THEJOURNAL OF BIOLOGICAL CHEMISTRY Q 1985 by The American Soeiety of Biological Chemists, Inc.

Vol. 260. No.6,Issue of March 25, pp. 3477-3483, 1985 Printed in U.S.A.

Reconstitution of Resolved Muscarinic Cholinergic Receptors with Purified GTP-binding Proteins*

(Received for publication, September 6, 1984)

Vincent A. FlorioS and PaulC. Sternweis

From the Department of Pharmacology, University of Texas Health Science Center at Dallas, Dallas, Texas 75235

The association of agonists with muscarinic recep- protein that mediates the stimulation of adenylate cyclase by tors in membranes from bovine brainaffected only hormones (4). Membranes from the cyc- variant of S49 lymwas slightly by guanine nucleotides. However, solubiliza- phoma cells, which lack functional Gs, do not display 8tion of these membranes with deoxycholate and sub- adrenergic stimulation of adenylate cyclase (5), and the affinsequent removal of detergent resulted in a preparation @adrenergic receptors in these membranes for agonists ity of of receptors with increased affinity for agonists and a is not alteredby guanine nucleotides. The addition of a crude large increase in response guanine nucleotides. to Chromatography of deoxycholate extracts of mem- preparation of Gs restored the sensitivity of adenylate cyclase affinity branes on DEAE-Sephacel resulted in the separation to stimulatory hormones and caused an increase in the of 8-adrenergic receptors for agonists; this higher affinity was of receptors from 96%of the guanine nucleotide-binding activity. Guanine nucleotides hadno effect on the reversed by the addition of guanine nucleotides (6). Hsia et al. (7) have recently provided evidence that the binding of agonists to these resolved receptors. The effect of guanine nucleotides was restored after effects of guanine nucleotides on enkephalin receptors that the addition of either oftwo purified guanine nucleo- mediate inhibition of adenylate cyclase are due to th6 inhibitide-binding proteinsfrom bovine brain. One of these tory G-protein, GI (8). Treatment of NG108-15 cells with proteins, presumably brain is composed of subunits islet-activating protein, the toxin from Bordetella pertussis GI, with the same molecular weights (a, 41,000; B, 35,000; that catalyzes ADP-ribosylation ofGI and attenuates recepy, 11,000) andfunctions as theinhibitoryguanine tor-mediated inhibition of adenylate cyclase (9), caused a nucleotide-bindingproteinisolated from liver. The decrease in the affinity of enkephalin receptors for agonists other protein, termed is a novel guanine nucleotideGo, binding protein that possesses a similar subunit com- and abolished the effects of guanine nucleotides. Muscarinic receptors mediate inhibition of adenylate cyposition (a,39,000; 8, 36,000; y, 11,000) but whose function is not yet known. Addition of either protein clase in heart (lo), brain (111, and other tissues (12). The effects of guanine nucleotides on the binding of muscarinic to the resolved receptor preparation increased agonist affinity by at least 10-20-fold, and low concentrations agonists in these tissues are well documented (Ref. 13 and of guanine nucleotides specificallyreversed this effect. references therein). We have chosen bovine brain as asource Reconstitution of receptors with the resolved sub- of muscarinic receptors and G-proteins for use in elucidation units of Go demonstrates that thefl subunit alone had of the mechanism of this modulation of agonist binding affinno effect on agonist binding, but that this subunit does ity by guanine nucleotides. appear to enhance the effects observed with the a subWe have recently reported the purification of two major Gunit alone. proteins from membranes of bovine brain andhave presented evidence pointing to the similarity of these proteins to the well-characterized G-proteins, Gs, GI, and transducin (14). The effect of guanine nucleotides in decreasing the affinity One of these proteins was tentatively identified as GI and with which agonists bind to receptors was first demonstrated possesses a , 8, and y subunits with molecular weights of for hepatic glucagon receptors (1).A large body of subsequent 41,000, 36,000and 11,000, respectively; this structure is idenwork demonstrated similar effects with a wide variety of tical to that GIpurified from rabbit liver. The other protein, of receptors, particularly those linked to adenylate cyclase (e.g. labeled Go, has identical p and y subunits but an a subunit Refs. 2 and 3). of only 39,000 Da. While the functional and structural relaSeveral lines of evidence have suggested that this modula- tionship of these two G-proteins to each other and to other tion of agonist affinity is mediated by regulatory proteins (G- G-proteins is not clear, they exist in large quantities (about yet proteins) that bind guanine nucleotides. Gs is the regulatory 1-2% of membrane protein from brain) and can be easily purified in the detergent sodium cholate. Furthermore, the * This work was supported by Searle ScholarsAward 83-5-102and Goa subunit (39,000 Da) can be obtained in an unliganded S-435.The costs of publication March of Dimes Basil OConnor Grant of this article were defrayed in partby the payment of page charges. andstable form. Therefore, these proteinsare likely and This article must therefore be hereby marked advertisement in convenient candidates to use for study of the interaction of G-proteins with the muscarinic receptor. accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. $ Supported by National Institutes of Health Postdoctoral TrainWe report here the solubilization of the muscarinic receptor ing Grant HD07190. from membranes of bovinebrain, its separation from the bulk The abbreviations used are: G-protein, any GTP-binding protein of GTP-binding activity in these membranes, and the subsewithin the family of homologous proteins composed minimally of Gs, quent reconstitution of this receptor with purified GI and Go G ,Go, and transducin; Ga and GI, the identified regulatory compoI nents of adenylate cyclase that mediate stimulation and inhibition, from the same source. respectively; Go,a new GTP-binding protein from membranes of
bovine brain; GTPyS, guanosine 5-(3-O-thio)triphosphate; QNB, 3quinuclidinyl benilate; GppNHp, guanyl-5-yl imidodiphosphate; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.
MATERIALS ANDMETHODS

The following reagents were purchased from Sigma: acetylcholine, ATP, atropine sulfate, bovine serum albumin, carbamylcholine, cho-

3477

3478

Muscarinic Receptors: Interaction with GTP-binding

Proteins

lesterol, dithiothreitol, d-tubocurarine, egg phosphatidylcholine, GDP, GMP, GTP, Hepes, hexamethonium, ITP, methacholine, nicotine, oxotremorine, and physostigmine. Cholic acid was obtained from Sigma and was purified on DEAE-cellulose as described by Ross and Schatz (15). Lubrol 12A9 was a gift from IC1 and was deionized prior to use. Deoxycholic acid (Aldrich, gold seal 99+%) was used without further purification. It should be noted that extraction efficiency, receptor stability, and the behavior of receptors on DEAESephacel were all changed when deoxycholate from Sigma was used. We were unable to achieve satisfactory resolution of receptors from G-proteins using the Sigma product. DEAE-Sephacel and Sephadex G-50 were obtained from Pharmacia. [3H]QNBand [%]GTPyS were purchased from New England Nuclear. GF/F filters and BA85 filters were obtained from Whatman and Schleicher & Schuell, respectively.

Preparation of Bovine Brain Membranes crude membranes from bovine brain were prepared as described (14). Briefly, cerebra were dissected to remove brain stem and large portions of white matter. The remaining tissue was homogenized in a blender with 4 volumes'of 10 m Tris-C1, pH 7.5, 10% sucrose. M After filtration through four layers of cheesecloth, membranes were collected by centrifugation at 20,000 X g for 30 min. Pellets were resuspended in the same buffer with a Potter-Elvehjemhomogenizer and collected by centrifugation a t 20,000 X g for 60 min. This step was repeated and then pellets were resuspended to about 20 mg/ml in the same buffer and stored at -80 "C. This preparation was the starting material for purification of G-proteins and resolution of the muscarinic receptor. Purification of G-proteins and Subunits from Bovine Brain The major G-proteins, GI and GO,were purified as described (14). In summary, Go and GI werecopurified through DEAE-Sephacel and Ultrogel AcA and partially resolved during chromatography through heptylamine-Sepharose, the final step for purification. The two proteins couldberesolved further by chromatography of Go- or GIenriched pools through the same heptylamine-Sepharose column under identical conditions. In this fashion, fractions of pure Goa (39,000 Da) and fractions enriched in GI (a = 41,000 Da, p, and y) could be obtained. While no G~ol(41,000) could be detected in the purified Goa, purified preparations highly enriched in GI still contained about 5-10% Goa (14). The purified proteins werepooled, concentrated by pressure filtration on an Amicon PM-30 membrane, and stored at 0 or -80 'C. GOand GI werequantitated by their ability to bind GTPyS; thus, 1 mol of binding sites indicated 1 mol of the a subunit or 1 mol of the multimeric protein. Purified p subunit was obtained from brain (14) by procedures originally described by Northup and colleagues (16). The preparation contained two forms of /3 subunits (Mr = 36,000 and M , = 35,000) and a potential y subunit (Mr = 11,000). The molar concentration of purified @ was calculated with an assumed M,of 35,000; the presence of a y subunit suggests this overestimates the actual concentration. Solubilization of Receptors
Brain membranes (70 ml, 1.4 g of protein) were rapidly thawed, mixed with 95 ml of Tris-C1, pH 8, 1.5 m NaEDTA, and 0.25 m M M atropine, and incubated at 30 "C for 15 min. The membranes were collected by centrifugation a t 90,000 X g for 30 min at 4 "C and resuspended with a Dounce homogenizer in 47 m Tris-C1, pH 8 , l . l M m NaEDTA, and 0.23 m atropine to a volume of 88 ml. Sodium M M deoxycholate (20.3 ml of 4% (w/v)), pH 8, was slowlyadded to a final concentration of 0.75%, and themixture was incubated at 0 "C for 30 min with occasional shaking. Particulate material was then removed by centrifugation a t 160,000 X g for 2 h. Atropine was included in these and subsequent steps to stabilize receptors from marked losses of binding activity during exposure to deoxycholate. Oxotremorine can also prevent these losses, and one of these ligands was always added to preparations of receptors during treatments with detergents.

allowed a rapid flow rate without loss of resolution.) The gel wasthen washed with 100 ml of the same solution; receptors were subsequently eluted by raising the NaCl concentration to 0.4 M. Fractions of 6 or 3 ml (during the wash and elution, respectively) were collected into siliclad tubes. Fractions near the receptor peak were collected into 0.5 ml of 30 mg/ml eggphosphatidylcholine and 3mg/ml cholesterol that had been sonicated in the wash solution; this further improved recovery of receptors. The peak of QNB-binding activity (about 18 ml)waspooled,mixed with 18 g of XAD-2 beads (Mallinckrodt Chemical Works), and incubated at 0 "C for 1 h with shaking. This procedure resulted in the removal of 95% of deoxycholate with a concomitant loss of 25% of the QNB binding. The treated pool was removed from the beads and subjected to pressure filtration on an Amicon PM-30 membrane. After a 2-3-fold concentration, aliquots were frozen at -80 'C. QNB-binding activity of this preparation was unchanged after repeated freezing and thawing. For smaller preparations, deoxycholate could be removed by gel filtration as described in the legend to Fig. 3.

Reconstitution of Receptors with G-proteins

The procedures described below utilized resolved receptors from which detergent had been removed by either gel filtration or XAD-2 bead treatment. Receptors that had been prepared by the latter M M procedure were gel-filtered into 20 m NaHepes, pH 8 , l m EDTA, 160 m NaCl before reconstitution with G-proteins; this step reM moved atropine and residual deoxycholate. Three methods were developed for reconstitution of the resolved receptor. The resultant preparations could be assayed directly for binding of QNB. Method 1: Gel Filtration (Used in Fig. 3)"Resolved receptors (1-2 pmol and 2-8 mg of protein in 0.5 ml) were mixed with 0.5-1.0 nmol of G-protein (either 0.5 ml of crude G-protein from early fractions of the DEAE column or 0.02-0.15 ml of purified G-protein in 20 m M M M Tris-CI, pH 8, 1 m NaEDTA, 1 m dithiothreitol, 1% sodium cholate, 100 m NaCI; this solution was also added to control tubes). M M Deoxycholate (0.5% final) and oxotremorine (1m final) were added, and themixture was allowedto standon ice for 1h. The mixture was M then diluted with 4 ml of 20 m NaHepes, pH 8, 1 m NaEDTA, 1 M m dithiothreitol, 160 m NaCl and applied to a 50-ml G-50 column M M which was developed in the same solution. Leading turbid fractions were pooled (turbidity corresponded well to QNB-binding capacity). Unfortunately, the recovery of receptor binding activity through this procedure was variable. Therefore, alternative procedures for reconstitution were also developed. Method 2: Sedimentation through Sucrose Gradients (Used in Fig. 4)"Receptors and G-proteins were mixed at 0 "C in a volume of 0.31.2 ml. Atropine (10 p~ final) and either deoxycholate (0.7% final) or cholate (0.4% final) were added. After 1h at 0 "C, the mixture was applied to the top of a step gradient of sucrose (30 ml of 5% (w/v), 5 ml of 40% (w/v)) in 20 m NaHepes, pH 8 , l m NaEDTA, 160 m M M M NaCI. The gradients were centrifuged for 3 hat 27,000 rpm in an SW 27 rotor. Receptors were harvested from the 5-40% interface and were resuspended with a syringe and needle and diluted in the solution above without sucrose. Recovery of receptors from this procedure was 50-80%. Method 3 Dilution (Used in Table II and Figs. 5 and 6)"G-proteins : were mixed with resolved receptors at 0 "C in a total volume of 0.51.0 ml; MgCI, was added to a concentration of 20 mM. Concentrations of cholate in the initial mixtures did not exceed 0.3%. After 1 h at 0 "C, the mixture was diluted slowly (over 5 min) with 5-10 ml (10fold dilution) of 20 m Hepes, pH 8, 1m NaEDTA, 160 m NaC1. M M M The omission of deoxycholate and muscarinic ligands from this procedure eliminates the need for a separation step and does not appear to reduce the efficiency of reconstitution. Recovery of receptors was approximately 100%.

Incorporation of Small Aliquots of Receptor into Phospholipid for Assay

Since binding of QNB could not be detected directly in samples containing deoxycholate (i.e. extracts and DEAE fractions), this method was devised to prepare small aliquots for assay by removal of detergent and concomitant incorporation of protein into phospholipid. Aliquots (50 or 100 p l ) of samples were mixed with 500 p1 of a lipid mixture containing egg phosphatidylcholine (3 mg/ml), cholesterol (0.3 mg/ml), and sodium cholate (3 mg/ml) that had been sonicated under nitrogen in Solution A (25 m NaHepes, pH 8, 2 M M M m MgC12, 1 m NaEDTA, and 100 m NaC1). The mixtures were M

Separation of Solubilized Muscarinic Receptors from G-proteins Extracts were supplemented with 20% glycerol, 80 m NaCl, and M 0.2% sodium cholate (final concentrations).Theseadditions were found to improve recovery of receptors through the following procedure. The mixture was applied to a column (3.2 X 5 cm) of DEAESephacel that had been equilibrated with 37.5 m Tris-C1, pH 8, 1 M m NaEDTA, 0.2 m atropine, 80 m NaCI, 0.65% sodium deoxyM M M cholate, 0.2% sodium cholate, and 20% glycerol. short, wide column (A

Muscarinic Receptors: Znteraction

applied to 5-ml columns of Sephadex G-50 (medium) that had been equilibrated with Solution A. The columns were washed with 1 1 ml . of Solution A, and the receptors were eluted with another 1.0 ml of the same solution. Eluted receptors could be assayed for QNB binding by vacuum filtration as described below. Recovery of receptors after extraction and detergent removal was 70-80% relative to original membranes, suggesting that thisprocedure does not result in significant loss of receptors. This treatment also provided for efficient removal of unbound ligands present in receptor preparations. Any carryover of ligand bound to receptors would not be sufficient to compete significantly with QNB during the binding assay.
Assay of QNB Binding

with GTP-binding Proteins

3479

Binding of QNB to membranes and reconstituted mixtures was measured by vacuum filtration, essentially as described by Yamamura and Snyder (17). Samples were incubated at 30 'C in 1 ml of 25 m M potassium phosphate, pH 7.5, 08 M NaEDTA, 10 mMMgC12, 230 .m m NaCl, 0 6 mg/ml bovine serum albumin, 4 m NaHepes, pH M . M 8, and the indicated concentrations of nucleotides and cholinergic ligands. ['HIQNB was present at a concentration of 0.19 nM for competition experiments and 3 nM for measurement of total receptor binding sites. After 1 h, samples were diluted with 3 ml of ice-cold 25 m potassium phosphate, pH 7.5, 5 m MgC12, 1 m EDTA, and M M M filtered through Whatman GF/F filters, followed by three washes with 5 ml of the same buffer. The filters were dried and counted in toluene containing 0.5% 2,5-diphenyloxazole with an efficiency of 55%.The nonspecific binding for 13H]QNBwas less than 5% of total binding for all preparations of receptors utilized. Assay of GTPyS Binding Binding of [%]GTPyS was assayed as described by Northup etal. (18).Samples to be assayed were diluted in 5 or more volumes of 20 m NaHepes, pH 8, 1 m NaEDTA, 1 m dithiothreitol, and 0.1% M M M Lubrol 12A9. The diluted samples (50pl) were added to 50 pl of 50 m NaHepes, pH 8, 100 m MgCL, 1 m EDTA, 200 m NaCl, 4 M M M M p~ GTPrS and["SIGTPyS (1-2 X lo6 cpm) in siliclad tubes. After 1 h at 30 'C, samples were diluted and filtered as described (18). Filters were dried, dissolved, and counted in Liquiscint (National Diagnostics).
Other Assays Protein concentration was determined by staining with Amido Black as described by Schaffner and Weissman (19)using bovine serum albumin as a standard. Phosphate concentration was determined by the method of Ames (20)after digestion of samples with perchloric acid.
RESULTS

competition with QNB was atropine > oxotremorine >> nicotine. Table I also shows that GTP could decrease the affinity of these binding sites for agonists, butnot antagonists. The effect of GTP in this preparation (!&"-fold increase in the IC, of agonists) was smaller than effects observed with muscarinic receptors from rat brain (21) and myocardium (22). Fig. ZA demonstrates this small effect of GTP on membrane a preparation from bovine brain. The low apparent affinity of these receptors for agonists and the small effects of GTP suggest that the majority of these receptors are notsubject to regulation by G-proteins. However, subsequent data demonstrate that regulation of these receptors by GTP is possible. If receptors were solubilized with deoxycholate and the detergent was then removed by gel filtration, the apparent affinity of receptors for agonists was increased markedly (Fig. 1B).Thus, the IC, for oxotremorine in the preparation that has been treated with detergent was 0.03 PM,while the K O for [3H]QNB was unchanged (not shown). The addition of 0.1 m GTP resulted in the reduction of the apparent affinity of M oxotremorine to anIC,, of 10 ~ C I M with no effect on the K D for [3H]QNB.Without the addition of detergent, no treatment of the membranes has been able to induce this result. Therefore, the process of solubilization and removal of detergent appears to promote the interaction of muscarinic receptors with a component sensitive to guanine nucleotide. Moreover, the

Effects of Guanine Nucleotide i Membranes and Reconstin tuted Extracts-Membranes from bovine brain bound [3H] QNB with high affinity. The KO for [3H]QNB was 250 p~ and was unaffected by the presence of 0.1 m GTP. Binding M in the presence of 50 WM atropine (nonspecific binding) was less than 5% of total binding. The muscarinic specificity of these sites is shown in Table I; thus, the order of potency for
TABLE I Effects of GTP on the binding of cholinergic ligands to muscarinic receptors in membranes from bovine brain Membranes from bovine brain were prepared and assayed as described (see "Materials and Methods"). Assays contained 80 pgof membrane protein with 79 fmol of binding sites for QNB, 0.19 p M ['HIQNB, and 0 1 m GTP where indicated. . M
Ligand
ICm

Log [ ~xotremorine]
FIG. 1 Effect of GTP on agonist binding to membranes and . reconstituted receptors. Brain membranes (60mg of protein) were incubated for 20 min at 30 "C in 6 ml of 20 m NaHepes, pH 7.5,5 M M M m MgC12, 1 m EDTA, 1 m dithiothreitol, and 100 p M oxotreM morine. After collection by centrifugation, 30 mg of membranes were washed twice at 4 "C in the absence of MgC12 and oxotremorine and suspended for assay ( A ) .The other 30 mg of membrane protein were suspended at 0 "C in 2 6 ml of the solution containing oxotremorine . but without MgC12. Deoxycholate (0.4 ml of5% (w/v)) was added, and extraction wasallowed to proceed for 20 min on ice.After centrifugation, 2 ml of the extract were mixedwith 200 pl of sonicated egg phosphatidylcholine (15 mg/ml) and applied to a 50-ml column of Sephadex G-50 for removal of detergent and oxotremorine. After M M M elution with 20 m NaHepes, pH 7.5, 1 m EDTA, and 160 m NaCl, cloudy fractions containing vesicles were pooled and assayed directly for binding of QNB by vacuum filtration ( B ) . The yield of binding sites for QNB in the pool of vesicles was 50% of the sites measured in the equivalent amount of original membrane. Assays contained 0.18 nM [3H]QNB.GTP (0)was included at 100 p ~ .

+GTP

-GTP
PM

Oxotremorine Carbachol Atropine Hexamethonium 1100 Nicotine 150 d-Tubocurarine

2.8 650 0.0055 0.0055 4500 1100 150

70 . 1900

5500

3480

Muscarinic Receptors: Interaction with GTP-binding Proteins

data suggest that a large proportion of the treated receptors tical in the two preparations (compare Tables I and 11), and were capable of this interaction. the ICs0 values were similar. It is of interest that the membranes used in Fig. 1 were The apparent affinity resolved receptors for the of agonist, incubated with oxotremorine prior to and during extraction. oxotremorine, was low (ICs0 10 p M ) and was not affected If the antagonist, atropine,was substituted for oxotremorine by guanine nucleotides(Fig. 3). If, however, some of the during the treatment, therecovery of binding sites for QNB GTPyS-binding activityfrom the DEAE fractions was added was increased slightly, but the expression of high affinity for oxotremorine was much less (ICs0 of 1 and 10 p~ in the TABLE I1 absence and presence of GTP, respectively). The difference Effects of purified G-protein and GTP on the binding of cholinergic between oxotremorine and atropine always observedin this is ligands to resolvedmuscarinic receptors from bovine brain crude experiment; however, the difference is not observed Resolved receptors were prepared by DEAE chromatography and when either ligand is utilized during reconstitutionof resolved treatment with XAD-2 beads, reconstituted with G-protein by Method 3 (dilution), and assayed for binding of QNB as described receptors. (see Materials and Methods). G-protein was added at a 500-1000Resolution of Receptors from G-proteins and Reconstitution fold molar excess over receptors. Assays contained about 40 fmol of of Guanine Nucleotide Effects-Fig. 2 demonstrates the sepa- binding sites, 0.19 nM [3H]QNB, and 0.1 m GTP when indicated. M ration of solubilized muscarinic receptors from G-proteins. The data were compiled from several experiments. Each ligand was Application of a deoxycholate extract of brain membranes to examined at least twice, except for hexamethonium and d-tubocurarDEAE-Sephacel resulted in retentionof the binding sites for ine; a ligand that showed a shift in affinity was included in each QNBandthemajority of the phospholipid; most of the experiment. The qualitative results were always the same, although GTPyS-binding activity androughly half of the protein was the degree of reconstitution was variable. The purified G-protein utilized was enriched in Gr as described under Materials and Methwashed through. Receptors and phospholipid were then eluted ods. by inclusion of 0.4 M NaCl in thewash solution. The yield of IC, receptors in these fractions was 35% of the original extract. Ligand G-protein G-protein No Plus Further washes with higher concentrations of NaCl released small amounts of protein and phospholipid but very little +GTP +GTP -GTP -GTP QNB-binding activity. The peakof eluted receptorsalso conPM tained about 5% of the original GTPyS-binding activity but Oxotremorine 10 15 0.25 20 essentially no Gs activity (not shown). Since and Go elute Carbachol GI 1000 1000 30 800 from DEAE slightly prior to Gs (14), most of the activity in Methacholine 450 450 120 700 30 30 4.5 45 the receptor peak is probably due to other proteins that bind Acetylcholine 0.010 0.010 0.010 0.010 Atropine guanine nucleotides. Hexamethonium 6500 6500 5500 5500 The peak of QNB-binding activitywas pooled, treated with Nicotine 1900 1900 1400 1800 XAD-2 beads, concentrated, and stored at -80 C (see Ma- d-Tubocurarine 60 60 120 60 terials and Methods). The overall yield of receptors from this procedure was 15-20% relative tothe original membranes; no significant purificationwas achieved. This preparation of resolved receptors was used in the reconstitutions with G-proteins from bovine brain described below. The KOof 0.2 nM for bindingof [3H]QNB to theseresolved receptors was similar tovalues obtained with untreated membranes. This preparation also displayed the same muscarinic specificity as the original membranes (Table 11);the order of - 2.0 potency forcholinergic agents todisplace [3H]QNB was iden1.0

: 0

o
12.0
10.0

4.2
1.0

z
0

8 .O
4 .O 6o

0.6

0.4 -

1:
,
-9

.T G P ; -

2.0

RECONSTITUTED 0 RECEPTOR

\.

,/~028
s
-4

1
-3

-8 -7 -6 -5 LOP [~xotremorine]

I2

I6 20 24 28 Fraction Number

32

36

40

FIG. 2. Resolution of muscarinic receptors from GTPySbinding activity by DEAE-Sephacel chromatography. Procedures and assays are described under Materials and Methods. Fraction volumes of either 6 or 3 ml were collected in fractions 1-17 and 18-38, respectively. Application of 0.4 M NaCl was begun during fraction 18. Fractions 21-38were supplemented with lipid as described; determinations of inorganic phosphate were corrected for these additions. Individual fractions of the initial flow-through were not collected; collection of fraction 1 coincides with the application of wash solution.

FIG. 3. Reconstitution of resolved muscarinic receptors with resolved G-protein from DEAE chromatography. Receptors were resolved from G-protein by DEAE chromatography. Fractions containing receptor (about 5 ml) were applied to a 25-ml column of Sephadex G-50 and eluted with 20 m NaHepes, pH 8, 1 m M M EDTA, 160 m NaCl, and 0.1 m oxotremorine. Turbid fractions M M were pooledand utilized for reconstitution. Lower, the resolved receptors (0.5 ml) were mixed with 0.5 ml of a crude fraction of G-protein from the DEAE flow-through and reconstituted by Method 1 (gel filtration) as described under Materials and Methods; upper, the resolved receptors were subjected tothe same treatmentsas for reconstitution with the exception of the addition of G-protein. Assays contained 0.19 nM [3H]QNB and100 p M GTP (0).

Muscarinic Receptors: Interaction with GTP-binding Proteins back to the receptors, the apparent affinity for oxotremorine was increased by about 100-fold in the absence of GTP (IC60 of 0.1 p ~ ) Addition of GTP resulted in the complete reversal . . of this effect (IC6,, of 10 p ~ ) Thus, chromatography on DEAE separated receptors from a component(s) necessary for the expression of high affinity binding of agonists. This component(s)co-fractionated with GTPyS-binding activity and could be used to restore the high affinity for agonists to resolved receptors. Reconstitution with Purified G-proteins-Two proteins with GTPyS-binding activity have been purified from bovine brain (14); these proteins have been designated GI and GO. The addition of either of theseproteins to resolved receptors resulted in an increase in the apparent affinity of receptors for agonists. Guanine nucleotides reversed this effect completely. Fig. 4 documents the displacement of t3H]QNB from isolated receptors by oxotremorine in the absence or presence of a GI-enriched preparation of purified G-protein from brain. In the presence of the GI, significant displacement was observed at low concentrations of oxotremorine (0.1 WM) and the IC, was decreased 15-fold. The IC, in the presence of GI plus GTP was the same as in theabsence of GI (10 p ~ ) . Fig. 5 shows the effect of the a subunit of Go on the affinity of receptors for oxotremorine. All preparations were supplemented with equal concentrations of the purified /3 subunit; this subunit, by itself, did not affect agonist binding (see below;Fig. 6B). In the absence of added a subunit, the displacement of QNB was similar in the absence or presence of GTP. The addition of a subunit in a 100-fold molar excess over receptors resulted in an increase in apparent agonist affinity, with significant displacement at 0.1 p M oxotremorine and a decrease of 4-fold in the ICw. A 10-fold higher concentration of 1y subunit resulted in a slightly greater increase in
25

3481

-7 -6 -5 Log [Oxotremorine]

-4

I -3

FIG. 5. Reconstitution of resolved receptors with purified GO.Receptors were prepared by DEAE chromatography and treatment with XAD-2 beads. The receptors (1.5 pmol) were mixed with 1 nmol of purified /3 subunit and the indicated amount of purified Goa subunit (39,000Da) in a total volume of 0.5 ml. Reconstitution was carried out by Method 3 (dilution) as described under "Materials and Methods." Binding assays contained 0.19 nM [3H]QNB and 100
pM

GTP (0).

15

t
ISOLATED RECEPTOR
0 -GTP 0 +GTP

lot

%, .

lot
5

RECONSTITUTED RECEPTOR

L 4 ,

FIG. 4. Reconstitution of resolved receptors with purified G-protein from brain. Receptors were prepared by DEAE chromatography and treatment with XAD-2 beads as described under "Materials and Methods." Resolved receptors (1.8 pmol, 5.5 mg of protein) in 0.95 ml were mixed with the purified preparation of G protein enriched inGI (1.6 nmol in 0.12 ml) or 0.12 ml of I%cholate as a detergent control. Atropine, MgC12, and cholate were added to final concentrations of 10 FM,20 mM, and 0.476, respectively. Incorporation of proteins into phospholipid was carried out by sedimentation through sucrose (Method 2, reconstitution, see "Materials and Methods"). Binding assays contained 0.19 nM ['HIQNB and 100 PM GTP (0).

agonist affinity with a 'I-fold decrease in IC5,,. In each case, GTP reversed the effect. The addition of a 100-fold 100 ~ L M excess of Go (i.e. 100-foldexcess of binding sites for GTPyS) is about the same as the number of binding sites for GTPyS remaining in the preparation of receptor. The proteins responsible for this residual binding activity either do not interact with the muscarinic receptor or are much less potent than Go for effecting the expression of high affinity for agonists. The increase in apparent binding affinity induced byGI and Go was specific for agonists of the muscarinic receptor (Table 11). GI and Go did not change the affinities observed for muscarinic (atropine) and nicotinic (&tubocurarine) antagonists or for nicotinic agonists (nicotine). The differences observed in the extent of shifts observed for different muscarinic agonists are due at least in part to variability in reconstitution obtained in separate experiments; no attempt has been made to quantitate effects of several agonists within the same experiment. The increased affinity for muscarinic agonists induced by Go and GI was specifically abolished by guanine nucleotides. The order of potency among various nucleotides for this effect was the same as reported for effects on muscarinic agonist binding to intact membranes (23). Thus, GTPyS (K,,, = IO-' M) was the most potent of all the nucleotides tested, followed by GppNHp MI, GTP M), GDP (3 x M), and ITP (10"j M ) . Neither GMP nor ATP had any effect at M (data not shown). Reconstitution with Resolved Components of Go-The purification procedure for the G-proteins from bovine brain yields the free homogeneousa subunit of Go. This a subunit (39,000Da polypeptide) binds guanine nucleotides, is stable at 0 "C in cholate, and interacts with the /3 subunit, which decreases its affinity for GTPyS (14). The effect of the resolved LY and fi subunits on agonist binding to isolated muscarinic receptors is shown in Fig. 6. Fig. 6B shows that the/3 subunit alone did notreconstitute guanine nucleotide modulation of agonist binding; no change in apparent agonist affinity has been found with addition of up to a 3000-fold molar excess of /3 subunit over receptors. Addition of the a39 subunit alone did yield an increase in agonist affinity (Fig. 6C); a 3-fold decrease in the IC6@ oxotremorine was obtained and was reversedby GTP. of

3482

Muscarinic Receptors: Interaction with Proteins GTP-binding

but still bind muscarinic ligands, to experimental constraints such as vesicle composition that do not allow all of the receptors to exist in a high affinity state, or to the presence of two independent populations of muscarinic receptor. In the latter case, only one class of receptors would be capable of regulation by G-proteins. While we have attempted to utilize [3H]pirenzepineto examine potential subclasses of muscarinic receptor, high nonspecific binding in reconstituted vesicles has not allowed reliable evaluation of this possibility. Furthermore, it is not known whether GI and Go interact with the same or different populations of muscarinic receptors. Experiments in which GI and GOwere added together or separately to receptors revealed no conclusive differences in the extent of Feconstitution achieved (data not shown). The observation that GI and Go can achieve similar extents of reconstitution within the same experiment suggests that the two proteins may interact with the same population of receptor. Log [Oxotremorine ] The availability of the purified G-proteins enabled us to evaluate their role in more detail. The action of the G-proteins FIG. 6. Reconstitution of resolved receptors with purified subunits. Receptors were prepared by DEAE chromatography and on receptors was reversed by guanine nucleotides with the treatment with XAD-2 beads. The receptors (1.6 pmol in 475 pl) were same order of potency that was reported for effects on agonist mixed with 90 pl of 1% cholate ( A ) ,70 pl of B subunit (2 nmol) and binding in membranes (23). Measurement of nucleotide bind20 pl 1% cholate (B),20 pl of Goa subunit (0.34 nmol) and 70 p1 1% cholate (C), and 70 p1 of subunit (2 nmol) and 20 pl of Goa subunit ing to the a subunit of GO also yielded the same order of a (0.34 nmol) ( D ) . Reconstitution was accomplished by Method 3 specificity and potency (14). This indicates that the subunits (dilution) as described under "Materials and Methods." Binding of GI and Go are thesites of action of guanine nucleotides on the regulation of affinity of the muscarinic receptors for assays contained 0.19 nM [3H]QNBand 100 p~ GTP (0). agonists. The ability to prepare the isolated j3 subunit of G-proteins This effect is smaller than those shown in Figs. 4 and 5, where both a and @ subunits were added. If the same amounts of (16) as well as the isolated a subunit of Go (14) has allowed purified a and #3 subunits were added together (Fig. 6D), the evaluation of the effects of these individual subunits on the decrease in IC60 (9-fold) of oxotremorine was significantly binding of agonists. The j3 subunit alone does not appear to greater than with the addition of the a subunit alone. In four modulate agonist binding (Fig. 6B). The a39subunit does such experiments, the decrease in IC, for oxotremorine was induce an increase in affinity for agonists (Fig. 6C), and the 2.8 k 0.5-fold in the absence of added j3 subunit and 7.1 f simultaneous addition of a and j3 produces a larger effect (Fig. 0.5-fold when the j3 subunit was included. Thus, the j3 subunit 6D). The effect of j3 subunit in enhancing the effect of a was probably not due to stabilization of (Y against denaturation, appears to enhance the effects of the a subunit. since the recoveryof GTPyS-binding activity from these reconstitutions was not alteredappreciably by the addition of DISCUSSION @ subunit (not shown). The significance of the reconstitution We have presented evidence for the functional interaction by the a! subunit alone is not totally clear. It is likely that the of two major guanine nucleotide-binding proteins from bovine brain with muscarinic receptors from the same source. The resolved receptor preparation contained significant amounts of the j3 subunit? Therefore, it is possible that both LY and j3 criteria we have used to measure this functional interaction subunits are required for modulation of agonist binding and were the ability of the G-proteins to increase the affinity of that the effect of (Y alone was due to cooperative action with muscarinic receptors for agonists and the specific reversal of endogenous j. The assays for the j subunit in these crude 3 3 this effect by low concentrations of guanine nucleotides. extracts have not proven sensitive enough to eliminate this Chromatography of extracts from brain membranes on possibility. While the j3 subunit certainly improves the interDEAE-Sephacel in deoxycholate and cholate resulted in the action of Goa with receptors, the determination of whether @ separation of receptors from 95% of the high affinity binding is a total requirement awaits further purification of the recepsites for guanine nucleotides. These resolved receptors bound tors. agonists with a low affinity that was not affected by guanine We have not observed any major differences between GI nucleotides (Figs. 3 upper, and 4, upper). The addition of and Go in this reconstitutive assay. A large excess of either either GI or Go resulted in an increase in theaffinity of these protein was required before any clear change in agonist affinreceptors for agonists (Figs. 4, lower, and 5) with no change ity was observed. The requirement for large amounts of Gin the affinity for 13H]QNBor other antagonists (Table 11). protein existed regardless of the method employed for reconThe increase in affinities for agonists could be reversed with stitution. This requirement did not seem to reflect inactivaguanine nucleotides. tion of the G-proteins during the procedures for reconstitution While it is clear that a portion of the muscarinic receptors since the recovery of GTPyS-binding activity is 50-100%. in these preparations hasbeen reconstituted with G-proteins, The maximal effects achieved with GO-or GI-enriched prepit is not clear that all of the receptors were responding. The arations (still containing about 5-10% Goa) are similar and shallow competition curves obtained with agonists in the occurred at approximately a 1000-fold excess of G-protein absence of GTP suggest the presence of at least two popula- over receptors. Therefore, it appears unlikely that the effect tions of receptor, even when apparent saturating concentrations of G-protein were present. It is not known whether the 2The behavior of G-proteins on DEAE during purification in sites with low affinity for agonists after reconstitution are due cholate results in variable quantities of the B subunits eluting with to defective receptors that no longer interact with G-proteins higher salt than required for elution of the GTPyS-binding activity.

Fduscarinic Receptors:

Interaction with GTP-binding Proteins

3403

of either G-protein was attributable to contamination by the other. An alternative explanation would be that the activity was due to the presence of a minor contaminant in both purified Go and GI. This, however, seems unlikely as an active contaminant would have to be present to the same extent in both preparations of G-proteins andbe a guanine nucleotidebinding protein with the same specificity as GO. The mechanism of reconstitution is not clear. Initial experiments with crude extracts suggested that anactivated receptor (ie. in the presence of agonist, oxotremorine) enhanced the interaction of G-proteins with the receptor during detergent treatments. This property was not observed after resolution of the receptors from the G-proteins; therefore, either oxotremorine or atropine could be used to stabilize the receptor with equivalent results. The difference in these experiments may indicate that there is another factor (lost during resolution of receptors) involved in efficient interaction of the G-proteins with receptor. Alternatively, the receptor has been altered duringresolution such that theefficiency of its reconstitution is no longer affected by the two different ligands. There areseveral reasons to expect a large requirement for G-protein during thesereconstitutions. The first is the physical state of ourpreparation. Although no morphological characterization of our reconstituted mixtures has been performed, the ratio of lipid to protein (2-32 by weight) is favorable for the formation of vesicles upon removal of detergent by dilution or gel filtration. If such vesicles were 5001000A in diameter, a typical size reported after the slow removal of ionic detergents (24), there would be an average of only one receptor for every 20-80 vesicles. If G-proteins partition equally among vesicles, a 20-80-fold excess of Gproteins over receptors would be necessary to approach conditions where each receptor had access to at least one molecule of G-protein. It is also possible that each receptor requires more than one molecule of G-protein for the expression of a high affinity binding site for agonists; this could be due to a stoichiometry of G-protein to receptor of greater than 1 or to kinetic constants thatrequire high concentrations of G-protein to effect a high proportion of complex formation. Evaluation of these possibilities will be contingent upon purification of the muscarinic receptor and reconstitution with highly concentrated receptor preparations. A final explanation for the high requirement for G-protein is the loss of reconstitutive activity during purification. In this case, both the Go and GI preparations would have to be equivalently susceptible to this loss of activity. While we have attempted to explain why the reconstitution may require large amounts of G-protein, we note that this might be predicted, as the native membranes from brain also contain 300-600 times as much G-protein as muscarinic receptor. The reconstitution of high affinity agonist binding by the addition of G-proteins to muscarinic receptors demonstrates the existence of an interaction between these two proteins. Why is this interaction not readily observed in membranes from bovine brain andwhy does solubilization and subsequent removal of detergent result in greatly increased interaction? a Although the 2-3-fold change in the ICeofor agonists that we observe in membranes is smaller than effects observed by others in the same tissue from other species, we have not been able to increase this effect of guanine nucleotides by treatment with agonists for the receptor or with GMP. A large number of possible explanations for the poor interaction of receptors and G-proteins in membranes exist. The procedure for membrane preparation may resuIt in the physical separation of G-proteins from receptors; therefore, a

receptor in a membrane particle may have access to fewer molecules of G-protein than itdoes in an intact cell. Solubilization of these receptors in the presence of agonist (Fig. 1) may then favor association with soluble G-proteins. It is also possible that the disruption of membrane structure by solubilization improves the interaction between receptors and Gproteins. It is possible that receptors and G-proteinsin brain are, for the most part, present in different cell types and, therefore, interact only after solubilization. However, the high quantity of GIand Go in brain makes this seem unlikely. Gs and GI also appear to be rather ubiquitous in that they are present in all cell types tested so far. An intracellular separation of receptors and G-proteins, however, remains a possibility with potentially interesting possibiIities for regulation. The evaluation of these possibilities awaits the cellular and subcellular localization of the proteins. The results presented here demonstrate that a substantial proportion of the muscarinic receptors in bovine brain can interact with regulatory G-proteins, in specific, two purified proteins, GI and Go, that exist in high quantity in this tissue (14). This suggests that atleast part of the effects of muscarinic agonists on cellular function are directed through these G-proteins. The procedures developed for isolation and reconstitution of the muscarinic receptor will allow more detailed analysis of its specificity and mechanism of interaction with various purified G-proteins.
Acknowledgments-Superb technical assistance during experimentation wasprovidedbyHowardCummings,Marcia Timko, and Barbara Creighton. We thank Dr. Murray Smigel forvery useful discussion of the manuscript and Nancy Bryant for skillful technical assistance in its preparation.
REFERENCES 1. Rodbell, M., Krans, H. M. J., Pohl, S. L., and Birnbaumer, L. (1971) J. Blol. Chem. 246,1872-1876 2. Ross, E M., Maguire, M. E., Sturgill, T. W., Biltonen, R. L.,and Gilman, . A. G. (1977) J.Bioi. Chem. 262,5761-5775 3. Tsai, B., and Lefiowitz, R. J. (1979) Mol. P h u r m o l . 1 6 , 6 1 4 8 4. Sternweis, P. C.. NorthuD, J. K., Smiael. M.D.. and Gilman. A. G. (1981) . . J. BioL Chem.~266,li517-11526 5. Rosa, E.M., Howlett, A. C ,Ferguaon, K. M., and Gilman, A. G. (1978) J. . Bwl. Chem. 263,6401-6412 6. Sternweis, P. C., and Gilman, A. G. (1979) J. Bid. Chem. 264,3333-3340 7. Hsia, J. A., Moss, J., Hewlett, E. L., and Vaughan, M.(1984) BioL Chem. J. 269,1086-1090 8. Bokoch, G. M.,Katada, T., Northup, J. K., Ui, M.,and Gilman, A. G. . (1984) J Biol. Chem. 269,3560-3567 9. Katada, T., Ui, M. (1982) J. Biol. Chem. 267,7210-7216 and 10. Watanabe, A. M., McConnaughey, M. M., Strawbridge, R. A,, Fleming, J. W., Jones, L. R., and Besch, H. R., Jr. (1978) J. Bid. Chem. 263,48334836 11. Onafi, P.. Olianas. M. C.. Neff. N. H.. and Costa.E. (1983) Mol. P h u r m o l . . . . . . 24,380-386 12. Lkhtstein, D., Boone. G., and Blume, A. (1979) J. Cyclic Nucleotide Res. 6.367-375 .,~.. 13. Sokolovsky, M., Gurwitz, D., and Kloog. T.(1983) Adu. Enzymol. 66,137196 14. Sternweis, P. C., and Robishaw, J. D. (1984) J. Biol. Chem. 269, 1380613813 15. Ross, E. M., and Schatz, G. (1978) Methods Enzymol. 63,222-229 16. Northup, J. K ,Smiget, M. D., Sternweis, P. C., and Gilman, A. G.(1983) . J.Biol. Chem. 268,11369-11376 17. Yamamura, H.I., and Snyder, S. H.(1974) Proc. Natl. Acod. Sci. U.S . A. 7 1 , 1725-1729 1 . Northup, J. X ,Smiael, M. D., and Gilman. A. G. (1982) J. Biol. Chem. 8 . . . 267,-11416-1142319. Schaffner, W., and Weissman, C. (1973) Anal. Biochem. S6, 502-504 20. Ames, B. N. (1966) Methcds Enzymol. 8,115-118 . . 21. Birdsall, N. J. M.. Bureen.A. S. V.. and Hulme.E C.(1978)Mol. Phrmocol. . - . 14,723-760 22. Berrie, C. P., Blrdsall, N. J. M., Burgen, A. S. V., and Hulme, E.C. (1979) Biochem. Biophys. Res. Commun. 8 7 , 1000-1005 23. Sokolovsky, M., Gurwitz, D., and Galron, R. (1980) Biochern. Biophys. Res. Commun. 94,487-492 24. Szoka, F., and Papahadjopoulos, D. (1980) Annu. Reu. Biophys. Bioeng. 9, 467-508
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