(Received for publication, October 20, 1995)
From the
To determine the regions of interleukin-8 (IL-8) that allow high
affinity and interleukin-8 receptor type 1 (IL8R1)-specific binding of
chemokines, we produced chimeric proteins containing structural domains
from IL-8, which binds to both IL8R1 and interleukin-8 receptor type 2
(IL8R2) with high affinity, and from GRO, which does not bind to
IL8R1 and binds to IL8R2 with reduced affinity. Receptor binding
activity was tested by competition of
I-IL-8 binding to
recombinant IL8R1 and IL8R2 cell lines. Substitution into IL-8 of the
GRO
sequences corresponding to either the amino-terminal loop
(amino acids 1-18) or the first
-sheet (amino acids
18-32) reduced binding to both IL8R1 and IL8R2. The third
-sheet of IL-8 (amino acids 46-53) was required for binding
to IL8R1 but not IL8R2. Exchanges of the second
-sheet (amino
acids 32-46) or the carboxyl-terminal
-helix (amino acids
53-72) had no significant effect. When IL-8 sequences were
substituted into GRO
, a single domain containing the second
-sheet of IL-8 (amino acids 18-32) was sufficient to confer
high affinity binding for both IL8R1 and IL8R2. The amino-terminal loop
(amino acids 1-18) and the third
-sheet (amino acids
46-53) of IL-8 had little effect when substituted individually
but showed increased binding to both receptors when substituted in
combination.
Individual amino acid substitutions were made at
positions where IL-8 and GRO sequences differ within the regions
of residues 11-21 and 46-53. IL-8 mutations L49A or L49F
selectively inhibited binding to IL8R1. Mutations Y13L and F21N
enhanced binding to IL8R1 with little effect on IL8R2. A combined
mutation Y13L/S14Q selectively decreased binding to IL8R2. Residues
Tyr
, Ser
, Phe
, and Lys
are clustered in and around a surface-accessible hydrophobic
pocket on IL-8 that is physically distant from the previously
identified ELR binding sequence. A homology model of GRO
,
constructed from the known structure of IL-8 by refinement
calculations, indicated that access to the hydrophobic pocket was
effectively abolished in GRO
. These studies suggest that the
surface hydrophobic pocket and/or adjacent residues participate in IL-8
receptor recognition for both IL8R1 and IL8R2 and that the hydrophobic
pocket itself may be essential for IL8R1 binding. Thus this region
contains a second site for IL-8 receptor recognition that, in
combination with the Glu
-Leu
-Arg
region, can modulate receptor binding affinity and IL8R1
specificity.
Interleukin-8 (IL-8) ()and the related GRO proteins
are members of a superfamily of proinflammatory cytokines that
stimulate neutrophil activation and chemoattraction (see (1, 2, 3) for review). These proteins contain
four conserved cysteine residues with a single intervening amino acid
between the first two cysteine residues and are designated the
C-X-C chemokines. IL-8 mediates the recruitment and activation
of neutrophils during inflammation and has been implicated in multiple
pathologic conditions involving chronic and acute inflammation and in
neutrophil-mediated
injury(4, 5, 6, 7, 8, 9, 10, 11) .
Two receptors for IL-8 have been identified by molecular cloning and
account for the observed effects of IL-8 and other C-X-C
chemokines on neutrophils(12, 13) . Although both are
seven-transmembrane, G-protein-coupled receptors, Type 1 and Type 2
IL-8 receptors differ in ligand specificity. The Type 1 receptors
(IL8R1) have restricted specificity and bind IL-8 exclusively with high
affinity(14, 15) . In contrast, the Type 2 receptors
(IL8R2) bind IL-8 with a similarly high affinity but also recognize
several other C-X-C chemokines with varying
affinities(14, 15) .
Structures for both the
crystal and solution forms of IL-8 have been
determined(16, 17) . IL-8 comprises five discrete
structural domains: an amino-terminal loop, three antiparallel
-sheets, and a carboxyl-terminal
-helix. The highest regions
of amino acid homology among the C-X-C chemokines occur at the
conserved cysteine residues and at other key structural residues,
suggesting that the basic structural elements of IL-8 are conserved
among family members (1, 2, 3) . The solution
structure for GRO
(18) and crystal structure for
neutrophil-activating peptide-2 (19) show similar organization.
Several studies have addressed the structure-activity relationships
required for C-X-C chemokine binding to neutrophils. A
conserved sequence that is essential for binding,
Glu-Leu
-Arg
(ELR), was identified
by scanning mutagenesis of IL-8 (20) and by amino-terminal
truncated analogs(21) . The exclusive binding of IL-8 to IL8R1
and the different affinities among C-X-C chemokines for
binding to IL8R2 suggest that a second site on IL-8 determines receptor
specificity. Hybrid proteins derived from IL-8 and IP10, a
C-X-C chemokine that lacks the ELR sequence and that does not
bind neutrophil IL-8 receptors, indicated that the ELR sequence as well
as IL-8 sequences for amino acids 10-22 and 30-34 are
required for neutrophil recognition(22) . Chimeric analysis of
IL-8 with the murine chemokine N51/KC indicated a site that enhances
binding to neutrophils is within amino acids 13-29(23) .
Receptor specificity was addressed directly on recombinant IL-8
receptors with chimeric proteins derived from IL-8 and GRO
or
rabbit IL-8 and demonstrated that IL8R1 specificity element(s) reside
between Cys
and Cys
of IL-8(24) . In
this study we have generated chimeric proteins containing domains from
IL-8 and GRO
and report that the second site for receptor binding
and specificity is localized in the region of a unique hydrophobic
surface pocket on IL-8. Through amino acid replacement studies we have
identified residues in and around the hydrophobic pocket that influence
binding of IL-8 to IL8R1 and IL8R2.
Figure 1:
Amino acid sequences of C-X-C
chemokines. Sequences for IL-8, GRO/MGSA, GRO/MIP2
, and
GRO
/MIP2
are aligned for maximal homology and numbered
according to the 72-amino acid form of IL-8 ( (1, 2, 3) and references therein). Underlined residues are the conserved amino acids selected for
domain boundaries.
Figure 2:
Cloning strategy for IL-8/GRO
chimeras. PCR primers are indicated for the construction of each
chimeric protein and correspond to oligonucleotide sequences listed in Table 1. Sequences derived from IL-8 are shown as open
boxes, and sequences from GRO
are shown as filled
boxes. Additional details are given under ``Experimental
Procedures.''
Chimeric proteins with
single domains of GRO substituted into IL-8 were used to test the
role of each
-sheet region. Replacement of amino acids
18-32, corresponding to the first
-sheet, reduced binding of
IL-8 to both IL8R1 and IL8R2 (Fig. 3, I18G32I). Exchange of
regions within amino acids 32-46 was not important for binding to
either IL8R1 or IL8R2 (Fig. 3, I32G46I). Substitution for amino
acids 46-53 reduced binding to IL8R1 by 10-fold without affecting
binding to IL8R2 (Fig. 3, I46G53I), confirming that the third
-sheet of IL-8 participates in IL8R1 specificity.
Figure 3:
Relative receptor binding activity of
IL-8/GRO chimeras with substitutions of individual structural
domains. Left panels, individual domains of GRO
inserted
into IL-8. Right panels, individual domains of IL-8 inserted
into GRO
. Competitive binding assays were performed on CHO-IL8R1 (upper panels) and CHO-IL8R2 cells (lower panels)
with 0.2 nM
I-IL-8 and 0.001-30 µg/ml
of test proteins. The IC
values for IL-8 were 0.037
± 0.006 µg/ml and 0.023 ± 0.005 µg/ml for IL8R1
and IL8R2, respectively. Relative potency was calculated as the ratio
of IC
values for IL-8 versus the IC
value of each chimera. Data are the mean ± S.E. of at
least three independent experiments.
In the
reciprocal experiment, individual domains of IL-8 were substituted into
GRO to examine whether any of these regions could enhance binding
to IL-8 receptors. Replacement of residues 18-32, corresponding
to the first
-sheet of IL-8, was sufficient to allow nearly
IL-8-like activity on both IL8R1 and IL8R2 (Fig. 5, G18I32G).
Two domains, corresponding to amino acids 1-18 and 32-46,
slightly enhanced binding to IL8R1 (Fig. 3, I18G and G32I46G).
Since the amino-terminal loop and the third
-sheet were both
important for IL8R1 binding of IL-8 but had little effect when
substituted into GRO
individually, a chimera was generated to test
whether simultaneous replacement of these regions of GRO
with
corresponding IL-8 sequences could confer IL-8-like activity. The
combined substitution of amino acids 1-18 and 46-53 allowed
high affinity binding of GRO
to both IL8R1 and IL8R2 (Fig. 3, I18G46I53G).
Figure 5:
Competitive inhibition studies with IL-8
mutations in the hydrophobic pocket. Proteins (0.001-100
µg/ml) were mixed with 1.0 nMI-IL-8 and
examined for their ability to bind to IL8R1 expressed in CHO cells. The
data are the mean ± S.D. of triplicate determinations of three
independent experiments.
, IL-8;
, F21N;
, L49F;
, V41F;
, D45R;
, L49S.
Figure 4:
Chemokine structures for IL-8 receptor
recognition. Left, surface hydrophobic pocket on IL-8. Right, GRO homology model. Surface profiles were
generated from the solution structure of IL-8 (17) and from a
homology model of GRO
(see ``Experimental Procedures'')
with a 1-Å sphere and are displayed in the same relative
orientation with the amino terminus to the top. Atoms are colored as follows: red, oxygen; blue, nitrogen; white, carbon.
A homology-based model of GRO was constructed
to compare the relative positions of the corresponding residues. The
predicted structure is shown in the same relative orientation as the
structure of IL-8 (Fig. 4). In IL-8, one side of the hydrophobic
pocket is formed by the Tyr
-Ile
strand.
In GRO
, little of this stretch of residues is conserved, and there
is a deletion of one residue. Tyr
and Phe
are
conservatively substituted with isoleucine in GRO
, and Ile
is conserved. Thus, the residues contributing to the hydrophobic
core appear to be conserved. However, the deletion corresponding to
Lys
in IL-8 reduces the length of the
Tyr
-Ile
strand, effectively shrinking
the hydrophobic pocket in GRO
. The substitution of Pro
by a leucine in GRO
affects the structure of this strand and
its contribution to the hydrophobic pocket. According to our homology
model, the hydrophobic pocket in GRO
is much smaller than that in
IL-8 and cannot accommodate a phenyl ring. In addition, none of the
gateway residues of IL-8 are conserved in GRO
.
Figure 6:
Neutrophil chemotactic activity of
IL-8/GRO chimeras. A, effect of the amino-terminal loop
residues 1-18. Chemotaxis assays were performed with IL-8
(
), GRO
(
), G18I (
), and I18G (
). B, effect of first
-sheet residues 18-32.
Chemotaxis assays were performed with IL-8 (
), GRO
(
),
I18G32I (
), and G18I32G (
). C, effect of third
-sheet residues 46-53. Chemotaxis assays were performed with
IL-8 (
), GRO
(
), I46G (
), I46G53I (
), and
G46I53G (
). Data are the mean ± S.D. of triplicate
determinations from a single experiment and are representative of at
least three independent experiments for each
chimera.
IL-8 variants with point mutations also stimulated chemotaxis with the same efficacy as IL-8 at optimal doses but displayed variable chemotactic potency. Y13L, which showed increased binding to IL8R1, was 2-3-fold more potent than IL-8 in chemotaxis assays (data not shown). L49F had a reduced chemotactic potency proportionate to reduced activity for IL8R1. However, L49A had only slightly reduced chemotactic activity despite a significantly decreased ability to bind to IL8R1 (data not shown). The Y13L/S14Q mutation decreased binding specifically to IL8R2 but had no effect on chemotaxis, consistent with the major role of IL8R1 in chemotaxis(25) .
The functional activity of mutant proteins
was further analyzed by intracellular signaling assays. Wu et al.(37) have demonstrated that IL-8 receptors signal via
G subunits. Therefore, the ability of IL8R1 and IL8R2
to reduce cAMP concentrations was determined in CHO cells as described
in Shyamala et al.(38) . IL-8 inhibited the
forskolin-induced elevation of cAMP levels in either CHO-IL8R1 or
CHO-IL8R2 cells (data not shown). The 11 mutated IL-8 proteins also
inhibited cAMP levels upon interaction with either IL8R1 or IL8R2 to
varying degrees (data not shown), confirming that these proteins are
functional agonists of IL8R1 and IL8R2.
By construction of IL-8/GRO chimeras we have
demonstrated that the amino-terminal loop (amino acids 1-18) and
the first
-sheet (amino acids 18-32) of IL-8 contain
residue(s) that are essential for high affinity, IL-8-like binding to
both IL8R1 and IL8R2. These domains encompass the regions identified by
IL-8/IP10 hybrids (22) and by IL-8/N51 chimeric proteins (23) as necessary for maximal binding to neutrophils. The third
-sheet of IL-8 contains residue(s) that confer IL8R1-specific
binding and are not required for binding to IL8R2. Complete analysis of
this region by point mutations indicated that L49A substitution is
responsible for the specific reduction of IL8R1 binding. Heinrich et al. (23) observed that a chimeric IL-8/N51 protein
containing this substitution (corresponding to I34N50I) exhibited
neutrophil binding properties similar to IL-8. That result may reflect
the participation of both IL8R1 and IL8R2 receptors in neutrophil
binding.
Schraufstatter et al.(24) have reported
that the carboxyl terminus of IL-8/GRO chimeric proteins affects
specificity of binding to IL8R2 but not to IL8R1. Chimeric I51G
behaved like IL-8 in its binding to IL8R1 but had 5-fold lower affinity
for IL8R2(24) . The present study demonstrates that the
carboxyl terminus of IL-8 beyond amino acid residue 53 does not define
receptor specificity for IL8R1 or IL8R2. Exchanges of the carboxyl
terminus did not affect neutrophil binding of chimeric IL-8/IP10 (22) or IL-8/N51(23) , and with truncated analogs of
IL-8 the removal of the terminal
-helix reduced but did not
prevent neutrophil receptor binding(21) .
We have identified
several variants of IL-8 with specifically altered binding affinity for
IL8R1 or IL8R2. Mutations at Tyr, Phe
, and
Leu
modified IL8R1 binding selectively. Recent studies by
Schraufstatter et al. (39) identified Tyr
as well as Lys
as determinants of the differential
IL8R1 affinity for human and rabbit IL-8. These key residues that
affect binding to IL8R1 surround a unique hydrophobic pocket on the
surface of IL-8. The increase in binding to IL8R1 by the replacement of
Tyr
with leucine indicates that the removal of the
aromatic ring provides increased access for receptor docking. Y13H also
decreased binding to IL8R1(39) , and other mutations at this
position altered binding to neutrophils(22) . The increased
binding to IL8R1 of F21N can also be explained as facilitating the
receptor interaction by the replacement of the aromatic group with
smaller amino acids. F21L had reduced binding to
neutrophils(22) . Substitutions at Leu
are not as
clear-cut. L49F decreases binding to IL8R1, which could be interpreted
as the large aromatic ring of phenylalanine obstructing the receptor
docking. L49A also decreases R1 binding, which might suggest that this
is due to an alteration of the hydrophobic pocket itself. The
replacement with an intermediately sized residue, L49S, moderately
increased binding. The double mutation Y13L/S14Q decreases binding to
IL8R2 selectively, indicating that IL8R2 recognition involves different
determinants than IL8R1 but is also localized near the hydrophobic
pocket on IL-8. Thus this region contributes to IL8R2 binding, and the
pocket itself may be essential for binding to IL8R1.
The
surface-accessible hydrophobic pocket on IL-8 is sufficiently large to
accommodate an aromatic ring structure and could accept a phenylalanine
or tyrosine side chain from IL8R1. The region of the hydrophobic
pocket, and particularly of residues 12-18 within the
amino-terminal loop, corresponds to the site of greatest structural
differences between IL-8 and other C-X-C chemokines (GRO
and NAP-2) that bind to IL8R2 but not to
IL8R1(18, 19) . The solution structure of GRO
indicates a hydrophobic pocket smaller than that on IL-8(18) .
For GRO
, homology-based structural modeling predicted that the
hydrophobic pocket is much smaller and more restricted in access,
consistent with the lack of IL8R1 binding and reduced IL8R2 binding.
Interleukin-8 interacts with two sites on IL-8 receptors; conserved
charged residues in extracellular domains 3 and 4 are essential for
IL-8 binding to IL8R1 or IL8R2(40) , and the amino-terminal
extracellular domains confer the ligand specificity profiles
characteristic of each receptor type(41, 42) . Our
data indicate that the hydrophobic pocket and/or surrounding residues
contribute to the binding of C-X-C chemokines to IL-8
receptors and serve as the second binding site on IL-8 to modify
receptor recognition in conjunction with the ELR sequence. Thus,
receptor extracellular domains 3 and 4 provide the primary interaction
with ELR (residues 4-6) of IL-8, and receptor amino-terminal
domains interact with a secondary binding site localized in or near the
surface hydrophobic pocket on IL-8 bounded by residues
Tyr, Lys
, Phe
, and
Arg
.