Structure-Function Studies of the Receptor Tyrosine Kinase MuSK

神经肌肉接头（NMJ）是一种高度特化的突触，其形成依赖于 MuSK（肌肉特异性激酶），一种由聚集蛋白（一种硫酸乙酰肝素）激活的受体酪氨酸激酶 来源于运动神经元的蛋白聚糖。MuSK激活导致肌肉蛋白重新分布， 突触后位点，包括乙酰胆碱受体（AChR）、rapsyn、ErbBs和MuSK本身。在 此外，聚集蛋白诱导的MuSK激活导致突触特异性的选择性转录上调。 基因在突触下核，并诱导逆行信号导致突触前分化。在 在成熟的NMJ中，从运动神经元释放的乙酰胆碱激活AChR并触发肌肉收缩。 MuSK的胞外域和胞浆区在信号转导过程中均发挥重要作用 导致了NMJ的形成。然而，这些信号的分子/结构机制 过程不被理解。本提案的目标是阐明结构机制 控制：（i）聚集蛋白对MuSK的激活;（ii）MuSK与AChR和rapsyn共聚簇，以及（iii）MuSK 募集非受体酪氨酸激酶Src和Src。本提案的具体目标是： 目标1。MuSK胞外域的结构和功能表征 目标二。MuSK与非受体之间相互作用模式的分子表征 酪氨酸激酶Src和Abl 为了实现这些目标，我们将采用X射线晶体学来确定三维 MuSK胞外域和胞质结构域的结构。然后，我们将描述的功能作用， 通过在M/SK缺陷型细胞中表达选择的MuSK突变体，从结构研究中鉴定的关键残基 肌管和测定聚集蛋白诱导的MuSK活化、AChR聚集以及Src和Abl的募集 关于MuSK 相关性：NMJ的形成是一个基本的生物学过程，对生物体至关重要。 发展通过揭示这种重要突触形成的生化机制， 我们将更好地了解，在分子水平上，这个过程中的缺陷如何引起神经肌肉 紊乱

The neuromuscular junction (NMJ) is a highly specialized synapse whose formation is dependent on MuSK (muscle-specific kinase), a receptor tyrosine kinase that is activated by agrin, a heparan-sulfate proteoglycan derived from motor neurons. MuSK activation results in the redistribution of muscle proteins to the postsynaptic site, including the acetylcholine receptor (AChR), rapsyn, ErbBs and MuSK itself. In addition, agrin-induced MuSK activation leads to selective transcriptional upregulation of synapse-specific genes in subsynaptic nuclei, and to induction of a retrograde signal leading to presynaptic differentiation. In mature NMJs, acetylcholine released from motor neurons activates AChRs and triggers muscle contraction. Both the ectodomain and cytoplasmic domain of MuSK play essential roles in signaling processes leading to formation of the NMJ. However, the molecular/structural mechanisms underlying these signaling processes are not understood. The goals of this proposal are to elucidate the structural mechanisms governing: (i) MuSK activation by agrin; (ii)MuSK co-clustering with the AChR and rapsyn, and (iii)MuSK recruitment of the non-receptor tyrosine kinases Src and Abl. The specific aims of this proposal are: Aim 1. Structural and functional characterization of the MuSK ectodomain Aim 2. Molecular characterization of the modes of interaction between MuSK and the non-receptor tyrosine kinases Src and Abl To achieve these aims, we will employ x-ray crystallography to determine the three-dimensional structures of the MuSK ectodomain and cytoplasmic domain. We will then characterize the functional roles of key residues identified from the structural studies by expressing select MuSK mutants in M/SK-deficient myotubes and assaying for agrin-induced MuSK activation, AChR clustering, and recruitment of Src and Abl to MuSK. Relevance: Formation of the NMJ is a fundamental biological process which is critical for organismal development. By revealing the biochemical mechanisms underlying the formation of this important synapse, we will better understand, at a molecular level, how defects in this process give rise to neuromuscular disorders.