Role Of The Helix-8 And C-Terminal Tail In Regulating Proteinase Activated Receptor 2 Signaling

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 (Ser(383-385), Ser(387)-Thr(392)) and the helix-8 palmitoylation site (Cys(361)) in signaling regulation downstream of the proteolytically activated GPCR, PAR2. We examined G alpha(q/11)-coupled calcium signaling, beta 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 (SLIGRLNH(2)) as well as activation with its endogenous trypsin revealed a tethered ligand. We find that alanine substitution of the membrane proximal serine residues (Ser(383-385)Ala) had no effect on SLIGRL-NH2- or trypsin-stimulated beta-arrestin recruitment. In contrast, alanine substitutions in the Ser387-Thr392 cluster resulted in a large (similar to 50%) decrease in beta-arrestin-1/-2 recruitment triggered by the activating peptide, SLIGRL-NH2, but was without an effect on trypsin-activated beta-arrestin-1/-2 recruitment. Additionally, we find that alanine substitution of the helix-8 cysteine residue (Cys(361)Ala) led to a large decrease in both G alpha(q/11) coupling and beta-arrestin-1/-2 recruitment to PAR2. Furthermore, we show that Gaq/ 11 inhibition with YM254890, inhibited ERK phosphorylation by PAR2 agonists, while genetic deletion of beta-arrestin-1/-2 by CRISPR/Cas9 enhanced MAPK activation. Knockout of beta-arrestins also enhanced Gaq/11-mediated calcium signaling. In line with these findings, a C-tail serine/threonine mutant that has decreased beta-arrestin recruitment also showed enhanced ERK activation. Thus, our studies point to multiple mechanisms that regulate beta-arrestin interaction with PAR2 and highlight differences in regulation of tethered-ligand- and peptide-mediated activation of this receptor.