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INTRODUCTION |
The risk for developing atherosclerosis and cardiovascular disease
is inversely related to plasma concentrations of high density lipoprotein (HDL)1
cholesterol (1). Although the mechanism of this protective effect
remains uncertain, it has been known for some time that HDL plays a
pivotal role in the transport of free cholesterol and cholesteryl
esters (CE) through the plasma. HDL participates in reverse cholesterol
transport (2), a process involving the uptake of free cholesterol from
peripheral tissue and its subsequent delivery (as free cholesterol or
CE) to steroidogenic (for hormone synthesis) and hepatic (for bile acid
synthesis) tissues. HDL provides CE to cells via the selective uptake
pathway in which HDL CE is taken into the cell without the
internalization and lysosomal degradation of the HDL particle (3-8).
Recent studies identified a cell surface receptor, scavenger receptor
BI (SR-BI), which binds HDL particles and mediates the selective uptake
of HDL CE in transfected cells (9). Immunochemical analysis of SR-BI in
rodents indicates that it is expressed most abundantly in the liver and
in steroidogenic cells of the adrenal gland and ovary (9-11), where
the selective uptake of HDL CE is greatest (4, 7). SR-BI expression is
regulated by gonadotropins and adrenocorticotropic hormone coordinately
with the selective uptake of HDL CE and steroidogenesis (10, 11). In
addition, antibody blocking experiments show that SR-BI is the receptor
responsible for the uptake of HDL CE and its delivery to the
steroidogenic pathway in adrenocortical cells (12). Inactivation of the
SR-BI gene in mice alters plasma HDL metabolism and reduces adrenal gland CE accumulation, results consistent with a major role for SR-BI
in cholesterol metabolism in vivo (13). Taken together, these studies indicate that SR-BI is a physiologically relevant receptor for the selective uptake of HDL CE.
The biochemical mechanism by which SR-BI mediates the selective uptake
of HDL CE is poorly understood. Given that CE transfer will occur to
some extent from HDL or microemulsion particles to protein-free
synthetic membranes (14), one hypothesis is that the role of SR-BI is
primarily to tether HDL in close apposition to the cell surface to
facilitate CE transfer from the particle to the plasma membrane. In the
present study, we test this hypothesis by comparing the selective
uptake of HDL CE mediated by mouse SR-BI (mSR-BI) with that mediated by
rat CD36 (rCD36), a closely related receptor that also binds HDL with
high affinity (15). SR-BI was originally defined as a class B scavenger
receptor (16-18) in a family that includes CD36, LIMPII, and SR-BII, a
form of SR-BI with an alternate C-terminal cytoplasmic tail (19). The amino acid sequence homology between SR-BI and CD36 polypeptides has
been reported to be 20-33%; however, when the amino acid sequences are properly aligned, the proteins are strikingly similar. In fact, the
predicted secondary structures and sizes of SR-BI and CD36 are nearly
identical. Each protein contains two transmembrane and two cytoplasmic
domains (the amino- and carboxyl-terminal domains) as well as a large
extracellular domain containing a cysteine-rich region and nine
putative sites for N-linked glycosylation. In addition, both
proteins are palmitoylated and localized to caveolae (20-22). The
results of the present study comparing mSR-BI with rCD36 suggest that
one component of HDL CE selective uptake is due to particle tethering.
However, SR-BI-mediated selective uptake is a much more efficient
process that combines tethering of the HDL particle to the cell surface
and facilitated HDL CE movement into the cell. Furthermore, comparison
of SR-BI, SR-BII, CD36, and chimeric receptors showed that the
extracellular domain of mSR-BI is essential for efficient HDL CE
uptake, but the C-terminal cytoplasmic tail also has a major influence
on the selective uptake process.
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EXPERIMENTAL PROCEDURES |
Plasmids and Sequencing--
PCR amplifications were performed
using a Perkin-Elmer DNA Thermal Cycler model 480 or 9700. Oligonucleotides were either purchased from Integrated DNA Technologies
or synthesized on a Beckman Oligo 1000 DNA synthesizer.
The mSR-BI coding region was amplified from pSR-BI77 (obtained
from M. Krieger, MIT) with the following primer pair:
5'-GACCGAATTCCAATTGCCGTCTCCTTCAGGTCCTGAGC-3' and
5'-GACCGGATCCAGATCTGCGGACAGGTGTGACATCTGG-3'. The resulting PCR
product was restricted with MfeI and BglII and
ligated into an EcoRI- and BglII-restricted pSG5
vector (Stratagene, Inc.) to produce pSG5(mSR-BI). The
cloning of pSG5(rCD36), which contains the rat CD36 coding
region, was described previously (23).
pSG5(SR-BII), pSG5(CD/SRTM), and
pSG5(SR/CD/SR) were obtained by "seamless cloning," a
technique recently developed by Stratagene, Inc. For construction of
pSG5(SR-BII), the following two primers were employed for the
amplification of pSG5(mSR-BI): 5'-AGTTACTCTTCACCTGAAGACACTATAAGCCC-3' and 5'-AGTTACTCTTCAAGGACCCTGGCTGCGCAGTTGGC-3'. The resulting PCR product was digested with Eam1104I and recircularized. This
generated a vector that was missing the portion of the SR-BI cDNA
encoding the wild type cytoplasmic tail leaving only the portion
encoding the cytoplasmic tail of SR-BII. For construction of
pSG5(CD/SRTM), primers 5'-AGTTACTCTTCATAGAAATAAGTAGTGGATGAGGATCC-3' and
5'-AGTTACTCTTCACAGGGTCACTTGGTTTCTGAACATTTC-3' and primers
5'-AGTTACTCTTCACTGATGCCCCAGGTTCTTCACTACGCG-3' and 5'-AGTTACTCTTCACTATAGCTTGGCTTCTTGCAGC-3' were employed to amplify pSG5(rCD36) and pSG5(mSR-BI), respectively. For construction of pSG5(SR/CD/SR), primers 5'-AGTTACTCTTCACTGATGCCCCAGGTTCTTCACTACGCG-3' and 5'-AGTTACTCTTCAAAGGAGCACCTGCTGCTTGATGAGGGAG-3' and primers 5'-AGTTACTCTTCACTTGAAGAAGGAACCATTGCTTTC-3' and
5'-AGTTACTCTTCACAGGGTCACTTGGTTTCTGAACATTTC-3' were employed to
amplify pSG5(mSR-BI) and pSG5(rCD36), respectively. The resulting PCR
products were digested with Eam1104I and ligated.
The "seamless cloning" technique was also employed to clone
pSG5(CD/SRT) and pSG5(SR/CDT). The strategy was amended to include a
different restriction enzyme, SapI (New England
Biolabs, Inc.). For construction of pSG5(CD/SRT), primers
5'-AGCCAGCTCTTCACAGCATAAAAGCAACAAACATCACTACTCC-3' and
5'-AGCCAGCTCTTCATAGAAATAAGTAGTGGATGAGGATGATCC-3' and primers 5'-AGTTACTCTTCACTATAGCTTGGCTTCTTGCAGC-3' and
5'-AGTTACTCTTCACTGCGCAGCCAGGAGAAATGC-3' were employed to amplify
pSG5(rCD36) and pSG5(mSR-BI), respectively. The resulting PCR products
were digested with Sap I and ligated. For construction of pSG5(SR/CDT),
primers 5'-AGCCAGCTCTTCATGAAGATGTCACACCTGTCCGC-3' and
5'-ACTGAGCTCTTCAAGCCTGGCTGCGCAGTTGGGC-3' were employed to amplify
pSG5(mSR-BI). The resulting PCR product was digested with Sap I and
ligated to a double-stranded oligonucleotide encoding the cytoplasmic
tail of CD36.
pSG5(CD36)X was constructed to disrupt the extracellular
domain of CD36 by inserting the 14-amino acid M45 monoclonal
antibody epitope (24) between amino acids 46 and 47. Primers,
5'-AGCCAGCTCTTCACTACCTCCTTTTGAGACAGAGACGAAGCTGACCTACAACGAATC-3' and
5'-AGCTAGCTCTTCATAGGCGATCCCTACTCCGATCCATGGACCTGCATGCCTC-3', each containing half of the M45 epitope encoding sequence, were employed to amplify pSG5(rCD36), and the resulting PCR product was
digested with SapI and recircularized.
All plasmids were prepared using Endotoxin-free Qiagen Maxi-prep kits
and sequenced throughout the coding region to confirm the correct
fragment insertion and to ensure that no point mutations had been
generated during the amplification process. DNA sequencing was
performed by the automated sequencing facility at SUNY Stony Brook.
Reactions were prepared using a dye termination cycle sequencing kit
and analyzed on an Applied Biosystems model 373 DNA (Perkin-Elmer Applied Biosystems).
Immunoblots with several antibodies to different regions of SR-BI and
CD36 were performed on lysates from COS-7 cells expressing chimeric
receptors. As expected, these experiments confirmed the presence of
each of the described regions of either mSR-BI or rCD36 in each of the
expressed chimeras (data not shown).
Cell Culture and Maintenance--
Stable clones of Ob17PY
fibroblasts transfected with pSG5(rCD36) or with pSG5 vector alone were
maintained in low glucose Dulbecco's modified Eagle's medium
supplemented with 4% (v/v) fetal bovine serum, 15 mM
Hepes, 33 µM biotin, 17 µM pantothenate, 200 units/ml penicillin, 50 µg/ml streptomycin, and 0.2 mg/ml geneticin (23). ldlA[mSR-BI] and ldlA cells (9) were grown in Ham's
F-12 medium supplemented with 5% (v/v) fetal bovine serum, 2 mM L-glutamine, 50 units/ml penicillin, 50 µg/ml streptomycin with or without 0.5 mg/ml geneticin, respectively.
All cells were kept in a 37 °C humidified 95% air, 5%
CO2 incubator.
Transient Transfection of COS-7 Cells--
COS-7 cells were
maintained in Dulbecco's modified Eagle's medium, 10% calf serum, 2 mM L-glutamine, 50 units/ml penicillin, 50 µg/ml streptomycin, and 1 mM sodium pyruvate. Cells were
seeded at a density of 2.0 × 106 in 10-cm tissue
culture dishes in 10 ml of fresh media. The cells were then incubated
until approximately 80-90% confluence (~18 h). Transfections were
performed with Fugene 6, a liposomal-like transfection reagent from
Boehringer Mannheim, as directed by the manufacturer. The following
day, the cells were trypsinized, resuspended in a total volume of 6 ml
with fresh medium, and added to six wells of a six-well plate. After
18-24 h, the cells were assayed for their ability to bind HDL
particles and mediate the uptake of CE.
Preparation of 125I/3H-Labeled
hHDL3--
Human HDL3 (hHDL3;
1.125 g/ml <
< 1.210 g/ml) labeled with
125I-dilactitol tyramine and [3H]cholesteryl
oleolyl ether was prepared as described (25, 26). The specific activity
of the 125I/3H-labeled hHDL3 ranged
from 15 to 86 dpm/ng of protein for 125I and from 1.1 to
8.2 dpm/ng of protein for 3H.
HDL Cell Association, Selective CE Uptake, and Apolipoprotein
Degradation--
HDL assays were performed ~48 h post-transfection.
Cells were washed once with serum-free medium, 0.5% BSA.
125I-Dilactitol tyramine-3H-cholesterol oleolyl
ether hHDL3 particles were added at a concentration of 10 µg protein/ml (unless otherwise indicated) in serum-free medium.
After incubation for 1.5 h, the HDL-containing media was removed,
and the cells were washed three times with 0.1% BSA in PBS (pH 7.4)
and one time with PBS (pH 7.4). The cells were lysed with 1.1 ml of 0.1 N NaOH and pipetted 18-20 times to fragment DNA. The
lysate was then processed to determine trichoroacetic acid soluble and
insoluble 125I radioactivity and organic
solvent-extractable 3H radioactivity as described (25, 26).
The values for the cell-associated HDL apolipoprotein, the endocytosed
and degraded HDL apolipoprotein, the total cell-associated HDL CE, and
the selective uptake of HDL CE were obtained as described previously (25, 26). Note that cell-associated HDL CE is derived from acid-insoluble 125I radioactivity values and is believed to
be primarily cell surface-bound, although it may include a small
fraction of HDL particles that have been endocytosed but not yet
degraded. In our experiments, endocytosed and degraded HDL is typically
about 5% of cell-associated HDL. Thus, error due to endocytosed but
not yet degraded HDL would be minimal.
Receptor Expression Levels--
COS-7 cells (in 35-mm wells)
were washed with 2 ml of cold PBS. Cells were removed from plates by
the addition of 1 ml PBS, 0.5 mm EDTA and incubation for 5-7 min at
room temperature. Cells were placed in a microcentrifuge tube and
centrifuged at 200 × g for 2-3 min and were
resuspended in 100 µl of PBS, 1% BSA. Anti-SR-BI primary antibody
356 (12) at a concentration of 0.48 mg/ml IgG or anti-CD36 39815 antiserum (27) at a 1:50 dilution was added to the cells and incubated
for 1 h at 4 °C. The cells were centrifuged at 200 × g for 2-3 min, and the supernatant was aspirated. Cells were washed with 0.5 ml of PBS, 1% BSA before incubation with secondary antibody (4 µl of fluorescein (Sigma) or
phycoerythrin-conjugated anti-rabbit antibody (Molecular Probes, Inc.,
Eugene, OR) in 300 µl of PBS, 1% BSA) for 30 min at 4 °C. Cells
were washed three times with 0.5 ml of PBS, 1% BSA and fixed with 0.5 ml of 1% formaldehyde in PBS, 1% BSA for 15 min at 4 °C with
gently shaking. Following incubation with fixative, the cells were
centrifuged for 2-3 min and resuspended in 0.5 ml PBS, 1% BSA.
Fluorescence intensities were measured using a Becton Dickinson
FACSAdvantage cell sorter or FACScan flow cytometer.
For immunoblot analysis, cells were washed twice with PBS (pH 7.4) and
lysed with 300 µl of Nonidet P-40 cell lysis buffer (11, 12)
containing 1 µg/ml pepstatin, 0.2 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 10 µg/ml aprotinin. Protein
concentrations were determined as described (28).
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RESULTS |
The Role of HDL Tethering in HDL CE Selective Uptake--
The high
degree of similarity between mSR-BI and rCD36 led us to ask whether
rCD36 was capable of binding HDL particles and mediating the selective
uptake of HDL CE. We first tested for HDL binding to rCD36 by
transiently transfecting COS-7 cells with a vector that could drive
high level receptor expression. Like rCD36, the mSR-BI coding region
was cloned into the pSG5 vector under the transcriptional control of
the SV40 promoter. pSG5(mSR-BI), pSG5(rCD36), or vector alone were
transfected into COS-7 cells using a liposomal mediated protocol that
gave high transfection efficiencies. Cell surface receptor levels were
monitored by flow cytometry using polyclonal antibodies to the
extracellular domains of mSR-BI (12) and rCD36 (27). This analysis
showed that 20-60% of the cells expressed the receptors (data not
shown). Western blot analysis also showed strong expression of both
receptors. mSR-BI transiently expressed in COS-7 cells co-migrated with
mSR-BI stably expressed in the ldlA[mSR-BI] cell (data not shown).
Similarly, rCD36 transiently transfected in COS-7 cells co-migrated
with rCD36 stably expressed in Ob17PY fibroblasts (23) (data not shown).
Fig. 1A shows that in
comparison to vector-transfected cells, mSR-BI-expressing COS-7 cells
bound HDL in a high affinity saturable manner. Nonlinear regression and
Scatchard analysis of these data indicated an apparent
Kd of 10 µg/ml HDL protein. This value is similar
to that previously reported for HDL binding to mSR-BI in murine Y1-BS1
adrenocortical cells that naturally express SR-BI (12). Similar results
were obtained with rCD36-expressing cells (Fig. 1B), which
showed an apparent Kd of 22 µg/ml HDL protein.
Thus, rCD36, like mSR-BI recognized HDL saturably and with high
affinity in transiently transfected COS-7 cells.

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Fig. 1.
Cell association of HDL mediated by mSR-BI
and rCD36. COS-7 cells transiently expressing SR-BI or rCD36 were
incubated at 37 °C for 1.5 h with the indicated concentrations
of 125I/3H-labeled HDL3, after
which the cells were processed to determine cell associated HDL
particles. Cell-associated HDL3 is expressed on the basis
of CE content. A, mSR-BI-expressing cells in comparison with
vector-transfected cells. B, rCD36-expressing cells in
comparison with vector-transfected cells. Values represent the mean of
triplicate determinations after subtraction of values obtained with the
addition of a 50-fold excess of unlabeled HDL3.
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In order to compare the capacities for HDL CE selective uptake, cells
expressing mSR-BI or rCD36 or cells transfected with vector alone were
incubated for 1.5 h with 10 µg/ml double-labeled (125I-apolipoprotein/3H-cholesteryl oleolyl
ether)human HDL3, after which HDL cell association (Fig. 2A) and selective uptake
of HDL CE (Fig. 2B) were determined. In contrast to the
efficient binding of HDL by rCD36 relative to mSR-BI, rCD36 mediated a
considerably reduced level of HDL CE selective uptake. In order to
compare the relative efficiencies of mSR-BI and rCD36 for selective
uptake, the contribution from vector-transfected cells was subtracted,
and the amount of selective uptake was expressed relative to the amount
of cell-associated HDL (Fig. 2C). In this way, HDL CE
selective uptake is normalized to the quantity of HDL particles bound
to the cell surface. This analysis showed that mSR-BI mediated HDL CE
selective uptake with an approximately 7-fold greater efficiency than
rCD36.

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Fig. 2.
Comparison of HDL CE selective uptake
mediated by SR-BI and CD36. COS-7 cells transiently expressing
SR-BI or CD36 were incubated at 37 °C for 1.5 h with
125I/3H-labeled HDL (10 µg of protein/ml),
after which the cells were processed to determine cell-associated HDL
CE (A) and HDL CE selective uptake (B). Values
represent the mean of triplicate determinations after subtraction of
the determinations with the addition of a 50-fold excess of cold
HDL3. C, the efficiency of HDL CE selective
uptake was determined by subtracting the values from vector-transfected
cells and normalizing the amount of HDL CE selective uptake to the
amount of cell-associated HDL particles.
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The reduced efficiency of HDL CE selective uptake with rCD36 suggested
that this level of selective uptake might be attributable only to
tethering of HDL particles on the cell surface. Alternatively, expression of rCD36 might indirectly alter HDL CE selective uptake by
perturbing plasma membrane structure or lipid domains to facilitate the
transfer of HDL CE to the plasma membrane independently of receptor-HDL
particle binding. To test whether rCD36-mediated HDL CE selective
uptake is due to HDL binding, we mutated the extracellular domain of
rCD36 to disrupt HDL binding by inserting the 14-amino acid monoclonal
M45 epitope within the N-terminal region of the extracellular domain
(rCD36-X). Flow cytometry of cells expressing rCD36 or rCD36-X showed
that both receptors were expressed on the cell surface to similar
extents (data not shown). Comparison of HDL cell association (Fig.
3A) and HDL CE selective uptake (Fig. 3B) showed that the epitope tag reduced HDL
binding and HDL CE selective uptake to the level seen with
vector-transfected cells. This result indicates that the HDL CE
selective uptake mediated by rCD36 requires cell surface HDL particle
binding. These data support the "tethering hypothesis" by showing
that as long as rCD36 can bind HDL particles close to the cell surface of COS-7 cells, HDL CE selective uptake will occur, albeit at a reduced
efficiency compared with SR-BI.

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Fig. 3.
Cell-associated HDL and HDL CE selective
uptake mediated by CD36 and CD36-X. COS-7 cells transiently
expressing CD36 or CD36-X were incubated at 37 °C for 1.5 h
with 125I/3H-labeled HDL (10 µg of
protein/ml), after which cells were processed to determine
cell-associated HDL CE (A) and HDL CE selective uptake
(B). Values represent the mean of triplicate determinations
after subtraction of values obtained with the addition of a 50-fold
excess of unlabeled HDL3.
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Role of the C-terminal Cytoplasmic Tail in HDL Binding and HDL CE
Selective Uptake--
mSR-BII is identical to mSR-BI except that it
contains a different C-terminal cytoplasmic tail as a result of
alternate splicing of SR-BI pre-mRNA (19, 29). The influence of the
alternate C-terminal tail on HDL binding and HDL CE selective uptake
was determined in transient transfection assays. Little or no
difference was seen in the ability to bind HDL particles (Fig.
4A), but COS-7 cells
expressing mSR-BII showed a greatly reduced ability to mediate selective uptake (Fig. 4B). When HDL CE selective uptake was
expressed on the basis of cell-associated HDL particles (Fig.
4C), mSR-BI-mediated HDL CE selective uptake with an
approximately 7-fold greater efficiency than mSR-BII. Therefore, rCD36
and mSR-BII both showed a similar low efficiency of HDL CE selective
uptake in comparison with mSR-BI.

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Fig. 4.
Cell-associated HDL and HDL CE selective
uptake mediated by SR-BI and SR-BII. COS-7 cells transiently
expressing SR-BI or SR-BII were incubated at 37 °C for 1.5 h
with 125I/3H-labeled HDL (10 µg of
protein/ml), after which cells were processed to determine
cell-associated HDL CE (A) and HDL CE selective uptake
(B). Values represent the mean of triplicate determinations
after subtraction of the determinations with the addition of a 50-fold
excess of cold HDL3. C, the efficiency of HDL CE
selective uptake was determined by subtracting the values from
vector-transfected cells and normalizing the amount of HDL CE selective
uptake to the amount of cell-associated HDL particles. The selective
uptake efficiency for CD36 is shown for comparison.
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The comparison of mSR-BI and mSR-BII suggests that the C-terminal
cytoplasmic tail has a significant influence on the selective uptake
process. To further test the role of the cytoplasmic domains, chimeric
receptors were constructed and expressed in COS-7 cells in comparison
with mSR-BI and rCD36. One set of chimeric receptors contained the
rCD36 extracellular domain with the C-terminal cytoplasmic tail (T) of
mSR-BI with (CD/SRTM) or without (CD/SRT) the C-terminal transmembrane
domain (M) of mSR-BI. Another chimeric receptor has the extracellular
domain of CD36 with both the N- and C-terminal tails and transmembrane
domains of mSR-BI (SR/CD/SR) (Fig. 5). When these chimeric receptors were expressed in COS-7 cells, each bound
HDL nearly as well as native rCD36 (Fig.
6A), and each showed a similar
level of HDL CE selective uptake as compared with rCD36 (Fig.
6B). Comparison of the selective uptake efficiencies of these chimeras (Fig. 6C) showed that they were no different
than CD36. Thus, the cytoplasmic tails and transmembrane domains of mSR-BI were not sufficient to confer efficient HDL CE selective uptake
activity on the rCD36 extracellular domain despite its ability to bind
HDL particles.

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Fig. 5.
Schematic diagram of mSR-BI, CD36, and
chimeric receptors. This diagram illustrates the wild type
structures of SR-BI and CD36 in comparison with the chimeric receptors.
The amino acid delineations of the chimeras are as follows: CD/SRT,
rCD36 amino acids 1-458 and mSR-BI amino acids 464-509; CD/SRTM,
rCD36 amino acids 1-435 and mSR-BI amino acids 436-509; SR/CD/SR,
rCD36 amino acids 44-435 and mSR-BI amino acids 1-41 and 436-509;
SR/CDT, mSR-BI amino acids 1-467 and rCD36 amino acids 459-472.
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Fig. 6.
Cell-associated HDL and HDL CE selective
uptake mediated by SR-BI, CD/36, and chimeric receptors. COS-7
cells transiently expressing mSR-BI, rCD36, CD/SRTM, SR/CD/SR, or
SR/CDT (Fig. 6) were incubated at 37 °C for 1.5 h with
125I/3H-labeled HDL (10 µg of protein/ml),
after which cells were processed to determine cell-associated HDL CE
(A) and HDL CE selective uptake (B). Values
represent the mean of triplicate determinations after subtraction of
values obtained with the addition of a 50-fold excess of unlabeled
HDL3. C, the efficiency of HDL CE selective
uptake was determined by subtracting the values from vector-transfected
cells and normalizing the amount of HDL CE selective uptake to the
amount of cell-associated HDL particles.
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To determine whether the C-terminal cytoplasmic tail of mSR-BI is
unique in facilitating HDL CE selective uptake in the context of SR-BI,
the C-terminal tail of SR-BI was replaced with that of rCD36 (Fig. 5,
SR/CDT). When expressed in COS-7 cells, this chimera bound
HDL (Fig. 6A) and mediated HDL CE selective uptake (Fig.
6B) similar to native mSR-BI. Comparison of the selective uptake efficiency for SR/CDT (Fig. 6C) showed that the rCD36
C-terminal cytoplasmic tail supported full HDL CE selective uptake
activity as seen with native mSR-BI.
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DISCUSSION |
Despite extensive studies of the HDL CE selective uptake process
over the past 18 years, the molecular mechanism by which hydrophobic
cholesteryl esters are transferred from the HDL particle to the plasma
membrane has remained elusive. The finding that mSR-BI mediates HDL CE
selective uptake provides a molecular link between a specific cell
surface receptor and a widespread, but poorly understood, biological
process. In the present study, we have examined the mechanism of
mSR-BI-mediated HDL CE selective uptake by comparing mSR-BI (and
mSR-BII) with the closely related class B scavenger receptor, rCD36,
and with chimeric receptors formed from mSR-BI and rCD36. The results
lead to three major conclusions.
First, it is clear that the high efficiency of mSR-BI-mediated HDL CE
selective uptake is due to more than simply tethering HDL particles on
the cell surface. Analysis of HDL binding and selective CE uptake
showed that CD36 can mediate HDL CE selective uptake with a reduced
efficiency. This component of selective uptake is probably due to
tethering HDL particles to the plasma membrane, since disruption of HDL
binding to rCD36 also disrupted HDL CE selective uptake (Fig. 3).
Furthermore, reduced levels of selective uptake similar to that seen
with rCD36 also were seen with mSR-BII and three chimeric receptors
containing the extracellular domain of rCD36. Taken together, these
results indicate that binding of HDL particles to a class B scavenger
receptor is sufficient to produce HDL CE selective uptake, presumably
by bringing the HDL particle in apposition to the plasma membrane. This
finding is consistent with earlier studies in cell-free systems showing
selective transfer of HDL CE to protein-free model membranes in a
collision-mediated process (14). Thus, HDL CE transfer to membranes may
occur at low levels in the absence of any receptor protein and is
accelerated by tethering HDL particles to a class B scavenger receptor.
The enhancement of HDL CE selective uptake that is specific to mSR-BI,
however, exceeds this tethering contribution by a factor of 7-fold. The
mechanism by which mSR-BI facilitates HDL CE transfer is presently
unclear, but it is interesting to note that the Arrhenius activation
energy for mSR-BI-dependent HDL CE selective uptake in
Y1-BS1 adrenocortical cells is very low (~9
kcal/mole).2 This suggests
that CE molecules must move through a nonaqueous pathway or channel
from the HDL particle to the plasma membrane. The high efficiency of
mSR-BI-mediated selective uptake may be due to the formation of a
nonaqueous pathway to permit HDL CE molecules to move efficiently into
the plasma membrane. We hypothesize from these data that
mSR-BI-mediated HDL CE selective uptake occurs through two steps: a
generalized HDL tethering component that is shared with other class B
scavenger receptors and an active facilitation of HDL CE uptake that is
unique to mSR-BI. We note that it is formally possible that rCD36 also
exhibits the facilitation of HDL CE uptake but does so at much reduced
efficiency. However, the quantitative equivalence of the HDL CE
selective uptake activity of rCD36, mSR-BII, and three chimeric
receptors is more likely the result of a clear dissociation between HDL
tethering and the facilitation of HDL CE uptake.
Second, comparison of mSR-BI and mSR-BII showed that mSR-BII mediated
HDL CE selective uptake with a low efficiency similar to that seen with
rCD36. Webb et al. (29) also noted that a stable CHO cell
line expressing mSR-BII mediated HDL CE selective uptake to a lesser
extent than a cell line expressing mSR-BI. Since mSR-BII and mSR-BI
differ only in the C-terminal cytoplasmic tail, this result suggests
that the tail is important for high efficiency HDL CE selective uptake
as mediated by mSR-BI. The C-terminal tail of mSR-BI might be
responsible for targeting the receptor to a plasma membrane domain or
for interactions with cytoplasmic proteins that are necessary for
selective uptake. With regard to the first possibility, mSR-BI has been
found in membrane caveolae (22), a location that might have functional consequences for cholesterol flux (31). However, both mSR-BII and CD36
have also been found in caveolae (20, 21, 29), suggesting that caveolar
localization per se may not explain the difference in
selective uptake efficiency of mSR-BI versus mSR-BII and
rCD36, unless there are uncharacterized differences in localization to
lipid domains within caveolar fractions. Another possibility is that
the mSR-BI C-terminal tail facilitates interactions with other membrane
or cytoplasmic proteins that are necessary for efficient HDL CE uptake.
If this is the case, these interactions must not be unique to the
mSR-BI tail, since the rCD36 C-terminal tail yields full HDL CE
selective uptake activity when swapped for the mSR-BI tail. Another
possibility is that the C-terminal tail of mSR-BI has no active role in
the selective uptake process but that the C-terminal tail of SR-BII is
in some way inhibitory, perhaps by altering the conformation of the
extracellular domain. Further studies will be necessary to resolve this point.
Third, the mSR-BI N- and C-terminal tails and transmembrane domains
were not sufficient to confer high efficiency HDL CE selective uptake
when expressed in a chimeric receptor with the extracellular domain of
CD36 (Fig. 6). This result indicates that the extracellular domain of
SR-BI does more than simply bind HDL with high affinity and tether the
particles close to the plasma membrane. The extracellular domain
appears to be required for the marked facilitation of HDL CE uptake
that occurs in addition to the component that derives from HDL cell
surface binding. One potential role of the extracellular domain is to
create a nonaqueous pathway or channel for HDL CE transfer either by
homomeric interactions or heteromeric interactions with other membrane
proteins. This activity of the mSR-BI extracellular domain might also
require the transmembrane domains of mSR-BI, although the transmembrane
domains of mSR-BI themselves were not adequate to support high
efficiency selective uptake when appended to the extracellular domain
of rCD36.
An interesting result in the present study is the finding that rCD36
mediates HDL CE selective uptake although with a much reduced
efficiency as compared with mSR-BI. This result raises the question of
whether lipoprotein selective lipid uptake mediated by CD36 may be of
physiological significance in some circumstances. Recent studies, for
example, show that CD36 mediates the entry of lipid activators into
macrophage from oxidized LDL. This results in transcriptional
activation of PPAR
and increased expression of CD36 (32). This
positive feedback pathway has been proposed to play a role in
macrophage foam cell formation in the vascular wall during
atherogenesis. Whether the selective uptake by CD36 of PPAR
activators from oxidized LDL contributes to this pathway remains to be
determined. Furthermore, selective uptake of HDL CE or LDL-CE by CD36
may contribute to foam cell formation once this receptor has been
up-regulated.
In summary, the present studies with mSR-BI, mSR-BII, CD36, and
chimeric receptors support the idea that HDL CE selective uptake
results from at least two steps. In the first, tethering the HDL
particle to the cell surface via a class B scavenger receptor leads to
a moderate increase in HDL CE uptake. The mechanism of this CE transfer
is not clear, but it presumably reflects an enhancement of the process
that can occur with model membranes in cell free systems as a result of
bringing the HDL particle close to the plasma membrane. The second step
is a marked facilitation of HDL CE selective uptake that is specific to
mSR-BI. This facilitation step requires the extracellular domain of
mSR-BI but can occur with the C-terminal cytoplasmic tail of either
mSR-BI or CD36. We speculate that this mSR-BI-specific step is
responsible for the formation of a nonaqueous pathway that permits the
movement of the hydrophobic CE down its concentration gradient from the HDL particle to the plasma membrane.