Characterization of porcine UL16-binding protein 1 endothelial cell surface expression

We studied dynamics of cell surface expression of proteolytically activated thrombin receptor (PAR-1) in human pulmonary artery endothelial cells (HPAEC). PAR-1 activation was measured by changes in cytosolic calcium concentration ([Ca2+]i) and HPAEC retraction response (determined by real-time transendothelial monolayer electrical resistance). [Ca2+]iincrease in response to thrombin was abolished by preexposure to 25 nM thrombin for >60 min, indicating PAR-1 desensitization, but preexposure to 25 nM thrombin for only 30 min or to 10 nM thrombin for up to 2 h did not desensitize PAR-1. Exposure to 10 or 25 nM thrombin decreased monolayer electrical resistance 40–60%. Cells preexposed to 10 nM thrombin, but not those preexposed to 25 nM thrombin, remained responsive to thrombin 3 h later. Loss of cell retractility was coupled to decreased cell surface PAR-1 expression as determined by immunofluorescence. Cell surface PAR-1 disappeared upon short-term (30 min) thrombin exposure but reappeared within 90 min after incubation in thrombin-free medium. Exposure to 25 nM thrombin for >60 min prevented rapid cycloheximide-insensitive PAR-1 reappearance. Cycloheximide-sensitive recovery of cell surface PAR-1 expression required 18 h. Therefore, both duration and concentration of thrombin exposure regulate the time course of recovery of HPAEC surface PAR-1 expression. The results support the hypothesis that initial recovery of PAR-1 surface expression in endothelial cells results from a rapidly mobilizable PAR-1 pool, whereas delayed recovery results from de novo PAR-1 synthesis. We conclude that thrombin itself regulates endothelial cell surface PAR-1 expression and that decreased surface expression interferes with thrombin-induced endothelial cell activation responses.

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Obesity-Linked Variants of Melanocortin-4 Receptor Are Misfolded in the Endoplasmic Reticulum and Can Be Rescued to the Cell Surface by a Chemical Chaperone

Endocrine Reviews ◽

10.1210/edrv.31.4.9985 ◽

2010 ◽

Vol 31 (4) ◽

pp. 605-605

Author(s):

Susana Granell ◽

Sameer Mohammad ◽

Ramanagouda Ramanagoudr-Bhojappa ◽

Giulia Baldini

Keyword(s):

Endoplasmic Reticulum ◽

Cell Surface ◽

Er Stress ◽

Binding Protein ◽

Intracellular Localization ◽

Specific Inhibitor ◽

Surface Expression ◽

Cell Surface Expression ◽

Chemical Chaperone ◽

Melanocortin 4 Receptor

Abstract Melanocortin-4 receptor (MC4R) is a G protein-coupled receptor expressed in the brain where it controls food intake. Many obesity-linked MC4R variants are poorly expressed at the plasma membrane and are retained intracellularly. We have studied the intracellular localization of four obesity-linked MC4R variants, P78L, R165W, I316S, and I317T, in immortalized neurons. We find that these variants are all retained in the endoplasmic reticulum (ER), are ubiquitinated to a greater extent than the wild-type (wt) receptor, and induce ER stress with increased levels of ER chaperones as compared with wt-MC4R and appearance of CCAAT/enhancer-binding protein homologous protein. Expression of the X-box-binding-protein-1 with selective activation of a protective branch of the unfolded protein response did not have any effect on the cell surface expression of MC4R-I316S. Conversely, the pharmacological chaperone 4-phenyl butyric acid (PBA) increased the cell surface expression of wt-MC4R, MC4R-I316S, and I317T by more than 40%. PBA decreased ubiquitination of MC4R-I316S and prevented ER stress induced by expression of the mutant, suggesting that the drug functions to promote MC4R folding. MC4R-I316S rescued to the cell surface is functional, with a 52% increase in agonist-induced cAMP production, as compared with untreated cells. Also direct inhibition of wt-MC4R and MC4R-I316S ubiquitination by a specific inhibitor of the ubiquitin-activating enzyme 1 increased by approximately 40% the expression of the receptors at the cell surface, and the effects of PBA and ubiquitin-activating enzyme 1 were additive. These data offer a cell-based rationale that drugs that improve MC4R folding or decrease ER-associated degradation of the receptor may function to treat some forms of hereditary obesity.

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Characterization of the biosynthesis and cell surface expression of avian metapneumovirus attachment glycoprotein

Virus Research ◽

10.1016/j.virusres.2009.10.022 ◽

2010 ◽

Vol 147 (2) ◽

pp. 189-194 ◽

Cited By ~ 1

Author(s):

Lizhong Luo ◽

Krista Nishi ◽

Li Liu ◽

Marta I. Sabara ◽

Yan Li

Keyword(s):

Cell Surface ◽

Surface Expression ◽

Cell Surface Expression ◽

Avian Metapneumovirus

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A foldable CFTRΔF508 biogenic intermediate accumulates upon inhibition of the Hsc70–CHIP E3 ubiquitin ligase

The Journal of Cell Biology ◽

10.1083/jcb.200410065 ◽

2004 ◽

Vol 167 (6) ◽

pp. 1075-1085 ◽

Cited By ~ 118

Author(s):

J. Michael Younger ◽

Hong-Yu Ren ◽

Liling Chen ◽

Chun-Yang Fan ◽

Andrea Fields ◽

...

Keyword(s):

Cystic Fibrosis ◽

Cell Surface ◽

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

Surface Expression ◽

Cell Surface Expression ◽

Folding Defect ◽

Cellular Components ◽

Soluble State

CFTRΔF508 exhibits a correctable protein-folding defect that leads to its misfolding and premature degradation, which is the cause of cystic fibrosis (CF). Herein we report on the characterization of the CFTRΔF508 biogenic intermediate that is selected for proteasomal degradation and identification of cellular components that polyubiquitinate CFTRΔF508. Nonubiquitinated CFTRΔF508 accumulates in a kinetically trapped, but folding competent conformation, that is maintained in a soluble state by cytosolic Hsc70. Ubiquitination of Hsc70-bound CFTRΔF508 requires CHIP, a U box containing cytosolic cochaperone. CHIP is demonstrated to function as a scaffold that nucleates the formation of a multisubunit E3 ubiquitin ligase whose reconstituted activity toward CFTR is dependent upon Hdj2, Hsc70, and the E2 UbcH5a. Inactivation of the Hsc70–CHIP E3 leads CFTRΔF508 to accumulate in a nonaggregated state, which upon lowering of cell growth temperatures, can fold and reach the cell surface. Inhibition of CFTRΔF508 ubiquitination can increase its cell surface expression and may provide an approach to treat CF.

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Cell Surface Expression of HIP, a Novel Heparin/Heparan Sulfate-binding Protein, of Human Uterine Epithelial Cells and Cell Lines

Journal of Biological Chemistry ◽

10.1074/jbc.271.20.11824 ◽

1996 ◽

Vol 271 (20) ◽

pp. 11824-11830 ◽

Cited By ~ 34

Author(s):

Larry H. Rohde ◽

JoAnne Julian ◽

Ari Babaknia ◽

Daniel D. Carson

Keyword(s):

Epithelial Cells ◽

Cell Surface ◽

Heparan Sulfate ◽

Cell Lines ◽

Binding Protein ◽

Surface Expression ◽

Cell Surface Expression ◽

Uterine Epithelial Cells

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Regulation of the cell-surface expression of an HTLV-I binding protein in human T cells during immune activation

Blood ◽

10.1182/blood-2002-07-2277 ◽

2003 ◽

Vol 101 (8) ◽

pp. 3085-3092 ◽

Cited By ~ 30

Author(s):

Manisha D. Nath ◽

Francis W. Ruscetti ◽

Cari Petrow-Sadowski ◽

Kathryn S. Jones

Keyword(s):

T Cells ◽

T Cell ◽

Cell Surface ◽

Cell Lines ◽

Cd4 T Cells ◽

Binding Protein ◽

Surface Expression ◽

Cell Surface Expression ◽

Type I ◽

Primary Cells

AbstractLittle is known about the requirements for human T-cell leukemia virus type I (HTLV-I) entry, including the identity of the cellular receptor(s). Recently, we have generated an HTLV-I surface glycoprotein (SU) immunoadhesin, HTSU-IgG, which binds specifically to cell-surface protein(s) critical for HTLV-I–mediated entry in cell lines. Here, expression of the HTLV-I SU binding protein on primary cells of the immune system was examined. The immunoadhesin specifically bound to adult T cells, B cells, NK cells, and macrophages. Cell stimulation dramatically increased the amount of binding, with the highest levels of binding on CD4+ and CD8+ T cells. Naive (CD45RAhigh, CD62Lhigh) CD4+ T cells derived from cord blood cells, in contrast to other primary cells and all cell lines examined, bound no detectable HTLV-I SU. However, following stimulation, the level of HTSU-IgG binding was rapidly induced (fewer than 6 hours), reaching the level of binding seen on adult CD4+ T cells by 72 hours. In contrast to HTLV-I virions, the soluble HTSU-IgG did not effect T-cell activation or proliferation. When incubated with human peripheral blood mononuclear cells in a mixed leukocyte reaction, HTSU-IgG inhibited proliferation at less than 1 ng/mL. These results indicate that cell-surface expression of the HTLV SU binding protein is up-regulated during in vitro activation and suggest a role for the HTLV-I SU binding proteins in the immunobiology of CD4+ T cells.

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