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Prepublished online as a Blood First Edition Paper on April 17, 2002; DOI 10.1182/blood-2001-11-0087.
CHEMOKINES
From the Gerontology Research Center, National
Institute on Aging, National Institutes of Health, Baltimore, MD.
The chemokine receptor, CCR5, is used as a human immunodeficiency
virus coreceptor in combination with CD4 during transmission and early
infection. CCR5 has been shown to be palmitoylated and targeted to
cholesterol- and sphingolipid-rich membrane microdomains termed
"lipid rafts." However, the role of cholesterol and lipid rafts on
chemokine binding and signaling through CCR5 remains unknown. We found
that cholesterol extraction by hydroxypropyl- Chemokine receptors have drawn much attention
since their description as human immunodeficiency virus (HIV)
coreceptors by several groups in 1996.1-4 Prior to that
time, HIV tropism was defined as either macrophage or T-cell tropic,
which corresponded to nonsyncytia- or syncytia-inducing viruses,
respectively. Today, the classification of HIV tropism is defined by
chemokine receptor usage of either CCR5, CXCR4, or both receptors,
although usage of other chemokine receptors has been
reported.2,5 Chemokine receptors are a family of 7 transmembrane-spanning G protein-coupled receptors that are
differentially expressed by a number of immune and nonimmune cell
populations. These receptors are quite specific and mediate immune cell
responses to a family of soluble chemoattractant molecules, termed
chemokines.6 CCR5 is the receptor for several CC
chemokines, including RANTES, macrophage inflammatory protein 1 "Lipid rafts" is a broad term for the collection of membrane
microdomains enriched in cholesterol, sphingolipids,
glycosylphosphatidylinositol-anchored proteins, and acylated signaling
molecules.12 Lipid rafts are believed to be important
signaling platforms enriched in many signaling proteins, including but
not limited to src kinases, G One important component in maintaining the higher lipid order of rafts
is cholesterol. Circular multimeric sugar molecules, known as
cyclodextrins, have been used to extract cholesterol from membranes,
thereby disrupting lipid rafts and increasing overall membrane
fluidity.20,21 The loss of cholesterol has been shown to
have profound effects on the ability of several G protein-coupled
receptors to bind their ligand. The oxytocin, cholecystokinin, galanin,
and Cell lines and reagents
BCD treatment
Fluorokine ligand staining Biotinylated MIP-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 × 106/mL. Cells (2 × 105) were
mixed with 20 µL of 3.0 µg/mL biotinylated MIP-1 or 5.0 µg/mL
biotinylated-soybean trypsin inhibitor (negative control) and then
incubated at 4°C for 1 hour. Fluorescein-conjugated avidin (10 µg/mL) was added (10-20 µL) to the cells and incubated for an
additional 30 minutes. at 4°C. After incubation, cells were washed
with 1 × RDF-1 buffer (R & D Systems) and then fixed with 2%
paraformaldehyde-PBS before being analyzed on a FACScan (Becton Dickinson).
Intracellular calcium mobilization Measurement of calcium mobilization by chemokine stimulation was performed as previously described.24 Briefly, untreated or BCD-treated CEM-R5 cells (8 × 106/mL) were incubated in PBS with Ca++ and Mg2+ containing 5 µM Fura-2 AM for 30 minutes at room temperature. The cells were subsequently washed and then resuspended at 1 × 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 (Perkin-Elmer, 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. Data were
collected every 0.96 seconds. RANTES and MIP-1 (Pepro Tech, Rocky
Hill, NJ) were tested at a final concentration of 1 µg/mL. Values
were graphed as 30-second interval averages minus the background change
in fluorescence ratio seen in untreated cells. The first
30-second interval of each set was set at 0. Error bars represent the
SD within each 30-second interval.
Flow cytometry CEM-R5 cells (1 × 106) in PBS containing 2% heat-inactivated FBS were added to 1 to 2 µg mAbs and incubated for 30 minutes on ice. Cells were washed with PBS, resuspended in 100 µL of 20 µg/mL GAM-AF488, and incubated on ice for 30 minutes. 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 minutes on ice. After incubation, the cells were then washed with PBS, resuspended in PBS containing 2% FBS, and then incubated an additional 30 minutes on ice before staining with mAbs. Permeabilization experiments were performed by using Caltag Laboratories Fix and Perm Cell Permeabilization Kit (Burlingame, CA).Immunomicroscopy CEM-R5 cells (1 × 106) were washed in cold PBS, resuspended in 100 µL PBS containing 2% FBS and 20 µg/mL CT-B Alexa Fluor 596, and then incubated on ice for 30 minutes. Cells were then washed with PBS and subsequently stained for CCR5 by using 2 µg CCR5 mAb, CTC5, 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 with the use of a cytospin centrifuge (Shandon, Pittsburgh, PA) at 1000 rpm for 2 minutes. Bound cells were layered with 30 µL 50% glycerol in PBS and covered with a glass coverslip. Images were acquired by Spot Advanced software on a Zeiss Axiovert S100 microscope under × 100 objective (Carl Zeiss, Thornwood, NY).Confocal microscopy Untreated or 10 mM BCD-treated CEM-R5 cells (1 × 106) were fixed in 1% paraformaldehyde in PBS for 30 minutes at 4°C, washed with PBS, and then resuspended in 100 µL PBS with or without 0.1% Triton X-100. Cells were then washed with PBS and subsequently stained for CCR5 by using 1 µg CCR5 mAb, 45531, for 30 minutes at 4°C. Cells were washed and resuspended in 100 µL GAM-AF488 plus 0.5 µg (10 µL) propidium iodide (PI) staining solution (BD Pharmingen, San Diego, CA) for 30 minutes at 4°C. The cells were washed with PBS and then fixed with 1% paraformaldehyde in PBS. The cells were cytospun onto slides as described above and then examined by a Zeiss LSM410 confocal microscope under × 63 objective under oil immersion. Images were processed by using Adobe Photoshop software for presentation.
Cholesterol extraction inhibits MIP-1 , followed by binding of an
avidin-fluorescein isothiocyanate (FITC) conjugate, and subsequent
examination by using flow cytometric analysis. Cholesterol extraction
from the cell membrane significantly impaired MIP-1 binding to cell
surface CCR5 (Figure 1A). The percentage
of positive cells for MIP-1 binding decreased from 84.5% for
untreated cells to 17.4% for BCD-treated cells. Positive cells are
defined by the M1 region with FITC fluorescence more than 98% of the
mouse IgG2a control. Treatment of these cells with 20 mM BCD for 1 hour
removed approximately 75% of total cellular cholesterol as measured by
the Amplex Red Cholesterol Assay Kit (Figure 1B). Cells treated with
BCD failed to demonstrate any levels of toxicity, as assessed by the
release of cytoplasmic lactate dehydrogenase into the supernatant or by
Trypan Blue exclusion (data not shown). Similar experiments were
attempted with biotinylated RANTES; however, this ligand exhibited very
low levels of binding. It seems quite possible that the biotinylation
of RANTES may have altered the secondary structure of this ligand or
masked some binding sites that normally interact with CCR5. These
results clearly demonstrate a requirement for cholesterol in the cell membrane for MIP-1 binding to CCR5.
We next examined alterations in the function of CCR5 by measuring
changes in intracellular calcium in response to ligand treatment of
BCD-treated CEM-R5 cells. Recombinant human MIP-1
Reloading T cells with cholesterol restores MIP-1 binding to BCD-treated T cells for various times was performed, followed by reloading of fresh cholesterol into the cell
membranes using chol-BCD complexes. We verified that the total cellular
cholesterol levels were restored to normal (or above normal) levels
through an assessment of total cholesterol by using the Amplex Red
cholesterol assay (data not shown). The results in Figure
3 demonstrate that MIP-1 binding is
rapidly lost with BCD treatment, suggesting that MIP-1 binding is
sensitive to cholesterol extraction. Reloading of cholesterol onto cell membranes restored MIP-1 binding to these treated cells (Figure 3A).
Overall, these results confirm that reloading T cells with cholesterol
restores MIP-1 binding activity and that cell death does not account
for the initial loss in ligand binding. Moreover, BCD itself does not
appear to exert any inhibitory activity as repletion of cholesterol
into cell membranes also involves the use of BCD as a vehicle for
cholesterol. Consequently, intracellular calcium mobilization by
MIP-1 was also restored to normal when BCD-treated cells were
reloaded with cholesterol (Figure 3B). To further verify the
specificity of cholesterol reloading, we also reloaded BCD-treated
cells with 4-cholesten-3-one, an oxidized form of cholesterol. These
cells did not regain the ability to bind MIP-1, unlike
cholesterol-reloaded cells (Figure 3C).
BCD treatment of T cells decreases mAb binding to CCR5 On the basis of the above results, we speculated that the loss in MIP-1 binding and subsequent signaling in BCD-treated T cells may be
due to changes in CCR5 conformation. In an effort to analyze
conformational changes in CCR5, we used a panel of anti-CCR5 antibodies
with well-characterized binding epitopes. These antibodies were
previously distinguished by their binding or lack of binding to
CCR5/CCR2 chimeras with interchanged N-termini or extracellular loops
(ECLs).26 More specifically, we analyzed the binding of 2 N-terminus-specific mAbs, CTC5 and 45502, an ECL2-specific mAb, 45531, and 2 multidomain-specific mAbs, 45523 and 45549 (Figure
4A). In the current analysis, we
attempted to determine if antibodies recognizing different regions of
the CCR5 molecule would exhibit distinct alterations in binding after
cholesterol extraction.
The results shown in Figure 4B demonstrate that all of our anti-CCR5
antibodies exhibited diminished binding to varying degrees on
BCD-treated CEM-R5 cells. Shifts in the anti-CCR5+
population curves can clearly be observed in Figure 4B. Table 1 expresses the mean fluorescence
intensity and percentage of positive gated T cells in this experiment.
The control mAbs, anti-CD4 and anti-CD45, did not show any change in
mean fluorescence intensity (MFI) and percentage positive. In terms of
percentage reduction in MFI, all our anti-CCR5 mAbs exhibited
a decrease in CCR5 binding, CTC5 (50.5), 45502 (24.7), 45523 (23.2), 45549 (40.0), and 45531 (85.7). As expected, BCD
treatment failed to decrease the overall binding of
HIV-1BaL gp120 to these cells, suggesting that CD4 remains
intact and at functional levels on the cell surface (data not shown).
Similarly, another study by Mañes et al27
demonstrated that HIV-1IIIB recombinant gp120 binding to
CD4+ MT2 cells was unaffected by BCD treatment. The
differences in epitope-specific losses in anti-CCR5 binding suggest
that the overall receptor conformation has been altered by the loss of cholesterol, whereas CD4 and CD45 epitopes remain unchanged.
To examine the specificity of cholesterol in these results, we once again reloaded BCD-treated T cells with chol-BCD and then examined anti-CCR5 mAb binding. Reloading T cells with cholesterol restored binding of the ECL2-specific mAb, 45531, to 100% of control, but the amino terminal-specific mAb, CTC5, binding was only restored to 87% (Figure 4C). Interestingly, anti-CD45 binding was not increased by reloading of cholesterol, suggesting that the changes in anti-CD45 binding seen with BCD treatment may be noncholesterol specific or may be affected in a way that cannot be restored by exogenous cholesterol. We next examined if BCD treatment of T cells rendered them more susceptible to anti-CCR5 mAb-induced receptor internalization. As the staining of cells occurred at 4°C in our assays, we would expect very little internalization within our treatment groups. However, to directly examine this question, we stained BCD-treated cells with mAbs, fixed, permeabilized, and then added GAM-AF488. BCD treatment demonstrated no significant difference in mAb detection between permeabilized and nonpermeabilized cells, indicating that very little internalization had occurred over the treatment periods (Figure 4D). Paraformaldehyde fixation prior to mAb staining enhances binding of some anti-CCR5 mAbs on BCD-treated cells To confirm that CCR5 conformation was being altered and not internalized during BCD treatments, we mildly fixed the CEM-R5 cells with paraformaldehyde immediately after the BCD treatments. The results shown in Figure 5 and Table 2 demonstrate an increase in the overall binding of the anti-CCR5 mAbs after BCD treatment of cells. The MFI of CTC5 on BCD-fixed cells increased 400% from the control-fixed population. More surprisingly, 45502 and 45549 MFI increased from 5.71 to 251 and from 5.8 to 241, respectively, resulting in a more than 40-fold increase in MFI after treatment/fixation. Interestingly, these 2 mAbs, 45502 and 45549, of all the CCR5 mAbs, have the least binding to control-fixed or unfixed CEM-R5 cells. In addition, there was also a 3-fold increase in the MFI of multidomain-specific mAb, 45523. These results were quite reproducible and specific, as there was no difference in alterations in the binding of the ECL2-specific mAb, 45531, between control-fixed and BCD-fixed cells. To visually confirm that the BCD-fixation treatments did not detect internalized receptors, we performed confocal microscopy on treated cells. We used the binding of a nonmembrane permeant DNA binding dye, PI, to evaluate membrane permeabilization on untreated, 10 mM BCD-treated, and 0.1% Triton X-100 treated CEM-R5 cells. We found that BCD treatment did not significantly increase the number of cells stained positive for PI, nor did it increase mAb 45531 staining of cytoplasmic receptors (Figure 5B). However, our positive control for permeabilization showed 100% cells nuclear stained with PI and cytoplasmic staining for 45531 mAb (Figure 5B).
MIP-1 binding to T cells corresponded to
CCR5 localization within lipid rafts rather than nonraft CCR5. If
cholesterol is essential to ligand binding, we suspected that lipid
rafts might be the preferred site for MIP-1 binding, presuming that
CCR5 outside of rafts may have less interaction with cholesterol. The
results in Figure 6 show
immunofluorescence microscopy images of CEM-R5 cells treated with
biotinylated MIP-1 followed by avidin-FITC, and then overlaid with
Alexa Fluor 596 CT-B conjugate. The ganglioside, GM1, has been
extensively used as a specific marker for lipid rafts and is recognized
by pentameric CT-B. A significant overlap in staining between MIP-1
and GM1 was observed on CEM-R5 cells, suggesting that functional CCR5
or higher affinity CCR5 preferentially localized to lipid rafts on the
T-cell surface. Dense patches of MIP-1 bound to the cell
surface would yield a brighter signal in raft areas in which CCR5 may
be more dense. However, we want to point out that diffuse binding of
MIP-1 in nonraft areas may not be as evident, given the diminished
signal of such staining in nonraft less dense areas of the
cell.
Anti-CCR5 staining partially colocalizes with CT-B labeled GM1 Previous raft isolation studies have shown only a partial partitioning of CCR5 to lipid raft fractions.16 We wanted to confirm and extend these results by using microscopy with our CEM-R5 cells. CT-B, as described earlier, recognizes ganglioside GM1 and partitions to lipid rafts. In our culture of CEM-R5 cells, about 25% of cells display a capped appearance for lipid rafts, visualized either as a crescent staining or a distinct uropod projection. In Figure 7, we present images of capped cells compared with noncapped cells. Panels A and B were stained for GM1 with CT-B and CCR5 with mAb, CTC5. In capped cells, CCR5 appears to colocalize with GM1 (Figure 7A), but in noncapped cells GM1 remains in distinct microdomains that do not colocalize with CCR5 (Figure 7B). Our results demonstrate that normal CCR5 localization in noncapped cells appears to be in non-GM1 areas, presumably outside of rafts. However, binding of MIP-1 seems to be specific to capped cells in domains
that are enriched in GM1. Together, these results support a role for cholesterol in the function and conformation of CCR5 in MIP-1 binding and signaling in T cells.
In this current report, we have provided evidence for an essential
role for cholesterol in structural conformation and ligand binding
activity of CCR5. MIP-1
Alterations in antibody binding to cell surface CCR5 after treatment with BCD provide interesting insights into the role of cholesterol in CCR5 conformational states. Although each of the antibodies used may be well characterized, very few antibodies to chemokine receptors recognize linear epitopes, borne out by the fact that most antibodies do not recognize denatured CCR5 in Western blot analysis.26 Lee et al26 found that even CTC5, which recognizes denatured CCR5 in Western blots, may not recognize a linear epitope because a 9-amino acid HA tag added to the N-terminus of CCR5 eliminates binding. The binding of 2 mAbs, 45502 and 45549, was dramatically increased (> 40-fold in MFI) when BCD-treated cells were fixed with paraformaldehyde. We suspect that these 2 mAbs recognize a form of CCR5 that has low levels of interaction with cholesterol. We believe that on any given cell membrane, CCR5 may exhibit different levels of cholesterol interaction. These antibodies may not only be recognizing different regions of the CCR5 molecule but also different conformation states based on their interactions directly with cholesterol or the interactions of CCR5 with cholesterol. For example, under normal conditions, there appears to be sufficient levels of cholesterol to maintain a high-cholesterol binding version of CCR5 (hi-chol-R5) in which the N-terminal-specific mAb, 45502, and the multidomain-specific mAb, 45549, would exhibit low levels of receptor binding and the ECL2-specific mAb, 44531, would exhibit a high level of binding. After BCD treatment, membrane fluidity is dramatically increased, thereby increasing CCR5 "floppiness" and decreasing binding of most of the antibodies. This finding would be supported by our data, demonstrating a significant loss in 45531 mAb binding that prefers hi-chol-R5. However, on paraformaldehyde fixation, the CCR5 molecule is no longer fluid and the low-chol-R5 version becomes preserved. This finding is supported by our results in Figure 5 and Table 2 that demonstrate a dramatic increase in binding of the low-chol-R5-recognizing mAbs, 45502 and 45549, even above that of the hi-chol-R5 mAb, 45531, under normal conditions. Overall, the conformational changes in CCR5 with BCD treatment are really too complex to be studied by simply examining changes in cell surface binding profiles by using several receptor-specific antibodies. However, we can conclude that the variations in epitope-specific antibody and ligand binding support the increased susceptibility to conformational changes of certain epitopes of CCR5 after cholesterol extraction. Our immunofluorescence images provide 2 important findings. First, CCR5
and GM1 appear to colocalize in capped cells but are in distinct
regions in noncapped cells. Cells exhibiting a "capped" phenotype
demonstrated colocalization of CCR5 and GM1 similar to previous results
using HELA and human breast carcinoma cells.17,27 These
results suggest that cellular polarization or capping of suspension
cells is required for CCR5 colocalization with GM1. Second, the
majority of bound MIP-1 Multiple palmitoylation of cysteine residues is believed to be a
mechanism to target transmembrane proteins to lipid rafts, likely
because of the highly ordered lipid-packing properties of cholesterol
and palmitoyl chains.12 CCR5 has been found to be
palmitoylated at cysteine residues 321, 323, and 324 within the
cytoplasmic tail, resulting in the formation of a fourth intracellular loop.17-19 Palmitoylation-deficient mutants of CCR5 that
retain a similar binding capacity to MIP-1 Transmission and early stages of HIV infection are predominated by CCR5-using viruses.29 Our results here have direct implications for the modulation of cholesterol in preventing HIV transmission. Recent studies have suggested that lipid rafts serve as the site for HIV fusion and infection based on interactions of gp120 with lipid raft components, such as glycolipids and CD4.30 The inhibitory effects of cholesterol depletion on HIV infectivity are believed to be due to the disruption of lipid rafts, thereby preventing recruitment of chemokine receptors into receptor assemblies with CD4.27,31 We believe that lipid rafts and cholesterol may be playing a more significant role in HIV infection than serving as sites for protein recruitment. On the basis of the data presented here, cholesterol is not only necessary for lipid raft integrity but also appears to be important for CCR5 conformational stability. Recent studies have also implicated cholesterol-dense lipid rafts as the sites for concentration of SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), which are responsible for initiating fusion of exocytic vesicles with the cell membrane.32 Given these receptor-raft associations, it is not surprising that the HIV fusion process would require cholesterol-enriched patches dense with chemokine receptors. Similarly, fusion of the human T-cell leukemia virus type 1 (HTLV-1) may also be mediated through lipid rafts, as 4 mAbs that recognize lipid raft proteins were also found to block HTLV-1 syncytium formation.33 Other studies have demonstrated that lipid rafts are the areas of the cell membrane where newly formed virions of HIV assemble and bud.34 Several other viruses, including influenza, polio, measles, and Sendai virus, may similarly bud from lipid rafts.35-38 In fact, recent studies suggest that the ability to create "pseudotyped" virus particles that contain envelope molecules and structural proteins from different viruses is a consequence of the preferential budding of many virus types from lipid rafts.39 Localized targeting of raft function by cholesterol extraction may prove to be effective in preventing HIV transmission. Overall, our results have numerous implications for chemokine receptor function outside of the HIV arena. Several inflammatory disease states have been associated with alterations in chemokine production and responses, including cardiovascular disease, asthma, multiple sclerosis, cancer, and arthritis.40 Modulation of the responses to chemokines could benefit individuals with many of these diseases. In addition, alterations in membrane cholesterol may hinder one's ability to mount an optimal immune response to a pathogenic or antigen challenge. Similarly, modifications of cholesterol as seen with cholesterol oxidation on immune cell populations may prevent optimal cellular recruitment into inflammatory sites or sites of vaccine administration. Our results with reloading 4-cholesten-3-one suggest that oxidized cholesterol may hinder the function of chemokine receptor by altering receptor conformation and inhibiting chemokine binding, similar to that seen with the galanin receptor.23 This finding may also apply to aging immune cells, as increases in lipid oxidation have been associated with process of aging.41 Restoration of immune cells with normal nonoxidized cholesterol may potentially improve dampened chemokine and antigen responses in aging cells that may possess higher levels of oxidized cholesterol than cells from their younger counterparts. Understanding the membrane mechanisms and requirements involved in chemokine receptor function may lead to a number of possible therapeutic interventions to modulate immune responses and HIV pathogenesis.
We thank Christa Morris, Dr Robert Wersto, and Francis J. Chrest of the NIA Flow Cytometry Laboratory, and Magdalena Juhazsova of the NIA Confocal Imaging Facility for their assistance. We also thank Dr Eric Schaffer, Dr Valeria Coelho, and Deborah Nguyen for helpful discussions.
Submitted November 29, 2001; accepted January 24, 2002.
Prepublished online as Blood First Edition Paper, April 17, 2002; DOI 10.1182/blood-2001-11-0087.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Dennis Taub, National Institute on Aging, 5600 Nathan Shock Dr, Baltimore, MD 21224; e-mail: taubd{at}grc.nia.nih.gov.
1. Alkhatib G, Combadiere C, Broder CC, et al. CC CKR5: a RANTES, MIP-1alpha, MIP-1beta receptor as a fusion cofactor for macrophage-tropic HIV-1. Science. 1996;272:1955-1958[Abstract]. 2. Choe H, Farzan M, Sun Y, et al. The beta-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates. Cell. 1996;85:1135-1148[CrossRef][Medline] [Order article via Infotrieve]. 3. Dragic T, Litwin V, Allaway GP, et al. HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC- CKR-5. Nature. 1996;381:667-673[CrossRef][Medline] [Order article via Infotrieve]. 4. Feng Y, Broder CC, Kennedy PE, Berger EA. HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science. 1996;272:872-877[Abstract]. 5. Bjorndal A, Deng H, Jansson M, et al. Coreceptor usage of primary human immunodeficiency virus type 1 isolates varies according to biological phenotype. J Virol. 1997;71:7478-7487[Abstract]. 6. Mackay CR. Chemokines: immunology's high impact factors. Nat Immunol. 2001;2:95-101[CrossRef][Medline] [Order article via Infotrieve]. 7. Cocchi F, DeVico AL, Garzino-Demo A, Cara A, Gallo RC, Lusso P. The V3 domain of the HIV-1 gp120 envelope glycoprotein is critical for chemokine-mediated blockade of infection. Nat Med. 1996;2:1244-1247[CrossRef][Medline] [Order article via Infotrieve]. 8. Howard OM, Korte T, Tarasova NI, et al. Small molecule inhibitor of HIV-1 cell fusion blocks chemokine receptor-mediated function. J Leukoc Biol. 1998;64:6-13[Abstract]. 9. Donzella GA, Schols D, Lin SW, et al. AMD3100, a small molecule inhibitor of HIV-1 entry via the CXCR4 co-receptor. Nat Med. 1998;4:72-77[CrossRef][Medline] [Order article via Infotrieve].
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2000;275:17221-17224 13. Holowka D, Baird B. Fc(epsilon)RI as a paradigm for a lipid raft-dependent receptor in hematopoietic cells. Semin Immunol. 2001;13:99-105[CrossRef][Medline] [Order article via Infotrieve]. 14. Viola A. The amplification of TCR signaling by dynamic membrane microdomains. Trends Immunol. 2001;22:322-327[CrossRef][Medline] [Order article via Infotrieve]. 15. Cheng PC, Cherukuri A, Dykstra M, et al. Floating the raft hypothesis: the roles of lipid rafts in B cell antigen receptor function. Semin Immunol. 2001;13:107-114[CrossRef][Medline] [Order article via Infotrieve]. 16. Manes S, Mira E, Gomez-Mouton C, et al. Membrane raft microdomains mediate front-rear polarity in migrating cells. EMBO J. 1999;18:6211-6220[CrossRef][Medline] [Order article via Infotrieve]. 17. Percherancier Y, Planchenault T, Valenzuela-Fernandez A, Virelizier JL, Arenzana-Seisdedos F, Bachelerie F. Palmitoylation-dependent control of degradation, life span and membrane expression of the CCR5 receptor. J Biol Chem. 2001;276:31936-31944 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
binding and conformational integrity of CC chemokine receptor
5
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Abstract
Introduction
(MIP-1
-aminobutyric acid receptors have all been found to require
cholesterol for their function.
