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(Received for publication, December 8, 1994) From the
We have demonstrated that the orphan receptor representing the
putative mouse (mu) homolog of the human (hu) interleukin-8 receptor
Two interleukin-8 receptors (IL-8R), (
The N51 and chimeric proteins were expressed in the baculovirus
system and purified as described previously(11) . 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-8R
Figure 1:
Binding of
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-8R
Figure 3:
Competition of
The original
analysis of the N51 and IL-8 chimeras on intracellular Ca
Volume 270,
Number 10,
Issue of March 10, 1995 pp. 4987-4989
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Binds N51
STRUCTURE-FUNCTION ANALYSIS USING N51/IL-8 CHIMERIC MOLECULES (*)
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(IL-8R) binds the mouse N51 cytokine, also known as KC. The
muIL-8R gene was constitutively expressed in NIH 3T3 cells
(NIH-muIL-8R). Cells and plasma membranes from the
NIH-muIL-8R 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 GRO
/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-8R.
) (A or I)
and (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 GRO/MGSA bind the huIL-8R with low affinity and
huIL-8R 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-8R(5, 6, 7, 8) . This
receptor shows a 71% similarity to the huIL-8R and 68% to the
huIL-8R. 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-8R prompted us to determine if this receptor
would bind I-N51. The muIL-8R 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-8R 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-8R.
Cloning and Expression of the Mouse IL-8 Receptor
The gene for the mouse homolog of the IL-8 receptor (muIL-8R, GenBank Accession number L13239) was obtained
by polymerase chain reaction (PCR) cloning. The open reading frame of
muIL-8R 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-8R, the muIL-8R 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-8R 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-8R (NIH-muIL-8R cells) were
independently pooled. Cells were maintained in selection medium at 37
°C in a humidified incubator (7% CO
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, 1 mM MgCl, and 2% BSA (HBSS-3).Binding Analysis
Plasma membranes were made by
washing the cells twice with PBS, once with lysis buffer (50 mM -glycerol phosphate, 50 mM Tris-base, 2 mM EGTA, 2 mM EDTA, 10 mM MgCl, 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 
g and the supernatant ultracentrifuged at 30,000 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. 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.
(muIL-8R) prompted us to determine whether this new
receptor was the N51 receptor. The muIL-8R was cloned by
PCR methods and stably expressed in NIH 3T3 cells (NIH-muIL-8R).
These cells were used to determine the binding of I-N51
to muIL-8R.The muIL-8R
Binding of Gene Product Binds N51I-N51 to NIH-muIL-8R cells and their plasma
membranes was temperature-, time-, and concentration-dependent (data
not shown). Binding analyses with NIH-muIL-8R 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.
I-N51 to plasma membranes prepared from NIH-muIL-8R
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
Plasma membranes from NIH-muIL-8RI-N51 Binding by Homologs
and Chimeras cells were
assayed for binding of I-N51 in the presence of four
homologs, one from mouse, MIP-2, and three human, GRO/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, GRO/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-8R gene product(7) .
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.
I-N51 binding
to plasma membranes prepared from NIH-muIL-8R 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).
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-8R. 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.
)
We thank Rolf-Peter Ryseck for guidance in cloning the muIL-8R, Karen Hartl for advice in binding assays, and
Heather Macdonald-Bravo and Edward C. O'Rourke for comments on
the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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