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* Department of Biochemistry, Meharry Medical College, Nashville, TN 37214;
Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville TN 37232; and
Departments of Medicine and
Cell Biology, Duke University Medical Center, Durham, NC 27710
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-arrestin 2 (arr-2) translocation. The lack of internalization by this receptor may be due to its reduced association with arr-2 and the adaptor protein-2. The C-tail-deleted and phosphorylation-deficient receptors were resistant to receptor phosphorylation, desensitization, arrestin translocation, and internalization. They also mediated greater phosphoinositide hydrolysis and exocytosis and sustained Ca2+ mobilization, but diminished chemotaxis. These data indicate that phosphorylation of the C-tails of CXCR1 and CXCR2 are required for arrestin translocation and internalization, but are not sufficient to explain the rapid internalization of CXCR2 relative to CXCR1. The data also show that receptor internalization is not required for chemotaxis. The lack of receptor phosphorylation was correlated with greater signal transduction but diminished chemotaxis, indicating that second messenger production, not receptor internalization, negatively regulates chemotaxis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To date, little is known about the mechanisms governing IL-8R activation and regulation. Studies with neutrophils and transfected cell lines have demonstrated that CXCR1 and CXCR2 undergo receptor phosphorylation, desensitization, and internalization upon activation by IL-8 (7, 8, 9, 10, 11). Although both receptors internalize via arrestin/dynamin-dependent mechanisms, CXCR2 internalizes more rapidly (
95% CXCR2 vs 10% CXCR1 in the first 5–10 min) and recovers more slowly (35% CXCR2 vs 100% of CXCR1 after 90 min) at the cell surface than CXCR1 (10, 11, 12, 13, 14, 15, 16). Recent studies in this laboratory have also shown that CXCR1, but not CXCR2, generates signals for receptor cross-phosphorylation and cross-desensitization (cross-regulation) (10). This distinction appears to be determined by the duration of activation of the receptor, which in the case of CXCR2 is terminated rapidly by phosphorylation of specific sites in the carboxyl terminus, followed by receptor internalization (10).
In the present study we sought to determine whether the cytoplasmic tails of the receptors alone are responsible for the differences in CXCR1- and CXCR2-induced leukocyte activation. For this purpose, wild-type, chimeric, phosphorylation-deficient, and carboxyl-terminus deletion mutants of CXCR1 and CXCR2 were produced and expressed in RBL-2H3 cells alone or along with -arrestin-2 (arr-2) and studied for their ability to undergo receptor phosphorylation, desensitization, and internalization. The results indicate that the cytoplasmic tails (C-tails) of the receptors are necessary for receptor phosphorylation and subsequent arrestin binding, but are not sufficient to account for differences in arrestin-mediated internalization and cellular activation. The data also suggest that receptor-mediated second messenger production, rather than receptor internalization, regulates leukocyte chemotaxis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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[32P]Orthophosphate (8500–9120 Ci/mmol), myo-2-[3H]inositol (24.4 Ci/mmol), [
-32]GTP (6000 Ci/mmol), and [125I]IL-8 were purchased from DuPont-NEN (Boston, MA). IL-8 (monocyte derived) and growth-related oncogene 
were purchased from PeproTech (Rocky Hill, NJ). Geneticin (G418) and all tissue culture reagents were purchased from Life Technologies (Gaithersburg, MD). 12CA5 mAb, protein G-agarose, and protease inhibitors were purchased from Roche (Indianapolis, IN). Anti-human IL-8RA (CXCR1) and IL-8RB (CXCR2) Abs were purchased from BD PharMingen (San Diego, CA). Mouse mAbs against arr-1 and arr-2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal mouse anti-adaptin protein-2 (anti-AP-2) was obtained from Transduction Laboratories (Lexington, KY). Indo-1 acetoxymethyl ester and pluronic acid were purchased from Molecular Probes (Eugene, OR). PMA, GDP, GTP, and ATP were purchased from Sigma-Aldrich (St. Louis, MO). All other reagents were from commercial sources. cDNAs encoding the chimeric mutants AB5 and BA5 were gifts from Dr. P. M. Murphy (National Institutes of Health, Bethesda, MD). cDNA encoding the tail-deleted mutant of CXCR2, 
CXCR2 (331T), was provided by Dr. A. Richmond (Vanderbilt University, New Orleans, LA). cDNAs encoding the green fluorescent protein (GFP)-tagged arr-2, AP-2, Src, and dynamin I (Dyn I) were gifts from Drs. M. G. Caron and R. J. Lefkowitz (Duke University, Durham, NC).
Construction of chimeric and deletion mutants of CXCR1 and CXCR2
CXCR1 and CXCR2 cDNAs possess a unique conserved NcoI restriction site located upstream of the sequences encoding the amino acid residues of the C-tails. The chimeric mutants ABt and BAt were made by ligating NcoI restriction fragments of CXCR1 and CXCR2 as described previously (Table I) (17). PCR was used to generate the phosphorylation-deficient mutants of CXCR1 (M8-A) and CXCR2 (M10-B) and the carboxyl-terminal truncated mutant of CXCR1 (CXCR1). For M8-A, M10-B, and CXCR1, a 5' oligonucleotide corresponding to the N terminus of CXCR1 or CXCR2 was used with a 3' oligonucleotide complementary to either the CXCR1 (M8-A) or the CXCR2 (M10-B) tail replacing serine and threonine residues with alanine, or a 3' oligonucleotide complementary to aa 318–325 in CXCR1, followed by a stop codon (CXCR1). The resulting PCR products as well as BA5, AB5, and CXCR2 were cloned into the eukaryotic expression vector pcDNA3. All receptors were sequenced to confirm the intended mutations and lack of secondary mutations.
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RBL-2H3 cells were maintained as monolayer cultures in Earle's modified Eagle's medium supplemented with 15% heat-inactivated FBS, 2 mM glutamine, penicillin (100 U/ml), and streptomycin (100 mg/ml) (18). RBL-2H3 cells (1 x 107 cells) were transfected by electroporation with 20 µg of pcDNA3 containing the receptor cDNAs, and geneticin-resistant cells were cloned into single cells by FACS analysis. Levels of protein expression were monitored by FACS analysis and Western blotting using specific Abs against each molecule. For transient transfection, RBL-2H3 cells (2 x 107 cells) stably expressing the receptors were electroporated with 30 µg of cDNAs encoding GFP vector or vector expressing GFP-tagged arr-2, AP-2, Src, or Dyn I. Levels of protein expression were monitored 24 h post-transfection by FACS analysis and Western blotting using either GFP or specific Abs against each molecules. Cells were sorted and subcultured overnight in 24-well plates (0.5 x 106 cells/well) in growth medium.
FACS analysis
For flow cytometric analysis, RBL cells were detached by Versene (Life Technologies, Rockville, MD) treatment, washed with HEPES-buffered HBSS, and resuspended in the same medium. Cells (1–5 x 106 cells) were incubated with anti-CXCR1 or anti-CXCR2 Abs (1 µg/ml) in a total volume of 400 µl of HEPES-buffered HBSS for 60 min at 4°C. The cells were then washed, incubated with FITC-anti-mouse IgG for 60 min at 4°C, washed, and analyzed for cell surface expression of the receptor on a FACScan cytometer (BD Biosciences, Mountain View, CA) (8, 18). Cells expressing GFP-tagged proteins were detached by Versene treatment and sorted as described above.
Radioligand binding assays and receptor internalization
Radioligand binding assays were conducted as described previously (19). 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 DMEM supplemented with 20 mM HEPES (pH 7.4) and 10 mg/ml BSA and incubated on ice for 2–4 h in the same medium (250 µl) containing [125I]IL-8 (0–10 nM). Reactions were stopped with 1 ml of ice-cold PBS containing 10 mg/ml BSA and washed three times with the same buffer. Then cells were solubilized with RIPA buffer (200 µl) and dried under vacuum, and bound radioactivity was counted (8, 20). Nonspecific radioactivity bound was determined in the presence of 500 nM unlabeled IL-8. The distribution constant (Kd) and binding capacity (Bmax) were determined using GraphPad radioligand binding data analysis (San Diego, CA). For receptor internalization, cells were incubated with ligand for 0–60 min at 37°C. Cells were then washed with ice-cold PBS, and [125I]IL-8 binding (0.1 nM) was conducted as described above.
PI hydrolysis and secretion, 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 FBS and 1 µCi/ml [3H]inositol. The generation of inositol phosphates and secretion of -hexosaminidase were determined as previously reported (18, 20). For calcium mobilization, cells (3 x 106) were removed, washed with HEPES-buffered saline, and loaded with 1 µM Indo-I/AM in the presence of 1 µM pluronic acid for 30 min at room temperature. Then the cells were washed and resuspended in 1.5 ml of buffer. The increase in intracellular calcium in the presence or the absence of ligands was measured as described previously (18, 20).
Chemotaxis
RBL-2H3 cells (n = 50,000) were incubated at 37°C with different concentrations of IL-8. Chemotaxis was assessed in 48-well microchemotaxis chambers using polyvinylpyrrolidone-free, 8-µm pore size membranes. Migration was allowed to continue for 3 h at 37°C in humidified air containing 5% CO2. The membrane was removed, and the upper surface was washed with PBS, scraped, fixed, and stained. The results are represented as the chemotactic index (mean number of cells per high power field for chemokine dilution/mean number of cells per high power field for medium) (21). The results are representative of three separate experiments.
GTPase activity
Cells were treated with the appropriate concentrations of stimulants, and membranes were prepared as previously described (20). GTPase activity using 10–20 µg of membrane preparations was determined as described previously (20).
Phosphorylation of receptors
Phosphorylation of receptors was performed as described previously (18, 20, 21). RBL cells (5 x 106) expressing the receptors were incubated with [32P]orthophosphate (150 µCi/dish) for 90 min. Then labeled cells were stimulated with the indicated ligands for 5 min at 37°C. Cells were then washed and solubilized in 1 ml of buffer (RIPA) containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS. Cells lysates were immunoprecipitated with specific Abs against the N terminus of CXCR1 or CXCR2, analyzed by SDS-PAGE, and visualized by autoradiography.
Confocal microscopy studies
Transfected cells were plated overnight onto 35-mm plastic dishes containing a centered, 1-cm, glass-bottom well. Cells were placed in 1 ml of MEM buffered with 20 mM HEPES and treated with IL-8 (100 nM) in the same medium. Images were collected with a laser scanning confocal microscope (LSM-410; Carl Zeiss, New York, NY) (22).
Coimmunoprecipitation and immunoblotting
RBL cells stably expressing the receptors were treated with or without IL-8 (100 nM) for 2 min, washed three times with ice-cold PBS, and solubilized in 1 ml of RIPA. The lysates were precleared for 1 h by addition of 20 µl of protein G-agarose beads. Supernatants were immunoprecipitated with 15 µl of anti-CXCR1 or anti-CXCR2 and protein G-agarose beads for 2 h. The beads were then washed with three times with 1 ml of ice-cold RIPA and immunoprecipitates were resolved by 10% SDS-PAGE, transferred to nitrocellulose membrane, and probed with mouse mAbs against arr2, AP-2, CXCR1, or CXCR2. Abs were detected with HRP-conjugated sheep anti-mouse Ab (Amersham Pharmacia Biotech, Piscataway, NJ) and ECL (Amersham Pharmacia Biotech).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine the role of the C-tails of CXCR1 and CXCR2 in IL-8-mediated leukocyte functions, four chimeric receptors, ABt, BAt, AB5, and BA5; two phosphorylation-deficient mutants, M8-A and M10-B, and two carboxyl tail-truncated mutants, CXCR1 and CXCR2, were made (Table I and Fig. 1) (17). ABt, BAt, BA5, M8-A, M10-B, CXCR1, and CXCR2, expressed in RBL cells (Fig. 1, B and C), bound IL-8 with affinities (Table II) similar to those of the wild-type CXCR1 and CXCR2. AB5 did not bind IL-8 or melanoma growth-stimulating activity, although surface expression of the receptor could be measured by FACS analysis (Table II and Fig. 1B)
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CXCR1, and CXCR2 stimulated dose-dependent PI hydrolysis (Fig. 2, A and B), secretion of -hexosaminidase (Fig. 2, C and D) and chemotaxis (Fig. 2, E and F). Cellular responses to the chimeric receptors BAt and BA5 (Fig. 2, B, D, and F) were equipotent to those of CXCR2, whereas ABt (Fig. 2, A, C, and E) induced greater PI hydrolysis and secretion, but lower chemotaxis, relative to CXCR1. The phosphorylation-deficient M8-A and M10-B and the tail-deleted mutants, CXCR1 and CXCR2, mediated much greater PI hydrolysis and secretion (Fig. 2, A–D), but 50% lower chemotaxis (Fig. 2, E and F) relative to CXCR1 and CXCR2.
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CXCR1, and CXCR2 stimulated a rapid and transient increase in free intracellular Ca2+ mobilization in response to IL-8 (Fig. 3). However, Ca2+ responses to M8-A, M10-B, CXCR1, CXCR2, and ABt were more sustained than those of CXCR1, CXCR2, BAt, and BA5.
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Desensitization of intracellular Ca2+ mobilization and GTPase activity
Ca2+ mobilization in response to a second dose of IL-8 (100 nM) was desensitized by prestimulation of the cells with the first dose of IL-8 (Table III). Responses to ABt, BAt, and BA5 were desensitized to an extent similar to that of the wild-type receptor (90%), whereas M8-A, M10-B, CXCR1, and CXCR2 were more resistant to desensitization (41, 36, 43, and 49%, respectively).
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CXCR1-, and CXCR2-mediated GTPase activity. Desensitization of receptor-mediated GTPase activity was lower and more sensitive to receptor phosphorylation than that of Ca2+ mobilization. This difference probably indicates that the desensitization of Ca2+ mobilization occurs at two levels: receptor/G protein coupling via receptor phosphorylation and modification of phospholipase C activity, whereas desensitization of GTPase activity occurs only at the level of receptor/G protein coupling (23).
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To study receptor phosphorylation, 32P-labeled cells expressing CXCR1, CXCR2, ABt, BAt, BA5, M8-A, M10-B, CXCR1, or CXCR2 were stimulated with either IL-8 (100 nM) or PMA (100 nM) for 5 min. The cell lysates were immunoprecipitated with specific Ab directed against the N terminus of either CXCR1 or CXCR2. As shown previously (8, 20) CXCR1 migrated as a single band of 70 kDa, whereas two forms of CXCR2 were observed: a slow (70 kDa) and a fast (45 kDa) migrating form. IL-8 and PMA mediated phosphorylation of ABt as well as CXCR1 (Fig. 5A, lanes 5 and 6 vs lanes 2 and 3). Phosphorylation of BAt and BA5 by IL-8 and PMA was also similar to that of CXCR2 (Fig. 5B, lanes 5 and 6 vs lane 2 and lanes 7 and 8 vs lane 3). M8-A (Fig. 5A, lanes 7–9), M10-B (Fig. 5B, lanes 10–12), and the C-tail-deleted mutants, CXCR1 and CXCR2 (data not shown), were resistant to receptor phosphorylation.
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CXCR1, and CXCR2
IL-8 (100 nM) induced a time-dependent internalization of CXCR1 and CXCR2 (Fig. 6). In agreement with previous reports (10, 15, 16) CXCR2 internalized more quickly than CXCR1 (95 vs 50% for CXCR2 and CXCR1, respectively, after 60 min). BAt and BA5 internalized as well as the wild-type CXCR2 (90% after 60 min; Fig. 6B), whereas ABt showed a marked decrease in internalization relative to CXCR1 (10% after 60 min; Fig. 6A). The phosphorylation-deficient mutants, M8-A, CXCR1, and CXCR2, were resistant to internalization, whereas M10-B showed an 20 internalization in response to IL-8 (Fig. 6, A and B). MGSA also induced an 15% internalization of M10-B vs 90% for CXCR2, BAt, and BA5 after 60 min (data not shown).
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arr in IL-8-mediated internalization of CXCR1 and CXCR2
RBL cells predominantly express arr-2 (12). To determine the mechanisms of internalization of CXCR1 and CXCR2, the receptors were stably coexpressed in RBL cells along with GFP-tagged arr-2 (GFP intensity, <101). Fluorescence microscopy was used to study the time course of IL-8-mediated receptor clearance from the cell surface. Upon exposure to IL-8, CXCR1, CXCR2, ABt, BAt, and BA5, but not M8-A, M10-B, CXCR1, and CXCR2, induced rapid translocation of arr-2 to the cell membrane (Fig. 7 and data not shown). However, arrestin-mediated internalization of the receptors into coated vesicles was shown for CXCR1, CXCR2, BAt, and BA5, but not ABT (Fig. 7, A and B). Vesicle formation and internalization were more rapid for CXCR2, BAt, and BA5 (12 min) relative to CXCR1 (35 min).
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arr-2 and adaptin-2
Since ABT translocated arrestin as well as CXCR1, it was determined whether the chimeric receptor bound arr. RBL cells stably expressing CXCR1 and ABt (Table II) were stimulated with IL-8 (100 nM) for 2 min, and cell lysates were immunoprecipitated with anti-CXCR1 and immunoblotted with anti-arr-2. As shown in Fig. 8A, IL-8 stimulation increased arr-2 (50 kDa) association to CXCR1 (lanes 1 and 2), but not ABt (lanes 2 and 4).
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was shown to be important for CXCR2 internalization (24, 25, 26, 27, 28). Since ABt expresses the C-tail of CXCR2, its role in ABt internalization was also assessed. CXCR1 and ABT RBL cells were treated as described above, immunoprecipitated with anti-CXCR1, and immunoblotted with anti-AP-2. As shown in Fig. 8B, IL-8 treatment induced AP-2 (102 kDa) association to CXCR1, but not ABt.
Role of Src, Dyn I, and AP-2 in ABt internalization
The tyrosine kinase Src, the GTPase protein Dyn I, and AP-2 are known to be important for arrestin-dependent internalization (24, 25, 26). To assess their roles in ABt internalization, GFP-tagged Src, Dyn I, or AP-2 was overexpressed in RBL expressing ABT. Cells (GFP intensity, 103) were collected and assayed for receptor internalization. As shown in Fig. 9A, AP-2, but not Src or Dyn I, increased receptor internalization to an extent similar to that of CXCR1. Overexpression of AP-2 had no effect on IL-8-mediated internalization of M8-A, M10-B, CXCR1, or CXCR2 (Fig. 9B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In other studies CXCR2 expressed in human embryonic kidney 293 (HEK 293) cells was reported to internalize via a phosphorylation/arrestin-independent mechanism (28). Binding of the adaptor protein AP-2 to the carboxyl-terminus, leucine-rich motif LLKIL was required for the phosphorylation-independent internalization of the receptor (28). The authors suggested that AP-2 could cause the rapid internalization of CXCR2 relative to CXCR1. Arguing against this hypothesis is the finding reported herein that CXCR1 bound AP-2 as well as CXCR2 upon IL-8 activation (Fig. 8B and data not shown). In addition, the chimeras BAt and BA5, which express the carboxyl tail of CXCR1 internalized as well as the wild-type CXCR2 (Fig. 6B).
Of interest is that the ABt chimera, despite its resistance to receptor internalization, induced arrestin translocation to the cell membrane as well as CXCR1 (Fig. 7A). Arrestin translocation and binding to the phosphorylated receptor are thought to be the initial requirement for internalization (29). The inability of arrestin to internalize the phosphorylated and desensitized receptor may be explained in several ways. First, it could be that the chimeric receptor was being recycled to the cell surface at a faster rate than the wild-type CXCR1, thus reducing the internalized fraction of the phosphorylated/arrestin bound receptor into clathrin-coated vesicles. Recycling of the receptor, however, would require rapid receptor dephosphorylation and resensitization. No decrease in receptor phosphorylation or the extent of desensitization of GTPase activity was observed in membranes from cells treated with IL-8 or PMA for up to 30 min (data not shown). Second, a conformational change in the C-tail of the receptor could have caused a decrease in the affinity of the phosphorylated receptor for arrestin. The resulting disassociation of the arrestin from the arrestin/receptor complex would thus prevent targeting of the receptor to the clathrin-coated vesicles. Supporting this hypothesis is that upon activation by IL-8, the amount of arr-2 coimmunoprecipitated with ABt was less than that of CXCR1 (Fig. 8A). Third, several adaptor proteins, including the tyrosine kinase Src, the GTPase protein Dyn I, and AP-2, are shown to be critical for receptor endocytosis (16, 25, 26). Thus, it is possible that the complex of phosphorylated receptor/arrestin lost its affinity for the adaptor proteins, resulting in a failure of receptor internalization. Indeed, the amount of AP-2 coimmunoprecipitated with ABt was less than that of CXCR1 (Fig. 8B). However, while overexpression of AP-2 increases the ability of IL-8 to induce ABt internalization, Src or Dyn I had no effect (Fig. 9).
Yang et al. (13) reported that agonist-stimulated receptor internalization is essential for chemokine receptor-mediated chemotaxis. However, the ABt chimera that is resistant to internalization showed only a modest decrease in receptor-mediated chemotaxis relative to the wild-type receptor (Figs. 2 and 3). Interestingly, the modest decrease in chemotaxis correlated with an increase in receptor-mediated PI hydrolysis, exocytosis, and sustained Ca2+ mobilization (Fig. 2). In addition, the phosphorylation-deficient receptors, which mediated greater activation of PI hydrolysis and Ca2+ mobilization, displayed lower chemotaxis responsiveness relative to native receptors (Fig. 2). Thus, it is likely that second messenger production rather than receptor internalization plays a negative feedback regulatory role in chemotaxis. Supporting this contention is that mutations of the formylpeptide receptor, which abolished receptor internalization, did not reduce fMLP-mediated chemotaxis (30, 31). Furthermore, mutations of chemoattractant receptors, including CXCR1, CXCR4, CCR1, CCR2, and protease-activated receptor-1, which enhanced cellular responses (i.e., PI hydrolysis, Ca2+ mobilization, and secretion), diminished chemotaxis (20, 21, 32, 33, 34). These studies further undermine the requirement for receptor endocytosis in chemotaxis and indicate that endocytosis and chemotaxis are two independent processes that are probably regulated by different pathways.
The data herein indicate that the cytoplasmic tails of CXCR1 and CXCR2 are necessary for IL-8-mediated receptor phosphorylation, desensitization, and internalization. However, the resistance of ABt to IL-8-induced internalization suggests that receptor phosphorylation and arrestin translocation, while necessary, are not sufficient for receptor internalization. Furthermore, the ability of BAt and BA5, which express the C-tail of CXCR1, to internalize as rapidly as the wild-type CXCR2 indicates that another motif(s) or specific conformational changes beyond the C-tails of the receptors modulate their rates of internalization, lengths of signaling, and, thus, biological activities. Despite its resistance to internalization, ABt mediated chemotaxis as well as CXCR1 and CXCR2. Thus, receptor internalization is not required for directional migration. Since M8-A, M10-B, CXCR1, and CXCR2 induced greater cellular responses (i.e., PI hydrolysis, Ca2+ mobilization), but decreased chemotaxis, these data further underscore the roles of these second messengers in down-regulating chemotaxis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ricardo M. Richardson, Department of Biochemistry, Meharry Medical College, 1005 Dr. D. B. Todd, Jr., Boulevard, Nashville, TN 37208. E-mail address: mrrichardson{at}mmc.edu
3 Abbreviations used in this paper: PI, phosphoinositide; AP-2, adaptor protein-2; arr-2, -arrestin 2; C-tail, cytoplasmic tail; CXCR1, IL-8R A; CXCR2, IL-8R B; Dyn I, dynamin I; G protein, GFP, green fluorescent protein; GTP-regulatory protein.
Received for publication October 7, 2002. Accepted for publication January 15, 2003.
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References |
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, stimulates phospholipase D activity. Blood 85:522.-arrestin1, and -arrestin2 for G protein-coupled receptors delineate two major classes of receptors. J. Biol. Chem. 275:17201.-Arrestin 1 interacts with the catalytic domain of the tyrosine kinase c-SRC: role of -arrestin1-dependent targeting of c-src in receptor endocytosis. J. Biol. Chem. 275:11312.-Arrestin-dependent formation of 2-adrenergic receptor-Src protein kinase complexes. Science 283:655.2-adrenergic receptor/-arrestin complex recruits the clathrin adaptor AP-2 during endocytosis. Proc. Natl. Acad. Sci. USA 96:3712.-adrenergic receptor function. FASEB J. 4:2881.[Abstract]
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M. W. Nasser, R. J. Marjoram, S. L. Brown, and R. M. Richardson Cross-Desensitization among CXCR1, CXCR2, and CCR5: Role of Protein Kinase C-{epsilon} J. Immunol., June 1, 2005; 174(11): 6927 - 6933. [Abstract] [Full Text] [PDF]
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 A. Strassburg, D. Droemann, G. van Zandbergen, H. Kothe, and K. Dalhoff Enhanced PMN response in chronic bronchitis and community-acquired pneumonia Eur. Respir. J., November 1, 2004; 24(5): 772 - 778. [Abstract] [Full Text] [PDF]
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J. Sai, G.-H. Fan, D. Wang, and A. Richmond The C-terminal domain LLKIL motif of CXCR2 is required for ligand-mediated polarization of early signals during chemotaxis J. Cell Sci., November 1, 2004; 117(23): 5489 - 5496. [Abstract] [Full Text] [PDF]
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 S. M. Youngerman, A. M. Saxton, S. P. Oliver, and G. M. Pighetti Association of CXCR2 Polymorphisms with Subclinical and Clinical Mastitis in Dairy Cattle J Dairy Sci, August 1, 2004; 87(8): 2442 - 2448. [Abstract] [Full Text] [PDF]
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R. A. Colvin, G. S. V. Campanella, J. Sun, and A. D. Luster Intracellular Domains of CXCR3 That Mediate CXCL9, CXCL10, and CXCL11 Function J. Biol. Chem., July 16, 2004; 279(29): 30219 - 30227. [Abstract] [Full Text] [PDF]
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J. J. Rose, J. F. Foley, P. M. Murphy, and S. Venkatesan On the Mechanism and Significance of Ligand-induced Internalization of Human Neutrophil Chemokine Receptors CXCR1 and CXCR2 J. Biol. Chem., June 4, 2004; 279(23): 24372 - 24386. [Abstract] [Full Text] [PDF]
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, Serine or threonine residues that are potential sites for receptor phosphorylation. ABt, CXCR1 expressing the C-tail of CXCR2; BAt, CXCR2 expressing the C-tail of CXCR1; AB5, CXCR1 expressing transmembrane domains (TMD) 5–7 and the C-tail of CXCR2; BA5, CXCR2 expressing TMD 5–7 and the C-tail of CXCR1; M8-A, CXCR1 with serines 337, 340, 341, 342, 346, and 347 and threonines 336 and 339 substituted for alanine; M10-B, CXCR2 with serines 334, 339, 342, 346, 347, 348, and 352 and threonines 351, 353, and 354 substituted for alanine; 
