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.

Sphingosine 1-phosphate elicits RhoA-dependent proliferation and MRTF-A mediated gene induction in CPCs

“…This finding is consistent with other evidence that CPCs do not fully differentiate into functional cardiomyocytes in culture . We also noted that mRNAs for LPAR 1 and LPAR 2 subtypes, and for the thrombin receptor PAR1, all of which can activate RhoA [51–53], were highly expressed in mouse CPCs (Fig. S.I.1A).…”

N-Glycosylation of Asparagine 130 in the Extracellular Domain of the Human Calcitonin Receptor Significantly Increases Peptide Hormone Affinity

“…It thus remains to be determined if CTR is unique or if there are other class B GPCRs for which their N-glycans modulate ligand binding and/or signaling. Notably, it was recently demonstrated that N-glycans in the class A GPCR PAR1 regulate its G-protein signaling bias 48 . Clearly GPCR N-glycans can modulate their pharmacology and it will be important to determine the structural basis of these effects.…”

New paradigms in chemokine receptor signal transduction: Moving beyond the two-site model

“…Dynamic interactions between ECL and TM residues may act as molecular switches regulating receptor activation, especially in the case of ECL2. While ECL2 displays high variability in sequence, length, and structure even among related receptor subtypes, an impressive number of family A receptors seem to utilize ECL2 in this capacity, including rhodopsin [109–112], the serotonin 5-HT4 receptor [113], the V(1a) vasopressin receptor (V1aR) [114], the cannabinoid receptor 1 (CB1) [115–117], the β 2 AR [118], the angiotensin II type 1a receptor (ATII1aR) [119, 120], the D2 dopamine receptor (D2R) [121], the C5a complement receptor (C5aR) [122], and protease activated receptor 1 (PAR-1) [123], among others [124, 125]. Considering CXCR4, we recently suggested that manipulation of ECL2 by CXCL12 draws ECL2 toward the orthosteric pocket, thereby moving TMs 2 and 3 closer to one another [13].…”