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Volume 270, Number 11, Issue of March 17, 1995 pp. 5786-5792
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Signal Transduction and Ligand Specificity of the Human Monocyte Chemoattractant Protein-1 Receptor in Transfected Embryonic Kidney Cells (*)

(Received for publication, December 6, 1994; and in revised form, January 9, 1995)

Scott J. Myers (1), (§),  Lu Min Wong (1),  (3),  Israel F. Charo (1) (3)(¶) (2)

From the  (1)Gladstone Institute of Cardiovascular Disease, San Francisco, California 94141-9100 and the (2)Department of Medicine and (3)Daiichi Research Center, University of California, San Francisco, California 94143

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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-1alpha, MIP-1beta, RANTES, interleukin 8 (IL-8), and Gro-alpha 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-1alpha/RANTES receptor also mediated inhibition of adenylyl cyclase activity but with a different pharmacological profile: MIP-1alpha (110 pM, IC), RANTES (140 pM), MIP-1beta (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 Galphai. 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).

INTRODUCTION

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), (^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 alpha, or C-X-C subfamily, these two cysteines are separated by 1 amino acid, whereas in the beta, or C-C branch, they are adjacent. The chemokines form dimers in solution, and while the structure of the monomeric form of the alpha- and beta-chemokines is quite similar(7) , the quarternary structures of the alpha- and beta-dimers are quite different(8) . Interleukin 8 (IL-8) and Gro-alpha are examples of C-X-C branch chemokines, and MCP-1, RANTES (regulated on activation, normal T expressed and secreted), and macrophage inflammatory protein 1alpha and 1beta (MIP-1alpha, MIP-1beta) 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-1alpha 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).

MATERIALS AND METHODS

Reagents

The chemokines MCP-1, MIP-1alpha, MIP-1beta, RANTES, IL-8, and Gro-alpha were obtained from R& Systems, Inc. (Minneapolis, MN). Indo-1 AM was purchased from Molecular Probes, Inc. (Eugene, OR). Pertussis toxin was purchased from List Biological Laboratories, Inc. (Campbell, CA). Lipofectamine, G418 sulfate, and MEM with Earle's balanced salt solution (MEM-EBSS) were obtained from Life Technologies, Inc. Fetal calf serum was obtained from Hyclone Laboratories, Inc. (Logan, UT). [2,8-^3H]Adenine and myo-[2-^3H]inositol were obtained from Du Pont NEN. Alumina, the Dowex resins 50W (200-400 mesh, hydrogen form) and AG1-X8 (100-200 mesh, formate form), 3-isobutyl-1-methylxanthine, forskolin, EGTA, Triton X-100, dimethyl sulfoxide (Me(2)SO), imidazole, sodium azide, trichloroacetic acid, bovine serum albumin (BSA; fraction V), cAMP, and ATP were purchased from Sigma. The human M1 muscarinic receptor (16) was a generous gift of Dr. Wolfgang Sadée, University of California, San Francisco, and the oxytocin receptor (17) was a generous gift of Dr. Michael Brownstein of the National Institutes of Health, Bethesda, MD. The expression vector pcDNA3 was purchased from Invitrogen Inc. (San Diego, CA). Restriction enzymes were purchased from Boehringer Mannheim.

Tissue Culture and Stable Transfections

Human embryo kidney (HEK)-293 (CRL 1573) cells were obtained from the American Type Culture Collection (Bethesda, MD) and were grown in minimal essential media with EBSS supplemented with 10% fetal calf serum and 1% penicillin/streptomycin, at 37 °C in a humidified 5% CO(2) atmosphere. cDNAs for the MCP-1 receptor (MCP-1RB) and the MIP-1alpha/RANTES receptor were cloned into the polylinker of the mammalian cell expression vector pcDNA3 (Invitrogen) and transfected into 293 cells (50-80% confluent) with a DNA/Lipofectamine (Life Technologies, Inc.) mixture according to the manufacturer's instructions. After selection for 2-3 weeks in the presence of G418 (0.8 mg/ml), colonies were picked and stable cell lines were screened by Northern blot analysis for receptor expression. In general, there was a strong correlation between the level of receptor expression as judged by Northern blot analysis and the strength of the receptor signals obtained in the functional assays. Transfected cells that failed to express the receptor on Northern blots were used as negative controls in the binding and signaling experiments.

Binding Assays

Equilibrium binding assays were performed using the method of Ernst et al.(18) . Briefly, varying amounts of I-labeled MCP-1 (DuPont NEN) were incubated with 6 times 10^6 cells resuspended in binding buffer (50 mM HEPES, pH 7.2, 1 mM CaCl(2), 5 mM MgCl(2), 0.5% BSA) in the presence or absence of 100-fold excess of unlabeled chemokines. Competition experiments were performed using 500 pMI-labeled MCP-1 and the indicated concentrations of unlabeled chemokines. Each data point was determined in triplicate. Equilibrium binding data were analyzed according to the method of Scatchard using the program ``LIGAND'' (19) (Biosoft, Ferguson, MO) on a Macintosh computer.

Calcium Fluorimetry

Transfected HEK-293 cells were grown until confluent, trypsinized briefly, washed with phosphate-buffered saline containing 1 mg/ml BSA (PBS-BSA), and resuspended in serum-free MEM-EBSS supplemented with 1 mg/ml BSA and 10 mM HEPES (pH 7.0) at a density of 2 times 10^7 cells/ml. The cells were incubated in the dark at 37 °C for 20 min in the presence of 5-10 µg/ml indo-1 AM. Nine volumes of PBS-BSA were added, and the cells were incubated for an additional 10 min at 37 °C, pelleted by centrifugation, and washed twice with 50 ml of the PBS-BSA solution. Washed, indo-1-loaded cells were then resuspended in Hank's balanced salt solution (1.3 mM Ca) supplemented with 1 mg/ml BSA at a density of approximately 0.5 times 10^6 cells/ml at room temperature. To measure intracellular calcium ([Ca](i)), 0.5 ml of the cell suspension was placed in a quartz cuvette in a Hitachi F-2000 fluorescence spectrophotometer. Chemokines dissolved in Hank's balanced salt solution-BSA were injected directly into the cuvette in 5 µl volumes. Intracellular calcium was measured by excitation at 350 nm and fluorescence emission detection at 490 nm (F1) and 410 nm (F2) wavelengths. The [Ca](i) was estimated by comparing the 490/410 fluorescence ratio after agonist application (R) to that of calibration ratios measured at the end of each run, according to the equation:

where R(max) and R(min) represent the fluorescence ratio under saturating (1.3 mM Ca) and nominally free (10 mM EGTA) calcium conditions, K(d) 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](i) levels in response to each concentration of agonist were determined by subtracting the base line from peak [Ca](i) 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.

Adenylyl Cyclase Assays

HEK-293 cells stably transfected with the MCP-1RB and the MIP-1alpha/RANTES receptors were grown until confluent in 24-well tissue culture dishes and labeled overnight with 2 µCi/ml of [^3H]adenine (25-30 Ci/mmol) in MEM-EBSS supplemented with 10% fetal calf serum. The next day, the cells were washed by incubation at room temperature with 0.5 ml of serum-free MEM-EBSS media supplemented with 10 mM HEPES, 1 mg/ml BSA, and 1 mM 3-isobutyl-1-methylxanthine for 5 min. After removal of the wash media, the cells were stimulated by addition of fresh media containing either chemokine alone, forskolin alone (10 µM), or chemokine plus forskolin, all in the presence of 1 mM 3-isobutyl-1-methylxanthine, for 20 min at room temperature. The incubation was terminated by replacement of the media with 1 ml of ice-cold 5% trichloroacetic acid, 1 mM cAMP, and 1 mM ATP. Following incubation at 4 °C for 30 min, the labeled [^3H]ATP and [^3H]cAMP pools were separated and quantitated by chromatography on Dowex 50W and neutral alumina columns, essentially as described(21, 22) . The 1-ml acid supernatant was loaded onto a 1-ml Dowex 50W column and the ATP pool eluted with 3 ml of H(2)O. The Dowex 50W columns were then placed over 1-ml alumina columns, and 10 ml of H(2)O was added to the Dowex resin and the eluant allowed to drip directly onto the neutral alumina. The cAMP pool was then eluted directly from the alumina with 5 ml of 0.1 M imidazole, 0.01 mM sodium azide. The [^3H]ATP and [^3H]cAMP fractions were counted by liquid scintillation spectroscopy. The cAMP pool for each sample was normalized to its own ATP pool and expressed as a ratio by the equation (cAMP counts/min/ATP counts/min) times 100. In each experiment full dose-response curves were generated and expressed as a percent of the forskolin control. All data points were determined in duplicate. In all experiments, the maximum inhibition of adenylyl cyclase activity mediated by the MCP-1RB or MIP-1alpha/RANTES receptor was 80 and 55%, respectively.

Phospholipase C Assays

Total inositol phosphate accumulation was determined essentially as described(23) . HEK-293 cells were grown until confluent in 24-well tissue culture dishes and labeled overnight with 2 µCi/ml [^3H]myo-inositol (23 Ci/mmol) in inositol-free MEM-EBSS supplemented with 10% dialyzed fetal calf serum. Following labeling, the media were removed, and the cells were incubated at room temperature for 5-10 min in 0.5 ml of serum-free MEM-EBSS media supplemented with 10 mM HEPES, 1 mg/ml BSA, and 10 mM LiCl. The washed cells were then incubated with chemokines for 1-30 min at room temperature in the presence of 10 mM LiCl. The incubation was terminated by removal of the incubation media and addition of 1 ml of ice-cold 20 mM formic acid. Plates were incubated at 4 °C for 30 min before the supernatants were applied to 1-ml Dowex AG1-X8 chromatography columns. Columns were washed with 8 ml of water followed by 5 ml of 40 mM sodium formate. Total [^3H]inositol phosphates were eluted with 5 ml of 2 M ammonium formate, 0.1 M formic acid and quantitated by liquid scintillation spectroscopy.

Pertussis Toxin Treatment

Pertussis toxin was dissolved in 0.01 M sodium phosphate (pH 7.0), 0.05 M sodium chloride and diluted into normal serum containing media at final concentrations of 0.1-100 ng/ml and incubated with cells overnight (14-16 h) at 37 °C. The conditions of the PT treatment of the 293 cells were identical for calcium fluorimetric and adenylyl cyclase experiments. In the adenylyl cyclase experiments, the PT was added at the same time as [^3H]adenine.

Analysis of Data

Full dose-response curves were generated in both the calcium fluorimetric assays and inhibition of adenylyl cyclase assays. All dose-response curves were then fit by a nonlinear least-squares program to the logistic equation:

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.

RESULTS

Binding of MCP-1 to MCP-1RB/293 Cells

A cell line stably expressing the MCP-1 receptor was produced by transfection of MCP-1RB into HEK-293 cells. Transfected cells bound I-labeled MCP-1 specifically and with high affinity (Fig. 1A). The closely related C-C chemokines MIP-1alpha, MIP-1beta, and RANTES, as well as the C-X-C chemokine IL-8 did not compete for binding. No specific binding was detected using transfectants that expressed little or no MCP-1RB on Northern blots (data not shown). Analysis of equilibrium binding data indicated a dissociation constant (K(d)) of 260 pM (Fig. 1B). This K(d) is in good agreement with that reported for the binding of MCP-1 to monocytes (2, 24) and THP-1 cells(25) . These data indicate that I-MCP-1 bound specifically and with high affinity to the MCP-1RB receptor expressed in 293 cells.

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 pMI-labeled MCP-1 and the indicated concentrations of unlabeled MCP-1, MIP-1alpha, MIP-1beta, 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.


Calcium Mobilization by MCP-1

MCP-1 stimulated robust calcium mobilization in the stably transfected MCP-1RB/293 cells in a specific and dose-dependent manner. Small but reproducible signals were seen with as little as 100 pM MCP-1, and the average EC from four full dose-response curves to MCP-1 was 3.4 nM (2.7-4.4 nM; Fig. 2, A and B). The MCP-1RB receptor was selectively activated by MCP-1 in that RANTES, MIP-1alpha, MIP-1beta, Gro-alpha, and IL-8 failed to stimulate significant calcium signals in these same cells, even when present at high concentrations (Fig. 2B). Furthermore, these chemokines also failed to block stimulation of the cells by MCP-1, indicating that they are unlikely to act as endogenous antagonists of the MCP-1RB receptor (data not shown). The MCP-1-dependent intracellular calcium fluxes were characterized by short lag times, followed by a rapid rise in [Ca](i) that returned to near basal levels within 80-90 s of the addition of MCP-1 (Fig. 2A). The cells demonstrated homologous desensitization in that they were refractory to activation by a second challenge with MCP-1 (Fig. 2C). These pharmacological, kinetic, and desensitization properties of MCP-1-stimulated calcium mobilization in the MCP-1RB/293 cells are similar to those reported previously in monocytes (25) and monocyte-like cell lines(15, 26) .

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-1alpha, MIP-1beta, RANTES, IL-8, and Gro-alpha 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.


Source of Calcium Mobilized

To determine the source of the intracellular calcium flux, the MCP-1RB/293 cells were challenged with MCP-1 in the presence or absence of extracellular calcium. The rise in cytoplasmic calcium was largely unchanged by the chelation of extracellular calcium with 3 mM EGTA (Fig. 3). Similar results were seen when the cells were washed and resuspended in calcium-free PBS supplemented with 1 mM EGTA, or when 5 mM Ni was added to the cuvette to block the influx of extracellular calcium (27, 28) (data not shown). The fall in cytoplasmic calcium to base line was slightly prolonged in the presence of extracellular calcium, suggesting that calcium influx may contribute to maintaining the response to MCP-1 after intracellular stores are depleted. These data suggest that the primary means of calcium mobilization in these transfected 293 cells is through release of intracellular calcium. Inositol(1, 4, 5) -triphosphate mobilizes intracellular calcium in response to activation of a wide spectrum of receptors, including many seven-transmembrane-domain receptors(23, 29) . Activation of the MCP-1 receptor in transfected 293 cells, however, induced little or no hydrolysis of phosphatidyl inositol (data not shown). In control experiments activation of the muscarinic or oxytocin receptor, co-transfected into these same 293 cells, led to a 5-9-fold increase in PI turnover.

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.


Adenylyl Cyclase Inhibition

Activation of the MCP-1 receptor resulted in a potent and dose-dependent inhibition of adenylyl cyclase activity. MCP-1 significantly reduced basal cAMP accumulation in these cells by 55% (p < 0.01, Student's t test) (Fig. 4A). Forskolin activation of adenylyl cyclase increased cAMP levels 16-fold, and co-addition of MCP-1 blocked this increase by 78%, with an IC of 90 pM (70-140 pM) (Fig. 4, A and B). The magnitude and potency of MCP-1 inhibition of adenylyl cyclase activity was independent of the forskolin concentration (3-30 µM; data not shown). MCP-1 neither stimulated nor inhibited cAMP formation in untransfected or pcDNA3 transfected 293 cell controls (data not shown). Together these results demonstrate that inhibition of adenylyl cyclase activity provides a sensitive and quantitative assay for MCP-1RB receptor activation in 293 cells. Virtually no activation of the MCP-1 receptor could be detected in this assay in response to high concentrations of RANTES, MIP-1alpha, MIP-1beta, IL-8, or Gro-alpha (Fig. 4B), which is consistent with our observations in the calcium fluorimetric assay (Fig. 2B) and in Xenopus oocytes(15) .

Figure 4: MCP-1RB and the MIP-1alpha/RANTES receptor mediate inhibition of adenylyl cyclase. HEK-293 cells expressing MCP-1RB (4, A and B) or the MIP-1alpha/RANTES (C) receptor were labeled with [^3H]adenine and stimulated with 10 µM forskolin in the presence or absence of chemokines. [^3H]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-1alpha, MIP-1beta, RANTES, IL-8, and Gro-alpha were inactive at doses up to 100 nM. C, the MIP-1alpha/RANTES receptor mediates inhibition of adenylyl cyclase in transfected 293 cells. MIP-1alpha and RANTES blocked the forskolin-stimulated accumulation of cAMP by 52.3 ± 2% and 54.9 ± 2%, respectively. The calculated IC values were MIP-1alpha = 110 pM (80-160 pM), RANTES = 140 pM (90-200 pM), MIP-1beta = 10 nM (4-30 nM), and MCP-1 = 820 nM. IL-8 and Gro-alpha 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-1alpha/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-1alpha/RANTES receptor was activated by multiple chemokines with varying degrees of potency. MIP-1alpha and RANTES were virtually equipotent in inhibiting adenylyl cyclase activity with IC values of 110 and 140 pM, respectively. MIP-1beta (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-1alpha and RANTES. The C-X-C chemokines IL-8 and Gro-alpha did not activate the MIP-1alpha/RANTES receptor.

Table 1compares the activation of the MCP-1 receptor and the MIP-1alpha/RANTES receptor by a variety of chemokines and demonstrates the specificity of the MCP-1RB receptor for MCP-1, and the MIP-1alpha/RANTES receptor for MIP-1alpha and RANTES. Neither of the C-X-C chemokines was active on either of the two cloned C-C chemokine receptors.

Inhibition of MCP-1 Receptor Activation by Pertussis Toxin

The MCP-1-induced mobilization of intracellular calcium, as well as the inhibition of adenylyl cyclase, was substantially blocked by pretreatment of cells with bordetella pertussis toxin (PT, Fig. 5, A and B). Dose-response studies indicated a similar degree of inhibition of these two pathways by pertussis toxin, as well as a component (approx20%) that was resistant to inhibition by up to 100 ng/ml of PT (Fig. 6). The effect of PT treatment was to reduce the magnitude of the MCP-1 inhibition of cAMP accumulation without significantly shifting the MCP-1 IC (Fig. 5B), a result consistent with the hypothesis that PT treatment functionally uncouples the MCP-1RB receptor from Galphai. These results also suggest that both the inhibition of adenylyl cyclase activity and the mobilization of intracellular calcium may be mediated through activation of the same G-protein in the 293 cells.

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 [^3H]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.


DISCUSSION

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 Galphai, 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. (^2)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 Galphai [see (33) for a review of G-protein coupling]. Similar results were obtained using the cloned MIP-1alpha/RANTES receptor, indicating that at least two of the receptors for C-C chemokines activate Galphai. 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 Galphai. 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 Galphai in leukocytes are not well understood. Although inhibition of adenylyl cyclase is the most thoroughly characterized effect, Galphai 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 Galphai-dependent activation of Ras and microtubule-associated protein kinase in fMLP stimulated neutrophils. Thus, activation of Galphai 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 beta dimers, released in conjunction with Galphai, activate the beta(2) 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-1alpha/RANTES receptor had an IC of approximately 100 pM for MIP-1alpha and RANTES, and 10 and 820 nM for MIP-1beta and MCP-1, respectively. Thus, MCP-1 had a selectivity of at least 9000-fold for the MCP-1 receptor, whereas MIP-1alpha and RANTES had a similar preference for the MIP-1alpha/RANTES receptor, as compared to MCP-1RB. It is likely, therefore, that under physiological conditions, MCP-1, MIP-1alpha, and RANTES act as specific agonists of MCP-1RB and the MIP-1alpha/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. (^3)

The IC for MCP-1-mediated inhibition of adenylyl cyclase was approximately 90 pM, which is well below the dissociation constant for binding (K(d) = 260 pM) and suggests that relatively few receptors must be occupied for efficient coupling to Galphai. 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 Galphai 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-1alpha/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-1alpha/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-1alpha/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 Galphai. Preliminary data indicate that MCP-1RA also couples via Galphai 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 Galphai, 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.

FOOTNOTES

*
This work was supported in part by the National Institutes of Health Grant HL52773 and the University of California TobaccoRelated Research Program 3RT-0354. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Pharmacology, Emory University, 1510 Clifton Rd., Atlanta, GA 30322-3090.

To whom correspondence should be addressed: Gladstone Institute of Cardiovascular Disease, P. O. Box 419100, San Francisco, CA 94141-9100. Tel.: 415-826-7500; Fax: 415-285-5632.

(^1)
The abbreviations used are: MCP-1, monocyte chemoattractant protein-1; IL, interleukin; RANTES, regulated on activation, normal T expressed and secreted; MIP, macrophage inflammatory protein; PT, pertussis toxin; MEM, minimal essential medium; EBSS, Earle's balanced salt solution; BSA, bovine serum albumin; PBS, phosphatebuffered saline.

(^2)
B. Bottazzi and S. Sozzani, unpublished results.

(^3)
I. F. Charo and J. Van Damme, unpublished observations.

ACKNOWLEDGEMENTS

We thank Susannah White for typing the manuscript; Marie Gipson, Amy Corder, and John Carroll for preparation of the figures; and Lewis DeSimone and Dawn Levy for editorial assistance.

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