Cross-Desensitization among CXCR1, CXCR2, and CCR5: Role of Protein Kinase C-1
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免疫学杂志 2005年第11期
Abstract
The IL-8 (or CXCL8) chemokine receptors, CXCR1 and CXCR2, activate protein kinase C (PKC) to mediate leukocyte functions. To investigate the roles of different PKC isoforms in CXCL8 receptor activation and regulation, human mononuclear phagocytes were treated with CXCL8 or CXCL1 (melanoma growth-stimulating activity), which is specific for CXCR2. Plasma membrane association was used as a measure of PKC activation. Both receptors induced time-dependent association of PKC, -1, and -2 to the membrane, but only CXCR1 activated PKC. CXCL8 also failed to activate PKC in RBL-2H3 cells stably expressing CXCR2. CXCR2, a cytoplasmic tail deletion mutant of CXCR2 that is resistant to internalization, activated PKC as well as CXCR1. Expression of the PKC inhibitor peptide V1 in RBL-2H3 cells blocked PKC translocation and inhibited receptor-mediated exocytosis, but not phosphoinositide hydrolysis or peak intracellular Ca2+ mobilization. V1 also inhibited CXCR1-, CCR5-, and CXCR2-mediated cross-regulatory signals for GTPase activity, Ca2+ mobilization, and internalization. Peritoneal macrophages from PKC-deficient mice (PKC–/–) also showed decreased CCR5-mediated cross-desensitization of G protein activation and Ca2+ mobilization. Taken together, the results indicate that CXCR1 and CCR5 activate PKC to mediate cross-inhibitory signals. Inhibition or deletion of PKC decreases receptor-induced exocytosis and cross-regulatory signals, but not phosphoinositide hydrolysis or peak intracellular Ca2+ mobilization, suggesting that cross-regulation is a Ca2+-independent process. Because CXCR2, but not CXCR2, activates PKC and cross-desensitizes CCR5, the data further suggest that signal duration leading to activation of novel PKC may modulate receptor-mediated cross-inhibitory signals.
Introduction
Chemokines are a large family of chemotactic cytokines that induce leukocyte chemoattraction and activation at sites of inflammation (1, 2). These functions are mediated via specific cell surface G protein-coupled receptors (GPCRs)3 (3). Chemokine receptors couple predominantly to Gi to activate phospholipase C and increase intracellular Ca2+, activation of phospholipase D (PLD), generation of diacylglycerides, and activation of protein kinase C (PKC) (4, 5). Chemokines and chemokine receptors are redundant in their interactions (4). Because individual receptors mediate multiple and distinct signaling pathways upon activation, cross-desensitization among multiple chemokines seems to play an important role in limiting their signal redundancy (6). CXCL8 activates two receptors, CXCR1 and CXCR2, to mediate cellular responses. CXCR1 is specific for CXCL8, whereas CXCR2 also interacts with CXCL1, epithelial cell-derived neutrophil attractant 78 (CXCL5), and neutrophil-activating peptide-2 (CXCL7). CXCR1, but not CXCR2, activates PLD, superoxide production, and cross-desensitization pathways (7, 8). Studies with leukocytes and transfected RBL-2H3 indicated that cross-desensitization requires sustained PKC activation, which, in the case of CXCR2, was prevented by rapid receptor phosphorylation and internalization (9, 10).
PKC is a family of at least 10 isoforms that can be divided into three groups: the classical PKCs (or calcium and diacylglycerol (DAG) dependent) , I, II, and ; the novel (or calcium independent) , , , and ; and the atypical (or calcium and DAG independent) and (11, 12). The specificity of many signal transduction pathways relies on the sequential activation of PKC by Ca2+ and DAG (13). Several leukocyte responses, including secretion and respiratory burst, are mediated through PKC-activating pathways (14, 15, 16, 17). In this study, human mononuclear phagocytes, murine peritoneal macrophages, and stably transfected RBL-2H3 cells were used to investigate the roles of different PKC isoforms in the ability of CXCR1, CXCR2, and CCR5 to mediate cross-regulatory signals. The data from this study demonstrate that CXCR1 and CCR5, but not CXCR2, activate PKC to cross-desensitize cellular responses and limit signal redundancy.
Materials and Methods
Radioligand binding assays and receptor internalization
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 in the same medium containing CCL5 or CXCL8 (100 nM) for 60 min. For radioligand binding, cells were placed on ice, washed three times with ice-cold PBS containing 10 mg/ml BSA, and incubated for 2–4 h in DMEM (250 μl) containing the radiolabeled ligand (0.1 nM). Reactions were stopped with 1 ml of ice-cold PBS, and cells washed five times. Cells were then solubilized with RIPA buffer (200 μl) and dried under vacuum, and bound radioactivity was counted. Nonspecific radioactivity bound was determined in the presence of 500 nM unlabeled ligand (10).
Results
CXCR1- and CXCR2-mediated PKC activation
To determine the ability of CXCR1 and CXCR2 to activate PKCs, mononuclear phagocytes isolated from blood were treated with 100 nM CXCL8, which activates CXCR1 and CXCR2, or with CXCL1, which is specific for CXCR2, for different periods of time. Plasma membrane association of PKC was determined by Western blotting using anti-PKC, -I-, -II-, and --specific Abs. As shown in Fig. 1, both CXCL8 and CXCL1 induced time-dependent association of PKC, -I, and II to the membrane. The time course of CXCL8-mediated PKC association, however, was more sustained than that of CXCL1. Interestingly, CXCL8, but not CXCL1, showed a robust increase in PKC association to the plasma membrane (1–5 min; Fig. 1A). Mouse peritoneal macrophages pretreated with murine CXCL1 also showed no increase in PKC association to the plasma membrane compared with untreated cells (data not shown).
Role of PKC in CXCR1- and CCR5-mediated cross-desensitization
To determine the role of PKC in CXCL8- and CCL5-induced cross-desensitization, V1 fragment or vector alone was expressed in ACCR5 and BCCR5 (RBL cells stably coexpressing CXCR2 and CCR5) (10) RBL cells. Ligand-induced GTPase activity in membrane was measured. As shown in Fig. 5, CXCL8 and CCL5 pretreatment of ACCR5 (Fig. 5, A and B) and BCCR5 (Fig. 5, C and D) cells homologously desensitized GTPase activity to the same agonist, relative to control or untreated cells. In ACCR5 cells expressing the vector alone (Fig. 5A), but not BCCR5 cells (Fig. 5C), and pretreated with CXCL8, CCL5-mediated GTPase activity was reduced by 45%. ACCR5 (Fig. 5A) and BCCR5 (Fig. 5C) pretreated with CCL5 also exhibited a 50–60% decrease in CXCL8-mediated GTPase activity. Expression of the V1 fragment in ACCR5 cells (Fig. 5B) significantly reduced CXCL8-mediated cross-desensitization of CCL5 (25%). CCL5-mediated cross-desensitization of CXCL8 was also diminished in ACCR5 (20%; Fig. 5B) and BCCR5 (7%; Fig. 5D) cells expressing the V1 fragment. As expected, CCL5-mediated GTPase activity was resistant to cross-desensitization by CXCL8 in BCCR5 cells that express the CXCR2 receptor (Fig. 5, C and D).
The effect of V1 on CXCL8- and CCL5-induced cross-desensitization of intracellular Ca2+ mobilization was also determined in ACCR5 cells expressing V1 vs control or vector alone. As shown in Table I, V1 had no effect on CXCL8- and CCL5-mediated homologous (CXCL8CXCL8 and CCL5CCL5) and heterologous (PMACXCL8 and PMACCL5) desensitization of Ca2+ mobilization. V1 expression, however, diminished CXCL8- and CCL5-mediated cross-desensitization of Ca2+ mobilization to each other by 70% (50 vs 16% for CXCL8CCL5) and 83% (41 vs 7% for CCL5CXCL8), respectively (Table I).
In vivo role of PKC in cross-desensitization between CCR5 and CXCR2
The role of PKC in cross-desensitization was also determined in murine peritoneal macrophages from PKC-deficient mice (PKC–/–) vs control animals (PKC+/+) (26). Because mice only expresses a human homologue of CXCR2, KC, the murine homologue of CXCL1 was used (27). CCL5 and CXCL1 homologously desensitized Ca2+ mobilization in response to a second dose of the same ligand in both PKC–/– and PKC+/+ (Fig. 7). CCL5-mediated cross-desensitization of Ca2+ mobilization to CXCL1, however, was markedly decreased in macrophages from PKC–/– (Fig. 7, right panel) relative to wild-type mice PKC+/+ (Fig. 7, left panel). CXCL1-mediated GTPS binding in membrane was also resistant to cross-desensitization by CCL5 in PKC–/– mice (Fig. 8B) relative to PKC+/+ control mice (Fig. 8A). CXCL1 pretreatment had no effect on CCL5-mediated Ca2+ mobilization or GTPS binding in both PKC–/– and PKC+/+ mice (Fig. 7 and data not shown).
Discussion
Most chemokines and their receptors have redundant specificity, in that many chemokines activate more than one chemokine receptor, and many chemokine receptors are activated by multiple chemokines. Studies with neutrophils and RBL cells coexpressing different combinations of chemokine receptors indicated that cross-desensitization among multiple chemokines may be important in limiting their signal redundancy at the site of inflammation and infection (6). To date, the molecular events underlying receptor cross-desensitization are poorly understood. One of the sites of cross-desensitization appears to be at the level of receptor/G protein coupling and involves cross-phosphorylation of the receptor by a PKC-dependent mechanism, followed by receptor uncoupling from its G protein (6, 9). The second one is an as yet unidentified modulation of a downstream component leading to decreased activation of phospholipase C (6, 9). CXCL8 activates CXCR1 and CXCR2, but only CXCR1 mediates receptor cross-phosphorylation and cross-desensitization (9, 10). This study was designed to investigate the roles of different PKC isoforms in the distinct ability of CXCL8 receptors to mediate cross-desensitization. The data from this study demonstrated that CXCR1, but not CXCR2, activates PKC to mediate receptor cross-desensitization and cross-internalization. This contention is supported by the following observations. First, in human mononuclear phagocytes, CXCL8, but not CXCL1, increased PKC association with the plasma membrane (Fig. 1). Second, in RBL cells stably coexpressing GFP-tagged PKC and either CXCR1 or CXCR2, CXCL8 activation of CXCR1, but not CXCR2, induced PKC translocation to the membrane (Fig. 2). Third, expression of the PKC inhibitor peptide V1 in ACCR5-RBL cells blocked PKC translocation to the plasma membrane and reduced CXCL8-mediated cross-desensitization of both intracellular Ca2+ mobilization and GTPase activity to CCL5 as well as cross-internalization of CCR5 (Table I and Figs. 3, 5, and 6).
Previous studies from our laboratory and others suggested that signal duration resulting in activation of PLD and sustained production of DAG is important for chemokine receptors to mediate leukocyte cytotoxicity and activate cross-inhibitory pathways (7, 9, 28, 29). The data presented in this study further support this contention. First, inhibition of PKC in RBL cells had no effect in CXCL8- or CCL5-mediated PI hydrolysis or peak intracellular Ca2+ mobilization, but diminished receptor-mediated exocytosis by 50% (Fig. 4 and Table I). Second, peritoneal macrophages from PKC–/– mice also showed no change in CCL5-induced peak intracellular Ca2+ mobilization (Fig. 7), but exhibited a significant decrease in superoxide production relative to PKC+/+ macrophages (data not shown). Third, the phosphorylation- and desensitization-resistant mutant of CXCR2, CXCR2, which generated longer signals (i.e., GTPase activity, PI hydrolysis, and Ca+2 mobilization) (9, 10) than CXCR2 and activated PLD, induced PKC translocation and cross-desensitization of the Ca2+ response and GTPase activity to CCR5 (Fig. 2 and data not shown).
Of interest is that PKC inhibition had no effect on the receptor homologous and heterologous desensitization of Ca2+ mobilization and G protein activation, but inhibited cross-desensitization (Table I and Figs. 5, 7, and 8). Homologous desensitization is mediated predominantly via GPCR kinase-dependent mechanisms (30, 31). The heterologous and cross-desensitizations of chemoattractant receptors, however, are both PKC dependent (6, 30, 31). Thus, the lack of effect of PKC inhibition on heterologous desensitization probably indicates that receptor heterologous and cross-desensitizations are two independent signaling events mediated via different PKC-dependent pathways. Heterologous phosphorylation and desensitization are early signaling events, which occur via transient activation of PKCs, whereas cross-desensitization requires prolonged receptor stimulation and sustained PKC activation. Supporting this contention is the finding that upon activation by CXCL8, both CXCR1 and CXCR2 induced transient activation of PKC, -1, and -2 and underwent heterologous desensitization (Fig. 1 and Table I). Furthermore, CXCR2, but not CXCR2, which is resistant to receptor internalization, mediated signal for cross-desensitization of CCR5 (Fig. 5) (9, 10).
Zhang et al. (32) demonstrated that CCR1 predominantly activates PKC to cross-desensitize the response to the opioid receptor. In RBL-2H3 cells, however, expression of the V1 fragment had no significant effect on CXCL8-mediated cross-desensitization of Ca2+ mobilization and GTPase activity to CCR5. One possible explanation could be that CCR1 and CXCR1 activate different novel PKC isoforms to mediate cross-desensitization. Second, as indicated by the authors, because PKC translocated predominantly to the Golgi in NIH-3T3 cells pretreated with PMA, its involvement in CCR1 cross-desensitization was not assessed (33). As shown in Fig. 2, however, both CXCL8 and PMA induced PKC translocation from the cytosol to the membrane in RBL-2H3 cells. PMA also induced PKC translocation to the membrane in human mononuclear phagocytes and alveolar macrophages (34, 35).
The data from this study, however, do not exclude the participation of other PKC isoforms in CXCL8-mediated receptor cross-desensitization. First, staurosporine reversed CXCL8-mediated cross-desensitization of CCR5 by 100%, whereas inhibition or deletion of PKC only caused an 80% decrease in receptor cross-desensitization (9, 36). Second, previous studies have shown that PKC and PKC are important for chemoattractant receptor-mediated activation and regulation of exocytosis and respiratory burst (14, 15, 16, 17). Thus, it is possible that sustained activation of both classical and novel PKCs by DAG is required for receptor to cause cross-phosphorylation, cross-desensitization, and cross-internalization of susceptible receptors as well as modification of downstream effector activities.
In summary, the results indicate that CXCR1 and CCR5, but not CXCR2, activate PKC to mediate cross-desensitization of intracellular Ca2+ mobilization and GTPase activity and cross-internalization. CXCR2, which generates longer signals relative to CXCR2, activates PKC and mediates cross-desensitization. Thus, chemokines interacting with different receptors, based on signal strength, may activate selective PKC isoforms to mediate receptor cross-regulation and limit signal redundancy at sites of inflammation.
Footnotes
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.
1 This work was supported by National Institutes of Health Grants AI38910 and CA92077 and U.S. Department of Veterans Affairs Grant 626/151.
2 Address correspondence and reprint requests to Dr. Ricardo M. Richardson, Julius L. Chambers Biomedical/Biotechnology Research Institute, North Carolina Central University, 1801 Fayetteville Street, Durham, NC 27707. E-mail address: mrrichardson{at}nccu.edu
3 Abbreviations used in this paper: GPCR, G protein-coupled receptor; DAG, diacylglycerol; GTPS, guanosine-5',0-(3-thiotriphosphate); IP, inositol phosphate; KC, keratinocyte-derived chemokine; PI, phosphoinositide; PKC, protein kinase C; PLD, phospholipase D.
Received for publication November 22, 2004. Accepted for publication March 15, 2005.
References
Locati, M., P. M. Murphy. 1999. Chemokines and chemokine receptors: biology and clinical relevance in inflammation and AIDS. Annu. Rev. Med. 50: 425-440.
Murphy, P. M., M. Baggiolini, I. F. Charo, C. A. Hebert, R. Horuk, K. Matsushima, L. H. Miller, J. J. Oppenheim, C. A. Power. 2000. International union of pharmacology. XXII. Nomenclature for chemokine receptors. Pharmacol. Rev. 52: 145-176.
Lefkowitz, R. J.. 2000. The superfamily of heptahelical receptors. Nat. Cell. Biol. 2: E133-E136.
Baggiolini, M., B. Dewald, B. Moser. 1997. Human chemokines: an update. Annu. Rev. Immunol. 15: 675-705.
Uhing, R. J., R. Snyderman. 1999. Chemoattractant stimulus-response coupling. J. I. Gallin, and R. Snyderman, eds. Inflammation: Basic Principles and Clinical Correlates 3rd Ed. 607-626 Lippincott Williams & Wilkins, Raven Press, Philadelphia. .
Ali, H., R. M. Richardson, B. Haribabu, R. Snyderman. 1999. Chemoattractant receptor cross-desensitization. J. Biol. Chem. 274: 6027-6030.(Mohd W. Nasser*, Robin J.)
The IL-8 (or CXCL8) chemokine receptors, CXCR1 and CXCR2, activate protein kinase C (PKC) to mediate leukocyte functions. To investigate the roles of different PKC isoforms in CXCL8 receptor activation and regulation, human mononuclear phagocytes were treated with CXCL8 or CXCL1 (melanoma growth-stimulating activity), which is specific for CXCR2. Plasma membrane association was used as a measure of PKC activation. Both receptors induced time-dependent association of PKC, -1, and -2 to the membrane, but only CXCR1 activated PKC. CXCL8 also failed to activate PKC in RBL-2H3 cells stably expressing CXCR2. CXCR2, a cytoplasmic tail deletion mutant of CXCR2 that is resistant to internalization, activated PKC as well as CXCR1. Expression of the PKC inhibitor peptide V1 in RBL-2H3 cells blocked PKC translocation and inhibited receptor-mediated exocytosis, but not phosphoinositide hydrolysis or peak intracellular Ca2+ mobilization. V1 also inhibited CXCR1-, CCR5-, and CXCR2-mediated cross-regulatory signals for GTPase activity, Ca2+ mobilization, and internalization. Peritoneal macrophages from PKC-deficient mice (PKC–/–) also showed decreased CCR5-mediated cross-desensitization of G protein activation and Ca2+ mobilization. Taken together, the results indicate that CXCR1 and CCR5 activate PKC to mediate cross-inhibitory signals. Inhibition or deletion of PKC decreases receptor-induced exocytosis and cross-regulatory signals, but not phosphoinositide hydrolysis or peak intracellular Ca2+ mobilization, suggesting that cross-regulation is a Ca2+-independent process. Because CXCR2, but not CXCR2, activates PKC and cross-desensitizes CCR5, the data further suggest that signal duration leading to activation of novel PKC may modulate receptor-mediated cross-inhibitory signals.
Introduction
Chemokines are a large family of chemotactic cytokines that induce leukocyte chemoattraction and activation at sites of inflammation (1, 2). These functions are mediated via specific cell surface G protein-coupled receptors (GPCRs)3 (3). Chemokine receptors couple predominantly to Gi to activate phospholipase C and increase intracellular Ca2+, activation of phospholipase D (PLD), generation of diacylglycerides, and activation of protein kinase C (PKC) (4, 5). Chemokines and chemokine receptors are redundant in their interactions (4). Because individual receptors mediate multiple and distinct signaling pathways upon activation, cross-desensitization among multiple chemokines seems to play an important role in limiting their signal redundancy (6). CXCL8 activates two receptors, CXCR1 and CXCR2, to mediate cellular responses. CXCR1 is specific for CXCL8, whereas CXCR2 also interacts with CXCL1, epithelial cell-derived neutrophil attractant 78 (CXCL5), and neutrophil-activating peptide-2 (CXCL7). CXCR1, but not CXCR2, activates PLD, superoxide production, and cross-desensitization pathways (7, 8). Studies with leukocytes and transfected RBL-2H3 indicated that cross-desensitization requires sustained PKC activation, which, in the case of CXCR2, was prevented by rapid receptor phosphorylation and internalization (9, 10).
PKC is a family of at least 10 isoforms that can be divided into three groups: the classical PKCs (or calcium and diacylglycerol (DAG) dependent) , I, II, and ; the novel (or calcium independent) , , , and ; and the atypical (or calcium and DAG independent) and (11, 12). The specificity of many signal transduction pathways relies on the sequential activation of PKC by Ca2+ and DAG (13). Several leukocyte responses, including secretion and respiratory burst, are mediated through PKC-activating pathways (14, 15, 16, 17). In this study, human mononuclear phagocytes, murine peritoneal macrophages, and stably transfected RBL-2H3 cells were used to investigate the roles of different PKC isoforms in the ability of CXCR1, CXCR2, and CCR5 to mediate cross-regulatory signals. The data from this study demonstrate that CXCR1 and CCR5, but not CXCR2, activate PKC to cross-desensitize cellular responses and limit signal redundancy.
Materials and Methods
Radioligand binding assays and receptor internalization
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 in the same medium containing CCL5 or CXCL8 (100 nM) for 60 min. For radioligand binding, cells were placed on ice, washed three times with ice-cold PBS containing 10 mg/ml BSA, and incubated for 2–4 h in DMEM (250 μl) containing the radiolabeled ligand (0.1 nM). Reactions were stopped with 1 ml of ice-cold PBS, and cells washed five times. Cells were then solubilized with RIPA buffer (200 μl) and dried under vacuum, and bound radioactivity was counted. Nonspecific radioactivity bound was determined in the presence of 500 nM unlabeled ligand (10).
Results
CXCR1- and CXCR2-mediated PKC activation
To determine the ability of CXCR1 and CXCR2 to activate PKCs, mononuclear phagocytes isolated from blood were treated with 100 nM CXCL8, which activates CXCR1 and CXCR2, or with CXCL1, which is specific for CXCR2, for different periods of time. Plasma membrane association of PKC was determined by Western blotting using anti-PKC, -I-, -II-, and --specific Abs. As shown in Fig. 1, both CXCL8 and CXCL1 induced time-dependent association of PKC, -I, and II to the membrane. The time course of CXCL8-mediated PKC association, however, was more sustained than that of CXCL1. Interestingly, CXCL8, but not CXCL1, showed a robust increase in PKC association to the plasma membrane (1–5 min; Fig. 1A). Mouse peritoneal macrophages pretreated with murine CXCL1 also showed no increase in PKC association to the plasma membrane compared with untreated cells (data not shown).
Role of PKC in CXCR1- and CCR5-mediated cross-desensitization
To determine the role of PKC in CXCL8- and CCL5-induced cross-desensitization, V1 fragment or vector alone was expressed in ACCR5 and BCCR5 (RBL cells stably coexpressing CXCR2 and CCR5) (10) RBL cells. Ligand-induced GTPase activity in membrane was measured. As shown in Fig. 5, CXCL8 and CCL5 pretreatment of ACCR5 (Fig. 5, A and B) and BCCR5 (Fig. 5, C and D) cells homologously desensitized GTPase activity to the same agonist, relative to control or untreated cells. In ACCR5 cells expressing the vector alone (Fig. 5A), but not BCCR5 cells (Fig. 5C), and pretreated with CXCL8, CCL5-mediated GTPase activity was reduced by 45%. ACCR5 (Fig. 5A) and BCCR5 (Fig. 5C) pretreated with CCL5 also exhibited a 50–60% decrease in CXCL8-mediated GTPase activity. Expression of the V1 fragment in ACCR5 cells (Fig. 5B) significantly reduced CXCL8-mediated cross-desensitization of CCL5 (25%). CCL5-mediated cross-desensitization of CXCL8 was also diminished in ACCR5 (20%; Fig. 5B) and BCCR5 (7%; Fig. 5D) cells expressing the V1 fragment. As expected, CCL5-mediated GTPase activity was resistant to cross-desensitization by CXCL8 in BCCR5 cells that express the CXCR2 receptor (Fig. 5, C and D).
The effect of V1 on CXCL8- and CCL5-induced cross-desensitization of intracellular Ca2+ mobilization was also determined in ACCR5 cells expressing V1 vs control or vector alone. As shown in Table I, V1 had no effect on CXCL8- and CCL5-mediated homologous (CXCL8CXCL8 and CCL5CCL5) and heterologous (PMACXCL8 and PMACCL5) desensitization of Ca2+ mobilization. V1 expression, however, diminished CXCL8- and CCL5-mediated cross-desensitization of Ca2+ mobilization to each other by 70% (50 vs 16% for CXCL8CCL5) and 83% (41 vs 7% for CCL5CXCL8), respectively (Table I).
In vivo role of PKC in cross-desensitization between CCR5 and CXCR2
The role of PKC in cross-desensitization was also determined in murine peritoneal macrophages from PKC-deficient mice (PKC–/–) vs control animals (PKC+/+) (26). Because mice only expresses a human homologue of CXCR2, KC, the murine homologue of CXCL1 was used (27). CCL5 and CXCL1 homologously desensitized Ca2+ mobilization in response to a second dose of the same ligand in both PKC–/– and PKC+/+ (Fig. 7). CCL5-mediated cross-desensitization of Ca2+ mobilization to CXCL1, however, was markedly decreased in macrophages from PKC–/– (Fig. 7, right panel) relative to wild-type mice PKC+/+ (Fig. 7, left panel). CXCL1-mediated GTPS binding in membrane was also resistant to cross-desensitization by CCL5 in PKC–/– mice (Fig. 8B) relative to PKC+/+ control mice (Fig. 8A). CXCL1 pretreatment had no effect on CCL5-mediated Ca2+ mobilization or GTPS binding in both PKC–/– and PKC+/+ mice (Fig. 7 and data not shown).
Discussion
Most chemokines and their receptors have redundant specificity, in that many chemokines activate more than one chemokine receptor, and many chemokine receptors are activated by multiple chemokines. Studies with neutrophils and RBL cells coexpressing different combinations of chemokine receptors indicated that cross-desensitization among multiple chemokines may be important in limiting their signal redundancy at the site of inflammation and infection (6). To date, the molecular events underlying receptor cross-desensitization are poorly understood. One of the sites of cross-desensitization appears to be at the level of receptor/G protein coupling and involves cross-phosphorylation of the receptor by a PKC-dependent mechanism, followed by receptor uncoupling from its G protein (6, 9). The second one is an as yet unidentified modulation of a downstream component leading to decreased activation of phospholipase C (6, 9). CXCL8 activates CXCR1 and CXCR2, but only CXCR1 mediates receptor cross-phosphorylation and cross-desensitization (9, 10). This study was designed to investigate the roles of different PKC isoforms in the distinct ability of CXCL8 receptors to mediate cross-desensitization. The data from this study demonstrated that CXCR1, but not CXCR2, activates PKC to mediate receptor cross-desensitization and cross-internalization. This contention is supported by the following observations. First, in human mononuclear phagocytes, CXCL8, but not CXCL1, increased PKC association with the plasma membrane (Fig. 1). Second, in RBL cells stably coexpressing GFP-tagged PKC and either CXCR1 or CXCR2, CXCL8 activation of CXCR1, but not CXCR2, induced PKC translocation to the membrane (Fig. 2). Third, expression of the PKC inhibitor peptide V1 in ACCR5-RBL cells blocked PKC translocation to the plasma membrane and reduced CXCL8-mediated cross-desensitization of both intracellular Ca2+ mobilization and GTPase activity to CCL5 as well as cross-internalization of CCR5 (Table I and Figs. 3, 5, and 6).
Previous studies from our laboratory and others suggested that signal duration resulting in activation of PLD and sustained production of DAG is important for chemokine receptors to mediate leukocyte cytotoxicity and activate cross-inhibitory pathways (7, 9, 28, 29). The data presented in this study further support this contention. First, inhibition of PKC in RBL cells had no effect in CXCL8- or CCL5-mediated PI hydrolysis or peak intracellular Ca2+ mobilization, but diminished receptor-mediated exocytosis by 50% (Fig. 4 and Table I). Second, peritoneal macrophages from PKC–/– mice also showed no change in CCL5-induced peak intracellular Ca2+ mobilization (Fig. 7), but exhibited a significant decrease in superoxide production relative to PKC+/+ macrophages (data not shown). Third, the phosphorylation- and desensitization-resistant mutant of CXCR2, CXCR2, which generated longer signals (i.e., GTPase activity, PI hydrolysis, and Ca+2 mobilization) (9, 10) than CXCR2 and activated PLD, induced PKC translocation and cross-desensitization of the Ca2+ response and GTPase activity to CCR5 (Fig. 2 and data not shown).
Of interest is that PKC inhibition had no effect on the receptor homologous and heterologous desensitization of Ca2+ mobilization and G protein activation, but inhibited cross-desensitization (Table I and Figs. 5, 7, and 8). Homologous desensitization is mediated predominantly via GPCR kinase-dependent mechanisms (30, 31). The heterologous and cross-desensitizations of chemoattractant receptors, however, are both PKC dependent (6, 30, 31). Thus, the lack of effect of PKC inhibition on heterologous desensitization probably indicates that receptor heterologous and cross-desensitizations are two independent signaling events mediated via different PKC-dependent pathways. Heterologous phosphorylation and desensitization are early signaling events, which occur via transient activation of PKCs, whereas cross-desensitization requires prolonged receptor stimulation and sustained PKC activation. Supporting this contention is the finding that upon activation by CXCL8, both CXCR1 and CXCR2 induced transient activation of PKC, -1, and -2 and underwent heterologous desensitization (Fig. 1 and Table I). Furthermore, CXCR2, but not CXCR2, which is resistant to receptor internalization, mediated signal for cross-desensitization of CCR5 (Fig. 5) (9, 10).
Zhang et al. (32) demonstrated that CCR1 predominantly activates PKC to cross-desensitize the response to the opioid receptor. In RBL-2H3 cells, however, expression of the V1 fragment had no significant effect on CXCL8-mediated cross-desensitization of Ca2+ mobilization and GTPase activity to CCR5. One possible explanation could be that CCR1 and CXCR1 activate different novel PKC isoforms to mediate cross-desensitization. Second, as indicated by the authors, because PKC translocated predominantly to the Golgi in NIH-3T3 cells pretreated with PMA, its involvement in CCR1 cross-desensitization was not assessed (33). As shown in Fig. 2, however, both CXCL8 and PMA induced PKC translocation from the cytosol to the membrane in RBL-2H3 cells. PMA also induced PKC translocation to the membrane in human mononuclear phagocytes and alveolar macrophages (34, 35).
The data from this study, however, do not exclude the participation of other PKC isoforms in CXCL8-mediated receptor cross-desensitization. First, staurosporine reversed CXCL8-mediated cross-desensitization of CCR5 by 100%, whereas inhibition or deletion of PKC only caused an 80% decrease in receptor cross-desensitization (9, 36). Second, previous studies have shown that PKC and PKC are important for chemoattractant receptor-mediated activation and regulation of exocytosis and respiratory burst (14, 15, 16, 17). Thus, it is possible that sustained activation of both classical and novel PKCs by DAG is required for receptor to cause cross-phosphorylation, cross-desensitization, and cross-internalization of susceptible receptors as well as modification of downstream effector activities.
In summary, the results indicate that CXCR1 and CCR5, but not CXCR2, activate PKC to mediate cross-desensitization of intracellular Ca2+ mobilization and GTPase activity and cross-internalization. CXCR2, which generates longer signals relative to CXCR2, activates PKC and mediates cross-desensitization. Thus, chemokines interacting with different receptors, based on signal strength, may activate selective PKC isoforms to mediate receptor cross-regulation and limit signal redundancy at sites of inflammation.
Footnotes
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.
1 This work was supported by National Institutes of Health Grants AI38910 and CA92077 and U.S. Department of Veterans Affairs Grant 626/151.
2 Address correspondence and reprint requests to Dr. Ricardo M. Richardson, Julius L. Chambers Biomedical/Biotechnology Research Institute, North Carolina Central University, 1801 Fayetteville Street, Durham, NC 27707. E-mail address: mrrichardson{at}nccu.edu
3 Abbreviations used in this paper: GPCR, G protein-coupled receptor; DAG, diacylglycerol; GTPS, guanosine-5',0-(3-thiotriphosphate); IP, inositol phosphate; KC, keratinocyte-derived chemokine; PI, phosphoinositide; PKC, protein kinase C; PLD, phospholipase D.
Received for publication November 22, 2004. Accepted for publication March 15, 2005.
References
Locati, M., P. M. Murphy. 1999. Chemokines and chemokine receptors: biology and clinical relevance in inflammation and AIDS. Annu. Rev. Med. 50: 425-440.
Murphy, P. M., M. Baggiolini, I. F. Charo, C. A. Hebert, R. Horuk, K. Matsushima, L. H. Miller, J. J. Oppenheim, C. A. Power. 2000. International union of pharmacology. XXII. Nomenclature for chemokine receptors. Pharmacol. Rev. 52: 145-176.
Lefkowitz, R. J.. 2000. The superfamily of heptahelical receptors. Nat. Cell. Biol. 2: E133-E136.
Baggiolini, M., B. Dewald, B. Moser. 1997. Human chemokines: an update. Annu. Rev. Immunol. 15: 675-705.
Uhing, R. J., R. Snyderman. 1999. Chemoattractant stimulus-response coupling. J. I. Gallin, and R. Snyderman, eds. Inflammation: Basic Principles and Clinical Correlates 3rd Ed. 607-626 Lippincott Williams & Wilkins, Raven Press, Philadelphia. .
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