Bone and Joint Institute


Role of the Helix-8 and C-Terminal Tail in Regulating Proteinase Activated Receptor 2 Signaling

Document Type


Publication Date



ACS Pharmacol. Transl. Sci.





First Page


Last Page


URL with Digital Object Identifier


The C-terminal tail of G-protein-coupled receptors (GPCR) contain important regulatory sites that enable interaction with intracellular signaling effectors. Here we examine the relative contribution of the C-tail serine/threonine phosphorylation sites (Ser383–385, Ser387–Thr392) and the helix-8 palmitoylation site (Cys361) in signaling regulation downstream of the proteolytically activated GPCR, PAR2. We examined Gαq/11-coupled calcium signaling, β-arrestin-1/-2 recruitment, and MAPK activation (p44/42 phosphorylation) by wild-type and mutant receptors expressed in a CRISPR/Cas9 PAR2-knockout HEK-293 cell background with both peptide stimulation of the receptor (SLIGRL-NH2) as well as activation with its endogenous trypsin revealed a tethered ligand. We find that alanine substitution of the membrane proximal serine residues (Ser383–385Ala) had no effect on SLIGRL-NH2- or trypsin-stimulated β-arrestin recruitment. In contrast, alanine substitutions in the Ser387–Thr392 cluster resulted in a large (∼50%) decrease in β-arrestin-1/-2 recruitment triggered by the activating peptide, SLIGRL-NH2, but was without an effect on trypsin-activated β-arrestin-1/-2 recruitment. Additionally, we find that alanine substitution of the helix-8 cysteine residue (Cys361Ala) led to a large decrease in both Gαq/11 coupling and β-arrestin-1/-2 recruitment to PAR2. Furthermore, we show that Gαq/11 inhibition with YM254890, inhibited ERK phosphorylation by PAR2 agonists, while genetic deletion of β-arrestin-1/-2 by CRISPR/Cas9 enhanced MAPK activation. Knockout of β-arrestins also enhanced Gαq/11-mediated calcium signaling. In line with these findings, a C-tail serine/threonine mutant that has decreased β-arrestin recruitment also showed enhanced ERK activation. Thus, our studies point to multiple mechanisms that regulate β-arrestin interaction with PAR2 and highlight differences in regulation of tethered-ligand- and peptide-mediated activation of this receptor.

Citation of this paper:

Role of the Helix-8 and C-Terminal Tail in Regulating Proteinase Activated Receptor 2 Signaling Pierre E. Thibeault and Rithwik Ramachandran.Pierre E. Thibeault and Rithwik Ramachandran

ACS Pharmacol. Transl. Sci. 2020, 3, 5, 868–882 Publication Date:August 10, 2020 Copyright © 2020 American Chemical Society