| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
(Received for publication, October 20, 1995)
From the
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
(GRO/MGSA), and I-IL-8 were prepared as
described(25) . Recombinant human GRO (MIP2) and
GRO (MIP2) were produced in yeast and purified by
chromatography on heparin-Sepharose and Sephacryl S-100 HR.
were identified by protein sequence
alignments shown in Fig. 1. DNA encoding chimeric IL-8/GRO
were generated by recombinant polymerase chain reaction (PCR) (26) and/or by ligation with synthetic oligonucleotides
encoding the desired amino acid as depicted in Fig. 2. A
synthetic IL-8 gene, il8syn(27) , and a MIP2 cDNA
clone, MIP540(28) , were used as initial templates for IL-8 and
GRO sequences. PCR primers and synthetic oligonucleotide linkers
are listed in Table 1. For G18I, sequences between the Asp718 and MluI restriction sites were generated with
oligonucleotides accX, Xmlu, Xacc, and mluX. For I18G, sequences
between Asp718 and BglII were from oligonucleotides
accZ, Zbgl, Zacc, and bglZ. Chimeric DNA were used as PCR templates in
some cases: G32I and I46G for G32I46G, I32G and G46I for I32G46I, and
G46I53G for I18G46I53G. Constructs were ligated, as Asp718/SalI fragments a transfer vector containing
the GAP promoter fused to the -factor leader (29) .
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.''
was built based on the NMR solution-derived structure of
IL-8(17) . The LOOK software program (Molecular Applications
Group, Mountain View, CA) was used to align the GRO and IL-8
sequences(33) . Then a three-dimensional model of GRO was
built by Levitt's automatic segment matching (34) and
further refined by restrained energy minimization and molecular
dynamics(35) .
Proteins proteins were designed to test the contribution of each
structural domain of IL-8 for binding to IL8R1 and IL8R2. Four
conserved amino acid residues were identified as structural domain
boundaries for IL-8: His
, Pro
,
Gly
, and Pro
(Fig. 1). To maintain the
overall C-X-C chemokine structure, sequences for complete
structural domains between these boundaries were interchanged between
IL-8 and GRO (Fig. 2). The corresponding chimeric
chemokines were produced in yeast using the -factor mating
pheromone secretion pathway (29) and purified to near
homogeneity by a single step enrichment/purification protocol. With the
exception of I32G, all proteins were expressed and recovered in yields
sufficient for testing. Truncated variant proteins were observed for
several of the mutants in which the amino-terminal sequences of
GRO were fused with carboxyl-terminal portions of IL-8 and varied
from 20 to 80% of the total chimera protein. Only the predicted
amino-terminal protein sequence was detected in these samples. The
carboxyl-terminal sequence of IL-8 reveals a Lys-Arg motif at residues
67 and 68 (Fig. 1). This sequence is a substrate site for the
yeast dibasic protease KEX2(36) , and cleavage at this position
can account for the observed reduction of approximately 5000 molecular
mass units. Although previous studies have suggested that the
-helix region is not essential for IL-8 recognition by neutrophil
receptors(21) , the utilization of a previously silent KEX2
yeast-processing site suggests an increase in the accessibility of this
region to protease attack and may indicate aberrant folding for this
series of chimeric proteins.
I-IL-8 binding to recombinant IL8R1 and IL8R2 cell lines.
GRO did not bind to IL8R1 and was 15-fold less potent than IL-8
for binding to IL8R2 (Table 3). IL-8 or GRO purified in
small scale by the procedure used for chimeric proteins had the same
potency as that purified in large scale by multiple chromatography
(data not shown). For IL-8/GRO chimeras, substitution of GRO
sequences onto the amino terminus of IL-8 (amino acids 1-18 or
more) resulted in a loss of affinity to both IL8R1 and IL8R2 (Table 3). Chimeric chemokines G32I, G46I, and G53I were even
less potent than GRO, which may reflect improper folding since
these proteins were prone to truncation as described above. In
substitutions at the carboxyl terminus, replacement of the -helix
residues 53-72 of IL-8 with the corresponding region of GRO
had no significant effect on receptor binding activity (Table 3,
I53G). In contrast, substitution of residues 46-72 of IL-8 with
the corresponding GRO sequence selectively reduced binding to
IL8R1 but not IL8R2 (Table 3, I46G), suggesting that residue(s)
within the third -sheet (amino acids 46-53) are involved in
specific binding to IL8R1 and not IL8R2.
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 nMI-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.
Residues within the
Third -Sheet reduced binding to IL8R1 without affecting
the binding to IL8R2, the role of individual amino acids in this region
was examined through point mutations to substitute GRO residues
into IL-8. Four amino acid residues differ between IL-8 and GRO in
this region: Arg
, Glu
, Leu, and
Asp
. The GRO amino acids were introduced into IL-8 to
test each residue individually and in combinations. The mutation of
IL-8 L49A, alone or in combination, selectively decreased the binding
to IL8R1 (Table 4). Neither the E48K replacement nor the
conservative substitution D52N affected binding to either receptor,
although the double mutation of E48K/D52N decreased binding to IL8R1 by
3-fold. R47K marginally decreased the binding to both IL8R1 and IL8R2
but had no impact on receptor binding in combination mutations. The
triple mutation R47K/E48K/D52N also had little effect on binding to
either receptor and no selectivity for IL8R1. No protein was expressed
for IL-8 mutations R47K/E48K and E48K/L49A/D52N, suggesting abnormal
folding and protein degradation. IL-8 R47K/L49A did not generate yeast
transformants, implying intolerance of the mutated protein by this host
cell. Consistent with the chimeric substitutions in the 46-53
region, substituting Leu decreased the binding to IL8R1
with no significant effect on binding to IL8R2. These data indicate
that Leu is the primary determinant within the third
-sheet for IL8R1 specificity of IL-8.
Residues within the
Amino-terminal Loop. IL-8 amino acids 13-15 were substituted
with comparable amino acids of GRO. No protein was expressed for
mutants containing the K15G mutation alone or in combinations,
suggesting that this alteration of the chemokine structure is not
tolerated. The mutation Y13L increased the binding to IL8R1 over 3-fold
without affecting IL8R2 binding (Table 4). S14Q marginally
decreased the binding to IL8R1, but the double mutation of Y13L/S14Q
significantly decreased the binding to IL8R2 over 4-fold with little
effect on IL8R1 (Table 4). These results are consistent with the
amino acid 1-18 chimeric substitutions and demonstrate that
Tyr and Ser contribute to the specificity of
IL-8 binding to both IL8R1 and IL8R2.
Homology
Model,
Ser, and Leu lie on a single face of the IL-8
molecule in a region that is unique and distant from both the conserved
ELR residues previously identified as essential for IL-8 binding to
IL-8 receptors and the strand of hydrophobic residues that participates
in the monomer-monomer interface of the dimer form of IL-8 (Fig. 4). These residues coincide with or are adjacent to a
surface-exposed hydrophobic pocket on IL-8 that consists of
Tyr, Phe
, Ile
, Val
,
Leu
, Leu, and Leu
(Fig. 4). This slot-like hydrophobic pocket is large
enough to accommodate a phenyl ring, which fits into the pocket as a
coin in a slot. The entrance to this pocket is flanked by
Tyr, Lys
, Phe, and
Arg.
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.
, Val, Asp
, and
Leu, corresponding to residues that are unique in IL-8.
F21N increased the binding affinity by 5.5- and 2.4- fold to IL8R1 and
IL8R2, respectively (Fig. 5). Mutation D45R had no effect on
IL8R1 and IL8R2 binding, indicating that this charged residue is not
critical for binding. The mutation V41F, which introduces a more bulky
hydrophobic group within the pocket, also had little effect on either
receptor. Three additional mutants, F21T, V41R, and V41K, did not
express the recombinant protein. At Leu, insertion of the
aromatic residue in L49F selectively decreased binding to IL8R1 by
6.2-fold with no significant effect on IL8R2. In contrast the
relatively conservative substitution of L49S had little effect on IL-8
receptor activity. Taken together with the observations for L49A and
for Y13L, these mutations reveal that key residues for determining
receptor-specific binding of IL-8 are clustered around the
surface-accessible hydrophobic pocket and suggest that increased access
to the pocket by removal of the aromatic residues at Tyr and Phe can enhance binding to IL8R1.
was less potent than IL-8 and had lower efficacy at optimal chemotaxis
concentrations (Fig. 6). Chimeric IL-8/GRO proteins
demonstrated neutrophil chemotactic activity consistent with relative
receptor binding activity ( Fig. 6and additional data not
shown). Proteins with characteristic binding properties of GRO, i.e. an absence of binding to IL8R1 and a reduced affinity
binding to IL8R2, displayed GRO-like chemotactic activity. An
IL-8-like chemotactic efficacy correlated with recognition by IL8R1,
and the relative potency paralleled the affinity for IL8R1 observed in
competitive binding assays (Fig. 6).
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.
) is also known as melanocyte growth-stimulating activity,
GRO/MGSA, and that GRO and GRO are also known as macrophage
inflammatory proteins MIP2 and MIP2, respectively); ELR,
Glu-Leu-Arg sequence; PCR,
polymerase chain reaction; CHO, Chinese hamster ovary.I-IL-8, Lawrence
Cousens for purification of GRO, Frank Masiarz and Scott
Chamberlain for protein sequencing, and Terry Calarco for preparation
of graphics.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  D. Wang, H. Wang, J. Brown, T. Daikoku, W. Ning, Q. Shi, A. Richmond, R. Strieter, S. K. Dey, and R. N. DuBois CXCL1 induced by prostaglandin E2 promotes angiogenesis in colorectal cancer J. Exp. Med., April 17, 2006; 203(4): 941 - 951. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M.-B. Huang, L. L. Jin, C. O. James, M. Khan, M. D. Powell, and V. C. Bond Characterization of Nef-CXCR4 Interactions Important for Apoptosis Induction J. Virol., October 15, 2004; 78(20): 11084 - 11096. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Rajagopalan and K. Rajarathnam Ligand Selectivity and Affinity of Chemokine Receptor CXCR1: ROLE OF N-TERMINAL DOMAIN J. Biol. Chem., July 16, 2004; 279(29): 30000 - 30008. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. De La Luz Sierra, F. Yang, M. Narazaki, O. Salvucci, D. Davis, R. Yarchoan, H. H. Zhang, H. Fales, and G. Tosato Differential processing of stromal-derived factor-1{alpha} and stromal-derived factor-1{beta} explains functional diversity Blood, April 1, 2004; 103(7): 2452 - 2459. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K.V. Brinda, N. Kannan, and S. Vishveshwara Analysis of homodimeric protein interfaces by graph-spectral methods Protein Eng. Des. Sel., April 1, 2002; 15(4): 265 - 277. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Suetomi, Z. Lu, T. Heck, T. G. Wood, D. J. Prusak, K. J. Dunn, and J. Navarro Differential Mechanisms of Recognition and Activation of Interleukin-8 Receptor Subtypes J. Biol. Chem., April 23, 1999; 274(17): 11768 - 11772. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Dealwis, E. J. Fernandez, D. A. Thompson, R. J. Simon, M. A. Siani, and E. Lolis Crystal structure of chemically synthesized [N33A] stromal cell-derived factor 1alpha , a potent ligand for the HIV-1 "fusin" coreceptor PNAS, June 9, 1998; 95(12): 6941 - 6946. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. J. Rollins Chemokines Blood, August 1, 1997; 90(3): 909 - 928. [Full Text] [PDF]
|
|
|
|
|
|
L. Wu, N. Ruffing, X. Shi, W. Newman, D. Soler, C. R. Mackay, and S. Qin Discrete Steps in Binding and Signaling of Interleukin-8 with Its Receptor J. Biol. Chem., December 6, 1996; 271(49): 31202 - 31209. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Ye, L. L. Kohli, and M. J. Stone Characterization of Binding between the Chemokine Eotaxin and Peptides Derived from the Chemokine Receptor CCR3 J. Biol. Chem., August 25, 2000; 275(35): 27250 - 27257. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. S. Mizoue, S. K. Sullivan, D. S. King, T. N. Kledal, T. W. Schwartz, K. B. Bacon, and T. M. Handel Molecular Determinants of Receptor Binding and Signaling by the CX3C Chemokine Fractalkine J. Biol. Chem., August 31, 2001; 276(36): 33906 - 33914. [Abstract] [Full Text] [PDF]
|
|
| ||||||
