Regulation of the Synaptic Localization of the nAChR

描述(申请人提供)：突触对于有效的突触传递是高度专门化的，突触前神经末梢中的递质释放位置与突触后神经递质受体的浓度一致。在发育中的神经肌肉连接处，一种称为集聚蛋白的神经源性因子指导着乙酰胆碱受体的突触后定位，我们的目标是了解集聚蛋白作用的分子机制。具体地说，我们将测试这一假设，即集聚蛋白通过诱导AChR的磷酸化并改变其与细胞内支架蛋白Rapsyn的联系来调节AChR的定位。我们有以下具体目的：(1)明确集聚蛋白诱导的β亚基磷酸化在AChR定位中的作用。我们将产生一个缺乏磷酸化位点的β亚基的转基因小鼠，然后确定这种突变是否会损害AChR的突触定位。我们还将确定磷酸化是否特定地调节突触后膜上受体的锚定、聚集和/或稳定。(2)明确Rapsyn介导的AChR定位的分子机制。在这里，我们将定义Rapsyn在AChR上的结合位点(S)，并确定结合是否受集聚蛋白调节。此外，我们还将研究集聚蛋白诱导的Rapsyn/AChR关联改变如何介导受体定位。这项工作与神经肌肉疾病直接相关，如先天性和自身免疫性肌无力综合征，在突触AChR水平严重不足，导致突触功能受损和肌肉衰弱。我们的工作将有助于确定受体定位的机制，并可能建议在肌无力综合征或其他形式的神经肌肉疾病患者中稳定受体定位的策略。

DESCRIPTION (provided by applicant): Synapses are highly specialized for efficient synaptic transmission, with the transmitter release sites in the presynaptic nerve terminal being aligned with postsynaptic concentrations of the neurotransmitter receptor. At the developing neuromuscular junction, a nerve-derived factor called agrin directs the postsynaptic localization of the acetylcholine receptor, and our goal is to understand the molecular mechanisms that underlie agrin's action. Specifically, we will test the hypothesis that agrin regulates AChR localization by inducing phosphorylation of the AChR and altering its association with the intracellular scaffolding protein, rapsyn. We have the following specific aims: (1) To define the role of agrin-induced phosphoryation of the beta subunit in AChR localization. We will generate transgenic mice with a beta subunit lacking the phosphorylation site, and then determine whether this mutation impairs the synaptic localization of the AChR. We will also determine whether phosphorylation specifically regulates the anchoring, clustering, and/or stabilization of the receptor in the postsynaptic membrane. (2) To define the molecular mechanism of rapsyn-mediated AChR localization. Here, we will define rapsyn's binding site(s) on the AChR and determine whether binding is regulated by agrin. Moreover, we will investigate how agrin-induced changes in rapsyn/AChR association mediate receptor localization. This work is directly relevant to neuromuscular disorders such as congenital and autoimmune myasthenic syndromes, where there are severe deficiencies in AChR levels at the synapse, leading to impaired synaptic function and debilitating muscle weakness. Our work will help define the mechanisms involved in receptor localization and may suggest strategies to stabilize its localization in patients with myasthenic syndromes or other forms of neuromuscular disease.