From the Departments of Physiology & Biophysics and
** Psychiatry, University of Illinois at Chicago, Chicago,
Illinois 60612, ¶ Millennium Pharmaceuticals Inc., Cambridge,
Massachusetts 02139, and the
Department of Pharmacology,
Vanderbilt University Medical Center, Nashville, Tennessee 37232
Received for publication, January 24, 2003, and in revised form, February 10, 2003
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ABSTRACT |
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Gs G proteins act as intracellular transducers to propagate a variety
of signals across the plasma membrane. Due to their interaction with
transmembrane receptors and lipid modification, G proteins are usually
associated with the plasma membrane. Recently, increasing evidence has
emerged that G proteins are also present at intracellular areas such as
Golgi apparatus, endoplasmic reticulum, cytoskeleton, and even the
nucleus (1-5). Moreover, some studies have also shown that activated
G Microtubules, a major component of the cytoskeleton, are involved in
many cellular functions including chromosome movements during mitosis,
intracellular transport, and the modulations of cell morphology. The
biological function of microtubules is based, in significant part, on
the ability of tubulin to polymerize and depolymerize. A heterodimer of
Certain G protein In the present study, functional domains in the
Gi Construction of
Chimeras--
Gt Expression and Purification of Chimera 3 and
Gi AlF Tubulin Iodination--
Ovine brain tubulin was made by two
assembly-disassembly cycles (22), in the presence of
microtubule-associated proteins, which were subsequently removed by
phosphocellulose chromatography. An aliquot of 100 µg of
PC-tubulin in 100 µl of PIPES buffer was applied to a 12 × 75 mm glass tube precoated with 100 µg IODO-GEN (Pierce), which
was dissolved in 100 µl of triethanolamine and dried in a ventilated
hood. 2 µCi of Na125I (Amersham Biosciences) was
then added, and the reaction was allowed to proceed with gentle
agitation for 15 min. The reaction was terminated by addition of 100 µl of PIPES buffer containing 4 mM dithiothreitol. The
free iodide was removed by ultrafiltration by loading the tubulin
suspension on a 3-ml P-6DG (Bio-Rad) desalting column twice. The
desalted 125I-tubulin was centrifuged at 11,500 rpm
for 10 min to remove denatured protein, and the supernatant containing
iodinated tubulin was used in protein binding experiments (14).
Binding of Tubulin to G Photoaffinity Labeling and Nucleotide Transfer--
Tubulin was
incubated with [32P]p3-1, 4-azidoanilido-p1-5'-GTP
(AAGTP) on ice for 30 mins in buffer (10 mM HEPES, pH 7.4, 5 mM MgCl2, 150 mM NaCl, 1 mM Transfection--
Plasmids were purified using the Qiagen Maxi
purification kit. COS-1 cells were split and plated in a 1:15
dilution to 10 cm plates the day before transfection. Cells were
transfected either with calcium phosphate or Lipofectin (Invitrogen).
For calcium phosphate transfection, 20 µg DNA was dissolved in 0.5 ml
of 0.2 M CaCl2 then added drop by drop to the
0.5 ml bubbling 2 × HBS (280 mM NaCl, 10 mM KCl, 1.5 mM Na2HPO4,
12 mM dextrose, 50 mM HEPES). The precipitates
were kept at room temperature for 30 min and were then applied to the
plates. After 6 h of transfection, the cells were washed with PBS
twice and changed to complete medium. Transfections with Lipofectin
were done according to the manufacturer's instruction.
Immunocytochemistry--
COS-1 cells were grown on cover slips
in 24-well plates containing Dulbecoo's modified Eagle's medium with
10% fetal bovine serum and 50 mg penicillin and streptomycin. Before
staining, the medium was removed, the cells were washed twice with PBS, fixed with freezing cold 100% methanol in
The quantification of images was done by assembling fluorescence images
from cells transfected with the indicated constructs as well as
non-transfected cells (examined by differential-interference contrast
microscopy). Images were counted by two individuals blind to
experimental condition, and the number and extent of processes for
His6 positive cells (and control cells) was determined.
Construction and Expression of
Gi Binding of Tubulin by G Chimera 3 Is Not Transactivated by AAGTP-Tubulin--
The above
data show that both chimera 1 and chimera 3 bind to tubulin with about
the same affinity as Gi Chimera 3 Blocks the Formation of Cellular
Outgrowths--
Previous studies, as well as data obtained in Figs. 2
and 3, suggested that G
Native or vector-transfected COS-1 cells extend moderate length
cellular processes that tend to be slightly shorter than the body of
the cell (Fig. 4 and Table I).
Transfection with Gi Data in this study suggest that distinct regions on G The Tubulin is able to transfer nucleotide (GTP or AAGTP) directly to
various G protein Insertion of Gt In COS-1 cells expressing
His6-Gi Microtubules are dynamic structures that undergo assembly and
disassembly within the cell. They function both to determine cell shape
and in a variety of cell movements, including some forms of cell
locomotion, the intracellular transport of organelles, and the
separation of chromosomes during mitosis. Several studies (11, 19) have
suggested G proteins might participate in modulation of the
cytoskeleton. Association between tubulin or microtubules and G Curiously, expression of chimera 3 blocks the extension of cellular
processes. Although it would be premature to suggest that Gi In summary, this study reveals that distinct regions on G,
Gi
1, and Gq
subunits bind
tubulin with high affinity, whereas transducin (Gt
) does
not. The interaction between tubulin and G
, which also
involves the direct transfer of GTP from tubulin to G
(transactivation), is not yet fully understood. This study, using
chimeras of Gi
and Gt
, showed that the
Gi
(215-295) segment converted Gt
to
bind to tubulin and this chimera (chimera 1) could be transactivated by
tubulin. Insertion of Gt
(237-270) into chimera 1 to
form chimera 2 resulted in a protein that, like Gt
, did
not bind tubulin. Thus, it was thought that the Gi
(237-270) domain was essential to modulate the binding of
Gi
1 to tubulin. Surprisingly, when domain
(237-270) of Gi
was replaced by Gt
(237-270) to form chimera 3, the chimera bound to tubulin with a
similar affinity (KD
120 nM) as
wild-type Gi
1. However, even though chimera
3 displayed normal GTP binding, it was not transactivated by
GTP-tubulin. Furthermore, when these chimeras were expressed in COS-1
cells, cellular processes in cells overexpressing Gi
1 or chimera 1 were more abundant and
longer than those in native cells. G
was seen throughout the length
of the process. Morphology of cells expressing chimera 2 was identical
to controls. Consistent with the role of Chimera 3 as a "dominant
negative" G
, cells transfected with chimera 3 had only few
truncated processes. This study demonstrates that although
Gi
(237-270) is not obligatory for the binding of
Gi
to tubulin, it is crucial for the transactivation of
G
by tubulin. These results also suggest that the
transactivation of G
by tubulin may play an important role in
modulating microtubule organization and cell morphology.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
s can be released from the plasma membrane to the cytoplasm (6-9).
Thus, it is possible that G proteins exist in several different
cellular locations and play roles in various physiologic processes.
- and
-tubulin is the basic building block of microtubules.
Tubulin is a GTP-binding protein, two GTP molecules are bound
noncovalently in exchangeable (E-site on
-tubulin) and
nonexchangeable (N-site on
-tubulin) sites. Both G
subunits and
tubulin have intrinsic GTPase activity but that of tubulin is activated
during the process of polymerization (10) or when complexed with G
(11).
subunits (Gs
,
Gi
1,1
and Gq
) bind to tubulin with high affinity
(KD
120 nM). Transducin, however, does not bind to tubulin. Complexes between Gs
or
Gi
and tubulin were co-immunoprecipitated from detergent
extracts of synaptic membrane (12-15). This binding appears to
activate the G protein due to a direct transfer of GTP from the E-site
in tubulin to G
(transactivation) (16-18). In addition, the
association of G
and tubulin induces GTPase activity in tubulin and
modulates microtubule polymerization dynamics in vitro
(11, 19). Agonists for certain G protein-coupled receptors induce
microtubule depolymerization and tubulin relocation to the plasma
membrane (20). These studies suggest that microtubules might be
involved in regulation of signal transduction, whereas G protein
signaling may modulate microtubule polymerization.
1 amino acid sequence that mediate the
interaction of Gi
with tubulin were investigated. We
report here that the 237-270 region of Gi
appears not only to contribute to the binding of Gi
to
tubulin but also is crucial for the transactivation of G
by tubulin. The transactivation of G
by tubulin may be an important factor for
the modulation of tubulin polymerization dynamics and cell shape by
hormone and neurotransmitters.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/Gi
1 chimera
A, (Gi
1 1-237/Gt
237-270/Gi
1 270-295/Gt
295-314/Gi
1 314-354), and
Gt
/Gi
1 chimera B,
(Gt
1-236/Gi
1 236-350) were
first constructed. These were used to construct chimera 3 by
replacing the chimera A C-terminal sequence (270-350) with the chimera
B Gi
1 (270-350) domain. To assemble DNA
fragments encoding chimera 3, both plasmids were digested with
HindIII and AsuII. A short
AsuII-HindIII DNA fragment from the expression vector encoding chimera B was ligated with a large DNA fragment derived
from the expression vector encoding chimera A. This new chimeric
Gt
/Gi
1 construct, chimera 3, was confirmed by restriction analysis and DNA sequencing. Chimera 1 and
chimera 2 had been previously described (21). In addition, all chimeras
and His6 tag Gi
1 were subcloned
into PcDNA3 vector (Invitrogen) for expression in the
mammalian cells.
1--
The Escherichia coli
BL21 cells transformed with the vectors harboring chimera 3 and
Gi
1 were grown in 2× YT medium with 100 µg/ml of ampicillin at room temperature up to
A600 of 0.5 and then induced with 30 µM isopropyl-1-thio-
-D-galactopyranoside at room temperature for 16-20 h. The cell pellet was resuspended in
1:20 of a cell culture volume of buffer containing 50 mM
Tris-HCl, pH 8.0, 50 mM NaCl, 5 mM
MgCl2, 50 µM GDP, 0.1 mM
phenylmethylsulfonyl fluoride, and 5 mM
-mercaptoethanol (buffer A) and disrupted by ultra-sonication
using eight 30 s pulses with 1 min breaks in between pulses. The
crude cell lysate was cleared by centrifugation at 100,000 × g for 60 min. The supernatant was collected and adjusted to
20 mM Tris-HCl, 500 mM NaCl, and 20 mM imidazole (binding buffer). The cell lysate was loaded
onto 5 ml of nickel-nitrilotriacetic acid column preliminary
equilibrated with the binding buffer. After washing the column with 10 bed volumes of binding buffer, the bound material was eluted with 20 mM Tris-HCl, 500 mM NaCl, and 100 mM imidazole and subjected to overnight dialysis against buffer A with 20% glycerol. The protein samples were directly applied
to 1 ml Mono Q column equilibrated with buffer A without GDP and
-mercaptoethanol. Proteins were eluted with 0-1 M NaCl gradients in buffer A. An increase in tryptophan fluorescence was
measured with excitation at 280 nm and emission at 340 nm to monitor
AlF
subunits, to determine the
activity of the purified protein as described by Skiba et
al. (21). The eluted proteins were supplemented with GDP,
-mercaptoethanol, and phenylmethylsulfonyl fluoride to a
concentration of 25 µM, 2 mM, and 0.1 mM, respectively, aliquoted and stored at
80 °C for
several months with no loss of functional activity (21).
--
Binding of
AlF
-GDP mimics the GTP
conformation of the molecule and changes in Trp fluorescence can be
used to monitor the "viability" of a G
construct. To measure this, 200 nM Gi
1 or the
chimeras were incubated at room temperature in 50 mM
Tris-HCl, pH 8.0, 50 mM NaCl, and 2 mM MgCl.
Fluorescence was measured in an Aminco-Bowman Series 2 Spectrometer
(SLM-Aminco) using excitation of 280 nm and emission at 340 nm.
Measurements were taken before and 5 min after the addition of 10 mM NaF and 30 µM AlCl3.
Fluorescence increases were expressed as a percent change from the
initial fluorescence: (
F (%) = (F
Fo)/Fo × 100, where Fo is the initial fluorescence and
F is the fluorescence after fluoride addition. This method
is described in Ref. 21.
--
Purified
Gi
1, chimera 3, bovine transducin, and
ovalbumin (Sigma) were applied to nitrocellulose membrane (Midwest
Scientific) in the amounts of 150, 100, 50, 25 ng, respectively.
Nitrocellulose was incubated with 10% bovine serum albumin in 100 mM PIPES buffer at room temperature for 2 h to block
nonspecific binding and then was incubated with 100 nM
iodinated tubulin in 100 mM PIPES buffer at room
temperature for 2 h. Nitrocellulose was then washed three times
with PIPES buffer. Binding of tubulin to G protein
subunits was detected by radioautography. The affinity of tubulin for chimera 3 was estimated by immobilizing chimera 3 upon nitrocellulose and
hybridizing with varying concentrations of 125I-tubulin.
This was quantified in a gamma counter (Beckman Gamma 9000). The
binding was dose-dependent and saturable (14). Immobilized chimeras (other than chimera 2) bound 0.3-0.5 mol of tubulin/1 mol of
G
.
-mercaptoethanol). Free AAGTP was removed with two
passes through a P6-DG column. The labeled tubulin was incubated with
equimolar concentrations of Gi
1, chimera 3, and Gt
at 30 °C for 30 min. UV irradiation, SDS gel
electrophoresis, and analysis of results were performed as described in
Ref. 16. AAGTP transferred from tubulin to G protein was quantified in a phosphorimaging device (Storm 840; Molecular Dynamics).
20 °C for 10 min, and washed again with PBS twice. The cells were then incubated with 5%
normal goat serum in PBS for 1 h and incubated in 1:100 dilution of primary antibody in blocking buffer for 1 h. Subsequently, the
cells were washed with PBS four times and incubated with 1:100 dilution
secondary antibodies labeled with fluorescein isothiocyanate or TRITC
(rhodamine) (EY Labs) in blocking buffer for 45 min. Finally,
the cells were washed with PBS four times and mounted on the slide with
polyvinyl alcohol mounting medium. The slides were air dried and
examined with a fluorescence microscope with a 100 watt mercury arc
lamp (Nikon, TE300). Images were collected with an interline
charge-coupled device camera (Model 1300, Roper Scientific, Trenton,
NJ) driven by IP lab software (Scanalytics Inc., Suitland, VA),
and assembled in Adobe Photoshop.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1-Transducin Chimeras--
To map
tubulin-binding sites on Gi
1 we constructed
Gi
1/Gt
chimeras where we
exchanged several corresponding regions of these two structurally
related proteins. The structures of chimeras are schematically
illustrated in Fig. 1. The recombinant
subunits were expressed in E. coli and purified using a
combination of affinity chromatography on nickel-nitrilotriacetic acid
agarose and Mono Q high pressure liquid chromatography. Both the GTP
binding capability and the AlF
1 and each of
the 3 chimeric proteins. For Gi
1,
Gt
, chimera 1, and chimera 2, the
AlF
F (% increase) was 55, 70, 50-55, and 60-70, respectively. For
chimera 3, the AlF
F
(% increase) was 50. The values given represent the increase in
fluorescence upon G
and G
chimera activation. Ranges are given
for multiple preparations (between two and six, depending upon the
protein). Where a single value is given, two preparations gave the same
results. Detected changes in intrinsic Trp fluorescence indicated that
Gi
1 and chimeras were fully
functionally active. Those values, which are dependent upon the number
of tryptophan residues, are similar to the values reported previously
for Gi
1, Gt
and
Gi
1/Gt
chimeras
(21).
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Fig. 1.
Model for
Gi 1/Gt
chimera constructs. All constructs contain His6
tag at the N-terminal.
-chimeric Proteins--
Previous
results had suggested that Gi
1 binds
tubulin with a KD of 120 nM, whereas the
affinity of Gt
for tubulin was too low to be measured
reliably (14). A site on Gt
thought to interact with
effectors was mapped to the residues 237-270 (21). Therefore, two
reciprocal chimeras containing the region 237-270 from
Gi
1 or Gt
(chimeras 1 and 2, respectively) were constructed. Fig. 2
shows that chimera 1 was virtually indistinguishable from
Gi
1 in its ability to bind
125I-tubulin, whereas chimera 2, similar to transducin, did
not bind tubulin in any measurable manner. The third chimera, chimera
3, was constructed to measure the contributions of regions 237-270 directly. The replacement of this region of
Gi
1 with the analogous region of transducin
resulted in a protein that bound tubulin to roughly the same extent as
Gi
1 (Fig. 2A). Scatchard analysis of tubulin binding to Gi
1 and chimera 3 reveals saturable binding and a KD of 120 and 123 nM, respectively.
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Fig. 2.
Specificity and affinity of
125I-tubulin binding to G . A,
125I-tubulin binding to G protein
subunits.
Gi
1, chimera 1, chimera 2, chimera 3, and
Gt
were applied to a nitrocellulose sheet in the amounts
indicated and air-dried at room temperature. Following this, tubulin
binding was assessed by overlay with 125I-tubulin and
autoradiography as described under "Materials and Methods." One of
three similar experiments is shown. B, saturation isotherm
and Scatchard plot for tubulin binding to
Gi
1 and chimera3. Data were derived from dot
blotting performed with a method similar to that for A. 100 ng of Gi
1 or chimera 3 were applied to each
spot. Triplicate nitrocellulose spots corresponding to the total
binding and nonspecific binding (determined in the presence of 100-fold
excess unlabeled tubulin) were cut out and counted in an LKB Rack gamma
counter. The graph on the left shows the
saturation isotherm for specific binding of 125I-tubulin to
chimera 3 (
) or Gi
1(
). On the
right are the Scatchard plots derived form these data. The
KD and Bmax for
Gi
1 were 121 nM and 386 fmol/ng,
respectively. For chimera 3, these data were 123 nM and 473 fmol/ng, respectively. Data were calculated from two similar
experiments.
1. Chimera 1 can be
transactivated via transfer of GTP from tubulin to a similar extent as
Gi
1. Approximately 50% of the AAGTP is
distributed to each protein during this process, and GTP in the milieu
has no access to either G
or tubulin once a complex has formed (19). Fig. 3 demonstrates that chimera 3 failed
to serve as a substrate for transactivation despite the fact that it
bound to tubulin. When tubulin and Gi
1 were
incubated along with chimera 3, transactivation of
Gi
1 by tubulin was substantially diminished.
Equimolar chimera 3 inhibits transactivation of Gi
by
49.9 ± 13%. When the concentration of chimera 3 was increased to
twice that of Gi
1, transfer of AAGTP to G
was blocked, and the AAGTP bound to tubulin was also decreased. These
data suggest that chimera 3 cannot be transactivated by tubulin, and it
inhibits the transfer of GTP from tubulin to Gi
1. Thus, chimera 3 may act as a dominant
negative to inhibit the transactivation of Gi
by
tubulin.
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Fig. 3.
Transactivation of G
constructs by tubulin. Freshly prepared
tubulin-[32P]AAGTP was incubated with equimolar
Gi
1, chimera 3, Gt
,
Gi
1 plus chimera 3, or twice molar
Gi
1 or chimera 3 as indicated. AAGTP
binding to tubulin or G
was made covalent by UV irradiation, and
samples were prepared for SDS-PAGE. [32P]AAGTP bound to
tubulin or G
(as a result of transactivation) was quantified by
phosphoimaging analysis. Images shown are from one of three similar
experiments.
might alter cellular projections containing microtubules (11, 17, 19). To test this, G
i1 and
chimeras 1-3 were expressed in COS-1 cells. The expression of each of
these chimeras, which was about 3-fold greater than the endogenous
Gi
, was identified with an antibody against the
His6 tag on the protein, and the expression level for each
one of the constructs appears to be comparable. This was determined by
both the immunostaining seen in the Fig.
4 and by Western blot (not shown). Fig. 4
also demonstrates the effect that the expression of the various
constructs has on the formation of cellular outgrowths (indicated by
arrows).
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Fig. 4.
G protein and tubulin dual staining of
transiently transfected COS-1 cells. COS-1 cells were transfected
with 10 µg of pcDNA3 coding for Gi 1,
chimera 1, chimera 2, and chimera 3, respectively, and grown in normal
Dulbecoo's modified Eagle's medium for 48 h. The cells were
fixed with
20 °C methanol and double stained with polyclonal
His6 tag antibody and fluorescein isothiocyanate goat anti
rabbit IgG and with
-tubulin antibody and rhodamine-coupled goat
anti-mouse IgM. G
constructs appear green and
microtubules are seen in red. The figure shows the
overlay of the tubulin and His6 tag antibodies. Areas of
tubulin/G
overlap appear yellow. The arrows
indicate cellular processes. Images shown are typical of five
experiments in which at least 60 cells of each type were
examined.
1 or chimera 1 increased
both the number of cells displaying processes and the mean length of
those processes (Table I). Thus, a moderate increase in the expression
of Gi
increases the length and extent of
microtubule-bearing cellular outgrowths. By contrast, chimera 2, which
did not bind to tubulin, did not significantly affect cellular
outgrowths on COS-1 cells. Both the number and size of processes in
chimera 2-transfected cells were similar to control. When cells were
transfected with chimera 3, cellular processes were sparse and
extremely short (Fig. 4 and Table I). The expression of chimera 3 prevented the transactivation of endogenous G
by tubulin and
prevented cells from sending out these microtubule-rich processes (Fig.
4 and Table I).
Number and extent of cell process formation in COS 1 cells expressing
various chimeric G protein constructs
20°C methanol and stained with
rabbit anti-His6 tag and mouse anti-
-tubulin (ICN). Forty to
forty-five cells were counted for each construct from 3 different
transfections. A process was defined as an extension from the cell body
longer than one third of the cell length. Values are mean ± SEM
for 40-45 cells from three separate transfection experiments. Both the
number of processes/cell and the average process length are
significantly (p < 0.001) less for cells transfected with chimera 3 than observed in control cell.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
mediate
the binding and the transactivation of G
protein and tubulin. Gi
1 (237-270) plays a crucial role for the
transactivation of Gi
by tubulin, even though it is not
a region required for the binding of those two molecules. Based on the
crystal structure of G
protein, this region includes the helix of
3, a loop, and the
5 strand. The (237-270) domain in
Gi
1 is one of the binding sites for
effectors such as adenylyl cyclase (23). Similarly, this domain in
transducin binds the phosphodiesterase
subunit allowing activation
of retinal phosphodiesterase (31). The importance of the domain
implies that the interaction of Gi
1 and
tubulin, which evokes transactivation of the later molecule, may play a role in modulating the physiology of signaling pathway and may orchestrate organization of cytoskeleton.
subunits of heterotrimeric G proteins are a family of proteins
of 39-52 kDa that display a similarity of about 45-80% at the amino
acid level. Previous studies (14) suggested that Gi
1 bound tubulin with high affinity
(KD = 120 nM) and Gi
was
transactivated by directly transferring GTP. Transducin neither bound
tubulin nor was its substrate for transactivation (14). In this study,
when the Gi
1 (215-295) segment replaced the
Gt
sequence, chimera 1 could bind to tubulin and was
transactivated by transfer of GTP from tubulin in vitro.
This suggests that the region 215-295 might be important for mediating
Gi
1 interaction with tubulin. However, in a
manner similar to transducin, chimera 2 did not bind tubulin in
vitro. Thus, we initially thought that the
Gi
1 (237-270) domain might play a key role
in rendering Gi
1 able to bind tubulin
in vitro. Strangely, chimera 3 bound to tubulin with the
same high affinity as wild-type Gi
1 did.
Nonetheless, it appears that chimera 3 inhibited the "productive"
association between Gi
1 and tubulin, which
allows for the transactivation of Gi
. Previous studies
(14, 17, 24) have shown that there may be multiple binding sites on the
tubulin dimer for Gi
1, and part of the
receptor interaction domain of G
may mediate this binding. Based on
data in this study, it is likely that the
Gi
1 (237-270) domain is involved in the
binding of Gi
and tubulin; however, there is an
additional site (or sites) for Gi
1
interaction with tubulin, and this is located within 1-215 and
295-354 regions of Gi
1. As shown in a solid
phase binding assay, replacement of Gi
237-270 with
Gt
237-270 in Gi
sequence did not
significantly affect the binding affinity of chimera 3 and tubulin
(KD
123 nM for either molecule),
implying that this second site(s) is crucial for the high affinity
binding of Gi
1 to tubulin.
-subunits (Gs
,
Gi
1, and Gq
) (13, 16, 19).
This phenomenon has been referred to as transactivation, and during the
process nucleotide is not released into the milieu but rather is
transferred directly from the tubulin to the G protein
-subunit
(19). It suggests that tubulin activates G proteins by directly
transferring GTP to G
, thus "bypassing" a
Gs
-coupled receptor (16). This interaction between G
and tubulin has led to a hypothesis that the cytoskeleton may
participate in the regulation of G protein-signaling pathways.
Colchicine, a microtubule depolymerizing agent, potentiated
-adrenoreceptor-stimulated cyclic AMP in lymphoma cells, where
activation of the
-adrenergic receptor was obligatory for
GTP-activation of adenylyl cyclase (25). In permeable C6 cells, tubulin
stimulates adenylyl cyclase activity by transactivating Gs
(16). Exogenous tubulin can also inhibit rat cerebral
cortex membrane adenylyl cyclase, apparently by transactivation of
Gi
1 (18, 19).
237-270 into Gi
did not
alter the GTP binding or the characteristics of binding between
Gi
and tubulin, but chimera 3 did not serve as a
substrate for transactivation by tubulin. Chimera 3 also inhibited GTP
transactivation of wild-type Gi
(Fig. 3). These results
suggest that the Gi
237-270 domain is essential to
transfer GTP from tubulin to Gi
. Thus, it appears that
chimera 3 acts as a dominant negative mutant of
Gi
1 with regard to the "fruitful"
interactions between Gi
and tubulin. The domain
identified might provide a useful tool to further study and
understanding the biologic role of interaction of Gi
and tubulin in cells.
1 and chimeras, it
appeared that these proteins were found throughout the cytosol.
This may be because the tagged His6 at the end of the
N-terminal of Gi
1 disrupted the
Gi
1 associated with plasma membrane
(7). Addition of GFP to the N-terminal of Gs
blocked its
association with plasma membrane (6). However, myristoylated and
non-myristoylated Gi
bind tubulin with equal affinity
and both activate tubulin GTPase, suggesting that they bind to tubulin
in a similar fashion (11). Thus, the physiologic potential of these
G
constructs may be apparent in these experiments. The considerable
difference in effect among these varying constructs of G
supports
this notion. Furthermore, cytosolic G
may be more relevant to the
modulation of microtubule dynamics than membrane-associated G
. In
Dictyostelium, G
appears cytosolic, and this may help to orient
cells in response to a polarizing signal (26).
has
been established for some time (14, 27, 28). In this study,
overexpression of His6-Gi
1 and
chimera 1 in COS-1 cells increases the length and number of cellular
processes, whereas chimera 2, which does not bind tubulin, had no
effect. This is consistent with the notion that the binding and
transactivation of Gi
1 by tubulin might have
a role in the regulation of microtubules. Previous studies also show
that G
stabilizes microtubules (4), whereas
Gi
1 increases microtubule dynamics by
increasing the frequency of microtubule catastrophe (11). Although
these data are from in vitro studies, they are consistent
with the possibility that G
may modulate the dynamics of
microtubules in cells. Note that an increase in microtubule dynamics is
not inconsistent with increased length and number of cellular processes
induced by increased expression of Gi
1 or
chimera 1. In fact, microtubules in areas of dynamic cellular
extension, such as growth cones, display more dynamic behavior (32). A
recent study (33) has demonstrated an increase in the association of
Gi
(and Gs
) with microtubules in cellular
processes induced by nerve growth factor and other "differentiating" agents in PC12 pheochromacytoma cells.
normally accelerates the extension of cellular
process thorough its interaction with tubulin, these results are
consistent with such a possibility. To draw more specific conclusions,
more studies in vitro and in vivo are required.
The role of G
transactivation by tubulin in this process is also
unclear. Although transactivation seems pertinent to the regulation of
adenylyl cyclase or phospholipase C by tubulin (16, 20), its role in
regulation of microtubule dynamics is unknown. Nevertheless, it is
noteworthy that activation of Gi
and Gs
has been linked to a rapid increase in microtubule depolymerization (29, 30).
mediate
the tubulin binding and the transactivation process.
Gi
1 219-295 is important to the binding of
Gi
1 and tubulin, and Gi
237-270 contributes to the transactivation by tubulin of
Gi
1. The data in this study implicate
Gi
1 transactivation by tubulin in the
regulation of microtubule organization. Such a regulation might provide
new insight into the relationship between hormone or neurotransmitter
action and cell morphology or other aspects of the dynamic cytoskeleton.
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ACKNOWLEDGEMENTS |
---|
We thank Ji Eun Oh and other members in the Rasenick Lab for advice and discussion.
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FOOTNOTES |
---|
* This work is supported by US Public Health Service grants MH 39595, MH 57391, and AG 15482 (to M. M. R.) and EY06062 and EY10291 (to H. E. H.).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.
§ These authors contributed equally to this work.
To whom correspondence should be addressed: Dept. of Physiology
& Biophysics, M/C 901, University of Illinois at Chicago, College of
Medicine, 835 S. Wolcott Ave., Chicago, IL 60612-7342. Fax:
312-996-1414; E-mail: raz@uic.edu.
Published, JBC Papers in Press, February 11, 2003, DOI 10.1074/jbc.M300841200
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ABBREVIATIONS |
---|
The abbreviations used are:
Gi,
subunit of the heterotrimeric Gi
protein;
Gt
, transducin;
PBS, phosphate-buffered saline;
PIPES, 1,4-piperazinediethanesulfonic acid;
TRITC, tetramethylrhodamine isothiocyanate.
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