Oxidised LDL internalisation by the LOX-1 scavenger receptor is dependent on a novel cytoplasmic motif and is regulated by dynamin-2.
ABSTRACT The LOX-1 scavenger receptor recognises pro-atherogenic oxidised low-density lipoprotein (OxLDL) particles and is implicated in atherosclerotic plaque formation, but this mechanism is not well understood. Here we show evidence for a novel clathrin-independent and cytosolic-signal-dependent pathway that regulates LOX-1-mediated OxLDL internalisation. Cell surface labelling in the absence or presence of OxLDL ligand showed that LOX-1 is constitutively internalised from the plasma membrane and its half-life is not altered upon ligand binding and trafficking. We show that LOX-1-mediated OxLDL uptake is disrupted by overexpression of dominant-negative dynamin-2 but unaffected by CHC17 or mu2 (AP2) depletion. Site-directed mutagenesis revealed a conserved and novel cytoplasmic tripeptide motif (DDL) that regulates LOX-1-mediated endocytosis of OxLDL. Taken together, these findings indicate that LOX-1 is internalised by a clathrin-independent and dynamin-2-dependent pathway and is thus likely to mediate OxLDL trafficking in vascular tissues.
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. Walker1and Sreenivasan Ponnambalam1,*
1Endothelial Cell Biology Unit, Leeds Institute of Genetics, Health & Therapeutics, University of Leeds, Clarendon Way, Leeds, LS2 9JT, UK
2Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Headington, UK
*Author for correspondence (e-mail: email@example.com)
Oxidised low-density lipoprotein (OxLDL) is a key initiating factor
in atherosclerosis that is recognised by vascular scavenger receptors
and triggers conversion of macrophages into lipid-laden foam cells
during plaque development, endothelial dysfunction and cellular
apoptosis (Lusis, 2000). Lectin-like oxidised low-density lipoprotein
receptor-1 (LOX-1, also known as OLR1 and LOX1) is a class E
scavenger receptor originally cloned as the major receptor for
OxLDL on endothelial cells (Sawamura et al., 1997). LOX-1 is also
expressed in macrophages, smooth muscle cells and platelets
(Aoyama et al., 2000; Chen et al., 2001a; Yoshida et al., 1998).
LOX-1 is a Type II membrane glycoprotein with an extracellular
C-type lectin-like ligand-binding domain (Chen et al., 2001b; Ohki
et al., 2005), which binds to a diverse range of ligands including
OxLDL, phosphatidylserine, apoptotic bodies, bacteria and platelets
(Kakutani et al., 2000; Murphy et al., 2006; Oka et al., 1998;
Shimaoka et al., 2001).
Genetic and biochemical evidence indicates a role for LOX-
1 in cardiovascular disease initiation and progression (Vohra et
al., 2006; Dunn et al., 2008). LOX-1 levels are elevated within
atherosclerotic plaques (Chen et al., 2000; Kataoka et al., 1999)
and LOX-1 allelic polymorphisms can confer an increased risk
of cardiovascular disease (Mango et al., 2003; Tatsuguchi et al.,
2003). Furthermore, studies on LOX-1-null mice support the view
that expression of the gene encoding LOX-1 accelerates the
initiation and progression of atherosclerotic plaques (Mehta et
al., 2007). LOX-1-mediated OxLDL binding in macrophages
stimulates the formation of lipid-laden cells that resemble the
foam cells found in atherosclerotic plaques (Smirnova et al.,
However, the molecular and cellular biology underlying LOX-
1-mediated OxLDL trafficking is not understood. The best
understood mechanisms of receptor-ligand internalisation are
clathrin-mediated endocytosis and caveolae- or lipid-raft-mediated
uptake (Conner and Schmid, 2003; Mayor and Pagano, 2007) as
candidates for LOX-1-mediated ligand uptake. However, the
epidermal growth factor receptor (EGFR/ErbB1) can undergo
regulated internalisation by both clathrin and lipid-raft-dependent
routes. Clathrin-mediated endocytosis may result in EGFR
recycling to the cell surface whereas lipid raft-mediated
internalisation is thought to result in EGFR degradation in
lysosomes (Sigismund et al., 2005). The route taken by EGFR is
dependent on activation caused by binding of EGF ligand
(Sigismund et al., 2005). Another receptor tyrosine kinase that is
degraded in response to ligand binding is vascular endothelial
growth factor receptor 2 (VEGFR2) (Ewan et al., 2006). Cellular
responses to ligands that activate intracellular signalling require
co-ordination with receptor-ligand trafficking, recycling or
degradation in the endosome-lysosome system to ensure spatial
and temporal regulation of physiological responses.
It is not known whether LOX-1 has a role as an OxLDL
trafficking receptor. Here, we have utilised a biochemical and
cellular approach to elucidate this mechanism: we have identified
a constitutive internalisation pathway for LOX-1 that is regulated
by a key GTPase in conjunction with recognition of a novel LOX-
1 cytoplasmic motif. In our model, extracellular OxLDL undergoes
The LOX-1 scavenger receptor recognises pro-atherogenic
oxidised low-density lipoprotein (OxLDL) particles and is
implicated in atherosclerotic plaque formation, but this
mechanism is not well understood. Here we show evidence for
a novel clathrin-independent and cytosolic-signal-dependent
pathway that regulates
internalisation. Cell surface labelling in the absence or presence
of OxLDL ligand showed that LOX-1 is constitutively
internalised from the plasma membrane and its half-life is not
altered upon ligand binding and trafficking. We show that LOX-
1-mediated OxLDL uptake is disrupted by overexpression of
dominant-negative dynamin-2 but unaffected by CHC17 or μ μ2
(AP2) depletion. Site-directed mutagenesis revealed a conserved
and novel cytoplasmic tripeptide motif (DDL) that regulates
LOX-1-mediated endocytosis of OxLDL. Taken together, these
findings indicate that LOX-1 is internalised by a clathrin-
independent and dynamin-2-dependent pathway and is thus
likely to mediate OxLDL trafficking in vascular tissues.
Supplementary material available online at
Key words: LOX-1, OxLDL, Endocytosis, Dynamin-2
Accepted 9 April 2008
Journal of Cell Science 121, 2136-2147 Published by The Company of Biologists 2008
Journal of Cell Science
Endocytosis of OxLDL via LOX-1
constitutive internalisation via LOX-1 and delivery to the endosome-
OxLDL endocytosis is LOX-1 dependent
HeLa cells were transfected with the full-length human LOX-1
cDNA containing an engineered FLAG-tag at the extreme C-
terminus (LOX-1-FLAG) (Fig. 1A) and LOX-1 expression assessed
by western blotting. HeLa cells transfected with the LOX-1-FLAG
construct expressed a ~40 kDa polypeptide that was detected by
anti-LOX-1 antibodies, but this species was absent in mock-
transfected cells (Fig. 1B). This LOX-1 polypeptide corresponds
to the molecular mass of human LOX-1 detected in vascular tissues
and leukocytes (Xie et al., 2004; Yoshida et al., 1998). This LOX-
1 polypeptide represents an N-glycosylated membrane protein,
because the mature protein is sensitive to tunicamycin and PNGase
F (see supplementary material Fig. S1). In addition, this LOX-1
molecule was detected on the surface of transfected cells (Fig. 1C)
and corresponds to the steady-state localisation of endogenous or
transfected LOX-1 mammalian orthologues from other studies
(Chen and Sawamura, 2005; Sawamura et al., 1997).
To assay for binding of OxLDL to LOX-1, fluorescent labelled
DiI-OxLDL was incubated with mock-transfected or LOX-1-FLAG-
expressing cells. DiI-OxLDL only bound to HeLa cells expressing
LOX-1-FLAG (Fig. 1C). To further confirm that binding of OxLDL
was LOX-1 dependent, LOX-1-FLAG-transfected cells were pre-
incubated with JTX92, an anti-human LOX-1 antibody that blocks
LOX-1 binding to ligand (Li et al., 2003). As expected, the JTX92
antibody blocked OxLDL binding to HeLa cells expressing LOX-
1 (Fig. 1C). By contrast, LOX-1 transfected cells incubated with
labelled OxLDL revealed accumulation of ligand in punctate
perinuclear structures within 1 hour of incubation (Fig. 1D). The
codistribution of labelled OxLDL and LOX-1 over time was
quantified (Fig. 2). OxLDL colocalised extensively with LOX-1
during the first 15 minutes of internalisation but after 60 minutes,
the colocalisation between OxLDL and LOX-1 had been reduced
to 24% of that at the starting point (t=0). This indicated that the
majority of LOX-1 and OxLDL dissociated from each other early
in the endocytic pathway. We attempted to elucidate the effects of
antibody labelling of cell surface LOX-1 and subsequent intracellular
fate of LOX-1 but this was unsuccessful (data not shown).
Ligand-independent endocytosis of LOX-1
To determine whether LOX-1 internalisation is dependent on
OxLDL binding, serum-starved LOX-1-FLAG-expressing cells
were incubated with or without a saturating concentration of
OxLDL (data not shown) at 4°C followed by cell surface labelling
and biochemical analysis. Cell surface proteins were biotinylated
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.
Journal of Cell Science
2138Journal of Cell Science 121 (13)
using sulfo-NHS-S-S-biotin before incubation at 37°C for 15 or 30
minutes to stimulate endocytosis. This was followed by treatment
with the non-permeable reducing agent MESNA to cleave the biotin
moiety from the remaining labelled cell surface proteins. Internalised
and biotinylated proteins were isolated from detergent-solubilised
cell lysates using neutravidin-agarose and analysed by western
blotting using anti-LOX-1 antibodies (Fig. 3A). After 30 minutes,
either 28% or 27% of cell surface LOX-1 is internalised in the
presence or absence of OxLDL, respectively (Fig. 3A). Thus LOX-
1 is constitutively internalised. The transferrin receptor TfR (Ajioka
and Kaplan, 1986) was used as a control because it also shows
constitutive internalisation in the absence of transferrin ligand
(Harding et al., 1983) (Fig. 3A).
To test whether ligand recognition affected LOX-1 turnover and
degradation, LOX-1-FLAG-transfected cells were incubated with
(or without) a saturating concentration of OxLDL followed by cell
surface biotinylation. After incubation at 37°C for different time
periods, cells were lysed and analysed for biotinylated LOX-1. Total
cellular biotinylated proteins were isolated after each incubation
time period and probed by western blotting using anti-LOX-1
antibodies. The results clearly show that the half-life of LOX-1 is
similar in either the presence or absence of OxLDL ligand (Fig.
3B). Six hours after cell surface biotinylation, 40% or 38% of
biotinylated LOX-1 remained in either the presence or absence of
OxLDL, respectively. These results, in conjunction with the finding
that LOX-1 and OxLDL are separated into different pathways along
the endocytic pathway (Fig. 2), indicate that LOX-1 is constitutively
internalised at the cell surface and uncoupled from ligand in
endosomes (supplementary material Fig. S2). A preliminary study
on LOX-1 recycling in comparison to TfR was inconclusive (data
LOX-1 internalisation is regulated by dynamin-2
The dynamin-2 protein is a mechanoenzyme and a GTPase that
regulates plasma membrane internalisation (Diatloff-Zito et al.,
1995; Herskovits et al., 1993; van der Bliek et al., 1993). The
K44A dominant-negative mutation locks the protein in a
membrane-associated GTP-bound state, thus blocking plasma
membrane scission, which depends on cycling through GTP and
GDP-bound states. Overexpression of dominant-negative
dynamin-2 inhibits transferrin internalisation by TfR (Damke et
al., 1994) (Fig. 4A). Overexpression of wild-type dynamin-2 did
not affect OxLDL internalisation and accumulation of labelled
ligand in punctate perinuclear structures (Fig. 4B). However,
overexpression of dominant-negative dynamin-2 blocked OxLDL
internalisation causing ligand clustering at the plasma membrane
Overexpression of dominant-negative dynamin-2 also caused
LOX-1 clustering at the plasma membrane in contrast to wild-
type dynamin-2 overexpression, which had no significant effects
(Fig. 4C). A 0.5 μm confocal section through a transfected cell
coexpressing LOX-1 and dominant-negative dynamin-2 in
transfected cells revealed substantial colocalisation with OxLDL
ligand at the plasma membrane positioned over the cell apex (Fig.
4C). By contrast, a similar analysis of transfected cells
overexpressing both LOX-1 and wild-type dynamin-2 proteins
showed LOX-1 accumulation at the plasma membrane, whereas
OxLDL accumulated in punctate perinuclear structures showing
significant divergence in post-plasma-membrane trafficking (Fig.
4C). This suggests that prolonged inhibition of endocytosis due
to expression of dominant-negative dynamin-2 caused LOX-1 to
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.
Journal of Cell Science
Endocytosis of OxLDL via LOX-1
become clustered at the plasma membrane. To verify that the
clustered LOX-1 was indeed arrested at the cell surface,
extracellular N-linked carbohydrates were labelled with a
(Bhattacharyya et al., 1991) (Fig. 4D). The LOX-1 glycoprotein
showed substantial colocalisation with Con-A-labelled N-linked
carbohydrates supporting the accumulation of glycosylated species
at the plasma membrane including the mature N-glycosylated
LOX-1 (Fig. 4D, arrow). Quantification showed that >90% of cells
expressing wild-type dynamin-2 protein showed OxLDL
concanavalin A (Con A)
accumulation in punctate perinuclear structures whereas <5% of
cells expressing dominant-negative dynamin-2 displayed
perinuclear OxLDL accumulation (Fig. 4E). Thus a functional
dynamin-2 GTPase activity is required for LOX-1-dependent
accumulation of OxLDL within the cell.
Hypertonic treatment blocks LOX-1-mediated OxLDL
To test for requirement for clathrin and/or cytosolic factors in LOX-
1-mediated OxLDL internalisation, LOX-1-transfected cells were
subjected to potassium depletion or hypertonic treatment. These
conditions disrupt clathrin-coated pit assembly and endocytosis
(Heuser and Anderson, 1989; Larkin et al., 1983). Both potassium
depletion or hypertonic treatment inhibited TfR-regulated transferrin
endocytosis (Fig. 5A). Potassium depletion caused a 55% reduction
in endocytosis of OxLDL (Fig. 5D). Similarly, hypertonic treatment
of cells caused a 62% reduction in OxLDL endocytosis (Fig. 5D).
These inhibitory effects on OxLDL endocytosis caused by either
potassium depletion or hypertonic treatment were reversible, thus
showing that such conditions were not causing cytotoxic effects
that irreversibly blocked clathrin-mediated uptake (Fig. 5C). To test
further whether potassium depletion and hypertonic treatment were
specific for clathrin-mediated uptake, a labelled ganglioside GM1
was used. GM1 can be internalised by both clathrin and caveolae
or raft-based pathways (Singh et al., 2003). Neither potassium
depletion nor hypertonic treatment prevented the internalisation of
fluorescent GM1 (data not shown), thus indicating that caveolae
or lipid-raft-linked internalisation pathways are relatively unaffected
under such conditions.
LOX-1-mediated internalisation by a clathrin-independent
To further characterise internalisation of LOX-1-OxLDL, we
investigated the colocalisation of internalised OxLDL with clathrin
and caveolin-1, which represent two major dynamin-2-regulated
pathways for plasma membrane uptake. Microscopy was carried
out on transfected HeLa cells expressing tagged LOX-1, which were
labelled on ice with OxLDL and then warmed to 37°C for a short
period (10 minutes) to allow internalisation. Analysis of labelled
OxLDL or LOX-1 with clathrin light chain or caveolin-1 indicated
increased codistribution with clathrin but not caveolin-1
(supplementary material Fig. S3). This was also similar to results
observed for TfR (supplementary material Fig. S3).
To further analyse LOX-1 endocytosis dependence on clathrin
and cytosolic factors, we carried out RNAi to deplete clathrin heavy
chain (CHC17) or the μ2 subunit (of the AP2 adaptor complex)
followed by endocytosis of labelled transferrin and OxLDL in
LOX-1-transfected HeLa cells (Fig. 6). Depletion of either CHC17
or μ2 blocked transferrin uptake (via TfR) and showed plasma
membrane transferrin accumulation (Fig. 6A, arrows in left
panels). However, analysis of the same cells overexpressing LOX-
1 showed that OxLDL internalisation was not blocked (Fig. 6A).
Western blotting revealed >90% depletion of CHC17 or μ2 using
RNAi (Fig. 6B). Quantification of internalised labelled transferrin
and OxLDL in controls, CHC17 or μ2-depleted cells showed that
whereas OxLDL uptake was relatively unaffected, transferrin
uptake was reduced by ~40% upon CHC17 depletion or ~50% upon
μ2 depletion (Fig. 6C). This suggests that CHC17 and μ2 are not
required for LOX-1-mediated OxLDL uptake. Thus LOX-1 and
OxLDL endocytosis must occur via a clathrin-independent
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).
Journal of Cell Science
2140Journal of Cell Science 121 (13)
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.
Journal of Cell Science
Endocytosis of OxLDL via LOX-1
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.
Journal of Cell Science
A novel cytoplasmic acidic motif within LOX-1 regulates
Short cytoplasmic motifs regulate receptor-mediated endocytosis
in eukaryotes: these include YxxΦ, NPxY and di-leucine-based
motifs (Bonifacino and Traub, 2003). Multiple sequence alignment
of the LOX-1 cytoplasmic domain from mammalian species (Fig.
7A) reveals a lack of conserved sequences that bear homology to
previously characterised endocytic signals. However, the existence
of highly conserved acidic residues was noticed including a
Journal of Cell Science 121 (13)
completely conserved di-aspartate motif (residues 4 and 5) and
another completely conserved aspartic acid residue (residue 16).
In addition, a partially conserved GL motif (residues 26-27) may
be a potential di-leucine-based motif (Fig. 7A). In human LOX-
1, the non-conserved aromatic residues such as a phenylalanine
(residue 3) and a tyrosine (residue 31) could be part of non-
conventional endocytosis motifs. Site-directed mutagenesis was
used to substitute each residue for alanine within tagged LOX-1
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
Journal of Cell Science
Endocytosis of OxLDL via LOX-1
The LOX-1 mutants were assessed for their ability to internalise
labelled OxLDL compared with wild-type LOX-1. Transfection and
expression of each LOX-1 mutant protein was verified by western
blot analysis and expression of an endogenous membrane protein
(TGN46): the different mutations did not affect LOX-1 expression,
stability or turnover (data not shown). All wild-type and LOX-1
mutants were localised to the plasma membrane (Fig. 7B;
supplementary material Fig. S4B). The LOX-1 mutants were
assessed for their ability to internalise labelled OxLDL compared
with wild-type LOX-1 (Fig. 7B). Transfected HeLa cells were
pulsed with labelled OxLDL and cells with internalised OxLDL
were quantified (Fig. 7C). Alanine substitution of LOX-1 residues
that caused the most significant effects on OxLDL uptake were D4
(40% inhibition), D5 (92% inhibition) and L6 (43% inhibition) (Fig.
7C). By comparison, other mutations around this site had little effect
on LOX-1-mediated OxLDL uptake.
Importantly, cells expressing LOX-1 containing either the D4A,
D5A or L6A mutations were not only unable to internalise OxLDL
but also displayed substantial colocalisation of OxLDL ligand with
clustered LOX-1 at the cell surface (Fig. 7B, arrows). This
morphology was qualitatively similar to that previously observed
in cells expressing dominant-negative dynamin-2 and LOX-1 (Fig.
4C). Again, the plasma membrane clusters of LOX-1-D4A, -D5A
or -L6A mutants showed substantial colocalisation with labelled
Con A used to detect N-glycosylated plasma membrane proteins
(Fig. 7B). The LOX-1-DD(4,5)AA double mutant displayed an
intermediate effect with ~50% inhibition in OxLDL uptake;
however, the LOX-1-D16A, GL(25,26)AA and Y31A mutants all
exhibited no significant effects on OxLDL uptake (supplementary
material Fig. S4). Thus, a partially conserved acidic tripeptide motif
(DDL) mediates constitutive internalisation of the LOX-1 membrane
Although the pro-atherogenic nature of OxLDL is well established,
relatively little is known about scavenger receptor-mediated lipid
particle internalisation, trafficking and processing during
atherosclerotic plaque formation and progression (Bobryshev,
2006). In this study on the human LOX-1 scavenger receptor, we
have delineated a clathrin-independent but dynamin-2-dependent
pathway that regulates the endocytosis and accumulation of
OxLDL. First, OxLDL internalisation via LOX-1 was inhibited
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.
Table 1. Mutational analysis of the LOX-1 cytoplasmic
LOX-1 cytoplasmic domain sequence
*Alanine-scanning mutagenesis of specific residues within the LOX-1
cytoplasmic domain is indicated by bold underlined lettering.
Journal of Cell Science
by the expression of a dynamin-2 mutant that blocks receptor-
ligand uptake. Second, hypertonic treatment completely blocked
uptake of OxLDL by LOX-1. Furthermore, site-directed
mutagenesis of the LOX-1 cytoplasmic domain identified a novel
partially conserved tripeptide motif (DDL) proximal to the N-
terminus (+4 to +6) that regulates efficient receptor-mediated
endocytosis. The di-aspartate sequence is completely conserved
in LOX-1 mammalian orthologues (Fig. 8), indicating the
functional significance of this motif. Finally, the receptor
undergoes constitutive internalisation and the binding of OxLDL
ligand does not significantly alter receptor trafficking or
Our results also show that LOX-1 undergoes constitutive
endocytosis and neither its rate of internalisation nor its half-life is
significantly altered upon binding OxLDL; the effects of antibody
crosslinking on receptor trafficking were not addressed in this study.
Intracellular LOX-1 staining diverges from internalised OxLDL
ligand after 30 minutes of trafficking along the endocytic pathway.
The LOX-1-OxLDL complex probably dissociates within the acidic
environment of the endosome, a feature common for other
lipoprotein particle receptors such as LDL-R (Davis et al., 1987).
One possibility is that LOX-1 is recycled, but our current assay is
currently not sensitive enough to detect this aspect of trafficking.
Alternatively, LOX-1 could be stationed in a different endosomal
compartment after uncoupling from bound OxLDL; in this case,
LOX-1 may have a relatively long half-life and thus might be
protected from degradation by lysosomal proteases.
The dynamin-2 GTPase regulates multiple plasma membrane
internalisation events including clathrin-dependent, caveolae-
dependent and clathrin/caveolin-independent steps (Conner and
Schmid, 2003; Mayor and Pagano, 2007). Depletion of either the
clathrin heavy chain (CHC17) or μ2 subunit of AP2 adaptor
complex blocked transferrin uptake, but OxLDL internalisation
via LOX-1 was not affected. In this model for OxLDL uptake via
LOX-1, a clathrin-independent pathway is utilised to effect
delivery of the receptor-ligand complex to the endosomal network
How does the LOX-1 cytoplasmic motif (DDL) compare with
other trafficking motifs for plasma membrane endocytosis?
Importantly, cytoplasmic signals for clathrin-independent uptake are
not well defined (Mayor and Pagano, 2007); our study is one of the
first to define a tripeptide cytoplasmic motif that regulates receptor-
ligand uptake by such a route. In contrast to the well-characterised
Journal of Cell Science 121 (13)
YxxΦ and di-leucine motifs that interact with the AP2 adaptor
complex to specify inclusion into clathrin-coated vesicles, the NPxY
motif found in LDL-R interacts with other clathrin-associated
adaptor proteins such as ARH and Dab2 (Bonifacino and Traub,
2003). It has also been noted that plasma membrane LDL-R and
EGFR appear to undergo endocytosis via different clathrin-dependent
pathways (Motley et al., 2003). The DDL motif does not resemble
such motifs and the RNAi depletion of clathrin heavy chain or μ2
(AP2) point to another cytosolic factor or ‘coat complex’ that
mediates LOX-1 recognition and endocytosis. Intriguingly,
hypertonic treatment is well known to perturb clathrin and AP2-
mediated endocytosis; this treatment also completely blocks LOX-
1-mediated ligand uptake. These findings suggest that LOX-1-
mediated endocytosis is dependent on recognition of this unique
tripeptide motif by cytosolic factors that are clathrin-independent
but still susceptible to perturbation by hypertonic conditions. In
support of this, it has been noted that the clathrin-independent
endocytosis of certain G-protein-coupled receptors (Cinar and
Barnes, 2001; Idkowiak-Baldys et al., 2006) and the Menkes’ disease
copper ATPase (ATP7A) (Lane et al., 2004) are also blocked by
The acidic motif (DDL) is required for LOX-1-mediated ligand
uptake (Fig. 7). Clusters of acidic residues can influence the
trafficking of proteins, such as the human cytomegalovirus
glycoprotein B (Tugizov et al., 1999) and the pseudorabies virus
Us9 envelope protein (Brideau et al., 1999). Recently, glutamate-
based motifs in the cytoplasmic domain of the potassium channel
Kir3.4 subunit have been shown to mediate the endocytosis and/or
recycling of membrane proteins via a clathrin-independent Arf6-
dependent pathway (Gong et al., 2007). Acidic sequences are also
responsible for basolateral sorting of the LDL-R and for targeting
the dendritic cell receptor DEC-205 (CD205) to late endosomes
(Matter et al., 1994; Mahnke et al., 2000).
LOX-1 belongs to one of several diverse classes of eukaryote
scavenger receptors that bind OxLDL, apoptotic bodies and
phospholipids (Murphy et al., 2005). LOX-1 is a scavenger receptor
whose levels are elevated in vascular tissues under pro-inflammatory
conditions; this molecule could play a major role in contributing
to lipid accumulation during atherosclerotic plaque formation
(Smirnova et al., 2004). Our results provide a model (Fig. 8) in
which LOX-1 undergoes constitutive endocytosis by a clathrin-
independent pathway and delivery to endosomes. In this way, LOX-
1 would act as a true ‘scavenging’ receptor, allowing the continuous
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.
Journal of Cell Science
Endocytosis of OxLDL via LOX-1
internalisation and accumulation of OxLDL in macrophages. Taken
together, our results show that LOX-1-dependent internalisation of
OxLDL is due to the recognition of a DDL motif at the cytoplasmic
N-terminus and internalisation requires dynamin-2 but not clathrin
or AP2. This work now provides a mechanism to account for lipid
accumulation in vascular tissues where LOX-1 levels are elevated
in response to pro-atherogenic stimuli.
Materials and Methods
Well-characterised affinity-purified sheep anti-LOX-1 and sheep anti-TGN46
antibodies have been previously described (Murphy et al., 2006; Towler et al., 2000).
Mouse monoclonal anti-LOX-1 blocking antibody (JTX92) was a kind gift from T.
Sawamura (National Cardiovascular Center Research Institute, Osaka, Japan).
Rabbit anti-clathrin light chain polyclonal and mouse anti-CHC17 (X22) monoclonal
antibodies were kindly provided by F. Brodsky (University of California, San
Francisco, CA). Anti-Myc monoclonal antibody was obtained from the mouse 9E10
hybridoma cell line from the European Cell and Animal Cultures Collection (ECACC,
Porton Down, UK). Mouse M2 anti-FLAG monoclonal antibody was from Sigma
(Poole, UK). Mouse anti-TfR monoclonal antibody (clone H68.4) was from
Invitrogen. Rabbit anti-caveolin-1 was from BD Transduction Laboratories (Oxford,
UK). Secondary antibodies conjugated to FITC or Cy5 were from Jackson
ImmunoResearch Laboratories (West Grove, PA); secondary antibodies conjugated
to Alexa Fluor 488 or Alexa Fluor 594 were from Invitrogen, (Amsterdam, The
Netherlands). All other reagents were purchased from Sigma (Poole, UK) unless
HeLa cells were maintained in a complete medium containing high glucose DMEM
supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine,
10% (v/v) fetal bovine serum and nonessential amino acids (Invitrogen). All cells
were grown at 37°C in a humidified atmosphere containing 5% (v/v) CO2.
Preparation and labelling of oxidised LDL
Human LDL particles were isolated from plasma, chemically modified to make
OxLDL and analysed using established procedures as described previously (Vohra
et al., 2007). OxLDL particles were labelled with a fluorescent dye, 1,1?-dioctadecyl-
3,3,3?,3?-tetramethylindocarbocyanine perchlorate (DiI; Invitrogen), also as described
previously (Vohra et al., 2007).
Gene manipulation, transfection and RNAi
Full length human LOX-1 was cloned into pCDNA3.1+(Invitrogen) in conjunction
with a FLAG tag at the C-terminus of LOX-1 as described previously (Murphy et
al., 2006). Alanine substitutions were introduced into the LOX-1 sequence by site-
directed mutagenesis using Quikchange (Stratagene). All mutations were confirmed
by DNA sequencing. A mammalian expression plasmid (pCMV5) containing either
rat wild-type dynamin-2 or dominant-negative (K44A) dynamin-2 cDNA fused to a
Myc tag at the N-terminus was a kind gift of H. McMahon (MRC Laboratory of
Molecular Biology, Cambridge, UK). HeLa cells were transfected with DNA plasmids
using a calcium phosphate method (Towler et al., 2000) and assayed 48 hours post
Synthetic oligonucleotide duplexes (Ambion) corresponding to the mRNAs
of clathrin heavy chain (GGGUGCCAGAUUAUCAAUUtt)
(GGUGUUUGAACCGAAGCUGtt) were used in conjunction with Oligofectamine
as specified by the manufacturer (Invitrogen, Amsterdam, The Netherlands).
Subconfluent HeLa cells were incubated with preformed lipid-siRNA complexes (100
nM siRNA) for 6 hours, after which fresh medium was added. After 36 hours, cells
were transferred onto coverslips and 60 hours after the initial siRNA transfection,
cells were subsequently transfected with LOX-1-FLAG. Following LOX-1
transfection after 12 hours, cells were processed for internalisation assays by
incubation with Alexa Fluor 488-transferrin (Invitrogen) and DiI-OxLDL for 15
minutes at 37°C, followed by chase at 37°C for 30 minutes with fresh media. Cells
were fixed and processed as previously described (Towler et al., 2000) using confocal
microscopy (see later).
Cellular proteins were analysed by western blotting using enhanced
chemiluminescence (Murphy et al., 2006). Primary antibodies used were sheep anti-
LOX-1 (0.5 μg/ml), sheep anti-TGN46 (0.1 μg/ml) or tissue culture supernatant of
mouse anti-transferrin receptor (1:5000). Levels of protein expression were evaluated
from multiple western blotting datasets (n?3) using AIDA (Advanced Image Data
Analyzer) 2.11 software (FujiFilm, Japan). For the RNAi experiments, cell lysates
(10 μg total protein per lane) were run on 3-8% Tris-acetate SDS-PAGE gels
(Invitrogen). Mouse monoclonal antibodies to clathrin heavy chain and the μ2 subunit
of AP2 (BD Biosciences, Oxford, UK) or actin (Sigma, Poole, UK) were used for
western blot detection.
Cells were plated on sterile glass coverslips and transfected as described previously
(Murphy et al., 2006) with the indicated plasmids. Cells were pulsed with 10 μg/ml
DiI-OxLDL in complete medium for 5 minutes at 37°C and then washed three times
with PBS. Cells were fixed and processed for immunofluorescence microscopy or
chased for the indicated time periods at 37°C before fixation and processing. To block
OxLDL binding, cells were incubated with 10 μg/ml LOX-1 blocking antibody JTX92
(Li et al., 2003) in complete medium for 30 minutes at 37°C and washed three times
before addition of labelled OxLDL. To monitor transferrin internalisation by
microscopy, 50 μg/ml tetramethylrhodamine-conjugated transferrin (Invitrogen) was
incubated with cells in the indicated buffers for 15 minutes at 37°C prior to washing,
fixation and processing. For GM1 uptake, 1 μM BODIPY FL C5-ganglioside GM1
(Invitrogen) was incubated with cells for 30 minutes at 37°C in the indicated buffers
prior to washing, fixation and processing.
Immunofluorescence was performed exactly as described previously (Towler et al.,
2000). Cells were fixed in 3% (w/v) paraformaldehyde in PBS and quenched with
50 mM ammonium chloride in PBS. Where stated, cells were permeabilised with
0.1% (v/v) Triton X-100 in PBS for 4 minutes. For experiments involving DiI-labelled
OxLDL, cells were permeabilised with 0.3% (v/v) Tween-20 in PBS for 4 minutes
(Lukas et al., 1998). All subsequent incubations were carried out in 0.2% (v/v) fish
skin gelatin in PBS. Coverslips were mounted in Fluoromount G (SouthernBiotech,
Birmingham, AL). To label N-linked carbohydrates attached to cell surface
glycoproteins, samples were incubated with 10 μg/ml FITC-conjugated Concanavalin
A (Sigma) in PBS containing 1 mM CaCl2for 5 minutes at 4°C immediately before
Microscopy and quantification
High-resolution images were collected using the DeltaVision Optical Restoration
Microscopy System (Applied Precision, Issaquah, WA) with an Olympus IX-70
epifluorescence microscope and 60? objective. 0.2-μm-thick optical sections were
collected and datasets deconvolved using the SoftWorX deconvolution algorithm.
Quantification of colocalisation was determined using the IMARIS software suite
(Bitplane AG, Zurich, Switzerland) on selected cell regions as described previously
(Herbert et al., 2005). Background was eliminated by excluding pixel values lower
than 10% of the maximum pixel intensity. Colocalised pixels were expressed as a
percentage of the total pixels selected.
For quantification of labelled OxLDL uptake, samples were analysed using a Zeiss
LSM 510 Meta confocal microscope attached to an Axiovert 200M inverted
microscope using pinhole settings to acquire confocal sections equal to <1 μm (Carl
Zeiss, Jena, Germany). The ImageJ (http://rsb.info.nih.gov/ij/) program was used in
such analyses. Differences in LOX-1 transfection levels were minimised by dividing
the total pixel intensity of intracellular labelled OxLDL by the total pixel intensity
of intracellular (cytosolic vesicles) plus plasma membrane-associated labelled
OxLDL, i.e. amount internalised as a percentage of the total cell-associated OxLDL.
Background pixel intensities were subtracted from all readings.
For quantification of accumulated labelled transferrin and OxLDL in RNAi-
treated cells and controls, cells were processed as previously described for
immunofluorescence microscopy. Images were captured in sequential scanning
mode using a Zeiss LSM-510 Meta confocal microscope and a Zeiss Plan
Apochromat 63? oil immersion lens (NA 1.4). The pinhole was set such that each
optical section was 1 μm thick. DIC contrast enhanced transmitted light images
were captured simultaneously. All the images were acquired at a 1024?1024 pixel
resolution with 4096 grey levels per pixel. Each channel image was then
exported as a 12-bit black and white TIFF into Metamorph 5.0 for quantification
(Molecular Devices, Sunnyvale, CA). The region of interest (ROI) within each
cell was traced using the MetaMorph Trace Region tool. The ROI outline was
transferred (copied) into each fluorescence channel image to quantify the
fluorescence intensity within the same region of the cell. The mean pixel intensity
was calculated for each channel in the ROI. To eliminate any background
fluorescence the pixel intensity was only measured in the brighter cellular regions
where pixel intensity was greater than 512 grey levels, i.e. 88% of the grey level
scale was measured, where pure black pixels=0 and pure white=4095. At least 30
cells were analysed per treatment. Raw data was exported into Excel, transferred
and presented using GraphPadPrism 5.0. The mock-transfected cells were used as
the baseline control and normalised as 100% transferrin or OxLDL uptake per cell
Cell surface biotinylation assay
These assays are based on a previously described procedure (Marijanovic et al., 2006).
Cells were seeded in six-well dishes and transfected with LOX-1-FLAG as described.
After 48 hours, cells were starved in serum-free medium for 2 hours, chilled on ice
and incubated with or without 10 μg/ml OxLDL in serum-free medium for 1 hour
at 4°C. Cells were then washed three times with PBS+ (PBS containing 0.7 mM
CaCl2, 0.5 mM MgCl2) and incubated with 0.3 mg/ml EZ-Link-Sulfo-NHS-S-S-biotin
(Pierce, Rockford, IL) in PBS+ for 30 minutes at 4°C to biotinylate cell surface
proteins. Cells were then washed three times with TBS to remove and quench unbound
Journal of Cell Science
2146Journal of Cell Science 121 (13)
biotin. To assess LOX-1 degradation, biotinylated cells were incubated at 37°C in
serum-free media for the indicated times and then lysed in 1% (v/v) NP-40, 50 mM
Tris-HCl pH 7.5, 150 mM NaCl. To assess LOX-1 internalisation, biotinylated cells
were incubated at 37°C in serum-free medium for the indicated times, then re-chilled
on ice and washed three times with PBS. Cell surface biotin labelling was removed
by three 10-minute incubations with ice-cold reducing buffer (100 mM MESNA, 50
mM Tris-HCl pH 8.6, 100 mM NaCl, 1 mM EDTA, 0.2% (w/v) BSA). Cells were
washed and excess reducing agent quenched by incubation with 120 mM
iodoacetamide in PBS for 10 minutes at 4°C before cell lysis. Biotinylated proteins
were recovered by incubation with 30 μl neutravidin-agarose (Pierce) overnight at
4°C. The isolated biotinylated proteins were washed three times with lysis buffer,
boiled in SDS-PAGE sample buffer and probed for LOX-1 using western blotting.
Hypertonic treatment and potassium depletion
For hypertonic treatment (Heuser and Anderson, 1989), cells were incubated with
complete medium containing 0.45 M sucrose for 15 minutes at 37°C; this was followed
by a 5 minute pulse with 10 μg/ml DiI-labelled OxLDL followed by a 55 minute
chase, in complete media with 0.45 M sucrose. Control incubations were carried out
in complete medium. Cells were then fixed and processed for immunofluorescence
microscopy. For potassium depletion (Hansen et al., 1993), cells were washed with
K+-free buffer (20 mM HEPES pH 7.4, 140 mM NaCl, 1 mM CaCl2, 1 mM MgCl2,
1 mg/ml D-glucose), incubated with hypotonic buffer (K+-free buffer diluted 1:1 with
water) for 5 minutes at 37°C, washed three times with K+-free buffer and incubated
with K+-free buffer for 30 minutes at 37°C. Cells were then pulsed with 10 μg/ml
labelled OxLDL for 5 minutes followed by a 55 minute chase, both in K+-free buffer
and at 37°C. Control cells were assayed in the same buffers containing 10 mM KCl.
Cells were then fixed and processed for immunofluorescence microscopy.
Quantification of OxLDL uptake
Internalisation of DiI-labelled OxLDL in potassium-depleted or hypertonic treated
cells was quantified using ImageJ as described above. In all other cases, quantification
in internalisation experiments was assessed using data from three separate experiments
(n?150 cells in total). Plasma membrane and intracellular punctate staining patterns
were compared. Cells pulsed and chased with labelled ligand displayed intracellular
punctate structures with little, if any, plasma membrane staining; the converse was
true where labelled ligand was bound but not internalised.
This work was supported by a British Heart Foundation project grant
(S.P., J.H.W. and S.H.V.), a BBSRC DTA PhD studentship (J.E.M.), a
Royal College of Surgeons Fellowship (R.S.V.) and the Wellcome Trust
(A.P.M.). We thank Gareth Howell for helpful discussions and technical
advice. We are grateful to T. Sawamura (National Cardiovascular Center
Research Institute, Osaka, Japan), H. McMahon (MRC Laboratory of
Molecular Biology, Cambridge, UK) and F. Brodsky (University of
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