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

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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.