Low-density lipoprotein endocytosis. I. Influence of the multivalent ligand cationized ferritin on normal and receptor-negative human fibroblasts.
Based upon the observation that the multivalent ligand cationized ferritin (CF) alters the cell surface distribution of anionic domains and significantly enhances the adsorptive endocytosis of 125I-labeled human serum albumin, these studies were undertaken to probe the influence of CF on receptor-mediated low-density lipoprotein (LDL) endocytosis and the nature of the mechanisms involved. A brief 1-min exposure of normal receptor upregulated fibroblasts to CF (0.2 mg/ml) resulted in a significant decrease (P less than 0.001) in the subsequent internalization and degradation of 125I-LDL. Studies with receptor downregulated normal fibroblasts indicated that CF pretreatment did not measurably influence 125I-LDL internalization and only slightly inhibited its degradation (P less than 0.05). In contrast, CF pretreatment of FH receptor-negative mutant skin fibroblasts resulted in a modest but significant increase in both 125I-LDL internalization and degradation (P less than 0.05). Scatchard analyses of binding data indicated that CF-pretreated upregulated normal fibroblasts exhibit a single class of LDL binding sites with an affinity, Kd = 24.7 +/- 4.1 nM, almost 10-fold lower than the affinity of binding sites in untreated controls, Kd = 3.2 +/- 0.06 nM. Increasing either the concentration or the duration of CF exposure resulted in additional inhibition of LDL internalization and degradation associated primarily with a decrease in the number of LDL binding sites without any further change in binding affinity. Total cellular LDL receptor-mediated binding, measured using an octylglucoside solubilization-filtration assay, confirmed the CF-induced decrease in high-affinity LDL binding. Pulse-chase experiments showed that CF had no direct influence on LDL degradation, nor did it influence targeting of the LDL-containing endosome toward exocytosis. Further, restoration of LDL receptor function to control values after CF pretreatment required de novo protein synthesis. The normal feedback inhibition of HMG-CoA reductase activity was nearly abolished by CF pretreatment. Additionally, CF pretreatment was found to induce not only a redistribution of surface anionic sites, but also a very rapid internalization of surface components labeled with 4,4'-[3H]diisothiocyano-1,2..diphenylethane-2,2'-disulfonic acid. It is concluded that the inhibitory influence of CF on LDL endocytosis is mediated via a decrease in the affinity and in the number of functional LDL receptors.
Edwards
E H
EH
Department of Pathology, University of Texas Health Science Center, San Antonio 78284.
Sprague
E A
EA
Schwartz
C J
CJ
D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D004705:Endocytosis / Q000187:drug effects*; D005293:Ferritins / Q000494:pharmacology*; D005347:Fibroblasts / Q000187:drug effects* / Q000502:physiology; D006801:Humans; D006903:Hydroxymethylglutaryl CoA Reductases / Q000378:metabolism; D006937:Hypercholesterolemia / Q000473:pathology; D008077:Lipoproteins, LDL / Q000172:deficiency / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D011973:Receptors, LDL / Q000378:metabolism*
D008077:Lipoproteins, LDL; D008565:Membrane Proteins; D011973:Receptors, LDL; D005293:Ferritins; D003513:Cycloheximide; D006903:Hydroxymethylglutaryl CoA Reductases