Orita S, Sasaki T, Komuro R, Sakaguchi G, Maeda M, Igarashi H, Takai Y
Shionogi Institute for Medical Science, Japan.
J Biol Chem. 1996 Mar 29;271(13):7257-60. doi: 10.1074/jbc.271.13.7257.
We previously isolated a new protein having two C2-like domains which interacted with Ca2+ and phospholipid and named Doc2 (Double C2). Because Doc2 was abundantly expressed in brain where it was highly concentrated on the synaptic vesicle fraction, we have examined here whether Doc2 is involved in Ca2+-dependent exocytosis from cultured PC12 cells. For this purpose, we took advantage of the growth hormone (GH) co-expression assay system of PC12 cells in which GH is stored in dense core vesicles and released in response to high K+ in an extracellular Ca2+-dependent manner. Northern and Western blot analyses indicated that Doc2 is present in PC12 cells. Overexpression of hemagglutinin-tagged Doc2 stimulated the Ca2+-dependent, high K+-induced release of co-expressed GH without affecting the basal release. In the PC12 cells transfected with a plasmid with the coding sequence of Doc2 in the antisense orientation, the high K+-induced release of co-expressed GH was inversely inhibited. The Doc2 mutant expressing an N-terminal fragment or a C-terminal fragment containing two C2-like domains inhibited the high K+-induced release of co-expressed GH. These results indicate that Doc2 enhances Ca2+-dependent exocytosis of dense core vesicles from PC12 cells.
我们之前分离出一种新的蛋白质，它具有两个与Ca2+和磷脂相互作用的C2样结构域，并将其命名为Doc2（双C2）。由于Doc2在大脑中大量表达，且高度集中在突触小泡组分上，我们在此研究了Doc2是否参与培养的PC12细胞中Ca2+依赖的胞吐作用。为此，我们利用了PC12细胞的生长激素（GH）共表达检测系统，在该系统中，GH储存在致密核心小泡中，并以细胞外Ca2+依赖的方式响应高K+而释放。Northern和Western印迹分析表明Doc2存在于PC12细胞中。血凝素标记的Doc2的过表达刺激了共表达的GH的Ca2+依赖的、高K+诱导的释放，而不影响基础释放。在用反义方向的Doc2编码序列质粒转染的PC12细胞中，高K+诱导的共表达GH的释放受到反向抑制。表达含有两个C2样结构域的N端片段或C端片段的Doc2突变体抑制了高K+诱导的共表达GH的释放。这些结果表明，Doc2增强了PC12细胞中致密核心小泡的Ca2+依赖的胞吐作用。
