title: N-linked glycosylation of protease-activated receptor-1 at extracellular loop 2 regulates G-protein signaling bias.
abstract: Protease-activated receptor-1 (PAR1) is a G-protein-coupled receptor (GPCR) for the coagulant protease thrombin. Similar to other GPCRs, PAR1 is promiscuous and couples to multiple heterotrimeric G-protein subtypes in the same cell and promotes diverse cellular responses. The molecular mechanism by which activation of a given GPCR with the same ligand permits coupling to multiple G-protein subtypes is unclear. Here, we report that N-linked glycosylation of PAR1 at extracellular loop 2 (ECL2) controls G12/13 versus Gq coupling specificity in response to thrombin stimulation. A PAR1 mutant deficient in glycosylation at ECL2 was more effective at stimulating Gq-mediated phosphoinositide signaling compared with glycosylated wildtype receptor. In contrast, wildtype PAR1 displayed a greater efficacy at G12/13-dependent RhoA activation compared with mutant receptor lacking glycosylation at ECL2. Endogenous PAR1 rendered deficient in glycosylation using tunicamycin, a glycoprotein synthesis inhibitor, also exhibited increased PI signaling and diminished RhoA activation opposite to native receptor. Remarkably, PAR1 wildtype and glycosylation-deficient mutant were equally effective at coupling to Gi and β-arrestin-1. Consistent with preferential G12/13 coupling, thrombin-stimulated PAR1 wildtype strongly induced RhoA-mediated stress fiber formation compared with mutant receptor. In striking contrast, glycosylation-deficient PAR1 was more effective at increasing cellular proliferation, associated with Gq signaling, than wildtype receptor. These studies suggest that N-linked glycosylation at ECL2 contributes to the stabilization of an active PAR1 state that preferentially couples to G12/13 versus Gq and defines a previously unidentified function for N-linked glycosylation of GPCRs in regulating G-protein signaling bias.
authors.affiliation: Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, CA 92093; Biomedical Sciences Graduate Program, School of Medicine, University of California, San Diego, La Jolla, CA 92093;
authors.forename: Antonio G
authors.initials: AG
authors.lastname: Soto
authors.affiliation: Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, CA 92093; Biomedical Sciences Graduate Program, School of Medicine, University of California, San Diego, La Jolla, CA 92093;
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Smith
authors.affiliation: Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, CA 92093;
authors.forename: Buxin
authors.initials: B
authors.lastname: Chen
authors.affiliation: Department of Immunology, Beckman Research Institute of the City of Hope, Duarte, CA 91010;
authors.forename: Supriyo
authors.initials: S
authors.lastname: Bhattacharya
authors.affiliation: Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, CA 92093; Biomedical Sciences Graduate Program, School of Medicine, University of California, San Diego, La Jolla, CA 92093;
authors.forename: Isabel Canto
authors.initials: IC
authors.lastname: Cordova
authors.affiliation: Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill, NC 27599.
authors.forename: Terry
authors.initials: T
authors.lastname: Kenakin
authors.affiliation: Department of Immunology, Beckman Research Institute of the City of Hope, Duarte, CA 91010;
authors.forename: Nagarajan
authors.initials: N
authors.lastname: Vaidehi
authors.affiliation: Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, CA 92093; joanntrejo@ucsd.edu.
authors.forename: JoAnn
authors.initials: J
authors.lastname: Trejo
mesh_terms: D000465:Algorithms; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D002522:Chlorocebus aethiops; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000235:genetics / Q000378:metabolism*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D018345:Mice, Knockout; D009154:Mutation; D011485:Protein Binding / Q000187:drug effects; D034622:RNA Interference; D044463:Receptor, PAR-1 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013917:Thrombin / Q000494:pharmacology; D013936:Thymidine / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
chemical_list: D044463:Receptor, PAR-1; D013917:Thrombin; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020742:rhoA GTP-Binding Protein; D013936:Thymidine
keywords: GPCR; RhoA; arrestin; endothelial; thrombin