Abstract
The hepatic asialoglycoprotein receptor (ASGP-R) internalizes desialylated glycoproteins via the clathrin-coated pit pathway and mediates their delivery to lysosomes for degradation. The human ASGP-R contains two subunits, H1 and H2. Cytoplasmic residues Cys(36) in H1, as well as Cys(54) and Cys(58) in H2 are palmitoylated (Zeng, F.-Y., and Weigel, P. H. (1996) J. Biol. Chem. 271, 32454). In order to study the function(s) of ASGP-R palmitoylation, we mutated these Cys residues to Ser and generated stably transfected SK-Hep-1 cell lines expressing either wild-type or nonpalmitoylated ASGP-Rs. Compared with wild-type ASGP-Rs, palmitoylation-defective ASGP-Rs showed normal ligand binding, intracellular distribution and trafficking patterns, and pH-induced dissociation profiles in vitro. However, continuous ASOR uptake, and the uptake of prebound cell surface ASOR were slower in cells expressing palmitoylation-defective ASGP-Rs than in cells expressing wild-type ASGP-Rs. Unlike native ASGP-Rs in hepatocytes or hepatoma cells, which mediate endocytosis via the clathrin-coated pit pathway and are almost completely inhibited by hypertonic medium, only approximately 40% of the ASOR uptake in SK-Hep-1 cells expressing wild-type ASGP-Rs was inhibited by hyperosmolarity. This result suggests the existence of an alternate nonclathrin-mediated internalization pathway, such as transcytosis, for the entry of ASGP-R.ASOR complexes into these cells. In contrast, ASOR uptake mediated by cells expressing palmitoylation-defective ASGP-Rs showed only a marginal difference under hypertonic conditions, indicating that most of the nonpalmitoylated ASGP-Rs were not internalized and processed normally through the clathrin-coated pit pathway. Furthermore, cells expressing wild-type ASGP-Rs were able to degrade the internalized ASOR, whereas ASOR dissociation was impaired and degradation was barely detectable in cells expressing nonpalmitoylated ASGP-Rs. We conclude that palmitoylation of the ASGP-R is required for its efficient endocytosis of ligand by the clathrin-dependent endocytic pathway and, in particular, for the proper dissociation and delivery of ligand to lysosomes.
Highlights
Lation plays a role in regulating specific protein functions [1,2,3,4,5]
Internalization of prebound ASOR was not detected in cells expressing nonpalmitoylated asialoglycoprotein receptor (ASGP-R), showing that there was a defect in the rapid endocytosis of surface-bound ASOR. These results show that palmitoylation plays a role in regulating the rapid internalization (Ͻ12 min) of wild-type ASGP-Rs through the clathrincoated pit pathway in transfected SK-Hep-1 cells
In order to elucidate the role of palmitoylation in ASGP-R function, we constructed stable cell lines expressing either the wild-type, or mutant ASGP-Rs incapable of palmitate attachment to either one or both receptor subunits
Summary
Lation plays a role in regulating specific protein functions [1,2,3,4,5]. For example, CHO cells expressing nonpalmitoylated mutant transferrin receptors show a significant increase in the rate of receptor internalization when compared with the wild-type receptor, indicating that palmitoylation inhibits the rate of transferrin receptor endocytosis [6]. ASOR uptake mediated by cells expressing palmitoylation-defective ASGP-Rs showed only a marginal difference under hypertonic conditions, indicating that most of the nonpalmitoylated ASGP-Rs were not internalized and processed normally through the clathrin-coated pit pathway.
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