Immunology, The University of Adelaide, North Terrace, South Australia; tDepartment of Immunology, Genentech. Inc., South San Francisco, California ...
Identification interleukin-8 intracellular BASSAM KEVIN
of G-protein receptors loops
B. DAMAJ, T. OGBORN,t
binding sites of the human by functional mapping of the
SHAUN R. McCOLL,* KULDEEP NEOTE,t NA SONGQING,t CAROUNE A. HEBERT,t AND PAUL H. NACCACHE’
Le Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUL and the Department of Medicine, Faculty of Medicine, Universit#{233} Laval, Sainte-Foy, Qu#{233}bec, Canada; 4Department of Microbiology and Immunology, The University of Adelaide, North Terrace, South Australia; tDepartment of Immunology, Genentech Inc., South San Francisco, California 94080-4980, USA; and tPfizer Central Research, Groton, Connecticut 06340-5196, USA
ABSTRACt’ Interleukin 8 (IL-8) is considered to be a major mediator of the inflammatory response. Recent evidence indicates that a direct physical association occurs between IL-8 receptors and the a subunit of guanine nucleotide regulatory protein (G1ct2) upon stimulation of human neutrophils by IL-8. In the present study, we identified by site-directed mutagenesis key residues within the three intracellular loops of the IL-8RA receptor involved in the interaction with G1a2. We first systematically mutated, in groups of two to four, all the residues in the three intracellular loops of the IL-8 type A receptor to alanine and analyzed the mutant receptors transiently expressed in 293 cells. Four residues in the second intracellular loop (Y136, L137, 1139, V 140) and one residue in the third intracellular loop (M241) were shown to be crucial for mediating calcium signaling in response to IL-8. Other residues in the second and third intracellular loops were also found to affect IL-8RA-mediated signaling, but to a lesser extent. These effects were not due to lower expression or low IL-8 binding affinities to the mutated receptors. Mutagenesis of the residues in the first intracellular loop had only weak effects on the mobilization of calcium induced by IL-8. We then used a coimmunoprecipitation protocol with antiGa2 antibodies to determine the involvement of the two regions defined above in Ga2 coupling to IL-8 type A receptors. Whereas the anti-Gia2 antibodies coimmunoprecipitated IL-8 receptors in the wildtype cells, this interaction was lost in cells expressing mutated receptors that affected intracellular calcium mobilization. The peptides corresponding to the regions of the type A receptor found to be critical for G1a2 coupling and induction of intracellular calcium mobilization were next introduced into cells expressing wild-type IL-8RA or IL-8RB to assess their role in coupling G1a2 to both IL-8 receptors. The results obtained in the latter experiments suggest that the same regions of the second intracellular loop (Yl 36,
1426
L137, 1139, V140) and of the third intracellular loop (M241) are critically involved in the coupling of both IL-8RA and IL-8 RB to Ga2 as well as to a downstream effector (or effectors) involved in calcium mobilization.-Damaj, B. B., McColl, S. R., Neote, K., Songqing, N., Ogborn, K. T., H#{233}bert,C. A., Naccache, P. H. Identification of G-protein binding sites of the human interleukin-8 receptors by functional mapping of the intracellular ioops. FASEB J. 10, 1426-1434 (1996) Key Words: neutrophils calcium mobilization tion Gro- NAP-2 C-X-C chemokines .
.
.
signal transduc-
.
8 (IL-8),2 ALSO KNOWN as neutrophil-activating peptide-1 (NAP-i), is a potent neutrophil agonist produced by many cell types in response to inflammatory stimuli (1-4). This cytokine, as well as two structurally related ligands, Gro-a , and NAP-2, are known to be highly, but not exclusively (e.g., 5-8), selective for neutrophils and to elicit various functional responses including chemotaxis, intracellular calcium mobilization, stimulation of phospholipase D activity, exocytosis, and the respiratory burst (1, 9-13). The cDNAs for the human neutrophil receptors that bind IL-8 have been cloned and sequenced (i4-i7). Two distinct, but related, receptors, designated type A (IL8RA) and type B (IL-8RB), have been identified. IL-8 binds to both human receptors with high affinity (Kd 1-4 nM), whereas NAP-2 and Gro-a bind with high INTERLEUKIN
tTo whom correspondence
and reprint
requests
should
be addressed,
at: Centre de Recherche en Rhumatologie et Immunologic, CHtJL, 2705 Blvd. Laurier, Ste-Foy, Quebec, GJV 4G2, Canada. 2Abbreviations: IL-8, interleukin-8; Gm-a, melanocyte growth-stimulatory activity (MCSA); IL-8RA, interleukin-8 type A receptor; Kj, dissociation constant; NAP-2, neutrophil-activating peptide-2; C-protein, guanine nucleotide binding protein; HBSS, Hank’s balanced salt solution; STM receptors; C-protein-linked receptors containing seven-transmembrane domains; FSB, FACS staining buffer.
0892.6638/96/0010-1
426/$01 .50. © FASEB
-
affinity only to IL-8RB (Kd=2 nM). These two receptors share 32% similarity in their amino-terminal extracellular domains, >98% similarity in their three intracellular domains, and an overall similarity of about 77%. The extracellular domains of the IL-8 receptors have been shown to be important for ligand binding (18). The amino acid sequences encoded by the eDNA clones of IL-8 receptors show that they belong to the superfamily of Gprotein-linked receptors that contain seven-transmembrane domains (STM receptors) (14, 15, 17, 19). This result is in accord with the known sensitivity of the functional responses induced by IL-8, NAP-2 and Groto pertussis toxin (1, 9, 10, 17, 20, 21). Furthermore, a physical association between the IL-8 receptors and the a2 subunit of G1 has recently been observed in human neutrophils and in 293 cells transfected with IL-8RA or IL-8RB receptors (22). Significant interest in the structural determinants of receptor-G-protein interactions has led to the recognition of the importance of the second and third intracellular loops of STM receptors for G-protein coupling as shown, for example, for the adrenergic, muscarinic, and angiotensin II receptors (23-26). Partial evidence has been obtained concerning the neutrophil chemoattractant receptors, and more specifically the formyl peptide receptor. Data indicate that the second intracellular loop of the formyl peptide-receptor interacts with G-proteins and that the role of the third intracellular loop of this receptor, which, like that of IL-8R, is much shorter than that of most other STM receptors, is less clear-cut (27-29). Although the extracellular portion of IL-8RA has been mapped and the segments responsible for the binding of IL-8 have been identified (18), little, if anything, is known about the functional topology of the intracellular segments of these receptors. In the present study, the three intracellular loops of IL-8RA were mapped by sitedirected mutagenesis for their ability to physically interact with the a2 subunit of G1 as well as for their involvement in the mobilization of calcium induced by IL-8.
MATERIALS
AND METHODS
Materials 1251-labeled IL-8 (specific activity 143-180 mCi/mg) was labeled using lactoperoxidase as previously described (30). The rabbit anti-G1a2 was raised against the LERIAQSDYI sequence of Ga2 (amino acids 159168). Goat anti-IL-8 was a gift from Mr. Henry J. Showell of Pfizer Central Research (Groton, Conn.). PVDF membranes were from Millipore (Bedford, Mass.). Rainbow prestained molecular weight markers and ECL developing kits were purchased from Amersham (Ontario, Canada). IL-8, 293 cells, 293 IL-8RB cells, and 293 IL-8RA cells were prepared and characterized as previously described (22). Pertussis toxin, pertussis toxin B oligomer, and cholera toxin were purchased from List Biological Laboratories Inc. (Campbell, Calif.). Indo-1/AM was obtained from Molecular Probes (Junction City, Oreg.). Hank’s balanced salt solution (HBSS), RPMI, penicillin, streptomycin, geneticin, and fetal bovine serum were purchased from GIBCO (Burlington, Ont., Can-
IDENTIFYING
C-PROTEIN
BINDING
SITES OF HUMAN
IL-B
ada). Protein A Sepharose was ordered from Pharmacia LKB (Uppsala, Sweden). All other reagents were from Sigma Chemical Company (St. Louis, Mo.).
Neutrophil
preparation
Blood was collected from healthy adult volunteers into tubes containing heparin. After 2% dextran sedimentation, neutrophils were purified under sterile conditions by centrifugation on Ficoll-Paque cushions. Residual erythrocytes were removed by hypotonic lysis and the cells were resuspended in HBSS containing 1.6 mM CaCl2, at a final concentration of 15 x 106 cells/mI. The entire procedure was carried out at room temperature.
Cell
culture
The 293 IL-8RA and IL-8RB cells were maintained in polystyrene tissue culture flasks containing RPM! 1640 medium supplemented with 10% fetal calf serum (FCS), 100 U penicillin/mi, 100 mg streptomycm/mI, and (800 mglml) geneticin. The cells were grown at 37#{176}C in a humidified atmosphere of 5% CO2 in air.
Peptides The following peptides used in these studies were synthesized by ANA Spec Incorporated (San Jose, Calif.) and were >98% pure as tested by
HPLC. LERIAQSDYI: corresponds to amino acids 159 to 168 of Ga2 and was used to raise the anti-G1a2 antibodies. HMGQKHRAMR: corresponds to amino acids 233 to 242 of IL-8RA. HMGQKHAAAA: corresponds to the mutated form of the HMGQKHRAMR peptide. DRYIAIVHAT: corresponds to amino acids 134 to 143 of IL-8RA. DRAAAAAHAT: corresponds to the mutated form of the DRYLAIVHAT peptide. TLTQKRHLVK: corresponds to the amino acids 145 to 154 of IL8RA. RTLFKAHMGQ: corresponds to the amino acids 227 to 236 of IL8RA. Mutagenesis
A diagram Fig. 1. positions order to Mutants
of IL-8 receptor
A
of the putative membrane topography of IL-8RA is shown in The intracellular loops are indicated, as are the amino acid of the amino and carboxyl termini of each of these loops, in help locate the positions of the different mutants produced. were prepared as previously described (18) using the dut-ung
coli CJ236 and a pUC-derived vector containing a cDNA insert coding for IL-8RA: pRK5B.IL-8r1.1. In some cases, the expression vector pcDNA3 (Invitrogen Corporation, San Diego, Calif.) containing the coding sequence of IL-8RA was used and site-directed mutagenesis was performed as described by Neve et al. (31), where PCR was used to incorporate mutations at the desired positions. Mutations were confirmed by DNA sequencing using the Sequenase version 2.1 kit and the manual sequencing or the Perkin Elmer Applied Biosystem Automated sequencer (model 373 DNA sequencer Stretch) in conjunction with the PRISM ready reaction dye-deoxy-terminator cycle sequencing kit (Perkin Elmer). After verification of the mutant DNA sequence, the mutated plasmid preparations were purified using a Qiagen plasmid Maxi Kit (Qiagen mc, Chatsworth, Calif.) and introduced into human fetal kidney 293 cells by the lipofectamine method. The cell cultures were incubated for 5 h in the presence of the pUC-derived vector containing no IL-8RA eDNA insert (sham) or mutant or wild-type DNA (5 ig of DNA/100 mm dish). The cells were then cultured overnight before analysis by flow cytometry, intracellular calcium measurements, and 1251-labeled IL-8 binding assays.
strain of Escherichia
Calcium mobilization Intracellular free calcium was measured Indo-1 as previously described (32,33).
uaing the fluorescent Cell suspensions (10
probe cells/nil)
1427
were loaded with Indo-1/AM (1 mM , 30 mm at 37#{176}C), washed, resuspended at 2 x 106 cells/mI in HBSS, and their fluorescence monitored in a spectrofluorometer (SLM 8000, SLM-Aminco, Urbana, Ill.) (excitation and emission wavelengths, 350 and 405 nm, respectively). The internal calcium concentrations were calculated as described in Tsien et al. (34). Each assay was individually calibrated.
Cell permeabilization Cell permeabilization was performed using saponin essentially as previously described (35). Briefly, 293 LL-8RA or 293 IL-8RB cells (50 x 106/ml) suspended in HBSS containing 0.1 M Hepes (pH 7.4) were incubated at room temperature for 30 mm with 0.01% saponin and Indo-1/pentapotassium salt (nonpermeant form) (1 mM) in the presence or absence of the indicated peptides (100 mg/mI) or antibodies (25 j.tg/ml). After incubation, the cells were washed with HBSS, resuspended at 2.5 x 106 cells/mI, and tested for calcium mobilization or C-protein coupling by coimmunoprecipitation. Flow
The expression of IL-8RA (sham, mutant, wild type) on 293 cells was assessed by staining the 293 cells with a monoclonal antibody (4C8) generated by immunizing mice with an 18 amino acid peptide corresponding to the amino-terminal sequence of IL-8RA (residues 2-19, SNITDPQMWDFDDLNFTG). Characterization of this antibody against the IL-8 RA receptor has been previously reported (36). Briefly, 106 cells/well were incubated at 4#{176}C for 30 mm with mAb 4C8 or with an isotype control diluted in FACS staining buffer (FSB) (1% FBS in phosphate-buffered saline + 0.02% sodium aside) or with FSB only. After washing twice with FSB, the cells were incubated at 4#{176}C for 30 mm with fluorescein isothiocyanate-conjugated goat anti-mouse F(ab)’2 diluted 1:500 in FSB. The cells were washed twice with FSB before FACScan analysis. To determine the percentage of positive cells, an analysis marker was set at a position that gave a reading of a maximum of 2% positive cells in the sample of sham-transfected cells stained with the same antibody.
IL-8 binding
assays
1251-labeled IL-8 binding assays were performed as previously described (30). Transfected 293 cells were incubated (1 x 106 cells/mI) with 1251-laheled IL-8 (0.5 nM) and varying concentrations of unlabeled IL-8 in a final volume of 250 ml. The cells were incubated at 4#{176}C for 1 h. The incubation was terminated by separating cells from the suspending media by centrifugation through a sucrose buffer. Nonspecific binding was determined in the presence of 300 nM unlabeled ligand. The binding data were curve-fitted with the computer program IGOR to determine the affinity (K,i) and the number of binding sites. The range of unlabeled IL-8 concentrations in the binding assays was 0.01-300 nM.
Gta2
immunoprecipitation
Cell suspensions (20 x 106 cells/sample) were incubated at 37#{176}C and, where indicated, stimulated with 100 nM !L-8 for 1 mm. Cells were lysed by adding an equal volume of immunoprecipitation buffer [50 mM Tris, pH 7.4; 0.1M NaCI; 0.25 mM phenylmethane sulfonyl fluoride (PMSF), 2 mM Na3VO4J containing 1% octyl$-D-glucopyranoside. The samples were vortexed and placed on ice for 30 mm. The insoluble material was removed by centrifugation at 12,000 x g for 15 mm. The supernatants were diluted with immunoprecipitation buffer to obtain a final octylb-3-D-glucopyranoside concentration of 0.413%. Protein ASephamse preincubated for 30 mm at 37#{176}C with the anti-G1a2 antibody was added and immunoprecipitations were earned out for 90 mm on ice. The immune complexes were recovered by centrifugation at 12,000 X g for 2 mm, washed three times with 50 mM Iris (pH7.4), 0.2 mM NaCl, 2 mM PMSF, 2 mM Na3VO4, and finally resuspended in SDS sample buffer (60 mM Tris, pH8.0; 2% SDS; 2% dithiothreitol; 10%glycerol; 0.01%bromophenol blue). Samples were boiled 10 mm before being subjected to SDS-PAGE on 15% gels.
1428
This was performed essentially as previously described (37). Briefly, electrophoretic transfer cells (Fisher Scientific Instruments, Ontario, Canada) were used to transfer proteins from the gels to Immobilon PVDF membranes. Nonspecific sites were blocked using 2% gelatin in TBSTween (25 mM Tris-HC1, pH 7.8, 190 mM NaCI, 0.15% Tween 20) for 1 h at 37#{176}C. The polyclonal goat anti-human IL-8 antibody was then incubated with the membranes for 1 h at 37#{176}C at a final concentration of 1/1000 in fresh blocking solution. The membranes were washed three times at room temperature in TBS-Tween and then incubated with a horseradish peroxidase-linked swine anti-goat IgG antibody for 45 mm at 37#{176}C at a final concentration of 1/10,000 in fresh blocking solution. The membranes were washed three times with TBS-Tween and the bands were revealed using the ECL Western blotting system.
RESULTS
analysis
cytometry
1251-Iabeled
Immunoblotting
Vol.10 October
1996
Mutagenesis
and
transfection
studies
of 293
cells
While the results of functional and coimmunoprecipitation experiments (22), as well as the pertussis toxin sensitivity of IL-8-induced cellular responses (1, 9, 10, 17, 20, 21), indicate that IL-8R, like the formylpeptide receptor (38), are physically coupled to a G-protein, no data are currently available concerning the identity of the IL-8R residues involved in these interactions. To address thisissue,we substitutedin groups of two to four all the amino acids of the three intracellular ioops of the IL-8 RA receptor with alanine. The mutated receptors were then analyzed by expression in 293 cells. The ability of the wild-type and mutant receptors to mediate an IL-8-induced mobilization of calcium in transfected293 cells was tested, and the results of these studies are summarized in Fig. 2. As previouslyreported (22),stimulation of
293
cells
transfected
with
wild-type
IL-8RA
by
IL-8
M), but not Gro-a or NAP-2 (10-v M), led to rapid (peak between 15 and 20 s) mobilization of intracellular calcium. On the other hand, sham-transfected cellsdid not respond to IL-8 (datanot shown). (10b010-7
Mutations
in the first
intracellular
loop
led
to a partial
inhibitionof IL-8-induced calcium mobilization. Substitution of VTD (73-75) and RS (71-72) by alanine reduced by 55% the cium induced
increases in the levels of cytoplasmic calby IL-8, whereas substitution of region
RYG (68-70) by alanine had less effect on these calcium transients(Fig.2).Substitutionto alanine of amino acids 134-140 of the second intracellular loops completely ablated intracellular calcium mobilization in response to IL8. The mutant receptors in which the DRY (134-136) or LAIV (137-140) sequences were substituted with alanine were essentially unable to produce a calcium spike in response to IL-8. The tripletmutations of the rest of the second
intracellular
loops
had
no significant
effect
abilityof IL-8 to mobilize calcium in 293 (Fig. 2). Finally, the triplet mutation scanning intracellular loop also revealed one specific region
essential
for
calcium
signaling.
on the
IL.-8RA cells of the third amino acid
Substitution
of
amino acids 239-242 (RAMR) to alanine prevented the cellsfrom responding to IL-8,whereas mutating the other
The FASEB Journal
DAMAJ E AL.
Extracellular NH2-
Intracellular
-COOK 350
Figure 1. Putative membrane topography of lL-8RA. The three intracellular i2, and i3, respectively. The amino acid positions at the amino and carboxyl three intracellular loops are indicated.
residuesof the thirdintracellular loop to alanine had little or no effect. Because the first round of mutants contained mutations of sequences of two to four amino acids, we created a second
panel
of mutants
in which
single
residues
were
replaced by alanine in order to assess the contributions of individualamino acids of the second intracellular ioop of IL-8RA. These single amino acid mutants were analyzed as above and the results of these studies are summarized in Fig. 3. Substitution of amino acids 134 (D), 135 (R), and 138 (A) of the second intracellular loop by alanine had no effect on the IL-8-induced responses. On the other hand, substitution of amino acids 136 (Y), 137 (L), 139 (I), and 140 (V) by alanine ablated the mobilization of calcium induced by IL-8. Substitution of amino acid 241 (M) of the third intracellular loop completely inhibited the calcium response induced by IL-8, whereas substitution of amino acid 242 (R) had no detectable effect.
On the other hibited
the
hand,
of the calcium shown). Analysis binding
substitution
mobilization
mobilization
of mutant
of 239
of calcium
induced
receptor
(R) partially
and
altered
by IL-8
expression
in-
the shape
(data
not
and
affinities
The levels of expression and the IL-8 binding affinities of the mutated receptors in the transiently transfected cells were measured and compared to cells transiently expressing wild-type IL-8RA. The data relative to the functionally
relevant
and
3 are
ences
could
amino summarized be noted
IDENTIFYING G-PROTEIN
acid
sequences
in Table in the
BINDING
identified
in Figs.
1. No significant
percentage
of positive
SITES OF HUMAN
IL-8
differanti-
2
loops are labeled ii, terinini of each of the
(as assessed by flow cytometry analyaffinities of the receptors or the number between the cells transfected with wildwith the various mutated forms of the refor 239 (R), where although receptor expression was comparable to that of the wild type receptors, no IL-8 binding could be detected. Furthermore, the Kd’s of the receptors of the transfected cells (about 2 nM) corresponded to those of the native receptors on human body-staining
cells
sis), the binding of receptors/cell type IL-8RA, or ceptors except
neutrophils
(39,
40).
Coimmunoprecipitation
of Gia2
and IL-8RA
The formation of a physical Gcx2 in human neutrophils
complex between IL-8RA and as well as in transfected 293 cells was recently demonstrated using a coimmunoprecipitation method (22). In this method, the IL-8/IL8RA/G-protein complex is first immunoprecipitated with an anti-G,a2 antibody and a Western blot is performed with an anti-IL-8antibody.The detectionof IL-8 on the Western blot is an indication for the presence of a complex of IL-8/IL-8RA/G-protein and hence a physical association of the G-protein with the receptor. As shown in Fig. 4, stimulation of wild-type 293 RA transfected cells with iO M IL-8 led to the rapid formation of this tnmolecular complex. The immunizing peptide used to raise the G1cx2antiserum prevented the immunoprecipitation of the IL-8RJG1a complex, thereby establishing the specificity of the immunoprecipitation. Control blots with the anti-Gcx2 antibodies demonstrated that equal amounts of G1a2 were immunoprecipitated and loaded in each lane (data not shown). This approach was then used to examine the effect
of the mutations
in the
second
intracellular
1429
#{182}20
#{182}00
80
60
40
20
1(31 0
-
CIII
S
0
0 0#{216}IA 0 >
1-
0 01
0 0
0 0
01 0
III
00
001
IA
711
0
r-
1-
5.
IA
III 0
IA
0
0 0
.j
IA
A
Al N
0101 N N
IA
I.-
I 0
N N
01 N
IA N
-j
..I
I 01 III N
N Al II
o x
(31 z
IA
01 N
II.
a0
0.
0
0
lar loops of IL-8RA identified above. Under these conditions, more than 90% of the cells were found to incorporate FITC-labeled antibodies, and 70% of the cells excluded Trypan blue after the removal of saponin (data not shown). The resealed cells were then stimulated for 1 mm with IL-8 and the coimmunoprecipitation protocol described in Fig. 4 was used to monitor the association of IL-8R with G1a2.As shown in Fig. 5A, B, C, respectively, stimulationof human neutrophils,293 IL-8 RA, or 293 IL-8RB cells with 10 M IL-8 led to rapid formation of the trimolecular complex between IL-8, IL-8R, and Ga2, and the coimmunoprecipitation was inhibited in the presence of the immunizing peptide (LERIAQSDYI). The peptide mapping strategy was then used to confirm the results
0. 0 0
0 -l
Figure 2. IL-8-induced calcium mobilization in 293 cells transfected with wild-type or mutated IL-8RA. The indicated amino acid sequences (identity and position) were replaced by alamne. WT, wild-type IL-8RA; sham, 293 cells transfected with vector alone. The data represent the mean ±SCM of the increases of the level of free cytoplasmic calcium induced by i07 M IL-8 as determined in three separate transfections, each carried out in triplicate.
loop of the IL-8RA,
identified as functionally important in the context of induction of intracellular calcium mobilization on the physical association between IL-8RA and GmU2. The results of these studies are illustrated in Fig. 4. Substitution of amino acids 138 (A), 239 (R), or 242 (R) to alanine had no effect on the IL-8-induced coupling of IL-8RA to G1a2. On the other hand, substitution of amino acids 136 (Y), 137 (L), 139 (L), and 140 (V) in the second intracellular loop, or M (241) in the third intracellular loop, by alanine resulted in complete inhibition of the detection of IL-8 in the anti-Gcz2 precipitates, thereby indicating a disruption of the physical coupling between IL-8RA and G1a2.
of the
experiments
identifying
the
amino
acid
se-
quences affecting the induction of calcium mobilization and the coupling of Gcx2 to IL-8RA, as well as to determine whether these sequences were involved in Ga2 coupling to IL-8 type B receptor in 293 cells and in neutrophils. As shown in Fig. 5, peptides DRYLAIVHAT and HMGQKHRAMR, which contained the LAIV (137140) and RAM (239-24 1) sequences of the second and the third intracellular loops, respectively, prevented formation of the IL-8RA/G1cx2 and IL-8RB/G1a2 complex in all three cell types. In contrast, the respective alaninesubstituted peptides had no significant effect on formation of the complex after ligation of IL-8. Peptide RTLFKAHMGQ inhibitedby about 50% (as determined by gel scanning) IL-8RA/Ga2 and IL-8RB/Ga2 complex formation, whereas the last peptide tested (TLTQKRHLVK) had no effect on the physical coupling in any of the three cell types. Control blots with the antiG1a2 antibodies demonstrated that equal amounts of G1a2
120 100
Effects of peptides loops of IL-8RA
derived
from
the intracellular
80 w
IAw
60
WI.-
Sequence alignment shows that the three intracellular loops of the two IL-8 receptor types are nearly identical except for one amino acid [amino acid 151 (H)] in the second intracellular loop. Because of the high similarity in the amino acid sequence of the intracellular loops of
both receptors,
we used
a domain-specific
peptide
Vol. 10 October
1996
20 5-
0 0
#{149} N 0
map-
ping strategy to evaluate the role of regions 136-140 and 239-242 of IL-8RA in G-protein coupling and calcium signaling in neutrophils as well as in 293 cells transfected with IL-8RA or IL-8RB receptors. Neutrophils or 293 cellspermanently expressing wildtype IL-8RA or RB were transiently permeabilized as previously described (22, 35) in the absence or presence of peptides derived from the sequences of the intracellu-
1430
40 a.
5.
-j LOOP II
.(
-
5.
IA
LOOP III
Figure 3. Effect of single-point mutations of IL-8RA on the mobilization of calcium induced by IL-8 in transfected 293 cells. The residues replaced by alanine are identified. WT, wild-type IL-8RA; sham, 293 cells transfected with vector alone. The data represent the mean sti4 of the increases of the level of free cytoplasmic calcium induced by 1O M IL-8 as determined in three separate transfections, each carried out in triplicate.
The FASEB Journal
DAMAJ
ET AL
TABLE 1. Expression levels, binding affinities (Kj), and receptor a numbers of the wild-type and matant IL-8RA transfected into 293 cells % Positive cells (n3)
Kd, nM (n=2)
Receptor number (n2)
A
95 94
1.51 2.0
66,541
Y (136)-. L (137)-*
A
93
2.0
66,370
A (138)-* 1(139)-.
A A
89 95
2.01
64,865
1.54
67,337
V (140)-4
A
94
1.48
66,974
R (239)-* M (241)-. R (242)-. SHAM
A6. #{176} A6. C A6
110 91 94
N/D
Wild typeb
3
65,221
N/D
0.90
33,600
0.58
52,800
N/D
N/D
were immunoprecipitated and loaded into each lane (data not shown). The effect of the peptides tested in the coimmunoprecipitation protocol on the mobilization of intracellular calcium induced by IL-8 in 293 IL-8RA and 293 IL-8RB cells was examined next. Neutrophils could not be used in this assay because the cells did not retain sufficient quantities of lndo-1 after permeabilization by saponin to enable reproducible assessment of calcium mobilization. As shown in Fig. 64, B, the resealed 293 cells remained capable of mobilizing calcium in response to IL8, i.e., they appear to have retained a functional transduction apparatus. As previously shown (22), the Ga2 immunizing peptide (LERIAQSDYI) inhibited the increases
in cytoplasmic
free
calcium
induced
by IL-8.
and are critical for the subsequent mobilization of calcium. Three basic important observations were made during this investigation. By examining the effects of sitedirected mutagenesis of IL-8RA, several regions of the three intracellular loops of IL-8RA were identified that appear to be crucial for coupling this receptor to G1a2, as well as for the mobilization of calcium that results from its occupation by IL-8. In the second part, we observed that these amino acids are in direct contact with G1a2. Finally, it was observed that the same regions of the three cytoplasmicloops are involved in the interactions of Ga2 with both types of IL-8 receptors. Because no structural data on the IL-8 receptor are available, we based our understanding of the receptor structure on what is known about the seven-transmembrane G-proteincoupled receptors to which IL-8 receptor superfamily belongs. Indirect approaches previously suggested the involvement of the intracellular loops in G-protein coupling to several seven-transmembrane receptors (23-26). These reportshave substantiated thattheroleofthefirst intracellular loop of seven-transmembrane receptors in G-protein coupling is minimal, whereas the second and third loops are Ga2
suspected to play a more important role in the physical coupling of these receptors to G-proteins. However, these reports have not directly identified the regions in these receptors
that physically
bind
to G-proteins.
In this study, the
IMMUNOPRECIPITATION WITH and-C12 Ab
In
addition, introduction of peptides DRYLAIVHAT and HMGQKHRAMR, which inhibited the formation of the IL-8fIL-8RJGa2 complex, also prevented the mobilization of calcium induced by IL-8 whereas their corresponding, alanine-substituted peptides were without effect in both cell types. As observed in the coimmunoprecipitation protocol (Figs. 4 and 5), peptide RTLFKAHMGQ partially inhibited the responses to IL-8, as it did the coimmunoprecipitation, whereas peptide TLTQKRHLVK was without effect.
8kD -
.
I
.
IC
-
E
DISCUSSION C) The involvement of heterotrimeric G-proteins of the Gm family in the transductionof the signals initiatedupon the binding of IL-8 to its receptors is supported by three lines of evidence: 1) the realization that the cloned IL-8R belong to the superfamily of seven-transmembrane G-protein-coupled receptors (14, 15, 17, 19); 2) the inhibition of IL-8-induced functional responses by pertussis toxin (1, 9, 10, 17, 20, 21); and 3) the recent demonstration of an IL-B-stimulated association between IL-8R and G1a2 in neutrophils and 293 cells expressing wild-type IL-8RA or IL-8RB (22). The present study extends these observations and defines, by functional mapping, the regions of the three cytoplasmic loops of IL-8R that interact with
IDENTIFYING G-PROTEIN BINDING SITESOF HUMAN
IL-S
r
r
0
z
-I Cl)
-s
-
Figure 4. Effect of selected mutations of IL-8RA on its physical association with Ga2 in transfected 293 cells. The results of immunoprecipitations carried out with anti-G1Ct2 antibodies in transiently transfected 293 IL-8RA cells are shown. Immune complexes were obtained and analyzed as described in Materials and Methods. The cells were stimulated with iO M IL-8 for 1 mm. The residues replaced by alanine are identified. The results are from a single experiment representative of at least three other independent determinations. The molecular weight marker is indicated on the left.
1431
IMMUNOPRECIPITATION WITH anti-Gte2 Ab
reminiscent
of the extracellular
loop
Leong et al. (18) that were expressed
I
but unable
I
to transduce
mutants
a calcium
ably were unable to couple to G-proteins. The ability of the mutated receptors
IL-8
-
-
-I
IL-8
with
+
+
+
+
+
+
++-
+
+
+
+
+
+
+--
-
DO
I
11
IL-8
Gia2.
PEPTIDE
000 I
IXCfl
XI
r>I
IL-8
o Figure 5. Effect of IL-8RA-derived peptides on the stimulation of the association between IL-8R and G1cx2 in human neutrophils, 293 IL-8RA, and 293 IL-8RB cells. Immune complexes were obtained and analyzed as described in Materials and Methods. The indicated cells were first loaded with the peptides identified as described in Materials and Methods and then stimulated with io M IL-8 for 1 mm. The G1a2 immunoprecipitating antibody at a dilution of 15 mI/mI was used as is or after neutralization with the LERIAQSDYI peptide (incubation with 100 mg/ml peptide for 30 mm at 37#{176}C). The results are from a single experiment representative of at least three other independent determinations. The molecular weight marker is indicated on the left.
inhibition of the coimmunoprecipitation by substituting amino acids 136, intracellular
loop and coupled with
137,
of IL-8RA and Ga2 139, or 140 of the
241 of the third intracellular the inhibitory effects of pep-
tides containing these sequences (but not their mutated counterparts), strongly argues for a direct role of these amino acids in a physical association between IL-8R and G1a2. The finding that the regions containing amino acids 136 (Y), 137 (L), 139 (I), 140 (V), and M (241) of IL-8R are the binding sites for G1cx2 may have significance extending beyond signal
transduction,
as 1) the hydrophobic
to
seven-transmembrane All the intracellular
more than forty proteins receptors (41). ioop mutants
nature
is conserved in many (41), and 2) these re-
of the amino acids in the second loop other seven-transmembrane receptors gions are common
Finally,
the
effects
of GTP
analogs
on the
binding
affinity of IL-8 to its receptors are relatively modest (less than twofold change in affinity) (19). Direct studies of the effects of guanine nucleotides on the binding characteristics of IL-8 to the mutants are needed to clarify these findings. Sequence alignment shows that the three intracellular loops of the IL-8 receptors are identical in the two human
0
loop to alanine,
the binding of IL-8 was measured on intact cells to membrane fractions. Second, the uncoupling was between IL-8 receptors and GiU2 only,
whereas the addition of nonhydrolysable GTP analogs to membrane fractions will activate indiscriminately all G-proteins, some of which are likely to be able to substitute for
-I
chemokine
to bind
cols followed in the present studies are fundamentally different from those used to demonstrate the regulation of the binding affinities by guanine nucleotides. First, in the present study, as opposed we observe
second
by
apparently intact affinity while loosing their coupling to Gia2 is an anomalous finding in view of the often-described regulation of the interaction of agonists with STM receptors by guanine nucleotides. However, the experimental proto-
P
8 kD
described
on the transfected cells, signal, and thus presum-
including
generated
several
in this study
receptors
(type A and B) except
for amino acid 151 (H)
in the second intracellular ioop. The demonstration that both IL-8 receptors associate with Ga2 (22) and the near identity of their three intracellular loops makes it likely that G1a2 interacts with the same amino acids of the three intracellular loops of both IL-8 receptors. The peptide mapping strategy illustrated in Figs. 5 and 6 strongly supports this deduction,
as the profiles
of inhibition of the mobilization of calcium and the coimmunoprecipitation of Ga2 and IL-8R were identical in 293 IL-8RA and 293 IL-8RB cells and in neutrophils that express both types of IL-8 receptors. These results indicate, therefore, that the signal transduction pathways activated by the two types the interaction transduction
of IL-8 with pathways
receptors
are indistinguishable
Ga2. However, coupled
although
to IL-8RA
up to
the signal
and
to IL-8RB
share several elements (42; Damaj et al., unpublished results), they are not identical. L’Heureux et al. (10) have demonstrated that, of the three major C-X-C chemokines, only IL-8 stimulated to a significant extent the activity of PLD in human neutrophils. Moreover, Magazin et al. (43) have
also reported
differences
in the biological
activities
of
Gro-J and IL-8 toward human neutrophils. The present results indicate that the basis for the differences in the functional effects mediated by the two IL-8 receptors lies downstream
(or in parallel)
to the interaction
with
G1a2 at
a
presently undetermined site. The only previously available data on the involvement of the intracellular regions of the IL-8 receptors in cell
(with the exception of 239 (R)) were able to bind IL-8 with near-normal affinities and were expressed at essentially the same levels as the wild-type receptors. The reasons underlying the lack of binding capacity of mutant 239 (R) are un-
activation concerned the implication of IL-8RB carboxyl terminus in mediating IL-8-induced chemotaxis (44). However, this report never tested whether the inhibition of chemotaxis obtained by the carboxy terminus deletions
known
were
1432
at present.
Vol. 10 October
The behavior
1996
of this
mutant
is, however,
The FASEBJournal
due
to uncoupling
of the receptor
from
G-proteins
or
DAMAJ E AL
3.293 tL-8R8 CELLS
A. 293 IL-8RACELLS
200
200 0
t3J
tsI 150
150
100
100
H
so
w
Mi U-0
O -
50
0 ‘C
.(
I
I
‘U -#{149}
4 4
1
‘C
‘C
0 0 I
0
-
0 0
I‘C
‘U
#{149}- ‘U 4
‘C
‘C
2
,-
-
‘U -
‘C
‘C 4
5
‘C
-
0
I ‘U a
‘C
0
#{149}- I z
0
-#{149}
I z
Papruoss
PSPTIOES
LOOP N
1
‘C ‘C .5
0
I
0 0
4
5
II
0
LOOP III LOOP N
LOOP
NI
Figure 6. Effect of IL-8RA derived-peptides on the mobilization of calcium induced by IL-8 in 293 IL-8RA and 293 IL-8RB cells. The indicated peptides were introduced into the cells as described in Materials and Methods. The peptides were introduced inside the cells using the saponin permeabilization protocol described in.Materials and Methods before stimulation with IL-8 (10 M. The calcium levels were monitored as described in Materials and Methods. IL-8 induced an increase in the levels of cytoplasmic free calcium of 155±15 nM in control 293 cells (mean±SEM, three detenninations). Mean ±SEM of the increases in the level of cytoplasmic free calcium induced by IL-8 in three independent experiments each carried out in duplicates.
to disruptions caused
of G-protein/IL-8 by conformational changes.
To our knowledge, identifies the amino in physical binding
rect method
interaction. 68-75 inhibited
to show such
Alanine substitutions to a moderate degree
induced
interactions
this is the first report that not only acids on the IL-8 receptors involved to G-proteins, but also applies a di-
(coimmunoprecipitation)
cal
calcium
receptor
by IL-8.
This
physi-
of amino acids the mobilization of
observation
is similar
to
the partial inhibition of phosphatidylinositol turnover coupled to human muscarinic cholinergic receptors (Hml) induced by alanine substitutions of equivalent regions These
of the first intracellular data
indicate
that
ioop of those receptors (24). the first loop plays a role in cou-
pling IL-8R to the mobilization of calcium, but that this role is only partial. The “DRY box” region suspected to play a dominant role in G-protein interaction with STM receptors, including formyl peptide receptors (45), was indeed action amino
involved
in the
functional
and
the
physical
inter-
of G1cz2 with the IL-8 receptors. However, only acid 136 (Y) of this region is involved in this in-
teraction. Moreover, acids [137 (L), 139
we have identified three other (I), and 140 (V)] that are also
amino impli-
cated in the interaction of IL-8R with Ga2. The potential involvement of the third intracellular loop in other G-protein-linked receptors has been a matter of controversy, plication of this
as some loop
studies in G-protein
cellular loop are not highly conserved among STM receptors. Thus, the contribution of the third intracellular loop to the coupling to G-proteins must be specifically assessed with each receptor type. Our results cate that only residue 241 (M) of the third
clearly
indi-
intracellular ioop of IL-8R plays a critical functional role in C-X-C chemokine signaling, being involved in the physical association between IL-8R and G1a2 as well as in the initiation of a calcium signal. However, the hydrophobic amino acids of the third ioop suspected to be involved in G-protein coupling to other STM receptor (23, 46, 47) did not significantly affect G-protein coupling to IL-8R. The data presented in this report complement the model of the proposed structure of the extracellular domains
of IL-8RA
receptor
proposed
We have demonstrated in 136 (Y), 137 (L), 139 (L), cellular loop, and M (241) are the keys for binding of ing
the
subsequent
juxtaposition
calcium
of extracellular
Leong et al. (18) would together
of the third
Thus, amino acids in close proximity, tact
by Leong
et al. (18).
this report that amino acids 140 (V) in the second intrain the third intracellular loop, Ga2 to IL-8R and for mediatsignaling. loops
be consistent
and sixth
The 1 and
hypothesized 2 proposed
with
transmembrane
the
by
bringing
domains.
136-140 and 239-241 would also be and together may form the G1a2 con-
site.
have reported the imcoupling to the formyl
peptide receptor by the use of specific domain-peptide mapping (29), whereas others reached opposite conclusions by the use of site-directed mutagenesis (27). In contrast to the second intracellular loop, it should be noted that the composition and length of the third intraIDENTIFYING G-PROTEIN BINDING SITESOF HUMAN IL-8
Supported in part by grants and fellowships from the Medical Research Council of Canada, the Arthritis Society of Canada, and the Fonds de la Recherche en Sante du Qu#{233}bec. The authors would like to thank Mr. Steve Leong (Department of Immunology, Genentech, Inc., South San Francisco, Calif.) for his expert help with the mutagenesis experiments.
1433
25.
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The FASEB Journal
Receivedforpubdkation February 27, 1996. Accepted for publication May 23, 1996.
DAMAJ Er AL
