From the Department of Chemistry, Indiana University, Bloomington, Indiana 47405-0001
Received for publication, December 12, 2000, and in revised form, January 30, 2001
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ABSTRACT |
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Eotaxin is a CC chemokine that
specifically activates the receptor CCR3 causing accumulation of
eosinophils in allergic diseases and parasitic infections. Twelve amino
acid residues in the N-terminal (residues 1-8) and N-loop (residues
11-20) regions of eotaxin have been individually mutated to alanine,
and the ability of the mutants to bind and activate CCR3 has been
determined in cell-based assays. The alanine mutants at positions
Thr7, Asn12, Leu13,
and Leu20 show near wild type binding affinity and
activity. The mutants T8A, N15A, and K17A have near wild type binding
affinity for CCR3 but reduced receptor activation. A third class of
mutants, S4A, V5A, R16A, and I18A, display significantly perturbed
binding affinity for CCR3 while retaining the ability to activate or
partially activate the receptor. Finally, the mutant Phe11
has little detectable activity and 20-fold reduced binding affinity relative to wild type eotaxin, the most dramatic effect observed in
both assays but less dramatic than the effect of mutating the corresponding residue in some other chemokines. Taken together, the
results indicate that residues contributing to receptor binding affinity and those required for triggering receptor activation are
distributed throughout the N-terminal and N-loop regions. This
conclusion is in contrast to the separation of binding and activation
functions between N-loop and N-terminal regions, respectively, that has
been observed previously for some other chemokines.
Chemokines (chemotactic cytokines) are a family of small (8-10
kDa) secreted proteins whose major function is to recruit leukocytes to
sites of injury or infection (1). Several diseases, including asthma
and other allergic disorders, can result from the overaccumulation of
leukocytes. There are two main classes of chemokines as defined by the
pattern of the first two of four conserved cysteine residues located
near the N-terminus (2). When the cysteines are adjacent, the
chemokines are classified as CC chemokines and typically attract monocytes, eosinophils, basophils, and T-lymphocytes. If a single residue separates the cysteines they are referred to as
CXC chemokines and typically attract neutrophils or
lymphocytes. There are also two minor classes, which include the C
chemokine lymphotactin (3) and the CX3C
chemokine fractalkine (4). The receptors for chemokines (designated CCR
or CXCR, according to the class of chemokine that activates
them) are seven transmembrane helix G-protein-coupled receptors located
in the leukocyte cell membrane (5). Chemokines of a certain class
typically bind to a subset of receptors from the corresponding receptor
class (2), although the determinants of receptor specificity are not
well understood. In addition to their signaling roles, chemokine
receptors, particularly CCR5, CXCR4, and CCR3 have been
shown to be co-receptors, in conjunction with CD4, for
HIV1 infection (6).
The CC chemokine eotaxin (7-9), and the related proteins eotaxin-2
(10, 11) and eotaxin-3 (12), are specific for the receptor CCR3, which
is the most abundant chemokine receptor found on the surface of
eosinophils, a terminally differentiated class of granulocytes (9, 13,
14). Through its interaction with CCR3, eotaxin facilitates the
recruitment of eosinophils to the sites of parasitic infection or
allergen stimulation (15). Eosinophil accumulation is a hallmark of
several human diseases, including Hodgkin's disease (15) and
inflammatory diseases of the lungs (asthma), heart (hypereosinophilic
syndrome), intestines (inflammatory bowl disease and gastroenteritis),
and skin (atopic dermatitis). The overexpression of eotaxin in many of
these diseased tissues, along with its ability to recruit eosinophils,
suggests a likely role for eotaxin in these inflammatory disorders (16,
17).
The three-dimensional structures of chemokines have been reviewed
recently (18). The structures of the monomeric subunits are largely
conserved and consist of a single turn of helix followed by a
three-stranded antiparallel Extensive mutational studies of several chemokines have led to a
proposed "two-step" model for receptor interaction in which the
N-loop of the chemokine initially binds to the receptor then the
N-terminal region of the chemokine docks with the receptor to induce a
conformational change, resulting in receptor activation (21, 23-25).
Recent papers describing the binding of receptor-derived peptides to a
groove located between the N-loop and Eotaxin, eotaxin-2, and eotaxin-3 differ from most chemokines in that
they are specific for only one receptor, CCR3. In addition, eotaxin
shares a high degree of sequence identity (~60%) with the MCP
proteins, which bind a different subset of receptors. This provides us
with a good model system to study chemokine/receptor interactions and
the determinants of receptor specificity. To this end, we have
performed alanine-scanning mutagenesis on the N-terminal and N-loop
regions of eotaxin to resolve which residues in these regions are
involved in receptor interactions. Herein we report the results of
these studies and their interpretation in light of the available data
for other chemokines.
Cell Lines--
The human osteosarcoma (HOS-CD4) cell line
stably transfected with CCR3 (29, 30) was acquired from the AIDS
Research and Reference Reagent Program and was maintained in
Dulbecco's modified Eagle's medium, 10% fetal bovine serum, and 1 µg/ml puromycin. The murine L1.2 pre-B cell line stably transfected
with CCR3 (31) was obtained as a generous gift from Dr. Osamu Yoshi
and was maintained in RPMI 1640, 10% fetal bovine serum, and 0.8 mg/ml
geneticin. All cell culture materials were purchased from Life
Technologies, Inc. (Rockville, MD).
Chemokines--
Site-directed mutagenesis was performed using
the QuikChange method (Stratagene, La Jolla, CA) and confirmed by DNA
sequencing. Wild type eotaxin and all eotaxin mutants were expressed
and purified as described previously for wild type eotaxin (28). In
brief, the pET30a-eotaxin plasmid was transformed into BL21(DE3) cells (Novagen, Santa Clarita, CA). Cells were grown at 37 °C to an A600 Calcium Mobilization Assay--
HOS-CD4-CCR3 cells were washed
with phosphate buffered saline, trypsinized, and resuspended in
complete medium, counted, spun down, and resuspended in complete
medium at a concentration of 1 × 107 cells/ml. Fura-2
and pluronic-127 (both from Molecular Probes, Eugene, OR) were added to
final concentrations of 2.5 µg/ml and 0.05%, respectively. The cells
were incubated for 20 min in the dark at room temperature, brought to a
total volume of 30 ml with complete medium, and incubated for another
20 min in the dark at 37 °C. The cells were then spun down and
washed twice with 30 ml of flux buffer (Hank's balanced salt solution
with 25 mM HEPES at pH 7.4), resuspended in the assay
buffer at 2 × 106 cells/ml, and stored in the dark on
ice until they were used (<2 h). Prior to the assay, 2 ml of cells
were placed in a quartz cuvette and incubated in the fluorometer at
37 °C with stirring for 10 min. The fluorescence ratio was
determined by exciting at 340 and 380 nm with emission monitored at 510 nm. Injections into the cuvette were done in a 10-µl volume. Data
were collected on a SLM-AMINCO 8100 spectrofluorometer (SLM Instruments).
Competitive Radioligand Binding Assay--
The competitive
binding assay was performed using previously reported procedures with
slight modifications (31). Briefly, 4 × 106 L1.2-CCR3
cells/ml in 25 mM HEPES, 1 mM
CaCl2, 5 mM MgCl2, 120 mM NaCl, 0.5% bovine serum albumin (protease-free; Sigma),
pH 7.6, were incubated with ~0.15 nM
125I-eotaxin (~2000 Ci/mmol; Amersham Pharmacia Biotech)
and increasing amounts of cold competitor protein in 250 µl for
1 h at room temperature. After the incubation period, the cells
were layered onto an ice-cold sucrose cushion (20% sucrose, 140 mM NaCl, 40 mM Tris, 0.4% bovine serum albumin
(protease-free; Sigma), pH 7.6) and centrifuged for 10 min at 1500 × g. Both liquid layers were carefully aspirated, the cells
were resuspended in scintillation fluid, and radioactivity was counted
on a Packard 1600 TR liquid scintillation counter. Using SigmaPlot, the
data were fit to the following equation: percent inhibition = (Bmax [L])/(IC50 + [L]),
in which Bmax is the maximum binding, [L] is
the cold competitor concentration, and IC50 is the midpoint
of the transition. All binding assays were performed in duplicate.
Selection of Residues for Alanine-scanning Mutation--
To assess
the contribution of each amino acid side chain in the N-terminal and
N-loop regions of eotaxin (residues 1-20) to receptor binding and
activation, we individually mutated each residue to alanine. The only
residues omitted from the scanning mutagenesis were glycine, proline,
and alanine (because of their unique structural properties) and the two
conserved cysteines residues (because of the requirement for the
structural integrity of the protein). Thus, the following twelve
residues were individually converted to alanine in this study:
Ser4, Val5, Thr7, Thr8,
Phe11, Asn12, Leu13,
Asn15, Arg16, Lys17,
Ile18, and Leu20 (Fig.
1). All of the mutants were expressed in
Escherichia coli and purified using the protocol we
have developed for wild type eotaxin with no modifications needed (28).
The mutants expressed at approximately wild type levels (~5 mg/liter
of culture).
Activity of Eotaxin and Mutants--
The ability of wild type
eotaxin and each mutant to activate CCR3 was determined using a calcium
mobilization assay performed on HOS cells expressing CCR3 on the
surface. The observed calcium flux for wild type eotaxin is shown in
Fig. 2A, and the corresponding dose-response curve is presented in Fig. 2B. The
concentration required for half-maximal eotaxin activity is ~7
nM, and maximum activity is seen for concentrations
exceeding ~30 nM.
The activity of each mutant protein was determined at a concentration
of 100 nM at which wild type eotaxin has maximal activity; the results are listed in Table I.
Substantial losses of activity (<30% wild type activity) resulted
from the substitutions at Val5 and Phe11, with
F11A showing a near complete loss of activity (within experimental error) at 100 nM concentration. A moderate loss of activity
(<80% wild type activity) was seen for substitutions at positions
Ser4, Thr8, Asn15,
Arg16, and Lys17, whereas substitution of
alanine at residues Thr7, Asn12,
Leu13, Ile18, and Leu20 produced
mutant proteins with near wild type activity at 100 nM.
Receptor Binding of Eotaxin and Mutants--
The losses in
activity observed in the calcium mobilization assay could potentially
result from a decreased affinity for the receptor. Alternatively, the
mutants might have wild type binding affinity but be unable to induce
the receptor conformational change required for transmembrane
signaling. To distinguish between these two possibilities, we measured
the ability of each mutant protein to inhibit binding of wild type
eotaxin to CCR3 using a competitive radioligand binding assay performed
using murine pre-B cells (L1.2) expressing CCR3 on their surface.
A representative competition binding curve for wild type eotaxin is
shown in Fig. 3. The concentration
required for 50% inhibition of 125I-labeled wild type
eotaxin binding (IC50) is 1.2 ± 0.2 nM.
Eotaxin mutants in which the residue Thr7,
Thr8, Asn12, Leu13,
Asn15, Lys17, or Leu20 was replaced
by alanine competed with labeled wild type eotaxin with
IC50 values in the range 0.5 to 3.5 nM (Table
I), i.e. within a factor of four of wild type
eotaxin. A curve for N15A is shown as an example in Fig. 3. In
contrast, a second group of mutants with alanine substitution at
Ser4, Val5, Phe11,
Arg16, or Ile18 had IC50 values in
the range 8.6 to 24.5 nM (Table I),
i.e. a decrease of more than 7-fold compared with
wild type eotaxin. The curve for F11A is shown in Fig. 3. The receptor
binding and activation data are summarized in Table I.
Previous chemokine mutational studies, and their interpretation
according to the prevailing two-step model for chemokine receptor activation, have suggested a separation of function between the N-loop
and N-terminal regions of chemokines (21, 23-25). The N-loop is
proposed to be important for providing the initial binding energy,
whereas the N-terminus is suggested to be required for triggering
receptor activation subsequent to binding. As discussed below, the
current calcium mobilization and radioligand binding results for
eotaxin mutants suggest that the functions of the N-terminal and N-loop
regions of eotaxin are not separated in a straightforward manner.
The following discussion is based on the premise that reductions in
receptor binding affinity or activation upon mutation of a particular
residue imply a role for that residue in the receptor interaction. As
in all mutational studies, an alternative possibility is that the
mutations induce a structural change in the protein, detrimentally
affecting its ability to interact with the receptor. Although we have
not experimentally excluded this possibility in the current study, it
appears relatively unlikely considering that the mutations have all
been made in regions of the protein that are known to be
conformationally flexible (32).
A graphical representation of the influence of alanine mutations on the
receptor binding and activity of eotaxin (Fig.
4) illustrates the classification of
mutants into four categories (groups 1-4 in Fig. 4). The
alanine mutants at positions Thr7, Asn12,
Leu13, and Leu20 (group 1) show near
wild type binding and activity suggesting that their side chains do not
play a major role in the mechanism of receptor activation. It is
important to note that small reductions in activity (less than
~5-fold increases in EC50 values) would not necessarily
be detected by measuring calcium flux at 100 nM concentration, so it remains possible that these mutations have subtle
influences on receptor activation.
INTRODUCTION
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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-sheet and a C-terminal
-helix (Fig.
1). The CC or CXC motif is contained in a long unstructured region (preceding the helical turn) that is covalently linked through a
pair of disulfide bonds to the
-sheet and the loop connecting
strands one and two (30s loop). Herein we refer to the region preceding
the CC or CXC motif as the N-terminal region and the region
between the CC or CXC motif and the helical turn as the
N-loop region (Fig. 1). The quaternary structures of chemokines vary
dramatically within the superfamily, with a range of monomeric, dimeric, and tetrameric structures seen, although there is convincing evidence that the monomer is sufficient for receptor activation (19,
20). The three-dimensional structures of eotaxin (21), eotaxin-2 (22),
and eotaxin-32 have recently
been determined and are all monomeric. The conserved nature of
the chemokine structure has revealed little information about the
mechanism of receptor specificity.
2-
3 hairpin of their
cognate chemokines (interleukin-8, fractalkine, eotaxin, and
eotaxin-2) have reinforced the importance of the N-loop region for
receptor binding (22, 26-28). However, the specific protein residues
in the chemokine that contribute to receptor interactions vary
significantly between chemokines.
EXPERIMENTAL PROCEDURES
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0.7, induced with 1 mM
isopropyl
-D-thiogalactopyranoside, and harvested 6 h later. Cells were resuspended in buffer (50 mM
Na2HPO4, 500 mM NaCl, 5 mM imidazole, pH 8.0) and lysed. The protein was loaded
onto a Ni2+-nitrilotriacetic acid-agarose affinity column
(Qiagen, Valencia, CA), washed with 50 mM
Na2HPO4, 500 mM NaCl, 30 mM imidazole, pH 8.0, and eluted with 50 mM
Na2HPO4, 500 mM NaCl, 200 mM imidazole, pH 8.0. The (His)6-tag was
removed by treatment with thrombin (1 µg thrombin/mg eotaxin in 20 mM Tris, pH 8.0, room temperature, overnight). The cleaved
protein was further purified on a Source 15 cation exchange column
(Amersham Pharmacia Biotech) equilibrated with 20 mM
Tris, pH 8.5, using a 0 to 2 M NaCl gradient.
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Fig. 1.
A ribbon structure of eotaxin (Protein Data
Bank accession number 1EOT) showing the locations of the N-terminal and
N-loop regions. Side chains are shown as dark gray
cylinders for the residues mutated in the current study. Several
of the residues that were found to be important for binding or
activation are labeled. The two disulfide bonds are displayed as
light gray cylinders.
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Fig. 2.
Calcium flux activity assay for wild type
eotaxin. A, a typical calcium transient observed upon
addition of 100 nM eotaxin to Fura-2-labeled HOS-CCR3
cells. B, a dose-response curve showing the concentration
dependence of the observed calcium flux signal. Data points and error
bars are the average and standard deviation, respectively, of
triplicate data, normalized to the average of the signals for the three
highest concentrations.
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Fig. 3.
Competitive radioligand binding data for wild
type eotaxin (circles), the N15A mutant
(triangles), and the F11A mutant
(squares). Data for the N15A and F11A mutants are
presented as typical examples of mutants with binding affinity within
4-fold of wild type and those with binding affinity reduced by greater
than 7-fold, respectively.
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Fig. 4.
Graphical representation of the observed
changes in CCR3 binding affinity and activation, relative to wild type
eotaxin, for the 12 mutants studied here. Mutants with similar
effects are grouped together as follows: Group 1, close to
wild type binding affinity and activity; Group 2, near wild
type binding affinity but <80% activity; Group 3, >7-fold
reduced binding affinity but partial or full receptor activation; and
Group 4 (Phe11), substantial losses in both
binding affinity and activity. Wild type is represented by
WT.
The mutants T8A, N15A, and K17A (group 2, Fig. 4) all have near wild type binding affinity for CCR3 yet have reduced activation as measured by calcium mobilization. These results are interesting, because they not only confirm the predicted importance of the N-terminus (Thr8) for receptor activation but also implicate residues in the N-loop (Asn15 and Lys17) in activation of CCR3 by eotaxin. In a recent NMR study of binding between eotaxin and peptides derived from the N-terminus of CCR3, residues Asn15 and Lys17 of eotaxin underwent chemical shift changes upon addition of peptide (28). These combined results suggest that the N terminus of CCR3 interacts with the N-loop residues Asn15 and Lys17 during the events that lead to receptor activation.
A third class of mutants, S4A, V5A, R16A, and I18A (group 3, Fig. 4), each possess significantly perturbed binding affinity for CCR3 while retaining the ability to activate or partially activate the receptor. The mutation at Ile18 is interesting, because it shows a 7-fold reduced binding affinity yet has wild type activity at 100 nM. This can be rationalized by noting that even with its weakened binding affinity, significant I18A should be able to bind CCR3 at the 100 nM concentration used in the activity assays (>90% predicted receptor occupancy), allowing the production of a near wild type signal. As noted above for the group 1 mutants, it remains possible that the I18A mutation has a small effect on the EC50 for receptor activation. Importantly, two of these four group 3 mutants with reduced binding affinity are located in the N-loop (R16A and I18A), whereas the other two are located in the N-terminus (S4A and V5A). This observation again challenges the hypothesis of binding and activation functions being partitioned between these two structural elements. The importance of the N-terminus in receptor binding is supported by the observation that eotaxin truncated by two N-terminal residues displays reduced receptor binding affinity and activation (33). NMR binding studies using receptor-derived peptides (28) support the involvement of the two N-loop residues in receptor binding; Ile18 is located at the base of the binding groove proposed in that study, and Arg16 is at the hydrophilic edge of the same groove.
In addition to their weakened receptor binding affinity, mutants at positions Ser4, Val5, and Arg16 also show reduced receptor activation in the calcium flux assay. It would not be unexpected for a mutation that affects receptor binding to also affect receptor activation. However, it is also possible for a mutation to affect both the initial binding step and the receptor-triggering event separately. Although it is difficult to interpret the influence of mutations at these positions on the receptor activation step, it is noteworthy that the three mutants all show approximately the same range of reduced binding (S4A, V5A, and R16A have IC50 values of 12.5, 9.8, and 12.2 nM, respectively) whereas V5A has a much greater effect than the other two mutations on eotaxin activity (23% of wild type for V5A versus 76% for S4A and 61% for R16A). This comparison suggests that the mutation at Val5 is likely to affect both the initial binding and the receptor-triggering steps separately.
Finally, the mutant Phe11 has little detectable activity
and 20-fold reduced binding affinity relative to wild type eotaxin, the
most dramatic effect observed in both assays. The substantial loss of
receptor interactions at this position is not surprising as several
mutational studies on other chemokines have implicated an aromatic
residue at this position as being key for receptor binding and
activation (19, 34-37). Of some interest in the case of F11A in
eotaxin is that the magnitude of the change of binding affinity is
smaller than is seen in several other chemokines. For the CC chemokines
MCP-1 and macrophage inflammatory protein-1, mutation of the
comparable aromatic position to alanine resulted in significant loss of
activity for the proteins and 100- and 1000-fold weaker receptor
binding affinity, respectively (34, 36). For the CC chemokine RANTES
(regulated on activation normal T cell expressed and secreted) mutation
of the aromatic residue to alanine resulted in 5000-fold weaker binding
affinity for CCR3 (37). In all studies, the aromatic residue was shown
to provide very important protein/receptor interactions.
Alanine-scanning mutagenesis has been performed previously on the CC chemokines MCP-1 and RANTES (34, 35, 37). Results from the RANTES experiments are difficult to compare, as the protein is able to bind and activate several receptors, and experiments were performed using cells that may have expressed multiple receptors. In the case of MCP-1, no individual residues in the N-terminus were identified as being important for binding or activation of the receptor CCR2b. However, N-terminal truncation resulted in a complete loss of ability for the truncated protein to activate the receptor while still retaining the ability to bind CCR2b (19). Thus, it was suggested that, for the case of MCP-1, the exact nature of the side chains in the N-terminus is unimportant for activation of the receptor and that the mere presence of a polypeptide chain is sufficient for receptor activation once the vital binding step has occurred (35). The only residue in MCP-1 found to be important for binding in either the N-terminal or N-loop regions was Tyr13 (corresponding to Phe11 in eotaxin) (34). These results contrast with those shown here for eotaxin, in which individual residues in the N-terminus are linked to binding and activation of the receptor, and several residues in the N-loop, in addition to Phe11, show involvement in receptor activation and binding. Thus, eotaxin displays a relatively diffuse or delocalized set of contributions to receptor binding and activation throughout the N-terminus and N-loop regions whereas MCP-1 has a more important single residue (Tyr13). These contrasting results for eotaxin and MCP-1, proteins similar in sequence that are specific for different receptors, provide some clues about the different mechanisms by which receptor specificity may be achieved.
Conclusions--
Several residues important for the binding and
activation of CCR3 by eotaxin have been identified. The location of
these residues demonstrates that the N-terminus and N-loop of eotaxin
are each involved in both signaling and receptor binding. Thus, the
results of this study demonstrate that, at least for some chemokines, the N-loop and N-terminus do not have separable receptor binding and
activation functions. The varying distributions of functionally important residues in different chemokines are likely to play an
important role in controlling chemokine/receptor specificity.
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ACKNOWLEDGEMENTS |
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We are indebted to Dr. Osamu Yoshie for the generous gift of L1.2 cells. We also thank Lisa Laws for assistance with tissue culture, Drs. John Richardson, Kristen Mayer, and Jiqing Ye for helpful discussions, and Dr. David Daleke for access to his fluorometer. The HOS-CD4-CCR3 cells were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, National Institutes of Health, and HOS-CD4 cells were obtained from Dr. Nathaniel Landau.
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FOOTNOTES |
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* This work was supported by Grant GM-55055 (to M. J. S.) from the National Institutes of Health, by the American Heart Association, National Affiliate (Established Investigator award), and by a fellowship (to M. R. M.) from the American Heart Association.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of
Chemistry, Indiana University, 800 E. Kirkwood Ave., Bloomington, IN 47405-0001. Tel.: 812-855-6779; Fax: 812-855-8300; E-mail:
mastone@indiana.edu.
Published, JBC Papers in Press, January 30, 2001, DOI 10.1074/jbc.M011202200
2 J. Ye, K. L. Mayer, M. R. Mayer, and M. J. Stone, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: HIV, human immunodeficiency virus; MCP, monocyte chemoattractant protein; R, receptor; HOS, human osteosarcoma.
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