Asialoglycoprotein receptor phosphorylation and receptor-mediated endocytosis in hepatoma cells. Effect of phorbol esters

R. J. Fallon, A. L. Schwartz

Research output: Contribution to journalArticle

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Abstract

The asialoglycoprotein (ASGP) receptor on Hep G2 cells undergoes constitutive recycling and ligand endocytosis in the presence of phorbol dibutyrate, at a 50% reduced rate relative to control cells (Fallon, R. J., and Schwartz, A. L. (1986) J. Biol. Chem. 261, 15081-15089). The relevance of receptor phosphorylation to these events was investigated by selective immunoprecipitation of surface receptors with polyclonal anti-human ASGP antiserum and pulse-chase labeling with [32P]orthophosphate to identify subcellular locations of initial receptor phosphorylation events as well as the eventual fate of phosphorylated receptor during recycling. The surface immunoprecipitation method recovers greater than 95% of surface ASGP receptors and only 5% or less of intracellular (brief[35S]methionine pulse-labeled) receptors. With this assay we detected low levels of ASGP receptor phosphorylation at the cell surface in control cells (0.1 mol of P/mol of R) which were rapidly (< 1 min) stimulated 20-fold by 400 nM phorbol dibutyrate addition (1.7 mol of P/mol of R). Staurosporine, a protein kinase C inhibitor, blocks this stimulation by phorbol. Receptor phosphorylation at early time points in the presence of phorbol esters was restricted to the plasma membrane. Subsequent chase in the presence of excess unlabeled phosphate and phorbol esters lowered [32P]ATP(i) specific activity by 68% at 1 h. Surface immunoprecipitation during this chase period showed the phosphorylated ASGP receptors were rapidly lost from the cell surface (t( 1/2 ) = 20 min). In contrast, examination of intracellular receptor during the pulse-chase experiment in phorbol dibutyrate-treated cells showed the presence of phosphorylated pool(s) of ASGP receptors which were detectable for 6 h of chase. Since no labeled receptor can be detected at the cell surface at this time, the described intracellular phosphorylated receptors are in a non-recycling pool.

Original languageEnglish (US)
Pages (from-to)13159-13166
Number of pages8
JournalJournal of Biological Chemistry
Volume263
Issue number26
StatePublished - Jan 1 1988

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Asialoglycoprotein Receptor
Phosphorylation
Phorbol Esters
Endocytosis
Hepatocellular Carcinoma
Immunoprecipitation
Recycling
Asialoglycoproteins
Phosphates
Staurosporine
Protein C Inhibitor
Hep G2 Cells
Protein Kinase Inhibitors
Methionine
Protein Kinase C
Cell membranes
Immune Sera
Labeling
Adenosine Triphosphate
Cell Membrane

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Asialoglycoprotein receptor phosphorylation and receptor-mediated endocytosis in hepatoma cells. Effect of phorbol esters. / Fallon, R. J.; Schwartz, A. L.

In: Journal of Biological Chemistry, Vol. 263, No. 26, 01.01.1988, p. 13159-13166.

Research output: Contribution to journalArticle

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abstract = "The asialoglycoprotein (ASGP) receptor on Hep G2 cells undergoes constitutive recycling and ligand endocytosis in the presence of phorbol dibutyrate, at a 50{\%} reduced rate relative to control cells (Fallon, R. J., and Schwartz, A. L. (1986) J. Biol. Chem. 261, 15081-15089). The relevance of receptor phosphorylation to these events was investigated by selective immunoprecipitation of surface receptors with polyclonal anti-human ASGP antiserum and pulse-chase labeling with [32P]orthophosphate to identify subcellular locations of initial receptor phosphorylation events as well as the eventual fate of phosphorylated receptor during recycling. The surface immunoprecipitation method recovers greater than 95{\%} of surface ASGP receptors and only 5{\%} or less of intracellular (brief[35S]methionine pulse-labeled) receptors. With this assay we detected low levels of ASGP receptor phosphorylation at the cell surface in control cells (0.1 mol of P/mol of R) which were rapidly (< 1 min) stimulated 20-fold by 400 nM phorbol dibutyrate addition (1.7 mol of P/mol of R). Staurosporine, a protein kinase C inhibitor, blocks this stimulation by phorbol. Receptor phosphorylation at early time points in the presence of phorbol esters was restricted to the plasma membrane. Subsequent chase in the presence of excess unlabeled phosphate and phorbol esters lowered [32P]ATP(i) specific activity by 68{\%} at 1 h. Surface immunoprecipitation during this chase period showed the phosphorylated ASGP receptors were rapidly lost from the cell surface (t( 1/2 ) = 20 min). In contrast, examination of intracellular receptor during the pulse-chase experiment in phorbol dibutyrate-treated cells showed the presence of phosphorylated pool(s) of ASGP receptors which were detectable for 6 h of chase. Since no labeled receptor can be detected at the cell surface at this time, the described intracellular phosphorylated receptors are in a non-recycling pool.",
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