The cytoplasmic coatomer protein COPI. A potential translational regulator.
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.
de La Vega
L A
LA
Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Stockert
R J
RJ
D020121:5' Untranslated Regions / Q000378:metabolism*; D037263:Asialoglycoprotein Receptor; D001692:Biological Transport; D020756:Coatomer Protein / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000737:chemistry; D011485:Protein Binding; D014176:Protein Biosynthesis; D016601:RNA-Binding Proteins / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000096:biosynthesis* / Q000235:genetics; D015534:Trans-Activators / Q000378:metabolism*
D020121:5' Untranslated Regions; D037263:Asialoglycoprotein Receptor; D020756:Coatomer Protein; D016601:RNA-Binding Proteins; D011956:Receptors, Cell Surface; D015534:Trans-Activators