scholarly journals CFTR-associated ligand is a negative regulator of Mrp2 expression

2017 ◽  
Vol 312 (1) ◽  
pp. C40-C46 ◽  
Author(s):  
Man Li ◽  
Carol J. Soroka ◽  
Kathy Harry ◽  
James L. Boyer
Keyword(s):  
Cell Surface ◽  
Protein Interactions ◽  
Posttranscriptional Regulation ◽  
Pdz Domain ◽  
Rat Hepatocytes ◽  
Surface Expression ◽  
Negative Regulator ◽  
Cell Surface Expression ◽  
Binding Motif ◽  
Sodium Hydrogen Exchanger

The multidrug resistance-associated protein 2 (Mrp2) is an ATP-binding cassette transporter that transports a wide variety of organic anions across the apical membrane of epithelial cells. The expression of Mrp2 on the plasma membrane is regulated by protein-protein interactions. Cystic fibrosis transmembrane conductance regulator (CFTR)-associated ligand (CAL) interacts with transmembrane proteins via its PDZ domain and reduces their cell surface expression by increasing lysosomal degradation and intracellular retention. Our results showed that CAL is localized at the trans-Golgi network of rat hepatocytes. The expression of CAL is increased, and Mrp2 expression is decreased, in the liver of mice deficient in sodium/hydrogen exchanger regulatory factor-1. To determine whether CAL interacts with Mrp2 and is involved in the posttranscriptional regulation of Mrp2, we used glutathione S-transferase (GST) fusion proteins with or without the COOH-terminal PDZ binding motif of Mrp2 as the bait in GST pull-down assays. We demonstrated that Mrp2 binds to CAL via its COOH-terminal PDZ-binding motif in GST pull-down assays, an interaction verified by coimmunoprecipitation of these two proteins in cotransfected COS-7 cells. In COS-7 and LLC-PK1 cells transfected with Mrp2 alone, only a mature, high-molecular-mass band of Mrp2 was detected. However, when cells were cotransfected with Mrp2 and CAL, Mrp2 was expressed as both mature and immature forms. Biotinylation and streptavidin pull-down assays confirmed that CAL dramatically reduces the expression level of total and cell surface Mrp2 in Huh-7 cells. Our findings suggest that CAL interacts with Mrp2 and is a negative regulator of Mrp2 expression.

Retargeting of the mitochondrial protein p32/gC1Qr to a cytoplasmic compartment and the cell surface

2001 ◽  
Vol 114 (11) ◽  
pp. 2115-2123
Author(s):  
Hans C. van Leeuwen ◽  
Peter O’Hare
Keyword(s):  
Cell Surface ◽  
Protein Interactions ◽  
Mitochondrial Protein ◽  
Physiological Role ◽  
Surface Expression ◽  
Transport Processes ◽  
Cell Surface Expression ◽  
Bacterial Proteins ◽  
Er Chaperone ◽  
Cellular Compartments

p32/gC1qR is a small acidic protein that has been reported to have a broad range of distinct functions and to associate with a wide array of cellular, viral and bacterial proteins. It has been found in each of the main cellular compartments including mitochondria, nucleus and cytoplasm and is also thought to be located at the plasma membrane and secreted into the extracellular matrix. The true physiological role(s) of p32 remains controversial because it has been difficult to reconcile all of the findings on protein interactions and the seemingly disparate observations on compartmentalisation. However, it has been proposed that p32 is somehow involved in transport processes connecting diverse cellular compartments and the cell surface. Here we show that native p32 appears to be localised mainly in the mitochondria and is not detectable on the cell surface. However, addition of a short tag to the N-terminus of p32 appears to block its mitochondrial targeting, resulting in redirection into a cytoplasmic vesicular pattern, overlapping with the endoplasmic reticulum. The redirection of p32 results in an alteration in and co-localisation with ER markers including calreticulin, a lumenal ER chaperone. Furthermore, we show both by immunofluorescence and cross-linking studies that this also results in cell-surface expression of p32. These results indicate that, at least under certain circumstances, p32 can be retargeted and may help to provide an explanation for the diverse observations on its localization.

scholarly journals Position 97 of HLA-B, a residue implicated in pathogenesis of ankylosing spondylitis, plays a key role in cell surface free heavy chain expression

2016 ◽  
Vol 76 (3) ◽  
pp. 593-601 ◽  
Author(s):  
Liye Chen ◽  
Hui Shi ◽  
Jack Yuan ◽  
Paul Bowness
Keyword(s):  
Ankylosing Spondylitis ◽  
Cell Surface ◽  
Heavy Chain ◽  
Protein Interactions ◽  
Hela Cells ◽  
Antigen Processing ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Expression Levels ◽  
Free Heavy Chain

ObjectiveAssociation of position 97 (P97) residue polymorphisms in human leucocyte antigen (HLA)-B, including HLA-B*27, with ankylosing spondylitis (AS) has recently been reported. We studied the effect of P97 variations on cell surface expression of the AS-associated HLA-B*27 and HLA-B*51, and the AS-protective HLA-B*7.MethodsFlow cytometry was used to measure surface expression of HLA-B*27 in C1R/HeLa cells expressing HLA-B*27 (N97) and six mutants at P97 (N97T, N97S, N97V, N97R, N97W and N97D). Transporter associated with antigen processing-deficient T2, tapasin-deficient 220, β2m-deficient HCT15 and endoplasmic reticulum aminopeptidase 1 or β2m-clustered regularly interspaced short palindromic repeats/Cas9-knockout HeLa cells were used to provide evidence for specific protein interactions. Surface expression of HLA-B*7/HLA-B*51 P97 mutants was also studied.ResultsMutation of HLA-B*27 P97 to the AS risk residue threonine increased cell surface free heavy chain (FHC) expression. Protective residues (serine or valine) and non-AS-associated residues (arginine or tryptophan) did not alter FHC expression. The N97D mutation reduced expression of conventional and FHC forms of HLA-B*27. Differences in FHC expression levels between HLA-B*27, HLA-B*27-N97T and HLA-B*27-N97D were dependent on the presence of functional β2m. HLA-B*7, which has an AS-protective serine at P97, expressed lower levels of FHC than HLA-B*27 or HLA-B*51. Introduction of asparagine at P97 of both HLA-B*7 and HLA-B*51 increased FHC expression.ConclusionsThe nature of P97 residue affects surface expression of HLA-B*27, B*7 and B*51, with AS-associated residues giving rise to higher FHC expression levels. The association of P97 amino acid polymorphisms with AS could be, at least in part, explained by its effect on HLA-B*27 FHC cell surface expression.

scholarly journals Low HLA-C expression at cell surfaces correlates with increased turnover of heavy chain mRNA.

1995 ◽  
Vol 181 (6) ◽  
pp. 2085-2095 ◽  
Author(s):  
J A McCutcheon ◽  
J Gumperz ◽  
K D Smith ◽  
C T Lutz ◽  
P Parham
Keyword(s):  
Cell Surface ◽  
Heavy Chain ◽  
Protein Interactions ◽  
Stop Codon ◽  
Mrna Level ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Protein Protein Interactions ◽  
Peptide Binding Groove ◽  
Beta 2 Microglobulin

In comparison with HLA-A and -B, the protein products of the HLA-C locus are poorly characterized, in part because of their low level of expression at the cell surface. Here, we examine how protein-protein interactions during assembly and regulation of the mRNA level affect cell surface expression of HLA-C. We find that intrinsic properties of the HLA-C heavy chain proteins do not correlate with low cell surface expression: HLA-C heavy chains associate and dissociate with beta 2-microglobulin (beta 2m) at rates comparable to those found for HLA-A and -B, and increased competition for beta 2m does not alter the surface expression of HLA-C. From studies of chimeric genes spliced from the HLA-B7 and -Cw3 genes, we find that chimeric proteins containing the B7 peptide-binding groove can have low cell surface expression, suggesting that inefficiency in binding peptides is not the cause of low cell surface expression for HLA-C. The surface levels of HLA-A, -B, or -C in cells transfected with cDNA can be similar, implicating noncoding regions of HLA-C heavy chain genes in the regulation of surface expression. We find that HLA-C mRNA is expressed at lower levels than HLA-B mRNA and that this difference results from faster degradation of the HLA-C message. Experiments examining chimeric B7/Cw3 and B7/Cw6 genes suggest that a region determining low expression of HLA-C is to be found between the 3' end of exon 3 and a site in the 3' untranslated region, approximately 600 bases downstream of the translation stop codon.

scholarly journals Activity and PI3-kinase dependent trafficking of the intestinal anion exchanger downregulated in adenoma depend on its PDZ interaction and on lipid rafts

2010 ◽  
Vol 299 (4) ◽  
pp. G907-G920 ◽  
Author(s):  
S. Lissner ◽  
L. Nold ◽  
C.-J. Hsieh ◽  
J. R. Turner ◽  
M. Gregor ◽  
...  
Keyword(s):  
Cell Surface ◽  
Lipid Rafts ◽  
Adaptor Proteins ◽  
Surface Expression ◽  
Pi3 Kinase ◽  
Cell Surface Expression ◽  
Binding Motif ◽  
Wild Type ◽  
Pdz Proteins ◽  
Nonionic Detergent

The Cl/HCO3 exchanger downregulated in adenoma (DRA) mediates electroneutral NaCl absorption in the intestine together with the apical Na/H exchanger NHE3. Lipid rafts (LR) modulate transport activity and are involved in phosphatidylinositol 3-kinase (PI3-kinase)-dependent trafficking of NHE3. Although DRA and NHE3 interact via PDZ adaptor proteins of the NHERF family, the role of LR and PDZ proteins in the regulation of DRA is unknown. We examined the association of DRA with LR using the nonionic detergent Triton X-100. DRA cofractionated with LR independently of its PDZ binding motif. Furthermore, DRA interacts with PDZK1, E3KARP, and IKEPP in LR, although their localization within lipid rafts is independent of DRA. Disruption of LR integrity resulted in the disappearance of DRA from LR, in a decrease of its surface expression and in a reduction of its activity. In HEK cells the inhibition of DRA by LR disruption was entirely dependent on the presence of the PDZ interaction motif. In addition, in Caco-2/BBE cells the inhibition by LR disruption was more pronounced in wild-type DRA than in mutated DRA (DRA-ETKFminus; lacking the PDZ binding motif)-expressing cells. Inhibition of PI3-kinase decreased the activity and the cell surface expression of wild-type DRA but not of DRA-ETKFminus; the partitioning into LR was unaffected. Furthermore, simultaneous inhibition of PI3-kinase and disruption of LR did not further decrease DRA activity and cell surface expression compared with LR disruption only. These results suggest that the activity of DRA depends on its LR association, on its PDZ interaction, and on PI3-kinase activity.

scholarly journals Towards a transcriptome-based theranostic platform for unfavorable breast cancer phenotypes

2016 ◽  
Vol 113 (45) ◽  
pp. 12780-12785 ◽  
Author(s):  
Andrey S. Dobroff ◽  
Sara D’Angelo ◽  
Bedrich L. Eckhardt ◽  
Fortunato Ferrara ◽  
Daniela I. Staquicini ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Surface ◽  
Antibody Fragment ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Binding Motif ◽  
Human Antibody ◽  
Surface Receptors ◽  
Cancer Phenotypes

Inflammatory breast carcinoma (IBC) is one of the most lethal forms of human breast cancer, and effective treatment for IBC is an unmet clinical need in contemporary oncology. Tumor-targeted theranostic approaches are emerging in precision medicine, but only a few specific biomarkers are available. Here we report up-regulation of the 78-kDa glucose-regulated protein (GRP78) in two independent discovery and validation sets of specimens derived from IBC patients, suggesting translational promise for clinical applications. We show that a GRP78-binding motif displayed on either bacteriophage or adeno-associated virus/phage (AAVP) particles or loop-grafted onto a human antibody fragment specifically targets orthotopic IBC and other aggressive breast cancer models in vivo. To evaluate the theranostic value, we used GRP78-targeting AAVP particles to deliver the human Herpes simplex virus thymidine kinase type-1 (HSVtk) transgene, obtaining simultaneous in vivo diagnosis through PET imaging and tumor treatment by selective activation of the prodrug ganciclovir at tumor sites. Translation of this AAVP system is expected simultaneously to image, monitor, and treat the IBC phenotype and possibly other aggressive (e.g., invasive and/or metastatic) subtypes of breast cancer, based on the inducible cell-surface expression of the stress-response chaperone GRP78, and possibily other cell-surface receptors in human tumors.

Cell surface expression by adult rat hepatocytes of a non-collagen glycoprotein present in rat liver biomatrix

1984 ◽  
Vol 152 (2) ◽  
pp. 402-414 ◽  
Author(s):  
Douglas C. Hixson ◽  
Maria DeLourdes Ponce ◽  
James P. Allison ◽  
Earl F. Walborg
Keyword(s):  
Cell Surface ◽  
Rat Liver ◽  
Rat Hepatocytes ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Adult Rat ◽  
Adult Rat Hepatocytes

scholarly journals Receptor sequestration in response to β-arrestin-2 phosphorylation by ERK1/2 governs steady-state levels of GPCR cell-surface expression

2015 ◽  
Vol 112 (37) ◽  
pp. E5160-E5168 ◽  
Author(s):  
Justine S. Paradis ◽  
Stevenson Ly ◽  
Élodie Blondel-Tepaz ◽  
Jacob A. Galan ◽  
Alexandre Beautrait ◽  
...  
Keyword(s):  
Steady State ◽  
Cell Surface ◽  
G Protein ◽  
Chemokine Receptors ◽  
Surface Expression ◽  
Negative Regulator ◽  
Scaffolding Protein ◽  
Cell Surface Expression ◽  
Reciprocal Effect

MAPKs are activated in response to G protein-coupled receptor (GPCR) stimulation and play essential roles in regulating cellular processes downstream of these receptors. However, very little is known about the reciprocal effect of MAPK activation on GPCRs. To investigate possible crosstalk between the MAPK and GPCRs, we assessed the effect of ERK1/2 on the activity of several GPCR family members. We found that ERK1/2 activation leads to a reduction in the steady-state cell-surface expression of many GPCRs because of their intracellular sequestration. This subcellular redistribution resulted in a global dampening of cell responsiveness, as illustrated by reduced ligand-mediated G-protein activation and second-messenger generation as well as blunted GPCR kinases and β-arrestin recruitment. This ERK1/2-mediated regulatory process was observed for GPCRs that can interact with β-arrestins, such as type-2 vasopressin, type-1 angiotensin, and CXC type-4 chemokine receptors, but not for the prostaglandin F receptor that cannot interact with β-arrestin, implicating this scaffolding protein in the receptor’s subcellular redistribution. Complementation experiments in mouse embryonic fibroblasts lacking β-arrestins combined with in vitro kinase assays revealed that β-arrestin-2 phosphorylation on Ser14 and Thr276 is essential for the ERK1/2-promoted GPCR sequestration. This previously unidentified regulatory mechanism was observed after constitutive activation as well as after receptor tyrosine kinase- or GPCR-mediated activation of ERK1/2, suggesting that it is a central node in the tonic regulation of cell responsiveness to GPCR stimulation, acting both as an effector and a negative regulator.

scholarly journals The CFTR-Associated Ligand Arrests the Trafficking of the Mutant ΔF508 CFTR Channel in the ER Contributing to Cystic Fibrosis

2018 ◽  
Vol 45 (2) ◽  
pp. 639-655 ◽  
Author(s):  
Emily Bergbower ◽  
Clement Boinot ◽  
Inna Sabirzhanova ◽  
William Guggino ◽  
Liudmila Cebotaru
Keyword(s):  
Cell Surface ◽  
Cell Biology ◽  
Pdz Domain ◽  
Coiled Coil ◽  
Surface Expression ◽  
26S Proteasome ◽  
Scaffolding Protein ◽  
Cell Surface Expression ◽  
Membrane Expression ◽  
Δf508 Cftr

Background/Aims: The CFTR-Associated Ligand (CAL), a PDZ domain containing protein with two coiled-coil domains, reduces cell surface WT CFTR through degradation in the lysosome by a well-characterized mechanism. However, CAL’s regulatory effect on ΔF508 CFTR has remained almost entirely uninvestigated. Methods: In this study, we describe a previously unknown pathway for CAL by which it regulates the membrane expression of ΔF508 CFTR through arrest of ΔF508 CFTR trafficking in the endoplasmic reticulum (ER) using a combination of cell biology, biochemistry and electrophysiology. Results: We demonstrate that CAL is an ER localized protein that binds to ΔF508 CFTR and is degraded in the 26S proteasome. When CAL is inhibited, ΔF508 CFTR retention in the ER decreases and cell surface expression of mature functional ΔF508 CFTR is observed alongside of enhanced expression of plasma membrane scaffolding protein NHERF1. Chaperone proteins regulate this novel process, and ΔF508 CFTR binding to HSP40, HSP90, HSP70, VCP, and Aha1 changes to improve ΔF508 CFTR cell surface trafficking. Conclusion: Our results reveal a pathway in which CAL regulates the cell surface availability and intracellular retention of ΔF508 CFTR.

scholarly journals Targeting CAL as a Negative Regulator of ΔF508-CFTR Cell-Surface Expression

2006 ◽  
Vol 282 (11) ◽  
pp. 8099-8109 ◽  
Author(s):  
Michael Wolde ◽  
Abigail Fellows ◽  
Jie Cheng ◽  
Aleksandr Kivenson ◽  
Bonita Coutermarsh ◽  
...  
Keyword(s):  
Cell Surface ◽  
Surface Expression ◽  
Negative Regulator ◽  
Cell Surface Expression ◽  
Δf508 Cftr

scholarly journals High-Content Imaging for Large-Scale Detection of Low-Affinity Extracellular Protein Interactions

2019 ◽  
Vol 24 (10) ◽  
pp. 987-999
Author(s):  
Laura Wood ◽  
Gavin J. Wright
Keyword(s):  
Cell Surface ◽  
Protein Interactions ◽  
Large Scale ◽  
Local Environment ◽  
Surface Expression ◽  
Extracellular Protein ◽  
Automated Image Analysis ◽  
Cell Surface Expression ◽  
High Content Imaging ◽  
Pathogen Invasion

Extracellular protein interactions coordinate cellular responses with their local environment and have important roles in pathogen invasion and disease. Due to technical challenges associated with studying binding events at the cell surface, the systematic and reliable identification of novel ligand–receptor pairs remains difficult. Here, we describe the development of a cell-based assay using large-scale transient transfections and high-content imaging (HCI) to detect extracellular binding events. We optimized the parameters for efficient transfection of human cells with cDNA plasmids encoding full-length cell surface receptors in 384-well plates. Using a range of well-characterized structurally diverse low-affinity cell surface interactions, we show that transfected cells probed with highly avid ligands can be used to successfully identify ligand–receptor pairs using an HCI platform and automated image analysis software. To establish the high-throughput potential of this approach, we also screened a pool of ligands against a collection of 2455 cell surface expression clones and found that known ligand–receptor interactions could be robustly and consistently detected across the library using this technology.

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