Open AccessRole of the Helix-8 and C-Terminal Tail in Regulating Proteinase Activated Receptor 2 Signaling
Author(s) -
Pierre E. Thibeault,
Rithwik Ramachandran
Publication year - 2020
Publication title -
acs pharmacology and translational science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.271
H-Index - 10
ISSN - 2575-9108
DOI - 10.1021/acsptsci.0c00039
Subject(s) - g protein coupled receptor , arrestin , phosphorylation , microbiology and biotechnology , serine , biology , receptor , alanine , signal transduction , protease activated receptor , biochemistry , chemistry , amino acid , thrombin , platelet , immunology
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 Gα q/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-NH 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-NH 2 - or trypsin-stimulated β-arrestin recruitment. In contrast, alanine substitutions in the Ser 387 -Thr 392 cluster resulted in a large (∼50%) decrease in β-arrestin-1/-2 recruitment triggered by the activating peptide, SLIGRL-NH 2 , 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 (Cys 361 Ala) led to a large decrease in both Gα q/11 coupling and β-arrestin-1/-2 recruitment to PAR2. Furthermore, we show that Gα q/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 Gα q/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.