chimeric g,,/gai2 proteins define domains on g,, that interact with

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemietry and Molecular Biology, Inc.

Val. 269, No. 34, Issue of August 26, pp. 2174t?-21754, 1994 Printed in U.S.A.

Chimeric G,,/Gai2 Proteins Define Domains on G,, That Interact with Tubulin for P-Adrenergic Activation of Adenylyl Cyclase*

(Received for publication, May 9, 1994)

Juliana S. PopovaS, Gary L. Johnsons, and Mark M. Rasenickfi From the Department of Physiology and Biophysics and the Committee on Neuroscience, University of Illinois, College of Medicine, Chicago, Illinois 60612-7342 and the 5National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206

Previous studies have demonstrated that dimeric tubulin, associated with synaptic membrane, is capable of activating the G-proteins G, and Gail via transfer of GTP. To clarify the mechanism of intracellular interaction between tubulin and G,, as it refers to adenylyl cyclase activation, wild type and chimeric GJG, proteins were transiently overexpressed in COS 1 cells. Effects of tubulin dimers with guanosine 5’-(P,y-imi- do)triphosphate (Gpp(NH)p) bound (tubulin-Gpp(NH)p) or Gpp(NH)p withlwithout isoproterenol on adenylyl cyclase were assessed in cells made permeable with saponin. In naive and wild type Gas-overexpressing COS 1 cells, the P-adrenergic agonist isoproterenol potenti- ated significantly the stimulatory effects of Gpp(NH)p and, to an even greater extent, tubulin-Gpp(NH)p on adenylyl cyclase. In COS 1 cells expressing the chimera G,i(sq)lB (Gai2 1-54, G, 62-394 amino acids), tubulin-Gp- p(NH)p was more potent than Gpp(NH)p in the presence of isoproterenol, but the maximal activity was equal. In chimera GdicsS, (GaB 1-356, G,, 357-392) tubulin-Gp- p(NH)p or Gpp(NH)p stimulated adenylyl cyclase activity 11-14 times above the control whether or not P-adrenergic receptor was activated, suggesting that G, chimera and the p-adrenergic receptor are uncoupled. The chimera Gai/s(Bpm) (G, 1-212, G,, 213-292) was nearly identical to native COS 1 cells, but isoproterenol poten- tiated Gpp(NH)p but not the tubulin-Gpp(NH)p response. The construct Goi(Bam),s/i(s8) (Goiz 1-212, G,, 213-356, G,, 357-392) was weakly responsive to Gpp(NH)p or tubulin-Gpp(NH)p and unresponsive to isoproterenol. In photoaffinity labeling studies with t~bulin-[~~P]azidoanilido-GTP (~U~UI~~-[~~P]AAGTP) , isoproterenol increased the amount of tubulin associated with membranes and the transfer of [s2P]AAGTP from tubulin to

slightly to G,s/i(38y These results suggest that regions between the 54th and 212th amino acids of G,, are important for guanine nucleotide transfer from tubulin, while the 1st to 54th amino acids of G,, are required for the ability of tubulin to activate adenylyl cyclase. We specu- late that the active G,, conformation provoked by nucleotide transfer from tubulin is stabilized by G,-tubulin

Goli(sl)/s, G,s, and Gai/s(B-), but not to Goli(Bam)/s/i(SB) and very

IBN9121540 and United States Public Health Service Grants MH39595 * This work was supported by National Science Foundation Grant

and GM30324. The costs of publication of this article were defrayed in part by the payment of page charges. This 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 Award from the American Heart Association of Metropolitan Chicago.

ll Recipient of Research Scientist Development Award MH 00699 from the National Institute of Mental Health. To whom correspondence should be addressed: Dept. of Physiology and Biophysics (IWC 901),

202, Chicago, IL 60612-7342. Tel.: 312-996-6641; Fax: 312-996-1414; University of Illinois College of Medicine, 901 South Wolcott Ave., Rm.

E-mail: [email protected].

interaction leading to extended stimulation of adenylyl cyclase.

Heterotrimeric GTP binding proteins (G-proteins)l couple a wide range of cell surface receptors to membrane-bound effector molecules including adenylyl cyclase, phospholipase C, and ion channels (1-7). When a G-protein is in its basal inactive state, the a subunit contains tightly bound GDP and is associated with the P-y subunit complex. Interaction with agonist- bound activated receptor triggers the release of bound GDP and its exchange for GTP. This leads to functional dissociation of G-protein from receptor and of a subunit from P-y, The activated GTP-bound a subunit interacts and regulates an effector, and it has been proven recently that P-y complexes in some cases may also have such activity (8). The a subunit has an intrinsic GTPase activity, which causes its functional dissociation from effector and reassociation with P-y. Thus G-proteins act as molecular switches that can be turned “on” and “off’ through the GTPase cycle. Intensive studies based on expression of mutant G, proteins or construction and expression of chimeric G, proteins have helped to elucidate regions of a subunit polypeptide involved in receptor recognition (9, lo), GTP binding and hydrolysis (11-14), guanine nucleotide-induced conformational changes (151, and effector interaction (16-18). It has been suggested that the a subunit amino terminus contains a regulatory region controlling 6-y subunit interactions and GDP dissociation (independent of GTPase and effector activation domains) (19). I t has been suggested that this region interacts negatively with the carboxyl-terminal effector enzyme domain (17). The carboxyl-terminal portion of G, has been found to include effector and receptor interaction sites (16). Extrapolation from the p21”“ crystal structure has supported the idea that five highly conserved discontinuous regions of the a subunit primary sequence are involved in guanine nucleotide binding and hydrolysis (7).

The cytoskeletal protein, tubulin, shares several features with G-proteins. I t binds GTP, self-assembles, and forms complexes with other proteins to build microtubules. I t hydrolyzes GTP, and at the end of the microtubule this causes instability

tein; COS 1 cells, monkey kidney epithelial cells; G,, adenylyl cyclase The abbreviations used are: G-protein, GTP-binding regulatory pro-

stimulatory G-protein; Gi, adenylyl cyclase inhibitory protein; Go, the predominant brain G-protein; Gt, transducin, retinal rod G, protein; G,, the a subunit of G-protein; G,, and Gmi, the a subunits of G, and G,, respectively; PC-tubulin, tubulin deprived of high molecular weight

phy; tubulin-Gpp(NH)p, tubulin liganded with Gpp(NH)p; tubulin- microtubule-associated proteins with phosphocellulose chromatogra-

AAGTP, tubulin liganded with AAGTP; Gpp(NH)p, guanosine 5’46,~-

AAGTP, P3(4-azidoanilido)-P1-5’-GTP Pipes, piperazine-NJV”bis(2- imid0)triphosphate; GTPyS, guanosine 5’-0-(3-thiotriphosphate);

ethanesulfonic acid); G, chimeric G protein the sequences of which are denoted by the numbers that correspond to the indicated G, subunit.

21748

lhbulin-G-protein Interaction for Adenylyl Cyclase Activation 21749 and dissociation of the complex formation (21). Like G-proteins tubulin is also a substrate for ADP-ribosylation by cholera and pertussis toxins (22).

Tubulin and some G-proteins have been shown to interact with each othe? (23-25). 1Z51-Tubulin was found to bind with high affinity to purified G,, and G,, proteins, but not to G,,, Gois, G,,, and transducin (24). It has been observed that Gail and G,, form complexes with synaptic membrane tubulin,' and a direct transfer of nucleotide from the exchangeable GTP bind- ingaite of tubulin to these proteins has been shown in synaptic membranes and in a purified system as well (23, 25). The importance of this interaction for the intracellular signal trans- duction has been also shown recently. In C6 glioma cells tubulin with hydrolysis-resistant GTP analog bound (Gpp(NH)p) has been found to activate adenylyl cyclase, bypassing the @-adrenergic receptor, which appears to be due to the direct transfer of nucleotide from tubulin to G,, (26). Although domains on tubulin required for interactions with G-proteins have been studied (251, the regions on G, proteins involved in interaction with tubulin are not yet known. Since Gail, Gai2, G,,, and Go all bind tubulin after SDS-polyaclylamide gel electrophoresis and transfer to nitrocellulose, some unique aspect of the native conformation of G,, and Gni1 should allow them to bind tubulin.

This study is directed to determine regions on G,, protein involved in nucleotide transfer and/or binding of tubulin. The relevance of this tubulin-G-protein interaction to adenylyl cyclase stimulation during the course of @-adrenergic receptor activation is assessed. Expression of different chimeric G,$Gai2 proteins in COS 1 cells is used to approach the problem, since tubulin does not bind and transfer nucleotide to G,, (24). Transfected COS 1 cells made permeable with saponin are used throughout the study (27) in order to preserve cell architecture almost intact to provide the right orientation between @-adrenergic receptor, G,, proteins, adenylyl cyclase, and tubulin.

EXPERIMENTAL PROCEDURES Construction of Plasmids-All methods used to construct plasmids

for expression of rat G-protein (Y subunits have been described previously (14, 17, 19, 28). In general, to construct the Gos,i,38) chimera a 1.26-kilobase HindIII-Aha11 fragment from rat G,. cDNA was ligated to a 640-base pair BglII-Hind111 G,, fragment. The chimera was generated using the conserved BamHI site in the G,, and G, cDNAs. The chimera G,icB,vdic3a, was constructed similarly to Goi/s(Bam) using the 3' BamHI fragment generated from G,,,i,,,,. The chimeric Gui(s4vs cDNA was assembled by ligating a 318-base pair EcoRI-Sau3Al G,, fragment and a 1191-base pair BamHI-Hind111 G,, fragment. All chimeric cDNAs were inserted into the Hind111 cloning site of the expression vector pCW1-neo (28), and the orientation of the inserts was verified by re- striction enzyme analysis and DNA sequencing.

Cell Culture and DNA-mediated Gene Dansfer-COS 1 cells were grown and maintained in Dulbecco's modified Eagle's medium and 10% Hyclone. Expression of G,, constructs in COS 1 cells, which express large T antigen for transient plasmid amplification (29), was performed according to the DEAE-dextran procedure described by Ausubel et al. (30). Transfected cells were screened for protein expression by immunoblotting and [32PlAAGTP labeling and used in experiments 65-80 h after transfection. The expression of mutant polypeptides generally varied by less than 20% for any given construct and for each construct within 30% of the wild type Gms. Thus, instead of showing data from representative experiments on adenylyl cyclase activity, we have calculated mean values from not less than three experiments to further decrease the possibility that the changes observed could be affected by differences in the level of construct expression.

Adenylyl Cyclase Assay in Saponin-permeable Cells-Adenylyl cyclase activity was studied in COS 1 cells made permeable by saponin pretreatment according to a modification of a procedure established previously (27). The cells were washed three times with complete Locke's solution (154 mM NaCI, 2.6 m~ KC1, 2.15 m~ KHPO,, 0.85 mM

K. Yan, F. Belga, and M. M. Rasenick, unpublished observations. ~~

KH,PO,, 10 m~ glucose, 2.2 mM CaCI,, 1.0 m~ MgCl,, pH 7.4) for 5 min at 37 "C. Saponin solution (140 mM potassium glutamate, pH 6.8,2 mM ATP, and saponin [lo0 pg/mll) was added for 150 s at room temperature. The plates were inverted, the saponin solution was drained, and the cells were removed from the dish by repeated squeezing of 140 mM potassium glutamate, pH 6.8, from an Eppendorf pipette onto the cells at close range to dislodge and disperse the cells. The cells were centrifuged at 100 x g for 5 min; the solution was aspirated and replaced with Hanks' buffer, pH 7.4. The volume was adjusted to get about 2 x lo6 celldl.0 ml, which were used immediately. 50 pl of cell suspension was pipetted into 1.5-ml microcentrifuge tubes and incubated with [32PlATP (to give 2 x 10' cpm), 0.5 mM ATP, 1 mM MgCl,, 0.5 mM 3-isobutyl-l- methylxanthine in Hanks' buffer, with or without (-)-isoproterenol, Gpp(NH)p, or tubulin-Gpp(NH)p at appropriate concentrations (as shown in legends) in a final volume of 150 pl for 10 min at 37 "C in a shaking water bath. Reactions were stopped with 400 p1 of ice-cold 15 m~ Hepes buffer pH 7.4, and the tubes were frozen immediately at -80 "C. After thawing, the broken cell preparations were boiled for 5 min in a heat block and then centrifuged at 15,000 x g for 10 min at 4 "C. Supernatants were removed, transferred into glass tubes, and 100 pl of stop solution (2% sodium lauryl sulfate, 45 m~ ATP, 1.3 mM 3'3'- CAMP), 50 pl of [3HlcAMP (0.02 pCi), and 1.0 ml of distilled H,O were added. The tubes were decanted over Dowex columns, and [32P]cAMP levels were measured according to the procedure described by Salomon (31). Control values ranged between 0.78 & 0.10 and 4.23 & 0.46 pmol of cAMP/mg of proteidmin depending on the construct expressed. Protein concentrations were determined by the method of Bradford (32) using bovine serum albumin as a standard.

Thbulin Preparations-Microtubule proteins were prepared by the method of Shelanski et al. (33). Briefly, microtubules were polymerized and pelleted by incubation of supernatant of chicken brain homogenates with 2.5 M glycerol, 1 mM GTP, 2 mM EGTA, 1 mM MgCl, in 100 mM Pipes, pH 6.9, at 37 "C followed by centrifugation at 100,000 x g . The microtubule pellet was depolymerized on ice for 1 h. Nucleotides were removed from tubulin by charcoal treatment as described by Rasenick and Wang (23). A second polymerization step was performed in the presence of 1 m~ GTP. This allowed an incorporation of 0.82-0.84 mol of GTP/mol of tubulin (23). Tubulin preparations were stored in aliquots at -80 "C and used less than 4 weeks after preparation. Tubulin-Gp- p(NH)p was prepared from tubulin-GTP by the removal of GTP by means of charcoal treatment as described above, through a third polymerization step in the presence of 150 1.1~ Gpp(NH)p, and incubated for 20 min at 37 "C. This tubulin preparation contained microtubule- associated proteins, which could be removed by phosphocellulose chro- matography with the eluting buffer of 100 mM Pipes, pH 6.9, 1 mM EGTA, 1 mM MgCI, (PC-tubulin). The resulting preparations were greater than 97% tubulin as estimated by Coomassie Blue staining. Prior to use, tubulin-guanine nucleotide preparations (tubulin- Gpp(NH)p or tub~lin-[~~PlAAGTP) were passed through P6-DG resin (Bio-Rad) columns twice in order to remove the excess of unbound nucleotide. After this procedure 0.4-0.6 mol of nucleotide were bound per mol of tubulin (23).

Photoafinity Labeling and Nucleotide D ~ ~ s ~ ~ ~ - [ ~ ' P ] A A G T P a s well as AAGTP were synthesized by the method of Pfeuffer (34). Tubulin- [32PlAAGTP was prepared from PC-tubulin incubated with 150 1.1~ [32PlAAGTP for 30 min on ice, and the excess of nucleotide was removed as described above.

COS 1 cells were made permeable with saponin solution as described above, washed three times with 140 mM potassium glutamate, pH 6.8, and incubated at 37 "C while attached to plates with indicated concentrations of (-)-isoproterenol, [3zPlAAGTP (for 3 rnin), or tubulin- [3zPlAAGTP (for 10 min) in Hanks' buffer, 0.5 mM MgC1,. The plates were then UV-irradiated for 5 min with a Spectroline W lamp (254 nm, 9 watts) on ice at a distance of 4 cm. The reaction was quenched with ice-cold 2 mM Hepes pH 7.4, 1 mM MgCl,, 4 m~ dithiothreitol, and then the cells were scraped, transferred into glass tubes, and sonicated for 5 s. The broken cell preparations were centrifuged for 10 min at 600 x g at 4 "C. The supernatants were decanted and centrifuged again at 100,000 x g for 30 min at 4 "c. cytosol supernatants were transferred to glass tubes, and membrane pellets were mixed with 10% trichloroacetic acid and centrifuged under the same conditions. They were then washed three times with 15 m~ Hepes pH 7.4 and dissolved in 3% SDS Laemmli sample buffer with 50 mM dithiothreitol. The cytosol supernatants were precipitated with 10% trichloroacetic acid for 30 min on ice, followed by centrifugation at 100,000 x g for 30 min. The remaining pellets were washed three times as indicated above and dissolved in 3% SDS Laemmli sample buffer with 50 mM dithiothreitol.

Samples were heated for 5 min at 90 "C, and 70 pg of protein from

21750 nbulin-G-protein Interaction for Adenylyl Cyclase Activation

FIG. 1. Adenylyl cyclase activity in permeable COS 1 cells. Enzyme activity was measured in control cells (mock, carrier cDNA only) (panel A ) and cells expressing wild type G,, (panel B ) or different GJG,, chimeric proteins; Gai(SIVs (panel C ) , G,,m3,) (panel D ) , Gage(Barn) (panel E), and Gui(BarnVs,i(3B) (panel F ) . The schematic diagram of each chimera is shown in the upper left corner of each panel. Three days after transfection with indicated pCW1-neo G, subunit constructs COS 1 cells were made permeable with saponin and assayed for adenylyl cyclase in the presence of indicated concentrations of Gpp(NH)p, Gpp(NH)p and 10 PM (-)-isoproterenol, tubulin-Gpp(NH)p, or tubulin-Gpp(NH)p and 10 PM (-)-isoproterenol as described under “Experi- mental Procedures.” Means of at least three experiments, each one done in trip- licate, are shown. Control values (pmols of cAMP/mg of proteidmin) for each kind of experiment were as follows: 0.78 f 0.10 (A) ; 2.31 f 0.35 ( B ) ; 3.00 f 0.50 (C); 3.19

( F ) . f 0.36 ( D l ; 2.64 * 0.29 ( E ) ; and 4.23 f 0.46

?.

Y

each sample (if not otherwise stated) were loaded and electrophoresed in SDS-polyacrylamide gels (10% acrylamide and 0.133% bisacrylam- ide) by the procedure of Laemmli (35). After electrophoresis gels were either stained and radioautographed (Coomassie Blue, Kodak XAR-5 film) or used for Western blotting followed by autoradiography.

Western Blotting-Immunoblot analysis was carried out by a modification of the procedure described by Wang et al. (24). Membrane proteins, resolved by SDS-polyacrylamide gel electrophoresis, were transferred to nitrocellulose using a semi-dry transfer apparatus (Bio-Rad). They were probed with antibodies specific for the carboxyl terminus of either G,, or Gmi, at a dilution of 1500 (36) or tubulin at a dilution of 1:lOOO. Biotinylated goat anti-rabbit IgG and streptavidin-alkaline phosphatase conjugate as the signal-generating system were used. Den- sitometry measurements of the G, bands was performed, and ratios of an expressed G, protein band to an endogenous band (as-S or as-L, for chimeras with a G,, COOH terminus) were calculated to assess the level of expression.

M~terials-[a-~~PlATP (650 Ci/mmol) was from ICN Biomedicals, Inc. (Irvine, CA). All nucleotides were from Boehringer Mannheim. p-Azidoaniline was synthesized by Dr. William Dunn (University of Illinois, Chicago). All other reagents were of analytical grade. Antibod- ies against G,, and Gmi, were provided by Dr. D. Manning, Philadelphia (antisera 1190 and 1521, respectively), and a polyclonal anti-tubulin antibody (code 65-095-1) was from ICN Biomedicals, Inc.

RESULTS

It has been demonstrated that transient expression of wild type and chimeric G, proteins in COS 1 cells provides a mechanism with which to observe the relationship between these

Y

0 - 8 - 7 - 6 LOG [DRUG] ( M )

D

5 ~ 1 1 1 1 1 1 z x 0 - 9 - 8 - 7 - 6

LOG [DRUG] ( M )

F

constructs, membrane receptors, and adenylyl cyclase (17, 19, 28,371. This experimental approach allows the study of regions of G, polypeptide chain directly involved in these interactions since signals are amplified due to overexpression of G, proteins. Schematic diagrams of the G,JGai2 chimeras used in the present study are shown in the upper left corners ofpanels C-F in Fig. 1. The G,, polypeptide is 394 amino acid residues, and G,, polypeptide is 354 amino acid residues. The G,i(54)ls chimera is encoded by the first 54 residues of G,, and amino acids 62-394 of Gas. The Gai/s(Bam) chimera is encoded by the first 212 amino acids of Gmi, and residues 235-394 of Go*. The Gas/i(38, chimera contains the first 356 amino acid residues of G,, and the last 36 amino acid residues of Goi2. The chimera G,i(Baml/s~i(S8) is a combination of Gai/s(Bam) and G,s,i(38) chimeric proteins. Western blotting and ADP-ribosylation experiments have shown that transfection of COS 1 cells with these G,, cDNAs (wild type or chimeric), while inserted in pCW1-neo plasmid, results in overexpression of the respective G, polypeptides. This event is cor- related with changes in intracellular CAMP level or membrane adenylyl cyclase activity, depending on the nature of the chimera expressed (16, 17, 19, 28).

The effect of tubulin-Gpp(NH)p on G,,-mediated adenylyl cyclase was evaluated in permeable naive COS 1 cells with or without concomitant p-adrenergic receptor activation. As shown in Fig. 1, panel A, in control COS 1 cells (mock, carrier

lhbulin-G-protein Interaction for Adenylyl Cyclase Activation 21751

A

a s - L - a.9-s-

B membranes cytosol

0 0 0) v

FIG. 2. Immunoblots of membrane preparations of COS 1 cells expressing wild type or mutant GJG,, polypeptides were probed with COOH-terminal anti-G, (panel A ) or anti-Gei, (panel B ) antisera. Lanes represent the various transfection conditions with carrier cDNA only (mock) or pCW1-neo expression plasmid with the indicated cDNA inserts. The experiments were done as described in the text. 70 and 35 pg of membrane protein were loaded and electrophoresed in the experiments shown in panels A and B, respectively. as-L indicates the immunoreactive large G,, molecular mass band (52 kDa), as-S the small Gas molecular mass band (45 kDa), and ai2 the Gei, molecular mass band (41 kDa). Both the small and the large G,, splice variants are expressed endogenously in COS 1 cells a t similar levels. The G,, cDNA used for mutation and expression codes for the large 394-amino acid G,, chain is shown. The results are representative of seven independent experiments.

cDNA only) Gpp(NH)p as well as tubulin-Gpp(NH)p slightly increased adenylyl cyclase activity when applied at higher concentrations (1 PM). No significant differences in their effects could be observed without P-adrenergic receptor stimulation. The addition of the P-adrenergic receptor agonist isoproterenol (10 PM) increased significantly enzyme activity in both cases; however, the effect was greater with tubulin-Gpp(NH)p. Satu- ration of the tubulin-Gpp(NH)p effect was not observed in the concentration range studied, and higher tubulin-Gpp(NH)p concentrations could not be used due to initiation of tubulin self-assembling processes (38). Nonetheless, it was obvious that tubulin-Gpp(NH)p was a more efficient stimulator of adenylyl cyclase activity than the free guanine nucleotide upon receptor activation.

When wild type G,, protein was overexpressed in COS 1 cells (Fig. 1, panel B ) , adenylyl cyclase followed the same activity patterns as the controls (Fig. 1, panel A), although enzyme activity was twice as high as that in naive COS 1 cells. At 10 1.1~ isoproterenol, saturable stimulation was observed, EC,, values being 7.08 nM and 8.41 nM for tubulin-Gpp(NH)p and Gpp(NH)p, respectively. These experiments showed that in permeable COS 1 cells in contrast to C6 glioma cells (26, 27, 39), tubulin-Gpp(NH)p was not able to bypass the constraint p-adrenergic receptor exerts on G, while not agonist activated, but tubulin-Gpp(NH)p was capable of potentiating agonist-stimulated adenylyl cyclase activity. Clearly, in COS 1 cells, activated P-adrenergic receptor and tubulin act in concert.

In order to find facets of G,, that interact functionally with tubulin, similar experiments were performed in COS 1 cells overexpressing several different chimeric Gu.JGei2 proteins. In cells expressing a chimeric G,, which substitutes the amino- terminal 54 amino acids of G,, to Gai2 (G,i(54Ks), adenylyl cyclase activity increased significantly upon tubulin-Gpp(NH)p or Gpp(NH)p addition (Fig. 1, panel C). Unlike the situation with G,, overexpression, Gpp(NH)p alone allowed stimulation of adenylyl cyclase, which exceeded that induced by tubulin- Gpp(NH)p. Further, while isoproterenol-activated adenylyl cyclase continued to be more sensitive to tubulin-Gpp(NH)p, maximal activity achieved by the two agents was equal. Gai(54Vs is efficiently coupled to receptors (17). A time course study showed an enhanced rate of adenylyl cyclase activation by GTPyS in membranes from G,,,,,,-transfected COS 1 cells due

GCY- wt 5 - -

I I I I

(-)-is0 FIG. 3. Transfer of [s2PlAAGTP from t~bulin-[~~PlAAGTP (T) to

G, (Ga) in permeable G,-overexpressing COS 1 cells and the effect of P-adrenergic receptor stimulation. Permeable cells were incubated with 1 p~ tubulin-["'PIAAGTP and either with (+) or without (-1 10 p~ isoproterenol as described under "Experimental Procedures." Radioautographs of membrane or cytosolic proteins resolved by SDS- polyacrylamide gel electrophoresis are shown. Results from one of two similar experiments are shown.

GCY- (-)-is0

FIG. 4. Effect of isoproterenol on the transfer of ["PIAAGTP from tubulit1-[~2P]AAGTP to different GAG,, chimeric proteins in permeable COS 1 cells. Permeable cells, expressing carrier Go, proteins (mock), overexpressing wild type G,, ( s ) or mutant GJG",, proteins (ildharn), i(ham)lsli(38), i(54)ls, or sli(38)) were incubated with 1 p~ tubulin-["PIAAGTP and either with (+I or without (-) 10 pbl isoproterenol as indicated in the text. Radioautograph of membranes resolved by SDS-polyacrylamide gel electrophoresis is shown. The results are representative of five independent experiments.

to accelerated GDP dissociation and faster GTPyS binding (17, 19). The present concentration-response experiments showed that in permeable COS 1 cells expressing Gni(54),,, while tubulin- Gpp(NH)p was more potent than Gpp(NH)p in stimulating enzyme activity upon receptor activation (EC,, values of 1.34 nM and 7.9 nM, respectively), it was less efficient when compared with wild type GOs-transfected cells (Fig. 1, panel B 1. At a 1 PM concentration there was no difference between tubulin-Gp- p(NH)p and Gpp(NH)p-evoked responses. Despite the fact that Gui(54)/s chimeric protein is a dominant Gus mutant whose activity is constitutively enhanced (17), i t responds less successfully to tubulin-Gpp(NH)p stimulation. As tubulin does not bind and transfer nucleotide to Gai2 (261, which represents the first part of Gui(54V, polypeptide, it is suggested that the 1st to 61st amino acids of G,, protein are needed to increase the efficiency of tubulin-Gpp(NH)p above that of Gpp(NH)p.

When another constitutively active chimeric G, protein, Gndi(3R, (Gus 1-356; Gai2 320-355) (4,6), was expressed, guanine nucleotide- or tubulin-guanine nucleotide-stimulated adenylyl cyclase activity increased 11-14 times above the control, whether or not the p-adrenergic receptor was activated (Fig. 1, panel D ) . The EC,, values were lower (in the presence of 10 PM isoproterenol, 1.58 and 1.85 nM for tubulin-Gpp(NH)p and Gpp(NH)p, respectively), but isoproterenol failed to further increase enzyme activity. These results complemented the finding showing a decreased time required to achieve maximal adenylyl cyclase activation by GTPyS in membranes from Chi- nese hamster ovary cells expressing (28) and confirmed that guanine nucleotide (and tubulin-guanine nucleotide)-activated adenylyl cyclase activities were reproducibly greater in G,,,,,,,,-expressing cells compared with wild type or G,,-expressing cells. I t seemed, however, that the substitution of the last 38 amino acids of Gus with the carboxyl-terminal 36 residues of

21752 lbbulin-G-protein Interaction for Adenylyl Cyclase Activation

uta/

FIG. 5 . Mechanism of tubulin-Gpp(NH)p potentiation of P-adrenergic receptor-triggered adenylyl cyclase activation. The transfer of nucleotide from tubulin to G , promotes conformational change of G,, leading to its functional dissociation from the receptor and p-y subunit and subsequent interaction and activation of adenylyl cyclase. The binding of tubulin to G,, slows down G,, functional dissociation from adenylyl cyclase, thus increasing the lifetime of the active enzyme conformation.

G,, (28) prevented the coupling of this chimeric G, protein to the P-adrenergic receptor.

Two other chimeric proteins, Gai/B(Bam) (G,, 1-212; Gas 235-

(17,19), were expressed. Both had been shown to be functional Go, polypeptides capable of increasing intracellular CGMP pro- duction in transfected COS 1 cells and S49cyc- cells (16,17,19). The G,,,,,, chimera was shown to behave as a functional wild type Gas with respect to receptor selectivity as well (17). The present study showed that isoproterenol (10 p ~ ) increased (al- beit weakly) Gpp(NH)p-stimulated adenylyl cyclase in Goi/s(Bam)- transfected COS 1 cells (Fig. 1, panel E ) . However, it failed to potentiate the effect of tubulin-Gpp(NH)p. In the absence of isoproterenol, no difference between Gpp(NH)p and tubulin- Gpp(NH)p was observed, similar to the situation for control COS 1 cells (Fig. 1, panel A). When the cells were transfected with the construct Gai(Bamydi(38), which is a combination of G,s(Bam) and GosEi(38), they were weakly responsive to 1 p~ Gp- p(NH)p or tubulin-Gpp(NH)p and unresponsive to isoproter enol (at 10 p ~ ) (Fig. 1, panel F) . The results obtained confirmed the importance of the COOH-terminal part of G,, polypeptide for the coupling to p-adrenergic receptor (16) and the signifi- cance of the region between the 1st and 212th amino acids of G,, for activation of the G-protein by tubulin-Gpp(NH)p.

Although responses to Gpp(NH)p and tubulin-Gpp(NH)p were weak in Goi/s(Bm) and G,,,,,,,,,-transfected COS 1 cells, it was not due to a lack of expression of the respective chimeric

394) and G,i(Bamydi(38) (G,iz 1-212; G,, 235-356; G,, 320-355)

G, proteins. Fig. 2 shows results of immunoblotting experiments using specific anti-Gas (panel A ) or anti-G,, (panel B ) antibodies, depending on the COOH-terminal region of the chimeric protein expressed. It can be seen from both anti-G,, and anti-G,, probed blots that G, proteins are expressed at similar level. Densitometry measurements (n = 7) indicated approximately 3-5-fold increased abundance of as-L (52 kDa) when wild type G,, was overexpressed, or of a band at 45 kDa, when the construct G,,(Bam) was expressed. Similar abundance of Gui(54ys (48 kDa) was obtained after transfection. Anti-G,, COOH-terminal antisera treatment also revealed a 3-4-fold increase, compared with G,,, when the constructs Gos,i(38) or G,(Bmys/i(38) were expressed (Fig. 2, panel B ) .

In order to visualize the transfer of nucleotide from tubulin to G,, proteins (wild type and chimeric) and to verify its coupling to P-adrenergic receptor stimulation, we used the hydrolysis- resistant photoaffinity GTP analogAAGTP (in its w3'P version), bound to tubulin (tub~lin-[~~PlAAGTP), in photolabeling experiments performed on permeable transfected COS 1 cells. 10 p~ isoproterenol increased the amount of tub~lin-[~~PlAAGTP bound to membranes and also the transfer of [32P]AAGTP from tubulin to Gas (Fig. 3). When autoradiograms were measured by densitometry, 8.0 * 2.2% more t~bulin-[~'PlAAGTP was found associated with membranes after isoproterenol stimulation ( n = 2). Isoproterenol increased [32P]AAGTP transfer from tubulin to Gas by 76 2 15% compared with controls. In cytosol, although more of the t~bulin-[~~PlAAGTP was found, it diminished after

mbulin-G-protein Interaction for Adenylyl Cyclase Activation 21753

p-adrenergic receptor stimulation by 10.3 f 3.2%, which can be accounted for by the isoproterenol-induced increase in tubulin binding to the membranes and the concomitant [32P]AAGTP transfer to Ge8. It had been shown previously that treatment with isoproterenol caused G,, to shift from the membrane-bound to the soluble compartment (40, 41). It was suggested that the redistribution ofG,, was due to conformational change that loos- ened attachment of that molecule to membranes and increased its turnover rate (41). As seen in Fig. 3, we were not able to detect an increase of G,,-[32PIAAGTP in cytosol after isoproterenol stimulation, although a small signal was observed when @-adrenergic receptors were not stimulated. An explanation of this finding could be the possibility that tubulin binding to G,, upon receptor stimulation slowed down G,, turnover rate, increasing the lifetime of its active conformation. Since such a release into the cytosol has been observed in other cell types (40) it is possible that different cell types display unique behavior in this regard.

Results shown in Fig. 4 confirmed again that tubulin transferred guanine nucleotide to G,, proteins and clearly showed that isoproterenol (10 1.1~) increased the transfer of [32PlAAGTP from tubulin to Gai(64),s, G,,, and Gogs(Bam) but not to Gai(Barn)/di(38)

(which was expected to lie below the small G,, band) and only slightly to The experimental approach used did not allow us to detect differences in labeling among the G, bands as the signals were always overlapping.

DISCUSSION

Tubulin-Gpp(NH)p appears to potentiate @adrenergic receptor activation of adenylyl cyclase in COS 1 cells through a direct interaction with Goa. The studies performed using different chimeric GJG,,, proteins are consistent with the suppositions that ( a ) the loss of the COOH-terminal part of G,, suppresses p-adrenergic potentiation of tubulin-guanine nucleotide stimulation of adenylyl cyclase activity, ( b ) the core G,, sequence, amino acids 54-212, is important for guanine nucleotide transfer from tubulin, and (c) the amino terminus of G,, (residues 1-54) is required for the ability of tubulin to activate adenylyl cyclase.

Fig. 5 is a schematic illustration of our current understand- ing of the molecular events leading to tubulin-guanine nucleotide potentiation of P-adrenergic receptor-triggered adenylyl cyclase activation. A key point in the widely accepted mechanism for G-protein coupled receptor-effector signal transduc- tion is that the GTPase cycle of the G-protein a subunit func- tions as a timer to control the maintenance of the activated conformation of the protein and thus the duration of adenylyl cyclase activation. This is the step at which we predict that tubulin is exerting its effect on adenylyl cyclase. Upon receptor stimulation, tubulin transfers Gpp(NH)p from its exchangeable site on its 0 subunit to G,,, thus driving a conformational switch in G,, tertiary structure. The latter leads to functional dissociation of G,, from the receptor and G,., and subsequent association with adenylyl cyclase. Whether the tubulin molecule interacts with activated G,, before or after G,, association with adenylyl cyclase, the net result is stabilization of the active G,, conformation and the Gm,-adenylyl cyclase complex, leading to increased stimulation of the enzyme activity. Recent findings support this idea. First, it has been shown in recon- stitution experiments (42) that when tubulin-guanine nucleotide binds to Gail, the nucleotide binding is stabilized in the complex. Second, purified G-protein pr was able to override the effect of Gail, perhaps by altering the interaction between tubulin and Gail (42).

In the living cell, the active guanine nucleotide is GTP and not Gpp(NH)p, and it is the GDP exchange for GTP that is triggered upon receptor activation. Mammalian cells contain high concentrations of intracellular GTP, estimated at approxi-

mately 0.5 m~ (43,441. Although tubulin is an abundant intracellular protein, its maximal concentration in mouse 3T3 cells has been calculated to be around 20 p ~ , ie. 25 times less than free GTP, and approximately 40% of it is polymerized (45). These facts appear to diminish the likelihood of tubulin affect- ing adenylyl cyclase through the GTP cycle of Go*. But there are at least two arguments to consider. First, the distribution of the pool of soluble tubulin throughout the cell (ie. is it uniform or heterogeneous?) is unknown. Tubulin represents a major com- ponent of certain membranes,’ and this membrane tubulin would be likely to have “favored access” to G, or Gi,. Second, the effect of tubulin-GTP appears to be longer lasting and more efficacious than that of GTP, even a t much lower levels of tubulin-G,, interaction.

It has been shown that GTPase-inhibiting mutations activate the a chain of G, and stimulate adenylyl cyclase in human pituitary tumors, thus bypassing the normal cell requirement for trophic hormone (46). Since CAMP stimulates the growth of some cultured cells (47, 48) and the effect of tubulin-guanine nucleotide on the adenylyl cyclase stimulatory cascade in COS 1 cells, although significant, has shown a requirement for receptor activation, it can be speculated that the data presented in this report describe a mechanism for enhanced adenylyl cyclase stimulation resulting in increased CAMP synthesis and protein kinase A activation. Perhaps information about changes in cell shape and the cytoskeleton is communicated to the cellular interior via such a mechanism. Efforts to understand this process are under way.

Acknowledgments-We thank Dr. D. Manning for providing the anti-G, antisera and Dr. W. Dunn for 4-azidoaniline. We are also grate- ful to M. Talluri for growing cells and technical assistance.

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