated it in solution assays showing that the FITC/Alexa647 ratio decreased as the pH decreased. Using flow cytometry in both RAW264.7 and BHK cell lines, we found that incubation of the apoAI pH indicator with ABCA1 expressing cells at 37 o C led to a large acidification of apoAI, which may be due to the uptake of apoAI into endosomes/lysosomes. However, incubation of the apoAI pH indicator with ABCA1 expressing cells at 21 o C or with an inhibitor of endocytosis, where the vast majority of apoAI is on the cell surface, still led to mild apoAI acidification. To elucidate the mechanism of ABCA1 effects on local acidification of the plasma membrane, we examined if ABCA1 regulates vacuolar ATPase (V-ATPase) proton pump, which is composed of a peripheral V 1 domain and an embedded V 0 domain. We observed that bafilomycin, the V-ATPase inhibitor, dose dependently inhibited ABCA1 mediated cholesterol efflux to apoAI, and also eliminated the pH shift of apoAI on plasma membrane. We showed V-ATPase was expressed on the cell surface by cell surface biotinylation. Although the total level of V 0 A1 subunit didn’t change in ABCA1 expressing cells, ABCA1 induced the cell surface level of the V 0 A1 subunit. Conclusion: Our results support that ABCA1 induces the levels of V-ATPase on the cell surface leading to local acidification of apoAI, which may promote apoAI unfolding and lipidation.

Abstract 549: ABCA1 Expression Promotes ApoAI Acidification on the Plasma Membrane via Recruitment of Vacuolar ATPase

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Shuhui Wang ◽  
Gregory Brubaker ◽  
Jonathan D Smith
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Vacuolar Atpase ◽  
Acidic Ph ◽  
Atpase Inhibitor ◽  
Ph Indicator ◽  
Cell Surface Biotinylation ◽  
Abca1 Expression ◽  
Helical Hairpin ◽  
Raw264.7 Cell

Objective: We have shown that ABCA1 mediates unfolding of apoAI N-terminal helical hairpin on the cell surface. Others have shown that acidic pH exposes apoAI’s hydrophobic surfaces. We postulated that ABCA1 may mediate the local acidification of the plasma membrane to promote apoAI unfolding and lipidation. Methods and Results: We found that apoAI unfolding in guanidine was facilitated at acidic pH (EC 50 for unfolding at 0.94 and 0.75 M guanidine at pH 7.5 and 5, respectively), and confirmed that acidic conditions promoted apoAI solubilization of and binding to egg PC:PS (8:2 mole ratio) liposomes. We generated a FITC/Alexa647-labeled fluorescent ratiometric apoAI pH indicator whose FITC/Alexa647 emission ratio decreased as pH decreased. Using flow cytometry in BHK and RAW264.7 cell lines, we found that incubation of the apoAI pH indicator with ABCA1 expressing cells at 37 o C led to substantial acidification of apoAI, which may be due to the uptake of apoAI into endosomes/lysosomes. However, incubation of the apoAI pH indicator with ABCA1 expressing cells at 21 o C or with an inhibitor of endocytosis, where apoAI is on the cell surface, still led to mild apoAI acidification. To elucidate the mechanism of ABCA1 effects on local acidification of the plasma membrane, we examined if ABCA1 regulates vacuolar ATPase (V-ATPase) proton pump, which is composed of a peripheral V 1 domain and an embedded V 0 domain. We observed that bafilomycin, the V-ATPase inhibitor, eliminated the pH shift of apoAI on plasma membrane, and dose dependently inhibited ABCA1 mediated cholesterol efflux to apoAI. Although ABCA1 did not alter total cellular levels of V-ATPase, we showed that ABCA1 increased the cell surface levels of the V 0 A1 and V 1 E1 subunits of V-ATPase by cell surface biotinylation and immunofluorescence. Moreover, inhibition of apoAI acidification by bafilomycin decreased apoAI unfolding on the cell surface. Conclusions: Our results support that ABCA1 induces the levels of V-ATPase on the cell surface leading to local acidification of apoAI, which promotes apoAI unfolding and lipidation.

scholarly journals TRAIL Induces Nuclear Translocation and Chromatin Localization of TRAIL Death Receptors

Cancers ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 1167 ◽  
Author(s):  
Ufuk Mert ◽  
Alshaimaa Adawy ◽  
Elisabeth Scharff ◽  
Pierre Teichmann ◽  
Anna Willms ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Nuclear Export ◽  
Nuclear Translocation ◽  
Nuclear Accumulation ◽  
Nuclear Trafficking ◽  
Surface Receptors ◽  
Tnf Superfamily ◽  
Cell Surface Biotinylation ◽  
Intracellular Compartments

Binding of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to the plasma membrane TRAIL-R1/-R2 selectively kills tumor cells. This discovery led to evaluation of TRAIL-R1/-R2 as targets for anti-cancer therapy, yet the corresponding clinical trials were disappointing. Meanwhile, it emerged that many cancer cells are TRAIL-resistant and that TRAIL-R1/-R2-triggering may lead to tumor-promoting effects. Intriguingly, recent studies uncovered specific functions of long ignored intracellular TRAIL-R1/-R2, with tumor-promoting functions of nuclear (n)TRAIL-R2 as the regulator of let-7-maturation. As nuclear trafficking of TRAIL-Rs is not well understood, we addressed this issue in our present study. Cell surface biotinylation and tracking of biotinylated proteins in intracellular compartments revealed that nTRAIL-Rs originate from the plasma membrane. Nuclear TRAIL-Rs-trafficking is a fast process, requiring clathrin-dependent endocytosis and it is TRAIL-dependent. Immunoprecipitation and immunofluorescence approaches revealed an interaction of nTRAIL-R2 with the nucleo-cytoplasmic shuttle protein Exportin-1/CRM-1. Mutation of a putative nuclear export sequence (NES) in TRAIL-R2 or the inhibition of CRM-1 by Leptomycin-B resulted in the nuclear accumulation of TRAIL-R2. In addition, TRAIL-R1 and TRAIL-R2 constitutively localize to chromatin, which is strongly enhanced by TRAIL-treatment. Our data highlight the novel role for surface-activated TRAIL-Rs by direct trafficking and signaling into the nucleus, a previously unknown signaling principle for cell surface receptors that belong to the TNF-superfamily.

scholarly journals The Glycosylphosphatidyl Inositol-Anchored Adhesion Molecule F3/Contactin Is Required for Surface Transport of Paranodin/Contactin-Associated Protein (Caspr)

2000 ◽  
Vol 149 (2) ◽  
pp. 491-502 ◽  
Author(s):  
Catherine Faivre-Sarrailh ◽  
France Gauthier ◽  
Natalia Denisenko-Nehrbass ◽  
André Le Bivic ◽  
Geneviève Rougon ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Adhesion Molecule ◽  
Neuroblastoma Cells ◽  
Surface Expression ◽  
Gpi Anchor ◽  
Transmembrane Glycoprotein ◽  
Cell Surface Biotinylation ◽  
Fibronectin Type Iii ◽  
Glycosylphosphatidyl Inositol

Paranodin/contactin-associated protein (caspr) is a transmembrane glycoprotein of the neurexin superfamily that is highly enriched in the paranodal regions of myelinated axons. We have investigated the role of its association with F3/contactin, a glycosylphosphatidyl inositol (GPI)-anchored neuronal adhesion molecule of the Ig superfamily. Paranodin was not expressed at the cell surface when transfected alone in CHO or neuroblastoma cells. Cotransfection with F3 resulted in plasma membrane delivery of paranodin, as analyzed by confocal microscopy and cell surface biotinylation. The region that mediates association with paranodin was mapped to the Ig domains of F3 by coimmunoprecipitation experiments. The association of paranodin with F3 allowed its recruitment to Triton X-100–insoluble microdomains. The GPI anchor of F3 was necessary, but not sufficient for surface expression of paranodin. F3-Ig, a form of F3 deleted of the fibronectin type III (FNIII) repeats, although GPI-linked and expressed at the cell surface, was not recovered in the microdomain fraction and was unable to promote cell surface targeting of paranodin. Thus, a cooperative effect between the GPI anchor, the FNIII repeats, and the Ig regions of F3 is required for recruitment of paranodin into lipid rafts and its sorting to the plasma membrane.

scholarly journals Endocytosis separates EGF receptors from endogenous fluorescently labeled HRas and diminishes receptor signaling to MAP kinases in endosomes

2016 ◽  
Vol 113 (8) ◽  
pp. 2122-2127 ◽  
Author(s):  
Itziar Pinilla-Macua ◽  
Simon C. Watkins ◽  
Alexander Sorkin
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Map Kinases ◽  
Fluorescent Protein ◽  
Growth Factor Receptor ◽  
Egf Receptors ◽  
Downstream Signaling ◽  
Transient Activation ◽  
Cell Surface Biotinylation ◽  
Signaling Axis

Signaling from epidermal growth factor receptor (EGFR) to extracellular-stimuli–regulated protein kinase 1/2 (ERK1/2) is proposed to be transduced not only from the cell surface but also from endosomes, although the role of endocytosis in this signaling pathway is controversial. Ras is the only membrane-anchored component in the EGFR–ERK signaling axis, and therefore, its location determines intracellular sites of downstream signaling. Hence, we labeled endogenous H-Ras (HRas) with mVenus fluorescent protein using gene editing in HeLa cells. mVenus-HRas was primarily located at the plasma membrane, and in small amounts in tubular recycling endosomes and associated vesicles. EGF stimulation resulted in fast but transient activation of mVenus-HRas. Although EGF:EGFR complexes were rapidly accumulated in endosomes together with the Grb2 adaptor, very little, if any, mVenus-HRas was detected in these endosomes. Interestingly, the activities of MEK1/2 and ERK1/2 remained high beyond the point of the physical separation of HRas from EGF:EGFR complexes and down-regulation of Ras activity. Paradoxically, this sustained MEK1/2 and ERK1/2 activation was dependent on the active EGFR kinase. Cell surface biotinylation and selective inactivation of surface EGFRs suggested that a small fraction of active EGFRs remaining in the plasma membrane is responsible for continuous signaling to MEK1/2 and ERK1/2. We propose that, under physiological conditions of cell stimulation, EGFR endocytosis serves to spatially separate EGFR–Grb2 complexes and Ras, thus terminating Ras-mediated signaling. However, sustained minimal activation of Ras by a small pool of active EGFRs in the plasma membrane is sufficient for extending MEK1/2 and ERK1/2 activities.

scholarly journals Cooh-Terminal Truncations Promote Proteasome-Dependent Degradation of Mature Cystic Fibrosis Transmembrane Conductance Regulator from Post-Golgi Compartments

2001 ◽  
Vol 153 (5) ◽  
pp. 957-970 ◽  
Author(s):  
Mohamed Benharouga ◽  
Martin Haardt ◽  
Norbert Kartner ◽  
Gergely L. Lukacs
Keyword(s):  
Cystic Fibrosis ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Cell Surface Biotinylation ◽  
Partially Unfolded ◽  
Cf Transmembrane Conductance Regulator

Impaired biosynthetic processing of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel, constitutes the most common cause of CF. Recently, we have identified a distinct category of mutation, caused by premature stop codons and frameshift mutations, which manifests in diminished expression of COOH-terminally truncated CFTR at the cell surface. Although the biosynthetic processing and plasma membrane targeting of truncated CFTRs are preserved, the turnover of the complex-glycosylated mutant is sixfold faster than its wild-type (wt) counterpart. Destabilization of the truncated CFTR coincides with its enhanced susceptibility to proteasome-dependent degradation from post-Golgi compartments globally, and the plasma membrane specifically, determined by pulse–chase analysis in conjunction with cell surface biotinylation. Proteolytic cleavage of the full-length complex-glycosylated wt and degradation intermediates derived from both T70 and wt CFTR requires endolysosomal proteases. The enhanced protease sensitivity in vitro and the decreased thermostability of the complex-glycosylated T70 CFTR in vivo suggest that structural destabilization may account for the increased proteasome susceptibility and the short residence time at the cell surface. These in turn are responsible, at least in part, for the phenotypic manifestation of CF. We propose that the proteasome-ubiquitin pathway may be involved in the peripheral quality control of other, partially unfolded membrane proteins as well.

Abstract 463: Transregulation and Plasma Membrane Recruitment of the Dopamine-1 (D1R) and Angiotensin Type 2 (AT2R) Receptors in Human Renal Proximal Tubule Cells (RPTC)

Hypertension ◽  
2013 ◽  
Vol 62 (suppl_1) ◽  
Author(s):  
Phuong B Opper ◽  
John J Gildea ◽  
Robin A Felder
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Sodium Reabsorption ◽  
Nucleotide Analogs ◽  
Proximal Tubule Cells ◽  
Membrane Recruitment ◽  
Cell Surface Biotinylation ◽  
Angiotensin Iii ◽  
Specific Antagonist ◽  
Renal Proximal Tubule Cells

Both the D1R and AT2R are key natriuretic receptors in that stimulation by dopamine agonists or AT2R agonist angiotensin III (AngIII) increases cAMP and cGMP, respectively, causing inhibition of sodium reabsorption. The interdependence of D1R and AT2R stimulation on receptor translocation has not been studied. We utilized normally coupled RPTC (nRPTC) and RPTC with defective D1R coupling to adenylyl cyclase (uRPTC). A 1 hr addition of the cell permeable cyclic nucleotide analogs 8Br-cGMP (1 mM) or 8Br-cAMP (1 mM) caused a similar recruitment of both D1R and AT2R, as measured by cell surface biotinylation and western blotting (see table below). Flow cytometry confirmed these results using fluorescently labeled extracellular epitope-specific D1R and AT2R antibodies. When 8Br-cAMP and 8Br-cGMP were added together, no added effects were seen. AngIII-alone increased plasma membrane recruitment of D1R (1.23±0.04 fold, n=6, p<0.05, VEH=0.63) and AT2R (1.65±0.10 fold, n=6, p<0.05, VEH=0.69) in nRPTC but not in uRPTC. The nRPTC response was completely blocked by the AT2R specific antagonist PD123319 or the D1R antagonist LE300. Inhibition of either PKA with adenosine-3’, 5’-cyclic monophosphorothioate Rp-Isomer (100 μM) or PKG with 8-(4-chlorophenylthio) guanosine-3’, 5’ -cyclic monophosphorothioate Rp-isomer (10 μM) completely inhibited the cell surface recruitment of both D1R and AT2R when stimulated with 8Br-cAMP or 8Br-cGMP, respectively. In conclusion, we found a novel AngIII-interdependent AT2R and D1R cell surface recruitment in human nRPTC cells. Furthermore, defects in this pathway in uRPTC can be bypassed by incubating the cells with either 8Br-cAMP or 8Br-cGMP.

scholarly journals MT1-MMP hemopexin domain exchange with MT4-MMP blocks enzyme maturation and trafficking to the plasma membrane in MCF7 cells

2006 ◽  
Vol 398 (1) ◽  
pp. 15-22 ◽  
Author(s):  
Susan J. Atkinson ◽  
Christian Roghi ◽  
Gillian Murphy
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Matrix Metalloproteinase ◽  
Matrix Metalloproteinase 2 ◽  
Mcf7 Cells ◽  
Matrix Metalloproteinase 1 ◽  
Cell Surface Biotinylation ◽  
Membrane Type ◽  
Hemopexin Domain

The hemopexin-like domain of membrane-type matrix metalloproteinase-1 (MT1-MMP) enables MT1-MMP to form oligomers that facilitate the activation of pro-matrix metalloproteinase-2 (pro-MMP-2) at the cell surface. To investigate the role of the MT1-MMP hemopexin domain in the trafficking of MT1-MMP to the cell surface we have examined the activity of two MT1–MT4-MMP chimaeras in which the hemopexin domain of MT1-MMP has been replaced with that of human or mouse MT4-MMP. We show that MT1-MMP bearing the hemopexin domain of MT4-MMP was incapable of activating pro-MMP-2 or degrading gelatin in cell based assays. Furthermore, cell surface biotinylation and indirect immunofluorescence show that transiently expressed MT1–MT4-MMP chimaeras failed to reach the plasma membrane and were retained in the endoplasmic reticulum. Functional activity could be restored by replacing the MT4-MMP hemopexin domain with the wild-type MT1-MMP hemopexin domain. Subsequent analysis with an antibody specifically recognising the propeptide of MT1-MMP revealed that the propeptides of the MT1–MT4-MMP chimaeras failed to undergo proper processing. It has previously been suggested that the hemopexin domain of MT4-MMP could exert a regulatory mechanism that prevents MT4-MMP from activating pro-MMP-2. In this report, we demonstrate unambiguously that MT1–MT4-MMP chimaeras do not undergo normal trafficking and are not correctly processed to their fully active forms and, as a consequence, they are unable to activate pro-MMP-2 at the cell surface.

scholarly journals Cytosolic Stress Reduces Degradation of Connexin43 Internalized from the Cell Surface and Enhances Gap Junction Formation and Function

2005 ◽  
Vol 16 (11) ◽  
pp. 5247-5257 ◽  
Author(s):  
Judy K. VanSlyke ◽  
Linda S. Musil
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Gap Junctions ◽  
Gap Junction ◽  
Intracellular Transport ◽  
Rat Kidney ◽  
Proteasome Inhibitors ◽  
Cell Surface Biotinylation ◽  
Normal Rat ◽  
And Function

The protein constituents of gap junctions, connexins, have a rapid basal rate of degradation even after transport to the cell surface. We have used cell surface biotinylation to label gap junction-unassembled plasma membrane pools of connexin43 (Cx43) and show that their degradation is inhibited by mild hyperthermia, oxidative stress, and proteasome inhibitors. Cytosolic stress does not perturb endocytosis of biotinylated Cx43, but instead it seems to interfere with its targeting and/or transport to the lysosome, possibly by increasing the level of unfolded protein in the cytosol. This allows more Cx43 molecules to recycle to the cell surface, where they are assembled into long-lived, functional gap junctions in otherwise gap junction assembly-inefficient cells. Cytosolic stress also slowed degradation of biotinylated Cx43 in gap junction assembly-efficient normal rat kidney fibroblasts, and reduced the rate at which gap junctions disappeared from cell interfaces under conditions that blocked transport of nascent connexin molecules to the plasma membrane. These data demonstrate that degradation from the cell surface can be down-regulated by physiologically relevant forms of stress. For connexins, this may serve to enhance or preserve gap junction-mediated intercellular communication even under conditions in which protein synthesis and/or intracellular transport are compromised.

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