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National Institute on Aging, National Institutes of Health, Gerontology Research Center, Baltimore, MD 21224
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-cyclodextrin (BCD)
inhibits stromal cell-derived factor 1
(SDF-1) binding to CXCR4
on T cell lines and PBMCs. Intracellular calcium responses to SDF-1,
as well as receptor internalization, were impaired in treated T cells.
Loss in ligand binding is likely due to conformational changes in CXCR4
and not increased sensitivity to internalization. SDF-1 binding and
calcium responses were effectively restored by reloading cholesterol.
Immunofluorescence microscopy revealed that SDF-1 binding occurred
in lipid raft microdomains that contained GM1. CXCR4 surface
expression, on the other hand, only partially colocalized with GM1.
HIV-1IIIB infection assays confirmed the functional loss of
CXCR4 in the cell lines tested, Sup-T1 and CEM-NKR-CCR5. These data
suggest that cholesterol is essential for CXCR4 conformation and
function and that lipid rafts may play a regulatory role in SDF-1
signaling. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(SDF-1,2 CXCL12) is
a member of the CXC chemokine family that specifically binds to CXCR4
present on T cells, B cells, and macrophages, as well as a number of
other immune and nonimmune cell subsets (2). HIV has
adapted to using CXCR4 as a coreceptor for infection. Viral tropism is
defined by usage of either CXCR4 (X4 viruses) and/or CCR5 (R5
viruses) (3). Chemokine receptors play a critical
role in viral fusion with the host membrane. Following binding to CD4,
conformational changes in the viral envelope surface protein
(env) triggers binding to CXCR4 and induces fusion of the
apposing membranes (4). SDF-1 has been shown to be able
to block X4 tropic-HIV fusion (5, 6). Molecules that block
CXCR4 interaction with env have also been demonstrated to be effective
in preventing infection (7, 8).
Lipid rafts are membrane microdomains enriched in cholesterol,
sphingolipids, GPI-anchored proteins, and acylated signaling molecules
on the plasma membrane of immune and nonimmune cells (9).
In T cells, lipid rafts are important sites of assembly for the TCR
signaling complex (10). CD4 and CD8, as well as their
cytoplasmic partner, Lck, have been demonstrated to be palmitoylated
and present in lipid rafts (11, 12, 13). Recent evidence has
established that CXCR4 localizes to lipid rafts, suggesting that these
membrane domains are the preferred sites for HIV entry
(14). Soluble HIV viral env protein, gp120, has been shown
to form trimolecular complexes with CD4 and CXCR4 within lipid rafts,
supporting the model that rafts serve as sites of HIV infection
(14). Also, the depletion of cholesterol, which disrupts
lipid rafts, has been shown to inhibit HIV infection and syncytium
formation (15). Cholesterol depletion has also been
demonstrated to inhibit T cell polarization and chemotaxis induced by
SDF-1 (16). Cholesterol may be more important to CXCR4
and chemokine receptors than simply maintaining raft integrity.
Previous studies with several other seven-transmembrane-spanning G
protein-coupled receptors, including the oxytocin, cholecystokinin,
galanin, and 
-aminobutyric acid receptors, have shown that
ligand binding is decreased with the removal of cholesterol by
-cyclodextrin (BCD) (17, 18, 19). Modulation of lipids,
especially cholesterol, in the cell membrane may be more likely to
affect proteins that have large portions within the cell membrane,
including chemokine receptors. We sought to determine whether
modulation of cholesterol content in cell membranes by the removal of
cholesterol might similarly affect the ability of CXCR4 to bind
SDF-1. Our data demonstrate that cholesterol is required for ligand
binding to CXCR4 and subsequent events, including calcium stimulation
and receptor internalization.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell lines and HIV-1IIIB were obtained through the AIDS Research and Reference Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, from Dr. A. Trkola (CEM-NKR-CCR5, referred to in this article as CEM-R5), Dr. A. Weiss (Jurkat clone E6-1), Dr. J. Hoxie (Sup-T1), and Dr. R. Gallo (HIV-1IIIB). Cells were grown in RPMI 1640 (Mediatech; Cellgro, Herndon, VA) supplemented with 10% heat-inactivated FBS (BioSource International, Rockville, MD), 10 mM HEPES, and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin) (cRPMI). mAbs to CXCR4, clone 12G5, were purchased from BD PharMingen (San Diego, CA) and clones, 44708.111, 44716.111, and 44717.111 were purchased from R&D Systems (Minneapolis, MN). Rabbit anti-human CXCR4 polyclonal Ab, AHP442, was purchased from Serotec (Raleigh, NC). Mouse IgG2a control, mouse IgG1 control, anti-CD4 (clone RPA-T4), and anti-CD45 (clone HI30) were purchased from BD Biosciences (San Diego, CA). Goat anti-mouse IgG (H + L) labeled with Alexa Fluor 488 (GAM-AF488) and cholera toxin subunit B labeled with Alexa Fluor 594 were purchased from Molecular Probes (Eugene, OR). Alexa Fluor 488 has a laser absorption and emission spectrum profile similar to fluorescein, while Alexa Fluor 594 has a profile similar to that of Texas Red.
BCD treatment
BCD, Trappsol (Cyclodextrin Technologies Development,
Gainesville, FL), was dissolved in PBS to the desired concentrations.
Cholesterol-loaded BCD (chol-BCD) was prepared as previously described
(15). Briefly, cholesterol (5-cholesten-3-ol;
3-hydroxy-5-cholestene; Sigma-Aldrich, St. Louis, MO) powder was
added to 240 mM BCD solution at 1.16 mg/ml, vortexed for 6 h, and
was subsequently syringe filter sterilized using a 0.22-µm filter
unit. In cholesterol extraction studies, up to 8 x
106 suspension cells were washed with PBS and
resuspended in 1 or 2 ml of 20 mM BCD in PBS or PBS alone as a control.
The cells were then incubated for 1 h at 37°C before being
washed with either PBS or RPMI 1640. In cholesterol-reloading
studies, cells were incubated with chol-BCD in PBS at a concentration
of 300 µM cholesterol for 30 min. To remove chol-BCD, cells were
washed with at least 10 volumes of PBS and resuspended in PBS for
further analysis.
Fluorokine ligand staining
Biotinylated SDF-1 (Fluorokine; R&D Systems) staining was
performed according to R&D Systems’ protocols, with slight
modifications. Briefly, control or treated cells were resuspended in
PBS at 4 x 106/ml. Fifty microliters of
cells was then mixed with 20 µl of 2.5 µg/ml biotinylated SDF-1
or 5 µg/ml biotinylated soybean trypsin inhibitor and then incubated
at 4°C for 1 h. Fluorescein-conjugated avidin (10 µg/ml) was
added (10–20 µl) to the cells and incubated for an additional 30 min
at 4°C. After incubation, cells were washed with 1x RDF-1 buffer
(R&D Systems) and then fixed with 2% paraformaldehyde in PBS before
being analyzed on a FACScan (BD Biosciences).
Intracellular calcium mobilization
Measurement of calcium mobilization by chemokine stimulation was
performed as previously described (20). Briefly, untreated
or BCD-treated CEM-NKR-CCR5 (CEM-R5) cells (8 x
106/ml) were incubated in PBS with
Ca2+ and Mg2+ containing 5
µM fura-2-acetoxymethyl ester (Molecular Probes) for 30 min at room
temperature. The cells were subsequently washed and then resuspended at
1 x 106/ml in PBS. A total of 2 ml of the
cell suspension was placed in a continuously stirred cuvette at room
temperature in an LS50B spectrophotometer (PerkinElmer, Wellesley, MA).
Fluorescence was monitored at 
ex1 = 340 nm,
ex2 = 380 nm, and em
= 510 nm. The data are presented as the relative ratio of fluorescence
excited at 340 and 380 nm. SDF-1 (PeproTech, Rocky Hill, NJ) was
tested at a final concentration of 1 µg/ml.
Flow cytometry
CEM-R5 cells (1 x 106) in PBS
containing 2% FBS were added to 1–2 µg of mAbs and incubated for 30
min on ice. Cells were washed with PBS, resuspended in 100 µl of 20
µg/ml GAM-AF488, and incubated on ice for 30 min. Cells were then
washed with PBS and fixed with 2% paraformaldehyde in PBS, followed by
analysis on a FACScan. For the prefixing experiments, cells were washed
with PBS after BCD treatment and then fixed with 2% paraformaldehyde
in PBS for 30 min on ice. After incubation, the cells were then washed
with PBS, resuspended in PBS containing 2% FBS, and then incubated an
additional 30 min on ice before staining with mAbs. For internalization
assays, BCD-treated CEM-R5 cells were incubated with or without 1
µg/ml SDF-1 at 37°C for 30 min, washed with PBS, and then fixed
with 2% paraformaldehyde in PBS. Remaining surface expression of
receptor was analyzed by flow cytometry using mIgG2a, anti-CD4 as a
control, or anti-CXCR4 mAb, 12G5. Percent internalization was
calculated as [(mean fluorescence intensity
(MFI)c -
MFIt)/MFIc ] x 100, where
MFIc = mean fluorescence intensity of 12G5
binding to cells incubated with PBS only and MFIt
= mean fluorescence intensity of cells after incubation with
SDF-1.
Immunomicroscopy
CEM-R5 cells (1 x 106) were washed in cold PBS, resuspended in 100 µl of PBS containing 2% FBS, and 20 µg/ml cholera toxin B Alexa Fluor 594 and then incubated on ice for 30 min. Cells were then washed with PBS and subsequently stained for CXCR4 with 2 µg of 12G5, followed by GAM-AF488. The cells were washed with PBS and then fixed with 1% paraformaldehyde in PBS. After staining, the cells were placed into cytospin funnels and spun onto glass slides using a cytospin centrifuge (Thermo Shandon, Pittsburgh, PA). Bound cells were layered with 30 µl of 50% glycerol in PBS and covered with a glass coverslip. Images were acquired by Spot Advanced software on a Zeiss Axiovert S100 microscope under x100 objective.
HIV infection
Sup-T1 and CEM-R5 cells were treated with BCD or untreated. Cells were washed with PBS, resuspended in cRPMI to a concentration of 4 x 106/ml, and then incubated with cRPMI-diluted HIV-1IIIB at 0.5 50% tissue culture-infective dose/cell for 90 min at 37°C. Cells were washed twice with PBS, resuspended in cRPMI, and cultured in triplicate wells of a 96-well tissue culture treated plate. HIV p24 was determined from supernatant collected on day 4 postinfection with a standard p24 ELISA kit (Zymed Laboratories, San Francisco, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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binding to T cells is significantly inhibited by
cholesterol extraction with BCD
To directly examine the role of cholesterol in SDF-1 binding to
CXCR4 on the surface of T cells, we treated fresh human PBMCs and three
human T cell lines, Jurkat clone E6-1, Sup-T1, and CEM-NKR-CCR5
(CEM-R5) with BCD. Treatment of cell lines with 20 mM BCD in PBS
removes 
75% of total cellular cholesterol within 1 h and is
nontoxic to cells, as measured by the release of cytoplasmic lactate
dehydrogenase and exclusion of trypan blue (data not shown). SDF-1
binding was found to be markedly reduced after BCD treatment of all the
cells tested (Fig. 1
A). The
effects were greatest on Jurkat cells, demonstrating nearly 100%
inhibition of SDF-1 binding. For subsequent studies, we used the
CEM-R5 cells which possess a moderate amount of SDF-1 binding under
normal conditions. A dose-dependent reduction in SDF-1 binding to
CEM-R5 cells was observed using increasing BCD treatment
concentrations, indicating specificity for cholesterol extraction (Fig. 1B).
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C demonstrate a significant loss of
SDF-1 binding after only 10 min of BCD treatment, indicating that
cholesterol extraction and the effects on CXCR4 are rapid. Reloading
cell membranes with cholesterol completely restored SDF-1 binding to
these treated cell populations (Fig. 1C). These results
confirm that reloading of cholesterol restores SDF-1 binding and
that cell death does not account for any loss in ligand binding.
Moreover, BCD itself does not exert any inhibitory activity as
repletion of cholesterol into cell membranes also involves BCD as a
vehicle for cholesterol.
BCD treatment inhibits SDF-1-induced intracellular calcium
response and receptor internalization
We next examined changes in CXCR4 signaling by measuring
intracellular calcium mobilization in response to SDF-1 binding on
BCD-treated CEM-R5 cells. A distinct rise in calcium mobilization with
recombinant SDF-1 treatment was observed in control CEM-R5 cells
(Fig. 2A). This rise was
significantly inhibited after BCD (64% inhibition) treatment of these
cells (Fig. 2A). Reloading of cholesterol onto BCD-treated
cells restored the calcium response to normal (Fig. 2A).
These results provide functional confirmation for the loss in surface
binding of SDF-1. The effects were cholesterol-specific, as
restoration of cholesterol levels on BCD-treated T cell membranes
restored the ability of SDF-1 to bind to and induce intracellular
calcium mobilization with these T cells.
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ligation of CXCR4 results in rapid receptor
internalization. To further demonstrate that SDF-1 interactions with
cell surface CXCR4 was inhibited on treated cells, we next examined
chemokine-induced receptor internalization following BCD treatment. In
CEM-R5 cells, 1 µg/ml recombinant SDF-1 at 37°C for 30 min
induces nearly 70% receptor internalization, as analyzed by mAb, 12G5,
surface binding (Fig. 2B). BCD-treated cells had a
significant loss in SDF-1-induced receptor internalization, detected
by mAb binding at the cell surface, resulting in only 39%
internalization. CD4 staining was analyzed as a nonspecific control for
internalization. These results confirm the loss in SDF-1 binding and
induction of calcium mobilization appears not to be due to receptor
internalization but rather an alteration in the CXCR4 receptor at the
plasma membrane. BCD treatment alters mAb binding to CXCR4
The loss in SDF-1 binding to cholesterol-deficient cells may be
attributable to changes in conformation of the receptor. We analyzed
the binding of a multidomain recognizing mAb, 12G5, as well as three
other anti-CXCR4 mAbs capable of neutralizing SDF-1-mediated
chemotaxis, to determine whether mAb-binding epitopes of CXCR4 were
altered on BCD-treated cells. We found that 12G5 binding was
significantly decreased by BCD (58.8% reduction in MFI, see Table I). Binding of control mAbs, anti-CD4
and anti-CD45, were not affected by BCD treatment. The binding of
three other anti-CXCR4 mAbs, 44708, 44716, and 44717 also decreased
following BCD treatment (Table II). To
show that CXCR4 remained at the surface after BCD treatment, we fixed
the cells with paraformaldehyde immediately after BCD treatment and
then analyzed mAb binding. The binding of 12G5, 44708, and 44716
actually increased on BCD-fixed cells and not on control-fixed cells
(Table II). On the other hand, the recovery of 44717 binding after
fixation did not reach 100% of the control cells. These findings
clearly indicate that the 12G5 binding epitope can be recovered and
enhanced by fixation on BCD-treated cells, suggesting once again that a
conformational alteration may have occurred. The results also suggest
that epitopes recognized by distinct CXCR4 mAbs are differentially
affected by BCD treatment. To demonstrate that the effects on 12G5
binding are cholesterol specific, we reloaded cholesterol onto
BCD-treated cells and analyzed mAb binding. Binding of 12G5 was
restored to >100% by reloading cholesterol onto BCD-treated cells
(Fig. 3A). As a control, mAb
binding to CD45 was not dependent on cholesterol levels.
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B) that AHP442
staining using FITC-goat anti-rabbit conjugate was significantly
lower than 12G5 (detected by goat anti-mouse AF488). Interestingly,
the binding of AHP442 to CEM-R5 cells was minimally affected by 10 mM
BCD treatment (MFI from 8.28 to 8.12), whereas the 12G5 values dropped
significantly (MFI from 161 to 112) (Fig. 3B). The minimal
change in AHP442 binding suggests that the recognized linear epitope is
not significantly affected by BCD and that the receptor remains on the
surface after BCD treatment.
SDF-1 binds to lipid raft regions but CXCR4 only partially
colocalizes with lipid rafts
Cholesterol is enriched in and essential to the formation of lipid
rafts. We speculated that SDF-1 may preferentially bind to CXCR4
within lipid rafts since cholesterol appears to be essential for
overall SDF-1 binding. We examined fluorescently labeled SDF-1
membrane binding to CEM-R5 and Sup-T1 cells labeled with cholera toxin
B subunit (CT-B)-Alexa Fluor 594 conjugate to detect GM1 enriched in
lipid rafts. We found that 25% of CEM-R5 cells exhibited a capped
or polarized phenotype for GM1. In these cells, the majority of
SDF-1 binding clearly colocalized with CT-B staining (Fig. 4A). Sup-T1 cells also
exhibited colocalization of SDF-1 and GM1, although most cells did
not display a capped phenotype (Fig. 4A). In contrast,
although staining with anti-CXCR4 (12G5) revealed that receptor
colocalization with GM1 also occurs in rafts but only on capped CEM-R5
cells (Fig. 4B), CXCR4 and GM1 do not colocalize and appear
as distinct patches on noncapped cells (Fig. 4C). These
findings strongly suggest that SDF-1 preferentially binds to
raft-associated CXCR4. Although it is possible that low levels of
SDF-1 binding occur outside of GM1-stained areas that are not
detected by microscopy.
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It has been previously demonstrated that BCD treatment of target
cells inhibits HIV-1 infection (14, 15). We next wished to
establish that BCD treatment would similarly inhibit HIV-1 infection in
our cell lines, correlating the SDF-1 binding results. BCD treatment
inhibited infection by R4-tropic HIV-1IIIB in
both Sup-T1 cells (84.7% inhibition) and CEM-R5 cells (47.2%
inhibition) (Fig. 5). These data
correspond with the loss in ligand binding (80% inhibition with
Sup-T1, 62% inhibition with CEM-R5 cells, Fig. 1A), calcium
mobilization, and internalization of CXCR4 seen with BCD treatment of T
cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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binding to its receptor, CXCR4, on the surface of T cells. It
is clear that the cholesterol molecule is important for proper function
of CXCR4 in binding to SDF-1. Reloading cholesterol onto cells
treated with BCD can rapidly restore SDF-1 binding. Paraformaldehyde
fixation restores anti-CXCR4 mAb binding to BCD-treated cells as
well, most likely due to the reformation of epitopes by cross-linking.
We have determined that the loss of SDF-1 binding is not due to
internalization of the receptor, but is likely due to a loss in
conformation since fixation following BCD treatment of cells restores
and improves mAb binding. This is further supported by the evidence
that binding of a polyclonal Ab to the amino terminus of CXCR4 did not
change upon BCD treatment. We have also established that conformational
changes in another chemokine receptor, CCR5, occur with cholesterol
depletion.3
Those findings with macrophage-inflammatory protein 1 binding
parallel these SDF-1 results.
Mañes et al. (14) proposed that lipid rafts serve as
the sites for assembly of gp120 with CD4 and CXCR4. They demonstrated
that CD4 binding to soluble gp120 is independent of cholesterol and
lipid rafts in cell membranes. We propose that CD4-bound viruses are
unable to interact with CXCR4 due to its conformational changes in the
absence of normal cholesterol, thus inhibiting infection (Fig. 6). Changes in receptor conformation
could also explain the loss in cell polarity and chemotaxis with
BCD-treated cells upon stimulation with SDF-1 (16).
Thus, not only do lipid rafts serve as platforms for HIV infection,
they provide cholesterol-enriched sites necessary for appropriate
receptor conformation to support chemokine binding. Our attempts to
directly measure the effects of BCD treatment on soluble CD4-gp120
complex binding to Sup-T1 and CEM-R5 cells were unsuccessful due to the
very low levels of binding detected with our methods. Nonetheless, we
believe that lipid rafts are the cell surface sites where CD4 localizes
and CXCR4 is most functional. Therefore, disruption of rafts would have
a 2-fold effect on HIV infection by dispersing CD4 from functional
CXCR4 and also changing the binding properties of CXCR4.
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One could propose that the integrity of lipid rafts, not the presence
of cholesterol alone, may be essential for chemokine receptor function.
Our results in no way demonstrate a direct or specific interaction of
cholesterol molecules with CXCR4 within the cell membrane. For example,
the packing of sphingolipids, gangliosides, or even GPI-anchored
proteins into highly ordered domains may be providing the appropriate
environment for SDF-1 binding and signaling. Additionally, the fact
that G proteins required for chemokine receptor signaling are localized
to lipid rafts on the cytoplasmic surface, predicts that raft
disruption would also inhibit signaling (21). Our
demonstration that SDF-1 binding preferentially occurs in lipid
rafts, despite the presence of CXCR4 outside of GM-stained areas on the
T cell surface, supports this model. Another possibility may be that
SDF-1 binding causes aggregation of receptor molecules to lipid
rafts similar to that mediated by gp120 (14).
Cholesterol appears to play a critical role in the function of many
GPCRs. The function of many multiple membrane-spanning proteins,
including the chemokine receptor family, is likely to be affected by
modulation of membrane lipids, especially cholesterol. We have
established that cholesterol directly participates in CXCR4 function by
preserving the functional conformation of the receptor. It seems
possible that the inability of SDF-1 to bind to non-raft-associated
CXCR4 may play a regulatory role in maintaining receptor activity and
the migratory potential of the cell. We believe that circulating T
cells possess predominantly non-raft-associated CXCR4 with low affinity
for chemokine ligands within the circulation. Upon a specific
activating signal or through selectin-integrin interactions with the
endothelial cell surface, these receptors are recruited to rafts within
the membrane, thus yielding a high-affinity, SDF-1 responsive cell
population. Studies are currently underway to directly examine this
question. We hope that these studies may lead to further understanding
of the dynamic interactions between lipids and receptor function and
regulation in immune cells.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: SDF-1, stromal cell-derived factor 1; GPCR, G protein-coupled receptor; BCD, hydroxypropyl--cyclodextrin; MFI, mean fluorescence intensity; CT-B, cholera toxin B subunit; chol-BCD, cholesterol-loaded BCD.
3 D. H. Nguyen and D. Taub. Cholesterol is essential for MIP-1 binding and conformational integrity of CC chemokine receptor 5. Submitted for publication.
Received for publication November 7, 2001. Accepted for publication February 13, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Y. Le, M. Honczarenko, A. M. Glodek, D. K. Ho, and L. E. Silberstein CXC Chemokine Ligand 12-Induced Focal Adhesion Kinase Activation and Segregation into Membrane Domains Is Modulated by Regulator of G Protein Signaling 1 in Pro-B Cells J. Immunol., March 1, 2005; 174(5): 2582 - 2590. [Abstract] [Full Text] [PDF]
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C.-S. Chung, C.-Y. Huang, and W. Chang Vaccinia Virus Penetration Requires Cholesterol and Results in Specific Viral Envelope Proteins Associated with Lipid Rafts J. Virol., February 1, 2005; 79(3): 1623 - 1634. [Abstract] [Full Text] [PDF]
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M. Wysoczynski, R. Reca, J. Ratajczak, M. Kucia, N. Shirvaikar, M. Honczarenko, M. Mills, J. Wanzeck, A. Janowska-Wieczorek, and M. Z. Ratajczak Incorporation of CXCR4 into membrane lipid rafts primes homing-related responses of hematopoietic stem/progenitor cells to an SDF-1 gradient Blood, January 1, 2005; 105(1): 40 - 48. [Abstract] [Full Text] [PDF]
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 C. M. Finnegan, S. S. Rawat, A. Puri, J. M. Wang, F. W. Ruscetti, and R. Blumenthal Ceramide, a target for antiretroviral therapy PNAS, October 26, 2004; 101(43): 15452 - 15457. [Abstract] [Full Text] [PDF]
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S. Campbell, K. Gaus, R. Bittman, W. Jessup, S. Crowe, and J. Mak The Raft-Promoting Property of Virion-Associated Cholesterol, but Not the Presence of Virion-Associated Brij 98 Rafts, Is a Determinant of Human Immunodeficiency Virus Type 1 Infectivity J. Virol., October 1, 2004; 78(19): 10556 - 10565. [Abstract] [Full Text] [PDF]
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 G. del Real, S. Jimenez-Baranda, E. Mira, R. A. Lacalle, P. Lucas, C. Gomez-Mouton, M. Alegret, J. M. Pena, M. Rodriguez-Zapata, M. Alvarez-Mon, et al. Statins Inhibit HIV-1 Infection by Down-regulating Rho Activity J. Exp. Med., August 16, 2004; 200(4): 541 - 547. [Abstract] [Full Text] [PDF]
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 J. D. van Buul and P. L. Hordijk Signaling in Leukocyte Transendothelial Migration Arterioscler Thromb Vasc Biol, May 1, 2004; 24(5): 824 - 833. [Abstract] [Full Text]
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 A. Vendeville, F. Rayne, A. Bonhoure, N. Bettache, P. Montcourrier, and B. Beaumelle HIV-1 Tat Enters T Cells Using Coated Pits before Translocating from Acidified Endosomes and Eliciting Biological Responses Mol. Biol. Cell, May 1, 2004; 15(5): 2347 - 2360. [Abstract] [Full Text] [PDF]
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 C. Gomez-Mouton, R. A. Lacalle, E. Mira, S. Jimenez-Baranda, D. F. Barber, A. C. Carrera, C. Martinez-A., and S. Manes Dynamic redistribution of raft domains as an organizing platform for signaling during cell chemotaxis J. Cell Biol., March 1, 2004; 164(5): 759 - 768. [Abstract] [Full Text] [PDF]
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W. Popik and T. M. Alce CD4 Receptor Localized to Non-raft Membrane Microdomains Supports HIV-1 Entry: IDENTIFICATION OF A NOVEL RAFT LOCALIZATION MARKER IN CD4 J. Biol. Chem., January 2, 2004; 279(1): 704 - 712. [Abstract] [Full Text] [PDF]
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E. G. Argyris, E. Acheampong, G. Nunnari, M. Mukhtar, K. J. Williams, and R. J. Pomerantz Human Immunodeficiency Virus Type 1 Enters Primary Human Brain Microvascular Endothelial Cells by a Mechanism Involving Cell Surface Proteoglycans Independent of Lipid Rafts J. Virol., November 15, 2003; 77(22): 12140 - 12151. [Abstract] [Full Text] [PDF]
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 Z. Qiuping, L. Qun, H. Chunsong, Z. Xiaolian, H. Baojun, Y. Mingzhen, L. Chengming, H. Jinshen, G. Qingping, Z. Kejian, et al. Selectively Increased Expression and Functions of Chemokine Receptor CCR9 on CD4+ T Cells from Patients with T-Cell Lineage Acute Lymphocytic Leukemia Cancer Res., October 1, 2003; 63(19): 6469 - 6477. [Abstract] [Full Text] [PDF]
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A. Fittipaldi, A. Ferrari, M. Zoppe, C. Arcangeli, V. Pellegrini, F. Beltram, and M. Giacca Cell Membrane Lipid Rafts Mediate Caveolar Endocytosis of HIV-1 Tat Fusion Proteins J. Biol. Chem., September 5, 2003; 278(36): 34141 - 34149. [Abstract] [Full Text] [PDF]
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F. C. Bender, J. C. Whitbeck, M. Ponce de Leon, H. Lou, R. J. Eisenberg, and G. H. Cohen Specific Association of Glycoprotein B with Lipid Rafts during Herpes Simplex Virus Entry J. Virol., September 1, 2003; 77(17): 9542 - 9552. [Abstract] [Full Text] [PDF]
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D. R. M. Graham, E. Chertova, J. M. Hilburn, L. O. Arthur, and J. E. K. Hildreth Cholesterol Depletion of Human Immunodeficiency Virus Type 1 and Simian Immunodeficiency Virus with {beta}-Cyclodextrin Inactivates and Permeabilizes the Virions: Evidence for Virion-Associated Lipid Rafts J. Virol., August 1, 2003; 77(15): 8237 - 8248. [Abstract] [Full Text] [PDF]
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S. Venkatesan, J. J. Rose, R. Lodge, P. M. Murphy, and J. F. Foley Distinct Mechanisms of Agonist-induced Endocytosis for Human Chemokine Receptors CCR5 and CXCR4 Mol. Biol. Cell, August 1, 2003; 14(8): 3305 - 3324. [Abstract] [Full Text] [PDF]
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Y.-H. Zheng, A. Plemenitas, C. J. Fielding, and B. M. Peterlin Nef increases the synthesis of and transports cholesterol to lipid rafts and HIV-1 progeny virions PNAS, July 8, 2003; 100(14): 8460 - 8465. [Abstract] [Full Text] [PDF]
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Y. Ganor, M. Besser, N. Ben-Zakay, T. Unger, and M. Levite Human T Cells Express a Functional Ionotropic Glutamate Receptor GluR3, and Glutamate by Itself Triggers Integrin-Mediated Adhesion to Laminin and Fibronectin and Chemotactic Migration J. Immunol., April 15, 2003; 170(8): 4362 - 4372. [Abstract] [Full Text] [PDF]
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 V. D. Dixit, R. Sridaran, M. A. Edmonsond, D. Taub, and W. E. Thompson Gonadotropin-Releasing Hormone Attenuates Pregnancy-Associated Thymic Involution and Modulates the Expression of Antiproliferative Gene Product Prohibitin Endocrinology, April 1, 2003; 144(4): 1496 - 1505. [Abstract] [Full Text] [PDF]
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J. J. von Lindern, D. Rojo, K. Grovit-Ferbas, C. Yeramian, C. Deng, G. Herbein, M. R. Ferguson, T. C. Pappas, J. M. Decker, A. Singh, et al. Potential Role for CD63 in CCR5-Mediated Human Immunodeficiency Virus Type 1 Infection of Macrophages J. Virol., March 15, 2003; 77(6): 3624 - 3633. [Abstract] [Full Text] [PDF]
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L. Ding, A. Derdowski, J.-J. Wang, and P. Spearman Independent Segregation of Human Immunodeficiency Virus Type 1 Gag Protein Complexes and Lipid Rafts J. Virol., February 1, 2003; 77(3): 1916 - 1926. [Abstract] [Full Text] [PDF]
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Y. Percherancier, B. Lagane, T. Planchenault, I. Staropoli, R. Altmeyer, J.-L. Virelizier, F. Arenzana-Seisdedos, D. C. Hoessli, and F. Bachelerie HIV-1 Entry into T-cells Is Not Dependent on CD4 and CCR5 Localization to Sphingolipid-enriched, Detergent-resistant, Raft Membrane Domains J. Biol. Chem., January 24, 2003; 278(5): 3153 - 3161. [Abstract] [Full Text] [PDF]
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M. Viard, I. Parolini, M. Sargiacomo, K. Fecchi, C. Ramoni, S. Ablan, F. W. Ruscetti, J. M. Wang, and R. Blumenthal Role of Cholesterol in Human Immunodeficiency Virus Type 1 Envelope Protein-Mediated Fusion with Host Cells J. Virol., October 11, 2002; 76(22): 11584 - 11595. [Abstract] [Full Text] [PDF]
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G. del Real, S. Jimenez-Baranda, R. A. Lacalle, E. Mira, P. Lucas, C. Gomez-Mouton, A. C. Carrera, C. Martinez-A., and S. Manes Blocking of HIV-1 Infection by Targeting CD4 to Nonraft Membrane Domains J. Exp. Med., August 5, 2002; 196(3): 293 - 301. [Abstract] [Full Text] [PDF]
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D. H. Nguyen and D. Taub Cholesterol is essential for macrophage inflammatory protein 1beta binding and conformational integrity of CC chemokine receptor 5 Blood, May 29, 2002; 99(12): 4298 - 4306. [Abstract] [Full Text] [PDF]
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, Untreated cells; 
,
BCD-treated cells. The data represents two separate experiments with
the error bars representing SD.
