A novel method for measuring protein expression at the cell surface

1993 ◽  
Vol 106 (4) ◽  
pp. 1201-1209
Author(s):  
W. Stoorvogel ◽  
V. Oorschot ◽  
B. Neve
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Protein Expression ◽  
Surface Expression ◽  
Surface Proteins ◽  
Proteinase K ◽  
Cell Surface Expression ◽  
Specific Cell ◽  
Quantitative Measurements ◽  
Dab Cytochemistry

All methods described in the literature that allow quantitative measurements of protein expression at the cell surface are applicable to subsets of surface-exposed proteins only. We developed a new method, involving 3,3′-diaminobenzidine (DAB) cytochemistry, which allowed determination of cell-surface expression of all plasma membrane proteins measured, in at least three different cell lines. Adherent cells were first brought into suspension by proteinase K and EDTA treatment at 0 degrees C removing many, but not all, surface-exposed proteins. Subsequently, horseradish peroxidase (HRP) was linked by means of its glycosyl residues to specific cell-surface-exposed sugar moieties using the multivalent lectin concanavalin A (ConA). The suspended cells were encapsulated by polymerized DAB, a process that was catalysed by plasma membrane-bound HRP. After cell lysis, and removal of nuclei and most of the DAB polymer by centrifugation, proteins were analysed by SDS-PAGE. Surface proteins encapsulated by non-pelleted DAB polymer were retained on top of the stacking gel. After 125I-labelling the cell surface, protease-resistant 125I-labelled proteins could be quantitatively coupled to DAB polymer. This process was completely dependent on the presence of ConA, HRP, DAB and H2O2. Surface 125I-labelled beta-Na+,K(+)-ATPase was resistant to proteinase K but could be completely removed using DAB cytochemistry. Intracellular ConA binding proteins were not affected. Other intracellular proteins, including endosomal asialoglycoprotein receptor and cation-independent mannose 6-phosphate/insulin-like growth factor II receptor were also not affected.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of aging on CD11b and CD69 surface expression by vesicular insertion in human polymorphonuclear leucocytes

1999 ◽  
Vol 97 (3) ◽  
pp. 323-329 ◽  
Author(s):  
J. M. NOBLE ◽  
G. A. FORD ◽  
T. H. THOMAS
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Surface Expression ◽  
Cell Surface Receptor ◽  
Cell Surface Expression ◽  
Elderly Subjects ◽  
Polymorphonuclear Leucocytes ◽  
Cd11b Expression ◽  
Cd69 Expression ◽  
Vesicle Exocytosis

The exocytosis of intracellular vesicles is an important function of the plasma membrane, which is responsible for hormone secretion, cell surface expression of antigens, ion transporters and receptors, and intracellular and intercellular signalling. Human aging is associated with many physiological and cellular changes, many of which are due to alterations in plasma membrane functioning. Alterations in vesicle externalization with age could account for many of these changes. We investigated whether alterations in vesicle exocytosis occur with increasing age by flow-cytometric determination of CD11b and CD69 expression on the surface of human polymorphonuclear leucocytes (PMN) stimulated with phorbol myristate acetate (PMA), a tumour promoter which binds to and activates protein kinase C (PKC) directly, or with formyl-Met-Leu-Phe (fMLP), which activates PKC indirectly via interactions with a cell surface receptor and G-protein, and subsequent inositol phosphate hydrolysis. Following stimulation with PMA, a decrease in the proportion of PMN expressing CD69 at high levels was observed in elderly compared with young subjects (young, 55.3%; elderly, 43.9%; P = 0.01). No aging-related differences in the proportion of PMN expressing CD11b (young, 73.7%; elderly, 68.4%; P = 0.15), or in the number of molecules of CD69 or CD11b expressed per cell, were observed. Stimulation with fMLP or low PMA concentrations resulted in full CD11b expression but minimal CD69 expression in both young and elderly subjects. Cells which expressed CD69 had no CD11b expression, while those cells expressing CD11b had minimal CD69 expression. Thus the PMA-induced expression of CD11b and CD69 in human PMN represents two separate processes, only one of which is affected in aging. CD11b expression appears to require a lesser degree of PKC stimulation compared with that required for CD69 expression. The age-associated reduction in PMA-stimulated CD69 expression may occur either at or distal to PKC activation. Such a decrease may contribute to the age-associated impairments in PMN function that contribute, in turn, to immunosenescence.

scholarly journals Mammalian PIG-L and its yeast homologue Gpi12p are N-acetylglucosaminylphosphatidylinositol de-N-acetylases essential in glycosylphosphatidylinositol biosynthesis

1999 ◽  
Vol 339 (1) ◽  
pp. 185-192 ◽  
Author(s):  
Reika WATANABE ◽  
Kazuhito OHISHI ◽  
Yusuke MAEDA ◽  
Nobuo NAKAMURA ◽  
Taroh KINOSHITA
Keyword(s):  
Cell Surface ◽  
Chinese Hamster ◽  
Surface Expression ◽  
Amino Acid Identity ◽  
Eukaryotic Cell ◽  
Surface Proteins ◽  
Ovary Cell ◽  
Cell Surface Expression ◽  
Expression Cloning

Glycosylphosphatidylinositol (GPI) is used as a membrane anchor by many eukaryotic cell-surface proteins. The second step of GPI biosynthesis is de-N-acetylation of N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI). We have previously cloned the rat PIG-L gene by expression cloning that complemented a mutant Chinese hamster ovary cell line defective in this step. Here we show that recombinant rat PIG-L protein purified from Escherichia coli as a complex with GroEL has GlcNAc-PI de-N-acetylase activity in vitro. The activity was not enhanced by GTP, which is known to enhance the de-N-acetylase activity of mammalian cell microsomes. As with other de-N-acetylases that act on the GlcNAc moiety, metal ions, in particular Mn2+ and Ni2+, enhanced the enzyme activity of PIG-L. The Saccharomyces cerevisiae YMR281W open reading frame encodes a protein (termed Gpi12p) with 24% amino acid identity with rat PIG-L. On transfection into mammalian PIG-L-deficient cells, this gene, GPI12, restored the cell-surface expression of GPI-anchored proteins and GlcNAc-PI de-N-acetylase activity. The disruption of the gene caused lethality in S. cerevisiae. These results indicate that GlcNAc-PI de-N-acetylase is conserved between mammals and yeasts and that the de-N-acetylation step is also indispensable in yeasts.

Erlotinib Antagonizes Efflux Via ABC Transporters and Decreases P-Gp Cell Surface Expression by Inhibiting SRC Kinase and mTOR Pathways in Acute Myeloid Leukemia (AML)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2564-2564 ◽  
Author(s):  
Elodie Lainey ◽  
Marie Sebert ◽  
Cyrielle Bouteloup ◽  
Carole Leroy ◽  
Sylvain Thepot ◽  
...  
Keyword(s):  
Cell Surface ◽  
Protein Expression ◽  
Atpase Activity ◽  
Abc Transporters ◽  
Ex Vivo ◽  
Synergistic Effects ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Drug Efflux ◽  
Hoechst 33342

Abstract Abstract 2564 Background: Erlotinib (Erlo) was originally developed as an epidermal growth factor receptor inhibitor, yet it also exerts antileukemic “off-target” effects, in vitro and in vivo in MDS and AML (Boehrer et al., Blood, 2008). In a preliminary pre-clinical study, we observed that Erlo increased chemosensitivity to current AML drugs in different AML cell lines and in ex vivo AML patient cells (n=3) (ASH 2010, 2163). Those first results suggested an implication of ABC-transporters in the potentiation of apoptosis. Here, we bring direct evidence for Erlo's ability to hinder efflux pumps and to decrease their expression on AML cells. Methods: Drug efflux via ABC-transporters (substrate: mitoxantrone-MTZ or doxorubicin-Dox), and specific efflux via P-gp (substrates: DioC23 and Rho-123), MRP (s: Calcein and CDCFDA) and BCRP (s: Hoechst 33342) were quantified by FACS following incubation with 10mM Erlo. Intracellular VP-16) content was quantified by Rapid Resolution Liquid Chromatography (RRLC). Biochemical inhibitors of the respective ABC-transporters (CSA (1μM), verapamil (Vera-10μM), MK571 (10μM), KO143 (500nM) served as positive controls. To assess chemosensitivity, 10mM Erlo was combined to AraC (100nM), Dox (100nM), or VP-16 (1mM) and apoptosis over-time (24, 48, 72h) quantified by DioC3(6)/PI staining. Assessment of sensitivity to the drug combinations listed above were carried out in KG-1 cells, and its more immature variant KG-1a and in ex vivo CD34+ marrow cells from AML patients (AML post MDS n=5, de novo AML n=5). P-gp's ATPase activity was quantified with the luminescence-based Pgp-Gloä Assay System. Surface expression of P-gp was determined by FACS analysis and total protein expression of MRP, BCRP and P-gp by immunoblot analysis. Functional relevance of signaling pathways was tested using the SRC inhibitor PP2 (10μM) and the mTOR inhibitor Rapamicin (10nM). Results: We found that I) Erlo inhibited efflux via P-gp, MRP and BCRP as demonstrated by increased intracellular retention of DioC23/Rho-123, Calcein/CDCFDA and Hoechst 33342, respectively, andby its ability to retain MTX (300nM) and Dox (200nM) intracellularly II) Inhibition of drug efflux was higher in KG-1 than in KG-1a cellss, in agreement with a lower expression of P-gp and BCRP on KG-1a as compared to KG-1 cells; III) Quantification of VP-16 by RRLC after incubation with or without Erlo showed the ability of Erlo to increase intracellular VP-16 contents by approximately 60%; IV) Erlo increased ATPase activity in a dose-dependant manner, supporting the notion that Erlo is a competitive inhibitor of P-gp; IV) Erlo combined to VP-16 induced synergistic effects on apoptosis in KG-1 cells, and to a lesser extent in KG-1a (48h KG-1: Erlo 20%, VP-16 38%, Erlo+VP16 78%, KG-1a 48h: Erlo 10%, VP-16: 12%, Erlo+VP16: 35%); V) 48h of incubation with Erlo reduced cell surface expression of P-gp in KG-1 cells by 50%, whereas total P-gp protein expression remained unchanged, suggesting that Erlo interferes exclusively with the protein form expressed on the cell surface, VI) Decrease of P-gp cell surface expression was recapitulated upon incubation with PP2 (10μM) or Rapamicin (10nM); VII) the combination of Erlo+VP-16 in 10 AML-patient samples induced synergistic effects on apoptosis in 5 of them and additive effects in 3 of them. Conclusions: We here confirm that Erlo increases sensitivity towards chemotherapeutic agents subjected to drug efflux via ABC-transporters and delineate the molecular pathways conveying these effects. Disclosures: Fenaux: Celgene: Honoraria, Research Funding.

scholarly journals GLUT8 Subcellular Localization and Absence of Translocation to the Plasma Membrane in PC12 Cells and Hippocampal Neurons

Endocrinology ◽  
2005 ◽  
Vol 146 (11) ◽  
pp. 4727-4736 ◽  
Author(s):  
Mathieu Widmer ◽  
Marc Uldry ◽  
Bernard Thorens
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Cell Surface ◽  
Pc12 Cells ◽  
Hippocampal Neurons ◽  
Osmotic Shock ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Intracellular Compartment ◽  
Early Endosomes

GLUT8 is a high-affinity glucose transporter present mostly in testes and a subset of brain neurons. At the cellular level, it is found in a poorly defined intracellular compartment in which it is retained by an N-terminal dileucine motif. Here we assessed GLUT8 colocalization with markers for different cellular compartments and searched for signals, which could trigger its cell surface expression. We showed that when expressed in PC12 cells, GLUT8 was located in a perinuclear compartment in which it showed partial colocalization with markers for the endoplasmic reticulum but not with markers for the trans-Golgi network, early endosomes, lysosomes, and synaptic-like vesicles. To evaluate its presence at the plasma membrane, we generated a recombinant adenovirus for the expression of GLUT8 containing an extracellular myc epitope. Cell surface expression was evaluated by immunofluorescence microscopy of transduced PC12 cells or primary hippocampal neurons exposed to different stimuli. Those included substances inducing depolarization, activation of protein kinase A and C, activation or inhibition of tyrosine kinase-linked signaling pathways, glucose deprivation, AMP-activated protein kinase stimulation, and osmotic shock. None of these stimuli-induced GLUT8 cell surface translocation. Furthermore, when GLUT8myc was cotransduced with a dominant-negative form of dynamin or GLUT8myc-expressing PC-12 cells or neurons were incubated with an anti-myc antibody, no evidence for constitutive recycling of the transporter through the cell surface could be obtained. Thus, in cells normally expressing it, GLUT8 was associated with a specific intracellular compartment in which it may play an as-yet-uncharacterized role.

Apical membrane and intracellular distribution of endogenous alpha 2A-adrenergic receptors in MDCK cells

1994 ◽  
Vol 267 (3) ◽  
pp. F347-F353 ◽  
Author(s):  
M. D. Okusa ◽  
K. R. Lynch ◽  
D. L. Rosin ◽  
L. Huang ◽  
Y. Y. Wei
Keyword(s):  
Cell Surface ◽  
De Novo ◽  
Mdck Cells ◽  
Specific Binding ◽  
Surface Expression ◽  
Surface Proteins ◽  
Cell Surface Expression ◽  
Surface Binding ◽  
Binding Studies ◽  
Apical Domain

The purpose of the current studies was to characterize the endogenous alpha 2-adrenergic receptor (AR) subtypes present in Madin-Darby canine kidney (MDCK) cells and to determine their level of expression and pattern of distribution. By saturation binding analysis with [3H]MK-912, MDCK cells expressed high levels of alpha 2-ARs with a maximum receptor density (Bmax) of 798 +/- 55 fmol/mg protein and an equilibrium dissociation constant (Kd) of 0.98 +/- 0.32 nM. Competitive binding studies using prazosin, oxymetazoline, phentolamine, and epinephrine to displace [3H]MK-912 demonstrated inhibition constant (Ki) values of 1,270 +/- 250, 5.0 +/- 0.4, 5.5 +/- 0.3, and 392 +/- 150 nM (n = 3), respectively. In Northern blot analysis we found that MDCK cells expressed transcripts encoding alpha 2A-AR and not alpha 2B-AR or alpha 2C-AR. Surface binding experiments suggested that approximately 60% of alpha 2A-ARs are distributed at the cell surface domain. Specific binding of [3H]MK-912 to soluble apical and basolateral surface proteins isolated by surface biotinylation indicated the expression of surface alpha 2A-ARs was limited to the apical domain of MDCK cells. No alpha 2A-ARs were detected on the basolateral surface. We conclude that endogenous alpha 2A-ARs are targeted to the apical domain of MDCK cells and that the intracellular compartment may contain ARs as a reservoir for de novo cell surface expression or, alternatively, may represent internalized receptors.

scholarly journals Molecular pathogenesis of human CD59 deficiency

2018 ◽  
Vol 4 (6) ◽  
pp. e280 ◽  
Author(s):  
Netanel Karbian ◽  
Yael Eshed-Eisenbach ◽  
Adi Tabib ◽  
Hila Hoizman ◽  
B. Paul Morgan ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Cell Surface ◽  
Chronic Inflammatory Demyelinating Polyneuropathy ◽  
Membrane Attack Complex ◽  
Surface Expression ◽  
Surface Proteins ◽  
Cell Surface Expression ◽  
Congenital Deficiency ◽  
Recurrent Strokes ◽  
And Function

ObjectiveTo characterize all 4 mutations described for CD59 congenital deficiency.MethodsThe 4 mutations, p.Cys64Tyr, p.Asp24Val, p.Asp24Valfs*, and p.Ala16Alafs*, were described in 13 individuals with CD59 malfunction. All 13 presented with recurrent Guillain-Barré syndrome or chronic inflammatory demyelinating polyneuropathy, recurrent strokes, and chronic hemolysis. Here, we track the molecular consequences of the 4 mutations and their effects on CD59 expression, localization, glycosylation, degradation, secretion, and function. Mutants were cloned and inserted into plasmids to analyze their expression, localization, and functionality.ResultsImmunolabeling of myc-tagged wild-type (WT) and mutant CD59 proteins revealed cell surface expression of p.Cys64Tyr and p.Asp24Val detected with the myc antibody, but no labeling by anti-CD59 antibodies. In contrast, frameshift mutants p.Asp24Valfs* and p.Ala16Alafs* were detected only intracellularly and did not reach the cell surface. Western blot analysis showed normal glycosylation but mutant-specific secretion patterns. All mutants significantly increased MAC-dependent cell lysis compared with WT. In contrast to CD59 knockout mice previously used to characterize phenotypic effects of CD59 perturbation, all 4 hCD59 mutations generate CD59 proteins that are expressed and may function intracellularly (4) or on the cell membrane (2). None of the 4 CD59 mutants are detected by known anti-CD59 antibodies, including the 2 variants present on the cell membrane. None of the 4 inhibits membrane attack complex (MAC) formation.ConclusionsAll 4 mutants generate nonfunctional CD59, 2 are expressed as cell surface proteins that may function in non–MAC-related interactions and 2 are expressed only intracellularly. Distinct secretion of soluble CD59 may have also a role in disease pathogenesis.

Tumor-specific cell surface expression of the -KDEL containing endoplasmic reticular heat shock protein gp96

1996 ◽  
Vol 69 (4) ◽  
pp. 340-349 ◽  
Author(s):  
Anne Altmeyer ◽  
Robert G. Maki ◽  
Anna M. Feldweg ◽  
Michael Heike ◽  
Vladimir P. Protopopov ◽  
...  
Keyword(s):  
Heat Shock ◽  
Cell Surface ◽  
Heat Shock Protein ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Specific Cell ◽  
Heat Shock Protein Gp96 ◽  
Specific Cell Surface

scholarly journals Intracellular Retention of Glycosylphosphatidyl Inositol-Linked Proteins in Caveolin-Deficient Cells

2002 ◽  
Vol 22 (11) ◽  
pp. 3905-3926 ◽  
Author(s):  
Federica Sotgia ◽  
Babak Razani ◽  
Gloria Bonuccelli ◽  
William Schubert ◽  
Michela Battista ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Lipid Rafts ◽  
Recombinant Expression ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Null Mouse ◽  
Tissue Samples ◽  
Caveolin 1 ◽  
Glycosylphosphatidyl Inositol

ABSTRACT The relationship between glycosylphosphatidyl inositol (GPI)-linked proteins and caveolins remains controversial. Here, we derived fibroblasts from Cav-1 null mouse embryos to study the behavior of GPI-linked proteins in the absence of caveolins. These cells lack morphological caveolae, do not express caveolin-1, and show a ∼95% down-regulation in caveolin-2 expression; these cells also do not express caveolin-3, a muscle-specific caveolin family member. As such, these caveolin-deficient cells represent an ideal tool to study the role of caveolins in GPI-linked protein sorting. We show that in Cav-1 null cells GPI-linked proteins are preferentially retained in an intracellular compartment that we identify as the Golgi complex. This intracellular pool of GPI-linked proteins is not degraded and remains associated with intracellular lipid rafts as judged by its Triton insolubility. In contrast, GPI-linked proteins are transported to the plasma membrane in wild-type cells, as expected. Furthermore, recombinant expression of caveolin-1 or caveolin-3, but not caveolin-2, in Cav-1 null cells complements this phenotype and restores the cell surface expression of GPI-linked proteins. This is perhaps surprising, as GPI-linked proteins are confined to the exoplasmic leaflet of the membrane, while caveolins are cytoplasmically oriented membrane proteins. As caveolin-1 normally undergoes palmitoylation on three cysteine residues (133, 143, and 156), we speculated that palmitoylation might mechanistically couple caveolin-1 to GPI-linked proteins. In support of this hypothesis, we show that palmitoylation of caveolin-1 on residues 143 and 156, but not residue 133, is required to restore cell surface expression of GPI-linked proteins in this complementation assay. We also show that another lipid raft-associated protein, c-Src, is retained intracellularly in Cav-1 null cells. Thus, Golgi-associated caveolins and caveola-like vesicles could represent part of the transport machinery that is necessary for efficiently moving lipid rafts and their associated proteins from the trans-Golgi to the plasma membrane. In further support of these findings, GPI-linked proteins were also retained intracellularly in tissue samples derived from Cav-1 null mice (i.e., lung endothelial and renal epithelial cells) and Cav-3 null mice (skeletal muscle fibers).

scholarly journals Bovine CD18 Is Necessary and Sufficient To Mediate Mannheimia (Pasteurella) haemolytica Leukotoxin-Induced Cytolysis

2002 ◽  
Vol 70 (9) ◽  
pp. 5058-5064 ◽  
Author(s):  
M. S. Deshpande ◽  
T. C. Ambagala ◽  
A. P. N. Ambagala ◽  
M. E. Kehrli ◽  
S. Srikumaran
Keyword(s):  
Cell Surface ◽  
Surface Expression ◽  
Surface Proteins ◽  
Polymorphonuclear Neutrophils ◽  
Cell Surface Expression ◽  
Target Cells ◽  
Dependent Manner ◽  
Β Subunit ◽  
Concentration Dependent Manner ◽  
Necessary And Sufficient

ABSTRACT Leukotoxin (Lkt) secreted by Mannheimia (Pasteurella) haemolytica is an RTX toxin which is specific for ruminant leukocytes. Lkt binds to β2 integrins on the surface of bovine leukocytes. β2 integrins have a common β subunit, CD18, that associates with three distinct α chains, CD11a, CD11b, and CD11c, to give rise to three different β2 integrins, CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1), and CD11c/CD18 (CR4), respectively. Our earlier studies revealed that Lkt binds to all three β2 integrins, suggesting that the common β subunit, CD18, may be the receptor for Lkt. In order to unequivocally elucidate the role of bovine CD18 as a receptor for Lkt, a murine cell line nonsusceptible to Lkt (P815) was transfected with cDNA for bovine CD18. One of the transfectants, 2B2, stably expressed bovine CD18 on the cell surface. The 2B2 transfectant was effectively lysed by Lkt in a concentration-dependent manner, whereas the P815 parent cells were not. Immunoprecipitation of cell surface proteins of 2B2 with monoclonal antibodies specific for bovine CD18 or murine CD11a suggested that bovine CD18 was expressed on the cell surface of 2B2 as a heterodimer with murine CD11a. Expression of bovine CD18 and the Lkt-induced cytotoxicity of 2B2 cells were compared with those of bovine polymorphonuclear neutrophils and lymphocytes. There was a strong correlation between cell surface expression of bovine CD18 and percent cytotoxicity induced by Lkt. These results indicate that bovine CD18 is necessary and sufficient to mediate Lkt-induced cytolysis of target cells.

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