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Differential effects of leupeptin, monensin and colchicine on ligand degradation mediated by the two asialoglycoprotein receptor pathways in isolated rat hepatocytes.

ABSTRACT: We have shown that degradation of asialo-orosomucoid (ASOR) in isolated rat hepatocytes occurs by two different intracellular pathways [Clarke, Oka & Weigel (1987) J. Biol. Chem. 262, 17384-17392] mediated by two subpopulations of cell surface galactosyl (Gal) receptors, designated State 1 or State 2 receptors. In the present study, several inhibitors were tested for their effects on ligand degradation by the State 1 or State 2 pathway. Leupeptin, monensin and chloroquine completely inhibited degradation of 125I-labelled ASOR in both pathways. Dose-response studies showed, however, that the State 2 pathway was more sensitive to leupeptin or monensin than the State 1 pathway. No differences were observed with chloroquine. For example, the onset of inhibition in the State 2 and State 1 pathways occurred at about 0.05 and 0.3 microM-leupeptin respectively, a 6-fold difference. At 3.5 microM-monensin, 125I-ASOR degradation in the State 2 pathway was completely blocked, whereas degradation in the State 1 pathway was essentially unaffected. Colchicine was observed to give the largest differential sensitivity between the two pathways. The State 2 degradation pathway was about 30-fold more sensitive to colchicine than the State 1 pathway. Lumicolchicine had no affect. The onset of inhibition of the rate of 125I-ASOR degradation in the State 2 and State 1 pathways occurred at approximately 0.1 and 3.0 microM-colchicine respectively. At very high concentrations (greater than 0.1 mM), the State 1 pathway could be completely inhibited. We conclude that intracellular 125I-ASOR processing or delivery to degradative compartments in both the State 1 and State 2 Gal receptor pathways requires low pH. Ligand delivery to the degradative compartment does not require microtubules in the State 1 pathway, consistent with the very rapid onset of degradation in this pathway. The State 2 degradation pathway does require microtubules.

SUBMITTER: Clarke BL

PROVIDER: S-EPMC1133258 | biostudies-other | 1989 Aug

REPOSITORIES: biostudies-other

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Project description:In isolated rat hepatocytes, a radiolabelled tyramine-cellobiose conjugate of asialo-orosomucoid, 125I-TC-AOM, was rapidly taken up by receptor-mediated endocytosis and proteolytically degraded in the lysosomes, where radioactive degradation products accumulated. Okadaic acid and other protein phosphatase inhibitors (microcystin-LR, calyculin A) strongly reduced the fraction of asialoglycoprotein (ASGP) receptors localized to the cell surface, and correspondingly inhibited the uptake of 125I-TC-AOM. In addition, the inhibitors suppressed 125I-TC-AOM degradation strongly (90% at 150 nM) and potently (half-maximal effect at 20 nM okadaic acid), indicating an involvement of protein phosphorylation, and of a protein phosphatase of type 2A, in the regulation of intracellular endocytic flux. The effects of okadaic acid on 125I-TC-AOM accumulation, as well as on degradation, could be eliminated by the protein kinase inhibitor genistein. Okadaic acid prevented the transfer of 125I-TC-AOM to a non-recycling endocytic compartment, causing its retention in a recycling compartment from which about one-third of the endocytosed 125I-TC-AOM could be returned to the cell surface and 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 125I-TC-AOM. Sucrose density gradient analysis and sedimentation studies indicated that okadaic acid caused accumulation of 125I-TC-AOM in light endosomes (1.11 g/ml), preventing its transfer to dense endosomes (1.14 g/ml) and lysosomes (1.18 g/ml). The lysosomes could be identified in density gradients by their contents of lysosomal marker enzymes and acid-soluble radioactivity, and by their sensitivity towards the lysosome-disrupting agent glycyl-L-phenylalanine-2-naphthylamide. By using endocytosed AOM-gold particles 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 and multivesicular endosomes. Whereas in control cells 4% of the AOM-gold was in small vesicles/tubules, 55% in multivesicular endosomes and 41% in lysosomes, the corresponding figures for okadaic acid-treated cells were 17%, 73% and 11%. Our results thus indicate that protein phosphatase inhibitors have two effects on ASGP endocytosis: (1) an early inhibition of ligand uptake, due to a reduction in the fraction of ASGP receptors at the cell surface, and (2) an inhibition of ASGP transfer from a recycling compartment consisting of light, small endocytic vesicles and multivesicular endosomes, to a non-recycling compartment consisting of dense multivesicular endosomes.(ABSTRACT TRUNCATED AT 400 WORDS)

Project description:The hepatic carbohydrate-recognizing asialoglycoprotein receptor (ASGR1) mediates the endocytosis/lysosomal degradation of desialylated glycoproteins following binding to terminal galactose/N-acetylgalactosamine. Human heterozygote carriers of ASGR1 deletions exhibit ∼34% lower risk of coronary artery disease and ∼10% to 14% reduction of non-HDL cholesterol. Since the proprotein convertase PCSK9 is a major degrader of the low-density lipoprotein receptor (LDLR), we investigated the degradation and functionality of LDLR and/or PCSK9 by endogenous/overexpressed ASGR1 using Western blot and immunofluorescence in HepG2-naïve and HepG2-PCSK9-knockout cells. ASGR1, like PCSK9, targets LDLR, and both independently interact with/enhance the degradation of the receptor. This lack of cooperativity between PCSK9 and ASGR1 was confirmed in livers of wildtype (WT) and Pcsk9-/- mice. ASGR1 knockdown in HepG2-naïve cells significantly increased total (∼1.2-fold) and cell-surface (∼4-fold) LDLR protein. In HepG2-PCSK9-knockout cells, ASGR1 silencing led to ∼2-fold higher levels of LDLR protein and DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate)-LDL uptake associated with ∼9-fold increased cell-surface LDLR. Overexpression of WT-ASGR1/2 primarily reduced levels of immature non-O-glycosylated LDLR (∼110 kDa), whereas the triple Ala-mutant of Gln240/Trp244/Glu253 (characterized by loss of carbohydrate binding) reduced expression of the mature form of LDLR (∼150 kDa), suggesting that ASGR1 binds the LDLR in both a sugar-dependent and -independent fashion. The protease furin cleaves ASGR1 at the RKMK103↓ motif into a secreted form, likely resulting in a loss of function on LDLR. Altogether, we demonstrate that LDLR is the first example of a liver-receptor ligand of ASGR1. We conclude that silencing of ASGR1 and PCSK9 may lead to higher LDL uptake by hepatocytes, thereby providing a novel approach to further reduce LDL cholesterol levels.

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Differential effects of leupeptin, monensin and colchicine on ligand degradation mediated by the two asialoglycoprotein receptor pathways in isolated rat hepatocytes.

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