Expression of G, in Escherichia coli · Expression of G, in Escherichia coli PURIFICATION AND PROPERTIES OF TWO FORMS OF THE PROTEIN* (Received for publication, July 20, 1988) Michael

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 264, No. 1, Issue of January 5, pp. 409418,1989 Printed in U.S.A.

Expression of G , in Escherichia coli PURIFICATION AND PROPERTIES OF TWO FORMS OF THE PROTEIN*

(Received for publication, July 20, 1988)

Michael P. Graziano, Michael FreissmuthS, and Alfred G. Gilman From the Department of Pharmacology, The University of Texas Southwestern Medical Center, Dallas, Texas 75235

Cloning of complementary DNAs that encode either of two forms of the (Y subunit of the guanine nucleotide- binding regulatory protein (G.) that stimulates adenylyl cyclase into appropriate plasmid vectors has al- lowed these proteins to be synthesized in Escherichia coli (Graziano, M. P., Casey, P. J., and Gilman, A. G. (1987) J. Biol. Chem. 262, 11375-11381). A rapid procedure for purification of milligram quantities of these proteins is described.

As expressed in E. coli, both forms of G, (apparent molecular weights of 45,000 and 52,000) bind guanosine 5’-(3-O-thio)triphosphate stoichiometrically. The proteins also hydrolyze GTP, although at different rates (i.e. 0.13-min” and 0.34-min” at 20 “C for the 45- and the 52-kDa forms, respectively). These rates reflect differences in the rate of dissociation of GDP from the two proteins. Both forms of recombinant G, have essentially the same kcat for GTP hydrolysis, -4. min-l.

Recombinant G, interacts functionally with G protein @-y subunits and with @-adrenergic receptors. The proteins can also be ADP-ribosylated stoichiometrically by cholera toxin. This reaction requires the addition of @-y subunits. Both forms of recombinant G, can reconstitute GTP-, isoproterenol + GTP-, guanosine 5’-(3-O-thio)triphosphate-, and fluoride-stimulated adenylyl cyclase activity in S49 cyc- membranes to maximal levels, although their specific activities for this reaction are lower than that observed for G. purified from rabbit liver. Experiments with purified bovine brain adenylyl cyclase indicate that the affinity of recombinant G, for adenylyl cyclase is 5-10 times lower than that of liver G. under these assay conditions; however, the intrinsic capacity of the recombinant protein to activate adenylyl cyclase is normal. These findings suggest that G,, when synthesized in E. coli, may fail to undergo a posttranslational modification that is crucial for high affinity interaction of the G protein with adenylyl cyclase.

Cell surface receptors for many hormones and neurotrans- mitters interact with and activate members of a group of

* This work was supported by United States Public Health Service grant GM34497, American Cancer Society Grant BC5551, and the Raymond and Ellen Willie Chair of Molecular Neuropharmacology. We also acknowledge support from the Perot Family Foundation and The Lucille P. Markey Charitable Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ‘‘aduertisement’’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Recipient of a Max Kade Foundation Postdoctoral Research Exchange Grant.

homologous guanine nucleotide-binding regulatory proteins or G proteins’; G proteins, in turn, control the activity of membrane-bound effector molecules such as adenylyl cyclase (for review, see 1, 2). G proteins share a heterotrimeric struc- ture, with subunits designated a, p, and y. The a subunits comprise a family of highly related yet distinct polypeptides with molecular masses of 40-46 kDa. The identity of the a subunit defines a given G protein oligomer. Molecular biological and immunological evidence indicates that there are two forms of the p subunit (molecular mass, 37 kDa) (3,4) and at least three forms of y (apparent molecular weight -8,000) (5- 7). Studies of purified proteins suggest that the G proteins undergo a reversible cycle of ligand-mediated subunit dissociation (1). In its inactive oligomeric state, the G protein a subunit is associated with GDP. An appropriate agonist- receptor complex catalyzes exchange of GTP for GDP; the resultant conformational change promotes dissociation of a . GTP from By. In this dissociated state, a . GTP modulates the status of an effector. Recent, albeit controversial, data suggest that the free Py subunit complex may also interact with and regulate effectors (8). Hydrolysis of bound GTP to GDP by the a subunit promotes reformation of the oligomer, and signal transmission is terminated.

G, mediates the hormonal stimulation of adenylyl cyclase by a variety of hormones (e.g. p-adrenergic agonists, glucagon, ACTH). Purification of the protein indicated that there were two major forms of the a subunit of G, (Gs,), with apparent molecular weights of 45,000 and 52,000 (9). Sequencing and expression of cDNA clones now suggest that four forms of G., can be produced by alternative splicing of a single precur- sor mRNA (10, 11). Electrophoretically resolvable forms of G,, differ by the inclusion or deletion of 15 amino acid residues that are encoded by exon 3 of the G., gene (12). In addition, two different species of each of these forms of the protein are produced by utilization of an alternative splice site during RNA processing, which results in the inclusion or deletion of

The abbreviations used are: G proteins, guanine nucleotide-binding regulatory proteins; G., the guanine nucleotide-binding regulatory protein that mediates stimulation of adenylyl cyclase; Gem, the (Y

subunit of G,; rGsa, recombinant Gem; GTPrS, guanosine 5’-(3-0- thioltriphosphate; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol. The buffers utilized were as follows. TEDP, 50 mM Tris. HCl, pH 8.0, 1 mM EDTA, 1 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride; TDPK, 50 mM Tris. HCl, pH 8.0, 1 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride, 10 mM KH~POI; HED, 50 mM NaHepes, pH 7.6, 1 mM EDTA, 1 mM DTT; HEDL, HED plus 0.1% Lubrol; HME, 20 mM NaHepes, pH 8.0, 2 mM MgC12, 1 mM EDTA; Mono Q buffer, 50 mM Tris. HCl, pH 8.0, 0.02 mM EDTA, 1 mM DTT; HPHT buffer, 10 mM Tris.HC1, pH 8.0, 1 mM DTT, 10 mM potassium phosphate, pH 8.0; Hepes, 4- (2-hydroxyethyl)-l-piperazineethanesulfonic acid GDPpS, guanosine 5’-0-(2-thiodiphosphate).

409

410 Purification of Recombinant G,,

a single serine residue at the carboxyl-terminus of this regionz In native membranes, the relative amount of the various forms of G,, varies. Some tissues contain only M, 45,000 or 52,000 proteins, while multiple forms coexist in most tissues (13). Studies of purified preparations of G,, indicate that M, 45,000 and 52,000 forms can both mediate hormonal stimulation of adenylyl cyclase (14); this suggestion was supported by recent studies of partially purified preparations of G., expressed in Escherichia coli (15). Several studies have suggested quantitative differences in the extent to which different forms of G,, can stimulate adenylyl cyclase (16, 17). Expression of individual forms of G., in E . coli permits unambiguous as- sessment of their activities and mechanistic evaluations of any differences that are observed.

In this report we present a method for the purification of a 45- and a 52-kDa form of G., following their expression in E. coli. As purified, both proteins bind and hydrolyze GTP and can mediate hormonal stimulation of adenylyl cyclase, albeit with quantitative differences. The nature and mechanistic basis of these differences are explored.

EXPERIMENTAL PROCEDURES

Construction of Gam Expression Vectors-Methods for construction of G., expression vectors are detailed in Maniatis et al. (18). The expression system of Tabor and Richardson (19), which utilizes the promoter for bacteriophage T7 RNA polymerase, was employed. Plasmid pT7-5 (15, 19) was sequentially cleaved in its multicloning site with EcoRI and BamHI and purified by agarose gel electrophoresis. The following two complementary oligonucleotides were synthesized, annealed, and then ligated with the cleaved plasmid (rbs = ribosomal binding sequence).

r h c

applied to a Pharmacia Mono Q HR 10/10 fast protein liquid chromatography (FPLC) column that had been equilibrated with Mono Q buffer. The column was washed with one volume of Mono Q buffer, and adsorbed protein was eluted at 4 ml/min with a 240-ml linear gradient of NaCl (0-350 mM) in Mono Q buffer; 4-ml fractions were collected.

rG., was purified to homogeneity by chromatography on a Bio-Rad HPHT column (a high performance liquid chromatography hydroxylapatite column); the column fittings were adapted to allow the column to be utilized with the FPLC system. Fractions from the Mono Q column that contained the peak of GTPyS-binding activity were pooled, diluted 4-fold with HPHT buffer, and applied to a Bio- Rad HPHT column equilibrated with the same buffer. The column was washed with one volume of HPHT buffer, and protein was eluted at a flow rate of 0.75 ml/min with a linear gradient of potassium phosphate (0-290 mM, pH 8) in HPHT buffer. Fractions (0.75 ml) that contained the peak of GTPyS-binding activity were pooled, and buffer was exchanged by sequential cycles of concentration and dilution into HED in a Centricon 30 microconcentrator (Amicon). Purified rG., (protein concentration > 1 mg/ml; phosphate concentration < 10 mM) was frozen in liquid N2 and stored at -80 "C.

Purification of Other Proteins-The resolution of By from purified oligomeric bovine brain G proteins (a mixture of Gi and Go) was achieved by methods described previously (15). Adenylyl cyclase was purified from Lubrol extracts of bovine brain membranes according to Pfeuffer et al. (21). Fractions from the first forskolin-Sepharose column were applied to a 0.5-ml Bio-Gel HTP column in order to concentrate the sample and remove the forskolin; protein was eluted with HED containing 0.05% Lubrol and 200 mM potassium phosphate, pH 8. As judged from silver-stained SDS-PAGE gels, more than 90% of the total protein was located in two bands (apparent molecular weights 120,000 and 150,000) that are associated with adenylyl cyclase activity. The specific activity of the purified preparation was 0.4-0.5 pmol of cyclic AMP/min. mg protein in the absence of forskolin (basal activity) and 2.5-3.1 pmol of cyclic AMP/min.mg

Ne01 Sma I 5 ' A A T T C T A m G G T T T A A C C A T G G C C C G G G G

I "U

3' 3' G A T T C C T C C A A A T T G G T A C C G G G C C C C C T A G 5 '

This plasmid will subsequently he referred to as NpT7-5. The NcoI-StuI fragment of pG.,.. or pGao.L (10) was ligated with

NpT7-5 after digestion of the plasmid with NcoI and SmaI. This construction places the initiation codon for G, 10 nucleotides down- stream from a consensus ribosomal-binding sequence (20). The use of these constructions (NpT7-5/G,, and NpT7-5/G,.L) results in a 2-3-fold increase in the levels of rG., that accumulate in E. coli as compared to previous results (15). This increase presumably reflects alterations in the nucleotide sequence directly upstream from the initiation codon.

Purification of rG.,-Nine liters of medium were inoculated with E. coli strain K38 harboring plasmids pGp1-2 (19) and either NpT7- 5/G.,.. or NpT7-5/GS,.L, and cells were grown, harvested, and lysed as previously described (15). All subsequent manipulations were performed at 4 "C. Lysates were centrifuged at 30,000 X g in a Beckman JA-14 rotor for 1 h. The resulting supernatants (1200-1300 ml) were pooled, and DEAE-Sephacel (100 ml of packed volume, equilibrated with TEDP) was added. After 15 min of gentle stirring, the mixture was centrifuged at 300 X g for 5 min, and the supernatant was discarded. The suspended resin was applied to a Buchner funnel fitted with a Whatman 4 filter and washed, under vacuum, with 1 liter of TEDP. Adsorbed protein was eluted from the resin (by gravity flow) with three 100-ml aliquots of TEDP containing 300 mM NaC1. The eluant was applied directly to a 100-ml column of hydroxylapatite (Bio-Gel HTP; Bio-Rad) (2.6-cm inner diameter), equilibrated with TDPK containing 300 mM NaCl. The column was washed with one volume of TDPK plus 300 mM NaC1, and adsorbed protein was eluted with a 500-ml linear gradient of NaCl (300-1500 mM) in TDPK. Fractions (8 ml) containing G, activity (Fig. 1) were pooled, and excess NaCl was removed by ultrafiltration in a 250-ml stirred cell (Amicon) fitted with a PM-30 membrane, followed by dilution with Mono Q buffer. The resulting concentrate (e20 mM NaC1) was

(SDS-polyacrylamide gel electrophoresis) are 45,000 and 52,000. The The apparent molecular weights of the two major forms of G.,

deduced molecular weights are 44,500 and 46,000. The electrophoretic behavior of the larger form appears to be anomalous (15).

protein in the presence of 100 p~ forskolin. G. was purified from cholate extracts of rabbit liver membranes

according to Sternweis et al. (14) and was generously supplied by Dr. Patrick Casey (this laboratory); the amount of G., was quantified by labeling with cholera toxin, as described in the following section.

Cholera Toxin-catalyzed ADP-ribosylation of G,-Cholera toxin- catalyzed ADP-ribosylation of purified rG., and rabbit liver G. was performed by a modification of the method of Schleifer et al. (22), as previously described (15). The extent of ADP-ribosylation was estimated by the use of [32P]NAD as a substrate for the reaction. Radiolabeled protein was purified electrophoretically, excised from the dried gel, and counted.

Intrinsic Fluorescence of rG,,-Measurements of the intrinsic fluorescence of rGsa were made with a SPEX Fluorolog 211 spectropho- tometer as previously detailed for purified bovine brain G proteins (23). Excitation and emission wavelengths were 290 and 340 nm, respectively. All measurements were performed at 20 "C.

Miscellaneous Procedures-Reconstitution of rG, with membranes prepared from the G,-deficient S49 lymphoma cell (cyc-) was performed (with no preincubation of the membranes with rG,) as described previously (24). Recombinant G., was stably reconstituted into S49 cyc- membranes by the procedure of Sternweis and Gilman (25), with minor modifications. rGa,.s or rGsa.L was mixed with a 3- fold excess of purified 07 and incubated with S49 cyc- membranes (1 mg/ml) over a range of 5-150 pmol rG.,/mg of membrane protein. The efficiency of incorporation of G., into the membranes, as judged by cholera toxin-catalyzed ADP-ribosylation, was 4040%; if the reconstitution was carried out in the absence of added By, only about 15% of the o( subunit was recovered in the membranes. However, the final specific activity of rG, was comparable whether the stable reconstitution was carried out in the absence or presence of By when this value was calculated based on the extent of protein incorporated into cyc- membranes. The specific activity of stably reconstituted G, was determined by assaying 50 pg of reconstituted membranes for adenylyl cyclase activity in the presence of 10 p M (-)-isoproterenol and 10 p~ GTP+ in a final volume of 100 pl (30 "C, 30 min incubation).

Reconstitution of rG., or liver G. with purified adenylyl cyclase

Purification of Recombinant G,, 411

was carried out as follows. rG., or G, (160 pg to 200 pg/ml) was activated by incubation for 1 h at 30 "C in a solution containing HEDL plus 10 mM MgSO, and 100 p~ GTPyS. Appropriate dilutions were then made in the same solution and were held on ice. Activated G., (10 pl) was then incubated for 15 min on ice with 10 pl of purified adenylyl cyclase (2-500 ng in HED containing 0.05% Lubrol). The adenylyl cyclase reaction was started by addition of 80 p1 of a prewarmed solution containing 20 mM NaHepes, pH 7.6, 1 mM EDTA, 25 mM MgC12, 0.625 mM [cz-~'P]ATP (20 cpm/pmol), and 10 pg of bovine serum albumin and carried out for 5 min at 30 'C. Basal activity, i.e. adenylyl cyclase activity in the absence of any added GsO, was not affected by GTPyS and MgSOl included in the GB-containing solution.

GTPyS binding was quantitated as previously described (26). GTPase activity was measured as outlined for bovine brain G proteins (27). The ka, for the GTPase reaction was measured by a modification of the method described by Higashijima et al. (27). Briefly, 50 nM rGB. was incubated with 1 p~ [Y-~'P]GTP (specific activity -10,000 cpm/pmol) in 50 mM NaHepes, pH 7.6, 5 mM EDTA, 1 mM DTT, and 0.1% Lubrol for 16 min at 20 "C. MgSO4 and GTP were then added to final concentrations of 10 mM and 100 PM, respectively. Aliquots were taken at time points and processed as detailed previously (27). [cY-~'P]GDP was prepared by the method of Johnson and Walseth (28). Binding of [cY-~'P]GDP was performed as previously described (29). SDS-polyacrylamide gel electrophoresis of proteins was performed by the method of Laemmli (30); sample buffer was modified to include 20 mM DTT. Protein was determined by staining with Amido Black (31) or by staining as described by Bradford (32). Both methods yielded quantitatively similar values.

RESULTS

Purification of rG,-Expression of rGs,.a in E. coli as described above allows the protein to accumulate to a concentration of 1-2 mg/liter of culture, based on immunoblotting. The quantity represents approximately 0.3% of total cell protein. Although variable, about 50% of the immunoreactive protein is recovered in the soluble lysate. Starting from 9 liters of culture, milligram quantities of purified G., are obtained in two days (Fig. 1, Table I). The first chromatographic step in the purification procedure is particularly useful and is unusual, in that it relies on the elution of rG,, from a hydroxylapatite column with NaCI. The vast majority of E. coli proteins remain adsorbed to the column at NaCl concentrations in excess of 1 M. G,, activity is labile during the early stages of the purification, and the final yield is significantly increased by minimizing the overall time involved. SDS- PAGE of the purified preparation reveals a single protein band with an apparent molecular weight of 45,000 (Fig. 2;

left). The purified protein comigrates with the 45-kDa form of G,, purified from bovine brain (data not shown). NH2- terminal amino acid analysis reveals that the initiating me-

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FIG. 1. Chromatography of recombinant G , ... Nine liters of E. coli K38 harboringplasmids pGpl-2 and NpT7-5/G,, were grown at 30 "C until ODW = 1.3-1.4; bacteria were then induced to express rGeo. After initial processing as described under "Experimental Pro- cedures,'' rG8,.e was purified from the DEAE-Sephacel eluate by sequential chromatography on Bio-Gel HTP (toppanel), Mono Q HR 10/10 (middle panel), and Bio-Rad HPHT (bottom panel) columns. Regions of each chromatogram denoted by the horizontal bar represent the fractions that were pooled for further chromatography. Values for protein, GTPyS-stimulated adenylyl cyclase activity, and GTPyS binding were determined as outlined in the legend to Table I and represent the mean of duplicate determinations from a single representative preparation.

TABLE I Purification of recombinant G8a.s

Nine liters of E. coli strain K38 transformed with plasmids pGpl-2 and NpT7-5/G.,.. were cultured, and recombinant G., was purified as described under "Experimental Procedures." For the assay of adenylyl cyclase activity, aliquots of each fraction were incubated with 20 p~ GTPyS, 5 mM MgC12, and 0.1% Lubrol for 45 min at 30 'C, diluted to an appropriate concentration with HME containing 20 p~ GTPyS and 0.1% Lubrol, and reconstituted with S49 cyc- membranes. Adenylyl cyclase activity was assayed for 30 min at 30 "C. Assays of GTPyS binding were performed at a final concentration of 2 p~ GTPyS. -

Adenylyl cyclase activity GTPrS binding activity

Fraction Volume Protein nmol cAMP.min"

nmol

motein" cAMP.min".mg nmol nmol . mg

protein"

rnl w ~~~~~~ ~ ~

Soluble lysate 1200 2820 1040" DEAE 876" Bio-Gel HTP 1218 Mono Q 678 HPHT

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19.6 350 1365

216" 0.07 136"

62.1 0.10

130 7.8

2.09 127 70 8.79

1.6 252 157 30 18.10

90 8 1

The instability of rG,, in these fractions makes accurate quantitation of the yield difficult. Estimates of rG., concentrations based on immunoblotting indicate that the overall yield of purified protein is 10-20% of that present in the soluble lysate. From multiple preparations, the yield of rG., has ranged from 1.2 to 1.6 mg for rGa,,.8 and 0.8 to 0.9 mg for rGar.-L. Other guanine nucleotide-binding proteins are present in the lysate. These are resolved on the Bio-Gel HTP column.

~~~

412 Purification of Recombinant G,, thionine is cleaved from rGSm.* and that the protein sequence is identical to that predicted from the bovine adrenal GSm+ cDNA (analysis performed for 13 cycles). Two-dimensional gel analysis of the purified preparation reveals a single protein species with a PI of approximately 6.5.

Guanine Nucleotide Binding and Hydrolysis by rG8-As shown in Table I, purified rGao binds GTPyS essentially

A B C

45 kDa+

D E

52 kDa, 45 kDa-

A B

FIG. 2. SDS-polyacrylamide gel electrophoresisof fractions obtained from the purification of rG,. Left panel, aliquots of fractions from the various stages in the purification of rGSa+ were resolved by SDS-PAGE on an 11% polyacrylamide gel, and proteins were visualized by staining with Coomassie Blue. Where applicable, fractions shown are aliquots of the pooled fractions identified in Fig. 1. Lane A, 25 pg of the DEAE-Sephacel eluate; lane E , 10 pg of the Rio-Gel H T P pool; lane C , 5 pg of the Mono Q HR10/10 pool; lanes D and E , 2 and 6 pg, respectively, of the Bio-Rad HPHT pool. Apparent molecular weights were determined from the migration of protein standards resolved in the same gel. Right panel, 8 pg each of rG.,.,, (lane A ) or rGSn." (lane B ) (Bio-Rad HPHT pool) was subjected to SDS-PAGE on an 11% polyacrylamide gel, and proteins were visualized by staining with Coomassie Blue. Apparent molecular weights were determined from the migration of protein standards resolved on the same gel.

stoichiometrically; values for various preparations have ranged from 16-22 nmol/mg protein. The interaction of rG.m.s with GTPyS was examined in more detail by determin- ing the association rate constant at various ligand concentrations. As shown in Fig. 3 (left), the time course of binding is independent of the concentration of GTPyS over a large concentration range. This behavior is similar to that observed for G, subunits purified from a number of sources (29) and is consistent with the fact that the rate of dissociation of bound GDP is the rate-limiting step in the reaction (1, 29). When maximal binding values are plotted as a function of GTPyS concentration (Fig. 3, right), the resultant curve can be fitted to an equation describing the interaction of GTPyS with a single class of binding sites with a Elmax of 20.2 nmol/mg protein, a value that approaches the theoretical value of 22.5 nmol/mg protein. However, the apparent K d (110 nM) grossly

3

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FIG. 4. GTP hydrolysis by purified rG"*. Left panel, rGM.s (5 p ~ ) was incubated on ice in the absence (0) and presence (W) of a 4- fold molar excess of purified bovine brain 6y in HEDL. After 15 min, the reaction was initiated by a 100-fold dilution into the same buffer containing 1 p~ [yR2P]GTP (specific activity 10,000 cpm/pmol) and 10 mM MgSO,, which had been warmed to 30 "C. At the times indicated, duplicate 50-pl aliquots were withdrawn and processed as outlined under "Experimental Procedures." Right panel, rGsa.* (250 nM) was incubated on ice with the indicated molar ratios of By in 10 pl of HEDL. After 15 min, the reaction was started by the addition of 40 p1 of HEDL (at 30 "C) containing 1.25 p~ [-y"'P]GTP (specific activity 8000 cpm/pmol) and 12.5 mM MgSO,, and incubation was continued for 10 min a t 30 "C. The solid line was drawn using the parameters calculated from a logistic equation (Hill equation). Data points shown represent the mean of duplicate determinations from three separate experiments.

FIG. 3. GTP-yS binding to rG-+.. lRf t panel, rGm.r was incubated a t 30 "C with ['"SIGTPyS and binding was determined as described under "Experimental Procedures." The concentrations of GTPyS were as follows: 0, 1 pM; W, 0.3 pM; 0, 0.1 pM; A, 32 nM; A, 12 nM; 0, 1.6 nM; 0, 0.65 nM. Data shown are the mean of triplicate determinations from a single experiment, which is representative of three similar experiments. The solid lines were drawn using parameters calculated from a nonlinear least squares fit to the equation R = E , (1 - e-bf) . Right panel, Emax values extrapolated from the time course were plotted as a function of GTP+ concentration. The curve was obtained by fitting the data to a saturation isotherm. Time ( min 1 [GTPy S] (nmol . I" 1


underestimates the affinity of rGan for GTPyS. In the presence of 10 mM MgS04, binding of GTPyS to rG,.s is essentially irreversible (data not shown). The apparent Kd observed in the experiment in Fig. 3 reflects the rate constant of several processes (e.g. the dissociation rate for prebound GDP, the association rate for GTPyS, and the rate of thermal inacti- vation of rGsn).

As shown in Fig. 4, rG, possesses an intrinsic GTPase activity. The steady-state rate of GTP hydrolysis is 0.3 mol/ min. mol rGsn.s a t 30 "C and is linear with time for a t least 10 min. A rate of 0.13 mol/min.mol rGSn., is observed at 20 "C. The rate of GTP hydrolysis can be inhibited in a concentration-dependent manner by the addition of purified bovine brain By (Fig. 4, right panel). Half-maximal inhibition occurs at, a @y/rGsc..s ratio of 3:1, and GTP hydrolysis is essentially completely inhibited at a By/rGsn.s ratio of 1O:l. The inhibition of steady-state hydrolysis of GTP reflects the ability of rG8n.s to interact with By and is due to a py-mediated decrease in the rate of dissociation of GDP (33). A large excess of By (e.g. 100 @y:l rG,) has no effect on the bat for the GTPase reaction (data not shown). Direct interaction of rGan with By can also be observed by sucrose density gradient centrifuga- tion (data not shown).

Cholera Toxin-catalyzed ADP-ribosylutwn of rGs,-G, can be ADP-ribosylated by cholera toxin; the reaction is also dependent on ADP-ribosylation factor, a 21-kDa GTP-binding protein (34). We have previously shown that partially

purified preparations of rGsn can be ADP-ribosylated by cholera toxin after reconstitution of the protein into cyc- mem- branes (15). Following similar reconstitution, purified rG, can be ADP-ribosylated in a cholera toxin-dependent fashion, although the stoichiometry of labeling is low (e.g. 0.07-0.1 mol of ADP-ribose/mol rGS,J (Fig. 5). Addition of purified bovine brain By to the reaction greatly increases the maximal extent of labeling (By has no effect on the time course of ADP-ribosylation; data not shown). The effect of by on ADP- ribosylation is concentration dependent; the half-maximal effect of By occurs at a py/rG, ratio of 2:1 and essentially stoichiometric labeling of rG8-.,, is seen at a By/rG, ratio of 101. The effect of Py on the ADP-ribosylation of rG, does not reflect stabilization of the protein during the assay. Ad- dition of a 10-fold excess of By to the reaction mixture after 40 min of incubation leads to a level of ADP-ribosylation that is 80% of that seen when By is present initially (Fig. 5). In the absence of cyc- membranes, stoichiometric cholera toxin- catalyzed ADP-ribosylation of rG,, was also dependent on both By and ADP-ribosylation factor (purified from bovine brain and generously supplied by Dr. Richard Kahn, National Institutes of Health, Bethesda, MD.; data not shown).

Purification of a 52-kDa Form of &*,-The identical procedure employed for the purification of rG,., can also be used to purify rGsn.L, a protein with an apparent molecular weight of 52,000 (Fig. 2; right) (10, 15). The purified protein binds GTPyS stoichiometrically and also serves as a substrate for

30 60 90 120 Time (min)

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FIG. 5. Cholera toxin-catalyzed ADP-ribosylation of rG,. Left panel, Twenty pmol of rG, was reconstituted with 600 pg of S49 cyc- membranes in a final volume of 500 pl containing 100 mM potassium phosphate, pH 8.0, 12 mM thymidine, 1 mM ATP, 0.1 mM GTP, 0.5 mM EDTA, 1 mM MgClz, 10 p M 13'P]NAD (specific activity 10,000 cpm/pmol), 0.003% Lubrol, and 62.5 pg/ml of activated cholera toxin. The mixture was incubated a t 30 "C in the absence (A) and presence (0) of a 10-fold molar excess of bovine brain By. After 40 min, the reaction mixture containing no By was split into two, and purified bovine brain fly was added to one incubation to achieve a 10-fold molar excess of py over rG, (0). A t the time points indicated, 50-pl aliquots were precipitated with trichloroacetic acid; proteins were resolved by SDS-PAGE and autoradiography was performed on the dried gels. The region of the dried gel that contained rG,.s was excised, and 'lP was quantitated by liquid scintillation counting. Right panel, (2 pmol/assay) was incubated under the conditions described above with 60 pg of S49 cyc- membranes and the indicated quantities of purified bovine brain By. Incubations were performed for 2 h in a final volume of 50 pl. The solid line was drawn by fitting the data points to a logistic equation (Hill equation). Photo inset shows the autoradiogram (Kodak XAR-5 film, 5-h exposure a t -80 "C with intensifying screen): lane A , rabbit liver G, (0.8 pmol in the 45-kDa band, 0.5 pmol in the 52-kDa band); lane B, 2 pmol of rG, incubated in the absence of cholera toxin; lane C, 2 pmol of rG,; lanes D-H represent 2 pmol of rG, in the presence of 0.6, 2, 6, 20, and 60 pmol of bovine brain fly, respectively; lane I, 60 pmol of bovine brain by in the absence of any added rG,.


cholera toxin-catalyzed ADP-ribosylation (data not shown). Reconstitution of Adenylyl Cyclase Activity by rG,-Purified

preparations of rGs,.8 and rG,,.L impart basal and isoproterenol-, GTPyS-, and AMF (10 pM A1Cl3, 10 mM MgC12, and 10 mM NaF)-stimulated adenylyl cyclase activity to ,949 cyc- membranes (Fig. 6; Table 11). The relative activities seen under these various conditions are typical of those observed when liver G. is assayed in this manner, and the maximal activity observed is typical of reconstituted cyc- membranes (25). However, the amount of rG,, required to stimulate adenylyl cyclase activity is excessive. Accordingly, we under- took a detailed analysis of the reconstitutive specific activity of rGs,. This specific activity is expressed as reconstituted adenylyl cyclase activity/quantity of G,, over a linear range of G,, concentrations. Specific activity is measured as a function of adenylyl cyclase concentration and extrapolated to an infinite concentration of this component (14).

The reconstitutive-specific activity of GTPyS-bound rG8Y.8 under the conditions employed in Fig. 6 approaches only 0.15 pmol/min.mg rGs,.s. If the concentration of catalyst is increased by raising the amount of S49 cyc- membranes in the reconstitution assay, a plot of the initial slope uersus catalyst concentration predicts a specific activity of 0.5 pmol CAMP/ min. mg rG,, at an infinite concentration of cyc- membranes

AMF 0

&

v. IN€ + GTP

GTP

1 I 1 I

100 200 300 400

rGo (ng1

FIG. 6. Reconstitution of adenylyl cyclase activity in S49 cyc- membranes by purified rG,. S49 cyc- membranes (60 pg) were reconstituted with serial dilutions of rG., under the conditions outlined in the legend to Table 11. Data shown represent the mean of duplicate determinations from a single experiment. B, 10 p~ GTP; A, 10 p~ isoproterenol (ZNE) plus 10 p~ GTP; 0, 10 p~ GTPyS; 0, AMF.

(data not shown); previously determined values for rabbit liver G, are approximately 20-fold higher. However, the apparent K d of rG,, for adenylyl cyclase is about 30 times larger than that of liver G, assayed under similar conditions. Hence, the modest range of variation in catalyst concentration that can be achieved by adding increasing amounts of S49 cyc- membranes may not suffice to estimate the extrapolated specific activity. The following approaches were thus taken to determine if the specific activity of the recombinant protein would approach values for G. purified from mammalian sources. First, rGs, was incorporated into cyc- membranes using the stable reconstitution protocol. Under these conditions, the specific activities were more than 10-fold higher than under standard assay conditions (Table 111). One possible explanation is that the interaction of rGsa with the catalyst may be limited by its poor ability to interact with the bilayer. Evidence for this interpretation is provided by the fact that negligible levels of [35S]GTPyS-labeled rG., were found in the membrane pellet after incubation of S49 cyc- membranes with rGB, for 0-20 min at 30 "C under standard assay conditions (data not shown). Under similar conditions, a significant fraction of liver G, is incorporated into cyc- membranes (35).

The ability of rGsa to interact with purified bovine brain adenylyl cyclase was next tested over a wide range of catalyst concentrations (Fig. 7, Table 111). It is evident that under these conditions the extrapolated specific activities for rabbit liver G, and the recombinant proteins are essentially the same and within the range reported for comparable experiments (14, 36). Interestingly, although rabbit liver G,, displays vir- tually identical apparent affinities in the cyc- reconstitution assay and in the measurements with purified adenylyl cyclase, the apparent dissociation constant for the recombinant proteins was markedly different under these conditions (Table 111).

With partially purified preparations of rG.,, Py subunits from bovine brain G proteins, but not bovine retinal trans- ducin, dramatically increased the reconstitutive activity of rGsa (15, 37). This effect is less striking with purified rG,,, where a 2-3-fold increase in reconstitutive activity (cyc- assay) is observed in the presence of a 10-fold molar excess of Py (data not shown). However, in parallel experiments the same molar excess of By has a slight inhibitory effect on the ability of rG,, . GTPyS to stimulate purified adenylyl cyclase; this is due to a depression of V,,, with no change in apparent K d . In addition, a 10-fold molar excess of Py has essentially no effect on the activity of stably reconstituted G., when assayed in the presence of GTPyS (data not shown). Taken together, these findings suggest that a significant portion of

TABLE I1 Reconstitution of adenylyl cyclase activity in S49 cyc- membranes by or rGsa.L

Quantities of rG., in a range where reconstitutive activity is linear with rG., concentration (50-200 ng, depending on the activation conditions) were diluted into HME containing 0.1% Lubrol and were reconstituted with 60 pg of S49 cyc- membranes. Where applicable, rG., was activated prior to dilution by incubation with 20 pM GTPyS plus 5 mM MgCl, for 30 min at 30 "C. Adenylyl cyclase activity was assayed for 30 min at 30 "C. Final concentrations of GDPBS, GTP, GTPyS, and isoproterenol (INE) were 10 p ~ . Data shown are the mean of duplicate determinations from a single exaeriment that was rearesentative of three such exaeriments.

Reconstitutive adenylyl cyclase activity

Activators present in assay nmol

CAMP. min" . mg ActivitylGDPBS activity rG.=.*-' d L . L - 1

CAMP. min" . mg nmol ActivitylGDPBS

activity

GDPOS 11 1.0 12 GTP

1.0 18 1.6 24

GTP + INE 2.0

73 6.6 46 3.8 GTPyS 136 12.4 76 AMF

6.3 79 7.2 55 4.6


TABLE I11 Specific activities of G,, under different assay conditions

The measurements of specific activities of G,, were carried out as outlined under “Experimental Procedures” and in the legend to Fig. 7. In order to account for depletion of G8a, the apparent dissociation constant, K d , was estimated by extrapolating to infinitely low catalyst concentration for experiments with purified adenylyl cyclase. The K d for the cyc- reconstitution assay was calculated by nonlinear, least-squares curve-fitting to saturation isotherms. The specific activities for the stable reconstitution experiments were calculated from the initial slope of the adenylyl cyclase activity in the presence of 10 PM isoproterenol and 10 p~ GTP-yS, where a plot of picomoles of cholera toxin-catalyzed ADP-ribosylation substrate versus adenylyl cyclase activity yields a saturation isotherm with an apparent K d of 4 to 6 nM. Results are means f S.E. for two to five experiments (ND = not determined).

- Cyc- reconstitution Stable Purified adenylyl cyclase

Protein reconstitution

Specific activity Specific activity K d Specific activity K d

pmol/min/mg nM pmol/min/mg pmol/min/mg nM

Liver G., 2.1 f 0.2 3.4 f 0.8 ND 11.0 f 0.3” 3.6 f 0.5

rGBOI-$ 0.14 f 0.02 89 f 12 1.74 f 0.31 12.7 f 0.8” 27.2 f 0.6

rGsa-L 0.09 f 0.02 166 f 20 1.86 f 0.35 12.9 f 0.8” 14.4 f 0.5

10.20.b

0.4“ -

a Values derived from extrapolation to infinite concentration of adenylyl cyclase. Value is from Ref. 14.

Adenylyl Cyclase Activity [Adenylyl Cyclase Activity] X 10 -1

(pmol cAMP.min” 1 (pmol CAMP” .min)

FIG. 7. Stimulation of purified bovine brain adenylyl cyclase by rabbit liver G. and rG”.. Assay conditions were as described under “Experimental Procedures.” Left panel, the specific activity of rG=.# (0) and rabbit liver G. (0) as a function of adenylyl cyclase concentration. Two sets of six saturation isotherms were generated, with basal adenylyl cyclase activities ranging from 0.8 to 260 pmol/min, a t seven logarithmically spaced concentrations of rG.,.s (45 ng/ml to 150 pg/ml) and rabbit liver G. (4.8 ng/ml to 16 pg/ml; protein mass expressed as the a subunit alone). The specific activity at each individual adenylyl cyclase concentration was calculated from the initial slope of each saturation curve. Enzyme activity was stimulated 8.4 f 0.9- and 7.1 f 0.5-fold by rG-.# and liver G., respectively. The solid lines were drawn using the parameters calculated by fitting the points to a rectangular hyperbola. Extrapolation to infinite adenylyl cyclase concentration yielded specific activities of 10.7 f 1.1 and 13.6 f 1.3 pmol of cAMP/min. mg of rabbit liver G., and rG-.@, respectively. Right panel, Lineweaver-Burk transformation of the data.

the apparent stimulation of reconstitutive activity observed in partially purified preparations of rG,, reflects stabilization of the free a subunit during activation. However, a very large excess of Pr in the presence of GTP+ and M$+ may still be able to facilitate interaction of a with the membrane via formation of the heterotrimeric complex.

Although the reconstitutive specific activities (measured in the presence of GTPyS) are essentially the same for rG8,.8 and rGs,.L (Table 111), the ratio of activities GTPIGDPBS or GTPIGTPrS are higher for rGsa.L than for rGsm.s (Table 11). This difference is examined in more detail in Fig. 8. In assays performed by reconstitution of rG., with either cyc- mem- branes (Fig. 8, left panel) or with purified adenylyl cyclase (Fig. 8, right panel), it is evident that the curve for rGea.L lies

above that for rGsm.s when GTP is present and that values are essentially identical in the presence of GDPPS. These differences in activities suggest that a larger fraction of the total rGsa exists in the active GTP-bound form in the presence of GTP with rG,,.t than with rGBU.8. The fraction of protein in the GTP-bound form is equal to kOff/kat + kff, where kff is the rate of dissociation of GDP and kcat is the catalytic rate for hydrolysis of GTP (27). As shown in Fig. 9, both forms of rG., have essentially identical values of kcat of -4 min” at 20 “C. This value is in good agreement with that determined previously for rabbit liver G, (38).

rGen.L binds GTPrS a t a rate approximately 2-3-fold faster than that for rG8,.s (Fig. 10, upperpanel). G, subunitspurified from bovine brain display an increase in intrinsic tryptophan


40:

0 400 800 500 1000

rGsa (w) FIG. 8. Comparison of the activity of rG”, and rG,L in the presence of GTP. Left panel, S49 cyc-

membrane reconstitution. rG.*.* ( 0 , O ) and rG,.L (0, W) were diluted into HEDL containing 10 mM MgSO, and either 100 p~ GDPBS (0,O) or 100 p~ GTP (0, W). Appropriate dilutions (10 pl) were mixed with 60 pg of S49 cyc- membranes in a final volume of 100 pl and adenyl cyclase was assayed. Maximal activity was calculated from saturation isotherms run in parallel with GTPyS-preactivated G, and amounted to 300 and 250 pmol/min.mg cyc- membrane protein for rGm.s and rG,.L, respectively. Rightpanel, stimulation of purified bovine brain adenylyl cyclase. Purified adenylyl cyclase (10 pl; 46 ng/assay) was incubated on ice for 15 min with 10 pl of appropriate dilutions of rGw.n (0 ,O) or rG,.L (0, W) as described for the cyc- reconstitution assay. Reactions were started by adding 80 pl of a solution containing 20 mM NaHepes, pH 7.6, 1 mM EDTA, 25 mM MgC12,0.625 mM [as2P]ATP (20 cpm/pmol), and 10 pg of bovine serum albumin and were carried out for 5 min at 30 “C. Basal enzymatic activity was 23 pmol/min and was not affected by GTP or GDPBS. V,,, as determined from saturation isotherms using GTPyS-activated rGeU.# and rG,.L, was 131 and 111 pmol/min, respectively.

0 b- 10 12 14 16 18 20

Time (min)

FIG. 9. Determination of kc., for the hydrolysis of GTP by rG=.# or rGa-L. rGma.8 (0) or rG,.L (0) (64 pmol of each) was mixed with 1 p M [y-32P]GTP (specific activity 8000 cpm/pmol) at 20 “C in 0.8 ml of HEDL. At the times indicated, 50-p1 aliquots were withdrawn and 32P was determined as described under “Experimental Procedures.” At 16 min, MgSOd and GTP were added to final concentrations of 10 mM and 100 pM, respectively. Values of kat shown represent the mean f S.D. of two determinations.

fluorescence upon binding and activation by GTPyS plus M e ; this change presumably reflects an altered conforma- tion of the activated protein (23). Upon binding GTPyS, both forms of rGea display a 20% enhancement of intrinsic tryptophan fluorescence (Fig. 10, lowerpanel). The k,, values for the GTPyS-induced fluorescence enhancement are indistinguishable from those observed for radioligand binding. In solution, the rate of binding of GTPyS to G protein a subunits and the steady-state rate of hydrolysis of GTP by G, subunits are limited by, and hence are equal to, the rate of dissociation of GDP (1). As would be predicted from the kinetics of GTPyS

30 t

” 6 12 18 24

Time (min)

FIG. 10. The binding of GTP-yS to rG.,,-* or rGa.-L. Upperpanel, rGSe.* (5 pmol) (0) or rG.-.L (4 pmol) (0) were incubated with 2 p~ [35S]GTPyS (specific activity 3000 cpm/pmol) at 20 “C in a final volume of 60 pl. At the times indicated, duplicate 50-pl aliquots were withdrawn and bound GTPyS was determined as outlined under “Experimental Procedures.” Lower panel, tryptophan fluorescence was determined by incubating 0.4 nmol of either rGem.8 or rGao.L in 1.25 ml of HEDL containing 10 mM MgSO, at 20 “C for 3 min in a 5 X 5-mm fluorescence cuvette fitted with a magnetic stir bar to provide continuous mixing. At t = 0 in the figure, GTPyS was added to a final concentration of 20 p ~ . The values shown for kapp in both the upper and lower panels were determined by fitting the data to the equation B = B, (I - e-kt) .

binding, the steady-state rate of hydrolysis of GTP by rGsa.L is 2-3-fold greater than that observed for rGaa.s (Table IV), as is the rate of dissociation of GDP. This difference between rGsa.L and rGea.8 in the rate of dissociation of GDP persists over a broad range of M e concentrations (3 nM to 100 mM) and temperatures (10-35 “C).


TABLE IV Rate constants for guanine nucleotide binding to rG,,

Values of kapp for GTP-yS binding were taken from the data shown in Fig. 10. The steady-state rate of GTP hydrolysis was determined at 20 "C, as described in the legend to Fig. 4. The rate of dissociation of GDP (k,ff) was determined at 20 "C using [CY-~'P]GDP; values for k,ff were determined by fitting the data to the equation describing a monoexponential decay.

Rate constant r L e rGso-L

kapp GTP-yS binding (min") Radioligand binding 0.13 & 0.01 0.34 +. 0.03 Fluorescence enhancement 0.13 & 0.01 0.38 f 0.01

Steady-state GTP hydrolysis (pmol 0.12 & 0.03 0.31 & 0.03

k,fr GDP (min") 0.16 f 0.01 0.36 f 0.02 Pi/pmol rG., ... min)

-

DISCUSSION

We have expressed complementary DNAs that encode two forms of G,, in E. coli. Purification in the absence of detergent yields milligram quantities of these proteins from 9-liter cul- tures. The purification procedure is rapid and can be carried out in less than 2 days. The use of batch chromatographic methods and high capacity resins should allow this protocol to be scaled up significantly.

Purified rG,, appears to be equivalent to the mammalian protein in almost all respects. After synthesis in E. coli, rGs, binds guanine nucleotides stoichiometrically. Rates of guanine nucleotide association, hydrolysis, and dissociation are normal. Characteristic changes in intrinsic tryptophan fluorescence are seen under activating conditions. rGs, interacts with @-y with high affinity; the protein is ADP-ribosylated by cholera toxin; the protein interacts functionally with @-adrenergic receptors in cyc- membranes. Studies in progress indicate that the interaction between rG., and @-adrenergic receptors reconstituted into phospholipid vesicles is extremely e f f i~ i en t .~ rG,, can also activate adenylyl cyclase, although it does so with a reduced specific activity when this value is assessed in common assay systems. However, the specific activity of the recombinant protein attains a value that is indistinguishable from that of G, purified from mammalian sources when it is assayed in solution in the presence of high concentrations of adenylyl cyclase.

We have attempted to determine if rGsu is a heterogeneous product or if the purified population of molecules simply has a uniformly poor affinity for adenylyl cyclase. Several argu- ments favor the latter hypothesis, including the observation of a single species of protein on one- and two-dimensional electrophoresis, amino-terminal amino acid sequencing, appropriate interactions with guanine nucleotides, and the @-y subunit complex, and a normal ability to activate adenylyl cyclase in the presence of high concentrations of the catalyst. The data of Fig. 7 fail to reveal heterogeneity of affinities for adenylyl cyclase. Hence, our working hypothesis is that rG,, lacks a post- (or co-) translational modification that is nec- essary for high affinity interaction with the enzyme. It is thus of interest that bovine brain Gi, and Go, have recently been found t o be myristoylated. However, no fatty acids were detected in bovine brain G., (39). On the other hand, ADP- ribosylated G,, displays isoelectric heterogeneity on two-dimensional gels, consistent with the presence of covalent modifications (40). Both forms of ADP-ribosylated G,, from S49 wild type cell membranes migrated as multiple spots with PI- values in the range of 5.5-6.0, whereas rG8,.a has an isoelectric point of about 6.5. Some of the difference is attributable to

R. Rubenstein; unpublished observation.

ADP-ribose. We are currently searching for covalent modifications of Gem.

During the purification of G. from rabbit liver, partial resolution of oligomers containing the 45- and the 52-kDa forms of the a subunit was achieved on a heptylamine-sepharose column. Studies of the kinetics of activation suggested that fractions enriched in the 52-kDa form displayed faster rates of activation than did fractions in which the 45-kDa form predominated (14). Likewise, experiments in native membranes (16, 17) suggest that the two forms may differ with respect to their kinetics of activation by GTP-yS. In this report, we demonstrate that significant differences do exist between the two forms of G., with respect to their kinetics of guanine nucleotide binding: the dissociation rate of GDP is 2-3-fold faster from rGsa.L (-0.4. min-') than from rGs,.8 (-0.15.min-'), while both forms have similar values of kat for the hydrolysis of GTP (-3.5 min-'). The fraction of G, that is GTP bound is defined by kOrf/kcat + k,fr (27). If the differences in kerf for GDP persist in the membrane and in the presence of (37, it is probable that more Gsa.~ than would be in the active GTP-bound form under basal, physio- logical conditions. This prediction is supported by the reconstituted adenylyl cyclase activities that were observed in both the cyc- reconstitution assay and the experiments with purified adenylyl cyclase. I t will also be of interest to examine receptor-stimulated rates of dissociation of GDP from the different forms of Gs,. I t is possible that the 52-kDa form of G,, may transduce information from receptor to effector more rapidly than does the 45-kDa form of the protein.

The possibility that G. serves to regulate the activity of effectors other than adenylyl cyclase is intriguing. Indirect evidence suggests that G, is in some way responsible for regulation of M$+ flux across the plasma membrane (41). Yatani et al. (42) have recently shown that G, purified from human erythrocytes can activate dihydropyridine-sensitive Ca2' channels in bovine cardiac sarcolemmal vesicles. Human erythrocytes contain primarily the 45-kDa form of G,, (43). The availability of individual species of G, will facilitate study of differential effects, if any, that these various polypeptides have in such systems.

Acknowledgments-We thank Drs. Patrick Casey and Tsutomu Higashijima for their helpful advice and discussions, Dr. Clive Slaugh- ter and Kim Orth for amino acid sequence determination, Dr. Richard Kahn for his gift of bovine brain ADP-ribosylation factor, Pamela Sternweis for her skillful technical assistance, and Wendy Deaner for her excellent editorial assistance.

REFERENCES 1. Gilman, A. G. (1987) Annu. Reu. Biochem. 56,615-649 2. Casey, P. J., and Gilman, A. G. (1988) J. Biol. Chem. 263,2577-

2580 3. Fong, H. K. W., Amatruda, T. T., Birren, B. W., and Simon, M.

I. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 3792-3796 4. Gao, B., Mumby, S., and Gilman, A. G. (1987) J. Biol. Chern.

262, 17254-17257 5. Hurley, J. B., Fong, H. K. W., Teplow, D. B., Dreyer, W. J., and

Simon, M. I. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 6948- 6952

6. Roof, D. J., Applebury, M. L., and Sternweis, P. C. (1985) J. Biol. Chem. 260,16242-16249

7. Evans, T., Fawzi, A., Fraser, E. D., Brown, M. L., and Northup, J. K. (1987) J. Biol. Chem. 262, 176-181

8. Logothetis, D. E., Kurachi, Y., Galper, J., Neer, E. J., and Cla- pham, D. E. (1987) Nature 325,321-326

9. Northup, J. K., Sternweis, P. C., Smigel, M. D., Schleifer, L. S., Ross, E. M., and Gilman, A. G. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 6516-6520

10. Robishaw, J. D., Smigel, M. D., and Gilman, A. G. (1986) J. Biol. Chem. 261,9587-9590

11. Bray, P., Carter, A., Simons, C., Guo, V., Puckett, C., Kamholtz,


J., Spiegel, A., and Nirenberg, M. (1986) Proc. Natl. Acad. Sci. Chem. 257 , 11416-11423 U, S. A. 83,8893-8897 27. Higashijima, T., Ferguson, K. M., Smigel, M. D., and Gilman, A.

12. Kozasa, T., Itoh, H., Tsukamoto, T., and Kaziro, Y. (1988) Proc. G. (1987) J. Biol. Chem. 262 , 757-761

13. Mumby, S. M., Kahn, R. A., Manning, D. R., and Gilman, A. G. Res. 10,135-168

14. Sternweis, P. C., Northup, J. K., Smigel, M. D., and Gilman, A. G. (1986) J. Biol. Chem. 261,7393-7399

15. Graziano, M. P., Casey, P. J., and Gilman, A. G. (1987) J. Bwl. 31. Schaffner, W., and Weissmann, C. (1973) Anal. Biochem. 56,

16. Kaslow, H. R., Farfel, Z., Johnson, G. L., and Bourne, H. R. 32. Bradford, M. M. (1976) Anal Bwchem. 72,248-254

17. Larner, A. E., and Ross, E. M. (1981) J. Biol. Chem. 256,9551- D., and Gilman, A. G. (1987) J. Biol. Chem. 262,762-766

18. Maniatis, T., Fritsch, E., and Sambrook, J. (1982) Molecular 6234

Natl. Acad. Sci. U. S. A. 85, 2081-2085 28. Johnson, R. H., and Walseth, T. F. (1979) Adu. Cyclic Nucleotide

(1986) Proc. Natl. Acad. Sci. U. S. A. 83, 265-269 29. Ferguson, K. M., Higashijima, T., Smigel, M. D., and Gilman, A.

G. (1981) J. Biol. Chem. 256, 11517-11526 30. Laemmli, U. K. (1970) Nature 227,680-685

Chem. 262,11375-11381 502-514

(1979) Mol. Phurmacol. 15,472-483 33. Higashijima, T., Ferguson, K. M., Sternweis, P. C., Smigel, M.

9557 34. Kahn, R. A., and Gilman, A. G. (1984) J. Biol. Chem. 259,6228-

Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, 35. Howlett, A. C., Sternweis, P. C., Macik, B. A., Van Arsdale, P. NY M., and Gilman, A. G. (1979) J. Biol. Chem. 254 , 2287-2295

19. Tabor, S., and Richardson, C. C. (1985) Proc. Natl. Acad. Sci. U. 36. Northup, J. K., Smigel, M. D., Sternweis, P. C., and Gilman, A.

20. Shine, J., and Dalgarno, L. (1974) Proc. Natl. Acad. Sci. U. S. A. 37. Casey, P. J., Graziano, M. P., and Gilman, A. G. (1988) Biochem-

21. Pfeuffer, E., Mollner, S., and Pfeuffer, T. (1985) EMBO J. 4, 38. Brandt, D. R., and Ross, E. M. (1986) J. Biol. Chem. 261,1656-

22. Schleifer, L. S., Kahn, R. A,, Hanski, E., Northup, J. K., Stern- 39. Buss, J. E., Mumby, S. M., Casey, P. J., Gilman, A. G., and weis, P. C., and Gilman, A. G. (1982) J. Biol. Chem. 257 , 20- Sefton, B. M. (1987) Proc. Natl. Acad. Sci. U. S. A. 8 4 , 7493- 23 7497

23. Higashijima, T., Ferguson, K. M., Sternweis, P. C., Ross, E. M., 40. Schleifer, L. S., Garrison, J. C., Sternweis, P. C., Northup, J. K., Smigel, M. D., and Gilman, A. G. (1987) J. Biol. Chem. 2 6 2 , and Gilman, A. G. (1980) J. Biol. Chem. 255,2641-2644

S. A. 82,1074-1078 G. (1983) J. Biol. Chem. 2 5 8 , 11369-11376

71,1342-1346 istry, in press

3675-3679 1664

752-756 41. Maguire, M. E., andErdos, J. J. (1980) J. Biol. Chem. 255,1030- 24. Sternweis, P. C., Northup, J. K., Hanski, E., Schleifer, L. S., 1035

Smigel, M. D., and Gilman, A. G. (1981) Ado. Cyclic Nucleotide 42. Yatani, A., Codina, J., Imoto, Y., Revees, J. P., Birnbaumer, L., Res. 14,23-36 and Brown, A. M. (1987) Science 238, 1288-1292

25. Sternweis, P. C., and Gilman, A. G. (1979) J. Biol. Chem. 254, 43. Codina, J., Hildebrandt, J. D., Sekura, R. D., Birnbaumer, M.,

26. Northup, J. K., Smigel, M. D., and Gilman, A. G. (1982) J. Biol. J. Biol. Chem. 259,5871-5866 3333-3340 Bryan, J., Manclark, R., Iyengar, R., and Birnbaumer, L. (1984)

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Expression of G, in Escherichia coli · Expression of G, in Escherichia coli PURIFICATION AND PROPERTIES OF TWO FORMS OF THE PROTEIN* (Received for publication, July 20, 1988) Michael