Degradation of asialoglycoproteins mediated by the galactosyl receptor system in isolated hepatocytes. Evidence for two parallel pathways

The ability of rat hepatocytes to degrade internalized surface-bound ¹²⁵I-asialoorosomucoid (ASOR) was determined by measuring the appearance of acid-soluble radioactive at 37°C. The degradation kinetics were biphasic in cells previously equilibrated at 37°C for 1 h or cultured for 24 h. Degradation began immediately and was linear for at least 20 min after which the rate increased to a steady state value 3-4 times greater than the initial rate. We previously showed that hepatocytes have two functionally distinct populations of galactosyl receptors that mediate ligand dissociation by two kinetically different pathways (Weigel, P.H., Clarke, B.L., and Oka, J.A. (1986) Biochem. Biophys. Res. Commun. 140, 43-50). The activity of one receptor population, designated State 2 galactosyl receptors, can be reversibly modulated by incubating cells between 22 and 37°C and is not expressed on the surface of freshly isolated cells. When ¹²⁵I-ASOR was prebound to freshly isolated cells at 4°C and degradation was assessed subsequently at 37°C, the kinetics were monophasic, not biphasic. Degradation of the surface-bound ¹²⁵I-ASOR began immediately and was >90% complete by 6 h. Freshly isolated cells were incubated at temperatures between 22 and 37°C, chilled to 4°C, allowed to pre-bind ¹²⁵I-ASOR, and then incubated at 37°C. As the State 2 galactosyl receptor population increased, the kinetics of degradation became progressively more biphasic and the rate of the delayed degradation process increased. This effect could be reversed in cells in culture or in suspension by down-modulating surface receptor activity at temperatures below 37°C; only the degradation process appearing after a 20-min lag was affected. Degradation in both pathways is an apparent first order process with identical rate constants (k = 0.006 min^-1, t( 1/2 ) = 116 min). We conclude that there are two separate pathways by which asialoglycoproteins are degraded. The major 'classic' pathway mediated by State 2 galactosyl receptors occurs after a 20-min lag and the minor pathway mediated by State 1 galactosyl receptors begins immediately with no detectable lag.