Cytosolic Gα_s Acts as an Intracellular Messenger to Increase Microtubule Dynamics and Promote Neurite Outgrowth^,

Abstract

It is now evident that Gα_s traffics into cytosol following G protein-coupled receptor activation, and α subunits of some heterotrimeric G-proteins, including Gα_s bind to tubulin in vitro. Nevertheless, many features of G-protein-microtubule interaction and possible intracellular effects of G protein α subunits remain unclear. In this study, several biochemical approaches demonstrated that activated Gα_s directly bound to tubulin and cellular microtubules, and fluorescence microscopy showed that cholera toxin-activated Gα_s colocalized with microtubules. The activated, GTP-bound, Gα_s mimicked tubulin in serving as a GTPase activator for β-tubulin. As a result, activated Gα_s made microtubules more dynamic, both in vitro and in cells, decreasing the pool of insoluble microtubules without changing total cellular tubulin content. The amount of acetylated tubulin (an indicator of microtubule stability) was reduced in the presence of Gα_s activated by mutation. Previous studies showed that cholera toxin and cAMP analogs may stimulate neurite outgrowth in PC12 cells. However, in this study, overexpression of a constitutively activated Gα_s or activation of Gα_s with cholera toxin in protein kinase A-deficient PC12 cells promoted neurite outgrowth in a cAMP-independent manner. Thus, it is suggested that activated Gα_s acts as an intracellular messenger to regulate directly microtubule dynamics and promote neurite outgrowth. These data serve to link G-protein signaling with modulation of the cytoskeleton and cell morphology.

Heterotrimeric G proteins, activated upon agonist binding to G protein-coupled receptors, play a vital role in propagating extracellular signals across the plasma membrane. Gα and βγ subunits undergo a functional dissociation upon activation, allowing them to regulate downstream effectors, such as adenylyl cyclase and ion channels. Signaling is terminated when the intrinsic GTPase of Gα subunits hydrolyzes GTP into GDP. Although most heterotrimeric G proteins are localized on the plasma membrane, numerous studies have suggested intracellular functions either in the cytosol or in conjunction with cytosolic organelles (1–6, 13, 14). Recently, a number of biochemical studies observed intracellular translocation of Gα_s proteins subsequent to activation by β-adrenergic agonists, cholera toxin, or direct binding of a hydrolysis-resistant GTP analog (7–9). More recently, taking advantage of an internal sequence Gα_s-GFP fusion protein, the translocation of Gα_s into the cytoplasm was directly observed in living cells upon stimulation with agonists (10, 11). This translocation of Gα_s from membrane to cytoplasm triggered by agonist appears to occur through lipid rafts on the plasma membrane (12).

The fate of internalized Gα_s is not well characterized. Two current studies have suggested that activated, internalized Gα_s could invoke developmental paradigms. In mouse oocytes, meiotic prophase was maintained (arrested) due to the prolonged, receptor-mediated activation of Gα_s, which assumed a cytosolic localization subsequent to internalization (13). Activated Gα_s was also invoked to explain prostaglandin E₂-mediated stimulation of colon cancer cell growth. It appeared that Gα_s associated with axin and allowed β-catenin to activate the proliferative state (14). Results from these studies suggest that Gα_s subunits may undertake some intracellular functions beyond the “traditional” pathway of G protein signaling.

Microtubules, a major component of the cytoskeleton, participate in many cellular activities, including chromosome movements during mitosis, intracellular transport, and the modulation of cell morphology. A heterodimer of α- and β-tubulin is the basic building block of microtubules, and both α- and β-tubulin bind GTP; this GTP is hydrolyzed to GDP in β-tubulin subunits by an intrinsic GTPase, which is activated by the association of a second microtubule subunit in the growing microtubule (15). GTP hydrolysis allows microtubules to depolymerize by weakening the bonds between tubulin subunits to decrease microtubule stability (16, 17). These cellular biologic functions of microtubules are dependent, in significant part, on the regulation of microtubule dynamics and stability. In non-mitotic cells, at least two populations of microtubules have been distinguished: short lived or dynamic microtubules (t_½ = 5–10 min) and long lived or stable microtubules (t_½ > 1 h) (18, 19). In many cell types, stable microtubules accumulate detyrosinated tubulin and acetylated tubulin due to post-translational modification. In contrast, dynamic microtubules contain predominantly tyrosinated tubulin (20). In cells, some microtubule-binding proteins, such as Tau protein or SCG40, can also modulate microtubule stability through direct interaction with microtubules. The interaction between Gα subunits and tubulin has been studied for more than 2 decades (21), and studies with purified proteins implicate Gα subunits as potential regulators (22, 23).

This study inquired whether activated Gα_s, released from the plasma membrane, regulates microtubule stability via direct interaction with microtubules. Gα_s binds to tubulin and acts as a GTPase-activating protein for that molecule. The resulting loss of the GTP cap confers an increase in dynamic instability of microtubules. One result of this is to potentiate neurite outgrowth in PC12 cells. This report shows that Gα_s serves as an intracellular messenger to regulate microtubule dynamics and does so in a cAMP-independent fashion. Thus, we demonstrate a direct link between heterotrimeric G protein signaling and modulation of the cytoskeleton.

EXPERIMENTAL PROCEDURES

Materials—Wild-type PC12 cells, wild-type rat Gα_s with hemagglutinin epitope (HA),3 tag and constitutively activated Gα_s^Q227L with HA tag were obtained from the American Type Culture Collection. Gα ^Q213L_s and the protein kinase A (PKA)-deficient PC12 cells (123.7) were generous gifts from John A. Wagner (Cornell University Medical College) (24) and Tarun Patel (Loyola University, Chicago, IL), respectively. Construction of Gα_s-GFP was described in a previous report (10). Monoclonal anti-α-tubulin and anti-HA antibodies were purchased from ICN Biomedicals (Costa Mesa, CA) and Berkeley Antibody Co. (Richmond, CA). Polyclonal antibody against detyrosinated tubulin was from Chemicon International Inc. (Temecula, CA). Monoclonal antibodies against acetylated and tyrosinated tubulin were from Sigma. Polyclonal antibody against Gα_s was purchased from PerkinElmer Life Sciences. All other biochemicals used were of the highest purity available.

Cell Culture and Transfection—PC12 cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum, 5% horse serum, and 1% antibiotic (penicillin and streptomycin). Cells were maintained in a 5% CO₂ incubator at 37 °C. Medium was changed every 3 days, and cells were passaged once per week.

PC12 cells in 12-well culture plates were transfected with Gα_s-GFP using GenePORTER™ transfection reagent (Gene Therapy Systems, Inc., San Diego, CA) according to the manufacturer's instructions.

Adenoviral Gene Transfer—Construction of recombinant adenoviruses Ad/Gα_s or Ad/Gα_s^Q227L was as follows. Rat Gα_s and Gα_s^Q227L cDNAs were cloned into PcDNA3 vector at the SalI site, and the entire cassette was excised and cloned into pADtrack-CMV shuttle vector (Quantum Biotechnologies, Inc.). The linearized shuttle vector and AdEasy vector (Quantum Biotechnologies) were then co-transformed into Escherichia coli strain BJ5183. Positive recombinant plasmid Ad/Gα_s or Gα_s^Q227L was selected, respectively. The virus was purified with CsCl banding and stored at –70 °C. Ten μl with 1 × 10⁵ or 4 × 10⁵ virus particles of Ad/Gα_s or Gα_s^Q227L was applied to each well of culture cells in a 12-well plate or to each 25-ml culture flask for infection. Greater than 90% of cells were infected in any given experiments.

Immunoprecipitation and Western Blot—PC12 cells infected with Ad/GFP, Ad/Gα_s, or Ad/Gα_s^Q227L were cultured for 40 h and then washed twice in PBS. Cells were lysed in 500 μl of lysis buffer (PBS, 0.5% Triton X-100, 5 mm EDTA, protease inhibitors) on ice for 30 min. The lysate was collected and cleared by centrifuging at 12,000 × g for 20 min at 4 °C. Protein concentration of supernatants was determined by the method of Bradford (Bio-Rad). After adjusting protein concentration to equal amounts for each sample, the supernatant (450 μl) was transferred to 1.5-ml microcentrifuge tubes and incubated with agarose beads coated with anti-mouse IgG for 1 h at 4 °C with continuous gentle inversion. The agarose beads were pulled down by centrifuging at room temperature and discarded. The lysate was then incubated with 5 μl of monoclonal antibody against HA for 20 h at 4 °C, and then the antibody/lysate mixture was incubated with agarose beads coated with anti-mouse IgG for 2 h at 4 °C with continuous gentle inversion. After the agarose beads were washed with lysis buffer three times, the 50 μl of SDS-PAGE sample buffer was added to the agarose beads. Fifteen μl of supernatant was applied onto 5–12% gradient SDS-PAGE, and the resolved proteins were analyzed on a Western blot using the polyclonal antibody against α-tubulin. The film was stripped with stripper buffer (100 mm β-mercaptoethanol, 62.5 mm Tris-HCl, 2% SDS, pH 6.7) and then detected with antibody against Gα_s to show sample loading. The Western blot was done as described previously (10). Tubulin bands in immunoblotting were quantified, and the integrated optical density of each band was measured and was expressed as a percentage of control.

Immunocytochemistry—PC12 cells grown on coverslips in 12-well plates were washed twice with PBS and fixed with cold 100% methanol (–20 °C) for 4 min after extraction with 0.2% (w/v) saponin in microtubule-stabilizing buffer (80 mm PIPES/KOH, pH 6.8, 1 mm MgCl₂, 1 mm EGTA, 30% (v/v) glycerol, 1 mm GTP). The coverslips were then incubated with PBSS buffer (PBS plus 0.01% saponin) containing 10% bovine serum albumin for 20 min, and then incubated in a 1:1000 dilution of anti-α-tubulin in PBSS buffer for 3 h. Subsequently, the coverslips were washed with PBSS four times and incubated with a 1:180 dilution of secondary antibodies labeled with TRITC in PBSS buffer for 40 min. These coverslips were washed with PBSS buffer four times and mounted on the slide with mounting medium. The slides were air-dried and examined by deconvolution microscopy.

Microscopy—Cells were observed using a Nikon diaphot digital fluorescence microscope equipped with a 100-watt mercury arc lamp. Images were acquired with an interline charge-coupled device camera (1300 YHS; Roper Scientific, Trenton, NJ) driven by IP Lab imaging software (Scanalytics, Inc., Suitland, VA) and processed with IP Lab and Adobe Photoshop 5.0 (Adobe Systems, Mountain View, CA). For deconvolution microscopy, images were captured with the Applied Precision, Inc. (Seattle) DeltaVision system built on an Olympus IX-70 base. Z-stacks were deconvolved using the Softworx software. Sections were captured every 200 nm. Typically, 15 iterations based on a measured point spread function, calculated from 1 μm fluorescent beads, were used. PC12 cells transfected with Gα_s-GFP were fixed, stained for tubulin, and shown as a volume projection of a z-series. Images were processed using Adobe Photoshop 5.0.

Quantification of the internalization of Gα_s-GFP was done as described previously (10). The mean gray value within the cytoplasm in fluorescence images was collected by selecting an area that corresponded to the maximal cytoplasmic region for each cell using Scion Image (Scion, Frederick, MD).

In order to quantify the colocalization between Gα_s and microtubules, images were imported to Volocity (Improvision Inc., Waltham, MA) for deconvolution and colocalization analysis. Measuring the degree of colocalization was done by “volocity quantitation,” as described by Manders et al. (25). The extent of overlap between Gα_s and microtubules was defined with Pearson's correlation.

Purification of PC-tubulin and His₆-tagged Gα_s—PC-tubulin was purified from sheep brain by two cycles of assembly and disassembly followed by phosphocellulose chromatography. The tubulin preparation made by two assembly-disassembly cycles contains microtubule-associated proteins. These microtubule-associated proteins were removed by phosphocellulose chromatography. His₆-tagged G_sα was expressed in the E. coli and purified as described earlier (26). Tryptophan fluorescence was determined with excitation at 280 nm and emission at 340 nm to monitor AlF^–₄-dependent conformational change of Gα_s (27). The eluted proteins were stored at –80 °C for several months with no loss of functional activity.

Pull-down Assay—Purified Gα_s with the His₆ tag was loaded with GDPβS or GTPγS, respectively, as described previously (28), and free nucleotides were removed by concentration/dialysis (Millipore Corp., Bedford, MA). The GDP- and GTP-bound Gα_s were incubated with a 1- or 3-fold molar ratio of PC-tubulin/Gα for 1 h, and complexes were incubated with 50 μl of Ni²⁺-nitrilotriacetic acid-agarose beads. After 2.5 h at 4 °C, the samples were centrifuged and washed with 50 mm Tris buffer. The samples were resuspended in 1× SDS sample buffer and separated on 12% SDS-polyacrylamide gels. The gels were stained with Coomassie Blue before drying.

Surface Plasmon Resononance Using BIAcore 1000—To determine Gα_s-tubulin affinity, amine groups on tubulin were cross-linked to a carboxymethyl dextran-coated CM5 BIAcore sensor chip (500–1000 resonance units). His₆-tagged Gα ^Q213L_s or wild-type His₆-tagged Gα_s in buffer (10 mm HEPES, 150 mm NaCl, 0.005% P-20, pH 6.9) was allowed to bind for 10 min at 25 °C, followed by 15 min of disassociation at a 10 μl/min flow rate, and kinetic curves were fit to a 1:1 association model after controlling for nonspecific binding in a sham-immobilized reference flow cell and buffer responses. K_D and B_max values were determined using equilibrium analysis for nine concentrations of Gα_s in duplicate. Control experiments using ovalbumin failed to show binding to tubulin. Statistical analysis was done using BIAEvaluation version 4.1 and GraphPad Prism version 4.0 software. BIAcore 1000 and sensor chips were obtained from GE Healthcare.

GTPase Assay—For steady-state assay of GTPase, PC-tubulin was allowed to bind GTP (23). The samples were then incubated with Gα_s-GDPβS or Gα_s-GTPγS or ovalbumin 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. 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 μl of each sample. The spots containing [³²P]GTP or [³²P]GDP were visualized with a UV lamp, and plates were exposed to film for autoradiography. Quantitative analysis was done by measuring the integrated optical density of each GDP spot. The integrated optical density represents the GTPase activity as measured by GDP formation.

To determine single-turnover tubulin GTPase activity, tubulin was loaded with [³²P]GTP, and unbound nucleotide was removed by gel filtration using a P6-DG column (Bio-Rad). Tubulin-GTP (0.6–0.9 mol of ³²P/mol of tubulin) was added to Gα ^Q213L_s-GTP in buffer (100 mm PIPES, 1 mm MgCl₂, 1 mm EDTA, pH 6.9) at 37 °C for 5–20 min, the reaction was stopped with 5% trichloroacetic acid, and released ³²P_i was quantified after charcoal extraction (29).

Measurement of Detergent-insoluble Microtubules—To measure microtubule mass, detergent-extracted cytoskeletons, free of unassembled tubulin, were prepared under microtubule-stabilizing conditions essentially as described by Solomon et al. (30, 31). The tubulin content of the cytoskeletons was measured with immunoblot. In brief, cells in a 25-ml flask were washed once with 37 °C PBS and once with extraction buffer (0.1 m PIPES, 1 mm MgSO₄, 2 mm EGTA, 0.1 mm EDTA, 2 m glycerol, pH 6.75). Cells were subsequently extracted twice for 8 min with 0.5 ml of extraction buffer containing 0.1% Triton X-100 and protease inhibitors. After excess extraction buffer was drained from each flask, 0.5 ml of lysis buffer (25 mm Na₂HPO₄, 0.4 m NaCl, 0.5% SDS, pH 7.2) was added for 3–5 min to solubilize the detergent-extracted cytoskeletons. In addition, the 0.5 ml of extraction buffer used to extract PC12 cells was centrifuged for 1 min to collect insoluble material that came off of the culture flask during extraction. This material was added back to the lysis mixture in lysis buffer. The viscous cytoskeletal lysate was boiled for 3 min and then centrifuged for l0 min (2000 × g) in a tabletop centrifuge, and the DNA-containing pellet was removed. The protein concentration of the extracted and cytoskeletal fractions was determined by the Lowry assay. Equal amounts of cytoskeletal protein fraction samples were loaded onto SDS-polyacrylamide gels, and the tubulin contents were determined by immunoblotting.

Quantification of Neurite Outgrowth—The extent of neurite outgrowth was quantitated in more than three independent experiments on living cells. About 100 transfected PC12 or PC12–123.7 cells were scored for each experiment. The morphological differentiation of cells was determined by the percentage of neurite-bearing cells. One individual, blinded to experimental conditions, scored a cell as neurite-bearing if a cell contained at least one slender projection that exceeded the cellular diameter in length.

Statistical Analysis—Data are from at least three different independent experiments and expressed as mean ± S.E. Significant differences (p < 0.05) were determined by a one-way analysis of variance using the Prism version 3.0 software package for statistical analysis (GraphPad Software Inc., San Diego, CA).

RESULTS

The GTP-bound Form of Gα_s Interacts Preferentially with Tubulin in Vitro and in Vivo—To test whether the activated (GTP-bound) or inactive (GDP-bound) form of Gα_s differentially binds to tubulin, purified His₆-tagged Gα_s was loaded with GTPγS or GDPβS and incubated with PC-tubulin. The results indicate that tubulin cosedimented with Gα_s-GTPγS but not Gα_s-GDPβS when proteins were incubated at equal molar concentrations (Fig. 1A and supplemental Fig. 2A). In the presence of a 3-fold molar excess of tubulin to Gα_s, both Gα_s-GDPβS and Gα_s-GTPγS bound tubulin. However, a strong preference was consistently shown for tubulin binding to the GTP form of Gα_s. To confirm that the activated form of Gα_s was binding to tubulin, we compared the affinity of wild-type Gα_s loaded with GDP to constitutively activated Gα ^Q213L_s using surface plasmon resononace. The results (Fig. 1B) showed that purified Gα ^Q213L_s bound strongly to immobilized tubulin, whereas wild-type Gα_s-GDP did not. Gα ^Q213L_s bound with a K_D of 102 ± 18 nm and B_max of 127 ± 6 resonance units, whereas wild-type Gα_s-GDP was insufficient to calculate their values (Fig. 1B).

FIGURE 1.

The GTP form of Gα_s is the binding partner for tubulin in vitro. A, purified His-Gα_s-GTPγS (5 μg) or His-Gα_s-GDPβS (5 μg) was incubated with tubulin for 2 h at room temperature followed by pull-down using nickel-agarose beads. After separation by SDS-PAGE, the gel was stained with Coomassie Blue. The data are representative of three independent experiments. B, varying concentrations of purified Gα ^Q213L_s-GTP (constitutively active) and wild-type Gα_s-GDP were allowed to bind tubulin immobilized to a dextran matrix, and the extent of binding was quantified using surface plasmon resonance. Data shown are representative of three independent experiments. The dashed lines indicate S.E. for hyperbolic best fit curves. C, co-immunoprecipitation of Gα_s with tubulin. PC12 cells were infected with adenovirus containing GFP alone, HA-Gα_s^Q227L-GTP, or HA-Gα_s (wild-type) cDNA. Thirty-six hours after infection, wild-type Gα_s-infected cells were treated with cholera toxin (CTX) for 1.5 h. Cells were lysed, and Gα_s was immunoprecipitated using an HA antibody. Western blots with monoclonal anti-tubulin antibody reveal the extent of tubulin complexed with the two forms of Gα_s. Lane 1, loaded tubulin. D, membranes were stripped and reprobed using a polyclonal anti-Gα_s antibody to reveal that comparable amounts of Gα_s were present in each sample. Results are representative of three experiments.

These in vitro results gave rise to the possibility that activated Gα_s interacts with microtubules and tubulin in intact cells. To test this, adenoviruses with Gα_s (Ad/Gα_s) or its activated mutant (Ad/Gα_s^Q227L) were constructed (supplemental Fig. 1). PC12 cells were infected with Ad/Gα_s or Ad/Gα_s^Q227L, and expressions of both constructs were approximately equal and 2.5-fold that of endogenous Gα_s (supplemental Fig. 2B). Immunoprecipitation was conducted with anti-HA polyclonal antibody. The cells expressing Ad/Gα_s^Q227L or Ad/Gα_s treated with cholera toxin showed a 4-fold increase in association between tubulin and Gα_s compared with that in cells infected with Ad/Gα_s (Fig. 1C and supplemental Fig. 3A). No changes in the level of Gα_s loaded were observed by Western blotting (Fig. 1D). Since activation of Gα_s, either by mutation or cholera toxin, can increase intracellular levels of cAMP and activates PKA, it is necessary to determine whether this affects Gα_s association with tubulin and microtubules. Cells were treated with Rp-cAMP (an inhibitor of PKA) prior and subsequent to infection with Ad/Gα_s^Q227L. Gα_s binding to tubulin was maintained in the presence of the cAMP inhibitor, suggesting that the interaction is independent of the cAMP/PKA pathway (supplemental Fig. 3B). Note that Gα ^Q213L_s and Gα_s^Q227L represent the same mutant in the short and long isoform of Gα_s, respectively, and they appear to be functionally identical.

Cholera Toxin Promotes Gα_s-GFP Localization on Cellular Microtubules—We previously developed a fully functional Gα_s-GFP fusion protein that couples to G protein-coupled receptors and activates adenylyl cyclase. We have used this construct to observe that activation of the fusion protein by receptor agonists or cholera toxin promoted Gα_s-GFP internalization (10). In order to determine if activated Gα_s associates with microtubules, PC12 cells were transfected with Gα_s-GFP. Transfected cells were treated with cholera toxin to activate Gα_s-GFP or forskolin to stimulate adenylyl cyclase and increase cAMP. Microtubules were visualized in saponin-extracted cells, which remove soluble unpolymerized tubulin. Deconvolved fluorescent images showed that either forskolin or cholera toxin treatment promoted neurite outgrowth (Fig. 2). However, cholera toxin activation of Gα_s-GFP, but not forskolin or vehicle treatments, resulted in a displacement of Gα_s-GFP from the plasma membrane. Compared with pretreatment values, the mean gray value of Gα_s in cytoplasm was increased by 60 ± 12% following cholera toxin treatment; however, no significant increase in cytoplasmic Gα_s was observed in cells treated with forskolin. Gα_s colocalization along microtubules was seen in cells treated with cholera toxin but not in those treated with forskolin (Fig. 2). Quantification of colocalization revealed that cholera toxin increased the Pearson's correlation from 0.397 ± 0.02 to 0.591 ± 0.02. However, forskolin treatment did not significantly change the Pearson's correlation relative to control (0.393 ± 0.02 versus 0.397 ± 0.09). These results suggest that activation of Gα_s induces Gα_s translocation to the cytoplasm, where it associates with microtubules.

FIGURE 2.

Activated, intracellular, Gα_s-GFP colocalizes with microtubules. PC12 cells expressing Gα_s-GFP were treated with vehicle (Control), cholera toxin (CTX), or forskolin and fixed with cold methanol after extraction with saponin. Microtubules were visualized with a α-tubulin antibody and a rhodamine-conjugated secondary antibody. Cells were examined with deconvolution microcopy. Yellow shows the overlay of Gα_s-GFP along microtubules. Tubulin (red) and Gα_s-GFP (green) are also shown in the image. Bar, 10 μm.

Constitutively Activated Gα_s Promotes Morphology Change in PC12 Cells—In order to determine whether activation of Gα_s is sufficient to induce morphology change, PC12 cells were infected with Ad/Gα_s or Ad/Gα_s^Q227L. Although activated Gα_s^Q227L significantly increased the number of cells bearing neuritis, wild-type Gα_s did not (Fig. 3). In an effort to clarify the role of PKA in this phenomenon, Gα_s and Gα_s^Q227L were expressed in a PKA-deficient PC12 cell line (123.7 cells), and expression of both constructs was to a similar extent (about 2.5-fold of endogenous Gα_s). Expression of Gα_s^Q227L in 123.7 cells induced neurite outgrowth, but this was not observed in cells expressing GFP alone or in cells infected with wild-type Gα_s virus (Fig. 3A). Expression of Gα_s^Q227L promoted neurite outgrowth in 35% of PKA-deficient 123.7 cells and 60% of native PC12 cells (Fig. 3B). These results suggest that PKA contributes to neurite outgrowth but is not required for Gα_s-induced neurite outgrowth. In conclusion, activated Gα_s can induce morphologic changes independently of PKA in PC12 cells, suggesting that Gα_s can signal independently of this canonical pathway to modulate cytoskeleton-related morphologic changes.

FIGURE 3.

Activated Gα_s promotes neurite outgrowth in PKA-deficient PC12 cells. Cells were infected with adenovirus containing both GFP and Gα_s or Gα_s^Q227L. A, images were acquired after PC12 123.7 cells were infected with indicated viruses. Fluorescence of GFP indicated infected cells expressing Gα_s. Differential interference contrast and fluorescent images are displayed. Bar, 20 μm. B, quantitative analysis of process formation in adenovirus-infected 123.7 cells. The morphology of PC12 cells was determined by the percentage of neurite-bearing cells. All values represent the mean ± S.E. (n = 50) from at least three different experiments. *, significant difference (p < 0.05) compared with cells expressing GFP only.

Gα_s-dependent Neurite Outgrowth in PC12 Cells Is cAMP-independent—In PC12 cells, the increase of intracellular cAMP initiated by stimulating adenylyl cyclase with forskolin or introducing membrane-permeable cAMP analogues initiates neurite outgrowth (32, 33). cAMP mediates its biological functions mainly through activation of PKA as well as Epac1 and Epac2, exchange factors that activate the small GTPases Rap1 and Rap2 (34). In order to differentiate between the actions of cAMP and those of activated, cytoplasmic Gα_s, the 123.7 cells were treated with neural growth factor (100 ng/ml), cholera toxin (100 ng/ml), or 8-bromo-cAMP (5 mm; a cell-permeable cAMP analogue) for 20 h. Neural growth factor promoted neurite outgrowth in PKA-deficient 123.7 cells (Fig. 4) to a similar rate and extent as it did in wild-type PC12 cells, consistent with previous reports (35). However, the concentration of 8-bromo-cAMP that induced cellular process formation in wild-type PC12 cells (24) did not induce morphologic changes in the 123.7 cells (Fig. 4). In addition, treatment of 123.7 cells with 8-CPT-cAMP (a specific Epac agonist) for 20 h did not result in cellular outgrowth (Fig. 4). Activation of endogenous Gα_s by cholera toxin promoted neurite outgrowth in the PKA-deficient 123.7 cells (Fig. 4). The failure of 8-bromo-cyclic AMP or 8-CPT-cAMP to promote process formation in PKA-deficient cells suggests that the effects of activated Gα_s are independent of PKA and Epac pathways.

FIGURE 4.

Neurite outgrowth promoted by activated Gα_s is cAMP- and Epac-independent. A, process formation in 123.7 cells. The cells were treated with 8-CPT-cAMP, 8-bromo-cyclic AMP, neural growth factor, and cholera toxin (as indicated) for 20 h, and differential interference contrast images are displayed. Bar, 20 μm. B, quantitative analysis of process formation in cells. The morphology of PC12 cells was determined by the percentage of neurite-bearing cells. All values represent the mean ± S.E. (n = 50). Data are from three experiments. *, a significant increase compared with control (p < 0.05).

Constitutively Activated Gα_s Decreases the Stability of Microtubules in Vitro and in PC12 Cells—The above experiments demonstrated that activated Gα_s interacts with tubulin, associates with microtubules, and alters cellular morphology in a cAMP-independent manner. The mechanism underlying these morphologic changes remains unclear. Microtubule stability, a determinant of cell morphology, is largely regulated by the hydrolysis of GTP on β-tubulin (36–38). We postulate that activated Gα_s increases the GTPase activity of tubulin, leading to decreased microtubule stability. Cells were extracted with detergent under microtubule-stabilizing conditions, and tubulin content was determined with immunoblotting. In PKA-deficient 123.7 cells, neither forskolin nor cholera toxin treatment altered the amount of total tubulin (Fig. 5A, bottom). However, detergent-insoluble microtubules decreased by ∼25% after cholera toxin treatment, whereas forskolin did not alter the microtubule mass in detergent-insoluble fractions (Fig. 5, A (top) and C). As an alternate approach, PKA-deficient 123.7 cells were infected with Ad/Gα_s or Ad/Gα_s^Q227L. The amount of total tubulin was unchanged by expression of either construct (Fig. 5, B (bottom) and C). Expression of Gα_s^Q227L reduced the detergent-insoluble fraction of tubulin by ∼45% compared with uninfected cells, whereas wild-type Gα_s had no effect (Fig. 5, B (top) and C). These data suggest that activated Gα_s decreases the stable pool of polymerized microtubules in intact cells and that this is cAMP-independent. An in vitro study further confirmed this finding (Fig. 5D). Activated Gα_s significantly depolymerized microtubules in a manner and to an extent similar to that of colchicine (Fig. 5D and supplemental Fig. 4A).

FIGURE 5.

Activated Gα_s decreases the pool of stable microtubules in PKA-deficient 123.7 PC12 cells. A, cholera toxin activation of Gα_s decreases the amount of detergent-insoluble microtubules in 123.7 cells. Cells were treated with cholera toxin or forskolin as indicated. Detergent-insoluble microtubules were obtained and detected by Western blot using anti-tubulin antibody. B, expression of activated Gα_s^Q227L decreases detergent-insoluble microtubules in 123.7 cells. Cells infected with virus encoding Gα_s or Gα_s^Q227L and detergent-insoluble microtubules were detected by Western blotting. C, quantitative analysis. The amount of detergent-insoluble tubulin detected in blots was quantified by scanning densitometry and expressed as a percentage of control. D, Gα ^Q213L_s (15 μm), boiled Gα ^Q213L_s (10 min at 95 °C, 15 μm), colchicine (5 μm), or Taxol (5 μm) was added to polymerized microtubules (15 μm tubulin) in G-PEM buffer for 1 h at 37 °C. The relative amount of tubulin in pellet was quantified. Results shown are normalized to control. All values represent the mean ± S.E. from three independent experiments. *, p < 0.05 versus control.

Activated Gα_s Decreases the Level of Acetylated Tubulin—Several post-translational modifications of α- and β-tubulin subunits are associated with altered microtubule dynamics and stability. Detyrosination (yielding detyrosinated tubulin) and acetylation of tubulin are common modifications associated with stable, long lived microtubules (18, 39–41). Elevated levels of these modified forms of tubulin are correlated with microtubule stability (18, 39–41). To investigate whether activated Gα_s alters post-translational modifications on tubulin, PC12 123.7 cells were infected with Ad/Gα_s or Ad/Gα_s^Q227L, and tubulin forms were analyzed by immunoblotting with specific antibodies. Expression of Gα_s^Q227L significantly reduced the level of acetylated tubulin (Ace-tubulin) by ∼25%, whereas total tubulin levels were unchanged (Fig. 6, A and B). No significant differences in tyrosinated or glutamylated tubulin were observed. These alterations in acetylated tubulin are consistent with the observed decrease in microtubule stability. Nonetheless, the relatively modest change in these post-translational tubulin modification suggest that the activation of GTPase by Gα_s and resulting dynamic instability may be a greater factor than the post-translational modification of tubulin.

FIGURE 6.

Constitutively active Gα_s^Q227L reduces microtubule-stabilizing modifications in PKA-deficient 123.7 PC12 cells. A, post-translational modification of tubulin in 123.7 cells. The cells were infected with adenoviruses and lysed, and proteins were analyzed by immunoblotting with anti-acetylated tubulin (Ace-tubulin), anti-detyrosinated tubulin (Glu-tubulin), anti-tyrosinated tubulin (Tyr-tubulin), or anti-α-tubulin antibodies. B, quantitative analysis. Levels of post-translationally modified tubulin were quantified using scanning densitometry of blots, and data were normalized to percentage of control. All values represent the mean ± S.E., n = 3. *, p < 0.05 versus control.

Activated Gα_s Increases the Intrinsic GTP Hydrolysis Activity of Tubulin—GTP hydrolysis by β-tubulin is considered a key element in increasing the dynamic behavior of microtubules (15, 36, 37, 42). To further understand the mechanism of Gα_s-induced changes in microtubule mass, we tested the effect of activated Gα_s on tubulin GTPase activity. Experiments revealed that tubulin alone has a very low intrinsic GTP hydrolysis activity (Fig. 7). When incubated at equimolar concentrations, Gα_s-GTPγS stimulated tubulin GTPase to a greater extent; the GDP production was increased 2-fold compared with that in groups incubated with Gα_s-GDPβS. (Fig. 7A and supplemental Fig. 4B). This effect was more pronounced when the Gα_s/tubulin ratio was increased (Fig. 7A and supplemental Fig. 4B).

FIGURE 7.

Activated Gα_s increases the intrinsic GTPase activity of tubulin. A, [³²P]GTP-tubulin (1.5 μm) was incubated with GTPγS-Gα_s or GDPβS-Gα_s for 20 min at room temperature. The samples were then treated with 1% SDS and subjected to thin layer chromatography on polyethyleneimine-cellulose plates. T, [³²P]GTP-tubulin alone. GT-1, [²³P]GTP-tubulin incubated with equimolar Gα_s-GTPγS. GD-1, [³²P]GTP-tubulin incubated with equimolar Gα_s-GDPβS. GT-4, [³²P]GTP-tubulin incubated with a 4-fold molar excess of Gα_s-GTPγS. GD-4, [³²P]GTP-tubulin incubated with a 4-fold molar excess of Gα_s-GDPβS. The blot image is shown in supplemental Fig. 4B. *, p < 0.05 versus GD-1 or GD-4. B, measurement of single-turnover tubulin GTPase activity. 200 nm tubulin-GTP was added to the indicated amounts of Gα ^Q213L_s, and released ³²P_i was quantified. Error bars, S.E. (six replicates). *, p < 0.001 versus basal. #, p < 0.001 versus 1:1 ratio.

Gα_s^Q227L, which lacks intrinsic GTPase activity, also increased the tubulin GTPase activity in a single-turnover assay, suggesting direct modulation of tubulin GTPase activity rather than alterations in tubulin GTP binding or GDP release (Fig. 7B). Thus, activated Gα_s promotes GTP hydrolysis in tubulin, and this could represent a mechanism underlying activated Gα_s-induced changes in microtubule polymerization and cell morphology.

DISCUSSION

The data in this report suggest that the activated, GTP-bound conformation of Gα_s interacts directly with intracellular microtubules. Association of activated Gα_s with microtubules activates β-tubulin intrinsic GTPase activity and increases the dynamic instability of microtubules, which may lead to cellular morphology change in PC12 cells. This process appears to be, in part, independent of the cAMP/PKA pathway. Furthermore, Epac does not participate in this process. Fig. 8 describes a scenario for this.

FIGURE 8.

Model for neurite outgrowth by the interaction of activated Gα_s with cellular microtubules. Cholera toxin (CTX)-activated Gα_s or constitutively activated Gα_s was internalized and associated with microtubules, primarily at their plus ends. Gα_s increased the intrinsic GTPase activity of β-tubulin, acting as a GTPase-activating protein for that molecule. The subsequent loss of the “GTP cap” increases the dynamic instability of microtubules. This increase in microtubule dynamics may contribute to neurite outgrowth induced by activated Gα_s. In this system, Gα_s acts as a second messenger, and cAMP (either through PKA or Epac) is not required for Gα_s-mediated neurite outgrowth. *, activated PKA.

Tubulin May Be an Effector for Activated Gα_s—G proteins can be activated upon GTP binding to the α subunit. The GTP confers on Gα_s the ability to carry the message to effector, which, in the case of Gα_s, is usually adenylyl cyclase. Interestingly, the accumulating evidence suggests that activated Gα_s is displaced from the plasma membrane into the cytoplasm (9–12, 43, 44), where it appears to localize microtubules (Fig. 2). A previous study has suggested that Gα_s might immunoprecipitate with microtubules in glomerulosa cells (45). Results from this report provide the first evidence that the activated form of Gα_s (the GTP form) associates with tubulin/microtubules with a K_D of 102 nm. In vitro, both Gα_s-GTPγS and mutationally activated Gα_s directly bind to tubulin, and in cells, either mutationally activated Gα_s or cholera toxin-activated Gα_s bound to tubulin/microtubules (Figs. 1 and 2). These data give rise to the possibility that tubulin may act as a cytosolic effector for activated Gα_s within a novel Gα_s signaling pathway.

Activated Gα_s Interacts with Microtubules to Increase GTPase Activity of β-Tubulin—Microtubules are polar structures formed by the head-to-tail association of tubulin heterodimers (46). The GTP cap model suggests that β-tubulin is exposed at microtubule plus ends, where a GTP “cap” helps to stabilize microtubules (38). However, a recent finding suggests that the GTP-tubulin also exists at other sites on microtubules (47). A recent modeling study predicts that Gα_s overlaps the GTP binding site on β-tubulin (48). Based on the above, we postulate that, in cells, activated Gα_s might bind to GTP β-tubulin at microtubule plus ends as well as other spots on the microtubules that contain GTP-tubulin. Fig. 2 in this study showed many colocalized spots between Gα_s and tubulin along the microtubule, supporting our postulation and consistent with the “GTP remnant model” for microtubules (47).

In microtubules, GTP in β-tubulin is normally hydrolyzed to GDP by an intrinsic GTPase (15). Here, we use different approaches to demonstrate that activated Gα_s is a GTPase-activating protein for tubulin and efficiently stimulates the GTP hydrolysis of tubulin (Fig. 7). GTP hydrolysis in β-tubulin allows microtubules to depolymerize by weakening the bonds between tubulin subunits (16, 17).

Note that Gα_s-GDPβS (nonactivated Gα_s) shows a weak ability to increase the GTPase activity of tubulin compared with the control group (tubulin alone). This may result from the weak but still extant binding between Gα_s-GDPβS and tubulin (see Fig. 1A). In addition, previous results from our laboratory revealed partial agonist activity of GDPβS for Gα_s in the activation of adenylyl cyclase (49). Since the presumed binding site for tubulin on Gα_s overlaps with that for adenylyl cyclase (48), a small effect of GDPβS on GTPase of tubulin is consistent.

The Effect of the Interaction of Activated Gα_s with Tubulin on Cell Morphology Is Independent of the cAMP Pathway—The action of cAMP in cells is mediated through the PKA and Epac. It is well documented that cAMP regulates actin dynamics (50). However, it appears that the cAMP pathway is not involved in regulation of microtubule dynamics, although some microtubule-associated proteins might be phosphorylated by PKA (51). Furthermore, results from this study showed that the activated Gα_s modulated microtubule stability in a manner independent of PKA. This is because of the following findings. (i) PKA inhibitors did not influence the binding between these two proteins (Fig. 1 and supplemental Fig. 3). (ii) Microtubule stability was decreased by activated Gα_s, in vitro, to the same extent as colchicine. (iii) Activated Gα_s identically increased microtubule dynamics in wild-type PC12 cells and in those deficient in PKA (see supplemental Fig. 5 and Figs. 5 and 6).

cAMP may stimulate PC12 cell differentiation with neurite-like process outgrowth (24, 35, 52, 53). The mechanism of cAMP promoting cellular process formation in PC12 cells is not clear. One study suggests that both PKA and Epac pathways are necessary and that neither PKA nor Epac alone is sufficient to induce the cellular morphologic change (52). Interestingly, activated Gα_s promoted neurite outgrowth in both wild-type and PKA-deficient PC12 cells, but neurite outgrowth was significantly less in PKA-deficient cells than in wild-type cells (Fig. 3). Furthermore, treatment of PKA-deficient cells with cAMP analog and specific Epac agonist did not induce neurite outgrowth (Fig. 4). Thus, it appears that cAMP pathways should not be necessary for neurite outgrowth induced by activated Gα_s.

Activated Gα_s Alters Cellular Morphology through the Increase of Microtubule Dynamics—Expression of the activated mutant, Gα_s^Q227L, did induce process formation in PC12 cells, whereas expression of wild-type Gα_s was not effective. Since cAMP pathways may not participate in the process, microtubule dynamics may be a potential contributor for neurite outgrowth. In the developing nervous system, the microtubule-destabilizing proteins SCG10 and stathmin are highly expressed (54). SCG10 accumulates in the central domain of the growth cone (55, 56), and overexpression of SCG10 in PC12 cells strongly enhances neurite outgrowth (57, 58). One study has also shown that depolymerization of the microtubule with colchicine significantly enhanced the neurite outgrowth (59). Stabilizing microtubules with Taxol or disrupting by stathmin inhibits both the neurite outgrowth and growth cone turning (60–62). Activated Gα_s is clearly able to stimulate the intrinsic GTPase of tubulin, which decreases the pool of the stable microtubules in cells. Taken together, these findings support our hypothesis that activated, translocated Gα_s increases microtubule dynamic instability and contributes to neurite outgrowth.

The overall effect of activated Gα_s on microtubule stability (Fig. 5) is great (about 40%). Although we do see a small but significant decrease in the amount of acetylated tubulin (consistent with an increase in dynamic instability), this change is probably not sufficient to explain the differences in microtubule stability seen in Fig. 5. As such, the increase in tubulin GTPase evoked by activated Gα_s may be a more significant factor to explain our findings than post-translational modification of tubulin.

Cells contain far more tubulin than Gα_s; thus, it is reasonable to ask whether there is a sufficient quantity of activated Gα_s to modulate microtubule dynamics. It is noteworthy, however, that Gα_s displays a heterogeneous distribution on the plasma membrane (10, 12), and this protein is concentrated in synaptic regions (63, 64). Furthermore, Gα_s traffics to and concentrates in lipid rafts prior to internalization (10, 12). Taken together, localized regions of high Gα_s concentration are likely extant, and it is this Gα_s that interacts with microtubules.

It is noteworthy that in addition to Gα_s, other purified G protein α subunits (Gα_i1 and Gα_q) bind to tubulin with affinity almost equal to that of Gα_s (22, 49, 65, 66). One previous study showed that overexpression of His₆-Gα_i1 increased cellular outgrowth in COS-1 cells (27). Since the NH-terminal His₆ prevents Gα_i1 protein association with Gβγ, the His₆-Gα_i1 is entirely cytosolic. However, unlike Gα_s, Gα_i1 and Gα_q are not released from cell membranes upon activation. Thus, G protein-mediated regulation of microtubule dynamics in cells appears unique to activated Gα_s.

The current study indicated that activated Gα_s associates with microtubules and regulates microtubule dynamics. The interplay between Gα_s and microtubules suggests that G proteins may act as intracellular messengers to alter the cytoskeleton, and this study implicates their role in modification of cellular differentiation and control of cell morphology.