Role of the C-terminal tail in regulating Proteinase Activated Receptor 2 (PAR2) signalling

Abstract

ABSTRACTThe C-terminal tail of G-protein-coupled receptors contain important regulatory sites that enable interaction with intracellular signalling 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 signalling regulation downstream of the proteolytically-activated GPCR, PAR2. We examined Gαq/11-coupled calcium signalling, 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. 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. Alanine substitutions in the Ser387-Thr392cluster resulted in a large (∼50%) decrease in β-arrestin-1/2 recruitment triggered by the activating peptide, SLIGRL-NH2,but was without 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 (∼50%) decrease in β-arrestin-1/-2 recruitment in response to both trypsin and SLIGRL-NH2. We further show that Gαq/11inhibition 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 signalling. In line with these findings, C-tail serine/threonine and cysteine residue mutants that have decreased β-arrestin recruitment also showed enhanced ERK activation. Thus, our studies point to multiple mechanisms that regulate β-arrestin interaction with PAR2 to regulate receptor-mediated signalling.