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Originally published In Press as doi:10.1074/jbc.M610289200 on January 4, 2007

J. Biol. Chem., Vol. 282, Issue 9, 6906-6915, March 2, 2007
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CXCR1 and CXCR2 Activation and Regulation

ROLE OF ASPARTATE 199 OF THE SECOND EXTRACELLULAR LOOP OF CXCR2 IN CXCL8-MEDIATED RAPID RECEPTOR INTERNALIZATION*

Mohd W. Nasser{ddagger}, Sandeep K. Raghuwanshi{ddagger}, Kimberly M. Malloy{ddagger}, Pavani Gangavarapu§, Joong-Youn Shim{ddagger}, Krishna Rajarathnam§, and Ricardo M. Richardson{ddagger}1

From the {ddagger}Julius L. Chambers Biomedical/Biotechnology Research Institute and the Department of Biology, North Carolina Central University, Durham, North Carolina 27707 and the §Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas 77555-1055

Received for publication, November 3, 2006 , and in revised form, December 26, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
CXCL8 (interleukin-8) interacts with two receptors, CXCR1 and CXCR2, to activate leukocytes. Upon activation, CXCR2 internalizes very rapidly relative to CXCR1 (~90% versus ~10% after 5 min). The C termini of the receptors have been shown to be necessary for internalization but are not sufficient to explain the distinct kinetics of down-regulation. To determine the structural determinant(s) that modulate receptor internalization, various chimeric and point mutant receptors were generated by progressively exchanging specific domains or amino acids between CXCR1 and CXCR2. The receptors were stably expressed in rat basophilic leukemia 2H3 cells and characterized for receptor binding, intracellular Ca2+ mobilization, phosphoinositide hydrolysis, phosphorylation, internalization, and MAPK activation. The data herein indicate that the second extracellular loop (2ECL) of the receptors is critical for the distinct rate of internalization. Replacing the 2ECL of CXCR2 with that of CXCR1 (B2ECLA) or Asp199 with its CXCR1 valine counterpart (BD199VA) delayed CXCR2 internalization similarly to CXCR1. Replacing Asp199 with Asn (BD199N) restored CXCR2 rapid internalization. Structure modeling of the 2ECL of the receptors also suggested that Asp199 plays a critical role in stabilizing and modulating CXCR2 rapid internalization relative to CXCR1. BD199N internalized rapidly but migrated as a single phosphorylated form like CXCR1 (~75 kDa), whereas B2ECLA and BD199VA showed slow and fast migrating forms like CXCR2 (~45 and ~65 kDa, respectively) but internalized like CXCR1. These data further undermine the role of receptor oligomerization in CXCL8 receptor internalization. Like CXCR1, BD199VA also induced sustained ERK activation and cross-desensitized Ca2+ mobilization to CCR5 relative to BD199N and CXCR2. Altogether, the data suggest that the 2ECL of the CXCL8 receptors is important in modulating their distinct rate of down-regulation and thereby signal length and post-internalization activities.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemokines are a family of structurally related peptides that regulate inflammation through cell-surface G protein-coupled receptors on leukocytes. These peptides mediate diverse biological and biochemical activities, including endothelial adhesion, directed migration, and activation of cytotoxic activities such as the respiratory burst and exocytosis (1, 2). Chemokines have been classified into four families (C, CC, CXC, and CX3C) based on the number and positions of the N-terminally conserved cysteine residues. Most chemokines activate more than one chemokine receptor, and many chemokine receptors are activated by multiple chemokines (3). CXCL8 activates two receptors, CXCR1 and CXCR2. CXCR1 is specific for CXCL8, whereas CXCR2 also interacts with CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, and CXCL7 (4). Upon activation, both receptors couple to pertussis toxin-sensitive G protein to mediate phosphoinositide (PI)2 hydrolysis, intracellular Ca2+ mobilization, chemotaxis, and exocytosis. CXCR1 (but not CXCR2) activates phospholipase D and mediates respiratory burst, suggesting that the two receptors may play different physiological roles (5, 6). Like many G protein-coupled receptors, both receptors become phosphorylated, desensitized, and internalized upon exposure to CXCL8. Over 95% of CXCR2 internalizes in the first 2-5 min of activation compared with ~10% of CXCR1 (7-10). CXCR2 also recovers more slowly (~35% after 90 min) to the cell surface than does CXCR1 (~100% after 90 min) upon removal of CXCL8 (7, 8, 11-14). This difference in receptor trafficking appears to be an important factor in the distinct ability of CXCR1 and CXCR2 to mediate leukocyte activation and regulation in response to CXCL8 (9, 10).

To date, the molecular basis for the differential regulation of the CXCL8 receptors remains unclear. CXCR1 and CXCR2 are highly homologous (77%) (15, 16). The most divergent regions are the N terminus, the fourth transmembrane domain (TMD), the second extracellular loop (ECL), and the C terminus (15-18). Although both receptors internalize via a phosphorylation/arrestin/dynamin-dependent mechanism, CXCR2 has been also shown to internalize via a phosphorylation-independent mechanism (19). This partial internalization of the receptors requires the interaction of the C-terminal dileucine motif LLKIL with scaffold proteins such as AP-2 (adaptor protein-2) (20). A previous study using phosphorylation-deficient and C-terminal deletion mutants of CXCR1 and CXCR2 indicates that the cytoplasmic tails of the receptors are necessary for receptor phosphorylation and subsequent arrestin binding but are not sufficient to account for the differences in receptor internalization and recycling (10). Recent studies in our laboratory (10, 14) and others (8, 13, 21, 22) also demonstrated that chimeric receptors in which the C terminus of CXCR1 is exchanged for that of CXCR2 and vice versa mediate cellular responses and internalize as well as the wild-type (WT) receptors. This suggests that domains other than the C termini modulate the rates of CXCR1 and CXCR2 trafficking in response to CXCL8.

In this work, we sought to determine the structural determinants of CXCR2 involved in rapid receptor internalization relative to CXCR1. To this end, chimeric and point mutant receptors with different regions or specific amino acids of CXCR2 replaced with their CXCR1 counterparts were generated and expressed in rat basophilic leukemia (RBL) 2H3 cells. The receptors were characterized for their pharmacological and physiological properties as well as their ability to undergo receptor internalization, phosphorylation, and desensitization in response to CXCL8 occupancy. The data herein indicate that the 2ECL of CXCR2 is critical for its rapid internalization relative to CXCR1. Point mutants and computational modeling studies further identified Asp199 of the 2ECL as a key modulator of CXCR2 rapid internalization.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—[32P]Orthophosphate (8500-9120 Ci/mmol), myo[2-3H]inositol (24.4 Ci/mmol), and 125I-labeled CXCL8 were purchased from PerkinElmer Life Sciences. CXCL8/interleukin-8 was prepared as described previously (23). Growth-regulated oncogene-{alpha} (CXCL1) was purchased from Pepro-Tech. Geneticin (G418) and all tissue culture reagents were purchased from Invitrogen. Monoclonal antibody 12CA5, protein G-agarose, and protease inhibitors were purchased from Roche Applied Science. Anti-human interleukin-8 receptor A (CXCR1) and interleukin-8 receptor B (CXCR2) antibodies were purchased from PharMigen. Phorbol 12-myristate 13-acetate was purchased from Sigma. All other reagents are from commercial sources. cDNAs encoding the chimeric mutants AB1, BA1, ABA, and BAB were generous gifts from Dr. Philip M. Murphy (National Institutes of Health). "A" refers to CXCR1, and "B" refers CXCR2. To facilitate the interpretation of the data, A and B were also employed throughout this work to designate the chimeric and point mutant receptors. The chimeras were excised from pCEP4 using NotI and XhoI and subcloned into the expression vector pcDNA3.

Construction of Chimeric and Point Mutants of CXCR1 and CXCR2—The CXCR1 and CXCR2 cDNAs possess a unique conserved BamHI restriction site located at the junction of the second ECL (2ECL) and 5TMD. This site is also conserved in chimeric mutants ABA and BAB and was used to generate the chimeric receptors (17). A4TMDB and B4TMDA were generated by PCR using ABA and BAB as templates, T7 pcDNA3 as the forward primer, and two reverse primers corresponding to the 2ECL of CXCR1 (A2ECL;5'-GGGCAGGATCCGTAACACCATCCGCCATTTTGCTGTGTCGTTGCCCAGGACCTCATAGCAAACTGGGCTGGAATTGTTTGGATGGACGGTCCTTCG-3') and that of CXCR2 (B2ECL;5'-AGGCAGGATCCGCAACAGCATCCGCCAGTTTGCTGTATTATTTCCCATGTCCTCATAGCAGGCTGGACTAACATTGGATGAGTAGTAAGCCTGGCG-3'). The BamHI site is underlined. For the generation of A2ECLB and B2ECLA, the same reverse primers were employed using CXCR1 (B2ECL) and CXCR2 (A2ECL) as templates, respectively. The resulting PCR products were digested with NotI and BamHI and ligated into pcDNA3-CXCR1 (A4TMDB and A2ECLB) or pcDNA3-CXCR2 (B4TMDA and B2ECLA) digested with the same restriction enzymes. Point mutations were introduced into pcDNA3-CXCR1 or pcDNA3-CXCR2 constructs using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. Forward and reverse primers were designed with single base changes to incorporate amino acid point mutations. The identity of all chimeric and point mutant constructs and the fidelity of all PCR-based coding sequences were verified by sequencing.

Cell Culture and Transfection—RBL-2H3 cells were maintained as monolayer cultures in Dulbecco's modified Eagle's medium supplemented with 15% heat-inactivated fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, and 100 mg/ml streptomycin (24). RBL-2H3 cells (1 x 107) were transfected by electroporation with 20 µg of pcDNA3 containing the receptor cDNAs, and Geneticin-resistant cells were cloned into single cells by fluorescence-activated cell sorter (FACS) analysis. The levels of protein expression were monitored by FACS analysis.

FACS Analysis—For flow cytometric analysis, RBL cells were detached by EDTA treatment, washed with HEPES-buffered Hanks' balanced salt solution, and resuspended in the same medium. Cells (1-5 x 106) were incubated with anti-CXCR1 or anti-CXCR2 antibody (1 µg/ml) in a total volume of 400 µl of HEPES-buffered Hanks' balanced salt solution for 60 min at 4 °C. The cells were then washed and incubated with fluorescein isothiocyanate-conjugated anti-mouse IgG for 60 min at 4 °C. The cells were washed again and analyzed for cell-surface expression of the receptor on a BD Biosciences FACScan cytometer (10).

Radioligand Binding Assays and Receptor Internalization—Radioligand binding assays were carried out as described previously (10). Briefly, RBL-2H3 cells were subcultured overnight in 24-well plates (0.5 x 106 cells/well) in growth medium. Cells were then rinsed with Dulbecco's modified Eagle's medium supplemented with 20 mM HEPES (pH 7.4) and 10 mg/ml bovine serum albumin and incubated on ice for 2-4 h in the same medium (250 µl) containing 125I-labeled CXCL8 (0.1-1 nM). Reactions were stopped with 1 ml of ice-cold phosphate-buffered saline (PBS) containing 10 mg/ml bovine serum albumin and washed three times with the same buffer. The cells were then solubilized with radioimmune precipitation assay buffer (200 µl) and dried under vacuum, and bound radioactivity was counted (25). Bound nonspecific radioactivity was determined in the presence of 500 nM unlabeled CXCL8. Kd and Bmax values were determined by GraphPad radioligand binding data analysis. For receptor internalization, cells were incubated with ligand for 0-60 min at 37 °C and washed with ice-cold PBS, and 125I-labeled CXCL8 (0.1-1 nM) binding was carried out as described above.

PI Hydrolysis and Calcium Measurement—RBL-2H3 cells were subcultured overnight in 96-well culture plates (50,000 cells/well) in inositol-free medium supplemented with 10% dialyzed fetal bovine serum and 1 µCi/ml [3H]inositol. Cells were then washed with HEPES-buffered saline supplemented with 100 mM LiCl2 in the presence of 0.1% bovine serum albumin and preincubated with 50 µl of the same buffer for 10 min at 37 °C. Cells were stimulated with 100 nM CXCL8 or CXCL1. Reactions were terminated 10 min later by addition of 200 µl of chloroform, methanol, and 4 N HCl (100:200:2); 75 µl of 0.1 N HCl; and 75 µl of chloroform. Total [3H]inositol phosphates in the aqueous phase were separated on columns of Dowex formate (24, 26). For calcium mobilization, cells (5 x 106) were washed with HEPES-buffered saline and loaded with 1 µM indo-1 acetoxymethyl ester in the presence of 1 µM pluronic acid for 30 min at room temperature. The cells were then washed with HEPES and resuspended in 1.5 ml of Siraganian buffer. Intracellular calcium increase in the presence or absence of ligands was measured as described previously (27).


Figure 1
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  		FIGURE 1.
Schematic representation and expression of WT CXCR1 and CXCR2 and the chimeric receptor mutants AB1, BA1, ABA, and BAB. A, gray traces represent CXCR1, and black traces represent CXCR2. B, FACS analysis of surface expression of WT and chimeric receptors in RBL-2H3 cells after staining with anti-CXCR1 (CXCR1, AB1, and ABA) or anti-CXCR2 (CXCR2, BA1, and BAB) antibody. C, competitive binding of 125I-labeled CXCL8 to CXCR1, CXCR2, AB1, BA1, ABA, and BAB in the presence of different concentrations of CXCL8 (0-100 nM). Experiments are representative of three independent determinations.

 
Phosphorylation of Receptors—Phosphorylation of receptors was performed as described previously (10, 25, 28). RBL-2H3 cells (5 x 106) expressing the receptors were incubated with [32P]orthophosphate (150 µCi/dish) for 90 min. The labeled cells were then stimulated with the indicated ligands for 5 min at 37 °C. Cells were washed with ice-cold PBS and solubilized in 1 ml of radioimmune precipitation assay buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS. Cell lysates were immunoprecipitated with specific antibodies against the N terminus of either CXCR1 or CXCR2, analyzed by SDS electrophoresis, and visualized by autoradiography.

ERK Activity—For ERK activity, RBL-2H3 cells (5 x 106) expressing the receptors were washed three times with PBS and then resuspended in PBS containing CXCL8 (100 nM) for different periods of time at 37 °C. The reactions were stopped with ice-cold PBS; cells were collected by centrifugation; and membranes were prepared and assayed for protein concentration as described previously (28). Membrane proteins (~50 µg) were resolved by 10% SDS-PAGE, transferred to a nitrocellulose membrane, and probed with antibody against either ERK1/2 or phospho-ERK1/2 (28). Detection was carried out with horseradish peroxidase-conjugated sheep anti-mouse antibody and by ECL.

Homology Modeling of Human CXCR2—Multiple sequence alignment of rhodopsin and human CXCR2 along with human CXCR1 was performed using ClustalW (align.genome.jp) using the BLO-SUM matrix. To prohibit any gap within the TMD core, a gap open penalty of 15.0 and a gap extension penalty of 0.1 were used. The calculated sequence alignment was validated by examining the alignment of the highly conserved amino acid residues, including the NLAXXD motif in TMD2, the (D/E)RY motif in TMD3, the CWXP motif in TMD6, and the NPXXY motif in TMD7 of G protein-coupled receptors (29).

To construct the seven transmembrane helices and the beta3-beta4 hairpin structure in the N-terminal region of the 2ECL of human CXCR2, the C-{alpha} coordinates of the recently published x-ray crystal structure of bovine rhodopsin (Protein Data Bank code 1U19) (30) were used as the template. The intracellular and extracellular loops (i.e. 2ECL, C-terminal region, third intracellular loop, and 3ECL) that are different in length from those of rhodopsin were constructed using the loop search tool implemented in the Insight II Homology module and the C-{alpha} distance matrix from a selected data set of the Protein Data Bank. The typical disulfide linkage between TMD3 and the 2ECL common to most G protein-coupled receptors was inserted between Cys119 and Cys196. After a short minimization, the resulting structure was subjected to a 500-ps molecular dynamics simulation at 300 K, during which the transmembrane backbone atoms and the H-bonds in the 2ECL beta3-beta4 hairpin structure were restrained. The receptor model was sampled by extracting a snapshot of the structure every 1 ps during the simulation. The averaged structure from the last 50 ps was energy-minimized with the restraints mentioned above first and then with the transmembrane backbone constraint only. Overall, three separate simulations were repeated for WT CXCR1 (Val190) and CXCR2 (Asp199) and the mutant forms (i.e. BD199VA, BD199N, and AV190DB). Energy minimization and molecular dynamics simulations were carried out on a Silicon Graphics Origin 350 work station using the consistent force field (31) implemented in Insight II (Version 2005, Accelrys, San Diego, CA). The cell multipole method (32) with a distance-dependent dielectric constant ({epsilon} = {epsilon}0r, with {epsilon}0 = 4.0) was used for summation of nonbonding interactions.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of CXCR1 and CXCR2 Chimeric Mutants in RBL-2H3 Cells—To determine the structural determinants involved in the rapid internalization of CXCR2 relative to CXCR1, four chimeric receptors with the N terminus (AB1 and BA1) or the region comprising the 4TMD and 2ECL (ABA and BAB) of CXCR1 exchanged for those of CXCR2 and vice versa were stably expressed in RBL-2H3 cells (Fig. 1, A and B). Competition binding of 125I-labeled CXCL8 to the chimeric receptors in the presence of unlabeled CXCL8 and Scatchard analysis demonstrated that AB1 and ABA (but not BA1 and BAB) bound CXCL8 with similar affinities compared with CXCR1 and CXCR2, respectively (Fig. 1C and Table 1). However, all receptors stimulated a rapid and transient increase in free intracellular Ca2+ mobilization and PI hydrolysis in response to both CXCL8 and CXCL1 (Fig. 2, A-C). Except for CXCR2, CXCL1 induced significantly lower PI hydrolysis relative to CXCL8 (Fig. 2C). No significant difference was found in CXCL8-induced PI hydrolysis mediated by AB1, ABA, and BA1 relative to CXCR1 and CXCR2 (3.5-4-fold over the basal level) (Fig. 2C). BAB-mediated PI hydrolysis in response to CXCL8 was significantly lower (~2.5-fold over the basal level) compared with WT receptor-mediated PI hydrolysis. Because BAB displayed no ligand binding properties (Table 1), it is difficult to determine whether this difference is due to a decreased affinity of the chimeric receptor for CXCL8 or an impaired ability to couple to Gi to activate phospholipase Cbeta.


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  		TABLE 1
Ligand binding affinity of WT and chimeric CXCR1 and CXCR2 expressed in RBL-2H3 cells

Data are the 125I-labeled CXCL8-binding sites/cell and the apparent binding affinity (Kd) values for WT and mutant receptors. ND, not determined.

 


Figure 2
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  		FIGURE 2.
Functional characterization of the WT and chimeric receptors. For intracellular Ca2+ mobilization, RBL-2H3 cells (5 x 106) stably expressing CXCR1, AB1, or ABA (A) or CXCR2, BA1, or BAB (B) were loaded with indo-1, and Ca2+ mobilization mediated by CXCL8 and CXCL1 (100 nM) was measured. For the generation of [3H]inositol phosphates, RBL-2H3 cells (50,000/well) were cultured overnight in the presence of myo[2-3H]inositol (1 µCi/ml) (C). Cells were preincubated for 10 min at 37 °C with HEPES-buffered saline containing 10 mM LiCl in a total volume of 200 µl and stimulated with CXCL8 or CXCL1 (100 nM) for 10 min. The supernatant was used to determine the release of [3H]inositol phosphates. Data are represented as the -fold stimulation over the basal level. The experiment was repeated three times with similar results. For CXCL8-mediated internalization, cells were treated with CXCL8 (100 nM) at different times, washed, and assayed for 125I-labeled CXCL8 (0.1 nM) binding (D). The values are presented as a percentage of the total, which is defined as the total amount of 125I-labeled CXCL8 bound to control (untreated) cells. The experiment was repeated five times with similar results.

 
We next measured the rate of internalization of the chimeric receptors upon CXCL8 (100 nM) pretreatment. CXCL8 induced time-dependent internalization of all four receptors (Fig. 2D). AB1 and ABA internalized as rapidly as CXCR2 (~90% after 5-10 min), whereas BA1 and BAB (~35% after 60 min) internalized far more slowly compared with CXCR1 (~55% after 60 min).

Role of the 4TMD and 2ECL of CXCR1 and CXCR2 in Receptor Internalization—To further delineate the region(s) of CXCR1 and CXCR2 involved in receptor internalization, four additional chimeras were constructed (Fig. 3A) by exchanging either the 4TMD (A4TMDB and B4TMDA) or the 2ECL (A2ECLB and B2ECLA). Except for B4TMDA, all chimeras were stably expressed in RBL-2H3 cells (Fig. 3B) and characterized. A4TMDB and B2ECLA (but not A2ECLB) bound CXCL8 with similar affinities compared with the WT receptors (Fig. 3C and Table 1). A4TMDB and B2ECLA mediated intracellular Ca2+ mobilization and PI hydrolysis in response to both CXCL8 and CXCL1 (Fig. 4, A and B). Like CXCR1, A4TMDB mediated greater responses to CXCL8, whereas like CXCR2, B2ECLA responded to both ligands with similar potency. Upon CXCL8 pretreatment, A4TMDB internalized as well as CXCR1 (~55% after 60 min) (Fig. 4C). Interestingly, B2ECLA also showed a slower time course of internalization (~50% after 60 min) (Fig. 4C).

Role of Receptor Glycosylation in CXCR2 Internalization—CXCR1 and CXCR2 express two N-glycosylation motifs in the 2ECL (Fig. 5A, boxed). Because the motifs are different between the two receptors, we constructed four chimeras in which the glycosylation sites of CXCR2 were converted to those of CXCR1 (BNNSSA and BN203DA) and vice versa (ASNVSB and AD194NB) (Fig. 5A). The mutants were stably expressed in RBL-2H3 cells and characterized. Except for ASNVSB, all receptor variants bound CXCL1 and CXCL8 to induce intracellular Ca2+ mobilization (Fig. 5B). Upon CXCL8 pretreatment, BNNSSA and BN203DA internalized like CXCR2, whereas AD194NB internalized like CXCR1 (Fig. 5C).

Identification of the Specific Residue(s) of the 2ECL Involved in Receptor Internalization—To identify the specific residue(s) of CXCR2 involved in rapid receptor internalization relative to CXCR1, we replaced all divergent amino acids in the 2ECL of CXCR2 with their CXCR1 counterparts and vice versa (Table 2). Except for BY188HA, all receptors were stably expressed in RBL-2H3 cells (Table 2). Except for AV190DB, the expressed receptors bound CXCL8 to induce intracellular Ca2+ mobilization similarly to the WT receptors. We next measured receptor internalization upon exposure to CXCL8 (60 min). As shown in Fig. 6A, the CXCR1 mutants AH179YB, AS183VB, AL191MB, and AV201LB internalized like CXCR1 (~55% after 60 min), whereas AP180SB (~40%), AV186AB (~40%), and AK197NB (~50%) were slightly more resistant to CXCL8-mediated internalization compared with CXCR1. All of the CXCR2 mutants internalized as rapidly as CXCR2, except for BD199VA, which displayed delayed internalization similarly to CXCR1 (~55% after 60 min) (Fig. 6B).


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  		TABLE 2
Expression and functional characterization of the 2ECL mutants of CXCR1 and CXCR2 expressed in RBL-2H3 cells

 


Figure 3
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  		FIGURE 3.
Schematic illustration and surface expression of the 2ECL (A2ECLB and B2ECLA) and 4TMD (A4TMDB and B4TMDA) chimeric mutants of CXCR1 and CXCR2. A, gray traces represent CXCR1, and black traces represent CXCR2. B, FACS analysis of surface expression of the chimeric receptors in RBL-2H3 cells after staining with anti-CXCR1 (A4TMDB and A2ECLB) or anti-CXCR2 (B4TMDA and B2ECLA) antibody. C, competitive binding of 125I-labeled CXCL8 to A2ECLB, A4TMDB, and B2ECLA in the presence of different concentrations of CXCL8 (0-100 nM). The data shown are representative of one of three experiments performed in triplicate.

 


Figure 4
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  		FIGURE 4.
Functional characterization and internalization of A2ECLB, A4TMDB, and B2ECLA. CXCL8- and CXCL1-induced intracellular Ca2+ mobilization (A), PI hydrolysis (B), and receptor internalization (C) were measured as described in the legend of Fig. 2. The experiments were repeated three times with similar results. 125IL-8, 125I-CXCL8.

 


Figure 5
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  		FIGURE 5.
Mutation and characterization of the glycosylation motifs of the 2ECL of CXCR1 and CXCR2. A, shown are the amino acid sequences of the 2ECL of CXCR1 and CXCR2 and the chimeric mutants with the glycosylation motifs (boxed) of CXCR1 replaced with those of CXCR2 (ASNVSB and AD194NB) and vice versa (BNNSSA and BN203DA). B and C, CXCL8- and CXCL1-induced intracellular Ca2+ mobilization and receptor internalization, respectively, were measured as described in the legend of Fig. 2. The experiments were repeated three times. 125IL-8, 125I-CXCL8.

 
To further determine the role of Asp199 in CXCR2 rapid internalization, we generated a CXCR2 mutant in which Asp199 was replaced with Asn (BD199N). BD199N was stably expressed in RBL-2H3 cells and was shown to bind CXCL8 as well as CXCR2 and BD199VA (Fig. 7A) to induce intracellular Ca2+ mobilization and internalization (Fig. 7B) (data not shown). BD199N internalization was identical to CXCR2 internalization, whereas BD199VA internalized like CXCR1 (Fig. 7B).

Role of Receptor Phosphorylation and Dimerization in CXCL8-mediated Receptor Internalization—It was reported previously that phosphorylated CXCR1 migrates as a single monomeric band of ~75 kDa, whereas CXCR2 shows two phosphorylated forms: a slow dimeric form (~65 kDa) and a fast monomeric form (~45 kDa) (10, 33-35). To assess the role of receptor phosphorylation and dimer formation in CXCL8-induced receptor internalization, 32P-labeled cells expressing CXCR1, CXCR2, ABA, BAB, A4TMDB, B2ECLA, BD199VA, and BD199N were stimulated with either CXCL8 (100 nM) or phorbol 12-myristate 13-acetate (100 nM) for 5 min. The cell lysates were immunoprecipitated with specific antibody directed against the N terminus of either CXCR1 (CXCR1, ABA, and A4TMDB) (Fig. 8A) or CXCR2 (CXCR2, B2ECLA, BD199VA, and BD199N) (Fig. 8B). The receptors were homologously phosphorylated by CXCL8 (lanes 2) and heterologously phosphorylated by phorbol 12-myristate 13-acetate (lanes 3). Phosphorylation by phorbol 12-myristate 13-acetate (lanes 3) was less compared with that by CXCL8 (lanes 2). CXCR2, ABA, A4TMDB, B2ECLA, and BD199VA displayed two forms of phosphorylated receptors (Fig. 8, A and B). The electro-phoretic mobilities of the two forms of A4TMDB (~70 and ~50 kDa) and B2ECLA and BD199V A(~70 and ~45 kDa) were slightly different from those of CXCR2 and ABA (~65 and ~45 kDa). BD199VA also showed greater phosphorylation of the dimeric form (~70 kDa) than the monomeric form (~45 kDa) (Fig. 8B). CXCR1 (Fig. 8A) and BD199N (Fig. 8B) migrated as a single phosphorylated band (~75 kDa).


Figure 6
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  		FIGURE 6.
CXCL8-mediated internalization of 2ECL point mutants of CXCR1 and CXCR2. RBL-2H3 cells (5 x 105/well) stably expressing CXCR1, CXCR2, or mutant receptors with divergent amino acids of the 2ECL of CXCR1 replaced with their CXCR2 counterparts (A) and vice versa (B) were treated with CXCL8 (100 nM) for 60 min, washed, and assayed for 125I-labeled CXCL8 binding. The values are presented as a percentage of the total, which is defined as the total amount of 125I-CXCL8 bound to control (untreated) cells. The data shown are representative of one of three experiments performed in triplicate. 125IL-8, 125I-CXCL8.

 


Figure 7
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  		FIGURE 7.
Expression and characterization of the BD199VA and BD199V mutants. A, FACS analysis of surface expression of CXCR2, BD199VA, and BD199N in RBL-2H3 cells after staining with anti-CXCR2 antibody. Inset, Kd values for BD199VA and BD199N. B, time course of CXCL8-induced receptor internalization determined as described in the legend of Fig. 2. The experiment was repeated four times with similar results. FITC, fluorescein isothiocyanate.

 


Figure 8
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  		FIGURE 8.
Phosphorylation of CXCR1, CXCR2, ABA, A4TMDB, B2ECLA, BD199VA, and BD199N. 32P-Labeled RBL-2H3 cells (5 x 106/60-mm plate) expressing CXCR1, ABA, or A4TMDB(A) or CXCR2, B2ECLA, BD199VA, or BD199N (B) were incubated for 5 min with or without stimulants as shown. Cells were lysed, immunoprecipitated with anti-CXCR1 (A) or anti-CXCR2 (B) antibody, and analyzed by SDS-PAGE and autoradiography. The results are from a representative experiment repeated three times. PMA, phorbol 12-myristate 13-acetate.

 


Figure 9
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  		FIGURE 9.
CXCR1-, CXCR2-, BD199VA-, and BD199N-induced ERK1/2 phosphorylation. RBL-2H3 cells stably expressing CXCR1 (A), CXCR2 (B), BD199VA (C), or BD199N (D) were stimulated with CXCL8 (100 nM) for 0-20 min. ERK1/2 phosphorylation and total ERK were determined by Western blotting using anti-phospho-ERK1/2 (pERK1/2) and anti-total ERK1/2 (tERK1/2) antibodies, respectively.

 
CXCR1-, CXCR2-, BD199VA-, and BD199N-mediated ERK Activation—CXCR1 and CXCR2 have been shown to mediate MAPK activation to different extents based on the cell lines used (36-38). To assess the ability of BD199VA and BD199N to activate MAPK in RBL-2H3 cells, we measured ERK1/2 phosphorylation in response to CXCL8. As shown in Fig. 9, CXCR1, CXCR2, BD199VA, and BD199N induced time-dependent phosphorylation of ERK1/2 upon activation by CXCL8 (100 nM). Maximum response was obtained at ~5 min. CXCR1 and BD199VA induced ERK1/2 phosphorylation to a greater extent relative to CXCR2 and BD199N (Fig. 9B).

Desensitization of BD199VA- and BD199N-mediated Intracellular Ca2+ Mobilization—We next studied the ability of BD199VA and BD199N to mediate and undergo desensitization by monitoring for CXCL8- and CXCL1-induced intracellular Ca2+ mobilization. As shown in Table 3, BD199VA-, BD199N-, CXCR1-, or CXCR2-mediated Ca2+ mobilization was desensitized by pretreatment of the cells with a first dose of CXCL8 or CXCL1 (100 nM). Homologous desensitization by CXCL8 was more potent (85-100%) than that by CXCL1 (47-84%).


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  		TABLE 3
Homologous desensitization of Ca2+ mobilization elicited by a first dose of CXCL8

RBL-2H3 cells (5 x 106/assay) expressing WT or mutant CXCR1 and CXCR2 were loaded with indo-1 and stimulated with CXCL8 or CXCL1 (100 nM). Cells were re-challenged 3 min later with a second dose of CXCL8 (100 nM), and peak intracellular Ca2+ mobilization was determined. Data are the means ± S.E. of three different experiments.

 
CXCR1 (but not CXCR2) has been shown to mediate receptor cross-desensitization in response to CXCL8 activation (14, 25, 27). To determine whether BD199VA and BD199N mediate cross-regulatory signals, RBL-2H3 cells stably coexpressing the RANTES (regulated on activation normal T cell expressed and secreted; CCL5) receptor CCR5 along with BD199VA (RBL-CCR5/BD199VA), BD199N (RBL-CCR5/BD199N), CXCR1 (RBL-CCR5/CXCR1), or CXCR2 (RBL-CCR5/CXCR2) were generated. CXCL8- and CCL5-mediated cross-desensitization of Ca2+ mobilization was measured. As shown in Table 4, pre-treatment of RBL-CCR5/CXCR1 or RBL-CCR5/BD199VA cells with a first dose of CXCL8 (100 nM) cross-desensitized CCL5 (100 nM)-induced Ca2+ mobilization by 56 and 41%, respectively. CCL5-mediated Ca2+ mobilization was resistant to cross-desensitization by CXCL8 in RBL-CCR5/CXCR2 (2%) and RBL-CCR5/BD199N (0%) cells. CCL5 activation of CCR5 cross-desensitized CXCL8-induced intracellular Ca2+ mobilization in all four cell lines (Table 4).


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  		TABLE 4
BD199VA- and BD199N-mediated cross-desensitization of intracellular Ca2+ mobilization elicited by CCR5

RBL-CCR5/CXCR1, RBL-CCR5/CXCR2, RBL-CCR5/BD199VA, or RBL-CCR5/BD199N cells (5 x 106/assay) were loaded with indo-1 acetoxymethyl ester and stimulated with a first dose of CXCL8 or CCL5 (100 nM). Cells were re-challenged 3 min later with a second dose of either CXCL8 or CCL5 (100 nM), and peak intracellular Ca2+ mobilization was determined. Values for cross-desensitization are represented as a percentage of inhibition of the Ca2+ response elicited in the absence (first dose) of pretreatment. Data are the means ± S.E. of three different experiments.

 
Computational Modeling of the 2ECL of CXCR1, CXCR2, BD199VA, and BD199N—Multiple sequence alignment and homology models of CXCR1 and CXCR2 were constructed based on the recent x-ray structure of rhodopsin (30). Molecular dynamics simulations of the ECL regions of WT CXCR1 and CXCR2 and mutants BD199VA, BD199N, and AV190DB were performed. The molecular interaction involving Asp199 of CXCR2 was measured by the H-bond formation with the neighboring residues (Table 5). As shown in Fig. 10B, Asp199 of CXCR2 forms H-bonds with Arg185, Thr186, and Arg208. The H-bonding network is similar to the one between the equivalent Asp190 of rhodopsin and Arg177, Tyr192, and Thr193 (Fig. 10A). When Asp199 was replaced with Val (BD199VA), the neighboring polar or charged residues (Arg185, Thr186, Tyr188, Met200, Asn202, and Arg208) could not interact with it to form the H-bonding network because of the highly hydrophobic nature of Val (Fig. 10C). In contrast, when Asp199 was replaced with Asn (BD199N), an H-bond could be formed with Thr186 and Arg185, but not with Arg208 (Fig. 10D). Apparently, the replacement was sufficient to regenerate the H-bonding network and to stabilize the receptor in a CXCR2-like conformation, thereby restoring rapid receptor internalization.


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  		TABLE 5
Comparison of H-bonding interactions for rhodopsin (Asp190), CXCR2 (Asp199), mutant BD199N (Asn199), and mutant AV190DB (Asp190)

H-bond acceptor and donor atoms are indicated by their positions from C-{alpha}. For example, Asp190 O-2{delta} indicates the second oxygen atom of the side chain carboxylate in Asp190.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
CXCL8 and its receptors CXCR1 and CXCR2 play a critical role in the pathophysiology of several inflammatory diseases, including multiple sclerosis, rheumatoid arthritis, and asthma. Upon activation, these receptors mediate similar as well as different cellular functions. This distinction correlates with the receptors' differing kinetics of internalization and recycling upon activation by CXCL8 (10). CXCR1 internalizes more slowly (50-60% after 60 min) and mediates leukocyte cytotoxicity (i.e. respiratory burst) and receptor cross-regulatory signals (6, 25). In contrast, CXCR2 internalizes rapidly (90-95% after 2-5 min) but mediates greater post-internalization signals (i.e. MAPK and protein kinase B (Akt) activation) (37). Attempts to determine the structural determinants responsible for the different kinetics of receptor down-regulation have focused predominantly on the intracellular domains of the receptor (13, 22). A recent study using tail-deleted and chimeric receptors in which the tail of CXCR1 was exchanged for that of CXCR2 and vice versa has shown that the C termini are important for receptor phosphorylation, desensitization, adaptor protein binding, and down-regulation but are not sufficient to explain the distinct kinetics of internalization of the two receptors in response to CXCL8 interaction (10). In this study, we employed a set of new (A4TMDB, B4TMDA, A2ECLB, and B2ECLA) and previously published (ABA, BAB, AB1, and BA1) (17) chimeric and point mutant receptors to further analyze domains upstream of the C termini of the two receptors that may modulate receptor down-regulation. The data herein indicate that the 2ECL modulates rapid internalization of CXCR2 relative to CXCR1. This contention is based on the following observations. First, the CXCR1-derived chimera ABA, expressing the 2ECL and 4TMD of CXCR2, internalized like CXCR2 (~90% in the first 5 min of activation) (Fig. 2D). Second, the CXCR2 chimera B2ECLA, expressing the 2ECL of CXCR1, internalized like CXCR1 (~50% after 60 min) (Fig. 4C). Third, the point mutant receptor BD199VA, with Asp199 of the 2ECL of CXCR2 replaced with the Val counterpart of CXCR1, showed decreased CXCL8-mediated internalization compared with CXCR2 (~45% versus 95% after 60 min) (Figs. 6B and 7B).


Figure 10
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  		FIGURE 10.
Computational modeling of the 2ECL of rhodopsin, CXCR2, CXCR1, and the receptor mutants BD199VA, BD199N, and AV190DB. Shown are receptor interactions involving Asp190 of rhodopsin (A) and its corresponding residues Asp199 of the 2ECL of CXCR2 (B), Val190 of CXCR1 (C), Val199 of BD199VA(D), Asn199 of BD199N (E), and Asp190 of AV190DB(F). The protein secondary structural elements are displayed as follows: {alpha}-helix, white; beta-sheet, yellow; turn, cyan; and random coil, purple. The color coding of receptor residue atoms is as follows: green, carbon; red, orange; and blue, nitrogen. H-bond-forming residues (in stick depiction) in association with Asp190 in rhodopsin (Tyr192, Thr193, and Arg177) (A), Asp199 in CXCR2 (Thr186, Arg208, and Arg185) (B), Asn199 in BD199N (Thr186 and Arg185) (E), and Asp190 in AV190DB (Arg199) (F) are shown by white dotted lines. No H-bond was formed in CXCR1 (C) and BD199VA(D). The residues within 3.0 Å of Val190 (CXCR1) or Val199 (BD199VA) are represented. TM, transmembrane domain.

 
Interestingly, when Asp199 was replaced with Asn instead of Val (BD199N), rapid internalization of CXCR2 (~90% after 5 min) was restored (Fig. 7B). These results further suggest that Asp199 of the 2ECL of CXCR2 plays a critical role in stabilizing the receptor in the conformation necessary for its rapid internalization upon binding to CXCL8. Homology modeling of the 2ECL of CXCR2 based on the crystal structure of rhodopsin also supports this contention. As shown in Fig. 10B, Asp199 may interact with Thr186, Arg185, and Arg208 to form an H-bonding network similar to the one formed between Asp190 and Arg177, Tyr192, and Thr193 of rhodopsin (Fig. 10A). When Asp199 was replaced with the corresponding hydrophobic Val of CXCR1, BD199VA (Fig. 10C), no H-bond could be formed, causing a conformational change in the 2ECL similar to that in CXCR1 (Fig. 10D). In contrast, the replacement of Asp199 with Asn, BD199N (Fig. 10E), partially restored the H-bounding network with Thr186 and R185, thereby the rapid kinetics of receptor internalization (Fig. 10D). Despite the differences in H-bonding patterns between CXCR1 and CXCR2, it cannot be ruled out that other factors may contribute to the differences in structure and rate of internalization. These receptors interact with several adaptor proteins (i.e. arrestins, AP-2, Src, and HIP) to mediate signal transduction (10, 19, 20). It could be that conformational changes induced by Asp199 alter receptor-adaptor protein complex formation and thereby receptor down-regulation (39).

A question addressed in this study is the role of receptor glycosylation in the distinct kinetics of CXCR1 and CXCR2 down-regulation. Both receptors express two glycosylation sites with different sequence motifs within the 2ECL (Fig. 5) (16, 17). It was shown previously that glycosylation of CXCR2 does not affect ligand binding, signaling, or trafficking abilities (40). Because replacing the 2ECL of CXCR2 with that of CXCR1 (B2ECLA) changed the rate of internalization (Fig. 4C), this raised the possibility that CXCR1-like glycosylation could influence a change in the kinetics of internalization. However, when the glycosylation motifs of the 2ECL of CXCR2 were replaced with those of CXCR1 (BNNSSA and BN203DA) or vice versa (AD194NB), no significant differences were found in CXCL8 binding to both receptors or in the rates of receptor internalization relative to the WT receptors (Fig. 5C) (data not shown). These observations suggest that receptor glycosylation plays no role in receptor internalization and further indicate that Asp199 is solely responsible for the rapid internalization of CXCR2.

Receptor oligomerization has been shown to play an important role in the activation and regulation of several chemokine receptors, including CCR2, CCR5, and CXCR4 (41-44). To date, contrasting results exist regarding the dimerization of CXCR1 and CXCR2 and their roles in receptor-mediated cellular functions. Previous studies from our group and others (9, 10, 33, 34) using native and epitope-tagged receptors have shown two migrating forms of CXCR2 (~40 and ~65 kDa) but one form of CXCR1 (~75 kDa). Trettel et al. (35) recently reported that CXCR2 (but not CXCR1) functions as a dimer and could affect the kinetics of receptor down-regulation. Wilson et al. (45) demonstrated, however, that both CXCR1 (~45 and ~75 kDa) and CXCR2 (~40 and ~65 kDa) are capable of forming dimers. In this study, because both CXCR1 and CXCR2 internalize in a phosphorylation-dependent fashion, we used CXCL8-induced receptor phosphorylation to assess the role of receptor dimerization in receptor internalization. The data herein clearly show one form of CXCR1 and further suggest that receptor internalization is independent of receptor dimerization (Figs. 4 and 6, 7 and 8). First, the chimeras B2ECLA and A4TMDB internalized like CXCR1 but displayed slow and fast migrating forms like CXCR2 (Figs. 4C and 8B). Second, BD199N migrated as a single slow form (~75 kDa) similarly to CXCR1 but internalized as rapidly as CXCR2 (~90% after 5 min) (Figs. 7B and 8B). Third, the related mutant BD199VA showed two forms (~75 and ~45 kDa) but internalized like CXCR1 (~45% after 60 min) (Figs. 7B and 8B). The reason for the contrast with the results reported by Wilson et al. (45) is unclear. One explanation could be that native CXCR1 predominantly exists as a dimer and that CXCR2 exists as an equal mixture of both dimer and monomer. Thus, because dimerization of the receptor occurs in the endoplasmic reticulum, it could be that overexpression of the receptors in human embryonic kidney 293 cells affects the rate of dimerization, resulting in two forms of CXCR1 (~75 and ~45 kDa). Against this hypothesis is that Tretell et al. (35) also used the human embryonic kidney 293 expression system in their study.

CXCR1 (but not CXCR2) has been shown to mediate receptor cross-regulatory signals (27). This distinction was attributed to the length of signaling, which, for CXCR2, is shortened by rapid receptor internalization (14). Indeed, mutant BD199VA, which displayed delayed CXCL8-mediated receptor internalization, cross-desensitized CCR5-mediated intracellular Ca2+ mobilization to CCL5 (Table 4). In contrast, BD199N, which internalized as rapidly as CXCR2, failed to cross-desensitize responses to CCL5. Like CXCR1, BD199VA also induced sustained ERK1/2 activation compared with BD199N and CXCR2 (Fig. 9). Sai et al. (46) have reported recently that delayed internalization of IL/AA mutant CXCR2 in HL-60 cells correlates with decreased ERK1/2 activation. These results are in contrast to the ones presented in this work and may well reflect differences between cell lines. Supporting this contention is that CXCR2-expressing mouse embryonic fibroblast cells deficient in beta-arrestin-1/2-/- expression also display decreased receptor internalization and sustained ERK1/2 and stress kinase p38 and JNK (c-Jun N-terminal protein kinase) activation relative to WT mouse embryonic fibroblasts (38). Furthermore, several phosphorylation- and internalization-resistant mutants of CCR5 and the follicle-stimulating hormone receptor expressed in RBL-2H3 and human embryonic kidney 293 cells, respectively, have been also shown to induce sustained ERK1/2 activation relative to the WT receptors (47, 48).

In all, the data herein provide new insights into the role of extracellular domains of the receptors in modulating signal length and ability to mediate a distinct set of cellular responses. A drawback is, however, that key chimeric or point mutants such as A2ECLB and AV190DB expressed but failed to bind or respond to ligand (Figs. 3B and 4A and Table 2). These are common limitations in studies using cellular and genetic approaches to elucidate the complexities of receptor activation and regulation. Despite these limitations, however, this study pointed out the importance of the 2ECL and, more specifically, Asp199 in the distinct ability of CXCR1 and CXCR2 to mediate and regulate inflammatory responses to CXCL8. As chimeras such as BAB and BA1 failed to show ligand binding but mediated functional responses (i.e. Ca2+ mobilization and PI hydrolysis) and underwent CXCL8-dependent internalization, the data further support the notion that distinct domains of the receptors may be involved in ligand interaction and receptor activation/regulation (17).


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants AI-38910 (to R. M. R.) and AI-069152 (to K. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Julius L. Chambers Biomedical/Biotechnology Research Inst., North Carolina Central University, 1801 Fayetteville St., Durham, NC 27707. Tel.: 919-530-6421; Fax: 919-530-7780; E-mail: mrrichardson{at}nccu.edu.

2 The abbreviations used are: PI, phosphoinositide; TMD, transmembrane domain; ECL, extracellular loop; WT, wild-type; RBL, rat basophilic leukemia; FACS, fluorescence-activated cell sorter; PBS, phosphate-buffered saline; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase. Back


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Philip M. Murphy for the generous gift of plasmid cDNAs expressing receptor chimeric mutants. We thank Ernest Johnson and James Crisp for technical support.



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 DISCUSSION
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