(Received for publication, February 23, 1996; and in revised form, March 26, 1996)
From the
The C-C chemokines human monocyte chemoattractant protein-1 and
-3 (MCP-1 and MCP-3) and mouse JE and FIC are potent activators of
monocytes. Several receptors for MCP-1 and MCP-3 have been cloned from
human monocytic cell lines, and one of these receptors, CCR2B, binds
both MCP-1 and MCP-3. Thus far, no murine receptors for JE or FIC have
been reported. We have cloned a novel murine C-C chemokine receptor,
designated mouse CCR2 (mCCR2), from the mouse monocyte cell line
WEHI265.1. The predicted 373-amino acid sequence of mCCR2 shows highest
identity (80%) with CCR2B. When stably expressed in human embryonic
kidney 293 cells, mCCR2 specifically bound I-JE with high
affinity. FIC was less potent than JE in competing
I-JE
binding to mCCR2-expressing cells, while three other mouse chemokines,
MIP-1
, C10, and N51/KC, did not compete. mccr2 mRNA
expression was detected in elicited peritoneal macrophages as well as
in several mouse organs. The cloning of mCCR2 provides an important
tool to investigate monocyte/macrophage responses to JE and FIC, to
identify other targets for their action, and potentially to study
models of CCR2 function in the mouse.
Chemokines are small secreted molecules that chemoattract and
activate specific leukocyte subpopulations in vitro and are
thought to be important for leukocyte trafficking in vivo(1, 2, 3) . The chemokine superfamily
has traditionally been divided into two subgroups, C-X-C or
C-C, based upon the presence or absence of an amino acid between the
first two cysteine residues of a conserved four-cysteine motif. In
general, C-X-C chemokines attract neutrophils, while C-C
chemokines attract mononuclear cells. Human monocyte chemoattractant
protein-1 (MCP-1), ()MCP-2, MCP-3, mouse JE, and mouse
FIC/MARC are structurally related (50-72% identity) C-C
chemokines that are potent in vitro chemoattractants and
activators of
monocytes(4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) . In vivo, MCP-1 has been implicated in monocytic infiltration
of tissues during several inflammatory diseases including
atherosclerosis (15, 16) and rheumatoid
arthritis(17) , while JE has been implicated in
macrophage-mediated tumor growth suppression in mice(18) .
Recent transgenic mouse models have suggested a role for JE in
monocyte/macrophage recruitment and in host responses to intracellular
pathogens(19, 20) . To further understand the
biological functions of MCP-1, MCP-3, JE, FIC, and related chemokines,
it will be important to identify and characterize the receptors that
bind and mediate their responses.
Three cDNAs, ccr1, ccr2A, and ccr2B, that encode receptors for MCP-1
and/or MCP-3, have been cloned from human monocytic cell
lines(21, 22, 23) . All three cDNAs encode
seven-transmembrane G protein-coupled receptors, and the alternatively
spliced CCR2A and CCR2B receptors differ only in their intracellular
carboxyl-terminal tails. When expressed in human embryonic kidney 293
(HEK293) cells, CCR2B binds and signals in response to MCP-1 and MCP-3
but not to other C-C chemokines such as MCP-2, RANTES, MIP-1, and
MIP-1
(24, 25) . When expressed in HEK293 cells,
CCR1 receptor binds and signals in response to MCP-3, MIP-1
, and
RANTES, but not to MCP-1(21, 26, 27) .
Because JE and FIC bind some of the same receptors on human monocytes
as MCP-1(13) , we predicted that murine receptors for JE and
FIC might resemble CCR1 and CCR2A/B. Two murine cDNAs homologous to ccr1 have been cloned, but thus far these receptors have only
been reported to bind and signal in response to mouse MIP-1
and
MIP-1
(28, 29) . We report here the molecular
cloning, RNA expression, and functional characterization of the first
murine receptor that binds JE and FIC.
Recombinant JE,
FIC, and N51/KC proteins were expressed using a baculovirus system and
purified as described previously(13, 33) . JE (20
µg) was radiolabeled by Bolton-Hunter to a specific activity of
2200 mCi/mmol (DuPont NEN Custom Iodination Laboratory). Recombinant
mouse MIP-1 and C10 were purchased from R& Systems.
Degenerate primers encoding the conserved
sequences NLAISDLL in transmembrane domain 2 and FLFWTPY in
transmembrane domain 6 amplified a PCR product from WEHI265.1 cDNA that
shared sequence similarity with ccr1, ccr2A, and ccr2B. The subcloned PCR product was used as a probe to
isolate four cDNA clones from a WEHI265.1 cDNA library. The longest
3.8-kb cDNA clone (pCA-4A) contained an open reading frame of 373 amino
acids that is highly homologous to CCR2B (80% identity; Fig. 1)
and to CCR2A (71% identity). The 373 residue sequence was also similar
but less homologous to CCR1 (56% identity), murine MIP-1R/mCCR1
(55% identity)(28, 29) , and murine
MIP-1
RL2/mCCR3 (55% identity) (28, 29) . Because
of the highest sequence homology to the two CCR2 receptors, we
designated this potential murine receptor mCCR2.
Figure 1: Sequence alignment of murine CCR2 (mCCR2) and human CCR2B (hCCR2B). The numbering of residues for the respective sequences is indicated on the right. Identical amino acids are shaded, and the seven hydrophobic domains are indicated by solid lines above the alignment. Conserved cysteines thought to be involved in extracellular disulfide bonding are indicated (*) above the respective residues.
Similar to other
cloned chemokine receptors, the mCCR2 sequence contains seven
hydrophobic regions suggesting a G-protein-coupled receptor. Several
other hallmarks of C-C chemokine receptors are also contained in the
mCCR2 sequence, including conserved extracellular cysteine residues,
the conserved sequence IFFIILLTIDRYLAIVHAVFAL from the middle of
transmembrane domain 3 to intracellular loop region 2, an extremely
basic intracellular loop region 3, and a serine/threonine-rich COOH
terminus(23, 34) . The predicted
NH-terminal extracellular region of mCCR2, like that of
mCCR1 and mCCR3, contains no predicted N-glycosylation sites,
unlike human CCR1, CCR2A, and CCR2B.
Figure 2:
Northern
blot analysis of mccr2. A, total RNA (10 µg) from
WEHI265.1 cells and 293/mCCR2 cells, and the indicated mouse organs was
blotted and hybridized with a probe specific for mccr2 (top). The blot was stripped and reprobed with a probe
specific for mouse -actin (bottom). The
mccr2-specific blot was exposed for 4 days at -70
°C, and the
-actin blot was exposed overnight at -70
°C. B, total RNA (10 µg) from WEHI265.1 cells and
thioglycollate-elicited macrophages was similarly blotted and
hybridized.
To determine the distribution of mccr2 expression in the mouse, Northern blot analyses were performed on total RNA extracted from multiple organs (Fig. 2A). Out of 12 organs analyzed by Northern blotting, mccr2 expression was detected in the kidney, lung, spleen, and thymus, although the mRNA levels were significantly lower than in WEHI265.1 cells. The low levels of mccr2 mRNA in the organs may represent expression in a specific subset of cells within the particular organs or in contaminating leukocytes such as monocytes/macrophages. Since mccr2 was cloned from the monocytic cell line WEHI265.1, we determined if mccr2 mRNA was also expressed in mouse mononuclear cells. When Northern blot analysis was performed on total RNA from thioglycollate-elicited peritoneal macrophages, mccr2 mRNA was detected at lower levels than in WEHI265.1 cells (Fig. 2B). Unlike the WEHI265.1 cells, the predominant mccr2 mRNA form in the elicited peritoneal macrophages is the smaller 2.8-kb species. The preferential expression of larger or smaller mRNAs in the WEHI265.1 cells and peritoneal macrophages suggests differential transcriptional regulation of mccr2 in different cell types.
Figure 3:
Binding of I-JE to 293/mCCR2
cells. A, 0.2-7.5 nM
I-JE was
bound to 293/mCCR2 cells in the presence or absence of unlabeled JE as
detailed under ``Experimental Procedures.'' The
difference between total and nonspecific binding is the specific
binding. Each data point is the mean of duplicate samples, and the
experiment shown is representative of three independent experiments.
Error bars depict the standard deviation for the mean values of total
and nonspecific binding data. B, Scatchard transformation of
the binding data.
To determine the
ligand binding specificity of mCCR2, competition binding analyses were
performed on 293/mCCR2 cells with 0.5 nMI-JE
and increasing concentrations of unlabeled chemokines (Fig. 4).
Both JE and FIC competed for
I-JE binding to mCCR2, with
JE competing approximately ten times more effectively than FIC. Two
other mouse C-C chemokines, MIP-1
(35) and
C10(36) , and the mouse C-X-C chemokine N51/KC (37, 38) did not effectively compete for
I-JE binding to mCCR2. These results suggest that mCCR2
preferentially binds JE and FIC among the mouse chemokines tested so
far and that mCCR2 is yet another chemokine receptor that binds more
than one ligand.
Figure 4:
Competition of I-JE binding
to 293/mCCR2 cells. 0.5 nM
I-JE was bound to
293/mCCR2 cells in the presence of increasing concentrations of the
indicated unlabeled chemokines as detailed under
``Experimental Procedures.'' Each data point is the
mean of duplicate samples, and the experiment shown is representative
of three independent experiments. Error bars depict the standard
deviation for the mean values of each data
point.
The sequence and binding specificity of mCCR2 make
it the most likely mouse species analog of CCR2B. mCCR2 binds JE with
high affinity and FIC with lower affinity, while CCR2B binds MCP-1 with
high affinity and MCP-3 with lower affinity. Interestingly, it has been
proposed by sequence similarities that JE is the mouse MCP-1 analog and
FIC the mouse MCP-3 analog(39, 40) . Since mCCR2 does
not bind MIP-1, while murine MIP-1
R/mCCR1 and murine
MIP-1
RL2/mCCR3 have not been reported to bind JE or FIC, mCCR2
most likely mediates distinct functions from the other two cloned
murine C-C receptors. mccr2 expression was detected in both
the WEHI265.1 monocytic cell line and in elicited peritoneal
macrophages, suggesting that mCCR2 will be important for mediating
mononuclear cell responses to JE and FIC. The generation of mice with a
targeted deletion of the mccr2 gene will be useful for testing
the role of mCCR2 in JE-mediated monocyte/macrophage recruitment and
host defense and may be useful for developing mouse models for
MCP-1/CCR2 functions.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U51717[GenBank].