Chemoattractant Receptors Activate Distinct Pathways for Chemotaxis and Secretion
1999; Elsevier BV; Volume: 274; Issue: 52 Linguagem: Inglês
10.1074/jbc.274.52.37087
ISSN1083-351X
AutoresBodduluri Haribabu, Doncho V. Zhelev, Bryan C. Pridgen, Ricardo M. Richardson, Hydar Ali, Ralph Snyderman,
Tópico(s)Neuropeptides and Animal Physiology
ResumoHuman 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 Gαiinhibitor. These receptors were chosen to represent the spectrum of Gi 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 Gi, whereas BLTR and PAFR utilize both Gi 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 Gi-mediated pathway. 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 Gαiinhibitor. These receptors were chosen to represent the spectrum of Gi 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 Gi, whereas BLTR and PAFR utilize both Gi 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 Gi-mediated pathway. formylmethionylleucylphenylalanine leukotriene B4receptor leukotriene B4 rat basophilic leukemia platelet-activating factor pertussis toxin phosphotidylinositol 3-kinase formyl receptor platelet-activating factor receptor Migration of leukocytes to sites of inflammation is mediated via the activation of G-protein-coupled chemoattractant receptors (1Snyderman R. Uhing R.J. Gallin J.I. Snyderman R. Inflammation: Basic Principles and Clinical Correlates. 3rd Ed. Lippincot Williams & Wilkins, Philadelphia1999: 607-626Google Scholar, 2Gerard C. Gerard N.P. Curr. Opin. Immunol. 1994; 6: 140-145Crossref PubMed Scopus (115) Google Scholar). Chemoattractants at low concentrations elicit shape change, pseudopod extension, and chemotaxis, and at higher doses; many of them also trigger degranulation and generation of superoxide anions (1Snyderman R. Uhing R.J. Gallin J.I. Snyderman R. Inflammation: Basic Principles and Clinical Correlates. 3rd Ed. Lippincot Williams & Wilkins, Philadelphia1999: 607-626Google Scholar, 3Baggiolini M. Kernen P. Deranleau D.A. Dewald B. Biochem. Soc. Trans. 1991; 19: 55-59Crossref PubMed Scopus (15) Google Scholar). Pathways leading to these activities have been shown to have different dose requirements, kinetics and regulation (4Snyderman R. Smith C.D. Verghese M.W. J. Leukocyte Biol. 1986; 40: 785-800Crossref PubMed Scopus (131) Google Scholar, 5Truett III, A.P. Verghese M.W. Dillon S.B. Snyderman R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1549-1553Crossref PubMed Scopus (73) Google Scholar), but the role of G-protein usage remains unknown.Formylpeptides (fMLP),1platelet-activating factor (PAF), and leukotriene B4(LTB4) are potent chemoattractants for neutrophils and to varying degrees also activate exocytosis and generation of superoxide anions (3Baggiolini M. Kernen P. Deranleau D.A. Dewald B. Biochem. Soc. Trans. 1991; 19: 55-59Crossref PubMed Scopus (15) Google Scholar, 4Snyderman R. Smith C.D. Verghese M.W. J. Leukocyte Biol. 1986; 40: 785-800Crossref PubMed Scopus (131) Google Scholar, 6Samuelsson B. Dahlen S.E. Lindgren J.A. Rouzer C.A. Serhan C.N. Science. 1987; 237: 1171-1176Crossref PubMed Scopus (1970) Google Scholar). These activities are mediated through G-protein-coupled receptors (FR, PAFR, and BLTR) (2Gerard C. Gerard N.P. Curr. Opin. Immunol. 1994; 6: 140-145Crossref PubMed Scopus (115) Google Scholar, 7Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (847) Google Scholar). G-protein usage of chemoattractant receptors was known to be different depending on cell types (1Snyderman R. Uhing R.J. Gallin J.I. Snyderman R. Inflammation: Basic Principles and Clinical Correlates. 3rd Ed. Lippincot Williams & Wilkins, Philadelphia1999: 607-626Google Scholar, 8Wu D. LaRosa G.J. Simon M.I. Science. 1993; 261: 101-103Crossref PubMed Scopus (332) Google Scholar, 9Kuang Y.N. Wu Y.P. Jiang H.P. Wu D.Q. J. Biol. Chem. 1996; 271: 3975-3978Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 10Arai H. Charo I.F. J. Biol. Chem. 1996; 271: 21814-21819Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Previous studies in RBL cells indicated that FR activated Gi, whereas PAFR utilized both Giand a Ptx-insensitive G-protein to activate phosphoinositide hydrolysis, calcium mobilization, and exocytosis (11Ali H. Richardson R.M. Tomhave E.D. Didsbury J.R. Snyderman R. J. Biol. Chem. 1993; 268: 24247-24254Abstract Full Text PDF PubMed Google Scholar, 12Ali H. Richardson R.M. Tomhave E.D. DuBose R.A. Haribabu B. Snyderman R. J. Biol. Chem. 1994; 269: 24557-24563Abstract Full Text PDF PubMed Google Scholar). However, the G-protein usage requirements for activating chemotaxisversus exocytosis in these cells were unknown. Therefore, we sought to determine the relationship between G-protein usageversus the subsequent responses activated by these receptors in a single cell line viz. RBL-2H3 cells. Epitope-tagged BLTR, FR, and PAFR were expressed in RBL-2H3 cells, and inhibitors of signaling through Gi proteins (Ptx) and the PI3 kinase pathway (wortmannin) were used to determine the role of these pathways in pseudopod extension, chemotaxis, phospholipase C activation, calcium mobilization, and exocytosis. These data demonstrate distinct G-protein usage among chemoattractant receptors and suggest that a Gi-mediated pathway, presumably involving βγ and PI3 kinase, is required for motility-related functions. On the other hand, stimulation of phospholipase C activity, calcium mobilization, and exocytosis can be mediated through activation of a Ptx-insensitive G-protein as well as through Gi.DISCUSSIONExpression of chemoattractant receptors that activate different G-proteins in a single cell line allowed the determination of the role of specific G-proteins in activating distinct biological responses. The data show that chemoattractant receptors may activate phospholipase C, calcium mobilization, and exocytosis through multiple G-proteins, but ligand-stimulated pseudopod extension and chemotaxis require the activation of a Ptx-sensitive G-protein and PI3 kinase.Comparison of biochemical responses activated by FR, BLTR, and PAFR, all of which activate Gi, but couple to varying degrees to a Ptx-insensitive G-protein, allowed clear distinctions to be made for the role of G-proteins in different activities. Previous studies have indicated that receptor occupancy, but not signaling through G-proteins, is essential for receptor phosphorylation (20Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1060) Google Scholar, 21Prossnitz E.R. Gilbert T.L. Chiang S. Campbell J.J. Qin S. Newman W. Sklar L.A. Ye R.D. Biochemistry. 1999; 38: 2240-2247Crossref PubMed Scopus (40) Google Scholar). Whereas the PAFR and BLTR were phosphorylated to similar levels in Ptx-treated cells, FR phosphorylation was inhibited. These data suggest that FR is likely phosphorylated by G-protein-coupled receptor kinases GRK2 or GRK3, because both require free βγ for translocation to membrane and activation (20Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1060) Google Scholar). Studies on in vitro phosphorylation of the C terminus of FR also support this contention (22Prossnitz E.R. Kim C.M. Benovic J.L. Ye R.D. J. Biol. Chem. 1995; 270: 1130-1137Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Lack of any affect, by Ptx, on ligand-induced receptor phosphorylation of PAFR and BLTR indicates that signal transduction through the Ptx-sensitive G-protein is not required for this event.Previous studies in neutrophils and in transfected cell lines indicated that all FR responses are completely Ptx-sensitive (1Snyderman R. Uhing R.J. Gallin J.I. Snyderman R. Inflammation: Basic Principles and Clinical Correlates. 3rd Ed. Lippincot Williams & Wilkins, Philadelphia1999: 607-626Google Scholar, 11Ali H. Richardson R.M. Tomhave E.D. Didsbury J.R. Snyderman R. J. Biol. Chem. 1993; 268: 24247-24254Abstract Full Text PDF PubMed Google Scholar, 23Verghese M.W. Charles L. Jakoi L. Dillon S.B. Snyderman R. J. Immunol. 1987; 138: 4374-4380PubMed Google Scholar). In contrast, both PAFR and BLTR can activate Ptx-insensitive G-proteins as well as Gi (7Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (847) Google Scholar, 23Verghese M.W. Charles L. Jakoi L. Dillon S.B. Snyderman R. J. Immunol. 1987; 138: 4374-4380PubMed Google Scholar, 24Amatruda III, T.T. Gerard N.P. Gerard C. Simon M.I. J. Biol. Chem. 1993; 268: 10139-10144Abstract Full Text PDF PubMed Google Scholar). Our previous observations in RBL cells showed that calcium mobilization was only marginally affected and phosphotidylinositol hydrolysis was partially inhibited by Ptx, which suggests that PAFR activate a Ptx-insensitive G-protein in RBL cells (12Ali H. Richardson R.M. Tomhave E.D. DuBose R.A. Haribabu B. Snyderman R. J. Biol. Chem. 1994; 269: 24557-24563Abstract Full Text PDF PubMed Google Scholar). PAFR activated sufficient levels of Ptx-insensitive G-protein in these cells to cause exocytosis. The current studies show that like PAFR, BLTR expressed in RBL cells could utilize a Ptx-insensitive G-protein to activate phosphotidylinositol hydrolysis and mobilize intracellular calcium. However, the most significant finding of the current study is that despite activating different G-proteins all three receptors displayed an absolute requirement for Gi to activate chemotaxis. This suggests that signal transduction pathways activated through Ptx-insensitive G-proteins are not sufficient, and the activation of a Gi-mediated event is essential for chemotaxis. Recent studies also indicate that βγ-mediated responses are essential for cell migration (25Neptune E.R. Bourne H.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14489-14494Crossref PubMed Scopus (238) Google Scholar, 26Arai H. Tsou C.L. Charo I.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14495-14499Crossref PubMed Scopus (148) Google Scholar, 27Neptune E.R. Iiri T. Bourne H.R. J. Biol. Chem. 1999; 274: 2824-2828Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). The data presented herein indicates that this βγ must come from the activation of a Ptx-sensitive G-protein, presumably Gi. The βγ released from a Ptx-insensitive G-protein by the same chemoattractant receptor was not sufficient for chemotaxis.The single cell manipulation assay has provided a means to correlate biochemical and motility-related cellular responses. The ability of RBL cells to extend a pseudopod similar to the one produced by leukocytes suggests molecular events in the migration of leukocytes and transfected RBL cells are related (19Zhelev D.V. Alteraifi A.M. Hochmuth R.M. Cell Motil. Cytoskeleton. 1996; 35: 331-344Crossref PubMed Scopus (22) Google Scholar). Whereas cell lines of non-lymphoid origin like Chinese hamster ovary cells and HEK 293 cells also undergo chemotaxis (7Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (847) Google Scholar, 25Neptune E.R. Bourne H.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14489-14494Crossref PubMed Scopus (238) Google Scholar), it is not known whether they use similar pathways or cytoskeletal processes as leukocytes for migration. In wortmannin-treated RBL cells, the low level of chemotactic responsiveness was associated with weak membrane ruffling and no pseudopod extension (Fig. 5). These results again underscore the dissociation of chemotaxis from the calcium release and exocytosis, which remain unaffected by wortmannin.The unique biochemical pathways activated by the βγ subunits of Gi proteins that mediate chemotaxis are at present unknown. The βγ induced PI3 kinase activity, and its related signaling pathway could be one of these events (28Hartwig J.H. Bokoch G.M. Carpenter C.L. Janmey P.A. Taylor L.A. Toker A. Stossel T.P. Cell. 1995; 82: 643-653Abstract Full Text PDF PubMed Scopus (606) Google Scholar, 29Knall C. Worthen G.S. Johnson G.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3052-3057Crossref PubMed Scopus (270) Google Scholar, 30Keely P.J. Westwick J.K. Whitehead I.P. Der C.J. Parise L.V. Nature. 1997; 390: 632-636Crossref PubMed Scopus (649) Google Scholar). Other studies also indicated that GTPγS could activate actin polymerization in soluble cell extracts of leukocytes in the presence of the low molecular weight G-protein CDC42 but not Rho or Rac (31Tapon N. Hall A. Curr. Opin. Cell Biol. 1997; 9: 86-92Crossref PubMed Scopus (690) Google Scholar, 32Zigmond S.H. Joyce M. Borleis J. Bokoch G.M. Devreotes P.N. J. Cell Biol. 1997; 138: 363-374Crossref PubMed Scopus (145) Google Scholar). Therefore it is possible that βγ dimers released from Gi, but not the Gq family of G-proteins, activate one or more low molecular weight G-proteins that may participate in leukocyte migration. In any case, the data clearly demonstrate that chemoattractant receptors may couple to individual or multiple G-proteins. To stimulate chemotaxis however, they must activate Gi. Moreover, distinct signaling pathways among G-proteins are likely to determine other cellular responses initiated by chemoattractant receptors. These data may explain numerous observations in neutrophils and other cells that different chemoattractant receptors display different abilities to trigger nonmotility-related functions (i.e. respiratory burst, mitogenesis) despite exhibiting similar chemotactic responses (1Snyderman R. Uhing R.J. Gallin J.I. Snyderman R. Inflammation: Basic Principles and Clinical Correlates. 3rd Ed. Lippincot Williams & Wilkins, Philadelphia1999: 607-626Google Scholar, 23Verghese M.W. Charles L. Jakoi L. Dillon S.B. Snyderman R. J. Immunol. 1987; 138: 4374-4380PubMed Google Scholar). The availability of a model system in which migration responses may be measured both at population and single cell levels will allow further investigation of the divergent pathways for motility and cytotoxic functions. Migration of leukocytes to sites of inflammation is mediated via the activation of G-protein-coupled chemoattractant receptors (1Snyderman R. Uhing R.J. Gallin J.I. Snyderman R. Inflammation: Basic Principles and Clinical Correlates. 3rd Ed. Lippincot Williams & Wilkins, Philadelphia1999: 607-626Google Scholar, 2Gerard C. Gerard N.P. Curr. Opin. Immunol. 1994; 6: 140-145Crossref PubMed Scopus (115) Google Scholar). Chemoattractants at low concentrations elicit shape change, pseudopod extension, and chemotaxis, and at higher doses; many of them also trigger degranulation and generation of superoxide anions (1Snyderman R. Uhing R.J. Gallin J.I. Snyderman R. Inflammation: Basic Principles and Clinical Correlates. 3rd Ed. Lippincot Williams & Wilkins, Philadelphia1999: 607-626Google Scholar, 3Baggiolini M. Kernen P. Deranleau D.A. Dewald B. Biochem. Soc. Trans. 1991; 19: 55-59Crossref PubMed Scopus (15) Google Scholar). Pathways leading to these activities have been shown to have different dose requirements, kinetics and regulation (4Snyderman R. Smith C.D. Verghese M.W. J. Leukocyte Biol. 1986; 40: 785-800Crossref PubMed Scopus (131) Google Scholar, 5Truett III, A.P. Verghese M.W. Dillon S.B. Snyderman R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1549-1553Crossref PubMed Scopus (73) Google Scholar), but the role of G-protein usage remains unknown. Formylpeptides (fMLP),1platelet-activating factor (PAF), and leukotriene B4(LTB4) are potent chemoattractants for neutrophils and to varying degrees also activate exocytosis and generation of superoxide anions (3Baggiolini M. Kernen P. Deranleau D.A. Dewald B. Biochem. Soc. Trans. 1991; 19: 55-59Crossref PubMed Scopus (15) Google Scholar, 4Snyderman R. Smith C.D. Verghese M.W. J. Leukocyte Biol. 1986; 40: 785-800Crossref PubMed Scopus (131) Google Scholar, 6Samuelsson B. Dahlen S.E. Lindgren J.A. Rouzer C.A. Serhan C.N. Science. 1987; 237: 1171-1176Crossref PubMed Scopus (1970) Google Scholar). These activities are mediated through G-protein-coupled receptors (FR, PAFR, and BLTR) (2Gerard C. Gerard N.P. Curr. Opin. Immunol. 1994; 6: 140-145Crossref PubMed Scopus (115) Google Scholar, 7Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (847) Google Scholar). G-protein usage of chemoattractant receptors was known to be different depending on cell types (1Snyderman R. Uhing R.J. Gallin J.I. Snyderman R. Inflammation: Basic Principles and Clinical Correlates. 3rd Ed. Lippincot Williams & Wilkins, Philadelphia1999: 607-626Google Scholar, 8Wu D. LaRosa G.J. Simon M.I. Science. 1993; 261: 101-103Crossref PubMed Scopus (332) Google Scholar, 9Kuang Y.N. Wu Y.P. Jiang H.P. Wu D.Q. J. Biol. Chem. 1996; 271: 3975-3978Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 10Arai H. Charo I.F. J. Biol. Chem. 1996; 271: 21814-21819Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Previous studies in RBL cells indicated that FR activated Gi, whereas PAFR utilized both Giand a Ptx-insensitive G-protein to activate phosphoinositide hydrolysis, calcium mobilization, and exocytosis (11Ali H. Richardson R.M. Tomhave E.D. Didsbury J.R. Snyderman R. J. Biol. Chem. 1993; 268: 24247-24254Abstract Full Text PDF PubMed Google Scholar, 12Ali H. Richardson R.M. Tomhave E.D. DuBose R.A. Haribabu B. Snyderman R. J. Biol. Chem. 1994; 269: 24557-24563Abstract Full Text PDF PubMed Google Scholar). However, the G-protein usage requirements for activating chemotaxisversus exocytosis in these cells were unknown. Therefore, we sought to determine the relationship between G-protein usageversus the subsequent responses activated by these receptors in a single cell line viz. RBL-2H3 cells. Epitope-tagged BLTR, FR, and PAFR were expressed in RBL-2H3 cells, and inhibitors of signaling through Gi proteins (Ptx) and the PI3 kinase pathway (wortmannin) were used to determine the role of these pathways in pseudopod extension, chemotaxis, phospholipase C activation, calcium mobilization, and exocytosis. These data demonstrate distinct G-protein usage among chemoattractant receptors and suggest that a Gi-mediated pathway, presumably involving βγ and PI3 kinase, is required for motility-related functions. On the other hand, stimulation of phospholipase C activity, calcium mobilization, and exocytosis can be mediated through activation of a Ptx-insensitive G-protein as well as through Gi. DISCUSSIONExpression of chemoattractant receptors that activate different G-proteins in a single cell line allowed the determination of the role of specific G-proteins in activating distinct biological responses. The data show that chemoattractant receptors may activate phospholipase C, calcium mobilization, and exocytosis through multiple G-proteins, but ligand-stimulated pseudopod extension and chemotaxis require the activation of a Ptx-sensitive G-protein and PI3 kinase.Comparison of biochemical responses activated by FR, BLTR, and PAFR, all of which activate Gi, but couple to varying degrees to a Ptx-insensitive G-protein, allowed clear distinctions to be made for the role of G-proteins in different activities. Previous studies have indicated that receptor occupancy, but not signaling through G-proteins, is essential for receptor phosphorylation (20Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1060) Google Scholar, 21Prossnitz E.R. Gilbert T.L. Chiang S. Campbell J.J. Qin S. Newman W. Sklar L.A. Ye R.D. Biochemistry. 1999; 38: 2240-2247Crossref PubMed Scopus (40) Google Scholar). Whereas the PAFR and BLTR were phosphorylated to similar levels in Ptx-treated cells, FR phosphorylation was inhibited. These data suggest that FR is likely phosphorylated by G-protein-coupled receptor kinases GRK2 or GRK3, because both require free βγ for translocation to membrane and activation (20Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1060) Google Scholar). Studies on in vitro phosphorylation of the C terminus of FR also support this contention (22Prossnitz E.R. Kim C.M. Benovic J.L. Ye R.D. J. Biol. Chem. 1995; 270: 1130-1137Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Lack of any affect, by Ptx, on ligand-induced receptor phosphorylation of PAFR and BLTR indicates that signal transduction through the Ptx-sensitive G-protein is not required for this event.Previous studies in neutrophils and in transfected cell lines indicated that all FR responses are completely Ptx-sensitive (1Snyderman R. Uhing R.J. Gallin J.I. Snyderman R. Inflammation: Basic Principles and Clinical Correlates. 3rd Ed. Lippincot Williams & Wilkins, Philadelphia1999: 607-626Google Scholar, 11Ali H. Richardson R.M. Tomhave E.D. Didsbury J.R. Snyderman R. J. Biol. Chem. 1993; 268: 24247-24254Abstract Full Text PDF PubMed Google Scholar, 23Verghese M.W. Charles L. Jakoi L. Dillon S.B. Snyderman R. J. Immunol. 1987; 138: 4374-4380PubMed Google Scholar). In contrast, both PAFR and BLTR can activate Ptx-insensitive G-proteins as well as Gi (7Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (847) Google Scholar, 23Verghese M.W. Charles L. Jakoi L. Dillon S.B. Snyderman R. J. Immunol. 1987; 138: 4374-4380PubMed Google Scholar, 24Amatruda III, T.T. Gerard N.P. Gerard C. Simon M.I. J. Biol. Chem. 1993; 268: 10139-10144Abstract Full Text PDF PubMed Google Scholar). Our previous observations in RBL cells showed that calcium mobilization was only marginally affected and phosphotidylinositol hydrolysis was partially inhibited by Ptx, which suggests that PAFR activate a Ptx-insensitive G-protein in RBL cells (12Ali H. Richardson R.M. Tomhave E.D. DuBose R.A. Haribabu B. Snyderman R. J. Biol. Chem. 1994; 269: 24557-24563Abstract Full Text PDF PubMed Google Scholar). PAFR activated sufficient levels of Ptx-insensitive G-protein in these cells to cause exocytosis. The current studies show that like PAFR, BLTR expressed in RBL cells could utilize a Ptx-insensitive G-protein to activate phosphotidylinositol hydrolysis and mobilize intracellular calcium. However, the most significant finding of the current study is that despite activating different G-proteins all three receptors displayed an absolute requirement for Gi to activate chemotaxis. This suggests that signal transduction pathways activated through Ptx-insensitive G-proteins are not sufficient, and the activation of a Gi-mediated event is essential for chemotaxis. Recent studies also indicate that βγ-mediated responses are essential for cell migration (25Neptune E.R. Bourne H.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14489-14494Crossref PubMed Scopus (238) Google Scholar, 26Arai H. Tsou C.L. Charo I.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14495-14499Crossref PubMed Scopus (148) Google Scholar, 27Neptune E.R. Iiri T. Bourne H.R. J. Biol. Chem. 1999; 274: 2824-2828Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). The data presented herein indicates that this βγ must come from the activation of a Ptx-sensitive G-protein, presumably Gi. The βγ released from a Ptx-insensitive G-protein by the same chemoattractant receptor was not sufficient for chemotaxis.The single cell manipulation assay has provided a means to correlate biochemical and motility-related cellular responses. The ability of RBL cells to extend a pseudopod similar to the one produced by leukocytes suggests molecular events in the migration of leukocytes and transfected RBL cells are related (19Zhelev D.V. Alteraifi A.M. Hochmuth R.M. Cell Motil. Cytoskeleton. 1996; 35: 331-344Crossref PubMed Scopus (22) Google Scholar). Whereas cell lines of non-lymphoid origin like Chinese hamster ovary cells and HEK 293 cells also undergo chemotaxis (7Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (847) Google Scholar, 25Neptune E.R. Bourne H.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14489-14494Crossref PubMed Scopus (238) Google Scholar), it is not known whether they use similar pathways or cytoskeletal processes as leukocytes for migration. In wortmannin-treated RBL cells, the low level of chemotactic responsiveness was associated with weak membrane ruffling and no pseudopod extension (Fig. 5). These results again underscore the dissociation of chemotaxis from the calcium release and exocytosis, which remain unaffected by wortmannin.The unique biochemical pathways activated by the βγ subunits of Gi proteins that mediate chemotaxis are at present unknown. The βγ induced PI3 kinase activity, and its related signaling pathway could be one of these events (28Hartwig J.H. Bokoch G.M. Carpenter C.L. Janmey P.A. Taylor L.A. Toker A. Stossel T.P. Cell. 1995; 82: 643-653Abstract Full Text PDF PubMed Scopus (606) Google Scholar, 29Knall C. Worthen G.S. Johnson G.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3052-3057Crossref PubMed Scopus (270) Google Scholar, 30Keely P.J. Westwick J.K. Whitehead I.P. Der C.J. Parise L.V. Nature. 1997; 390: 632-636Crossref PubMed Scopus (649) Google Scholar). Other studies also indicated that GTPγS could activate actin polymerization in soluble cell extracts of leukocytes in the presence of the low molecular weight G-protein CDC42 but not Rho or Rac (31Tapon N. Hall A. Curr. Opin. Cell Biol. 1997; 9: 86-92Crossref PubMed Scopus (690) Google Scholar, 32Zigmond S.H. Joyce M. Borleis J. Bokoch G.M. Devreotes P.N. J. Cell Biol. 1997; 138: 363-374Crossref PubMed Scopus (145) Google Scholar). Therefore it is possible that βγ dimers released from Gi, but not the Gq family of G-proteins, activate one or more low molecular weight G-proteins that may participate in leukocyte migration. In any case, the data clearly demonstrate that chemoattractant receptors may couple to individual or multiple G-proteins. To stimulate chemotaxis however, they must activate Gi. Moreover, distinct signaling pathways among G-proteins are likely to determine other cellular responses initiated by chemoattractant receptors. These data may explain numerous observations in neutrophils and other cells that different chemoattractant receptors display different abilities to trigger nonmotility-related functions (i.e. respiratory burst, mitogenesis) despite exhibiting similar chemotactic responses (1Snyderman R. Uhing R.J. Gallin J.I. Snyderman R. Inflammation: Basic Principles and Clinical Correlates. 3rd Ed. Lippincot Williams & Wilkins, Philadelphia1999: 607-626Google Scholar, 23Verghese M.W. Charles L. Jakoi L. Dillon S.B. Snyderman R. J. Immunol. 1987; 138: 4374-4380PubMed Google Scholar). The availability of a model system in which migration responses may be measured both at population and single cell levels will allow further investigation of the divergent pathways for motility and cytotoxic functions. Expression of chemoattractant receptors that activate different G-proteins in a single cell line allowed the determination of the role of specific G-proteins in activating distinct biological responses. The data show that chemoattractant receptors may activate phospholipase C, calcium mobilization, and exocytosis through multiple G-proteins, but ligand-stimulated pseudopod extension and chemotaxis require the activation of a Ptx-sensitive G-protein and PI3 kinase. Comparison of biochemical responses activated by FR, BLTR, and PAFR, all of which activate Gi, but couple to varying degrees to a Ptx-insensitive G-protein, allowed clear distinctions to be made for the role of G-proteins in different activities. Previous studies have indicated that receptor occupancy, but not signaling through G-proteins, is essential for receptor phosphorylation (20Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1060) Google Scholar, 21Prossnitz E.R. Gilbert T.L. Chiang S. Campbell J.J. Qin S. Newman W. Sklar L.A. Ye R.D. Biochemistry. 1999; 38: 2240-2247Crossref PubMed Scopus (40) Google Scholar). Whereas the PAFR and BLTR were phosphorylated to similar levels in Ptx-treated cells, FR phosphorylation was inhibited. These data suggest that FR is likely phosphorylated by G-protein-coupled receptor kinases GRK2 or GRK3, because both require free βγ for translocation to membrane and activation (20Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1060) Google Scholar). Studies on in vitro phosphorylation of the C terminus of FR also support this contention (22Prossnitz E.R. Kim C.M. Benovic J.L. Ye R.D. J. Biol. Chem. 1995; 270: 1130-1137Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Lack of any affect, by Ptx, on ligand-induced receptor phosphorylation of PAFR and BLTR indicates that signal transduction through the Ptx-sensitive G-protein is not required for this event. Previous studies in neutrophils and in transfected cell lines indicated that all FR responses are completely Ptx-sensitive (1Snyderman R. Uhing R.J. Gallin J.I. Snyderman R. Inflammation: Basic Principles and Clinical Correlates. 3rd Ed. Lippincot Williams & Wilkins, Philadelphia1999: 607-626Google Scholar, 11Ali H. Richardson R.M. Tomhave E.D. Didsbury J.R. Snyderman R. J. Biol. Chem. 1993; 268: 24247-24254Abstract Full Text PDF PubMed Google Scholar, 23Verghese M.W. Charles L. Jakoi L. Dillon S.B. Snyderman R. J. Immunol. 1987; 138: 4374-4380PubMed Google Scholar). In contrast, both PAFR and BLTR can activate Ptx-insensitive G-proteins as well as Gi (7Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (847) Google Scholar, 23Verghese M.W. Charles L. Jakoi L. Dillon S.B. Snyderman R. J. Immunol. 1987; 138: 4374-4380PubMed Google Scholar, 24Amatruda III, T.T. Gerard N.P. Gerard C. Simon M.I. J. Biol. Chem. 1993; 268: 10139-10144Abstract Full Text PDF PubMed Google Scholar). Our previous observations in RBL cells showed that calcium mobilization was only marginally affected and phosphotidylinositol hydrolysis was partially inhibited by Ptx, which suggests that PAFR activate a Ptx-insensitive G-protein in RBL cells (12Ali H. Richardson R.M. Tomhave E.D. DuBose R.A. Haribabu B. Snyderman R. J. Biol. Chem. 1994; 269: 24557-24563Abstract Full Text PDF PubMed Google Scholar). PAFR activated sufficient levels of Ptx-insensitive G-protein in these cells to cause exocytosis. The current studies show that like PAFR, BLTR expressed in RBL cells could utilize a Ptx-insensitive G-protein to activate phosphotidylinositol hydrolysis and mobilize intracellular calcium. However, the most significant finding of the current study is that despite activating different G-proteins all three receptors displayed an absolute requirement for Gi to activate chemotaxis. This suggests that signal transduction pathways activated through Ptx-insensitive G-proteins are not sufficient, and the activation of a Gi-mediated event is essential for chemotaxis. Recent studies also indicate that βγ-mediated responses are essential for cell migration (25Neptune E.R. Bourne H.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14489-14494Crossref PubMed Scopus (238) Google Scholar, 26Arai H. Tsou C.L. Charo I.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14495-14499Crossref PubMed Scopus (148) Google Scholar, 27Neptune E.R. Iiri T. Bourne H.R. J. Biol. Chem. 1999; 274: 2824-2828Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). The data presented herein indicates that this βγ must come from the activation of a Ptx-sensitive G-protein, presumably Gi. The βγ released from a Ptx-insensitive G-protein by the same chemoattractant receptor was not sufficient for chemotaxis. The single cell manipulation assay has provided a means to correlate biochemical and motility-related cellular responses. The ability of RBL cells to extend a pseudopod similar to the one produced by leukocytes suggests molecular events in the migration of leukocytes and transfected RBL cells are related (19Zhelev D.V. Alteraifi A.M. Hochmuth R.M. Cell Motil. Cytoskeleton. 1996; 35: 331-344Crossref PubMed Scopus (22) Google Scholar). Whereas cell lines of non-lymphoid origin like Chinese hamster ovary cells and HEK 293 cells also undergo chemotaxis (7Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (847) Google Scholar, 25Neptune E.R. Bourne H.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14489-14494Crossref PubMed Scopus (238) Google Scholar), it is not known whether they use similar pathways or cytoskeletal processes as leukocytes for migration. In wortmannin-treated RBL cells, the low level of chemotactic responsiveness was associated with weak membrane ruffling and no pseudopod extension (Fig. 5). These results again underscore the dissociation of chemotaxis from the calcium release and exocytosis, which remain unaffected by wortmannin. The unique biochemical pathways activated by the βγ subunits of Gi proteins that mediate chemotaxis are at present unknown. The βγ induced PI3 kinase activity, and its related signaling pathway could be one of these events (28Hartwig J.H. Bokoch G.M. Carpenter C.L. Janmey P.A. Taylor L.A. Toker A. Stossel T.P. Cell. 1995; 82: 643-653Abstract Full Text PDF PubMed Scopus (606) Google Scholar, 29Knall C. Worthen G.S. Johnson G.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3052-3057Crossref PubMed Scopus (270) Google Scholar, 30Keely P.J. Westwick J.K. Whitehead I.P. Der C.J. Parise L.V. Nature. 1997; 390: 632-636Crossref PubMed Scopus (649) Google Scholar). Other studies also indicated that GTPγS could activate actin polymerization in soluble cell extracts of leukocytes in the presence of the low molecular weight G-protein CDC42 but not Rho or Rac (31Tapon N. Hall A. Curr. Opin. Cell Biol. 1997; 9: 86-92Crossref PubMed Scopus (690) Google Scholar, 32Zigmond S.H. Joyce M. Borleis J. Bokoch G.M. Devreotes P.N. J. Cell Biol. 1997; 138: 363-374Crossref PubMed Scopus (145) Google Scholar). Therefore it is possible that βγ dimers released from Gi, but not the Gq family of G-proteins, activate one or more low molecular weight G-proteins that may participate in leukocyte migration. In any case, the data clearly demonstrate that chemoattractant receptors may couple to individual or multiple G-proteins. To stimulate chemotaxis however, they must activate Gi. Moreover, distinct signaling pathways among G-proteins are likely to determine other cellular responses initiated by chemoattractant receptors. These data may explain numerous observations in neutrophils and other cells that different chemoattractant receptors display different abilities to trigger nonmotility-related functions (i.e. respiratory burst, mitogenesis) despite exhibiting similar chemotactic responses (1Snyderman R. Uhing R.J. Gallin J.I. Snyderman R. Inflammation: Basic Principles and Clinical Correlates. 3rd Ed. Lippincot Williams & Wilkins, Philadelphia1999: 607-626Google Scholar, 23Verghese M.W. Charles L. Jakoi L. Dillon S.B. Snyderman R. J. Immunol. 1987; 138: 4374-4380PubMed Google Scholar). The availability of a model system in which migration responses may be measured both at population and single cell levels will allow further investigation of the divergent pathways for motility and cytotoxic functions. We thank Dr. Silvano Sozzani and Dr. Margarith Verghese for many helpful discussions.
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