Signaling by MuSK, a component of the Agrin receptor

描述（由申请人提供）：我们未能理解聚集蛋白如何刺激MuSK酪氨酸磷酸化，这可能是我们理解神经肌肉突触信号传导中最根本和最明显的差距。我们发现Lrp 4是LDL受体家族的一员，对于聚集蛋白刺激MuSK酪氨酸磷酸化是必不可少的，这表明Lrp 4是聚集蛋白受体复合物的关键组分，并且可能是聚集蛋白长期寻找的受体。本文所述的实验旨在确定Lrp 4是否结合聚集蛋白和/或MuSK以及Lrp 4如何介导聚集蛋白应答和突触分化。MuSK一旦被激活，如何刺激突触后分化也同样知之甚少。Dok-7最近被鉴定为衔接蛋白，其被募集到酪氨酸磷酸化的MuSK，并且其对于突触分化是必需的。此外，Dok-7的突变是神经肌肉疾病的主要原因，称为先天性肌无力综合征（CMS）。本文描述的实验旨在鉴定和研究Dok-7招募的蛋白质，这对于了解Dok-7如何调节突触分化以及Dok-7中的突变如何导致CMS至关重要。由于几个原因，拟议的研究具有临床相关性。首先，Dok-7的突变导致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下游基因的突变也可能导致CMS，因此确定Dok-7下游的途径同样重要。第三，由于其他突触基因（例如AChR亚基，rapsyn，MuSK，AChE）的突变也会导致CMS，因此选择性干扰肌肉中Lrp 4功能的突变也可能导致CMS。最后，由于20%的重症肌无力患者对AChR或MuSK的自身抗体呈血清阴性，因此这些个体可能携带对其他突触蛋白（可能是Lrp 4）的自身抗体。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纤维之间复杂的信号交换，导致高度特化的突触后膜和高度分化的神经末梢的形成。负责这个复杂的分化程序的信号和机制知之甚少，但需要神经源性配体，聚集蛋白，和受体酪氨酸激酶，MuSK。这种信号通路的缺陷是导致各种先天性神经肌肉疾病的原因，并可能导致或促成神经退行性疾病，如ALS。本文描述的实验旨在确定Lrp 4和Dok-7（该信号通路的两个新发现的组分）如何介导聚集蛋白反应性，导致MuSK磷酸化并刺激突触分化。

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.