©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Interleukin-8 Receptor
THE ROLE OF THE CARBOXYL TERMINUS IN SIGNAL TRANSDUCTION (*)

Adit Ben-Baruch (1)(§), Kathleen M. Bengali (2), Arya Biragyn (3), Jim J. Johnston (4), Ji-Ming Wang (2), Jin Kim (5), Anan Chuntharapai (5), Dennis F. Michiel (1), Joost J. Oppenheim (1), David J. Kelvin(¶) (1)

From the (1) Laboratory of Molecular Immunoregulation, Biological Response Modifiers Program, (2) PRI/DynCorp, Frederick Cancer Research and Development Center, the (3) Preclinical Evaluation Laboratory, Biological Response Modifiers Program, and the (4) Laboratory of Experimental Immunology, Biological Response Modifiers Program, NCI, National Institutes of Health, Frederick, Maryland 21702-1201 and (5) Genentech, Inc., South San Francisco, California 94080

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Two interleukin-8 (IL-8) receptors, 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.

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 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.


INTRODUCTION

IL-8() 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) .

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/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 Cby coupling to Gor Gand to interact with recombinant Gproteins, 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.

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 -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) .

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 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.




EXPERIMENTAL PROCEDURES

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 pCRII 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 10cells/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 10cells/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.).

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) .

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 10cells/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.

For each IL-8 concentration tested, cells migrating through to the underside of the filter were counted in three high power fields ( 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).

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 (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.

Using the chemotaxis index, migration potencies of cells transfected with WT and truncated IL-8R 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.


RESULTS

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.

Cells transfected with the truncated receptors 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).

In contrast to the 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.


DISCUSSION

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 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.

This study is the first to present data suggesting a role for the C` domain of IL-8R 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.

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 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.

The Scatchard analysis shows that cells transfected with 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.

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 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).

These results suggest that a region existing in 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.

The involvement of the membrane proximal portion of the C` in signal transduction by IL-8R 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.

The possible involvement of the C` of IL-8R 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.

Although we suggest that the membrane proximal portion of the C` of IL-8R 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.

In conclusion, we have identified a membrane proximal region of the IL-8R 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)

The results of 2-3 experiments done for each cell line are presented.



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.

Current address: The Siebens-Drake Research Institute, London, Ontario, N6G 2V4 Canada.

§
To whom correspondence and reprint requests should be addressed: LMI, BRMP, NCI-FCRDC, Bldg. 560, Rm. 31-19, Frederick, MD 21702-1201. Tel.: 301-846-1347; Fax: 301-846-1673.

The abbreviations used are: IL-8, interleukin-8; IL-8R, 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.


ACKNOWLEDGEMENTS

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.


REFERENCES
  1. Baggiolini, M., Dewald, B., and Moser, B. (1994) Adv. Immunol. 55, 97-179 [Medline] [Order article via Infotrieve]
  2. Oppenheim, J. J., Zachariae, C. O. C., Mukaida, N., and Matsushima, K. (1991) Annu. Rev. Immunol. 9, 617-648 [CrossRef][Medline] [Order article via Infotrieve]
  3. Murphy, P. M., and Tiffany, H. L. (1991) Science 253, 1280-1283 [Medline] [Order article via Infotrieve]
  4. Holmes, W. E., Lee, J., Kuang, W. J., Rice, G. C., and Wood, W. I. (1991) Science 253, 1278-1280 [Medline] [Order article via Infotrieve]
  5. Cerretti, D. P., Kozlosky, C. J., Vanden Bos, T., Nelson, N., Gearing, D. P., and Beckmann, M. P. (1993) Mol. Immunol. 30, 359-367 [CrossRef][Medline] [Order article via Infotrieve]
  6. Lee, J., Horuk, R., Rice, G. C., Bennett, G. L., Camerato, T., and Wood, W. I. (1992) J. Biol. Chem. 267, 16283-16287 [Abstract/Free Full Text]
  7. Murphy, P. M. (1994) Annu. Rev. Immunol. 12, 593-633 [CrossRef][Medline] [Order article via Infotrieve]
  8. Probst, W. C., Snyder, L. A., Schuster, D. I., Brosius, J., and Sealfon, S. C. (1992) DNA Cell Biol. 11, 1-20 [Medline] [Order article via Infotrieve]
  9. Gerard, N. P., and Gerard, C. (1991) Nature 349, 614-617 [CrossRef][Medline] [Order article via Infotrieve]
  10. Boulay, F., Tardif, M., Brouchon, L., and Vignais, P. (1990) Biochemistry 29, 11123-11133 [Medline] [Order article via Infotrieve]
  11. Neote, K., DiGregorio, D., Mak, J. Y., Horuk, R., and Schall, T. J. (1993) Cell 72, 415-425 [Medline] [Order article via Infotrieve]
  12. Charo, I. F., Myers, S. J., Herman, A., Franci, C., Connolly, A. J., and Coughlin, S. R. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2752-2756 [Abstract]
  13. Savarese, T. M., and Fraser, C. M. (1992) Biochem. J. 283, 1-19 [Medline] [Order article via Infotrieve]
  14. Kelvin, D. J., Michiel, D. F., Johnston, J. A., Lloyd, A. R., Sprenger, H., Oppenheim, J. J., and Wang, J.-M. (1993) J. Leukocyte Biol. 54, 604-612 [Abstract]
  15. Kupper, R. W., Dewald, B., Jakobs, K. H., Baggiolini, M., and Gierschik, P. (1992) Biochem. J. 282, 429-434 [Medline] [Order article via Infotrieve]
  16. Wu, D., LaRosa, G. J., and Simon, M. I. (1993) Science 261, 101-103 [Medline] [Order article via Infotrieve]
  17. Weiss, E. R., Kelleher, D. J., Wai Woon, C., Soparkar, S., Osawa, S., Heasley, L. E., and Johnson, G. L. (1988) FASEB J. 2, 2841-2848 [Abstract/Free Full Text]
  18. Dohlman, H. G., Thorner, J., Caron, M. G., and Lefkowitz, R. J. (1991) Annu. Rev. Biochem. 60, 653-688 [CrossRef][Medline] [Order article via Infotrieve]
  19. O'Dowd, B. F., Hnatowich, M., Regan, J. W., Leader, W. M., Caron, M. G., and Lefkowitz, R. J. (1988) J. Biol. Chem. 263, 15985-15992 [Abstract/Free Full Text]
  20. Cotecchia, S., Exum, S., Caron, M. G., and Lefkowitz, R. J. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2896-2900 [Abstract]
  21. Shapiro, R. A., Palmer, D., and Cislo, T. (1993) J. Biol. Chem. 268, 21734-21738 [Abstract/Free Full Text]
  22. Konig, B., Arendt. A., McDowell, J. H., Kahlert, M., Hargrave, P. A., and Hofmann, K. P. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 6878-6882 [Abstract]
  23. Schreiber, R. E., Prossnitz, E. R., Ye, R. D., Cochrane, C. G., and Bokoch, G. M. (1994) J. Biol. Chem. 269, 326-331 [Abstract/Free Full Text]
  24. Bommakanti, R. K., Klotz, K.-N., Dratz, E. A., and Jesaitis, A. J. (1993) J. Leukocyte Biol. 54, 572-577 [Abstract]
  25. Pin, J. P., Joly, C., Heinemann, S. F., and Bockaert, J. (1994) EMBO J. 13, 342-348 [Abstract]
  26. Joshia, S. M., Cyr, C. R., Chu, V., Grumet, M., Gardner, J. P., and Kris, R. M. (1994) Biochem. Biophys. Res. Commun. 199, 626-632 [CrossRef][Medline] [Order article via Infotrieve]
  27. O'Dowd, B. F., Hnatowich, M., Caron, M. G., Lefkowitz, R. J., and Bouvier, M. (1989) J. Biol. Chem. 264, 7564-7569 [Abstract/Free Full Text]
  28. Morrison, D. F., O'Brien, P. J., and Pepperberg, D. R. (1991) J. Biol. Chem. 266, 20118-20123 [Abstract/Free Full Text]
  29. Lefkowitz, R. J. (1993) Cell 74, 409-412 [Medline] [Order article via Infotrieve]
  30. Ali, H., Richardson, R. M., Tomhave, E. D., Didsbury, J. R., and Snyderman, R. (1993) J. Biol. Chem. 268, 24247-24254 [Abstract/Free Full Text]
  31. Takano, T., Honda, Z., Sakanaka, C., Izumi, T., Kameyama, K., Haga, K., Haga, T, Kurokawa, K., and Shimizu, T. (1994) J. Biol. Chem. 269, 22453-22458 [Abstract/Free Full Text]
  32. Sprenger, H., Lloyd, A. R., Lautens, L. L., Bonner, T. I., and Kelvin, D. J. (1994) J. Biol. Chem. 269, 11065-11072 [Abstract/Free Full Text]
  33. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467 [Abstract]
  34. Remes, J. J., Petaja-Pepo, U. E., Tuukkanen, K. J., and Rajaniemi, H. J. (1993) Exp. Cell Res. 209, 26-32 [CrossRef][Medline] [Order article via Infotrieve]
  35. Leong, S. R., Kabakoff, R. C., and Hébert, C. A. (1994) J. Biol. Chem. 269, 19343-19348 [Abstract/Free Full Text]
  36. Munson, P. J. (1983) Methods Enzymol. 92, 543-576 [Medline] [Order article via Infotrieve]
  37. Falk, W. R., Goodwin, R. H., Jr., and Leonard, E. J. (1980) J. Immunol. Methods 33, 239-247 [CrossRef][Medline] [Order article via Infotrieve]
  38. Grob, P. M., David, E., Warren, T. C., DeLeon, R. P., Farina, P. R., and Homon, C. A. (1990) J. Biol. Chem. 265, 8311-8316 [Abstract/Free Full Text]
  39. LaRosa, G. J., Thomas, K. M., Kaufmann, M. E., Mark, R., White, M., Taylor, L., Gray, G., Witt, D., and Navarro, J. (1992) J. Biol. Chem. 267, 25402-25406 [Abstract/Free Full Text]
  40. Gayle, R. B., III, Sleath, P. R., Srinivason, S., Birks, C. W., Weerawarna, K. S., Cerretti, D. P., Kozlosky, C. J., Nelson, N., Vanden Bos, T., and Beckmann, M. P. (1993) J. Biol. Chem. 268, 7283-7289 [Abstract/Free Full Text]
  41. Hébert, C. A., Chuntharapai, A., Smith. M., Colby, T., Kim, J., and Horuk, R. (1993) J. Biol. Chem. 268, 18549-18553 [Abstract/Free Full Text]
  42. Chuntharapai, A., Lee, J., Burnier, J., Wood, W. I., Hébert, C., and Kim, K. J. (1994) J. Immunol. 152, 1783-1789 [Abstract/Free Full Text]
  43. Moser, B., Schumacher, C., von Tscharner, V., Clark-Lewis, I., and Baggiolini, M. (1991) J. Biol. Chem. 266, 10666-10671 [Abstract/Free Full Text]
  44. Parruti, G., Peracchia, P., Sallese, M., Ambrosini, G., Masini, M., Rotilio, D., and De Blasi, A. (1993) J. Biol. Chem. 268, 9753-9761 [Abstract/Free Full Text]
  45. Ahuja, S. K., and Murphy, P. M. (1993) J. Biol. Chem. 268, 20691-20694 [Abstract/Free Full Text]
  46. Horuk, R. (1994) Immunol. Today 15, 169-174 [CrossRef][Medline] [Order article via Infotrieve]

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