(Received for publication, December 6, 1994; and in revised form, January 9, 1995)
From the
We have examined the ligand specificity and signal transduction
pathways of a recently cloned receptor for monocyte chemoattractant
protein-1 (MCP-1). In human 293 cells stably transfected with the MCP-1
receptor, MCP-1 bound specifically with high affinity (K = 260 pM) and induced
a rapid mobilization of calcium from intracellular stores. The closely
related chemokines MIP-1
, MIP-1
, RANTES, interleukin 8
(IL-8), and Gro-
were inactive at concentrations as high as 300
nM. Activation of the MCP-1 receptor potently inhibited
adenylyl cyclase with an IC
= 90 pM.
Activation of the MIP-1
/RANTES receptor also mediated inhibition
of adenylyl cyclase activity but with a different pharmacological
profile: MIP-1
(110 pM, IC
), RANTES (140
pM), MIP-1
(10 nM), and MCP-1 (820 nM).
Mobilization of intracellular calcium and inhibition of adenylyl
cyclase were blocked by pertussis toxin, suggesting that the MCP-1
receptor coupled to G
i. These results demonstrate that the MCP-1
receptor binds and signals in response to picomolar concentrations of
MCP-1 in a highly specific manner. Signaling was manifested as
mobilization of intracellular calcium and inhibition of adenylyl
cyclase and was mediated by a pertussis toxin-sensitive G-protein(s).
The molecular basis for the selective recruitment of monocytes
into sites of inflammation and early atherosclerotic lesions is
incompletely understood, but may involve locally generated cytokines
that mediate leukocyte chemotaxis and binding. Monocyte chemoattractant
protein 1 (MCP-1), ()is a potent monocyte agonist (1, 2) and is a member of a rapidly growing family of
chemotactic cytokines known as the
chemokines(3, 4, 5, 6) . The
chemokine family can be divided into two subfamilies, based on the
arrangement of the first 2 of 4 conserved cysteines. In the
, or
C-X-C subfamily, these two cysteines are separated by 1 amino
acid, whereas in the
, or C-C branch, they are adjacent. The
chemokines form dimers in solution, and while the structure of the
monomeric form of the
- and
-chemokines is quite
similar(7) , the quarternary structures of the
- and
-dimers are quite different(8) . Interleukin 8 (IL-8) and
Gro-
are examples of C-X-C branch chemokines, and MCP-1,
RANTES (regulated on activation, normal T expressed and secreted), and
macrophage inflammatory protein 1
and 1
(MIP-1
,
MIP-1
) are C-C chemokines. MCP-2 and MCP-3 are recently described
homologs of MCP-1 and are also potent monocyte
chemoattractants(9, 10) . In general, chemokines in
the C-X-C family are neutrophil-specific, whereas C-C
chemokines are monocyte-specific agonists. Recent data indicate that T
lymphocytes of the memory phenotype (CD45RO
) also
undergo chemotaxis in response to MCP-1, indicating a possible role for
MCP-1 in cell-mediated immunity(11) .
MCP-1 induces monocyte chemotaxis at subnanomolar concentrations and also activates host defense mechanisms such as superoxide production (12) and the oxidative burst(13) . MCP-1 also up-regulates the adhesion molecule Mac-1 (CD11b/CD11c)(14) , and this up-regulation may contribute to the tissue extravasation of monocytes at sites of inflammation. It is unclear how MCP-1 and other chemokines induce chemotaxis and activation of adhesion receptors; and this question constitutes an important area of investigation in leukocyte biology.
We have recently cloned two alternatively spliced
seven-transmembrane-domain receptors that mediate MCP-1dependent
calcium mobilization in Xenopus oocytes(15) . In the
present study, we examined the ligand specificity and signal
transduction pathways of one of these MCP-1 receptors and compared it
with the recently cloned receptor for MIP-1 and RANTES. Our
results demonstrate that the MCP-1 receptor binds and signals in
response to picomolar concentrations of MCP-1 in a highly specific
manner. Signaling in 293 cells is manifested as both calcium
mobilization and inhibition of adenylyl cyclase and is mediated via
activation of a pertussis toxin (PT)-sensitive G-protein(s).
where R and R
represent the fluorescence ratio under saturating (1.3 mM Ca
) and nominally free (10 mM EGTA)
calcium conditions, K
is the dissociation constant
of calcium for indo-1, R is the fluorescence ratio, and Sf2/Sb2 is the fluorescence ratio of free and bound indo-1 dye
at 410 nm(20) . For quantitation of the calcium responses, full
MCP-1 dose-response curves were generated in each experiment and the
results were expressed as a percent of the maximum calcium signal (at
300 nM MCP-1) measured in that experiment. The changes in
[Ca
]
levels in response to each
concentration of agonist were determined by subtracting the base line
from peak [Ca
]
levels, which
were determined by averaging 5 s of data prior to agonist addition and
surrounding the peak response, respectively. In experiments done to
determine the role of extracellular calcium, 3 mM EGTA was
added 60-90 s prior to MCP-1. Subsequent lysis of the cells with
Triton X-100 caused no change in indo-1 fluorescence, indicating that
EGTA had reduced the extracellular calcium concentration below that of
intracellular basal levels (approximately 70-100 nM).
All experiments were performed at room temperature, and each data point
was determined in duplicate.
where n and EC represent the Hill
coefficient and the agonist concentration that elicited a half-maximal
response, respectively, and were derived from the fitted curve. Curve
fitting was done with the computer program ``Prism'' (by
Graph Pad, San Diego, CA). All results shown represent the mean
± S.E. All data points were determined in duplicate. The 95%
confidence intervals (CI) of the EC
and IC
values, when given, were calculated from the log EC
and IC
values, respectively.
Figure 1:
Binding of I-MCP-1 to the recombinant MCP-1RB receptor. HEK-293
cells stably transfected with MCP-1RB were incubated with 500 pM
I-labeled MCP-1 and the indicated concentrations of
unlabeled MCP-1, MIP-1
, MIP-1
, RANTES, or IL-8, as described
under ``Materials and Methods.'' A, competition. B, Scatchard analysis. The calculated dissociation constant (K
) is 260 pM. All data points
were determined in triplicate, and error bars represent
standard deviations. Data shown are representative of four
experiments.
Figure 2:
MCP-1RB receptor-mediated calcium
mobilization. Stably transfected 293 cells were loaded with indo-1 AM,
and intracellular calcium levels were measured as described under
``Materials and Methods.'' A, intracellular calcium
flux as a function of MCP-1 concentration (nM). Calcium
transients peaked at 4-8 s after addition of MCP-1 and returned
to base line within 90 s of activation. B, MCP-1 stimulated
calcium mobilization with an EC of 3.4 nM (2.7-4.4). MIP-1
, MIP-1
, RANTES, IL-8, and
Gro-
had no appreciable effect on calcium mobilization (n = 2-3). The average maximal peak calcium
concentration was 673 ± 13 nM. Results are the mean
± S.E. of four separate experiments and are expressed as a
percent of the maximal calcium response to MCP-1. C, MCP-1
desensitized the cells to a second addition of
MCP-1.
Figure 3: MCP-1RB mobilizes intracellular calcium. MCP-1RB stably transfected 293 cells were loaded with indo-1 AM, and changes in intracellular calcium concentrations in response to MCP-1 (100 nM) were measured as described in Fig. 1. EGTA (3 mM) was added to the cuvette 60-90 s prior to the addition of MCP-1. The results shown are representative traces from one of four experiments in the absence and eight experiments in the presence of EGTA.
Figure 4:
MCP-1RB and the MIP-1/RANTES receptor
mediate inhibition of adenylyl cyclase. HEK-293 cells expressing
MCP-1RB (4, A and B) or the MIP-1
/RANTES (C) receptor were labeled with
[
H]adenine and stimulated with 10 µM forskolin in the presence or absence of chemokines.
[
H]cAMP pools were measured as described under
``Materials and Methods.'' A, cAMP accumulation in
MCP-1RB transfected cells. MCP-1(100 nM) inhibited basal cAMP
accumulation by 55 ± 4.3%. Forskolin stimulated a 16.5 ±
2.1-fold increase in cAMP accumulation over untreated cells, and this
was blocked by 78.4 ± 1.8% by MCP-1. The inhibition of cAMP
accumulation was significant at p < 0.01, in both cases. B, inhibition of adenylyl cyclase by MCP-1RB. The IC
for MCP-1 was 90 pM (66-143 pM).
MIP-1
, MIP-1
, RANTES, IL-8, and Gro-
were inactive at
doses up to 100 nM. C, the MIP-1
/RANTES receptor
mediates inhibition of adenylyl cyclase in transfected 293 cells.
MIP-1
and RANTES blocked the forskolin-stimulated accumulation of
cAMP by 52.3 ± 2% and 54.9 ± 2%, respectively. The
calculated IC
values were MIP-1
= 110 pM (80-160 pM), RANTES = 140 pM (90-200 pM), MIP-1
= 10 nM (4-30 nM), and MCP-1 = 820 nM. IL-8
and Gro-
did not inhibit adenylyl cyclase at up to 1
µM. The results shown are the mean ± S.E. of three
separate experiments. Each data point was determined in duplicate.
Where no S.E. bars are shown, they are smaller than the symbol
size.
In similar experiments the
MIP-1/RANTES receptor (30, 31) was stably
transfected into 293 cells and also found to mediate potent and
dose-dependent inhibition of adenylyl cyclase activity (Fig. 4C). Unlike the MCP-1RB receptor, however, the
MIP-1
/RANTES receptor was activated by multiple chemokines with
varying degrees of potency. MIP-1
and RANTES were virtually
equipotent in inhibiting adenylyl cyclase activity with IC
values of 110 and 140 pM, respectively. MIP-1
(IC
= 10 nM) and MCP-1 (IC
= 820 nM) also inhibited adenylyl cyclase
activity, though only at much higher concentrations, and neither
blocked cAMP accumulation to the same extent as MIP-1
and RANTES.
The C-X-C chemokines IL-8 and Gro-
did not activate the
MIP-1
/RANTES receptor.
Table 1compares the activation of
the MCP-1 receptor and the MIP-1/RANTES receptor by a variety of
chemokines and demonstrates the specificity of the MCP-1RB receptor for
MCP-1, and the MIP-1
/RANTES receptor for MIP-1
and RANTES.
Neither of the C-X-C chemokines was active on either of the
two cloned C-C chemokine receptors.
Figure 5:
Pertussis toxin inhibits MCP-1RB
signaling. MCP-1RB stably transfected 293 cells were incubated
overnight (16 h) with PT. Cells were loaded with indo-1 AM for calcium
fluorimetry (A) or labeled with
[H]adenine for adenylyl cyclase assays (B), as described under ``Materials and Methods.'' A, the peak [Ca
]
flux in response to 100 nM MCP-1 was reduced to 21
± 5% of control by PT. B, inhibition of adenylyl
cyclase by MCP-1 was blocked by PT in a dose-dependent
manner.
Figure 6: Inhibition of calcium mobilization and adenylyl cyclase by pertussis toxin. HEK-293 cells stably expressing MCP-1RB were activated by 100 nM MCP-1 in the presence of the indicated concentrations of PT. Calcium mobilization and adenylyl cyclase activity were equally blocked by increasing concentrations of PT. Approximately 80% inhibition was achieved with 1 ng/ml of PT, and 20% of each response was resistant to 100 ng/ml of PT. Results shown are the mean ± S.E. for three experiments. Each data point was determined in duplicate. Where no S.E. bars are shown, they are smaller than the symbol.
We have previously described two alternatively spliced forms
of the MCP-1 receptor, designated MCP-1RA and MCP-1RB, which differ
only in their carboxyl-terminal tails(15) . Each of these
receptors confers comparable MCP-1-dependent signaling when
microinjected into Xenopus oocytes. In this paper, we report
the functional expression of one of these receptors, MCP-1RB, in stably
transfected HEK-293 cells. The cloned receptor binds and signals in
response to subnanomolar concentrations of MCP-1 in a highly specific
manner. Signaling is mediated by one or more pertussis toxin-sensitive
G-proteins, most likely Gi, and is manifested by a rapid rise in
cytoplasmic calcium and potent inhibition of adenylyl cyclase.
Qualitatively similar signaling was observed in 293 cells expressing
MCP-1RA. These studies, the first to demonstrate the ligand specificity
and signal transduction pathways of the cloned MCP-1 receptor in
mammalian cells, provide strong support for the identification of
MCP-1RB as a high-affinity, specific receptor for MCP-1.
MCP-1
induced a rapid rise in intracellular calcium in indo-1-loaded 293
cells that were stably transfected with MCP-1RB. The kinetics of this
response were similar to those seen with MCP-1 activation of
monocytes(27) , THP-1 cells(26) , and MonoMac 6
cells(15) . The stable cell line also demonstrated
dose-dependent homologous desensitization of calcium mobilization in
response to MCP-1, which is consistent with published data on the
response of monocytes (13) and MonoMac 6 cells (15) to
MCP-1. The relative contributions of extracellular and intracellular
calcium stores to this calcium flux has been controversial. Using
fura-2-loaded human monocytes, Sozzani et al.(27, 32) reported that extracellular calcium was
required to detect calcium fluxes in response to MCP-1. More recently
these investigators have found that examination of adherent, single
monocytes using morphological techniques indicates significant
mobilization of intracellular calcium. ()In the present
study, several lines of evidence support the conclusion that the
initial rise in cytoplasmic calcium after activation of the MCP-1
receptor in 293 cells is almost exclusively due to the release of
intracellular calcium stores. First, chelation of extracellular calcium
with EGTA (3 mM to 10 mM) had little effect on the
rise and peak levels of the calcium transients, but did hasten the
return to base-line calcium levels. Second, the same result was
obtained when the transfected cells were incubated in calcium-free
media, supplemented with 1 mM EGTA. Finally, virtually
identical results were obtained in the presence of 5 mM Ni
, which blocks the influx of extracellular
calcium (28) . We conclude, therefore, that when transfected
into 293 cells the MCP-1 receptor mobilizes calcium primarily from
intracellular stores.
Seven-transmembrane-domain receptors couple
via heterotrimeric G-proteins to effect a wide spectrum of cellular
responses, and so it was of interest to determine the coupling
mechanism(s) of the MCP-1 receptor. Activation of the receptor led to
profound inhibition of adenylyl cyclase, suggesting coupling via one of
the isoforms of Gi [see (33) for a review of
G-protein coupling]. Similar results were obtained using the
cloned MIP-1
/RANTES receptor, indicating that at least two of the
receptors for C-C chemokines activate G
i. Moreover, pertussis
toxin blocked both the calcium mobilization as well as the inhibition
of adenylyl cyclase induced by MCP-1. The similarity in the pertussis
toxin dose-response curves for calcium mobilization and inhibition of
adenylyl cyclase suggests that both may be downstream consequences of
coupling to G
i. These studies are the first demonstration of
adenylyl cyclase inhibition by chemokine receptors, and are consistent
with reports that leukocyte chemotaxis to IL-8(3) ,
fMLP(34) , and MCP-1 (27) is sensitive to inhibition by
pertussis toxin.
The downstream effects of activation of Gi in
leukocytes are not well understood. Although inhibition of adenylyl
cyclase is the most thoroughly characterized effect, G
i has also
been implicated in the activation of potassium channels(35) ,
as well as in the induction of mitosis(36) . Recent studies by
Worthen et al.(37) have demonstrated a
G
i-dependent activation of Ras and microtubule-associated protein
kinase in fMLP stimulated neutrophils. Thus, activation of G
i may
activate a complex array of intracellular signals that ultimately lead
to leukocyte activation and chemotaxis.
Studies of the IL-8 receptor
by Wu et al.(38) have described a pertussis
toxin-sensitive signal transduction pathway in which dimers,
released in conjunction with G
i, activate the
isoform of phospholipase C to generate inositol (1, 4, 5) -triphosphate. Cellular activation
via this pathway would be expected to result in a pertussis
toxin-sensitive mobilization of intracellular calcium. We have found,
however, that 293 cells stably expressing the recombinant MCP-1
receptor hydrolyze little, if any, PI when challenged with MCP-1. In
control experiments, we demonstrated that Gq-coupled receptors,
co-transfected into this cell line, increased total inositol phosphates
5-9-fold upon activation. The failure to detect PI turnover in
the MCP-1RB transfected cells, as well as in freshly isolated human
monocytes(32) , suggests that the MCP-1 receptor may mobilize
intracellular calcium via a novel mechanism that is independent of
inositol(1, 4, 5) -triphosphate.
MCP-1RB
was remarkably specific for MCP-1. In the cyclase assay the IC for inhibition by MCP-1 was 90 pM, whereas closely
related chemokines were ineffective at up to 1 µM. In
contrast, the MIP-1
/RANTES receptor had an IC
of
approximately 100 pM for MIP-1
and RANTES, and 10 and 820
nM for MIP-1
and MCP-1, respectively. Thus, MCP-1 had a
selectivity of at least 9000-fold for the MCP-1 receptor, whereas
MIP-1
and RANTES had a similar preference for the
MIP-1
/RANTES receptor, as compared to MCP-1RB. It is likely,
therefore, that under physiological conditions, MCP-1, MIP-1
, and
RANTES act as specific agonists of MCP-1RB and the MIP-1
/RANTES
receptor, respectively. Although our data suggest that MCP-1 is the
sole agonist for MCP-1RB, preliminary studies indicate that MCP-3, a
very closely related chemokine(10) , is also a potent agonist
of both the ``A'' and ``B'' forms of the MCP-1
receptor. (
)
The IC for MCP-1-mediated
inhibition of adenylyl cyclase was approximately 90 pM, which
is well below the dissociation constant for binding (K
= 260 pM) and suggests that relatively few
receptors must be occupied for efficient coupling to G
i. In
contrast, very high receptor occupancy was required to elicit peak
intracellular calcium fluxes (EC
= 2-4
nM). It is interesting to note, in this regard, that the
EC
for monocyte chemotaxis to MCP-1 is
subnanomolar(2) . Thus, the induction of chemotaxis, which is
the hallmark function of MCP-1, is optimal at MCP-1 concentrations that
provide for efficient coupling/signaling through G
i but are
insufficient to elicit maximal intracellular calcium fluxes and
subsequent receptor desensitization. This suggests that modest
increases in intracellular calcium are sufficient to initiate and
support monocyte chemotaxis. The high levels of intracellular calcium
detected at nanomolar concentrations of MCP-1 may serve to stop
monocyte migration by desensitizing the receptor and up-regulating
adhesion molecules(13) .
In considering possible mechanisms
for providing specificity in leukocyte responses, it is interesting to
note that MCP-1 is synthesized and secreted in vitro by a
number of different cells in response to a variety of different
cytokines (39) or oxidatively modified
lipoproteins(40) . The remarkable specificity of the cloned
receptor for MCP-1, coupled with the fact that only monocytes,
basophils, and a subset of T lymphocytes respond to MCP-1, provides for
an effective means of limiting the spectrum of infiltrating leukocytes
in areas where MCP-1 is abundant. Consistent with this notion are the
observations that early atherosclerotic lesions have a predominately
monocytic infiltrate(41) , and that MCP-1 is abundant in these
lesions(42, 43) . In contrast, the MIP-1/RANTES
receptor binds and signals in response to multiple chemokines and may
serve to mediate more complex inflammatory reactions. Once activated,
however, the MCP-1 and MIP-1
/RANTES receptors appear to use
similar signal transduction pathways.
In summary, these data are the
first pharmacological and signal transduction studies of the cloned
MCP-1 receptor in mammalian cells. MCP-1RB signals in response to MCP-1
in a highly specific manner and mediates the release of intracellular
calcium in a pertussis toxin-sensitive manner. Activation of MCP-1RB,
as well as the MIP-1/RANTES receptor, leads to a dose-dependent
inhibition of adenylyl cyclase, which is consistent with the hypothesis
that the C-C chemokine receptors couple to G
i. Preliminary data
indicate that MCP-1RA also couples via G
i to raise cytoplasmic
calcium, and studies are in progress comparing the kinetics and MCP-1
dose-response curves of MCP-1RA and MCP-1RB in 293 cells. The
downstream effects of G
i, or other second messengers that
ultimately lead to chemotaxis in leukocytes, are unknown, but the
availability of stable mammalian cell lines that express these
receptors in a functional state provides a powerful model system for
addressing these questions.