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

分子筛蛋白介导的内吞作用(CME)调节多种营养和信号受体、离子通道和其他质膜蛋白的内化和细胞表面水平。此外，在神经末梢突触小泡(SVS)的生物发生和再循环以及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中，网状蛋白途径也是必需的。笼蛋白包裹的囊泡的形成涉及一个时空调节的蛋白质-蛋白质和蛋白质-脂相互作用网络，该网络以笼蛋白和异四聚体适配器复合体AP2为主要相互作用中心。我们已经开发出一组新的小分子抑制剂，在体外和活细胞中强烈干扰网状蛋白的功能。此外，我们正在获得有条件的AP2µ基因敲除小鼠。该项目现在的目标是进一步表征脑血栓停对内吞作用和生长因子诱导的细胞信号的影响，以及对SVS的外内吞周期的影响。此外，正在进行的努力旨在确定提高体内应用的效力和特异性的新的停滞剂。我们还计划设计和鉴定能够选择性地破坏货物特异的内吞适配器ARH和？-arrestins与AP2？的顶端结合的模拟多肽化合物。最后，我们将使用条件性AP2n基因敲除小鼠来研究AP2功能的持续丧失对SV膜的CME以及包括化学神经传递在内的其他信号过程的影响。

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.