(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
(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
.
Two interleukin-8 receptors (IL-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
.
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
nM
I-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.
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 (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
.
Figure 1:
Binding of 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 nM
I-N51 in the presence of increasing amounts of N51,
GRO, MIP-2, IL-8, and NAP-2.
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 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-8R
cells by the N51 and
IL-8 chimeras. Binding was done with 4 nM
I-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-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.