©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Receptor Recognition and Specificity of Interleukin-8 Is Determined by Residues That Cluster Near a Surface-accessible Hydrophobic Pocket (*)

(Received for publication, October 20, 1995)

Mary Ellen Wernette Hammond (§) Venkatakrishna Shyamala Michael A. Siani (¶) Carol A. Gallegos Paul H. Feucht Janine Abbott Gena Reza Lapointe Mehrdad Moghadam Hamid Khoja Joan Zakel Patricia Tekamp-Olson

From the From Chiron Corporation, Emeryville, California 94608

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 beta-sheet (amino acids 18-32) reduced binding to both IL8R1 and IL8R2. The third beta-sheet of IL-8 (amino acids 46-53) was required for binding to IL8R1 but not IL8R2. Exchanges of the second beta-sheet (amino acids 32-46) or the carboxyl-terminal alpha-helix (amino acids 53-72) had no significant effect. When IL-8 sequences were substituted into GRO, a single domain containing the second beta-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 beta-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^14, 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^4-Leu^5-Arg^6 region, can modulate receptor binding affinity and IL8R1 specificity.


INTRODUCTION

Interleukin-8 (IL-8) (^1)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 beta-sheets, and a carboxyl-terminal alpha-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 GROalpha (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^4-Leu^5-Arg^6 (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 GROalpha or rabbit IL-8 and demonstrated that IL8R1 specificity element(s) reside between Cys^7 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.


EXPERIMENTAL PROCEDURES

Materials

Recombinant human IL-8, GROalpha (GRO/MGSA), and I-IL-8 were prepared as described(25) . Recombinant human GRObeta (MIP2alpha) and GRO (MIP2beta) were produced in yeast and purified by chromatography on heparin-Sepharose and Sephacryl S-100 HR.

Construction of Chimeric Chemokines

Corresponding domains of IL-8 and GRO 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 MIP2beta 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 alpha-factor leader (29) .


Figure 1: Amino acid sequences of C-X-C chemokines. Sequences for IL-8, GRO/MGSA, GRObeta/MIP2alpha, and GRO/MIP2beta 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.''





Construction of Mutant Chemokines

All constructs were generated by PCR according to Shyamala and Ames (30) with the exception that vent polymerase was used in the place of Taq polymerase. The DNA template was either the native IL-8 or il8syn(27) . Amino acid substitutions were introduced by overlap PCR using the sense and antisense mutated primers in combination with appropriate end primers as listed in Table 2(31) . The PCR-amplified fragment was digested with Asp718/XhoI and ligated into the transfer vector.



Protein Expression and Purification

Expression cassettes for yeast secretion were transferred as BamHI restriction fragments into vector pAB24 (29) and introduced into Saccharomyces cerevisiae strain MB2-1 by electroporation. Chimeric and mutant chemokines were purified from 50-200 ml of yeast culture broth by batch adsorption on S-Sepharose FF (Pharmacia Biotech Inc.) after adjustment to pH 5.5 with 50 mM sodium acetate and eluted in 20 mM HEPES, pH 8.3, 1 M NaCl to a final concentration of 0.2-2 mg/ml. SDS-polyacrylamide gel electrophoresis on 18% Tris/glycine gels (Novex) indicated 80-95% purity. Protein concentrations were estimated by Coomassie-stained polyacrylamide gels and by BCA (Pierce) protein assays. Amino acid composition and amino-terminal sequencing were performed on selected proteins and agreed with predicted protein sequences.

Binding Assays

Competitive binding assays for the chimeric proteins were performed on CHO-IL8R1 and CHO-IL8R2 cells essentially as described(25) . Assays were performed in triplicate and data were analyzed by GraFit(32) .

Chemotaxis Assays

Assays were performed in triplicate on freshly isolated human neutrophils as described(25) . Chemotaxis to f-Met-Leu-Phe (100 nM) was measured as a positive control for each experiment.

Homology Modeling

A homology-based model of GRO 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) .


RESULTS

Chimeric IL-8/GRO Proteins

Chimeric IL-8/GRO 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^18, 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 alpha-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 alpha-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.

Receptor Binding Activity of Chimeras

Receptor binding activity of chimeric proteins was measured by competition of 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 alpha-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 beta-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 beta-sheet region. Replacement of amino acids 18-32, corresponding to the first beta-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 beta-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 beta-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 beta-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. bullet, IL-8; , F21N; , L49F; up triangle, V41F; circle, D45R; box, L49S.



Point Mutations of IL-8 to GRO Residues within the Third beta-Sheet

Since the replacement of IL-8 amino acids 46-53 with GRO 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 beta-sheet for IL8R1 specificity of IL-8.



Point Mutations of IL-8 to GRO Residues within the Amino-terminal Loop

The chimera data demonstrated that IL-8 amino acids 1-18 of the amino-terminal loop are important for binding of IL-8 to both IL8R1 and IL8R2. Within this region there are several amino acid differences; particularly, the five amino acids of IL-8 at positions 13-17 are replaced by only four different residues in GRO. 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^14 contribute to the specificity of IL-8 binding to both IL8R1 and IL8R2.

Comparison of IL-8 Structure and GRO Homology Model

Based upon the established structure of IL-8(16, 17) , residues Tyr, Ser^14, 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.

IL-8 Mutations within the Hydrophobic Pocket Region

Additional point mutations of IL-8 were designed to test the role of the key hydrophobic pocket and flanking residues in the recognition of IL-8 by IL8R1 and IL8R2. Mutations were introduced at positions Phe, 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.

Neutrophil Chemotaxis

The functional activation of IL-8 receptors was assessed in neutrophil chemotaxis assays. GRO 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 (circle), GRO (bullet), G18I (box), and I18G (). B, effect of first beta-sheet residues 18-32. Chemotaxis assays were performed with IL-8 (circle), GRO (bullet), I18G32I (up triangle), and G18I32G (). C, effect of third beta-sheet residues 46-53. Chemotaxis assays were performed with IL-8 (circle), GRO (bullet), I46G (times), I46G53I (down triangle), 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.


DISCUSSION

By construction of IL-8/GRO chimeras we have demonstrated that the amino-terminal loop (amino acids 1-18) and the first beta-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 beta-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/GROalpha chimeric proteins affects specificity of binding to IL8R2 but not to IL8R1. Chimeric I51Galpha 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 alpha-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 (GROalpha and NAP-2) that bind to IL8R2 but not to IL8R1(18, 19) . The solution structure of GROalpha 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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Chiron Corp., 4560 Horton St., Emeryville, CA 94608. Tel.: 510-601-2939; Fax: 510-658-0329.

Current address: Gryphon Sciences, 250 E. Grand Ave. #90, South San Francisco, CA 94080.

(^1)
The abbreviations used are: IL-8, interleukin-8; IL8R1, interleukin-8 receptor type 1; IL8R2, interleukin-8 receptor type 2; GRO, growth related protein (note that GROalpha is also known as melanocyte growth-stimulating activity, GRO/MGSA, and that GRObeta and GRO are also known as macrophage inflammatory proteins MIP2alpha and MIP2beta, respectively); ELR, Glu^4-Leu^5-Arg^6 sequence; PCR, polymerase chain reaction; CHO, Chinese hamster ovary.


ACKNOWLEDGEMENTS

We thank Lauri Goda for DNA synthesis, Jean Joh and Guilin Wang for DNA sequencing, Susan Hilt for providing CHO-IL8R1 and CHO-IL8R2 cells, Kathryn Collins for I-IL-8, Lawrence Cousens for purification of GRO, Frank Masiarz and Scott Chamberlain for protein sequencing, and Terry Calarco for preparation of graphics.


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