Analysis of Familial Hypercholesterolemia Revealed the Pathway for Receptor Mediated Endocytosis of LDL Particles

There is no better example of the synergistic relation between basic molecular cell biology and medicine than the story of the discovery of the LDL receptor (LDLR) pathway for delivering cholesterol to cells. The series of elegant and Nobel Prize-winning studies leading to this discovery served as sources of insight into the mechanisms underlying LDL metabolism, the functions and properties of several key organelles, cellular systems for co-ordinately regulating complex metabolic pathways, and new approaches for treating atherosclerosis.

Some of these experiments compared LDL metabolism in normal human cells and in cells from patients with familial hypercholesterolemia (FH), a hereditary disease that is marked by elevated plasma LDL cholesterol and is now known to be caused by mutations in the LDLR gene. In patients who have one normal and one defective copy of the LDLR gene (heterozygotes), LDL cholesterol is increased about twofold. Those with two defective LDLR genes (homozygotes) have LDL cholesterol levels that are from fourfold to sixfold as high as normal. FH heterozygotes commonly develop cardiovascular disease about 10 years earlier than normal people do, and FH homozygotes usually die of heart attacks before reaching their late 20s. I

Here, we illustrate how analysis of the cellular defects underlying familial hypercholesterolemia can illuminate normal cellular processes. First let's consider typical cell-culture experiments in which the interactions of LDL with normal and FH homozygous cells were examined as a function of LDL concentration, which defined the high-affinity LDL receptor, and incubation temperature, which established the temperature dependence of LDL uptake. In these experiments, purified LDL was first labeled by the covalent attachment of radioactive 125I to the side chains of tyrosine residues in apoB-100 on the surfaces of the LDL particles. Cultured cells from normal persons and FH patients were incubated for several hours with the labeled LDL. Investigators then de termined how much LDL was bound to the surfaces of cells, how much was internalized, and how much of the apoB-100 component of the LDL was degraded by enzymatic hydrolysis to individual amino acids. The degradation of apoB-100 was detected by the release of 125I-tyrosine into the culture medium.

We can see from the results shown in Figure 18-14a that, compared with normal cells, homozygous FH cells are clearly defective in the binding and internalization of added LDL and in the degradation of apoB-100 at the normal physiologic temperature of 37 °C. The homozygous cells exhibit essentially no activity. Heterozygous cells exhibit about half the activity of normal cells. The shape of the binding curve for normal cells is consistent with a receptor that has a high affinity for LDL and is saturable. Note also that the curves for LDL internalization and degradation have the same shape as the binding curve. Moreover, when the experiments were performed with normal cells at 4 °C, LDL binding was observed, but internalization and degradation were inhibited (Figure 18-14b). Low temperature does not normally inhibit the binding of molecules to cell-surface receptors, but it does inhibit processes, such as the internalization and subsequent degradation of molecules, that depend on membrane trafficking (Chapter 17). Thus these results suggest that LDL first binds to cell-surface receptors and is subsequently internalized and degraded. One final feature of these results is worth noting. After cells were incubated for 5 hours at 37 °C, the amounts of internalized LDL and hydrolyzed apoB-100 were substantially greater than those of surface-bound LDL. This result indicates that each receptor molecule bound and mediated the internalization of more than one LDL particle in the incubation period. In other words, the LDL receptor is recycled.

Pulse-chase experiments with normal cells and a fixed concentration of 125I-labeled LDL helped to further define the time course of events in receptor-mediated cellular LDL processing. These experiments clearly demonstrate the order of events: surface binding of LDL ^ internalization ^ degradation (Figure 18-15). The results of electron microscopy studies with LDL particles tagged with an electron-dense label revealed that LDL first binds to clathrin-coated endocytic pits that invaginate and bud off to form coated vesicles and then endosomes (see Figure 17-27). Findings from further experiments showed that the LDL receptor recognizes apoB-100 and one or two closely related apolipopro-teins; thus binding by this receptor is highly specific for LDL. Binding is also pH dependent: strong binding of LDL occurs at the pH of extracellular fluid (7.4); weak or no binding occurs at the lower pH («4.5-6) found in some intracellular organelles (e.g., endosomes and lysosomes). Because of this property, the LDL receptor releases bound LDL within in-tracellular vesicles and can be recycled to the cell surface.

A variety of mutations in the gene encoding the LDL receptor can cause familial hypercholesterolemia. Some mutations prevent the synthesis of the LDLR protein; others prevent proper folding of the receptor protein in the ER,

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125I-LDL concentration
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