From the Departments of Physiology and Biophysics and
Psychiatry, University of Illinois, Chicago, Illinois 60612 and
the ¶ Department of Molecular, Cellular, and Developmental
Biology, University of California,
Santa Barbara, California 93106
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ABSTRACT |
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G proteins serve many functions involving the
transfer of signals from cell surface receptors to intracellular
effector molecules. Considerable evidence suggests that there is an
interaction between G proteins and the cytoskeleton. In this report, G
protein Microtubules, a major component of the cytoskeleton, are involved
in a variety of cellular functions including chromosome movements
during mitosis, intracellular transport, and the modulation of cell
morphology. In general, the biological function of microtubules is
based in significant part on the ability of tubulin to polymerize and
depolymerize. In living cells, microtubules exist in both dynamic and
stable populations, with each population called upon to carry out
distinct cellular functions (1, 2). Proper control of microtubule
dynamics is essential for many microtubule-dependent processes.
Microtubule ends can interconvert between slow elongation and rapid
shortening, a process called dynamic instability, because of the
presumed gain and loss of a small region of tubulin-liganded GTP at the
microtubule end (3-5). Tubulin dimers bind 2 mol of GTP/mol of
tubulin, one exchangeable (the
E-site1 in Several microtubule-associated proteins are known to regulate
microtubule dynamics by stabilizing microtubules (8, 9). Stabilization
of microtubules by microtubule-associated proteins is achieved, in
part, by suppressing the rate and extent of microtubule shortening and
by suppressing the catastrophe frequency and increasing the rescue
frequency (6, 10-12). It is noteworthy that the catastrophe frequency
observed in cells is much higher than that observed in vitro
with microtubules composed of pure tubulin (13), suggesting the
possible control of the process by additional cellular factors (14-17).
Studies have demonstrated that microtubule polymerization and stability
are also affected by second messenger-activated protein kinases,
suggesting the possibility that microtubule dynamics may be regulated
by extracellular signals through G proteins (for review see Ref. 18;
also Refs. 19 and 20). G proteins act as arbiters of cellular
signaling, and they may associate in cells directly with microtubules
(21-26). Heterotrimeric G proteins are composed of Tubulin Preparations--
Tubulin for all studies except the
dynamic instability analysis was purified from fresh sheep brain by
cycles of assembly and disassembly (33) followed by phosphocellulose
chromatography (34). The resulting tubulin preparations were more than
97% pure as determined by Coomassie Blue staining of
SDS-polyacrylamide gels (not shown). The tubulin was stored in liquid
nitrogen and used within 2 weeks. Bovine brain tubulin was used for
dynamic instability analyses as described elsewhere (10). Tubulin
liganded with GTP, GppNHp, or [ G Protein Purification--
Recombinant Gi1 GTP Hydrolysis--
Tubulin was allowed to bind
[ Microtubule Assembly--
Tubulin-GTP or tubulin-GppNHp in PEM
buffer (100 mM PIPES, 2 mM EGTA, 1 mM MgCl2, pH 6.9) was preincubated with or
without G Electron Microscopy--
Fifteen µl of the microtubule sample
was placed on a Formvar-coated nickel grid. After 10-15 s, the grids
were rinsed with 10 drops of 2% uranyl acetate for negative staining,
blotted dry with a filter paper, and viewed in a JEOL 100S electron microscope.
Microtubule Dynamics by Video Microscopy--
Tubulin (12 µM) was mixed with Strongylocentrotus
purpuratus flagellar seeds in 80 mM PIPES, 0.8 mM Mg2+, 1 mM EGTA, pH 6.8 (PME
buffer), containing 275 µM GTP in the absence or presence
of Gi1 Gi1
To distinguish between these possibilities, we used a mutated form of
Gi1 Gi1 Tubulin Exchanges Nucleotide in the G Microtubules Polymerized in the Presence of Gi1 Specificity of G Protein Gi1
Gi1 In the present study, the subunits Gi1
, Gs
, and
Go
are shown to activate the GTPase activity of tubulin,
inhibit microtubule assembly, and accelerate microtubule dynamics.
Gi
inhibited polymerization of tubulin-GTP into
microtubules by 80-90% in the absence of exogenous GTP. Addition of
exogenous GTP, but not guanylylimidodiphosphate, which is resistant to
hydrolysis, overcame the inhibition. Analysis of the dynamics of
individual microtubules by video microscopy demonstrated that
Gi1
increases the catastrophe frequency, the frequency
of transition from growth to shortening. Thus, G
may play a role in
modulating microtubule dynamic instability, providing a mechanism
for the modification of the cytoskeleton by extracellular signals.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-tubulin) and
the other nonexchangeable (in
-tubulin). GTP bound to the
exchangeable site becomes hydrolyzed upon incorporation of the tubulin
into the microtubule. This hydrolysis creates a microtubule consisting
largely of GDP-tubulin, but a small region of GTP-liganded tubulin,
called a "GTP cap," remains at the end. The loss of the cap results
in a transition from growth to shortening (called a catastrophe),
whereas the reacquisition of the GTP cap results in a transition from
shortening to growing (called a rescue) (6). The GTPase activity of
tubulin is normally low, and hydrolysis of the E-site GTP requires
activation. This activation normally occurs when the tubulin dimer
binds to the end of a growing microtubule. It is thus suggested that
one tubulin dimer might act as a GTPase activator for another during
polymerization (7).
and
subunits. G
subunits bind GTP and display various levels of
intrinsic GTPase activity. Certain G protein
subunits
(Gi1
, Gs
, and Gq
) bind to
tubulin with high affinity (27-30). This binding appears to activate
the G proteins in association with a direct transfer of GTP from the
E-site in tubulin to G
(transactivation) (29, 31). In addition to
activating G
, the association between G
and tubulin induces a
GTPase activity in tubulin similar to that seen after the
self-association of tubulin dimers during the formation of a
microtubule (32). Recent studies have also shown that
G
1
2 binds to microtubules and promotes microtubule assembly in vitro (26). These studies indicate
that G proteins may modulate microtubule polymerization dynamics and cytoskeletal organization or function. In the present study, the modulation of microtubule assembly and dynamics by G protein
subunits was investigated. We report here that
subunits of G proteins activate the intrinsic GTPase of tubulin (i.e. they
act as a GTPase activating protein for tubulin), and the GTP hydrolysis modulates microtubule assembly and dynamics in vitro.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]GTP was
prepared by removing exchangeable nucleotide from the tubulin by
charcoal treatment followed by incubation with 0.5 mM GTP,
0.5 mM GppNHp, or 0.1 mM
[
-32P]GTP (31). The samples were then desalted twice
on centrifugal gel filtration columns using P6-DG resin (Bio-Rad) as
described previously (31). After desalting, 0.5-0.8 mol of guanine
nucleotide was bound/mol of tubulin. Protein concentration was
determined by the method of Bradford using bovine serum albumin as a
standard (35).
,
Gs
, or Go
were produced in
Escherichia coli using constructs provided by Dr. Maurine
Linder (Washington University, St. Louis, MO). The vector used
contained Gi1
, Gs
, or Go
cDNA preceded by a nucleotide sequence encoding a
His6-amino acid stretch as an affinity tag under the
control of a T7 promoter. E. coli was grown and harvested,
and G proteins were purified over a Qiagen nickel column with a
subsequent MonoQ high pressure liquid chromatography step (36). The
Q204L mutant of Gi1
, a generous gift from Drs. J. Hepler and A. G. Gilman (University of Texas Southwestern Medical
Center, Dallas, Texas), was expressed in E. coli and
purified as described (36). Bacteria containing myristol transferase
and Gi1
(a gift from Dr. M. Linder) were used to express
myristoylated Gi1
, which was then purified as described
earlier (37).
-32P]GTP, and unbound nucleotide was removed by gel
filtration using a P6-DG column (Bio-Rad). The samples were then
incubated with or without G
at 30 °C for 30 min and treated with
1% SDS at room temperature for 15 min. Nucleotide analysis was done by
thin layer chromatography on polyethyleneimine cellulose plates (32,
38). Two µl of a 10 mM solution of GTP and GDP were
spotted 1.5 cm apart on a polyethyleneimine cellulose thin layer plate,
followed by 2-5 µl of each sample. The chromatograms were developed
in 0.35 M NH4HCO3. The spots
containing GTP or GDP were visualized with a UV lamp, and plates were
exposed to film for autoradiography. Quantitative analysis was done
using a Molecular Dynamics PhosphorImager system.
at 30 °C for 30 min. Polymerization was then initiated
by adding 30% glycerol and an additional 2 mM
MgCl2 and incubating at 37 °C for 45 min to 1 h.
The extent of microtubule assembly was quantified after pelleting the
microtubule polymers by centrifugation at 150,000 × g
for 20 min at 37 °C. Pellets were resuspended in 4 °C PEM buffer,
and protein concentrations in the pellet and supernatant fractions were
determined (35). Before testing the effect of G proteins on microtubule
assembly, free nucleotide was separated from G protein
subunits,
and the buffer was changed to PEM by passage of the proteins through a
rapid spin column (Bio-Gel P6DG, Bio-Rad). Alternatively, when G
concentrations were low, a buffer control was performed to avoid a
reduction in protein concentration by gel filtration.
and incubated for 25 min at 37 °C for assembly
to reach steady state. The seed concentration was adjusted to achieve
3-6 seeds/microscope field. 2.5 µl of the microtubule suspension was
prepared for video microscopy, and the dynamics of individual
microtubules were recorded at 37 °C as described previously (10).
Under the experimental conditions used, microtubule growth occurred
predominantly at the plus ends of the seeds as determined by the growth
rates, the number of microtubules that grew, and the relative lengths
of the microtubules at the opposite ends of the seeds (6, 10, 39-41).
Microtubule length changes were measured in real time at 3-6 s
intervals until microtubules underwent complete depolymerization to the
axoneme seed or until the microtubule end became obscured. The length changes undergone by a particular microtubule as a function of time
were used to create a "life history" plot. The growing and shortening rates were determined by least squares regression analysis of the data points for each growing or shortening phase. The reported mean growing and shortening rates represent the mean values for all
growing and shortening events observed for a particular reaction condition. We considered a microtubule to be in a growing phase if the
microtubule increased in length by >0.2 µm at a rate >0.15 µm/min
and in a shortening phase if the microtubule shortened in length by
>0.2 µm at a rate >0.3 µm/min. Length changes equal to or less
than 0.2 µm over the duration of 6 data points were considered as
attenuation phases. A total of 22-25 microtubules was analyzed for
each experimental condition. The catastrophe frequency was determined
by dividing the number of catastrophes by the sum of the total time
spent in the growing plus attenuated states for all microtubules for a
particular condition. The rescue frequency was calculated by dividing
the total number of rescue events by the total time spent in the
shortening states for all microtubules for a particular condition.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Activates the Intrinsic GTPase of
Tubulin--
Tubulin binds to Gi1
and Gs
with a Kd of approximately 130 nM
coupled with a transactivation of G
in which 25-50% of E-site
tubulin-bound GTP is transferred directly to the G
(28, 29).
Gi1
binding to tubulin in vitro also
activates GTP hydrolysis (32). Both tubulin and Gi1
have
intrinsic GTPase activities. Because the intrinsic GTPase activity of
tubulin is very low, two possibilities exist to explain the higher rate
of GTP hydrolysis. One possibility is that Gi
hydrolyzes
the E-site-bound GTP after transfer to the Gi
. The
second possibility is that Gi
activates the GTPase of
tubulin by inducing a conformational change in the tubulin, similar to
the way in which tubulin dimers activate neighboring GTPase activity
during microtubule polymerization.
with a single amino acid substitution,
Gln204
Lys (Q204LGi1
), with
incapacitated GTPase activity. However, the ability of the mutated
Q204LGi1
to bind GTP is unaltered (42). The mutated
Q204LGi1
, or wild-type Gi1
, was incubated with tubulin-[
-32P]GTP under conditions in which
tubulin does not polymerize, and the extent of GTP hydrolysis was
determined by thin layer chromatography. As shown in Fig.
1, the tubulin-bound
[
-32P]GTP was poorly hydrolyzed in the absence of
Gi1
(10.1 ± 1.9%, n = 10). In the
presence of Gi1
, 71.8 ± 3.4% (n = 10) of the E-site-bound GTP was hydrolyzed. This hydrolysis could be a
combination of that occurring in the tubulin E-site and in
Gi1
. When Q204LGi1
was added to the
tubulin, 49 ± 3% (n = 3) of the bound GTP was hydrolyzed. Because Q204LGi1
cannot hydrolyze GTP, the
tubulin must have been responsible for the GTP hydrolysis. Because in the presence of Gi1
, 71.8% of the E-site GTP was
hydrolyzed, approximately 23% of the GTP must have been hydrolyzed by
Gi1
. Myristoylated Gi1
was also tested
for its ability to activate tubulin GTPase. The amino terminus of
Gi1
is myristoylated in vivo, a modification
that is important for association of Gi1
with membranes
and G
(43). We found that 80.4 ± 2.3% (n = 7) of the tubulin-bound [
-32P]GTP was hydrolyzed by
myristoylated Gi1
. The slightly increased potency of
myristoylated Gi1
to activate tubulin GTPase as compared with Gi1
may suggest an enhanced ability of
myristoylated Gi1
to bind to tubulin. The results
indicate that Gi1
may act as a GTPase activating protein
for tubulin.
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Fig. 1.
Activation of tubulin-GTPase by
Gi1 .
Tubulin-[
-32P]GTP (1.25 µM) in PEM
buffer, made by incubating phosphatidylcholine tubulin (Tub)
with 0.1 mM [
-32P]GTP followed by
desalting (as described under "Experimental Procedures") was
incubated with Gi1
, myristoylated (Myr)
Gi1
, or a GTPase-deficient mutant of Gi1
(Gi1
Q204L) (2.5 µM) at 30 °C for 30 min. The samples were then treated with 1% SDS and subjected to thin
layer chromatography on polyethyleneimine cellulose plates. One of
three similar experiments is shown.
Inhibits Microtubule Assembly in a
GTP-dependent Manner--
Gi has been shown
previously to inhibit microtubule polymerization (44). This inhibition
might occur by binding of the Gi to tubulin and
sequestering it, making the tubulin unavailable for polymerization.
Tubulin with GTP in the E-site (1.5 mg/ml) polymerizes into
microtubules in the absence of exogenous GTP as shown in Table
I. Assembly of the tubulin-GTP in the
presence of 0.75 mg/ml of Gi1
resulted in ~85%
inhibition of assembly, and exogenous GTP overcame the ability of
Gi1
to inhibit assembly in a GTP
concentration-dependent manner. To determine whether inhibition of microtubule assembly by Gi1
was the result
of hydrolysis of the E-site GTP by Gi1
, we prepared
tubulin with GppNHp (a hydrolysis-resistant GTP analog) in the E-site.
As also shown in Table I, in the absence of exogenous nucleotide,
Gi1
reduced the extent of microtubule polymerization by
approximately 85%, and exogenous GppNHp did not restore microtubule
polymerization. Thus, it appears that GTP hydrolysis resulting from the
association of tubulin and Gi1
plays a critical role in
modulating microtubule assembly. When the microtubule pellet was
analyzed by SDS-gel electrophoresis, Coomassie Blue staining did not
reveal incorporation of Gi1
in microtubules. However,
some incorporation of G
into the microtubule fraction was observed
by Western blotting using a Gi1
antibody (data not
shown).
Comparison of the effect of Gi1 on microtubule assembly
induced by GTP or GppNHp
(as described in the Fig. 2 legend) followed by
polymerization in the presence of GTP or GppNHp as indicated. Assembly
was quantified by centrifuging the polymer at 150,000 × g and represented as % of control (assembly in the absence
of Gi1
was considered 100%). Values represent mean ± S.E. of at least three experiments.
-Tubulin
Complex--
Addition of exogenous GTP to the
tubulin-Gi1
complex with either GTP or GppNHp in the
E-site reversed the ability of Gi1
to inhibit
polymerization (Table II). Furthermore,
addition of exogenous GppNHp to the tubulin-Gi1
complex
with GTP in the E-site, inhibited microtubule polymerization. These
results indicate that exogenous GTP and GppNHp can exchange with either
GppNHp or GTP in the tubulin E-site when complexed with
Gi1
. The GTPase-deficient Gi1
variant,
Q204LGi1
, also inhibited microtubule polymerization in a
manner similar to Gi1
(by 74.5 ± 9.5%),
suggesting that GTP hydrolysis in G
does not cause the inhibition of
microtubule assembly.
Effect of GTP or GppNHp on Gi1-mediated inhibition of
microtubule assembly
, whereas
GppNHp was added to Gi1
-preincubated tubulin-GTP samples.
Samples were subjected to polymerization at 37 °C and quantified as
in Table I. Values represent mean ± S.E. of two experiments.
Have
Typical Morphology--
Electron microscopic analysis of the polymers
formed in the presence of Gi1
and excess GTP or GppNHp
indicated that they were normal microtubules. Gi1
blocked the formation of microtubules regardless of the nucleotide
bound to the tubulin (Fig. 2,
B and E). The addition of 50 µM GTP
reversed the Gi1
-mediated inhibition of microtubule
assembly, and microtubules were formed (Fig. 2C), whereas
the addition of 50 µM GppNHp did not (Fig.
2F).
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Fig. 2.
Electron microscopy of microtubules formed in
the absence of G (A and
D) or in the presence of G
(B, C, E, and F).
A-C, assembly carried out in the presence of GTP.
D-F, assembly carried out in the presence of
GppNHp. Note that in D, some microtubule bundling
occurred. Tub, tubulin.
Subunits for Inhibition of Microtubule
Assembly and GTPase Activity--
Gs
binds to tubulin
with an affinity similar to that of Gi1
(28). Thus, it
was predicted that Gs
would also inhibit microtubule assembly. In the presence of Gs
, microtubule assembly
was reduced to 22% (21.8 ± 10.5%) of the control (Fig.
3A). Although
Go
does not bind to tubulin with an affinity as high as
that of Gi1
or Gs
(28), Go
inhibited microtubule polymerization similarly to Gi1
and Gs
(by 85%). These results are consistent with the possibility that there is a preferential interaction of Go
with oligomeric tubulin or microtubules as compared with dimeric
tubulin (44). The retinal G protein transducin (Gt
),
which does not bind to tubulin or microtubules, did not inhibit
microtubule assembly (Fig. 3A). Furthermore, the GTPase
activity of tubulin was activated by Gs
(73.8 ± 3.8%) and Go
(93 ± 2.7%) but not by
Gt
(28.5 ± 2.5%) (Fig. 3B). The
activation of tubulin GTPase by Gi1
was maximal at a
G
:tubulin ratio of 1:1.
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Fig. 3.
Gs and
Go
activate tubulin GTPase and
inhibit microtubule assembly. A, tubulin
(Tub)-GTP (1.25 mg/ml) was incubated with Gs
,
Go
, or Gt
(0.5 mg/ml) at 30 °C for 30 min. Samples were then polymerized as described under "Experimental
Procedures." Microtubule pellets were resuspended in PEM buffer and
pellets, and supernatants were analyzed for protein content.
B, tubulin-[
-32P]GTP (2 µM)
was incubated with Gs
, Go
, or
Gt
(3 µM) at 30 °C for 30 min. Samples
were then treated with 1% SDS and subjected to polyethyleneimine
cellulose thin layer chromatography as described under "Experimental
Procedures." The autoradiogram of the plate is shown. One of three
similar experiments is shown.
Increases Microtubule Dynamic Instability by
Increasing the Catastrophe Frequency--
In an effort to determine
how G
modulates microtubule polymerization dynamics, we measured the
dynamics of individual microtubules at steady state in
vitro, in the presence or absence of Gi1
, by video
microscopy. Microtubules can alternate between phases of growing and
shortening and also spend a small fraction of time in an attenuated
(paused) state, neither growing nor shortening detectably, a behavior
called dynamic instability (10). The transition frequencies among the
growing, shortening, and attenuated states are thought to be important
in the regulation of microtubule dynamics in cells (13, 14, 45). Life
history traces of several microtubules in the absence (panel
A) or presence (panel B) of Gi1
are
shown in Fig. 4. Addition of
Gi1
(4 µM) visually increased the
catastrophe frequency. The dynamic instability parameters were
determined quantitatively from such life history plots. As shown in
Table III, Gi1
did not
alter the rates of microtubule growth or shortening. However, 4 µM Gi1
significantly reduced the average
length that microtubules grew per individual growth event (1.5 ± 0.2-0.9 ± 0.2 µm). Gi1
also reduced the
percentage of total time that microtubules spent in the growing phase
and increased the percentage of total time they spent in the shortening phase.
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Fig. 4.
Effect of Gi1
on microtubule dynamic instability at plus ends at
steady state. Life history traces of length changes at the plus
ends of individual microtubules with time are shown in the absence
(A) or presence (B) of 4 µM
Gi1
.
Effects of Gi1 on the dynamics of individual microtubules
concentration.
Tubulin is 12 µM throughout. All values are ±S.E.
significantly increased the catastrophe frequency
(by 2.6-fold in the presence of 4 µM Gi1
).
The catastrophe frequency per micrometer of length grown was determined
by dividing the total number of catastrophic events by total length
increase during growing events. Gi1
also increased the
catastrophe frequency per micrometer of length grown.
Gi1
had no effects on the rescue frequency (transition
from shortening to the growing or attenuated state) per unit of time or
per unit of length shortened (Table III). Dynamicity is a parameter
that reflects the overall dynamics of the microtubules (the total
detectable tubulin dimer addition and loss at a microtubule end
including the time spent in the attenuated state) (39).
Gi1
(4 µM) increased the dynamicity by
44%. Thus, Gi1
increases the dynamic behavior of the
microtubules primarily by increasing the catastrophe frequency.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits of G proteins
(Gi1, Gs, and Go) were shown to
activate the GTPase activity of tubulin, indicating that G
may serve
as a GTPase activating protein for tubulin. In addition, G
inhibited
microtubule assembly and increased microtubule dynamic instability
in vitro. The assembly of tubulin into microtubules was
blocked by G
(80-90%), regardless of whether GTP or GppNHp was
bound in the tubulin E-site. In addition, the addition of exogenous
GTP, but not the addition of the hydrolysis-resistant GppNHp, overcame
the inhibition of microtubule polymerization by G
. A model for how
G
might interact with tubulin and how exogenous GTP might overcome
the interaction is presented in Fig. 5A. In this model, G
is
suggested to bind to tubulin and activate the intrinsic GTPase of
tubulin in a manner similar to that in which GTP hydrolysis occurs in
tubulin during formation of a microtubule. However, unlike the
formation of microtubules from tubulin dimers, G
dissociates from
the tubulin-G
complex subsequent to GTP hydrolysis.
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Fig. 5.
Model for the effects of G
on microtubule assembly and dynamics. A, a scheme for
tubulin-G
interaction for the regulation of microtubule assembly.
The binding of G
to tubulin-GTP inhibits microtubule polymerization
and promotes GTP hydrolysis, suggesting that the binding of G
to
tubulin induces a conformation in tubulin similar to that occurring
during microtubule formation. G
dissociates from the tubulin-G
complex after GTP hydrolysis. Addition of exogenous GTP, but not
GppNHp, restores microtubule polymerization, indicating that the
formation of the tubulin-G
complex is required for the inhibition of
microtubule polymerization. B, possible mechanism for the
regulation of microtubule dynamics in vivo by G
. The
binding of G
to the end of a microtubule induces hydrolysis of GTP
and subsequent loss of the stabilizing cap, resulting in the transition
to microtubule depolymerization (a catastrophe). GTP hydrolysis
destabilizes the tubulin-G
complex, and G
dissociates from
tubulin and is now ready for another cycle of interaction with the
microtubule ends.
Gi1 altered microtubule dynamics by increasing the
catastrophe frequency (the frequency of switching from growing to
shortening; see Table III). Microtubules are composed of an unstable
tubulin-GDP core and a stable tubulin-GTP or tubulin-GDP-Pi
cap at the microtubule ends (46, 47). Microtubules grow for as long as
they maintain a GTP cap, but loss of the cap exposes the labile
tubulin-GDP core, and the microtubules rapidly shorten. These data are
consistent with the possibility that Gi1
activates
tubulin GTPase at the microtubule ends, thus resulting in loss of the
GTP cap (Fig. 5B). Alternatively, Gi1
might
increase the catastrophe frequency by reducing the effective tubulin
concentration, thereby binding to and sequestering soluble tubulin.
However, this sequence appears unlikely because Gi1
did
not reduce the individual microtubule growth rate (Table III).
It is suggested that G is released from microtubules after binding
and subsequent hydrolysis of the E-site GTP. The released G
could be
recycled for further interaction with newly growing microtubules,
reducing the G
concentration required to exert this effect. In fact,
4 µM Gi1
, a concentration 3-fold lower than the tubulin concentration (12 µM), increased the
catastrophe frequency 2.6-fold (Table III).
Although G proteins are usually confined to the plasma membrane,
translocation of activated G from the membrane to the cytosol has
been observed (48-51). Furthermore, whereas G proteins are normally
associated with second messenger-generating enzymes, or ion channels,
results from several laboratories suggest that G proteins may be
involved in cell growth and differentiation, perhaps through their
association with cytoskeletal components (21-26). For example, an
association of G
and G
with the microtubule cytoskeleton has
been reported (21, 24-26). Furthermore, an association of
Go
and -
(or -
) with spindle microtubules
suggests that G protein subunits may play some role in regulating the
assembly and disassembly of the mitotic spindle (23, 24). The
-adrenergic receptor kinase (known as
ARK or GRK2), which
mediates agonist-dependent phosphorylation and
desensitization of G protein coupled receptors, has been shown to
associate with microtubules and to phosphorylate tubulin in an
agonist-dependent manner (19, 20). Taken together, these
data suggest a link between microtubules and G protein-mediated signaling that may regulate cell division and differentiation.
G proteins, particularly Go and G
, are abundant at
the growth cone membrane of neurons (52). Growth cones at the growing tips of developing neurites are highly specialized organelles that
respond to a variety of extracellular signals to achieve neuronal
guidance and target recognition. Coordinated assembly of microtubules
in concert with actin filaments and neurofilaments is required for
growth cone motility and neurite outgrowth (53, 54). Activation of a G
protein coupled receptor has been shown to collapse the growth cone
cytoskeleton (55). Because some G
appears to be released from the
membrane subsequent to hormone or neurotransmitter activation (48-51),
it is possible that these proteins participate in localized regulation
of the cytoskeleton. Thus, microtubule dynamics at growth cones could
be mediated by Go
and Gi
. Based on
observations in this report as well as the emerging results from
various laboratories, it is reasonable to postulate that extracellular
signals orchestrate G proteins (both G
and G
) and mobilize
them to bind to microtubules. Such a process is likely to provide a
venue by which extracellular signals modify cell form and growth.
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ACKNOWLEDGEMENTS |
---|
We thank Madhavi Talluri, Amy Moss, and Dmitriy Shchepin for excellent technical assistance and Jaclyn Holda for critically reading the manuscript.
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FOOTNOTES |
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* This work was supported by Public Health Service Grants MH 39595 and AG 15482 (to M. M. R.) and NS13560 (to L. W.).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.
§ Current address: Dept. of Biological Sciences, University of Texas, El Paso, TX 79968; E-mail: sukla{at}utep.edu.
** To whom correspondence should be addressed: Dept. of Physiology & Biophysics, m/c 901, University of Illinois College of Medicine, 835 S. Wolcott Ave., Chicago, IL 60612-7342. Tel.: 312-996-6641; Fax: 312-996-1414; E-mail: raz{at}uic.edu.
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ABBREVIATIONS |
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The abbreviations used are: E-site, exchangeable site; GppNHp, guanylylimidodiphosphate; PIPES, 1,4-piperazinediethanesulfonic acid.
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