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, Ser³⁸⁷–Thr³⁹²) and the helix-8 palmitoylation site (Cys³⁶¹) 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-NH₂) as well as activation with its endogenous trypsin revealed a tethered ligand. We find that alanine substitution of the membrane proximal serine residues (Ser^383–385Ala) 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 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.

中文翻译：

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Helix-8 和 C 末端尾部在调节蛋白酶激活受体 2 信号传导中的作用

G 蛋白偶联受体 (GPCR) 的 C 末端尾部包含重要的调节位点，可以与细胞内信号传导效应器相互作用。在这里，我们检查了 C 尾丝氨酸/苏氨酸磷酸化位点（Ser ^383-385、Ser ^387- Thr ³⁹²）和 helix-8 棕榈酰化位点（Cys ³⁶¹）在蛋白水解激活的 GPCR、PAR2 下游信号调节中的相对贡献. 我们检查了 CRISPR/Cas9 PAR2 敲除 HEK-293 细胞中表达的野生型和突变受体的Gα _q/11偶联钙信号传导、β-arrestin-1/-2 募集和 MAPK 激活（p44/42 磷酸化）两种肽刺激受体的背景（SLIGRL-NH ₂) 以及用其内源性胰蛋白酶激活揭示了一个系留配体。我们发现膜近端丝氨酸残基（Ser ^383-385 Ala）的丙氨酸取代对 SLIGRL-NH ₂ - 或胰蛋白酶刺激的 β-抑制蛋白募集没有影响。相比之下，Ser ³⁸⁷ –Thr ³⁹²簇中的丙氨酸取代导致激活肽 SLIGRL-NH ₂触发的 β-arrestin-1/-2 募集大量减少（~50%），但对胰蛋白酶激活的 β-arrestin-1/-2 募集。此外，我们发现 helix-8 半胱氨酸残基（Cys ³⁶¹ Ala）的丙氨酸取代导致 Gα _q/11偶联和 β-arrestin-1/-2 募集到 PAR2。此外，我们表明YM254890 抑制Gα _q/11，抑制了 PAR2 激动剂的 ERK 磷酸化，而 CRISPR/Cas9 基因缺失 β-arrestin-1/-2 增强了 MAPK 激活。敲除 β-抑制蛋白也增强了 Gα _q/11介导的钙信号传导。与这些发现一致，降低 β-抑制蛋白募集的 C 尾丝氨酸/苏氨酸突变体也显示出增强的 ERK 激活。因此，我们的研究指出了调节 β-抑制蛋白与 PAR2 相互作用的多种机制，并突出了在调节系留配体和肽介导的该受体激活方面的差异。