From the Laboratory of Molecular Neurobiology, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
Received for publication, February 10, 2003
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ABSTRACT |
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The G Heterotrimeric G proteins act as a molecular switch that conveys
signals from G protein-coupled receptors
(GPCRs)1 in the cell membrane
to intracellular downstream effectors, and they are divided into four
major families, Gs, Gi, Gq, and
G12, based on sequence homology of their Recently, we demonstrated that G Materials--
Agents obtained and commercial sources were as
follows: thrombin and LPA, Sigma; rabbit polyclonal
anti-G Plasmid Constructions--
cDNAs of wild-type
G Cell Culture, Transfection, and Measurement of Activities of
G We reported previously that active forms of G subunits of the G12
family of heterotrimeric G proteins, defined by G
12 and
G
13, have many cellular functions in common, such as
stress fiber formation and neurite retraction. However, a variety of G
protein-coupled receptors appear to couple selectively to
G
12 and G
13. For example, thrombin and
lysophosphatidic acid (LPA) have been shown to induce stress fiber
formation via G
12 and G
13, respectively.
We recently showed that active forms of G
12 and
G
13 interact with Ser/Thr phosphatase type 5 through its tetratricopeptide repeat domain. Here we developed a novel assay to measure the activities of G
12 and
G
13 by using glutathione S-transferase-fused
tetratricopeptide repeat domain of Ser/Thr phosphatase type 5, taking
advantage of the property that tetratricopeptide repeat domain strongly
interacts with active forms of G
12 and G
13. By using this assay, we identified that thrombin
and LPA selectively activate G
12 and G
13,
respectively. G
12 and G
13 show a high
amino acid sequence homology except for their N-terminal short
sequences. Then we generated chimeric G proteins
G
12N/13C and G
13N/12C, in which the
N-terminal short sequences are replaced by each other, and
showed that thrombin and LPA selectively activate G
12N/13C and G
13N/12C, respectively.
Moreover, thrombin and LPA stimulate RhoA activity through
G
12 and G
13, respectively, in a
G
12 family N-terminal sequence-dependent
manner. Thus, N-terminal short sequences of the G12
family determine the selective couplings of thrombin and LPA receptors
to the G
12 family.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
subunits (1).
The G12 family is defined by G
12 and
G
13, and they share 67% amino acid sequence identity
(2). Active forms of G
12 and G
13 induce several biological responses via activation of Rho, a major downstream common target for G
12 and G
13, including
stimulation of stress fiber formation and focal adhesion assembly (3),
induction of neurite retraction (4), induction of apoptosis (5),
transformation of fibroblasts (6), activation of phospholipase D (7),
inhibition of Ca2+-dependent exocytosis (8),
and activation of c-Jun N-terminal kinase (9). Recently, several
proteins that interact with both G
12 and
G
13 have been identified, including p115, a Rho guanine nucleotide exchange factor (10), cadherin (11), and radixin (12).
Therefore, G
12 and G
13 are thought to
transduce mutual downstream signaling through these effectors. In
contrast, a variety of GPCRs appear to selectively couple to
G
12 or G
13 (13). For example, thrombin
and lysophosphatidic acid (LPA) have been shown to induce stress fiber
formation via G
12 and G
13, respectively, by using dominant negative mutants of G
12 and
G
13 (13). However, this receptor-G protein coupling is
not directly evaluated.
12 and
G
13 interact with Ser/Thr phosphatase type 5 (PP5)
through its tetratricopeptide repeat (TPR) domain and stimulate its
phosphatase activity (14). In this study, we developed a novel assay to
evaluate the activities of G
12 and G
13 by
using glutathione S-transferase (GST)-fused TPR domain of
PP5 (GST-TPR), taking advantage of the property that GST-TPR strongly
interacts with active forms of G
12 and G
13. By using this assay, we showed selective
activations of G
12 by thrombin and G
13 by
LPA, and found that N-terminal short sequences of G
12
and G
13 determine these selective activations.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
12 and anti-G
13 antibodies and
mouse monoclonal anti-RhoA antibody, Santa Cruz Biotechnology, Inc.;
horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G
antibody and horseradish peroxidase-conjugated swine anti-rabbit
immunoglobulin G antibody, DAKO; and Chemiluminescence ECL Western
blotting system, Amersham Biosciences. GST-fused proteins were
purified from Escherichia coli as described previously (8, 14). The TPR domain used for GST-TPR is a splicing variant of rat PP5
that contains residues 9-122 following a new short coding region (12 amino acids; SRALGMGQLPAP) (14). The nucleotide sequence of this TPR
variant is available from GenBankTM/EMBL/DDBJ under
accession number AB101661.
12 and G
13 (G
12WT and
G
13WT), and their constitutively active mutants of
G
12 (G
12Q229L, G
12QL) and
G
13 (G
13Q226L, G
13QL) were
obtained as described previously (4, 14). To generate chimeric cDNA
of G
13 substituted with the N-terminal short sequence of
G
12 (G
12N/13C), cDNAs encoding the
N-terminal part of G
12 (amino acids 1-37) and
the C-terminal part of G
13 (amino acids 31-377) were
combined by using a BglII site generated by PCR-mediated
mutagenesis, and then it was subcloned into
BamHI/NotI sites of pcDNA3 vector. Chimeric
cDNA of G
12 substituted with the N-terminal short
sequence of G
13 (G
13N/12C) was generated
by combining cDNAs encoding the N-terminal part of
G
13 (amino acids 1-30) and the C-terminal part of
G
12 (amino acids 38-379), using a XbaI site
generated by PCR-mediated mutagenesis, and it was subcloned into
HindIII/NotI sites of pcDNA3 vector.
12, G
13, and RhoA--
293T cells were
cultured in Dulbecco's modified Eagle's medium containing 10%
fetal bovine serum, 4 mM glutamine, 100 units/ml penicillin, and 0.1 mg/ml streptomycin under humidified air containing 5% CO2 at 37 °C. 1 × 106 cells were
seeded in a 60-mm dish and transfected with 2 µg of cDNA using
LipofectAMINE Plus (Invitrogen) according to the manufacturer's instructions. After transfection, medium was replaced to
Dulbecco's modified Eagle's medium containing 1% fetal bovine serum
and incubated for 12-15 h. To examine the activities of
subunits,
cells transfected with G
12WT, G
12QL,
G
13WT, G
13QL, G
12N/13C, or
G
13N/12C were stimulated with either thrombin (1.5 units/ml) or LPA (1 µM) for the indicated times, rinsed
once with phosphate-buffered saline, and lysed with 500 µl of the
ice-cold cell lysis buffer (20 mM Hepes, pH 8.0, 2 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, 0.5% Triton X-100, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml
leupeptin) containing 3 µg of GST-TPR. In the case of
AlF
12 and anti-G
13 antibodies (1:200
dilution, both). To examine the endogenous RhoA activity, cells
transfected with G
12WT, G
13WT,
G
12N/13C, or G
13N/12C were stimulated
with either thrombin (1.5 units/ml) or LPA (1 µM) for 1 min, and cell lysates were incubated with GST-fused Rho-binding domain
of Rhotekin as described previously (8), and bound proteins were
immunoblotted with anti-RhoA antibody (1:100 dilution). The primary
antibodies were detected by using horseradish peroxidase-conjugated
secondary antibodies and the ECL detection kit. Densitometric analyses
were performed by using NIH Image software, and the amounts of active
forms of
subunits and RhoA were normalized to the total amounts of
subunits and RhoA in cell lysates, respectively.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
12 and
G
13, but not G
q and G
i2,
interact with PP5 through its TPR domain (14). To develop a method to
measure the activities of G
12 and G
13, we
examined the ability of GST-TPR to pull-down active forms of
G
12 and G
13. As shown in Fig.
1, GST-TPR strongly precipitated G
12WT and G
13WT in the presence of
AlF
subunits (15), and their constitutively active forms,
G
12QL and G
13QL, but precipitation of
G
12WT and G
13WT in the absence of
AlF
12 and
G
13 can be detected by the GST-TPR pull-down assay.
View larger version (21K):
[in a new window]
Fig. 1.
Pull-down assay for active forms of
G 12 and
G
13 by using GST-TPR. 293T
cells were transfected with an empty vector (lanes 1 and
5) or with a vector for G
12WT (lanes
2 and 3), G
12QL (lane 4),
G
13WT (lanes 6 and 7), or
G
13QL (lane 8). Cells were treated
(lanes 3 and 7) or not treated (lanes
1, 2, 4, 5, 6, and
8) with AlF
subunits and total amounts of
subunits in cell lysates were
determined by immunoblotting with polyclonal antibodies against
G
12 (lanes 1-4) and G
13
(lanes 5-8). The results shown are representative of three
independent experiments that yielded similar results.
Thrombin and LPA have been shown to induce stress fiber formation via
G12 and G
13, respectively, by using
dominant negative mutants of G
12 and G
13
(13). Therefore, we measured the thrombin and LPA-induced
G
12 and G
13 activations by using this
GST-TPR pull-down assay. Because the expression levels of endogenous
G
12 and G
13 were extremely low in a
variety of cell lines including 293T, COS-7, and Swiss 3T3 cells (Fig.
1 and data not shown), it was unable to detect the endogenous
activities of G
12 and G
13 by using
GST-TPR pull-down assay. Then we transfected 293T cells with
G
12WT or G
13WT and treated the cells with
either thrombin or LPA. Thrombin rapidly activated
G
12WT, the activation level reaching a maximum at 1 min
and gradually decreasing to the basal level within 10 min. However, LPA
had no effect on G
12WT activity (Fig.
2A). On the other hand, LPA
rapidly activated G
13WT, whereas thrombin had no effect
on G
13WT activity (Fig. 2B). Thus, thrombin
and LPA receptors selectively couple to and activate G
12
and G
13, respectively.
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G12 and G
13 show a high amino acid
sequence homology (67%) except for their N-terminal short sequences in
which the amino acid identity is only 16% (2). This limited homology
prompted us to speculate that the N-terminal short sequences determine the specificities of G
12 and G
13 for
thrombin and LPA, respectively, and then we generated chimeric
subunits G
12N/13C and G
13N/12C, in which
the N-terminal sequence is replaced each other (Fig. 3A). Because
anti-G
12 and anti-G
13 antibodies used
here are raised against their N-terminal amino acids,
G
12N/13C and G
13N/12C can be recognized
with respective antibodies. 293T cells expressing G
12N/13C or G
13N/12C were treated with
either thrombin or LPA, and activities of chimeric proteins were
measured by GST-TPR pull-down assay. Similar to G
12,
G
12N/13C was strongly activated by thrombin but not by
LPA (Fig. 3, B and D), though it is mostly
composed of G
13. On the other hand,
G
13N/12C, which is mostly composed of
G
12, was strongly activated by LPA but not by thrombin
(Fig. 3, C and E). These results indicate that
N-terminal short sequences of G
12 and G
13
determine their specificities for thrombin and LPA, respectively.
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Activation of G12 or G
13 increases RhoA
activity (8, 16). To next evaluate whether selective couplings of
thrombin and LPA receptors to G
12 and G
13
are functional, we examined the effects of thrombin and LPA on RhoA
activity in the cells expressing G
12,
G
13, or chimeric
subunits. Thrombin and LPA activated RhoA strongly via G
12WT and
G
13WT, respectively (Fig. 4A), the specificities
corresponding with the results shown in Fig. 2. With respect to
chimeric
subunits, thrombin and LPA activated RhoA strongly via
G
12N/13C and G
13N/12C, respectively (Fig.
4, B and C). Thus, thrombin and LPA receptors
functionally couple to G
12 and G
13,
respectively, and these functional couplings are determined by the
N-terminal short sequences of G
12 and
G
13.
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Gohla et al. (3) showed that LPA stimulated
[-32P]GTP azidoanilide-labeling of both
G
12 and G
13 in the isolated membranes, and Ponimaskin et al. (17, 18) showed that thrombin also
stimulated binding of [35S]GTP
S to both
G
12 and G
13 in the membranes, indicating
that thrombin and LPA receptors have the ability to stimulate both G
12 and G
13 in vitro. However,
thrombin- and LPA-induced stress fiber formations via RhoA are mediated
specifically by G
12 and G
13, respectively
(3, 13), suggesting that both receptors selectively use G proteins in
intact cells. In this study, we developed a novel assay to
evaluate the activities of G
12 and G
13.
By using this assay, we directly demonstrated that thrombin and LPA
selectively activate G
12 and G
13,
respectively, in intact cells. These results taken together indicate
that thrombin and LPA receptors can potently couple to both
G
12 and G
13 in vitro, but they
show selective usage of G proteins in vivo. We further demonstrated that N-terminal short sequences of G
12 and
G
13 determine the selective couplings of thrombin and
LPA receptors to G
12 and G
13,
respectively. C-terminal domains of
subunits are well known to play
an important part in specifying receptor interactions of G proteins
(1). However, G
12 and G
13 share very high
amino acid sequence identity in the C-terminal region (2), suggesting
that C-terminal domains of G
12 and G
13 do not contribute to the coupling selectivity of thrombin and LPA receptors.
Recently, Waheed et al. (19) showed that G12
resides in lipid raft fraction, whereas G
13 is not
associated with lipid rafts in COS-7 cells and NIH 3T3 cells, and that
the N-terminal sequence of G
12 including a
palmitoylation site is important for the lipid raft localization of
G
12. Moreover, they showed that the targeting of
G
12 to lipid raft is mediated by Hsp90, a chaperone that
specifically associates with G
12 but not
G
13 (20). Although intracellular localizations of
thrombin and LPA receptors are not yet known, selective targeting of G
proteins and receptors to either lipid raft fraction or other fraction may determine the selective coupling. G
12 and
G
13 mostly display mutual downstream signal
transductions, such as RhoA activation and serum response factor
activation (2, 16). Recently, a variety of downstream effectors or
interacting proteins of the G
12 family have been
identified, including Rho guanine nucleotide exchange factors,
cadherin, and PP5, and they interact with both G
12 and
G
13 (10, 11, 14), suggesting that downstream signal transduction pathways of G
12 and G
13 are
highly overlapped. On the other hand, stress fiber formations induced
by a variety of GPCRs are selectively mediated by either
G
12 or G
13 (13), suggesting that
receptor-G protein coupling is selective. G
12 and
G
13 are highly expressed in brain, and it has been shown that G
12 is mainly expressed in somata of the neurons,
but G
13 is predominantly localized in the neuropil of
central neurons, indicating that G
12 and
G
13 exert their effects at the different area in the
cell (21). In the light of this knowledge, different localizations of
receptors and G proteins in the cells may be important for their
selective coupling.
In conclusion, we have developed a novel assay to evaluate the
activities of G12 and G
13, and we
directly demonstrated selective couplings of thrombin and LPA receptors
to G
12 and G
13, respectively, dependent
on the N-terminal short sequences of G proteins. This study will
contribute not only to the understanding of the activation mechanism of
the G
12 family, but will also help to elucidate the
signaling mechanism of heterotrimeric G proteins.
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FOOTNOTES |
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* This work was supported in part by grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by a grant from Takeda Science Foundation.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.
Recipient of Research Fellowships from the Japan Society for the
Promotion of Science for Young Scientists.
§ To whom correspondence should be addressed: Laboratory of Molecular Neurobiology, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan. Tel.: 81-75-753-4547; Fax: 81-75-753-7688; E-mail: mnegishi@pharm.kyoto-u.ac.jp.
Published, JBC Papers in Press, February 19, 2003, DOI 10.1074/jbc.M301409200
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ABBREVIATIONS |
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The abbreviations used are: GPCR, G protein-coupled receptor; LPA, lysophosphatidic acid; PP5, Ser/Thr phosphatase type 5; TPR, tetratricopeptide repeat; GST, glutathione S-transferase; GST-TPR, GST-fused TPR domain of PP5; WT, wild-type.
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