Sadakata Tetsushi, Washida Miwa, Furuichi Teiichi
Laboratory for Molecular Neurogenesis, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako, Saitama, Japan.
BMC Neurosci. 2007 Apr 12;8:25. doi: 10.1186/1471-2202-8-25.
Ca2+-dependent activator protein 2 (CAPS2/CADPS2) is a secretory vesicle-associated protein involved in the release of neurotrophin. We recently reported that an aberrant, alternatively spliced CAPS2 mRNA that lacks exon 3 (CAPS2Deltaexon3) is detected in some patients with autism. Splicing variations in mouse CAPS2 and their expression and functions remain unclear.
In this study, we defined 31 exons in the mouse CAPS2 gene and identified six alternative splicing variants, CAPS2a-f. CAPS2a is an isoform lacking exons 22 and 25, which encode part of the Munc13-1-homologous domain (MHD). CAPS2b lacks exon 25. CAPS2c lacks exons 11 and 22. CAPS2d, 2e, and 2f have C-terminal deletions from exon 14, exon 12, and exon 5, respectively. On the other hand, a mouse counterpart of CAPS2Deltaexon3 was not detected in the mouse tissues tested. CAPS2b was expressed exclusively in the brain, and the other isoforms were highly expressed in the brain, but also in some non-neural tissues. In the brain, all isoforms showed predominant expression patterns in the cerebellum. In the developing cerebellum, CAPS2b showed an up-regulated expression pattern, whereas the other isoforms exhibited transiently peaked expression patterns. CAPS2 proteins were mostly recovered in soluble fractions, but some were present in membrane fractions, except for CAPS2c and 2f, both of which lack the PH domain, suggesting that the PH domain is important for membrane association. In contrast to CAPS2a and 2b, CAPS2c showed slightly decreased BDNF-releasing activity, which is likely due to the C-terminal truncation of the PH domain in CAPS2c.
This study indicates that, in mouse, there are six splicing variants of CAPS2 (CAPS2a-f), and that these are subdivided into two groups: a long form containing the C-terminal MHD and a short form lacking the C-terminal MHD. These results demonstrate that the splicing variations correlate with their expression patterns and intracellular distribution, and affect BDNF release; however, whether or not the short forms possess activities other than BDNF release, for example as natural dominant-negative isoforms, remains to be determined.
钙离子依赖激活蛋白2（CAPS2/CADPS2）是一种与分泌囊泡相关的蛋白，参与神经营养因子的释放。我们最近报道，在一些自闭症患者中检测到一种异常的、缺失外显子3的可变剪接CAPS2 mRNA（CAPS2Deltaexon3）。小鼠CAPS2的剪接变异及其表达和功能仍不清楚。
在本研究中，我们确定了小鼠CAPS2基因中的31个外显子，并鉴定出6种可变剪接变体，即CAPS2a-f。CAPS2a是一种缺失外显子22和25的异构体，这两个外显子编码Munc13-1同源结构域（MHD）的一部分。CAPS2b缺失外显子25。CAPS2c缺失外显子11和22。CAPS2d、2e和2f分别从外显子14、外显子12和外显子5开始有C端缺失。另一方面，在测试的小鼠组织中未检测到CAPS2Deltaexon3的小鼠对应物。CAPS2b仅在脑中表达，其他异构体在脑中高表达，但在一些非神经组织中也有表达。在脑中，所有异构体在小脑中均表现出主要的表达模式。在发育中的小脑中，CAPS2b表现出上调的表达模式，而其他异构体则表现出瞬时峰值表达模式。CAPS2蛋白大多在可溶性组分中回收，但除了缺乏PH结构域的CAPS2c和2f外，一些也存在于膜组分中，这表明PH结构域对膜结合很重要。与CAPS2a和2b相比，CAPS2c的脑源性神经营养因子（BDNF）释放活性略有降低，这可能是由于CAPS2c中PH结构域的C端截短所致。
本研究表明，在小鼠中，CAPS2有6种剪接变体（CAPS2a-f），这些变体可分为两组：一种是含有C端MHD的长形式，另一种是缺乏C端MHD的短形式。这些结果表明，剪接变异与其表达模式和细胞内分布相关，并影响BDNF释放；然而，这些短形式是否具有除BDNF释放之外的其他活性，例如作为天然显性负性异构体，仍有待确定。
