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First published online 10 June 2008
doi: 10.1242/jcs.020917

Journal of Cell Science 121, 2136-2147 (2008)
Published by The Company of Biologists 2008
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Oxidised LDL internalisation by the LOX-1 scavenger receptor is dependent on a novel cytoplasmic motif and is regulated by dynamin-2

Jane E. Murphy1, Ravinder S. Vohra1, Sarah Dunn1, Zoe G. Holloway2, Anthony P. Monaco2, Shervanthi Homer-Vanniasinkam1, John H. Walker1 and Sreenivasan Ponnambalam1,*

1 Endothelial Cell Biology Unit, Leeds Institute of Genetics, Health & Therapeutics, University of Leeds, Clarendon Way, Leeds, LS2 9JT, UK
2 Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Headington, UK


Figure 1
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  		Fig. 1. OxLDL binding to HeLa cells expressing LOX-1. (A) Domain structure of the human LOX-1-FLAG construct. CD, cytoplasmic domain; TMD, transmembrane domain. (B) Equal quantities of total cell lysate (30 µg) from mock-transfected and LOX-1-FLAG transfected cells were analysed by western blotting using sheep anti-LOX-1 antibodies or sheep antibodies that recognise a ubiquitous Golgi membrane glycoprotein and loading control (TGN46). (C) Mock and LOX-1-FLAG transfected HeLa cells were incubated with labelled DiI-OxLDL ligand (red) prior to cell fixation, processed and stained with a mouse anti-FLAG antibody. Ligand binding was also performed after pre-incubation with the JTX92 LOX-1 blocking antibody. Cell surface LOX-1 was detected by the mouse anti-FLAG and FITC-conjugated anti-mouse IgG antibodies (green). (D) HeLa cells transfected with LOX-1-FLAG were incubated with DiI-OxLDL (red) in DMEM prior to washing and chasing for different time periods before fixation. Nuclei were stained with DAPI (blue). Scale bar: 20 µm.

 

Figure 2
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  		Fig. 2. OxLDL colocalisation with LOX-1. (A) HeLa cells transfected with LOX-1-FLAG were incubated with labelled OxLDL (red) and mouse anti-FLAG antibodies on ice (1 hour) and then warmed to 37°C for the indicated times before fixation. Cells were permeabilised and antibodies bound to LOX-1 detected using FITC-conjugated anti-mouse IgG antibodies (green). Boxes display enlarged areas with colocalisation appearing yellow. Scale bar: 10 µm. (B) Amount of colocalisation of labelled OxLDL and LOX-1 was calculated (n=3 separate experiments in each of which five cells were quantified, values are mean ± s.e.m.). The P values calculated compared with values at t=0 minutes are indicated by asterisks, *P<0.05; **P<0.01. Values were expressed as a percentage of the colocalisation at t=0 minutes.

 

Figure 3
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  		Fig. 3. Internalisation and half-life of LOX-1. Serum-starved LOX-1-FLAG transfected HeLa cells were incubated with or without saturating OxLDL levels for 1 hour and biotinylated as described in Materials and Methods. (A) Cells were incubated at 37°C for 0, 15 or 30 minutes before cell surface biotin cleavage followed by cell lysis. (B) Biotinylated cells were incubated at 37°C for different time periods before lysis. Biotinylated proteins were isolated and analysed by western blotting with purified anti-LOX-1 antibodies. The blot in A was reprobed with mouse anti-TfR (control). The amount of biotinylated LOX-1 was quantified using densitometry (mean ± s.e.m. of three experiments) and compared with total cell surface LOX-1 (t=0 minutes) to quantify percentage of LOX-1 internalised (A) or remaining (B).

 

Figure 4
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  		Fig. 4. Dynamin-2 regulates LOX-1-mediated OxLDL internalisation. HeLa cells cotransfected with tagged LOX-1 and Myc-tagged wild-type (WT) dynamin-2 or Myc-tagged K44A dominant-negative dynamin-2 were incubated with (A) rhodamine-conjugated transferrin (red) for 15 minutes or (B) pulsed with OxLDL (red) for 5 minutes followed by chase at 37°C (for 55 minutes) before fixation and labelling with mouse anti-Myc and FITC-conjugated anti-mouse IgG antibodies (green). Panels in A represent whole cell projections and nuclei are stained with DAPI (blue). Images in B represent 0.5 µm optical sections through the cell nucleus (middle) or at the cell apex (top). Asterisks indicate cell nuclei. (C) HeLa cells cotransfected with tagged LOX-1 and Myc-tagged WT dynamin-2 or Myc-tagged dominant-negative dynamin-2 were pulsed with labelled OxLDL (red, i) followed by fixation and incubation with sheep anti-LOX-1 and Cy5-conjugated anti-sheep IgG antibodies (displayed as green, ii) and mouse anti-Myc and FITC-conjugated anti-mouse IgG antibodies (not shown). (iii) Merged images with colocalisation of labelled OxLDL and LOX-1 shown as yellow. (iv) Z-axis image view of cells shown in panel iii. Images represent projected stacks of whole cells. (D) Labelling of cell surface glycoproteins and LOX-1 on HeLa cells transfected with LOX-1 and dominant-negative dynamin-2 using FITC-conjugated Con A (Con A, green, i) followed by sheep anti-LOX-1 and Alexa Fluor 594-conjugated anti-sheep IgG antibodies (red, ii) and mouse anti-Myc and Alexa Fluor 633-conjugated anti-mouse IgG antibodies (not shown). Con A labelling was performed on ice prior to fixation and processing for microscopy. (iii) Merged image with colocalisation appearing yellow. (iv) Z-axis image view of cells shown in panel iii. Images represent projected stacks of whole cells. Scale bars: 10 µm. (E) The percentage of WT or DN dynamin-2 transfected cells with internalised labelled OxLDL was evaluated. Data represent the mean ± s.e.m. (n=3 experiments). Comparison to control to calculate P values, *P<0.001.

 

Figure 5
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  		Fig. 5. LOX-1-dependent OxLDL endocytosis is inhibited by potassium depletion. (A) HeLa cells were incubated with rhodamine-conjugated transferrin (red) for 15 minutes under control conditions or in potassium-free or hypertonic buffers as described in Materials and Methods. The plasma membrane was labelled with FITC-conjugated Con A (green) immediately before fixation. (B) LOX-1 expressing HeLa cells were pulsed with labelled OxLDL (red) for 5 minutes and chased at 37°C (for 55 minutes) in control, potassium-free or hypertonic buffers before labelling the plasma membrane with FITC-conjugated Con A (green) and fixation. (C) Addition of exogenous 10 mM KCl or addition of normal media for 1 hour reverses the inhibitory effects (washout) on labelled OxLDL uptake. Scale bars: 10 µm. Asterisks indicate cell nuclei. (D) The amount of internalised OxLDL under these conditions was again calculated as described in Materials and Methods (n=3 separate experiments in each of which five cells were quantified, values are mean ± s.e.m.). Comparison with control to calculate P values, *P<0.05.

 

Figure 6
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  		Fig. 6. Clathrin and AP2-independent uptake of OxLDL by LOX-1. Following RNAi, LOX-1 was expressed in transfected HeLa cells and labelled transferrin and OxLDL uptake was monitored (see Materials and Methods) by microscopy (A) and quantified (C). (A) HeLa cells subjected to RNAi through mock treatment (mock), a control scrambled siRNA duplex (scrambled), a siRNA duplex specific for the clathrin heavy chain (CHC17) or a siRNA duplex specific for the µ2 subunit of the AP2 adaptor complex (µ2) on cells expressing LOX-1-FLAG. After 12 hours, cells were incubated with Alexa Fluor 488-transferrin and DiI-OxLDL for 15 minutes followed by 30 minute chase and then fixation and confocal laser-scanning microscopy. Arrows (left hand panels) indicate plasma membrane transferrin accumulation in cells (*) where clathrin or AP2-mediated uptake is inhibited. In right panels, transverse z-axis sections are also shown to visualise intracellular staining; small arrows denote endosomes containing labelled OxLDL. Scale bars: 10 µm. (B) Western blotting to demonstrate depletion of endogenous protein levels after RNAi treatment using CHC17 siRNA (lane 1), µ2 siRNA (lane 2), scrambled siRNA (lane 3) and mock-transfected cells (lane 4). (C) Quantification of uptake of labelled transferrin and OxLDL ligands in LOX-1-transfected HeLa cells (n=30, error bars indicate s.e.m.) was carried out as described in Materials and Methods.

 

Figure 7
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  		Fig. 7. The LOX-1 cytoplasmic domain contains a novel aspartate-based endocytic motif. (A) Multiple sequence alignment of mammalian LOX-1 cytoplasmic domains. Completely conserved amino acid residues are indicated by an asterisk, conservative amino acid substitutions are indicated by a colon and predominantly conserved amino acid substitutions are indicated by a full stop. LOX-1 wild-type and mutant proteins were expressed and OxLDL uptake monitored (see Materials and Methods) by microscopy (A) and quantified (B). (A) Fixed cells were labelled with mouse anti-FLAG and FITC-conjugated anti-mouse IgG antibodies. Arrows indicates areas of clustered LOX-1 and OxLDL ligand at the plasma membrane. (C) The uptake of labelled OxLDL in LOX-1-transfected HeLa cells were quantified (see Materials and Methods). The percentage of transfected cells with internalised OxLDL was counted (n=3 separate experiments, 50 cells from each experiment, mean ± s.e.m.). Comparison with control LOX-1 WT was used to calculate P values, *P<0.01.

 

Figure 8
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  		Fig. 8. Model for trafficking of the LOX-1-OxLDL complex. The findings indicate that LOX-1 constitutively cycles between the plasma membrane (PM) and endosomes (E) in the absence of ligand. LOX-1 and LOX-1-OxLDL complexes are internalised via a dynamin-2 and clathrin-independent mechanism, which may involve the recruitment of cytosolic factors. The majority of LOX-1 dissociates from OxLDL early in the endocytic pathway and may recycle to the plasma membrane, whereas the OxLDL traffics to later endocytic compartments (L). It is likely that some LOX-1 does not dissociate from OxLDL and traffics to later endocytic compartments with subsequent degradation in a late endocytic or lysosomal compartment. Thus, LOX-1 is able to mediate the continuous uptake of OxLDL into the cell.

 

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© The Company of Biologists Ltd 2008