The long-term objective of our studies is to elucidate the mechanism of clathrin-coated vesicle assembly and sorting, and the role of accessory factors in this phenomenon. Assembly and subsequent fission of the sorting coat requires, in addition to clathrin and the AP-2 adaptor heterotetramer that contacts both cargo molecules and clathrin directly, the participation of several so-called endocytic accessory proteins. Recent work suggests that accessory proteins containing a phosphotyrosine-binding (PTB) domain, a protein-protein interaction module commonly found in signal transduction components, play an important role in cargo selection at the clathrin bud site. In studies performed during the previous grant cycle, we discovered two PTB domain proteins, Disabled-2 (Dab2) and the autosomal recessive hypercholesterolemia (ARH) protein, each display functional attributes of an endocytic-sorting adaptor. Our identification of ARH as an endocytic constituent is significant because it represents the first demonstration that a defective clathrin adaptor can lead to human disease. Like many endocytic accessory proteins, Dab2 and ARH function, in part, by engaging the principal coat components AP-2 and clathrin, and a biochemical approach will be used to characterize novel AP-2- and clathrin-binding determinants in molecular detail. We will also test the hypothesis that Dab2 and ARH are functionally redundant and ascertain whether the normal low density lipoprotein (LDL) uptake seen in ARH patient fibroblasts is due to the activity of Dab2. This will be achieved using ARH-null cells in conjunction with siRNA knockdown techniques to eliminate both proteins simultaneously. Additional studies will be aimed at dissecting how the different functional domains of these two proteins dictate their endocytic function. This will center upon structural studies, in vitro biochemical assays for lattice assembly, live cell imaging, and reintroduction of mutant proteins into ARH/Dab2 null cells. In this way, we can determine how Dab2 and ARH directly influence LDL receptor sorting at the clathrin bud site. Once the contributions of Dab2 and ARH have been delineated, we will begin to study the possible regulatory inputs that could modulate endocytic activity of these accessory proteins. The study is designed to broaden our understanding of the general molecular mechanisms that govern cargo selection at intracellular sorting stations, as well as to provide a detailed understanding of the nature and function of the proteins responsible for LDL receptor uptake in clathrin-coated vesicles.
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