From the Krebs Institute, University of Sheffield,
Western Bank, Sheffield S10 2TN, United Kingdom,
§ Laboratory of Host Defenses, NIAID, National Institutes of
Health, Bethesda, Maryland 20892, and
LeukoSite, Inc.,
Cambridge, Massachusetts 02142
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ABSTRACT |
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CCR1 and CCR3 are seven-transmembrane domain G
protein-coupled receptors specific for members of the CC chemokine
subgroup of leukocyte chemoattractants. Both have been implicated in
the inflammatory response, and CCR3, through its expression on
eosinophils, basophils, and Th2 lymphocytes, may be especially
important in allergic inflammation. CCR1 and CCR3 are 54% identical in
amino acid sequence and share some ligands but not others. In
particular, macrophage inflammatory protein 1 (MIP-1
) is a ligand
for CCR1 but not CCR3, and eotaxin is a ligand for CCR3 but not CCR1.
To map ligand selectivity determinants and to guide rational antagonist design, we analyzed CCR1:CCR3 chimeric receptors. When expressed in
mouse pre-B cells, chimeras in which the N-terminal extracellular segments were switched were both able to bind both MIP-1
and eotaxin, but in each case, binding occurred via separate sites. Nevertheless, neither MIP-1
nor eotaxin were effective agonists at
either chimeric receptor in either calcium flux or chemotaxis assays.
These data are consistent with a multi-site model for chemokine-chemokine receptor interaction in which one or more subsites
determine chemokine selectivity, but others are needed for receptor
activation. Agents that bind to the N-terminal segments of CCR1 and
CCR3 may be useful in blocking receptor function.
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INTRODUCTION |
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Chemokines and their seven-transmembrane domain G protein-coupled receptors constitute a large and highly differentiated signaling system involved in multiple biologic processes, including development, hematopoiesis, angiogenesis, and regulation of specific leukocyte trafficking (1-3). Together the system is capable of supporting host defense and repair functions but may also act as an amplifier of inappropriate inflammation in diseases such as asthma. Moreover, many components of the chemokine system are exploited as pro-microbial factors (3, 4). For example, diverse chemokine receptors can be exploited as cell entry factors by HIV-11 (5).
As the chemokine system expanded by gene replication, some functions
were conserved, but new ones were added, allowing functional back-up to
grow apace with functional diversification. This is illustrated nicely
by the receptors CCR1 and CCR3. These receptors are more closely
related in sequence to each other than to other chemokine receptors
(54% amino acid identity) and have overlapping but nonidentical
functional specificities (6-12). Both bind multiple chemokines, but
only members of the CC subgroup of chemokines, including RANTES and
MCP-3. However, each receptor also binds a selective ligand, eotaxin in
the case of CCR3 and MIP-1 in the case of CCR1. Another differential
feature is that CCR3 is an HIV-1 coreceptor, whereas this activity has
not been found for CCR1 (13-17).
CCR3 is the only known eotaxin receptor, whereas several MIP-1
receptor subtypes have been identified (18-20). Eotaxin is a major
activator of eosinophils, basophils, and Th2 lymphocytes, acting by
binding to CCR3 (21-23), which has suggested that eotaxin and CCR3 are
major factors regulating allergic inflammation. Consistent with this,
mice rendered deficient in eotaxin by gene targeting exhibit reduced
eosinophilic inflammation in response to both allergen challenge of the
airway and cornea (24).
Compared with CCR3, CCR1 appears to be expressed at higher levels on lymphocytes and monocytes and at lower levels on eosinophils (25, 26). In mice it is an important neutrophil chemotactic receptor, but this function may not be expressed in humans (26, 27). Mice lacking CCR1 have increased susceptibility to Aspergillus infection, reduced granulomatous responses to Schistosome egg challenge, and reduced pneumonitis in a pancreatitis-induced pneumonitis model (27, 28).
Because of their roles in inflammation, identifying agents that specifically block CCR1 and CCR3 function may be therapeutically useful, and information about the ligand binding site may help to develop the most efficacious blocking agents. Also, CCR3 binding agents may be useful as anti-HIV agents. Eotaxin itself has this property, whereas RANTES and MCP-3 are much less potent (17). We have previously analyzed a series of chimeric CCR1:CCR3 receptors to map determinants of the HIV-1 coreceptor activity at CCR3(17). Here we use chimeric receptors to map chemokine selectivity determinants for CCR1 and CCR3.
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EXPERIMENTAL PROCEDURES |
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Construction of Chimeric Receptors-- Construction of DNA-encoding chimeric receptors was accomplished by overlap extension polymerase chain reaction using the p4 cDNA-encoding CCR1 and the clone 3 cDNA-encoding CCR3 as templates, as described previously (7, 9, 17). The sequence encoding the FLAG epitope (Eastman Kodak Co.) was inserted between the first two codons by inclusion in the 5' oligonucleotide primer. Chimeric DNA was then ligated into the vector pcDNA3 (Invitrogen, CA) at the HindIII (5') and XhoI (3') sites, and sequences were confirmed by double-stranded DNA sequencing.
Establishment of Stable Transfectants-- The murine pre-B cell lymphoma cell line 4DE4 was generously provided by L. Staudt and maintained as described previously (29). Plasmid DNA (10 µg) was introduced into 3 × 106 cells by electroporation in 0.5 ml of Hanks' buffered saline solution (250 V, 940 µF). After 48 h in culture, the medium was supplemented with G418 (Life Technologies, Inc.) at 0.6 mg/ml until the flask achieved confluency, after which the concentration of G418 was raised to 1 mg/ml, and the cells were cloned by limiting dilution. Individual clones were then screened for receptor expression by radioligand binding and chemokine-induced calcium release assays.
Intracellular Calcium Release--
4DE4 cells were resuspended
at 107 cells/ml in phosphate-buffered saline and were
loaded with 2.5 µM Fura-2 (Molecular Probes, OR) for 30 min at 37 °C in the dark. Cells were then washed twice in Hanks'
buffered saline solution and resuspended at 1.5 × 106/ml. Cells were then stimulated with various doses of
either eotaxin or MIP-1 in a continuously stirred cuvette at
37 °C in a fluorometer (Photon Technology, Inc., Piscataway, NJ). We
used the recombinant BB10010 variant of human MIP-1
, a generous gift
of L. Czaplewski (British Biotech Inc., Oxford, UK) and recombinant
human eotaxin purchased from Peprotech (Rocky Hill, NJ). Data were
recorded every 200 ms as the relative ratio of fluorescence emitted at 510 nm after sequential stimulation at 340 and 380 nm.
Chemotaxis Assays-- These were carried out as described previously using microchemotaxis chambers (Neuroprobe, Cabin John, MD) (29). Results obtained were expressed as a chemotactic index.
Radioligand Binding Assays--
125I-MIP-1 and
125I-eotaxin were purchased from NEN Life Science Products.
The specific activities were 2200 Ci/mmol. Cells were washed once in
binding buffer (Hanks' buffered saline solution containing 1% bovine
serum albumin and 0.05% NaN3) and resuspended in the same
buffer at 2-3 × 106 cells/ml. 100 µl of this
suspension were added to duplicate Eppendorf tubes containing 0.25 nM labeled ligand and varying concentrations of cold
competing ligand in a final volume of 200 µl. Ligand binding was
allowed to proceed at room temperature for 1 h, after which 500 µl of binding buffer adjusted to 0.5 M NaCl was added,
and the cells were pelleted by centrifugation at 10,000 × g for 5 min. the supernatant was aspirated, and the cell pellet was cut from
the tube and counted in a gamma counter. Nonspecific binding was
typically 20-40% of the total counts. The data were fit to a curve,
and the apparent binding affinity and receptor density were estimated
using the program LIGAND.
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RESULTS AND DISCUSSION |
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Differential Chemokine Specificities of Wild Type CCR1 and CCR3-- We have previously analyzed HIV-1 specificity determinants for CCR3 using a panel of CCR1:CCR3 chimeric receptors transiently expressed in NIH 3T3 cells. This system, which depends on vaccinia virus activation of a vaccinia promoter in the plasmid vector pSC59, allows high levels of receptor expression; however it could not be used for the present study because vaccinia interrupts G protein signaling in these cells.2 We therefore subcloned the chimeric DNA inserts into the mammalian expression vector pcDNA3 and cloned stably transfected cell lines in a mouse pre-B cell lymphoma cell line.
Untransfected cells did not exhibit specific binding of MIP-1
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Mapping of Ligand Specificity Determinants to the N-terminal Extracellular Segments of CCR1 and CCR3-- Having established the receptor parameters for wild type CCR1 and CCR3 in our system, we next tested the importance of the N-terminal segments of each receptor in determining differential ligand selectivity. We focused on this region because the corresponding region has been shown to be a ligand selectivity determinant for several other chemokine receptors, including CXCR1, CXCR2, Duffy and CCR2 (31-37).
One chimera contained amino acids 1-32 from CCR1 joined to amino acids 33-355 of CCR3 and was named CHI1. The reciprocal chimera contained amino acids 1-32 of CCR3 fused to amino acids 33-356 of CCR1 and was named CHI2. The junctions were based on the hydropathy plot of the receptors. When expressed in pre-B cells, both chimeras exhibited chimeric ligand recognition, specifically binding both 125I-MIP-1
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Independence of MIP-1 and Eotaxin Binding Sites in CCR1:CCR3
Chimeric Receptors--
Heterologous competition binding was carried
out to examine the relationship of the MIP-1
and eotaxin binding
sites on CHI1 and CHI2. Neither chemokine could compete for binding to
the site labeled by the other on either chimera, even when 1 µM heterologous chemokine was used (data not shown),
suggesting that the sites are independent, as they are on wild type
CCR1 and wild type CCR3. Independence of binding sites has also been
reported for interleukin-8 and GRO
on CXCR1:CXCR2 chimeric receptors
and for MCP-1 and MIP-1
on chimeric receptors (33-36). In the case
of CCR2, a pseudo-tethered N-terminal domain binds MCP-1 with affinity
similar to the full-length wild type receptor, suggesting that the bulk
of the binding energy between CCR2 and MCP-1 is conferred by
interactions occurring solely between MCP-1 and this domain (36). Also,
there is direct structural evidence from spectroscopy studies for
binding of interleukin-8 to the N-terminal segment of CXCR1 (38).
Ligand selectivity determinants have been mapped to the second
extracellular loop of CCR5 using CCR2:CCR5 chimeras and to the third
extracellular loop of CCR1 using CCR1:CCR2 chimeras; however, direct
binding sites have not been identified yet (39, 40). Thus, in some cases chemokines may bind to receptors at a single extracellular domain, and this domain could differ for different chemokines binding
to the same receptor. However, in other cases, ligands appear to share
sites on the same receptor.
Requirement for Other Receptor Domains for Receptor Activation-- With several exceptions, there is a fairly good correlation between the rank order of chemokine binding affinity for individual receptors and among different receptors versus the rank orders when the same chemokines are evaluated as agonists. Creation of chimeric receptors allows one to test whether chemokine binding specificity determinants and chemokine receptor activation determinants map to the same domains.
Unlike wild type CCR1 and wild type CCR3, we did not observe activation of either CHI1- or CHI2-expressing stable cell lines in calcium flux and chemotaxis assays when stimulated with either MIP-1Conclusions--
The results of the present study are consistent
with a multi-site model for chemokine-chemokine receptor interaction in
which one or more subsites determine chemokine selectivity, but others are needed for receptor triggering. In this model, ligand is envisioned to bind first via a docking domain, which may be the N-terminal segment
for eotaxin and MIP-1 in the case of CCR3 and CCR1, respectively, and then to be presented to a second activation site on the receptor. The first well documented example in support of this model was the C5a
receptor, which binds a peptide chemoattractant similar in size to the
chemokines (43). Since then, additional examples have been reported
based on the study of chimeric CCR1:CXCR2, CCR2:CCR5, and CXCR1:CXCR2
receptors (33-36).
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ACKNOWLEDGEMENT |
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We are grateful to the British Heart Foundation for their financial support of this project.
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FOOTNOTES |
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* 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: Leukocyte Biology, Imperial College School of Medicine, Division of Biomedical Sciences, Exhibition Road, London, SW7 2AZ, UK. Tel.: 0171-594-3124; E-mail: j.pease{at}ic.ac.uk.
The abbreviations used are:
HIV, human
immunodeficiency virus; MIP, macrophage inflammatory protein 1.
2 G. Alkhatib, M. Locati, P. M. Murphy, and E. A. Berger, unpublished observations.
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REFERENCES |
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