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J. Biol. Chem., Vol. 278, Issue 36, 34299-34308, September 5, 2003

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G Mediates the Interplay between Tubulin Dimers and Microtubules in the Modulation of G_q Signaling^*

Juliana S. Popova  and Mark M. Rasenick   ¶

From the Departments of Physiology and Biophysics and Psychiatry, College of Medicine, University of Illinois, Chicago, Illinois 60612-7342

Received for publication, February 19, 2003 , and in revised form, May 5, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Agonist stimulation causes tubulin association with the plasma membrane and activation of PLC1 through direct interaction with, and transactivation of, G_q. Here we demonstrate that G interaction with tubulin down-regulates this signaling pathway. Purified G, alone or with phosphatidylinositol 4,5-bisphosphate (PIP₂), inhibited carbachol-evoked membrane recruitment of tubulin and G_q transactivation by tubulin. Polymerization of microtubules elicited by G overrode tubulin translocation to the membrane in response to carbachol stimulation. G sequestration of tubulin reduced the inhibition of PLC1 observed at high tubulin concentration. G₁₂ interacted preferentially with tubulin-GDP, whereas G_q was transactivated by tubulin-GTP. Prenylation of the ₂ polypeptide was required for G/tubulin interaction. Both confocal microscopy and coimmunoprecipitation studies revealed the spatiotemporal pattern of G/tubulin interaction during carbachol stimulation of neuroblastoma SK-N-SH cells. In resting cells G localized predominantly at the cell membrane, whereas tubulin was found in well defined microtubules in the cytosol. Within 2 min of agonist exposure, a subset of tubulin translocated to the plasma membrane and colocalized with G. Fifteen min post-carbachol addition, tubulin and G colocalized in vesicle-like structures in the cytosol. G/tubulin colocalization increased after pretreatment of cells with the microtubule-depolymerizing agent, colchicine, and was inhibited by taxol. Taxol also inhibited carbachol-induced PIP₂ hydrolysis. It is suggested that G/tubulin interaction mediates internalization of membrane-associated tubulin at the offset of PLC1 signaling. Newly cytosolic G/tubulin complexes might promote microtubule polymerization attenuating further tubulin association with the plasma membrane. Thus G protein-coupled receptors might evoke G and G to orchestrate regulation of phospholipase signaling by tubulin dimers and control of cell shape by microtubules.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Binding of agonists to G protein-coupled receptors causes receptors to interact with specific G protein - heterotrimers, which, in turn, triggers the replacement of GDP for GTP on the subunit and the functional dissociation and activation of both and subunits. G and G are then able to activate effectors and initiate signal transduction.

Tubulin is a cytoskeletal protein that forms microtubules (1, 2) and regulates G protein-mediated signaling (3–5) through binding and hydrolysis of GTP. Transfer of GTP from tubulin to the subunit of G_i1, G_s, and G_q (transactivation) leads to G coupling to, and regulation of, adenylyl cyclase and phospholipase C1 (PLC1)¹ (4–8).

Tubulin translocates to the plasma membrane to regulate these G proteins. Activation by G_q-coupled muscarinic receptors (3, 9) or glutamate receptors (mGluR1) (10) causes microtubule depolymerization and association of tubulin with plasma membrane proteins in living cells. Upon binding to the plasma membrane tubulin transactivates G_q, which, in turn, activates PLC1 (7). However, continued accumulation of tubulin at the membrane inhibits PLC1 as that tubulin binds to the PLC1 substrate, phosphatidylinositol 4,5 bisphosphate (PIP₂) (9). Because the product of PLC1-directed PIP₂ hydrolysis, inositol 1,4,5-trisphosphate (IP₃), mobilizes stored calcium that evokes microtubule depolymerization (11), feedback inhibition of PLC1 may be caused by increased concentration of tubulin dimers in regions close to the plasma membrane (9).

Both G protein and subunits regulate the assembly of the microtubule cytoskeleton in vitro. Although G activates tubulin GTPase and increases microtubule dynamics (12), G promotes microtubule polymerization and stabilizes microtubules in vitro (13). Post-translational isoprenylation of G appears important to this process, because the mutant 1₂(C68S), which is isoprenylation-deficient, and 11, which is farnesylated, rather than geranylgeranylated, do not support microtubule formation.

Although this G effect on in vitro microtubule assembly has been shown, the potential impact of a tubulin-G interaction in the cell and its effect on intracellular signaling has not been investigated. Interaction of G and PIP₂ with the G protein receptor kinase 2 (GRK2) assists GRK2 membrane translocation and the subsequent phosphorylation of the activated -adrenergic receptor (14). G might be similarly instrumental in translocation of tubulin to its membrane-associated signaling partners. Regulated interactions of tubulin with either G or G might differentially affect cellular signaling, because these subunits reciprocally regulate tubulin polymerization. By promoting microtubule polymerization in response to a signal, G might sequester cytosolic tubulin and inhibit tubulin involvement in the regulation of intracellular signaling.

This report investigates the interaction of tubulin with G and the effect of this interaction on the interplay between agonist-evoked PLC1 signaling and microtubule assembly. It is suggested that agonist stimulation recruits tubulin to the membrane, where it transactivates G_q. Subsequent to PLC1 activation, tubulin sequesters with G, and this complex internalizes to the cytosol. In vitro membrane association/microtubule polymerization experiments suggest that such complexes might seed microtubule polymerization. Data from transactivation, coimmunoprecipitation and cross-linking experiments in vitro, as well as those from confocal immunofluorescence microscopy or coimmunoprecipitation in cells, indicate a direct functional relationship between G protein-coupled receptor signaling and the dynamics of the microtubule cytoskeleton. This report provides first direct evidence for agonist-evoked and G-mediated cross-regulation between G protein signaling and the dynamics of the microtubule cytoskeleton. Such a mechanism could be instrumental in the regulated reorganization of cytoskeletal elements or other cellular rearrangements leading to synapse formation, cell motility, and cell shape.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cell Culture—Sf9 cells were maintained in Sf-900 II SFM media as described previously (7). SK-N-SH neuroblastoma cells were grown in Dulbecco's modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum according to standard procedures (7).

Baculovirus-directed Protein Expression in Sf9 Cells—Sf9 cells were simultaneously infected with baculoviruses bearing m₁ muscarinic receptor, G_q, and/or PLC1 cDNA as described previously (7). The construction of these recombinant baculoviruses was described earlier (15–17). Membranes were prepared from cells collected 60 h post-infection. Membrane Preparation and Western Blotting—Sf9 or SK-N-SH cells were sonicated in ice-cold 20 mM Hepes, pH 7.4, 1 mM MgCl₂, 100 mM NaCl, 1 mM DTT, 0.3 mM phenylmethylsulfonyl fluoride, and membrane pellets were prepared as described (7). Protein concentration was measured by the Bradford dye-binding assay (18) with bovine serum albumin as a standard. Expression of receptors, G proteins, and PLC1, was determined by immunoblotting. Membrane proteins transferred to polyvinylidene difluoride membranes (0.45 µm, Millipore Corp.) were probed with antisera specific for the m₁ muscarinic receptor (#71, from G. Luthin), G_q/11 (#0945, from D. Manning, Philadelphia, PA), or PLC1 (K-32-3, monoclonal, from S. G. Rhee, Bethesda, MD) at a dilution of 1:500. Goat anti-rabbit or anti-mouse IgGs were used as secondary antisera, respectively, followed by ECL detection of the corresponding protein bands. Expression levels were estimated by densitometry of the bands (Storm 840, Amersham Biosciences). They varied by no more than 10% for a given recombinant protein. Receptor binding studies using [³H]QNB as a ligand were performed to monitor m₁ muscarinic receptor expression (7). When coexpressed with G_q and PLC1 in the Sf9 cells, m₁ muscarinic receptor density was estimated at 240 fmol/mg of membrane protein (7). SK-N-SH cells have a high density of m₃ muscarinic receptors (500 fmol/mg of membrane protein) coupled to phosphoinositide turnover (19–21).

Purification of Proteins—Microtubule proteins were isolated as described previously (22). Microtubule-associated proteins were removed by phosphocellulose chromatography, and the remaining pure tubulin fraction was termed PC-tubulin (23). PC-tubulin was aliquoted and stored in liquid nitrogen until used.

Replacement of GTP in -tubulin was performed as described previously (5). Briefly, GTP was removed from PC-tubulin by charcoal pretreatment followed by incubation of tubulin with 150 µM guanine nucleotide (GDP or [³²P]AAGTP) for 30 min on ice. Prior to use, these samples were passed through Bio-Gel P6DG desalting columns (Bio-Rad) twice to remove the unbound nucleotide. This procedure yields 0.4–0.6 mol of guanine nucleotide bound per mol of tubulin dimer. Tubulin-guanine nucleotide concentrations used throughout the study were based on the protein concentration.

G₁₂, purified from Sf9 cell membranes, was kindly provided by Dr. T. Kozasa (24). G₁1, purified from bovine retina, was a generous gift from Dr. H. E. Hamm. Mutant G₁₂(C68S) was generously provided by Drs. J. Hepler and A. G. Gilman. Purified G subunits were also kindly provided by Dr. P. Casey. G preparations were exchanged for the buffer used in the experiments (in most cases 100 mM Pipes buffer, pH 6.9, 2 mM EGTA, 1 mM MgCl₂ (buffer A)) through a rapid spin column (Bio-Gel P6DG, Bio-Rad) immediately prior to use (25).

Microtubule Assembly—PC-tubulin (1.5 mg/ml) was incubated with SK-N-SH membranes (0.3 mg/ml) and/or G (0.13 mg/ml), both in the presence and absence of 100 µM carbachol in polymerization buffer (100 mM Pipes, 2 mM EGTA, 3 mM MgCl₂, 1 mM GTP, pH 6.9). (When tested, G was preincubated with tubulin for 30 min on ice.) The assembly reaction was carried out for 30 min at 37 °C in a shaking water bath. Centrifugation at 150,000 x g for 30 min at 37 °C was performed, followed by separation of the pellets and the supernatants. Pellets were resuspended in identical amounts of cold polymerization buffer, kept on ice for 1 h, and centrifuged at 20,000 x g for 15 min at 4 °C, to separate membranes from depolymerized tubulin polymer. Samples were subjected to SDS-PAGE and immunoblotting with a monoclonal anti--tubulin antibody (DM1A, Sigma) and ECL detection (Amersham Biosciences). The results were analyzed in a Storm 840 imaging system (Amersham Biosciences). When tested, carbachol had no effect on tubulin polymerization in the absence of SK-N-SH membranes.

Photoaffinity Labeling with AAGTP—Cell membranes were incubated with the indicated concentrations of tubulin-[³²P]AAGTP in the presence or absence of carbachol and/or G (at a protein ratio with tubulin of 1:5) and/or PIP₂ in buffer A as described previously (26). (When tested, PIP₂ was evaporated under a stream of nitrogen, sonicated for 5 min in buffer A on ice, and preincubated with tubulin for 15 min at 4 °C in a bath sonicator.) Following incubation, tubes were UV-irradiated, and the reaction was quenched with ice-cold Pipes buffer, 1 mM MgCl₂, 4 mM DTT. After centrifugation at 20,000 x g for 15 min, the membrane pellets were washed with buffer A and dissolved in SDS Laemmli sample buffer with 50 mM DTT as described (27). SDS-PAGE of the samples was performed, and the gels were either stained (Coomassie Blue) or subjected to Western blotting, followed by autoradiography (Kodak XAR-5 film) or phosphor image analysis (Storm 840, Amersham Biosciences).

Membrane Association of Tubulin in SK-N-SH Cells—SK-N-SH cells were pretreated with 330 nM colchicine or 330 nM taxol, as described. Cells were collected and washed three times with PBS, and aliquots of 1 x 10⁷ cells were distributed in plastic tubes on ice. Carbachol (100 µM) was added, and the samples were incubated for the indicated time periods at 37 °C with constant shaking. Samples were transferred on ice and immediately sonicated, as described (3). Each sample was centrifuged at 600 x g at 4 °C (P₁ pellets). Supernatants were centrifuged at 20,000 x g at 4 °C (P₂ pellets), and the P₂ pellets were washed thoroughly with buffer. SDS-PAGE of the P₂ pellet and cytosolic fractions was followed by immunoblotting, using anti--tubulin antibody (DM1A, Sigma) and ECL detection (Amersham Biosciences). The results were analyzed in a Storm 840 image system (Amersham Biosciences).

Immunoprecipitation—Purified G subunits (G₁₂, G₁₁, and G₁₂ (68S)) and PC-tubulin in a GTP- or GDP-bound form, or devoid of nucleotide, as well as extracts from SK-N-SH neuroblastoma cells, were tested for tubulin/G coimmunoprecipitation. Purified G subunits and the different PC-tubulin species were tested at a protein ratio of 1:2. When tested, SK-N-SH membranes were extracted with 1% sodium cholate in buffer A for 1 h at 4 °C with constant stirring. Samples were centrifuged at 20,000 x g for 15 min at 4 °C, and the isolated membrane extracts (0.5 mg/ml membrane protein) were incubated with the indicated tubulin species (1 µM). Incubations were carried out in buffer A for 30 min at 24 °C and constant shaking. After preclearing (Pansorbin, Calbiochem), each sample was incubated overnight with appropriate specific antiserum or preimmune serum (1:20 dilution) at 4 °C with constant shaking. Note, that dimeric tubulin has a very low GTPase activity, which is activated upon polymerization at temperatures higher than 24 °C. In addition, no tubulin (or possible aggregates), or G, were immunoprecipitated without specific antibodies. Immune complexes were precipitated with Pansorbin, and each immunoprecipitate was subjected to SDS-PAGE, followed by immunoblotting and ECL detection of the protein bands. Polyclonal anti-tubulin (raised against the -tubulin C-terminal region of 422–431 amino acids) (3) and anti-G₁ (Santa Cruz Biotechnologies) antisera were utilized. No cross-reactivity between these antisera was observed.

Chemical Cross-linking—The method of Tucker and Goldstein (28) was used. Briefly, PC-tubulin, in its GDP-bound form, and purified G₁₂ subunits (at a protein ratio of 2:1) were incubated in buffer A for 30 min at 25 °C. The cross-linking agent EDC (Pierce) was added to the reaction mixture at the final concentration of 0.20 mM (concentration that limits nonspecific cross-linking), and the incubation was carried out for 2 h at 25 °C (28). The reaction was quenched by the addition of 2x SDS sample buffer. Proteins were separated by SDS-PAGE, followed by immunoblotting and ECL detection of the protein bands. Monoclonal anti--tubulin antibody (DM1A, Sigma) and polyclonal anti-G antiserum (Santa Cruz Biotechnologies) were used for immunodetection.

PLC₁ Assay—20 µg of Sf9 membrane protein was incubated with a [³H]PIP₂ substrate mixture (30 µM final concentration) as described (7). 10 µl of each GTPS, tubulin-GDP, G₁₂, and carbachol was added at appropriate concentrations to a final volume of 120 µl as indicated. When tested, GTPS was preincubated with the membranes for 30 min on ice. G₁₂ subunits were also preincubated with tubulin for 30 min on ice before the experiment. The tubes were incubated for 15 min at 37 °C with constant shaking, as described previously (7). [³H]inositol trisphosphate ([³H]IP₃) production was measured as described (29).

Analysis of Phosphoinositide Hydrolysis in SK-N-SH Cells—SK-N-SH neuroblastoma cells were grown in 6-well plates in DMEM supplemented with 10% fetal bovine serum and 50 units/ml penicillin-streptomycin. 24 h before the experiment, inositol-free DMEM supplemented with 2 µCi/well myo-[³H]inositol was added. The cells were washed three times with Locke's buffer, containing 10 mM LiCl, and incubated for 1 h with or without 330 nM taxol in the same buffer. After triplicate wash with Locke's buffer, 100 µM carbachol was added and the cells were incubated for 30 min at 37 °C. Carbachol effects were routinely controlled for by addition of 10 µM atropine. The reaction was stopped with ice cold 10% trichloroacetic acid, and the cells were scraped from wells with a rubber policeman and transferred to tubes. After sonication (as described above) and centrifugation at 20,000 x g for 15 min (4 °C), the supernatants were extracted with water-saturated ether and neutralized with 1 M NH₄HCO₃. Ion exchange chromatography (Dowex AG 1-X8 resin, formate form, Bio-Rad) of the samples was performed as described (29). Total [³H]inositol phosphates were quantified by liquid scintillation counting. The inositol phosphate content of SK-N-SH cells at the start of the experiment (0% increase) was 0.98 ± 0.31 x 10³ dpm per 10⁶ cells.

Microscopy—SK-N-SH neuroblastoma cells were plated onto glass coverslips in 12-well culture plates at a density of 1 x 10⁵. Where indicated, cells were pretreated for 1 h with 330 nM colchicine or taxol. After a PBS wash the cells were treated for the indicated times with 100 µM carbachol, 10 µM atropine, or both. After washing with PBS buffer the cells were immediately fixed in –20 °C methanol for 3 min and washed three times, 10 min each, in PBS, containing 0.1% Triton X-100. The cells were blocked for 40 min in PBS, containing 5% milk, and washed in PBS. Subsequently, the cells were incubated for 1 h with a polyclonal rabbit anti-G antiserum (Santa Cruz Biotechnologies) at a dilution of 1:100. Following a PBS wash, a secondary FITC (fluorescein isothiocyanate)-conjugated goat anti-rabbit antiserum (EY Labs) was applied for 1 h at a dilution of 1:100. The cells were washed and incubated for 1 h in blocking buffer, containing 10 µg/ml rabbit IgG. Subsequently a monoclonal anti--tubulin antibody (DM1A, Sigma) was applied for 1 h at a dilution of 1:500. Following a PBS wash, secondary Texas Red-conjugated goat anti-mouse antibody (Jackson ImmunoResearch Laboratories) (1:100 dilution) was applied for 1 h, followed by washing and mounting of the coverslips. Images were acquired using a laser scanning confocal microscope (Ziess LSM 510) equipped with a x63 water immersion objective. A 488-nm beam from an argon-krypton laser was used for the excitation of FITC, whereas a 543-nm beam was used for Texas Red excitation. Emission from FITC was detected through a BP505 filter, whereas emission from Texas Red was detected through a LP560 filter. Areas of antibody colocalization appeared in yellow. Differential interference contrast images of the cells were regularly acquired as well. Coverslips were examined at random. For each experimental condition a total of 80 randomly selected cells over six consecutive experiments were evaluated for G and tubulin distribution and colocalization. Final image composites were created using Adobe Photoshop 6.0. No specific FITC or Texas Red labeling was observed in cells treated with mouse or rabbit preimmune serum instead of anti-tubulin or anti-G antisera, respectively. Texas Red labeling was not observed when the anti-tubulin antibody was preincubated overnight at 4 °C with PC-tubulin (1:1 ratio), and FITC labeling was not detected when the anti-G antiserum was preincubated with purified G, both conditions tested at the same antibody dilutions (1:100) afterward.

Materials—[-³²P]GTP was from ICN Biomedicals, Inc. [³²P]AAGTP and AAGTP were synthesized as described (26). p-Azidoaniline was synthesized by Dr. W. Dunn (University of Illinois at Chicago). [³H]PIP₂ was from American Radiolabeled Chemicals Inc. [³H]QNB was from Amersham Biosciences. GTPS, GTP, and GDP were from Roche Applied Science. Carbachol, atropine sulfate, and PIP₂ were from Sigma Chemical Co. The cross-linking agent EDC was from Pierce, and Bio-Gel P6-DG was from Bio-Rad. P11 cellulose phosphate was from Whatman. All other reagents were of analytical grade.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Microtubule Stabilization Inhibits Agonist-induced PLC₁ Activation—To verify the physiological significance of tubulin regulation of PLC1 signaling in vivo, SK-N-SH neuroblastoma cells were treated with the microtubule-stabilizing agent taxol before stimulating the cells with the muscarinic receptor agonist, carbachol (Fig. 1). Because taxol increases the formation of microtubule polymers and inhibits microtubule dynamics, the pool of dimeric tubulin available for regulation of PLC1 is decreased. As seen in Fig. 1, the normal 6-fold increase in phosphoinositide hydrolysis elicited by carbachol (610 ± 67% (±S.D.)) was reduced by half in taxol-pretreated cells (350 ± 26% (±S.D.)).

View larger version (21K): [in this window] [in a new window]   FIG. 1. Taxol-evoked stabilization of microtubules inhibits PLC signaling in SK-N-SH neuroblastoma cells. Myo-[3H]inositol-prelabeled SK-N-SH cells were treated for 1 h with 330 nM taxol (where indicated). Carbachol (100 µM) was added (where indicated), the samples were incubated for 30 min at 37 °C, and the total inositol phosphate production was measured as described. Values are means ± S.D. of three independent experiments performed in triplicate. **, significantly different from control cells (p < 0.001); *, significantly different from carbachol-treated cells (p < 0.01), Student's t test.

Agonist-evoked Membrane Association of Tubulin Is Not Assisted by G Subunits—It has been previously shown that both G and G subunits interact with tubulin and these interactions reciprocally regulate microtubule polymerization in vitro (12, 13). Although we have shown that interaction of tubulin with certain G subunits, including G_q, regulates intracellular signaling (4–8), the role of G in such membrane-located signaling events has not been evaluated. Because G subunits appear to regulate the translocation of signaling enzymes to their membrane-located signaling targets (30–32), we initially aimed to determine whether G also modifies the recruitment of tubulin to the plasma membrane in response to carbachol stimulation (3).

Membranes from SK-N-SH neuroblastoma cells were incubated with tubulin-[³²P]AAGTP in the presence or absence of carbachol and/or purified G subunits (Fig. 2). As seen previously (7), association of tubulin with SK-N-SH membranes increased by 133 ± 11% (±S.D.) after carbachol stimulation. However, when G complexes were present in the medium, the carbachol-induced increase in tubulin association with the membrane was blocked. At the concentration used, G had no effect on the "basal" association of tubulin with the membrane.

View larger version (29K): [in this window] [in a new window]   FIG. 2. G prevents carbachol-induced membrane association of tubulin. SK-N-SH neuroblastoma cell membranes were incubated with tubulin-[32P]AAGTP (1 µM), carbachol (100 µM), and/or G (20 µg/ml) for 5 min, followed by UV irradiation as described under "Experimental Procedures." Membranes were washed and subjected to SDS-PAGE and phosphorimaging analysis. Densitometric values are means ± S.D. of four independent experiments with similar results. *, significantly different from the control, p < 0.05, Student's t test. A representative experiment is shown on the top. While tubulin is recruited to the membrane in response to carbachol, the addition of G inhibits this process.

Concomitant Binding of G and PIP₂ to Tubulin Prevents Tubulin Association with the Plasma Membrane—Tubulin binds PIP₂ (7), which appears to act as a membrane anchor (9). Coordinated binding of PIP₂ and G to the pleckstrin homology domain of GRK2 synergistically enhances GRK2 membrane association in response to agonist stimulation (14). To test if PIP₂ and G similarly affected tubulin, membranes from Sf9 cells containing recombinant m₁ muscarinic receptors, G_q and PLC1, were incubated with tubulin-[³²P]AAGTP in the presence or absence of carbachol, G, and/or PIP₂ (Fig. 3). Because G_q is overexpressed in this system, we initially tested whether exogenous G associated with the membranes. Less than 10% of the added G subunits bound to the membranes. Carbachol stimulation did not alter this.

View larger version (29K): [in this window] [in a new window]   FIG. 3. G inhibits membrane recruitment of tubulin and this is potentiated by PIP2. Membranes from Sf9 cells containing m1 muscarinic receptors, Gq, and PLC1 were incubated with tubulin-[32P]AAGTP (1 µM), with or without carbachol (100 µM), and/or (20 µg/ml), and/or PIP2 (30 µM), for 5 min at 23 °C, as described under "Experimental Procedures." Membranes were UV-irradiated, washed, and subjected to SDS-PAGE, immunoblotting with anti-G antiserum, and phosphorimage analysis. The phosphor image (A) of the G immunoblot (B) of a representative of four identical experiments with similar results is shown. G inhibits tubulin association and the resulting transactivation of Gq. When PIP2 was present, that inhibition is nearly complete.

Stimulation of m₃ muscarinic receptors by carbachol caused tubulin to associate with the membrane and transactivate G_q (182 ± 35% (±S.D.) increase in [³²P]AAGTP-labeled G_q) (Fig. 3A). G inhibited this process by 77 ± 15% (±S.D.), and G_q transactivation was completely blocked in the presence of PIP₂ and G. PIP₂ also decreased the amount of purified G that associated with the membrane (Fig. 3B). Thus, unlike GRK2/PIP₂/G (14), tubulin/PIP₂/G complexes did not translocate to the membrane in response to agonist stimulation.

G₁₂ Subunits Decrease Agonist-evoked Membrane Association of Tubulin by Promoting Tubulin Polymerization—G subunits attenuate the carbachol-induced association of tubulin with the plasma membrane. Because carbachol stimulation also appears to cause microtubule depolymerization (3, 7), a possible mechanism for G inhibition of these processes is G-induced microtubule assembly (13). To test this, tubulin was incubated first with G₁₂ subunits and, then, with SK-N-SH membranes under conditions that allow microtubule polymerization. Microtubule polymerization increased more than 2-fold (120 ± 13% (±S.D.) increase above control) in the presence of G₁₂ subunits (Fig. 4). The majority of tubulin was either in microtubules or soluble tubulin dimers. Small amounts of tubulin associated with the SK-N-SH membranes. This might be due to nonspecific membrane association of G-bound tubulin dimers, because small amounts of G routinely associated with the membranes independent of carbachol stimulation. Carbachol stimulation tripled the amount of membrane-associated tubulin (199 ± 37% (±S.D.) increase above control), whereas the microtubule mass correspondingly decreased (254 ± 43% (±S.D.)). However, microtubule polymerization, but not membrane association of tubulin, was observed in the presence of carbachol and G₁₂ subunits. Thus, G₁₂ appeared to drive tubulin toward polymerization and away from association with the plasma membrane in response to agonist stimulation. This is consistent with the microtubule-polymerizing role of G seen with pure components (13). It is also consistent with the observation that taxol-induced microtubule polymerization inhibits membrane-located PLC1 signaling (Fig. 1).

View larger version (18K): [in this window] [in a new window]   FIG. 4. G12 inhibits agonist-evoked membrane recruitment of tubulin due to increased microtubule polymerization. Redistribution of tubulin between microtubules and membranes was studied during the course of carbachol stimulation of SK-N-SH membranes both in the presence and absence of purified G12. PC-tubulin (1.5 mg/ml) was incubated for 30 min at 37 °C (conditions that allow microtubule assembly) in the presence of SK-N-SH membranes (0.3 mg/ml), with or without carbachol (100 µM), and/or G (0.13 mg/ml), as described under "Experimental Procedures." Membranes and microtubules were pelleted by centrifugation at 37 °C. Pellets were resuspended in cold polymerization buffer and kept on ice for 1 h to depolymerize microtubules. Tubulin was then separated from the membranes by centrifugation at 4 °C. Both tubulin supernatants and pelleted membranes were subjected to SDS-PAGE, immunoblotting with anti-tubulin antibody, and ECL detection of the protein bands. A representative of three identical experiments with similar results is shown. Numerical values are given under "Results." Note that, although carbachol stimulation triggers tubulin association with membranes, the addition of G causes microtubule polymerization and an increase in the microtubule pellet even after carbachol exposure.

G Reverses the Inhibition of PLC₁ Caused by High Concentration of Tubulin Dimers—High, millimolar, concentrations of tubulin inhibit PLC1 presumably through receptor-independent association of tubulin with PIP₂/PLC1 sites at the cell membrane (7, 9). Diminished access of PLC1 to its substrate, PIP₂, and/or obstruction of G_q coupling to PLC1, appear to be responsible for this inhibition (9). We wanted to determine whether G binding of tubulin dimers would also block this process. Tubulin-GDP was used in these experiments, because it did not transactivate G_q and, thus, did not activate PLC1 (3).

PLC1 activation was evaluated in the presence and absence of carbachol, GTPS, tubulin-GDP, and G₁₂ subunits in Sf9 membranes containing recombinant m₁ muscarinic receptors, G_q, and PLC1 (Fig. 5). Both carbachol and GTPS increased IP₃ generation. When applied together, they significantly increased the activation of PLC1 (90 ± 10% (±S.D.) above the control). (Note that coexpression of G_q and PLC1 in the Sf9 cells increased significantly the basal PLC1 activity. This decreased the percent agonist stimulation of PLC1, because m₁ muscarinic receptors were moderately expressed (240 fmol/mg of membrane protein).) At the concentrations tested, tubulin-GDP or G₁₂ did not affect the basal activity of PLC1. G₁₂ also did not change carbachol activation of PLC1 regardless of the presence of GTPS. This suggested that added G₁₂ did not directly affect membrane PLC1 signaling. However, carbachol-activated PLC1 was inhibited by 64 ± 4% (±S.D.) by tubulin-GDP (note that tubulin with GTP-bound transactivates G_q (3)). When tubulin-GDP was preincubated with G₁₂ subunits, this inhibition was reversed by 60 ± 13% (±S.D.). Thus, the binding of tubulin to G₁₂ subunits appeared to prevent its membrane association and involvement in the regulation of PLC1. A mechanism for control of PLC1 inhibition by high tubulin concentrations was suggested by these experiments.

View larger version (32K): [in this window] [in a new window]   FIG. 5. G12 reverses the inhibition of PLC1 at high tubulin concentrations. Sf9 membranes, containing recombinant m1 muscarinic receptors, Gq, and PLC1 were assayed for PLC1 activity as described under "Experimental Procedures." Effects of GTPS (10 µM), carbachol (100 µM), tubulin-GDP (100 µg/ml), and G (50 µg/ml) on IP3 generation were tested as indicated. The IP3 production of control samples (no additions) was 0.59 ± 0.04 nmol/min/mg of protein (±S.D.). Values are means ± S.D. of three independent experiments performed in duplicate. *, significantly different from the control samples; **, significantly different from the samples treated with GTPS plus carbachol; ***, significantly different from the samples treated with GTPS plus carbachol plus tubulin-GDP. p < 0.05 for all samples, Student's t test. While tubulin-GDP inhibits carbachol and GTPS stimulated PLC1, G capture of tubulin is able to restore PLC1 activation.

G₁₂ Subunits Preferentially Interact with Tubulin-GDP— G_q interacts with, and is transactivated by, tubulin-GTP (3). G subunits decorate brain microtubules (13), which consist of tubulin-GDP (to promote microtubule polymerization, G must interact with tubulin at some time). We wanted to test directly whether G₁₂ bound preferentially to tubulin-GDP. Purified G₁₂ was incubated with tubulin-GTP, tubulin-GDP, or tubulin dimers, which had been stripped of nucleotide. Tubulin/G₁₂ coimmunoprecipitation was tested with both anti-tubulin (Fig. 6A) and anti-G₁ antisera (Fig. 6B). In both cases, preferential coimmunoprecipitation of G₁₂ subunits with tubulin-GDP was observed. Coimmunoprecipitation of G₁₂ with tubulin-GDP was 157 ± 28% (±S.D.) higher that that with tubulin-GTP. This corresponded to the coimmunoprecipitation of tubulin-GDP with G₁₂, which was 198 ± 32% (±S.D.) higher than that of tubulin-GTP. Similarly, when extracts from SK-N-SH neuroblastoma membranes were incubated with GTP- or GDP-liganded tubulin, preferential coimmunoprecipitation of G₁₂ with tubulin-GDP was detected. G₁₂/tubulin-GDP coimmunoprecipitation was 136 ± 25% (±S.D.) higher than that of G₁₂/tubulin-GTP (Fig. 6C). Thus, although G_q interacted with, and was activated by, tubulin-GTP, G₁₂ subunits interacted preferentially with the GDP-bound form of tubulin.

View larger version (17K): [in this window] [in a new window]   FIG. 6. Prenylated G12 binds preferentially tubulin-GDP. A and B, PC-tubulin with the indicated nucleotide were incubated with purified G (at a protein ratio of 2:1) as described under "Experimental Procedures." Immunoprecipitation was carried out with anti-G1 (A) or anti-tubulin antiserum (B). Samples were subjected to SDS-PAGE, immunoblotting with either anti-tubulin (A) or anti-G1 (B) antisera, and ECL detection of the protein bands. C, extracts of SK-N-SH membranes (0.5 mg/ml) were incubated with 1 µM PC-tubulin in either GDP- or GTP-bound forms as indicated. Immunoprecipitation with anti-tubulin antiserum and immunodetection with anti-G1 antiserum were performed as in A and B. As also seen in A and B, preferential interaction of G12 with the GDP-bound form of tubulin is apparent. D, tubulin-GDP was incubated with either G12, G11, or G12(68S) as indicated. Immunoprecipitation was performed with anti-tubulin antiserum and immunodetection with anti-G1 antiserum. Tubulin binds to G12 but not to G11 or the prenylation-deficient G12(68S). The examples shown in A–D are representative of six identical experiments with similar results. Although not shown, each experimental condition described (A–D) included control samples containing preimmune serum instead of the antiserum used for immunoprecipitation. Neither G12 nor tubulin precipitation was observed in the absence of antibody.

It has also been shown that G₁₂ subunits in which the ₂ polypeptide is isoprenylated promote microtubule assembly (13). To evaluate if this lipid modification is important for G interaction with tubulin-GDP dimers, as it is for their microtubule binding, coimmunoprecipitation experiments of tubulin-GDP with G₁₂, G₁₁, and G₁₂(68S) were performed (Fig. 6D). The mutant G₁₂(68S) is unable to undergo post-translational modification and, thus, does not carry a geranylgeranyl moiety (33). G₁₂, but not G₁₂(68S) or G₁1 (which is farnesylated rather that geranylated), coimmunoprecipitated with the GDP-bound tubulin dimers. These results suggested that geranylgeranylation of the subunit is important for its interaction with the GDP-bound form of dimeric tubulin.

Chemical Cross-linking of G and Tubulin—The interaction between G₁₂ and tubulin-GDP was verified by chemical cross-linking (Fig. 7). Tubulin-GDP was incubated with G₁₂ or G₁1, followed by cross-linking of the proteins with the agent EDC, SDS-PAGE, and immunoblotting with anti-G or anti-tubulin antisera. A band consistent with the electrophoretic mobility of cross-linked G₁₂ dimers (molecular mass of 40–42 kDa), and a band, consistent with that of tubulin-G₁₂ complexes (molecular mass of 140 kDa), were detected with the anti-G antibody (Fig. 7, lane a). Immunoblotting with the anti-tubulin antibody revealed the presence of both tubulin monomers (at 50 kDa) and cross-linked tubulin dimers (at 100 kDa) (Fig. 7, lane c). A band at 140 kDa was also recognized, confirming the interaction between tubulin and G₁₂. Under the chosen experimental conditions, (28) higher order molecular weight complexes were not detected by either antiserum. When G₁₁ subunits were tested, labeling at 140 kDa was barely visible (Fig. 7, lanes b and d). These experiments confirmed tubulin-GDP interaction with G protein 1₂ subunits. It also indicated that the stoichiometry of this interaction was 1:1.

View larger version (14K): [in this window] [in a new window]   FIG. 7. G12 cross-linking to tubulin-GDP. To test the interaction of G12 with tubulin-GDP, purified G12 subunits (0.63 mg/ml) were incubated with PC-tubulin in the GDP-bound form (protein:protein ratio of 1:2) for 30 min at 25 °C. Interacting partners were cross-linked with the agent EDC as described under "Experimental Procedures." Purified G11 subunits were employed as a control. Protein complexes were separated by SDS-PAGE, followed by immunoblotting and ECL detection of the protein bands. Monoclonal anti--tubulin antibody (DM1A) and polyclonal anti-G antiserum were used for immunodetection. A representative experiment of four identical ones with similar results is shown. Suggested identity of the cross-linked protein species is indicated. It is evident that, although G12 forms complexes with tubulin-GDP, G11 does not.

Agonist-regulated Spatiotemporal Pattern of Tubulin-G Colocalization in Neuroblastoma SK-N-SH Cells—Because tubulin and G interact with each other, they would be expected to colocalize at specific cellular locations. In addition, if tubulin/G interaction is signal-regulated, spatiotemporal colocalization should exist in the living cell. To test this, SK-N-SH cells were studied by confocal microscopy before and after carbachol stimulation (Fig. 8). Tubulin is shown to regulate PLC1 signaling in this neuroblastoma cell line (3).

View larger version (31K): [in this window] [in a new window]   FIG. 8. Colocalization of tubulin and G. SK-N-SH cells were treated for the indicated times with 100 µM carbachol as described under "Experimental Procedures." The cells were fixed and incubated with anti-G antiserum and monoclonal anti--tubulin antibody (DM1A). The anti-G antiserum was recognized with a secondary FITC-conjugated antiserum (green), and the anti-tubulin antibody was recognized with a secondary Texas Red-conjugated antiserum (red). Microtubules are seen in samples prior to carbachol treatment and 15 min after the drug application but are not observed at the 2-min point. Areas of tubulin/G colocalization appear in yellow. Carbachol-induced tubulin/G colocalization is greatest at the cell membrane after 2 min and in vesicle-like structures in the cytosol after 15 min.

In unstimulated SK-N-SH cells, tubulin and G colocalized sporadically in the cytoplasm (Fig. 8, 0 min). G, but not tubulin, was seen at the membrane of the resting cells. Two minutes after carbachol stimulation, microtubules depolymerized and tubulin translocated to the cell membrane (Fig. 8, 2 min). G and tubulin colocalized at the membrane. Fifteen minutes subsequent to carbachol exposure, tubulin and G were colocalized in vesicle-like structures in the cytosol (Fig. 8, 15 min). Diminished association of tubulin with the plasma membrane was seen at this time point. As previously reported (3, 7, 9), carbachol-induced redistribution of tubulin was inhibited by atropine.

These findings were corroborated by coimmunoprecipitation studies of carbachol-treated SK-N-SH cells (Fig. 9). Two minutes after carbachol stimulation G/tubulin complexes were immunoprecipitated from detergent-extracted SK-N-SH membranes (increase by 61 ± 10% (±S.D.) compared with control untreated cells). Fifteen minutes later such complexes were observed in the cytosol (increase by 44 ± 17% (±S.D.) compared with control cells). Thus, both confocal microscopy and coimmunoprecipitation experiments in cells indicated agonist-evoked interaction between tubulin and G at the plasma membrane as well as translocation of these complexes to the cell cytosol at the offset of signaling.

View larger version (18K): [in this window] [in a new window]   FIG. 9. Tubulin coimmunoprecipitation with G during the course of carbachol stimulation of SK-N-SH cells. SK-N-SH cells were treated for the indicated times with 100 µM carbachol as in Fig. 8. Membrane and cytosol fractions were immediately separated as described under "Experimental Procedures." Extracted membrane and cytosol fractions were subjected to immunoprecipitation with either (A) anti-G (membrane extracts) or (B) anti-tubulin (cytosol fractions) antisera. Immunoblotting was performed, with monoclonal anti--tubulin (membrane extracts) or polyclonal anti-G (cytosol fractions) antibodies as described. Representative experiments of four with similar results are shown. Increased G/tubulin coimmunoprecipitation from membrane extracts was observed 2 min after carbachol exposure of the cells. Fifteen minutes later G/tubulin coimmunoprecipitation increased from the cytosol. These data appeared to match those from immunocytochemical experiments in Fig. 8. When 10 µM atropine was applied before carbachol, no time-related changes in G/tubulin coimmunoprecipitation were detected.

Microtubule Depolymerization Increases Membrane Recruitment of Tubulin and Its Colocalization with G—We used compounds that would either increase or decrease tubulin dimer concentrations to evaluate tubulin translocation and colocalization with G in response to agonist stimulation. As previously observed (9), colchicine depolymerized SK-N-SH cell microtubules and increased the amount of membrane-associated tubulin by 44 ± 11% (±S.D.) (Fig. 10B). These biochemical data correlated well with images showing colchicine-induced redistribution of tubulin toward the plasma membrane regardless of carbachol stimulation (Fig. 10, A and C, center panels). Tubulin/G colocalization in the cytosol was also readily observed. To the contrary, taxol stabilized the microtubule network and decreased membrane-associated tubulin by 27 ± 17% (±S.D.) (Fig. 10B). Images from these experiments showed taxol-bundled microtubules and no tubulin colocalization with membrane G (Fig. 10, A and C, bottom panels). In addition, the G-containing endocytic vesicles, observed after 2 min of carbachol stimulation, also lacked tubulin. These findings corroborated the notion that tubulin dimers translocated to the plasma membrane in response to carbachol stimulation might subsequently sequester through G-containing endocytic vesicles.

View larger version (23K): [in this window] [in a new window]   FIG. 10. Microtubule stabilization prevents agonist-evoked translocation of tubulin and colocalization with membrane G. SK-N-SH cells were pretreated for 1 h with or without 330 nM of the microtubule-depolymerizing agent, colchicine, or the microtubule-stabilizing agent, taxol. A, representative images of control and drug-treated cells, as indicated. After the treatment the cells were fixed and incubated with DM1A anti--tubulin antibody and secondary Texas Red-conjugated antibody. Cells were examined by confocal microscopy as described under "Experimental Procedures." Note the extensive microtubule network of control cells, depolymerized tubulin along the cell membrane in colchicine pretreated cells and microtubule bundles in taxol-pretreated cells. B, membrane fractions were prepared from cells similar to those shown in A and membrane-associated tubulin was detected by immunoblotting. A representative experiment of three with similar results is shown. Colchicinepretreatment increased membrane-associated tubulin, whereas taxol pretreatment did not. C, cells pretreated with the indicated agents were subsequently treated for 2 min with either 100 µM carbachol or vehicle. Cells were fixed and incubated with anti-G antiserum and DM1A anti--tubulin antibody. The secondary antibody for anti-G was FITC-conjugated (green), whereas that for anti--tubulin was Texas Red-conjugated (red). Areas of tubulin/G colocalization appear in yellow. An arrow denotes such an area in the control cells at 2 min post-carbachol. Carbachol stimulation does not evoke tubulin/G colocalization at the plasma membrane in taxol-pretreated cells.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
G heterodimers perform many diverse intracellular regulatory functions. Adenylyl cyclases and PLC isozymes, ion channels, and kinases are directly regulated by G protein subunits (for reviews see Refs. 34 and 35). The membrane association of G_s, G_q, and G_z requires G subunits (36–38). G subunits may also act as "molecular levers" for activated receptors to pry open the G guanine nucleotide binding pocket and thus, release the GDP (38). G is involved in the targeting of cytosolic GRKs, phosphoinositide 3-kinase (PI3K), as well as PLC1, to the membrane of the cell (30–32). G isoform selectivity for this process has been demonstrated (36, 39). G₁₂, but not G₁₁ subunits, bind to microtubules and promote microtubule assembly (13) (Fig. 4). G subunits regulate the migration of the centrosome around the nucleus and hence, the orientation of the mitotic spindle, in embryos of Caenorhabditis elegans (40). This report suggests that, in the cell, G interaction with tubulin is signal-regulated and responsible for both down-regulation of tubulin involvement in PLC1 signaling and remodeling of the microtubule network. Cross-regulation between intracellular signaling and cytoskeletal reorganization is proposed.

The ability of G₁₂ subunits to affect tubulin regulation of PLC1 appeared related to their preferential interaction with tubulin-GDP. Both coimmunoprecipitation and chemical cross-linking experiments showed the specificity of this interaction (Figs. 6 and 7). This was in contrast to the G subunits G_i1, G_s, and G_q, which interacted preferentially with tubulin-GTP (3–5, 7–9, 25, 41). Such preferred interactions suggest a mechanism by which receptor activation evokes membrane association of tubulin-GTP leading to G_q transactivation and initiation of PLC1 signaling. This is followed by internalization of tubulin-GDP/G complexes at the offset of signaling. They also explain the accelerated microtubule dynamics caused by purified G_i1 (12) and the increased microtubule polymerization promoted by G₁₂ (13) (Fig. 4). Chemical cross-linking also indicated that the ratio of tubulin/G binding was 1:1 (Fig. 7). EMAP, an echinoderm MAP, which has significant sequence homology with G (42), binds to both microtubules and tubulin dimers (43). The ratio of dimeric tubulin/EMAP binding is 1:1.

Down-regulation of tubulin involvement in PLC1 signaling by G subunits was supported by the following observations. G subunits attenuated agonist-evoked membrane association of tubulin and G