Kinetics of internalization and recycling of the asialoglycoprotein receptor in a hepatoma cell line.

Abstract

The presence of a large number of functional asialoglycoprotein receptors in the human hepatoma cell line Hep 6 2 has allowed us to determine the parameters which define ligand binding to the surface receptor, ligand receptor internalization, and receptor recycling.We have developed a simple kinetic model which encompasses all of these processes.These cells contain 226,000 functional asialoglycoprotein receptors, and at 37 "C uptake at a half-saturating concentration (2 pg/ ml) of '251-asialoorosomucoid (ASOR) is linear for several hours at a rate of 15,000 molecules/cell/min.Neither the rate of '261-ASOR binding at 4 "C nor the rate of uptake at 37 "C was altered under conditions in which protein synthesis was completely inhibited.Thus each receptor recycles a ligand, on the average, every 16 min.The rate of binding of '251-ASOR to cells at 37 "C, independent of internalization, was measured in the presence of 10-12 m~ azide.The rate of internalization at 37 "C was determined from cells, prebound with ligand at 4 "C, by following EDTA-resistant cellassociated "%ASOR.The distribution of functional receptors within the cell was assessed from ligand binding at 4 "C and ligand uptake at 37 "C in cells pretreated with trypsin to remove all surface receptors.In growing Hep G2 cells 87% of the receptors are on the cell surface, and over 88% of these are unoccupied with ligand.During steady state internalization of '"1-ASOR, the number of surface ASOR receptors decreases 17%.At 2 pg/ml of '261-ASOR, an unoccupied surface receptor requires a mean time of 8.7 min to bind a ligand, and a mean time of 2.2-2.3 min is required to internalize a surface receptor-ligand complex.An additional 4.2 min is required for ligand to dissociate from an internalized receptor and for the receptor to return to the cell surface.We have been able to determine or calculate most of these parameters of receptor recyclindby two or more independent sets of measurements.Many serum glycoproteins and peptide hormones are bound to the cell surface by specific protein receptors.The subsequent fate of the ligand and of the receptor is dependent on the rates and extents of the following reactions: the binding of