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 asialoorosomucoid ( Although its function in vivo is unknown, the hepatic ASGP-R 1 can mediate the clearance of injected asialoglycoproteins from the circulation via the clathrin-coated pit pathway (1-6). The human ASGP-R is a hetero-oligomer composed of a major subunit (H1) and a minor subunit (H2) that are encoded by two different genes. The two subunits are glycoproteins that are highly homologous to each other, with the exception that an 18-aa cytoplasmic insert is found only in the 50-kDa H2 but not in the 46-kDa H1 (7). The 42-kDa rat homologue of the major subunit H1 is designated RHL1 (8). Similar to the minor subunit H2, the rat homologue of H2 is encoded by a single gene. However, two distinct proteins of ϳ50 and 60 kDa can be separated by SDS-PAGE due to differences in glycosylation, and they are, therefore, designated as RHL2 and RHL3, respectively (9). The mouse ASGP-R is also composed of a major subunit, MHL-1 (10), and a minor subunit, MHL2 (11). In all species examined, each ASGP-R subunit contains a short 40-to 60-aa NH 2 -terminal cytoplasmic domain, a single-pass transmembrane domain, an ϳ80-aa extracellular stalk region, and a ϳ130-aa carbohydrate recognition domain that is capable of recognizing a terminal galactose or N-acetylgalactosamine residue found on the carbohydrate chains of naturally occurring desialylated glycoproteins or neoglycoproteins (12)(13)(14). High affinity ligand binding by the human ASGP-R, with K d values in the nanomolar range, requires the formation of hetero-oligomeric ASGP-R complexes that are composed of both H1 and H2 subunits (15) as well as ligands containing carbohydrate chains with two or more branches (12).The cDNAs of three naturally occurring H2 splice variants, designated H2a, H2b, and H2c (see Fig. 1), have been isolated from human liver and HepG2 hepatoma cells (7,16). Relative to H2c, the H2a cDNA contains a 57-nt cytoplasmic insert and a 15-nt insert near the junction between the transmembrane domain and the ectodomain (7). H2a is not part of native ASGP-R complexes, because the 5-aa sequence, encoded by the 15-nt insert, serves as a cleavage signal that results in proteolysis and the secretion of the entire H2a ectodomain (17). H2b lacks the 5-aa insert and is, therefor...

Endocytic Function, Glycosaminoglycan Specificity, and Antibody Sensitivity of the Recombinant Human 190-kDa Hyaluronan Receptor for Endocytosis (HARE)

“…one cell surface equivalent per 7-9 min) was comparable to that determined in primary rat LECs (17). The apparently slower rate of rHARE recycling in SK-Hep-1 cell lines (ϳ20 min) is likely due to a decreased capacity of the coated pit pathway in this latter cell line (9,43,44), rather than to intrinsic differences between the recombinant rHARE and hHARE. The affinities of the smaller rHARE and hHARE were also very similar, with K d values of 4.1 and 7.2 nM, respectively.…”

Nonpalmitoylated Human Asialoglycoprotein Receptors Recycle Constitutively but Are Defective in Coated Pit-mediated Endocytosis, Dissociation, and Delivery of Ligand to Lysosomes

“…ASOR conjugated to Alexa Fluor 488 (fl-ASOR) was prepared using the Alexa Fluor 488 Protein Labeling Kit from Molecular Probes according to the manufacturer's instructions. The generation and purification of the polyclonal rabbit anti-H1 and goat anti-H2 IgGs have been described previously (32). Sheep antirabbit IgG Fab' fragment conjugated to Cy3 was from Sigma.…”

“…125 I-ASOR, iron-saturated human 125 Itransferrin, and 125 I-anti-H1 and -H2 antibodies were prepared as described previously (32). ASOR conjugated to Alexa Fluor 488 (fl-ASOR) was prepared using the Alexa Fluor 488 Protein Labeling Kit from Molecular Probes according to the manufacturer's instructions.…”

“…Cells were then solubilized in the latter buffer containing 1% Triton-X 100, 0.1% SDS, and 5 mM EDTA, and the lysates were subjected to reducing SDS-PAGE. The proteins were electrotransferred to nitrocellulose and the H1 and H2 subunits were detected by Western analysis using subunit-specific polyclonal antibodies (32). Images of the Western blots were captured digitally, and the density of the protein bands was quantified using a Fluorchem™ 8000 Imaging System from Alpha Innotech Corp.…”

H2, the Minor Subunit of the Human Asialoglycoprotein Receptor, Trafficks Intracellularly and Forms Homo-oligomers, but Does Not Bind Asialo-orosomucoid

“…A BamHIEcoRI H2 cDNA fragment, excised from the parental plasmid H2/pcDNA3.1 (26), was inserted into corresponding sites in pcDNA4/HisMax version C plasmid to create pH2/HisMax. The pH2/ HisMax plasmid was used to create H2 stable cell lines in SK-Hep-1 cells as well as for transient expression of H2/HisMax fusion protein in COS-7 cells.…”