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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM030218-08
Application #
3277856
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1982-02-01
Project End
1990-12-31
Budget Start
1989-02-01
Budget End
1990-12-31
Support Year
8
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Texas Medical Br Galveston
Department
Type
Schools of Medicine
DUNS #
041367053
City
Galveston
State
TX
Country
United States
Zip Code
77555
Weigel, P H; Oka, J A (1998) The dual coated pit pathway hypothesis: vertebrate cells have both ancient and modern coated pit pathways for receptor mediated endocytosis. Biochem Biophys Res Commun 246:563-9
Haynes, P A; Oka, J A; Weigel, P H (1994) The rat hepatic lectin 1 subunit of the rat asialoglycoprotein receptor is a phosphoprotein and contains phosphotyrosine. J Biol Chem 269:33146-51
Haynes, P A; Medh, J D; Weigel, P H (1994) Inhibition of tyrosine phosphorylation in the rat hepatic lectin 1 subunit of the rat asialoglycoprotein receptor prevents ATP-dependent receptor inactivation in permeabilized hepatocytes. J Biol Chem 269:33152-8
Weigel, P H (1993) Endocytosis and function of the hepatic asialoglycoprotein receptor. Subcell Biochem 19:125-61
Weigel, P H; Oka, J A (1993) Regulation of asialoglycoprotein receptor activity by a novel inactivation/reactivation cycle. Receptor reactivation in permeable rat hepatocytes is mediated by fatty acyl coenzyme A. J Biol Chem 268:27186-90
Medh, J D; Weigel, P H (1991) Reconstitution of galactosyl receptor inactivation in permeabilized rat hepatocytes is ATP-dependent. J Biol Chem 266:8771-8
Weigel, P H; Oka, J A (1991) Coated pits and asialoglycoprotein receptors redistribute to the substratum during hepatocyte adhesion to galactoside surfaces. Biochem Biophys Res Commun 180:1304-11
McAbee, D D; Lear, M C; Weigel, P H (1991) Total cellular activity and distribution of a subpopulation of galactosyl receptors in isolated rat hepatocytes are differentially affected by microtubule drugs, monensin, low temperature, and chloroquine. J Cell Biochem 45:59-68
McAbee, D D; Clarke, B L; Oka, J A et al. (1990) The surface activity of the same subpopulation of galactosyl receptors on isolated rat hepatocytes is modulated by colchicine, monensin, ATP depletion, and chloroquine. J Biol Chem 265:629-35
Herzig, M C; Weigel, P H (1990) Surface and internal galactosyl receptors are heterooligomers and retain this structure after ligand internalization or receptor modulation. Biochemistry 29:6437-47

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