Receptor mediated endocytosis (RME) is s system for the movement into the cell of macromolecules. One of the best characterized systems utilizing RME is the hepatic receptor for asialoglycoproteins (ASGP-R). At present there is no complete picture of the structure, function, intracellular movement and modulation of any one particular cell surface receptor. The ASGP-R is well suited for these studies since we have purified receptor, full length receptor cDNA anti-recep r antibodies, purified ligands, anti-ligand antibodies and a continuous human hepatoma cell line which expresses abundant ASGP-R. In order to examine the intracellular movement and the events which mediate the role of organelle fusion in the recycling pathways, we shall define the role of endoproteases in these processes. These and other elements essential for fusion will be characterized using an in vitro fusion assay which relys on the fusion of endocytotic vesicles with plasma membrane vesicles. As receptor phosphorylation is important in the regulation of receptor function and movement during RME, we shall examine the number and kinetics of phosphorylation sites on the ASGP-R, in basal and in hyperphosphorylated states. The role of each phosphoaminoacid will be evaluated by in vitro mutagenesis and transfection studies. The effect of ASGP-R phosphorylation on receptor-ligand interactions and, receptor movement within the RME pathways will be characterized. In addition, modulation of the cytoplasmic kinase(s) responsible for ASGP-R phosphorylation and down regulation will be characterized and purified. These studies will substantially increase our understanding of endocytosis and intracellular sorting of cell surface receptors.
