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J Biol Chem, Vol. 273, Issue 46, 30183-30188, November 13, 1998
From the Division of Biology, 147-75, California Institute of
Technology, Pasadena, California 91125
We constructed a double mutant version of the Hundreds of seven-transmembrane receptors activate heterotrimeric
G proteins and transduce signals across cell membranes in eukaryotic
cells. The stimulated receptors catalyze the exchange of GTP for GDP
bound to G protein We recently reported that a mutant version of Go Materials--
Purified bovine retinal transducin Expression and Purification of His6-tagged
Go Membrane Preparation from Baculovirus-infected Sf9
Cells--
Sf9 cells were grown and infected with recombinant
baculoviruses encoding either m2 MAChR or NFPR (9-11). Infected cells
were centrifuged and resuspended at <107 cells/ml in
HME/PI buffer (20 mM NaHepes, pH 8.0, 2 mM
MgCl2, 1 mM EDTA, 0.1 mM
phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, and 10 µg/ml
leupeptin). The cell suspension was homogenized by 10 strokes in a
glass homogenizer followed by passing through a 27 gauge hypodermic
needle several times. The homogenate was briefly centrifuged at
3,000 × g for 10 min, and then the supernatant was
collected and centrifuged at 30,000 × g for 30 min.
The pellet was washed once with HME/PI, and the final pellet was
resuspended in HME/PI at a protein concentration of 5 mg/ml.
Synthesis of XTP Nucleotide Binding of Purified Go Radioligand Binding of Receptors--
The ligand binding assays
of membrane-bound receptors were performed as described (9-11). The
total concentration of m2 MAChR and the affinities of NFPR were
determined by incubating membranes with 2 nM
[3H]QNB or various concentrations of
fML[3H]P for 1 h in 20 mM Tris-HCl, pH
7.4, 12.5 mM MgCl2, and 1 mM EDTA
at 30 °C in a final volume of 0.5 ml. Nonspecific binding was
defined as binding that was not displaced by 10 µM
atropine for m2 MAChR or 10 µM fMLP for NFPR. Unbound
ligands were removed by filtration through Whatman GF/C filters and
washing four times using ice-cold binding buffer. The amount of bound
radioactivity was determined by scintillation counting.
Binding of Go Cell Culture and Transfection--
COS-7 cells were cultured in
Dulbecco's modified Eagle's medium containing 10% fetal calf serum.
1 × 105 cells/well were seeded in 12-well plates 1 day before transfection. All transfection assays contained a total
amount of 1 µg of DNA, and pCIS encoding Stimulation of XTP High Affinity Ligand Binding of N-formyl Peptide Receptor Induced
by Go Binding of Go Dominant-negative Effect of Go
To test if Go Go Because cells lack xanthine nucleotides, Go Because Go In the previous report (6), we showed that the single Go The recombinant baculovirus encoding
m2 MAChR was a generous gift from Dr. E. Ross. We thank members of Dr.
Simon's lab for helpful discussions, and Dr. Tau-Mu Yi for comments on
the manuscript.
*
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.
The abbreviations used are:
XDP, xanthine
diphosphate; XTP, xanthine triphosphate; MAChR, muscarinic cholinergic
receptor; NFPR, N-formyl peptide receptor; TRH, thyrotropin-releasing hormone; NDK, nucleotide diphosphate kinase; IP3, inositol 1,4,5-trisphosphate; PLC, phospholipase C; GTP
Interaction of the Xanthine Nucleotide Binding Go
Mutant
with G Protein-coupled Receptors*
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
subunit of Go that was regulated by xanthine nucleotides instead of
guanine nucleotides (GoX). We investigated the interaction between
GoX and G protein-coupled receptors in vitro. First, we
found that the activated m2 muscarinic cholinergic receptor (MAChR)
could facilitate the exchange of XTP
S for XDP in the GoX
heterotrimer. Second, the GoX complex was able to induce the
high affinity ligand-binding state in the N-formyl peptide
receptor (NFPR). These experiments demonstrated that GoX was able to
interact effectively with G protein-coupled receptors. Third, we found that the empty form of GoX, lacking a bound nucleotide and , formed a stable complex with the m2 muscarinic cholingeric receptor associated with the plasma membrane. Finally, we investigated the
interaction of GoX with receptor in COS-7 cells. The empty form of
GoX bound tightly to the receptor and was not activated because XTP
was not available intracellularly. We tested the ability of GoX to
inhibit the activities of several different G protein-coupled receptors
in transfected COS-7 cells and found that GoX specifically inhibited
Go-coupled receptors. Thus the modified G proteins may act as
dominant-negative mutants to trap and inactivate specific subsets of receptors.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
subunits. Activated GTP-bound subunits and
free subunits regulate a variety of cellular effectors including
enzymes and ion-channels (1-3). Signaling is normally initiated by the
binding of agonist to receptor, which stabilizes the receptor in an
active conformation. Receptors function to stimulate the dissociation
of GDP bound to the G protein subunits. The subsequent binding of
GTP to the empty subunit promotes the conformational change of G
and dissociation of the subunits. The G protein subunit in
the nucleotide-free state appears to be an important intermediate in
the activation. From studies of rhodopsin and transducin, it has been
postulated that the empty G protein (nucleotide-free) forms a stable
complex with the receptor (4). Both empty forms of Gi and Go subunits have been purified under harsh conditions (1 M
(NH4)2SO4 and 20% glycerol), and
they were unstable (5).
, GoX
(GoD273N/Q205L), was regulated by xanthine nucleotides, not by
guanine nucleotides (6). GoX bound
XDP1 and XTP instead of GDP
or GTP. GoX bound G protein subunits only in the presence of
XDP, and XTP stimulated dissociation of the GoX heterotrimer.
XTP-bound GoX underwent a conformational change similar to the
activated wild-type Go. In the present study, we investigated the
interaction between GoX and G protein-coupled receptors. We found
that GoX mutant proteins retained the receptor binding specificity
of the wild-type Go and were able to interact with Go-coupled
receptors, such as the m2 muscarinic cholinergic receptor (MAChR),
N-formyl peptide receptor (NFPR), and thrombin receptor, but
not with m1 MAChR or thyrotropin-releasing hormone (TRH) receptor which
do not couple to wild-type Go. Because the concentrations of XDP and
XTP are relatively low in vivo (7), GoX mutant proteins
are essentially nucleotide-free unless exogenous xanthine nucleotides
are provided. Thus, GoX provides an excellent model to study the
receptor interaction of empty G protein subunits. Consistent
with the previously reported studies on the empty form of transducin
(4), our data are most readily interpreted as showing that "empty"
GoX can form a stable complex with appropriate receptors on the membrane.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
were
generous gifts from Dr. O. Nakanishi (Division of Biology, Caltech).
Xanthine and guanine nucleotides were from Sigma. All the radioactive
ligands including [35S]ATPS,
[35S]GTPS, [3H]QNB, and
fML[3H]P were from NEN Life Science Products.
--
Both wild-type Go and mutant GoX were subcloned into
the Escherichia coli expression vector pET-15b (Novagen)
with His6 tag at the N terminus. These clones were used to
transform the E. coli strain BL21(DE3), and proteins were
expressed. Expression and purification of these proteins was described
previously (6, 8). After harvesting the culture, cell extracts were
resuspended in the binding buffer (5 mM imidazole, 0.5 M NaCl, 160 mM Tris-HCl, pH 7.9, and 1 mM Me). The His6-tagged proteins were
purified from Ni2+-NTA column according to the protocol
provided by Novagen. Purified proteins were stored in TED buffer (20 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 1 mM dithiothreitol) with 0.1 mM
MgCl2 and 0.1 mM nucleotide diphosphate (GDP or
XDP as appropriate).
S--
XTPS was synthesized from XDP and
ATPS with nucleotide diphosphate kinase (NDK) as described
previously (12). To produce 35S-labeled XTPS, the
reaction contained 10 µM XDP, 1 µM
[35S]ATPS, and 10 units of NDK (Sigma) in 100 µl of
NDK buffer (1 mM MgCl2, 5 mM
dithiothreitol, and 20 mM Tris-HCl, pH 8.0). The mixture
was incubated at room temperature for 2 h. The resulting concentration of [35S]XTPS was about 1 µM (1 µCi/pmol). The radiochemical purity of XTPS
was monitored by TLC on Avicel/PEAE plates (Analtech) in 0.07 N HCl.
--
Binding of
[35S]GTPS and [35S]XTPS to the
recombinant Go or the mutant proteins was performed as described
(6). The binding reaction contained 0.5 µg of purified protein in TED
buffer, with 0.1 mM MgCl2, 1 µM
ATP, and 0.1 µM GTPS or XTPS (20,000 cpm/pmol). For
the time course experiments, 20-µl aliquots were withdrawn from a
200-µl reaction, diluted 10-fold with ice-cold TED buffer containing
0.1 mM MgCl2, filtered through 45-µm
nitrocellulose, washed, and dried. The amount of bound radioactivity
was determined by scintillation counting.
on Sf9 Cell Membranes--
0.2 µg of
purified Go or Go mutant proteins were incubated with 100 µg of
Sf9 cell membranes in TED buffer of a final volume of 100 µl
at room temperature for 1 h. The membranes were centrifuged and
subjected to Western blot using antibodies against Go.
-galactosidase was used
to maintain a constant amount of DNA. To each well, 1 µg of DNA was
mixed with 5 µl of LipofectAMINE (Life Technologies, Inc.) in 0.5 ml
of Opti-MEM (Life Technologies, Inc.), and 5 h later, 0.5 ml of
20% fetal calf serum in Dulbecco's modified Eagle's medium was added
to the medium. After 48 h, cells were assayed for inositol
phosphate levels as described previously (13, 14).
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
S Binding of GoX by Activated M2 Muscarinic
Receptor--
To test if GoX could interact with G protein-coupled
receptors, we investigated the receptor-stimulated nucleotide binding of GoX. Activated G protein-coupled receptors are known to
facilitate the binding of GTPS to G protein subunits. It has
been reported that recombinant m2 MAChR from Sf9 cells
stimulated the binding of GTPS to wild-type Go 2-3-fold in
response to muscarinic agonists (9, 10). We infected Sf9 cells
with recombinant baculoviruses encoding m2 MAChR and prepared
membranes. The concentration of receptor was about 20 pmol/mg of
membrane protein, determined from [3H]QNB binding. We
have previously shown that GoX mutant proteins bind only xanthine
nucleotides, but not guanine nucleotides. In this experiment, we
reconstituted purified GoX with Sf9 cell membranes containing
m2 MAChR in the presence of XDP and G protein subunits purified
from bovine retina, and followed agonist-dependent stimulation of XTPS binding to GoX. We found that carbachol accelerated the XTPS binding of GoX, in a fashion similar to the
acceleration of GTPS binding observed with wild-type Go (Fig.
1a). In control experiments
using wild-type Sf9 cell membranes, both atropine and carbachol
had no effect on the XTPS binding of GoX (Fig. 1a).
subunits were required for the carbachol-dependent stimulation of nucleotide binding (Fig. 1b), suggesting that
only the trimeric complex of GoX with can be activated to
exchange XDP for XTPS by the ligand-bound receptors.
View larger version (21K):
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Fig. 1.
M2 MAChR stimulated XTP S binding of
GoX. a, 0.5 µg of purified GoX was incubated
with 1 µg of , 100 µg of m2 MAChR membranes, or control
Sf9 cell membranes and with 10 µM XDP in TEDM
buffer (20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol, and 1 mM MgCl2)
for 30 min at room temperature, and then the mixture was diluted
10-fold with TEDM buffer containing 0.1 µM
[35S]XTPS (20,000 cpm/pmol) and 100 µM
carbachol or 2 µM atropine at time 0. 20-µl aliquots
were withdrawn and assayed for the bound nucleotides at the indicated
times. b, 0.5 µg of purified wild-type Go or GoX
were subjected to the similar nucleotide binding assay as in
panel a with GTPS or XTPS under indicated conditions.
Only data at 20 min were shown as the percentage of maximum
binding.
X--
Another well documented indication of receptor-G
protein interaction is that GTP or GTPS inhibits the high affinity
binding of G protein-coupled receptors to their agonists. NFPR
receptors expressed in Sf9 cells are known to be in the low
ligand affinity state (~60 nM fMLP) (11), presumably
because of the lack of mammalian Gi-like G proteins in Sf9
cells, whereas NFPR in neutrophils and NFPR expressed in mouse L cells
exhibited high affinity ligand binding (0.5-3 nM fMLP)
(15, 16). Therefore, we reconstituted GoX with NFPR from Sf9
cells and investigated whether GoX could induce the high affinity
ligand binding state in NFPR receptors. Sf9 cell membranes
containing NFPR receptors were prepared as described under
"Experimental Procedures." The expression level of NFPR was about
20 pmol/mg of membrane protein, determined by fML[3H]P
binding. We incubated the NFPR with wild-type Go or mutant GoX in
the presence of and varieties of nucleotides and determined their affinities for the agonist fMLP. As expected, NFPR expressed in
Sf9 cells showed low ligand affinity binding of fMLP (~100 nM), and GoX alone did not affect ligand binding (Fig.
2a). More interestingly, NFPR
exhibited high affinity ligand binding (~10 nM) when
GoX, , and XDP were present (Fig. 2a). Both
and XDP were required to induce the high ligand affinity state of NFPR,
and XTP inhibited the fMLP binding of the receptors (Fig. 2b). In the control experiments, wild-type Go
heterotrimer was also found to convert the NFPR to the high affinity
ligand binding state, which was inhibited by GTPS (Fig.
2b). These experiments demonstrated that the heterotrimeric
complex of GoX can interact efficiently with NFPR.
View larger version (22K):
[in a new window]
Fig. 2.
High affinity ligand binding of NFPR induced
by Go X. a, 10 µg of NFPR membranes or wild-type
Sf9 cell membranes were incubated with various concentrations of
fML[3H]P for 1 h in 20 mM Tris-HCl, pH
7.4, 12.5 mM MgCl2, and 1 mM EDTA
at 30 °C in a final volume of 0.5 ml, in the presence of 0.1 µg of
GoX, 0.2 µg of , and 100 µM XDP, or GoX
alone. The amount of bound radioligand was then determined. Nonspecific
binding was defined as binding in the presence of 10 µM
cold fMLP, which was less than 10% of total ligand binding, and was
subtracted before analyzing. b, NFPR was incubated with 50 nM fML[3H]P and various reagents under
the same conditions as panel a.
X with M2 Muscarinic Receptor on Sf9 Cell
Membranes--
The previous two experiments showed that GoX
heterotrimer could interact with the G protein-coupled receptors
efficiently and that the interaction was similar to the interaction
between wild-type Go and receptors. To investigate receptor interaction of GoX more directly, we studied binding of GoX to receptor containing Sf9 cell membranes. Purified wild-type Go or
GoX were incubated with Sf9 cell membranes containing m2
MAChR in the presence of different reagents. The membranes were then
pelleted and subjected to Western blotting using antibodies against
Go to see if Go remained bound to the membrane. In the control
experiments using wild-type Sf9 cell membranes without m2 MAChR,
both wild-type Go and GoX did not remain associated with the
membrane. However, wild-type Go was bound to membrane when it was
coincubated with . Similarly, GoX stayed on the membrane when
in complex with in the presence of XDP (data not shown). These
experiments using wild-type Sf9 cell membranes showed that Go
bound to the membranes only in the complex form, presumably
because facilitates membrane association. In the experiments
using membranes containing m2 MAChR, we found somewhat surprisingly
that GoX bound to receptor-containing membranes even in the absence
of carbachol and without (Fig. 3,
lane 12), whereas wild-type Go did not (Fig. 3,
lane 1), suggesting GoX alone was able to bind to
receptor. Interestingly, both XDP and XTP abolished the interaction
between GoX and m2 MAChR-containing membranes and released GoX
from the membrane fraction (Fig. 3, lanes 7 and 8), whereas GDP or GTP had no effect (data not shown),
suggesting that the nucleotide-free form of GoX can recognize and
bind to Go-mediated receptor. As expected, GoX stayed on the
membrane when both XDP and were present (Fig. 3, lane
10), and XTP promoted dissociation of the GoX complex
(Fig. 3, lane 9). In the case of wild-type Go, the
binding pattern was the same between membranes with or without the
receptors, and XDP or XTP had no effect on binding (Fig. 3, lanes
1-5). In a titration experiment, quantitation of GoX revealed
that the amount of GoX bound to the membrane increased linearly
until it reached saturation, and the level of saturation was
proportional to the amount of receptor incubated in the reaction (Fig.
4, a-c). Furthermore, similar
experiments using Sf9 cell membranes containing NFPR were also
performed, and the results were similar (data not shown). These
experiments indicated that the empty form of GoX, without a bound
nucleotide and , could form a stable complex with receptor. In
summary, these data suggest that GoX with XDP bound and bind
to membranes, whereas the XTP form is found to be cytoplasmic. The
nucleotide-free form is able to bind to Go-mediated receptors.
View larger version (26K):
[in a new window]
Fig. 3.
Binding of Go X to m2 MAChR on Sf9
cell membranes. 0.2 µg of wild-type Go (with 100 µM GDP) or GoX were incubated with 100 µg of m2
MAChR membranes in TED buffer of a final volume of 100 µl at room
temperature for 1 h with indicated reagents. The membrane then was
centrifuged and subjected to Western blot using antibodies against
Go. All nucleotide concentrations were 100 µM, and the
amount of was 0.5 µg. Lane 13 shows the total
amount of Go used in each assay.
View larger version (32K):
[in a new window]
Fig. 4.
Titration of Go X bound to m2 MAChR on
Sf9 cell membranes. The binding assays were done under the
same conditions described in Fig. 3 legend. a, indicated
amount of GoX was incubated with 100 µg of m2 MAChR membranes in
TED buffer. b, the relative intensities of bands in
panel a were quantitated. c, quantitated binding
of 0.2 µg of GoX with indicated amount of m2 MAChR membranes.
Wild-type Sf9 cell membranes were used to maintain a constant
amount of membranes of 200 µg in each binding reaction.
X on Receptor Activation in COS-7
Cells--
Because our experiments suggested that empty GoX was
able to bind to the receptor in vitro, we went on to test
for this interaction in intact cells. Indeed, we found that GoX was
able to interact with receptors and inhibit their activities in COS-7
cells consistent with the observation that GoX did not dissociate
from the receptors without xanthine nucleotides. Thrombin receptors are
known to couple with G proteins from both the Gi and Gq families (17). In COS-7 cells transfected with the thrombin receptor, endogenous Gq is
activated by the addition of thrombin and stimulates PLC isoforms to
elevate cellular IP3 concentration. Inhibition of receptor
activation in transfected cells by wild-type G proteins was observed
before (18). Thus if cells are cotransfected with both the thrombin
receptor and wild-type Go subunit, the activation of Gq is inhibited
because of the competition of Go for the receptor or endogenous G
protein subunits (Fig.
5a). We cotransfected thrombin
receptor and GoX to determine whether GoX could compete with
endogenous Gq for the receptors. Indeed, we found that GoX inhibited
Gq activity stimulated by thrombin, and the inhibition was proportional
to the amount of GoX cDNA used in the transfection (Fig.
5a). Because GoX in the absence of XDP does not interact with and does not affect the -stimulated PLC activity
in COS-7 cells (6), the inhibition by GoX of Gq activation
stimulated by the thrombin receptor must come from the competitive
binding of GoX to the receptor. Similar experiments were performed
with m1 MAChR and TRH receptor which were known to couple only to the Gq family of G proteins and not to the Go family (10, 19, 20).
GoX had no effect on the activation of m1 MAChR or TRH receptor
(Fig. 5, b and c). On the other hand, wild-type
Go, which can compete for endogenous , inhibited both m1 MAChR
and TRH receptor stimulation as expected. In COS-7 cells, the
activation of thrombin receptor, m1 MAChR, and TRH receptor share the
same Gq pathway downstream of the receptor. Because GoX inhibited only the thrombin receptor activity, but had no effect on m1 MAChR or
TRH receptor, we concluded that GoX inhibited thrombin receptor stimulation by competitive binding to the receptor.
View larger version (45K):
[in a new window]
Fig. 5.
The negative inhibitory effects of Go X on
receptor-stimulated PLC activation in COS-7 cells. 1 × 105 cells/well were seeded in a 12-well plate and then were
transfected with cDNAs encoding the indicated G proteins (GoDN
designates GoD273N) and thrombin receptor (a), m1 MAChR
(b), TRH receptor (c), or m2 MAChR
(d). In panels a-c, the amount of receptor
cDNA used in each well was 0.25 µg, and the amount of Go
cDNA was 0.75 µg/well unless otherwise indicated. In panel
d, the amount of both m2 MAChR and G15 cDNA was 0.2 µg/well and that of Go was 0.6 µg/well. The total amount of
cDNA for each well was adjusted to 1.0 µg by the addition of
CMV-LacZ cDNA. After cells were labeled with
[3H]inositol overnight, they were incubated in the medium
containing 0.1 unit/ml thrombin (a), 1 µM
carbachol (b and d), or 1 µM TRH
(c) before levels of inositol phosphates were
determined.
X could bind to other Go-coupled receptors in cells, we
looked into the interaction between GoX and m2 MAChR. Because m2
MAChR couples only to the Gi family of G proteins and not to the Gq
family (9, 21), we could not assay their interaction in the same way as
the thrombin receptor by monitoring PLC activities in COS-7 cells
transfected with the receptor and GoX. Therefore, we constructed an
artificial pathway by cotransfecting both m2 MAChR and G15 into
COS-7 cells. G15 is known as a promiscuous G protein that can be
activated by all kinds of G protein-coupled receptors, and G15 also
activates PLC isoforms (21). In cells cotransfected with both m2
MAChR and G15, we were able to activate endogenous PLC isoforms
by the addition of the muscarinic agonist carbachol. We found that this
m2 MAChR stimulation pathway could also be inhibited by GoX (Fig.
5d). All these experiments suggested that GoX was able to
interact with G protein-coupled receptors in cells and retained the
receptor specificity of wild-type Go; it coupled with thrombin
receptor and m2 MAChR, but not with m1 MAChR or TRH receptor.
Furthermore, GoX exhibited dominant-negative inhibitory effects
against these receptors in cells.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
X (GoD273N/Q205L) was the first reported mutant of
heterotrimeric G protein subunits that bound xanthine nucleotides, not guanine nucleotides (6). It bound only in the presence of
XDP and could be activated by XTP. We continued to study the interaction of GoX with G protein-coupled receptors in this report. The interaction of G proteins and their receptors is best demonstrated in two experiments: agonist-stimulated GTPS binding of G protein subunits and inhibition of high affinity ligand binding of the receptors by GTPS. To test if GoX can interact with G
protein-coupled receptors and be activated by their agonists, we
reconstituted purified GoX with Sf9 cell membranes containing
m2 MAChR or NFPR. First, we found that binding of XTPS to GoX was
stimulated by the muscarinic agonist carbachol in the presence of m2
MAChR. In similar experiments using wild-type Go, GTPS binding
was also stimulated by carbachol. In both cases, was required
for the carbachol-dependent nucleotide binding, suggesting
that only GoX heterotrimer could interact with the receptors
effectively. Second, we tested GoX to determine whether it could
induce the high affinity state in NFPR receptors expressed in
Sf9 cells. The NFPR expressed in these cells is known to be in
the low affinity state probably because of lack of mammalian Gi-like
proteins in Sf9 cells (11). In our experiments, we found that
GoX could convert NFPR into the high affinity state in the presence
of and XDP, and this effect was inhibited by XTP. These two
experiments demonstrated that GoX, when in complex with and
XDP, could interact with G protein-coupled receptors effectively and be
activated by the agonists.
X provides an excellent
model to study empty G protein subunits. The empty form of G is
an important intermediate in receptor activation and has long been
proposed to form a stable complex with activated receptors. However
stable interaction between empty G proteins and their receptors was
only reported in the transducin-rhodopsin system. Empty transducin
apparently formed a stable complex with light-activated rhodopsin and
stayed on the rod outer segment membrane. Interestingly, deactivation
of the rhodopsin did not lead to the dissociation of transducin from
the complex (4). In this report, we showed that empty GoX was able
to bind to the receptor on the membrane in the absence of
subunits and without agonists, and the interaction could be abolished
by either XDP or XTP. The amount of GoX associated on the membranes
with m2 MAChR was proportional to the amount of receptor at saturation. Interestingly, binding of GoX alone did not convert the receptor to
the high ligand affinity conformation, which required the complex. Therefore, the binding of GoX alone to the receptor is not
functional in contrast with the binding in the presence of and XDP.
X appears to form a stable complex with the receptor, we
tested whether GoX could inhibit receptor activation in cells. In
transfected COS-7 cells, we showed that GoX was able to inhibit
thrombin receptor or m2 MAChR stimulated PLC activities via the Gq
or G15 pathway, but had no effect on m1 MAChR or TRH receptor
stimulation. Because both thrombin receptor and m2 MAChR are known to
couple with wild-type Go, and m1 MAChR and TRH receptor only couple
with Gq, we interpret the data to mean that GoX retained the
receptor specificity of wild-type Go and was able to interact with
Go-coupled receptors in cells. The inhibitory binding of GoX enables
us to specifically block Go-coupled receptors in certain systems. This
could be a useful means to analyze different receptor-stimulated signal
transduction pathways, and could perhaps be useful in drug screening
associated with G protein-coupled receptors.
mutant,
GoD273N, lost the ability to bind either guanine nucleotides or
xanthine nucleotides and could not bind under any conditions. Surprisingly, GoD273N can still bind to receptors. In transfected COS-7 cells, we found that GoD273N inhibited thrombin receptor and
m2 MAChR activation, in a fashion similar to GoX (Fig. 5, a and d). GoD273N also retained the same
receptor specificity as wild-type Go; i.e. it had no
effect on m1 MAChR or TRH receptor stimulated pathways (Fig. 5,
b and c). In the Sf9 cell membrane binding
assay, it only bound to the m2 MAChR membranes, not to the control
wild-type Sf9 cell membranes. However in contrast to GoX,
GoD273N was not released from the m2 MAChR membranes by XDP or XTP,
consistent with its inability to bind nucleotides (data not shown). The
reason that GoD273N mutant proteins do not bind xanthine nucleotides
is not clear. Apparently it must have a structure similar to that of
the empty Go which enables it to bind receptors, but the structure
is probably not stable locally around the nucleotide binding pocket.
Nevertheless, GoD273N may also be useful as a dominant-negative
inhibitor of receptor functions.
ACKNOWLEDGEMENTS
FOOTNOTES
S, guanosine 5'-O-(3-thiotriphosphate); XTPS, xanthine 5'-O-(3-thiotriphosphate); [3H]QNB, quinuclidinylbenzilate; fML[3H]P, formyl-methionyl-leucyl-phenylalanine.
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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Y.-L. Wu, S. B. Hooks, T. K. Harden, and H. G. Dohlman Dominant-negative Inhibition of Pheromone Receptor Signaling by a Single Point Mutation in the G Protein {alpha} Subunit J. Biol. Chem., August 20, 2004; 279(34): 35287 - 35297. [Abstract] [Full Text] [PDF]
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 A. A. Roy, K. E. Lemberg, and P. Chidiac Recruitment of RGS2 and RGS4 to the Plasma Membrane by G Proteins and Receptors Reflects Functional Interactions Mol. Pharmacol., September 1, 2003; 64(3): 587 - 593. [Abstract] [Full Text] [PDF]
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P. Molinari, C. Ambrosio, D. Riitano, M. Sbraccia, M. C. Gro, and T. Costa Promiscuous Coupling at Receptor-Galpha Fusion Proteins. THE RECEPTOR OF ONE COVALENT COMPLEX INTERACTS WITH THE alpha -SUBUNIT OF ANOTHER J. Biol. Chem., April 25, 2003; 278(18): 15778 - 15788. [Abstract] [Full Text] [PDF]
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A. Gille, K. Wenzel-Seifert, M. B. Doughty, and R. Seifert GDP Affinity and Order State of the Catalytic Site Are Critical for Function of Xanthine Nucleotide-selective Galpha s Proteins J. Biol. Chem., February 28, 2003; 278(10): 7822 - 7828. [Abstract] [Full Text] [PDF]
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D. S. Feldman, A. M. Zamah, K. L. Pierce, W. E. Miller, F. Kelly, A. Rapacciuolo, H. A. Rockman, W. J. Koch, and L. M. Luttrell Selective Inhibition of Heterotrimeric Gs Signaling. TARGETING THE RECEPTOR-G PROTEIN INTERFACE USING A PEPTIDE MINIGENE ENCODING THE Galpha s CARBOXYL TERMINUS J. Biol. Chem., August 2, 2002; 277(32): 28631 - 28640. [Abstract] [Full Text] [PDF]
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C. H. Berlot A Highly Effective Dominant Negative alpha s Construct Containing Mutations That Affect Distinct Functions Inhibits Multiple Gs-coupled Receptor Signaling Pathways J. Biol. Chem., May 31, 2002; 277(23): 21080 - 21085. [Abstract] [Full Text] [PDF]
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B. Yu |