Endocytosis of Apolipoprotein A-V by Members of the Low Density Lipoprotein Receptor and the Vps10p Domain Receptor Families

Abstract

Apolipoprotein A-V (apoA-V) is present in low amounts in plasma and has been found to modulate triacylglycerol levels in humans and in animal models. ApoA-V displays affinity for members of the low density lipoprotein receptor (LDL-R) gene family, known as the classical lipoprotein receptors, including LRP1 and SorLA/LR11. In addition to LDL-A binding repeats, the mosaic receptor SorLA/LR11 also possesses a Vps10p domain. Here we show that apoA-V also binds to sortilin, a receptor from the Vsp10p domain gene family that lacks LDL-A repeats. Binding of apoA-V to sortilin was competed by neurotensin, a ligand that binds specifically to the Vps10p domain. To investigate the biological fate of receptor-bound apoA-V, binding experiments were conducted with cultured human embryonic kidney cells transfected with either SorLA/LR11 or sortilin. Compared with nontransfected cells, apoA-V binding to SorLA/ LR11-and sortilin-expressing cells was markedly enhanced. Internalization experiments, live imaging studies, and fluorescence resonance energy transfer analyses demonstrated that labeled apoA-V was rapidly internalized, co-localized with receptors in early endosomes, and followed the receptors through endosomes to the trans-Golgi network. The observed decrease of fluorescence signal intensity as a function of time during live imaging experiments suggested ligand uncoupling in endosomes with subsequent delivery to lysosomes for degradation. This interpretation was supported by experiments with 125 I-labeled apoA-V, demonstrating clear differences in degradation between transfected and nontransfected cells. We conclude that apoA-V binds to receptors possessing LDL-A repeats and Vsp10p domains and that apoA-V is internalized into cells via these receptors. This could be a mechanism by which apoA-V modulates lipoprotein metabolism in vivo.APOA5 is localized in the apolipoprotein APOA4/APOC3/ APOA1 gene cluster on human chromosome 11q23 (1). Transgenic mice expressing human apolipoprotein A-V (apoA-V) have decreased plasma triacylglycerol (TG) 2 levels, whereas APOA5 knock-out mice show increased plasma TG levels. Genetic variation in the human APOA5 locus correlate with changes in plasma lipoprotein levels (2-4), and a common polymorphism in APOA5 is significantly associated with increased risk for the metabolic syndrome (5, 6). Mutations in the APOA5 gene, leading to truncated apoA-V devoid of lipidbinding domains, have been demonstrated to cause severe hyperlipidemia if present in patients in the homozygous state (7).The mechanism whereby apoA-V exerts its effect on plasma TG levels is unknown. Animal studies and in vitro experiments have given rise to two different hypotheses for the function of apoA-V in vivo, inhibition of VLDL-TG production (8) or modulation of lipoprotein lipase (LPL) activity (8 -11). Adenovirusmediated gene transfer of human apoA-V into apoE-deficient mice decreased plasma TG levels as well as total plasma cholesterol levels without affecting LPL activity (12). In vitro experiments support a role fo...

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