Studies on the mechanism of signal transduction of Arc, a gene involved in synaptic plasticity

为了确定可卡因和甲基苯丙胺对行为产生持久影响的基因表达的变化，我们在实验室对药物诱导的即刻早期基因Arc进行了鉴定，并利用双杂交文库筛选了Arc的相互作用伙伴。我在体内发现了一种新的SH3蛋白，SH3P13与Arc结合。SH3P13由两个选择性剪接产物13L和13S组成，并与参与内吞机制的动力素结合。原代培养的海马神经元免疫组织化学结果显示，Dynamin和SH3P13蛋白均在神经元中表达，并共同定位于突触。此外，免疫电子显微镜显示SH3P13定位于突触后棘。它激活了动力素的GTP酶，该酶调节多巴胺受体介导的内吞作用。因此，我们研究了SH3P13在COS-7细胞中的过表达是否调节了动力蛋白依赖的多巴胺D2受体的内化。出乎意料的是，共表达SH3P13几乎完全抑制了多巴胺刺激后D2受体的封存。这些结果表明，SH3P13通过与内源性动力蛋白结合蛋白(S)竞争，负性调节多巴胺受体介导的内吞作用。可卡因或甲基苯丙胺诱导的ARC蛋白可能通过与SH3P13结合来调节动力蛋白依赖性的多巴胺受体内化。研究SH3P13在Arc对多巴胺依赖的内吞作用中的调节作用可能有助于深入了解SH3P13和Arc蛋白的生理功能。

To define the changes in gene expression that may underlie long-lasting behavioral effects by cocaine and methamphetamine, drug-induced immediate early gene Arc has been characterized and proteins are screened the two-hybrid library to identify interacting partners of Arc in our laboratory. I found a novel SH3 protein, SH3P13 bound to Arc in vivo. SH3P13 consists of twoalternative splicing products 13L and 13S, and binds to dynamin that is involved in endocytotic machinery. immunohistochemistry of primary hippocampal neurons showed that both dynamin and SH3P13 proteins were expressed in neurons and co-localized at synapses. Furthermore, immunoelectron microscopy reveal that SH3P13 was localized to postsynaptic spines. It activated the GTPase of dynamin, which is known to regulate dopamine receptor-mediated endocytosis. Therefore, we examined whether overexpression of SH3P13 in COS-7 cells regulates dynamin-dependent internalization of dopamine D2 receptor. Unexpectedly, coexpression of SH3P13 almost completely inhibited sequestration of D2 receptor after dopamine stimulation. These results suggest that SH3P13 negatively regulates dopamine receptor mediated-endocytosis by competing with endogenous dynamin-binding protein(s) in COS-7 cells. Arc protein induced by cocaine or methamphetamine might regulate dynamin-dependent internalization of dopamine receptor via binding to SH3P13. The study of the regulation of SH3P13 on the dopamine-dependent endocytosis by Arc may provide an insight into physiological function of SH3P13 and Arc protein.