Tubulin stimulates adenylyl cyclase activity in rat striatal membranes via transfer of guanine nucleotide to Gs protein

BRAIN RESEARCH ELSEVIER

Brain Research 704 (199.5) 23-30

Research report

Tubulin stimulates adenylyl cyclase activity in rat striatal membranes via transfer of guanine nucleotide to Gs protein Shinichi Hatta a,*, Hiroki Ozawa b, Toshikazu

Saito b, Norihito Amemiya

a, Hideyo Ohshika a

aDepartment of Pharmacology, School of Medicine, Sapporo Medical University, Sapporo 060, Japan h Department of Neuropsychiatry, School of Medicine, Sapporo Medical Uniuersiry, Sapporo 060, Japan Accepted 8 August 1995

Abstract Previous studies of rat cerebral cortex and rat C6 glioma cells have demonstrated that dimeric tubulin is capable of activating the G proteins Gs and Gil via transfer of guanine nucleotide from tubulin to Gscu and Gilcu. To provide further information regarding cytoskeletal modulation of adenylyl cyclase, the present study examined effects of tubulin on the activation of the enzyme in rat striatal membranes. Tubulin, prepared from rat brain by polymerization with the hydrolysis-resistant GTP analog 5’-guanylylimidodiphosphate (GppNHp) caused significant activation of adenylyl cyclase by u 130%. Furthermore, tubulin-GppNHp activated SKF 38393-sensitive adenylyl cyclase and potentiated forskolin-stimulated activity of the enzyme. When tubulin, polymerized with the hydrolysis-resistant photoaffinity GTP analog [ 32p]p3 (4-azidoanilido)-p’-S-GTP ([ 32P]AAGTP), was incubated with striatal membranes, AAGTP was transferred from tubulin to Gscu as well as Gicu with the extents of nucleotide transfers being 7.6 k 0.8% and 17.8 f 1.4% of AAGTP originally bound to tubulin, respectively. These results indicate that, in rat striatum, the tubulin dimer participates in the stimulatory

regulation of adenylyl cyclase by transferring guanine nucleotide to Gsa, regulation of neuronal signal transduction. Keywords;

supporting the hypothesis that tubulin contributes to the

Tubulin; G protein; Adenylyl cyclase; Dopamine receptor; Signal transduction

1. Introduction

Guanine nucleotide-binding regulatory proteins (G proteins) are a family of proteins involved in mediating signal transduction between membrane-associated receptors and intracellular effecters, such as adenylyl cyclase, cyclic GMP phosphodiesterase, and phospholipases, as well as the modulation of activities of various ion channels [2,11]. They function as a heterotrimeric complex consisting of (Y, p, and y subunits. The (Y subunits, upon interaction with agonist-bound activated receptors, exchange GDP for GTP, dissociate from the P-y subunits, and associate with appropriate effector molecules. Hydrolysis of the bound GTP by the (Y subunit results in reformation of the inactive, trimeric complex. The CYsubunit appears responsible then for the transduction of extracellular signals from specific receptors

* Corresponding author. Department of Pharmacology, School of Medicine, Sapporo Medical University, South-l, West-17, Chuo-ku, Sapporo 060, Japan. Fax: (81) (11) 612-5861. 0006-8993/95/$09.50

0 1995 Elsevier Science B.V. All rights reserved

SSDI 0006-8993(95)01073-4

to specific effecters. The Py subunits, on the other hand, appear to inactivate the (Y subunit and may anchor the complex to membranes. Recent evidence suggests that the j?r subunits in some cases may be involved as well in the regulation of effector molecules [9]. The receptor-mediated stimulation and inhibition of adenylyl cyclase are mediated by two distinct G proteins, termed Gs and Gi, respectively. Although receptor-G protein-adenylyl cyclase complexes can reconstitute hormone-sensitive signaling systems in vitro [4,22], it is likely that the regulation of adenylyl cyclase in vivo is substantially more complicated. Not only are these components presented to one another in an orientation dictated by the plasma membrane, but also elements of the cytoskeleton, particularly tubulin, may regulate the signal transduction process [38]. Tubulin, the primary constituent of microtubules, is a GTP-binding protein with significant functional similarities and amino acid sequence homology to the signal transducing G proteins [24,25,38,47]. Recent studies appear to indicate that, in neuronal systems, one locus of interaction between cytoskeletal components and the

24

S. Hatta et al. /Brain

adenylyl cyclase system is between tubulin and G proteins, which are involved in the regulation of that enzyme [38,52]. Tubulin dimers have been demonstrated to interact with some G proteins which mediate neuronal signal transduction. ‘251-Tubulin was found to bind with high affinity to purified Gs LYand Gil (Y proteins, but not to Gi2 cr, Gi3 a, Go (Y, and Gt (Y [49]. Furthermore, the tubulin dimer, when polymerized with the hydrolysis-resistant GTP analogs S-guanylylimidodiphosphate (GppNHp) or P’ (4azidoanilidol-p’-S-GTP (AAGTP), has been shown to cause stimulation and inhibition of adenylyl cyclase in rat cerebral cortex membranes, and a direct transfer of nucleotide from the exchangeable GTP-binding site of tubulin to Gscu and Gicu (presumably Gil) is suggested as a mechanism of this regulation [17,37,40]. Thus, the interaction and nucleotide transfer between tubulin and G proteins appears to represent a form of intracellular regulation of neuronal adenylyl cyclase. However, the only studies which have characterized the tubulin-G protein interactions in the regulation of adenylyl cyclase in brain tissue are those conducted with rat cerebral cortex [17,18,37], although the interaction of tubulin with G proteins was shown in studies using rat C6 glioma cells [52] and the purified G protein (Y subunit [40,49]. Thus, it is not known whether such tubulin-G protein interaction occurs in other regions of brain as well. To provide further information regarding cytoskeletal modulation of adenylyl cyclase signal transduction, therefore, the present study, using rat striatum, examined effects of tubulin on the stimulatory regulation of adenylyl cyclase. It has been demonstrated that the Gs-adenylyl cyclase system in the striatum differs in several respects (e.g. the sensitivity to GTP [8], forskolin [43], and Ca*+/calmodulin [27], and the amount and/or types of Gs [5,13]) from that in the cortex. Furthermore, striatal dopaminergic signal transduction plays an important role in the coordination of various motor behaviors, and deficits in this system are characteristic correlates of aging [26], which is known to be associated with changes in the brain cytoskeletal system [34,35]. The data presented here indicate that the tubulin dimer is capable of activating adenylyl cyclase in synaptic membranes prepared from rat striatum. This stimulatory effect of tubulin on striatal adenylyl cyclase appears to involve the transfer of guanine nucleotide from tubulin to striatal membrane Gs, as has been suggested by the findings with the rat cerebral cortex [18].

2. Materials and methods 2.1. Tissue preparation Male Wistar rats (180-220 g> were used for the experiments. The striatum was dissected as described by Heffner et al. [20], and striatal membranes were prepared as described previously [16,19] and stored at -80°C until use.

Research 704 (1995) 23-30

2.2. Tub&n

preparation

Tubulin (GppNHp-liganded tubulin and [ 32P]AAGTPliganded tubulin) were prepared from rat brains by the method of Shelanski et al. [45] with modifications as described by Rasenick and Wang [37]. Briefly, microtubules were polymerized from homogenates of ten rat brains. After the first polymerization, tubulin pellets were resuspended in 1 ml of ice-cold buffer [lo0 mM piperazine-N,N’-bis(2-ethanesulfonic acid) (pH 6.9), 2 mM EGTA, 1 mM MgCl,] and incubated on ice for 1 h to allow for complete depolymerization. Three milligrams of charcoal (Norit A) was added for 10 min, and the solution was intermittently vortex mixed and centrifuged at 20,000 X g for 10 min. Charcoal extraction was repeated again to ensure that the tubulin was stripped of bound nucleotide [lo]. A second polymerization step was performed using either GppNHp or [ 32P]AAGTP (150 PM). Nucleotides were added and the solutions were incubated for 40 min at 37°C and then centrifuged for 20 min at 110,000 X g at 37°C in a Hitachi 65P ultracentrifuge. Microtubule pellets were rinsed in 2 mM HEPES (pH 7.4), 1 mM MgC12, and 5% glycerol, resuspended in the same buffer, and stored at -80°C. Before use, preparations were centrifuged at 110,000 X g for 20 min to remove any tubulin aggregates and centrifuged-dialyzed in Amicon ” Centricon-10” filters to remove the unbound nucleotide. The final preparations were comprised of _ 97% tubulin as estimated by Coomassie blue staining in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. The percentage of GppNHp-liganded tubulin in the tubulin preparation was 90.6 + 2.4% as estimated by tubulin polymerization with [“HlGppN~p. The concentration of tubulin-GppNHp used for the experiments was corrected by these values. 2.3. Adenylyl cyclase assay Striatal membranes were thawed and resuspended in a buffer containing 20 mM HEPES (pH 7.5), 5 mM MgCl,, 1 mM dithiothreitol (DDT), and 0.3 mM phenylmethylsulfonyl fluoride (PMSF). Adenylyl cyclase activity in striatal membranes was assayed as described previously [16,19]. Membranes (30-40 pg) were incubated with or without the indicated protein (tubulin or IgG) or nucleotide for 10 min at 30°C in 100 ~1 of medium containing 15 mM HEPES (pH 7.5), 0.05 mM ATP, [ cr-a*P]ATP ( - 2 X 10h cpm/tube), 5 mM MgCl,, 1 mM EGTA, 1 mM DTT, 0.05 mM cyclic AMP, 60 mM NaCl, 0.25 mg/ml of bovine serum albumin, 0.5 mM 3-isobutyl-l-methylxanthine, 1 U/ml of adenosine deaminase, and a nucleotide triphosphate-regenerating system consisting of 0.5 mg of creatine phosphate, 0.14 mg of creatine phosphokinase, and 15 U/ml of myokinase. The reaction was stopped by the addition of 0.1 ml of a solution containing 2% SDS, 1.4 mM cyclic AMP, and 40 mM ATP, and the cyclic [ 32P]AMP formed was isolated by the method of Salomon

S. Hatta et al/Brain

Research 704 (1995) 23-30

[41]. In certain experiments, adenylyl cyclase activity was assayed in the presence of SKF 38393 or forskolin with tubulin or nucleotide.

240 &220-

2.4. Photoafinity

labeling

-

GPPNHP

-

T"-GPPNHP

t;.$ 2: 200-

was synthesized and purified by the [32P]AAGTP method of Pfeuffer [31]. [32P]AAGTP photoaffinity labeling was performed as described previously [16,18]. Striatal membranes were washed and resuspended in 2 mM HEPES (pH 7.4)/5 mM MgCl,. Membrane suspensions (2-3 mg of protein/ml) were incubated with 0.1 PM [ 32P]AAGTP or the indicated concentrations of tubulin-[ 32P]AAGTP for 5 min at 3O”C, and the reaction was terminated by dilution with 1 ml of the above ice-cold buffer followed by centrifugation at 15,000 X g for 10 min to remove unbound [ 32P]AAGTP. Where noted, membrane suspensions were incubated with guanine nucleotide for 5 min at 3O”C, washed and resuspended in the above buffer before incubation with tubulin-[ 32P]AAGTP. Membranes were washed again and resuspended in the same buffer followed by 5 min of UV photolysis with a Spectroline UV lamp (254 nm, 9W) on ice at a distance of 3 cm. The reaction was quenched with 1 ml of ice-cold 2 mM HEPES (pH 7.4)/5 mM MgC1,/4 mM DTT, followed by centrifugation at 15,000 X g for 10 min. Membrane pellets were dissolved in 3% SDS Laemmli sample buffer [23] with 50 mM DTT and electrophoresed in 10% SDS-polyacrylamide gels by the procedure of Laemmli [23]. After electrophoresis, gels were stained with Coomassie blue, dried and autoradiographed with Kodak XAR-5 film. The developed autoradiographs were analyzed by laser densitometer (Model SLR-2D/lD, Biomed Instrument, Inc.). 2.5. Materials [ w~*P]ATP (30 Ci/mmol; 1 Ci = 37 GBq) was purchased from Du Pont-New England Nuclear. [a-32P]GTP (400 Ci/mmol) and [8-3H]GppNHp (19.4 Ci/mmol) were from Amersham. GTP and GppNHp were from Boehringer Mannheim. R(+)-SKF 38393 hydrochloride was from Funakoshi. Forskolin was from Calbio-Chem. pAzidoaniline was from Aldrich. All other reagents used were of analytical grade. Protein was determined by the Coomassie blue binding method [3] with bovine serum albumin as a standard. Data were analyzed for statistical significance using a two-tailed Student’s t-test. Values of P < 0.05 were taken to indicate significance.

(Dr -oE !gn180urn xg :a

**

160-

ZE 8%40-

// //

100~----7//. co

7 8.0

I

-Log

x 7.0

I

of adenylyl

cyclase by GppNHp

'I' 6.0

8

I 5.0

w

Fig. 1. GppNHp-dependent and tubulin-GppNHp-dependent activation of adenylyl cyclase activity in rat striatal membranes. Adenylyl cyclase was assayed with the indicated concentrations of GppNHp or tubulin-GppNHp (Tu-GppNHp). Tubulin was balanced with bovine IgG to yield a constant amount of added protein. The values are mean k SEM of eight experiments. * P < 0.05 and * * P < 0.01 indicate significant differences compared with the basal activity.

cyclase, GppNHp (lo-’ N lop5 M) significantly activated adenylyl cyclase to N 174% of basal activity (Fig. 1). To examine effects of GppNHp-liganded tubulin (tubulinGppNHp) on adenylyl cyclase activity, similar experiments were done on striatal membranes with the indicated concentration of tubulin-GppNHp instead of GppNHp. Tubulin-GppNHp (lo-’ N lo- 5 M) produced significant activation of the enzyme to - 130% of basal activity, although the potency of tubulin-GppNHp activating the enzyme was lower than that of GppNHp (Fig. 1). A stimulatory effect of tubulin-GppNHp on adenylyl cyclase was also observed in D, dopaminergic regulation of the enzyme (Fig. 2). It has been documented that D, dopaminergic receptors are linked to stimulation of adenylyl cyclase through Gs in striatum [2]. SKF 38393, a D, dopamine receptor agonist, did not increase adenylyl cyclase activity in the absence of GppNHp (10e6 M) or tubulin-GppNHp (1O-6 M). On the other hand, in the presence of GppNHp or tubulinGppNHp, significant stimulation of the enzyme activity with SKF 38393 was observed. Furthermore, tubulinGppNHp activated SKF 38393-sensitive adenylyl cyclase with a potency comparable to that of GppNHp. The SKF 38393-induced activation of the enzyme was suppressed to the basal level by addition of SCH 23390, a D, receptor antagonist (10e5 M) (data not shown). 3.2. Potentiation of forskolin-stimulated by GppNHp and tubulin-GppNHp

3. Results 3.1. Activation lin-GppNHp

25

adenylyl

cyclase

and tubu-

When striatal membranes were incubated with the indicated concentrations of GppNHp and assayed for adenylyl

Forskolin, a diterpene, is known to interact with the catalytic subunit of adenylyl cyclase with high affinity in the presence of activated G protein (as) and with low affinity in the absence of (YS [42]. As shown in Fig. 3,

S. Hatta et al. /Brain Research 704 (1995) 23-30

26

A

140= e E 5

--D130-

B

a

B

b

c

d

No Addition

-

GPPNHP

--O--

Tu-GppNHp

Tubulin

)

Es

:

GSH B

903

8 .i:. co

1 6.0 -

8 ”

I 7.0

c 6.0

9

I 5.0

8

7 4.0

Log [SKF 393931 (M)

Fig. 2. Activation of SKF 38393.sensitive adenylyl cyclase activity by GppNHp or tubulin-GppNHp in striatal membranes. Adenylyl cyclase was assayed with the indicated concentrations of SKF 38393 in the absence (No addition) or presence of GppNHp and tubulin-GppNHp (Tu-GppNHp). The concentration of GppNHp and tubulin-GppNHp used was 10e6 M. Tubulin was balanced with bovine IgG to yield a constant amount of added protein. The values are expressed as a percentage of the corresponding control activity and are shown as mean + S.E.M. of six experiments. * P < 0.05 and * * P < 0.01 indicate significant differences compared with the corresponding control.

GppNHp (lop5 M) effectively potentiated activation of adenylyl cyclase induced by forskolin at a low concentration (10e6 M) (- 135% of the forskolin-stimulated activity). This appears to represent the interaction of activated Gs (CXS) and the catalytic subunit of adenylyl cyclase. Similar to results obtained with GppNHp, tubulin-GppNHp (lop5 M) caused significant potentiation of the forskolin-

=

300-

;

? ?Basal ? ?FSK

f

260-

c

la

FSK/GppNHp

W

FSK/Tu-GppNHp

X

x E

100

$ 60

1

Fig. 3. Potentiation by GppNHp or tubulin-GppNHp of forskolin-stimuIated adenylyl cyclase activity in striatal membranes. Adenylyl cyclase activity was determined in the presence of forskolin (FSK, lo-’ M) without or with GppNHp (10e5 Ml and tubulin-GppNHp (Tu-GppNHp, 10m5 Ml. Tubulin was balanced with bovine IgG to yield a constant amount of added protein. The basal activity was 130.43 k 4.51 pmol/mg protein/min. The values are expressed as a percentage of the basal activity and are shown as mean k S.E.M. of six experiments. * P < 0.05 and * * P < 0.01 indicate significant differences compared with the activity of forskolin alone.

ES’

:

G 32

)

G 32 )

Fig. 4. [‘*PI AAGTP photoaffinity labeling (A) and transfer of [“*PI AAGTP from tubulin to G proteins (B) in striatal membranes. Striatal membranes were incubated with 0.1 PM [‘*PI AAGTP for 5 min (A) or were incubated with 10 pg (lanes b-d) of tubulin-[32P] AAGTP for 5 min (B). Tubulin-[32P] AAGTP was irradiated for 5 min before incubation with membranes (lane c). Membranes were incubated with lo-” M GppNHp for 5 min at 30°C before incubation with tubulin-[ s2 P] AAGTP (lane d). In lane a, 10 pg of tubulin-[“PI AAGTP was UV-irradiated without incubating membranes. After incubation, the membranes were washed and subjected to photoaffinity labeling as described in Materials and methods. After UV irradiation, quenching and washing, 30 pg of each membrane was subjected to SDS-PAGE and autoradiography. The autoradiographs are representative of five similar experiments. GsH and GsL (Gs) refer to the 52.kDa and 45-kDa cholera toxin substrates, respectively. Gi refers to the GTP-binding pertussis toxin substrates clustered around 40 kDa. These include Gi and Go. G,? refers to a 32-kDa AAGTP-binding protein that appears to be particularly abundant in rat brain. It is not yet clear whether this species functions in signal transduction.

stimulated stimulated

enzyme activity).

3.3. Transfer protein

activity

of guanine

(N 113%

nucleotide

of the forskolin-

from

tub&in

to G

In previous studies on the rat cerebral cortex [17,18,37], a direct transfer of guanine nucleotide from the exchangeable GTP binding site of tubulin to Gs and Gi has been demonstrated as the mechanism for tubulin-dependent stimulation or inhibition of adenylyl cyclase. To examine whether guanine nucleotide could be transferred from tubulin to G proteins in rat striatal membranes, we used the hydrolysis-resistant photoaffinity GTP analog ([ 32P])AAGTP, bound to tubulin (tubulin-[ 32P]AAGTP) in photoaffinity labeling experiments. Representative autoradiographs for the photoaffinity labeling of striatal membranes with [ 32P]AAGTP are shown in Fig. 4A. AAGTP labeling corresponding to Gs CYor Gi (Y in striatal membranes was identified based on AAGTP binding to G proteins previously described [16]. G,, is a major AAGTP-binding protein in rat brain and human brain [16,29]. It is not a substrate for ADP-ribosylation by pertussis or cholera toxin [16] and does not appear to be a proteolytic fragment of Gs or Gi [36]. However, it is not yet clear whether this species functions in signal transduction. Addition of tubulin-[ 32P]AAGTP to striatal membranes for 5 min followed by a wash and UV irradiation resulted

S. Hatta et al. /Brain Research 704 (1995) 23-30

in AAGTP incorporation into Gscu and Gicu (7.6 + 0.8% and 17.8 k 1.4% of [32P]AAGTP originally bound to tubulin, respectively), although the major portion of labeling was observed on Gia (Fig. 4B). In contrast to the result obtained with [32P]AAGTP, no significant labeling of the nucleotide on G,, was observed when striatal membranes were incubated with tubulin-[32 PIAAGTP. AAGTP incorporation into G proteins could be blocked by UV irradiation of tubulin-[ 32P]AAGTP to covalently incorporate AAGTP into tubulin before incubation with striatal membranes, or by exposing the striatal membranes to an excess amount of GppNHp (10m4 M) to saturate the nucleotide binding sites of the G protein before incubation with tubulin-[32P]AAGTP (Fig. 4B). These results are consistent with previous reports on cerebral cortex membranes [17,18,37]. Thus, they appear to indicate transfer of AAGTP to Gscu and Gicu from tubulin molecules.

4. Discussion Adenylyl cyclase activity is known to be coordinately regulated by stimulatory and inhibitory signals acting through Gs and Gi, respectively. Previous studies have suggested that tubulin dimers in synaptic membranes may associated with CYsubunits of Gs and Gi, and that they activate those G proteins by transferring GTP from the exchangeable site of tubulin to the (Y subunit of the G proteins [40,48,49]. As a functional consequence of activation of Gs and Gi, adenylyl cyclase activity is stimulated and inhibited, respectively [37,52]. In a previous study on rat cerebral cortex [18], we have demonstrated that tubulin activates adenylyl cyclase under conditions optimal for stimulation of the enzyme [30,51], i.e., in the presence of 5 mM MgCl, and 1 mM EGTA at 30°C. This stimulatory effect of tubulin was suggested to be due to a direct transfer of guanine nucleotide from tubulin to Gs. Although guanine nucleotide was also transferred to Gicw from tubulin, the nucleotide transfer to Gs (Y, which occurred only under conditions optimal for the enzyme stimulation, appeared to elicit an increase in the relative activation of Gs to Gi, leading to net stimulation of adenylyl cyclase [18]. In contrast, under conditions in which inhibition of adenylyl cyclase was favored [16,30,37,51], i.e., in the presence of 1 mM MgCl, at 23°C guanine nucleotide was transferred from tubulin only to Gicr, and tubulin caused inhibition of the enzyme but did not produce stimulation [17]. In this study under experimental conditions in which stimulation of adenylyl cyclase was favored, addition of tubulin-GppNHp to striatal membranes resulted in significant activation of adenylyl cyclase (- 130% of basal activity), although the potency of tubulin-GppNHp was relatively lower than that of GppNHp (N 174%). These results indicate that tubulin-GppNHp is capable of activating adenylyl cyclase in striatal membranes in accord with

27

the findings in cerebral cortex membranes [18]. The stimulatory effect of tubulin-GppNHp on striatal adenylyl cyclase was also evident with respect to SKF 38393-sensitive adenylyl cyclase (Fig. 2) and forskolin-stimulated activity of the enzyme (Fig. 3). A photoaffinity labeling study with tubulin-[‘32 P] AAGTP indicated that AAGTP was transferred from tubulin to Gsa and Gia during the brief incubation of striatal membranes with tubulin-[ 32P]AAGTP (Fig. 4B). Previous studies [18,37,39,40] have suggested that the transfer of guanine nucleotide is not due to a release of the nucleotide from tubulin with subsequent rebinding by the cx subunit of G proteins. The extents of nucleotide transfers from tubulin to Gs (Y and Gi (Y (7.6 k 0.8% and 17.8 f 1.4%, respectively) were comparable to those observed in cerebral cortex membranes [18]. Although the major portion of labeling was observed on Gi (Y, the nucleotide transfer to Gscu from tubulin may have caused an increase in .the relative activation of Gs to Gi, which is related to the activation of adenylyl cyclase [15,16], as suggested in our previous study on cerebral cortex [18]. As a functional consequence of this, adenylyl cyclase activity may‘ be expressed in a stimulatory state. It is likely, therefore, that the tubulin dimer participates in the regulation of adenylyl cyclase in the rat striatum by transferring guanine nucleotide to the I_Ysubunit of G protein, consistent with the hypothesis proposed in studies with rat cerebral cortex [17,18,37,40]. We observed recently that tubulin-GppNHp reduced the agonist binding affinities for the D, and D, dopamine receptors, i.e., tubulin-GppNHp caused a rightward shift of the dopamine competition curve for [3H] SCH 23390 binding and [3H]spiperone binding in striatal membranes in a manner similar to those observed with GppNHp (Hatta et al., unpublished observations). These findings may also support the nucleotide transfer from tubulin to striatal G proteins and the consequent activation of G proteins. Thus, the present study indicates that the contribution of the tubulin dimer to the regulation of adenylyl cyclase is not limited to the region of the cerebral cortex but that it appears to function in other regions of the central nervous system as well. In the present study, we demonstrate that tubulinGppNHp activated the D, dopaminergic agonist SKF 38393-sensitive adenylyl cyclase (Fig. 2). This result is analogous to a previous finding [18] showing activation of the P-adrenergic agonist isoproterenol-sensitive adenylyl cyclase by tubulin-GppNHp in cerebral cortex membranes. Both P-adrenergic and D, dopaminergic receptors are known to couple to Gs [21. Thus, it might be possible that, through transfer of guanine nucleotide to Gs, the tubulin dimer participates in the stimulatory regulation of adenylyl cyclase by other Gs-coupled receptors as well. Furthermore, an apparent difference in the potency of tubulinGppNHp for activating adenylyl cyclase in the absence (Fig. 1) and the presence of SKF 38393 (Fig. 2) may imply that tubulin-GppNHp more efficiently interacts and trans-

28

S. Hatta et al. /Brain

Research 704 (1995123-30

fers guanine nucleotide to Gscr, which couples with agonist-occupied receptors. Such unique accessibility of tubulin-GppNHp to Gs when coupled to P-adrenergic receptors has also been demonstrated in previous studies [l&32,50]. The precise physiological roles of the tubulin molecule in the regulation of signal transduction still remain to be established. In the striatum, dysfunctional dopaminergic neurotransmissions have been observed in several neuropathological conditions, including schizophrenia, Parkinson’s, Huntington’s, and Alzheimer’s diseases. In addition, D, dopaminergic receptor activity, i.e., the density of D, receptor and D, receptor-stimulated adenylyl cyclase, declines with aging [12,21]. Some of those conditions, e.g., Alzheimer’s disease or aging, have been reported to be associated with alterations in brain cytoskeletal systems [14,34,35,44]. It is tempting to speculate that alterations in the cytoskeleton lead to changes in the interaction between the cytoskeletal system and the signal transduction system, resulting in altered responsiveness of adenylyl cyclase [7,28]. Our previous study on age-related alterations in tubulin function and the adenylyl cyclase system may be provide some support for such a possibility [17]. In addition, we recently observed an age-related decrease in tubulin-dependent stimulation of adenylyl cyclase in rat striaturn (Hatta et al., unpublished observation). The transfer of guanine nucleotide from tubulin to the (Y subunit of G protein is suggested to consist of two phases, the interaction or association between tubulin and G protein and, subsequently, the direct transfer of the nucleotide from one to the other [40,49]. Although the precise molecular details of these processes still remain to be established, several molecular features in the process of interaction and transfer of guanine nucleotide between tubulin and G proteins have been reported. It has been suggested that tubulin-G protein complex formation occurs at regions of tubulin which are likely to be involved in binding to other tubulin dimers during the process of microtubule polymerization, since the ability of tubulin to bind to Ga is decreased upon tubulin polymer formation, and binding of GLX to tubulin decreases tubulin polymerization [48]. It has been demonstrated that the domains on tubulin involved in the physical interaction between tubulin and G proteins are distinct from those required for the transfer of the nucleotide from tubulin to G proteins [40]. Recently, Popova et al. [32], using different chimeric Gscu/Gi2cu proteins expressed in COS-1 cells, suggested that the regions between the 54th and 212th amino acids of GSLV are important for guanine nucleotide transfer from tubulin, while the 1st to the 54th amino acids of Gsa are involved in the regulation of the tubulin efficiency for activating adenylyl cyclase. Furthermore, Roychowdhury and Rasenick have demonstrated in reconstitution experiments [39] that formation of a complex between tubulin and Gicr changes the GTP-binding characteristics of both molecules and, consequently, tubulin-Gi (Y interaction stabilizes nucleotide binding in the complex. These findings may indi-

cate that tubulin not only transfers nucleotide to G proteins leading to the activation of G proteins, but also stabilizes nucleotide binding through formation of a tubulin-G protein complex [32,39]. Tubulin represents a major component of the synaptic membrane [1,48]. Since microtubules have not been observed to associate with membranes [46], it is likely that much of this tubulin is present in dimer form. In addition, in cerebral cortex, Gs (Y or Gil c~ appears to exist in complexes with tubulin [6]. It is feasible to assume, therefore, that interaction and complex formation between tubulin and G protein favorably occurs and, as a functional consequence of this, tubulin modulates the adenylyl cyclase activity by transferring guanine nucleotide to Gs and Gi. In this regard, it is noteworthy that tubulin is capable of transferring GTP to Gila under conditions in which Gilcr is incapable of binding GTP available in the medium [40]. Since tubulin interacts with Gs and Gil with high affinity, studies concerning participation of tubulin in signal transduction have thus far focused mainly upon the adenylyl cyclase system. Most recently, tubulin has been demonstrated to activate Gq, which mediates activation of phospholipase C, through the complex formation and the nucleotide transfer between them, as is the case with Gs and Gil [33]. Therefore, it may be possible that membrane tubulin participates in the intracellular regulation of phospholipase C as well as adenylyl cyclase. Thus, the ability of the tubulin dimer to bind and transfer guanine nucleotide to the G proteins makes it a relevant candiate as an endogenous modulator in the neuronal signal transduction system. Modulation of signal transduction by the cytoskeletal protein, tubulin, and the various implications of that modulation deserve further investigation.

Acknowledgements This work was supported by a Grant-in-Aid from the Sapporo Medical University Foundation for Research Promotion, the Japan Foundation for Neuroscience and Mental Health, and the Scientific Research Fund of the Ministry of Education, Science and Culture of Japan.

References 111Bhattacharyya,

B., Sackett, D.L. and Wolff, _I., Tubulin, hybrid dimers and tubulin S: stepwise charge reduction and polymerization, J. Bid. Chem., 260 (1985) 10208-10216. 121Birnbaumer, L., Abramowitz, J. and Brown, A.M., Receptor-effector coupling by G proteins, Biochim. Biophys. Acta, 1031 (1990) 163-224. [31 Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72 (1976) 248-254. GTPase [41 Brandt, D.R. and Ross, E.M., Catecholamine-stimulated cycle: multiple sites of regulation by /3-adrenergic receptor and

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