Tyrosine phosphorylation of the asialoglycoprotein receptor

Journal Article · Sun Feb 25 00:00:00 EST 1990 · Journal of Biological Chemistry; (USA)

OSTI ID:7027314

Fallon, R J ^[1]

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1. Washington Univ. School of Medicine, St. Louis, MO (USA)

The asialoglycoprotein (ASGP) receptor undergoes constitutive endocytosis through the coated pit/coated vesicle pathway in hepatocytes. Studies on HepG2 cells have shown that the receptor is phosphorylated at serine under control conditions and following protein kinase C stimulation. This study examined whether the ASGP receptor could also serve as a substrate for a tyrosine kinase in HepG2 cells. 32P labeling was performed in membrane preparations, in permeabilized cells at 4 degrees C, and in intact cells at 37 degrees C. The phosphorylated ASGP receptor was isolated by immunoprecipitation, hydrolyzed in 6 N HCl at 110 degrees C, and analyzed by two-dimensional high voltage electrophoresis. The receptor isolated from a membrane preparation incubated in vitro with (gamma-32P)ATP incorporated radiolabel predominantly (greater than 90%) into phosphotyrosine. ASGP receptor phosphorylation at both tyrosine and serine was detected in intact cells incubated with phosphatase inhibitors for 60 min at 37 degrees C. The presence of both phenylarsine oxide (20 microM) and sodium orthovanadate (200 microM) was required for tyrosine phosphorylation. Use of these inhibitors together resulted in a 16.4-fold increase in phosphorylation of the immunoprecipitated ASGP receptor, whereas phosphorylation of total HepG2 membrane proteins was not significantly augmented by this procedure. Selective proteolytic digestion of ASGP receptors in isolated vesicles demonstrated that the phosphorylation site identified in these studies is located at tyrosine 5 in the cytoplasmic tail.

• OSTI ID: 7027314
• Journal Information: Journal of Biological Chemistry; (USA), Vol. 265:6; ISSN 0021-9258
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
GLYCOPROTEINS
RECEPTORS
PHOSPHORYLATION
BIOLOGICAL PATHWAYS
CELL MEMBRANES
ELECTROPHORESIS
ENZYME INHIBITORS
IMMUNOASSAY
LIVER CELLS
MAN
PHOSPHORUS 32
TRACER TECHNIQUES
TYROSINE
AMINO ACIDS
ANIMAL CELLS
ANIMALS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOASSAY
CARBOXYLIC ACIDS
CELL CONSTITUENTS
CHEMICAL REACTIONS
DAYS LIVING RADIOISOTOPES
HYDROXY ACIDS
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
MAMMALS
MEMBRANE PROTEINS
MEMBRANES
NUCLEI
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
PHOSPHORUS ISOTOPES
PRIMATES
PROTEINS
RADIOISOTOPES
SOMATIC CELLS
VERTEBRATES
550201* - Biochemistry- Tracer Techniques