Functional analysis of cell signaling events following inhibition of clathrin/ AP2-mediated endocytosis

网格蛋白介导的内吞作用（CME）调节各种营养和信号受体、离子通道和其他质膜蛋白的内化和细胞表面水平。此外，网格蛋白途径是神经末梢突触囊泡（SV）的生物发生和再循环所必需的，并通过调节表面活性神经递质受体的数量来调节突触后反应。网格蛋白包被囊泡的形成涉及蛋白质-蛋白质和蛋白质-脂质相互作用的时空调节网络，其以网格蛋白和异四聚体适配器复合物AP 2为中心作为主要的相互作用枢纽。我们已经开发了一组新的小分子抑制剂，在体外和活细胞中急性干扰网格蛋白功能。此外，我们正在获得条件性AP 2 µ敲除小鼠。该项目现在的目标是进一步表征进站口对内吞作用和生长因子诱导的细胞信号传导以及SV的外吞-内吞循环的影响。此外，正在进行的努力旨在鉴定用于体内应用的增加的效力和特异性的新的进站。我们还计划设计和鉴定能够选择性破坏货物特异性内吞衔接子ARH和β-抑制蛋白与AP 2 β的顶部位点的缔合的拟肽化合物。最后，我们将使用条件性AP 2n基因敲除小鼠研究AP 2功能持续丧失对SV膜CME和其他信号传导过程（包括化学神经传递）的影响。

Clathrin-mediated endocytosis (CME) regulates the internalization and cell surface levels of a variety of nutrient and signaling receptors, ion channels, and other plasma membrane proteins. Moreover, the clathrin pathway is required for the biogenesis and recycling of synaptic vesicles (SVs) at nerve terminals and in scaling postsynaptic responses by regulating the number of surface-active neurotransmitter receptors. Clathrin-coated vesicle formation involves a spatiotemporally regulated network of protein-protein and protein-lipid interactions that are centered around clathrin and the heterotetrameric adaptor complex AP2 as major interaction hubs. We have developed pitstops as a novel group of small molecule inhibitors that acutely interfere with clathrin function in vitro and in living cells. Moreover, we are in the process of obtaining conditional AP2µ knockout mice. The project now aims at further characterizing the effect of pitstops on endocytosis and growth factor-induced cell signaling as well as on exo-endocytic cycling of SVs. Moreover, ongoing efforts are directed at identifying novel pitstops of increased potency and specificity for in vivo applications. We also plan to devise and identify peptidomimetic compounds capable of selectively disrupting association of the cargo-specific endocytic adaptors ARH and ß-arrestins with the top site of AP2ß. Finally, we will use conditional AP2n knockout mice to study the effect of sustained loss-of-function of AP2 on CME of SV membranes and on other signaling processes including chemical neurotransmission.