Connecdenn, a novel DENN domain-containing protein of neuronal clathrin-coated vesicles functioning in synaptic vesicle endocytosis
Connecdenn, a novel DENN domain-containing protein of neuronal clathrin-coated vesicles functioning in synaptic vesicle endocytosis
复制标题
DOI：
10.1523/jneurosci.4608-06.2006
复制
发表时间：
2006-12-20
影响因子：
5.3
通讯作者：
McPherson, Peter S.
中科院分区：
文献类型：
--
作者：
Allaire, Patrick D.;Ritter, Brigitte;McPherson, Peter S.
网格蛋白包被的囊泡（CCV）负责多种物质的内吞作用，包括突触囊泡膜。我们现在描述了一种新的CCV蛋白，称为connecdenn，它包含一个N端DENN（在肿瘤细胞与正常细胞中差异表达）结构域，一个在多种功能不相关的蛋白中发现的特征不明确的蛋白模块，以及一个C端肽基序结构域，其中包含三个不同的基序，用于结合网格蛋白接头蛋白2（AP-2）的α-耳。 Connecdenn 与 AP-2 共免疫沉淀并部分共定位，核磁共振和肽竞争研究表明所有三个 α-耳结合基序都有助于 AP-2 相互作用。此外，connecdenn 含有多个 Src 同源 3 (SH3) 结构域结合基序，并与突触 SH3 结构域蛋白 intersectin 和内亲素 A1 共免疫沉淀。有趣的是，connecdenn 在神经元 CCV 上富集，并且存在于神经元的突触前区室中。此外，connecdenn 与 CCV 膜具有独特的稳定结合，因为与大多数其他 CCV 蛋白不同，它能抵抗 Tris 和高盐缓冲液的提取，但在纯化的突触小泡上未检测到它。总之，这些观察结果表明 connecdenn 在突触小泡周期的内吞肢体上发挥作用。因此，通过C端肽基序结构域的过度表达来破坏connecdenn与其结合伙伴的相互作用，或者通过慢病毒递送小发夹RNA来敲低connecdenn，都会导致培养的海马神经元中突触小泡内吞作用的缺陷。因此，我们将 connecdenn 鉴定为在突触小泡内吞作用中发挥作用的内吞机制的一个组成部分，这为含 DENN 结构域的蛋白质在内吞作用中的作用提供了第一个证据。
Clathrin- coated vesicles ( CCVs) are responsible for the endocytosis of multiple cargo, including synaptic vesicle membranes. We now describe anewCCVprotein, termed connecdenn, that contains an N- terminalDENN( differentially expressed in neoplastic versus normal cells) domain, a poorly characterized protein module found in multiple proteins of unrelated function and a C- terminal peptide motif domain harboring three distinct motifs for binding the alpha-ear of the clathrin adaptor protein 2 ( AP- 2). Connecdenn coimmunoprecipitates and partially colocalizes with AP- 2, and nuclear magnetic resonance and peptide competition studies reveal that all three alpha-earbinding motifs contribute to AP- 2 interactions. In addition, connecdenn contains multiple Src homology 3 ( SH3) domain- binding motifs and coimmunoprecipitates with the synaptic SH3 domain proteins intersectin and endophilin A1. Interestingly, connecdenn is enriched on neuronal CCVs and is present in the presynaptic compartment of neurons. Moreover, connecdenn has a uniquely stable association with CCV membranes because it resists extraction with Tris and high- salt buffers, unlike most other CCV proteins, but it is not detected on purified synaptic vesicles. Together, these observations suggest that connecdenn functions on the endocytic limb of the synaptic vesicle cycle. Accordingly, disruption of connecdenn interactions with its binding partners through overexpression of the C- terminal peptide motif domain or knock down of connecdenn through lentiviral delivery of small hairpin RNA both lead to defects in synaptic vesicle endocytosis in cultured hippocampal neurons. Thus, we identified connecdenn as a component of the endocytic machinery functioning in synaptic vesicle endocytosis, providing the first evidence of a role for a DENN domain- containing protein in endocytosis.