ABSTRACT
Biotin regulation of asialoglycoprotein receptor expression and insulin receptor activity has been established in two human hepatoblastoma cell lines, Hep G2 and HuH-7. Second messenger cGMP mimics the effect of biotin on asialoglycoprotein receptor expression at the translational level. Metabolic labeling and subsequent immunoprecipitation indicate that the loss of insulin receptor activity during biotin deprivation was due to suppression of receptor synthesis. Evidence for posttranscriptional regulation of insulin receptor synthesis was provided by rapid biotin induction of receptor synthesis without an increase in gene transcript number. Addition of a cGMP-dependent protein kinase (cGK) inhibitor prevented biotin induction of the insulin and asialoglycoprotein receptors, suggesting that protein phosphorylation propagates the cGMP signal transduction cascade. Coatomer protein COPI was recently identified as the trans-acting factor that regulates in vitro translation of the asialoglycoprotein receptor. Biotin repletion of the culture medium resulted in the hyperphosphorylation of alpha-COP, which was prevented by simultaneous addition of the cGK inhibitor. These findings suggest that the end point of this cGMP signal cascade is modulated by cGK and that a phosphorylation reaction governs the expression of both receptor proteins.
Subject(s)
Cyclic GMP-Dependent Protein Kinases/metabolism , Receptor, Insulin/genetics , Receptors, Cell Surface/genetics , Asialoglycoprotein Receptor , Biotin/pharmacology , Coatomer Protein/metabolism , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , Hepatoblastoma , Humans , Liver Neoplasms , Phosphorylation , Protein Biosynthesis/drug effects , Protein Biosynthesis/physiology , Second Messenger Systems/drug effects , Second Messenger Systems/physiology , Signal Transduction/drug effects , Signal Transduction/physiology , Tumor Cells, CulturedABSTRACT
Expression of the asialoglycoprotein receptor (ASGR) by the human hepatocellular carcinoma cell lines HepG2 and HuH-7 in response to intracellular cGMP concentrations was previously shown to be regulated at the translational level (1). Stable transfection of COS-7 cells with deletion constructs encoding the asialoglycoprotein receptor H2b subunit localized the cGMP-responsive cis-acting element to the mRNA 5'-untranslated region. Resolution by anion exchange chromatography of an S-100 isolated from human liver resulted in the partial purification of an RNA-binding protein specific to this cis-acting element. Northwestern analysis using the 5'-untranslated region as probe indicated that a 140-kDa protein was the potential RNA-binding protein. Sequence of tryptic peptides suggested that the 140-kDa protein was the alpha-COP subunit of coatomer protein COPI, usually associated with trans-Golgi network membrane traffic. Immunoblot analysis confirmed the presence of alpha-COP in the Mono-Q fraction as well as that of a second coatomer subunit, beta-COP. Antibody induced gel retardation supershift confirmed the identification of the RNA-binding proteins as alpha- and beta-COP. Although the RNA recognition motif appears to reside solely in alpha-COP, antibody-induced supershift strongly indicated that the entire coatomer complex was the trans-acting factor. Depletion of S-100 with the antibody to beta-COP confirmed that the coatomer was the sole protein binding to the ASGR mRNA 5'-untranslated region in liver cytosol and responsible for inhibition of in vitro translation of the asialoglycoprotein receptor.
