©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Orphan Mouse Receptor Interleukin (IL)-8R Binds N51
STRUCTURE-FUNCTION ANALYSIS USING N51/IL-8 CHIMERIC MOLECULES (*)

(Received for publication, December 8, 1994)

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 orphan receptor representing the putative mouse (mu) homolog of the human (hu) interleukin-8 receptor beta (IL-8Rbeta) binds the mouse N51 cytokine, also known as KC. The muIL-8Rbeta gene was constitutively expressed in NIH 3T3 cells (NIH-muIL-8Rbeta). Cells and plasma membranes from the NIH-muIL-8Rbeta clone showed binding of I-N51 that was displaced by unlabeled N51. Other related cytokines were assayed for their ability to displace I-N51. MIP-2 and GROalpha/MGSA competed as well as N51 for the receptor, but huIL-8 and NAP-2 did not compete at all. Chimeric molecules between IL-8 and N51 were used to extend the binding analysis. The segment between the conserved cysteines 2 and 3, named domain I; cysteines 3 and 4, domain II; and cysteine 4 and the C terminus, domain III of IL-8 were replaced by the corresponding domains of N51 and vice versa. When studying the binding of I-N51 and the hybrid molecules to the receptor, we observed that chimeras of N51 containing either domain I, II, or III of IL-8 were agonists of N51, and chimeras of IL-8 containing domain II or III of N51 were partial agonists of N51. These results demonstrate that domain I of N51 does not confer binding specificity and suggest that the region from the third cysteine to the C terminus of the N51 molecule is more important for binding to muIL-8Rbeta.


INTRODUCTION

Two interleukin-8 receptors (IL-8R), (^1)alpha (A or I) and beta (B or II) have been cloned from human neutrophils (1, 2, 3, 4) . The deduced amino acid sequences of the receptors indicate that they belong to the superfamily of seven transmembrane receptors which couple to G nucleotide-binding proteins. These huIL-8 receptors present an overall similarity of 77%, with their N and C termini being significantly different, whereas their central portions are almost identical(1, 2) . When expressed in mammalian cells, IL-8 appears to bind both receptors with high affinity; the homologs NAP-2 and GROalpha/MGSA bind the huIL-8Ralpha with low affinity and huIL-8Rbeta with high affinity(3, 4) . Recently, a number of reports have described the cloning of a mouse orphan receptor representing the putative homolog of the huIL-8Rbeta(5, 6, 7, 8) . This receptor shows a 71% similarity to the huIL-8Rbeta and 68% to the huIL-8Ralpha. We have demonstrated previously that N51, the protein product of the mouse immediate early gene N51(9) also known as KC(10) , was biologically active on human neutrophils and bound to the huIL-8 receptors(11) . Therefore, the cloning of muIL-8Rbeta prompted us to determine if this receptor would bind I-N51. The muIL-8Rbeta was constitutively expressed in NIH 3T3 cells, and binding analysis of I-N51 to plasma membranes from these cells was performed. This report is the first demonstration that the muIL-8Rbeta binds I-N51. In addition, chimeric molecules between IL-8 and N51 were used to demonstrate that the amino acid sequence from the third cysteine to the C terminus in N51 is important for binding to muIL-8Rbeta.


MATERIALS AND METHODS

Cloning and Expression of the Mouse IL-8 Receptor beta

The gene for the mouse homolog of the IL-8 receptor beta (muIL-8Rbeta, GenBank Accession number L13239) was obtained by polymerase chain reaction (PCR) cloning. The open reading frame of muIL-8Rbeta was amplified by PCR using mouse genomic DNA (Stratagene) and oligonucleotide primers corresponding to the sense 5` and antisense 3` ends, including restriction sites for BamHI and HindIII, respectively. The sense oligonucleotide was 5`-TCGTAGGATCCGAAGAGGACATGGGAGAATTCAAGGTGG-3` and the antisense oligonucleotide was 5`-GATCAAGCTTCTAGAGGGTAGTAGAGGTGTTTGCTG-3`. The mouse genomic DNA was denatured for 10 min at 95 °C, briefly placed on ice, and then added to a PRC reaction containing the GeneAmp® Core Reagents (Perkin Elmer). The PCR incubation 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 HindIII and BamHI, subcloned into Bluescript (Stratagene), and sequenced using Sequenase version 2.0 DNA sequencing kit (U. S. Biochemical Corp.). To obtain cell lines constitutively expressing the muIL-8Rbeta, the muIL-8Rbeta fragment from the Bluescript vector was subcloned into a vector containing a neomycin gene (pMexneo; (12) ) using the restriction sites KpnI and NotI. The pMexneo-muIL-8Rbeta and pMexneo plasmids were each transfected into NIH 3T3 cells by the Ca phosphate precipitation method, as described previously(13) . Cells were selected for 2 weeks in Dulbecco's modified minimum essential medium containing antibiotics (100 µg/ml penicillin and 50 µg/ml streptomycin) supplemented with 10% fetal calf serum in the presence of 400 µM G418. The selected clones transfected with pMexneo alone (NIH-pMexneo cells) and those with pMexneo-muIL-8Rbeta (NIH-muIL-8Rbeta cells) were independently pooled. Cells were maintained in selection medium at 37 °C in a humidified incubator (7% CO(2) and 93% air). For receptor binding experiments, cells were detached from the plate by washing twice in PBS and then incubated in HBSS (Hanks' balanced salt solution supplemented with 25 mM HEPES, pH 7.5) containing 0.2% BSA and 5 mM EDTA. Cells were washed twice in HBSS supplemented with 1 mM CaCl(2), 1 mM MgCl(2), and 2% BSA (HBSS-3).

Binding Analysis

Plasma membranes were made by washing the cells twice with PBS, once with lysis buffer (50 mM beta-glycerol phosphate, 50 mM Tris-base, 2 mM EGTA, 2 mM EDTA, 10 mM MgCl(2), pH 7.4) in the presence of protease inhibitors (50 µg/ml each of soybean trypsin inhibitor, aprotinin, and leupeptin and 10 µg/ml of pepstatin) and subjected to nitrogen cavitation at 500 p.s.i. for 30 min. The lysed cells were centrifuged for 10 min at 100 times g and the supernatant ultracentrifuged at 30,000 times g for 15 min. The membrane pellets were resuspended in 250 mM sucrose, 50 mM Tris-base, 2 mM EGTA, 20 mM MgCl pH 7.4, to a concentration of 3-8 mg/ml.

The N51 and chimeric proteins were expressed in the baculovirus system and purified as described previously(11) . I-N51 was custom-iodinated by the Bolton-Hunter method to a specific activity of 50.7 µCi/µg (DuPont NEN). The standard binding assay was performed in tubes formatted in a 96-well box, with continuous shaking on a DIGIT shaker for 30 min at 25 °C, in a volume of 100 µl of HBSS-3, with 80 µg of plasma membranes, 4 nMI-N51, and 100-fold excess N51. Binding assays performed under different conditions are described in the figure legends. Binding was stopped by diluting the reaction with 2 ml of PBS and filtering it through glass fiber filters that were preincubated in 0.3% polyethyleneimine. The filters were washed twice with 2 ml of PBS and counted in a counter. Binding in the absence and presence of 100-fold excess N51 represents total and nonspecific binding, respectively, and the difference between them is referred to as specific binding. Data were analyzed by least square nonlinear curve fit for a model of multiple ligand binding sites using Cricket Graph and KaleidaGraph software.


RESULTS AND DISCUSSION

We have shown previously that N51 is biologically active on human neutrophils via the IL-8R(s) (11) and that it has the capacity to recruit mouse neutrophils in vivo(14) . Therefore, the report of the cloning of a putative mouse homolog of the huIL-8Rbeta (muIL-8Rbeta) prompted us to determine whether this new receptor was the N51 receptor. The muIL-8Rbeta was cloned by PCR methods and stably expressed in NIH 3T3 cells (NIH-muIL-8Rbeta). These cells were used to determine the binding of I-N51 to muIL-8Rbeta.

The muIL-8Rbeta Gene Product Binds N51

Binding of I-N51 to NIH-muIL-8Rbeta cells and their plasma membranes was temperature-, time-, and concentration-dependent (data not shown). Binding analyses with NIH-muIL-8Rbeta plasma membranes were similar to those obtained with the intact cells, and the assays presented in this paper were done with plasma membranes at room temperature. Fig. 1A shows that binding with increasing concentrations of I-N51 is saturable and it approximates maximal binding at 5 nM. The data plotted according to Scatchard (inset) indicate the presence of two classes of binding sites, one with an apparent K of 1 nM and the other with an apparent K of 9 nM. Both apparent K values are representative of high affinity receptors. The presence of two binding affinities for a single receptor may reflect its complex interactions with the guanidine nucleotide-binding proteins or other effector molecules.


Figure 1: Binding of I-N51 to plasma membranes prepared from NIH-muIL-8Rbeta cells. A, binding was done for 30 min with increasing concentrations of I-N51 in the absence and presence of 100-fold excess unlabeled N51. The difference between the total and nonspecific binding is the specific binding. Inset, Scatchard plot of the same data. B, binding of 4 nMI-N51 in the presence of increasing amounts of N51, GRO, MIP-2, IL-8, and NAP-2.



Competition of I-N51 Binding by Homologs and Chimeras

Plasma membranes from NIH-muIL-8Rbeta cells were assayed for binding of I-N51 in the presence of four homologs, one from mouse, MIP-2, and three human, GROalpha/MGSA, IL-8, and NAP-2, and the results are presented in Fig. 1B. Two of these, IL-8 and NAP-2, did not compete at all, whereas the other two, GROalpha/MGSA and MIP-2, competed as well as or better than N51. The lack of competition by IL-8 is consistent with a recent report by Suzuki and collaborators which showed the absence of binding of I-IL-8 to Chinese hamster ovary cells expressing the muIL-8Rbeta gene product(7) .

We previously reported on the biological activities of chimeras between N51 and IL-8 on human neutrophils(11) . Sequences between the second and third cysteines, the third and fourth cysteines, and those after the fourth cysteine constituting the C terminus were referred to as domains I, II, and III, respectively, as indicated in Fig. 2. The chimeras were named according to the parental/donor molecule and the number of the exchanged domain(11) .


Figure 2: Comparison of N51 and IL-8 and designation of domains. The amino acid sequence of N51 and IL-8 are aligned according to the 4 conserved cysteines.



Plasma membranes from NIH-muIL-8Rbeta cells were assayed for I-N51 binding in the presence of increasing concentrations of the chimeric molecules (Fig. 3A). The competition of I-N51 binding by N51/IL-8I, N51/IL-8II, and N51/IL-8III is comparable with that by N51. The IL-8 and IL-8/N51I molecules, at concentrations 100-fold greater than I-N51, do not compete at all (Fig. 1B and data not shown). In contrast, the IL-8/N51II and IL-8/N51III chimeras can efficiently compete I-N51 binding. The results indicate that the N51 molecule can support one of the IL-8 domains without having its binding properties significantly altered and that either domain II or III of N51 can convert IL-8 to a more N51-like molecule.


Figure 3: Competition of I-N51 binding to plasma membranes prepared from NIH-muIL-8Rbeta cells by the N51 and IL-8 chimeras. Binding was done with 4 nMI-N51 and increasing concentrations of N51, N51/IL-8I, N51/IL-8II, and N51/IL-8III (A) and IL-8/N51II and IL-8/N51III (B).



The original analysis of the N51 and IL-8 chimeras on intracellular Ca flux on human neutrophils led us to hypothesized that domains II and III were important for the binding of N51(11) . Our data indicate that indeed N51 binding is determined more by domains II and III rather that domain I and demonstrate that N51 binds with high affinity to the muIL-8Rbeta. This differs to that observed for IL-8 binding to its human receptor, where the region comprising domain I has been reported to be essential(11, 15, 16) . These results suggest that the use of different domains to recognize a receptor may be one mechanism by which the many chemokines attain 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. Tel.: 609-252-5744; Fax: 609-252-6051.

(^1)
The abbreviations used are: IL-8, interleukin-8; IL-8R, interleukin-8 receptor; PCR, polymerase chain reaction; HBSS, Hanks' balanced salt solution.


ACKNOWLEDGEMENTS

We thank Rolf-Peter Ryseck for guidance in cloning the muIL-8Rbeta, Karen Hartl for advice in binding assays, and Heather Macdonald-Bravo and Edward C. O'Rourke for comments on the manuscript.


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