(Received for publication, July 19, 1996, and in revised form, November 13, 1996)
From the Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226, Japan
G protein and
subunits (G
and G
)
form a complex that is involved in various signaling pathways. We
reported that the C-terminal 10 amino acids of G
are required for
association with G
(Yamauchi, J., Kaziro, Y., and Itoh, H. (1995)
Biochem. Biophys. Res. Commun., 214, 694-700). To evaluate
further the significance of the C-terminal region of G
in the
formation of a G
complex and its signal transduction, we
constructed several C-terminal mutants and expressed them in human
embryonal kidney 293 cells. The mutant lacking the C-terminal 2 amino
acids (
C2) failed to associate with G
, whereas deletion of the
C-terminal amino acid (
C1), replacement of Trp at
2 position by
Ala (W339A), and addition of six histidines ((His)6) at the
C terminus did not affect the association with G
. We also studied
the effect of these mutations on the activation of mitogen-activated
protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and
c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK).
Co-expression of the
C2 or (His)6 mutant with G
did
not activate MAPK/ERK at all, whereas the
C1 or W339A mutant showed
the MAPK/ERK activation. The JNK/SAPK activity was stimulated by the
W339A,
C2, or (His)6 mutant, but not by the
C1
mutant. These results suggest that the C-terminal region of G
participates differentially in the signaling for MAPK/ERK and JNK/SAPK
activations in mammalian cells.
Heterotrimeric guanine nucleotide-binding regulatory proteins (G
proteins)1 mediate signals from a variety
of cell surface receptors to effector molecules (1-5). G proteins are
composed of ,
, and
subunits. Binding of ligands to G
protein-coupled receptors stimulates the dissociation of G
and
G
, which regulate, independently or cooperatively, a variety of
effector molecules.
Normally, G and G
associate tightly and function as a complex.
G
contains seven WD repeating units, each of which consists of
approximately 40 amino acids and ends mainly with Trp-Asp (WD) (6).
Recently, Wall et al. (7) and Lambright et al.
(8) reported the x-ray crystallographic structure of heterotrimer of
G
i1
1
2 and
G
i/t
1
1, respectively, and
the structure of G
1
1 was analyzed by
Sondek et al. (9). These reports have revealed that the
structure of G
in the trimeric complex is not very much different
from that in the dimeric complex. The WD repeat provides a rigid
scaffold of
-propeller structure, which is composed of seven
-propeller blades containing four antiparallel
-sheets. The N
terminus of G
forms an
-helical coiled coil structure with the N
terminus of G
, and the remainder of G
stretches along the
-propeller blades, forming multiple interaction sites with G
. On
the other hand, the N-terminal
-helix of G
binds with a
-propeller blade of G
, and a region designated switch II of G
fits into the top of the
-propeller.
In mammalian cells, G has been shown to modulate the activities
of adenylyl cyclases (10), phospholipase C
isozymes (11, 12),
phosphatidylinositol 3-kinase
(13), inward rectifier potassium
channels (14, 15), and N-type and P/Q-type calcium channels (16, 17).
Furthermore, it has been reported that G
binds directly with the
C-terminal region of the pleckstrin homology domain to regulate
-adrenergic receptor kinases (18, 19) and Tec family
protein-tyrosine kinases (20, 21). More recently, G
was shown to
stimulate the activities of the mitogen-activated protein kinase
subfamilies MAPK/ERK (22-25) and JNK/SAPK (26). These studies have
established a critical role of G
in the intracellular signal
transduction, although little is known about the region involved in the
effector regulation.
In the present study, we demonstrated that a few amino acids at the C
terminus of G are involved in the complex formation with G
.
Furthermore, we found that mutations in C-terminal amino acids
influence differentially the stimulation of MAPK/ERK and JNK/SAPK
activities.
Rabbit polyclonal anti-Go
antibody was produced against amino acids 94-108 of G
o
and purified by a peptide affinity column. Rabbit polyclonal antibody
(06-238) that recognizes a peptide spanning amino acids 127-139
identical in G
1 and G
2 was purchased from
Upstate Biotechnology, Inc. Mouse monoclonal antibodies (M2 and 12CA5)
against FLAG epitope (8 amino acids, EYKEEEEK) and HA epitope (9 amino
acids, YPYDVPDYA) were from Eastman Kodak Co. and Boehringer Mannheim,
respectively. Rabbit anti-mouse Ig antibody (55480) was from
Cappel.
Rat Go-1 cDNA (27,
28) was inserted into mammalian expression vector pCMV5 (29). cDNAs
of bovine G
1 (30) and bovine G
2 (31, 32)
were generously provided by M. I. Simon (California Institute of
Technology) and T. Nukada (Tokyo Institute of Psychiatry), respectively. Wild type G
1 and G
2 were
subcloned into pCMV5 as described before (25). To construct mutants of
G
, the coding region of G
1 was amplified by PCR using
5
sense primer containing an EcoRI recognition site and 3
antisense primer containing a HindIII site. PCR was carried
out for 30 cycles, each at 94 °C for 1 min, 50 °C for 2 min, and
72 °C for 3 min, using Pfu polymerase (Stratagene). The
following oligonucleotides were used as PCR primers: primer 1, 5
-CCGGAATTC
AGTGAACTTGACCAGTTA-3
; primer 2, 5
-TTAAAAGCTTGCGGCCG
GCTGTCCCAGGATCCCGT-3
; primer 3, 5
-CGCCAAGCT
GATTTTGAGGAAGCT-3
; primer 4, 5
-CACCAAGCTTTGCGCA
CCAGATTTTGAGGAAGCT-3
; and primer 5, 5
-CCCAAGCTTCTCGAG
GTTAGCGATTTTGAGGAAGCTGTCCCA-3
(start and stop codons are underlined). Primer 1 was used to
construct all G
mutants as a 5
sense primer. Primers 2, 3, 4, and 5 were used for the
C6,
C2,
C1, and W339A mutants, respectively.
PCR products were double-digested by EcoRI and
HindIII and inserted into pCMV5. cDNAs for
G
1 tagged with six histidines ((His)6) at
the C terminus, the N-terminal 38-amino acid deletion mutant (
N38),
and G
2 tagged with FLAG epitope at the N terminus
(FLAG-G
) were prepared and inserted into pCMV5 as described
previously (33). The DNA sequence of amplified inserts was confirmed by the dideoxy method and the chemiluminescence detection system (New
England Biolabs). The pLNCX-RafFH6 plasmid, which expresses c-Raf-1
with FLAG epitope and six histidines at the C terminus, was a gift from
M. McMahon (DNAX Research Institute) (25). Escherichia coli
expression plasmids of six-histidine-tagged MEK and kinase-negative GST-fused MAPK were kindly provided by E. Nishida (Kyoto University) (34, 35). The SR
-HA-ERK2 and SR
-HA-JNK1 plasmids, which express
ERK2 and JNK1, respectively, with HA epitope at the N terminus, and the
E. coli expression plasmid of GST-c-Jun (amino acids 1-79)
were kindly provided by M. Karin (University of California, San
Diego).
Human embryonal kidney (HEK) 293 cells were maintained in Dulbecco's modified Eagle's medium (Sigma) containing 1 µg/ml kanamycin (Sigma) with 10% heat-inactivated fetal bovine serum (Life Technologies, Inc.). The cells were cultured at 37 °C in humidified atmosphere containing 10% CO2. Plasmid DNAs were transfected into HEK 293 cells by the calcium phosphate precipitation technique. The final amount of transfected DNA per 60-mm dish was adjusted to 30 µg by adding empty vector pCMV5. The medium was replaced 24 h after transfection, and the cells were harvested at 48 h posttransfection.
Cell Lysis and ImmunoprecipitationTo analyze the
association of G mutants with FLAG-G
, cells were transfected with
10 µg of G
and 10 µg of G
DNAs. The transfected cells were
washed twice with phosphate-buffered saline and suspended in lysis
buffer A (20 mM HEPES-NaOH (pH 7.5), 5 mM
MgCl2, 150 mM NaCl, 1 mM
phenylmethanesulfonyl fluoride, 1 µg/ml leupeptin, 1 mM
EDTA, and 1% Lubrol-PX) in a total volume of 600 µl and incubated for 10 min on ice. To analyze the interaction of G
complex with G
o, 10 µg of G
, 10 µg of G
, and 10 µg of
G
o DNAs were transfected. The transfected cells were
lysed in lysis buffer A containing 100 µM GDP. The cell
lysates were centrifuged at 14,000 rpm for 10 min at 4 °C in a
microcentrifuge. The supernatants were incubated at 4 °C for 1 h with mouse anti-FLAG antibody (1 µg) and mixed gently at 4 °C
for 1 h with protein A-Sepharose CL-4B (Pharmacia Biotech Inc.),
which was preabsorbed with rabbit anti-mouse Ig antibody (1 µg). The
immune complexes were precipitated by centrifugation and washed four
times with lysis buffer A.
Samples were boiled in Laemmli sample buffer (50 mM Tris-HCl (pH 6.8), 2% SDS, 30 mM
dithiothreitol, and 10% glycerol). The boiled samples were separated
by SDS-polyacrylamide gel electrophoresis, and the proteins were
transferred to BA81 nitrocellulose membranes (Schleicher & Schuell).
After blocking the membranes, the separated proteins were immunoblotted
with rabbit anti-common G or G
o antibody. The bound
antibodies were visualized by the enhanced chemiluminescence detection
system using anti-Ig antibody conjugated with horseradish peroxidase as
a secondary antibody (Amersham Life Science Inc.).
The cells were transfected with cDNAs of
Go, G
mutants, and FLAG-G
and lysed in buffer A. The cell lysates were used for immunoprecipitation with mouse anti-FLAG
antibody as described above, and the complexes were incubated in buffer
A containing 500 µM GDP or GTP
S at 30 °C for 2 h. The reaction was stopped by chilling on ice. The complexes were
washed with buffer A, boiled in Laemmli sample buffer, and subjected to
immunoblot analysis using rabbit anti-G
o or anti-common
G
antibody.
The MEK kinase activity of Raf was measured
as described previously (25). The cells were transfected with 10 µg
of pLNCX-RafFH6, 10 µg of G DNA, and 10 µg of G
DNA. The
transfected cells were starved in the serum-free medium containing 1 mg/ml bovine serum albumin for 24 h. The cells were lysed in 600 µl of lysis buffer B (20 mM HEPES-NaOH (pH 7.5), 3 mM MgCl2, 100 mM NaCl, 2 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM EGTA, 1 mM Na3VO4, 10 mM NaF, 20 mM
-glycerophosphate, and 0.5% Nonidet P-40). After
centrifugation at 14,000 rpm for 10 min in a microcentrifuge, the
epitope-tagged c-Raf-1 was immunoprecipitated from aliquots of the
supernatants with mouse anti-FLAG antibody (1 µg) and protein
A-Sepharose CL-4B preabsorbed with rabbit anti-mouse Ig antibody (1 µg). The immunoprecipitates were washed twice with lysis buffer B and
twice with reaction buffer A (20 mM HEPES-NaOH (pH 7.5), 5 mM MgCl2, 0.5 mM MnCl2, 0.2 µg/ml aprotinin, 0.1 µg/ml leupeptin, and 0.1 mM
EGTA). The precipitates were incubated at 30 °C for 20 min in
reaction buffer A with 3.3 µg of recombinant MEK, 6.6 µg of
recombinant kinase-negative MAPK, 200 µM ATP, and 5 µCi
of [
-32P]ATP. Recombinant (His)6 MEK and
kinase-negative GST-fused MAPK were produced in E. coli and
purified as described before (34, 35). The reaction was stopped by
adding 4 × Laemmli sample buffer and heating at 95 °C for 5 min. The boiled samples were separated by SDS-polyacrylamide gel
electrophoresis, and the incorporation of radioactive phosphate into
the MAPK was measured by an imaging analyzer (Fuji BAS 2000).
The activities of MAPK/ERK and JNK/SAPK were measured as described
before (36). Cells were transfected with 5 µg of SR-HA-ERK2 or 5 µg of SR
-HA-JNK1 together with 10 µg of G
and 10 µg of G
DNAs or 5 µg of G
and 5 µg of G
DNAs, respectively. The
transfected cells were lysed in lysis buffer C (20 mM
HEPES-NaOH (pH 7.5), 3 mM MgCl2, 100 mM NaCl, 1 mM dithiothreitol, 1 mM
phenylmethanesulfonyl fluoride, 1 µg/ml leupeptin, 1 mM
EGTA, 1 mM Na3VO4, 10 mM NaF, 20 mM
-glycerophosphate, and 0.5%
Nonidet P-40). Aliquots of the supernatants were mixed with mouse
anti-HA antibody (1 µg). HA-ERK2 or HA-JNK1 was precipitated with
protein A-Sepharose CL-4B and washed twice with lysis buffer C and
twice with reaction buffer B (20 mM HEPES-NaOH (pH 7.5), 10 mM MgCl2, 0.1 mM
phenylmethanesulfonyl fluoride, 0.1 µg/ml leupeptin, 0.1 mM EGTA, 0.1 mM Na3VO4,
1 mM NaF, and 2 mM
-glycerophosphate). The
washed immunoprecipitates were incubated in reaction buffer B with 7.5 µg of myelin basic protein (Sigma) for ERK2 assay or 1.5 µg of
affinity-purified GST-c-Jun (amino acids 1-79) (36) for JNK1 assay, 20 µM ATP, and 5 µCi of [
-32P]ATP at
30 °C for 20 min, and the reaction was stopped by adding 4 × Laemmli sample buffer. The boiled samples were subjected to SDS-polyacrylamide gel electrophoresis, and the radioactivity incorporated into myelin basic protein or GST-c-Jun was measured by an
imaging analyzer (Fuji BAS 2000).
In a previous study, we found that the C-terminal region of G
is involved in the complex formation with G
(33). To identify which
amino acid residue(s) within the last 10 amino acids in the C-terminal
region is required for the association with G
, we constructed
several C-terminal deletion mutants that lack the last six (
C6), two
(
C2), and one (
C1) amino acid(s) (Fig. 1). Since
G
tagged with FLAG epitope at the N terminus can be
co-immunoprecipitated with G
, FLAG-G
was utilized to analyze the
complex formation with the C-terminal mutants of G
(33). The G
mutants and FLAG-G
were expressed at a similar level in HEK 293 cells (Fig. 2B). Fig. 2A shows
that the
C6 and
C2 mutants failed to associate with FLAG-G
,
whereas the
C1 mutant could form a G
complex. Next, we
constructed a mutant (W339A) in which the second amino acid (Trp) from
the C terminus was replaced by Ala (Fig. 1). The W339A mutant could
form a complex with FLAG-G
(Fig. 2A), suggesting that the
presence of an amino acid residue at the
2 position of G
appeared
to be important for the complex formation with G
. The addition of
six histidines ((His)6) to the C terminus of G
was not
inhibitory to the interaction of G
with G
(Fig. 2A).
The results of x-ray crystal structure analysis suggest that the C
terminus of G may be located near the N terminus of G
(7, 8). We
analyzed the effect of the C-terminal mutations of G
on the
interaction with G
. Addition of the FLAG sequence to the N terminus
of G
has no effect on either the association with G
or the
formation of a G
complex (33). In the presence of FLAG-G
,
the
C1, W339A, and (His)6 mutants retained their full
ability to interact with G
o (Fig. 3).
Furthermore, we examined the effect of the C-terminal mutations on the
GTP-dependent dissociation of G
from G
. The cells
were transfected with cDNAs of G
o, C-terminal
mutants of G
, and FLAG-G
. The cells were lysed, and the ternary
complexes were immunoprecipitated with anti-FLAG antibody. The immune
complexes were incubated with a buffer containing GDP or GTP
S (Fig.
4). The ternary complexes formed with G
mutants of
C1, W339A, and (His)6 showed the
GTP
Sdependent dissociation similar to the one formed
with wild type G
.
G has been shown to stimulate the MAPK/ERK signaling pathway
(22-25). To explore the effect of the C-terminal mutations of G
on
MAPK/ERK activation, cDNAs of G
mutants, wild type G
, and
HA-ERK2 were co-transfected into HEK 293 cells. HA-ERK2 was immunoprecipitated with anti-HA antibody, and the kinase activity was
assessed using myelin basic protein as a substrate. It has been shown
that MAPK/ERK activation by Gi-coupled receptors is mediated by G
, whereas the activation by Gq/11
coupled receptors is mediated by Gq/11 (22-25). Carbachol
stimulated the HA-ERK2 activity 2- and 5-fold in HEK 293 cells
transfected with m2-muscarinic and m1-muscarinic acetylcholine
receptors, respectively (data not shown). Crespo et al. (22)
and Faure et al. (23) demonstrated that the ERK2 activity is
stimulated 4- and 2-fold, respectively, by overexpression of G
in
COS cells. In HEK 293 cells, overexpression of G
induces a weak
phosphorylation of endogenous ERK2 (25). As shown in Fig.
5A, co-expression of wild type G
and G
activated HA-ERK2 about 1.5-fold, whereas the
C1 and W339A mutants
had a relatively weak effect on ERK2 activation, and the
C2,
C6, and (His)6 mutants could not activate ERK2 at all.
Furthermore, we examined the effect of the C-terminal mutations of G
on MEK kinase activity of c-Raf-1. Cells were co-transfected with
cDNAs of various G
mutants, wild type G
, and RafFH6, which is
c-Raf-1 tagged with FLAG epitope and six histidines. The RafFH6 was
immunoprecipitated with anti-FLAG antibody, and its MEK kinase activity
was assayed using recombinant MEK and recombinant kinase-negative MAPK.
The MEK kinase activity of RafFH6 was stimulated more than 1.5-fold by
wild type G
and G
. On the other hand, the activation of RafFH6 by
the
C1 and W339A mutants was less potent than that by the wild type
G
, and the
C2,
C6, and (His)6 mutants failed to
activate RafFH6 (Fig. 5B). Effects of the C-terminal
mutations on the stimulation of c-Raf-1 activity were comparable to
those on the stimulation of ERK2 activity. Transfection of G
or G
alone fails to activate ERK2 (see Fig. 8A, and Refs. 22, 23, and 25) and c-Raf-1 (see Fig. 8B). It is noteworthy that the (His)6 mutant could stimulate neither ERK2 nor c-Raf-1.
Since the N-terminal region of c-Raf-1 associates physically with the
C-terminal region of G (37), we tested whether the C-terminal
mutants of G
are able to interact with c-Raf-1. As shown in Fig.
6, the (His)6 mutant could not bind with
c-Raf-1, but other mutants could bind. It is likely that six histidine residues added at the C terminus of G
may sterically inhibit the
association of G
with c-Raf-1. It is suggested that the association of G
with c-Raf-1 by itself is not sufficient for activation of
c-Raf-1, although the association may be required for its
activation.
It has recently been reported that the signaling from G protein-coupled
receptors to JNK/SAPK involves G and that overexpression of
G
enhances JNK/SAPK activity (26). We examined the effect of the
G
C-terminal mutants on JNK/SAPK stimulation. The cells were
co-transfected with cDNAs of each G
mutant and wild type G
together with HA-JNK1. After lysis, HA-JNK1 was immunoprecipitated, and
its kinase activity was assayed using GST-c-Jun as a specific substrate. Wild type G
and G
increased the activity of JNK1 approximately 5-fold (Fig. 7A). In contrast
to the results of MAPK/ERK and c-Raf-1 activations (Fig. 5), the
C1
mutant had little ability to stimulate JNK1, whereas the
C2,
C6,
and (His)6 mutants showed moderate stimulatory effect. The
stimulatory effect of the W339A mutant on JNK1 was almost
indistinguishable from that of the wild type G
(Fig. 7A).
Although the
C2 and
C6 mutants had little ability to associate
with G
, they activated JNK1 significantly. In order to examine
whether G
may activate JNK1 in the absence of interaction with G
in the cells, we utilized a N-terminal deletion mutant of G
. Since
the N-terminal region of G
is essential to form an
-helical
coiled coil structure with the N terminus of G
, the deletion of this
region of G
completely prevents the dimer formation with G
(9,
33, 38). As shown in Fig. 7B, the
N38 mutant, which lacks
N-terminal 38 amino acids, stimulated the activity of JNK1
approximately 3-fold. Furthermore, we transfected with G
or G
alone and measured the JNK1 activity. Fig. 8C
shows that the transfection of G
alone caused the activation of
JNK1. These results suggested that the overexpression of G
alone can stimulate the JNK1 activity in the cells.
The mutant (C1) lacking the C-terminal amino acid of G
retained the full ability to associate with G
and to form a ternary complex with G
(Figs. 2, 3, 4). Although the C-terminal Asn-340 of
G
1 participates in the specific interaction with Asn-62
of G
1 (corresponding to Asn-59 of G
2)
(9), the deletion of Asn-340 of G
1 did not affect the
association with G
2. The interaction does not seem to be
necessary for the complex formation. Removal of the two amino acid
residues from the G
C terminus dramatically abolished the binding of
G
with G
(Fig. 2). Since G
associates with G
at multiple
sites (9), it was unexpected that the truncation of the last two amino
acid residues resulted in the loss of the ability to form a G
complex. We first thought that the Trp-339 may be important for the
complex formation, but the replacement of Trp-339 by Ala (W339A) did
not show any effect on the association of G
with G
(Fig. 2). It
appears that the presence of an amino acid residue at the
2 position
of G
is important for the G
complex formation.
Genetic studies of the pheromone response pathway in
Saccharomyces cerevisiae suggested that two regions of the
Ste4 protein (S. cerevisiae G) may be involved in the
effector activation (39). The first region is localized in the
-helical structure of the N terminus and far from the binding sites
with G
(7, 8, 39). If this region is involved in the binding with an effector, it is unclear how the activation of effector by G
is
inhibited by G
. The second region is found in the third WD repeat
(39). We made a substitution mutant using Gly at Val-135 that was well
conserved between mammalian G
and yeast Ste4. However, the V135G
mutant of G
retained the ability to stimulate the MAPK/ERK and
JNK/SAPK activities to an extent similar to that of the wild type G
in HEK 293 cells (data not shown). It is possible that the V135G
mutation may affect other signaling pathways in mammalian cells or that
other mutations in the second region of G
may affect ERK/MAPK and
JNK/SAPK activities.
We found differential effects of the C-terminal mutations of G on
the MAPK/ERK and JNK/SAPK pathways in mammalian cells. The
C1 mutant
of G
, together with G
, could stimulate the activity of c-Raf-1
and MAPK/ERK but showed only a slight activation of JNK/SAPK. On the
other hand, the (His)6 mutant of G
failed to stimulate
c-Raf-1 and MAPK/ERK activities in the presence of wild type G
, and
the mutant retained the ability to activate JNK/SAPK (Figs. 5 and 7).
The C-terminal amino acid residue of G
is localized on the outside
of a G
complex (9). It is possible that the C terminus of G
is
involved in binding with effector molecule(s). We speculate that the
differential effects of these mutations on the activation of MAPK/ERK
and JNK/SAPK might be due to the difference of their direct
effector(s). The putative effector molecule(s) of G
in the
MAPK/ERK and JNK/SAPK cascades are as yet unidentified, although
phosphatidylinositol 3-kinase has been reported to be involved in the
pathway from G
to MAPK/ERK (40). Phosphatidylinositol 3-kinase
(13) may be a candidate for its effector. It has been reported that
calcium ion is important in Gi-coupled receptor-mediated
stimulation of JNK/SAPK (41). The direct regulation of phospholipase
C
isozymes by G
(11, 12) may be involved in the signaling
pathway.
Recently, Coria et al. (42) reported that the C-terminal
region of yeast Ste4 is essential in triggering the yeast pheromone response cascade. Ste4 has four extra amino acid residues at the C
terminus compared with mammalian G. The extra amino acid residues may be important for the function of Ste4.
It has been shown that overexpression of Ste4 alone causes an increased
response to the pheromone in yeast (43-45). We found that the C2
and
C6 mutants, which were unable to associate with FLAG-G
, could
stimulate JNK/SAPK but not MAPK/ERK activity (Figs. 5 and 7).
Therefore, we examined whether G
alone can stimulate the activity of
JNK/SAPK in HEK 293 cells. It was observed that the transfection of
G
alone could stimulate JNK/SAPK to the same extent as
co-transfection of G
and G
(Fig. 8C). As shown in Fig.
7B, G
-incompetent G
mutant (
N38), which lacks
N-terminal
-helical structure for coiled coil interaction with G
,
has the ability to activate JNK/SAPK in the cells. Taken together, it is suggested that G
plays an essential role in the JNK/SAPK pathway in HEK 293 cells. Coso et al. (26) have reported that
overexpression of G
alone does not stimulate the JNK/SAPK activity
in COS cells. The discrepancy may be due to the difference of cell
type.
In the course of this study, two groups have reported the regions of
G involved in the interaction with effector. Yan and Gautam (46),
using a yeast two-hybrid system, showed that the N-terminal 100-amino
acid fragment of G
associated with adenylyl cyclase type II and G
protein-coupled inward rectifier potassium channel 1. Zhang et
al. (47) demonstrated that the G
mutation in the C-terminal
region prevented the stimulation of phospholipase C
2 in COS cells.
We obtained C-terminal mutants of G
that retained the ability to
interact with G
and G
yet exhibited dramatic decreases in the
MAPK/ERK or JNK/SAPK activation in mammalian cells. Further biological
studies using these mutants should throw more light on the role of G
in the distinct cellular signaling pathways.
We thank M. I. Simon, T. Nukada, M. McMahon, E. Nishida, and M. Karin for supplying the plasmids. We also thank S. Mizutani, K. Tago, and K. Nishida for advice regarding kinase assays.