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Iron loading of isolated rat hepatocytes inhibits asialoglycoprotein receptor dynamics and induces formation of rat hepatic lectin-1 [correction of leptin-1] (RHL-1) oligomers.

ABSTRACT: The major subunit [rat hepatic lectin-1 (RHL-1)] of the asialoglycoprotein (ASGP) receptor mediates endocytosis of the iron-binding protein lactoferrin (Lf) by isolated rat hepatocytes, yet iron loading of cultured adult rat hepatocytes increases the binding and endocytosis of Lf while greatly inhibiting the uptake of desialylated ligand. In the present study, we determined whether the iron-induced Lf-binding site is RHL-1 and examined the nature of the iron-induced block in ASGP receptor endocytic function. Isolated rat hepatocytes increased their non-haem iron content from 70 to 470 p.p. b. following incubation with ferric ammonium citrate (<=100 microgram/ml). These conditions blocked internalization of 125I-asialo-orosomucoid (ASOR) by approximately 90% but increased 125I-Lf endocytosis by 40%. ASOR and anti-RHL-1 sera blocked the binding and endocytosis of 125I-Lf on control cells but not on iron-loaded cells, indicating that the iron-induced Lf-binding site on hepatocytes is not RHL-1. Iron-loading of hepatocytes in the presence or absence of excess ASOR did not significantly alter the number of active ASGP receptors on the cell surface. In contrast, iron-loading decreased the number of active intracellular receptors by 40% and blocked the uptake of 125I-ASOR prebound to the cells by approximately 80%. Under these conditions, we found an iron-dependent evolution of 88 and 140 kDa RHL-1-containing, beta-mercaptoethanol-sensitive multimers that constituted up to 34 and 23%, respectively, of total immunodetectable RHL-1. We propose that iron-induced formation of cystinyl-linked RHL-1-containing multimers inhibits ASGP receptor movement between cell surface and interior and disrupts acylation of intracellular receptors.

SUBMITTER: McAbee DD

PROVIDER: S-EPMC1219410 | biostudies-other | 1998 May

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)

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Iron loading of isolated rat hepatocytes inhibits asialoglycoprotein receptor dynamics and induces formation of rat hepatic lectin-1 [correction of leptin-1] (RHL-1) oligomers.

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