Recycling of the Hepatic Asialoglycoprotein Receptor in Isolated Rat Hepatocytes

We recently reported that the dissociation of internalized receptor-1261-asialo-orosomucoid (ASOR) complexes by isolated hepatocytes is a biphasic process; most complexes dissociate rapidly but 25-50% dissociate slowly (Oka, J. A., and Weigel, P. H. J. Biol. Chern. 258, 10253-10262). Cells were allowed to endocytose a pulse of surface-bound '''I-ASOR, and were washed and then incubated at 37 "C in the presence or absence of ethylene glycol bis(&aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA). Without EGTA, very little intact ASOR appeared in the medium. With EGTA present, a large amount of intracellular ligand appeared undegraded in the medium in a time-dependent manner. N-Acetylgalactosamine, but not ASOR, in the medium also caused release of intact '261-ASOR. Within 15 min, more than 50% and by completion at least 80% of the internalized ligand in the slow dissociation compartment was released into the medium. If cells containing internalized ligand were incubated at 37 "C for increasing times before the addition of EGTA, then progressively less ligand accumulated in the medium. Experiments at 18 "C, a temperature at which neither degradation nor slow dissociation occurred, demonstrated that in the presence of EGTA the intracellular free 1251-ASOR pool did not change. The amount of receptor-bound ligand in the slowly dissociating pool decreased and the amount of intact ligand in the medium increased by essentially equal amounts. The temperature dependence for the return of internal '261-ASOR to the cell surface was similar to that for endocytosis, with a cut-off temperature of about 12 "C. We conclude that a normal part of the endocytic process involves the return of receptor-ligand complexes to the cell surface from an internal slowly dissociating pool. This might reflect either an obligatory step or a reversible statistically random step in the endocytic/recycling pathway.