©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
N51 Competes I-Interleukin (IL)-8 Binding to IL-8R but Not IL-8R
STRUCTURE-FUNCTION ANALYSIS USING N51/IL-8 CHIMERIC MOLECULES (*)

(Received for publication, September 5, 1995)

Julia N. Heinrich (§) Rodrigo Bravo (¶)

From the Department of Molecular Biology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have demonstrated that the mouse chemokine N51, also known as KC, can compete for I-human interleukin-8 (IL-8) binding to NIH 3T3 cells expressing the human IL-8 receptor beta (NIH-IL-8Rbeta) but not the IL-8 receptor alpha (NIH-IL-8Ralpha). In addition, we used the chimeras between N51 and IL-8 described previously (Heinrich, J. N., O'Rourke, E. C., Chen, L., Gray, H., Dorfman, K. S., and Bravo, R.(1994) Mol. Cell. Biol. 14, 2849-2861; Heinrich, J. N., and Bravo, R.(1995) J. Biol. Chem. 270, 4987-4989) to evaluate possible contributions of equivalent domains from each chemokine to binding and specificity. Specifically, the amino acid sequences between cysteines 2 and 3 or between cysteines 3 and 4 or the alpha-helical C-terminal end (domains I, II, and III, respectively) of one of the chemokines was exchanged with the corresponding sequence of the other and vice versa. Chimeras of IL-8 containing either domain II or III of N51 behaved similarly, but not identically, to IL-8 in competing I-IL-8 binding with both NIH-IL-8Ralpha cells and NIH-IL-8Rbeta cells. The IL-8 chimera containing domain I of N51 did not compete. On the other hand, N51 competes I-IL-8 binding with NIH-IL-8Rbeta but not NIH-IL-8Ralpha cells. The N51 chimera containing domain I of IL-8 was an agonist with NIH-IL-8Ralpha cells and was an even more potent agonist with NIH-IL-8Rbeta cells. On the latter cells it was more potent than either IL-8 or N51. The N51 chimera containing domain II of IL-8, compared with N51, was a partial agonist with NIH-IL-8Ralpha cells but was equivalent to N51 with NIH-IL-8Rbeta cells. However, N51 chimera containing domain III of IL-8 was a partial agonist with both cells. The results are consistent with the observations we originally made with human neutrophils and the NIH mouse IL-8Rbeta cells, i.e. domain I confers binding specificity for IL-8 and domains II and III of IL-8 and N51 may be interchangeable but they are not equivalent. Although we originally hypothesize that domains II and III confer binding specificity to N51, these results emphasize the role of domain III.


INTRODUCTION

The last decade has seen the emergence of a superfamily of genes whose protein products are proinflammatory cytokines and which are collectively called chemokines (for reviews see (3) and (4) ). In general, the chemokine genes code for secreted peptides of about 70-100 amino acids containing 4 conserved cysteines. The first two cysteines are either separated by one amino acid or adjacent to each other, and this distinction, which is correlated with functional properties (see below), is used to designate the members into two classes, the ``C-X-C'' or alpha-family and the ``C-C'' or beta-family, respectively. For members whose three-dimensional structure has been determined, namely platelet factor-4, interleukin-8 (IL-8), (^1)GROalpha or melanoma growth-stimulating activity (GROalpha/MGSA), and macrophage inflammatory protein-1beta (MIP-1beta), the cysteine motif appears to restrict the secondary structure to forming two disulfide bonds, one between the first and third cysteines and another between the second and fourth cysteines, resulting in similar tertiary structures. It is believed that all the members share this tertiary configuration, since the disulfide bonds are essential for the biological activity of chemokines for which this property has been tested (for reviews see Refs. 5 and 6). Recently, the identification of lymphotactin containing only 2 of the conserved cysteines (7) indicates that other families of chemokines may exist.

There are at least 15 human chemokines described so far, 8 in the ``C-X-C'' family including platelet factor-4, IL-8, GROalpha/MGSA, GRObeta, GRO, ENA-78, and -IP-10 and 7 in the ``C-C'' family including monocyte chemotactic protein-1 (MCP-1), MCP-2, MCP-3, RANTES, MIP-1beta, and I309(4) . The numbers are certain to increase from both the identification of the corresponding human homolog known in other species, like the newest ``C-C'' member from guinea pig, exotaxin(8) , and the direct finding of novel human members. The identification of corresponding homologs between species is complicated by two important observations. First, not all species may possess the same chemokines. For example, IL-8 has not been identified in mice. Second, there can be considerable variation in what represents a homolog in a particular species. For example, mouse MIP-2 was considered originally the homolog of human GROalpha/MGSA (51% identity); now mouse N51/KC is accepted as the true homolog (67% identity).

Generally observed biological activities of ``C-X-C'' and ``C-C'' chemokines are the activation of neutrophils and monocytes, respectively. Early studies showed that a number of the ``C-X-C'' chemokines, including ENA-78(9) , MIP-2, neutrophil-activating peptide-2 (NAP-2), and GROalpha/MGSA could desensitize human neutrophils to IL-8 and that MIP-2, NAP-2, and GROalpha could compete for I-IL-8 binding to human neutrophils(3, 9, 10) . These observations stimulated several investigations to determine whether specific residues in the chemokine promotes receptor binding specificity(11, 12, 13) . We demonstrated that N51 is biologically active on human neutrophils via the IL-8R and then performed structure and function analysis of N51 by comparing the biological activities of chimeras between N51 and IL-8(1) . Our data indicated that the amino acid sequences between cysteines 3 and 4 (domain II) and the alpha-helical C-terminal end (domain III) are important for the binding of N51.

Two human IL-8Rs, IL-8Ralpha and IL-8Rbeta, have been cloned, and their deduced amino acid sequences are typical of a seven-transmembrane G-coupled receptor(14, 15) . The receptors have been expressed in a number of cell lines and shown to bind IL-8 with high affinity. In addition, a number of the other ``C-X-C'' members, including GROalpha/MGSA, rabbit IL-8, and NAP-2, appear to show a low affinity for IL-8Ralpha and a high affinity for IL-8Rbeta(16, 17, 18) .

Here we have investigated whether N51 and chimeras between N51 and IL-8 can compete for I-IL-8 binding to the IL-8Rs expressed independently in NIH 3T3 cells (NIH-IL-8Ralpha cells and NIH-IL-8Rbeta cells). Our data demonstrate that N51 competes I-IL-8 binding for IL-8Rbeta but not for IL-8Ralpha. It also shows that the domains most important in conferring binding specificity are domain III of N51 and domain I of IL-8.


MATERIALS AND METHODS

Cloning and Expression of the Human IL-8 Receptors

The polymerase chain reaction (PCR) cloning of the human IL-8 receptors alpha and beta (IL-8Ralpha and IL-8Rbeta, respectively) and their expression in NIH 3T3 cells was performed as described previously for the mouse IL-8Rbeta (2) with appropriate modifications. Briefly, human genomic DNA (Stratagene) and the oligonucleotide primers derived from the human IL-8Ralpha (sense 5`-AACCAGAATTCAAACTGAAGAGGACATG-3` and antisense 5`-TCGATAAGCTTCAGAGGTTGGAAGAGACATTG-3`) and IL-8Rbeta (sense 5`-TCAGGGAATTCGTTTACCTCAAAAATGGAAG-3` and antisense 5`-AGGCAAAGCTTCTTAGAGAGTAGTGGAAGTG-3`) sequences (14, 15) were used for the PCR cloning. The oligonucleotide primers corresponding to the sense 5` and antisense 3` ends included the enzyme restriction sites for EcoRI and HindIII, respectively. The human genomic DNA was denatured for 10 min at 95 °C, briefly placed on ice, and then added to a PCR reaction containing the GeneAmp core reagents (Perkin-Elmer). The PCR was 50 cycles of 30 min at 95 °C, 1 min at 60 °C, and 1 min at 72 °C. The PCR product was digested with EcoRI and HindIII, subcloned into Bluescript (Stratagene), and sequenced using Sequenase version 2.0 DNA sequencing kit (U. S. Biochemical Corp.).

To generate cell lines stably expressing the receptors, NIH 3T3 cells were transfected with either pMexneo alone, pMexneo-IL-8Ralpha, or pMexneo-IL-8Rbeta and selected in Dulbecco's modified minimum essential medium containing antibiotics (100 µg/ml penicillin and 50 µg/ml streptomycin), 10% bovine serum, and 0.8 mg/ml G418. The generated cell lines, NIH-pMexneo, NIH-IL-8Ralpha, and NIH-IL-8Rbeta, represented a mixed population of the G418-resistant colonies. For binding analysis cells were detached from the plates by incubation with Hanks' balanced salt solution (HBSS) with phenol red, supplemented with 25 mM HEPES (pH 7.5), 0.2% bovine serum albumin (BSA, fatty acid-free, Sigma), and 5 mM EDTA (HBSS-B1) until they could be harvested by gentle scraping (15 min). The cells were pelleted by centrifugation at 250 times g for 10 min, washed twice in HBSS supplemented with 25 mM HEPES (pH 7.5), 1 mM CaCl(2), 1 mM MgCl(2) containing 2% BSA (HBSS-B2), resuspended to 5-15 times 10^6 cells/ml with HBSS-B2 containing 0.2% BSA (HBSS-B3), and cooled on ice for 30 min.

Binding Analysis

Binding was performed with 0.5-1.0 times 10^6 cells using 5 ng (3 nM) of I-IL-8 in the absence and presence of competitor in a total volume of 200 µl of HBSS-B3 for 1 h at 4 °C in tubes formatted in a 96-well box with continuous shaking on a DIGIT shaker. The binding reaction was stopped by diluting it with 1 ml of ice-cold HBSS-B3 and filtering it through glass fiber filters that were preincubated with 1% polyethyleneimine and 1% Prosil-28 (PRC Inc., Gainesville, FL). The filters were washed twice with 3 ml of ice-cold phosphate-buffered saline or HBSS-B2 containing 0.05% BSA and counted in a counter. The counts obtained from the binding reactions done in the absence and presence of 200-fold excess IL-8 represent total and nonspecific binding, respectively, and their difference corresponds to specific binding.

The N51 and the chimeric proteins were expressed in the baculovirus system and purified as described previously(1) . IL-8 (R & D Systems) was iodinated by the Bolton-Hunter method to a specific activity of 97.4 mCi/µg (DuPont NEN Custom Iodination Laboratory) as described (1) . The competition curve was performed with a 0.1-200-fold excess of competitor as compared with 3 nMI-IL-8. Data were analyzed by least square nonlinear curve fit for a model of single and multiple sites using KaleidaGraph software.


RESULTS AND DISCUSSION

We have reported previously that baculovirus-expressed N51 binds human neutrophils via the IL-8R(s) and that a N51 chimera containing domain I of IL-8 (N51/IL-8I) made N51 more IL-8-like. Our observations suggested that the high affinity receptor for N51 is the IL-8Rbeta(1) . To extend our findings we decided to investigate the ability of N51 and chimeras between N51 and IL-8 to compete for I-IL-8 binding to the human IL-8Ralpha and IL-8Rbeta independently expressed in NIH 3T3 cells. The chimeras between IL-8 and N51, illustrated in Fig. 1, have been described previously(1) .


Figure 1: Domains exchanged between N51 and IL-8 and comparison of the amino acid sequence of the chimeras. The first and sixth lines show the sequences of domains I, II, and III from N51 and IL-8, respectively. N51 residues and those in common with IL-8 are white are on a black background while those unique for IL-8 are black on a white background.



The competition curves for I-IL-8 binding to the NIH-IL-8Ralpha cells in the presence of increasing concentrations of IL-8, N51, and the chimeras are presented in Fig. 2. The competition with IL-8 shows a sigmoidal curve with an apparent K(d) of 5.5 nM. The IL-8 chimeras containing domain II or III from N51, IL-8/N51II and IL-8/N51III, respectively, retained their ability to compete I-IL-8 (Fig. 2A). However, IL-8/N51II is a significantly weaker competitor than IL-8. On the other hand, N51 was unable to compete for I-IL-8 binding to the NIH-IL-8Ralpha cells, but the N51 chimera containing domain I of IL-8, N51/IL-8I, competes efficiently, with a curve that is similar to IL-8 (Fig. 2B). The other chimeras, N51/IL-8II and N51/IL-8III, weakly competed when present at high concentrations (Fig. 2B). These results show that the chimeras that possess domain I of IL-8 such as IL-8/N51III, N51/IL-8I, and to a lesser extent IL-8/N51II are agonists, while those that lack domain I but have domain II or III of IL-8 such as N51/IL-8II and N51/IL-8III are at best partial agonists in NIH-IL-8Ralpha cells. This indicates that for binding of IL-8 to IL-8Ralpha, domain I is the major determinant. Recently, it has been shown that tyrosine 13 and lysine 15 are essential for high affinity binding of IL-8 to the IL-8Ralpha(19) ; these residues are not present in N51 but are in domain I of IL-8.


Figure 2: Competition of I-IL-8 binding to NIH-IL-8Ralpha cells. Binding was done with 3 nMI-IL-8 for 1 h at 4 °C in the absence and presence of up to 200-fold increasing concentrations of: A, IL-8, IL-8/N51II, or IL-8/N51III; and B, N51, N51/IL-8I, N51/IL-8II, or N51/IL-8III.



Fig. 3shows the competition of I-IL-8 binding to the NIH-IL-8Rbeta cells by increasing concentrations of either IL-8, N51, or the different chimeras. The competition by IL-8 shows a sigmoidal curve with an apparent K(d) of 8.7 nM. In contrast to the results obtained with NIH-IL-8Ralpha cells the capacity of IL-8, IL-8/N51II, and IL-8/N51III to compete the binding of I-IL-8 to NIH-IL-8Rbeta are very similar (Fig. 3A). This would indicate that IL-8Ralpha and IL-8Rbeta differentially recognize the N51 domains. This is supported by the fact that N51 did not compete I-IL-8 binding to IL-8Ralpha (Fig. 3A) but efficiently competed the binding to IL-8Rbeta (Fig. 3B). Interestingly, the competition curve of N51/IL-8I shows that this chimera is a stronger competitor than both IL-8 and N51, and that of chimera N51/IL-8III indicates that this molecule is a weak competitor (Fig. 3B). The results presented in Fig. 3B also show that N51 can compete I-IL-8 binding to IL-8Rbeta as efficiently as IL-8. The observation that chimera N51/IL-8III is a weak partial agonist would indicate that domain III of N51 may be contributing significantly to the binding specificity of N51. On the other hand, chimera N51/IL-8I is more potent than either IL-8 or N51, suggesting that domain I of IL-8 and domain III of N51 may independently contribute to binding.


Figure 3: Competition of I-IL-8 binding to NIH-IL-8Rbeta cells. Binding was done with 3 nMI-IL-8 for 1 h at 4 °C in the absence and presence of up to 200-fold increasing concentrations of: A, IL-8, IL-8/N51II, or IL-8/N51III; and B, N51, N51/IL-8I, N51/IL-8II, or N51/IL-8III.



The results presented here are consistent with and extend our previous observations using human neutrophils(1) . First, we have demonstrated that N51 can only displace I-IL-8 binding from beta receptor-containing cells but not from the alpha receptor-containing cells. This provides a simple explanation of why with human neutrophils N51 cannot displace all bound I-IL-8 and confirms our speculation that the high affinity receptor for N51 in human neutrophils is the IL-8Rbeta(1, 2) . Second, the ability of chimera N51/IL-8I to compete with I-IL-8 binding to both the NIH-IL-8Ralpha cells and NIH-IL-8Rbeta cells is consistent with our previous finding that N51/IL-8I mimics IL-8 on human neutrophils. In addition, domain I does include sequences that are reported to confer high affinity binding of IL-8 to its receptors (5, 12, 13, 19, 20) . Third, the competition we obtained with the chimeras was all anticipated, except for the observation that N51/IL-8III is a partial agonist in NIH-IL-8Rbeta cells. One possible explanation is that NIH-IL-8Rbeta cells may be permitting us to see the partial agonist activity of N51/IL-8III we previously saw in desensitization assays with human neutrophils(1) . From our observations with human neutrophils we suggested that domains II and III of N51 and IL-8 were interchangeable but not equivalent and that they were more important in conferring N51 binding specificity than IL-8, which depends on domain I(1) . These are supported by our analysis of binding of I-N51 and competition with the chimeras to NIH 3T3 cells expressing the mouse homolog of the IL-8Rbeta(2) . The present results may suggest that domain III has a greater role than domain II in N51 binding specificity. This report supports the notion that the use of different ligand domains to recognize a receptor may be one mechanism by which the many chemokines attain their biological specificity.


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.

§
Present address: Agricultural Research Division, American Cyanamid, P. O. Box 400, Princeton, NJ 08543-0400.

To whom correspondence should be addressed: Dept. of Molecular Biology, Bristol-Myers Squibb Pharmaceutical Research Inst., P. O. Box 4000, Princeton, NJ 08543-4000. Tel.: 609-252-5744; Fax: 609-252-6051.

(^1)
The abbreviations used are: IL, interleukin; MGSA, melanoma growth-stimulating activity; MIP, macrophage inflammatory protein; MCP, monocyte chemotactic protein; NAP, neutrophil-activating peptide; PCR, polymerase chain reaction; HBSS, Hanks' balanced salt solution; BSA, bovine serum albumin; GRO, growth-related gene; ENA, epithelial cell-derived neutrophil-activiting protein; -IP, interferon-inducible protein; RANTES, regulated on activation normal T-cell expressed and secreted.


ACKNOWLEDGEMENTS

We thank R.-P. Ryseck for guidance in cloning the human IL-8Rs and E. C. O'Rourke and Heather Macdonald-Bravo for valuable comments.


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