(Received for publication, September 16, 1994; and in revised form, December 5, 1994)
From the
The key event in receptor-catalyzed activation of heterotrimeric
G proteins is binding of GTP, which leads to subunit dissociation
generating GTP-bound subunits and free
complexes. We
have previously identified a mutation that abolished GTP binding in
G
(S47C) and demonstrated that the mutant retained the
ability to bind
and could act in a dominant negative fashion
when expressed in Xenopus oocytes (Slepak, V. Z., Quick, M.
W., Aragay, A. M., Davidson, N., Lester, H. A., and Simon, M. I.(1993) J. Biol. Chem. 268, 21889-21894). In the current work,
we investigated the effects of the homologous mutant of G
(S48C) upon signaling pathways reconstituted in transiently
transfected COS-7 cells. We found that expression of the G
S48C mutant prevented stimulation of phospholipase C (PLC)
2
by free
subunit complexes. This effect of G
S48C was not readily reversible in contrast to the inhibitory
effect of wild-type G
, which could be reversed upon
activation of the cotransfected muscarinic M2 receptor, presumably by
release of
from the G protein heterotrimer. Coexpression of
G
S48C or the wild-type G
also
dramatically decreased G
-mediated stimulation of PLC by
C5a in the cells transfected with cDNAs encoding C5a receptor and
G
. Activation of PLC via endogenous G
or
G
in the presence of
1C adrenergic receptors was
similarly attenuated by coexpression of G
or
G
S48C. Pertussis toxin treatment of the transfected
cells enhanced the inhibition of the receptor-stimulated PLC by
wild-type G
subunits but did not influence the effects
of the dominant negative mutant. The enhancement of the wild-type
G
inhibitory effect by pertussis toxin can be
explained by stabilization of G
binding to
as a result of ADP-ribosylation, while G
S48C mutant
binds
irreversibly even without pertussis toxin treatment.
Therefore, a feasible mechanism to rationalize the attenuation of the
G
and G
-mediated activation of PLC by
cotransfected G
is the competition between
G
and G
or G
for the
complexes, which are necessary for the G protein coupling
with receptors. These experiments provide new evidence for the role of
in the integration of signals controlling phosphoinositide
release through different G
families.
Many receptors of hormones, neuromediators, and growth factors
transmit signals via heterotrimeric G proteins. Receptor-induced
activation leads to dissociation of G protein subunits, generating
subunits charged with GTP and free
complexes. In
recent years, G protein-mediated signaling has proven to be far more
complex than just a combination of ``linear'' pathways from
specific receptors to their effectors. The number of cloned genes
encoding G protein subunits, effectors, and receptors includes hundreds
of members(1, 2, 3, 4) .
Furthermore, in many signaling pathways, activity of effectors is
modulated not solely by G
subunits as was traditionally thought
but also by G
complexes(5, 6, 7, 8, 9, 10, 11) .
One of the ways to define the specific links within the network of G
proteins, multiple receptors, and effectors is by blocking the
signaling circuits by expressing dominant negative mutants of different
G
subunits in vivo. Mutations of glycine residues in the
conservative sequence DVGGQR of the G
subunits were found to
reduce GTP binding and activation of the G proteins. Expression of
these mutants inhibited pathways controlled by G
and
G
(12, 13, 14, 15) . We
identified another interesting mutation that abolished GTP binding in
G
-G
S47C and demonstrated that the
mutant retained the ability to bind
and could suppress G
protein-mediated signal transduction in Xenopus oocytes (16) .
In the current work, we studied the effects of
expression of the G, G
, and their
dominant negative mutants G
S48C and G
S47C on reconstituted signaling pathways in transiently
transfected COS-7 cells. We found that G
can attenuate
activation of PLC (
)by hormone receptors coupled to the
enzyme via members of the G
family and that the most likely
molecular mechanism for this inhibition is competition for
subunits. Our data provide further evidence for the interdependence of
G protein-mediated signaling pathways and the important role of
subunits in controlling these interactions.
Substitution of serine 47 for cysteine in G abolished GTP binding, but the mutant retained its interaction
with
. Due to the apparent inability to release
upon the hormonal activation, this mutant behaved as dominant negative
in the G
-mediated signaling pathway reconstituted in Xenopus oocytes(16) . To obtain a similar mutant of
G
, we replaced the homologous Ser-48 with Cys by
modifying the cDNA of G
. We tested this mutant to
determine if it would inhibit pertussis toxin-sensitive pathways of PLC
stimulation in mammalian cells. In these pathways, PLC is apparently
activated by free
complexes (8, 9, 10) that are released from G protein
heterotrimers upon hormonal stimulation. Fig. 1demonstrates
that in the COS-7 cells cotransfected with cDNAs for
,
, and
PLC
2, there is a 4-6-fold stimulation of phosphoinositide
hydrolysis compared with cells expressing PLC
2 alone.
Cotransfection of both wild-type G
cDNA and the
G
S48C mutant abolished the
-induced PLC
activity, apparently due to sequestration of free
. However,
the behavior of mutant and the wild-type G
subunits was different
with respect to receptor-mediated activation of PLC. In the cells
cotransfected with muscarinic M2 receptors (Fig. 1B),
carbachol stimulation resulted in an increase of inositol phosphates
released in the presence of
and wild-type
G
. In contrast, no ligand-induced activity was
observed in cells cotransfected with the S48C mutant of
G
, presumably because it bound
irreversibly.
Figure 1:
Influence of G and
its S47C mutant on stimulation of PLC
2 by free
. A, cells were cotransfected with cDNAs encoding phospholipase
2 (0.2 µg), G
subunit (
, 0.2 µg),
G
(0.2 µg) (
2 or
5 were used in different
experiments), and wild-type (WT) or mutant G
(0.3 µg). Total amount of DNA was adjusted to 1.0 µg by
CMV-LZ cDNA. Cells were then labeled with
[
H]inositol, and levels of inositol phosphates
were determined. Transfection, labeling, and analysis of inositol
phosphate release were performed as previously described(15) . B, cells were cotransfected also with cDNA for muscarinic M2
receptor, and levels of released inositol phosphates were determined
after incubation of the cells with indicated concentrations of
carbachol.
PLC 2 can be stimulated by two pathways:
pertussis toxin-sensitive (by
subunits released upon the
activation of G proteins (members of the G
family)) and
pertussis toxin-insensitive (by G
subunits of the G
family). If both pathways share the same pool of
subunits, the dominant negative mutants of G
family
subunits would not only block the stimulation of PLC by
but
also prevent its activation through G
family
subunits. To test this idea, we cotransfected COS-7 cells with cDNAs
encoding the C5a receptor and G
, reconstituting a
pathway for G
-mediated stimulation of endogenous PLC by
C5a (Fig. 2). Coexpression of either wild-type G
subunit or its mutant S48C in the system did not have a
significant effect on the basal level of inositol phosphate release but
markedly reduced the C5a stimulation. Similar inhibitory effects were
observed upon cotransfection with other pertussis toxin-sensitive
G
subunits such as G
, G
, or the
G
S47C mutant (data not shown). Western analysis
demonstrated that introduction of these proteins did not change the
level of G
expression (Fig. 2B). This
suggested, that the inhibitory effect of G
proteins is
due to specific interaction with components of the reconstituted
signaling pathway and not because of interference with
transcription/translation machinery of the transfected cells. The
maximal level of inhibition of the C5a receptor-PLC pathway by
G
was around 70%. It is possible that the inhibitory
effect could have been larger if all of the transfected cells could
take up and express all three transfected cDNAs. The effect of
G
was proportional to the amount of protein expressed
in the cells, i.e. higher concentrations of G
caused stronger inhibition of the C5a-induced PLC activity (Fig. 3). This observation suggests that inhibition occurred due
to competition between G
and G
subunits
for the interaction with other protein components involved in the C5a
induction of PLC activity.
Figure 2:
Inhibition of C5a-stimulated PLC activity
by G and its S48C mutant in transiently transfected
COS-7 cells. Cells were transfected with C5a receptor (0.2 µg) and
G
(0.2 µg) cDNAs together with cDNAs (0.6 µg)
corresponding to wild-type (WT) G
, its S48C
mutant, or pCMV-LZ (control). A, levels of inositol phosphates
released were determined with no ligand or in the presence of 0.25
µM C5a. B and C, Western blot analysis
of the transfected COS-7 cells. Cells were treated the same way as
those for the PLC assay with exception that
[
H]inositol was omitted from the media. On the
day of the PLC assay, they were harvested and subjected to Western
analysis with antibodies raised against G
(B)
or G
(C).
Figure 3:
Inhibition of C5a-stimulated PLC activity
by different amounts of G S48C mutant. Cells were
transfected with C5a receptor (0.2 µg) and G
(0.2
µg) cDNAs and indicated amounts of G
S48C mutant
cDNA. The total amount of DNA per transfection was adjusted to 1.0
µg with pCMV-LZ. Levels of inositol phosphates were determined with
no ligand (openbar) or in the presence of 0.25
µM of C5a (graybars). Inset,
Western analysis of identically treated cells with antibodies raised
against G
(G
S48C) and G
.
Both G and G
have been shown to couple to the C5a
receptor(21, 22, 23) . It is possible that
G
sequesters
, which may be necessary for
coupling of C5a receptor with G
. Alternatively,
G
and G
may compete for interaction with the
receptor. Coupling of G
with its cognate receptors can be
abolished after ADP-ribosylation with pertussis toxin. We found,
however, that ADP-ribosylation of wild-type G
promoted
even further inhibition of the C5a-induced PLC activation, while cells
expressing only C5a and G
were insensitive to the
toxin (Fig. 4). This observation implies that G
attenuates the C5a-induced activation of PLC by competing with
the G
not on the receptor but rather by competing for
. It is known that ADP-ribosylation stabilizes association of
the
and
subunits(24) . In the pertussis
toxin-treated cells, the wild-type G
binds
stronger and therefore competes with G
for
more efficiently. Further support for this notion comes from
cotransfecting G
and C5a receptor together with the
S48C mutant of G
. Treatment of these cells with
pertussis toxin did not lead to any further inhibition because the
mutant has been shown to bind
irreversibly even without
pertussis toxin treatment(13) . To ensure that both wild-type
and the mutant G
were ADP-ribosylated equally, we
treated the membrane preparations of the COS-7 cells with pertussis
toxin in the presence of [
P]NAD. Fig. 4(inset) demonstrates that both proteins were
labeled to a similar extent, and, as in the case with
G
(13) , GTP
S did not influence labeling
of the S48C mutant but drastically reduced the modification of the
wild-type G
. These experiments suggest that the
mechanism for the attenuation of hormone-activated PLC by G
is based upon sequestering of
that can be
``shared'' with G
. It is unlikely that
competition occurs at the effector level because previous evidence
argues against the interaction of G
family proteins with
PLC(10, 18, 19) . The possibility that
G
causes the inhibition of PLC indirectly by
activation of a different effector is also ruled out because the
G
dominant negative mutant, which cannot bind GTP and
be activated, is more potent in attenuation of hormone-stimulated PLC
than wild-type G
.
Figure 4:
Influence of pertussis toxin (PTX) on inhibition of C5a-stimulated PLC by G or its S48C mutant in transfected COS-7 cells. Cells were
transfected with C5a receptor (0.2 µg), G
(0.2
µg) cDNAs and cDNAs (0.6 µg) corresponding to wild-type
G
, its S48C mutant, or pCMV-LZ (control). Levels of
inositol phosphates were determined after treatment with (blackbars) or without (graybars) 200
µg/ml pertussis toxin for 4 h at 37 °C prior to addition of
0.25 µM C5a. Inset, influence of 100 µM GTP
S on pertussis toxin-catalyzed ADP-ribosylation of
wild-type G
and the S48C mutant. Cell membranes were
obtained and treated with pertussis toxin in the presence of
[
P]NAD, 10 µM GDP, and with or
without 0.1 mM GTP
S as described under
``Experimental Procedures.'' Proteins were then resolved by
SDS electrophoresis. Gels were stained with Coomassie Blue, dried, and
exposed to x-ray film.
If it is binding of
that is responsible for the interference of G
with the
G
(G
)-mediated signaling, we
would expect that G
would inhibit ligand induction
through receptors that do not interact with G
, such as
1C adrenergic receptors(25) . This was indeed the case; in
the cotransfected COS-7 cells,
1C adrenergic receptor-PLC
coupling, which is apparently mediated by endogenous
G
(19) , was inhibited by G
and
G
(Fig. 5). As found with the C5a receptor,
this inhibition was significantly enhanced by pertussis toxin
treatment. Therefore, it is quite likely that endogenous
is
a limiting factor for the coupling of G
or
G
with their cognate receptors.
Figure 5:
Effect of G and
G
on stimulation of PLC by
1C adrenergic receptor
in transiently transfected COS-7 cells. Cells were transfected with
1C-adrenergic receptor (0.4 µg) cDNA and cDNAs (0.6 µg)
corresponding to wild-type G
, G
, or
pCMV-LZ. Stimulation of inositol phosphate release by 10 µM norepinephrine (NE) was determined after (blackbars) or with no (graybars) treatment
with pertussis toxin (PTX).
Recently
published crystal structures of transducin subunit complexes with
GTP
S (26) and GDP (27) show that the hydroxyl
group of serine 43, which is homologous to serine 48 in
G
, coordinates with Mg
found in the
GTP binding pocket. The conversion of the -OH to -SH in a
Ser
Cys mutant apparently does not cause a major disruption of
the overall structure of G
protein, since G
S48C
can still bind
. Because the serine residue is conserved in
the G
family, we introduced the Ser
Cys mutation into
different G
subunits to use them for inhibition of specific
signaling pathways. However, the mutations introduced in G
(S47C), G
(S56C) resulted in a null phenotype
(data not shown). The mutants were expressed in cells at the same level
as wild-type proteins according to Western analysis but failed to
reveal any functional activity. They did not stimulate PLC and did not
inhibit hormone-stimulated PLC or the stimulation of the enzyme by free
complexes. It is noteworthy that introduction of other
putative dominant negative mutations, G203T and G204A, into
G
and G
also resulted in a null
phenotype, (
)while the homologous mutations G203T and G204A
in G
(12, 13, 14, 15) or
G
(16) proved to be functional. Interestingly,
mutants G203T and G204A of recombinant G
had very
similar biochemical properties, yet only G203T G
behaved as a dominant negative in vivo while G204A
resulted in null phenotype(14) . At this point, we do not
understand exactly why the mutants of G
family
subunits were inactive. However, these results suggest that there are
local differences in structure and activity even in highly conserved
regions of G
subunits. Detailed comparison of the crystal
structures of G
and G
subunits will shed
light on the differences between these highly homologous proteins.
Cross-talk between the different G protein-mediated signaling
pathways has been previously
demonstrated(28, 29, 30) ; for instance,
adenylate cyclase type IV was found to integrate signals coming through
G, G
, and G
(31) . Here, we
demonstrate that G
-like proteins can alter the
pathways regulating PLC via G
family G proteins. Can this
occur in vivo? Some indirect evidence supports the existence
of such mechanisms. For example, in vivo most of the signaling
through C5a receptor is pertussis toxin sensitive, whereas in vitro C5a receptor couples to pertussis toxin-insensitive
G
. In neutrophils where G
is the most
abundant G
protein, these contradictory observations can be
reconciled if a mechanism similar to the one shown on Fig. 4took place, i.e. ADP-ribosylation increased
affinity of G
for
, thus preventing coupling
of receptor to G
. Therefore, the apparent pertussis toxin
sensitivity of G
-mediated signaling can be explained as a
result of depleting the pathway of
. In light of such a
possibility, the interpretation of the experiments on pertussis toxin
treatment of cells must be done with care; ADP-ribosylation not only
can uncouple G
from its cognate receptor, but it can shift
the equilibrium
+
=
i
toward the heterotrimer, thus reducing the available
pool of
subunits and affecting other pathways. This
mechanism implies that
is a limiting factor for G
(G
)-mediated signaling. Recent data show that
can be bound by other proteins such as effectors PLC
(8, 9, 10) , PLA2(6) , inositol
kinase(32) , and K
channel(33, 34) , ras-related
proteins(35) , phosducin(36) , receptor kinase, and
other proteins containing pleckstrin homology
domains(37, 38) , calmodulin(39) , etc.
Therefore, the GDP-bound G
subunits compete for the binding to
not only with different G
but also with these
``other'' proteins. It is clear that relative affinities of
the different G
subunits for
complexes are critical for
specific channeling of signals. Another important notion is that
regulation of the expression level of the G protein subunits could
provide additional diversity to signal transduction pathways in various
cells.