From the § Departments of Cellular and Molecular
Pharmacology and Medicine and Cardiovascular Research Institute, and
the Division of Pulmonary and Critical Care Medicine,
University of California, San Francisco, California 94143
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ABSTRACT |
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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 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 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 G One answer is that G 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 G 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 G 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.
Abundant evidence indicates that receptor activation of all
trimeric G proteins causes dissociation of G
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 G
, we tested the hypothesis that chemotaxis also requires activated
subunits
(G
i) of Gi proteins. HEK293 cells were
stably cotransfected with the Gi-coupled receptor for
interleukin-8, CXCR1, and with a chimeric G
, G
qz5, which resembles G
i in susceptibility to activation by
Gi-coupled receptors but cannot regulate the
G
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
G
i but not that of G
qz5. We infer that
chemotaxis does not require activation of G
i. Because chemotaxis is mediated by G
subunits released when
Gi-coupled receptors activate G
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
G
i.
INTRODUCTION
Top
Abstract
Introduction
References
subunit (G
), bound to GTP, and a free G
subunit (5). For
example, the
i subunits of Gi proteins
directly mediate inhibition of adenylyl cyclase, while the
subunits of these proteins mediate opening of K+ channels
and stimulation of phospholipase C
(6).
subunits of HEK293 cells include
s,
q,
i1,
i2, and
i3, but not
o or
z (7, 8). In this model we found that chemotaxis
requires receptors that activate Gi and that release of
free
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 G
from GTP-bound G
. Accordingly, it is
reasonable to ask why the G
released from
s·GTP
or
q·GTP could not mimic the chemotactic effect of
G
released from
i·GTP.
i itself makes the
difference, by activating an essential downstream signal distinct from
those triggered by G
. A second possibility is that Gi
proteins are simply more abundant than Gs or
Gq, and accordingly release more G
upon activation. A
third possibility is that inhibitory signals generated by the
subunits of Gs or Gq block the chemotactic
response to free
. To test these possibilities, which are not
mutually exclusive, we assessed chemotaxis of HEK293 cells expressing
different combinations of receptors and G
proteins. Our results show
that chemotaxis requires a receptor that can activate Gi
but does not require G
i itself. We propose that
chemotaxis requires not only G
, but also a signaling function of
Gi-coupled receptors that is distinct from activation of
G
i.
EXPERIMENTAL PROCEDURES
q and
G
q-Q205L, tagged with the EE epitope, were as described
(10). Mutagenesis was verified by DNA sequencing. EE-tagged
G
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.
constructs
in pcDNA1. Clones stably expressing both CXCR1 and the respective
G
constructs were selected and maintained in both G418 and
hygromycin (200 µg/ml). Cell lines were propagated as described
(9).
RESULTS
from G
·GTP. If so, why does receptor activation of Gi elicit chemotaxis,
but release of G
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 G
. 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
G
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
G
q can inhibit chemotaxis, we cotransfected cells
expressing recombinant CXCR1 with a cDNA (10) encoding mutationally
activated G
q (G
q-Q205L). Expression of
G
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
G s and
G
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
G
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 Gq cannot inhibit chemotaxis, we must ask
why the release of G
from receptor-activated Gq does
not mediate chemotaxis. The simplest explanation would be that
chemotaxis requires G
i·GTP, as well as G
.
Accordingly, we asked whether CXCR1 can elicit chemotaxis when it
activates a G protein containing a chimeric G
, G
qz5
(11), which cannot regulate activity of a direct effector of
G
i, adenylyl cyclase. G
qz5 is identical to G
q except that its C-terminal five amino acids are
replaced by the corresponding sequence of G
z, a G
that responds to stimulation by Gi-coupled receptors but is
not inhibited by treatment with pertussis toxin (16). Expression of
recombinant G
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
i is required for chemotaxis, however, because
G
z can mimic the inhibitory effect of G
i
on adenylyl cyclase (16).
We have shown that ligand-bound Gi-coupled receptors can
use Gqz5 to activate the phosphoinositide pathway
usually regulated by G
q (11). Cells that co-expressed
G
qz5 and CXCR1 migrated toward IL-8 (Fig.
2A). Chemotaxis was mediated by a
G protein containing G
qz5, rather than by endogenous
Gi, as shown by the inability of pertussis toxin to prevent
chemotaxis of G
qz5-expressing cells; the toxin
completely blocked chemotaxis toward IL-8 in control cells expressing
CXCR1 alone (Fig. 2B).
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This result strongly suggests that chemotaxis in HEK293 cells does not
require activated Gi, although it does require
activation of a Gi-coupled receptor. Controls indicated
that IL-8 did indeed activate G
qz5, but not
G
i. In G
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
Gq in HEK293 cells is unknown, it is probably lower than
that of the exogenous G
qz5 stably expressed in these
cells. Thus, it is possible that activation of Gq does
release G
, but that the amount of membrane-bound
G
q·
available for receptor activation in cells
transfected only with the m3AChR cDNA, unlike the presumably larger
amount of G
qz5·
in transfected cells, cannot
release sufficient amounts of G
in response to receptor stimulation.
To test this possibility, we increased the amount of available
Gq by stably transfecting a cDNA encoding
recombinant G
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 G
q was indeed
overexpressed and responsive to receptor stimulation in these cells.
Thus, expression of exogenous G
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 G
qz5 and CXCR1 (Fig.
3B). Moreover, recombinant G
q and
G
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 G
proteins.
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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 q family,
i.e. IL-8 stimulated phospholipase C in CXCR1-expressing
COS-7 cells if they coexpressed
14,
15,
or
16, but did not do so in cells expressing CXCR1 alone
or in combination with
q or
11. In the same study (17), CXCR1 mediated Gi- and
G
-dependent activation of phospholipase C, but only
in the presence of the
2 isoform of the phospholipase. In view of
this latter result, we suspect that HEK293 cells lack the
2 isoform
of the enzyme, because CXCR1 alone does not stimulate phosphoinositide
accumulation in these cells (Fig. 2C).
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DISCUSSION |
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Our experiments with HEK293 cells pose an intriguing twofold
paradox. First, as reported earlier (9), liberation of G from
G
is required for chemotaxis of these cells; nonetheless, even
though activation of any trimeric G protein releases G
, 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
G
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
G
isoforms, may generate a G
i-independent signal
in addition to G
, may be susceptible to novel regulatory
controls, or may promote co-localization in the plasma membrane of
appropriate effectors with the G
liberated by receptor
activation. These explanations are not mutually exclusive.
By choosing among polypeptides encoded by five G and 11 G
genes,
mammalian cells could express many different G
isoforms (6). Does
a specific G
isoform mediate chemotaxis? If so, the responsible
G
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 G
and G
isoforms in vitro (6) and in
regulating neuronal Ca2+ channels of intact cells (18, 19).
Shared specificity for one G
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 G
2 as an
essential component of Gi-mediated stimulation of
phospholipase C
in differentiated HL60 cells (20), and
5 and
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 G
dimer, except for the relative
weakness of those containing G
1 for stimulating any
effector (6, 22, 23).
Does chemotaxis require a receptor to generate a third kind of signal,
independent of Gi and in addition to the signal(s) relayed by free G
? 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 G
-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 Gi
and G
; 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 G
i·GTP and G
, 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 Gi and G
, 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 G, but not G
i. In this scenario, the
Gi-coupled receptor promotes liberation of G
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: G
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 C
, the effector of
G
q·GTP, accelerates receptor-stimulated exchange of
GTP for GDP bound to G
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 G
. In this regard, protein domains located in the
third intracellular loops (ic3 domains) of m2- and m3-muscarinic
receptors associate with free G
(but not with G
complexed
to G
·GDP) and G
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 -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 G (but not
G
i·GTP) mediates both responses, neither response is
elicited by the G
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.
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ACKNOWLEDGEMENTS |
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We thank Israel Charo, Mark von Zastrow, and members of the Bourne laboratory for useful advice and review of the manuscript.
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FOOTNOTES |
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* 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.
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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REFERENCES |
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