Tubulin, Gq, and Phosphatidylinositol 4,5-Bisphosphate Interact to Regulate Phospholipase Cbeta 1 Signaling*

(Received for publication, August 5, 1996, and in revised form, December 17, 1996)

Juliana S. Popova Dagger §, James C. Garrison , Sue Goo Rhee par and Mark M. Rasenick Dagger **Dagger Dagger

From the Dagger  Department of Physiology and Biophysics and the Committee on Neuroscience, University of Illinois College of Medicine, the ** Department of Psychiatry, University of Illinois College of Medicine, Chicago, Illinois 60612, the  Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908, and the par  Laboratory of Cell Signaling, NHLBI, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

The cytoskeletal protein, tubulin, has been shown to regulate adenylyl cyclase activity through its interaction with the specific G protein alpha  subunits, Galpha s or Galpha i1. Tubulin activates these G proteins by transferring GTP and stabilizing the active nucleotide-bound Galpha conformation. To study the possibility of tubulin involvement in Galpha q-mediated phospholipase Cbeta 1 (PLCbeta 1) signaling, the m1 muscarinic receptor, Galpha q, and PLCbeta 1 were expressed in Sf9 cells. A unique ability of tubulin to regulate PLCbeta 1 was observed. Low concentrations of tubulin, with guanine nucleotide bound, activated PLCbeta 1, whereas higher concentrations inhibited the enzyme. Interaction of tubulin with both Galpha q and PLCbeta 1, accompanied by guanine nucleotide transfer from tubulin to Galpha q, is suggested as a mechanism for the enzyme activation. The PLCbeta 1 substrate, phosphatidylinositol 4,5-bisphosphate, bound to tubulin and prevented microtubule assembly. This observation suggested a mechanism for the inhibition of PLCbeta 1 by tubulin, since high tubulin concentrations might prevent the access of PLCbeta 1 to its substrate. Activation of m1 muscarinic receptors by carbachol relaxed this inhibition, probably by increasing the affinity of Galpha q for tubulin. Involvement of tubulin in the articulation between PLCbeta 1 signaling and microtubule assembly might prove important for the intracellular governing of a broad range of cellular events.


INTRODUCTION

Heterotrimeric GTP-binding proteins (G proteins) couple a wide range of cell surface receptors to membrane-associated effector molecules, including adenylyl cyclase, PLCbeta 1,1 and ion channels. Receptor activation leads to GTP binding by, and activation of, G protein alpha  subunits. The activated alpha  subunit, and in some instances the beta gamma dimer of the G protein, transmits the receptor-generated signal to the effector molecule (1, 2). Many hormones and neurotransmitters elicit intracellular responses by activating receptors linked to Galpha q-mediated stimulation of PLCbeta 1 (3-8). Although isoforms of PLCbeta have been demonstrated to be activated by G protein alpha  and beta gamma subunits, PLCbeta 1 appears to be activated by Galpha q (9-11). PLCbeta 1 hydrolyzes the lipid precursor phosphatidylinositol 4,5-bisphosphate (PIP2) to generate two second messengers, diacylglycerol and inositol 1,4,5-trisphosphate (IP3). Diacylglycerol and IP3 trigger a variety of cellular functions by activating protein kinase C and mobilizing stored calcium (12).

The cytoskeletal GTP-binding protein tubulin activates or inhibits adenylyl cyclase through its interaction with Galpha s or Galpha i1 (13-16). Tubulin binds to these proteins and activates them via direct transfer of GTP. Both guanine nucleotide transfer and the stabilization of the active Galpha conformation are thought responsible for the sustained G protein activation by tubulin (15, 16). Complexes of dimeric tubulin with Galpha s and Galpha i1 exist in the synaptic membrane (17), and these complexes provide the physical framework for the interface between G protein-mediated signal transduction and the cytoskeleton. Previous work has focused on the regulation of adenylyl cyclase by tubulin, but the possibilities that other G protein-mediated signaling enzymes suffered similar regulation waranted investigation.

Although an association of PLC with the turkey erythrocyte cytoskeleton has been reported (18), the effect of tubulin on PLCbeta 1 signaling has not been examined. The current study was designed to determine whether tubulin plays a role in the regulation of PLCbeta 1, and, conversely, how phosphoinositides might affect the ability of tubulin to assemble into microtubules or to interact with G proteins. This was approached using Sf9 cells expressing various combinations of m1 muscarinic receptors, Galpha q, and PLCbeta 1. It was determined that tubulin does indeed bind to Galpha q and activates that molecule via the direct transfer of GTP. It is also demonstrated that PLCbeta 1 may enjoy a dual regulation by dimeric tubulin, the molecule inhibiting PLCbeta 1 by binding to the enzyme substrate PIP2. This represents another arena in which cell structure and cell signaling share a complex regulatory liaison.


EXPERIMENTAL PROCEDURES

Baculovirus-directed Protein Expression in Sf9 Cells, Membrane Preparation, and Western Blotting

Sf9 cells were infected in different combinations with baculoviruses bearing the m1 muscarinic receptor, Galpha q, or PLCbeta 1 cDNAs at a ratio of 1:1:1 and multiplicity of infection of 5. The construction of the recombinant baculoviruses was described earlier (19-21). Cells were harvested after 65 h, sonicated in ice-cold 20 mM Hepes, pH 7.4, 1 mM MgCl2, 100 mM NaCl, 1 mM DTT, 0.3 mM phenylmethylsulfonyl fluoride. After centrifugation at 500 × g, the supernatant was collected and centrifuged at 100,000 × g for 30 min at 4 °C. The membrane pellet was washed, resuspended in the same buffer, and frozen in aliquots in liquid nitrogen. Protein concentrations were determined by the Bradford dye-binding assay (22), using bovine serum albumin as a standard. The expression of recombinant proteins was verified by immunoblotting. Membrane proteins transferred to nitrocellulose were probed with antisera specific for the m1 muscarinic receptor (number 71, from G. Luthin, Philadelphia), Galpha q/11 (number 0945, from D. Manning, Philadelphia) or PLCbeta 1 (anti-holoenzyme) at a dilution of 1:500. Biotinylated goat anti-rabbit IgG and streptavidin-alkaline phosphatase conjugate were used for detection, and a Molecular Dynamics densitometer was used to assess expression levels, which varied by no more than 10% for a given recombinant polypeptide. Receptor binding studies using [3H]QNB (0.02-4.00 nM) as a ligand were also performed to assess the number and affinity of the m1 muscarinic receptors (23). The nonspecific binding, measured in the presence of 1 µM atropine, was less than 10%. Saturation isoterms were analyzed using the LUNDON 1 program.

Purification of PLCbeta 1

cDNA encoding the entire rat PLCbeta 1 amino acid sequence was cloned into the pSC11 vaccinia virus expression vector and HeLa cells were infected with recombinant virus as described (24). The enzyme was extracted from the membrane with 2 M KCl for 2 h. The extracted PLCbeta 1 was consecutively subjected to preparative HPLC on phenyl-5PW (150 × 21.5-mm column), analytical HPLC on heparin-5PW (75 × 7.5-mm column), and ion-exchange HPLC on Mono-Q FPLC (60 × 7-mm column) chromatography as described (24).

Tubulin Preparations

Microtubule proteins were prepared as described (25). Microtubule-associated proteins were removed by phosphocellulose chromatography (PC-tubulin) as described (26). Tubulin-GppNHp or tubulin-[32P]AAGTP were prepared from PC-tubulin by the removal of GTP by means of charcoal pretreatment, followed by incubation in the presence of 150 µM of GppNHp (or [32P]AAGTP) for 30 min on ice as described (27). Prior to use, tubulin-guanine nucleotide preparations were passed through P6-DG columns twice in order to remove unbound nucleotide. After this procedure 0.4-0.6 mol of nucleotide were bound per mol of tubulin. This remained stable even after five desalting steps (27). Tubulin-guanine nucleotide concentrations used throughout the study were based on the protein concentration. [32P]AAGTP and AAGTP were synthesized as described (28).

PLCbeta 1 Assay

20 µg of Sf9 cell membranes, containing indicated recombinant proteins, and 40 µl of substrate mixture (PIP2 at a final concentration of 30 µM (1 µCi of [3H]PIP2), evaporated under a stream of nitrogen and sonicated on ice for 5 min in 20 mM Tris maleate, pH 6.8, 6 mM MgCl2, 4 mM ATP, EGTA, and CaCl2 to give a final concentration of 50 nM Ca2+ (29) and deoxycholate to give a final concentration of 1 mM) were incubated in a Branson water bath sonicator for 15 min at 4 °C. Ten µl of GppNHp, tubulin-GppNHp, or tubulin, with or without 10 µl of carbachol, were added at appropriate concentrations to a final volume of 80 µl, and the tubes were incubated for 15 min at 37 °C with constant shaking. Aqueous and lipid phases were separated as described (30), and [3H]inositol trisphosphate ([3H]IP3) in the aqueous phase was quantified by liquid scintillation counting.

Reconstitution of Recombinant Galpha q and PLCbeta 1

Sf9 cells were infected with Galpha q baculovirus and extracted after nitrogen cavitation (500 p.s.i. for 20 min) with 0.1% CHAPS in 20 mM Hepes, pH 7.5, 1 mM EDTA, 3 mM MgCl2, 150 mM NaCl, 0.1 mM phenylmethylsulfonyl fluoride, and 2 µg/ml each of aprotinin, leupeptin, pepstatin, and benzamidine at 4 °C for 1 h with constant stirring. The tubes were centrifuged at 100,000 × g for 30 min at 4 °C. Galpha q-containing extracts (protein concentrations as indicated) were reconstituted into lipid vesicles (phosphatidylethanolamine:PIP2 at a 4:1 molar ratio) by means of sonication in 20 mM Tris maleate, pH 6.8, 6 mM MgCl2, 30 mM NaCl, 120 mM KCl, and EGTA and CaCl2 to give a final concentration of 50 nM Ca2+ (29). The PIP2 final concentration was 30 µM (1 µCi of [3H]PIP2). 40 µl of this mixture was transferred to each experimental tube; 20 µl of purified recombinant PLCbeta 1 (protein concentration as indicated) in 100 µg/ml bovine serum albumin was added, and the tubes were sonicated in a Branson water bath sonicator for 15 min at 4 °C as described above. Ten µl of GppNHp, tubulin-GppNHp, or tubulin, stripped of nucleotide, was added at appropriate concentrations to a final volume of 90 µl. The samples were incubated for 10 min at 30 °C with constant shaking. The reaction was stopped and [3H]IP3 was quantified as described above.

Photoaffinity Labeling and Nucleotide Transfer

Sf9 cell membranes, expressing recombinant proteins, were incubated with 1 µM tubulin-[32P]AAGTP in the presence or absence of 1 mM carbachol in 100 mM Pipes buffer, pH 6.9, 2 mM EGTA, 1 mM MgCl2 (buffer A) for 10 min at 23 °C with constant shaking. The tubes were UV-irradiated for 4 min on ice, and the reaction was quenched with ice-cold Hank's buffer, 1 mM MgCl2, 4 mM DTT. After centrifugation at 100,000 × g for 15 min, the membrane pellets and concentrated supernatants (Amicon, Centriprep 10) were dissolved in 3% SDS Laemmli sample buffer with 50 mM DTT. Membrane (70 µg of protein) and supernatant fractions (20 µg of protein) were subjected to SDS-PAGE (10% acrylamide and 0.133% bisacrylamide) as described (31). Gels were either stained (Coomassie Blue) or subjected to Western blotting, followed by autoradiography (Kodak XAR-5 film). The radioactivity of the bands was measured with a PhosphorImager (Molecular Dynamics).

Immunoprecipitation

Sf9 cells were infected separately with baculoviruses bearing either the m1 muscarinic receptor, Galpha q, or PLCbeta 1 cDNA. Each of the three different membrane preparations was extracted with 1% sodium cholate in buffer A for 1 h at 4 °C with constant stirring. The tubes were centrifuged at 20,000 × g for 15 min at 4 °C. Membrane extracts (0.5 mg/ml membrane protein) were incubated with 1 µM tubulin-[32P]AAGTP, as described above. After UV irradiation and preclearing (Calbiochem Standardized Pansorbin cells, manufacturer's instruction), each membrane extract was incubated overnight with appropriate specific or nonspecific antiserum (1:20 dilution) at 4 °C with constant shaking. Immune complexes were precipitated with pansorbin cells, and 100 µg of protein of each immunoprecipitate were subjected to SDS-PAGE and autoradiography. The antisera used showed no cross-reactivity to tubulin.

Gel Filtration Assay (32)

Homogeneous PIP2 micelles were prepared by suspending 100 µg of PIP2 in 0.5 ml of buffer A and sonicating on ice for 5 min. Tubulin was incubated with PIP2 micelles in a Branson water bath sonicator for 15 min at 4 °C, followed by an additional 45 min on ice. The samples were run on a 0.7 × 50-cm column of Ultrogel AcA 34 at 4 °C. The column was equilibrated with buffer A (flow rate = 20 ml/h), and fractions of 0.5 ml were collected and assayed for protein. The data are given in arbitrary units since lipids quench the Bradford dye-binding assay (32).

Electron Microscopy

Samples were taken from tubulin polymerization reactions (2 mg/ml final protein concentration) carried out in buffer A, containing 3 mM MgCl2, 1 mM GTP, and 30% glycerol, for 30 min at 37 °C with constant shaking (26). PIP2, when added, was sonicated in buffer A as described. 10 µl of the reaction mixture was applied to a carbon/Formvar-coated grid for about 10 s. The grids were stained with several drops of 1% uranyl acetate and air-dried. Observations were made in a JEM 100CX (JEOL) electron microscope. For microtubule length determinations, at least 100 assembled structures were measured in multiple fields from two different grids representing two different experiments.

Materials

[alpha -32P]GTP was from ICN Biomedicals, Inc. [3H]PIP2 was from American Radiolabeled Chemicals Inc. [3H]QNB was from Amersham Corp. GppNHp, GDP, and GDPbeta S were from Boehringer Mannheim. Carbachol, atropine sulfate, PIP2, phosphatidylcholine, and phosphatidylethanolamine were from Sigma. p-Azidoaniline was synthesized by Dr. William Dunn (University of Illinois, Chicago). Ultrogel AcA 34 was from Pharmacia Biotech Inc. P6-DG was from Bio-Rad. All other reagents were of analytical grade.


RESULTS AND DISCUSSION

Receptor-independent Regulation of PLCbeta 1 by Tubulin

Simultaneous baculovirus-mediated expression of Galpha q and PLCbeta 1 was performed in Sf9 cells, and the effects of GppNHp and tubulin with GppNHp bound (tubulin-GppNHp) on Galpha q-regulated PLCbeta 1 activity were studied in membranes prepared from infected cells (Fig. 1). GppNHp increased the already high basal PLCbeta 1 activity in a concentration-dependent and saturable manner (Fig. 1A), confirming the functional coupling between the recombinant Galpha q and PLCbeta 1. GppNHp (at 100 µM) did not affect PLCbeta 1 activity in membranes from cells expressing only PLCbeta 1 (Table I), thus indicating that neither Gbeta gamma nor any Galpha resident in Sf9 cell membranes is capable of activating the expressed PLCbeta 1. Expressed Galpha q did not couple to any endogenous PLC activity. Endogenous PLC activity was also undetectable in the presence or absence of GppNHp. Tubulin-GppNHp had been shown to activate adenylyl cyclase in membranes from either COS 1 or C6 glioma cells (16, 17) in a manner similar to GppNHp. Surprisingly, it appeared that in membranes prepared from Sf9 cells expressing Galpha q and PLCbeta 1 tubulin-GppNHp inhibited PLCbeta 1 under the same conditions that GppNHp stimulated the enzyme (Fig. 1B).


Fig. 1. Regulation of receptor-independent PLCbeta 1 activity by tubulin. Sf9 cells were infected with baculoviruses bearing Galpha q and PLCbeta 1 cDNAs and cultured as described under "Experimental Procedures." Cells were harvested at 65 h postinfection, and membranes were prepared as described. 20 µg of Sf9 cell membranes containing the indicated recombinant proteins and 40 µl of substrate mixture were incubated for 15 min at 4 °C. GppNHp or tubulin-GppNHp (TubGppNHp) were added at appropriate concentrations to a final volume of 80 µl and incubated for 15 min at 37 °C as described. A, concentration response to GppNHp. B, comparison of the effects of GppNHp and tubulin-GppNHp, both at a concentration of 5 µM, on receptor-independent PLCbeta 1 activity. The experiments shown are representative of four with similar results. Each value is mean ± S.D. of duplicate determinations.
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Table I.

Effect of GppNHp on IP3 generation in membranes from normal and infected Sf9 cells

Sf9 cells were infected with the indicated baculovirus construct as described. After 65 h cells were harvested, membranes prepared, and assayed for IP3 production as indicated under "Experimental Procedures." Assays were performed for 15 min in the presence and absence of GppNHp as indicated. The experiment shown is representative of two with similar results. Each value is the mean ± S.D. of duplicate determinations.
Baculovirus-directed expression GppNHp added IP3 production

nmol/min/mg protein
None (normal Sf9 cells) None 0.14  ± 0.01
100 µM 0.15  ± 0.02
Galpha q only None 0.15  ± 0.03
100 µM 0.16  ± 0.03
PLCbeta 1 only None 0.60  ± 0.01
100 µM 0.57  ± 0.02

To study this further, extracts of Sf9 cells expressing Galpha q were reconstituted with purified recombinant PLCbeta 1 and exogenous phospholipid vesicles and assayed under conditions similar to those used for Sf9 cell membranes (Fig. 2). The effects of tubulin-GppNHp, nucleotide-free tubulin, and GppNHp were compared. Although GppNHp stimulated PLCbeta 1 activity at concentrations higher than 1 µM, 30 nM tubulin-GppNHp activated the enzyme. However, at higher tubulin-GppNHp concentrations inhibition of PLCbeta 1 was observed. Nucleotide-free tubulin did not activate but only inhibited PLCbeta 1 at concentrations higher than 300 nM.


Fig. 2. Effects of GppNHp, tubulin-GppNHp, and tubulin, stripped of nucleotide, on PLCbeta 1 activity in a reconstituted system. Extracts of Sf9 cells expressing Galpha q (28 µg/ml final protein concentration) were reconstituted into phospholipid vesicles with 5 ng of PLCbeta 1 and incubated with the indicated concentrations of GppNHp, tubulin-GppNHp (Tub-GppNHp), or tubulin (Tub), stripped of nucleotide, for 10 min at 30 °C as described under "Experimental Procedures." Data points are the means of duplicate determinations and are representative of three similar experiments. AlF4- (10 mM NaF and 30 µM AlCl3) increased PLCbeta 1 activity 6-fold (26.9 ± 5.3 pmol/min).
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Tubulin Modulation of m1 Muscarinic Receptor Activation of PLCbeta 1

Involvement of tubulin in receptor-triggered PLCbeta 1 regulation was studied with membranes from Sf9 cells expressing m1 muscarinic receptors, Galpha q, and PLCbeta 1. The m1 muscarinic receptor expression level was estimated by receptor binding studies with [3H]QNB as a ligand. When all three recombinant proteins were expressed, the m1 muscarinic receptor binding capacity (Bmax) was 240 ± 21 fmol/mg membrane protein and Kd = 0.162 ± 0.010 nM (n = 3). The ability to construct a complete, receptor-activated and G protein-mediated system allowed a comparison of the effects of GppNHp, tubulin-GppNHp, and tubulin, denuded of exchangeable nucleotide, on carbachol-stimulated PLCbeta 1 activity (Fig. 3). Although GppNHp was able to activate PLCbeta 1 without receptor stimulation, the effect was potentiated by carbachol (Fig. 3A). The response of PLCbeta 1 to GppNHp (up to 1 mM) under these conditions was concentration-dependent, saturable, and potentiated by carbachol throughout the concentration range (50.5 ± 9.2% at 100 µM GppNHp). However, the effect of tubulin-GppNHp on PLCbeta 1 activity was again biphasic, stimulatory at the lower (30 nM) and inhibitory at the higher concentrations of tubulin (Fig. 3A). Carbachol potentiated the tubulin-GppNHp-evoked PLCbeta 1 activation. At 30 nM, tubulin-GppNHp was more efficacious than GppNHp, independent of the addition of carbachol. The effect of carbachol was receptor-mediated since, at tubulin concentrations (30 nM) that stimulated PLCbeta 1, atropine inhibited carbachol-induced PLCbeta 1 activity by about 80% (Fig. 3B). However, the m1 muscarinic receptor stimulation was able to overcome the inhibitory effect of higher tubulin concentrations, resulting in an effective PLCbeta 1 activation. Note that the highest tubulin concentrations used were below those where tubulin-GppNHp forms polymers. To clarify the mechanism of this dual regulation of PLCbeta 1 by tubulin, the effect of nucleotide-free tubulin on enzyme activity was studied (Fig. 3C). Tubulin (without nucleotide) inhibited PLCbeta 1 activity in a concentration-dependent manner. No enzyme activation was observed at any tested tubulin concentration. Furthermore, unlike tubulin-GppNHp, nucleotide-free tubulin did not potentiate the PLCbeta 1 activation induced by carbachol. Tubulin-GDP or tubulin-GDPbeta S (at 30 nM) also failed to potentiate carbachol-triggered PLCbeta 1 activation or to affect the basal enzyme activity. Thus, it appears that tubulin activates PLCbeta 1 only when GTP or GTP analog occupies the exchangeable GTP-binding site.2


Fig. 3.

Effect of m1 muscarinic receptor stimulation on PLCbeta 1 activity regulated by tubulin. Sf9 cells expressing recombinant m1 muscarinic receptors, Galpha q, and PLCbeta 1 were harvested 65 h after infection, and membranes were prepared as described under "Experimental Procedures." PLCbeta 1 activity was assayed using 20 µg of membrane protein. A, effect of carbachol (carb) on GppNHp or tubulin-GppNHp (Tub-GppNHp) regulated PLCbeta 1 activity. Indicated concentrations of carbachol, GppNHp, or tubulin-GppNHp were added to the membranes, and PLCbeta 1 activity was assayed as described above. PLCbeta 1 activity was 1.11 ± 0.03 nmol/min/mg protein in the presence of carbachol alone. B, atropine inhibition of carbachol-induced activation of PLCbeta 1 in the presence of tubulin-GppNHp. PLCbeta 1 activity was studied in the presence of 30 nM of tubulin-GppNHp, 1 µM carbachol, and 100 nM atropine as indicated. Control activity was 0.90 nmol IP3/min/mg protein. C, comparison of the effects of tubulin-GppNHp and nucleotide-free tubulin on PLCbeta 1 activity in the presence and absence of carbachol. Each experiment shown is representative of three with similar results. Values are mean ± S.D. of duplicate determinations.


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Activation of Galpha q by Tubulin and Recruitment of Tubulin to the Plasma Membrane

To study directly the ability of tubulin-guanine nucleotide to interact with Galpha q, the transfer of the hydrolysis-resistant photoaffinity GTP analog, [32P]AAGTP, from tubulin to Galpha q, was examined in Sf9 cells expressing the m1 muscarinic receptor, Galpha q and PLCbeta 1. Nucleotide transfer from tubulin appears to be responsible for the activation of Galpha s and Galpha i1, and this has been observed in permeable cells, membrane preparations, and reconstituted systems (13-17). It has been shown that Galpha proteins receive [32P]AAGTP from tubulin under conditions when Galpha is incapable of binding the free nucleotide. This appears to be due to the formation of a complex between tubulin and Galpha . Under these conditions, tubulin retains the nucleotide without releasing it to the media (15). Carbachol (1 mM) increased (36 ± 7%) the binding of tubulin-[32P]AAGTP to the membrane, and there was commensurate loss (45 ± 8%) of tubulin-[32P]AAGTP from the supernatant (Fig. 4). Transfer of [32P]AAGTP from tubulin to Galpha q was also potentiated (42 ± 9%) by carbachol. Atropine blocked carbachol-induced binding of tubulin to the membrane as well as the increase in [32P]AAGTP transfer.3 The manner in which the activated m1 muscarinic receptor increases tubulin association with the membrane to regulate PLCbeta 1 is currently under study. The increase in tubulin-GppNHp activation of PLCbeta 1 brought by carbachol may be explained partially by this facilitated association of tubulin with the membrane.


Fig. 4. Muscarinic receptor-triggered binding of tubulin-[32P]AAGTP to the membrane and the transfer of [32P]AAGTP to Galpha q. Membranes were prepared from Sf9 cells expressing recombinant m1 muscarinic receptors, Galpha q, and PLCbeta 1 and incubated with 1 µM tubulin-[32P]AAGTP in the presence or absence of 1 mM carbachol for 10 min at 23 °C as described under "Experimental Procedures." The tubes were UV-irradiated, and the membrane pellets and concentrated supernatants were subjected to SDS-PAGE as described. An autoradiogram of one of five independent experiments with similar results is shown.
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These findings, together with the results in Figs. 2 and 3, support the idea that nucleotide transfer from tubulin to Galpha q is a potential mechanism for PLCbeta 1 activation. Carbachol had no effect on the membrane association of tubulin, nucleotide transfer to Galpha q, or PLCbeta 1 activity in Sf9 cells expressing only Galpha q and PLCbeta 1. No effect of carbachol, guanine nucleotide, or tubulin-guanine nucleotide was observed in control Sf9 cells when measuring PIP2 hydrolysis or photoaffinity labeling.

Tubulin, Galpha q, and PLCbeta 1 Form a Complex

To understand better the mode of interaction of tubulin-GppNHp with the members of PLCbeta 1 signaling cascade, coimmunoprecipitation studies with tubulin-[32P]AAGTP were performed. As seen in Fig. 5, both Galpha q antiserum and, to a lesser extent, PLCbeta 1 antiserum coimmunoprecipitated tubulin when membrane extracts of cells infected with viruses for Galpha q or PLCbeta 1, respectively, were studied. In the membrane extracts from cells expressing Galpha q, [32P]AAGTP-labeling of Galpha q was also observed. The source of [32P]AAGTP was tubulin. No tubulin-[32P]AAGTP coimmunoprecipitation was observed when extracts from normal Sf9 cells were tested with anti-Galpha q or anti-PLCbeta 1 antisera. Thus, it is suggested that the formation of a complex between Galpha q, PLCbeta 1, and tubulin-GppNHp might be responsible for the higher efficacy of tubulin-GppNHp, compared with GppNHp, during the m1 muscarinic receptor stimulation.


Fig. 5. Coimmunoprecipitation of tubulin with Galpha q and PLCbeta 1. Membrane preparations of Sf9 cells, expressing only m1 muscarinic receptors, Galpha q, or PLCbeta 1 were extracted with 1% sodium cholate. Membrane extracts (0.5 mg/ml membrane protein) were incubated with 1 µM tubulin-[32P]AAGTP, as described under "Experimental Procedures." After UV irradiation each membrane extract was incubated overnight with appropriate specific or nonspecific antiserum as indicated. Immunoprecipitates were subjected to SDS-PAGE and autoradiography. An autoradiogram from one of four independent experiments with identical results is shown. Tub, tubulin.
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PIP2 Binding to Tubulin

In order to clarify the inhibitory action of tubulin on PLCbeta 1, the interaction between tubulin and the PLCbeta 1 substrate, PIP2, was studied. It has been shown previously that phosphatidylinositol inhibits microtubule assembly (34) and that the binding of profilin (an actin-binding protein) to PIP2 micelles inhibits PLCgamma 1-directed PIP2 hydrolysis (35). To test the possibility that tubulin binds PIP2, a gel filtration assay was performed. Two peaks of tubulin were resolved, one represented a small portion of oligomeric tubulin, and the second represented dimeric tubulin (Fig. 6). When the same amount of tubulin was incubated with PIP2 micelles and passed over an Ultrogel 34 column, an increase in the higher molecular weight peak was observed along with a corresponding decrease in the dimeric tubulin peak. When tested under the same conditions, phosphatidylcholine micelles did not shift the mobility of tubulin. Since PIP2 forms micelles of about 93 kDa in aqueous solutions (36), the increase in the higher molecular weight fraction could be attributed to either PIP2-induced tubulin oligomerization or to a binding of tubulin to PIP2 micelles, as has been observed in the case of profilin (32). To resolve this, polymerization of tubulin in the absence (Fig. 7A) or presence (Fig. 7B) of PIP2 micelles was studied by electron microscopy. Tubulin failed to polymerize properly in the presence of PIP2 (Fig. 7B). Microtubules were considerably shorter (1.7 ± 1.0 µM, compared with 10.2 ± 4.8 µM in the absence of PIP2) and 3-4 times thicker (75-100 nm versus 25 nm in the absence of PIP2). These results suggest a direct interaction between tubulin and PIP2 and support the hypothesis that increased concentrations of dimeric tubulin might prevent access of PLCbeta 1 to its substrate. Since increased activation of PLCbeta 1 might evoke localized increases in calcium which could increase tubulin dimer concentration, this might represent a feedback mechanism for the regulation of receptor-G protein-activated phospholipase C. It is noteworthy in this regard that phosphorylation of PLCgamma 1 by EGF receptor tyrosine kinase appears to overcome the inhibitory effect of profilin binding to PIP2, resulting in an effective activation of the enzyme (35). Similarly, m1 muscarinic receptor stimulation might reverse tubulin-evoked inhibition of PLCbeta 1 by increasing the affinity of Galpha q for tubulin.


Fig. 6. Interaction between tubulin and PIP2. Gel filtration assay for the binding of tubulin to PIP2 micelles was performed. Tubulin was incubated with PIP2 micelles in a Branson water bath sonicator for 15 min at 4 °C, followed by an additional 45 min on ice. The samples were then applied to and eluted from a 0.7 × 50 cm column of Ultrogel AcA 34 at 4 °C as described under "Experimental Procedures." Chromatograms of 0.7 mg of tubulin alone (open circles) or with PIP2 at a molar ratio of 1:14 (closed circles) are shown. When chromatographed alone, PIP2 micelles at the same concentration did not show detectable 280 nm absorbance in any fraction collected.
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Fig. 7. Effect of PIP2 on microtubule polymerization. Electron microscopy of polymers formed by PC-tubulin in the absence (A) and presence (B) of PIP2. Samples were taken from tubulin polymerization reactions (2 mg/ml final protein concentration) carried out for 30 min at 37 °C as described under "Experimental Procedures." The molar ratio between tubulin and PIP2, when added, was 1:6. Reaction mixtures were applied to carbon/Formvar-coated grids and prepared for electron microscopy as described under "Experimental Procedures." Grids were examined on a JEM 100CX (JEOL) electron microscope. Magnification = 7000 ×.
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Feedback Inhibition of PLCbeta 1 by Tubulin

It is suggested that the amount of dimeric tubulin normally accessible to the membrane is low; thus, initially, stimulation of Galpha q by tubulin would predominate. It is possible, however, that tubulin bound to PLCbeta 1 and/or PIP2 might not bind to Galpha q. The observed muscarinic receptor-triggered increase in Galpha q affinity for tubulin might be able to overcome such inhibition. It is also possible that the down-regulation of PLCbeta 1 signaling would channel heterotrimeric Gq to another signaling pathway (37, 38). Although complexes of tubulin with Galpha q, similar to those with Galpha i1 or Galpha s (17), have not yet been shown in the membrane, clearly the possibility exists for a system in which membrane-associated tubulin, through reversible interaction with other membrane proteins and/or lipids, could achieve a dual regulation of phospholipase C signaling within the cell. It has also been suggested that the activation of G protein alpha  subunits by tubulin dimer may provide an interface between G protein signaling through cAMP and G protein signaling through calcium (39). These dynamic interactions between G proteins and tubulin dimers at the synapse may also modify synaptic microarchitecture.

A noteworthy feature of this study is that tubulin regulates PLCbeta 1 signaling while the PLCbeta 1 substrate, PIP2, may regulate microtubule assembly. These reciprocal events could be involved both in intracellular signaling and the control of spindle morphogenesis and reorganization of microtubule arrays during the cell cycle. Since alterations in the metabolism of phosphoinositides (40, 41) or the expression of m1 muscarinic receptors (42) or G proteins (43, 44) have been implicated in cellular transformation, this regulatory mechanism might prove valuable in the control of cell proliferation as well.


FOOTNOTES

*   This study was supported by National Institutes of Health Grants MH 339595 (to M. M. R.) and CA 40042 (to J. C. G.) and the Council for Tobacco Research Grant 4089 (to M. M. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   Recipient of a Senior Fellowship from the American Heart Association of Metropolitan Chicago.
Dagger Dagger    Recipient of a Research Scientist Development Award from the National Institute of Mental Health. To whom correspondence should be addressed: Dept. of Physiology and Biophysics, M/C 901, University of Illinois, 835 S. Wolcott, Chicago, IL 60612-7342. E-mail: raz{at}uic.edu.
1   The abbreviations used are: PLCbeta 1, phospholipase Cbeta 1; GppNHp, guanosine 5'-(beta ,gamma -imido)triphosphate; AAGTP, P3(4-azidoanilido)-P1-5'-GTP; tubulin-GppNHp, dimeric tubulin with GppNHp bound; GDPbeta S, guanyl-5'-yl thiophosphate; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; Pipes, 1,4-piperazinediethanesulfonic acid; DTT, dithiothreitol; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; QNB, L-quinuclidinyl [phenyl-4(n)]benzilate, muscarinic receptor antagonist; PIP2, phosphatidylinositol 4,5-bisphosphate.
2   No baculovirus encoding G protein beta gamma subunit was included in these experiments. However, since the level of receptor expression is moderate, carbachol- and GTP-dependent activation of PLCbeta 1 suggest that the expressed m1 muscarinic receptors are coupling to expressed Galpha q and endogenous Sf9 Gbeta gamma (33).
3   J. S. Popova, J. C. Garrison, S. G. Rhee, and M. M. Rasenick, unpublished observations.

Acknowledgments

We thank Gary Luthin, David Manning, and Elliot Ross for their generous gifts of material and Richard Green and Heidi Hamm for advice and criticism on this manuscript. Madhavi Talluri is thanked for technical assistance and Sukla Roychowdhury for advice with electron microscopy. Meira Liang is thanked for constructing the baculovirus containing Galpha q and Miller Jones is thanked for Galpha q preparations.


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