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• W2000288557 abstract "It is now evident that Gαs traffics into cytosol following G protein-coupled receptor activation, and α subunits of some heterotrimeric G-proteins, including Gαs 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 Gαs directly bound to tubulin and cellular microtubules, and fluorescence microscopy showed that cholera toxin-activated Gαs colocalized with microtubules. The activated, GTP-bound, Gαs mimicked tubulin in serving as a GTPase activator for β-tubulin. As a result, activated Gαs 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 Gαs 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 Gαs or activation of Gαs with cholera toxin in protein kinase A-deficient PC12 cells promoted neurite outgrowth in a cAMP-independent manner. Thus, it is suggested that activated Gαs 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. It is now evident that Gαs traffics into cytosol following G protein-coupled receptor activation, and α subunits of some heterotrimeric G-proteins, including Gαs 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 Gαs directly bound to tubulin and cellular microtubules, and fluorescence microscopy showed that cholera toxin-activated Gαs colocalized with microtubules. The activated, GTP-bound, Gαs mimicked tubulin in serving as a GTPase activator for β-tubulin. As a result, activated Gαs 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 Gαs 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 Gαs or activation of Gαs with cholera toxin in protein kinase A-deficient PC12 cells promoted neurite outgrowth in a cAMP-independent manner. Thus, it is suggested that activated Gαs 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 (1Charlie N.K. Schade M.A. Thomure A.M. Miller K.G. Genetics.. 2006; 172: 943-961Google Scholar, 2Denker B.M. Saha C. Khawaja S. Nigam S.K. J. Biol. Chem... 1996; 271: 25750-25753Google Scholar, 3Kehlenbach R.H. Matthey J. Huttner W.B. Nature.. 1994; 372: 804-809Google Scholar, 4Drmota T. Novotny J. Gould G.W. Svoboda P. Milligan G. Biochem. J... 1999; 340: 529-538Google Scholar, 5Ahnert-Hilger G. Schafer T. Spicher K. Grund C. Schultz G. Wiedenmann B. Eur. J. Cell Biol... 1994; 65: 26-38Google Scholar-6Holtje M. von Jagow B. Pahner I. Lautenschlager M. Hortnagl H. Nurnberg B. Jahn R. Ahnert-Hilger G. J. Neurosci... 2000; 20: 2131-2141Google Scholar, 13Freudzon L. Norris R.P. Hand A.R. Tanaka S. Saeki Y. Jones T.L. Rasenick M.M. Berlot C.H. Mehlmann L.M. Jaffe L.A. J. Cell Biol... 2005; 171: 255-265Google Scholar, 14Castellone M.D. Teramoto H. Williams B.O. Druey K.M. Gutkind J.S. Science.. 2005; 310: 1504-1510Google Scholar). Recently, a number of biochemical studies observed intracellular translocation of Gαs proteins subsequent to activation by β-adrenergic agonists, cholera toxin, or direct binding of a hydrolysis-resistant GTP analog (7Ransnas L.A. Svoboda P. Jasper J.R. Insel P.A. Proc. Natl. Acad. Sci. U. S. A... 1989; 86: 7900-7903Google Scholar, 8Rasenick M.M. Wheeler G.L. Bitensky M.W. Kosack C.M. Malina R.L. Stein P.J. J. Neurochem... 1984; 43: 1447-1454Google Scholar-9Levis M.J. Bourne H.R. J. Cell Biol... 1992; 119: 1297-1307Google Scholar). More recently, taking advantage of an internal sequence Gαs-GFP fusion protein, the translocation of Gαs into the cytoplasm was directly observed in living cells upon stimulation with agonists (10Yu J.Z. Rasenick M.M. Mol. Pharmacol... 2002; 61: 352-359Google Scholar, 11Hynes T.R. Mervine S.M. Yost E.A. Sabo J.L. Berlot C.H. J. Biol. Chem... 2004; 279: 44101-44112Google Scholar). This translocation of Gαs from membrane to cytoplasm triggered by agonist appears to occur through lipid rafts on the plasma membrane (12Allen J.A. Yu J.Z. Donati R.J. Rasenick M.M. Mol. Pharmacol... 2005; 20: 1452-1461Google Scholar).The fate of internalized Gαs is not well characterized. Two current studies have suggested that activated, internalized Gαs could invoke developmental paradigms. In mouse oocytes, meiotic prophase was maintained (arrested) due to the prolonged, receptor-mediated activation of Gαs, which assumed a cytosolic localization subsequent to internalization (13Freudzon L. Norris R.P. Hand A.R. Tanaka S. Saeki Y. Jones T.L. Rasenick M.M. Berlot C.H. Mehlmann L.M. Jaffe L.A. J. Cell Biol... 2005; 171: 255-265Google Scholar). Activated Gαs was also invoked to explain prostaglandin E2-mediated stimulation of colon cancer cell growth. It appeared that Gαs associated with axin and allowed β-catenin to activate the proliferative state (14Castellone M.D. Teramoto H. Williams B.O. Druey K.M. Gutkind J.S. Science.. 2005; 310: 1504-1510Google Scholar). Results from these studies suggest that Gαs 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 (15Carlier M.F. Didry D. Valentin-Ranc C. J. Biol. Chem... 1991; 266: 12361-12368Google Scholar). GTP hydrolysis allows microtubules to depolymerize by weakening the bonds between tubulin subunits to decrease microtubule stability (16Cassimeris L. Cell Motil. Cytoskeleton.. 1993; 26: 275-281Google Scholar, 17Albrert B. Bray D. Lewis J. Raff M. Roberts K. Watson J.D. Molecular Biology of the Cell.Garland Publishing, Inc., New York. 1994; 37: 805-816Google Scholar). 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 (t½ = 5–10 min) and long lived or stable microtubules (t½ > 1 h) (18Webster D.R. Gundersen G.G. Bulinski J.C. Borisy G.G. Proc. Natl. Acad. Sci. U. S. A... 1987; 84: 9040-9044Google Scholar, 19Schulze E. Kirschner M. J. Cell Biol... 1987; 104: 277-288Google Scholar). 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 (20Bulinski J.C. Gundersen G.G. BioEssays.. 1991; 13: 285-293Google Scholar). 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 (21Roychowdhury S. Rasenick M.M. FEBS J.. 2008; 275: 4654-4663Google Scholar), and studies with purified proteins implicate Gα subunits as potential regulators (22Wang N. Rasenick M.M. Biochemistry.. 1991; 30: 10957-10965Google Scholar, 23Roychowdhury S. Rasenick M.M. Biochemistry.. 1994; 33: 9800-9805Google Scholar).This study inquired whether activated Gαs, released from the plasma membrane, regulates microtubule stability via direct interaction with microtubules. Gαs 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 Gαs 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 PROCEDURESMaterials—Wild-type PC12 cells, wild-type rat Gαs with hemagglutinin epitope (HA), 3The abbreviations used are: HA, hemagglutinin; GFP, green fluorescent protein; Ad, adenovirus; TRITC, tetramethylrhodamine isothiocyanate; 8-CPT-cAMP, 8-(4-chlorophenylthio)-adenosine-3′:5′-cyclic monophosphate; PKA, protein kinase A; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid; PC, phosphocellulose; GDPβS, guanyl-5′-yl thiophosphate; GTPγS, guanosine 5′-3-O-(thio)triphosphate. tag and constitutively activated GαsQ227L with HA tag were obtained from the American Type Culture Collection. Gα Q213Ls and the protein kinase A (PKA)-deficient PC12 cells (123.7) were generous gifts from John A. Wagner (Cornell University Medical College) (24Ginty D.D. Glowacka D. Bader D.S. Hidaka H. Wagner J.A. J. Biol. Chem... 1991; 266: 17454-17458Google Scholar) and Tarun Patel (Loyola University, Chicago, IL), respectively. Construction of Gαs-GFP was described in a previous report (10Yu J.Z. Rasenick M.M. Mol. Pharmacol... 2002; 61: 352-359Google Scholar). 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 Gαs 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 Gαs-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/Gαs or Ad/GαsQ227L was as follows. Rat Gαs and GαsQ227L 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/Gαs or GαsQ227L 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/Gαs or GαsQ227L 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/Gαs, or Ad/GαsQ227L 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 Gαs to show sample loading. The Western blot was done as described previously (10Yu J.Z. Rasenick M.M. Mol. Pharmacol... 2002; 61: 352-359Google Scholar). 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 Gαs-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 Gαs-GFP was done as described previously (10Yu J.Z. Rasenick M.M. Mol. Pharmacol... 2002; 61: 352-359Google Scholar). 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 Gαs 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. (25Aoki C. Go C.G. Wu K. Siekevitz P. Brain Res... 1992; 596: 189-201Google Scholar). The extent of overlap between Gαs and microtubules was defined with Pearson's correlation.Purification of PC-tubulin and His6-tagged Gαs—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 Gsα was expressed in the E. coli and purified as described earlier (26Linder M.E. Gilman A.G. Methods Enzymol... 1991; 195: 202-215Google Scholar). Tryptophan fluorescence was determined with excitation at 280 nm and emission at 340 nm to monitor AlF–4-dependent conformational change of Gαs (27Chen N.F. Yu J.Z. Skiba N.P. Hamm H.E. Rasenick M.M. J. Biol. Chem... 2003; 278: 15285-15290Google Scholar). The eluted proteins were stored at –80 °C for several months with no loss of functional activity.Pull-down Assay—Purified Gαs with the His6 tag was loaded with GDPβS or GTPγS, respectively, as described previously (28Zera E.M. Molloy D.P. Angleson J.K. Lamture J.B. Wensel T.G. Malinski J.A. J. Biol. Chem... 1996; 271: 12925-12931Google Scholar), and free nucleotides were removed by concentration/dialysis (Millipore Corp., Bedford, MA). The GDP- and GTP-bound Gαs 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 Gαs-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 Q213Ls or wild-type His6-tagged Gαs 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. KD and Bmax values were determined using equilibrium analysis for nine concentrations of Gαs 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 (23Roychowdhury S. Rasenick M.M. Biochemistry.. 1994; 33: 9800-9805Google Scholar). The samples were then incubated with Gαs-GDPβS or Gαs-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 Q213Ls-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 32Pi was quantified after charcoal extraction (29Mejillano M.R. Shivanna B.D. Himes R.H. Arch. Biochem. Biophys... 1996; 336: 130-138Google Scholar).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. (30Solomon F. Magendantz M. Salzman A. Cell.. 1979; 18: 431-438Google Scholar, 31Drubin D.G. Feinstein S.C. Shooter E.M. Kirschner M.W. J. Cell Biol... 1985; 101: 1799-1807Google Scholar). 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).RESULTSThe GTP-bound Form of Gαs Interacts Preferentially with Tubulin in Vitro and in Vivo—To test whether the activated (GTP-bound) or inactive (GDP-bound) form of Gαs differentially binds to tubulin, purified His6-tagged Gαs was loaded with GTPγS or GDPβS and incubated with PC-tubulin. The results indicate that tubulin cosedimented with Gαs-GTPγS but not Gαs-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 Gαs, both Gαs-GDPβS and Gαs-GTPγS bound tubulin. However, a strong preference was consistently shown for tubulin binding to the GTP form of Gαs. To confirm that the activated form of Gαs was binding to tubulin, we compared the affinity of wild-type Gαs loaded with GDP to constitutively activated Gα Q213Ls using surface plasmon resononace. The results (Fig. 1B) showed that purified Gα Q213Ls bound strongly to immobilized tubulin, whereas wild-type Gαs-GDP did not. Gα Q213Ls bound with a KD of 102 ± 18 nm and Bmax of 127 ± 6 resonance units, whereas wild-type Gαs-GDP was insufficient to calculate their values (Fig. 1B).These in vitro results gave rise to the possibility that activated Gαs interacts with microtubules and tubulin in intact cells. To test this, adenoviruses with Gαs (Ad/Gαs) or its activated mutant (Ad/GαsQ227L) were constructed (supplemental Fig. 1). PC12 cells were infected with Ad/Gαs or Ad/GαsQ227L, and expressions of both constructs were approximately equal and 2.5-fold that of endogenous Gαs (supplemental Fig. 2B). Immunoprecipitation was conducted with anti-HA polyclonal antibody. The cells expressing Ad/GαsQ227L or Ad/Gαs treated with cholera toxin showed a 4-fold increase in association between tubulin and Gαs compared with that in cells infected with Ad/Gαs (Fig. 1C and supplemental Fig. 3A). No changes in the level of Gαs loaded were observed by Western blotting (Fig. 1D). Since activation of Gαs, either by mutation or cholera toxin, can increase intracellular levels of cAMP and activates PKA, it is necessary to determine whether this affects Gαs association with tubulin and microtubules. Cells were treated with Rp-cAMP (an inhibitor of PKA) prior and subsequent to infection with Ad/GαsQ227L. Gαs 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 Q213Ls and GαsQ227L represent the same mutant in the short and long isoform of Gαs, respectively, and they appear to be functionally identical.Cholera Toxin Promotes Gαs-GFP Localization on Cellular Microtubules—We previously developed a fully functional Gαs-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 Gαs-GFP internalization (10Yu J.Z. Rasenick M.M. Mol. Pharmacol... 2002; 61: 352-359Google Scholar). In order to determine if activated Gαs associates with microtubules, PC12 cells were transfected with Gαs-GFP. Transfected cells were treated with cholera toxin to activate Gαs-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 Gαs-GFP, but not forskolin or vehicle treatments, resulted in a displacement of Gαs-GFP from the plasma membrane. Compared with pretreatment values, the mean gray value of Gαs in cytoplasm was increased by 60 ± 12% following cholera toxin treatment; however, no significant increase in cytoplasmic Gαs was observed in cells treated with forskolin. Gαs 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 Gαs induces Gαs translocation to the cytoplasm, where it associates with microtubules.FIGURE 2Activated, intracellular, Gαs-GFP colocalizes with microtubules. PC12 cells expressing Gαs-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 Gαs-GFP along microtubules. Tubulin (red) and Gαs-GFP (green) are also shown in the image. Bar, 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Constitutively Activated Gαs Promotes Morphology Change in PC12 Cells—In order to determine whether activation of Gαs is sufficient to induce morphology change, PC12 cells were infected with Ad/Gαs or Ad/GαsQ227L. Although activated GαsQ227L significantly increased the number of cells bearing neuritis, wild-type Gαs did not (Fig. 3). In an effort to clarify the role of PKA in this phenomenon, Gαs and GαsQ227L 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 Gαs). Expression of GαsQ227L in 123.7 cells induced neurite outgrowth, but this was not observed in cells expressing GFP alone or in cells infected with wild-type Gαs virus (Fig. 3A). Expression of GαsQ227L 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 Gαs-induced neurite outgrowth. In conclusion, activated Gαs can induce morphologic changes independently of PKA in PC12 cells, suggesting that Gαs can signal independently of this canonical pathway to modulate cytoskeleton-related morphologic changes.FIGURE 3Activated Gαs promotes neurite outgrowth i" @default.
• W2000288557 created "2016-06-24" @default.
• W2000288557 creator A5026181487 @default.
• W2000288557 creator A5036010034 @default.
• W2000288557 creator A5063049053 @default.
• W2000288557 creator A5070959736 @default.
• W2000288557 creator A5078793776 @default.
• W2000288557 date "2009-04-01" @default.
• W2000288557 modified "2023-10-17" @default.
• W2000288557 title "Cytosolic Gαs Acts as an Intracellular Messenger to Increase Microtubule Dynamics and Promote Neurite Outgrowth" @default.
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