Signaling by MuSK, a component of the Agrin receptor

描述(由申请者提供)：我们未能理解AGRIN如何刺激麝香酪氨酸磷酸化，这可能是我们对神经肌肉突触信号理解中最根本和最明显的差距。我们发现，Lrp4是低密度脂蛋白受体家族中的一员，是agin刺激麝香酪氨酸磷酸化所必需的，这表明Lrp4是agin受体复合体的关键成分，也可能是agin长期寻找的受体。这里描述的实验旨在确定Lrp4是否与Agin和/或Musk结合，以及Lrp4如何介导Agin反应和突触分化。马斯克一旦被激活，如何刺激突触后分化也同样鲜为人知。DOK-7最近被确定为一种接头蛋白，它被招募到酪氨酸磷酸化的麝香中，对突触分化至关重要。此外，DOK-7的突变是神经肌肉疾病的主要原因，被称为先天性肌无力综合征(CMS)。这里描述的实验旨在识别和研究招募到DOK-7的蛋白质，这将是理解DOK-7如何调节突触分化以及DOK-7突变如何导致CMS的关键。建议的研究具有临床相关性，原因有几个。首先，DOK-7的突变会导致CMS，因此了解DOK-7的工作原理将有助于更好地了解这种疾病，并可能导致新的治疗策略。其次，DOK-7下游基因的突变同样可能导致CMS，因此识别DOK-7下游的途径同样重要。第三，由于其他突触基因(如AChR亚单位、Rapsyn、Musk、AChE)的突变也会引起CMS，选择性地干扰肌肉中Lrp4功能的突变也可能是CMS的原因。最后，由于20%的重症肌无力患者对AChR或穆斯克自身抗体呈血清阴性，这些人可能携带对其他突触蛋白的自身抗体，可能是Lrp4。公共卫生相关性：神经肌肉突触的形成需要运动神经元和发育中的肌肉纤维之间进行复杂的信号交换，从而形成高度专门化的突触后膜和高度分化的神经末梢。负责这一复杂分化程序的信号和机制知之甚少，但需要神经衍生的配体AGRIN和受体酪氨酸激酶穆斯克。该信号通路的缺陷导致多种先天性神经肌肉疾病，并可能导致或促成神经退行性疾病，如肌萎缩侧索硬化症。这里描述的实验旨在确定该信号通路的两个新发现的成分Lrp4和DOK-7是如何介导农业蛋白反应，导致穆斯克磷酸化和刺激突触分化的。

DESCRIPTION (provided by applicant): Our failure to understand how Agrin stimulates MuSK tyrosine phosphorylation is perhaps the most fundamental and glaring gap in our understanding of signaling at the neuromuscular synapse. We found that Lrp4, a member of the LDL receptor family, is essential for Agrin to stimulate MuSK tyrosine phosphorylation, suggesting that Lrp4 is a key component of an Agrin receptor complex and potentially the long-sought receptor for Agrin. The experiments described here are designed to determine whether Lrp4 binds Agrin and/or MuSK and how Lrp4 mediates Agrin responsiveness and synaptic differentiation. How MuSK, once activated, stimulates postsynaptic differentiation is similarly poorly understood. Dok-7 has recently been identified as an adaptor protein, which is recruited to tyrosine phosphorylated MuSK and which is essential for synaptic differentiation. Further, mutations in Dok-7 are a major cause of neuromuscular disorders, termed congenital myasthenic syndromes (CMS). The experiments described here are designed to identify and study the proteins that are recruited to Dok-7, which will be critical to understand how Dok-7 regulates synaptic differentiation and how mutations in Dok-7 cause CMS. The proposed studies are clinically relevant for several reasons. First, mutations in Dok-7 cause CMS, so understanding how Dok-7 works will contribute to a better understanding of this disease and may lead to novel therapeutic strategies. Second, mutations in genes that are downstream from Dok-7 may likewise cause CMS, so identifying the pathway downstream from Dok-7 is likewise important. Third, because mutations in other synaptic genes (e.g. AChR subunits, rapsyn, MuSK, AChE) also cause CMS, mutations that interfere with the function of Lrp4 selectively in muscle may also be responsible for CMS. Finally, because 20% of patients with myasthenia gravis are sero- negative for auto-antibodies to AChR or MuSK, these individuals presumably carry auto-antibodies to other synaptic proteins, possibly Lrp4. PUBLIC HEALTH RELEVANCE: The formation of neuromuscular synapses requires a complex exchange of signals between motor neurons and developing muscle fibers leading to the formation of a highly specialized postsynaptic membrane and a highly differentiated nerve terminal. The signals and mechanisms responsible for this complex differentiation program are poorly understood but require the neurally-derived ligand, Agrin, and the receptor tyrosine kinase, MuSK. Defects in this signaling pathway are responsible for a variety of congenital neuromuscular disorders and could underlie or contribute to neurodegenerative diseases such as ALS. The experiments described here are designed to determine how Lrp4 and Dok-7, two newly discovered components of this signaling pathway mediate Agrin responsiveness, lead to MuSK phosphorylation and stimulate synaptic differentiation.