Our goal is to define the molecular details and mechanisms of receptor mediated endocytosis and receptor reutilization (recycling). Endocytosis of many hormones and growth factors may play an important role in the ability of these regulatory molecules to control cellular metabolism and growth. We are studying the rat hepatic galactosyl (asialoglycoprotein) receptor because this system offers an excellent opportunity to determine the details of receptor function during endocytosis, at both the cellular and molecular levels. The receptor binds a variety of experimentally useful ligands, is recycled both in vitro and in vivo and is one of the most active endocytic systems known. The receptor has been purified, characterized and has been studied extensively in intact animals and in isolated hepatocytes. We propose to answer several currently important questions about endocytosis and recycling of this receptor in isolated cells including; whether the cell surface and the large intracellular pools of receptor are functionally equivalent, whether receptor recycling occurs even in the absence of ligand and whether covalent modification of the receptor is responsible for the energy-dependent reversible modulation of total cell receptor activity. Active and inactive receptors will be isolated and characterized to determine the chemical basis for the observed receptor inactivation. In particular, we will determine whether the receptor is modified in intact cells by phosphorylation and the functional significance of this modification. We will biochemically and morphologically identify and characterize the sub-cellular compartments and organelles in which the fast and slow receptor-ligand dissociation processes, receptor/ligand segregation and receptor recycling occur. A series of novel chemical affinity derivatives, capable of specifically tagging the receptor with a radioactive or EM recorder group, will be synthesized for these studies. In addition, radio-active, electron dense and photoaffinity derivatives as well as polyclonal and monoclonal antibodies specific for the receptor will be used to monitor the cellular location and function of the receptor during endocytosis and recycling. Organic synthetic chemistry, electron microscopy, protein chemistry and general biochemical and cell biology techniques will be used in these studies.
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