Involvement of Gβγ subunits of Gi protein coupled with S1P receptor on multivesicular endosomes in F-actin formation and cargo sorting into exosomes
Involvement of Gβγ subunits of Gi protein coupled with S1P receptor on multivesicular endosomes in F-actin formation and cargo sorting into exosomes
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DOI：
10.1074/jbc.m117.808733
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发表时间：
2018-01-05
影响因子：
4.8
通讯作者：
Nakamura, Shun-ichi
中科院分区：
文献类型：
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作者：
Kajimoto, Taketoshi;Mohamed, Nesma Nabil Ibrahim;Nakamura, Shun-ichi
外切体通过将货物分子运送到受体细胞，在细胞间的通讯中发挥着关键作用。然而，外体多囊泡内小体(MVE)的发生机制一直是内小体研究领域的谜团之一。尽管神经酰胺和1-磷酸鞘氨醇(S1P)等鞘磷脂代谢物在MVE的形成和成熟中起着重要作用，但其详细的分子机制仍不清楚。在这里，我们发现Rho家族的GTP酶，包括CDC42和rac1，在外体MVEs上被结构性激活，并受S1P信号的调节，通过荧光共振能量转移(FRET)的构象变化来测量。此外，我们还检测到S1P信号诱导的丝状肌动蛋白(F-肌动蛋白)的形成。Gβγ亚基的选择性抑制剂M119强烈地抑制了血管上F-肌动蛋白的形成和血管腔内小泡内货物的分选，这两种作用都可以通过同时表达结构性活性的CDC42和rac1来完全挽救。我们的结果揭示了外体MVE成熟的机制，并有助于理解持续激活S1P受体和随后的下游G蛋白信号与Gβγ亚基/Rho家族GTP酶调节的MVE上F-肌动蛋白形成的生理相关性，从而将货物分类到外体内的小泡中。
Exosomes play a critical role in cell-to-cell communication by delivering cargo molecules to recipient cells. However, the mechanism underlying the generation of the exosomal multivesicular endosome (MVE) is one of the mysteries in the field of endosome research. Although sphingolipid metabolites such as ceramide and sphingosine 1-phosphate (S1P) are known to play important roles in MVE formation and maturation, the detailed molecular mechanisms are still unclear. Here, we show that Rho family GTPases, including Cdc42 and Rac1, are constitutively activated on exosomal MVEs and are regulated by S1P signaling as measured by fluorescence resonance energy transfer (FRET)based conformational changes. Moreover, we detected S1P signaling- induced filamentous actin (F-actin) formation. A selective inhibitor of G beta gamma subunits, M119, strongly inhibited both F-actin formation on MVEs and cargo sorting into exosomal intralumenal vesicles of MVEs, both of which were fully rescued by the simultaneous expression of constitutively active Cdc42 and Rac1. Our results shed light on the mechanism underlying exosomal MVE maturation and inform the understanding of the physiological relevance of continuous activation of the S1P receptor and subsequent downstream G protein signaling to G beta gamma subunits/Rho family GTPases-regulated F-actin formation on MVEs for cargo sorting into exosomal intralumenal vesicles.