Cross-Desensitization Among Receptors for Platelet Activating Factor and Peptide Chemoattractants: Evidence for Independent Regulatory Pathways

Cross-desensitization among receptors for peptide chemoattractants have been shown to involve two independent processes, receptor phosphorylation and inhibition of phospholipase C (PLC) activation. Receptors for lipid chemoattractants, i.e. platelet activating factor (PAF) and leukotriene B4, did not inhibit the responses of peptide chemoattractant receptors, suggesting distinct signaling pathways. To examine cross-desensitization between receptors for lipid and peptide chemoattractants, cDNA encoding the PAF receptor (PAFR) was co-expressed into RBL-2H3 cells with cDNAs encoding receptors for either formylated peptides (FR), a product of the fifth component of complement (C5aR) or interleukin-8 A (IL-8RA). PAFR was homologously phosphorylated and desensitized by PAF, and cross-phosphorylated and cross-desensitized by fMet-Leu-Phe, C5a, and IL-8. In contrast, the receptors for peptide chemoattractants were neither cross-phosphorylated nor cross-desensitized by PAF. Staurosporine blocked cross-phosphorylation and cross-desensitization of the PAFR by peptide chemoattractants. Truncation of the cytoplasmic tail of PAFR (mPAFR) abolished its homologous and cross- phosphorylation. mPAFR was also resistant to cross-desensitization by peptide chemoattractants at the level of PLC activation. Interestingly, mPAFR mediated a sustained Ca2+ mobilization in response to PAF and was more active in inducing GTPase activity, phosphoinositide hydrolysis, secretion, and phospholipase D activation than the wild type PAFR. In contrast to PAFR, stimulation of the mPAFR cross-phosphorylated and cross-desensitized responses to IL-8RA. As expected, FR, which is resistant to cross- phosphorylation by C5aR and IL-8RA, was not phosphorylated by mPAFR. However, unlike C5aR and IL-8RA, mPAFR did not inhibit the ability of FR to activate PLC. Blocking Ca2+ influx inhibited mPAFR-mediated sustained Ca2+ response, phospholipase D activation and secretion, but not phosphoinositide hydrolysis and cross-phosphorylation and cross-desensitization of IL-8RA. The data herein suggest that cross-desensitization of PAFR by peptide chemoattractants is solely due to receptor phosphorylation. The PAFR and the peptide chemoattractant receptors do not cross-regulate each other at the level of PLC, suggesting distinct regulatory pathways

Multiple Signaling Pathways of Human Interleukin-8 Receptor A: Independent Regulation by Phosphorylation

Interleukin-8 (IL-8) receptor A (CXCR1) couples to a pertussis toxin- sensitive G protein to mediate phospholipase Cβ (PLCβ) activation and cellular responses. Responses to CXCR1 are attenuated by prior exposure of neutrophils to either IL-8, a cleavage product of the fifth component of complement (C5a) or n-formylated peptides (formylmethionylleucylphenylalanine, fMLP). To characterize the role of receptor phosphorylation in the regulation of the CXCR1, a phosphorylation- deficient mutant, M2CXCR1, was constructed. This receptor, stably expressed in RBL-2H3 cells, coupled more efficiently to G protein and stimulated enhanced phosphoinositide hydrolysis, cAMP production, exocytosis, and phospholipase D activation, and was resistant to IL-8-induced receptor internalization. The rate and total amount of ligand stimulated actin polymerization remained unchanged, but interestingly, chemotaxis was decreased by ~30% compared with the wild type receptor. To study the role of receptor phosphorylation in cross-desensitization of chemoattractant receptors, M2CXCR1 was coexpressed with cDNAs encoding receptors for either fMLP (FR), C5a (C5aR), or platelet-activating factor (PAFR). Both C5aR and PAFR were cross-phosphorylated upon M2CXCR1 activation, resulting in attenuated guanosine 5\u27-3\u27-O-(thio)triphosphate (GTPγS) binding in membranes. In contrast, FR and M2CXCR1 were resistant to cross- phosphorylation and cross-inhibition of GTPγS binding by other receptors. Despite the resistance of M2CXCR1 to cross-phosphorylation and receptor/G protein uncoupling, its susceptibility to cross-desensitization of its Ca2+ response by fMLP and C5a, was equivalent to CXCR1. Regardless of the enhancement in certain receptor functions in M2CXCR1 compared with the wild type CXCR1, the mutated receptors mediated equivalent PLCβ3 phosphorylation and cross-desensitization of Ca2+ mobilization by FR, C5aR, and PAFR. The results herein indicate that phosphorylation of CXCR1 regulates some, but not all of the receptors functions. While receptor phosphorylation inhibits G protein turnover, PLC activation, Ca2+ mobilization and secretion, it is required for normal chemotaxis and receptor internalization. Since phosphorylation of CXCR1 had no effect on its ability to induce phosphorylation of PLCβ3 or to mediate class-desensitization, these activities may be mediated by independently regulated pathways

Thrombin Primes Responsiveness of Selective Chemoattractant Receptors at a Site Distal to G Protein Activation

To define the molecular basis of human chemoattractant receptor regulation, rat basophilic leukemia RBL-2H3 cells, which are thrombin- responsive, were transfected to stably express epitope-tagged receptors for C5a, interleukin-8 (IL-8), formylpeptides (e.g. N-formylmethionyl-leucyl- phenylalanine (fMLP)), and platelet-activating factor (PAF). Here we demonstrate that both thrombin and a synthetic peptide ligand for the thrombin receptor (sequence SFLLRN) caused phosphorylation and heterologous desensitization of the receptors for C5a, IL-8, and PAF but not that for formylpeptides as measured by agonist-stimulated [35S]guanosine 5\u27-3-O- (thio)triphosphate binding to membranes. Consistent with the PAF receptor phosphorylation, both thrombin and thrombin receptor peptide inhibited phosphoinositide hydrolysis, Ca2+ mobilization, and degranulation stimulated by PAF. Unexpectedly, despite heterologous desensitization at the level of receptor/G protein activation, there was enhancement (\u27priming\u27) by thrombin of subsequent activities stimulated by C5a and IL-8 as well as fMLP. The priming effect of thrombin was blocked by its inhibitor, hirudin. However, two other activators of the thrombin receptor, the peptide SFLLRN and trypsin, stimulated Ca2+ mobilization in RBL-2H3 cells but did not cause priming. In addition, SFLLRN and the thrombin receptor antagonist peptide FLLRN both inhibited thrombin-induced Ca2+ mobilization but not priming. Furthermore, the proteolytically active γ-thrombin, which does not stimulate the tethered ligand thrombin receptor and caused little or no Ca2+ mobilization in RBL-2H3 cells, effectively primed the response to fMLP. These data demonstrate that heterologous receptor phosphorylation and attenuation of G protein activation are not, by themselves, sufficient for the inhibition of biological responses mediated by C5a and IL-8. Moreover, thrombin appears to utilize mechanism(s) independent of its tethered ligand receptor to selectively prime phospholipase C-mediated biological responses of the C5a, IL-8, and formylpeptide receptors but not PAF. Because C5a, IL- 8, and formylpeptide activate phospholipase Cβ2, whereas PAF stimulates a different phospholipase C, the striking selectivity of thrombin\u27s priming may be mediated via its ability to enhance receptor-mediated activation of phospholipase Cβ2

Role of Phospholipase Cβ3 Phosphorylation in the Desensitization of Cellular Responses to Platelet-Activating Factor

Platelet-activating factor (PAF) stimulates a diverse array of cellular responses through receptors coupled to G proteins that activate phospholipase C (PLC). Truncation of the cytoplasmic tail of the receptor to remove phosphorylation sites (mutant PAF receptor, mPAFR) results in enhancement of PAF-stimulated responses. Here we demonstrate that PAF or phorbol 12- myristate 13-acetate (PMA) pretreatment inhibited wild type PAFR-induced PLC- mediated responses by ~90%, whereas these responses to the phosphorylation- deficient mPAFR were inhibited by ~50%, despite normal G protein coupling, suggesting a distal inhibitory locus. PAF and PMA, as well as a membrane permeable cyclic AMP analog, stimulated phosphorylation of PLCβ3. A protein kinase C (PKC) inhibitor blocked phosphorylation of PLCβ3 stimulated by PAF and PMA but not by cAMP. Activation of protein kinase A (PKA) by cAMP did not result in inhibition of Ca2+ mobilization stimulated by PAF. In contrast, cAMP did inhibit the response to formylpeptide chemoattractant receptor. These data suggest that homologous desensitization of PAF-mediated responses is regulated via phosphorylation at two levels in the signaling pathway, one at the receptor and the other at PLCβ3 mediated by PKC but not by PKA. Phosphorylation of PLCβ3 by PKA could explain the inhibition of formylpeptide chemoattractant receptor signaling by cAMP. As PAF and formylpeptide chemoattractant receptors activate PLC via different G proteins, phosphorylation of PLCβ3 by PKC and PKA could provide distinct regulatory control for classes of G protein-coupled receptors

Multiple Signaling Pathways of Human Interleukin-8 Receptor A: Independent Regulation by Phosphorylation

Interleukin-8 (IL-8) receptor A (CXCR1) couples to a pertussis toxin- sensitive G protein to mediate phospholipase Cβ (PLCβ) activation and cellular responses. Responses to CXCR1 are attenuated by prior exposure of neutrophils to either IL-8, a cleavage product of the fifth component of complement (C5a) or n-formylated peptides (formylmethionylleucylphenylalanine, fMLP). To characterize the role of receptor phosphorylation in the regulation of the CXCR1, a phosphorylation- deficient mutant, M2CXCR1, was constructed. This receptor, stably expressed in RBL-2H3 cells, coupled more efficiently to G protein and stimulated enhanced phosphoinositide hydrolysis, cAMP production, exocytosis, and phospholipase D activation, and was resistant to IL-8-induced receptor internalization. The rate and total amount of ligand stimulated actin polymerization remained unchanged, but interestingly, chemotaxis was decreased by ~30% compared with the wild type receptor. To study the role of receptor phosphorylation in cross-desensitization of chemoattractant receptors, M2CXCR1 was coexpressed with cDNAs encoding receptors for either fMLP (FR), C5a (C5aR), or platelet-activating factor (PAFR). Both C5aR and PAFR were cross-phosphorylated upon M2CXCR1 activation, resulting in attenuated guanosine 5\u27-3\u27-O-(thio)triphosphate (GTPγS) binding in membranes. In contrast, FR and M2CXCR1 were resistant to cross- phosphorylation and cross-inhibition of GTPγS binding by other receptors. Despite the resistance of M2CXCR1 to cross-phosphorylation and receptor/G protein uncoupling, its susceptibility to cross-desensitization of its Ca2+ response by fMLP and C5a, was equivalent to CXCR1. Regardless of the enhancement in certain receptor functions in M2CXCR1 compared with the wild type CXCR1, the mutated receptors mediated equivalent PLCβ3 phosphorylation and cross-desensitization of Ca2+ mobilization by FR, C5aR, and PAFR. The results herein indicate that phosphorylation of CXCR1 regulates some, but not all of the receptors functions. While receptor phosphorylation inhibits G protein turnover, PLC activation, Ca2+ mobilization and secretion, it is required for normal chemotaxis and receptor internalization. Since phosphorylation of CXCR1 had no effect on its ability to induce phosphorylation of PLCβ3 or to mediate class-desensitization, these activities may be mediated by independently regulated pathways

Differential Cross-Regulation of the Human Chemokine Receptors CXCR1 and CXCR2. Evidence for Time-Dependent Signal Generation

Neutrophils and transfected RBL-2H3 cells were used to investigate the mechanism of cross-regulation of the human interleukin-8 (IL-8) receptors CXCR1 and CXCR2 by chemoattractants. In neutrophils, Ca2+ mobilization by the CXCR2-specific chemokine, growth-related oncogene α (Groα), was desensitized by prior exposure to the chemoattractants N-formylated peptides (fMLP) or a complement cleavage product (C5a). In contrast, growth-related oncogene α did not desensitize the latter receptors. To investigate this phenomenon, CXCR2 was stably expressed in RBL-2H3 cells and mediated phosphoinositide hydrolysis, Ca2+ mobilization, chemotaxis, and secretion. In cells co-expressing CXCR2 and receptors for either C5a (C5aR) or fMLP (FR), CXCR2 was cross-phosphorylated and cross-desensitized by C5a and fMLP. However, neither C5aR nor FR was cross-phosphorylated or cross-desensitized by CXCR2 activation, although CXCR1 did mediate this process. Receptor internalization induced by IL-8 was more rapid and occurred at lower doses with CXCR2 than CXCR1, although both receptors mediated equipotent chemotaxis and exocytosis in RBL. Truncation of the cytoplasmic tail of CXCR2 (331T) prolonged its signaling relative to CXCR2, increased its resistance to internalization, and induced phospholipase D activation. 331T was resistant to homologous phosphorylation and cross-phosphorylation but not cross- desensitization of its Ca2+ mobilization by fMLP or C5a, indicating an inhibitory site distal to receptor/G protein coupling. In contrast to CXCR2, stimulation of 331T cross-desensitized Ca2+ mobilization by both FR and C5aR. CXCR2 and the mutant 331T induced phospholipase C β3 phosphorylation to an extent equivalent to that of CXCR1. Taken together, these results suggest that CXCR1 and CXCR2 bind IL-8 to produce a group of equipotent responses, but their ability to generate other signals, including receptor internalization, cross-desensitization, and phospholipase D activation, are very different. The latter phenomena apparently require prolonged receptor activation, which in the case of CXCR2 is precluded by rapid receptor phosphorylation and internalization. Thus, receptors coupling to identical G proteins may trigger different cellular responses dependent on the length of their signaling time, which can be regulated by receptor phosphorylation

Chemoattractant Receptors Activate Distinct Pathways for Chemotaxis and Secretion. Role of G-Protein Usage

Human leukocyte chemoattractant receptors activate chemotactic and cytotoxic pathways to varying degrees and also activate different G-proteins depending on the receptor and the cell-type. To determine the relationship between G-protein usage and the biological and biochemical responses activated, receptors for the chemoattractants formyl peptides (FR), platelet- activating factor (PAFR), and leukotriene B4 (BLTR) were transfected into RBL-2H3 cells. Pertussis toxin (Ptx) served as a Gα(i) inhibitor. These receptors were chosen to represent the spectrum of G(i) usage as Ptx had differential effects on their ability to induce calcium mobilization, phosphoinositide hydrolysis, and exocytosis with complete inhibition of all responses by FR, intermediate effects on BLTR, and little effect on PAFR. Ptx did not affect ligand-induced phosphorylation of PAFR and BLTR but inhibited phosphorylation of FR. In contrast, chemotaxis to formylmethionylleucylphenylalanine, leukotriene B4, and platelet-activating factor was completely blocked by Ptx. Wortmannin, a phosphotidylinositol 3- kinase inhibitor, also completely blocked ligand-induced chemotaxis by all receptors but did not affect calcium mobilization or phosphoinositide hydrolysis; however, it partially blocked the exocytosis response to formylmethionylleucylphenylalanine and the platelet-activating factor. Membrane ruffling and pseudopod extension via the BLTR was also completely inhibited by both Ptx and wortmannin. These data suggest that of the chemoattractant receptors studied, G-protein usage varies with FR being totally dependent on G(i), whereas BLTR and PAFR utilize both G(i) and a Ptx- insensitive G-protein. Both Ptx-sensitive and -insensitive G-protein usage can mediate the activation of phospholipase C, mobilization of intracellular calcium, and exocytosis by chemoattractant receptors. Chemotaxis, however, had an absolute requirement for a G(i)-mediated pathway

Chemoattractant Receptor-Induced Phosphorylation of L-Selectin

The selectin adhesion molecules and chemoattractant receptors synergistically regulate leukocyte migration into lymphoid tissues and sites of inflammation, but little is known about how these families of receptors modulate each other\u27s function. In this study, L-selectin was found to be phosphorylated in lymphoblastoid cell lines, and phosphorylation was enhanced by phorbol ester (phorbol 12-myristate 13-acetate (PMA)) treatment. Interactions between L-selectin and chemoattractant receptors were therefore examined using transfected rat basophilic leukemia cell lines (RBL-2H3) that expressed human L-selectin along with human leukocyte chemoattractant receptors. L-selectin was rapidly phosphorylated in cells treated with chemoattractants, thrombin, IgE receptor agonists, or PMA. Pertussis toxin or the protein kinase C inhibitor, staurosporine, completely blocked chemoattractant receptor-induced phosphorylation of L-selectin. PMA-induced phosphorylation was on serine residues within the cytoplasmic tail of L- selectin that have been well conserved during recent evolution. Although L- selectin phosphorylation was not essential for basal levels of adhesion through L-selectin in transformed cell lines, the rapid increase in ligand binding activity of L-selectin that occurs following leukocyte activation was blocked by staurosporine. These results demonstrate that L-selectin can be phosphorylated following engagement of chemoattractant receptors and suggest that this may be a physiologically relevant mechanism for the synergistic regulation of these receptors during leukocyte migration

Differential Regulation of Formyl Peptide and Platelet-Activating Factor Receptors: Role of Phospholipase Cβ3 Phosphorylation by Protein Kinase A

Formylated peptides (e.g. n-formyl-Met-Leu-Phe (fMLP)) and platelet- activating factor (PAF) mediate chemotactic and cytotoxic responses in leukocytes through receptors coupled to G proteins that activate phospholipase C (PLC). In RBL-2H3 cells, fMLP utilizes a pertussis toxin (ptx)-sensitive G protein to activate PLC, whereas PAF utilizes a ptx- insensitive G protein. Here we demonstrate that fMLP, but not PAF, enhanced intracellular cAMP levels via a ptx-sensitive mechanism. Protein kinase A (PKA) inhibition by H-89 enhanced inositol phosphate formation stimulated by fMLP but not PAF. Furthermore, a membrane-permeable cAMP analog 8-(4- chlorophenylthio)-cAMP (cpt-cAMP) inhibited phosphoinositide hydrolysis and secretion stimulated by fMLP but not PAF. Both cpt-cAMP and fMLP stimulated PLCβ3 phosphorylation in intact RBL cells. The purified catalytic subunit of PKA phosphorylated PLCβ3 immunoprecipitated from RBL cell lysate. Pretreatment of intact cells with cpt-cAMP and fMLP, but not PAF, resulted in an inhibition of subsequent PLCβ3 phosphorylation by PKA in vitro. These data demonstrate that fMLP receptor, which couples to a ptx-sensitive G protein, activates both PLC and cAMP production. The resulting PKA activation phosphorylates PLCβ3 and appears to block the ability of G(βγ) to activate PLC. Thus, both fMLP and PAF generate stimulatory signals for PLCβ3, but only fMLP produces a PKA-dependent inhibitory signal. This suggests a novel mechanism for the bidirectional regulation of receptors which activate PLC by ptx-sensitive G proteins