N-Linked Glycosylation Regulates Human Proteinase-activated Receptor-1 Cell Surface Expression and Disarming via Neutrophil Proteinases and Thermolysin

Xiao, Yu; Morice, Alyn H.; Compton, Steven J.; Sadofsky, Laura R.

doi:10.1074/jbc.m110.204271

Cited by 9 publications

(7 citation statements)

References 35 publications

(65 reference statements)

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“…PAR surface expression, cleavage and regulation of signaling (e.g. protease-induced disarming) can also be affected by the factors such as N-linked glycosylation [53], [54]. Those studies are comparable with our present study, because the same source of trypsin was used; however, the sources of both PR3 and elastase were different.…”

Section: Discussionsupporting

confidence: 87%

PR3 and Elastase Alter PAR1 Signaling and Trigger vWF Release via a Calcium-Independent Mechanism from Glomerular Endothelial Cells

et al. 2012

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Neutrophil proteases, proteinase-3 (PR3) and elastase play key roles in glomerular endothelial cell (GEC) injury during glomerulonephritis. Endothelial protease-activated receptors (PARs) are potential serine protease targets in glomerulonephritis. We investigated whether PAR1/2 are required for alterations in GEC phenotype that are mediated by PR3 or elastase during active glomerulonephritis. Endothelial PARs were assessed by flow cytometry. Thrombin, trypsin and agonist peptides for PAR1 and PAR2, TFLLR-NH2 and SLIGKV-NH2, respectively, were used to assess alterations in PAR activation induced by PR3 or elastase. Endothelial von Willebrand Factor (vWF)release and calcium signaling were used as PAR activation markers. Both PR3 and elastase induced endothelial vWF release, with elastase inducing the highest response. PAR1 peptide induced GEC vWF release to the same extent as PR3. However, knockdown of PARs by small interfering RNA showed that neither PAR1 nor PAR2 activation caused PR3 or elastase-mediated vWF release. Both proteases interacted with and disarmed surface GEC PAR1, but there was no detectable interaction with cellular PAR2. Neither protease induced a calcium response in GEC. Therefore, PAR signaling and serine protease-induced alterations in endothelial function modulate glomerular inflammation via parallel but independent pathways.

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Section: Discussionsupporting

confidence: 87%

PR3 and Elastase Alter PAR1 Signaling and Trigger vWF Release via a Calcium-Independent Mechanism from Glomerular Endothelial Cells

et al. 2012

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“…Interestingly, PAR-1 was observed as a broad migration band with a molecular mass in control siRNA-transfected cells. It has been reported that this broad migration band is due to N-linked glycosylation 41 . After treatment with siRNA the protein expression of PAR-1 was markedly reduced, and the flow-induced mRNA expressions of KLF2- and Nrf2-targeted genes were significantly inhibited (Fig.…”

Section: Resultsmentioning

confidence: 84%

PAR-1 is a novel mechano-sensor transducing laminar flow-mediated endothelial signaling

Kim

Han

Nam

et al. 2018

Sci Rep

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Recent studies have indicated that protease-activated receptor-1 (PAR-1) is involved in cytoprotective and anti-inflammatory responses in endothelial cells (ECs). However, the role of PAR-1 in laminar flow-mediated atheroprotective responses remains unknown. Herein, we investigated whether PAR-1 regulates laminar flow-mediated mechanotransduction in ECs. Confocal analysis showed that PAR-1 was internalized into early endosomes in response to laminar flow. In addition, flow cytometry analysis showed that cell surface expression of PAR-1 was reduced by laminar flow, suggesting that PAR-1 was activated in response to laminar flow. Depletion of PAR-1 using human PAR-1 siRNA inhibited unidirectional laminar flow-mediated actin stress fiber formation and cellular alignment as well as atheroprotective gene expressions in HUVECs. Moreover, PAR-1 knockdown inhibited laminar flow-stimulated eNOS phosphorylation, and inhibited the phosphorylations of Src, AMPK, ERK5 and HDAC5. Furthermore, PAR-1 depletion inhibited laminar flow-mediated anti-inflammatory responses as demonstrated by reduced TNFα-induced VCAM-1 expression and by monocyte adhesion to HUVECs, and prevented laminar flow-mediated anti-apoptotic response. An investigation of the role of PAR-1 in vasomotor modulation using mouse aortic rings revealed that acetylcholine-induced vasorelaxation was diminished in PAR-1 deficient mice compared to littermate controls. Taken together, these findings suggest that PAR-1 be viewed as a novel pharmacologic target for the treatment of vascular diseases, including atherosclerosis.

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“…As shown in Figure 1A, the NT of the D 2 receptor contains three potential N‐linked glycosylation sites (N 5 , N 17 , N 23 ) and the NT of the D 3 receptor contains two potential N‐linked glycosylation sites (N 12 , N 19 ). Roles of glycosylation in the NT of GPCRs have been reported for their correct expression on the cell surface (He et al ., 2002; Xiao et al ., 2011); hence, acceleration of receptor internalization when the potential glycosylation sites were mutated (Tansky et al ., 2007). As the N‐terminal deletion mutants of D 2 receptors are missing one or two potential glycosylation sites, the involvement of glycosylation in the cell surface expression, internalization and signalling was studied.…”

Section: Resultsmentioning

confidence: 99%

The N‐terminal region of the dopamine D₂ receptor, a rhodopsin‐like GPCR, regulates correct integration into the plasma membrane and endocytic routes

Cho

Min

Jung

et al. 2012

British J Pharmacology

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BACKGROUND AND PURPOSEFunctional roles of the N-terminal region of rhodopsin-like GPCR family remain unclear. Using dopamine D2 and D3 receptors as a model system, we probed the roles of the N-terminal region in the signalling, intracellular trafficking of receptor proteins, and explored the critical factors that determine the functionality of the N-terminal region. EXPERIMENTAL APPROACHThe N-terminal region of the D2 receptor was gradually shortened or switched with that of the D3 receptor or a non-specific sequence (FLAG), or potential N-terminal glycosylation sites were mutated. Effects of these manipulations on surface expression, internalization, post-endocytic behaviours and signalling were determined. KEY RESULTSShortening the N-terminal region of the D2 receptor enhanced receptor internalization and impaired surface expression and signalling; ligand binding, desensitization and down-regulation were not affected but their association with a particular microdomain, caveolae, was disrupted. Replacement of critical residues within the N-terminal region with the FLAG epitope failed to restore surface expression but partially restored the altered internalization and signalling. When the N-terminal regions were switched between D2 and D3 receptors, cell surface expression pattern of each receptor was switched. Mutations of potential N-terminal glycosylation sites inhibited surface expression but enhanced internalization of D2 receptors. CONCLUSIONS AND IMPLICATIONSShortening of N-terminus or mutation of glycosylation sites located within the N-terminus enhanced receptor internalization but impaired the surface expression of D2 receptors. The N-terminal region of the D2 receptor, in a sequence-specific manner, controls the receptor's conformation and integration into the plasma membrane, which determine its subcellular localization, intracellular trafficking and signalling properties.

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N-Linked Glycosylation Regulates Human Proteinase-activated Receptor-1 Cell Surface Expression and Disarming via Neutrophil Proteinases and Thermolysin

Cited by 9 publications

References 35 publications

PR3 and Elastase Alter PAR1 Signaling and Trigger vWF Release via a Calcium-Independent Mechanism from Glomerular Endothelial Cells

PR3 and Elastase Alter PAR1 Signaling and Trigger vWF Release via a Calcium-Independent Mechanism from Glomerular Endothelial Cells

PAR-1 is a novel mechano-sensor transducing laminar flow-mediated endothelial signaling

The N‐terminal region of the dopamine D₂ receptor, a rhodopsin‐like GPCR, regulates correct integration into the plasma membrane and endocytic routes

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N-Linked Glycosylation Regulates Human Proteinase-activated Receptor-1 Cell Surface Expression and Disarming via Neutrophil Proteinases and Thermolysin

Cited by 9 publications

References 35 publications

PR3 and Elastase Alter PAR1 Signaling and Trigger vWF Release via a Calcium-Independent Mechanism from Glomerular Endothelial Cells

PR3 and Elastase Alter PAR1 Signaling and Trigger vWF Release via a Calcium-Independent Mechanism from Glomerular Endothelial Cells

PAR-1 is a novel mechano-sensor transducing laminar flow-mediated endothelial signaling

The N‐terminal region of the dopamine D2 receptor, a rhodopsin‐like GPCR, regulates correct integration into the plasma membrane and endocytic routes

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The N‐terminal region of the dopamine D₂ receptor, a rhodopsin‐like GPCR, regulates correct integration into the plasma membrane and endocytic routes