Dissection of Thrombospondin-4 Domains Involved in Intracellular Adaptive Endoplasmic Reticulum Stress-Responsive Signaling
Dissection of Thrombospondin-4 Domains Involved in Intracellular Adaptive Endoplasmic Reticulum Stress-Responsive Signaling
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DOI：
10.1128/mcb.00607-15
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发表时间：
2016-01-01
影响因子：
5.3
通讯作者：
Molkentin, Jeffery D.
中科院分区：
文献类型：
--
作者：
Brody, Matthew J.;Schips, Tobias G.;Molkentin, Jeffery D.
血小板反应蛋白是一个应激诱导的分泌性糖蛋白家族，是组织重塑的基础。我们最近报道了血小板反应蛋白-4（Thbs 4）具有关键的细胞内功能，通过激活转录因子6 α（Atf 6 α）调节适应性内质网（ER）应激途径。在本研究中，我们解剖了Thbs 4的结构域，介导与ER蛋白，如BiP（Grp 78）和Atf 6 α的相互作用，以及介导ER应激反应激活的结构域。在功能上，Thbs 4定位于ER和ER后囊泡，并从心肌细胞中主动分泌，III型重复序列（T3 R）和TSP-C结构域也是如此，而LamG结构域定位于高尔基体。我们还突变了全长Thbs 4的T3 R结构域内的主要钙结合基序，导致ER滞留和分泌阻断。T3 R和TSP-C结构域以及野生型Thbs 4和钙结合突变体与Atf 6 α相互作用，诱导适应性ER应激反应，并引起细胞内囊泡扩张。相反，相关的分泌性寡聚糖蛋白Nell 2的过表达，它只缺乏T3 R和TSP-C结构域，没有引起这些影响。最后，Atf 6 α的缺失废除了Thbs 4诱导的囊泡扩张。综上所述，这些数据确定了Thbs 4的关键细胞内功能结构域，其以前被认为仅具有细胞外功能。
Thrombospondins are a family of stress-inducible secreted glycoproteins that underlie tissue remodeling. We recently reported that thrombospondin-4 (Thbs4) has a critical intracellular function, regulating the adaptive endoplasmic reticulum (ER) stress pathway through activating transcription factor 6 alpha (Atf6 alpha). In the present study, we dissected the domains of Thbs4 that mediate interactions with ER proteins, such as BiP (Grp78) and Atf6 alpha, and the domains mediating activation of the ER stress response. Functionally, Thbs4 localized to the ER and post-ER vesicles and was actively secreted from cardiomyocytes, as were the type III repeat (T3R) and TSP-C domains, while the LamG domain localized to the Golgi apparatus. We also mutated the major calcium-binding motifs within the T3R domain of full-length Thbs4, causing ER retention and secretion blockade. The T3R and TSP-C domains as well as wild-type Thbs4 and the calcium-binding mutant interacted with Atf6 alpha, induced an adaptive ER stress response, and caused expansion of intracellular vesicles. In contrast, overexpression of a related secreted oligomeric glycoprotein, Nell2, which lacks only the T3R and TSP-C domains, did not cause these effects. Finally, deletion of Atf6 alpha abrogated Thbs4-induced vesicular expansion. Taken together, these data identify the critical intracellular functional domains of Thbs4, which was formerly thought to have only extracellular functions.