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• W2021985622 abstract "Pertussis toxin inhibits chemotaxis of neutrophils by preventing chemoattractant receptors from activating trimeric G proteins in the Gi subfamily. In HEK293 cells expressing recombinant receptors, directional migration toward appropriate agonist ligands requires release of free G protein βγ subunits and can be triggered by agonists for receptors coupled to Gi but not by agonists for receptors coupled to two other G proteins, Gs and Gq. Because activation of any G protein presumably releases free Gβγ, we tested the hypothesis that chemotaxis also requires activated α subunits (Gαi) of Gi proteins. HEK293 cells were stably cotransfected with the Gi-coupled receptor for interleukin-8, CXCR1, and with a chimeric Gα, Gαqz5, which resembles Gαi in susceptibility to activation by Gi-coupled receptors but cannot regulate the Gαi effector, adenylyl cyclase. These cells, unlike cells expressing CXCR1 alone, migrated toward interleukin-8 even after treatment with pertussis toxin, which prevents activation of endogenous Gαi but not that of Gαqz5. We infer that chemotaxis does not require activation of Gαi. Because chemotaxis is mediated by Gβγ subunits released when Gi-coupled receptors activate Gαqz5, but not when Gq- or Gs-coupled receptors activate their respective G proteins, we propose that Gi-coupled receptors transmit a necessary chemotactic signal that is independent of Gαi. Pertussis toxin inhibits chemotaxis of neutrophils by preventing chemoattractant receptors from activating trimeric G proteins in the Gi subfamily. In HEK293 cells expressing recombinant receptors, directional migration toward appropriate agonist ligands requires release of free G protein βγ subunits and can be triggered by agonists for receptors coupled to Gi but not by agonists for receptors coupled to two other G proteins, Gs and Gq. Because activation of any G protein presumably releases free Gβγ, we tested the hypothesis that chemotaxis also requires activated α subunits (Gαi) of Gi proteins. HEK293 cells were stably cotransfected with the Gi-coupled receptor for interleukin-8, CXCR1, and with a chimeric Gα, Gαqz5, which resembles Gαi in susceptibility to activation by Gi-coupled receptors but cannot regulate the Gαi effector, adenylyl cyclase. These cells, unlike cells expressing CXCR1 alone, migrated toward interleukin-8 even after treatment with pertussis toxin, which prevents activation of endogenous Gαi but not that of Gαqz5. We infer that chemotaxis does not require activation of Gαi. Because chemotaxis is mediated by Gβγ subunits released when Gi-coupled receptors activate Gαqz5, but not when Gq- or Gs-coupled receptors activate their respective G proteins, we propose that Gi-coupled receptors transmit a necessary chemotactic signal that is independent of Gαi. As it migrates to a site of infection or tissue injury, an inflammatory cell must detect a chemokine gradient and organize its cytoskeleton to move in the right direction (1Baggiolini M. Dewald B. Moser B. Adv. Immunol. 1994; 55: 97-179Crossref PubMed Scopus (2258) Google Scholar, 2Murphy P.M. Annu. Rev. Immunol. 1994; 12: 593-633Crossref PubMed Scopus (1122) Google Scholar, 3Premack B.A. Schall T.J. Nat. Med. 1996; 2: 1174-1178Crossref PubMed Scopus (572) Google Scholar, 4Gerard C. Gerard N.P. Curr. Opin. Immunol. 1994; 6: 140-145Crossref PubMed Scopus (115) Google Scholar). The signaling pathways responsible for this complex cellular response are poorly understood. Pertussis toxin, which specifically prevents receptor-dependent activation of Gi proteins, blocks chemotactic migration of neutrophils; we therefore infer that Gi proteins play essential roles in mediating the chemotactic signal. Activation of G proteins by serpentine receptors releases two potential stimulators of downstream signals, an α subunit (Gα), bound to GTP, and a free Gβγ subunit (5Hamm H.E. J. Biol. Chem. 1998; 273: 669-672Abstract Full Text Full Text PDF PubMed Scopus (930) Google Scholar). For example, the αi subunits of Gi proteins directly mediate inhibition of adenylyl cyclase, while the βγ subunits of these proteins mediate opening of K+ channels and stimulation of phospholipase Cβ (6Clapham D.E. Neer E.J. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 167-203Crossref PubMed Scopus (699) Google Scholar).To identify the G protein subunit that mediates chemotaxis, we have begun to study chemotaxis in a cell line, HEK293, 1The abbreviations used are: HEK, human embryonal kidney; IL-8, interleukin 8; CXCR1, interleukin 8 receptor type A; m3AChR, m3-muscarinic acetylcholine receptor; GRK, G protein receptor kinase. 1The abbreviations used are: HEK, human embryonal kidney; IL-8, interleukin 8; CXCR1, interleukin 8 receptor type A; m3AChR, m3-muscarinic acetylcholine receptor; GRK, G protein receptor kinase.which is amenable to stable transfection with normal and mutant receptors and other signaling proteins. Endogenous Gα subunits of HEK293 cells include αs, αq, αi1, αi2, and αi3, but not αo or αz (7Law S.F. Yasuda K. Bell G.I. Reisine T. J. Biol. Chem. 1993; 268: 10721-10727Abstract Full Text PDF PubMed Google Scholar, 8Lounsbury K.M. Schlegel B. Poncz M. Brass L.F. Manning D.R. J. Biol. Chem. 1993; 268: 3494-3498Abstract Full Text PDF PubMed Google Scholar). In this model we found that chemotaxis requires receptors that activate Gi and that release of free βγ is essential (9Neptune E.R. Bourne H.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14489-14494Crossref PubMed Scopus (238) Google Scholar). Receptors that activate two other G proteins, Gs and Gq, could not mediate chemotaxis in HEK293 cells. Abundant evidence indicates that activation of these two G proteins, like that of Gi, involves dissociation of Gβγ from GTP-bound Gα. Accordingly, it is reasonable to ask why the Gβγ released from αs·GTP or αq·GTP could not mimic the chemotactic effect of Gβγ released from αi·GTP.One answer is that Gαi itself makes the difference, by activating an essential downstream signal distinct from those triggered by Gβγ. A second possibility is that Giproteins are simply more abundant than Gs or Gq, and accordingly release more Gβγ upon activation. A third possibility is that inhibitory signals generated by the α subunits of Gs or Gq block the chemotactic response to free βγ. To test these possibilities, which are not mutually exclusive, we assessed chemotaxis of HEK293 cells expressing different combinations of receptors and Gα proteins. Our results show that chemotaxis requires a receptor that can activate Gibut does not require Gαi itself. We propose that chemotaxis requires not only Gβγ, but also a signaling function of Gi-coupled receptors that is distinct from activation of Gαi.RESULTSAbundant evidence indicates that receptor activation of all trimeric G proteins causes dissociation of Gβγ from Gα·GTP. If so, why does receptor activation of Gi elicit chemotaxis, but release of Gβγ from activated Gq or Gsdoes not (9Neptune E.R. Bourne H.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14489-14494Crossref PubMed Scopus (238) Google Scholar, 12Arai H. Tsou C.L. Charo I.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14495-14499Crossref PubMed Scopus (148) Google Scholar)? One trivial explanation is that the second messengers synthesized in response to activation of Gs and Gq actually inhibit the chemotactic response that would otherwise be elicited by release of Gβγ. Fig.1 shows that this explanation could account for the failure of activated Gs, but not that of activated Gq, to mediate chemotaxis. Forskolin, which reproduces the stimulation of cAMP accumulation that would result from activation of Gαs, completely inhibited the chemotactic response to IL-8 in HEK293 cells expressing the recombinant IL-8 receptor, CXCR1 (Fig. 1 A); this result is in accord with previous observations (13Harvath L. Robbins J.D. Russell A.A. Seamon K.B. J. Immunol. 1991; 146: 224-232PubMed Google Scholar, 14Dai Y. Holgate S.T. Church M.K. Shute J.K. J. Leukocyte Biol. 1994; 56: 776-783Crossref PubMed Scopus (15) Google Scholar, 15Elferink J.G.R. VanUffelen B.E. Gen. Pharmacol. 1996; 27: 387-393Crossref PubMed Scopus (77) Google Scholar) that cAMP inhibits the chemotactic response of neutrophils and other cells. To test whether activated Gαq can inhibit chemotaxis, we cotransfected cells expressing recombinant CXCR1 with a cDNA (10Wilson P.T. Bourne H.R. J. Biol. Chem. 1995; 270: 9667-9675Crossref PubMed Scopus (94) Google Scholar) encoding mutationally activated Gαq (Gαq-Q205L). Expression of Gαq-Q205L increased basal phosphoinositide accumulation more than 30-fold (result not shown), but had no effect whatever on chemotaxis toward IL-8 (Fig. 1 B).If activated Gαq cannot inhibit chemotaxis, we must ask why the release of Gβγ from receptor-activated Gq does not mediate chemotaxis. The simplest explanation would be that chemotaxis requires Gαi·GTP, as well as Gβγ. Accordingly, we asked whether CXCR1 can elicit chemotaxis when it activates a G protein containing a chimeric Gα, Gαqz5(11Conklin B.R. Farfel Z. Lustig K.D. Julius D. Bourne H.R. Nature. 1993; 363: 274-276Crossref PubMed Scopus (601) Google Scholar), which cannot regulate activity of a direct effector of Gαi, adenylyl cyclase. Gαqz5 is identical to Gαq except that its C-terminal five amino acids are replaced by the corresponding sequence of Gαz, a Gα that responds to stimulation by Gi-coupled receptors but is not inhibited by treatment with pertussis toxin (16Wong Y.H. Conklin B.R. Bourne H.R. Science. 1992; 255: 339-342Crossref PubMed Scopus (228) Google Scholar). Expression of recombinant Gαz with CXCR1 conferred on the cells the ability to migrate toward IL-8 even after treatment with pertussis toxin (result not shown); this result did not speak to the question of whether αi is required for chemotaxis, however, because Gαz can mimic the inhibitory effect of Gαion adenylyl cyclase (16Wong Y.H. Conklin B.R. Bourne H.R. Science. 1992; 255: 339-342Crossref PubMed Scopus (228) Google Scholar).We have shown that ligand-bound Gi-coupled receptors can use Gαqz5 to activate the phosphoinositide pathway usually regulated by Gαq (11Conklin B.R. Farfel Z. Lustig K.D. Julius D. Bourne H.R. Nature. 1993; 363: 274-276Crossref PubMed Scopus (601) Google Scholar). Cells that co-expressed Gαqz5 and CXCR1 migrated toward IL-8 (Fig.2 A). Chemotaxis was mediated by a G protein containing Gαqz5, rather than by endogenous Gi, as shown by the inability of pertussis toxin to prevent chemotaxis of Gαqz5-expressing cells; the toxin completely blocked chemotaxis toward IL-8 in control cells expressing CXCR1 alone (Fig. 2 B).Figure 2Effect of overexpressed Gαqz5 on chemotactic and second messenger responses to IL-8. A and B. Migration assays were performed as described (9Neptune E.R. Bourne H.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14489-14494Crossref PubMed Scopus (238) Google Scholar) on cells expressing either CXCR1 with vector (squares, panels A andB) or CXCR1 with Gαqz5 (open triangles, panel A). Cells were incubated without (filled squares,panel B) or with (other symbols, panels A and B) 500 μg/ml pertussis toxin for 4 h at 37 °C. The cells were subsequently washed and assayed as described. C, accumulation of of inositol phosphates in response to the indicated concentrations of IL-8 was measured. CXCR1 alone, diamonds; CXCR1 plus Gαqz5, squares. Cells were treated with (open symbols) or without (filled symbols) pertussis toxin, as described for panels A and B. D, IL-8 (10 nm) inhibition of cAMP accumulation stimulated by forskolin (200 μm) in CXCR1-expressing cells. Cells expressed CXCR1, with or without Gαqz5 and were treated with or without pertussis toxin, as indicated in the figure. Values represent percent inhibition by IL-8 of the forskolin-stimulated cAMP response. Forskolin alone increased cAMP more than 100-fold. Values represent the mean ± S.E. of six determinations for panels A andB and three determinations for panels C and D. Similar results were obtained in three or more independent experiments.View Large Image Figure ViewerDownload (PPT)This result strongly suggests that chemotaxis in HEK293 cells does not require activated Gαi, although it does require activation of a Gi-coupled receptor. Controls indicated that IL-8 did indeed activate Gαqz5, but not Gαi. In Gαqz5-expressing cells, the chemokine stimulated accumulation of phosphoinositides, even after treatment with pertussis toxin (Fig. 2 C). In contrast, the chemokine inhibited cAMP accumulation in the same cells only if they had not been treated with pertussis toxin (Fig. 2 D),i.e. only when endogenous Gi was accessible to activation by CXCR1.We considered a potential quantitative explanation for the previously reported (9Neptune E.R. Bourne H.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14489-14494Crossref PubMed Scopus (238) Google Scholar) failure of a Gq-coupled receptor, the m3AChR, to mediate chemotaxis in HEK293 cells. Although the cellular content of Gαq in HEK293 cells is unknown, it is probably lower than that of the exogenous Gαqz5 stably expressed in these cells. Thus, it is possible that activation of Gq does release Gβγ, but that the amount of membrane-bound Gαq·βγ available for receptor activation in cells transfected only with the m3AChR cDNA, unlike the presumably larger amount of Gαqz5·βγ in transfected cells, cannot release sufficient amounts of Gβγ in response to receptor stimulation.To test this possibility, we increased the amount of available Gαq by stably transfecting a cDNA encoding recombinant Gαq into HEK293 cells already expressing the m3AChR. Carbachol, the m3AChR ligand, failed to elicit chemotaxis even in the doubly transfected cells (Fig.3 A). The negative inference, that a Gq-coupled receptor cannot elicit chemotaxis, was supported by control observations (Fig. 3, B andC) indicating that recombinant Gαq was indeed overexpressed and responsive to receptor stimulation in these cells. Thus, expression of exogenous Gαq allowed greater agonist-stimulated accumulation of phosphoinositides than that observed either in cells expressing the m3AChR alone or in pertussis-toxin treated cells expressing Gαqz5 and CXCR1 (Fig.3 B). Moreover, recombinant Gαq and Gαqz5 were expressed to nearly identical extents in the two types of cell (Fig. 3 C), as indicated by immunoblots with monoclonal antibodies against epitopes inserted into both Gα proteins.Figure 3Chemotaxis and signaling in cells overexpressing the m3AChR ± wild type Gαq. A, migration assays were performed on cells expressing CXCR1 alone (filled squares), the muscarinic acetylcholine receptor, type 3 (m3AChR, abbreviated MR3) alone (open triangles), or the m3AChR plus wild type Gαq (filled triangles). Concentrations of IL-8 (squares) or carbachol (triangles) are indicated on the abscissa.B, inositol phosphate accumulation on cells expressing (as indicated) the m3AChR alone, the m3AChR with wild type Gαq (qwt), or CXCR1 with Gαqz5(qz5) and treated with or without carbachol or IL-8, as indicated. C, immunoblots, using a monoclonal antibody against the EE epitope) of lysates from cells expressing m3AChR alone (lane 1), m3AChR and epitope-labeled wild type Gαq (lanes 2–4), or CXCR1 and epitope-labeled Gαqz5 (lanes 5–7). Lysates were undiluted (—) or diluted 1:2 or 1:4, as indicated.View Large Image Figure ViewerDownload (PPT)The failure of CXCR1 alone to mediate activation of phosphoinositide accumulation by IL-8 (Fig. 2 C) is consistent with results of a previous study (17Wu D. LaRosa G.J. Simon M.I. Science. 1993; 261: 101-103Crossref PubMed Scopus (332) Google Scholar), in which recombinant CXCR1 was found to activate some but not all members of the αq family,i.e. IL-8 stimulated phospholipase C in CXCR1-expressing COS-7 cells if they coexpressed α14, α15, or α16, but did not do so in cells expressing CXCR1 alone or in combination with αq or α11. In the same study (17Wu D. LaRosa G.J. Simon M.I. Science. 1993; 261: 101-103Crossref PubMed Scopus (332) Google Scholar), CXCR1 mediated Gi- and Gβγ-dependent activation of phospholipase C, but only in the presence of the β2 isoform of the phospholipase. In view of this latter result, we suspect that HEK293 cells lack the β2 isoform of the enzyme, because CXCR1 alone does not stimulate phosphoinositide accumulation in these cells (Fig. 2 C).DISCUSSIONOur experiments with HEK293 cells pose an intriguing twofold paradox. First, as reported earlier (9Neptune E.R. Bourne H.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14489-14494Crossref PubMed Scopus (238) Google Scholar), liberation of Gβγ from Gαβγ is required for chemotaxis of these cells; nonetheless, even though activation of any trimeric G protein releases Gβγ, receptors that activate G proteins other than Gi do not mediate chemotaxis. Second, even though receptors coupled to Gi are required to mediate chemotaxis of these cells, signaling by Gαi itself is not required, at least in HEK293 cells. Here we discuss four speculative ways to resolve these paradoxes: Gi-coupled receptors may activate a specific subset of Gβγ isoforms, may generate a Gαi-independent signal in addition to Gβγ, may be susceptible to novel regulatory controls, or may promote co-localization in the plasma membrane of appropriate effectors with the Gβγ liberated by receptor activation. These explanations are not mutually exclusive.By choosing among polypeptides encoded by five Gβ and 11 Gγ genes, mammalian cells could express many different Gβγ isoforms (6Clapham D.E. Neer E.J. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 167-203Crossref PubMed Scopus (699) Google Scholar). Does a specific Gβγ isoform mediate chemotaxis? If so, the responsible Gβγ dimer must possess specificity not only for a subset of G protein coupled receptors but also for the specific downstream effector(s) of chemotaxis. In both respects the evidence from other G protein-mediated signaling pathways is inconclusive. Receptors can select among Gβ and Gγ isoforms in vitro (6Clapham D.E. Neer E.J. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 167-203Crossref PubMed Scopus (699) Google Scholar) and in regulating neuronal Ca2+ channels of intact cells (18Kleuss C. Scherubl H. Hescheler J. Schultz G. Wittig B. Nature. 1992; 358: 424-426Crossref PubMed Scopus (332) Google Scholar, 19Kleuss C. Scherubl H. Hescheler J. Schultz G. Wittig B. Science. 1993; 259: 832-834Crossref PubMed Scopus (332) Google Scholar). Shared specificity for one Gβγ isoform has not been reported, however, for any group of G protein-coupled receptors, including those that couple to Gi. With respect to effectors, circumstantial evidence implicates Gγ2 as an essential component of Gi-mediated stimulation of phospholipase Cβ in differentiated HL60 cells (20Iiri T. Homma Y. Ohoka Y. Robishaw J.D. Katada T. Bourne H.R. J. Biol. Chem. 1995; 270: 5901-5908Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), and γ5 and γ12 are reported to colocalize in cultured cells with vinculin and F actin, respectively (21Ueda H. Saga S. Shinohara H. Morishita R. Kato K. Asano T. J. Cell Sci. 1997; 110: 1503-1511Crossref PubMed Google Scholar). Nonetheless, experiments in several laboratories have failed to show significant specificity of any Gβγ dimer, except for the relative weakness of those containing Gγ1 for stimulating any effector (6Clapham D.E. Neer E.J. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 167-203Crossref PubMed Scopus (699) Google Scholar, 22Iniguez-Lluhi J.A. Simon M.I. Robishaw J.D. Gilman A.G. J. Biol. Chem. 1992; 267: 23409-23417Abstract Full Text PDF PubMed Google Scholar, 23Logothetis D.E. Kim D.H. Northup J.K. Neer E.J. Clapham D.E. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5814-5818Crossref PubMed Scopus (111) Google Scholar).Does chemotaxis require a receptor to generate a third kind of signal, independent of Gαi and in addition to the signal(s) relayed by free Gβγ? G protein receptor kinases (GRKs) and arrestins, two potential candidates for generators of such a signal, are involved in agonist-dependent desensitization and endocytosis of receptors. In a Gβγ-dependent fashion, GRKs bind to and are activated by agonist-stimulated receptors; activated GRKs phosphorylate residues on the cytoplasmic face of receptors (24Freedman N.J. Lefkowitz R.J. Rec. Prog. Horm. Res. 1996; 51: 319-351PubMed Google Scholar) and could, hypothetically, phosphorylate downstream effectors. Receptor phosphorylation by GRKs markedly enhances agonist-dependent association of receptors with arrestins, which act negatively, by competing with G proteins, to damp receptor signaling (25Ferguson S.S. Barak L.S. Zhang J. Caron M.G. Can. J. Physiol. Pharmacol. 1996; 74: 1095-1110Crossref PubMed Scopus (318) Google Scholar). Arrestins also mediate association of receptors with other proteins, including components of the endocytotic machinery of clathrin-coated pits (26Goodman Jr., O.B. Krupnick J.G. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Crossref PubMed Scopus (1154) Google Scholar). This more positive role of arrestins could serve as an analog for association with and activation of a hypothetical downstream effector of chemotaxis. No member of either the GRK or the arrestin families, however, has yet been reported to interact specifically with chemotactic or Gi-coupled receptors.One piece of evidence indirectly suggests that chemotactic receptors may generate a signal separate from those mediated by Gαiand Gβγ; a C-terminal truncation of CXCR1 markedly inhibited the chemotactic response but did not alter the receptor's ability to trigger agonist-dependent inhibition of adenylyl cyclase or stimulation of the mitogen-activated protein kinase pathway (9Neptune E.R. Bourne H.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14489-14494Crossref PubMed Scopus (238) Google Scholar), responses mediated by Gαi·GTP and Gβγ, respectively (5Hamm H.E. J. Biol. Chem. 1998; 273: 669-672Abstract Full Text Full Text PDF PubMed Scopus (930) Google Scholar, 27Neer E.J. Smith T.F. Cell. 1996; 84: 175-178Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). C-terminal tails of receptors are implicated as sites that contribute to binding of GRKs and arrestins, and might very well bind to other target molecules as well (25Ferguson S.S. Barak L.S. Zhang J. Caron M.G. Can. J. Physiol. Pharmacol. 1996; 74: 1095-1110Crossref PubMed Scopus (318) Google Scholar).A third possibility is that, rather than generating a signal distinct from those mediated by Gαi and Gβγ, chemotactic Gi-coupled receptors are susceptible to a kind of regulatory control that does affect other receptors. For example, we could imagine that signaling by these receptors is enhanced or attenuated by a molecule that accumulates asymmetrically at the front or the back, respectively, of a cell migrating up a gradient of chemoattractant.A fourth speculation, perhaps the most interesting, could resolve the paradox created by dependence of chemotaxis on Gi-coupled receptors and Gβγ, but not Gαi. In this scenario, the Gi-coupled receptor promotes liberation of Gβγ in microdomains of the cell that contain critical downstream effectors of chemotaxis; the receptor could do so by associating with a scaffolding protein that sequesters effectors of chemotaxis in a signaling complex. The chemotrophic pheromone response of Saccharomyces cerevisiae furnishes a relevant precedent: Gβγ mediates this response by promoting formation of a signaling complex assembled by a scaffolding protein, STE5p (28Leberer E. Thomas D.Y. Whiteway M. Curr. Opin. Genet. Dev. 1997; 7: 59-66Crossref PubMed Scopus (188) Google Scholar). Other data raise the possibility that G protein-coupled receptors participate in signaling complexes containing both G protein subunits and effectors. For example, experiments in a reconstituted system using pure receptor, effector, and G protein suggest that all three components participate in a membrane-bound functional complex: phospholipase Cβ, the effector of Gαq·GTP, accelerates receptor-stimulated exchange of GTP for GDP bound to Gαq (29Biddlecome G.H. Berstein G. Ross E.M. J. Biol. Chem. 1996; 271: 7999-8007Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). Similarly, a recently identified scaffold protein, inaD, assembles downstream proteins involved in G protein-dependent phototransduction (including a phospholipase C) at specific subcellular locations in the retina of fruit flies (30Tsunoda S. Sierralta J. Sun Y. Bodner R. Suzuki E. Becker A. Socolich M. Zuker C.S. Nature. 1997; 388: 243-249Crossref PubMed Scopus (551) Google Scholar). Note that formation of the postulated chemotaxis signaling complex might depend on both the activated receptor and Gβγ. In this regard, protein domains located in the third intracellular loops (ic3 domains) of m2- and m3-muscarinic receptors associate with free Gβγ (but not with Gβγ complexed to Gα·GDP) and Gβγ and the ic3 domain appear to form a ternary complex with a receptor kinase, GRK2 (31Wu G. Benovic J.L. Hildebrandt J.D. Lanier S.M. J. Biol. Chem. 1998; 273: 7197-7200Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). The unusually large ic3 domains of muscarinic receptors may be analogs of the hypothetical scaffolding proteins that associate with Gi-coupled receptors.This fourth proposal for resolving the paradox raises an interesting question. Do all Gi-coupled receptors share the proposed ability of chemotactic receptors to organize signaling complexes that are required for chemotaxis? We and others have tested several Gi-coupled receptors, not previously identified as “professional” chemotactic receptors, for ability to mediate directional migration of cultured cells toward the appropriate ligand; the D2 dopamine receptor and the μ- and δ-opioid receptors do mediate chemotaxis, albeit not as efficiently as CXCR1 (9Neptune E.R. Bourne H.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14489-14494Crossref PubMed Scopus (238) Google Scholar, 12Arai H. Tsou C.L. Charo I.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14495-14499Crossref PubMed Scopus (148) Google Scholar). It is not clear whether this result can be generalized to include all, or even most, Gi-coupled receptors.The paradox we have described is paralleled, nonetheless, by similar paradoxes in two other responses to agonists for Gi-coupled receptors: stimulation of the mitogen-activated protein kinase pathway and opening of K+ channels. Even though Gβγ (but not Gαi·GTP) mediates both responses, neither response is elicited by the Gβγ liberated by activating receptors that activate G proteins other than Gi (32Dascal N. Cell. Signalling. 1997; 9: 551-573Crossref PubMed Scopus (266) Google Scholar). 2L. Y. Jan, personal communication. Possible resolutions of these paradoxes include those we have outlined for resolving the paradox in chemotactic signaling.Finally, our findings in HEK293 cells provide a starting point for dissecting the molecular basis of chemotactic signaling in neutrophils and other professionally chemotactic cells. Gi-coupled receptors and G protein subunits can serve as probes for identifying the critical but so far elusive effectors that harness the actin cytoskeleton to effect directional migration. As it migrates to a site of infection or tissue injury, an inflammatory cell must detect a chemokine gradient and organize its cytoskeleton to move in the right direction (1Baggiolini M. Dewald B. Moser B. Adv. Immunol. 1994; 55: 97-179Crossref PubMed Scopus (2258) Google Scholar, 2Murphy P.M. Annu. Rev. Immunol. 1994; 12: 593-633Crossref PubMed Scopus (1122) Google Scholar, 3Premack B.A. Schall T.J. Nat. Med. 1996; 2: 1174-1178Crossref PubMed Scopus (572) Google Scholar, 4Gerard C. Gerard N.P. Curr. Opin. Immunol. 1994; 6: 140-145Crossref PubMed Scopus (115) Google Scholar). The signaling pathways responsible for this complex cellular response are poorly understood. Pertussis toxin, which specifically prevents receptor-dependent activation of Gi proteins, blocks chemotactic migration of neutrophils; we therefore infer that Gi proteins play essential roles in mediating the chemotactic signal. Activation of G proteins by serpentine receptors releases two potential stimulators of downstream signals, an α subunit (Gα), bound to GTP, and a free Gβγ subunit (5Hamm H.E. J. Biol. Chem. 1998; 273: 669-672Abstract Full Text Full Text PDF PubMed Scopus (930) Google Scholar). For example, the αi subunits of Gi proteins directly mediate inhibition of adenylyl cyclase, while the βγ subunits of these proteins mediate opening of K+ channels and stimulation of phospholipase Cβ (6Clapham D.E. Neer E.J. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 167-203Crossref PubMed Scopus (699) Google Scholar). To identify the G protein subunit that mediates chemotaxis, we have begun to study chemotaxis in a cell line, HEK293, 1The abbreviations used are: HEK, human embryonal kidney; IL-8, interleukin 8; CXCR1, interleukin 8 receptor type A; m3AChR, m3-muscarinic acetylcholine receptor; GRK, G protein receptor kinase. 1The abbreviations used are: HEK, human embryonal kidney; IL-8, interleukin 8; CXCR1, interleukin 8 receptor type A; m3AChR, m3-muscarinic acetylcholine receptor; GRK, G protein receptor kinase.which is amenable to stable transfection with normal and mutant receptors and other signaling proteins. Endogenous Gα subunits of HEK293 cells include αs, αq, αi1, αi2, and αi3, but not αo or αz (7Law S.F. Yasuda K. Bell G.I. Reisine T. J. Biol. Chem. 1993; 268: 10721-10727Abstract Full Text PDF PubMed Google Scholar, 8Lounsbury K.M. Schlegel B. Poncz M. Brass L.F. Manning D.R. J. Biol. Chem. 1993; 268: 3494-3498Abstract Full Text PDF PubMed Google Scholar). In this model we found that chemota" @default.
• W2021985622 created "2016-06-24" @default.
• W2021985622 creator A5043192139 @default.
• W2021985622 creator A5063887149 @default.
• W2021985622 creator A5087239458 @default.
• W2021985622 date "1999-01-01" @default.
• W2021985622 modified "2023-10-04" @default.
• W2021985622 title "Gαi Is Not Required for Chemotaxis Mediated by Gi-coupled Receptors" @default.
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