Labeling of the Pr Subunit Complex of Transducin with an ...

Vol. 266, No. 17, Issue of dune 15, pp. 11017-11024,1991 Printed in U . S . A .

THEJOURNAI. OF BIOLOGICAL CHEMISTRY 81:)

1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Labeling of the Pr Subunit Complexof Transducin with an Environmentally Sensitive Cysteine Reagent USE OF FLUORESCENCESPECTROSCOPYTOMONITORTRANSDUCINSUBUNITINTERACTIONS* (Received for publication, July 5, 1990)

William J. Phillips and Richard A. Cerione From the Department of Pharmacology, College of Veterinary Medicine, Cornell Uniuersity, Ithaca, NewYork 14853

In this study, we have examined the interactionsof protein1-coupled signaling. As is the case for a number of the fly subunit complex of the retinal GTP-binding important biological signaling pathways, the phototransducprotein transducin (ByT) with its a subunit (aT)using tionsystem involves the interactions of three membranefluorescence spectroscopic approaches. The BYT sub- associatedproteins.Theseproteinsarethephotoreceptor, unit complex was covalently labeled with 2-(4’-malrhodopsin ( M ,-37,000), the heterotrimeric GTP-binding proeimidylanilino)napthalene-6-sulfonic acid(MIANS), tein (G protein), transducin (with the three subunits desigan environmentally sensitive fluorescent cysteine re- nated aso(1‘ (Mr-39,000), (3 (Mr-36,000), and YT ( M r-8,000)) agent. The formation of the MIANS ByTcomplexes (two (l),andtheheterotrimeric cyclic GMP phosphodiesterase to five MIANS adducts per ByT) resulted in 2-3-fold (PDE), with the subunits designated as cup^^ ( M , -85,000), enhancements in the MIANS fluorescence, and 20-25(31.1,E ( M , -85,000), and YI’W (Mr-14,000) (2, 3). The photonm blue shifts in the fluorescence emission maxima, transduction signaling cascade is initiated by the absorption relative to theemission for identical concentrationsof MIANS-labeled MIANS complexes. Theaddition of of light by rhodopsin which promotes the interaction of the aT.GDP to these MIANS ByT complexes resulted in an photoreceptor with holotransducin. This interaction results additionalenhancementinthe MIANS fluorescence in the exchange of a tightly bound molecule of GDP on the a,r subunit for GTP. Itcommonly has been proposed that the (typically ranging from 20 to 40%) and a 5-10-nm binding of GTP to aT results in the dissociation of the L Y T . blue shift in the wavelength for maximum emission. These fluorescence changes were specifically elicited G T P species both from rhodopsin and the P-yT subunit comby the GDP-bound form of aTand were not observed plex (the latter complex stays intact under nondenaturing upon the addition of purified aT-guanosine 5‘-0-(3- conditions). Thus, presumably it is a free cuT.GTP species thiotriphosphate) (GTPyS) complexes to the MIANS that couples to the effector enzyme (PDE) and mediates the @yTspecies. Conditions which resulted in the activation stimulation of cyclic GMP hydrolysis. The aT.GTP-stimuof the aT*GDP subunit (Le. the additionof A1F4- or the lated enzyme activity persists until the bound GTPhydrois addition of rhodopsin-containing vesicles and GTPyS) lyzed to GDP. The GTPase activity represents the deactivaresulted ina reversal of the aT.GDP-induced enhance- tion of the cu’r subunit and ultimately results in the reassociament of the MIANS ByTfluorescence. Thus theMIANS tion of an wr.GDP species with the P-yT complex to reform @ Y ~fluorescence provided a spectroscopic monitor for the holotransducin complex, thereby returning the signaling transducin-subunit association and transducin-activa- system to its starting point. tion. Based on the results from studies using this specThere have been a number of lines of evidence that have troscopic read-out, it appears that the association of suggested that G protein-cu subunits dissociate from their (3-y the aT*GDPspecies with the ByT subunit complex to form the holotransducin molecule is rapid and does not subunit complexes during the G protein-activation event (4). limit the rate of the rhodopsin-stimulated activationof This has been especially well documented for the G, and Gi holotransducin. However, either the dissociation of the proteins in hydrodynamic studieswhere the activation of the activated aTsubunit from the ByT complex, or a confor- G proteins was elicited by nonhydrolyzable G T P analogs or mational change inByT which occurs as a result of the byA1F4- ( 5 , 6). In addition, fluorescence studies monitoring subunit dissociation event, appears to be slow relative the changes in the intrinsic tryptophan fluorescence of CXT, that accompany the rhodopsin-stimulated activation-deactito the G protein-subunit association event. vation cycle of transducin, have suggested that one molecule of P-y.,. can act catalytically to promote the rhodopsin-stimulated activation of at least 20 molecules of aT (7). This ability The phototransduction cascade that is responsible for ver- of P-yT to act catalytically presumably reflects the continual tebrate vision represents an excellent model system for study- dissociation of (3-pr from activated aT species and the reassociation of this subunitcomplex with GDP-boundaT subunits. ing the molecular mechanisms underlying receptor/G Similarresults were observedwhen measuringrhodopsinstimulated GTPase activity(1). * This research was supported by National Institutes of Health Grants EY06429 and GM40654 and grantsfrom the PEWBiomedical Research Scholars Program and theCornell Biotechnology Institute, which is supported by the New York State Science Foundation, the LJnited States Army, and a consortium of industries. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore he hereby marked “aduerlisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

’ The abbreviations used

are: G proteins, GTP-binding proteins; subunit of transducin; By.l, thesubunit complex of transducin; HEPES,N-2-hydroxylethylpiperazine-N’-2-ethanesulfonic acid; GTPyS, guanosine 5’-0-(3-thiotriphosphate); ROS, rod outer segment;DTT,dithiothreitol;MIANS,2-(4’-maleimidylanilino)napthalene-6-sulphonicacid; PDE, phosphodiesterase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonic acid; SDS, sodium dodecyl sulfate.

w,

11017

N

11018

Studies with

a Fluorescent-labeled

Despite the correlation between G protein subunit association-dissociationeventsandtheactivation-deactivation cycles of these transducers, the interactions of G protein-a subunits with their Py subunit complexes havenot been directly examined in well defined systems, particularly under conditions where GTP-binding and/or GTPase activitywere occurring at the same time. Thus, we have set out todevelop fluorescence spectroscopic approaches for monitoring the association-dissociation equilibria for the subunits of the retinal G protein, transducin. Specifically, we have taken advantage of the relative ease in purifying milligram quantities of the PYTsubunit complex and the fact that the cysteineresidues of PYT can be modified without deleteriouseffects on the normal functioningof this complex. In this studywe describe T complexes that are labeled the preparation of P ~ subunit with 2-(4'-maleimidylanilino)napthalene-6-sulfonic acid (MIANS), an environmentally sensitive, fluorescent cysteine reagent.We show thattheMIANS fluorescence of the MIANS-labeled ByT complex is enhanced upon the interaction of this subunit complex with the a T . GDP species, and that the MIANS adducts serve as reporter groups for monitoring the association of the transducin subunits inreal time and simultaneously with rhodopsin-stimulated G T P binding and GTPase activities. MATERIALS AND METHODS

Subunit Complex of Transducin bed volume column was poured and equilibrated with ByT storage buffer (see above). The reaction mixture was run through the resin and the column was washed with 4 ml of the storage buffer followed by a 4-ml wash with PYT storage buffer containing 20 mM sodium phosphate. Finally, the MIANS / j y was ~ eluted from the column in buffer supplemented with 0.1 M phosphate. The eluted protein was completely resolved from free MIANS by this procedure. In order to achieve different extents of incorporation of MIANS into ByT, either the time period for the reaction or the concentration of MIANS in the reaction mixturewas varied. The stoichiometry of MIANS incorporation into PYTwas calculated using a molar extinction coefficient of 17,000 for MIANS (13) and a subunit M,of 43,000 for ByT. Fluorescence Spectroscopy-All fluorescence measurements were done using an SLM 8000 spectrofluorometer in the photon-counting mode. Generally the samples were diluted to 0.15 ml with HMD buffer (20 mM HEPES, pH 7.5, 5 mM MgCl,, 1 mM DTT) at the concentrations stated in the figure legends at 23 "C in 0.2-ml quartz cuvettes. Measurement of the guanine nucleotide exchange reaction of (YT was done as previously described (7). In all cases, the MIANS fluorescence of theMIANS PyT complexes, andthetryptophan fluorescence of the (YT subunit, were corrected for any background fluorescence contributed by other protein components present in the cuvette. All time-dependent fluorescence acquisitions were done at one determination per second, and generally additions to the cuvette were done while continuously monitoring thefluorescence. RESULTS

Characterization of the MIANS-labeled byT Subunit-Previous studies have suggested that the PYT subunit complex contains a total of six reactive cysteine residues (14).We have found that at least 5 of these cysteine residues can be covalently modified with the fluorescent cysteine reagent, MIANS. Fig. 1 (spectra 1-4) shows the intrinsic tryptophan fluorescence emission for the native PYTsubunit complex (spectrum 1), and for different MIANS-labeledPYTcomplexes containing two (spectrum 2 ) , four (spectrum 3 ) , and five (spectrum 4 ) MIANS groups per ByT. The intrinsic tryptophanemission of the ByT complex decreasesas the number of MIANS groups increases; this decrease in tryptophan emission is most likely the result of an energy transfer quenching of the tryptophan emission(emission maximum = 330 nm) by theMIANS moieties (absorption maximum = 322 nm).

Purification of Retinal Proteins-Frozen dark-adapted bovine retina were purchased from Hormel (Austin, MN). Rod outer segment (ROS) membranes were prepared as previouslydescribed (8), and were used directly or were used as a source for the purification of rhodopsin and transducin. Rhodopsin was purified under dimred light by first washing the ROS membranes (5 X) with hypotonic buffer (10 mM HEPES, pH 7.5, 6 mM MgCl,, 1 mM DTT) then solubilizing the rhodopsin with 20 mM CHAPS insolubilization buffer (50 mM Tris-acetate, pH 7.0, 1 mM CaCl,, 1 mM MnC12,0.1 M NaC1) (9). The soluble rhodopsin was then purified using concanavalin ASepharose chromatography (lo), and the bound protein was eluted with solubilization buffer supplemented with 0.3 M a-methylmannoside. The highly purified rhodopsin (1-5 mg/ml) was stored at -70 "C protected from light. Transducin was purified from ROS membranes as previously described (8, 9). Briefly, ROS membranes were exposed to room light, washed (5 X) with isotonic buffer (hypotonic buffer plus 0.15 M NaCl), and thenwashed (5 X ) with hypotonicbuffer. The transducin was then eluted from the membranes by suspending the pelletsin hypotonicbuffer supplementedwith 0.1 mM GTP(or GTPyS). The supernatants were collected and the subunits ((YT and flyr) were separated by Blue Sepharose chromatography (11).The subunits (generally 0.2-0.5 mg/ml) were stored at -70 "C in 10 mM HEPES, pH 7.5, 6 mM MgCl,, 1 mM DTT, 25% glycerol; the (YT subunit also contained 0.5 M KC1. Reconstitution of Rhodopsin-stimulated GTPase Actiuity-Purified rhodopsin was reconstituted into phospholipid vesicles under dim red light as previously described (7). The GTPase activity of aT.GDP was measured as follows: purified CYT.GDP andByT were mixed with rhodopsin vesicles in room light and diluted to50 pl with water. The assay was then initiated by the additionof assay buffer (final concentration, 20 mM HEPES, pH 7.5, 2 mM MgCl,, 0.7 p M GTP[r3*P]), and the assay was continued for 10 min a t 23 "C. The amount of "'Pi Emission wavelength (nrn) released was determined by extraction with a solution of isobutanol FIG. 1. Characterization of the MIANS-labeled DYT comand benzene (1:l) and ammoniummolybdate as previously described (12). Controlswere always performed where the GTPase activityfor plexes. A , purified PYT was labeled with MIANS a t three different wI. in the presence of PYT, but in the absence of rhodopsin, was stoichiometries of incorporationand purified by hydroxylapatite chromatography as described under "Materials and Methods." The measured. These values were generallyless than 10% of the total fluorescence emission of the labeled PyT (0.48 /.LMprotein) complexes counts and have been subtracted from the total. Modification of PYTwith 2-(4'-Maleimidylanilino)napthalene-6-sul- was then scanned for both intrinsic tryptophan and MIANS fluorescence. Spectra 1-4 are the tryptophan emission spectra (excitation phonic acid (MIANS)-Purified flyT was dialyzed into modification buffer(20 mM HEPES, pH 7.5, 5 mM MgCl,, 0.15 M NaCl, 20% 280 nm) of the unlabeled PYT(spectrum 1) and PyT that was labeled with MIANS a t stoichiometries of two (spectrum 2), four (spectrum glycerol) to remove the DTT. Thedialyzed BYT (0.5 ml of 0.1-0.5 mg/ ml 0-y~)was then mixed with MIANS (0.1 M stock solution made in 3 ) , and five (spectrum 4 ) MIANSper PYT. Spectra 5-7 arethe dimethylformamide) toa final concentration of 0.5-1 mM. The reac- corresponding MIANS emission spectra (excitation 322 nm) of the labeled P ~ complexes, T i.e. spectrum 5 corresponds to spectrum 2, tion generally proceeded for 1 h a t room temperature a t which point theMIANS (3y.r complexwas resolved from the free unreacted spectrum 6 to spectrum 3, and spectrum 7 to spectrum 4. Spectrum 8 MIANS using hydroxylapatite chromatography. Generally a 0.5-ml is the fluorescence emission of DTT-reacted MIANS (0.5 /.LMfinal).

Studies with a Fluorescent-labeled Py Subunit Complex Fig. 1 also shows the fluorescence emission spectrum (excitation 322 nm) for a DTT-reacted MIANSmoiety (spectrum 8) and the emission spectra for the MIANS @ y complexes ~ containing two (spectrum 7), four (spectrum 6), and five (spectrum 5 ) MIANS groups per @?T. The emission spectra for the MIANS conjugates (spectra 5, 6, and 7) are blueshifted by -20-25 nm relative to the emission spectrum for the DTT-reacted MIANSprobe (spectrum 8 ) , and their total fluorescence emission is enhanced by as much as 2-3-fold when compared to thefluorescence for similar concentrations of DTT-reacted MIANS. This suggests thattheMIANS adducts of the @yT complex are in morehydrophobic (buried) environments compared to the free MIANS probe. This is particularly the case for @TT complexes which contain five MIANS groups. Specifically, these complexes show a significantly enhanced MIANS fluorescence relative to the DTTreacted MIANS. This may either be due to the fifth MIANS moiety being much more buried than the others, or it may reflecta conformational state where the average environments of all of the MIANS groups are more buried in @YT complexes containing five MIANS groups compared to @YT complexes containing 5 four MIANS moieties (see spectrum 5). The levels of incorporation of MIANS into the@yTsubunit complex were consistently greater than those previously reported for the reaction of N-ethylmaleimide with ByT (ie. 2 NEM/@yT (14)). In order to determine the general locations of these MIANS adducts,we compared the tryptic fragments of two different preparationsof the MIANS-labeledByT (Fig. 2). Fung and Nash (15) have shown that trypsin cleaves the native @YT subunit complex once on the@ subunit, generating a 26-kDa carboxyl-terminal fragment and a 14-kDa aminoterminal fragment, while leaving the yT subunit intact. Fig. 2 shows the sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles, as visualized by protein staining (lanes I 4 ) and fluorescence (lanes 5-8), of two different MIANSof trypsin (lanes1,3, labeled @YT preparations in the absence 5, 7) or following trypsin treatment (lanes 2, 4, 6, 8). The results indicate that at the higher level of MIANS incorporation (ie.four MIANS groups per @YT; lunes I , 2,5, 6), both the 26- and 14-kDa tryptic fragmentsof the @ subunit, aswell 1 2 3 4

5 6

7

of Transducin

11019

as the subunit, are labeled with the MIANS probe. At the lower level of MIANS incorporation (i.e. two MIANS groups per @yT;lanes 3, 4, 7, 8 ) , a similarpattern of labeling is observed although the extent of labeling of both the 14-kDa YT subunit appears to be diminished. tryptic fragment and the In allcases, most of the MIANSlabel is presenton the larger tryptic fragment which is consistent with the fact that there are many morecysteineresidues (ie. 10) on the carboxylterminal fragment of the @ subunit compared to the aminoterminal fragment (which contains 4 cysteine residues) (16). The results in Fig. 3 show that the MIANS-labeled @ y ~ , which contains five MIANS groups per subunitcomplex, still was fully activein itsabilitytopromotethe rhodopsinstimulated GTPase activity in the purified cyT subunit. We havenot observed anydetectable differencesin the doseresponse profiles for the native- and labeled-@yTcomplexes in these experiments, consistent with the notion that the cysteine residues on the@yTcomplex are notdirectly involved ~ in (YT/@YT interactions nor in the interactions of @ y with rhodopsin (14). In all of the studies outlined below, we have ~ that were fully labeled with MIANS (i.e. used @ ycomplexes e four MIANS moieties per @yT) since these ultimately provided the most sensitive read-outsfor the interactions of the @yTcomplex with (Y~GDP. Fig. 4shows the fluorescenceemission spectra for the MIANS-labeled ByT complex (containing four MIANS groups per @YT) in the absence (spectrum I ) , and in the presence (spectrum Z ) , of the CYT.GDPspecies. Forthe conditions shown in Fig. 4, the interactionof ( YGDP ~ . with the MIANSlabeled @YT resulted in a20-30% enhancement of the MIANS fluorescence and a 5-10-nm blue shift in the emission maximum. We examined whether this enhancement in the MIANS fluorescence was specifically induced by the interactionof the MIANS @YT with an (YTsubunit which was in an inactive (Le. GDP-bound) conformation. No enhancement of the MIANS @yT complexwasobservedwhen the purified N T . G T P ~ S species (prepared as described under “Materials and Methods’’) was substituted for the(YT.GDP complex (datanot shown). Moreover, we found that inall casesthe enhancement was reversed when (YT.GDP was activated by AlF,- (see Fig. exchange 4, spectrum 3) or following the rhodopsin-stimulated of the bound GDPfor G T P (see Fig. 8, below). In the exper-

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FIG 2. Trypsin treatment of MIANS ByT complexes. Two preparations of MIANS ByT complexes with stoichiometries of incorporation of two and four MIANS groups per Byl were treated with either buffer or trypsin (0.01 mg/ml for 30 min a t room temperature) and theproteolysis was stopped by the additionof SDS samplebuffer. The samples were then electrophoresed through a 10% SDS-polyacrylamide gel and either stained with Coomassie dye (lanes I - 4 ) , or the fluorescent protein bands were photographed using a UV transilluminator to excite the fluorophores (lanes 5 - 8 ) . Lane I is MIANS (3r.r that has four MIANS groups per Byr, and lane 2 is the result of trypsin treatment. Lanes 5 and 6 show the fluorescence associated with the protein bands in lanes I and 2, respectively. Lanes 3, 4, 7, and 8 are the same as above except that the pyT complex had two MIANS groups per ByT.

0

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t P Y T l (nM) FIG. 3. Effect of the MIANS modification of BYT on the rhodopsin-dependent GTPase activity of ~ T * G D P .The MIANS-labeled 0r.r complex (containing five MIANS groupsper was prepared by incubating highly purified Br.r with 1 mM MIANS for 1 h a t room temperature as described under “Materials and Methods.” The MIANS ByT was then assayed for its ability to promote the rhodopsin-stimulated GTPaseactivity in the NT subunit. The final concentrations of NT.GTP and rhodopsin in the assay solutions were 1.6 PM and 4 nM, respectively, and the levels of B ~ T are shown on the abscissa. The hydrolysis of (y-’“P]GTP was measured as described under “Materials and Methods.” Each point is the mean of duplicates (& range) thathave been correctedfor background activity, i.e. activity in theabsence of added NT. ByT),

Studies with

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a Fluorescent-labeled Pr Subunit Complex of Trarzsducin

400 450 Emisslon Wavelength (nm)

500

FIG. 4. Effect of ~ T - G Don P thefluorescence of MIANS &T. MIANS-labeled P ~ T(8.4 pmol;four MIANS groups per P ~ T was ) diluted to 0.15 ml with HMD buffer (20 mM HEPES, pH 7.5, 5 mM MgCl,, 1 mM DTT). The MIANSfluorescence emission was scanned from 350 to 500 nm (excitation322 nm) andcorrected for background emission (ie. in the absence of added MIANS P ~ T (spectrum ) I). Spectrum 2 shows the results obtained when CYT.GDP (11.5 pmol) wasadded to a cuvette containing the MIANS ByT complex and incubated a t room temperature for 5 min. Spectrum 3 was obtained when A1F4- (5 mM NaF/20 FM AlClJ was added toa cuvette containing both MIANS &T and ~ T . G D Pand then incubated for 5 min a t room temperature.

,L o

1 100 10 IMgCI21 (mW

FIG. 6. Effect of MgCl, on the aT.GDP-dependent enhancement of MIANS @YT fluorescence. MIANS PyT, prepared as de-

scribed in the legend to Fig. 3, was diluted to 0.145 ml (final concentration, 0.26 FM) with HD buffer (20 mM HEPES, pH 7.5, 1 mM DTT) with increasing concentrations of MgC12 except for the 0 mM MgC1, sample which contained 2 mM EDTA. For each treatment, the MIANS fluorescence emission a t 420 nm was determined (excitation 322 nm), and then CYT.GDP (0.68 FM final) was added to the cuvette and mixed while continuously monitoring the MIANS fluorescence a t 420 nm. The new level of fluorescence emission was determined, and the data expressed are as the percentage of increase in the MIANS emission after CYT.GDPaddition a t each [MgCl,]. Each point is a single determination at the MgC1, concentrations shown.

in the absence of added MgC1, (and 1 mM EDTA), the a T . GDP complex elicited a 15% increase in the fluorescence of the MIANS-labeledPyT. As the levels of MgC1, were increased to a final concentrationof 5 mM, the enhancementincreased to a final extentof -35%. Higher levels of MgCle (5-100 mM) then inhibited the ability of the aT.GDP complex to elicit this enhancement. In the absence of the aT.GDP species, MgC12 hadno effect ontheMIANS fluorescence of the MIANS-labeled PyT subunit complex (data not shown). We havefoundessentiallyidentical dose-responseprofiles for MgC12, when monitoring its effects on rhodopsin-stimulated GDP-GTP exchange or on rhodopsin-stimulated GTPase activity (data not shown), as those obtained when monitoring the (YT.GDP-induced enhancement of the MIANS P-yT fluo0.0 1.o 2.0 3.0 rescence. These rhodopsin-stimulated activities are the direct [ W G W (wM) outcome of the coupling of light-activated rhodopsin to holoFIG. 5. Titration of the aT-GDP-dependent enhancementof (i.e. the aT.PyT complex). Taken together, these transducin MIANS @YT fluorescence. MIANS ByT, prepared as described in the legend to Fig. 3 , was diluted to 0.15 ml (final concentration, 0.39 different findingssuggest that the association of aT.GDP with PyT, and/or the conformation of PyT within the holop ~ with ) HMD buffer in the presence of increasing concentrations of nr.GDP as shown on the abscissa. The samples were then incutransducin complex, is highly sensitive to MgCL bated at room temperature for 5 min and the steady state MIANS Kinetics of the L I T . GDP-induced Changes in the Fluoresfluorescence emission at 420 nm was determined (excitation 322 nm). cence of the MZANS P-yT-It was of interest to examine how T h e data are plotted as the percentage of enhancement of MIANS fluorescencerelative t,o the fluorescenceemission of MIANS PYT the rateof the aT.GDP-induced enhancementof the MIANS of G protein-activation determined in the presence of an equivalent amount of U T storage ByT fluorescence compared to the rate (i.e. as induced by A1F4- or by the exchange of GDP for GTP). buffer. The points represent data from one experiment that hasbeen repeated three times with similarresults. Fig. 7 shows that the aT.GDP-induced enhancement of the MIANS PyT fluorescence was immediate, suggesting that the iment shown in Fig. 4, the MIANSPyT fluorescence observed association of these subunits occurred within the timeperiod . plus A1F4- was slightly reduced of mixing, i.e. a few seconds. The addition ofAlF4- to this in the presence of L Y T GDP (by less than 10%)relative to the fluorescence of the MIANS mixture then resulted in a decay of the MIANS fluorescence which most likely reflected the A1F4--induced dissociation of PyT (alone). However, this difference was not consistently speciesfrom the MIANS-labeled P ~ T observed and in many cases the MIANSP-yTfluorescence was the CYT.GDP(A~F~-) complex. We consistently find that this fluorescence decay essentially identicalfor these two conditions. Overall, these results suggest that changes in the fluores- requires several seconds for completion and appears to represent a slower eventthanthesubunit association event cence of the MIANS-labeled PyT complex would provide a convenient monitor for ( Y T . / ~ Ysubunit ~ interactions. Fig. 5 (which is reflected by the immediate enhancement of the shows the results of titrating the fluorescence enhancement MIANS fluorescence). In the experimentshown in Fig. 7, the BYT of the MIANS-labeled ByT complex (0.3 FM) with increasing addition of AlF4- to the mixtureof (YT.GDP and MIANS amounts of the aT.GDP species (over a range of 0.1-3 p M ) . did notcompletely reversethe aT.GDP-induced enhancement This titration profile can be fit well assuming a single class of the MIANS fluorescence, suggesting that some of the (YT. of wI'. GDP binding sites on the MIANS-labeled ByT complex GDP/MIANS ByT complexes did not respond to this activating ligand. However, in other experiments, we have found with a Kd value of -0.3 FM. The extent of the aT. GDP-induced fluorescence enhance- that AlF4- will completely reverse the aT.GDP-induced enment was highly dependent onMgC12 (see Fig. 6). Specifically, hancement of the MIANS PyT fluorescence (e.g. Fig. 8).

Studies with a

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Subunit Complex of Transducin

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excitation = 322 nm emission = 420nm

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Time (sed (L FIG.7. The effect ofAlF,- on the interaction of aT.GDP with MIANS ByT. MIANS / 3 - y ~complex, prepared as described in the legend to Fig. 3, was diluted to 0.145 ml (final [MIANS /3yT] = a,GDP GTP AI F ; 0.24 p ~ with ) HMD buffer, and the MIANS fluorescence was continII 0 1 0 0 200 300 uously monitored a t 420 nm (excitation322 nm) at one determination Time (sec) per second. At the time indicated on the trace, CCT.GDP (0.67 pM, final) was added andmixed while continuously monitoring theemisFIG. 8. Fluorescence monitoring of the rhodopsin-dependsion of MIANS fluorescence. A1F4- (5 mM NaF/20 p~ AIC1:I, final ent guanine nucleotide exchange of (YTSGDP using the MIANS concentrations), was then added and mixed at theindicated time with &T subunit complex. A , the dependence of the rhodopsin-stimucontinuous fluorescence monitoring. lated GDP-GTP exchange reaction of cq.GDP on MIANS/ 3 - y ~(prepared as described in the legend to Fig. 3) was determined by moniFig. 8 shows the results obtained when the MIANS 6-y~was toring the intrinsic tryptophan fluorescence of ~xT.GDPin absence or presence of added MIANS P-~T.The upper trace shows the trypreconstituted with purified rhodopsin (inserted into phospha-tophan fluorescence emission of CCT.GDP(0.68 p M ) and light-actitidylcholine vesicles) and thepurified (YT.GDP complex. The vated rhodopsin vesicles (0.034 p ~ a t) one determination per second resultspresentedin Fig. 8A (lowerpanel) illustratethe (excitation, 280 nm; emission, 335 nm). GTP (0.67 W M ) was added as changes thatoccurred in the net tryptophan fluorescence from indicated and the contents of the cuvette were mixed while continthe ( Y subunit ~ upon the addition of G T P (compare with Ref. ually monitoring the fluorescence emission. The lower panel repreBr.r (0.75 p ~ was ) sents an identical experiment except that MIANS 7). There was an immediate enhancement in the intrinsic included in the incubation. Thetraces shown were corrected for the tryptophan emission, thatcanbeattributedtotheGTP tryptophan fluorescence contributed by rhodopsin and the MIANS binding event (compare with Ref. 7 and 17), followed by a ljj-yT complex. B, monitoring the MIANSfluorescence of MIANS P-yT slower decay in thefluorescence emissionthat directly reflects during the activation/deactivationcycle of (YT.GDP. Theupper panel G T P hydrolysis. When the MIANS-labeled 6-y~ was omitted is the MIANSfluorescence (excitation, 322 nm; emission, 420 nm) of from the reconstitution, this GTP-induced fluorescence en- an incubation that included light-activated rhodopsin vesicles (0.034 ) MIANS / ~ Y T(0.37 p ~ ) The . fluorescence was continually hancement-decay sequence was completely eliminated (Fig. p ~ and ) monitored a t one determination per second, cuT.GDP (0.68 p ~ was 8A, upperpanel). These findings areconsistent with the then added at the time indicated, and the contents of the cuvette previous indications (e.g.Fig. 3) that theP-yT subunit complex were mixed. GTP-yS (3.3 p ~ and ) AIF,- (5 mM NaF/20 p~ ALCI:+, promotesrhodopsin-stimulatedguanine nucleotide (GDP- final concentrations) were added and mixed at the indicated times. G T P ) exchange on aT and that the MIANS-labeled P - ~ can T In the lower panel, an identical protocol was used except that GTP ) added instead of GTPyS. All incubations in A and B (0.67 p ~ was substitute for native P-yT in fulfilling this function. The results presented in the lower panel of Fig. 8B show were performed at room temperature.

the changesoccurring in the MIANSemission of the labeled PYT species during the rhodopsin-stimulated G T P bindingGTPase cycle of (YT. Upon the addition of the purified OIT. GDP spectra toa mixture of the rhodopsin-containing phosphatidylcholine vesicles and the MIANS P-yT, there was an immediate increase in the MIANSfluorescence, indicative of the rapid association of the G protein subunits. The addition of GTP then elicited a decrease in the fluorescence of the MIANS P-yT species. However, the decay intheMIANS fluorescence was transient and in fact it was followed by a slight rise in the MIANS fluorescence. After 180 s (i.e. at a time where all of the aTG T P species have been converted to a ~GDP . complexes as a result of G T P hydrolysis) the final level of the MIANSfluorescence waswithin 70% of the initial (aT.GDP-induced) enhanced state. The most likely explanation for these results is that either the GTP-induced dissocia-

tion of NT from MIANSP-yT, or the returnof the MIANSP-yT conformation to its initial resting state(which occurs following the dissociation of the CYTsubunit), occurs on a time scale comparable to GTP hydrolysis and is slow relative to the association of CYT.GDPwith the MIANS flyrrspecies(see “Discussion”). It is not clear whether there is any significance in the apparent differences between the extents of enhancement of the MIANS fluorescence observed upon the initial addition of aT.GDP, and the final level of MIANS fluorescence which is attained when all of the aT.G T P species have been convertedback to cq.GDP complexes, as anoutcome of G T P hydrolysis.However, we consistentlyfindthatafter G T P hydrolysis is complete, the addition of AlF,- will completelyreverse theaT.GDP-inducedenhancement of the MIANS fluorescence, presumably by eliciting the irreversible

11022

Studies with a Fluorescent-labeled

dissociation of any existingCYT.GDP/MIANS PyT complexes. The upper panelin Fig. 8B shows the resultsof an identical experiment as that presented in thelower panel, except that GTPyS was substituted for GTP. Unlike the case for GTP, the additionof GTPyS completely reversed the enhancement of the MIANS fluorescence which resulted from the initial formation of an (YT.GDP/MIANS PYTcomplex. This reversal again required several seconds for completion. However,after this period of time the additionof AlFq- had no furthereffect on the MIANS fluorescence. These results were as expected if the addition of GTPyS to the reconstituted rhodopsin/aT. complete conversion GDP/MIANS PYTsystem resulted in the of ~ T . G D P t o t h eactivated aT.GTPyS species, since the activated (YT.GTPyS species should essentially irreversibly dissociate from the MIANS-labeled PyT complexes. DISCUSSION

Subunit Complex

of Transducin

nucleotide exchangeor GTPase activity within the aT subunit, similar to the case reported for N-ethylmaleimide labeling of the PYT complex(14). Thus,ourstrategy was to use the environmentally sensitivefluorescence of the MIANS adducts as reportergroups for changes in thelocal environment (conformation) of the MIANS-reactive sites, which occur in response to the interactions of the labeled PyT complex with the (YT.GDP species. In the present studies we report that the fluorescence of the MIANS labels on the ByT complex is enhanced, and the wavelength of the emission maximum is blue-shifted, upon the interaction of an (YT.GDP species with theMIANS-labeled PyT. This enhancement is specifically elicited by the GDP-boundform of the OIT subunit and is not observed when the O(T is activated by GTP, nonhydrolyzable G T P analogs, or by AlFq-. These results indicate that the changes in the MIANS fluorescence specifically reflect the association of the aT.GDP subunit with the PyT complex to forma holotransducin molecule. Conditions that cause an activation of the G protein, and thuselicit the dissociation of the (YT subunit from PyT, result in a decay in the emission of the MIANSgroups, returning thefluorescence of the MIANS adducts from their enhanced state back toward their initial basal state. The enhancement of the MIANS emission that is induced by the CYT.GDPspecies, coupled with the blue shift in the wavelengthfor themaximum emission,suggests that the MIANS adducts are less accessible to solvent within the(YT. GDP/MIANS PyT complex relative to the accessibility of these adducts in the free PyT complex. The reduced accessibility of the reporter groups either could be due to a direct burying of one or more of the MIANS moieties on the PyT complex upon the bindingof the (YT.GDP subunit,or it could reflect a conformational change in the PYT subunit complex that is induced upon the association of the LYT.GDP species. We favor the latter explanationgiven that we have not found the MIANS-labeledPYT,nor byT complexes that were labeled with other cysteine reagents, such as N-ethylmaleimide, acrylodan, or dansyl azidirine (data not shown), to have been inhibited in their abilities to couple to (YT.GDP subunitsor to support rhodopsin-stimulated guanine nucleotide exchange or GTPase activities within the CYTsubunit. If the MIANSreactive amino acid side chains were within the binding domain of the o(T subunit ( i e . on the PyT subunit complex) it might havebeen expectedthatthe modification of these residues would have significantly influenced the abilityof the LYT subunit to couple to the 8-y~ complex. Fluorescence titrationexperiments,monitoringthe (YT. GDP-induced changes in the MIANS emission, suggest that the interactionof the a T .GDP complex with thePYTcomplex can be described by a dissociation constant in the submicro. the binding of the transducin molar range (-0.3 p ~ ) Thus, aTsubunit to the&T subunit complex appears tooccur with a n affinity similar to that reported for the binding of the a, subunit to a biotinylated Py complex ( i e . K d -0.35-0.4 p M ) but weaker than that for the interaction of the aisubunit (aql) with the biotinylated Py (& -0.02 p M ) (25). Given that theconcentration of transducinin rod outersegments is estimated to be -0.3 mM, these titration results also suggest thatactivatingguanine nucleotides (e.g. GTP,GTPyS), which are felt to elicit the complete dissociation of the (YT subunit from @-yT, must cause as much as a three order of magnitude reduction in the affinity of CYTfor P ~ T . '

The association-dissociation equilibrium for heterotrimeric G protein subunits is an important feature of the activation/ deactivation cycles of these transducer proteins.A number of lines of evidence have suggested that the by subunit complexes of G proteins play an essential role in promoting the functional coupling of the G protein-a subunits to their specific cell surface receptors, such thata tightly boundmolecule of GDP (on thea subunit) canbe exchanged for GTP (1,18). Following the guanine nucleotide exchange event, the GTPbound a subunits dissociate from both the receptor proteins and from the P-y subunit complexes (1,19) andgo on tocouple to specific effector proteins. Various procedures have been usedtoexaminetheassociation of different G protein-a subunits with the Py subunit complexes. For example, the ability of P-y complexes to promote the pertussis toxin-catalyzed ADP-ribosylation of the ai and a, subunits has served as a convenient read-out for G protein-subunit association (20-22). Sternweis and colleagues have examined the interactions of different G protein-a subunits with thePy subunit complexes from bovinebrain membranes bothby monitoring the enhanced abilities of the associated, holo-G proteins to bind to phospholipid vesicles, relative to the binding of the isolated subunitsto vesicles (23),and by theabilities of different purified a subunits to bind toa Py affinity support (24). Recently, Kohnken and Hildebrandt (25) developed an approach for studying the interactionsof the a subunits of Gi ( a 4 1or ) Go (a39) with by subunit complexes by binding the a subunits toa biotinyl. Py complex and then isolating the holoG protein complexes by immunoprecipitation witha streptavidin-agarose resin. However, while these various approaches have provided the effective means for characterizing G protein-subunit interactions, they do not provide a continuous assay system for obtaining rate information for subunit association-dissociation events. In the present work we set out to establisha fluorescence spectroscopic assay for the interactions of the transducin-a subunit ( i e . aT)with the ByT subunit complex. Specifically, the PyT complex was labeled with an environmentally sensitive fluorescentcysteine reagent, 2-(4'-maleimidylanilino) napthalene-6-sulfonic acid (MIANS). We have been able to label P-yTwith the MIANSprobe with stoichiometries ranging fromtwo to five MIANS groups per PYT. In all cases the labeling occurs predominantly on the 26-kDa carboxyl-terminal tryptic fragment of the P subunit, althoughat thehigher stoichiometries some labeling of the 14-kDa amino-terminal tryptic fragment of the P subunit, as well as labeling of the The concentration of rhodopsin in rod outer segments has been YT subunit, occurs. However, even at the highest levels of estimatedtobe 3 mM, whilethemolarratios of rhodopMIANS incorporation, the PYT subunit complex appears to sin:transducin:PDE is -1OO:lO:l (26). This then suggests that the be fully capable of promoting rhodopsin-stimulated guanine concentration of transducin is -0.3 mM.

Studies with a Fluorescent-labeled Based on the resultsof these fluorescence studies, it seems likely that MgCl, alsohas a significant influence on the interactions of the (YTsubunit with thePyT subunit complex. Specifically, we find that with increasinglevels of MgClz (up t o 5 mM) there is a steady increase in the extent of the aT.

Pr Subunit Complex of

+

PYT

11023

from these experiments indicated that a single ByT complex can rapidly promotetherhodopsin-stimulatedbinding of GTPyS to many o(T subunits, such that the dissociation of PYT from an activated CYTsubunit must occur within 1-2 s (and cannot require several seconds for completion).

Enhanced fluorescence

CXT.GDP MIANS

Tramducin

A aT.GDP - MIANS P’YT

Enhanced fluorescence

ff+

+ MIANS

t

P’YT

l3

(activated) MIANS

BYT

Overall, the results described above are consistent with the GDP-induced enhancement in the MIANSPYT fluorescence, whereas at MgC1, levels above 5 mM, a marked decline in the following scheme. The CY^. GDP-inducedconformational . enhancement of the MIANS change within the MIANS PyT complex (indicated by P’YT), extent of the 0 1 ~GDP-induced fluorescence is observed (see Fig. 6). Magnesium shows essen- which results in an enhanced MIANS fluorescence, is sugtially identicaleffects on the rhodopsin-stimulated GDP-GTPgested to occur immediately upon the formationof the holoexchange reaction within theCYTsubunit (as monitored by an transducin molecule (step 1).The dissociation of an activated enhancement of the intrinsic tryptophan fluorescence of the (YT* subunit from MIANS B’YT (step 2) also is suggested to occur immediately and to precede the slower relaxation of the subunit), and on the rhodopsin-stimulated GTPase activity (which is a direct outcome of rhodopsin-stimulated GTP- P’YT complex back to its original conformation ( i e . step 3). of PyT with We would further suggest that itis the relatively slow rate for binding toaT)(data not shown). The interactions step 3 which prevents us fromobserving a complete decay in CYTare believed t o be essential for both of these rhodopsinstimulated activities(1).Thus, one possible interpretation of the fluorescence of the MIANS PyT species, following the these data is that MgCl, exerts effects directly on theassoci- rhodopsin- and GTP-induced activation of the 0 1 ~subunit. ation of aT. GDP withPyT, such that atlower levels of MgC1, Specifically, if step 3 occurs on a timescale comparable to of CYT.GDP complexes, (5 mM), subunit dissociation is due to GTPhydrolysis, would result in a rapid reformationof more favored. The latter suggestion has been made for other CYT.GDP/MIANS &T complexes and a relatively rapid return of theMIANS fluorescence toitsenhancedstate.Inthe G proteins, and in particular for transducin, based on the future, we plan to examine further the relationship between findings from gel filtration experimentswhich show that Mg2+ will elicit the dissociation of the CYT subunit from the PyT the dissociation of the G protein subunits,which accompanies of the flyT subunit complex (27). However, it also is possible that the the activationof the OITsubunit, and the relaxation species back to its initial conformational state. One aim, in promotion by relatively low levels of MgC12 of the aT.GDPPYTfluorescence reflects particular, will be to develop resonance energy transfer apinduced enhancement in the MIANS a furthermodulation(by M e ) of the PYT conformation proaches, using CYTand byT subunits labeled with appropriate within the holotransducin complex. If this were the case it donor-acceptor pairs, as a complimentary method for moniwould have interesting functional implications, given that the toring G protein-subunit association-dissociation events. The MgCl,-induced changes in the abilityof (YT.GDP to elicit an combination of thesedifferent fluorescencespectroscopic enhancement in theMIANS PYT fluorescence are closely read-outs, performed in conjunction with rapid mixing techcorrelated with the ability of holotransducin to productively niques, should then make it possible to accurately measure the ratesof G protein-subunit association anddissociation as couple to light-activatedrhodopsin. in the aTand ByT Theactivation of the CYT subunit,as elicited by A1F4-, well as the ensuing conformational changes complexes, and to compare these rates to those for other key nonhydrolyzable G T P analogs, or rhodopsin plus GTP, reevents in the G protein-signaling cycle. sults in thereversal of the aT.GDP-induced enhancementof the MIANSPYTfluorescence (see Figs. 7 and 8B). This decay Acknowledgments-We wish to thank Linda Griswold and Cindy in the MIANS fluorescence, whichaccompanies the activation Westmiller for their expert secretarial assistance and Samuel Wong of the aT subunit, most likely reflects the dissociation of the for his technical assistance. activated aTfrom the MIANSPYTcomplex, since it has been well established that the activation of transducin results in REFERENCES the dissociation of its subunits (1,7). The decay in the MIANS 1. Fung, B. K.-K. (1983) J . Biol. Chem. 258, 10495-10502 &T fluorescence appears to be slow relative to both the aT. 2. Baehr, W., Devlin, M. J., and Applebury, M. L. (1979) J. Bid. GDP-induced enhancement in the MIANS fluorescence and Chern. 254, 11669-11677 3. Kohnken, R. E., Eadie, D. M., Revzin, A,, and McConnell, D. G. the rhodopsin- and GTP-induced enhancement in the tryp(1981) J . Bid. Chem. 256, 12502-12509 tophan emission of CYT(which directly reflects the activation 4. Gilman, A. G. (1987) Annu. Reu. Biochern. 56, 615-650 event) (for example, see Figs. 7 and 8B). This then suggests 5. Northup, J. K., Smigel, M. D., Sternweis, P. C., and Gilman, A. that either the dissociation of the activated o ~ Tsubunit from G. (1983) J . Bid. Chem. 258,11369-11376 the MIANSPYTcomplex, or the return of the MIANS ByT to 6. Codina, J., Hildebrandt, J. D., Birnbaumer, L., and Sekura, R. its original conformational state ( i e . the PyT conformation (1984) J . Biol. Chem. 259, 11408-11418 7. Phillips, W. J., and Cerione, R. A. (1988) J . Bid. Chern. 263, which existedpriortotheaddition of aT.GDP), is slow 15498-15505 relative to the associationof O(T.GDP with MIANSByT. We 8. Gierschik, P., Simons, C., Woodard, C., Somers, R., and Spiegel, favor the latter explanation based on our earlier studies of A. M. (1984) FEBS Lett. 172,321-325 the activation-deactivation cycle of transducin, monitoring 9. Phillips, W. J., Trukawinski, S., and Cerione, R. A. (1989) J . changes in the tryptophanemission of CYT(7, 17). The results Bid. Chern. 264,16679-16688

11024

Studies with Fluorescent-labeled a

10. Litman, B. J. (1982) Methods Enzymol. 81, 150-153 11. Pines, M., Gierschik, P., Milligan, G., Klee, W., and Spiegel, A. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 4095-4099 12. Avron, M. (1960) Biochim. Biophys. Acta 40, 257 13. Haugland, R. P. (1989) in Handbook of Fluorescent Probes and Research Chemicals,p. 25, Molecular Probes, Inc., Eugene, OR 14. Ho, Y.-K., and Fung, B. K.-K. (1984) J. Biol. Chem. 259, 66946699 15. Fung, B. K.-K.,andNash, C. R. (1983) J. Biol. Chem. 258, 10503-10510 16. Fong, H. K. W., Hurley, J. B., Hopkins, R. S., Miake-Lye, R., Johnson, M. S., Doolittle, R. F., and Simon, M. I. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 2162-2166 17. Guy, P. M., Koland, J. G., and Cerione, R. A. (1990) Biochemistry 29,6954-6964 18. Florio, V. A,, and Sternweis, P. C. (1985) J . Bid. Chem. 260, 3477-3483

By

Subunit Complex

of Transducin

19. Ransnas, L. A,, andInsel, P. A. (1988) J . Bid. Chem. 263,1534815353 20. Casey, P. J., Graziano, M. P., and Gilman,A. G. (1989) Biochemistry 28, 611-616 21. Huff, R. M., and Neer, E. J. (1986) J. Bid. Chem. 261, 11051110 22. Katada, T., Oinuma, M., and Ui, M. (1986) J . Biol. Chem. 261, 8182-8191 23. Sternweis, P. C. (1986) J . Biol. Chem. 261, 631-637 24. Pang, I.-H., and Sternweis, P. C. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 7814-7818 25. Kohnken, R. E., and Hildebrandt, J. D. (1989) J. Biol. Chem. 264, 20688-20696 26. Stryer, L., and Bourne, H. R. (1986) Annu. Reu. Cell Biol. 2,391419 27. Deterre, P., Bigay, J., Pfister, C., and Chabre, M. (1984) FEBS Lett. 178, 228-232