An in vivo Method for Determination of Endosomal Distribution of Both Ligand and Asialoglycoprotein Receptor in Rat Liver

Abstract

Alcoholic liver injury is a significant medical problem throughout the world. Protein trafficking pathways, including endocytosis, appear to be especially susceptible to the deleterious effects of alcohol. Using the asialoglycoprotein receptor (ASGPR) as a model, we have studied ethanol-induced alterations in the process of receptor-mediated endocytosis (RME). The ASGPR, a hepatocyte-specific receptor, binds proteins that have lost their terminal sialic acid moiety and have exposed galactose or N-acetylgalactosamine moieties [1]. During RME many extracellular ligands are bound by specific cell surface receptors, such as the ASGPR, and internalized via a clathrin-coated pit pathway [2]. In the endocytotic pathway, the lumen of the endosome becomes acidic allowing the receptor-ligand complexes to dissociate. The separated receptors and ligands can either be targeted for degradation in lysosomes or recycled back to the cell surface. Endosomal trafficking from the cell surface to the final destination in the cell is an extremely complex process that is still not completely understood. Our previous studies using the ASGPR model have identified several ethanol-induced alterations during the process of RME. Altered receptor-ligand uncoupling and endosomal acidification as well as decreased ligand binding, internalization, and degradation are some of the observed alterations [3-9]. For the current study, we wanted to examine the trafficking/distribution of both ligand and receptor in liver endosomes. To accomplish this we labeled asialoorosomucoid (ASOR), a ligand for the ASGPR, with either Texas-Red (TR-ASOR) or radioactive iodine (125I-ASOR). We exposed rats in vivo to the labeled ligand, and then assessed both ligand and ASGPR content in two isolated liver endosome populations. The early (EE) consist of endosomes from the periphery of the cell, including endosomes just entering the pathway and those containing receptor and/or ligand being recycled back to the cell surface. The late (LE) contain more distal endosomes which are destined for degradation in lysosomes. The purpose of the work described here was to gather information on the viability of this method for isolating endosome fractions and for assessing both ligand and receptor content in the isolated endosome fractions. Ultimately, our goal is to use this method to further investigate the affect of ethanol on the trafficking of ligand and receptor between endosome fractions.