Galpha i Is Not Required for Chemotaxis Mediated by Gi-coupled Receptors*

Enid R. NeptuneDagger §, Taroh Iiri§, and Henry R. Bourne§parallel

From the § Departments of Cellular and Molecular Pharmacology and Medicine and Cardiovascular Research Institute, and the Dagger  Division of Pulmonary and Critical Care Medicine, University of California, San Francisco, California 94143

    ABSTRACT
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Abstract
Introduction
References

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 beta gamma 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 Gbeta gamma , we tested the hypothesis that chemotaxis also requires activated alpha  subunits (Galpha i) of Gi proteins. HEK293 cells were stably cotransfected with the Gi-coupled receptor for interleukin-8, CXCR1, and with a chimeric Galpha , Galpha qz5, which resembles Galpha i in susceptibility to activation by Gi-coupled receptors but cannot regulate the Galpha i 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 Galpha i but not that of Galpha qz5. We infer that chemotaxis does not require activation of Galpha i. Because chemotaxis is mediated by Gbeta gamma subunits released when Gi-coupled receptors activate Galpha qz5, 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 Galpha i.

    INTRODUCTION
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Abstract
Introduction
References

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 (1-4). 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 alpha  subunit (Galpha ), bound to GTP, and a free Gbeta gamma subunit (5). For example, the alpha i subunits of Gi proteins directly mediate inhibition of adenylyl cyclase, while the beta gamma subunits of these proteins mediate opening of K+ channels and stimulation of phospholipase Cbeta (6).

To identify the G protein subunit that mediates chemotaxis, we have begun to study chemotaxis in a cell line, HEK293,1 which is amenable to stable transfection with normal and mutant receptors and other signaling proteins. Endogenous Galpha subunits of HEK293 cells include alpha s, alpha q, alpha i1, alpha i2, and alpha i3, but not alpha o or alpha z (7, 8). In this model we found that chemotaxis requires receptors that activate Gi and that release of free beta gamma is essential (9). 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 Gbeta gamma from GTP-bound Galpha . Accordingly, it is reasonable to ask why the Gbeta gamma released from alpha s·GTP or alpha q·GTP could not mimic the chemotactic effect of Gbeta gamma released from alpha i·GTP.

One answer is that Galpha i itself makes the difference, by activating an essential downstream signal distinct from those triggered by Gbeta gamma . A second possibility is that Gi proteins are simply more abundant than Gs or Gq, and accordingly release more Gbeta gamma upon activation. A third possibility is that inhibitory signals generated by the alpha  subunits of Gs or Gq block the chemotactic response to free beta gamma . To test these possibilities, which are not mutually exclusive, we assessed chemotaxis of HEK293 cells expressing different combinations of receptors and Galpha proteins. Our results show that chemotaxis requires a receptor that can activate Gi but does not require Galpha i itself. We propose that chemotaxis requires not only Gbeta gamma , but also a signaling function of Gi-coupled receptors that is distinct from activation of Galpha i.

    EXPERIMENTAL PROCEDURES

Materials-- Recombinant IL-8, forskolin, and rat collagen type 1 were obtained as described (9). The modified Boyden chamber was purchased from Neuroprobe, and the polycarbonate filters were procured from Poretics. Pertussis toxin was obtained from List Biologicals.

Plasmid Constructs-- Wild type Galpha q and Galpha q-Q205L, tagged with the EE epitope, were as described (10). Mutagenesis was verified by DNA sequencing. EE-tagged Galpha qz5 in pcDNA1 was obtained from Bruce Conklin, Gladstone Institute, San Francisco General Hospital (11). The interleukin 8 receptor type A (hereafter termed CXCR1) subcloned into pcDNA3 was obtained from Israel Charo, Gladstone Institute, San Francisco General Hospital.

Cell Culture and Transfection-- HEK293 cell lines stably expressing CXCR1 and the m3-muscarinic acetylcholine receptor (m3AChR) were generated as described (9) and maintained in G418 (800 µg/ml). For double stable transfectants, a vector containing a hygromycin resistance cassette was cotransfected with the various Galpha constructs in pcDNA1. Clones stably expressing both CXCR1 and the respective Galpha constructs were selected and maintained in both G418 and hygromycin (200 µg/ml). Cell lines were propagated as described (9).

Assays-- Assays of chemotaxis, cAMP accumulation, and inositol phosphate accumulation were performed as described (9).

Immunoblots-- Subconfluent cells (5 × 106) were lysed in RIPA buffer containing 5% Nonidet P-40, 2.5% deoxycholate, 250 mM Tris (7.4), 750 mM NaCl, and 12.5 mM MgCl2 for 30 min at 4 °C. Lysates were diluted as noted in RIPA buffer, separated on a 12% SDS gel, and transferred to polyvinylidene difluoride membranes. Membranes were probed with EE monoclonal antibody as described (10). ECL (NEN Life Science Products) was used to visualize immunoreactive bands.

    RESULTS

Abundant evidence indicates that receptor activation of all trimeric G proteins causes dissociation of Gbeta gamma from Galpha ·GTP. If so, why does receptor activation of Gi elicit chemotaxis, but release of Gbeta gamma from activated Gq or Gs does not (9, 12)? 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 Gbeta gamma . 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 Galpha s, completely inhibited the chemotactic response to IL-8 in HEK293 cells expressing the recombinant IL-8 receptor, CXCR1 (Fig. 1A); this result is in accord with previous observations (13-15) that cAMP inhibits the chemotactic response of neutrophils and other cells. To test whether activated Galpha q can inhibit chemotaxis, we cotransfected cells expressing recombinant CXCR1 with a cDNA (10) encoding mutationally activated Galpha q (Galpha q-Q205L). Expression of Galpha q-Q205L increased basal phosphoinositide accumulation more than 30-fold (result not shown), but had no effect whatever on chemotaxis toward IL-8 (Fig. 1B).


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Fig. 1.   Effect of concurrent activation of Galpha s and Galpha q on chemotaxis toward IL-8. Migration assays were performed in a 48-well Boyden chamber, as described (9), on cells stably expressing: CXCR1 alone (filled and open squares; panel A) or CXCR1 plus vector (open squares; panel B) or plus Galpha q-Q205L (open circles; panel B). Forskolin (open squares, panel A) was present at 200 µM. Values represent the mean ± S.E. of six determinations. Similar results were obtained in three or more independent experiments.

If activated Galpha q cannot inhibit chemotaxis, we must ask why the release of Gbeta gamma from receptor-activated Gq does not mediate chemotaxis. The simplest explanation would be that chemotaxis requires Galpha i·GTP, as well as Gbeta gamma . Accordingly, we asked whether CXCR1 can elicit chemotaxis when it activates a G protein containing a chimeric Galpha , Galpha qz5 (11), which cannot regulate activity of a direct effector of Galpha i, adenylyl cyclase. Galpha qz5 is identical to Galpha q except that its C-terminal five amino acids are replaced by the corresponding sequence of Galpha z, a Galpha that responds to stimulation by Gi-coupled receptors but is not inhibited by treatment with pertussis toxin (16). Expression of recombinant Galpha z 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 alpha i is required for chemotaxis, however, because Galpha z can mimic the inhibitory effect of Galpha i on adenylyl cyclase (16).

We have shown that ligand-bound Gi-coupled receptors can use Galpha qz5 to activate the phosphoinositide pathway usually regulated by Galpha q (11). Cells that co-expressed Galpha qz5 and CXCR1 migrated toward IL-8 (Fig. 2A). Chemotaxis was mediated by a G protein containing Galpha qz5, rather than by endogenous Gi, as shown by the inability of pertussis toxin to prevent chemotaxis of Galpha qz5-expressing cells; the toxin completely blocked chemotaxis toward IL-8 in control cells expressing CXCR1 alone (Fig. 2B).


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Fig. 2.   Effect of overexpressed Galpha qz5 on chemotactic and second messenger responses to IL-8. A and B. Migration assays were performed as described (9) on cells expressing either CXCR1 with vector (squares, panels A and B) or CXCR1 with Galpha qz5 (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 Galpha qz5, 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 Galpha qz5 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 and B and three determinations for panels C and D. Similar results were obtained in three or more independent experiments.

This result strongly suggests that chemotaxis in HEK293 cells does not require activated Galpha i, although it does require activation of a Gi-coupled receptor. Controls indicated that IL-8 did indeed activate Galpha qz5, but not Galpha i. In Galpha qz5-expressing cells, the chemokine stimulated accumulation of phosphoinositides, even after treatment with pertussis toxin (Fig. 2C). In contrast, the chemokine inhibited cAMP accumulation in the same cells only if they had not been treated with pertussis toxin (Fig. 2D), i.e. only when endogenous Gi was accessible to activation by CXCR1.

We considered a potential quantitative explanation for the previously reported (9) failure of a Gq-coupled receptor, the m3AChR, to mediate chemotaxis in HEK293 cells. Although the cellular content of Galpha q in HEK293 cells is unknown, it is probably lower than that of the exogenous Galpha qz5 stably expressed in these cells. Thus, it is possible that activation of Gq does release Gbeta gamma , but that the amount of membrane-bound Galpha q·beta gamma available for receptor activation in cells transfected only with the m3AChR cDNA, unlike the presumably larger amount of Galpha qz5·beta gamma in transfected cells, cannot release sufficient amounts of Gbeta gamma in response to receptor stimulation.

To test this possibility, we increased the amount of available Galpha q by stably transfecting a cDNA encoding recombinant Galpha q into HEK293 cells already expressing the m3AChR. Carbachol, the m3AChR ligand, failed to elicit chemotaxis even in the doubly transfected cells (Fig. 3A). The negative inference, that a Gq-coupled receptor cannot elicit chemotaxis, was supported by control observations (Fig. 3, B and C) indicating that recombinant Galpha q was indeed overexpressed and responsive to receptor stimulation in these cells. Thus, expression of exogenous Galpha q allowed greater agonist-stimulated accumulation of phosphoinositides than that observed either in cells expressing the m3AChR alone or in pertussis-toxin treated cells expressing Galpha qz5 and CXCR1 (Fig. 3B). Moreover, recombinant Galpha q and Galpha qz5 were expressed to nearly identical extents in the two types of cell (Fig. 3C), as indicated by immunoblots with monoclonal antibodies against epitopes inserted into both Galpha proteins.


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Fig. 3.   Chemotaxis and signaling in cells overexpressing the m3AChR ± wild type Galpha q. 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 Galpha q (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 Galpha q (qwt), or CXCR1 with Galpha qz5 (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 Galpha q (lanes 2-4), or CXCR1 and epitope-labeled Galpha qz5 (lanes 5-7). Lysates were undiluted (---) or diluted 1:2 or 1:4, as indicated.

The failure of CXCR1 alone to mediate activation of phosphoinositide accumulation by IL-8 (Fig. 2C) is consistent with results of a previous study (17), in which recombinant CXCR1 was found to activate some but not all members of the alpha q family, i.e. IL-8 stimulated phospholipase C in CXCR1-expressing COS-7 cells if they coexpressed alpha 14, alpha 15, or alpha 16, but did not do so in cells expressing CXCR1 alone or in combination with alpha q or alpha 11. In the same study (17), CXCR1 mediated Gi- and Gbeta gamma -dependent activation of phospholipase C, but only in the presence of the beta 2 isoform of the phospholipase. In view of this latter result, we suspect that HEK293 cells lack the beta 2 isoform of the enzyme, because CXCR1 alone does not stimulate phosphoinositide accumulation in these cells (Fig. 2C).

    DISCUSSION

Our experiments with HEK293 cells pose an intriguing twofold paradox. First, as reported earlier (9), liberation of Gbeta gamma from Galpha beta gamma is required for chemotaxis of these cells; nonetheless, even though activation of any trimeric G protein releases Gbeta gamma , 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 Galpha i 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 Gbeta gamma isoforms, may generate a Galpha i-independent signal in addition to Gbeta gamma , may be susceptible to novel regulatory controls, or may promote co-localization in the plasma membrane of appropriate effectors with the Gbeta gamma liberated by receptor activation. These explanations are not mutually exclusive.

By choosing among polypeptides encoded by five Gbeta and 11 Ggamma genes, mammalian cells could express many different Gbeta gamma isoforms (6). Does a specific Gbeta gamma isoform mediate chemotaxis? If so, the responsible Gbeta gamma 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 Gbeta and Ggamma isoforms in vitro (6) and in regulating neuronal Ca2+ channels of intact cells (18, 19). Shared specificity for one Gbeta gamma 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 Ggamma 2 as an essential component of Gi-mediated stimulation of phospholipase Cbeta in differentiated HL60 cells (20), and gamma 5 and gamma 12 are reported to colocalize in cultured cells with vinculin and F actin, respectively (21). Nonetheless, experiments in several laboratories have failed to show significant specificity of any Gbeta gamma dimer, except for the relative weakness of those containing Ggamma 1 for stimulating any effector (6, 22, 23).

Does chemotaxis require a receptor to generate a third kind of signal, independent of Galpha i and in addition to the signal(s) relayed by free Gbeta gamma ? 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 Gbeta gamma -dependent fashion, GRKs bind to and are activated by agonist-stimulated receptors; activated GRKs phosphorylate residues on the cytoplasmic face of receptors (24) 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 (25). Arrestins also mediate association of receptors with other proteins, including components of the endocytotic machinery of clathrin-coated pits (26). 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 Galpha i and Gbeta gamma ; 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 (9), responses mediated by Galpha i·GTP and Gbeta gamma , respectively (5, 27). 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 (25).

A third possibility is that, rather than generating a signal distinct from those mediated by Galpha i and Gbeta gamma , 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 Gbeta gamma , but not Galpha i. In this scenario, the Gi-coupled receptor promotes liberation of Gbeta gamma 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: Gbeta gamma mediates this response by promoting formation of a signaling complex assembled by a scaffolding protein, STE5p (28). 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 Cbeta , the effector of Galpha q·GTP, accelerates receptor-stimulated exchange of GTP for GDP bound to Galpha q (29). 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 (30). Note that formation of the postulated chemotaxis signaling complex might depend on both the activated receptor and Gbeta gamma . In this regard, protein domains located in the third intracellular loops (ic3 domains) of m2- and m3-muscarinic receptors associate with free Gbeta gamma (but not with Gbeta gamma complexed to Galpha ·GDP) and Gbeta gamma and the ic3 domain appear to form a ternary complex with a receptor kinase, GRK2 (31). 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 delta -opioid receptors do mediate chemotaxis, albeit not as efficiently as CXCR1 (9, 12). 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 Gbeta gamma (but not Galpha i·GTP) mediates both responses, neither response is elicited by the Gbeta gamma liberated by activating receptors that activate G proteins other than Gi (32).2 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.

    ACKNOWLEDGEMENTS

We thank Israel Charo, Mark von Zastrow, and members of the Bourne laboratory for useful advice and review of the manuscript.

    FOOTNOTES

* This work was supported in part by postdoctoral fellowships from the Howard Hughes Medical Institute and the Robert Wood Johnson Foundation (to E. R. N.) and National Institutes of Health Grants GM-27800 and CA-54427 (to H. R. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Fourth Dept. of Internal Medicine, University of Tokyo School of Medicine, Bunkyo-ku, Tokyo 112, Japan.

parallel To whom correspondence should be addressed: S-1212, Box 0450, University of California Medical Center, San Francisco, CA. Tel.: 415-476-8161; Fax: 415-476-5292; E-mail: h_bourne{at}quickmail.ucsf.edu.

The 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.

2 L. Y. Jan, personal communication.

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