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(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
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) ( 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 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
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
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
Figure 4:
Expression of the murine MIP-1
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
Figure 6:
Predicted amino acid sequence of the mJE
and mMIP-1
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
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
Figure 8:
Chromosomal localization of the JE and
MIP-1
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
Figure 9:
PCR analysis of P1 bacteriophage genomic
clones. mJE and mMIP-1
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
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, The second receptor cloned in this
study signaled well in response to low nanomolar concentrations of
murine MIP-1 The mJE and mMIP-1 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, ( The mJE and mMIP-1 The amino-terminal domains represent
the areas of greatest sequence divergence between the mJE and
mMIP-1 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
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](mMIP1
Volume 271,
Number 13,
Issue of March 29, 1996 pp. 7551-7558
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Receptors
EVIDENCE FOR TWO CLOSELY LINKED C-C CHEMOKINE RECEPTORS ON
CHROMOSOME 9 (*)
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
)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) ., 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. (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.
Reagents
Recombinant chemokines were obtained
from R & D Systems, Inc. (Minneapolis, MN). Initial experiments
used full-length mouse (9) and rat JE purified from
supernatants of stably transfected Chinese hamster ovary cells (kindly
provided by T. Yoshimura, National Cancer Institute, Frederick, MD, and
B. Rollins, Dana Farber Cancer Institute, Boston, MA). Murine JE
expressed in Escherichia coli (R & D Systems, Inc.) was
used in subsequent experiments. No differences in activity were
observed between the JE expressed in E. coli versus mammalian
cells. LipofectAMINE and G418 sulfate were from Life Technologies, Inc.
Restriction enzymes were from Boehringer Mannheim. Ca
was obtained from Amersham. All other
reagents were obtained from Sigma.
Tissue Culture, Calcium Fluorimetry, and Stable
Transfections
WEHI 3, WEHI 274.1, and WEHI 265.1 cells were
obtained from the American Type Culture Collection (Bethesda, MD) and
were grown in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum (Hyclone Laboratories, Logan,
UT) and 1% penicillin/streptomycin, at 37 °C in a humidified 5%
CO
atmosphere. P388D1 cells, obtained from the American
Type Culture Collection, were grown in RPMI 1640 containing 10% fetal
calf serum and antibiotics. All other cell lines were grown in
Dulbecco's modified Eagle's medium with 10% fetal calf
serum plus antibiotics. For calcium fluorimetry, cells were grown to
log phase, loaded with the calcium-specific dye Indo-1 AM (Molecular
Probes, Eugene, OR), and assayed by spectrofluorimetry for changes in
the concentration of intracellular calcium
([Ca]
) in response to
chemokines as described(22) . To generate stable cell lines
that expressed murine chemokine receptors, human embryonic kidney
(HEK)-293 cells, obtained at passage 36 from the American Type Culture
Collection, were grown in modified Eagle's medium/Earle's
balanced salt solution with 10% fetal calf serum and antibiotics.
Murine receptors were cloned into the polylinker of the mammalian
expression vector pcDNA3 (Invitrogen, San Diego, CA) and transfected
into HEK-293 cells (50-80% confluent) with a DNA/lipofectAMINE
mixture according to the manufacturer's instructions. Cells were
grown in the presence of G418 (0.8 mg/ml) for 3-6 weeks.
Surviving colonies were assayed by Northern blot, and clones expressing
high levels of receptor RNA were expanded for further studies.cDNA Library Construction and Screening
WEHI 274.1
cells were grown to log phase, and responsiveness to JE was confirmed
by demonstrating an intracellular calcium flux in Indo-1-loaded cells.
Total RNA was harvested from 5 
10
cells using the
Trizol reagent (Life Technologies, Inc.) following the
manufacturer's instructions, and poly(A) RNA was
selected using oligo(dT)-cellulose (Pharmacia Biotech Inc.). A cDNA
library was constructed from 5.0 µg of poly(A)
RNA
using the Lambda ZapII cDNA Kit (Stratagene) according to the
manufacturer's instructions. Analysis of the library revealed an
average insert size of 2.0 kb. Approximately 1.5
10
plaque-forming units were screened with a P-labeled
probe from the 5` end of the human MCP-1 receptor (base pairs
227-512 of the MCP-1R cDNA), in a hybridization solution of 30%
deionized formamide, 2
PIPES, 5 Denhardt's, 5%
dextran sulfate, 0.5% SDS, and 100 µg/ml salmon sperm DNA at 42
°C. Filters were washed in 0.1 SSC/0.1% SDS at 55 °C
until background counts stopped decreasing on control filters. Positive
clones were subjected to three rounds of plaque purification, and
phagemids (pBluescript SK) containing inserts were
excised according to the manufacturer's instructions. Ten
positive clones were characterized, which contained inserts ranging in
size from
2.0 to 4.0 kb. We also screened a mouse spleen cDNA
library cloned in Lambda Zap (Stratagene) with a probe derived from the
full-length MCP-1 receptor cDNA, using the same hybridization
conditions as above. Fifteen positive clones were obtained from 1.0
10 plaque-forming units. Inserts were sequenced
using fluorescently labeled dideoxynucleotides(25) .Calcium Efflux Assay
The calcium efflux assay was
performed as described(21) . Briefly, cDNAs were cloned
downstream of the SP6 promoter of pcDNA3, the plasmid was linearized,
and complementary RNA (cRNA) was transcribed using SP6 RNA polymerase.
The size and concentration of the transcription product were confirmed
by gel electrophoresis. Xenopus laevis oocytes were injected
with 25 ng of cRNA in a total volume of 50 nl per oocyte 1 day after
harvesting. After incubation in modified Barth's medium (21) for 2 days at 16 °C, the oocytes were loaded with Ca
(50 mCi/ml, Amersham) for 3 h, washed
for 1 h, and placed in groups of seven into wells of a 24-well plate in
a volume of 0.5 ml. Expression of recombinant receptors at the oocyte
surface was confirmed using an enzyme-linked immunosorbent assay, as
described(26) . The
Ca
efflux
following addition of agonist was determined by collecting samples of
the medium at 10-min intervals and counting
emissions in a liquid
scintillation counter. Agonists were applied to the oocytes in
Barth's medium for 10 min. Uninjected oocytes were used as
controls.Chemokine Binding Assay
Binding studies were
performed using membranes prepared from stable cell lines.
Approximately 5 10 stably transfected HEK-293 cells
were harvested by incubation with 1 mM EDTA in
phosphate-buffered saline, washed once with phosphate-buffered saline,
and resuspended in 25 ml of a hypotonic solution (10 mM Hepes,
pH 7.2, 0.2 mM CaCl, 1 mM MgCl, 0.1% bovine serum albumin, 2 mM EDTA, 1
µM leupeptin, 1 mM phenylmethylsulfonyl fluoride,
0.7 µg/ml pepstatin, and 1 µg/ml DNase). The resuspended cells
were homogenized using a Dounce homogenizer and sonicated for 45 s
(Branson Model 450 sonicator, output level 5, Danbury, CT), centrifuged
at 1000 g for 10 min, and the supernatants collected.
Membranes were obtained by centrifugation of the supernatants at 48,000
g for 30 min at 4 °C. The membrane pellet was
resuspended in binding buffer (50 mM Hepes, pH 7.4, 1 mM CaCl, 5 mM MgCl, 0.5% bovine
serum albumin). Approximately 2.5 µg of cell membranes in 0.3 ml of
binding buffer were added to 12 75 mm polypropylene tubes
containing murine JE that had been radiolabeled with I by
the Bolton-Hunter procedure (DuPont NEN) to a specific activity of 2200
Ci/mmol. After incubation for 90 min at 27 °C with gentle shaking,
the membranes were collected onto a glass fiber filter (presoaked in
0.3% polyethyleneimine, 0.2% bovine serum albumin for 1 h), using a
SKATRON filtration harvester (Sterling, VA), set at a vacuum pressure
of 500 mm Hg. Unbound ligand was removed by the addition of 4 ml of
wash buffer (10 mM Hepes, 0.5 M NaCl, 0.5% bovine
serum albumin) over an interval of 10 s. Bound
I-JE was
quantitated by counting
emissions. Essentially identical results
were obtained using intact transfected HEK-293 cells, but with a higher
level of nonspecific binding. In the case of the mMIP-1
receptor,
binding was performed using I-Bolton-Hunter-labeled
mMIP
and stably transfected HEK-293 cells. Dissociation constants
were calculated by the method of Scatchard using the program
LIGAND(27) . Binding data were plotted, and IC values were determined using the program Prism (by Graph Pad, San
Diego, CA).
Southern and Northern Blot Analysis
For Southern
blots, 10 µg of mouse genomic DNA was digested with restriction
enzymes, fractionated by size on a 0.6% agarose gel, and transferred to
Zetaprobe-GT (Bio-Rad) nylon membranes according to the
manufacturer's instructions. The membrane was sequentially
hybridized with probes specific for the murine MIP-1 and JE
receptors. These probes were derived from the 3`-untranslated regions
of the corresponding cDNAs, and their specificities were confirmed by
hybridization to increasing concentrations of both cDNAs spotted onto
nitrocellulose membranes. Prior to reprobing, the membrane was stripped
by incubation in 0.4 N NaOH at 45 °C for 30 min, followed
by washing in 0.1 SSC/0.1% SDS at 65 °C for 2 h. The
membrane was also hybridized with a probe that detects both receptors
(corresponding to nucleotides 335-620 of the mJE receptor cDNA,
which encode the highly conserved region from transmembrane domain (TM)
1 through TM3). Probes were labeled with [P]dCTP
to a specific activity of >8.0
10
cpm/µg and
used at a concentration of 4.0 10 cpm/ml.
Hybridizations were performed in 50% deionized formamide, 5
SSC, 5 Denhardt's, 1.0% SDS, and 100 µg/ml denatured
salmon sperm DNA at 42 °C for 16 h. The membrane was washed in 0.1
SSC/0.1% SDS at 55 °C for 3 h and exposed to x-ray film
with intensifying screens for 1-3 days at -80 °C. 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.P1 Clones
Murine genomic bacteriophage P1 clones
containing each of the two receptors were obtained using PCR primer
pairs. A 129 ES mouse genomic library (average insert size of 85 kb)
was screened by Genome Systems (St. Louis, MO) using primer pairs
specific for each receptor. The mMIP-1 receptor (MIP-1-R)
primers were located at nucleotides 1015-1033 (sense) and
1144-1164 (antisense) of the cDNA and the JE receptor primers at
nucleotides 1360-1378 (sense) and 1593-1612 (antisense) of
the cDNA. Of five positive clones for the mMIP-1 receptor, four
also produced a specific product using the JE receptor primer pair. We
further characterized two of the double positive clones by PCR and
confirmed that both primer pairs amplified a specific product from each
P1 clone. The PCR reactions included 0.19 µg of P1 DNA template,
100 pM concentration of each primer, and 2.5 units of Pfu DNA polymerase (Stratagene) in a volume of 100 µl. The PCR
conditions were 95 °C for 5 min, followed by 30 cycles of 95 °C
for 30 s, 50 °C for 60 s, and 72 °C for 2 min. PCR products
were separated on a 1.8% agarose gel and stained with ethidium bromide. Chromosomal Localization
The genes for mJE-R and
mMIP-1-R were mapped by linkage analysis of an interspecific
backcross of (C57BL/6J Mus spretus) F1
C57BL/6J mice, constructed as described previously(28) . The
cross was previously typed for several hundred restriction fragment
length variants (RFLVs) and simple sequence repeat length
polymorphisms(28) . To identify informative RFLVs, DNAs from
the parental strains were digested with various restriction enzymes and
subjected to Southern hybridization analysis as described above. A
survey of restriction enzymes revealed an RFLV for the mJE receptor
gene with the enzyme PvuII. Following digestion with PvuII, DNA from parental strain C57BL/6J exhibited a 6.2-kb
hybridizing fragment, DNA from M. spretus exhibited 3.8-kb and
1.6-kb fragments, and DNA from F1 hybrids exhibited all three fragments
(not shown). A survey of restriction enzymes revealed an RFLV for the
mMIP-1 receptor gene using the enzyme HindIII; thus,
following digestion, DNA from strain C57BL/6J exhibited bands of 16 kb
and 7.2 kb, DNA from M. spretus exhibited a band of 6.4 kb,
and DNA from F1 mice exhibited all three bands. The RFLVs were then
examined in a set of backcross mice, and the segregation patterns were
compared with those of previously typed markers.Chimeric Receptors
The amino termini of the two
murine receptors were interchanged by taking advantage of a conserved EcoRV site located within the putative first intracellular
loop of each receptor (residue 92 of the mJE-R and residue 69 of the
mMIP-1-R). These chimeric receptors, designated mJE/mMIP-1-R
and mMIP-1/mJE-R, were expressed and assayed for signaling in Xenopus oocytes as described above.
Cloning and Expression of Murine Chemokine
Receptors
To identify cell lines expressing the mJE receptor, we
screened murine monocyte/macrophage cell lines for responsiveness to
mJE and related C-C chemokines. As shown in Fig. 1, mJE and, to
a lesser extent, hMCP-1 induced a transient intracellular calcium flux
in WEHI 274.1 cells. Calcium fluxes were not observed in WEHI 265.1,
WEHI 3, or P388D1 cells (data not shown). To clone the mJE receptor, we
constructed a cDNA library from the WEHI 274.1 cells and screened this
library with a probe complementary to a highly conserved region of the
human MCP-1R. A 2.9-kb cDNA clone was obtained that conferred mJE- and
hMCP-1-dependent signaling, when expressed in Xenopus oocytes,
as assayed by Ca
efflux (Fig. 2).
This receptor was specific for JE/MCP-1, as no calcium efflux was
observed in response to the closely related C-C chemokines mMIP-1
,
mMIP-1, hRANTES, hMIP-1, or hMIP-1 (Fig. 2A). In addition, HEK-293 cells stably expressing
this cDNA also underwent a robust intracellular calcium flux in
response to mJE (1-30 nM) (Fig. 3A),
thus confirming that this receptor signals in mammalian cells. Human
MCP-1 also elicited a calcium flux, although at higher concentrations
than mJE (Fig. 3B). A small but reproducible signal was
seen in response to mMIP-1 (100 nM), but not to
hMIP-1, hMIP-1, or hRANTES (Fig. 3C). We
therefore refer to this cDNA clone as the mJE receptor.
, 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) .
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.
Binding of Radiolabeled Chemokines
Radiolabeled JE
bound with high affinity to membranes prepared from HEK-293 cells
expressing the putative JE receptor (Fig. 5A). Analysis
of these binding data by the method of Scatchard revealed a
dissociation constant (K
) of 46 pM. In
competition binding assays using 150 pMI-labeled JE, we observed very similar IC
values for unlabeled JE (195 pM) and human MCP-1 (210
pM) (Fig. 5B). HEK-293 cells expressing the
mMIP-1
receptor bound I-mMIP1
with high
affinity (K = 640 pM) (Fig. 5C). In competition assays, we found that
unlabeled hMIP-1, as well as mMIP-1, competed efficiently
with I-mMIP-1
for binding to the receptor (data not
shown).
),
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.
Sequence Similarity of the Murine and Human C-C Chemokine
Receptors
The murine JE receptor cDNA encoded a protein of 373
amino acids (Fig. 6). Hydropathy analysis of the predicted amino
acid sequence revealed seven putative transmembrane domains and an
extracellular amino terminus of 50 residues. The mJE receptor was
closely related to the MCP-1 receptor, being 75% identical overall at
the amino acid level. The carboxyl-terminal tail of the mJE receptor
was 81% identical with the corresponding region of the ``B''
form of the human hMCP-1 receptor (21) and did not resemble the
type ``A'' receptor tail(21) . The second novel
murine cDNA, the mMIP-1 receptor, encoded a seven-transmembrane
domain receptor of 354 amino acids, that was more closely related to
the hMCP-1R (71% identity) than to the hMIP-1/RANTES-R (Table 1)(19, 20) . The two murine receptors
were 72% identical overall. However, the transmembrane (TM) domains and
intracellular loops, particularly in the region of the second and third
TMs, were almost identical. Conversely, the amino termini, the second
and third extracellular loops, and the carboxyl-terminal tails were
less well conserved between the two murine receptors (Fig. 6),
suggesting that these regions may be involved in determining chemokine
specificity.
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.
Chromosomal Localization of mJE and mMIP-1
Hybridization of murine genomic DNA with probes
specific for the mJE and mMIP-1
Receptors receptors revealed single bands in
each lane of the Southern blot, suggesting that each receptor is
encoded by a single copy gene (Fig. 7). Rehybridization of this
same blot with a probe derived from a highly conserved portion of the
coding region of these two receptors (TM1 to TM3) failed to reveal
additional bands, suggesting the absence of other closely related
receptors (data not shown).
receptors.
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) .
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) .
receptors. We conclude, therefore, that these two receptors
are closely linked on mouse chromosome 9.
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).
Expression of the JE Receptor and the MIP-1
We examined a number of
hematopoietic and nonhematopoietic murine cell lines for expression of
the mJE receptor and the mMIP-1 Receptor
in Murine Monocytic Cell Lines receptor. As shown in Fig. 10, the mJE receptor mRNA was expressed by monocytic WEHI
274.1 cells and at lower levels by WEHI 265.1 and WEHI 3 cells. P388D1
cells, a macrophage-like line, and a variety of non-myeloid cell lines
did not express detectable levels of the mJE receptor mRNA. Conversely,
the mMIP-1 receptor message was expressed at high levels by P388D1
cells, at much lower levels by WEHI 265.1 cells, but was not detectable
in the other cell lines tested (Fig. 10). These results are
consistent with the observations that WEHI 274.1 cells respond to mJE (Fig. 1), and that P388D1 cells respond to hRANTES and
mMIP-1 (Table 2).
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).
Receptor Chimeras
To investigate the role of the
amino terminus in ligand recognition and signaling, we constructed
chimeric receptors in which the amino termini, along with TM1, were
interchanged between the mJE receptor and the mMIP-1 receptor.
These chimeric receptors were expressed in Xenopus oocytes and
assayed for signaling in response to various chemokines. As shown in Fig. 11, the chimeric mJE/mMIP-1 receptor (amino terminus
and TM1 from the mJE receptor spliced onto the mMIP-1 receptor)
signaled well in response to mMIP-1, and hRANTES, but did not
signal in response to mJE or hMCP-1. This chimeric receptor, therefore,
retained the same ligand specificity as the wild-type mMIP-1
receptor. In contrast, the complementary mMIP-1/mJE receptor
chimera failed to signal to any of the C-C chemokines (Fig. 11).
Enzyme-linked immunosorbent assays confirmed that this chimera was
expressed on the surface of microinjected oocytes at levels comparable
to, or higher than, the wild-type receptors (data not shown). These
data indicate, therefore, that the amino-terminal domain of the mJE
receptor, but not the mMIP-1 receptor, is critically involved in
receptor activation and signal transduction.
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.
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. 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.)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. 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) . 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. 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.
R).)-R, macrophage
inflammatory protein 1 receptor; kb, kilobase(s); PCR, polymerase
chain reaction; PIPES, 1,4-piperazinediethanesulfonic acid.))
We thank Yu-Rong Xia for assistance in the chromosomal
localization studies, Susannah White for typing the manuscript, Amy
Corder and John Carroll for preparation of the figures, and Gary Howard
for editorial assistance.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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