Abstract
The effects of the acidotropic agent, NH4Cl, on the recycling and turnover of low density lipoprotein (LDL) receptors were analyzed in human skin fibroblasts using ligand binding assays, [35S]methionine pulse-chase experiments, and electron microscopy. NH4Cl did not prevent receptor internalization but caused a marked redistribution of LDL receptors to intracellular sites (endosomes) that was completely dependent on the presence of apolipoprotein-B- or -E-containing ligands. Maximal inhibition of recycling was observed at LDL concentrations that only partially saturated receptors, suggesting that the receptors function as oligomers. In contrast, full receptor occupancy by the multivalent, apolipoprotein-E-containing beta-very low density lipoprotein was required for the same effect. The intracellular accumulation was reversible and the majority of receptors returned to the cell surface when NH4Cl was removed after short treatments. The rate of degradation of LDL receptors was greatly accelerated in the presence of NH4Cl and ligand, with a t1/2 of about 2 h (approximately 6 times faster than receptor degradation in the absence of NH4Cl). Neither the redistribution nor the accelerated loss of immunoprecipitable LDL receptors was observed in an LDL receptor internalization-defective mutant cell line. We conclude that NH4Cl inhibited the recycling specifically of occupied receptors, thereby accelerating their degradation, probably in endosomes.
Highlights
From the Medical Research Council/University of Cape Town, Research Unit for the Cell Biology of Medical Biochemistry, University of Cape Town Medical School, Cape Town, South Africa
The main conclusions of the present work are: (i) The recycling of occupied low density lipoprotein (LDL) receptors in fibroblasts is inhibited through the prevention of receptor-ligand dissociation by NH&l and this results in a redistribution of receptors between the cell surface and endosomes. (ii) The inhibition of LDL
Receptor recycling by NH,Cl requires only partial receptor occupancy by LDL, suggesting the existence of functional receptor oligomers. (iii) Inhibition of receptor recycling is followed by rapid receptor degradation that probably occurs in endosomes
Summary
From the Medical Research Council/University of Cape Town, Research Unit for the Cell Biology of Medical Biochemistry, University of Cape Town Medical School, Cape Town, South Africa. LDL Receptor Assays-For surface binding of lZ51-LDL at 4 “C, cells were incubated for 2 h in DMEM/LPDS
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