Tyrosine phosphorylation of the asialoglycoprotein receptor

Robert Fallon

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

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 °C, and in intact cells' at 37 °C. The phosphorylated ASGP receptor was isolated by immunoprecipitation, hydrolyzed in 6 N HCl at 100 °C, and analyzed by two-dimensional high voltage electrophoresis. The receptor isolated from a membrane preparation incubated in vitro with [γ-32P]ATP incorporated radiolabel predominantly (>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 °C. The presence of both phenylarsine oxide (20 μM) and sodium orthovanadate (200 μM) 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.

Original languageEnglish (US)
Pages (from-to)3401-3406
Number of pages6
JournalJournal of Biological Chemistry
Volume265
Issue number6
StatePublished - Feb 25 1990
Externally publishedYes

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Asialoglycoprotein Receptor
Phosphorylation
Tyrosine
Hep G2 Cells
Serine
Coated Vesicles
Membranes
Phosphotyrosine
Vanadates
Endocytosis
Electrophoresis
Phosphoric Monoester Hydrolases
Immunoprecipitation
Protein-Tyrosine Kinases
Labeling
Protein Kinase C
Digestion
Hepatocytes
Membrane Proteins
Adenosine Triphosphate

ASJC Scopus subject areas

  • Biochemistry

Cite this

Tyrosine phosphorylation of the asialoglycoprotein receptor. / Fallon, Robert.

In: Journal of Biological Chemistry, Vol. 265, No. 6, 25.02.1990, p. 3401-3406.

Research output: Contribution to journalArticle

Fallon, Robert. / Tyrosine phosphorylation of the asialoglycoprotein receptor. In: Journal of Biological Chemistry. 1990 ; Vol. 265, No. 6. pp. 3401-3406.
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