Signaling by MuSK, a component of the Agrin receptor

描述（由申请人提供）：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传导的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的一员，对Agrin刺激麝香酪氨酸磷酸化至关重要，这表明Lrp4是Agrin受体复合物的关键组分，可能是长期寻找的Agrin受体。本文描述的实验旨在确定Lrp4是否与Agrin和/或MuSK结合，以及Lrp4如何介导Agrin反应性和突触分化。MuSK一旦被激活，如何刺激突触后分化也同样不为人所知。Dok-7最近被确定为一种衔接蛋白，它被招募到酪氨酸磷酸化的MuSK中，并且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，称为先天性肌无力综合征（CMS）。本文描述的实验旨在识别和研究被募集到Dok-7的蛋白质，这对于理解Dok-7如何调节突触分化以及Dok-7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突变导致CMS，因此了解Dok-7如何工作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下游的基因突变也可能导致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（如AChR亚基、rapsyn、MuSK、AChE）的突变也会导致CMS，因此在肌肉中选择性干扰Lrp4功能的突变也可能是CMS的原因。最后，由于20%的重症肌无力患者血清AChR或MuSK自身抗体呈阴性，这些患者可能携带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纤维之间复杂的信号交换，从而形成高度特化的突触后膜和高度分化的神经末梢。这种复杂分化程序的信号和机制目前尚不清楚，但需要神经来源的配体Agrin和受体酪氨酸激酶MuSK。这一信号通路的缺陷是多种先天性神经肌肉疾病的原因，并可能是ALS等神经退行性疾病的基础或促成因素。本文所述的实验旨在确定Lrp4和Dok-7这两个新发现的信号通路成分如何介导Agrin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.