(Received for publication, September 5, 1995)
From the
We have demonstrated that the mouse chemokine N51, also known as
KC, can compete for I-human interleukin-8 (IL-8) binding
to NIH 3T3 cells expressing the human IL-8 receptor
(NIH-IL-8R
) but not the IL-8 receptor
(NIH-IL-8R
). In
addition, we used the chimeras between N51 and IL-8 described
previously (Heinrich, J. N., O'Rourke, E. C., Chen, L., Gray, H.,
Dorfman, K. S., and Bravo, R.(1994) Mol. Cell. Biol. 14,
2849-2861; Heinrich, J. N., and Bravo, R.(1995) J. Biol.
Chem. 270, 4987-4989) to evaluate possible contributions of
equivalent domains from each chemokine to binding and specificity.
Specifically, the amino acid sequences between cysteines 2 and 3 or
between cysteines 3 and 4 or the
-helical C-terminal end (domains
I, II, and III, respectively) of one of the chemokines was exchanged
with the corresponding sequence of the other and vice versa.
Chimeras of IL-8 containing either domain II or III of N51 behaved
similarly, but not identically, to IL-8 in competing
I-IL-8 binding with both NIH-IL-8R
cells and
NIH-IL-8R
cells. The IL-8 chimera containing domain I of N51 did
not compete. On the other hand, N51 competes
I-IL-8
binding with NIH-IL-8R
but not NIH-IL-8R
cells. The N51
chimera containing domain I of IL-8 was an agonist with NIH-IL-8R
cells and was an even more potent agonist with NIH-IL-8R
cells. On
the latter cells it was more potent than either IL-8 or N51. The N51
chimera containing domain II of IL-8, compared with N51, was a partial
agonist with NIH-IL-8R
cells but was equivalent to N51 with
NIH-IL-8R
cells. However, N51 chimera containing domain III of
IL-8 was a partial agonist with both cells. The results are consistent
with the observations we originally made with human neutrophils and the
NIH mouse IL-8R
cells, i.e. domain I confers binding
specificity for IL-8 and domains II and III of IL-8 and N51 may be
interchangeable but they are not equivalent. Although we originally
hypothesize that domains II and III confer binding specificity to N51,
these results emphasize the role of domain III.
The last decade has seen the emergence of a superfamily of genes
whose protein products are proinflammatory cytokines and which are
collectively called chemokines (for reviews see (3) and (4) ). In general, the chemokine genes code for secreted
peptides of about 70-100 amino acids containing 4 conserved
cysteines. The first two cysteines are either separated by one amino
acid or adjacent to each other, and this distinction, which is
correlated with functional properties (see below), is used to designate
the members into two classes, the ``C-X-C'' or
-family and the ``C-C'' or
-family, respectively.
For members whose three-dimensional structure has been determined,
namely platelet factor-4, interleukin-8 (IL-8), (
)GRO
or melanoma growth-stimulating activity (GRO
/MGSA), and macrophage
inflammatory protein-1
(MIP-1
), the cysteine motif appears to
restrict the secondary structure to forming two disulfide bonds, one
between the first and third cysteines and another between the second
and fourth cysteines, resulting in similar tertiary structures. It is
believed that all the members share this tertiary configuration, since
the disulfide bonds are essential for the biological activity of
chemokines for which this property has been tested (for reviews see
Refs. 5 and 6). Recently, the identification of lymphotactin containing
only 2 of the conserved cysteines (7) indicates that other
families of chemokines may exist.
There are at least 15 human
chemokines described so far, 8 in the ``C-X-C''
family including platelet factor-4, IL-8, GRO/MGSA, GRO
,
GRO
, ENA-78, and
-IP-10 and 7 in the ``C-C'' family
including monocyte chemotactic protein-1 (MCP-1), MCP-2, MCP-3, RANTES,
MIP-1
, and I309(4) . The numbers are certain to increase
from both the identification of the corresponding human homolog known
in other species, like the newest ``C-C'' member from guinea
pig, exotaxin(8) , and the direct finding of novel human
members. The identification of corresponding homologs between species
is complicated by two important observations. First, not all species
may possess the same chemokines. For example, IL-8 has not been
identified in mice. Second, there can be considerable variation in what
represents a homolog in a particular species. For example, mouse MIP-2
was considered originally the homolog of human GRO
/MGSA (51%
identity); now mouse N51/KC is accepted as the true homolog (67%
identity).
Generally observed biological activities of
``C-X-C'' and ``C-C'' chemokines are the
activation of neutrophils and monocytes, respectively. Early studies
showed that a number of the ``C-X-C'' chemokines,
including ENA-78(9) , MIP-2, neutrophil-activating peptide-2
(NAP-2), and GRO/MGSA could desensitize human neutrophils to IL-8
and that MIP-2, NAP-2, and GRO
could compete for
I-IL-8 binding to human
neutrophils(3, 9, 10) . These observations
stimulated several investigations to determine whether specific
residues in the chemokine promotes receptor binding
specificity(11, 12, 13) . We demonstrated
that N51 is biologically active on human neutrophils via the IL-8R and
then performed structure and function analysis of N51 by comparing the
biological activities of chimeras between N51 and IL-8(1) . Our
data indicated that the amino acid sequences between cysteines 3 and 4
(domain II) and the
-helical C-terminal end (domain III) are
important for the binding of N51.
Two human IL-8Rs, IL-8R and
IL-8R
, have been cloned, and their deduced amino acid sequences
are typical of a seven-transmembrane G-coupled
receptor(14, 15) . The receptors have been expressed
in a number of cell lines and shown to bind IL-8 with high affinity. In
addition, a number of the other ``C-X-C'' members,
including GRO
/MGSA, rabbit IL-8, and NAP-2, appear to show a low
affinity for IL-8R
and a high affinity for
IL-8R
(16, 17, 18) .
Here we have
investigated whether N51 and chimeras between N51 and IL-8 can compete
for I-IL-8 binding to the IL-8Rs expressed independently
in NIH 3T3 cells (NIH-IL-8R
cells and NIH-IL-8R
cells). Our
data demonstrate that N51 competes
I-IL-8 binding for
IL-8R
but not for IL-8R
. It also shows that the domains most
important in conferring binding specificity are domain III of N51 and
domain I of IL-8.
To generate cell lines stably
expressing the receptors, NIH 3T3 cells were transfected with either
pMexneo alone, pMexneo-IL-8R, or
pMexneo-IL-8R
and selected in Dulbecco's modified
minimum essential medium containing antibiotics (100 µg/ml
penicillin and 50 µg/ml streptomycin), 10% bovine serum, and 0.8
mg/ml G418. The generated cell lines, NIH-pMexneo, NIH-IL-8R
, and
NIH-IL-8R
, represented a mixed population of the G418-resistant
colonies. For binding analysis cells were detached from the plates by
incubation with Hanks' balanced salt solution (HBSS) with phenol
red, supplemented with 25 mM HEPES (pH 7.5), 0.2% bovine serum
albumin (BSA, fatty acid-free, Sigma), and 5 mM EDTA (HBSS-B1)
until they could be harvested by gentle scraping (15 min). The cells
were pelleted by centrifugation at 250
g for 10 min,
washed twice in HBSS supplemented with 25 mM HEPES (pH 7.5), 1
mM CaCl
, 1 mM MgCl
containing
2% BSA (HBSS-B2), resuspended to 5-15
10
cells/ml with HBSS-B2 containing 0.2% BSA (HBSS-B3), and cooled on ice
for 30 min.
The N51 and the chimeric proteins were expressed
in the baculovirus system and purified as described
previously(1) . IL-8 (R & D Systems) was iodinated by the
Bolton-Hunter method to a specific activity of 97.4 mCi/µg (DuPont
NEN Custom Iodination Laboratory) as described (1) . The
competition curve was performed with a 0.1-200-fold excess of
competitor as compared with 3 nMI-IL-8. Data
were analyzed by least square nonlinear curve fit for a model of single
and multiple sites using KaleidaGraph software.
We have reported previously that baculovirus-expressed N51
binds human neutrophils via the IL-8R(s) and that a N51 chimera
containing domain I of IL-8 (N51/IL-8I) made N51 more IL-8-like. Our
observations suggested that the high affinity receptor for N51 is the
IL-8R(1) . To extend our findings we decided to
investigate the ability of N51 and chimeras between N51 and IL-8 to
compete for
I-IL-8 binding to the human IL-8R
and
IL-8R
independently expressed in NIH 3T3 cells. The chimeras
between IL-8 and N51, illustrated in Fig. 1, have been described
previously(1) .
Figure 1: Domains exchanged between N51 and IL-8 and comparison of the amino acid sequence of the chimeras. The first and sixth lines show the sequences of domains I, II, and III from N51 and IL-8, respectively. N51 residues and those in common with IL-8 are white are on a black background while those unique for IL-8 are black on a white background.
The competition curves for I-IL-8 binding to the NIH-IL-8R
cells in the
presence of increasing concentrations of IL-8, N51, and the chimeras
are presented in Fig. 2. The competition with IL-8 shows a
sigmoidal curve with an apparent K
of 5.5
nM. The IL-8 chimeras containing domain II or III from N51,
IL-8/N51II and IL-8/N51III, respectively, retained their ability to
compete
I-IL-8 (Fig. 2A). However,
IL-8/N51II is a significantly weaker competitor than IL-8. On the other
hand, N51 was unable to compete for
I-IL-8 binding to the
NIH-IL-8R
cells, but the N51 chimera containing domain I of IL-8,
N51/IL-8I, competes efficiently, with a curve that is similar to IL-8 (Fig. 2B). The other chimeras, N51/IL-8II and
N51/IL-8III, weakly competed when present at high concentrations (Fig. 2B). These results show that the chimeras that
possess domain I of IL-8 such as IL-8/N51III, N51/IL-8I, and to a
lesser extent IL-8/N51II are agonists, while those that lack domain I
but have domain II or III of IL-8 such as N51/IL-8II and N51/IL-8III
are at best partial agonists in NIH-IL-8R
cells. This indicates
that for binding of IL-8 to IL-8R
, domain I is the major
determinant. Recently, it has been shown that tyrosine 13 and lysine 15
are essential for high affinity binding of IL-8 to the
IL-8R
(19) ; these residues are not present in N51 but are
in domain I of IL-8.
Figure 2:
Competition of I-IL-8
binding to NIH-IL-8R
cells. Binding was done with 3 nM
I-IL-8 for 1 h at 4 °C in the absence and
presence of up to 200-fold increasing concentrations of: A,
IL-8, IL-8/N51II, or IL-8/N51III; and B, N51, N51/IL-8I,
N51/IL-8II, or N51/IL-8III.
Fig. 3shows the competition of I-IL-8 binding to the NIH-IL-8R
cells by increasing
concentrations of either IL-8, N51, or the different chimeras. The
competition by IL-8 shows a sigmoidal curve with an apparent K
of 8.7 nM. In contrast to the results
obtained with NIH-IL-8R
cells the capacity of IL-8, IL-8/N51II,
and IL-8/N51III to compete the binding of
I-IL-8 to
NIH-IL-8R
are very similar (Fig. 3A). This would
indicate that IL-8R
and IL-8R
differentially recognize the
N51 domains. This is supported by the fact that N51 did not compete
I-IL-8 binding to IL-8R
(Fig. 3A)
but efficiently competed the binding to IL-8R
(Fig. 3B). Interestingly, the competition curve of
N51/IL-8I shows that this chimera is a stronger competitor than both
IL-8 and N51, and that of chimera N51/IL-8III indicates that this
molecule is a weak competitor (Fig. 3B). The results
presented in Fig. 3B also show that N51 can compete
I-IL-8 binding to IL-8R
as efficiently as IL-8. The
observation that chimera N51/IL-8III is a weak partial agonist would
indicate that domain III of N51 may be contributing significantly to
the binding specificity of N51. On the other hand, chimera N51/IL-8I is
more potent than either IL-8 or N51, suggesting that domain I of IL-8
and domain III of N51 may independently contribute to binding.
Figure 3:
Competition of I-IL-8
binding to NIH-IL-8R
cells. Binding was done with 3 nM
I-IL-8 for 1 h at 4 °C in the absence and
presence of up to 200-fold increasing concentrations of: A,
IL-8, IL-8/N51II, or IL-8/N51III; and B, N51, N51/IL-8I,
N51/IL-8II, or N51/IL-8III.
The
results presented here are consistent with and extend our previous
observations using human neutrophils(1) . First, we have
demonstrated that N51 can only displace I-IL-8 binding
from
receptor-containing cells but not from the
receptor-containing cells. This provides a simple explanation of why
with human neutrophils N51 cannot displace all bound
I-IL-8 and confirms our speculation that the high
affinity receptor for N51 in human neutrophils is the
IL-8R
(1, 2) . Second, the ability of chimera
N51/IL-8I to compete with
I-IL-8 binding to both the
NIH-IL-8R
cells and NIH-IL-8R
cells is consistent with our
previous finding that N51/IL-8I mimics IL-8 on human neutrophils. In
addition, domain I does include sequences that are reported to confer
high affinity binding of IL-8 to its receptors (5, 12, 13, 19, 20) .
Third, the competition we obtained with the chimeras was all
anticipated, except for the observation that N51/IL-8III is a partial
agonist in NIH-IL-8R
cells. One possible explanation is that
NIH-IL-8R
cells may be permitting us to see the partial agonist
activity of N51/IL-8III we previously saw in desensitization assays
with human neutrophils(1) . From our observations with human
neutrophils we suggested that domains II and III of N51 and IL-8 were
interchangeable but not equivalent and that they were more important in
conferring N51 binding specificity than IL-8, which depends on domain
I(1) . These are supported by our analysis of binding of
I-N51 and competition with the chimeras to NIH 3T3 cells
expressing the mouse homolog of the IL-8R
(2) . The present
results may suggest that domain III has a greater role than domain II
in N51 binding specificity. This report supports the notion that the
use of different ligand domains to recognize a receptor may be one
mechanism by which the many chemokines attain their biological
specificity.