Actinin-1 binds to the C-terminus of A2B adenosine receptor (A2BAR) and enhances A2BAR cell-surface expression

2016 ◽  
Vol 473 (14) ◽  
pp. 2179-2186 ◽  
Author(s):  
Ying Sun ◽  
Wenbao Hu ◽  
Xiaojie Yu ◽  
Zhengzhao Liu ◽  
Robert Tarran ◽  
...  
Keyword(s):  
Cell Surface ◽  
Protein Interactions ◽  
Adenosine Receptor ◽  
Point Mutations ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Protein Protein Interactions ◽  
Physiological Conditions ◽  
A2b Adenosine Receptor ◽  
C Terminus

A2BAR (A2B adenosine receptor) has been implicated in several physiological conditions, such as allergic or inflammatory disorders, vasodilation, cell growth and epithelial electrolyte secretion. For mediating the protein–protein interactions of A2BAR, the receptor's C-terminus is recognized to be crucial. In the present study, we unexpectedly found that two point mutations in the A2BAR C-terminus (F297A and R298A) drastically impaired the expression of A2BAR protein by accelerating its degradation. Thus we tested the hypothesis that these two point mutations disrupt A2BAR's interaction with a protein essential for A2BAR stability. Our results show that both mutations disrupted the interaction of A2BAR with actinin-1, an actin-associated protein. Furthermore, actinin-1 binding stabilized the global and cell-surface expression of A2BAR. By contrast, actinin-4, another non-muscle actinin isoform, did not bind to A2BAR. Thus our findings reveal a previously unidentified regulatory mechanism of A2BAR abundance.

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.

Abstract 468: Identification of a New Component of the Na v 1.5 Complex: αB-crystallin Interacts with Na v 1.5 and Regulates Ubiquitination and Internalization of Cell Surface Na v 1.5

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Yuan Huang ◽  
Zhijie Wang ◽  
Yinan Liu ◽  
Qiuyun Chen ◽  
Qing k. Wang
Keyword(s):  
Cell Surface ◽  
Protein Interactions ◽  
Sodium Current ◽  
Surface Expression ◽  
Expression Level ◽  
Cell Surface Expression ◽  
Protein Protein Interactions ◽  
Interacting Protein ◽  
Binding Partner ◽  
Αb Crystallin

Mutations in cardiac Na + channel Na v 1.5 cause cardiac arrhythmias and sudden death. The cardiac Na + channel functions as a protein complex, however, its complete components remain to be fully elucidated. A yeast two-hybrid screen identified a new candidate Na v 1.5-interacting protein, αB-crystallin. GST-pull-down, co-immunoprecipitation and immunostaining analyses validated the interaction between Na v 1.5 and αB-crystallin. Overexpression of αB-crystallin significantly increased peak sodium current ( I Na ) density, and the underlying molecular mechanism is the increased cell surface expression level of Na v 1.5 via reduced internalization of cell surface Na v 1.5 and ubiquitination of Na v 1.5. Knockout of αB-crystallin expression significantly decreased the cell-surface expression level of Na v 1.5. αB-crystallin interacted with Nedd4-2, however, a catalytically inactive Nedd4-2-C801S mutant reduced the interaction between αB-crystallin and Nedd4-2 and also blocked the increase of peak I Na density by αB-crystallin. Na v 1.5 mutation V1980A at the interaction site for Nedd4-2 eliminated the effect of αB-crystallin on reduction of Na v 1.5 ubiquitination and increases of I Na density. The data suggest that the interactions between αB-crystallin and functionally active Nedd4-2 and between αB-crystallin and Na v 1.5 are critical to increased I Na density by αB-crystallin. Multiple mutations in αB-crystallin have been associated with human diseases. Two mutations, R109H and R151X, eliminated the effect of αB-crystallin on I Na . This study identifies αB-crystallin as a new binding partner for Na v 1.5. αB-crystallin interacts with Na v 1.5 and increases I Na by modulating the expression level and internalization of cell surface Na v 1.5 and ubiquitination of Na v 1.5, which requires the protein-protein interactions between αB-crystallin and Na v 1.5 and between αB-crystallin and functionally active Nedd4-2.

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 C-terminal region regulates the functional expression of human noradrenaline transporter splice variants

2006 ◽  
Vol 401 (1) ◽  
pp. 185-195 ◽  
Author(s):  
Chiharu Sogawa ◽  
Kei Kumagai ◽  
Norio Sogawa ◽  
Katsuya Morita ◽  
Toshihiro Dohi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Surface ◽  
Splice Variants ◽  
Functional Expression ◽  
Surface Expression ◽  
Terminal Region ◽  
Site Directed Mutagenesis ◽  
Cell Surface Expression ◽  
C Terminus ◽  
And Function

The NET [noradrenaline (norepinephrine) transporter], an Na+/Cl−-dependent neurotransmitter transporter, has several isoforms produced by alternative splicing in the C-terminal region, each differing in expression and function. We characterized the two major isoforms of human NET, hNET1, which has seven C-terminal amino acids encoded by exon 15, and hNET2, which has 18 amino acids encoded by exon 16, by site-directed mutagenesis in combination with NE (noradrenaline) uptake assays and cell surface biotinylation. Mutants lacking one third or more of the 24 amino acids encoded by exon 14 exhibited neither cell surface expression nor NE uptake activity, with the exception of the mutant lacking the last eight amino acids of hNET2, whose expression and uptake resembled that of the WT (wild-type). A triple alanine replacement of a candidate motif (ENE) in this region mimicked the influences of the truncation. Deletion of either the last three or another four amino acids of the C-terminus encoded by exon 15 in hNET1 reduced the cell surface expression and NE uptake, whereas deletion of all seven residues reduced the transport activity but did not affect the cell surface expression. Replacement of RRR, an endoplasmic reticulum retention motif, by alanine residues in the C-terminus of hNET2 resulted in a similar expression and function compared with the WT, while partly recovering the effects of the mutation of ENE. These findings suggest that in addition to the function of the C-terminus, the common proximal region encoded by exon 14 regulates the functional expression of splice variants, such as hNET1 and hNET2.

scholarly journals Functional Analysis of the Murine Cytomegalovirus Chemokine Receptor Homologue M33: Ablation of Constitutive Signaling Is Associated with an Attenuated Phenotype In Vivo

2007 ◽  
Vol 82 (4) ◽  
pp. 1884-1898 ◽  
Author(s):  
Ruth Case ◽  
Emma Sharp ◽  
Tau Benned-Jensen ◽  
Mette M. Rosenkilde ◽  
Nicholas Davis-Poynter ◽  
...  
Keyword(s):  
Cell Surface ◽  
Salivary Glands ◽  
Surface Expression ◽  
Receptor Activation ◽  
Murine Cytomegalovirus ◽  
Cell Surface Expression ◽  
Transmembrane Receptors ◽  
C Terminus ◽  
The Impact

ABSTRACT The murine cytomegalovirus (MCMV) M33 gene is conserved among all betaherpesviruses and encodes a homologue of seven-transmembrane receptors (7TMR) with the capacity for constitutive signaling. Previous studies have demonstrated that M33 is important for MCMV dissemination to or replication within the salivary glands. In this study, we probed N- and C-terminal regions of M33 as well as known 7TMR signature motifs in transmembrane (TM) II and TM III to determine the impact on cell surface expression, constitutive signaling, and in vivo phenotype. The region between amino acids R340 and A353 of the C terminus was found to be important for CREB- and NFAT-mediated signaling, although not essential for phosphatidylinositol turnover. Tagging or truncation of the N terminus of M33 resulted in loss of cell surface expression. Within TM II, an F79D mutation abolished constitutive signaling, demonstrating a role, as in other cellular and viral 7TMR, of TM II in receptor activation. In TM III, the arginine (but not the asparagine) residue of the NRY motif (the counterpart of the common DRY motif in cellular 7TMR) was found to be essential for constitutive signaling. Selected mutations incorporated into recombinant MCMV showed that disruption of constitutive signaling for a viral 7TMR homologue resulted in a reduced capacity to disseminate to or replicate in the salivary glands. In addition, HCMV UL33 was found to partially compensate for the lack of M33 in vivo, suggesting conserved biological roles of the UL33 gene family.

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.

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