(Received for publication, June 22, 1995; and in revised form, September 19, 1995)
From the
At least 30 G protein-linked receptors stimulate
phosphatidylinositol 4,5-bisphosphate phosphodiesterase (phospholipase
C, PLC
) through G protein subunits to release intracellular
calcium from the endoplasmic reticulum (Clapham, D. E.(1995) Cell 80, 259-268). Although both G
[Medline]
and G
G protein
subunits have been shown to activate purified PLC
in
vitro, G
q has been presumed to mediate the pertussis
toxin-insensitive response in vivo. In this study, we show
that G
plays a dominant role in muscarinic-mediated
activation of PLC
by employing the Xenopus oocyte
expression system. Antisense nucleotides and antibodies to G
q/11
blocked the m3-mediated signal transduction by inhibiting interaction
of the muscarinic receptor with the G protein. Agents that specifically
bound free G
subunits (G
-GDP and a
-adrenergic
receptor kinase fragment) inhibited acetylcholine-induced signal
transduction to PLC
, and injection of G
subunits into
oocytes directly induced release of intracellular Ca
.
We conclude that receptor coupling specificity of the
G
q/G
heterotrimer is determined by G
q; G
is the predominant signaling molecule activating oocyte PLC
.
Muscarinic acetylcholine (ACh) ()receptors are
heptahelical G protein-linked receptors widely dispersed in a variety
of tissues including neurons of the central and peripheral nervous
system, heart, smooth muscle, and exocrine
glands(1, 2) . The five muscarinic receptor subtypes
(referred to as m1-m5) can be grouped into two broad categories
of signal transduction. Stimulation of m2 and m4 subtypes inhibits
adenylyl cyclase activity (3) and only weakly activates
phosphoinositide turnover(4) ; activation of m1, m3, and m5
receptors strongly induces phosphoinositide hydrolysis through a
pertussis toxin (PTX)-insensitive G protein(4, 5) .
Convincing biochemical and functional evidence established that
members of the Gq/11 family of G proteins activate
PLC
(6, 7, 8, 9, 10, 11) ,
but not PLC
or PLC
isoforms(7) . Equally strong
evidence demonstrated that PLC
enzymes are activated by free G
protein
complexes (12, 13, 14, 15, 16) . Thus
muscarinic receptor activation releases G
-GTP and G
,
both of which can stimulate PLC
(17) .
The goal of this
project was to identify which G protein subunits activate PLC
following stimulation of the m3 receptor in Xenopus laevis oocytes. The Xenopus oocyte PLC
has been cloned and
is unique, containing 33-64% amino acid identity to mammalian
PLC
isoforms(18) . Experiments with antisense
oligonucleotides designed to block synthesis of members of the
G
q-11 family of G proteins in Xenopus oocytes decreased
the peak m3 receptor-mediated calcium (Ca
) release as
measured by the Ca
-sensitive chloride current
(I
). Specific G
q function-blocking antibodies also
abrogated the m3 receptor-mediated response. Direct injection of
G
into oocytes increased intracellular Ca
[Ca
]
and
injection of specific G
-binding agents, G
-GDP, and a
-adrenergic receptor kinase (
ARK) fragment (19) attenuated the muscarinic receptor-mediated response in a
dose-dependent manner. We conclude that the m3 muscarinic signal
requires G
q for specificity, but the majority of the signal is
transduced by the G
dimer.
For experiments
determining the time course of the antisense oligonucleotide effects,
all oocytes were injected with antisense common1 or sense
oligonucleotides (0.8 mg/ml) on day 0 and groups of cells assayed for
the m3-mediated response during the subsequent 7 days. In order to rule
out an effect of varying levels of m3 receptor expression, the m3 mRNA
transcript was always injected 2 days prior to voltage clamp.
Constituitively active Gq (G
qQ209L DNA) was subcloned into
Bluescript SK+ (Promega) and translated into capped cRNA using the
Megacript kit (Ambion). Thus, for cells measured on day 0, mRNA for the
muscarinic receptor had been injected 2 days prior to day 0 and cells
were voltage clamped the same day as antisense or sense
oligonucleotides were injected. In contrast, oocytes measured on day 7
were injected with oligonucleotides on day 0 and mRNA for m3 receptors
on day 5.
We found no appearance of nonspecific effects of oligonucleotide injection on oocytes at concentrations below 1.0 mg/ml (see Fig. 1C). However, at antisense and sense oligonucleotide concentrations greater than 1.5 mg/ml, oocytes showed visible signs of deterioration (loss of pigmentation of animal pole, loss of intracellular contents, and deterioration of the resting membrane potential, n = 26), illustrating that, with higher oligonucleotide concentrations, global effects occurred that were not specific to G protein function.
Figure 1:
G antisense oligonucleotides
decrease the m3-mediated response. A, oocytes were injected
with 40 ng/oocyte of common1 G
oligonucleotides 4 days prior to
voltage clamp (holding potential, -70 mV) and 5 µM ACh added to the bath (arrow). The endogenous (no m3
expression) ACh-induced response was less than 0.3 µA. The
m3-expressing, sense-injected oocyte responded to ACh with a 5.2-µA
increase in current (exogenous, sense). The m3-expressing, antisense
nucleotides (common1)-injected ACh-induced current increased to 0.6
µA (exogenous, antisense). The delay in the response in the
antisense nucleotide-injected oocyte was likely due to diffusion of ACh
in the bath. B, oocytes injected with antisense nucleotides
common to the G
q family (q/11-com) displayed 70% smaller
ACh-stimulated I
than sense controls (2.7 ± 0.3
and 0.8 ± 0.2 µA in G
q sense- and antisense-injected
cells, respectively). In G
11 antisense nucleotide-injected
oocytes, peak ACh-induced I
decreased 76% compared to
sense-injected cells (0.7 ± 0.2 and 2.9 ± 0.5 µA,
respectively). I
in oocytes injected with antisense
specific to G
q alone were reduced by 81% from sense controls (2.6
± 0.5 and 0.5 ± 0.2 µA in sense- and
antisense-injected cells, respectively). The ACh-induced responses in
oocytes injected with sense and antisense nucleotides to other G
protein subunits were not statistically different then water-injected
oocytes (H
O). Average n = 33 oocytes/group. C, the optimal dose for
inhibition of the ACh-induced current with antisense common1 was 0.8
mg/ml (pipette concentration). The antisense nucleotides targeted to a
common region of G
q and G
11 maximally inhibited the ACh
current at 0.6 mg/ml. Sense oligonucleotides (0.6 mg/ml) to the same
region did not significantly alter the m3-mediated response. Average n = 15 oocytes/group.
After SDS electrophoresis (24) and
transfer onto a poly(vinylidene fluoride) membrane, proteins were
immunoblotted according to published procedures(25) .
Visualization was accomplished with I-labeled goat
anti-rabbit IgG-F(ab`)
fragments and autoradiography. For
G
q staining, three different antibodies to G
q were used. Two
were raised to the C-terminal amino acids (CILQLNLKEYNLV) of G
q
from two different sources, Z811 (26) and CQ2(27) , and
the third antibody was raised to the internal portion of G
q, W082
(EVDVEKVSAFENPYVDAIK) (28) . Block of Z811 antibody by the
epitope peptide was performed by preincubation of antibodies with 50
µM C-terminal peptide of G
q followed by 10-fold
dilution for immunoblotting. Bovine brain G protein subunits were
isolated as described previously(29) . They were shown to be
active by their ability to bind GTP
S(29) , and G
subunits were able to bind immobilized G
and vice
versa(30) .
Specific antisense oligonucleotides for
the individual G subtypes were injected into oocytes using the
protocol described above. Only those cells receiving antisense
nucleotides to the G
q family (G
q/11-com) or to its specific
members (G
q or G
11) exhibited a decrease in the ACh-induced
response when compared to water- and sense-injected cells (Fig. 1B). A 70-84% decline in the ACh-induced
current was measured in oocytes injected with antisense
oligonucleotides designed to G
11 and G
q, respectively. We
could not distinguish between G
q and G
11 subunits in the ACh
response, since injection of antisense oligonucleotides to either
G
q or G
11 resulted in statistically indistinguishable
suppression of the m3 response. The antisense nucleotides may have
interacted with both G
11 and G
q mRNAs, since they have a high
identity, or both G
q and G
11 may participate in the m3
response. Such an interaction has been described for the
thyrotropin-releasing hormone coupling to G proteins(40) . The
dose-response relation illustrates similar efficacies of common1
antisense and G
q/11 common antisense (Fig. 1C).
The same concentration of sense oligonucleotides did not alter peak
I
when compared to water-injected cells (n = 65). Thus, antisense oligonucleotides directed to members
of the G
q family, specifically G
q and G
11, blocked the
m3 muscarinic receptor mediated response.
Figure 2:
Immunoblots of Gq family proteins in
oocyte membranes. A, human embryonic kidney (A293 or HEK) and
oocyte membranes were immunoblotted with antibodies specific to members
of the G
q family of proteins. The first two lanes show
immunostaining with antibody Z811 in HEK (H) and oocytes (O) membranes. This recognition was inhibited by the
C-terminal peptide (lane 3). The remaining lanes illustrate
immunostaining of both HEK and oocyte membranes with the C-terminal
G
q family antibody, CQ2 and the G
q-specific antibody, W082
(designed to an internal portion of G
q). B, the level of
protein recognized by G
q antibodies increased 3-fold in oocytes
expressing the exogenous Xenopus oocyte G
q clone. Protein
levels were quantified by densitometry and normalized to water-injected
oocytes.
Figure 3:
Gq-family antibodies blocked the
m3-mediated response. A, the m3-mediated I
response was inhibited 83% by Z811 antibody (mean peak
I
= 0.2 ± 0.1 µA) at a dose of 70
µg/ml. Preimmune control sera at the same concentration did not
significantly decrease the ACh-induced response. Average n = 36 oocytes/group. B, the mean peak I
was 3.1 ± 0.3 and 3.6 ± 0.6 µA for preimmune-
and vehicle-injected oocytes, respectively. Antibody CQ2 blocked the
ACh response by 77% (mean peak I
= 0.7 ±
0.3 µA). Injection of activated PTX followed by incubation in
PTX-containing medium did not alter the response in control or
antibody-injected cells (mean peak I
= 3.1
± 0.7 and 0.3 ± 0.1 µA for PTX-treated cells injected
with KCl or Z811, respectively). Average n = 34
oocytes/group.
Figure 4:
G-GDP blocked the m3-mediated
response. A, injection of purified G
-GDP (14 nM calculated intracellular concentration) 5 min prior to
voltage-clamp attenuated the m3-mediated response at all ACh
concentrations. Higher doses of G
-GDP (1.75 µM intracellular concentration) completely blocked the ACh-induced
response. Injection of a fragment of the G
-binding protein
ARK (200 µM) greatly attenuated the ACh-induced
response. The amplitude of the peak current (vehicle) did not increase
with higher doses of ACh (100 µM). Average n = 5 oocytes/group. B, G
subtypes bound to GDP
equally inhibited the I
response. G
i-1 (17
nM), G
i-2 (10 nM), G
i-3 (25 nM)
(Iniguez-Lluhi et al., 1992), and G
q (10 nM)
(Hepler et al., 1993) were recombinant proteins. G
o/i (10
nM) was purified from bovine brain and consisted predominantly
of G
o. Average n = 18
ooyctes/group.
To
rule out the possibility that G-GDP interacted directly with
oocyte PLC
, thereby blocking access of G
-GTP to the PLC
molecule, we injected oocytes with the G
-binding region of
ARK. The
ARK fragment has been used to bind G
and
block activation of the muscarinic-gated K channel(19) .
Injection of this
ARK fragment (200 µM) abolished the
1 µM ACh-induced response (Fig. 4A, n = 5). With higher doses of ACh (10 µM, n = 7), more than 75% of the response was blocked compared to
buffer-injected cells (mean peak I
= 0.7 µA, n = 10). Thus, agents known to bind free G
(G
-GDP and
ARK fragment) blocked the ACh-induced current in
m3-expressing oocytes.
Figure 5:
G alone released
[Ca
]
. A, an
increase in I
was elicited following injection (arrow) of 60 nM (estimated final concentration)
purified G
into a single oocyte. B, the mean
amplitude of the G
(700 nM estimated final
concentration) response was 2.2 ± 0.3 µA. Injection of the
holomultimeric G protein complex (700 nM estimated final
concentration) did not cause a significant increase in I
(0.4 ± 0.1 µA). Boiled G
failed to induce a
response (0.2 ± 0.1 µA) as did G
co-injected with
heparin (10 µg/ml, 0.3 ± 0.2 µA) or EGTA (10
mM, 0.1 ± 0.0 µA). Average n = 14
oocytes/group
Activated brain
G-GTP
S (85 nM) resulted in a small increase in
I
when injected into oocytes (average peak = 0.5
µA, n = 18), less than one fourth the amplitude of
the G
response. G
and especially G
q subunits bind
poorly to GTP
S in vitro(8) , therefore excess
GTP
S was included in the solution (10 times > GTP
S versus G
). Injection of equal concentrations of GTP
S
alone increased I
to the same extent as
G
-GTP
S (n = 17). Confocal imaging of oocytes
following injection of 0.5 µM GTP
S revealed
Ca
waves near the injection site (data not shown, n = 6; see also (4) ). The Ca
release by GTP
S and subsequent activation of I
was likely due to activation of the endogenous oocyte G protein
subunits. These results indicate that the small increase in I
with G
-GTP
S may be due to the presence of free
GTP
S, rather than any direct activation by the exogenous G
protein subunit.
To further examine the possible role of Gq on
intracellular Ca
release, we overexpressed the
constitutively active G
q subunit in oocytes(63) . ACh was
applied to oocytes 3 days after coinjection of constitutively active
G
q mRNA and the m3 receptor mRNA (n = 30) or the
m3 receptor mRNA alone (n = 20). The ACh-induced change
in I
averaged 6-fold larger in oocytes expressing the
m3 receptor alone compared to oocytes coexpressing m3 and
constitutively active G
q. This effect was similar to the effect of
G
-GDP injection (Fig. 4B). Thus, contrary to what
might be expected if G
q were the direct activator of phospholipase
C
, I
responses were inhibited by the expressed
G
q. Although several interpretations are possible, such as the
induction of crosstalk, this result can be explained by trapping of
free G
by overexpressed G
q. This is consistent with our
observations that active G
q-GTP
S can bind G
, albeit
with lower affinity. (
)If constitutively active G
q
activated phospholipase C
, we would also expect a reproducible
shift in membrane potential as intracellular calcium levels activate
I
. Coexpression of G
q and the m3 receptor changed
the membrane potentials of the oocytes only slightly (average of
-36 ± 3 mV and -45 ± 3 mV, for m3 and m3
+ G
q, respectively; these values were not significantly
different in a Student's paired t test). These results
do not support the hypothesis that constitutively active G
q is the
major direct activator of oocyte phospholipase C
.
Figure 6:
G subunits diffused freely in the
cytoplasm, but G
subunits did not. A, fluorescently
labeled G
recorded at 15 s and 5 min after injection
demonstrates that the hydrophobic G
subunit does not readily
diffuse in oocyte cytoplasm. In contrast, G
fluoresced throughout
the oocyte only 15 s after injection. B, the site of high
G
concentration coincided with the region of intracellular
Ca
release.
Our results indicate that the G protein subunit G
alone may activate PLC
to initiate the cascade for intracellular
Ca
release; the G
q subunit couples to the m3
muscarinic receptor providing specificity for the activated pathway.
This conclusion is based on experiments that isolated the function of
endogenous oocyte G
q and G
subunits using: 1) antisense
nucleotides to block protein production, 2) antibodies to block protein
interactions, 3) direct injection of activated G protein subunits, and
4) specific G
binding compounds (G
-GDP and
ARK
fragment) that compete with G
's ability to interact
with other molecules.
Antisense oligonucleotides have been used
previously to identify the involvement of both G and G
subunits in inhibition of Ca
voltage-dependent
channels(37, 47, 48) . However, antisense
oligonucleotide block alone cannot distinguish which G protein subunit
was required for interaction with the muscarinic receptor or with the
effector, PLC
. Antisense oligonucleotide (G
q) treatment of
the oocytes effectively suppressed the m3 muscarinic signal by 80%,
while reducing the protein levels immunostained by G
q/11
antibodies by only 40%. These results may be explained by a population
of stained, but inactive, G
q present in oocytes, or by the known
nonlinearity of signal transduction. Signal transduction steps between
the muscarinic receptor and the measured I
include the
G protein, PLC
enzyme, InsP
generation, release of
Ca
, and Ca
-dependent activation of
I
. The requirement of G
q/11 for the signal appears
to lie in its ability to couple to the muscarinic receptor. In this
model the specificity for the pathway lies in that G
/receptor
interaction. This suggestion is supported by the fact that antibodies
that inhibit binding of G
q to the receptor, but do not interfere
with the interaction with PLC
(27) , blocked the m3 signal.
The addition of exogenous G
-binding proteins has been used
previously to block the function of the
G
(42, 43, 44, 49) .
Complete block of the m3 response by factors that bind free G
(G
-GDP and
ARK fragment) suggests that most, if not all, of
the m3 muscarinic signal is transduced via G
.
The results
are in contrast with the common perception, based on reconstitution of
PLC subtypes with purified G
or G
subunits, that
both may transduce the signal(17, 50) , but G
q/11
is responsible in PTX-insensitive pathways(10, 11) .
We find that G
transduces the signal even when coupled to the
PTX-insensitive G
q subunit. Depending on the cell type and assays
used, laboratories have concluded that several different G
activate PLC
including G
o (51, 52) ,
G
i-1(53) , G
i-2 and
G
i-3(54, 55) ,
G
q(36, 40, 56, 57, 58) ,
G
11(40, 59) , and G
s(60) . Our
finding that the endogenous activator of oocyte PLC
is G
may explain these variations since there is apparently little
specificity of G
interactions with effector
proteins(43, 61) . We cannot rule out the possibility
that G
interacts with oocytes PLC
in a unique manner,
however the oocyte expression system has been used frequently to
identify receptor/G protein
specificity(51, 52, 53, 60) . The
results of this investigation are a cautionary reminder that the
interpretation of such experiments is not simple. Overexpression of
G
subunits in mammalian cell lines is another common approach to
identifying receptor/G protein specificity(9, 40) .
However, overexpressed G
subunits may bind endogenous G
,
thereby increasing the available G
that is activated
following the appropriate receptor stimulation. Overexpression studies
determine whether a G protein subunit is a component of the
receptor-activated pathway, but they cannot identify which G protein
subunit (G
or G
) interacts with PLC
.
Other
investigators have speculated that PTX-sensitive stimulation of
PLC is via G
, because in vitro experiments show
no direct effect of G
i-1, G
i-2, G
i-3, or G
o on
PLC
(62) , while the PTX-insensitive stimulation of
PLC
is through the G
q/11 family of G
subunits. In
contrast to earlier reports showing large, transient increases in
I
with injection of purified G
-GTP
S into
oocytes(52) , we found no evidence for specific activation of
the PLC pathway by injection of similar concentrations of
G
-GTP
S. We cannot completely exclude G
q/11 as carrying a
portion of the m3 signal to PLC
, but all activation that we
measured with G
-GTP
S could be ascribed to the activity of
free GTP
S, which activates intrinsic G protein pathways. Our
results demonstrate that even in the presence of activated G
q
following stimulation of the m3 muscarinic receptor, inhibition of
G
blocked signal transduction to oocyte PLC
.
The
simplest interpretation of the results is that both G and
G
subunits are necessary for m3 muscarinic signal
transduction; G
q/11 provides the specificity of the signal through
its interaction with the receptor and G
freed during
activation, transduces the signal to the effector. These experiments do
not exclude other interpretations. If G
or G
subunits do
not dissociate in the membrane following activation, but rather form a
macromolecular complex with the receptor and effector enzyme, these
experimental results may be explained by a constrained, activated
heterotrimer with activator sites on G
accessible to
inhibitory proteins. In any case, signal transduction in intact
membranes does not appear to behave solely as predicted from
experiments with purified subunits in solution.