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.

FIGURE 1.

Binding of ApoA-V-DMPC to sortilin studied by SPR. CM5 sensor chips with immobilized sortilin (17 fmol/mm²) in the active flow cells were used for binding studies. An equivalent amount of immobilized bovine serum albumin was used in a reference flow cell. Identical injections of apoA-V-DMPC disks were made with or without pre/co-injections of NT (846 nm), RAP (7.7 fmol/mm²), or heparin (10 units/ml).

FIGURE 2.

Neurotensin causes dissociation of apoA-V-DMPC disks and chylomicrons from sortilin. Sensor chip surfaces were prepared as in Fig. 1. A, apoA-V-DMPC was injected to a response corresponding to a bound mass of 0.53 ng/mm². Subsequently, 30 μl of NT (846 nm) was injected (diluted in running buffer), and the dissociation of apoA-V-DMPC from sortilin was recorded. B, identical injections of chylomicrons were made with and without co-injections of NT (846 nm).

FIGURE 3.

ApoA-V-DMPC disks are bound and internalized by HEK293 cells overexpressing SorLA or sortilin. ApoA-V-DMPC-488 disks (green) were incubated with HEK293 cells transfected with the different receptors. The top two panels show binding to the cell surface after incubation at 4 °C (time 0), and the middle panels show binding after incubation at 37 °C for 30 min. The bottom panels show loss of binding to cells expressing prosortilin and hampered internalization to cells expressing receptor mutants modified in the internalization motif. Double staining with monoclonal anti-SorLA and anti-sortilin and Alexa Fluor 568-labeled secondary antibodies (red) was applied for the images showing 30-min internalization and prosortilin-transfected cells. wt, wild type.

FIGURE 4.

Fluorescence resonance energy transfer results showing receptor-apoA-V FRET interactions in vesicular regions in the cytosol of SorLA- and sortilin-expressing HEK293 cells. Energy transfer is registered as blue regions in the FRET analysis images. As donor-acceptor FRET pair, Alexa 488 and 568 were used. The constant behavior of the apparent FRET probability, E_app, as a function of acceptor signal level within ROIs of size 20–25 pixels, is indicative of specific interactions. A, analyses of SorLA-HEK cells; B, analyses of sortilin-HEK cells.

FIGURE 5.

The endocytic route for apoA-V visualized by markers for lysosomes, late endosomes, and the trans-Golgi network. HEK-SorLA and HEK-sortilin cells were stained for intracellular markers for lysosomes (Lamp-1), late endosomes (MPR300), and TGN (TGN46), all shown in red, after internalization of apoA-V488 (green). Co-localization is shown in yellow (white arrows).

FIGURE 6.

Degradation of ¹²⁵I-labeled apoA-V-DMPC disks in cells expressing SorLA and sortilin. ApoAV-DMPC disks labeled with ¹²⁵I were incubated at 37 °C with HEK293 cells expressing SorLA and sortilin; as a control, wild type HEK293 cells were used. Degradation of the tracer were measured at two different time points, 45 min (gray bars) and 90 min (black bars).