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Abstract
The functional human hepatic asialoglycoprotein receptor (ASGP-R) is a hetero-oligomer composed of two subunits, designated H1 and H2, which are highly homologous. Despite their extensive homology, the major H1 subunit is stably expressed by itself, whereas in the absence of H1 most of the H2 subunits are degraded in the ER. In this study, we were able to investigate the capability of the minor ASGP-R subunit, H2, to function independently of H1, because it was apparently stabilized by fusing its NH(2) terminus with an epitope tag. We could thus create stable cell lines in hepatoma-derived SK-Hep-1 cells that expressed the H2 subunit alone. H2 was expressed on the cell surface and was internalized, predominantly through the clathrin-coated pit pathway. Since the internal pool of H2 was also able to traffick to the cell surface, we conclude that H2 recycles between the surface and intracellular compartments, similar to the constitutive recycling of hetero-oligomeric ASGP-R complexes. However, the rate of H2 recycling and internalization was approximately 25-33% that of H1. Similar to H1, the H2 polypeptides were also able to self-associate to form homo-oligomers, including trimers and tetramers. However, unlike H1, which can bind the ligand asialo-orosomucoid (ASOR) when overexpressed in COS-7 cells, H2 failed to bind or endocytose ASOR. In summary, the H2 subunit of the human ASGP-R contains functional, although weak, signal(s) for endocytosis and recycling and has the ability to oligomerize. H2 homo-oligomers, however, do not create binding sites for desialylated glycoproteins, such as ASOR, that contain tri- and tetra-antennary N-linked oligosaccharides. Nonetheless, these results raise the intriguing possibility that naturally occurring H2 homo-oligomers may exist in human hepatocytes and have an as yet undiscovered function.
Highlights
The ASGP-R1 is an endocytic recycling receptor localized to the sinusoidal face of the hepatocyte plasma membrane where it could be responsible for the removal and degradation of potentially deleterious [1] circulating glycoconjugates [2,3,4,5,6,7,8]
We were able to study the characteristics of the H2 subunit in the absence of the H1 subunit, because masking its NH2 terminus with an epitope tag apparently stabilized the expression of H2
The native H2 polypeptides derived from pcDNA3.1 and the H2/HisMax fusion proteins derived from pcDNA4/HisMax were expressed in COS-7 cells and the H2 proteins were detected by immunoblotting
Summary
The ASGP-R1 is an endocytic recycling receptor localized to the sinusoidal face of the hepatocyte plasma membrane where it could be responsible for the removal and degradation of potentially deleterious [1] circulating glycoconjugates [2,3,4,5,6,7,8]. Cells were washed with ice-cold PBS and incubated at 4 °C for 1 h with 0.5 ml Buffer 1/BSA per well containing 1 g/ml 125I-ASOR or affinity-purified anti-H2 125I-IgG. H2/HisMax Is Localized at the Plasma Membrane in the Absence of H1—The H2/HisMax fusion cDNA was transfected into SK-Hep-1 cells, to create cell lines that stably expressed H2 at levels similar to native hepatoma cell lines.
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