(Received for publication, August 24, 1995; and in revised form, December 20, 1995)
From the
We have isolated cDNA clones that encode two closely related,
murine C-C chemokine receptors. Both receptors are members of the
G-protein-coupled, seven-transmembrane domain family of receptors and
are most closely related to the human monocyte chemoattractant protein
1 receptor. Expression of each of the receptors was detected in murine
monocyte/macrophage cell lines, but not in nonhematopoietic lines.
Expression of these receptors in Xenopus oocytes revealed that
one receptor signaled in response to low nanomolar concentrations of
murine JE, whereas the second receptor was activated by murine
macrophage inflammatory protein (MIP) 1 and the human chemokines
MIP-1
and RANTES. Binding studies revealed high affinity binding
of radiolabeled mJE to the mJE receptor and murine MIP-1
to the
second receptor. Chromosomal localization indicated that the two
receptor genes were clustered within 80 kilobases of each other on
mouse chromosome 9. Creation of receptor chimeras suggested that the
amino terminus was critically involved in mediating signal transduction
and ligand specificity of the mJE receptor, but not the mMIP-1
receptor. The identification and cloning of two functional murine
chemokine receptors provides important new tools for investigating the
roles of these potent cytokines in vivo.
Leukocyte trafficking plays an important role in immune system
surveillance and chronic inflammation. Locally produced chemoattractant
cytokines, known as chemokines, are thought to play a critical role in
this directed migration (see (1, 2, 3, 4) for recent reviews).
Human monocyte chemoattractant protein 1 (hMCP-1) ()and its
murine homolog, JE (mJE), are members of the C-C family of chemokines,
in which the first two of four conserved cysteines are adjacent to each
other. Other C-C chemokines include macrophage inflammatory protein
1
and 1
(MIP-1
, MIP-1
) and RANTES (regulated on
activation, normal T cell-expressed). In general, C-C chemokines are
potent monocyte and lymphocyte chemoattractants. A C-C chemokine that
is a chemoattractant for eosinophils, eotaxin(5) , has recently
been described, as well as a novel lymphocyte chemokine containing two,
rather than four cysteines, known as lymphotaxin(6) .
The
murine JE gene was originally identified by virtue of its dramatic
induction in murine fibroblasts by platelet-derived growth factor and
other growth factors(7) . Characterization of the gene by
Rollins et al.(8) revealed important similarities to
known cytokines such as macrophage colony-stimulating factor,
interferon , and interleukin (IL) 6. Murine JE and hMCP-1 are 62%
identical over their amino-terminal domains, but mJE extends an
additional 49 amino acids beyond the carboxyl end of hMCP-1. This
carboxyl-terminal extension, which is extensively glycosylated, is not
required for the chemoattractant activity of mJE(9) . Further,
mJE and hMCP-1 have similar chemoattractant activity for human
monocytes(9) . Murine JE is thus a structural and functional
analog of hMCP-1.
MCP-1 has been implicated in the pathogenesis of diseases characterized by monocytic infiltrates, including psoriasis(10) , pulmonary fibrosis (11) , rheumatoid arthritis(12) , and atherosclerosis(13, 14) . In mice, mJE has been shown to be up-regulated by infusion of minimally oxidized low density lipoproteins (15) and thus may play a role in the accumulation of monocyte/macrophages in early atherosclerotic lesions. A possible role for mJE in tumor suppression in vivo was suggested by Rollins et al.(16) who found that expression of hMCP-1 or mJE in Chinese hamster ovary cells suppressed the ability of the cells to form tumors in nude mice.
Human receptors for IL-8 (Type A and Type
B(17, 18) ) and a single receptor that binds both
RANTES and MIP-1 (19, 20) have been cloned and
shown to be members of the seven-transmembrane domain superfamily of
receptors. We have recently reported the cloning and expression of two
alternatively spliced forms of the human MCP-1 receptor, which differ
only in their terminal carboxyl tails (21) and which couple to
G
in a pertussis toxin-sensitive manner(22) .
To investigate further the roles of chemokines in vivo,
considerable effort has been focused recently on the cloning of their
murine receptors. In contrast to the situation in the human, a single
receptor appears to exist for murine IL-8(23) . Extramedullary
myelopoiesis, as well as a decreased neutrophil response after
injection of thioglycolate, was noted in mice in which the IL-8
receptor was deleted by homologous recombination(23) . These
studies suggest that IL-8, and perhaps other chemokines, are involved
in the regulation of myelopoiesis. Gao and Murphy (24) have
very recently reported the cloning of a murine MIP-1
receptor, as
well as two orphan receptors. In this paper, we report the cloning and
functional expression of a murine JE/MCP-1 receptor, as well as a
second, closely related receptor that signals in response to
mMIP-1
, hMIP-1
, and hRANTES.
For Northern blots, 10 µg of total RNA was size-fractionated on
a 1.0% agarose, 0.66 M formaldehyde gel, transferred to a
nylon membrane (Hybond-N, Amersham), and stained with 0.03% methylene
blue in 0.3 M sodium acetate, pH 5.2, to visualize ribosomal
RNAs. The filter was sequentially hybridized with P-labeled receptor-specific probes described above. The
probe concentration was 1.0
10
cpm/ml, and
hybridizations were at 42 °C overnight in the above hybridization
mixture, except that 5% dextran sulfate was included and PIPES (0.8 M NaCl, 20 mM PIPES, pH 6.5) replaced the SSC. The
membrane was stripped by boiling for 10 min in water containing 0.5%
SDS.
Figure 1: Calcium mobilization in WEHI 274.1 cells by mJE and hMCP-1. WEHI 274.1 cells were loaded with Indo-1 AM and challenged with mJE (30 nM) or hMCP-1 (100 nM) at the time indicated by the arrow.
Figure 2: Expression of the mJE receptor in Xenopus oocytes. A, specificity of the mJE receptor. All chemokines were used at a final concentration of 100 nM. B, dose-response curves for mJE and hMCP-1. All data points were determined in triplicate. The data shown are representative of three experiments.
Figure 3: Calcium mobilization in 293 cells stably transfected with the mJE receptor cDNA. Dose-response curves to mJE (A), hMCP-1 (B), and other C-C chemokines (100 nM) (C).
Screening of
a mouse spleen library yielded a second cDNA clone that also hybridized
strongly to the hMCP-1 receptor probe. In contrast to the mJE receptor,
however, the receptor encoded by this cDNA signaled in response to
mMIP-1, hRANTES, and hMIP-1
, but did not respond to mJE,
hMCP-1, mMIP-1
, or hMIP-1
(Fig. 4). We therefore refer
to this receptor as the murine MIP-1
(mMIP-1
) receptor. The
response of this receptor to murine, but not human MIP-1
, is
intriguing, as the human MIP-1
/RANTES (C-C CKR-1) receptor
responds equally well to both human and murine MIP-1
(19) .
Figure 4:
Expression of the murine MIP-1
receptor in Xenopus oocytes. A, specificity of the
mMIP-1
receptor. The indicated chemokines were used at final
concentrations of 100 nM. B, dose-response curves for
mMIP-1
and hMIP-1
. All data points were determined in
triplicate. The data shown are representative of three similar
experiments.
Figure 5:
Binding of chemokines to the cloned
receptors. A, radiolabeled mJE was added at the indicated
concentrations to membranes prepared from HEK-293 cells stably
expressing the mJE receptor. Nonspecific binding was determined by the
addition of a 100-fold excess of unlabeled JE. Specific binding was
determined by subtraction of the nonspecific binding from the total
binding. The dissociation constant (K),
determined by Scatchard analysis, was 46 ± 18 pM. Shown
is one of three similar experiments. Very similar results were obtained
using intact HEK-293 cells. B, competition of mJE and hMCP-1
for the mJE receptor. Radiolabeled mJE (150 pM) was added to
membranes prepared from HEK-293 cells stably expressing the mJE
receptor. Unlabeled mJE and hMCP-1 were added at the indicated
concentrations. The IC
values were 195 pM for mJE
and 210 for hMCP-1. C, radiolabeled mMIP-1
was added at
the indicated concentrations to HEK-293 cells stably expressing the
mMIP-1
receptor. Nonspecific binding was determined by the
addition of a 100-fold excess of unlabeled mMIP-1
. The apparent K
was 640
pM.
Figure 6:
Predicted amino acid sequence of the mJE
and mMIP-1 receptors. The murine receptors are shown aligned with
the human C-C chemokine receptors. Gaps inserted to optimize
the alignments are indicated by dashes. The seven predicted
transmembrane domains are indicated by the horizontal bars and numbers.
Figure 7:
Southern blot analysis of murine chemokine
receptor genes. Mouse genomic DNA (10 µg) was digested with HindIII (lane 1), EcoRI (lane 2), BamHI (lane 3), or XbaI (lane 4)
and hybridized under conditions of high stringency with radiolabeled
probes specific for the 3`-untranslated regions of the mJE and
mMIP-1 receptors.
The chromosomal locations of the two
genes were determined by linkage analysis of an interspecific backcross
involving the parental mouse strains C57BL/6J and M. spretus as described previously (28) . Receptor-specific probes
were used to identify informative RFLVs of the genes upon Southern
hybridization. The segregation of the RFLV was examined in 65 (C57BL/6J
M. spretus) F1
C57BL/6J backcrossed mice. DNA
from these mice has been typed previously for over 200 genetic markers
spanning all chromosomes except the Y chromosome(28) . The mJE
receptor RFLV exhibited linkage with a number of markers on the distal
portion of mouse chromosome 9, the nearest proximal marker being the
microsatellite marker D9Mit19 (1 recombinant out of 65
animals) and the nearest distal marker being the random cDNA RFLV D9Ucla3 (1 recombinant out of 65 animals) (Fig. 8). The
linkage was highly significant, as both markers exhibited logarithm of
the odds scores exceeding 17.3. Analysis of the segregation of an RFLV
for the mMIP-1
receptor gene revealed complete co-segregation with
the mJE receptor RFLV (no recombination out of 65 animals). These
results indicate that the genes for the mJE receptor and the
mMIP-1
receptor are tightly linked on mouse chromosome 9 (Fig. 8). The results indicate the following order of markers
typed on distal chromosome 9, with distances given in centimorgans
± S.E.: centromere-(D9Ucla2, D9Mit36) ( 5.9
± 3.3 centimorgans)-2Mit6 h2 (1.5 ± 1.5
centimorgans)- D9Mit19 (1.5 ± 1.5
centimorgans)-(Jer, Mip1ar) (1.5 ± 1.5
centimorgans)-D9Ucla3 (1.5 ± 1.5
centimorgans)-D9Ucla5. We designate the symbols Jer and Mip1ar for the JE receptor and MIP-1
receptor,
respectively. This region of murine chromosome 9 is syntenic to human
chromosome 3p21(29) .
Figure 8:
Chromosomal localization of the JE and
MIP-1 receptors. Restriction fragment length variants (RVLPs)
between C57BL/6J and M. spretus mice were used to map the
murine receptors as described under ``Materials and
Methods.'' Both receptors map to adjacent locations near the
distal part of murine chromosome 9. The distances, in centimorgans,
between the chromosome 9 markers mapped in this cross are
indicated(28) .
Further evidence that the two
receptors are closely linked was obtained by screening a murine 129ES
genomic library constructed in P1 bacteriophage clones (average insert
size of 85 kb). Using PCR primer pairs specific for each receptor (see
``Materials and Methods''), we obtained two independent P1
clones that amplified the predicted products for both receptors (Fig. 9). In addition, Southern analysis of one P1 clone (clone
5340) produced the same hybridization pattern with receptor-specific
probes as that observed with total genomic DNA (data not shown, see Fig. 7), indicating that this P1 clone contains both the mJE and
mMIP-1 receptors. We conclude, therefore, that these two receptors
are closely linked on mouse chromosome 9.
Figure 9:
PCR analysis of P1 bacteriophage genomic
clones. mJE and mMIP-1 receptor-specific primers were used to
amplify the indicated DNA templates. Lane 1, mMIP-1
receptor cDNA; lane 2, mJE receptor cDNA; lane 3, P1
clone 5203 and mMIP-1
receptor primers; lane 4, P1 clone
5203 and mJE receptor primers; lane 5, P1 clone 5340 and
mMIP-1
receptor primers; lane 6, P1 clone 5340 and mJE
receptor primers. No bands were seen when mJE receptor primers were
used with the mMIP-1
receptor cDNA as the template and vice versa
(not shown).
Figure 10:
Northern blot analysis of chemokine
receptor expression by murine cell lines. Total RNA (10 µg/lane)
from the indicated cell lines was electrophoresed in a 1% agarose gel
and hybridized with radiolabeled probes specific for the mJE receptor
and the mMIP-1 receptor. WEHI 274.1 and 265.1 are monocytic cell
lines. WEHI 3 and P388D1 are macrophage cell lines. NS1 is a myeloma
cell line. B16.F10 is a melanoma cell line. BALB/c, L929, and NIH3T3
are fibroblasts. Y1 is an adrenal tumor cell line. Exposure times were
2 days at -80 °C. The positions of 18 S and 28 S ribosomal
RNAs are indicated to the left. Methylene blue staining of the
filter revealed intact ribosomal bands of approximately equal intensity
in all lanes, except for the BALB/c lane, in which the amount of RNA
was reduced (data not shown).
Figure 11: Expression of chimeric C-C chemokine receptors in Xenopus oocytes. The indicated chemokines were used at final concentrations of 200 nM.
Murine JE has been implicated in models of disease
characterized by prominent monocyte/macrophage infiltrates, but the
mechanisms of monocyte activation and directed migration induced by mJE
are not well understood. To gain insight into this phenomenon, and as a
first step in genetic modification of the mJE receptor gene, we have
cloned its cDNA from a murine monocytic cell line. Several lines of
evidence support the conclusion that this cDNA encodes a murine JE
receptor. First, injection into Xenopus oocytes of cRNA
obtained from this clone conferred mJE/hMCP-1-dependent activation at
low nanomolar concentrations. We have confirmed these results in
transfected mammalian cells. In both Xenopus oocytes and
HEK-293 cells expressing the JE receptor, calcium is mobilized much
more efficiently by mJE as compared to hMCP-1. Similar results were
obtained using wild-type WEHI 274.1 murine monocytes. Second, these
responses are specific for mJE/hMCP-1, as other closely related
chemokines failed to induce signals. Third, I-labeled JE
bound with high affinity to HEK-293 cells transfected with this cDNA.
Fourth, Northern blot analysis revealed high levels of expression of
the receptor mRNA in monocytic cell lines that responded to mJE and
little or no mRNA in lines that failed to respond to mJE. Finally,
sequencing of the cDNA revealed a putative seven-transmembrane domain
receptor with a predicted amino acid sequence that was 72% identical
with the hMCP-1 receptor. We conclude, therefore, that this cDNA
encodes a murine JE receptor.
The second receptor cloned in this
study signaled well in response to low nanomolar concentrations of
murine MIP-1 and also bound this chemokine with high affinity. It
is likely, therefore, that mMIP-1
is the natural ligand for this
receptor. The mMIP-1
receptor also signaled in response to human
MIP-1
and thus represents the first example of a cloned receptor
activated by MIP-1
. In addition, hMIP-1
competed well with
radiolabeled mMIP-1
in receptor binding assays. Whether or not
MIP-1
is a natural ligand for this receptor remains unclear,
however, because the murine form of MIP-1
was not efficient at
receptor activation. Similarly, this receptor was activated by human
RANTES, and it will be interesting to determine if murine RANTES is a
functional ligand.
The mJE and mMIP-1 receptors are almost
completely identical in the putative transmembrane domains, as well as
in the first extracellular loop. In addition, the second and third
intracellular loops are nearly identical, suggesting that both
receptors may couple to the same or very similar G-proteins.
Interestingly, the murine MIP-1
receptor is more closely related
to the MCP-1 receptor than to C-C CKR-1, the human receptor that binds
and signals in response to MIP-1
and RANTES (19) . These
data suggest that the MIP-1
receptor is a novel receptor and not
simply a murine homolog of the human MIP-1
/RANTES receptor. Based
on primary sequence identity, it may in fact represent the murine form
of a human MCP-1 receptor homolog. This receptor does not, however,
signal in response to human or murine MCP-1. The mJE receptor signals
primarily in response to mJE and hMCP-1 and, in this regard, is very
reminiscent of the ligand specificity of the hMCP-1 receptor. We have
recently found that hMCP-3, but not hMCP-2, is a functional ligand for
the human MCP-1 receptor(30) . It remains to be determined if
the MARC/fic protein(31) , which appears to be the murine
homolog of hMCP-3, activates the mJE receptor.
The cloned mJE
receptor bound mJE and hMCP-1 in a comparable manner, yet signaled much
more efficiently in response to mJE as compared to hMCP-1. High
affinity binding of the ligand to the receptor thus appears to be
necessary, but not sufficient, to initiate signaling. These data are
consistent with a model in which one portion of the receptor binds the
ligand with high affinity, while a second receptor domain interacts
with the chemokine to initiate signaling. Recent work in our laboratory
on the binding of hMCP-1 to its receptor supports such a model, ()as does published work on the C5a receptor(32) .
It should be noted that hMCP-1 and mJE have been found to be equipotent
in inducing chemotaxis of human monocytes(9) . Thus, unlike the
murine receptor, the human MCP-1 receptor may not distinguish between
human and murine MCP-1. Studies are currently in progress in our
laboratory comparing the binding and signaling properties of the human
and murine MCP-1 receptors.
The mJE and mMIP-1 receptors appear
to have arisen by gene duplication and may represent the first two
members of a family of receptor genes clustered on chromosome 9. The
evidence for this hypothesis includes the high degree of identity
between these two receptors at the DNA sequence level, their
co-segregation in a genetic cross, and their co-localization on a P1
bacteriophage clone. This area of mouse chromosome 9 is syntenic to
human chromosome 3p21, where the hMIP-1
/RANTES (20) and
hMCP-1 (
)receptor genes are found in close proximity. This
region does not correspond to any mutations with obvious relevance to
these receptors. In addition, Gao and Murphy (24) have very
recently identified a murine MIP-1
receptor distinct from the
receptors described in this paper, as well as two additional closely
related murine receptors without identified ligands, all of which map
to mouse chromosome 9. Other chemokine receptors have been localized to
human chromosome 2q34-q35 in the case of the human type A and B IL-8
receptors (33) and to chromosome 19q13.3 for the formyl peptide
and C5a receptors(34) .
The amino-terminal domains represent
the areas of greatest sequence divergence between the mJE and
mMIP-1 receptors. The amino termini of the receptors for
thrombin(35) , thyrotropin(36) , C5a(37) , and
IL-8 (38) participate in the binding of their respective
ligands. Taken together, these observations suggest that divergence of
this domain in the mJE and mMIP-1
receptors may contribute to
their different agonist specificities. To test this hypothesis, we
constructed two chimeric receptors in which the amino-terminal domains
were exchanged between the mJE receptor and the mMIP-1
receptor.
Analysis of the signaling properties of these two chimeras in Xenopus oocytes indicated that the amino terminus of the mJE
receptor, but not the mMIP-1
receptor, was critical for signaling.
This result is in agreement with recent results obtained using the
human MCP-1 receptor,
and suggests that distinct mechanisms
of ligand binding have evolved within the C-C chemokine receptor
family. Since the first extracellular loop of the mMIP-1
receptor
and mJE receptor are identical, it is likely that the second and third
extracellular loops of the mMIP-1
-R will be found to mediate
ligand binding and specificity.
In summary, we have cloned two novel
murine receptors that appear to define a family of C-C chemokine
receptors clustered on chromosome 9. Through the construction of
receptor chimeras, we have demonstrated that signaling of the mJE
receptor, but not the mMIP-1 receptor, is critically dependent
upon ligand interaction with the receptor amino terminus. The
identification of the murine JE receptor represents an important step
in the creation of genetically modified mice to probe the role of
JE/MCP-1 in models of human disease.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank(TM)/EMBL Data Bank with accession number(s) U47035 [GenBank](JE-R) and U47036 [GenBank](mMIP1R).