(Received for publication, April 25, 1995; and in revised form, August 14, 1995)
From the
The Duffy antigen (DARC) is a promiscuous chemokine receptor
that also binds Plasmodium vivax. DARC belongs to a family of
heptahelical chemokine receptors that includes specific (IL-8RA) and
shared (IL-8RB) IL-8 receptors. Ligand binding specificity of IL-8
receptors was localized to the amino-terminal extracellular (E1)
domain. To determine the basis for promiscuous chemokine binding by
DARC, a chimeric receptor composed of the E1 domain of DARC and
hydrophobic helices and loops from IL-8RB (DARC/IL-8RB)
was constructed. Scatchard analysis of stable transfectants
demonstrated that the DARC
/IL-8RB chimeric receptor bound
IL-8 and melanoma growth stimulating activity (MGSA) with K
values almost identical to the native
receptors. The hybrid receptor also bound RANTES, MCP-1, and
MGSA-E
A (which binds DARC, but not IL-8RB), but not
MIP-1
, similarly to DARC. Ligand binding to DARC transfectants was
unaltered by anti-Fy3, but inhibited by Fy6, which binds an epitope in
the E1 domain. The epitope recognized by Fy3 was localized to the third
extracellular loop by analysis of insect cells expressing chimeric
receptors composed of complementary portions of DARC and IL-8RB. These
findings implicate the E1 domain of DARC in multispecific chemokine
binding.
Interleukin-8 (IL-8) and melanoma growth stimulating
activity (MGSA) are members of a bipartite family of structurally
related chemoattractant cytokines, known as chemokines(1) . The
chemokines are characterized by the presence of four conserved cysteine
residues and have been classified into two separate groups, dependent
on whether the first two conserved cysteine residues are separated by
an intervening amino acid (C-X-C) or whether they are adjacent
(C-C)(1) . The C-X-C class members include IL-8, MGSA,
and neutrophil-activating protein-2, while the C-C class includes
RANTES, monocyte chemoattractant protein (MCP-1), and the macrophage
inhibitory protein (MIP-1) proteins. C-X-C chemokines are
strong chemoattractants and neutrophil agonists(2) , which
up-regulate adhesion molecules (3, 4) that mediate
granulocyte adherence to endothelial cells lining post-capillary venues (4) . By contrast, C-C chemokines, such as RANTES, MCP-1, and
the MIP-1 proteins, are chemoattractants for leukocytes involved in
more chronic inflammatory processes(1) .
Receptors for pro-inflammatory chemokines are members of a superfamily that have seven transmembrane-spanning helices and are coupled to guanine nucleotide-binding proteins (G-proteins)(5) . Complementary DNAs (cDNA) encoding a specific IL-8 receptor (IL-8RA) (6) and a shared receptor that binds both IL-8 and MGSA (IL-8RB) (7) have been molecularly cloned. These receptors do not bind to members of the C-C branch. Similarly, specific receptors for C-C chemokines have been cloned, but these do not bind members of the C-X-C branch(8, 9) . Recently, a promiscuous chemokine receptor (DARC), which binds C-X-C chemokines as well as C-C chemokines, was identified in erythrocytes(10, 11, 12) . This receptor, known as DARC, is identical to the Duffy blood group antigen(13) , which is a binding protein for the malarial parasite Plasmodium vivax(14) . However, since greater than 95% of Africans in endemic areas and approximately 70% of African-Americans lack expression of the Duffy blood group antigen(15) , this has raised some doubts about the possible physiologic significance of this receptor. These doubts have been somewhat allayed by the recent finding that DARC is ubiquitously expressed by endothelial cells lining post-capillary venules (16) in individuals who are positive or negative for the blood group antigen(46) . In essence, these findings provide a novel perspective into the potential relevance of DARC which is so clearly expressed in an anatomic site central to chemokine-induced leukocyte trafficking.
Although both the IL-8 receptors and DARC bind chemokines, their functions in normal and pathologic chemokine physiology are probably very different. For example, DARC lacks a DRY motif in the second intracellular loop(17) , which has been associated with G-protein coupling(18) , and stimulation of DARC transfectants with chemokine fails to reveal evidence of cytosolic mobilization of free calcium ions (19) , a characteristic feature of all other G-protein-linked chemokine receptors.
To delineate sequences responsible for the divergence between the ligand binding specificities of DARC and IL-8 receptors, chimeric receptors were analyzed for chemokine binding. The binding specificity of a hybrid receptor composed of the amino-terminal extracellular domain of DARC and the membrane-spanning helices and loops of IL-8RB resembled that of DARC. While a monoclonal antibody (mAb) to an epitope in the amino-terminal extracellular domain of DARC blocked chemokine binding, a mAb to an epitope in the extracellular loops had no effect. Thus, these data demonstrate that the amino-terminal domain of DARC contributes sequences critical for determining the repertoire of ligands bound.
Wild type and chimeric
chemokine receptors were also expressed in insect cells using a
baculovirus expression system using strategies detailed above. DNA
segments encoding DARC, IL-8RB, DARC/IL-8RB (DARC aa
1-66; IL-8RB aa 27-355), IL-8RB
/DARC (IL-8RB
aa 1-47; DARC aa 67-338), DARC
/IL-8RB (DARC
aa 1-131; IL8RB aa 117-355), and
DARC
/IL-8RB (DARC aa 1-209; IL-8RB aa
208-355) were cloned into the pVL 1393 baculovirus transfer
vector utilizing BamHI and NotI sites in the
polylinker. Constructs were co-transfected along with linearized AcMNPV
DNA (Invitrogen, San Diego) into Sf21 cells and infectious recombinant
baculovirus was isolated from culture supernatants. Infected Sf21 cells
were stained with monoclonal antibodies to the appropriate chemokine
receptor amino terminus, as described above, or with the Fy3 monoclonal
antibody.
To identify the receptor domains responsible for the
promiscuous chemokine binding of DARC, we made transfectants that
express chimeric receptors composed of complementary portions of DARC
and IL-8RB. Complementary DNA encoding DARC, IL-8RB, and a hybrid
receptor containing the amino-terminal extracellular domain of DARC and
transmembrane-spanning helices and loops of IL-8RB
(DARC/IL-8RB) were prepared by DNA amplification. cDNA
templates encoding the wild type receptors were reverse-transcribed
from total RNA and amplified using primers corresponding to 5` sense
and 3` antisense sequences following reverse transcription. These
cDNAs, in turn, served as templates for the amplification of receptor
domains for the construction of chimeras, as shown in Fig. 1.
The amino-terminal extracellular domain of DARC was amplified using an
upstream primer in the 5` non-coding region and a downstream primer (Ch1) corresponding to sequences complementary to those
encoding seven amino acid residues located in the proximal portion of
the amino-terminal extracellular domain at the interphase of the first
transmembrane-spanning helix of DARC (residues 62-69) and IL-8RB
(residues 27-34). Similarly, a segment encoding the
transmembrane-spanning helices and loops of IL-8RB was amplified using
an upstream primer (Ch2) containing sequences complementary to
those present in the downstream DARC primer (Ch1) and a
downstream primer in the 3`-untranslated regions. These two overlapping
segments served as templates in a PCR reaction containing a 5` sense
DARC primer and a 3` antisense IL-8RB primer, thereby generating a
hybrid cDNA encoding a chimeric receptor. Clones encoding each receptor
were verified by nucleotide sequence analysis, subcloned into an
episomal vector, and introduced into K562 cells by lipofection.
Following selection in hygromycin, transfectant pools were analyzed for
receptor expression by flow cytometry using the appropriate monoclonal
antibodies to detect DARC and DARC
/IL-8RB (Fy6) and the
IL-8RB (10H2). As shown in Fig. 2, transfectants with high level
receptor expression were enriched by multiple rounds of
fluorescence-activated cell sorting. Transfectants with high level
expression were used for analysis of ligand binding.
Figure 1:
Schematic
representation of wild type IL-8RB and DARC and the chimeric receptor
(DARC/IL-8RB) composed of the amino-terminal extracellular
domain of DARC and the predicted loops and transmembrane-spanning
helices of IL-8RB.
Figure 2:
Flow cytometric analysis of wild type and
chimeric chemokine receptor expression by K562 transfectants. Prior to
chemokine binding experiments, K562 transfectants were stained with
monoclonal antibodies to the amino terminus of the respective receptors
(Fy6, DARC and DARC/IL-8RB; 2H10, IL-8RB) as described
under ``Experimental Procedures'' ( . . . , specific
antibody, -, myeloma protein control). All of the transfectants
(IL-8RB, top panel; DARC, middle panel; and
DARC
/IL-8RB, bottom panel) showed high level
expression of their respective receptors.
To identify the
receptor domains in DARC that give rise to the promiscuous chemokine
binding of the molecule, we compared the ligand binding profile and
receptor binding affinity of the DARC/IL-8RB chimera with
those of the parent receptors, DARC and IL-8RB. To facilitate direct
comparison of binding affinities, the receptors were stably expressed
in K562 cells, and receptor competition curves were generated over a
wide concentration range of unlabeled chemokines (MGSA, IL-8,
MGSA-E
A) (Fig. 3). Scatchard analysis of
I-MGSA displacement binding demonstrated that DARC
transfectants bound MGSA with a K
of 21
nM, which is consistent with that previously
reported(26) . Equally, IL-8RB transfectants bound MGSA at high
affinity, 9.8 nM, similar to that observed in
neutrophils(27) . Scatchard analysis of
I-MGSA
binding to DARC
/IL-8RB transfectants showed that the
hybrid receptor bound ligand with an affinity of 18 nM akin to
that observed for DARC. Receptor binding experiments with
I-IL-8 were virtually identical to those obtained with
MGSA (K
DARC 20 nM, IL-8RB 2.2
nM, and DARC
/IL-8RB 5.5 nM).
Figure 3:
Inhibition of binding of I-MGSA to K562 cells transfected with DARC (A),
IL-8RB (B), and DARC
/IL-8RB (C) by
unlabeled MGSA, IL-8 and E
A. Cells (1
10
cells/ml) were incubated for 1 h at 4 °C with
I-MGSA (0.2 nM) in the presence of increasing
concentrations of unlabeled MGSA, IL-8, and E
A. The
incubation was terminated by removing aliquots from the cell suspension
and separating cells from buffer by centrifugation through a
silicone/paraffin oil mixture as described previously(25) .
Nonspecific binding was determined in the presence of 1 µM unlabeled ligand. The binding data were curve-fit with the
computer program IGOR to determine receptor binding affinity (K
).
The
ligand binding profile of the DARC/IL-8RB chimeric
receptor was further analyzed by receptor binding studies with an MGSA
mutant, E
A, that has previously been demonstrated to bind
DARC, but not IL-8RB(21) . Scatchard analysis of displacement
of
I-MGSA by MGSA-E
A revealed a K
of 28 nM for the hybrid receptor and 27
nM for DARC. In contrast, the K
for
MGSA-E
A binding to IL-8RB was 476 nM, which was
consistent with that previously reported (21) (Fig. 3).
Although DARC and IL-8RB have virtually identical binding specificities
for C-X-C chemokines, they are not highly related at the level
of primary structure (17, 28) and DARC, in addition,
binds chemokines of the C-C
type(10, 11, 12) . The binding site on
chemokines recognized by DARC is probably different from that
recognized by IL-8RB, since mutation of the ELR motif does not abrogate
binding to DARC(21) , as is observed for the
IL-8RB(29, 30) .
The promiscuous binding repertoire
of the DARC/IL-8RB chimeric receptor was further
demonstrated by displacement of
I-RANTES binding with
C-X-C and C-C chemokines. IL-8 and RANTES were capable of
displacing the binding of
I-RANTES to K562 transfectants
expressing the hybrid receptor and DARC (Fig. 4). As expected,
cells transfected with the IL-8RB did not bind radiolabeled RANTES
(data not shown). Since the C-C chemokine RANTES was able to
specifically bind to the DARC
/IL-8RB receptor chimera, we
determined the affinity of RANTES binding to the hybrid by Scatchard
analysis (Fig. 5). Scatchard analysis of
I-RANTES
binding to the hybrid receptor yielded a linear plot consistent with a
single class of high affinity binding sites with a K
of 5.0 nM compared to a K
of 7.0
nM for binding to DARC (data now shown). Competition of
I-MCP-1 binding with unlabeled MCP-1 resulted in
augmentation of radioligand binding, an effect that has been previously
reported with chemokines(31) .
Figure 4:
Inhibition of I-RANTES
binding to K562 cells transfected with DARC, and
DARC
/IL-8RB. Cells were incubated for 1 h at 40 °C
with
I-RANTES, as indicated above, in the absence of
cross competing ligand (solid boxes) or with 100 nM
concentrations of unlabeled IL-8 (open boxes) or RANTES (cross-hatched boxes).
Figure 5:
Scatchard analysis of I-RANTES binding to K562 cells transfected with
DARC
/IL-8RB. Cells (1
10
cells/ml)
were incubated with
I-labeled RANTES (0.2 nM) in
the presence and absence of increasing concentrations of unlabeled
RANTES. The Scatchard plot shown here is representative of three
separate determinations. Binding shown represents specific binding.
Nonspecific binding was around 2% of total
I-RANTES added
and has been subtracted.
These findings implicate the amino-terminal extracellular domain of DARC as a critical region in determining the promiscuous ligand binding repertoire. Additional studies were performed to gain direct insight into the role of the extracellular loops in receptor binding. In the context of the Duffy blood group antigen, the monoclonal antibody Fy6 is directed against an epitope in the amino-terminal extracellular domain and is sensitive to proteolysis with chymotrypsin. A second antibody type, designated Fy3, recognizes a chymotrypsin-insensitive epitope expressed at the cell surface, which has not yet been located on the receptor.
Since the
Fy3 epitope apparently resides on a domain of DARC other than the
amino-terminal extracellular domain, the effect of the monoclonal
antibody on the ability of the receptor to bind labeled chemokine
ligand was determined. As shown in Fig. 6, the binding of I-MGSA to DARC-positive erythrocytes was inhibited by
preincubation of target cells with Fy6 (50% inhibition at 100
pM). In contrast, preincubation of the erythrocytes with
increasing concentrations of Fy3 (up to 175 µM final
concentration) resulted in chemokine binding levels virtually identical
to that observed in reactions containing equal amounts of a control
monoclonal antibody to IL-8RB, which does not bind to DARC, as well as
a non-immune mouse myeloma protein (data not shown).
Figure 6:
Inhibition of MGSA binding to DARC by
anti-Fy6 and anti-Fy3 monoclonal antibodies to the human Duffy blood
group antigen. Erythrocytes (1 10
cells in 500
µl) were preincubated with increasing concentrations of anti-Fy6 or
anti-Fy3 for 1 h at 4 °C. The cells were then incubated for another
hour with 0.25 nM
I-labeled MGSA in a final
volume of 600 µl. The binding reactions were terminated as
described previously. Nonspecific binding was determined by addition of
100 nM unlabeled IL-8 to the binding reactions. The effect of
an antibody to IL-8RB on MGSA binding to erythrocytes was also
determined.
The absence of
inhibition of chemokine binding by the Fy3 monoclonal antibody, a
sterically large molecule, suggests that the domain(s) that harbor the
epitope may have an inconspicuous role in ligand binding. The region of
DARC that harbors the Fy3 epitope was further investigated by testing
Sf21 cells that express chimeric receptors for binding to Fy3. As shown
in Fig. 7, flow cytometric analysis confirmed that Sf21 cells
infected with a recombinant baculovirus encoding DARC reacted with Fy3,
as well as Fy6 (panel A). Sf21 cells infected with the virus
encoding DARC/IL-8RB lacked reactivity with Fy3 but
expressed the chimeric receptor on the cell surface, as evidenced by
binding of the DARC
domain to Fy6 (panel B).
Infection of Sf21 cells with the IL-8RB
/DARC virus
resulted in cell surface expression of a chimeric receptor that bound
Fy3, localizing this epitope to the extracellular loops of DARC (panel C). The Fy3 epitope was further localized by parallel
analysis of chimeric receptors containing the amino-terminal
extracellular domain and first extracellular loop of DARC
(DARC
/IL-8RB) and the amino-terminal extracellular
domain, first, and second extracellular loops of DARC
(DARC
/IL-8RB). Sf21 cells infected with recombinant
baculoviruses encoding DARC
/IL-8RB (panel D)
and DARC
/IL-8RB (panel E) expressed chimeric
receptors on the plasma membrane that bound Fy6 but not Fy3. These data
indicate that sequences in the predicted third extracellular loop are
required for expression of the Fy3 epitope. Sf21 cells expressing
IL-8RB also lacked binding to Fy3 (data not shown). The finding that a
monoclonal antibody to the amino-terminal extracellular domain of DARC
blocks ligand binding, whereas a monoclonal antibody to the third
extracellular loop has no effect, further implicates the former in
ligand binding and supports the finding of promiscuous chemokine
binding to the chimeric receptor that contains the amino-terminal
extracellular domain of DARC.
Figure 7: Analysis of insect cells expressing chimeric receptors for epitopes recognized by anti-Fy6 and anti-Fy3. Sf21 cells were infected with recombinant baculoviruses encoding DARC (panel A) or chimeric receptors composed of complementary segments of DARC and IL-8RB (panels B-E, see diagram). Cells stained with anti-Fy6, anti-Fy3, and a control mouse myeloma protein by indirect immunofluorescence on day 3 were analyzed by flow cytometry.
In contrast to other chemoattractant
receptors, such as the N-formyl peptide receptor (32) and the C5a receptor(33, 34) which are
also members of the serpentine receptor family, the amino-terminal
extracellular domain of the IL-8 receptors have been shown to be major
determinants in determining ligand binding specificity(35) .
The repertoire and affinity of chemokine binding by DARC and the IL-8RB
were analyzed to determine the domain(s) responsible for promiscuous
chemokine binding. A chimeric receptor composed of the amino-terminal
extracellular domain of DARC and the transmembrane-spanning helices and
loops of IL-8RB were efficiently expressed on the cell surface and
found to bind MGSA and IL-8, as well as RANTES, at high affinity.
Moreover, this hybrid receptor bound MGSA-EA, a mutant form
of MGSA that lacks high affinity binding to IL-8RB but retains high
affinity binding to DARC. Together, these findings indicate that
sequences that confer a promiscuous ligand binding repertoire are
localized to the amino-terminal extracellular domain of DARC.
The ligand binding pockets of the vast majority of seven transmembrane-spanning receptors, aside from receptors for glycoprotein hormones(36, 37) , and chemokines(5, 38) , involve hydrophobic segments predicted to be localized to intramembranous regions(32, 39) . Mutational analysis of receptors for C5a and N-formyl peptides, both potent chemoattractants for neutrophils, revealed that ligand binding domains involved sequences in the transmembrane-spanning helices(34, 40) . A two-site model of ligand binding, which includes transmembrane-spanning loops and the amino-terminal extracellular domain, has been proposed for the C5a receptor(33, 34) . These data are based on the finding that antagonists of C5a binding fail to inhibit the binding of peptides from the carboxyl terminus of C5a and that truncation of the amino-terminal extracellular domain of the receptor, and also protease digestion, abolish the binding of intact C5a, but not binding and receptor activation by carboxyl-terminal peptides(33, 34) .
Based on primary structure, DARC is as closely related to the endothelin receptors as it is to the IL-8RB. The three non-allelic forms of endothelins are potent vasoactive agents secreted by endothelial cells of veins and arteries that have 21 amino acid residues and two intrachain disulfide bonds(41) . Two receptors of different ligand binding specificity, designated type A and B, have been cloned(42, 43) . Analysis of the domains involved in endothelin binding reveals that the endothelin type A receptor, which is expressed primarily by smooth muscle cells, has a ligand binding pocket that includes sequences in transmembrane helices I, II, III, and VII. In contrast, the endothelin type B receptor, which binds all three endothelins at similar affinity and is expressed by endothelial cells, has a ligand binding pocket that involves sequences from transmembrane-spanning helices IV-VI(44) .
A similar
receptor chimera approach has been used to define the domain(s) of the
type B receptor for IL-8 that confer high affinity binding of
MGSA(35) . Analysis of ligand binding characteristics of
chimeras composed of reciprocal portions of IL-8RA and IL-8RB revealed
that the amino-terminal extracellular domain was sufficient to confer
high affinity binding to both IL-8 and MGSA to the
IL-8RB/IL-8RA chimeric receptor. The reciprocal chimera,
IL-8RA
/IL-8RB, demonstrated high affinity binding limited
to IL-8, indicating that the extracellular loops did not contribute to
ligand binding specificity.
It is not clear whether the amino terminus of receptors for IL-8 contain all of the sequences necessary for high affinity chemokine binding. However, at least for the IL-8RA it has been demonstrated that residues in the amino-terminal extracellular domain and each of the extracellular loops, primarily cysteine residues, are required for high affinity ligand binding(45) . While several reports have used genetic approaches to study structure-function relationships in heptahelical receptors, this is the first demonstration of domains involved in chemokine binding using both genetic techniques and monoclonal antibodies to multiple topologic regions of the receptor.
The extracellular loops of chemokine receptors could function as a second contact point for chemokine binding that does not impart ligand specificity or as a polypeptide scaffold that interacts with the amino-terminal extracellular domain to generate a conformation of the latter permissive for high affinity binding. The finding that the binding of an Fy3 immunoglobin macromolecule to the third predicted extracellular loop of DARC failed to alter chemokine binding provides independent evidence to support the interpretation that the loops contribute to the overall conformation required to form a binding pocket, but not as a second contact point for binding. Thus, based on our findings and the above studies, we postulate that although the amino-terminal extracellular domain of DARC contains the primary structural motifs required for promiscuous chemokine binding, the extracellular loops of this receptor may also play a role through a cooperative interaction to form an extracellular structure with the appropriate conformation for binding to pro-inflammatory chemokine ligands. Since occupation of the binding pocket antagonizes the association of the receptor with the P. vivax polypeptide ligand(13) , these studies also may provide valuable insight into the pathophysiology of malarial invasion of erythroid cells.