Function of Acetylcholine Receptor Phosphorylation

描述(由申请人提供)： Agin刺激培养的肌管中乙酰胆碱受体(AChR)的聚集和酪氨酸磷酸化。这些数据导致了一种假说，即AChR的酪氨酸磷酸化可能是AChR和其他突触后蛋白聚集在神经肌肉突触上的关键和必要步骤。利用胚胎干细胞的定点突变和同源重组，我培育出了突变小鼠，它们在AChR-β的大细胞内环中携带酪氨酸残基突变。我的初步结果表明，AChR的酪氨酸磷酸化虽然本身不是形成突触所必需的，但它是形成正常AChR大小、密度和形态的突触所必需的。然而，AChR-酪氨酸磷酸化导致AChR结构改变的方式尚不清楚。此外，这些突触扰动对神经肌肉传递的功能影响尚不清楚。利用分子生物学、细胞生物学、显微镜和电生理学，我提出了一系列实验来解决这些问题，希望进一步了解AChR-酪氨酸磷酸化在突触形成中的作用。

DESCRIPTION (provided by applicant): Agrin stimulates the clustering and tyrosine phosphorylation of the acetylcholine receptor (AChR) in cultured myotubes. These data have led to the hypothesis that tyrosine phosphorylation of the AChR may be a critical, and possibly a requisite step to cluster AChRs and other postsynaptic proteins at neuromuscular synapses. Using site-directed mutagenesis and homologous recombination in embryonic stem (ES) cells, I have generated mutant mice that bear mutations in tyrosine residues within the large intracellular loop of AChR-¿. My preliminary results indicate that tyrosine phosphorylation of AChR-¿, while not necessary to form synapses per se, is required to form synapses of normal AChR size, density and morphology. The manner by which AChR-¿ tyrosine phosphorylation leads to changes in AChR architecture, however, is unknown. Further, the functional effects of these synaptic perturbations on neuromuscular transmission are not understood. Using molecular biology, cell biology, microscopy and electrophysiology I propose a set of experiments to address these questions in hopes of further understanding the role of AChR-¿ tyrosine phosphorylation in synapse formation.