Figure 1.
Two-dimensional diagram of human CXCR1 receptor.
The positions of the residues (Leucine 128 and Valine 247) that were targeted for mutagenesis in TM3 and TM6 of the CXCR1 are indicated by gray and black filled circles respectively. The putative disulfide bridge, formed by Cys110/Cys 187 is indicated by “S-S”.
Figure 2.
FACS analysis of CXCR1 WT and mutants.
HEK 293 cells were transiently transfected with CXCR1 or its mutants. Cells were incubated with FITC-conjugated mouse anti-human CD181 (CXCR1) antibody. Specificity of signal was confirmed by staining the cells with mouse IgG1 isotype control. (A) Transfected HEK 293 cell population stained positive for this anti-CXCR1 antibody (M2 region). (B) Fluorescence of positively stained cells was quantified by FACS (± SEM, with CXCR1 WT set to 100%).
Figure 3.
Binding of 125I-labeled IL-8 to COS-7 cells expressing WT CXCR1 or its mutants.
The CXCR1 mutants were transiently transfected in COS-7 cells. Maximal binding of 125I-IL-8 to CXCR1 WT and mutants (A) and competition binding experiments (B and C) were performed as described under “Materials and Methods”. Competitive binding studies were conducted using 125I-labeled IL-8 and various unlabelled ligands as described under “Materials and Methods”. Representative maximal binding experiments for L128 and V247 mutants are shown in Fig. 3A, performed in triplicates. Mock was transfected with pSG5 plasmid. Representative competitive binding experiments for L128 and V247 mutants are shown in Fig. 3(B) and Fig. 3(C), performed in triplicates. Nonspecific binding was determined by adding 250 nM unlabeled IL-8. Curve fitting was done using GraphPad Prism data analysis program, and the affinity constant (Kd) for CXCR1 mutants is shown in Table 1.
Table 1.
Summary of ligand binding assay of CXCR1 WT and mutants1.
Figure 4.
CXCR1 mutants coupled to Gα15.
(A) Activation of PLCβ2 by CXCR1 through Gα15 proteins. COS-7 cells were cotransfected with equal amounts of cDNA (0.3 µg per well per component) encoding either pSG5, PLCβ2, Gα15, CXCR1 wild type (WT), Gα15 plus WT, or PLCβ2 plus WT. Inositol phosphates were measured 1 hours after treatment in the presence (shaded bars) or absence (open bars) of IL-8 (40 nM). Data are mean ± SEM of replicate wells. ** P<0.01, Gα15+WT (+IL-8) vs. Gα15+WT (−IL-8). (B) CXCR1 mutants coupled to Gα15. Basal and IL-8-stimulated inositol phosphate (IP) accumulation in COS-7 cells transiently co-transfected with WT (0.3 µg) or mutant CXCR1 (0.3 µg) and Gα15 (0.3 µg). Data for mutants are summarized from 3–7 experiments, each performed in triplicate, and are expressed as a percentage of WT CXCR1 baseline determined in parallel. The results are mean ± SEM. ** P<0.01, vs. WT+ Gα15 (in the absence of IL-8).
Figure 5.
Constitutively active mutants of CXCR1.
(A) Basal and IL-8-stimulated inositol phosphate (IP) accumulation in COS-7 cells transiently co-transfected with WT or mutant CXCR1 and Gα15. IP production was determined in detail as described under “Materials and Methods”. Data are mean ± SEM of replicate wells from a representative experiments performed in triplicate. **, P<0.01, vs. WT+ Gα15 (in the absence of IL-8). (B) Calcium signaling capacity in COS-7 cells transfected with CXCR1 WT and V247N mutant. The increase in intracellular calcium concentration ([Ca2+]i) in COS-7 cells transfected with Gα15 and CXCR1 WT or V247N mutant was measured using BD calcium assay kit. The cells were stimulated with or without IL-8 (40 nM). Values represent the mean (± the SEM) increase of [Ca2+]i (n = 4). **, P<0.01, vs. WT+ Gα15 (in the absence of IL-8).
Figure 6.
CXCR1 and mutants coupled to Gαi.
(A) COS-7 cells were cotransfected with equal amounts of cDNA (0.1 µg per well per component) encoding Gαi2, Gβ1, Gγ2, PLCβ2, the WT CXCR1 or its mutants. (B) COS-7 cells were cotransfected with equal amounts of encoding Gαqi5 (qi5) and the WT CXCR1 or its mutants. The release of inositol phosphates, induced by 40 nM IL-8, was measured 1 hour after the treatment in the presence or absence of pertussis toxin (PTX) (100 ng/ml) for 18 hrs. Data are mean ± SEM of replicate wells from a representative experiment. **, P<0.01, vs. WT+ qi5 (in the absence of IL-8).
Figure 7.
V247N mutant constitutively activates Gαi.
(A and B) Surface expression of CXCR1 WT and V247N on COS-7 cells (A) and TSA-201 cells (B). Cells were incubated with mouse anti human CD128a (CXCR1) antibody at 4°C overnight. After washing three times, the cells were incubated with the secondary antibody (DyLight 549-conjugated goat anti-mouse IgG(H+L)) at RT for 1 hr and counterstained with Dapi. Expression of CXCR1 or the mutants was observed using a confocal microscope. The pink color represents the surface expression of CXCR1 WT or mutant with Dapi shown in blue. (C) Inhibition of forskolin-stimulated cAMP production in COS-7 cells. COS-7 cells were transfected with CXCR1 WT or V247N mutant, as well as Gαi2, Gβ1, and Gγ2 expression vector. IL-8 significantly inhibited forskolin-induced stimulation of cAMP formation in transfected cells whereas V247N mutation inhibited forskolin-induced cAMP levels even in the absence of IL-8. Transfected cells were used for determination of cAMP levels in the absence (open bars) or in the presence of IL-8 (closed bars). The results shown are representative. (D) Inhibition of hCG-stimulated cAMP accumulation by V247N mutant. TSA-201 cells were transfected with CXCR1 WT or V247N mutant, as well as LH receptor expression vector. The TSA-201 cells were pretreated with 0.5 mM IBMX for 30 min followed by treatment with hCG (10−7 M) in the presence or absence of IL-8 (40 nM) for another 30 min. Following incubation with hCG in the presence or absence of IL-8, cells were lysed and intracellular cAMP measured using a RIA kit as described in “Experimental Procedures”. Data presented are the mean ± SEM for three assays with each condition performed in triplicate.
Figure 8.
Chemotaxis assay of CXCR1 WT and its constitutively active mutants.
Chemotaxis assays were performed as described in Materials and Methods, using HEK293 cells expressing constitutively active mutants (V247A and V247N) or the WT CXCR1. Data presented are the mean ± SEM for three assays with each condition performed in triplicate. *, P<0.05, V247A vs. WT (+IL-8). **, P<0.01, V247N vs. WT (+IL-8).