INHIBITION OF ASIALOGLYCOPROTEIN ENDOCYTOSIS AND DEGRADATION IN RAT HEPATOCYTES BY PROTEIN PHOSPHATASE INHIBITORS

In isolated rat hepatocytes, a radiolabelled tyramine-cellobiose conju gate of asialo-orosomucoid, I-125-TC-AOM, was rapidly taken up by rece ptor-mediated endocytosis and proteolytically degraded in the lysosome s, where radioactive degradation products accumulated. Okadaic acid an d other protein phosphatase inhibitors (microcystin-LR, calyculin A) s trongly reduced the fraction of asialoglycoprotein (ASGP) receptors lo calized to the cell surface, and correspondingly inhibited the uptake of I-125-TC-AOM. In addition, the inhibitors suppressed I-125-TC-AOM d egradation strongly (90 % at 150 nM) and potently (half-maximal effect at 20 nM okadaic acid), indicating an involvement of protein phosphor ylation, and of a protein phosphatase of type 2A, in the regulation of intracellular endocytic flux. The effects of okadaic acid on I-125-TC -AOM accumulation, as well as on degradation, could be eliminated by t he protein kinase inhibitor genistein. Okadaic acid prevented the tran sfer of I-125-TC-AOM to a non-recycling endocytic compartment, causing its retention in a recycling compartment from which about one-third o f the endocytosed I-125-TC-AOM could be returned to the cell surface a nd detached from its receptor in the presence of EGTA. ASGP receptors recycled extensively both in the presence and absence of okadaic acid, as indicated by a sustained uptake of (TC)-T-125-AOM. Sucrose density gradient analysis and sedimentation studies indicated that okadaic ac id caused accumulation of I-125-TC-AOM in light endosomes (1.11g/ml), preventing its transfer to dense endosomes (114 g/ml) and lysosomes (1 .18 g/ml). The lysosomes could be identified in density gradients by t heir contents of lysosomal marker enzymes and acid-soluble radioactivi ty, and by their sensitivity towards the lysosome-disrupting agent gly cyl-L-phenylalanine-2-naphthylamide, By using endocytosed AOM-gold par ticles as an ultrastructural endocytic marker, it could be shown that the light endosomes accumulating ASGP in the presence of okadaic acid had the morphological appearance of small endocytic vesicles/tubules a nd multivesicular endosomes. Whereas in control cells 4% of the AOM-go ld was in small vesicles/tubules, 55% in multivesicular endosomes and 41% in lysosomes, the corresponding figures for okadaic acid-treated c ells were 17%, 73 % and 11%. Our results thus indicate that protein ph osphatase inhibitors have two effects on ASGP endocytosis: (1) an earl y inhibition of ligand uptake, due to a reduction in the fraction of A SGP receptors at the cell surface, and (2) an inhibition of ASGP trans fer from a recycling compartment consisting of light, small endocytic vesicles and multivesicular endosomes, to a non-recycling compartment consisting of dense multivesicular endosomes. This late step may repre sent the detachment of multivesicular endosomes from a continuous tubu lovesicular network, a process apparently subject to regulation by a g enistein-sensitive protein kinase and a protein phosphatase of type 2A .