©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Role of Tyr and Lys of Interleukin-8 in the High Affinity Interaction with the Interleukin-8 Receptor Type A(*)

Ingrid U. Schraufstätter (§) , Min Ma , Zenaida G. Oades , Diana S. Barritt , Charles G. Cochrane

From the (1) Department of Immunology, Scripps Research Institute, La Jolla, California 92037

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Interleukin-8 (IL-8) has at least two binding regions for both the A and the B type IL-8 receptors. This study defines an important region between Cys and Cys that, together with the Glu-Leu-Arg sequence of the NH terminus, accounts for the high affinity binding of IL-8 to the IL-8 A receptor on leukocytes. Utilizing rabbit IL-8 that shares 82% sequence identity with human IL-8, but has 200-fold lower binding affinity for the IL-8 A receptor, residues of the human homologue were sequentially exchanged into the rabbit molecule. Replacement of rabbit His and Thr with Tyr and Lys of the human molecule converted the low affinity binding of the rabbit IL-8 to the high affinity binding of human IL-8 as shown by both competitive binding and by Ca mobilization. As a corollary, replacement of the Tyr and Lys of the human IL-8 with His and Thr of the rabbit IL-8 reduced binding activity of this mutated human IL-8 200-fold. The site of interaction on the IL-8 receptor type A for the Tyr and Lys sequence was found to be in the NH-terminal region of this receptor. A structural pattern of the binding between IL-8 and the A type IL-8 receptor is proposed.


INTRODUCTION

IL-8() is a stimulatory peptide for neutrophils that appears to play an important role in the inflammatory process (1, 2) . It shows high affinity binding for two receptors on neutrophils (3, 4, 5) : the specific A-type receptor, which has a Kof 10 M for human IL-8 but binds rabbit IL-8, MGSA, and NAP2 with poor affinity (>3 10 M in all cases) (6, 9) and the less selective B-type receptor, which binds human and rabbit IL-8 and MGSA with nanomolar affinities (6, 9) and NAP2 with 10 M affinity. It has been shown that IL-8 binds to both A and B receptors in part through a Glu-Leu-Arg sequence of the NH terminus (7, 8) . The binding of IL-8 to its two receptors is, however, more complex. While the amino-terminal Glu-Leu-Arg sequence is necessary for high affinity binding, it is not sufficient. Both rabbit IL-8 and MGSA contain this sequence but bind poorly to the A receptor (6, 9) , showing an affinity that is 150- and 200-fold lower than that of human IL-8. Additional binding sites have been postulated; for the IL-8 B receptor the carboxyl-terminal -helix appears to be involved (8, 9) , and for the IL-8 A receptor a second site has been postulated to be located between amino acids 7 and 50 (9) . Several locations in this area, the loop between amino acids 8 and 16, and the turns at residues 17-21 and 30-35 are surface-exposed (10, 11) , and as such they are possible regions of interaction with the receptor.

We now report that mutation of amino acids 13 and 15 from the rabbit (His-Ser-Thr) to the human sequence (Tyr-Ser-Lys) confers the high affinity of human IL-8 for the human IL-8 A receptor to this mutated form of rabbit IL-8. The site of interaction of this region of IL-8 with the IL-8 type A receptor appears to be in the amino terminus of the receptor as shown by studies with a chimeric receptor construct.


EXPERIMENTAL PROCEDURES

Materials

The cDNA representing the IL-8 A receptor was obtained by PCR amplification of human neutrophil RNA and cloned into the EcoRI site of the stable mammalian expression vector pSFFV.neo as described previously (9, 12) . The cDNA that encodes the human IL-8 B receptor was kindly provided by Philip Murphy (NIH) and similarly cloned into pSFFV.neo (9) . Reagents and enzymes for the manipulation of DNA were from New England Biolabs, Life Technologies, Inc., Boehringer Mannheim, or U. S. Biochemical Corp. Oligonucleotides were purchased from Operon. I-IL-8 was prepared as described previously (9) .

Construction of Rabbit IL-8 Mutants

Rabbit and human IL-8 were expressed in Escherichia coli as fusion proteins with glutathione S-transferase as described previously (9) . r/h MUT1, r/h MUT2 and the h/r mutants 1-3 were generated by introducing the respective base changes into the oligonucleotide used for PCR amplification and verified by sequencing with the Sequenase version 2.0 kit (U. S. Biochemical Corp.). r/h MUT3 was introduced by the method of Kunkel (13) using the rabbit IL-8 cDNA cloned into M13p19. Single-stranded DNA template was prepared in the dut ung strain of E. coli CJ 236. Mutagenesis was conducted in the E. coli strain DH5aF`, selected by DNA sequencing and subcloned into the pGEX-2T plasmid (Pharmacia Biotech Inc.). The mutants described show the sequences shown in Fig. 1.


Figure 1: Sequence comparison between human and rabbit IL-8 and mutants.



Amino acids differing from the rabbit IL-8 sequence are underlined. Protein was purified and thrombin cleaved as described (14) . IL-8 was quantified by enzyme-linked immunosorbent assay using a goat anti-rabbit IL-8 polyclonal antibody that cross-reacts with human IL-8 in a sandwich enzyme-linked immunosorbent assay using biotinylated second antibody (15) .

Construction of IL-8 Receptor A/B and B/A Chimerae

The chimeric receptors were constructed by using the natural CelII site in the first transmembrane domain shared by both the A and the B type IL-8 receptors, as described previously for a rabbit/human receptor chimera (16, 17) . Polymerase chain reaction-amplified cDNA was digested with CelII and EcoRI and ligated into pSFFV.neo, resulting in a receptor that switches from the A to the B or from the B to the A receptor sequence at Asn in the IL-8 A receptor.

Receptor Expression

HL60 cells were transfected with ScaI-cut receptor DNA by electroporation (18) , and mouse L-cells were transfected with lipofectamine (Life Technologies, Inc.). Both cells were selected in the presence of 0.6 mg/ml of G418 (Life Technologies, Inc.). Receptor expression was verified by fluorescence-activated cell sorting analysis using a polyclonal rabbit antibody raised against a fusion protein of the first 44 amino acids of the IL-8 A receptor with glutathione S-transferase. The antibody was produced by biweekly injections of 100 µg of this antigen. 200 µl of 10 cells/ml in 2% fetal calf serum/phosphate-buffered saline were incubated with a 1:100 dilution of antibody for 30 min on ice. After three washes, the cells were incubated for 30 min with a 1:250 dilution of fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Tago). The cells were washed once more in 2% fetal calf serum/phosphate-buffered saline and analyzed on FACSCAN.

Binding Studies

I-IL-8 binding, competition with mutant proteins, and Ca mobilization were performed as described previously (9) . Untransfected mouse L-cells or HL60 cells showed no Ca response when stimulated with 10 M IL-8.


RESULTS AND DISCUSSION

Since rabbit IL-8 showed a 200-fold lower affinity for the IL-8 A receptor than its human homologue and since it is 82% identical to the human sequence, we decided to mutate amino acids that differ in the two molecules from the rabbit sequence back to the human sequence.

An increase in affinity of these mutants should define amino acids in the human sequence that are important for high affinity binding to the A receptor.

In r/h MUT2 His and Thr in the rabbit IL-8 were replaced with Tyr and Lys of the human sequence; in r/h MUT3, Val of the rabbit sequence was converted to Ser of the human sequence, and in r/h MUT1 the last four differing amino acids starting with Gln in the carboxyl-terminal -helix were replaced by the human sequence.

These mutants were used to assess their affinity for the transfected IL-8 A receptor in competition binding experiments with human IL-8. When His and Thr in the rabbit IL-8 were replaced with Tyr and Lys of the human sequence (r/h MUT2), the binding affinity rose to that of human IL-8 for the IL-8 A receptor as shown in Fig. 2 A. The other two mutants, r/h MUT1 and r/h MUT3 behaved like wild-type rabbit IL-8 in competition binding experiments with this receptor (Fig. 2 A).


Figure 2: Competition binding between I-IL-8 and various mutants in L-cells expressing the IL-8 A receptor. I-IL-8 binding and competition with mutant proteins were performed as described previously (9). A, competition binding with humanized rabbit mutants. , human IL-8; , rabbit IL-8; r/h MUT1; , r/h MUT2; ▾ r/h MUT3. The mean ± S.D. of three experiments in duplicate is shown. B, competition binding with human IL-8 mutated to rabbit sequence. , human IL-8; , h/r MUT1; , h/r MUT2, , h/r MUT3



In order to verify this observation we replaced the Tyr and Lys in the human sequence with the respective amino acids in the rabbit molecule (h/r MUT3). To assess the relative importance of each amino acid, Tyr (h/r MUT2) and Lys (h/r MUT1) were mutated individually, and the competition experiment with I-IL-8 was repeated. The double mutation had the low affinity of rabbit IL-8 (Fig. 2 B). While replacement of Lys with Thr (h/r MUT1) only resulted in a 12-fold decrease in affinity compared with human wild-type IL-8, replacement of Tyr with His of the rabbit sequence decreased the affinity 150-fold (Fig. 2 B). Thus a single amino acid substitution caused a major loss of affinity.

Results obtained by competitive binding were paralleled by cell function as assessed by determinations of Ca mobilization in indo-1-labeled cells (Fig. 3). As in the binding experiments r/h MUT2 behaved like human wild type IL-8 in L-cells expressing the IL-8 A receptor, while neither one of the other two rabbit mutants, r/h MUT1 and r/h MUT3, increased the capacity to mobilize Ca.


Figure 3: Comparison of Ca mobilization in indo-1-labeled mouse L-cells carrying the IL-8 A receptor after stimulation with rabbit IL-8 or r/h MUT2. Ca mobilization was measured on an SLM 8000 fluorometer as described previously (9). The stimulus was added at 10 s ( arrow).



Two areas of interaction between the IL-8 A receptor and IL-8 have been described. These are the third extracellular loop and the amino terminus of the receptor. The third extracellular loop of the A receptor, specifically Asp and Arg are necessary for high affinity ligand binding (19) . This area is conserved throughout the receptor family (3, 4, 20) and has been proposed to interact with the Glu-Leu-Arg sequence of IL-8. Domain swapping between the IL-8 A and B receptors has shown that the amino terminus determines whether the receptor behaves like the A type receptor, which specifically binds human IL-8, or like the B type IL-8 receptor, which interacts with high affinity with MGSA as well as with IL-8 (17, 18) . Systematic mutations of the first 30 amino acids of the IL-8 A receptor failed to define any consensus sequence involved, but individual substitution by alanines of Thr, Pro, and Tyr decreased the affinity, and the presence of Cys is absolutely necessary for ligand interaction, presumably because of its function in maintaining structure (21) . A high affinity for rabbit IL-8, as described previously for MGSA, depended on the presence of the amino terminus of the IL-8 B receptor; when we exchanged the first 57 amino acids of the human IL-8 A receptor with B receptor sequence, rabbit IL-8 replaced human IL-8 with high affinity at a Kof 8.1 10 M, which is close to the Kof the B receptor for human IL-8 (5 10 M).

The reverse chimera that places the NH terminus of the IL-8 A receptor onto the B receptor sequence at the same location in the first transmembrane spanning domain behaves like the IL-8 A receptor. This A/B IL-8 receptor chimera shows poor binding competition for rabbit IL-8 but high affinity binding when r/h MUT2 with Tyr and Lys was used (Fig. 4). These results suggest that the region of amino acids 13-15 in human IL-8 interacts with the amino terminus of the IL-8 A receptor, which indicates that there are at least two areas of interaction between IL-8 and the A receptor.


Figure 4: Competition binding between human I-IL-8 and human, rabbit, or r/h MUT2 and MGSA in HL60 cells expressing an A/B receptor chimera. The mean ± S.D. of three experiments in duplicate is shown. , human IL-8; , rabbit IL-8; , r/h MUT2 and MGSA.



In Fig. 5the regions on IL-8 important for high affinity binding to the IL-8 A receptor are highlighted. Amino acids 4-6 of IL-8 (Glu-Leu-Arg) bind to the receptor, presumably to Glu and Arg in the third extracellular loop, while the region in the IL-8 molecule including Tyr, Ser, and Lys interacts with the amino terminus of the IL-8 A type receptor. Alternatively, the Tyr and Lys sequence may affect the conformation of IL-8 in a way that increases the affinity for the amino terminus of the A receptor. The structural analysis of IL-8 (11) shows that the area of amino acids 8-18 forms a loop in a series of nonclassical turns and that the structure of MGSA differs significantly from that of IL-8 in that area. In agreement with this, r/h MUT2 had no effect on binding competition for the IL-8 B receptor, suggesting that this region is of little importance to that receptor. A 6-fold decrease in affinity for the combination of the two IL-8 receptors as expressed on neutrophils has recently been described for a human IL-8 molecule in which Tyr was replaced with Thr (22) . Alternatively a threonine substitution in this location may have a smaller effect on the IL-8 A receptor individually. The one amino acid substitution from Tyr to His in this location had a much greater effect in our system where we looked specifically at IL-8 type A receptor binding. This specific effect of a single amino acid may explain why systematic substitution of all amino acids of the NH terminus of the IL-8 A receptor with alanines failed to define a receptor ligand binding site (21) . As shown in Fig. 5 both Tyr and Lys of the IL-8 molecule are surface-exposed. These two areas, i.e. the Glu-Leu-Arg sequence and the loop including Tyr-Ser-Lys form opposite ends on the same face of the IL-8 molecule. The two regions lie 23 Å apart, which would allow the molecule to span the distance between the amino terminus and the adjacent third extracellular loop of the receptor. While the NMR structure of IL-8 shows a dimer, IL-8 is functional in its monomeric form at physiological concentrations (23, 24) . Both the NH-terminal Glu-Leu-Arg sequence and the Tyr-Lys region would be surface-exposed independently from the monomeric or dimeric state of the molecule.


Figure 5: Schematic representation of the two areas of human IL-8 important for high affinity binding to the IL-8 A receptor: the ELR consensus sequence (amino acids 4-6) at the amino terminus and the region around amino acids 13 and 15. The precise location of Glu was not assigned in the data bank.



Complex interactions with seven-membrane-spanning receptors have been described previously for small hydrophobic peptide ligands (25) , amines (26) , and the photon-induced cis-trans conversion of retinal in the rhodopsin system (27) . These ligands interact primarily with residues in the hydrophobic transmembrane domains. While the larger, hydrophilic IL-8 molecule seems to interact with more surface-exposed amino acids on the receptor, the interaction clearly involves more than one area of importance.

Additional interaction with interhelical domains has not been evaluated at this point. Understanding the precise nature of the interactions between IL-8 and its receptors will provide an essential basis for the development of therapeutic inhibitors of the IL-8-stimulated reactions in the inflammatory process.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants A127506 and HL 23584. 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: Dept. of Immunology, Scripps Research Inst., 10666 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-554-8254; Fax: 619-554-6123.

The abbreviations used are: IL-8, interleukin-8; r/h, rabbit/human; h/r, human/rabbit; MGSA, melanoma growth-stimulatory activity; NAP2, neutrophil activating peptide 2.


ACKNOWLEDGEMENTS

We thank Dr. Jian Yao for assistance with the computer modeling of the IL-8 molecule.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.