Receptor-mediated endocytosis by the asialoglycoprotein receptor: Effect of ethanol administration on endosomal distribution of receptor and ligand

Using the asialoglycoprotein receptor (ASGP-R) and a representative ligand, asialoorosomucoid (ASOR), we have previously shown ethanol-induced impairment of endosomal acidification, receptor recycling and ligand binding, internalization, and degradation. In the current study, we further investigated ethanol-induced alterations in receptor/ligand trafficking by labeling endosomes in vivo with either Texas-Red-ASOR or ¹²⁵I-ASOR, and then assessing the receptor/ligand content of endosomes. We assessed two fractions after both 5 and 25 min of labeling: 'early endosomes' (EEs; endosomes from the cell periphery) and 'late endosomes' (LEs; endosomes farther into the cell interior). At both time points, significantly more ligand was found in EE fractions isolated from chow- and pair-fed controls (3:1, EE to LE, respectively). However, endosomes isolated from ethanol-fed animals showed a shift over time toward a more equal ligand distribution between endosome fractions (P≤0.05). Analysis of the ASGP-R content revealed a distribution pattern between the endosome fractions similar to that observed for ligand distribution. Impairment of receptor-ligand dissociation was assessed in endosome fractions by determining bound/free ligand ratios. Analysis showed that most of the ligand present in both endosome fractions was free (56-99%), although more was bound to receptor in EE vs LE of both control and ethanol animals (P≤0.05). At 5 min, more ligand remained bound in endosomes from ethanol-fed animals compared with control endosomes (P≤0.05), and the same pattern was observed at the latter time point. These results suggest that delayed dissociation may cause the receptor-ligand complexes to travel farther into the cell interior, which may impair proper trafficking of the ligand to lysosomes and alter the receptor recycling.