(Received for publication, July 27, 1995; and in revised form, October 12, 1995)
From the
Human interleukin-8 receptors A (IL-8RA) and B (IL-8RB) are
seven-transmembrane domain (TMD) neutrophil chemokine receptors with
similar sequences (77% amino acid identity) and similar G protein
selectivity, but markedly different selectivity for CXC chemokines.
IL-8RB is selective for IL-8, growth-related oncogene (GRO
)
and neutrophil-activating peptide-2 (NAP-2), whereas IL-8RA is
selective only for IL-8. To identify selectivity determinants, we made
eight chimeric receptors exchanging: 1) the three main regions of
sequence divergence between IL-8RA and IL-8RB (the N-terminal segment
before TMD1, the region from TMD4 to the end of the second
extracellular (e2) loop, and the C-terminal tail), and 2) the
N-terminal segment of CC chemokine receptor 1, which does not bind CXC
chemokines. Chimeras were tested by direct
I-IL-8,
I-GRO
, and
I-NAP-2 binding,
heterologous competition binding, and calcium flux assays using human
embryonic kidney 293 cells stably transfected with receptor DNAs. The
following results were obtained: 1) chimeric receptors had binding
sites for IL-8, GRO
and NAP-2 distinct from those on IL-8RA and
IL-8RB; 2) IL-8, GRO
and NAP-2 bound to overlapping but distinct
sites that mapped differentially to multiple domains on IL-8RB; 3) high
affinity radioligand binding and high agonist potency were separable
functions for IL-8, GRO
and NAP-2, suggesting that the
determinants of high affinity binding may not be critical for receptor
activation; and 4) determinants of GRO
and NAP-2 selectivity were
found in both the N-terminal segment before TMD1 and the region from TMD4 to the end of the e2 loop of IL-8RB, and
functioned independently of each other. Stated reciprocally, the
N-terminal segment of IL-8RA was not a dominant selectivity
determinant. These data suggest that both narrow and broad spectrum
chemokine antagonists can be developed to block functions mediated by
IL-8RB.
Interleukin-8 (IL-8), ()growth-related oncogene
(GRO
) and neutrophil-activating peptide-2 (NAP-2) are members of
the CXC branch of the chemokine superfamily of leukocyte
chemoattractants and activating factors(1) . All three
molecules are relatively selective for neutrophils, binding to the same
G
-coupled 7-transmembrane domain (7TMD) receptor named
interleukin-8 receptor B (IL-8RB; (2) and (3) ). While
it is thought that IL-8, GRO
, and NAP-2 act beneficially in host
defense and tissue repair, there is so far little direct evidence. On
the contrary, IL-8 has great pathologic potential, shown most clearly
by the ability of intravenous infusions of anti-IL-8 antibodies to
block lung ischemia-reperfusion injury (4) and
endotoxin-induced pleurisy (5) in rabbit models. Thus, IL-8
antagonists could be useful clinically in disorders that have an acute
inflammatory component, and substantial efforts are already under way
to develop them. The aim of the present study was to obtain basic
information about the nature of IL-8, GRO
, and NAP-2 binding sites
on IL-8RB that could be used to guide the development of antagonists.
By analogy with the structure of rhodopsin, the membrane-spanning
segments of all 7TMD receptors are thought to be arranged in a circle,
causing the extracellular domains to bunch together (reviewed in (6) ). Very small ligands such as catecholamines bind mainly to
TMD determinants, and very large glycoprotein ligands such as
thyroid-stimulating hormone bind mainly to the unusually long
N-terminal segment of their respective receptors(6) . Small
peptide ligands such as the neurokinins appear to bind to sites in the
extracellular loops and TMDs(6) . Chemokines are intermediate
in size, 70 amino acids, and chemokine receptors have relatively
small N-terminal segments(1, 3) . Since the potential
cumulative binding surface of the chemokine receptor ectodomains is
comparable in size to that of the ligand and since receptor binding is
sensitive to truncation of both N- and C-terminal residues of
IL-8(7) , it is likely that chemokine binding determinants are
broadly distributed on multiple domains of the receptors. C5a is a
non-chemokine chemotactic peptide for leukocytes that is similar in
size to chemokines(3) . It has been shown to bind to at least
two sites on a specific 7TMD receptor that is similar in size,
organization and sequence to IL-8RB(8, 9) . One site
is present on the N-terminal extracellular segment before TMD1, and the
second is within the TMDs.
The first experiments aimed at
identifying receptor regions important for chemokine binding by IL-8RB
took advantage of the fact that a second high affinity 7TMD neutrophil
IL-8 receptor exists (IL-8RA) that is 77% identical in amino acid
sequence to IL-8RB, yet binds neither GRO nor
NAP-2(10, 11, 12) . Most of the divergent
residues are clustered in three regions: 1) the N-terminal segment
before TMD1, 2) the region from TMD4 to the end of the second
extracellular (e2) loop, and 3) the C-terminal cytoplasmic tail (Fig. 1). Using transiently transfected mammalian cells and
human chemokines, both LaRosa et al.(13) and Gayle et al.(14) reported that a chimeric receptor in which
the N-terminal segment of rabbit IL-8RA (15) replaced the
corresponding region of human IL-8RB (chimera AB1) bound
I-IL-8 with high affinity, but GRO
(13, 14) and NAP-2 (13) competed weakly for binding,
mimicking results for human (11, 12) and rabbit wild
type IL-8RA(15) . Thus, the N-terminal segment appeared to be a
dominant determinant of receptor subtype selectivity. The two groups
reported conflicting results for the reciprocal chimera BA1; LaRosa et al.(13) reported high affinity
I-IL-8 binding that was effectively competed by unlabeled
GRO
and NAP-2 mimicking wild type IL-8RB, whereas Gayle et al.(14) reported that BA1 did not bind
I-IL-8,
even though BA1 expression on the cell surface was confirmed by
detection of an epitope tag. Neither study addressed direct binding of
either
I-GRO
or
I-NAP-2 or receptor
activation, and the approach taken was not designed to identify the
determinants of IL-8 selectivity. Moreover, the reported inability of
human GRO
and NAP-2 to compete effectively for
I-human IL-8 binding sites on the rabbit counterpart of
IL-8RB raised the possibility that the properties of cross- and
same-species IL-8 receptor chimeras could be fundamentally
different(16) .
Figure 1: Sequence alignment of IL-8RA, IL-8RB and amino acids 1-34 of CC CKR1. Vertical bars indicate identical residues for each adjacent sequence position. Dots indicate gaps that were inserted to optimize the alignment. The location of predicted membrane-spanning segments I-VII are noted. Boxes enclose consensus sequences for N-linked glycosylation. Arabic numbers correspond to the sequence of IL-8RA and are left justified. Arrows designate the aa position at which the sequences between receptors were switched using either polymerase chain reaction techniques or conserved restriction sites.
In this paper we have addressed all of these concerns, by investigating both the agonist selectivity and ligand binding selectivity for wild type human IL-8RA and IL-8RB, and for a series of eight chimeric IL-8 receptors. We present a substantially expanded interpretation of the importance of the N-terminal segment in human IL-8 receptor subtype determination that is based on new findings regarding the complexity of CXC chemokine binding sites.
The numbers in the names of the first five chimeras listed denote
the TMD where the switch occurs. In AB1 and BA1 all the divergent
residues in the exchanged regions precede aa 37 of IL-8RA and aa 46 of
IL-8RB. Thus, the switched divergent residues correspond to those
studied in LaRosa et al.(13) and Gayle et
al.(14) . The fidelity of the cloned expression plasmids
was verified by DNA sequencing. Human embryonic kidney (HEK) 293 cells
(10), grown to log phase in Dulbecco's modified
Eagle's medium with 10% fetal bovine serum, were electroporated
with 20 µg of plasmid DNA as described previously(19) .
Multiple hygromycin-resistant colonies were picked and expanded in 150
µg/ml hygromycin.
Figure 2:
Chemokine selectivity of wild type IL-8
receptors. HEK 293 cells stably transfected with plasmids encoding
human IL-8RA (a-d) or human IL-8RB (e-h)
were analyzed for their agonist selectivity (a and e), and ligand binding selectivity to I-IL-8 (b and f),
I-GRO
(c and g), and
I-NAP-2 (d and h). At
the top of each column of panels is the name of the corresponding IL-8
receptor tested and a schema illustrating its sequence composition (white, IL-8RA sequence; black, IL-8RB sequence). The
code for the symbols used is given at the upper left of the
figure. The average number of binding sites per cell calculated using
either
I-IL-8 and unlabeled IL-8 or
I-NAP-2
and unlabeled NAP-2 was between 1 and 2
10
for
IL-8RA and IL-8RB.
Figure 3:
Chemokine selectivity of chimeric
receptors switched in TMD1. HEK 293 cells stably transfected with the
following chimeras, AB1 (a-d), BA1 (e-h),
or CC CKR1/IL-8RB (i-l), were analyzed for their agonist
selectivity (a, e, and i), and ligand
binding selectivity to I-IL-8 (b, f,
and j),
I-GRO
(c, g, and k), and
I-NAP-2 (d, h, and l). At the top of each column of panels is the name of the
corresponding chimeric receptor and a schema illustrating its sequence
composition (white, IL-8RA sequence; black, IL-8RB
sequence; diamond, CC CKR1 sequence). The code for the symbols
used is given at the upper left of the figure. The average number of
binding sites per cell calculated using either
I-IL-8 and
unlabeled IL-8 or
I-NAP-2 and unlabeled NAP-2 was between
1 and 2
10
for AB1 and CC CKR1/IL-8RB, but 40,000
for BA1. Specific binding of
I-MIP-1
or
I-RANTES was not detected for CC
CKR1/IL-8RB.
Figure 4:
Mapping of GRO and NAP-2 determinants
in residues 51-360 of IL-8RB. HEK 293 cells stably transfected
with chimeras ABA (a-d), AB7 (e-h), or
AB5 (i-l) were analyzed for their agonist selectivity (a, e, and i) and ligand binding affinity to
I-IL-8 (b, f, and j),
I-GRO
(c, g, and k), and
I-NAP-2 (d, h, and l). At the
top of each column of panels is the name of the corresponding chimera
and a schema illustrating its sequence composition (white,
IL-8RA sequence; black, IL-8RB sequence). The average number
of binding sites per cell calculated using either
I-IL-8
and unlabeled IL-8 was between 1 and 2
10
for ABA,
AB7, and AB5.
Figure 5:
Ligand binding and agonist selectivity of
the chimeric IL-8 receptors. HEK 293 cells stably transfected with the
following chimeras, BAB (a-d) and BA5 (e-h), were analyzed for their agonist selectivity (panels a and e) and ligand binding affinity to I-IL-8 (b and f),
I-GRO
(c and g), and
I-NAP-2 (d and h). At the top of each
column of panels is the name of the corresponding IL-8 receptor tested
and a schema illustrating its sequence composition (white,
IL-8RA sequence; black, IL-8RB sequence). The code for the
symbols used is given at the upper left of the figure. The
average number of binding sites per cell calculated using
I-IL-8 and unlabeled IL-8 was between 1 and 2
10
for BA5.
The relative binding affinities of IL-8,
GRO, and NAP-2 for IL-8RA and IL-8RB correlated well with their
relative potencies as agonists. In the case of IL-8RA,
I-IL-8 bound with high affinity (K
= 3.6 nM; Fig. 2b), whereas
I-GRO
and
I-NAP-2 binding was not detectable (Fig. 2, c and d). Nevertheless, an interaction of GRO
and
NAP-2 with IL-8RA could be inferred by their ability to compete weakly
for the
I-IL-8-labeled sites on IL-8RA (Fig. 2b). In the case of IL-8RB,
I-IL-8,
I-GRO
, and
I-NAP-2 all were strong
ligands, binding with similar affinities (Fig. 2, f-h, and Table 1). These affinities were also
similar to the binding affinity for
I-IL-8 on IL-8RA (Table 1).
The rank order of competition by unlabeled CXC
chemokines for the radiolabeled binding sites on IL-8RB varied
considerably for the three different radioligands (Fig. 2, f-h, and Table 1). For example, unlabeled NAP-2
competed poorly for the I-IL-8-labeled sites on IL-8RB,
whereas both NAP-2 and IL-8 were very effective in competing for the
I-NAP-2-labeled sites on IL-8RB. These findings suggest
that the sets of binding sites on IL-8RB for IL-8, GRO
and NAP-2
overlap but are not identical. The unlabeled CC chemokines MIP-1
and MCP-1 did not compete for binding at any of the CXC
chemokine-labeled sites (Fig. 2, f-h).
These
results confirm previous reports based on direct IL-8 and GRO
binding (11, 12) and provide new information about
direct NAP-2 binding and the relative potency of all three chemokines
on both receptor subtypes. To investigate the structural basis of the
markedly different selectivities found for IL-8RA and IL-8RB, we made
chimeric receptors in which the main regions of divergence were
switched.
From these results we infer that
independent determinants of GRO and NAP-2 agonist selectivity are present in the IL-8RB-specific sequences both N-terminal and C-terminal to the switch point in TMD1.
LaRosa et al.(13) and Gayle et al.(14) did not report the agonist selectivity of their
chimeras. To compare more directly the properties of human-human AB1
and BA1 with those of rabbit-human chimeras, we carried out ligand
binding experiments.
In fact, the rank order of competition for the I-IL-8 binding site on human-human AB1 was identical to
that reported for rabbit-human AB1 and mimicked that for wild type
human IL-8RA (compare Fig. 2b and Fig. 3b, and Table 1). To address the apparent
incompatibility between the indirect ligand binding and signal
transduction results for GRO
and NAP-2, we carried out direct
I-GRO
(Fig. 3c) and
I-NAP-2 (Fig. 3d) binding with AB1, and
found that both radioligands were able to bind with high affinity. The
rank orders of competition determined with unlabeled CXC chemokines
indicate clearly that the sets of binding sites for
I-IL-8,
I-GRO
, and
I-NAP-2 on AB1 are overlapping but distinct from each
other, and distinct from the corresponding sites on IL-8RB (compare Fig. 3, panels b-d, and Fig. 2, panels
f-h; see also Table 1).
Chimera BA1 also had
binding properties distinct from both wild type receptors. Despite the
fact that IL-8 was its most potent agonist, similar in potency to wild
type IL-8RB (EC = 10 and 5 nM,
respectively; Fig. 2e and 3e), we could not
demonstrate specific binding of
I-IL-8 to it (Fig. 3f). This matches the report of Gayle et al. for rabbit-human BA1 (14) but differs from that of LaRosa et al.(13) . In contrast,
I-GRO
(Fig. 3g) and
I-NAP-2 (Fig. 3h) both bound to BA1 with high affinity. As for
AB1, competition binding analysis with unlabeled CXC chemokines
distinguished the binding sites for
I-GRO
and
I-NAP-2 on BA1 from those on IL-8RB (compare panels g and h in Fig. 2and Fig. 3; see also Table 1).
To test further the importance of IL-8RB regions
C-terminal to TMD1 for GRO and NAP-2 selectivity, we replaced
residues 1-50 of IL-8RB with the corresponding residues of the CC
chemokine restricted receptor CC CKR1 (amino acids 1-34), making
chimera CC CKR1/IL-8RB. The switched residues comprise the N-terminal
segments of the respective receptors (2, 18) . The
sequence of residues 1-34 of CC CKR1 differs markedly from the
corresponding sequence of IL-8RA and IL-8RB, but all three have a high
content of acidic residues and several invariant residues are present (Fig. 1). This chimera is also useful for mapping determinants
of IL-8 selectivity in IL-8RB, which cannot be meaningfully analyzed in
chimeras made from the two IL-8 receptors.
As for AB1, IL-8,
GRO, and NAP-2 all had high equipotent agonist activity for CC
CKR1/IL-8RB (Fig. 3i). In contrast, the agonists for CC
CKR1 (MIP-1
, RANTES, and MCP-3) lacked agonist activity for CC
CKR1/IL-8RB (Fig. 3i). The agonist rank order was IL-8
= GRO
NAP-2 (EC
10 nM in
each case), similar to that for wild type IL-8RB and chimera AB1 (Table 1). This further suggested that IL-8RB sequence C-terminal
to TMD1 contains determinants of GRO
and NAP-2 agonist selectivity
that can operate independently of GRO
and NAP-2 determinants in
the N-terminal segment of IL-8RB. Furthermore, it indicated that the
same region of IL-8RB contains determinants of IL-8 agonist
selectivity.
As we found for AB1 and BA1, all of the binding sites
on CC CKR1/IL-8RB were distinct from those on the wild type receptors (Fig. 3, j-l). The least unusual was the NAP-2
site (Fig. 3l). I-NAP-2 bound to CC
CKR1/IL-8RB with high affinity (K
= 1
nM). However, the rank order of competition for
I-NAP-2 binding was NAP-2 > GRO
> IL-8 for CC
CKR1/IL-8RB (Fig. 3l; Table 1) as compared to
IL-8
GRO
> NAP-2 for IL-8RB (Table 1). Binding of
I-GRO
to CC CKR1/IL-8RB was much more anomalous. A
high percentage of total radioligand added bound to cells expressing CC
CKR1/IL-8RB; however, unlabeled GRO
did not compete for binding
even when present in 500-fold molar excess (Fig. 3k).
Paradoxically, unlabeled IL-8 competed for the
I-GRO
site on CC CKR1/IL-8RB, but not very effectively (Fig. 3k). NAP-2 did not compete for
I-GRO
binding, even though
I-NAP-2
bound with high affinity (Fig. 3, k and l).
The IL-8 binding site on CC CKR1/IL-8RB was even more unusual and was
reminiscent of the IL-8 binding site on BA1. Even though IL-8 was a
potent agonist for CC CKR1/IL-8RB,
I-IL-8 binding was not
detectable (Fig. 3, i and j). However, a
binding interaction of IL-8 with CC CKR1/IL-8RB could be inferred by
its ability to compete for the
I-GRO
and
I-NAP-2 sites (Fig. 3, k and l).
Taken together, the results suggest that the determinants of high
affinity I-IL-8 binding differ from the determinants of
high agonist activity for IL-8, and that the N-terminal segment of
IL-8RB contains determinants of high affinity
I-IL-8
binding. The latter conclusion is consistent with the results of Suzuki et al.(21) , who reported that a chimeric receptor
composed of the N-terminal segment of rabbit IL-8RA and the sequence
C-terminal to TMD1 of the mouse IL-8 receptor homologue bound
I-human IL-8 with high affinity, whereas the wild type
mouse IL-8 receptor homologue did not(22, 23) . The
former conclusion was not tested by Suzuki et
al.(21) .
The most direct test of the importance of the
N-terminal segment in chemokine selectivity and receptor function is to
remove it; however, experiments with truncated receptors have been
non-informative so far ((14) ). ()Mutagenesis of the
IL-8RB-specific residues in chimera BA1 will be needed to locate more
precisely the GRO
and NAP-2 selectivity determinants in this
region. Additional mutagenesis of the invariant residues and acidic
residues in this region may also provide important insights for the
structural basis of chemokine selectivity.
IL-8 was a potent agonist for
chimera AB7, whereas GRO and NAP-2 were not agonists, suggesting
that the IL-8RB-specific residues present in the C-terminal cytoplasmic
tail do not determine GRO
and NAP-2 agonist selectivity (Fig. 4e). The region from the BamHI site to
the NcoI site contains 10 amino acid differences between
IL-8RA and IL-8RB that were not selectively tested in any of the
chimeras presented so far (Fig. 1). In chimera AB5 this region
was switched together with the C-terminal cytoplasmic tail (Fig. 4i). As we observed for ABA and AB7, IL-8 was a
strong agonist for AB5 but GRO
completely lacked agonist activity.
However, NAP-2 remained a strong agonist (Fig. 4i).
Taken together, the results of these functional tests suggest that
independent determinants of NAP-2 agonist selectivity are present in
three separate regions of IL-8RB: 1) the N-terminal segment, 2) the
region from TMD4 to the end of the e2 loop, and 3) among the 10
IL-8RB-specific amino acids from the BamHI and NcoI
sites, but are not present in the C-terminal cytoplasmic tail. In
contrast, independent determinants of GRO agonist selectivity are
present in the first two of these three regions.
When binding
experiments were carried out, the results for ABA and AB7 were very
much in line with those expected based on the signal transduction
results. In the case of ABA, I-IL-8,
I-GRO
, and
I-NAP-2 all bound with high
affinity (Fig. 4, b-d, and Table 1). The
apparent affinities of
I-GRO
and
I-NAP-2 on ABA were similar to each other and to the
corresponding affinities on IL-8RB, and correlated well with the
relative potency of GRO
and NAP-2 as agonists for both ABA and
IL-8RB (Table 1). The apparent affinity of ABA for
I-IL-8 was
200-fold greater than for
I-GRO
and
I-NAP-2 on ABA and for
I-IL-8,
I-GRO
, and
I-NAP-2 on wild type IL-8RB, which correlated well with
the high agonist activity of IL-8 for ABA relative to IL-8RB (Table 1).
In the case of AB7, only I-IL-8
binding was detectable and the apparent affinity was similar to that
observed for wild type IL-8RA and IL-8RB (Fig. 4f);
unlabeled GRO
and NAP-2 competed very ineffectively for
I-IL-8 binding to AB7 (Fig. 4, g and h). This also correlated well with the signal transduction
results for AB7 (Fig. 4e).
In contrast, even though
NAP-2 was a potent agonist for AB5, we were unable to detect I-NAP-2 binding to it (Fig. 4l). A
binding interaction of NAP-2 with AB5 consistent with its agonist
activity could be inferred by the ability of unlabeled NAP-2 to compete
for
I-IL-8 binding to AB5 (Fig. 4j). This
is the third chimera of those presented so far in which direct binding
of a radiolabeled agonist was not demonstrable.
I-GRO
binding to AB5 was not detectable, nor did
unlabeled GRO
compete significantly for
I-IL-8
binding (Fig. 4, j and k). These results were
concordant with GRO
's lack of agonist activity for AB5 (Fig. 4i).
The results presented so far can be
summarized as follows. 1) Complementary chimeras do not have
complementary functional properties; 2) chimeric receptors have sets of
chemokine binding sites that are distinct from those on wild type
IL-8RA and IL-8RB; 3) high affinity I-chemokine binding
and high chemokine potency are separable functions in chimeric
receptors; 4) multiple independent regions of IL-8RB can determine
agonist selectivity for GRO
and NAP-2; and 5) the sets of binding
sites for IL-8, GRO
, and NAP-2 on IL-8RB are overlapping but
distinct.
To test these conclusions further, we made two additional
chimeras, BA5 and BAB, the complements of AB5 and ABA, respectively.
IL-8, GRO, and NAP-2 were equipotent agonists for both chimeras,
supporting conclusion 4 (Fig. 5, a and e). The
agonist selectivities of BAB and BA5 support conclusion 1; the agonist
selectivity of BAB was identical to that of ABA (Fig. 4a and Fig. 5a), whereas the agonist selectivity of
BA5 and AB5 were also similar except for the inability of AB5 to
respond to GRO
(Fig. 4i and Fig. 5e). The rank order of competition for each of the
labeled sites on BA5 was distinct from IL-8RB, supporting conclusion 2 (Fig. 5, f-h, and Table 1). Binding of
radiolabeled IL-8, GRO
, and NAP-2 was not detectable for BAB, even
though all three unlabeled proteins were highly potent and effective
agonists (Fig. 5, a-d), supporting conclusion 3.
It is important to note that BA5 contained IL-8RB-specific sequence in
both the N-terminal segment and the region from TMD4 to the end of the
e2 loop, i.e. in two of the regions that contain independent
determinants of GRO
and NAP-2 agonist selectivity shown in
chimeras BA1, BAB, and ABA. The combined chemokine binding and agonist
selectivity found for BA5 is the most like wild type IL-8RB of the
eight chimeras tested (compare panels f-h in Fig. 2and Fig. 5, and Table 1). Gayle et al.(14) reported competition binding with unlabeled IL-8 and
GRO
and
I-IL-8 to a rabbit-human BA5, made from
rabbit IL-8RA and human IL-8RB, that had properties similar to those
shown for human-human BA5 in Fig. 5f.
I-GRO
and
I-NAP-2 binding and receptor
activation were not reported in their study (14) .
Figure 6:
Desensitization of calcium transients in
HEK 293 cells stably transfected with IL-8RB, BA5, and CC CKR1/IL-8RB.
Ratio fluorescence was monitored from FURA-2 loaded cells before and
during sequential addition of chemokines at the times indicated by the arrows. The concentrations of the CXC chemokines used were 10
nM each for IL-8 and GRO and 25 nM for NAP-2.
Each row of tracings corresponds to the receptor indicated at the right. The tracings are from a single experiment
representative of at least two separate
experiments.
The present work illustrates the complexity of CXC chemokine
interactions with IL-8RB. The major findings are as follows. 1) IL-8,
GRO, and NAP-2 selectivity determinants appear to be broadly but
differentially distributed on multiple domains of IL-8RB; and 2) low
affinity IL-8, GRO
, and NAP-2 binding sites appear to be capable
of mediating efficient receptor activation.
The first conclusion is
best illustrated by two examples. First, the efficacy of competition by
unlabeled NAP-2 varied considerably for the I-IL-8-,
I-GRO
-, and
I-NAP-2-labeled sites on
IL-8RB; second, three separate regions of IL-8RB can independently
confer NAP-2 agonist activity to IL-8RA
IL-8RB chimeric receptors
(BA1, ABA, AB5, and AB7). Furthermore, two of these three regions can
independently confer GRO
selectivity to IL-8RA
IL-8RB
chimeric receptors. Thus, in contrast to two previous reports
suggesting that the N-terminal segment of the IL-8 receptors is a
dominant determinant of receptor subtype
selectivity(13, 14) , our data would indicate that the
N-terminal segment of IL-8RB, but not IL-8RA, is dominant.
Although the backbone structures of chemokines are very similar,
differences in the side chains and/or in the composition of the
unordered N-terminal sequence prior to the first conserved cysteine
must account for the complexity of binding to IL-8RB, as well as for
the dramatic and unpredictable differences in chemokine selectivity
observed for the CC chemokine
receptors()(3, 18, 25) . Given the
likelihood that 7TMD receptors probably lack independently folding
domains, it should not be surprising that ``artificial''
receptors such as the chimeric receptors we tested have ligand binding
sites that are in some cases strikingly and unpredictably different
from those of the wild type receptors. Nevertheless, all of the
chimeric receptors tested were restricted to interactions with CXC
chemokines and failed to interact with CC chemokines, a characteristic
shared with wild type IL-8RA and IL-8RB.
In five instances we
observed that chimeric receptors could be efficiently activated by one
or more CXC chemokines, whereas direct binding to the chimera by the
corresponding iodinated protein could not be demonstrated. The examples
include IL-8 in the case of the chimeras BA1 and CC CKR1/IL-8RB; NAP-2
in the case of chimera AB5; and IL-8, GRO, and NAP-2 in the case
of chimera BAB. The extent to which chemical modification of the
ligands by iodination, and changes in on rates and off rates, account
for these surprising results has not been determined yet. Based on the K
values determined by heterologous competition
binding that were available for chimeras BA1 and CC CKR1/IL-8RB, we
estimate that the affinity in each of these cases is reduced at least
20-fold relative to the corresponding affinity determined on IL-8RB.
This implies that the determinants of high affinity binding for
I-IL-8,
I-GRO
, and
I-NAP-2 by the wild type receptors may differ from those
responsible for high agonist potency, at least for calcium
mobilization, the function that we tested directly. It is possible that
high affinity binding may be essential for some other function of
IL-8RB, such as neutrophil chemotaxis.
Functionally significant low
affinity chemokine binding sites have been proposed for two wild type
chemokine receptors. First, endothelial cells migrate and proliferate
in response to IL-8 presumably by binding to a specific receptor
(reviewed in (1) ); however, I-IL-8 binds to
endothelial cells with low affinity(26) . Second, the CC
chemokines MIP-1
, RANTES, and MIP-
are strong agonists for a
CC chemokine receptor named CC CKR5, but direct binding of the
corresponding radioligands has not been demonstrated.
Our findings imply that distinct antagonists could be
developed that block either high affinity binding but not receptor
activation, or receptor activation but not high affinity binding, or
both high affinity binding and receptor activation. The most useful
antagonists clearly would be those that block receptor activation.
Since the sets of binding sites for IL-8, GRO, and NAP-2 on IL-8RB
are overlapping but distinct, both narrow and broad spectrum chemokine
antagonists could conceivably be developed that act specifically at
IL-8RB or more broadly at both IL-8RA and IL-8RB.
Our results provide a broad foundation for future investigations of the structural basis of chemokine selectivity for IL-8RB as well as the mechanism of receptor activation. Site-directed mutagenesis of IL-8RB will be important for identifying functionally critical residues both within the set of residues that are different between IL-8RA and IL-8RB as well as within the set of conserved residues, as these could have different functional significance in different sequence contexts. Extensive site-directed mutagenesis studies for IL-8RA, testing both IL-8 binding and IL-8-induced calcium flux responses have been reported (27, 28) . Both functions were insensitive to alanine substitution for each of the amino acids in the N-terminal segment, with the exception of the cysteine at position 30(27, 28) . The C30A phenotype probably reflects an important role of this cysteine in the overall folding of the receptor. It would be surprising if the highly acidic nature of the N-terminal segment of the IL-8 receptors were not functionally important, however evidence is currently lacking. Systematic alanine-scanning mutagenesis of all the amino acids in the ectodomains of IL-8RA has shown that amino acids Cys-187, Arg-199, Arg-203, Asp-265, and Cys-277 in the predicted second and third extracellular loops were sensitive to mutation for both IL-8 binding and signal transduction(28) . All of these positions are conserved in the sequence of IL-8RB and therefore are unlikely to discriminate receptor subtype selectivity.
In summary, our results illustrate the complex nature of CXC
chemokine binding sites and show that the inferences drawn for them
from the properties of chimeric chemokine receptors can differ for
direct or indirect binding or signal transduction data sets. IL-8R
subtype selectivity is not simply determined by the N-terminal
segments, but instead is determined by multiple regions on IL-8RB that
can function independently in IL-8RAIL-8RB chimeric contexts.
Future studies using CXC chemokine mutants, the chimeric receptors we
have described and site-directed mutagenesis may help to infer a more
detailed model of chemokine binding to IL-8 receptors, which can be
tested by more direct physical and chemical methods.