(Received for publication, May 5, 1995; and in revised form, May 15, 1995)
From the
We report the cloning and characterization of a novel basophil
CC chemokine receptor, K5-5, from the human immature basophilic
cell line KU-812. The predicted protein sequence of K5-5 shows
only 49% identity to the macrophage inflammatory protein-1/RANTES
receptor (CC CKR-1) and 47% identity to monocyte chemotactic protein-1
receptor (b form), suggesting that this cDNA encodes a novel member of
the CC chemokine receptor family. Analysis of K5-5 mRNA
expression indicates that it is restricted to leukocyte-rich tissues.
In addition, we have shown significant levels of K5-5 mRNA in
human basophils, which were up-regulated by treatment with
interleukin-5. The CC chemokines, macrophage inflammatory
protein-1
, RANTES, and monocyte chemotactic protein-1 were able to
stimulate a Ca
-activated chloride channel in Xenopus laevis oocytes injected with K5-5 cRNA, whereas
no signal was detected in response to monocyte chemotactic protein-2,
macrophage inflammatory protein-1
, or the CXC chemokine,
interleukin-8. Taken together, these results indicate for the first
time the presence of a CC chemokine receptor on basophils, which
functions as a ``shared'' CC chemokine receptor and may
therefore be implicated in the pathogenesis of basophil-mediated
allergic diseases.
Basophils have been implicated in the pathogenesis of a number
of allergic inflammatory reactions including asthma, allergic rhinitis,
and atopic dermatitis in addition to other inflammatory pathologies
such as parasitic infections and inflammatory bowel
disease(1, 2, 3, 4, 5) .
Recent reports have demonstrated that local production of a number of
chemotactic peptides and activating factors including RANTES, monocyte
chemotactic protein-1 (MCP-1), ()macrophage inflammatory
protein-1
(MIP-1
), and interleukin-8 (IL-8) by a variety of
stimulated cell types, can influence the recruitment of basophils to
inflammatory sites and the subsequent release of mediators such as
histamine and
peptidoleukotrienes(6, 7, 8, 9) .
These chemotactic peptides are members of a group of at least 15
structurally related proinflammatory mediators known as chemokines,
which have been divided into two families based on the spacing of the
first two of four conserved cysteines, namely CXC chemokines
or CC chemokines (10, 11) .
The specific effects of
chemokines on the target cell are mediated by receptors, which belong
to the serpentine family of seven-transmembrane (7-TM),
G-protein-coupled receptors(12) . To date, at least five human
chemokine receptors have been identified by cDNA cloning, of which four
have highly homologous sequences at the protein level. The two IL-8
receptors (A and B) identified both bind the CXC chemokine
IL-8, with a 4 nM dissociation constant (K). However, the IL-8 receptor B is
promiscuous in that it binds to IL-8 and other CXC chemokines
such as neutrophil-activating peptide-2 and Gro
/melanocyte
growth-stimulating activity at equal high
affinity(13, 14) . The third member of this receptor
family has been shown to bind the CC chemokines MIP-1
and RANTES
and has been called CC CKR-1(15, 16) . More recently a
specific receptor for MCP-1 has been identified(17) . The fifth
human chemokine receptor, found mainly on the surface of erythrocytes,
is a promiscuous chemokine receptor (which binds both CXC and
CC chemokines) and has been shown to be identical to the Duffy
antigen(18) . In addition to these receptors, there is a
functional promiscuous CC chemokine receptor encoded by human
cytomegalovirus open reading frame US28(15, 19) . Although the latter two
proteins both appear to be 7-TM receptors based on hydrophobicity
plots, they show less than 30% amino acid identity to the other
chemokine receptors.
It is likely that still more as yet
unidentified chemokine receptors exist to accommodate the growing
number of chemokines identified year by year despite the ligand
promiscuity observed in the known receptors. For example, specific high
affinity receptors for MIP-1 and RANTES, distinct from CC CKR-1,
have been proposed to exist on basophils, eosinophils, and monocytes
based on the results of cross-desensitization experiments in response
to various CC chemokines (20, 21, 22, 23, 24) . In
an attempt to identify novel basophil chemokine receptors, we have used
the technique of reverse transcriptase-polymerase chain reaction with
degenerate oligonucleotide primers (25) that corresponded to
conserved amino acid sequences found in the IL-8 receptors, and in CC
CKR-1, betweeen transmembrane domains 3 and 4 and within transmembrane
domain 7. Since human basophils are difficult to obtain at high purity
in large numbers, we used a human immature basophilic cell line, KU812,
derived from a patient with chronic myelogenous leukemia in blast
crisis(26) . These cells express a number of basophil-related
intracellular proteins, and both RANTES and IL-8 are able to induce
chemotaxis of these cells(27) . Cloning and sequencing of PCR
products revealed a novel 7-TM receptor-related sequence, K5-5,
which was subsequently used to obtain a full-length cDNA from a human
spleen cDNA library.
A
full-length cDNA encoding the MIP1/RANTES receptor (CC CKR-1) was
obtained by reverse transcriptase-PCR from the human eosinophilic cell
line EOL-3 (32) using specific primers based on the published
sequence (15) and subcloned into the EcoRV site of
pcDNAI(33) . A full-length cDNA encoding the MCP-1 receptor b
was obtained from a human hypodense eosinophil
ZAPII cDNA library (
)and subcloned as an XhoI/KpnI fragment
into pcDNA1. Linearized DNA was prepared from CC CKR-1/pcDNAI by XbaI digestion and from MCP-1RB/pcDNA1 by HindIII
digestion as described above.
Capped cRNA transcripts were generated
from 1 µg of linearized DNA in a 100-µl reaction volume
containing 20 µl of 5 transcription buffer (200 mM Tris-HCl, pH 7.5, 30 mM MgCl
, 10 mM
spermidine, and 50 mM NaCl), 4 µl of NTP mix (10 mM ATP, UTP, and CTP, 3 mM GTP), 4 µl of 0.75 M
dithiothreitol, 2.5 µl of RNAsin, 0.5 µl of GTP (10
mM), 4 µl of 10 mM CAP analog
(m
G(5`)ppp(5`)G) and 2.5 µl of T7 RNA polymerase
(Promega) for K5-5 and CC CKR-1 or SP6 RNA polymerase (Promega)
for MCP-1 receptor b. After 1.5 h at 37 °C, 4 µl of RQ1 DNase
(Promega) was added, and the reaction mixture was incubated for a
further 15 min at 37 °C. The reaction mixture was then extracted
twice with 0.1 M Tris-HCl, pH 8.0, saturated phenol/chloroform
(1:1 v/v) and once with chloroform. cRNA was precipitated overnight at
-20 °C after addition of 0.1 volume of 3 M sodium
acetate, pH 5.5, and 2.5 volumes of ethanol. cRNA was recovered by
centrifugation, washed in 70% ethanol, and resuspended in sterile water
at 0.5 µg/µl.
Oocytes were harvested from adult female Xenopus laevis by a modification of the method of Bertrand et al.(34) . Oocytes were defollicullated by
incubation in 0.2% (w/v) collagenase (Sigma) in 50 ml of OR2 medium
(82.5 mM NaCl, 2.5 mM KCl, 1 mM
NaHPO
, 15 mM HEPES, pH 7.6) in a
spinner flask under slow agitation for 2 h at room temperature. Oocytes
were rinsed carefully with OR2 followed by MBS (88 mM NaCl, 1
mM KCl, 0.33 mM Ca(NO
)
, 0.41
mM CaCl
, 0.82 mM MgSO
, 2.4
mM NaHCO
, 10 mM HEPES, pH 7.6) and
allowed to recover for at least 1-2 h in MBS before selecting
stage V-VI oocytes. Selected oocytes were incubated in MBS-supplemented
penicillin/streptomycin (100 units/ml) overnight at 18 °C before
injection.
Oocytes were microinjected using an Inject + Matic air pump (Gabay) using needles made from Drummond calibrated 6-µl capillaries. cRNA (25 ng in 50 nl) was injected into the cytoplasm. Oocytes were individually transferred to wells of a 96-well flat bottom culture dish and incubated in MBS for 24-72 h.
Electrophysiological recordings were made 3 days after injection in
oocytes superfused with OR2 medium (containing 2 mM CaCl and 1 mM MgCl
) at room temperature under
voltage-clamped conditions using two microelectrodes (1-2
megaohms, both filled with 3 M KCl), with the membrane
potential routinely clamped at -100 mV using a Gene Clamp 500
instrument (Axon).
Test chemokines were dissolved in distilled water and then diluted to a final concentration of 1 µM in OR2. Fifty microliters of each chemokine was applied directly onto voltage-clamped oocytes for 6 s, and the current induced was monitored on a Tektronix 5113 dual beam storage oscilloscope linked to an IBM-PC. When multiple chemokines were tested on a single oocyte, a recovery time of 2 min was allowed between each application.
In order to isolate novel chemokine receptor-like sequences,
we have used a reverse transcriptase-PCR strategy. Degenerate
oligonucleotide primers corresponding to the intracellular loop between
transmembrane domains 3 and 4 with the peptide sequence RYLAIVH and to
transmembrane domain 7 with the consensus peptide sequence
CLNP(I/L/M/V)(L/I)Y(A/V)F were designed based on the published sequence
of the human IL-8 receptors and CC CKR-1. These regions are well
conserved between chemotactic peptide receptors. In view of the
pharmacological evidence for the existence of novel chemokine receptors
in basophils(20) , we chose the human immature basophilic
leukemia cell line, KU-812, as a source of RNA. This cell line has
previously been shown to have basophil-like properties(26) .
The resultant PCR products of approximately 500-550 bp from this
reaction were gel-purified, subcloned into Bluescript II
SK, and sequenced. Most of the sequences analyzed
encoded the previously described human homologue (35) of the
bovine neuropeptide Y receptor(36) . The ligand of the human
receptor remains unknown. We also detected a number of clones that
showed 60% homology at the DNA level to the recently cloned
MIP1
/RANTES receptor (CC CKR-1). One of these clones,
TM(2-7)5-5 was subsequently used to screen 5
10
plaque-forming units of a human spleen cDNA library in
GT11. This resulted in the isolation of clone K5-5, which
contained the same sequence as that identified by reverse
transcriptase-PCR. K5-5 contained a 1676-bp cDNA insert of which
182 bp are 5`-untranslated sequence. The first 88 bases are
pyrimidine-rich, suggesting the presence of an unspliced
intron(37) . The identification of an incompletely spliced
transcript is not unusual (particularly in lymphocytes) and may
represent an important mechanism for translational regulation in
vivo(38) . Translation of the longest open reading frame
of 1080 bases predicts a protein of 360 amino acids (Fig. 1).
There are a total of three potential N-glycosylation
sites(39) ; the first is located in the N terminus
extracellular domain (Asn
) and the other two in the
extracellular loop between transmembrane domains 4 and 5 (Asn
and Asn
, respectively). It is unlikely that the
first site on Asn
is glycosylated in
vivo(40) . There are two intracellular consensus sequences
for protein kinase C phosphorylation(41) , located in the
second intracellular loop on Ser
and in the C-terminal
cytoplasmic domain on Thr
. There are also three potential
intracellular sites for casein kinase-II phosphorylation (42) on Ser
, Ser
, and
Ser
, respectively. The carboxyl-terminal domain also
contains a total of 9 Ser and Thr residues, which may be important
sites for regulation of receptor activity by phosphorylation with, for
example, G-protein-coupled receptor kinases(43) . Alignment of
the deduced amino acid sequence with the other chemokine receptors
shows that K5-5 has 49% identity to CC CKR-1 (over 356 amino
acids), 46% identity to the MCP-1 receptor b form (over 360 amino
acids), and 42% (over 302 amino acids) and 41% (over 295 amino acids)
identity to IL-8 receptors A and B, respectively (Fig. 2).
Interestingly, the greatest divergence occurs in the N-terminal
extracellular domain, which has been shown to control ligand binding
specificity in the IL-8 receptors(44) . In this region, the
amino acid identity is reduced significantly to 30% over 40 amino acids
with the MCP-1 receptor and 42% over 26 amino acids for CC CKR-1.
Figure 1:
cDNA sequence and deduced
amino acid sequence of clone K5-5. Potential N-glycosylation sites are marked with an asterisk.
The nucleotide sequence of clone TM(2-7)5-5 obtained by
reverse transcriptase-PCR is underlined. The sequence of
K5-5 has been submitted to the GenBank/EMBL/DDBJ
data bases and has the accession number
X85740.
Figure 2: Alignment of amino acid sequences encoding human chemokine receptors. IL-8 RA(5) , IL-8 RB(6) , CC CKR-1(7) , MCP-1 receptor b(9) , and K5-5 are shown. The putative transmembrane-spanning domains are underlined. The numbering of residues is based on the IL-8 RA sequence.
Northern blot analysis indicated that K5-5 hybridized to an
approximately 4.0-kb mRNA species expressed at high levels in thymus
and in peripheral blood leukocytes and to a lesser extent in spleen (Fig. 3a). In order to define specific leukocyte
populations expressing K5-5 mRNA, we analyzed FACS-purified
peripheral blood leukocytes and a number of leukocyte cell lines by
reverse transcriptase-PCR. Expression of mRNA was evident in the KU812
cell line as well as in unstimulated or interleukin-2-stimulated
peripheral blood T cells, B cells, and monocytes (Fig. 3b). We could also detect high levels of mRNA for
K5-5 in human platelets. ()The size of the cloned cDNA
shown in Fig. 1is considerably smaller than the transcript size
of 4.0 kb indicated by Northern blotting. This probably reflects
priming from adenine-rich sequences other than the poly(A) tail in the
3`-untranslated region.
Figure 3: Analysis of K5-5 receptor mRNA expression. a, Northern blot analysis of human tissues. Lane1, spleen; lane2, thymus; lane3, prostate; lane4, testis; lane5, ovary; lane6, small intestine; lane7, colon; lane8, peripheral blood leukocytes. b, reverse transcriptase-PCR analysis of peripheral blood leukocytes and some human leukocytic cell lines. Lane1, molecular weight markers (1-kb ladder; Life Technologies, Inc.); lane2, IL-2-stimulated peripheral blood T cells (48 h); lane3, untreated peripheral blood T cells; lane4, Jurkat cells; lane5, MOLT-4 cells; lane6, tonsillar B cells; lane7, peripheral blood B cells; lane8, pulmonary macrophages; lane9, peripheral blood monocytes; lane10, KU812 cells; lane11, EOL-3 cells.
The human immature basophilic cell line
KU812, in which K5-5 was originally identified, has previously
been shown to undergo chemotaxis in response to RANTES and IL-8
following pretreatment with IL-5 or phorbol myristate
acetate(27) . An analysis of the chemokine receptor profile in
these cells by reverse transcriptase-PCR indicates the presence of
K5-5 and the IL-8 receptor B only (Fig. 4a). By
analogy, freshly purifed human basophils were shown to express IL-8
receptor B mRNA with barely detectable levels of K5-5 mRNA (Fig. 4b). However, after stimulation for 15 min with
IL-5 (10 ng/ml), there was significant up-regulation of K5-5 mRNA
and weak expression of MCP-1 receptor mRNA in basophils (Fig. 4c). RANTES, MCP-1, and MIP-1 are known to
induce chemotaxis, histamine release, and Ca
mobilization in basophils. However, since RANTES and MIP-1
have not been reported to activate the MCP-1 receptor, and the other
known receptor for MIP-1
/RANTES (CC CKR-1) does not appear to be
expressed on these cells under these conditions, it was possible that
K5-5 encoded a novel CC chemokine receptor, especially in view of
the homology of K5-5 to known chemokine receptors.
Figure 4: Expression of chemokine receptor mRNA by reverse transcriptase PCR in KU812 cells and peripheral blood basophils. a, KU812 cells. Lane1, 1-kb ladder markers; lane2, K5-5; lane3, MCP-1 receptor b; lane4, CC CKR-1; lane5, 0 DNA control; lane6, IL-8 RA; lane7, IL-8 RB; lane8, glyceraldehyde-3-phosphate dehydrogenase. b, basophils (unstimulated); c, IL-5-stimulated basophils. Lanes1, molecular mass markers; lanes2, K5-5; lanes3, IL-8 RA; lanes4, IL-8 RB; lanes5, MCP-1 receptor b; lanes6, CC CKR-1; lanes7, glyceraldehyde-3-phosphate dehydrogenase.
Therefore,
to determine if K5-5 encodes a functional chemokine receptor, we
transiently expressed full-length K5-5 cRNA in X. laevis oocytes. The ability of various chemokines to induce
Ca mobilization in oocytes and thus stimulate a
Ca
-activated chloride channel, was assayed using the
voltage clamp technique(34, 45) . Signalling by the CC
CKR-1 and the MCP-1 receptor b was examined in parallel. Results on
individual oocytes, tested 3 days after microinjection of cRNA, are
shown in Fig. 5. Of the chemokines tested, only CC chemokines
MIP-1
, MCP-1, and RANTES were able to induce a chloride current in
the oocytes injected with K5-5 cRNA (Fig. 5a).
The amplitude of the transient current induced varied from oocyte to
oocyte but was consistently highest in response to MIP-1
ranging
from 50 to 2200 nA (n = 29). In some, but not all
oocytes that responded to MIP-1
, we also detected a chloride
current on subsequent application of MCP-1 and to a lesser extent with
RANTES, ranging from 30 to 1500 nA and 80 to 1000 nA, respectively. No
oocyte response was detected on application of IL-8, MCP-2, MIP-1
,
or buffer alone. The transient current induced by MIP-1
or MCP-1
was comparable with that seen with oocytes injected with cRNA encoding
the CC CKR-1 or the MCP-1 receptors (Fig. 5, b and c). In order to show that the chemokine-induced responses
observed in K5-5-injected oocytes were specific, we tested the
same chemokines on oocytes microinjected with the cRNA for an unrelated
7-TM receptor, the neurokinin-2 receptor(46) . These oocytes
failed to respond to any of the chemokines tested while maintaining a
robust response to 1 µM neurokinin-A (Fig. 5d). These results indicate that K5-5 can
function as a promiscuous CC chemokine receptor. In basophils it is
likely that K5-5 is the functional MIP-1
receptor since we
were unable to detect mRNA coding for the previously identified
MIP-1
/RANTES receptor (CC CKR-1) in either unstimulated or
IL-5-stimulated cells.
Figure 5: Current induced in voltage-clamped oocytes on stimulation with different chemokine ligands. Chemokines (1 µM) were applied for 6 s at 2-min intervals. Results of individual oocytes tested are shown. The scalemarker is positioned at the start of the application. a, K5-5 cRNA oocytes i-iv; b, CC CKR-1 cRNA; c, MCP-1 receptor b cRNA; d, neurokinin-2 receptor cRNA oocytes i-iv.
In summary, we report the cloning and
functional expression of a novel CC chemokine receptor, K5-5,
from the immature basophilic cell line KU812. Using
electrophysiological studies in oocytes we have identified MIP-1,
MCP-1, and RANTES as ligands for this receptor. The presence of
K5-5 mRNA in basophils, T cells, and monocytes is consistent with
the finding that MIP-1
, MCP-1, and RANTES have been previously
shown to exert a diverse range of activities on these cell types,
including histamine release, chemotaxis, and Ca
mobilization in basophils (7, 47) and chemotaxis
in T cells(48, 49, 50) and
monocytes(51) . However, since we and others have now shown
that these cell types express several different chemokine receptors
with overlapping specificities it is not clear if different ligands
acting at the same receptor induce different signaling pathways or
whether the activation of distinct receptors accounts for the diversity
in cellular responses to a given chemokine. Although K5-5 shows
promiscuity in the Xenopus oocyte system, it is possible that in vivo the ligand specificity is determined by the cell
background in which the receptor is expressed, for example, in terms of
coupling to relevant G-proteins or regulation of receptor-mediated
signal transduction by a kinase/phosphatase regulatory pathway. Hence
in the future, it will be necessary to characterize postreceptor
signaling pathways in order to define the precise function of these
receptors in different leukocyte populations and their relevance in
inflammatory diseases.
Note Added in Proof-Subsequent to the acceptance of this manuscript for publication we were advised that the MCP-2 used in these studies was not fully active. We have also been made aware of the existence of a novel eosinophil chemokine receptor CC CKR-3. We therefore suggest that K5-5 be designated CC CKR-4.