THEJOURNAL 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 onG,, That Interact with Tubulin for P-Adrenergic Activationof Adenylyl Cyclase* (Received forpublication, May 9, 1994)
Juliana S. PopovaS, Gary L. Johnsons, andMark M. Rasenickfi From the Department of Physiology and Biophysics and the Committee on Neuroscience, University of Illinois, College of Medicine, Chicago, Illinois 60612-7342and the 5National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206 Previous studies have demonstrated that dimeric tu- interaction leading to extended stimulation of adenylyl bulin, associated with synaptic membrane, iscapable of cyclase. 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 cyHeterotrimeric GTP binding proteins (G-proteins)l couple a clase activation, wild type and chimeric GJG, proteins were transiently overexpressed in COS 1 cells. wide range of cell surface receptors to membrane-boundeffecEffects of tubulin dimers with guanosine 5’-(P,y-imi- tor molecules including adenylyl cyclase, phospholipase C, and do)triphosphate (Gpp(NH)p) bound (tubulin-Gpp(NH)p)ion channels (1-7). When a G-protein is in its basal inactive state, thea subunit contains tightlybound GDP andis associor Gpp(NH)p withlwithout isoproterenol on adenylyl cyclase were assessed in cells made permeable with ated with the P-y subunit complex. Interaction with agonistsaponin. In naive and wild type Gas-overexpressingCOS bound activated receptor triggers the release of bound GDPand 1 cells, the P-adrenergic agonist isoproterenol potenti- its exchange for GTP. This leads to functional dissociation of ated significantly the stimulatory effects of Gpp(NH)p G-protein from receptor and of a subunit from P-y, The actiand, to an even greater extent, tubulin-Gpp(NH)p on ad- vated GTP-bound a subunit interacts and regulates effector, an enylyl cyclase. In COS 1 cells expressing the chimera and it has been proven recently that P-y complexes in some G,i(sq)lB(Gai21-54, G, 62-394 amino acids), tubulin-Gp- cases may also have such activity (8). The a subunit has an p(NH)p was more potent than Gpp(NH)pin thepresence of isoproterenol, but themaximal activity was equal.In intrinsic GTPase activity, which causes its functionaldissociachimera GdicsS, (GaB 1-356, G,, 357-392) tubulin-Gp- tion from effector and reassociation with P-y. Thus G-proteins p(NH)por Gpp(NH)pstimulated adenylyl cyclaseactiv- act as molecular switches that can be turned “on” and “off’ ity 11-14 times abovethe control whether or not P-adre- through the GTPase cycle. Intensive studies based on expresnergic receptor was activated, suggesting that G, sion of mutant G, proteins or construction and expression of regions of a subchimera and the p-adrenergic receptor are uncoupled. chimeric G, proteins have helped to elucidate The chimera Gai/s(Bpm) (G, 1-212, G,, 213-292) was nearly unit polypeptide involved in receptor recognition (9, lo), GTP identical to native COS 1cells, but isoproterenol poten- binding and hydrolysis (11-14), guanine nucleotide-induced tiated Gpp(NH)p but not the tubulin-Gpp(NH)p re- conformational changes (151, and effector interaction (16-18). sponse. Theconstruct Goi(Bam),s/i(s8) (Goiz1-212, G,, 213-356, It has been suggested that the a subunit amino terminusconG,, 357-392) was weakly responsiveto Gpp(NH)p or tu- tains a regulatory region controlling 6-y subunit interactions bulin-Gpp(NH)pand unresponsive to isoproterenol. In and GDPdissociation (independent of GTPase andeffector acphotoaffinity labeling studies with t~bulin-[~~P]azidoativation domains) (19). It has been suggested that this region nilido-GTP ( ~ U ~ U I ~ ~ - [ ~ ~ P ] A isoproterenol AGTP), in- interacts negatively with the carboxyl-terminal effector encreased the amount of tubulin associated with memzyme domain (17). The carboxyl-terminal portion ofG, has branes and the transfer of [s2P]AAGTP from tubulin to been found to include effector and receptor interaction sites Goli(sl)/s,G,s, and Gai/s(B-), but not to Goli(Bam)/s/i(SB)and very slightly to G,s/i(38yThese results suggest that regions be- (16). Extrapolation from the p21”“ crystal structure has suptween the 54th and 212th aminoacids of G,, are impor- ported the idea that five highly conserved discontinuous retant for guanine nucleotide transfer from tubulin, while gions of the a subunit primary sequence areinvolved in guathe 1st to 54th amino acids of G,, are required for the nine nucleotide binding and hydrolysis (7). The cytoskeletal protein, tubulin, shares several features ability of tubulin to activate adenylyl cyclase.We speculate that the active G,, conformation provokedby nucle- with G-proteins. I t binds GTP, self-assembles, and forms comotide transfer from tubulin is stabilized by G,-tubulin plexes with other proteins to build microtubules. I t hydrolyzes GTP, and at the endof the microtubule this causes instability
* This work was supported by National Science Foundation Grant IBN9121540 and UnitedStates Public Health Service Grants MH39595 and GM30324. The costsof 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 MentalHealth. To whomcorrespondence should be addressed: Dept. of Physiology and Biophysics (IWC 901), University of Illinois Collegeof Medicine, 901 South Wolcott Ave., Rm. 202,Chicago,IL60612-7342.Tel.:312-996-6641;Fax:312-996-1414; E-mail:
[email protected].
The abbreviationsused are: G-protein, GTP-bindingregulatory protein; COS 1 cells, monkey kidney epithelial cells; G,, adenylyl cyclase 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 highmolecularweight microtubule-associatedproteinswithphosphocellulosechromatography; tubulin-Gpp(NH)p,tubulinligandedwithGpp(NH)p;tubulinAAGTP, tubulin liganded with AAGTP; Gpp(NH)p, guanosine 5 ’ 4 6 , ~ 5’-0-(3-thiotriphosphate); imid0)triphosphate; GTPyS, guanosine AAGTP, P3(4-azidoanilido)-P1-5’-GTP Pipes, piperazine-NJV”bis(2ethanesulfonic 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 Adenylyl forCyclase Activation and dissociation of the complex formation (21). Like G-proteins tubulin is also a substratefor ADP-ribosylationby cholera and pertussis toxins (22). Tubulin and some G-proteins have been shown t o interact with each othe? (23-25). 1Z51-Tubulin was found t o 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 complexeswith synaptic membrane tubulin,' and a direct transfer of nucleotide from the exchangeable GTP bindingaite of tubulin to these proteins has been shownin synaptic membranes and in a purified system as well (23, 25). The importance of this interaction for the intracellular signal transduction has been also shown recently. In C6 glioma cellstubulin with hydrolysis-resistant GTP analog bound (Gpp(NH)p) has been found to activate adenylyl cyclase, bypassing the @-adrenergicreceptor, 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 Goall bind tubulin afterSDS-polyaclylamidegel electrophoresis and transfer to nitrocellulose, some unique aspect of the native conformation of G,, and Gni1should allow them to bind tubulin. This study is directed to determine regions onG,, 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 @-adrenergicreceptor 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 andtransfer nucleotide to G,, (24). Transfected COS 1 cells made permeable with saponin are used throughout the study (27) in order t o preserve cell architecture almost intact to provide the right orientation between @-adrenergic receptor, G,, proteins, adenylyl cyclase, and tubulin. EXPERIMENTALPROCEDURES 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-kilobaseHindIII-Aha11fragment 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 a1191-basepair 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 restriction enzyme analysis and DNA sequencing. 1 cells were Cell Culture and DNA-mediated Gene Dansfer-COS grown and maintained in Dulbecco's modified Eagle's mediumand 10% Hyclone. Expression ofG,, constructs in COS 1 cells, which express large Tantigen 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 [32PlAAGTPlabeling 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 beaffected by differences in thelevel of construct expression. Adenylyl Cyclase Assay in Saponin-permeable Cells-Adenylylcyclase activity was studied in COS 1 cells made permeable by saponin pretreatment according to a modification of a procedure established previously (27). The cellswere 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.
21749
KH,PO,, 10 m~ glucose, 2.2mM 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 150s 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, froman 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 andreplaced with Hanks' buffer, pH 7.4. The volume was adjusted to get about 2 x lo6 celldl.0 ml, which were used immediately. 50 plof cell suspension was pipetted into 1.5-mlmicrocentrifuge tubes and incubated with [32PlATP (to give 2 x 10' cpm), 0.5 mM ATP, 1 mM MgCl,, 0.5 mM 3-isobutyl-lmethylxanthine 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 400p1of ice-cold 15 m~ Hepes buffer pH7.4, and the tubes were frozen immediately a t -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), 50pl 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 weremeasured according to the procedure describedby Salomon (31). Control values ranged between 0.78& 0.10 and 4.23 & 0.46 pmol of cAMP/mg ofproteidmin 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-Gpp(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 microtubuleassociated proteins, which could be removedby phosphocellulose chromatography 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 (tubulinGpp(NH)p or tub~lin-[~~PlAAGTP) were passed through P6-DG resin (Bio-Rad) columns twice in order toremove 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 Gwell TP as as AAGTP were synthesized by the method of Pfeuffer (34). Tubulin[32PlAAGTPwas prepared from PC-tubulin incubated with 150 1.1~ [32PlAAGTP for min 30 on ice,and theexcess of nucleotide was removed as described above. COS 1cells weremade 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 SpectrolineW 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, 1mM MgCl,, 4 m~ dithiothreitol, and then the cells werescraped, 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 werethen 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 minon 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)F (panel ) . The schematicdiagram of eachchimerais 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 presenceof 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 describedunder“ExperimentalProcedures.”Means of atleast three experiments, eachone done in triplicate, are shown. Control values (pmols of cAMP/mg of proteidmin) for each kind of experiment were asfollows: 0.78 f 0.10 ( A ) ;2.31 f 0.35 ( B ) ;3.00 f 0.50 (C);3.19 f 0.36 ( D l ;2.64 * 0.29 ( E ) ;and 4.23 f 0.46
0
- 7
-8
-6
LOG [DRUG] ( M )
Y
Y
D
5
~
1
1
1
1
1
z
x
0
- 9
-8
LOG [DRUG]
- 7
-6
(M)
F
(F).
each sample (if not otherwise stated) were loaded and electrophoresed constructs, membrane receptors, and adenylyl cyclase (17, 19, in SDS-polyacrylamide gels (10% acrylamide and 0.133% bisacrylam- 28,371. This experimental approach allows the studyof regions ide) by the procedure of Laemmli (35). After electrophoresis gels were either stained and radioautographed (Coomassie Blue, Kodak XAR-5 of G, polypeptide chain directly involved in these interactions since signals areamplified due tooverexpression of G, proteins. film) or used for Western blotting followed by autoradiography. Western Blotting-Immunoblot analysis was carried out by a modi- Schematic diagrams of the G,JGai2 chimeras used in thepresfication of the procedure described by Wang et al. (24). Membrane pro- ent study areshown in the upper left corners ofpanels C-F in teins, resolved by SDS-polyacrylamide gel electrophoresis, were trans- Fig. 1. The G,, polypeptide is 394 amino acid residues, andG,, ferred to nitrocellulose using a semi-dry transfer apparatus (Bio-Rad). polypeptide is 354 amino acid residues. The G,i(54)lschimera is They were probed with antibodiesspecific for the carboxyl terminus of encoded by the first54 residues of G,, and amino acids62-394 either G,, or Gmi,at a dilution of 1500 (36) or tubulin at a dilution of of Gas. The Gai/s(Bam) chimera is encoded by the first 212 amino 1:lOOO. Biotinylatedgoatanti-rabbit IgG andstreptavidin-alkaline phosphatase conjugateas the signal-generating system were used. Denacids of Gmi,and residues 235-394 of Go*.The Gas/i(38, chimera sitometry measurements of the G, bands was performed, and ratiosof contains thefirst 356 amino acid residues of G,, and the last 36 an expressed G, protein band toa n endogenous band(as-S or as-L, for amino acid residues of Goi2. The chimera G,i(Baml/s~i(S8) is a comchimeras with G,, a COOH terminus) were calculated t o assess thelevel bination of Gai/s(Bam) and G,s,i(38)chimeric proteins. Westernblotof expression. ting andADP-ribosylation experiments haveshown that transM~terials-[a-~~PlATP (650 Ci/mmol) was from ICN Biomedicals, Inc. (Irvine, CA). All nucleotideswere from BoehringerMannheim. fection of COS 1 cells with these G,, cDNAs (wild type or p-Azidoaniline was synthesized by Dr. William Dunn (University of chimeric), while inserted inpCW1-neo plasmid, results inoverIllinois, Chicago).All other reagents wereof analytical grade. Antibodexpression of the respective G, polypeptides. This eventis cories againstG,, and Gmi,were provided by Dr. D. Manning, Philadelphia related with changes in intracellular CAMPlevel or membrane (antisera 1190 and 1521, respectively), and a polyclonal anti-tubulin adenylyl cyclase activity, depending on the nature of the chiantibody (code 65-095-1) was from ICN Biomedicals, Inc. mera expressed (16, 17, 19, 28). RESULTS The effect of tubulin-Gpp(NH)p on G,,-mediated adenylyl
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
cyclase was evaluated in permeable naive COS 1cells with or without concomitant p-adrenergic receptor activation. As shown in Fig. 1,panel A, in control COS 1 cells (mock, carrier
1
lhbulin-G-protein Interaction Adenylyl for
membranes
B
A
Cyclase Activation
21751 cytosol
-
as- L a.9-s-
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 v effect of P-adrenergicreceptor stimulation. Permeable cells were incubated with 1 p~ tubulin-["'PIAAGTP and either with(+) or without FIG.2. Immunoblots of membrane preparations of COS 1 cells (-1 10 p~ isoproterenol a s described under "ExperimentalProcedures." expressing wild type or mutant GJG,, polypeptides were Radioautographs of membrane or cytosolic proteins resolved by SDSprobed with COOH-terminal anti-G, (panel A ) or anti-Gei, polyacrylamide gel electrophoresisare shown. Results from one of two (panel B ) antisera. Lanes represent the various transfection condi- similar experiments are shown. tions 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 wereloaded and electrophoresed in the experiments shown in panels A and B, respecGCYtively. as-L indicates the immunoreactive large G,, molecular mass band (52kDa), as-S the small Gas molecular mass band (45kDa), and (-)-is0 ai2 the Gei, molecular mass band (41 kDa). Both the small and the large G,, splice variants are expressed endogenously in COS 1cells a t similar levels. The G,, cDNA used for mutation and expression codes for the large 394-aminoacid G,, chain is shown. The results are representative of seven independent experiments. 0
0
0)
cDNA only) Gpp(NH)p as well as tubulin-Gpp(NH)p slightly FIG.4. Effect of isoproterenol on the transfer of ["PIAAGTP increased adenylyl cyclase activity when applied at higher con- from tubulit1-[~2P]AAGTP to different GAG,, chimeric proteins centrations (1 PM). No significant differences in their effects in permeable COS 1 cells. Permeable cells, expressing carrier Go, could be observed without P-adrenergic receptor stimulation. proteins (mock),overexpressing wild type G,, ( s ) or mutant GJG",, or sli(38)) were incubated The additionof the P-adrenergic receptor agonist isoproterenol proteins (ildharn), i(ham)lsli(38), i(54)ls, with 1 p~ tubulin-["PIAAGTP and either with(+I or without (-) 10 pbl (10PM) increased significantly enzyme activity in both cases; isoproterenol as indicated in the text. Radioautograph of membranes however, the effect was greater withtubulin-Gpp(NH)p. Satu- resolved by SDS-polyacrylamide gel electrophoresis is shown. The reration of the tubulin-Gpp(NH)p effect was not observed in the sults are representativeof five independent experiments. concentration rangestudied,andhigher tubulin-Gpp(NH)p concentrations could not be used due to initiation of tubulin to accelerated GDP dissociation and faster GTPyS binding (17, self-assembling processes (38).Nonetheless, it was obvious that 19). The present concentration-response experiments showed while tubulintubulin-Gpp(NH)p wasa more efficient stimulator of adenylyl that inpermeable COS 1cells expressing Gni(54),,, cyclase activity than the free guanine nucleotide upon receptor Gpp(NH)p was more potent thanGpp(NH)p in stimulating enzyme activity upon receptor activation (EC,, values of 1.34 nM activation. When wild type G,, protein was overexpressed in COS 1cells and 7.9 nM, respectively), it was less efficient when compared (Fig. 1, panel B ) , adenylyl cyclase followed the same activity with wild type GOs-transfectedcells (Fig. 1, panel B 1. At a 1 PM patterns as the controls (Fig. 1, panel A), although enzyme concentration therewas no difference betweentubulin-Gpactivity was twice as high as that in naive COS 1cells. At 10 1.1~ p(NH)p andGpp(NH)p-evoked responses. Despite the fact that is a dominant Gusmutant whose activisoproterenol, saturable stimulation wasobserved, EC,, values Gui(54)/s chimeric protein being 7.08 nM and 8.41 nM for tubulin-Gpp(NH)p and ity isconstitutively enhanced (17),i t responds less successfully Gpp(NH)p, respectively. These experimentsshowed that inper- to tubulin-Gpp(NH)p stimulation. As tubulin does not bind and meable COS 1 cells in contrast toC6 glioma cells (26, 27, 39), transfer nucleotide to Gai2(261, which represents the first part polypeptide, it is suggested that the1st to 61stamino tubulin-Gpp(NH)p was not able tobypass the constraintp-ad- of Gui(54V, renergic receptor exerts on G, while not agonist activated, but acids ofG,, protein are needed to increase the efficiency of tubulin-Gpp(NH)p wascapable of potentiating agonist-stimu- tubulin-Gpp(NH)p above that of Gpp(NH)p. When another constitutivelyactivechimeric G, protein, lated adenylyl cyclase activity. Clearly, in COS 1cells, activated Gndi(3R, (Gus1-356; Gai2320-355) (4,6), wasexpressed, guanine P-adrenergic receptor and tubulin act inconcert. In order tofind facets of G,, that interact functionally with nucleotide- or tubulin-guanine nucleotide-stimulated adenylyl 11-14 times above the control, tubulin, similar experiments were performed in COS 1 cells cyclase activityincreased overexpressing several different chimeric Gu.JGei2proteins. In whether or not thep-adrenergic receptor was activated(Fig. 1, cells expressing a chimeric G,, which substitutes the amino- panel D ) . The EC,, values were lower (in the presence of 10 PM terminal 54 aminoacids of G,, to Gai2(G,i(54Ks), adenylyl cyclase isoproterenol, 1.58 and 1.85 nM for tubulin-Gpp(NH)p and or Gpp(NH)p, respectively),but isoproterenol failed to further inactivity increased significantlyupontubulin-Gpp(NH)p Gpp(NH)p addition (Fig. 1, panel C).Unlike the situation with crease enzyme activity. These results complemented the findG,, overexpression, Gpp(NH)p aloneallowed stimulation of ad- ing showing a decreased time required to achieve maximal enylyl cyclase, which exceeded that induced by tubulin- adenylyl cyclase activation by GTPyS in membranesfrom Chiconfirmed Gpp(NH)p. Further, while isoproterenol-activated adenylyl cy- nesehamster ovary cells expressing(28)and clasecontinued to be more sensitiveto tubulin-Gpp(NH)p, that guaninenucleotide (and tubulin-guaninenucleotide)-actimaximal activity achievedby the two agents was equal.Gai(54Vsvated adenylyl cyclase activities were reproducibly greater in is efficiently coupled to receptors (17). A time course study G,,,,,,,,-expressing cells compared with wild type or G,,-expressshowed an enhanced rate of adenylyl cyclase activation by ing cells. It seemed, however, that the substitutionof the last GTPyS in membranesfrom G,,,,,,-transfected COS 1cells due 38 amino acids of Guswith the carboxyl-terminal 36 residuesof
21752
lbbulin-G-protein Interaction for Adenylyl Cyclase Activation
uta/
FIG.5 . Mechanism of tubulin-Gpp(NH)ppotentiationof P-adrenergic receptor-triggered adenylyl cyclase activation. The transfer of nucleotide fromtubulin to G , promotes conformationalchange of G,, leading to its functional dissociationfrom 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 G, proteins. Fig. 2 shows results of immunoblotting experithe P-adrenergic receptor. ments using specific anti-Gas (panel A ) or anti-G,, (panel B ) Two other chimeric proteins, Gai/B(Bam) (G,, 1-212; Gas235antibodies, depending on the COOH-terminal region of the chi394) and G,i(Bamydi(38)(G,iz1-212;G,,235-356; G,, 320-355) meric protein expressed. It can be seen from both anti-G,, and (17,19), were expressed. Both had been shown to be functional anti-G,, probed blots that G, proteins are expressed at similar Go, polypeptides capable of increasing intracellular CGMP pro- level. Densitometry measurements ( n = 7) indicated approxiduction in transfected COS 1cells and S49cyc- cells (16,17,19). mately 3-5-fold increased abundance of as-L (52 kDa) when chimera was shown to behave as afunctional wild wild type G,, was overexpressed, or of a band at 45 kDa, when The G, type Gas with respect to receptor selectivity as well (17). The the construct G,,(Bam) was expressed. Similar abundance of ) (al- Gui(54ys present study showed that isoproterenol (10 p ~ increased (48 kDa) was obtained after transfection. Anti-G,, beit weakly) Gpp(NH)p-stimulatedadenylyl cyclase in Goi/s(Bam)COOH-terminal antisera treatment also revealed a 3-4-fold or transfected COS 1cells (Fig. 1,panel E ) . However, it failed to increase, compared with G,,, when the constructs Gos,i(38) potentiate the effect of tubulin-Gpp(NH)p. In the absence of G,(Bmys/i(38) were expressed (Fig. 2, panel B ) . In order to visualize the transfer of nucleotide fromtubulin to isoproterenol, no difference between Gpp(NH)p and tubulinG,, proteins (wild type and chimeric) and to verify its coupling Gpp(NH)p was observed, similar to the situation for control COS 1 cells (Fig. 1,panel A). When the cells were transfected t o P-adrenergic receptor stimulation, we used the hydrolysiswith the construct Gai(Bamydi(38), which isa combination of resistant photoaffinityGTP analogAAGTP (in its w3'P version), G,s(Bam)and GosEi(38), they were weakly responsive to 1 p~ Gp- bound to tubulin (tub~lin-[~~PlAAGTP), photolabeling in experip(NH)p or tubulin-Gpp(NH)p and unresponsive to isoproter ments performed on permeable transfected COS 1 cells. 10 p~ ) 1,panel F ) . The results obtained confirmed isoproterenol increased the amount of tub~lin-[~~PlAAGTP enol (at 10 p ~ (Fig. the importance of the COOH-terminal part of G,, polypeptide bound to membranes and also the transfer of [32P]AAGTPfrom for the coupling to p-adrenergic receptor (16) and the signifi- tubulin to Gas(Fig. 3). When autoradiograms were measured by cance of the region between the 1st and 212th amino acids of densitometry, 8.0 * 2.2% more t~bulin-[~'PlAAGTP was found associated with membranes after isoproterenol stimulation ( n = G,, for activation of the G-protein by tubulin-Gpp(NH)p. Although responses t o Gpp(NH)p and tubulin-Gpp(NH)p 2). Isoproterenol increased [32P]AAGTPtransfer from tubulin to were weak in Goi/s(Bm) and G,,,,,,,,,-transfected COS 1cells, Gasby 76 2 15% compared with controls. In cytosol, although it was not due to a lack of expression of the respective chimeric more of the t~bulin-[~~PlAAGTP was found, it diminished after
mbulin-G-protein Interaction for Adenylyl Cyclase Activation 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 shiftfrom the membrane-bound to the soluble compartment (40, 41). It was suggested that the redistribution ofG,, was due conformational to changethat loosened attachmentof that molecule to membranes and increased its turnover rate (41).As seen inFig. 3, we were notable todetect an increase ofG,,-[32PIAAGTP in cytosol after isoproterenol stimulation, although a small signal was observed when @-adrenergic receptors were not stimulated. An explanation of this to G,, upon finding could be thepossibility that tubulin binding receptor stimulation slowed down G,, turnover rate, increasing the lifetime of its active conformation. Since such a release into the cytosol has beenobserved in othercell types (40) it ispossible 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 increased ) the transfer of [32PlAAGTP that isoproterenol (10 1.1~ 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 labelingamong 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 1cells through a direct interaction with Goa. The studies performed using different chimeric GJG,,, proteins are consistentwith 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 importantfor guanine nucleotide transfer from tubulin, and (c) the amino terminus ofG,, (residues 1-54) is required for the ability of tubulin to activate adenylyl cyclase. Fig. 5 is a schematic illustration of our current understanding 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 transduction is that the GTPasecycle of the G-protein a subunit functions 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 its effect on adenylyl cyclase. Upon receptor tubulin is exerting stimulation, tubulin transfersGpp(NH)p from its exchangeable site on its 0 subunit to G,,, thus driving a conformational switch in G,, tertiary structure. The latter leads tofunctional dissociation ofG,, 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 isstabilization of the active G,, conformation and the Gm,-adenylyl cyclase complex, leading toincreased stimulation of the enzyme activity. Recent findings support this idea. First, it has been shown in reconstitution 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, perhapsby 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-
21753
mately 0.5 m~ (43,441.Although tubulin is an abundant intracellular protein, its maximal concentration in mouse 3T3 cells has been calculated t o 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 affecting adenylyl cyclase through theGTP cycle of Go*.But there are at least two arguments toconsider. First, the distribution of the pool of soluble tubulin throughout the cell (ie. is it uniform or heterogeneous?) is unknown. Tubulin represents a major component of certain membranes,’ and this membrane tubulin would be likely to have“favored access” t o 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 thea chain of G, and stimulateadenylyl cyclase in human pituitary tumors, thus bypassing the normal cell requirement for trophic hormone (46). Since CAMPstimulates thegrowth 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 canbe speculated that the data presented in this report describeamechanism for enhancedadenylyl cyclase stimulation resulting in increased CAMPsynthesis and protein kinase A activation. Perhaps information about changes incell shape and thecytoskeleton is communicated to the cellular interior via such a mechanism. Efforts to understand this process are under way. Acknowledgments-We thank Dr. D. Manning forproviding the anti-G, antisera and Dr. W. Dunn for 4-azidoaniline.We are also grateful to M. Talluri for growing cells and technical assistance. REFERENCES 1. Gilman, A. G. (1987) Annu. Reu. Biochem. 56,6155649 2. Hepler, J. R., and Gilman, A. G. (1992) Dends Biochem. Sci. 17,383-387 3. Kaziro, Y., Itoh, H., Kozasa, T., Nakafuku, M., and Satoh,T. (1991)Annu. Reu. Biochem. 60,349-400 4. Bourne, H. R., Sanders, D. A., andMcCormick, F. (1991)Nature 349,117-127 5. Simon, M. I., Strathmann, M. P., and Gautam, N. (1991) Science 252,802-808 6. Johnson. G. L.. Dhanasekaran. N..GuDta. S . K.. Lowndes. J. M.. Vaillancourt. R. R.]’and Ruoho, A. E. (1991) J. d l l . Biochem. 47, 136-146 7. Spiegel,A.M.,Shenker,A,,andWeinstein, L. S. (1992) Endocl: Reu. 13, 536-565 8. Federman, A. D., Conklin, B. R., Schrader, K. A,, Reed, R. R., and Bourne,H. R. (1992) Nature 356, 159-168 9. Sullivan, K A,, Miller, R. T., Masters, S. B., Beiderman, B., Heideman, W., and Bourne, H. R. (1987) Nature 330,758-760 10. Rall, T.,and Harris, B. A. (1987) FEES Lett. 224,365-371 11. Graeiano, M. E?, and Gilman, A. G. (1989) J . B i d . Chem. 264, 15475-15482 12. Freissmuth, M., and Gilman, A. G. (1989) J . B i d Chem. 264,21907-21914 13. Masters, S. B., Miller, R. T., Chi, M.-H., Chang,E-H., Beiderman, B.,Lopez, N. G., and Bourne, H. R. (1989) J . Bid. Chem. 264, 15467-15474 14. Woon, C. W., Heasley, L., Osawa, S., and Johnson, G.L. (1989) Biochemistry 28,45474551 15. Miller, R. T.,Masters, S . B., Sullivan, K. A,, Beiderman, B., and Bourne, H.R. (1988) Nature 334, 712-715 16. Masters, S. B., Sullivan, K. A., Miller, R. T., Beiderman, B., Lopez, N. G., Ramachandran, J., and Bourne, H. R. (1988) Science 2 4 1 . 4 4 8 4 5 1 17. Osawa, S., Heasley, L.E., Dhanasekaran, N., Gupta, S . K., Woon, C. W., Berlot, C., and Johnson, G. L. (1990) Mol. Cell. Biol. 10, 2931-2940 18. Itoh, H., and Gilman, A. G. (1991) J. Bid. Chem. 266, 16226-16231 19. Osawa, S.,Dhanassekaran, N., Woon, C. W., and Johnson,G. L. (1990) Cell 63, 697-706 20. Gelfand, V. I., and Bershadsky, A. D. (1991) Annu. Reu. Cell Biol. 7, 93-116 21. Erickson, H. P., and OBrien, E. T.(1992)Annu. Reu. Biophya Biomol. Struct. 21, 145-165 22. Amir-Zaltsman, Y., Ezra, Z., Scherson, T., Littauer, U., and Salomon,Y. (1982) EMBO J . 1, 181-186 23. Rasenick, M. M., and Wang, N. (1988) J. Neurochem. 5 1 , 3 4 0 1 4 4 1 3 24. Wang, N., Yan, K., and Rasenick, M. M. (1990) J. Biol. Chem. 265,1239-1242 25. Roychowdhury, S., Wang, N., and Rasenick, M. M. (,1993) Biochemistry 32, 49554961 26. Yan, K., and Rasenick,M. M. (1990) inBiology ofCeZlular Dansducing Signals (Vanderhoek, J. Y., ed) pp. 163-172, Plenum Publishing Corp., New York 27. Rasenick, M. M., and Kaplan, R. S. (1986) FEES Lett. 207, 296-301 28. Woon, C. W., Soparkar, S.,Heasley, L., and Johnson, G. L. (1989)J. Biol. Chem. 264,5687-5693 29. Gluzman, Y. (1981) Cell 23, 175-182 30. Ausubel, F. M., Brent, R., Kingston,R. E., Moore, D. D., Smith, J. A,, Seidman, ~
~~~
21754
Tubulin-G-protein Interaction
J. G., and Strull, K. (1987)Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York 31. Salomon, Y. (1979)Adu. Cyclic Nucleotide Res. 10, 35-55 32. Bradford, M. M. (1976)Anal. Biochem. 72, 248-254 33. Shelanski, M., Gaskin, F., and Cantor, C. (1973)Proc. Natl. Acad.Sei. U. S. A . 70,765-768 34. Pfeuffer, T.(1977)J. Biol. Chem. 252, 7224-7234 35. Laemmli, U. K. (1970)Nature 227, 680-685 36. Carlson, K. E., Brass, L. F., and Manning, D.R. (1989)J. Biol. Chem. 264, 13298-13305 37. Osawa, S., and Johnson, G . L. (1991)J. Biol. Chem. 266,4673-4676 38. Gaskin, F., Cantor, C. R., andShelanski, M. L. (1974)J . Mol. Biol. 89, 737-758 39. Rasenick, M. M., Lazarevic, M., Watanabe, M., and Hamm, H. E. (1993)Meth-
for Adenylyl Cyclase Activation ods (Orlando) 5,252-257 40. Ransnas, L. A,, Svobada, P., Jasper, J. R., and Insel, P. A. (1989)Proc. Natl. Acad. Sci. U.S. A . 86, 7900-7903
41. Levis, M. J., and Bourne, H. R. (1992)J. Cell Biol. 119, 1297-1307 42. Roychowdhury, S., and Rasenick, M. M. (1994)Biochemistry, in press 43. Franklin, T.J., and Twose, P. A. (1977)Eur. J . Biochem. 77, 113-117 44. Brostrom, C. O., Bocckino, S. B., and Brostrom, M. A. (1983)J. Biol. Chem. 258,14390-14399 45. Hiller, G., and Weber, K. (1978)Cell 14, 795-804 46. Landis, C. A,, Masters, S. B., Spada, A., Pace, A. M., Bourne, H. R., and Vallar, L. (1989)Nature 340,692696 47. Dumont, J. E., Jauniaux, J. C., and Roger, P. P. (1989)Dends Biochem. Sci. 14, 67-71 48. Rozengurt, E. (1986)Science 234,161-166
