N-Linked Glycosylation of Protease-activated Receptor-1 Second Extracellular Loop: A CRITICAL DETERMINANT FOR LIGAND-INDUCED RECEPTOR ACTIVATION AND INTERNALIZATION

Antonio G Soto,Joann Trejo

doi:10.1074/jbc.m110.111088

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, thrombin-activated 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.

Highlights

Thrombin is a coagulant serine protease generated at sites of vascular injury and in thrombotic disease
This study reveals for the first time distinct functions for Protease-activated receptor-1 (PAR1) N-linked glycosylation at the N terminus versus ECL2 that are critical for the regulation of PAR1 signaling and trafficking
We have identified the major sites of PAR1 N-linked glycosylation and defined a function for this posttranslational modification in the regulation of receptor activation and trafficking

Summary

EXPERIMENTAL PROCEDURES

Reagents and Antibodies—Human ␣-thrombin was obtained from Enzyme Research Laboratories (South Bend, IN). PAR1 Immunoprecipitation and Immunoblotting—HeLa cells stably expressing FLAG-tagged PAR1 wild type or mutants were plated at 5 ϫ 105 cells per well in 6-well culture dishes and grown overnight at 37 °C. Thrombin Cold Cleavage Assay—HeLa cells expressing FLAG-tagged PAR1 wild type or mutants were plated at 5 ϫ 105 cells per well in 6-well culture dishes and grown overnight, washed with serum-free DMEM, and placed on ice for 30 min. Cells were incubated with cold DMEM with or without 30 nM thrombin, and incubated for 15 min on ice. Cells were lysed, and PAR1 was immunoprecipitated with M2 anti-FLAG antibody and immunoblotted as described above. Phosphoinositide Hydrolysis—HeLa cells stably expressing FLAG-tagged PAR1 wild type or mutants were plated at 1 ϫ 105 cells per well in a fibronectin-coated 24-well culture dish and grown overnight at 37 °C.

RESULTS

Findings

DISCUSSION