Cell surface assembly of lipoprotein(a) in primary cultures of baboon hepatocytes.

There is a link between diabetes mellitus and hypertension, but the underlying mechanisms are poorly understood. The epithelial Na + channel ENaC plays an important role in blood pressure control; ENaC mutations cause Liddle’s syndrome, an inherited form of hypertension. Previous work suggests that ENaC abundance is increased in diabetes mellitus, but the underlying mechanisms are unclear. Here we tested the effect of glucose on ENaC regulation. In Ussing chamber experiments using mouse kidney collecting duct cells (mCCD) and primary cultures of human lung epithelia, elevated glucose increased ENaC-mediated short-circuit current by 2-3 times in a dose-dependent manner from 100mg/dl to 400mg/dl of glucose. This was caused by an increase in ENaC abundance at the cell surface. We hypothesized that hyperglycemia might enhance ENaC cell surface abundance by altering activity of Nedd4-2, an E3 ubiquitin-protein ligase that binds to PY motifs within ENaC. Consistent with this hypothesis, we found that mutation of the PY motifs abolished ENaC stimulation by elevated glucose. Moreover, using a biotinylation assay, we found that elevated glucose (300 mg/dl) slowed ENaC endocytosis and reduced its degradation in the endocytic pathway. These changes in trafficking are explained by our finding that glucose reduced ENaC binding to Nedd4-2, and hence, reduced ENaC ubiquitination. O-GlcNAcylation plays a role in insulin signaling and glucose toxicity due to increased O-GlcNAcylation of target proteins. To test a role for O-GlcNAcylation in ENaC stimulation by glucose, we used 6-Diazo-5-oxo-l-norleucine (DON) to inhibit O-GlcNAcylation. DON abolished ENaC stimulation by elevated glucose. Using anti-O-GlcNAc antibody, we found that Nedd4-2 is a substrate for O-GlcNAcylation, and this modification was increased by elevated glucose. DON also reversed the reduction in binding of Nedd4-2 to ENaC at high glucose levels. Together, our data suggest a model in which hyperglycemia stimulates ENaC through O-GlcNAcylation of Nedd4-2, increasing ENaC abundance at cell surface thus increasing epithelial sodium absorption.

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Basolateral Na(+)-independent Cl(-)-HCO3- exchange in primary cultures of rat IMCD cells

AJP Renal Physiology ◽

10.1152/ajprenal.1992.263.3.f401 ◽

1992 ◽

Vol 263 (3) ◽

pp. F401-F410 ◽

Cited By ~ 1

Author(s):

J. A. Kraut ◽

D. Hart ◽

E. P. Nord

Keyword(s):

Steady State ◽

Cell Surface ◽

Primary Culture ◽

Anion Exchange ◽

Collecting Duct ◽

Primary Cultures ◽

Disulfonic Acid ◽

Absolute Requirement ◽

Ethanesulfonic Acid ◽

Base Load

The role of anion exchange in the regulation of intracellular pH (pHi) under base load and steady-state conditions was investigated in confluent monolayers of rat inner medullary collecting duct (IMCD) cells in primary culture using the pH-sensitive fluoroprobe 2,7-bis(carboxyethyl)-5(6')-carboxyfluorescein (BCECF). Recovery of pHi after imposition of a base load induced either by replacement of HCO3-/CO2 by N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) at the same extracellular pH (pHo) or deletion of Cl- from a HCO3-/CO2-buffered solution had an absolute requirement for Cl-, was Na+ independent, and was inhibited approximately 90% by 50 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). When pHo was decreased by lowering HCO3- concentration in the constant presence of 5% CO2, the rate of decrement in pHi was significantly blunted in the absence of Cl-. Imposition of a positive or negative diffusion potential of equal but opposite magnitude did not modify the anion exchange rate, confirming the electroneutrality of the process. Under steady-state conditions, pHi of cells bathed in a HCO3-/CO2-buffered solution was 7.33 +/- 0.06, significantly lower than that of cells bathed in a nominally HCO3-/CO2-free buffer (7.50 +/- 0.04), indicating that under physiological conditions the pathway functions as a base extruder. In studies performed on cells grown on permeable supports, the anion exchange pathway was found to be confined exclusively to the basolateral-equivalent cell surface. In summary, confluent monolayers of rat IMCD cells in primary culture possess a Na(+)-independent, DIDS-inhibitable electroneutral Cl(-)-HCO3- exchange pathway that is confined to the basolateral cell surface. The transporter is an important determinant of steady-state pHi and is the predominant mechanism whereby the cell recovers from imposed elevations in pHi.

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Differential Expression and Functional Characterization of Luteinizing Hormone Receptor Splice Variants in Human Luteal Cells: Implications for Luteolysis

Endocrinology ◽

10.1210/en.2008-1382 ◽

2009 ◽

Vol 150 (6) ◽

pp. 2873-2881 ◽

Cited By ~ 30

Author(s):

Rachel E. Dickinson ◽

Alan J. Stewart ◽

Michelle Myers ◽

Robert P. Millar ◽

W. Colin Duncan

Keyword(s):

Cell Surface ◽

Chorionic Gonadotropin ◽

Human Chorionic Gonadotropin ◽

Splice Variants ◽

Primary Cultures ◽

Surface Expression ◽

Full Length ◽

Luteinizing Hormone Receptor ◽

Cell Surface Expression ◽

Truncated Form

The human LH receptor (LHR) plays a key role in luteal function and the establishment of pregnancy through its interaction with the gonadotropins LH and human chorionic gonadotropin. We previously identified four splice variants of the LHR in human luteinized granulosa cells (LGCs) and corpora lutea (CL). Real-time quantitative PCR revealed that expression of the full-length LHR (LHRa) and the most truncated form (LHRd) changed significantly in CL harvested at different stages of the ovarian cycle (P < 0.01, ANOVA). LHRa expression was reduced in the late luteal CL (P < 0.05). Conversely, an increase in LHRd expression was observed in the late luteal CL (P < 0.01). Chronic manipulation of human chorionic gonadotropin in LGC primary cultures supported the in vivo findings. LHRd encodes a protein lacking the transmembrane and carboxyl terminal domains. COS-7 cells expressing LHRd were unable to produce cAMP in response to LH stimulation. COS-7 cells coexpressing LHRd and LHRa also failed to generate cAMP in response to LH, suggesting that this truncated form has a negative effect on the signaling of LHRa. Immunofluorescence staining of LGC and COS-7 cells implied that there is a reduction in cell surface expression of LHRa when LHRd is present. Overall, these results imply expression of LHR splice variants is regulated in the human CL. Furthermore, during functional luteolysis a truncated variant could modulate the cell surface expression and activity of full-length LHR.

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Periplasmic depolymerase provides insight into ABC transporter-dependent secretion of bacterial capsular polysaccharides

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1801336115 ◽

2018 ◽

Vol 115 (21) ◽

pp. E4870-E4879 ◽

Cited By ~ 5

Author(s):

Sean D. Liston ◽

Stephen A. McMahon ◽

Audrey Le Bas ◽

Michael D. L. Suits ◽

James H. Naismith ◽

...

Keyword(s):

Cell Surface ◽

Abc Transporter ◽

Pectate Lyase ◽

Capsular Polysaccharides ◽

Ph Optimum ◽

Pectate Lyases ◽

Surface Assembly ◽

Binding Groove ◽

Biochemical Analyses ◽

Vi Antigen

Capsules are surface layers of hydrated capsular polysaccharides (CPSs) produced by many bacteria. The human pathogenSalmonella entericaserovar Typhi produces “Vi antigen” CPS, which contributes to virulence. In a conserved strategy used by bacteria with diverse CPS structures, translocation of Vi antigen to the cell surface is driven by an ATP-binding cassette (ABC) transporter. These transporters are engaged in heterooligomeric complexes proposed to form an enclosed translocation conduit to the cell surface, allowing the transporter to power the entire process. We identified Vi antigen biosynthesis genetic loci in genera of theBurkholderiales, which are paradoxically distinguished fromS.Typhi by encoding VexL, a predicted pectate lyase homolog. Biochemical analyses demonstrated that VexL is an unusual metal-independent endolyase with an acidic pH optimum that is specific for O-acetylated Vi antigen. A 1.22-Å crystal structure of the VexL-Vi antigen complex revealed features which distinguish common secreted catabolic pectate lyases from periplasmic VexL, which participates in cell-surface assembly. VexL possesses a right-handed parallel β-superhelix, of which one face forms an electropositive glycan-binding groove with an extensive hydrogen bonding network that includes Vi antigen acetyl groups and confers substrate specificity. VexL provided a probe to interrogate conserved features of the ABC transporter-dependent export model. When introduced intoS. Typhi, VexL localized to the periplasm and degraded Vi antigen. In contrast, a cytosolic derivative had no effect unless export was disrupted. These data provide evidence that CPS assembled in ABC transporter-dependent systems is actually exposed to the periplasm during envelope translocation.

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The discrepancy between presenilin subcellular localization and γ-secretase processing of amyloid precursor protein

The Journal of Cell Biology ◽

10.1083/jcb.200104045 ◽

2001 ◽

Vol 154 (4) ◽

pp. 731-740 ◽

Cited By ~ 108

Author(s):

Philippe Cupers ◽

Mustapha Bentahir ◽

Katleen Craessaerts ◽

Isabelle Orlans ◽

Hugo Vanderstichele ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Cell Surface ◽

Amyloid Precursor Protein ◽

Primary Cultures ◽

Brefeldin A ◽

Precursor Protein ◽

Subcellular Compartment ◽

Marker Proteins ◽

Intermediate Compartment ◽

The Relationship

We investigated the relationship between PS1 and γ-secretase processing of amyloid precursor protein (APP) in primary cultures of neurons. Increasing the amount of APP at the cell surface or towards endosomes did not significantly affect PS1-dependent γ-secretase cleavage, although little PS1 is present in those subcellular compartments. In contrast, almost no γ-secretase processing was observed when holo-APP or APP-C99, a direct substrate for γ-secretase, were specifically retained in the endoplasmic reticulum (ER) by a double lysine retention motif. Nevertheless, APP-C99-dilysine (KK) colocalized with PS1 in the ER. In contrast, APP-C99 did not colocalize with PS1, but was efficiently processed by PS1-dependent γ-secretase. APP-C99 resides in a compartment that is negative for ER, intermediate compartment, and Golgi marker proteins. We conclude that γ-secretase cleavage of APP-C99 occurs in a specialized subcellular compartment where little or no PS1 is detected. This suggests that at least one other factor than PS1, located downstream of the ER, is required for the γ-cleavage of APP-C99. In agreement, we found that intracellular γ-secretase processing of APP-C99-KK both at the γ40 and the γ42 site could be restored partially after brefeldin A treatment. Our data confirm the “spatial paradox” and raise several questions regarding the PS1 is γ-secretase hypothesis.

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Cell Surface Assembly of HIV gp41 Six-Helix Bundles for Facile, Quantitative Measurements of Hetero-oligomeric Interactions

Journal of the American Chemical Society ◽

10.1021/ja301099s ◽

2012 ◽

Vol 134 (36) ◽

pp. 14642-14645 ◽

Cited By ~ 11

Author(s):

Xuebo Hu ◽

Piyali Saha ◽

Xiaoyue Chen ◽

Dogeun Kim ◽

Mahesh Devarasetty ◽

...

Keyword(s):

Cell Surface ◽

Quantitative Measurements ◽

Surface Assembly ◽

Hiv Gp41

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New insights in glycosphingolipid function: "glycosignaling domain," a cell surface assembly of glycosphingolipids with signal transducer molecules, involved in cell adhesion coupled with signaling

Glycobiology ◽

10.1093/oxfordjournals.glycob.a018822 ◽

1998 ◽

Vol 8 (10) ◽

pp. xi-xviii ◽

Cited By ~ 191

Author(s):

S.-i. Hakomori ◽

K. Handa ◽

K. Iwabuchi ◽

S. Yamamura ◽

A. Prinetti

Keyword(s):

Cell Adhesion ◽

Cell Surface ◽

Signal Transducer ◽

Surface Assembly ◽

A Cell

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Studies on the role of specific cell surface receptors in the removal of lipoprotein (a) in man.

Journal of Clinical Investigation ◽

10.1172/jci110896 ◽

1983 ◽

Vol 71 (5) ◽

pp. 1431-1441 ◽

Cited By ~ 144

Author(s):

F Krempler ◽

G M Kostner ◽

A Roscher ◽

F Haslauer ◽

K Bolzano ◽

...

Keyword(s):

Cell Surface ◽

Cell Surface Receptors ◽

Specific Cell ◽

Lipoprotein A ◽

Surface Receptors ◽

Specific Cell Surface

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Cloning and characterization of a myoblast cell surface antigen defined by 24.1D5 monoclonal antibody

Development ◽

10.1242/dev.105.4.723 ◽

1989 ◽

Vol 105 (4) ◽

pp. 723-731 ◽

Cited By ~ 1

Author(s):

H.J. Gower ◽

S.E. Moore ◽

G. Dickson ◽

V.L. Elsom ◽

R. Nayak ◽

...

Keyword(s):

Skeletal Muscle ◽

Monoclonal Antibody ◽

Cell Surface ◽

Human Skeletal Muscle ◽

Muscle Development ◽

Early Stage ◽

Primary Cultures ◽

Skeletal Muscle Development ◽

Mouse Muscle ◽

Human Skeletal Muscle Cells

Monoclonal antibody 24.1D5 reacts specifically with an epitope expressed on the cell surface of mononucleate myoblasts in primary cultures of human skeletal muscle cells, but not with either multinucleate myotubes or fibroblasts. Polypeptides of 60 and 100 X 10(3) Mr were identified by immunoblotting with the McAb. Human muscle cDNAs encoding the 24.1D5 epitope were used to study further the structure and expression of 24.1D5 during skeletal muscle development. Two mRNA species of 3.0 and 2.5 kb were identified in primary cultures of human skeletal muscle and in mouse muscle cell lines. The levels of both transcripts decreased during myotube formation in vitro and were similarly decreased during myogenesis in the mouse embryo. 24.1D5 mRNAs were expressed by multipotential cells and myoblast derivatives of the mouse embryonic cell line C3H10T1/2, suggesting that 24.1D5 is expressed at an early stage during skeletal muscle development.

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Corneal epithelial-specific cell surface antigen recognized by a monoclonal antibody

Development ◽

10.1242/dev.94.1.163 ◽

1986 ◽

Vol 94 (1) ◽

pp. 163-172

Author(s):

Marian G. Langer ◽

C. V. Sundarraj ◽

Nirmala Sundarraj

Keyword(s):

Epithelial Cells ◽

Cell Surface ◽

Primary Cultures ◽

Enzyme Linked Immunosorbent Assay ◽

Specific Cell ◽

Immunoperoxidase Technique ◽

Igg Antibody ◽

Corneal Epithelial Cells ◽

Corneal Epithelial ◽

Human Corneal Epithelial Cells

Monoclonal antibodies, specific against cell surface differentiation antigens of human corneal epithelial cells, were developed using epithelial cells resected from human corneas as the immunogens. One of these antibodies reacted specifically with corneal epithelial cells and not with epithelial cells of other tissues when tested by an indirect immunoperoxidase technique. Nonidet P-40 extracts of different subcellular fractions of human corneal epithelial cells were tested for their reactivity against this antibody using an enzyme-linked immunosorbent assay. The results indicated that the antigen recognized by this antibody is associated with the plasma membrane. This was further verified by immuno-electron-microscopic analysis using ferritin-conjugated anti-mouse IgG antibody. This antigen was not detectable in the corneal epithelial cells in primary cultures nor in the epithelial cells from early stages of developing cornea (12 to 18 weeks in utero) but was present in the epithelial cells in the corneas of an 8-month-old infant. Therefore, this surface-associated antigen identified in the present study is a developmentally regulated marker of human corneal epithelium.

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