The role of palmitoylation in signalling, cellular trafficking and plasma membrane localization of protease-activated receptor-2.
The role of palmitoylation in signalling, cellular trafficking and plasma membrane localization of protease-activated receptor-2.
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棕榈酰化在蛋白酶激活受体-2的信号传导，细胞运输和质膜定位中的作用。
DOI：
10.1371/journal.pone.0028018
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Hooper JD
中科院分区：
文献类型：
--
作者：
Adams MN;Christensen ME;He Y;Waterhouse NJ;Hooper JD
蛋白水解酶激活受体-2(PAR2)是一种G蛋白偶联受体(GPCR)，其氨基末端结构域被类胰酶丝氨酸蛋白酶酶解激活。这种不可逆的激活机制通过内化和降解导致受体快速脱敏。我们已经探索了棕榈酸酯的翻译后加成棕榈酰化在PAR2信号转导、运输、细胞表面表达和脱敏中的作用。使用棕榈酰化抑制剂2-溴棕榈酸酯的实验表明，棕榈酸酯加成在前列腺癌细胞株内源性表达的PAR2的运输中起着重要作用。使用两种方法的棕榈酸酯标记支持了这一点，这表明CHO-K1细胞稳定表达的PAR2是棕榈酰化的，并且棕榈酰化发生在半胱氨酸361上。棕榈酰化是最佳的PAR2信号转导所必需的，因为钙离子通量分析表明，棕榈酰化缺陷的PAR2对胰酶激动剂的反应比野生型受体的∼低9倍，通过突变受体诱导的最大信号减少约33%。共聚焦显微镜、流式细胞仪和细胞表面生物素化分析表明，细胞表面有效表达PAR2需要棕榈酰化。我们还表明，受体棕榈酰化发生在高尔基体内，并需要有效的激动剂诱导的Rab11a介导的PAR2到细胞表面的运输。受体脱敏也需要棕榈酰化，因为激动剂诱导的β-arrestin募集以及受体内吞和降解在CHO-PAR2-C361a细胞中显著减少。这些数据为PAR2的生命周期提供了新的见解，并表明棕榈酰化对于有效的信号传递、运输、细胞表面定位和该受体的降解至关重要。
Protease-activated receptor-2 (PAR2) is a G protein coupled receptor (GPCR) activated by proteolytic cleavage of its amino terminal domain by trypsin-like serine proteases. This irreversible activation mechanism leads to rapid receptor desensitization by internalisation and degradation. We have explored the role of palmitoylation, the post-translational addition of palmitate, in PAR2 signalling, trafficking, cell surface expression and desensitization. Experiments using the palmitoylation inhibitor 2-bromopalmitate indicated that palmitate addition is important in trafficking of PAR2 endogenously expressed by prostate cancer cell lines. This was supported by palmitate labelling using two approaches, which showed that PAR2 stably expressed by CHO-K1 cells is palmitoylated and that palmitoylation occurs on cysteine 361. Palmitoylation is required for optimal PAR2 signalling as Ca2+ flux assays indicated that in response to trypsin agonism, palmitoylation deficient PAR2 is ∼9 fold less potent than wildtype receptor with a reduction of about 33% in the maximum signal induced via the mutant receptor. Confocal microscopy, flow cytometry and cell surface biotinylation analyses demonstrated that palmitoylation is required for efficient cell surface expression of PAR2. We also show that receptor palmitoylation occurs within the Golgi apparatus and is required for efficient agonist-induced rab11a-mediated trafficking of PAR2 to the cell surface. Palmitoylation is also required for receptor desensitization, as agonist-induced β-arrestin recruitment and receptor endocytosis and degradation were markedly reduced in CHO-PAR2-C361A cells compared with CHO-PAR2 cells. These data provide new insights on the life cycle of PAR2 and demonstrate that palmitoylation is critical for efficient signalling, trafficking, cell surface localization and degradation of this receptor.
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