(Received for publication, September 18, 1995; and in revised form, December 27, 1995)
From the
The C-C chemokines are major mediators of chemotaxis of
monocytes and some T cells in inflammatory reactions. The pathways by
which the C-C chemokine receptors activate phospholipase C (PLC) were
investigated in cotransfected COS-7 cells. The C-C chemokine receptor-1
(CKR-1), the MCP-1 receptor-A (MCP-1Ra), and MCP-1Rb can reconstitute
ligand-induced accumulation of inositol phosphates with PLC 2 in a
pertussis toxin-sensitive manner, presumably through G
released from the G
proteins. However, these three
receptors demonstrated different specificity in coupling to the
subunits of the G
class. While none of the receptors can
couple to G
q/11, MCP-1Rb can couple to both G
14 and G
16,
but its splicing variant, MCP-1Rb, cannot. Since MCP-1Ra and -b differ
only in their C-terminal intracellular domains, the C-terminal ends of
MCP-1Rs determine G protein coupling specificity. CKR-1 can couple to
G
14 but not to G
16, suggesting some of the C-C chemokine
receptors, unlike the C-X-C chemokine receptors, discriminate against
G
16, a hematopoietic-specific G
subunit. The intriguing
specificity in coupling of the G
class of G proteins
implies that the chemokines may be involved in some distinct functions in vivo. The commonality of the chemokine receptors in
coupling to the G
-G
-PLC
2 pathway provides a
potential target for developing broad spectrum anti-inflammatory drugs.
Chemokines are a large family of small (8-10 kDa),
inducible, secreted, proinflammatory cytokines, which are produced by
various cell types. Members of the chemokine family share 20-90%
homology in their amino acid sequences. The sequences usually have four
conserved cysteine residues except lymphotactin. On the basis of the
positions of the cysteine residues, the chemokine family can be divided
into three subfamilies: the C-X-C or family, the C-C or
family, and the C or
family. The
family includes IL-8, (
)GRO (growth-related oncogene), NAP-2, ENA-78, platelet
factor 4, IP-10, and GCP-2, while the
family includes macrophage
chemotactic protein (MCP)-1, -2, and -3, RANTES (regulated upon
activation, normal T cell expressed and secreted), macrophage
inflammatory protein (MIP)-1
and -1
, I309, and C10 (for
reviews see (1, 2, 3) ). The newly discovered
family has only one member, lymphotactin. Lymphotactin, unlike
other chemokines, has only two conserved cysteine residues(4) .
The exact physiological and pathophysiological functions of these
factors are not yet clearly defined; however, it is generally believed
that their main function is recruitment and activation of leukocytes at
the site of inflammation.
Two receptors, IL-8RA and IL-8RB, have
been cloned for the C-X-C chemokine family(5) . We have
characterized the G protein-coupled pathways for these two receptors by
using the cotransfection assay(6) . Recently, three receptors
for the C-C chemokines were also cloned: CKR-1(7, 8) ,
MCP-1Ra, and MCP-1Rb(9) . CKR-1 binds to MIP-1, RANTES,
MIP-1
, and MCP-1 with varying affinities. However, only MIP-1
and RANTES can induce biological effects at physiological
concentrations(7) . MCP-1Ra and -b are two alternative splicing
variants, and they differ only in their C-terminal intracellular
domains(9) . MCP-1Rb binds to MCP-1 and MCP-3 but not to MIPs,
RANTES, or MCP-2(9, 10) . The C-C chemokine receptors
share about 50% sequence homology among themselves and less than 30%
homology with the C-X-C chemokine receptors(2) .
CKR-1 and
MCP receptors have typical structural characteristics of G
protein-coupled receptors, and they induce cytosolic Ca efflux(7, 9) , presumably through activation of
phospholipase C (PLC). Five cDNAs that encode the
subunits of the
G
class have been characterized, G
,
G
11, G
14, G
15, and G
16(11) , all of which
can activate all isoforms of PLC
, PLC
1-4, to
stimulate the release of inositol phosphates (IPs) (12, 13, 14, 15, 16, 17) .
COS-7 cells contain G
and G
11 but not G
14,
G
15, or G
16(15) . The expression of G
15 and
G
16 was detected only in hematopoietic cells (G
15 may be the
mouse counterpart of human
G
16)(18, 19, 20) , while G
14 is
expressed in some lineage of hematopoietic cells as well as other cell
types(19) . Many receptors, including the IL-8 receptors, were
found to couple to some of the
subunits of the G
class to activate PLC. Recently, the G
subunits of G
proteins were also found to activate specific isoforms of PLC
.
The G
-linked pathway may account for the PTX-sensitive
activation of PLC mediated by the IL-8 receptors in mature
leukocytes(6) .
Since the C-C chemokines play important
roles in chemotaxis of monocytes and some T cells, we characterized the
G protein-coupled signal transduction pathways for the three C-C
chemokine receptors by the cotransfection assay system in COS-7 cells.
We found that the C-C chemokine receptors showed different specificity
in coupling to the G subunits of the G
class, while
the receptors can all couple to the G
proteins to activate
PLC
2 through G
.
We tested in cotransfected COS-7 cells whether the newly
cloned C-C chemokine receptors, including the MCP-1Ra, MCP-1Rb, and
CKR-1, can couple to the subunits of the G
class of G
proteins. We have previously shown that receptors that can couple to
G
or G
11 gave ligand-induced accumulation of IPs
in COS-7 cells transfected with the receptor cDNAs(21) . Thus,
to test whether these C-C chemokine receptors can couple to
G
or G
11, we transfected the cDNAs corresponding
to each of the C-C chemokine receptors into COS-7 cells and determined
ligand-induced accumulation of IPs. There was little MIP-1
-induced
accumulation of IPs in cells expressing CKR-1, and neither was there
MCP-1-induced accumulation of IPs in cells expressing MCP-1Ra or
MCP-1Rb (Fig. 1A). These results indicate that these
receptors cannot couple to endogenous G
or G
11.
To test whether the receptors can couple to other members of the
G
class, we cotransfected COS-7 cells with each of the
receptor cDNAs and the cDNA corresponding to G
14 or G
16. We
and others have previously found that all the receptors tested in the
cotransfection assay, including
1A, -B, and -C(21) ,
2-adrenergic receptors(22) , the m2-muscarinic receptor,
D1-dopamine receptor, V2,V1a-vasopressin receptor, A2a-adenosine
receptor, µ-opioid receptor, 5-1a, 1c/2c serotonin receptors,
and thrombin receptor (17) can couple to G
16. However,
neither CKR-1 nor MCP-1Ra can couple to G
16, since cells
coexpressing G
16 and CKR-1 or MCP-1Ra showed little ligand-induced
accumulation of IPs (Fig. 1A). Interestingly, MCP-1Rb,
the alternative splicing variant of MCP-1Ra, gave ligand-dependent
release of IPs when coexpressed with G
16 (Fig. 1A), suggesting that MCP-1Rb can couple to
G
16. The activation of G
16 by MCP-1Rb was insensitive to PTX (Fig. 1B). Furthermore, these C-C chemokine receptors
demonstrated different selectivity in coupling to G
14; CKR-1 and
MCP-1Rb can couple to G
14, while MCP-1Ra cannot (Fig. 1A). The concentration-dependent responses to
ligand indicate a mean effective concentration (EC
) for
MCP-1Rb-mediated activation of G
16 and G
14 of about 7
nM.
Figure 1:
Coupling of the C-C chemokine receptors
to the subunits of the G
class. A, COS-7
cells were cotransfected with the cDNA (0.25 µg) encoding the C-C
chemokine receptors and the cDNA (0.25 µg) corresponding to
G
14, G
16, or Lac Z (
-galactosidase, as a
control) as indicated in the figure. Forty-eight hours after
transfection, MIP-1
-induced (7 nM) accumulation of IPs in
cells expressing CKR-1 and MCP-1-induced (20 nM) accumulation
of IPs in cells expressing MCP-1Ra or -B were determined 30 min after
addition of ligands. B, concentration-dependent accumulation
of IPs to MCP-1 was determined in cells coexpressing MCP-1Rb and
G
14 or G
16 in the presence (closed symbols) or
absence (open symbols) of PTX. PTX (500 ng/ml) was added 4 h
before the PLC assay. C and D, COS-7 cells were
cotransfected with the G
16 cDNA (0.25 µg) and cDNA (0.25
µg) encoding one of the C-C chemokine receptors. The cells were
lysed in SDS sample buffer 48 h after transfection. The proteins were
separated on 12% SDS-polyacrylamide gel and electroblotted onto a
nitrocellulose membrane. The G
16 (C) and G
14 (D) proteins were detected with antibodies specific to
G
16 and G
14, respectively.
We have demonstrated in many of our reports (6, 15, 21, 23) that coexpression of
one protein does not significantly affect the expression of others.
Nonetheless, we determined the expression of G16 and G
14 in
cells cotransfected with cDNA encoding CKR-1, MCP-1Ra, or MCP-1Rb to
eliminate the possibility that the inabilities of CKR-1 and MCP-1Ra to
couple to G
16 or G
14 were the results of lower expression
levels of the proteins. As shown by Fig. 1, C and D, the expression levels of G
16 and G
14 were similar
regardless of the nature of the coexpressed receptors. We also
determine the receptor levels by using
I-labeled MCP-1 or
MIP-1
. The cells transfected with CKR-1, MCP-1Ra, or MCP-1Rb all
show about 525-650 fmol of ligand-binding sites/1
10
cells, and the affinities are around 4.5 nM for
CKR-1, 2.2 nM for MCP-1Ra, and 1.7 nM for MCP-1Rb.
Therefore, the inabilities of CKR-1 and MCP-1Ra to couple to G
16
or G
14 are not due to variations in expression levels.
The
responses to the C-C chemokines (including MCP-1, MIP-1, and
RANTES) in monocytic phagocytes were found to be mostly PTX-sensitive (1, 2, 3) , yet the signal transduction
pathways mediated by the
subunits of the G
class are
PTX-resistant(11) . PTX is a bacterial toxin, which modifies
the C-terminal Cys residues of the G
and G
subunits. The modification prevents interactions between
receptors and G proteins. Recently, we proposed a novel pathway to
explain the PTX sensitivity; receptors interact with PTX-sensitive G
proteins to release the G
subunits, which then activate PLC
2(6, 23) . Since there are abundant G
proteins (predominantly G
2 as well as some
G
3) (24, 25) and the PLC
2 proteins
in leukocytes(26) , the G
-G
-PLC
2
pathway is likely to occur in vivo. To test whether the C-C
chemokine receptors can couple to endogenous PTX-sensitive G proteins
of COS-7 cells to activate PLC
2, we transfected COS-7 cells with
cDNA encoding PLC
2 and cDNA encoding each of the C-C chemokine
receptors. COS-7 cells contain endogenous G
2 but not
G
proteins, and they contain endogenous PLC
1 but
not PLC
2 as determined by specific antibodies(14) . The
accumulation of IPs in response to varying concentrations of MCP-1 or
MIP-1
was determined. All three receptors can induce activation of
PLC
2 with EC
of 0.5 nM for CKR-1 and 3
nM for MCP-1Ra and MCP-1Rb (Fig. 2). In addition, we
found that the ligand-induced responses in cells coexpressing the
receptors and PLC
2 were PTX-sensitive (Fig. 2). Thus, we
conclude that all three C-C chemokine receptors can couple to
endogenous PTX-sensitive G proteins, presumably the G
2
protein, to activate PLC
2 via G
(the G
subunits cannot directly activate PLC
(14) ). The
finding that MCP receptors can inhibit adenylyl cyclase activity in
A293 human kidney cells expressing the receptor confirms our notion
that the receptor can couple to the G
proteins(27) . The inability of the chemokine receptors
to activate endogenous PLC
(Fig. 1A) or
recombinant PLC
1 (data not shown) is consistent with our previous
observation that G
could not activate PLC
1 in the
cotransfection system(6, 21) . In addition, we found
that MCP-1 could not activate PLC in cells expressing CKR-1 and that
MIP-1
could not induce IP formation in cells expressing MCP
receptors.
Figure 2:
Activation of PLC 2 by the C-C
chemokine receptor in transfected COS-7 cells. COS-7 cells were
cotransfected with the PLC
2 cDNA (0.25 µg) and cDNA (0.25
µg) corresponding to CKR-1 (A), MCP-1Ra (B,
squares), and MCP-1Rb (B, triangles). Ligand-induced
accumulation of IPs was determined 30 min after addition of ligands
(MIP-1
in A, MCP-1 in B) in the presence (closed symbols) or absence (open symbols) of PTX.
PTX (500 ng/ml) was added 4 h before the PLC
assay.
Although the C-C chemokine receptors can couple to the
G-G
-PLC
2 pathway, these receptors
demonstrate interesting specificity in coupling to the
subunits
of the G
class. While none of the three receptors couples
to G
/11, MCP-1Rb can couple to both G
16 and
G
14, but its splicing variant MCP-1Ra cannot couple to either
G
14 or G
16. CKR-1 couples to G
14 but not to G
16.
The differences between MCP-1Ra and MCP-1Rb in G protein coupling
indicate that the C-terminal intracellular domains are critical in
determining the G protein coupling specificity, since these two
receptors differ only in the C-terminal ends(9) . Moreover, the
finding further supports our previous notion, drawn from our study of
the
1B-adrenergic receptor, that different receptor sequences are
required for activation of different G
subunits of the G
class(28) . The study of the
1B-adrenergic receptor
indicates that the
1B-adrenergic sequences required for activation
of G
14 are located in the third intracellular loop, whereas the
sequences required for activation of G
16 do not appear to be
localized within the third inner loop. This report, however, points out
that the sequences in the C-terminal intracellular domain are critical
for activation of both G
14 and G
16. We interpret the apparent
discrepancy to suggest that G protein-interacting sequences on
different receptors may be located at different sites or that there
exist multiple G protein-interacting sites on a receptor so that
alteration of any one of them abolishes the ability of the receptor to
couple to the G protein. In this report we did not account for the
influences of different G
subunits on the coupling of these
receptors to different G
subunits because there were no
significant differences observed for different G
subunits in
interaction with G
2 or in regulation of PLC
2 (15) or of adenylyl cyclase activities(29) .
Furthermore, the same system (COS-7 cells) was used in the studies;
thus, the differences in the coupling of these chemokine receptors to
different G
subunits cannot be attributed to G
.
The
physiological relevance of the pathways mediated by G,
G
16, and G
14 is not clear. All these G
subunits were
found in various hematopoietic cells. Although more systematic studies
of the expression of these G
subunits are needed, previous studies
suggest that there are very abundant G
subunits with
the majority of G
2 and some G
3 in
leukocytes (24, 25) and that the levels of the
G
subunits increase along with
differentiation(18) . G
16 and PLC
2 was detected only
in hematopoietic cells. G
16 was detected in neutrophils,
monocytes, lymphocytes, and erythrocytes as well as various
hematopoietic progenitor cells, and its expression in HL-60 promyeloid
cells decreases by 90% after differentiation(18) . These
results, in addition to the findings that responses to chemokines in
mature leukocytes were mostly PTX-sensitive, suggest that the
G
-linked pathway may be the predominant one in
chemokine-mediated effects in mature leukocytes, such as chemotaxis and
activation of leukocytes. If this hypothesis is correct, the activation
of PLC
2 by G
would be an excellent target for
developing broad spectrum anti-inflammatory drugs, because all the
known chemokine receptors, including the C-C chemokine receptors, can
couple to the G
-G
-PLC
2 pathway. The fact
that PLC
2 is expressed only in hematopoietic cells may limit
potential side effects.
Recently, some evidence indicates that
chemokines may be directly or indirectly involved in the regulation of
hematopoiesis; MIP-1 inhibits proliferation of the hematopoietic
stem cells(30) , and the IL-8 receptor-null mice have expanded
populations of neutrophils and B cells, in addition to their reduced
abilities to respond to inflammatory stimuli(31) . The
G
16-linked pathway may play a role in hematopoiesis as well as in
other hematopoietic functions, although there is a lack of evidence.
Nevertheless, regulation of expression levels by differentiation,
specificity in interactions between receptors and G proteins and
between G proteins and effectors, and diversity of molecular natures of
receptors, G proteins, and effectors in leukocytes underlie the
molecular basis for the complex function of signal transduction
networks in the hematopoietic system. Alternative splicing further
expands the signal-processing capabilities of eukaryotic cells.