title: The minor subunit splice variants, H2b and H2c, of the human asialoglycoprotein receptor are present with the major subunit H1 in different hetero-oligomeric receptor complexes.
abstract: The hepatic asialoglycoprotein receptor (ASGP-R) is an endocytic receptor that mediates the internalization of desialylated glycoproteins and their delivery to lysosomes. The human ASGP-R is a hetero-oligomeric complex composed of H1 and H2 subunits. There are three naturally occurring H2 splice variants, designated H2a, H2b, and H2c, although the expression of the H2c protein had not been reported. Following deglycosylation of purified ASGP-R, we detected the H2b and H2c proteins in HepG2 and HuH-7 hepatoma cells, using an antibody directed against a COOH-terminal peptide common to all H2 isoforms (anti-H2-COOH) and another antibody against a 19-amino acid cytoplasmic insert found only in H2b (anti-H2-Cyto19). H1 and both H2b and H2c were co-purified by affinity chromatography, using asialo-orosomucoid (ASOR)-, anti-H1-, or anti-H2-COOH-Sepharose, whereas only H1 and H2b were immunoprecipitated with anti-H2-Cyto19. These results indicate that H2b and H2c are not present in the same ASGP-R complexes with H1. Similar to the H2b isoform, H2c was also palmitoylated, indicating that the 19-residue cytoplasmic insert does not regulate palmitoylation. Stably transfected SK-Hep-1 cell lines expressing ASGP-R complexes containing H1 and either H2b or H2c had similar binding affinities for ASOR and endocytosed and degraded ASOR at similar rates. The pH dissociation profiles of ASOR.ASGP-R complexes were also identical for complexes containing either H2b or H2c. We conclude that the H2b and H2c isoforms are both functional but are not present with H1 in the same hetero-oligomeric ASGP-R complexes. This structural difference between two functional subpopulations of ASGP-Rs may provide a molecular basis for the existence of two different pathways, designated State 1 and State 2, by which several types of recycling receptors mediate endocytosis.
authors.affiliation: Department of Biochemistry & Molecular Biology, and The Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190, USA.
authors.forename: Jasper H N
authors.initials: JH
authors.lastname: Yik
authors.forename: Amit
authors.initials: A
authors.lastname: Saxena
authors.forename: Paul H
authors.initials: PH
authors.lastname: Weigel
mesh_terms: D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D037263:Asialoglycoprotein Receptor; D001483:Base Sequence; D019556:COS Cells; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004705:Endocytosis; D006031:Glycosylation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011956:Receptors, Cell Surface / Q000737:chemistry*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D037263:Asialoglycoprotein Receptor; D018076:DNA, Complementary; D009841:Oligonucleotides; D020033:Protein Isoforms; D011956:Receptors, Cell Surface; D012313:RNA