ANALYSIS OF RECEPTOR REGULATION OF HUMAN LIVER CELLS

  • Fallon, Robert, (PI)

Project: Research project

Description

The asialoglycoprotein (ASGP) receptor is a transmembrane glycoprotein
specific to hepatocytes which has been used as a model system to explore
receptor biosynthesis, trafficking and distribution within endosomal
compartments. In addition, the ASGP receptor in vivo has been used as a
target for liver-specific gene transformation suggesting a potential
application to the therapy of inherited metabolic diseases. This proposal investigates the mechanisms regulating the cell surface
expression and bioactivity of this model cell surface receptor. The
specific aims of this project focus on receptor phosphorylation and its
relationship to the regulation of receptor trafficking and down-regulation. This hypothesis was suggested by data on the ASGP and other receptors which
are phosphorylated during internalization and down-regulation (beta
adrenergic receptor, insulin, epidermal growth factor receptor). Prior
studies from this laboratory identified an intracellular population of
phosphorylated ASGP receptors which does not recycle to plasma membrane.
The physiologic role and regulation of this intracellular population of
receptors will be investigated in the Hep G2 cell line. The experimental approaches to this question will include whole cell
studies in Hep G2 cells, membrane preparations, and mammalian cell lives
transfected with wild-type ASGP receptor cDNA and that mutated at the
identified phosphorylation sites. Several novel techniques in whole cells
(selective immunoprecipitation of surface receptors, density-shift analysis
of isolated endosomal vesicles, and biotinylation of cell surface
receptors) will address the subcellular location of the initial
phosphorylation event, the routing of phosphorylated molecules in
intracellular compartments. Tryptic phosphopeptide and phosphoamino acid
analysis will localize the site of phosphorylation on the receptor
polypeptide. Site-directed mutagenesis will then be employed to
specifically alter the amino acid at this site, creating phosphorylation-
negative ASGP receptor mutants which will be transfected into suitable
surrogate cells. These cell lines expressing mutated and non mutated
receptor will be analyzed trafficking pathways. The combination of these
independent methodological approaches in this model system should yield
information on the relevance of phosphorylation to receptor regulation in
hepatocytes.
StatusFinished
Effective start/end date2/1/9012/31/94

Funding

  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $51,626.00
  • National Institutes of Health
  • National Institutes of Health: $25,516.00

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Asialoglycoprotein Receptor
Phosphorylation
Liver
Hep G2 Cells
Down-Regulation
Cell Membrane
Biotinylation
Phosphopeptides
Metabolic Diseases
Cell Surface Receptors
Site-Directed Mutagenesis
Immunoprecipitation
Epidermal Growth Factor Receptor
Population
Hepatocytes
Complementary DNA
Insulin
Amino Acids
Cell Line
Genes

Keywords

  • Medicine(all)