Amino Acid Residues Critical for Endoplasmic Reticulum Export and Trafficking of Platelet-activating Factor Receptor
Amino Acid Residues Critical for Endoplasmic Reticulum Export and Trafficking of Platelet-activating Factor Receptor
复制标题
DOI：
10.1074/jbc.m109.066282
复制
发表时间：
2010-02-19
影响因子：
4.8
通讯作者：
Nakamura, Motonao
中科院分区：
文献类型：
--
作者：
Hirota, Nobuaki;Yasuda, Daisuke;Nakamura, Motonao
在G蛋白偶联受体的跨膜结构域（TM）中有几个残基是保守的。在这里，我们证明了一个保守的脯氨酸，Pro（247），在血小板活化因子受体（PAFR）的TM 6是必需的内质网（ER）的出口和运输后激动剂诱导的内化。PAFR保守残基的丙氨酸取代突变体，包括P247 A，保留在ER中。因为PAFR拮抗剂Y-24180作为药理学伴侣来拯救ER滞留，所以这种滞留是由于PAFR的错误折叠。甲基氨基甲酰（mc）-PAF，PAFR激动剂，并没有增加细胞表面的P247 A的表达，即使另一个ER保留的突变体，D 63 A，有效地贩运。在mc-PAF处理的P247 A表达细胞中观察到早期内体中受体的信号传导和积累，表明P247 A被mc-PAF运输到细胞表面，然后由于异常运输而从表面消失，例如增强的内化、再循环缺陷和/或加速降解。用分选酶A介导的标记细胞表面蛋白的方法证实了异常运输。这些结果表明，保守的脯氨酸TM 6是至关重要的细胞内运输的PAFR。
Several residues are conserved in the transmembrane domains (TMs) of G-protein coupled receptors. Here we demonstrate that a conserved proline, Pro(247), in TM6 of platelet-activating factor receptor (PAFR) is required for endoplasmic reticulum (ER) export and trafficking after agonist-induced internalization. Alanine-substituted mutants of the conserved residues of PAFRs, including P247A, were retained in the ER. Because a PAFR antagonist, Y-24180, acted as a pharmacological chaperone to rescue ER retention, this retention is due to misfolding of PAFR. Methylcarbamyl (mc)-PAF, a PAFR agonist, did not increase the cell surface expression of P247A, even though another ER-retained mutant, D63A, was effectively trafficked. Signaling and accumulation of the receptors in the early endosomes were observed in the mc-PAF-treated P247A-expressing cells, suggesting that P247A was trafficked to the cell surface by mc-PAF, and thereafter disappeared from the surface due to aberrant trafficking, e.g. enhanced internalization, deficiency in recycling, and/or accelerated degradation. The aberrant trafficking was confirmed with a sortase-A-mediated method for labeling cell surface proteins. These results demonstrate that the conserved proline in TM6 is crucial for intracellular trafficking of PAFR.