From the
Two interleukin-8 (IL-8) receptors,
The capacity of the mutated
receptors to convey functional signals was evaluated by comparing the
chemotaxis index of cells expressing the C`-truncated receptors to the
index of cells expressing the wild type receptor. The results indicate
that while cells expressing
IL-8
The two IL-8Rs contain seven transmembrane domains separated by
three extracellular and three intracellular loops
(3, 4) , as shown also to be the case for receptors for
other chemoattractants ( e.g. receptors for C5a, fMLP,
MIP-1
Extensive research was performed during the last few years to
identify the domains of the seven transmembrane receptors (7TMR) which
interact with GTP-binding proteins (G proteins). Domains that show a
relatively low degree of conservation between 7TMR are believed to
determine the activation of specific G proteins by a unique stimulus.
The greatest degree of sequence and size heterogeneity resides within
the third intracellular (3i) loop and the carboxyl terminus of 7TMR
(13, 17) , and both regions were shown to be involved in
coupling or activation of G proteins
(18, 19, 20, 21, 22, 23, 24, 25, 26) .
In the adrenergic and the muscarinic receptors, the 3i loop couples the
G protein
(19, 20, 21) and consists of a long
stretch of amino acids ( e.g. 54 amino acids in the human
The involvement of the carboxyl terminus (C`) in the coupling and
the activation of G protein in adrenergic receptors and in rhodopsin
was shown to be mediated by the membrane proximal region of the C`
(19, 20, 22) . It is believed that this region
constitutes a putative fourth cytoplasmic loop due to anchoring to the
lipid membrane via palmitoylcysteines
(19, 22, 27, 28) . Another
characteristic of the C` which points to its importance for signaling
is the fact that in most of the 7TMR studied, the serine and threonine
residues in the C` are phosphorylated during desensitization of the
receptors. In some of the receptors, specific kinases (G
protein-coupled receptor kinases) were shown to phosphorylate this
region, thus facilitating the binding of arrestin-like proteins which
inhibit receptor-G protein coupling
(18, 29) .
The
importance of the C` in signaling and coupling of G protein in
chemotactic receptors is presently unknown; however, experimental and
structural features found within this family suggest that this region
may be important for the regulation of signaling events. In the case of
the human fMLP receptor, peptides corresponding to amino acids
308-336 (residing in the C`) eliminate signaling by the receptor
(23, 24) . One of the peptides (amino acids
308-322) is located in the membrane proximal region
(23) ,
in a similar position to the palmitoylated cysteines in the adrenergic
receptors and in rhodopsin. Since this region in the fMLP receptor does
not contain cysteine residues, it probably regulates signaling by a
different mechanism. The importance of the C` for signaling in
chemotactic receptors is further supported by the fact that numerous
serine and threonine residues are located in this region.
Phosphorylation of these residues might be a major mechanism of
desensitization of the receptors. Indeed, fMLP and C5a receptors were
shown to be phosphorylated during desensitization
(30) . In a
recent paper by Takano et al. (31) , it was also shown
that C` phosphorylation sites of platelet-activating factor receptor
play a critical role in desensitization.
The purpose of this study
was to determine which regions of IL-8R
The Langmuir protein-ligand binding model was used to
estimate the number of receptors per cell and binding affinities for
each receptor type in the study. Bound counts were first corrected for
nonspecific binding, converted to picomole concentrations, and then fit
directly to the model by nonlinear least squares regression. This
procedure yields parameter estimates that are less biased than those
that are estimated from the traditional Scatchard plot slope and
intercept
(36) .
For each IL-8
concentration tested, cells migrating through to the underside of the
filter were counted in three high power fields (
The data are
presented as the mean and standard errors (S.E.) of chemotaxis indices
of 3-4 experiments. The baseline level of the number of
transfected cells migrating was in a similar range for all the cells
tested (
Using the chemotaxis index,
migration potencies of cells transfected with WT and truncated
IL-8R
Cells
transfected with the truncated receptors
In contrast to the
Studies of IL-8Rs have defined their biological effects,
their binding specificities, and the intracellular events elicited upon
their activation. Less is known of the structure-function relationship
of these receptors. Studies of the N` portion of IL-8Rs show that it is
the major determinant of receptor subtype specificity
(39, 40) . Mutagenesis of the extracellular domains of
IL-8R
This study is
the first to present data suggesting a role for the C` domain of
IL-8R
The results of the FACS analysis show that all of the mutated
receptors were expressed in a high percentage of the transfected cells
(Fig. 2). The reason for the two-peak pattern of fluorescence
observed in
The Scatchard analysis shows that cells
transfected with
Since all the truncated receptors are expressed
and no major differences in binding affinities were found between them,
they were tested for their ability to transduce signals in response to
IL-8. Chemotaxis was considered to be the most appropriate assay to
estimate signal transduction since it is the end result of a cascade of
intracellular processes that are activated by a specific interaction of
ligand-receptor. For that purpose, a chemotaxis assay for 293 cells has
been developed, and, to our knowledge, this is the first time that the
migratory response of these cells is described. The chemotaxis assays
showed that
These results suggest
that a region existing in
The involvement of the membrane proximal portion of the C`
in signal transduction by IL-8R
The possible
involvement of the C` of IL-8R
Although we
suggest that the membrane proximal portion of the C` of IL-8R
In conclusion, we have identified a membrane proximal
region of the IL-8R
The results
of 2-3 experiments done for each cell line are presented.
We thank Dr. Gregory Alvord and Matthew Fivash for
their assistance in the statistical analysis of the data. We also thank
Dr. Dan Longo for reviewing the manuscript. The secretarial support of
Roberta Unger is gratefully acknowledged.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
and
, have been
identified and cloned. Both receptors are thought to transduce signals
by coupling to GTP-binding proteins. The aim of this study is to
determine whether the carboxyl terminus (C`) of IL-8 receptor
(IL-8R
) is involved in signaling in response to IL-8. We have
constructed a number of IL-8R
genes that encode truncated forms of
the IL-8R
. The deletions consisted of amino acids 349-355,
336-355, 325-355, and 317-355 (termed
2,
3,
4, and
5, respectively). 293 human embryonic kidney cells
were transfected with the wild type IL-8R
(
1) and with these
mutants. Cells transfected with the mutated receptors expressed the
receptors and bound IL-8 with the same high affinity as cells
transfected with the wild type receptor.
1,
2,
3, and
4 were
chemoattracted in response to IL-8, cells expressing
5 did not
migrate in response to IL-8 stimulation. Therefore, the data suggest
that amino acids 317-324 are involved in signaling by IL-8R
.
(
)
is a member of the chemokine family
of proinflammatory chemotactic cytokines. IL-8 was initially
characterized as a potent chemoattractant of neutrophils and was later
shown to also activate neutrophils to degranulate, adhere, and exhibit
a respiratory burst
(1, 2) . Two types of human IL-8
receptors have been cloned and sequenced
(3, 4) . These
receptors, IL-8 receptors
and
(IL-8R
and IL-8R
(known also as IL-8RA and IL-8RB, respectively)), share 77% amino acid
homology
(3, 5, 6, 7) but differ in
their binding characteristics; while IL-8R
binds only IL-8 with
high affinity and GRO
and NAP-2 with low affinity, IL-8R
binds all three chemokines with high affinity
(5, 6) .
/RANTES (C-C CKR1), and MCP-1)
(1, 8, 9, 10, 11, 12) .
The amino terminus (N`) of the molecule is extracellular, while the
carboxyl terminus (C`) is located in the cytoplasmic region of the cell
(3, 4) . These receptors belong to the GTP-binding
protein (G protein)-coupled receptor family
(7, 13, 14, 15) . Both IL-8Rs were shown
to activate phospholipase C
by coupling to
G
or G
and to interact with
recombinant G
proteins, followed by the release of
1
and
2 subunits and activation of phospholipase C
(16) . These findings suggest that several G proteins can
interact with the IL-8Rs, and that various regions in the receptors may
be responsible for activation processes which follow. Yet, it is still
unknown which segment(s) of IL-8Rs mediate these processes.
-adrenergic receptor, 75 amino acids in the mouse m1
muscarinic receptor)
(17, 19, 20, 21) .
In other 7TMR, such as the bovine rhodopsin, the 3i loop is very short
(22 amino acids)
(22) but is still capable of activating
various G proteins
(17) . It is believed that a longer and more
complex 3i loop enables the receptor molecules to discriminate among
different but highly homologous G proteins
(8, 17) .
are involved in signal
transduction. The structural similarity of IL-8R
with other
chemotactic receptors, such as fMLP receptor, suggested that the C` of
IL-8R
may have an important role in mediating a specific signal in
response to IL-8. In order to test whether the C` of IL-8R
is
involved in transducing signals, deletions of the C` were constructed
(Fig. 1, ) so that in each mutated receptor the
additional deleted segment contained a cluster of serines/threonines
and/or a consensus sequence between human IL-8R
, human IL-8R
,
and rabbit IL-8R. These mutated receptors were compared to a control
wild type (WT) receptor for their expression (determined by monoclonal
antibodies), binding of IL-8 (determined by Scatchard analysis), and
transducing a signal in response to IL-8 (determined by chemotaxis). In
this report, we shed light on the functional roles of the C` of
IL-8R
and provide evidence for the involvement of the membrane
proximal domain of the C` in signal transduction.
Figure 1:
Schematic
presentation of WT IL-8R (
1) and four C`-truncated mutants
(
2,
3,
4, and
5). -, transmembrane domains.
Extracellular and intracellular sides of the plasma membrane are
indicated.
,
, threonine and serine residues, respectively,
in the carboxyl terminus. aa, amino
acids.
Materials
The reagents were obtained from
commercial sources, as follows: restriction endonucleases, ligation
reagents, and DOTAP transfection reagent were obtained from Boehringer
Mannheim; polymerase chain reaction (PCR) kit was purchased from
Perkin-Elmer Cetus; Reagent kit for DNA sequencing was from U. S.
Biochemicals; SP6, T3, and T7 primers were obtained from Stratagene;
vectors (pCRII and pRc/CMV) and supercompetent cells XL-1
blue were purchased from Invitrogen; amp/IPTG/X-GAL plates were from
Advanced Biotechnologies, Inc.; amp plates were obtained from Digene;
low gelling temperature agarose (type XI), bovine serum albumin
(fraction V) and monoclonal antibodies against human CD3 (Isotype:
mouse IgG1) were purchased from Sigma; Dulbecco's modified
Eagle's medium, RPMI 1640, Dulbecco's phosphate-buffered
saline without calcium or magnesium were obtained from BioWhittaker,
Inc.; Fetal calf serum was from Hyclone Laboratories; Hepes buffer was
purchased from Applied Scientific; Geneticin (G418) was purchased from
Life Technologies, Inc.;
I-IL-8 (2200 Ci/mmol) was
purchased from DuPont NEN; IL-8 (72 amino acids) was from Pepro Tech;
FITC-conjugated goat anti-mouse IgG was obtained from Tago; 10-µm
polycarbonate filters for chemotaxis were purchased from Poretics;
mouse collagen type IV was from Collaborative Biomedical Products;
Diff-Quik kits for fixation and staining of cells on chemotaxis filters
were obtained from Biochemical Sciences.
Primers
To assess the role of the C` of IL-8R
in signal transduction, the gene for the wild type (WT) receptor and
four C`-truncated genes were constructed by PCR using five different
primers for the 3` end of the IL8R
gene and one primer for the 5`
end. The primers were made by a DNA Synthesizer, Advanced
Biotechnologies, Inc. shows which amino acids were deleted
in each of the receptors. A schematic presentation of the various
receptors is given in Fig. 1.
Constructs
Each of the receptor DNAs (WT and four
truncated constructs, Fig. 1) was produced by PCR. The template
DNA for the PCR consisted of the IL-8R gene ligated to pRc/CMV.
The IL-8R
open reading frame is encoded entirely in the third exon
(32) . The final mutated DNA fragments were ligated to
pCR
II vector, the constructs were transformed into
supercompetent cells, and clones were selected on amp/IPTG/X-GAL
plates. Selected clones were sequenced by the chain termination method
(33) , using T3 or T7 primers. DNA from clones having the
suitable coding sequence at the 3` end of the IL-8R
coding region
was shuttled into an expression vector (see below).
Expression Vector
The above mentioned constructs
were subjected to HindIII/ XbaI digest, followed by
electrophoresis on 0.8% agarose gel. The relevant piece of DNA coding
for the WT or truncated receptors was purified from the gel by using
low gelling temperature agarose, Type XI. That piece was then ligated
to the pRc/CMV expression vector previously digested by
HindIII/ XbaI. The resulting constructs were then
transformed into supercompetent cells, and subclones were selected on
amp plates. The subclones chosen for transfection represent the WT
receptor and four truncated receptors which were named 1 through
5, respectively (, Fig. 1). Full-length
sequencing of selected subclones was performed using SP6, T7, and two
additional primers (made specifically for the IL-8R
gene). No
mutations were detected in
1,
2,
3, and
4. In
5, a point mutation due to an error in the PCR process was
detected. This mutation resulted in a change in amino acid residue 38
from serine to proline. The changed amino acid is located in the N`
extracellular domain of the receptor, and it did not interfere with
binding of IL-8 (based on a comparison of the IL-8 binding
affinity and the number of binding sites/cell of cells transfected with
5 to those of cells transfected with
2,
3, and
4;
). It is important to note that in G protein-coupled
receptors, ligand binding was shown to be essential for transducing
signals, but the N` domain was not found to have an active role in G protein coupling or signaling
(17, 18, 34, 35) .
Cell Cultures and Transfections
293 human
embryonal kidney cells (a generous gift from Dr. P. Gray, ICOS Corp.)
were grown as monolayers in growth medium (Dulbecco's modified
Eagle's medium with 10% fetal calf serum, penicillin (100
units/ml), and streptomycin (100 µg/ml)). Cells were grown to
approximately 75% confluency in an atmosphere of 95% air, 5% COat 37°C. Transfections with 5-20 µg of DNA were
done with DOTAP transfection reagent, according to the
manufacturer's instructions. The resulting transfected cells were
given the same names as the receptors with which they were transfected
(
1-
5). Control transfections were done with the vector
(pRc/CMV) alone. Stably transfected cell lines were produced by adding
G418 (800 µg/ml) to cultures (for maintenance of selection
pressure) from day 4 after transfection and on.
FACS Analysis
Stable 293 transfectants were
trypsinized, washed twice in cell sorter buffer (CSB =
phosphate-buffered saline containing 1% fetal calf serum, 0.02%
NaN, and 25 m
M Hepes) and resuspended at the
concentration of 4
10
cells/ml in CSB. 25 µl of
cells were aliquoted into a 96-well microtiter plate with a u-shaped
bottom and mixed with 100 µl of monoclonal mouse anti-human
IL-8R
antibodies (IgG
, 8.6 mg/ml, diluted 1:10000 in
CSB). Baseline staining resulted from adding 100 µl of CSB to the
cells instead of anti-human IL-8R
antibodies. Following a 30-min
incubation at 4°C, the cells were washed three times in CSB, and 20
µl of FITC-conjugated goat anti-mouse IgG (1:10 dilution in CSB)
were added for 30 min at 4°C. The cells were washed twice in CSB
and resuspended in 300 µl of CSB. At least 10,000 events of live
and fresh cells were analyzed by FACS(Epics Profile, Coulter Corp.).
Scatchard Analysis
Stable 293 transfectants were
trypsinized, washed twice in cold bovine serum albumin medium (BSA
medium = RPMI 1640 containing 1% bovine serum albumin and 25
m
M Hepes buffer) and resuspended at the concentration of 2.5
10
cells/ml in ice cold BSA medium. Samples of 200
µl were incubated with 10 dilutions of
I-IL-8 in the
presence or in the absence of unlabeled IL-8. In all dilutions,
unlabeled IL-8 was in at least a 100 molar excess as compared to
labeled
I-IL-8. Following an incubation of 2 h at
4°C, the cells were spun for 2 min in a Microfuge. The supernatant
was removed and the cells were then resuspended in 200 µl of
ice-cold BSA medium and layered on a cushion of 800 µl of 10%
sucrose in phosphate-buffered saline. Following an additional spin at
4°C, the supernatant was removed and the counts remaining in the
pellet were assayed in a 1272 CliniGamma
-counter (Pharmacia
Biotech Inc.).
Chemotaxis Assays
The migration of 293 stable
transfectants was assessed by a 48-well microchemotaxis chamber
technique
(37) . The lower compartment of the chamber was loaded
with aliquots of BSA medium or of each of the different IL-8
concentrations (diluted in BSA medium). The upper compartment of the
chamber was loaded with a 50-µl cell suspension (5 10
cells/ml in BSA medium) of 293 cells which were previously
trypsinized and washed twice in BSA medium. The two compartments were
separated by a polycarbonate PVPF filter, 10-µm pore size, coated
with 20 µg/ml mouse collagen type IV for 2 h at 37°C. The
chamber was incubated for 4.5-6 h at 37°C in humidified air
with 5% CO
. At the end of the incubation period, the filter
was removed, fixed, and stained with a Diff-Quik kit.
400) by light
microscopy (after coding the samples) in triplicate. Since the results
of several experiments were combined in order to evaluate the migratory
response, and since variation in potency of migration were observed
between different experiments, the response to each of the IL-8
concentrations is shown as a chemotaxis index. The chemotaxis
index in each experiment was evaluated by calculating the following
ratio: chemotaxis index = the mean of the number of cells
migrating to a specific IL-8 concentration/the mean of the number of
cells migrating to BSA medium (= 0 ng/ml IL-8).
1-
5), with a mean of 6.7, ranging from
2-18 cells per high power field. Due to the large size of the
cells and to limitations at the size of the high power field, the
maximal response was limited to 70-80 cells per high power field.
The p values of migration in response to each of the IL-8
concentrations in comparison to migration in response to BSA medium
were calculated based on the actual numbers of migrating cells using
Student's t test.
were analyzed. Analysis of variance (ANOVA) was used to
assess trends across increasing concentrations of IL-8 and to compare
receptor type profiles to BSA medium as well as to each other. Analyses
of variance were followed with post hoc analyses and
Student's t tests. Efforts were made to control type I
compounding error rates. In some analyses, one-tailed strategies were
used to test directional hypotheses. However, for simplicity,
probabilities from two-tailed tests are presented, as results from
these and other procedures led to similar conclusions.
Expression of WT and Truncated
IL-8R
Anti-IL-8R antibodies were used to exclude the
possibility that truncation of different portions of IL-8R
resulted in an improper processing of the receptor, giving rise to a
product that is not expressed on the cell surface. These assays showed
that a high percentage of cells transfected with the WT receptor, as
well as of cells transfected with all the truncated receptors, are
expressing the receptor on the cell surface (Fig. 2). Yet,
differences in the pattern of fluorescence and in intensity could be
detected between the different receptors. The WT receptor (
1) and
the two longer mutants (
2,
3) yielded a similar pattern of
two-peak intensity of fluorescence. The two shorter mutants (
4 and
5) showed only one peak of fluorescence intensity, and the
5-transfected cells demonstrated a higher intensity of
immunofluorescence than
4-transfected cells (Fig. 2). In
contrast, the antibodies did not react with control cells transfected
with the vector alone (data not shown).
Figure 2:
Anti-IL-8R FACS analysis of cells
transfected with WT IL-8R
(
1) and four C`-truncated mutants
(
2,
3,
4, and
5).
1,
2,
3,
4,
and
5, cells stained with monoclonal antibodies (mouse
IgG
) directed against human IL-8R
followed by a
secondary staining step with FITC-conjugated goat anti-mouse IgG
antibodies. Baseline, baseline fluorescence of cells stained
only with FITC-conjugated goat anti-mouse IgG antibodies. The level of
this fluorescence was identical in all the cells studied. As a control
for the primary antibody, staining with anti-CD3 (mouse
IgG
) was performed followed by a secondary staining step
with FITC-conjugated goat anti-mouse IgG antibodies. The fluorescence
level in this case was identical with baseline fluorescence (data not
shown). A, fluorescence of
1-,
2-, and
3-transfected cells. B, fluorescence of
4- and
5-transfected cells. The staining pattern of each cell line is
from a representative experiment of at least five assays done for
each.
Ligand Binding by WT and Truncated
IL-8R
Binding of IL-8 is a prerequisite for transducing a
signal by any of the receptors. We evaluated the IL-8 binding affinity
of each of the receptors and the number of binding sites/cell on each
of the receptor-transfected cell lines (WT and truncated receptors).
Control cells transfected with the vector alone did not show any
detectable binding of IL-8 (data not shown). Scatchard analysis showed
that all the receptor-transfected cell lines (WT and truncated mutants)
bound IL-8 with a comparable and high binding affinity (approximately
10
M) (). A comparison
between the receptor-transfected cells showed differences in the
numbers of binding sites/cell. The highest number of binding sites/cell
was manifested on cells expressing the WT receptor (
1) with an
average of 80,350 binding sites/cell (). The average
number of binding sites/cell on each of the four cell lines transfected
with the truncated receptors (
2,
3,
4, and
5) was
3-fold lower than that of cells expressing the WT receptor
(). The four cell lines transfected with the mutated
receptors (
2-
5) showed on average an equivalent number
of binding sites/cell ().
Signal Transduction by WT and Truncated
IL-8R
Migration in response to IL-8 was used as a
functional readout of signal transduction. Chemotaxis assays were
performed in two separate sets of experiments, one covering IL-8
concentrations of 0.001-100 ng/ml and the other ranging from
10-1000 ng/ml (Figs. 3 and 4, respectively), because of space
limitations of the chemotaxis chambers. Migration of 293 cells
transfected with the WT receptor (1) was optimal in response to
10-100 ng/ml (Figs. 3 and 4). Neutrophils have also been shown to
respond optimally to the same concentrations of IL-8
(38) .
1-transfected 293 cells did not migrate in response to IL-8
concentrations of 0.001-0.01 ng/ml (Fig. 3), and their
migration was suboptimal in response to high concentrations of IL-8
(250-1000 ng/ml; Fig. 4). This gave rise to the typical
bell-shaped dose-response curve of migration observed with many
chemotactic stimuli
(38) . No migration in response to IL-8 was
detected with cells transfected with the vector only (data not shown).
Figure 3:
Chemotaxis in response to IL-8 of cells
transfected with WT IL-8R (
1) and four C`-truncated mutants
(
2,
3,
4, and
5): 0.001-100 ng/ml IL-8. The
chemotaxis of the different transfected cell lines was evaluated in
response to increasing concentrations of IL-8 in the range of
0.001-100 ng/ml. The data are expressed as chemotaxis index
values (defined under ``Experimental Procedures'') and are
the mean (±S.E.) of 3-4 experiments performed for each of
the cell lines. * = p < 0.001; ** = p < 0.01; *** = p < 0.05. A statistical
analysis of the differences in migration potency between cells
transfected with
2,
3,
4, and
5 and cells
transfected with the WT receptor (
1) was done in IL-8
concentrations of 1-100 ng/ml and resulted in the following p values (see ``Experimental Procedures''):
1
versus
2, p = 0.001;
1 versus
3, p = 0.3470;
1 versus
4,
p = 0.0001;
1 versus
5, p = 0.0001.
Figure 4:
Chemotaxis in response to IL-8 of cells
transfected with WT IL-8R (
1) and four C`-truncated mutants
(
2,
3,
4, and
5): 10-1000 ng/ml IL-8. The
chemotaxis of the different transfected cell lines was evaluated in
response to increasing concentrations of IL-8 in the range of
10-1000 ng/ml. The data are expressed as chemotaxis index values
(defined under ``Experimental Procedures'') and are the mean
(±S.E.) of 3-4 experiments performed for each of the cell
lines. * = p < 0.001; ** = p <
0.01; *** = p < 0.05. A statistical analysis of the
differences in migration potency between cells transfected with
2,
3,
4, and
5 and cells transfected with the WT receptor
(
1) was done in IL-8 concentrations of 10-250 ng/ml and
resulted in the following p values (see ``Experimental
Procedures):
1 versus
2, p = 0.0322;
1 versus
3, p = 0.1383;
1
versus
4, p = 0.0108;
1 versus
5, p = 0.0001.
Checkerboard-like analysis indicated that chemotaxis, and not
chemokinesis, was the major contributor to the migratory response of
the WT receptor-transfected cells. In this analysis, IL-8 and cells
were loaded in three different combinations into the two compartments
of the chemotaxis chamber. The chemotaxis assay with cells in the upper
compartment, resuspended in BSA medium, and 50 ng/ml IL-8 in the lower
compartment, resulted in a chemotactic index of 20.8. When cells in the
upper compartment were resuspended in 50 ng/ml IL-8 along with 50 ng/ml
IL-8 in the lower compartment, the chemotaxis index decreased to 4.7.
When cells in the upper compartment were resuspended in 50 ng/ml IL-8,
and only BSA medium was in the lower compartment, the chemotaxis index
was only 0.81. These results indicate that the cells are migrating in
response to a gradient of IL-8 as a chemotactic ligand. Similar results
were obtained at IL-8 concentrations of 10 and 100 ng/ml.
2,
3, and
4
demonstrated optimal migration in response to 10-100 ng/ml IL-8
(Figs. 3 and 4). These three truncated receptors are therefore
functional and transduce a signal in response to IL-8. The potency of
migration of
2-transfected cells, and to an even greater extent of
4-transfected cells, was significantly reduced as compared to WT
receptor-transfected cells. While a bell-shaped curve of migration was
detected in
2- and
3-transfected cells,
4-transfected
cells showed a change in the dose-response curve, and their migratory
response was not inhibited by high concentrations of IL-8
(Fig. 4).
2-,
3-, and
4-transfected cells, cells transfected with
5 did not migrate
in response to IL-8 (Figs. 3 and 4). The inability of these cells to
migrate, even in response to very high concentrations of IL-8,
indicates that this truncated receptor is not transducing signals which
are necessary for a functional chemotactic response. The data therefore
suggest that a region of the C` of IL-8R
is important for
IL-8-induced chemotactic response. This region exists in
4 and not
in
5 and consists of amino acids 317-324 in the C` of
IL-8R
.
has identified the residues mediating IL-8 binding
(35, 41) . On the other hand, the exact function of the
intracellular domains has not yet been characterized.
in regulating functional chemotactic responses. This was
achieved by constructing four C`-truncated IL-8R
(Fig. 1)
which were transfected into 293 cells. Prior to studying their ability
to transduce signals in response to IL-8, we tested whether the
truncated receptors were expressed on the cell surface and bind IL-8.
1,
2, and
3 is unclear, but it may reflect
the existence of two populations of cells with different receptor
numbers expressed on each. However, this pattern does not result from
the cell cycle phase of the cells because every cell line tested shows
a very specific and reproducible pattern of staining and the same
pattern was observed when cells were stained at S or post-S phase (50%
or 100% confluency respectively; data not shown). It is worth noting
that, in our hands, 293 cells transfected with IL-8R
also show a
two-peak pattern of fluorescence when stained with anti-IL-8R
antibodies. Moreover, Chuntharapai et al. (42) reported a similar two-peak pattern in 293 cells
transfected with IL-8R
.
1-
5 bind IL-8 with a similar very high
affinity (). As expected, only one type of receptor is
expressed on the surface of the transfected cells, showing an affinity
ranging from 0.099 n
M to 0.258 n
M ().
This affinity is comparable to the reported affinity of IL-8Rs
expressed on neutrophils (11 p
M to 2 n
M)
(14) . The number of binding sites/cell is reduced on cells
transfected with the truncated receptors as compared to the WT
transfected cells. A comparison between cells transfected with the
different mutated receptors shows that they all express the same
average number of binding sites/cell (). It should be
mentioned, however, that in cells expressing
1,
2, and
3
the number of binding sites/cell might actually be an average between
two different populations (see the discussion about FACS results).
These data document that the transfected 293 cells presumably express
the mutated IL-8R
in an appropriate configuration and in an
accessible manner.
1 (WT),
2,
3, and
4 receptors can
mediate a significant migratory response to IL-8 and that only the
shortest receptor (
5) was ineffective in transducing signals as
manifested by the inability of cells transfected with this receptor to
migrate in response to IL-8 (Figs. 3 and 4).
4 and missing in
5 is involved in
signal transduction. It is important to note that cells transfected
with
5 have the same affinity for the ligand and the same number
of binding sites/cell as the
4-transfected cells. Moreover,
5-transfected cells show an even higher fluorescence intensity
than those transfected with
4, both showing only a one-peak
fluorescence pattern. Therefore, the inability of
5 to transduce a
signal, when compared to
4 which does transduce, is not a
consequence of an improper expression of the receptor or due to an
inability to bind IL-8. We therefore suggest that a region of the C` of
IL-8R
has a role in regulating signal transduction. This region
resides between amino acids 317 and 324 and is located at the membrane
proximal portion of the carboxyl terminus of the receptor. This does
not necessarily imply that this domain is physically coupled to G
protein, but instead might have a role in making the coupling possible.
Further investigations will be necessary in order to evaluate whether
the membrane proximal domain is actually the site of G protein
coupling.
resembles that of other 7TMR
(
-adrenergic receptor,
-adrenergic
receptor, rhodopsin, and receptors for fMLP, glutamate, and neurokinin
A)
(19, 20, 22, 23, 24, 25, 26) .
Formation of a putative fourth intracellular loop by palmitoylcysteines
was suggested in
-adrenergic receptor and in rhodopsin
(19, 22, 27, 28) but not in the case of
the receptors for fMLP and glutamate. Since cysteine residues do not
exist in the C` of IL-8R
, formation of the putative fourth
intracellular loop cannot be assumed to have a role in coupling to G
protein. The mechanism of signaling by IL-8R
might therefore
resemble more that of fMLP and glutamate receptors. This is further
supported by the fact that IL-8R
, like fMLP and glutamate
receptors, has a very short 3i loop and C` and therefore resembles
structurally more the fMLP and glutamate receptors than the
-adrenergic receptor and rhodopsin.
in signaling is further supported
by two characteristics of the migratory response of
4-transfected
cells. ( a) These cells show a significant migration to IL-8,
but the potency of the response is significantly reduced when compared
to WT receptor-transfected cells. This suggests that the region that
was deleted in
4 (amino acids 325-335) also has a role in
transducing signals or stabilization of G protein-receptor
interactions. In the absence of this region, the migratory response is
attenuated, but not completely abolished (in contrast to the effect of
deleting amino acids 317-324, as indicated by the results with
5-transfected cells). However, a more complex mode of interaction
between various regions of the C` of IL-8R
and between molecules
regulating signaling may be involved in the signaling process, as
indicated by the fact that
2-transfected cells, but not
3-transfected cells, exhibited a reduced potency of migration.
( b)
4-transfected cells show a persistent response to
high doses of IL-8. This is in contrast to
1 (WT)-,
2-, and
3-transfected cells which show a decreased responsiveness to
higher IL-8 concentrations. The region deleted in
4-transfected
cells may therefore be involved in G protein coupling in the WT
receptor and in regulating the level of responsiveness to various
ligand concentrations. The presence of serine and threonine residues in
1-,
2-, and
3-transfected cells and their absence in
4-transfected cells indicate that these residues have a
significant role in controlling the responsiveness to various IL-8
concentrations. This may imply that the mechanism regulating the
unresponsiveness is similar to the one regulating desensitization and
is mediated by G protein-coupled receptor kinases that phosphorylate
serine and threonine residues. One should bear in mind, however, that
desensitization is the outcome of sequential stimulation of the
receptor by the ligand and that this process was not analyzed in our
assays. Nevertheless, since IL-8Rs were shown to be desensitized in
polymorphonuclear cells by an overdose of IL-8
(43) and since G
protein-coupled receptor kinases were shown to be expressed in
peripheral blood lymphocytes
(44) , it is reasonable to propose
that the cells transfected with the
4 mutated receptor may be more
difficult to desensitize due to the lack of phosphorylation sites.
Studies are currently in progress in order to identify the role of
these serine and threonine residues in desensitization.
is
involved in signaling, the involvement of other intracellular domains
in transducing signals cannot be ruled out. The third and the second
intracellular loops seem to be additional candidates for regulating G
protein coupling. The third intracellular loop was shown to mediate G
protein coupling in several 7TMR
(19, 20, 21, 22) . The second
intracellular loop was found to couple G protein in fMLP receptor,
which is structurally and functionally related to IL-8 receptors. It is
also important to note that in IL-8 receptors the second intracellular
loop contains a DRY motif (a triplet of Asp-Arg-Tyr)
(1, 3, 4, 45) that was shown to be
highly conserved in other 7TMR and has been implicated to be important
for making possible the signal transduction, by other 7TMR
(1, 7, 45, 46) . Since several G protein
coupling sites may exist in IL-8 receptors, we intend to further
broaden our study and to investigate the role of other intracellular
regions in signaling. The role of the various intracellular regions of
IL-8R
in signaling is also being studied in our laboratory at
present.
that is essential for functional chemotactic
responses to IL-8. It will be important to establish whether this
region binds intracellular signaling molecules and whether it could be
a possible target for therapeutic intervention.
Table:
Amino acids deleted in c`-truncated
IL-8R receptors
Table: Scatchard plot
analysis for IL-8 binding of cells transfected with WT IL-8R
(
1) and four C`-truncated mutants (
2,
3,
4,
5)
, type A receptor for IL-8; IL-8R
, type B receptor for
IL-8; N`, amino terminus domain of the receptor; C`, carboxyl terminus
domain of the receptor; G protein, GTP-binding protein; BSA, bovine
serum albumin; PCR, polymerase chain reaction; WT, wild type; 3i, third
intracellular; 7TMR, seven transmembrane receptors; FITC, fluorescein
isothiocyanate.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.