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

Abstract

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.

WITHDRAWN:Staphylococcus aureus-serineprotease-like protein B (SplB) activates PAR2 and induces endothelial barrier dysfunction

“…As expected, neither trypsin nor SplB was able to induce ERK phosphorylation in cells lacking PAR2 ( Figure 2E). In recent work, we demonstrated that PAR2-mediated ERK phosphorylation in HEK293 cells occurs downstream of Gα q/11 signaling (56), and this is consistent with the observations that SplB stimulation of PAR2 does not induce Gα q/11 signaling.…”

Molecular mechanisms regulating Proteinase‐Activated Receptors (PARs)

“…In keeping with other Rhodopsin family GPCRs, a highly conserved amphipathic H8 [4,48] is seen within the PAR C-terminal domain (Figure 1), though this structure was not resolved in the PAR1 crystal structure. In PAR1 and PAR2, the H8 is critically stabilized through anchoring to the plasma membrane by palmitoylation of the C-terminal cysteine residues [49][50][51][52] as seen in a large number of other GPCRs [53,54]. PAR3 has an unusually short C-terminal tail (13 amino acids), while the PAR4…”

“…Similar interactions appear to be involved in PAR activation of -arrestins and G proteins. Destabilization of the H8 by mutation of cysteine and other amino acids in PAR1 [49], PAR2 [52] and PAR4 [55] to alanine negatively impact G q/11 coupling, and in the case of PAR2 and PAR4, -arrestin-1 and -2 recruitment. Additional intracellular interactions are also likely for PAR interaction with G proteins and -arrestins.…”

“…The C-terminal GPCR kinase (GRK) phosphorylated tail of GPCRs is well established as a site for initial -arrestin interactions [63,64], and such serine and threonine GRK phosphorylation sites are present in PAR1, PAR2 and PAR4 (Figure 1) [65]. Mutation of serine and threonine clusters impacts desensitization and trafficking of PAR1 [66,67] and PAR2 [52,68,69]. Activated PAR1 is rapidly phosphorylated on sites within the receptor C-terminus by a number of different GRKs [70,71] and desensitized by β-arrestin-1 recruitment [72].…”

“…1), though this structure was not resolved in the PAR1 crystal structure. In PAR1 and PAR2, the H8 is critically stabilized through anchoring to the plasma membrane by palmitoylation of the C-terminal cysteine residues [48][49][50][51] as seen in a large number of other GPCRs [52,53]. PAR3 has an unusually short C-terminal tail (13 amino acids), while the PAR4 C-tail lacks the H8 cysteine residue but does possess an H8 motif that regulates receptor-mediated signaling [54].…”