Recycling of the asialoglycoprotein receptor: biochemical and immunocytochemical evidence

One of the best documented systems of receptor-mediated endocytosis is the clearance of asialoglycoproteins (ASGP) from the blood plasma by liver parenchymal cells. There are 200 000-500 000 ligand binding sites per cell, which makes this system favourable for molecular studies of receptor function. By using both biochemical and immunocytochemical approaches, we have obtained evidence for receptor recycling. We have also localized the intracellular site at which the endocytosed receptor and ligand dissociate. The human hepatoma cell Hep G2 contains abundant ASGP receptors (approximately 225 000 per cell). In growing cells approximately 85% of the functional receptors are on the cell surface and the remaining 15% are internal. The maximal rate of ligand uptake in this cell system at 37 degrees C is approximately 30 000 molecules per cell per minute. Each functional receptor can therefore bind and internalize more than 50 ligand molecules during a 6 h period (in the absence of new receptor synthesis), or one ligand each 8 min. To follow both ligand and receptor during their common endocytosis and to visualize the compartment in which the dissociation of ligand from receptor occurs, we have used our recently developed double-labelling immunocytochemical electron microscopic techniques with purified antibodies against ASGP ligand and ASGP receptor. In normal rat hepatocytes, both ligand and receptor are taken up from the sinusoidal cell surface in clathrin-coated vesicles. Both receptor and ligand are associated with the membrane of small clathrin-coated vesicles close to the cell surface. Larger vesicles, farther removed from the surface, contain ligand accumulated within the lumen. The membranes of these larger vesicles contain little receptor, but receptor was concentrated in detached vesiculotubular extensions, which were largely free of ligand. These vesicles represent the compartment of uncoupling of receptor and ligand (CURL) during their common endocytosis. Ligand contained within the vesicle lumen is then transferred to multivesicular bodies and lysosomes; the tubular extensions may carry receptor back to the cell surface.