G Protein alpha  Subunits Activate Tubulin GTPase and Modulate Microtubule Polymerization Dynamics*

Sukla RoychowdhuryDagger §, Dulal Panda, Leslie Wilson, and Mark M. RasenickDagger parallel **

From the Departments of Dagger  Physiology and Biophysics and parallel  Psychiatry, University of Illinois, Chicago, Illinois 60612 and the  Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, California 93106

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha  subunits Gi1alpha , Gsalpha , and Goalpha are shown to activate the GTPase activity of tubulin, inhibit microtubule assembly, and accelerate microtubule dynamics. Gialpha 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 Gi1alpha increases the catastrophe frequency, the frequency of transition from growth to shortening. Thus, Galpha 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

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 beta -tubulin) and the other nonexchangeable (in alpha -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).

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 alpha  and beta gamma subunits. Galpha subunits bind GTP and display various levels of intrinsic GTPase activity. Certain G protein alpha  subunits (Gi1alpha , Gsalpha , and Gqalpha ) 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 Galpha (transactivation) (29, 31). In addition to activating Galpha , the association between Galpha 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 Gbeta 1gamma 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 alpha  subunits was investigated. We report here that alpha  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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 [alpha -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 [alpha -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).

G Protein Purification-- Recombinant Gi1alpha , Gsalpha , or Goalpha were produced in Escherichia coli using constructs provided by Dr. Maurine Linder (Washington University, St. Louis, MO). The vector used contained Gi1alpha , Gsalpha , or Goalpha 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 Gi1alpha , 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 Gi1alpha (a gift from Dr. M. Linder) were used to express myristoylated Gi1alpha , which was then purified as described earlier (37).

GTP Hydrolysis-- Tubulin was allowed to bind [alpha -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 Galpha 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.

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 Galpha 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 alpha  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 Galpha concentrations were low, a buffer control was performed to avoid a reduction in protein concentration by gel filtration.

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 Gi1alpha 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

Gi1alpha Activates the Intrinsic GTPase of Tubulin-- Tubulin binds to Gi1alpha and Gsalpha with a Kd of approximately 130 nM coupled with a transactivation of Galpha in which 25-50% of E-site tubulin-bound GTP is transferred directly to the Galpha (28, 29). Gi1alpha binding to tubulin in vitro also activates GTP hydrolysis (32). Both tubulin and Gi1alpha 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 Gialpha hydrolyzes the E-site-bound GTP after transfer to the Gialpha . The second possibility is that Gialpha 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.

To distinguish between these possibilities, we used a mutated form of Gi1alpha with a single amino acid substitution, Gln204 right-arrow Lys (Q204LGi1alpha ), with incapacitated GTPase activity. However, the ability of the mutated Q204LGi1alpha to bind GTP is unaltered (42). The mutated Q204LGi1alpha , or wild-type Gi1alpha , was incubated with tubulin-[alpha -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 [alpha -32P]GTP was poorly hydrolyzed in the absence of Gi1alpha (10.1 ± 1.9%, n = 10). In the presence of Gi1alpha , 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 Gi1alpha . When Q204LGi1alpha was added to the tubulin, 49 ± 3% (n = 3) of the bound GTP was hydrolyzed. Because Q204LGi1alpha cannot hydrolyze GTP, the tubulin must have been responsible for the GTP hydrolysis. Because in the presence of Gi1alpha , 71.8% of the E-site GTP was hydrolyzed, approximately 23% of the GTP must have been hydrolyzed by Gi1alpha . Myristoylated Gi1alpha was also tested for its ability to activate tubulin GTPase. The amino terminus of Gi1alpha is myristoylated in vivo, a modification that is important for association of Gi1alpha with membranes and Gbeta gamma (43). We found that 80.4 ± 2.3% (n = 7) of the tubulin-bound [alpha -32P]GTP was hydrolyzed by myristoylated Gi1alpha . The slightly increased potency of myristoylated Gi1alpha to activate tubulin GTPase as compared with Gi1alpha may suggest an enhanced ability of myristoylated Gi1alpha to bind to tubulin. The results indicate that Gi1alpha may act as a GTPase activating protein for tubulin.


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Fig. 1.   Activation of tubulin-GTPase by Gi1alpha . Tubulin-[alpha -32P]GTP (1.25 µM) in PEM buffer, made by incubating phosphatidylcholine tubulin (Tub) with 0.1 mM [alpha -32P]GTP followed by desalting (as described under "Experimental Procedures") was incubated with Gi1alpha , myristoylated (Myr) Gi1alpha , or a GTPase-deficient mutant of Gi1alpha (Gi1alpha 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.

Gi1alpha 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 Gi1alpha resulted in ~85% inhibition of assembly, and exogenous GTP overcame the ability of Gi1alpha to inhibit assembly in a GTP concentration-dependent manner. To determine whether inhibition of microtubule assembly by Gi1alpha was the result of hydrolysis of the E-site GTP by Gi1alpha , 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, Gi1alpha 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 Gi1alpha 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 Gi1alpha in microtubules. However, some incorporation of Galpha into the microtubule fraction was observed by Western blotting using a Gi1alpha antibody (data not shown).

                              
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Table I
Comparison of the effect of Gi1alpha on microtubule assembly induced by GTP or GppNHp
Tubulin-GTP or tubulin-GppNHp was preincubated with or without Gi1alpha (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 Gi1alpha was considered 100%). Values represent mean ± S.E. of at least three experiments.

Tubulin Exchanges Nucleotide in the Galpha -Tubulin Complex-- Addition of exogenous GTP to the tubulin-Gi1alpha complex with either GTP or GppNHp in the E-site reversed the ability of Gi1alpha to inhibit polymerization (Table II). Furthermore, addition of exogenous GppNHp to the tubulin-Gi1alpha 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 Gi1alpha . The GTPase-deficient Gi1alpha variant, Q204LGi1alpha , also inhibited microtubule polymerization in a manner similar to Gi1alpha (by 74.5 ± 9.5%), suggesting that GTP hydrolysis in Galpha does not cause the inhibition of microtubule assembly.

                              
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Table II
Effect of GTP or GppNHp on Gi1alpha -mediated inhibition of microtubule assembly
Experimental protocol was similar to that as described in Table I except that GTP (at indicated concentrations) was added to the samples in which tubulin-GppNHp was preincubated with Gi1alpha , whereas GppNHp was added to Gi1alpha -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.

Microtubules Polymerized in the Presence of Gi1alpha Have Typical Morphology-- Electron microscopic analysis of the polymers formed in the presence of Gi1alpha and excess GTP or GppNHp indicated that they were normal microtubules. Gi1alpha 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 Gi1alpha -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 Galpha (A and D) or in the presence of Galpha (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.

Specificity of G Protein alpha  Subunits for Inhibition of Microtubule Assembly and GTPase Activity-- Gsalpha binds to tubulin with an affinity similar to that of Gi1alpha (28). Thus, it was predicted that Gsalpha would also inhibit microtubule assembly. In the presence of Gsalpha , microtubule assembly was reduced to 22% (21.8 ± 10.5%) of the control (Fig. 3A). Although Goalpha does not bind to tubulin with an affinity as high as that of Gi1alpha or Gsalpha (28), Goalpha inhibited microtubule polymerization similarly to Gi1alpha and Gsalpha (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 (Gtalpha ), which does not bind to tubulin or microtubules, did not inhibit microtubule assembly (Fig. 3A). Furthermore, the GTPase activity of tubulin was activated by Gsalpha (73.8 ± 3.8%) and Goalpha (93 ± 2.7%) but not by Gtalpha (28.5 ± 2.5%) (Fig. 3B). The activation of tubulin GTPase by Gi1alpha was maximal at a Galpha :tubulin ratio of 1:1.


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Fig. 3.   Gsalpha and Goalpha activate tubulin GTPase and inhibit microtubule assembly. A, tubulin (Tub)-GTP (1.25 mg/ml) was incubated with Gsalpha , Goalpha , or Gtalpha (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-[alpha -32P]GTP (2 µM) was incubated with Gsalpha , Goalpha , or Gtalpha (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.

Gi1alpha Increases Microtubule Dynamic Instability by Increasing the Catastrophe Frequency-- In an effort to determine how Galpha modulates microtubule polymerization dynamics, we measured the dynamics of individual microtubules at steady state in vitro, in the presence or absence of Gi1alpha , 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 Gi1alpha are shown in Fig. 4. Addition of Gi1alpha (4 µM) visually increased the catastrophe frequency. The dynamic instability parameters were determined quantitatively from such life history plots. As shown in Table III, Gi1alpha did not alter the rates of microtubule growth or shortening. However, 4 µM Gi1alpha significantly reduced the average length that microtubules grew per individual growth event (1.5 ± 0.2-0.9 ± 0.2 µm). Gi1alpha 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 Gi1alpha 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 Gi1alpha .

                              
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Table III
Effects of Gi1alpha on the dynamics of individual microtubules
Dynamic instability parameters were determined from life history plots of individual microtubules. The reported mean growing and shortening rates represent the mean values for all growing and shortening events observed for 22-25 microtubules at each Gi1alpha concentration. Tubulin is 12 µM throughout. All values are ±S.E.

Gi1alpha significantly increased the catastrophe frequency (by 2.6-fold in the presence of 4 µM Gi1alpha ). 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. Gi1alpha also increased the catastrophe frequency per micrometer of length grown. Gi1alpha 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). Gi1alpha (4 µM) increased the dynamicity by 44%. Thus, Gi1alpha increases the dynamic behavior of the microtubules primarily by increasing the catastrophe frequency.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the present study, the alpha  subunits of G proteins (Gi1, Gs, and Go) were shown to activate the GTPase activity of tubulin, indicating that Galpha may serve as a GTPase activating protein for tubulin. In addition, Galpha inhibited microtubule assembly and increased microtubule dynamic instability in vitro. The assembly of tubulin into microtubules was blocked by Galpha (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 Galpha . A model for how Galpha might interact with tubulin and how exogenous GTP might overcome the interaction is presented in Fig. 5A. In this model, Galpha 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, Galpha dissociates from the tubulin-Galpha complex subsequent to GTP hydrolysis.


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Fig. 5.   Model for the effects of Galpha on microtubule assembly and dynamics. A, a scheme for tubulin-Galpha interaction for the regulation of microtubule assembly. The binding of Galpha to tubulin-GTP inhibits microtubule polymerization and promotes GTP hydrolysis, suggesting that the binding of Galpha to tubulin induces a conformation in tubulin similar to that occurring during microtubule formation. Galpha dissociates from the tubulin-Galpha complex after GTP hydrolysis. Addition of exogenous GTP, but not GppNHp, restores microtubule polymerization, indicating that the formation of the tubulin-Galpha complex is required for the inhibition of microtubule polymerization. B, possible mechanism for the regulation of microtubule dynamics in vivo by Galpha . The binding of Galpha 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-Galpha complex, and Galpha dissociates from tubulin and is now ready for another cycle of interaction with the microtubule ends.

Gi1alpha 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 Gi1alpha activates tubulin GTPase at the microtubule ends, thus resulting in loss of the GTP cap (Fig. 5B). Alternatively, Gi1alpha might increase the catastrophe frequency by reducing the effective tubulin concentration, thereby binding to and sequestering soluble tubulin. However, this sequence appears unlikely because Gi1alpha did not reduce the individual microtubule growth rate (Table III).

It is suggested that Galpha is released from microtubules after binding and subsequent hydrolysis of the E-site GTP. The released Galpha could be recycled for further interaction with newly growing microtubules, reducing the Galpha concentration required to exert this effect. In fact, 4 µM Gi1alpha , 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 Galpha 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 Galpha and Gbeta gamma with the microtubule cytoskeleton has been reported (21, 24-26). Furthermore, an association of Goalpha and -beta (or -beta gamma ) 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 beta -adrenergic receptor kinase (known as beta 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 Goalpha and Gbeta , 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 Galpha 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 Goalpha and Gialpha . 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 Galpha and Gbeta gamma ) 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.

    ACKNOWLEDGEMENTS

We thank Madhavi Talluri, Amy Moss, and Dmitriy Shchepin for excellent technical assistance and Jaclyn Holda for critically reading the manuscript.

    FOOTNOTES

* 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.

    ABBREVIATIONS

The abbreviations used are: E-site, exchangeable site; GppNHp, guanylylimidodiphosphate; PIPES, 1,4-piperazinediethanesulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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