N-Linked Glycosylation of Protease-activated Receptor-1 Second Extracellular Loop

Abstract

Protease-activated receptor-1 (PAR1) contains five N-linked glycosylation consensus sites as follows: three residing in the N terminus and two localized on the surface of the second extracellular loop (ECL2). To study the effect of N-linked glycosylation in the regulation of PAR1 signaling and trafficking, we generated mutants in which the critical asparagines of the consensus sites were mutated. Here, we report that both the PAR1 N terminus and ECL2 serve as sites for N-linked glycosylation but have different functions in the regulation of receptor signaling and trafficking. N-Linked glycosylation of the PAR1 N terminus is important for transport to the cell surface, whereas the PAR1 mutant lacking glycosylation at ECL2 (NA ECL2) trafficked to the cell surface like the wild-type receptor. However, activated PAR1 NA ECL2 mutant internalization was impaired compared with wild-type receptor, whereas constitutive internalization of unactivated receptor remained intact. Remarkably, thrombinactivated PAR1 NA ECL2 mutant displayed an enhanced maximal signaling response compared with wild-type receptor. The increased PAR1 NA ECL2 mutant signaling was not due to defects in the ability of thrombin to cleave the receptor or signal termination mechanisms. Rather, the PAR1 NA ECL2 mutant displayed a greater efficacy in thrombin-stimulated G protein signaling. Thus, N-linked glycosylation of the PAR1 extracellular surface likely influences ligand docking interactions and the stability of the active receptor conformation. Together, these studies strongly suggest that N-linked glycosylation of PAR1 at the N terminus versus the surface of ECL2 serves distinct functions critical for proper regulation of receptor trafficking and the fidelity of thrombin signaling.

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

“…Interestingly, unlike in previous studies in HeLa (24), there is still a very high level of cell surface expression of hPAR 1 when removing all glycosylation sites on the N terminus. The ECL2 single glycosylation mutant N250Q displayed the lowest level of cell surface expression of hPAR 1 among all of the other single glycosylation mutants; furthermore, removing all glycosylation sequons on ECL2 resulted in similar low levels of receptor cell surface expression to the non-glycosylation mutant, where all glycosylation sequons had been deleted.…”

“…Removal of a single N-terminal glycan resulted in observable receptor cytosolic retention, but removal of all glycosylation sites produced a receptor that could mainly be seen in the cytosol with minimal cell surface expression. Therefore, we conclude that N-linked glycosylation of hPAR 1 at all sites might be required for the optimal receptor cell surface expression (24,25).…”

“…therefore be more important for receptor optimal cell surface expression. Glycosylation on ECL2 of PAR 2 is also important for enhancing receptor expression but not regulating receptor signaling; more importantly, the ECL2 region is extremely sensitive to mutations, and mutating residues in and around the glycosylation sequon, even neutral mutations such as alanine (Ala) in the ECL2 glycosylation sequon, alters receptor structure and function (19,24). The PAR 1 glycosylation study on HeLa cells (24) reported that N-terminal N-linked glycosylation of PAR 1 versus ECL2 N-linked glycosylation is critical for the regulation of PAR 1 signaling and trafficking.…”

The CXC Chemokine Receptor 4 Ligands Ubiquitin and Stromal Cell-derived Factor-1α Function through Distinct Receptor Interactions

“…1A). The multiple bands that were detectable can be likely attributed to the heterogeneity of receptor species that are generated by differential glycosylation in the Golgi apparatus (31). The band at ϳ37 kDa in cells transfected with CXCR7 may represent unprocessed receptor or a proteolytically processed fragment not observed with CXCR4.…”

Activated Protein C N-Linked Glycans Modulate Cytoprotective Signaling Function on Endothelial Cells

“…Glycosylation of membrane signaling receptors and soluble ligands is a common mechanism by which signaling networks can be regulated and can influence multiple aspects of signal transduction, including ligand recognition and affinity (29), intracellular trafficking, and receptor activation (30,31). In this study, the role of N-linked glycosylation of APC in modulating EPCR-dependent APC endothelial cell signaling via PAR1 was examined.…”