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(Received for publication, April 10, 1996, and in revised form, June 25, 1996)
and
From the 
Gladstone Institute of Cardiovascular
Disease, the § Department of Medicine, and the
¶ Daiichi Research Center, University of California,
San Francisco, California 94141-9100
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
ABSTRACT 
Monocyte chemoattractant protein-1 (MCP-1) is a
member of a family of chemotactic cytokines that induce directed
migration of leukocytes via activation of seven-transmembrane domain
receptors. To identify G-proteins that couple to the two forms of the
MCP-1 receptor, as well as to related chemokine receptors, we have
performed cotransfection experiments in mammalian cells. In COS-7
cells, the type A and type B MCP-1 receptors coupled to
G
i, Gq, and G16, whereas
the macrophage inflammatory protein-1/RANTES (regulated on
activation, normal T cell-expressed and secreted) receptor (C-CR1)
coupled to Gi and Gq but failed to couple
to G16. In HEK-293 cells, however, the MCP-1 receptors
and C-CR1 coupled to Gq but failed to couple to
G16. In contrast, the interleukin-8 and C5a receptors
did not couple to Gq in either COS-7 or HEK-293 cells
but did couple to G16. Exchange of intracellular loops
between the MCP-1 and interleukin-8 receptors to create chimeric
receptors revealed that the third loop of the MCP-1 receptor accounted
for virtually all of the coupling to Gq. We conclude
that the MCP-1 and related chemokine receptors couple to multiple
G-proteins, that coupling is cell type-specific, and that the third
intracellular loop of the C-C type receptors mediates Gq
coupling.
INTRODUCTION
Chemokines (chemotactic cytokines) are low
molecular weight proteins that are closely related in both primary
amino acid sequence and tertiary structure (1, 2, 3). The chemokine family
can be subdivided into two groups based on the presence or absence of
an amino acid between the first two cysteines. In general,
C-X-C family members are neutrophil agonists, and C-C
members are mononuclear cell and basophil agonists. Interleukin-8
(IL-8)1 is the most thoroughly
characterized member of the C-X-C or -chemokine family;
other members include growth regulatory gene, neutrophil-activating
peptide-2, and platelet factor 4. IL-8 is a potent polymorphonuclear
chemoattractant and agonist and has been implicated in neutrophil
infiltration in the lung, liver, and spleen (4). Monocyte
chemoattractant protein-1 (MCP-1) is the most extensively studied
member of the C-C or 
-chemokine family; other members include RANTES
(regulated on activation, normal T cell-expressed and secreted),
macrophage inflammatory proteins 1 and 1 (MIP-1, MIP-1),
and eotaxin (5). MCP-1 is secreted by numerous cell types, including
endothelial cells, epithelial cells, and hematopoietic cells, and is a
potent chemoattractant for monocytes and CD45RO+
lymphocytes (6). In vivo, MCP-1 has been implicated as an
important factor in mediating monocytic infiltration in early
atherosclerosis, as well as in a number of chronic inflammatory
diseases (7, 8).
Because of the likely importance of the chemokines in a wide range of
diseases, attention has recently been focused on the receptors that
mediate chemokine responses. Several of these receptors have been
cloned. In 1991, Gerard and Gerard cloned the receptor for the human
complement fragment C5a and showed that it was a member of the
G-protein-coupled, seven-transmembrane domain receptor family (9). Two
highly homologous seven-transmembrane domain receptors for IL-8 have
been cloned (IL-8RA and IL-8RB) and shown to be products of different
genes (10, 11). Signaling by the IL-8 receptor in neutrophils was
sensitive to inhibition by pertussis toxin (PTX), indicating that the
receptor coupled at least in part to Gi (1, 12). A
receptor for the C-C chemokines RANTES and MIP-1, C-CR1, was cloned
by Neote et al. (13) and Gao et al. (14), and we
have recently cloned two alternatively spliced forms of the MCP-1
receptor (MCP-1RA and MCP-1RB) which differ only in their
carboxyl-terminal tails (15). Signaling studies of the C-C chemokine
receptors in transfected HEK-293 cells revealed potent,
agonist-dependent inhibition of adenylyl cyclase and
mobilization of intracellular calcium, consistent with receptor
coupling to Gi (16). In these studies, the calcium
response was not, however, completely blocked by PTX, suggesting the
involvement of additional G-proteins.
To identify PTX-resistant G-proteins that couple to the MCP-1 receptor,
we have performed cotransfection experiments in COS-7 and HEK-293
cells. In this paper, we report that the C-C chemokine receptors, but
not the IL-8 or C5a receptors, coupled to Gq and that
virtually all of this coupling could be attributed to the third
intracellular loop of the receptor. The MCP-1 receptors, but not C-CR1,
were found to couple to G16 in COS-7 cells, but none of
the C-C chemokine receptors coupled to G16 in HEK-293
cells. These results suggest important differences in coupling between
the C-X-C and C-C chemokine receptors.
EXPERIMENTAL PROCEDURES
The chemokines MCP-1, MIP-1,
RANTES, and IL-8 were obtained from R&D Systems, Inc. (Minneapolis).
Lipofectamine, Opti-MEM, DMEM, and MEM with Earle's balanced salt were
obtained from Life Technologies, Inc. PTX was purchased from List
Biological Laboratories, Inc. (Eugene, OR). Fetal calf serum was
obtained from Hyclone Laboratories (Logan, UT).
myo-[2-3H]Inositol was obtained from DuPont
NEN.
cDNAs of the MCP-1 receptors (types A
and B) and C-CR1 were cloned as described (15). These constructs and
the IL-8R construct include the prolactin signal sequence followed by a
flag epitope joined to the receptor sequence (17). The expression
vector for the human M1 muscarinic receptor was a generous gift from
Dr. Wolfgang Sadee, University of California, San Francisco, and all of
the G-protein constructs were kindly provided by Dr. Bruce R. Conklin,
Gladstone Institute of Cardiovascular Disease. The beta adrenergic
receptor kinase 1 (ARK1) cDNA was a generous gift from Dr.
Robert J. Lefkowitz, Duke University. The C5a receptor expression
vector and the IL-8 receptor (type A) were generous gifts of Dr. Craig
Gerard, Children's Hospital, Boston, and Dr. William I. Woods,
Genentech, South San Francisco, respectively. Chimeric receptors that
exchanged intracellular loop 2 and 3 were created by overlapping
polymerase chain reaction (18), as shown in Fig. 6.
Cell Culture and Transfection
COS-7 cells and human embryo kidney (HEK)-293 (CRL 1573) cells were obtained from the American Type Tissue Culture Collection (Bethesda, MD) and were grown in DMEM and MEM, respectively, with Earle's balanced salt solution supplemented with 10% fetal calf serum, streptomycin (100 µg/ml), and penicillin (100 IU/ml) at 37 °C in 5% CO2. cDNAs were transfected with Lipofectamine according to the manufacturer's instructions. Briefly, cells were seeded in 24-well plates at a density of 4 × 104 cells/well and grown overnight. The cells were then washed with phosphate-buffered saline (PBS), and 0.3 µg of DNA mixed with 1.5 µl of Lipofectamine in 0.25 ml of Opti-MEM was added to each well. The total amount of DNA was maintained constant by adding DNA from an empty vector. After 5 h at 37 °C, the medium was replaced with the medium containing 10% fetal calf serum.
Inositol Phosphate (IP) Formation AssayApproximately
24 h after transfection, cells were labeled for 20-24 h with
myo-[2-3H]inositol (2 µCi/ml) in
inositol-free medium containing 10% dialyzed fetal calf serum. Labeled
cells were washed with inositol-free DMEM containing 10 mM
LiCl and incubated at 37 °C for 1 h with inositol-free DMEM
containing 10 mM LiCl and the indicated agonist. IP
formation was assayed as described (19). After incubation with
agonists, the medium was aspirated, and cells were lysed by addition of
0.75 ml of ice-cold 20 mM formic acid (30 min). Supernatant
fractions were loaded onto AG 1-X8 Dowex columns (Bio-Rad), followed by
immediate addition of 3 ml of 50 mM NH4OH. The
columns were then washed with 4 ml of 40 mM ammonium
formate followed by elution with 2 M ammonium formate.
Total IPs were quantitated by counting emissions.
The
surface expression of the MCP-1 receptor C-CR1 and the IL-8RA was
assessed by ELISA (20). Briefly, cells were split into 24-well plates
at 5 × 104 cells/well. One day later, cells were
transfected as described above. After 48 h, cells were fixed with
4% paraformaldehyde in PBS for 15 min. Plates were washed twice with
PBS and then incubated with 1 µg/ml of the M1 antibody directed
against the Flag epitope (IBI, Kodak) in DMEM containing 10 mM Hepes and 0.1% bovine serum albumin for 1 h at
room temperature. After washing with PBS, plates were incubated with
horseradish peroxidase-conjugated second antibodies (Bio-Rad; 1:1,000
dilution in DMEM/Hepes/bovine serum albumin) for 30 min at room
temperature. After additional washing with PBS, the plates were
developed using the peroxidase chromogenic substrate 2, 2
-aminobis(3-ethylbenzthiazinoline-6-sulfonic acid (1 mg/ml) in
citrate/phosphate buffer, pH 4.0, with 0.03% hydrogen peroxide.
Absorbance at 450 nm was read after 5-30 min on an ELISA plate reader
(Vmax, Molecular Devices, Menlo Park, CA).
RESULTS
We have shown previously that MCP-1RB and C-CR1 couple via
Gi to mediate agonist-dependent inhibition of
adenylyl cyclase and intracellular calcium release in stably
transfected HEK-293 cells (16). In these studies, signaling was not
completely blocked by pretreatment of the cells with PTX, suggesting
that the receptors also coupled to additional PTX-resistant G-proteins,
such as Gq or G16. To elucidate further
the signal transduction pathways of these -chemokine receptors, we
transiently transfected COS-7 cells with cDNAs encoding MCP-1RA,
MCP-1RB, and C-CR1 and measured the hydrolysis of phosphoinositide (PI)
induced by the appropriate ligands (Fig. 1). Activation
of either form of the MCP-1 receptor resulted in a similar degree of
hydrolysis of PI, and this was blocked by approximately 50% by
pretreatment of the cells with PTX. In contrast, little or no PI
hydrolysis was detected in response to activation of C-CR1 by either
MIP-1 or by RANTES. Cell surface expression of each of these
epitope-tagged receptors was quantitated by ELISA, which revealed that
C-CR1 was expressed essentially as well as MCP-1RB (see Fig. 4). The M1
muscarinic receptor couples exclusively via PTX-resistant
Gq to hydrolyze PI, and as expected, signaling by M1 was
not blocked by PTX pretreatment. These results indicate that both
isoforms of the MCP-1 receptor couple to PTX-sensitive as well as
PTX-resistant G-proteins to mediate IP release.
/RANTES receptor (C-CR1), or the M1 muscarinic receptor (M1R).
Cells were loaded for 24 h with
myo-[3H]inositol in the presence or absence of
100 ng/ml PTX and incubated in the presence of 10 mM LiCl
for 1 h at 37 °C with MCP-1 (100 nM), MIP-1 (100 nM), or carbacol (200 µM). Total
[3H]inositol phosphate was measured as described under
``Experimental Procedures.'' Each data point was determined in
triplicate, and the data shown are the means (±S.D.) of three
independent experiments. The asterisks (*) indicate
p < 0.05 versus control.
Hydrolysis of PI by Gi-coupled receptors is thought to
involve activation of phospholipase C by the 
subunit of the
G-protein complex (21). To determine if this mechanism was part of the
signal transduction pathway, we cotransfected the cDNA for ARK1,
which binds to and inactivates subunits (22), with the cDNA
for the MCP-1 receptor in COS-7 cells. In the presence of ARK1,
MCP-1-induced IP release was blocked to approximately the same extent
as by pretreatment of the cells with PTX (Fig. 2). These
data are consistent with a model in which activation of Gi
releases the associated subunit to activate phospholipase C and
provides further evidence that both forms of the MCP-1 receptor couple,
at least in part, to Gi. As expected, overexpression of
ARK1 did not affect carbacol-induced IP release in the cells
transfected with the Gq-coupled M1 muscarinic receptor.
Similar results were obtained by cotransfection with transducin, which
also binds and inactivates free dimers (23, 24) (data not
shown).
ARK1 on MCP-1-induced IP release
in COS-7 cells. COS-7 cells were transiently transfected with
cDNAs encoding either MCP-1RA, MCP-1RB, or the M1 muscarinic
receptor (M1R) in the presence of 1 µg/ml DNA of plasmids encoding
either ARK1 or vector alone. The cells were incubated with agonists,
and IP release was measured, as described in the legend to Fig. 1. Each
data point was determined in triplicate, and the data shown are the
means (±S.D.) of three independent experiments. The
asterisks (*) indicate p < 0.05 versus control.
To identify G-proteins that couple to C-C chemokine receptors in a
PTX-resistant manner, we cotransfected G-subunits and receptors in
COS-7 cells. Signaling by both forms of the MCP-1 receptor was enhanced
significantly by coexpression of Gq and
G16 (Fig. 3). PI hydrolysis mediated by
C-CR1 was potentiated by Gq but not by
G16. The chimeric G-protein Gqi5 has the
carboxyl-terminal five amino acids of Gi, which bind to
the receptor, spliced onto Gq (25). Cotransfection of
Gqi5 also significantly potentiated signaling by each of
the C-C chemokine receptors, consistent with coupling to
Gi. In contrast, IL-8RA did not induce PI turnover in
COS-7 cells unless it was cotransfected with G16 or
Gqi5. Furthermore, IL-8RA signaling was not enhanced by
Gq. Using an ELISA assay, we determined that
cotransfection of the G-proteins did not alter the surface expression
of any of the chemokine receptors (Fig. 4). We did note,
however, that MCP-1RA was expressed at the cell surface at
significantly lower levels than the other receptors. These results
suggest that both forms of the MCP-1 receptor couple efficiently to
Gq, G16, and Gi, whereas the
IL-8RA receptor has a preference for G16 and
Gi.
To confirm these findings in a second cell type, we coexpressed the
chemokine receptors and G-proteins in HEK-293 cells. In contrast to the
results found in COS-7 cells, MCP-1-induced IP release was enhanced by
cotransfection with Gq and Gqi5 but not
by G16 (Fig. 5). Similar results were
obtained using C-CR1. Like the IL-8 receptor, the human C5a receptor
signaled poorly in the absence of exogenous G16, and
signaling was not enhanced by coexpression of Gq.
Cotransfection of Gqi5 augmented signaling by the C5a
receptor, as reported previously (26).
To identify the domain(s) of the MCP-1 receptor which bind to
Gq, we replaced the second and third intracellular loops
of MCP-1RB with the corresponding regions of the IL-8RA (Fig.
6). As shown in Fig. 7, replacement of
the 23-amino acid third intracellular loop of MCP-1RB with that of
IL-8RA resulted in a chimera (MM8) which was phenotypically identical
to IL-8RA in terms of signaling. Thus, MCP-1-dependent
signaling was detected only in the presence of cotransfected
G16, and the receptor failed to couple to
Gq. Moreover, the complementary construct in which the
third intracellular loop of MCP-1RB was substituted into IL8-RA (88M)
resulted in a receptor in which signaling in response to IL-8 was
indistinguishable from that of MCP-1RB. Note that exchange of this loop
resulted in changes in only 14 amino acid residues since the carboxyl
ends of the loops are virtually identical in the MCP-1 and IL-8
receptors (Fig. 6). In contrast, substitution of the second
intracellular loop of IL-8RA into MCP-1RB (M8M) had no effect on
MCP-1-dependent signaling. Thus, in the context of the
receptor, the third intracellular loop of MCP-1RB was both necessary
and sufficient for coupling to Gq. The mechanism of
G16 coupling was considerably more complex in that
substitution of the third intracellular loop of C-CR1 (which fails to
couple to G16) into MCP-1RB did not change coupling to
G16 (data not shown).
q to the third
intracellular loop of the MCP-1 receptor. COS-7 cells were
transiently transfected with cDNAs encoding the wild-type and
chimeric receptors and the indicated G-proteins, as described in the
legend to Fig. 3. The cells were incubated with the indicated agonists
(100 nM), and IP release was measured, as described in the
legend to Fig. 1. Each data point was determined in triplicate, and the
data shown are the means (±S.D.) of three independent experiments. The
asterisks (*) indicate p < 0.05 versus receptor only.
DISCUSSION
In this paper we have used transient transfection of COS-7 and
HEK-293 cells to investigate the signal transduction pathways of the
MCP-1 and C-CR1 receptors. We have found that both forms of the MCP-1
receptor couple to at least three different G-proteins,
Gq, G16, and Gi, in COS-7
cells. The MIP-1/RANTES receptor C-CR1 couples to Gq
and Gi but fails to couple to G16. The
chimeric G-protein Gqi5 binds to Gi-coupled
receptors via its carboxyl end and activates phospholipase C via its
Gq portion (25). Since activation of phospholipase C by
Gq is generally more efficient than activation via
Gi, this construct was used as a measure of Gi
coupling of each of the receptors. As expected, the MCP-1 receptors and
C-CR1 coupled well to Gqi5 to effect PI turnover. In
contrast to the C-C chemokine receptors, the IL-8 and C5a receptors
coupled to G16 and Gqi5 but failed to
couple to Gq. Through the use of chimeric MCP-1/IL-8
receptors, we have shown that Gq coupling is determined
completely by the third intracellular loop of the C-C chemokine
receptors. We conclude that the chemokine receptors couple to multiple
G-proteins, that receptor/G-protein pairings are highly cell
type-specific, and that coupling to Gq may distinguish
C-C from C-X-C chemokine receptors.
The type A and type B MCP-1 receptors are alternatively spliced
variants of a single gene and differ only in their cytoplasmic tail
(15). We had found previously that MCP-1RB coupled to Gi to
inhibit adenylyl cyclase and mobilize intracellular calcium in
transfected HEK-293 cells, but we were unable to study the signaling of
MCP-1RA because cell surface expression was extremely poor in our
stably transfected cell lines (16). In the current study we have shown
that both forms of the receptor couple identically to
Gq, Gi, and G16. Receptor
coupling to endogenous Gq probably accounts for the
portion of PI turnover (approximately 50%) which is resistant to
PTX.
Significant differences in G-protein coupling were found among the
three C-C chemokine receptors and the IL-8 and C5a receptors. In COS-7
cells, both forms of the MCP-1 receptor, but not C-CR1, coupled to
G16. Previous work by several laboratories has
demonstrated that a large number of receptors, including the
2-adrenergic receptor, M2-muscarinic receptor, D1
dopamine receptor, µ-opioid receptor, and thrombin receptor, couple
to G16 in transiently transfected COS-7 cells (27). We
next examined C-C chemokine receptor coupling in a second cell type,
HEK-293. In contrast to COS-7 cells, the MCP-1 receptors failed to
couple to G16 in transfected HEK-293 cells. The C5a
receptor, previously shown to couple to G16 in this cell
type (26), was used as a positive control. In HEK-293 cells, therefore,
the C-C chemokine receptors coupled to Gq, but not
G16, whereas the C5a receptor coupled to
G16 but failed to couple to Gq.
It is likely that the promiscuous coupling of receptors to
G16 in COS-7 cells was due, at least in part, to the
high levels of G-protein expression achieved following transfection. In
this regard, Chabre et al. (28) reported that the
2A-adrenergic receptor preferentially interacted with
endogenous Gi but also coupled (with a higher
EC50) to transfected Gs and
Gq. It is also possible that selective expression of
G-protein subunits may account for the differences in coupling
observed between the COS-7 and HEK-293 cells. Kleuss et al.
(37) (CK832) found that in pituitary cells the 3 subtype was
required for coupling to the somatostatin receptor, whereas the 4
subtype was required for coupling to the muscarinic receptor. It is not
yet known which subunits are present in COS-7 cells
versus HEK-293 cells, nor is it known which subunits
are present in hematopoietic progenitor cells versus mature
mononuclear cells. Taken together, these data indicate that chemokine
receptor coupling is highly cell type-dependent and that in
the case of recombinant receptors and G-proteins, signal transduction
questions should be examined in multiple cell types.
The dramatic difference in coupling to Gq between the
C-C and C-X-C receptors provided an opportunity to identify
the receptor site(s) interacting with Gq. Extensive
investigation of the adrenergic receptors has implicated the extended
third intracellular loop, the second intracellular loop, and the
carboxyl tail as contributors to the binding of G-proteins (for review,
see Ref. 29). The high degree of amino acid sequence conservation in
the third loop between the MCP-1 and C-CR1 receptors suggested that
this domain might be critically involved in Gq coupling.
Consistent with this notion was the fact that the IL-8 and C5a
receptors, which fail to couple to Gq, bore little
resemblance in the third loop to the C-C chemokine receptors. We
therefore created a number of chimeric receptors that interchanged the
second and third intracellular loops between the IL-8 receptor and
MCP-1RB. Analysis of these constructs by agonist-dependent
PI turnover revealed that virtually all of the Gq
coupling could be attributed to the third intracellular loop of the
MCP-1 receptor. Comparison of the amino acid sequence in the third loop
of the MCP-1 and IL-8 receptors further suggested that
Gq coupled to the amino-terminal portion of the loop, as
the last 8 residues are either identical or conservatively substituted.
We used a similar approach in an attempt to identify the receptor
binding site for G16. Substitution of the third loop
from C-CR1, which fails to couple to G16, into MCP-1B
(resulting in the chimera MMC) did not, however, diminish coupling to
G16 (data not shown). We conclude, therefore, that
although the third intracellular loop of the MCP-1 receptor is crucial
for coupling to Gq, it may not be for G16
coupling. Experiments are currently in progress to determine if the
second intracellular loop of the MCP-1 receptor interacts with
G16.
Kuang et al. have recently reported that MCP-1RB, but not
MCP-1RA, coupled to G16 (30), and thus concluded that
the carboxyl-terminal tail of the receptor was critically involved in
G16 interactions. Our data do not support this
conclusion, as we found that MCP-1RA and MCP-1RB coupled similarly to
G16 in COS-7 cells, and neither form of the receptor
coupled to G16 in HEK-293 cells. The failure of Kuang
et al. to demonstrate MCP-1RA coupling to G16
was probably due to the relatively lower levels of surface expression
of MCP-1RA, compared with MCP-1RB, in transfected cells (Fig. 4). Kuang
et al. also reported that neither of the MCP-1 receptors
coupled to Gq (30). Two lines of evidence from the
present study indicate that both MCP-1RA and MCP-1RB are indeed
Gq-coupled. First, in the absence of exogenous
G-proteins, both forms of the receptor mediate
agonist-dependent PI turnover in COS-7 cells. Pretreatment
of the cells with PTX blocked only 50% of the
MCP-1-dependent PI turnover. This result raised the
possibility that Gq, which is PTX-resistant and is
endogenously expressed in COS-7 cells, coupled to the MCP-1 receptor.
Second, in cotransfection experiments, we have directly shown that both
forms of the MCP-1 receptor, as well as C-CR1, couple to
Gq to enhance PI turnover. Similar results were obtained
using transfected HEK-293 cells.
Cotransfection experiments are useful in that they reveal
receptor/G-protein interactions that occur under conditions in which
one or both are present at high concentrations. The coupling of
G-proteins to receptors under physiological conditions may be much more
stringent. Pretreatment of leukocytes with PTX abolishes the
chemotactic response to MCP-1, IL-8, and formyl peptides almost
completely, suggesting that coupling to Gi is necessary and
critically involved in mediating cell migration (31, 32, 33).
G16 is present in hematopoietic progenitor cells, and
its level in HL-60 cells falls dramatically after these cells are
terminally differentiated (34). These data raise the intriguing
possibility that PTX-resistant signaling pathways, and in particular
coupling to G16, may be important in leukocyte
maturation. MIP-1 and IL-8 have been recently shown to play roles in
hematopoiesis (35, 36).
In summary, we have shown that the MCP-1 and MIP-1/RANTES receptors
couple to multiple G-proteins in transfected COS-7 and HEK-293 cells
and that receptor/G-protein interactions are highly cell type-specific.
The two forms of the MCP-1 receptor couple to the same G-proteins and
in HEK-293 cells fail to couple to G16. Coupling to
Gq distinguishes the C-C chemokine receptors from the
IL-8 and C5a receptors, as does coupling to G16 in
HEK-293 cells. Virtually all of the Gq coupling of the
MCP-1 receptor, and most likely C-CR1 as well, can be attributed to the
third intracellular loop. Since C-C chemokine receptors are found
predominately in mononuclear cells, it is interesting to speculate that
Gq coupling may be particularly relevant in the setting
of chronic inflammation. In contrast, since the IL-8 and C5a receptors
are expressed well on polymorphonuclear leukocytes, G16
coupling may be more important in acute inflammation. Pharmacological
inactivation of leukocyte-specific G-proteins may thus provide an
alternative and more specific means of treating inflammation.
FOOTNOTES
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.
ARK, beta adrenergic receptor kinase; DMEM, Dulbecco's minimal
essential medium; ELISA, enzyme-linked immunosorbent assay; IP,
inositol phosphate; MCP-1, monocyte chemoattractant protein-1; MIP,
macrophage inflammatory protein; RANTES, regulated on activation,
normal T-expressed and secreted; MEM, minimal essential medium; PBS,
phosphate-buffered saline; PI, phosphoinositide; PTX, pertussis
toxin.
Acknowledgments
We thank Dr. Bruce R. Conklin for helpful discussions and Drs. Robert Pitas and Robert Mahley for careful readings of the manuscript. We thank John Carroll for preparation of the figures and Gary Howard for editorial assistance.
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Y. Su, S. K. Raghuwanshi, Y. Yu, L. B. Nanney, R. M. Richardson, and A. Richmond Altered CXCR2 Signaling in {beta}-Arrestin-2-Deficient Mouse Models J. Immunol., October 15, 2005; 175(8): 5396 - 5402. [Abstract] [Full Text] [PDF]
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B. Lagane, S. Ballet, T. Planchenault, K. Balabanian, E. Le Poul, C. Blanpain, Y. Percherancier, I. Staropoli, G. Vassart, M. Oppermann, et al. Mutation of the DRY Motif Reveals Different Structural Requirements for the CC Chemokine Receptor 5-Mediated Signaling and Receptor Endocytosis Mol. Pharmacol., June 1, 2005; 67(6): 1966 - 1976. [Abstract] [Full Text] [PDF]
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R. A. Colvin, G. S. V. Campanella, J. Sun, and A. D. Luster Intracellular Domains of CXCR3 That Mediate CXCL9, CXCL10, and CXCL11 Function J. Biol. Chem., July 16, 2004; 279(29): 30219 - 30227. [Abstract] [Full Text] [PDF]
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R. K. H. Lo and Y. H. Wong Signal Transducer and Activator of Transcription 3 Activation by the {delta}-Opioid Receptor via G{alpha}14 Involves Multiple Intermediates Mol. Pharmacol., June 1, 2004; 65(6): 1427 - 1439. [Abstract] [Full Text] [PDF]
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C. Liu, G. Sandford, G. Fei, and J. Nicholas G{alpha} Protein Selectivity Determinant Specified by a Viral Chemokine Receptor-Conserved Region in the C Tail of the Human Herpesvirus 8 G Protein-Coupled Receptor J. Virol., March 1, 2004; 78(5): 2460 - 2471. [Abstract] [Full Text] [PDF]
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M. C. Jimenez-Sainz, B. Fast, F. Mayor Jr., and A. M. Aragay Signaling Pathways for Monocyte Chemoattractant Protein 1-Mediated Extracellular Signal-Regulated Kinase Activation Mol. Pharmacol., September 1, 2003; 64(3): 773 - 782. [Abstract] [Full Text] [PDF]
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A. Amara, A. Vidy, G. Boulla, K. Mollier, J. Garcia-Perez, J. Alcami, C. Blanpain, M. Parmentier, J.-L. Virelizier, P. Charneau, et al. G Protein-Dependent CCR5 Signaling Is Not Required for Efficient Infection of Primary T Lymphocytes and Macrophages by R5 Human Immunodeficiency Virus Type 1 Isolates J. Virol., February 15, 2003; 77(4): 2550 - 2558. [Abstract] [Full Text] [PDF]
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D. Chodniewicz and D. V. Zhelev Chemoattractant receptor-stimulated F-actin polymerization in the human neutrophil is signaled by 2 distinct pathways Blood, February 1, 2003; 101(3): 1181 - 1184. [Abstract] [Full Text] [PDF]
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