A Monoclonal Antibody against the Rod Outer ... - Semantic Scholar

Vol. 264,No.19,Isaue of July 5,PP. 11475-11482, 1989 Printed in U.S. A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY

0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

A Monoclonal Antibody against theRod Outer Segment Guanyl Nucleotide-binding Protein, Transducin, Blocks the Stimulatory and Inhibitory G Proteins of Adenylate Cyclase* (Received for publication, November 1,1988)

Heidi E. Hamm, Dusanka Deretic, MariaR. Mazzoni, Chris Ann Moore,Joseph 5. Takahashi$, and Mark M. Rasenick From the Department of Physiology and Biophysics and Committee on Neuroscience, University of Illinois College of Medicine, Chicago, Illinois 60680 and the $ Department of Neurobiology and Physiology, Northwestern University, Evanston,Illinois 60208

GTP-binding proteins have been implicated as trans- receptor G protein transducin (G,)’ belongs to this class of ducers of a variety of biological signaling processes. proteins, which appears to have similar structure, function, These proteins share considerable structural as well as and regulatory roles in different tissues and organisms (Gilfunctional homology. Due to these similarities, it was man, 1987; Hurley, 1987; Spiegel, 1987; Stryer and Bourne, thought that a monoclonal antibody that inhibits the 1986). In hormone- or neurotransmitter-sensitive adenylate light activationof the rod outer segment GTP-binding cyclase systems, the GTP-binding proteins, G, and Gi, couple protein, transducin (GJ, might exert some functional receptors for stimulatory or inhibitory agonists to the adeneffect upon the G proteins that regulate the adenylate ylate cyclase enzyme (Rodbell, 1980). The natureof receptorcyclase system. Antibody 4A, raised against thea sub- G protein interaction as well as the mechanism of G protein unit of Gt, cross-reacted (by hybridization on nitrocel- activation (or inhibition) of adenylate cyclase or phosphodilulose) with purified a subunits of other G proteins (Gi esterase are still unknown. Yet there are several similarities and G., regulatory guanylnucleotide-binding proteins between the two systems (Stein et al., 1983). Reconstitution that mediate inhibition and stimulation of adenylate studies show functional exchangeability between the a subcyclase, respectively) as long as they were not dena- units of these proteins (Rasenick et al., 1981; Bitensky et al., tured. This antibody, which interferes with rod outer 1982; Cerione et al., 1985a,1985b,1986), and all three a segment cGMP phosphodiesterase activation by block- subunits interact with structurally similar 35- and 36-kDa /3 ing interaction between rhodopsin and Gt, also inter- subunits (Gierschik et al., 1985; Pines et al., 1985). Structural fered with actionsof both the stimulatory and inhibi- studies also show that they arerelated proteins (Manning and tory G proteinsof adenylate cyclase from rat cerebral Gilman, 1983) and are substrates for toxin-mediated ADP cortex membranes. Effects of monoclonal antibody ribosylation (Abood et al., 1982; West et al., 1985; Gill and (mAb) 4A were dose-dependent and not reversed by Wollakis, 1985). Protein- andcDNA-sequencing studies have washing. mAb 4A also blocked the Gi-mediated inhi- confirmed the existence of a multigene family of related GTPbition of adenylate cyclase in the cyc- variant of 549 binding proteins (Hurleyet al., 1984; Yatsunami and Khorana, lymphoma and indoing so raised thelevel of adenylate 1985; Lochrie et al., 1985; Tanabe et al., 1985; Medynski et cyclase activity in both the cyc- variant and the 549 al., 1985; Nukada et al., 1986a, 1986b; Itoh et al., 1986; Robwild type. There wasno effect of mAb 4A on adenylate ishaw et al., 1986; Jones and Reed, 1987; Suki et al., 1987; cyclase activity of the resolved catalytic subunit.These Matsuoka et al., 1988; Fong et al., 1988; Dietzel and Kujan, results suggest that the well known sequence homolo- 1987; Nakafuku et al., 1987). gies among the G proteins involved in cellular signal Recently, a series of monoclonal antibodies has been gentransduction may extend to the sites that interact witherated against ROS at; several of these antibodies (4A, 7A, other members of signal-transducing cascades (recep- 7B, 7C, and 7D) block light-activated GTP binding to G, and tors and effector molecules). Therefore, antibody 4A phosphodiesterase activation by the G .GTP complex, may serve as a useful tool to probe the similarities and whereas others (4H and 4C) do not affect light activation of differences among the varioussystems. Gt or phosphodiesterase (Hamm andBownds, 1984;Hamm et al., 1987). Antibody 4A blocks Gtactivation by interfering with its interaction with photoexcited rhodopsin. It elutes holo G, a& from the ROS membrane in the dark, blocking the tight light-induced binding of G, to metarhodopsin I1 A variety of GTP-binding proteins that participate in bio- (Hamm et al., 1987). The antigenic site of mAb 4A on ROS logical signal transduction have been identified. The photo* This work was supported by United States Public Health Service Grants EY06062 (to H. E. H.) and MH39595 (to M . R.), M . National Science Foundation Grant BNS 87-19758(to M. M. R.), National Science Foundation Research Opportunities for Women Research Career Advancement Award (to H. E. H.),and Department of Health and Human Services Research Scientist Development Award MHO0669 (to M.M. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: G,, rod outer segment guanyl nucleotide-binding protein, transducin;G. and Gi, regulatory guanyl nucleotide-binding proteins that mediate stimulation and inhibition of adenylate cyclase, respectively; at, a., and ai, a (GTP binding) subunits ofG,,G., and Gi, respectively; ROS, rod outer segment; mAb, monoclonalantibody;Gpp(NH)p,guanosine 5’-(P,y-imid0)triphosphate;GTP+, guanosine 5’-0-(3-thiotriphosphate); AAGTP, P3-(4-azido-anilido)-P1-5’-GTP; HEPES, 4-(2-hydroxylethyl)-1-piperazineethanesulfonicacid; EGTA, [ethylenebis(oxyethylenenitri1o)ltetraaceticacid; SDS, sodium dodecyl sulfate; GDPBS, guanosine 5’-0-2-(thio)diphosphate.

11475

11476

Antibody Blockade of Adenylate Cyclase G Proteins

at was localized to amino acids 311-328 near the C-terminal by tryptic and chymotryptic mapping (Deretic and Hamm, 1987) and peptide competition (Hamm etal., 1988). In this paper, we examine the structural and functional homology of the epitopes of blocking (4A, 7A, 7C) and nonblocking antibodies (4H) in other GTP-binding proteins. We demonstrate immunological recognition in dot blotting experiments between these antibodies and G, and Gi. Further, we demonstrate a functional blockade of both stimulated and inhibited adenylate cyclase by antibody 4A. EXPERIMENTALPROCEDURES

M~teriak-[a-~'P]ATP (800 Ci/mmol; 1 Ci = 37 GBq) was purchased from Du Pont-New England Nuclear. [o-~'P]GTPwas purchased from Amersham Corp. GTP, Gpp(NH)p, and GTPyS were from Boehringer Mannheim. Immunological Assays-Monoclonal antibodies used in this study were characterized in Witt et al. (1984) and Hamm et ~ l (1987) . and screened for effects on G, function (Hamm and Bownds, 1984;Hamm et QL, 1987). Antibodies were purified on protein A affinity columns and resuspended at 10 mg/ml in frog Ringer's solution (110 mM NaC1, 5 mM KC1,lO mM HEPES, pH7.5,5 mM MgC12). All antibodies used in this study were specific for at and did not recognize Pyt in Western blots. The slight cross-reactivity of flyt in dot blots (see Fig. 1) is from 5% contamination of by with a. at and Pyk were purified to greater than 95% purity from bovine rod outer segments. Blotting was performed as follows. Purified proteins were diluted in TBS (50 mM Tris-HC1, pH 8.5, 150 mM NaCl), and subsequent dilutions (1 p l ) were spotted onto dry nitrocellulose. Dots were air-dried for 10 min, and nitrocellulose was then incubated overnight in TBS containing 3% ovalbumin (OTBS) to block nonspecific protein binding. Dot blots were then incubated with 0.025 mg/ml purified mAb 4A or mAb 4H overnight at room temperature followed by sequential rinses with TBS, TBScontaining 0.1% Nonidet P-40, and OTBS. Nitrocellulose was then incubated with IO6 cpm/ml 'Z51-proteinA for 3 h, rinsed again, dried, and exposed to film overnight at room temperature. Preparation of Membranes-Rat brain cerebral cortex synaptic membranes were prepared as described in Rasenick and Bitensky (1980). Rat pineal glands were homogenized, and the membranes were washed three times in homogenate buffer (5 mM MgCl,, 1 mM EGTA, 1 mM dithiothreitol, 10 mM Tris-HC1, pH 7.4).C6glioma cell membranes were prepared as per Rasenick and KapIan (1986). S49 wildtype and cyc- membranes were the gift of J. Codina (Baylor College of Medicine). All membranes were stored under liquid N, until use. Zncubotion of Membranes with Antibodies-Membranes were incubated for 30 min on ice with specific or control antibody in Ringer's solution. In most experiments, commercially available rabbit IgG was present in control samples at thesame concentration as the specific antibodies. In a few experiments, ascites fluid of mice injected with the parent myeloma cellline, NS1, was purified on protein A affinity columns and processed identically to mAb4A. This preparation, called "NS1,"was used as an additional control for contaminant proteins in ascites fluid which copurify during affinity purification of the antibody. Adenylate Cyclase Assays-Adenylate cyclase of rat cerebral cortex synaptic membranes and C6 membranes was measured according to Salomon (1979)as modified byHatta et al. (1986).S49 cyc- membrane adenylate cyclase wasmeasured according to Hildebrandt et al. (1982). Membranes were thawed and resuspended in a buffer containing 20 mM HEPES (pH 7.5), 1 mMMgC1, 1 mM dithiothreitol, and 0.3 mM phenylmethylsulfonyl fluoride and were incubated with or without indicated antibody as noted. Membranes (10-20 pg) were incubated at 23 "C in 100 p1 of medium containing 15 mM HEPES (pH 7.5); 0.05 mM ATP; [L~-~'P]ATP (-5 X lo5 cpm/tube); 1 mMMgC12; 1 mM dithiothreitol; 0.05 mM cyclic AMP; 60 mM NaCl; 0.25 mg/ml bovine 1unit/ml adenserum albumin; 0.5 mM 3-isobutyl-1-methylxanthine; osine deaminase; a nucleoside triphosphate-regenerating system consisting of 0.5 mM creatine phosphate, 0.14 mg of creatine phosphokinase, and 15 units of myokinase/ml; and other reagents as indicated in the text. The reaction, which is linear for 30 min, was stopped after 10 min by the addition of0.1mlof a solution containing 2% SDS, 1.4 mM cyclic AMP, and 40 mM ATP, and the cyclic [32P]AMP formed was isolated by the method of Salomon (1979). In some

experiments, adenylate cyclase assays were carried out at 30 "C in the presence of 5 mMMgC12. Under these conditions, stimulatory rather thaninhibitory effects of Gpp(NH)p are favored. Pineal membrane adenylate cyclase was measured in 50 pl of reaction mixture containing (final concentrations) 42mM Tris-HC1, pH 8.0,6 mM MgC12,0.7 mM EGTA, 1.0 mM 3-isobutyl-1-methylxanthine, 0.2 mM dithiothreitol, 1 mM ATP, 5-25 pg of protein (10 pl of homogenate), and drug as indicated. Reactions were stopped by adding 250 pl of 50 mM sodium acetate buffer, pH 6.2, and placing the tubes in boiling water for 3 min. Cyclic AMP formed was measured by radioimmunoassay using components obtained from Becton Dickinson. Blanks (without enzyme) of the cyclase reaction mixture were added to the cyclic AMP radioimmunoassay standard curves to correct for cross-reactivity of the antibody to ATP. Under the conditions used, enzyme activity was proportional to time and enzyme concentration. Protein was determined by the Coomassie Blue binding method (Bradford, 1976) with bovine serum albumin as a standard. Preparation and Assay of Adenylate Cyclase Catalytic MoietyAdenylate cyclase catalytic moiety, resolved from G proteins, was prepared from bovine cerebral cortex by the method of Bender and Neer (1983) and stored in aliquots a t -80 "C until use. Adenylate cyclase assays were performed with 20 pgof this preparation as described above except that the assay contained 10 mM MgCl, and 75 mM sucrose in 100 pl, and theassay temperature was 30 "C. Gel Electrophoresis-This was performed according to Laemmli (1970) on SDS-10% polyacrylamide gels. RESULTS

Immunological Cross-reactivity among GTP-binding Proteins-Monoclonal antibody 4A blocks light activation of ROS phosphodiesterase by interfering with G, binding to and activation by rhodopsin (Hamm et al., 1987). The antigenic site of antibody 4A on ROS at was localized to a,-311-328 by tryptic andchymotryptic mapping (Deretic and Hamm,1987) and competition of antibody binding by synthetic peptides (Hamm et al., 1988). A comparison (see Fig. 7) of the deduced amino acid sequence of ROS at with the homologous sequences of a, (Nukada et al., 1986a; Robishaw et al., 1986) and ai (Nukada et al., 1986b; Itoh et al., 1986) reveals that enough homology exists in this region that monoclonal antibodies to at might be expected to cross-react with other G proteins. In early experiments designed to test whether mAb 4A could also block GTP-binding proteinsin brain, we noticed that when synaptic membranes were incubated for a few min with mAb4A, the membranes precipitated, whereas in the presence of nonspecific IgG, no precipitation occurred. This suggested that mAb 4A may be binding the homologous G proteins and cross-linking the membranes. To test for mAb 4A cross-reactivity with brain G proteins, a series of immunoprecipitation and protein-blotting experimentswas carried out. Although weak cross-reactivity with cholera toxin- and pertussis toxin-ADP-ribosylated bands was sometimes seen on protein blots, the results were not consistent (data not shown). In protein-blotting experimentsusing SDS-polyacrylamide gel electrophoresis, proteins become denatured and lose conformation-sensitive antigenic sites. It is known from tryptic digest studies that the mAb 4A epitope is partially conformation-sensitive (Deretic and Hamm, 1987). Thus, although denatured GTP-binding proteins might bind mAb 4A, they would do so with significantly lower affinity. To determine whether anti-at antibodies would interact with native nonSDS-treated (Y subunits, we applied purified native at,as,or various ai subtypes (ail,ai2,or ai3) onto nitrocellulose membranes andprobed antibody 4A binding to these proteins. Fig. 1shows that mAb 4A reacts strongly with at and cross-reacts with ai subtypes and weakly with as.There is no crossreactivity with control proteins. Denaturation of these proteins by treatment of the dot blotwith 2% SDS eliminated ae

of Adenylate Cyclase G Proteins

Antibody Blockade

and (yi cross-reactivity. Thus, it appears that there is a conformation-sensitive recognition of related GTP-binding proteins. Functional Cross-reactivitybetween Antibody4A and Other G Proteins-The immunological cross-reactivity ofmAb4A with other G proteins raised the possibility of similar effects on the function of homologous G proteins. To test whether mAb4A might have functional effects on the G proteins of the brain synaptic adenylate cyclase system, brain synaptic membranes were preincubated with mAb 4A. Fig. 2 shows that antibody 4A inhibited basal as well as Gpp(NH)p-stimulated or Gpp(NH)p-inhibited adenylate cyclase activity by 67, 54, and 56%, respectively. The NaF-stimulated adenylate cyclase was almost completely inhibited by mAb 4A. Forskolin- and Mnz+-stimulated adenylate cyclase activities were inhibited by 66 and 64%, respectively. mAb 4H, a monoclonal

*10 Ong

I

I

100 ng

50 25 12 6 3

1

2

3

4

5

6

7

FIG. 1. Antibody 4A recognizes GTP-binding proteins in native dot blots. Dilutions of purified proteins from the following sources were spotted onto nitrocellulose: at and Byt, bovine ROS; recombinant a. expressed in E. coli from cloned cDNA (ra.) (Graziano et al., 1987); three nl subtypes, purified from bovine brain ( a i l and ai2) and human erythrocytes (a& and trypsin inhibitor (TI) (Worthington). Amounts of proteins in each dot (in ng) are labeled. Dot blots were reacted with mAb 4A (1:250 dilution), and bound antibody was detected using '"I-protein A as described under "Experimental Procedures."

11477

antibody that binds to at but does not block its function (Hamm et al., 1987) also had no effect on adenylate cyclase activity (see Fig. 3). nzAb 4A Blockade of Adenylate Cyclase Actiuity in Other Ttssues-To assess how general this finding is, we examined mAb4A effects in two other tissue types: rat pineal membranes and C6 glioma cell membranes. Tables I and I1 show that similar results are obtained in these tissues. In pineal membranes, mAb4A partially blocks basal as well as 8adrenergic receptor-mediated stimulation of adenylate cyclase activity. Guanine nucleotide-, fluoride-, and forskolin-mediated but not Mn2+-mediatedstimulation of adenylate cyclase activity is also partially blocked by mAb 4A (Table I). In C6 gliomamembranes, there is a similar blockade of basal and stimulated (including Mn2+)adenylate cyclase activity by mAb 4A (Table 11). Specificity of the Antibody Inhibition-These results raised the possibility that mAb 4A was interacting with the synaptic membrane adenylate cyclase system. To test whether the antibody binding was specific, a battery of antibodies was employed. In the photoreceptor system, monoclonal antibodies 4A, 7A, 7B, 7C, and 7D, which bind to the same epitope, inhibit Gt activation by light, whereas antibodies binding to another region of at(e.g. mAb 4H) have no effect (Hamm et al., 1987; Deretic and Hamm, 1987). The same specificity applies to theeffect of these antibodies on adenylate cyclase, as shown in Fig. 3. Antibodies 4A,7A, and 7C blocked Gpp(NH)p-stimulatedadenylate cyclase activity by 25-30%, whereas antibody 4H and nonspecific IgG had no effect. Dose Dependence of mAb 4A Inhibition of Adenylate Cyclase-Effects ofmAb4A in inhibiting the photoreceptor phosphodiesterase are dose-dependent, saturable, and stable to washing with buffer. The mAb 4A-mediated inhibition of synaptic membrane adenylate cyclase activity is also dependent on mAb 4A concentration. Incubation of different concentrations ofmAb4A with synaptic membranes results in a dose-dependent inhibition of adenylate cyclase (Fig.4). Basal activity is depressed by 65% with no saturation of the inhibition up to 4 mg/ml mAb 4A. Under the conditions of this assay, adenylate cyclase activity is inhibited when Gpp(NH)p

300

250

FIG.2. mAb 4A blocks adenylate cyclase activity in cerebral cortex synaptic membranes. 840 pg of synaptic membrane protein was incubated with 675 pgofmAb4A or control IgG for 30 min on ice in a volume of 200 pl. Subsequent to this, the membrane suspension was diluted to 500 p l , and 8.6 pg of membrane protein was added to individual assay tubes in the presence of the indicated compound. Adenylate cyclase assays were then carried out a t 30 "C in the presence of 5 mM MgC12. Mean S.E. for one of five similar experiments are represented.

200 L

150

100

n 50

0

BASAL

0.1uM GppNHp

lOOuM QQRNHR

20mM NaF

1I 1OmM Mn

10~M FORSKOLIN

Antibody Blockude of Adenylate Cyclase G Proteins

11478 TABLEI

175

Effect of mAb 4A on rat pineal membrane adenylatecyclase activity Washed pineal membranes (158 pg of protein) were incubated with mAb 4A orNS1 control IgG(1.25mg)purifiedfrom the parent

myeloma cell line in a volumeof250plfor30min at 30 "C. The membrane-antibodysuspension(10pl)wasassayedforadenylate cyclase, and reactionswere carried out at 30 "C for 10 min. Numbers represent the results of one of three similar experiments (mean f S.E. of triplicates). Adenylate cyclase activity Addition

Hz0 GTP, 10 pM Isoproterenol, 10 p~ GTP + isoproterenol 615 NaF, 10 mM Forskolin, 100 p~ MnC12,5 mM

NS1 mAb 4A pretreatment pretreatment pmol cAMP/mg proteinlmin 198 f 25.6 104 f 4.41 164 f 6.06 112 f 6.51 279 f 11.9 151 f 3.72 f 51.3 354 f 20.3 622 f 47.2 1219 f 111 1255 f 91.8 795 f 92.9 272 f267 33.6 f 14.6

150 9)

c.

3

C

h.-

125

C

9)

c.

Q

P

100 c

!i

-d 5

75

0

t

50

25 0

TABLEI1 Effect of mAb 4A on C6 glioma membrane adenylate cyclase activity C6 membranes (0.3mg of protein) were incubated with mAb 4A or control IgG (0.6 mg) in a volume of 300 pl for 20 min (10 min 30 "C,

10 min ice). Following this, the membrane-antibody suspensionwas diluted to 900 pl, and 25-pl aliquots were added to tubes containing the indicated activators and the adenylate cyclase reaction cocktail (with 5 mM MgCl,). Reactions were carried out at 30 "C for 10 min. Numbers represent the results of one of three similar experiments (mean f S.E. of triplicates).

.5

1.o

2.0

3.0

4.0

MAb 4 A (mdrnl)

FIG. 4. Concentration dependence of mAb 4A effects. Indicatedconcentrations ofmAb4Awere incubatedwith 700 pg of synaptic membraneson ice for 30 min in a volume of 150 pl. Following dilution to 510 pl, 17.4 pg of protein was added to triplicate assay tubes in the presence or absence of 0.1 p~ Gpp(NH)p and assayed for adenylate cyclase in the presence of 1 mM MgClz for 10 min at 23 "C. Mean f S.E. for one of three similar experiments are represented.

Adenylate cyclase activity Addition

Effect of mAb 4Aon Resolved Catalytic Subunit-Since mAb 4A influences basal and Mn2+-stimulated adenylate cyclase in synaptic membranes, its effects may be directly on the catapmol cAMPlmg proteinlmin lytic subunit. To test this possibility, the catalyticmoiety was 8.94 f 0.63 11.2 f 0.66 Hz0 17.9 f 0.31 resolved from GTP-binding proteins by cholate-ammonium 25.3 f 1.8 GTP, 10 pM 12.5 f 0.46 12.4 f 1.48 Isoproterenol, 100 p M sulfatetreatment(BenderandNeer, 1983). The resolved 26.5 f 0.8 30.6 f 1.85 GTP + isoproterenol catalytic unit is stimulated by Mn2+ andby forskolin, but it 13 -+ 1.0 NaF, 10 mM 30 f 0.03 is nolonger influenced by fluoride,indicating that it has been 16.8 f 0.58 25.1 f 0.42 Forskolin, 10 pM separated effectively from G proteins (Table 111). Antibody 10.8 f 0.67 MnC12,5 mM 31.5 f 0.76 4A has nosignificant effecton Mn2+- and forskolin-stimulated activity in theresolved adenylate cyclase catalytic moiety. Effect of mAb4A on Gpp(NH)p-inhibited Adenylate Cyclose-The above data indicate thatG proteins are thelikely 50r targets of mAb 4A for the effects of that antibody on synaptic membrane adenylate cyclase. Thus, attempts were made t o study effects of both G, and Gi inthesemembranes. Gpp(NH)p inhibits neuronal membrane adenylatecyclase a t low concentrationsandstimulatesthat enzyme a t higher or inhibitioncanbe concentrations(althoughstimulation favored by varying assay conditions). When antibody 4Amediated depression of adenylate cyclase activity is monitored (under assay conditions favoring the "biphasic Gpp(NH)p response"), antibody 4A depressesbasaladenylate cyclase IgG 4A 7A 7c 4H activity by 63% (Fig. 5A). With increasing Gpp(NH)p conof the response FIG. 3. Antibody specificity of mAb 4A effects. Synaptic centrations in the assay, the inhibitory portion membranes(750 pg) andantibodies(750 pg) were incubated in a curve is bluntedby antibody 4A, suggesting that mAb 4A can volume of 150 pl for 30 min on ice. Membrane-antibody suspensions block Gi effects on adenylatecyclase. The stimulatory portion were then dilutedto 575 pl, and 16 pg of membrane protein/tube was of the curve isrelatively unchanged (see inset, Fig. 5). When assayed for adenylate cyclase in the presence of 1 mM EGTA and 5 adenylate cyclase activity (at M reaches nadir its mM MgCl, for 10 min at 30 "C. Mean f S.E. for one of two similar Gpp(NH)p), themAb 4A inhibitory effect is a 58% depression experiments are represented. which diminishes to 51% at a Gpp(NH)p concentration of M. 1 PM Gpp(NH)p (see Fig. 5). is presentintheassayat Gpp(NH)p-induced Override of mAb4A Blockade ofG,Antibody 4A further depresses the inhibited activityby 60%. Gpp(NH)p-stimulated adenylate cyclase activity was simi- stimulated Adenylate Cyclase-The results presented in Fig. larly inhibited. The effects of mAb 4A were not reversed by 5A raise the possibility that Gpp(NH)p (or any stable GTP analog) might activate stimulatory G proteins and preclude washing (not shown). I&

pretreatment pretreatment

,.

mAb 4A


11479

TABLE I11 Effect of mAb 4A on Mn'+-stimulated resolved catalytic subunit 19 pg of resolved adenylate cyclase catalytic subunit was incubated with 20 pg of mAb 4A or control IgG for 30 min on ice. Following this, ATP and other assay additionswere made, and adenylate cyclase activity was determined.Means f S.E. for one of three similar experiments are depicted. ND, not determined.

A.

Adenylate cyclase activity Addition

I&

pretreatment

4A

mAb pretreatment

pmol cAMP/mg proteinlmin

Hz0 NaF, 20 mM MnS04, 10 mM Forskolin, M,

+ MnSOl

1.2 +- 0.06 1.1 f 0.11 6.3 f 0.22 32.7 f 1.41

ND ND 5.6 f 0.49 37.8 f 0.51

TABLEIV Effect of mAb 4A on S49 cyc- adenylate cyclase activity S49 cyc- membranes (250 pgof protein) were incubatedwith control IgG or mAb 4A (350 pg) in a volume of 100 p l for 30 min on ice. Following this, membrane suspensions were diluted to 0.4 mg/ ml, and aliquots of 25 pl were assayed for adenylate cyclase as described under "Experimental Procedures." Numbers represent the results of one of three similar experiments (mean f S.E. of triplicates). B.

Adenylate cyclase activity 400 I

Addition

IgG pretreatment

mAb4A pretreatment

pmol cAMP/mg proteinlmin

300.

Hz0 ~ p p ( ~ ~ ) pM, Fluoride, 20 mM MnS04, 10 mM Forskolin, M

3.9 f 0.22 4.6 f 0.36 3.9 f 0.16 18.5 f 0.56 24.9 f 1.04

5.3 f 0.23 5.3 f 0.49 4.2 f 0.59 22.7 f 0.48 67.2 f 0.95

species counterbalance one another. We wished to examine the effects of mAb 4A on Gi independent ofG.. The S49 mouse lymphoma cyc- variant does not contain a functional G, (Johnson et al., 1980; Harris et al., 1986) and is therefore a useful preparation to examinespecifically the effect of mAb l o o k . 4A on Gi in the absence of other G proteins. Table IV shows Of 9 8 7 6 5 4 that Gpp(NH)p or fluoride alone does not stimulate cyc-log GppNHp (M) adenylate cyclase activity. There is no significant effect of mAb 4A on basal, Gpp(NH)p-, and fluoride-stimulated activFIG. 5. Effect of mAb 4A on Gpp(NH)p-stimulated adenylate cyclase activity under stimulatory and inhibitory assay ity. Manganese stimulates adenylatecyclase activity 4.7-fold, by mAb 4A of MnS04conditions. 300 pg of synaptic membranes and 450 pg of mAb 4A and there isa small (21%) stimulation were incubated in 200 p1 for 30 min on ice. Following dilution of the stimulated activity. Forskolin stimulates cyc- adenylate cysuspension, 12.7 pg of membrane protein was assayed for adenylate clase activity 6-fold. mAb 4A causes a further 2.7-fold encyclase. A , inhibitory conditions. Membranes were incubated in the hancement of forskolin stimulation, perhaps by blocking Gipresence of the indicated Gpp(NH)p concentrations for 10 min at mediated inhibition of adenylate cyclase. 23 "C, 1 mM MgC12. B , stimulatoryconditions.Membranes were Effect of mAb 4A onGTPyS Activation of Gi-To examine incubated in the presence of 5 mM MgClz and 1 mM EDTA for 10 furtherthe effect of mAb 4A on Gi, forskolin-stimulated min at 30 "C. Data are themean f S.E. for one of three representative experiments. adenylate cyclase was measured (Fig. 6 A ) .In this experiment, adenylate cyclase levels were elevated 600% over basal levels mAb 4A from exerting its inhibitory effect. To examine this byforskolin treatment.Preincubation of membraneswith further, synaptic membranes were assayed for adenylate cy- mAb 4A resulted in an additional327% stimulation of adenclase activity at 30 "C in the presence of 5 mM MgC12, condi- ylate cyclase. GTPyS concentrations from IO-' to M tions under which Gpp(NH)p treatment causes stimulation caused a dose-dependent inhibition of forskolin-stimulated rather than inhibition of the enzyme. When adenylatecyclase adenylate cyclase. Inthepresence of mAb 4A, forskolinis assayed under these conditions, the inhibitory effects of stimulated activitywas inhibited by GTPyS at a similar level antibody are overcome by increasing Gpp(NH)p concentra- (70.5-37.6 pmol/mg/min = 53% in the presence of mAb 4A; tion (Fig. 5 B ) . The Gpp(NH)p override of antibody 4A inhi- 21.5-12.3 = 57% in the control). The potency of GTPyS was bition is Gpp(NH)p dose-dependent. mAb 4A inhibition of unaltered. Thus, mAb 4A potentiates the forskolin stimulaadenylate cyclase is decreased from 32% in the absence of tion of adenylate cyclase, probably by blocking G, activity, Gpp(NH)pto7%inthe presence of M Gpp(NH)p. but the fact that GTPyS can still inhibit adenylate cyclase Effect of mAb 4A on S49 Lymphoma cyc- AdenylateCyclase suggests that some population of Gi molecules is not blocked Activity-In synaptic membranes, mAb 4A appears to block by the antibody. activity of both G, and Gi, and the activities of those two EffectofmAb4A on Adenylate Cyclase Activity of S49

Antibody Blockadeof Adenylate Cyclase G Proteins

11480

PERCENT STIMULATION BY MAb 4 A A. 515-

E. WT

"

"20

QTPYS.

lo-%

321.1

144

326

146

Q T P Y S . f0"M

313

1.0

QTPYS.

304

117

1 6 %

2501

125b' 9

0

7

logs are able to override the mAb 4A blockade of G., similar to the override seen in synaptic membranes (Fig. 5 B ) .

6

-log GTP) S (MI

FIG. 6. Effects of mAb 4A on forskolin-stimulated adenylate cyclase in 549 lymphoma cell membranes. A , adenylate cyclase activity in cyc- S49. Membranes (250 pg) prepared from S49 cyc- cells were incubated with mAb 4A or IgG (350 p g ) in a volume of 100 p1 for 30 min on ice. Membrane suspensions were diluted, and 10-pg aliquots of membrane proteins were assayed for adenylate cyclase in the presence of the indicated concentration of Gpp(NH)p as described under "Experimental Procedures." B , adenylate cyclase activity in wild-type S49.S49 wild-type membranes prepared and incubated as thecyc- membranes were assayed for adenylate cyclase. Mean _+ S.E. for one of two similar experiments are represented.

LymphomaWild-typeMembranes-In wild-type S49 cells, preactivation with forskolin increased adenylate cyclase levels, and pretreatmentwith mAb 4A caused a 144% additional stimulation of adenylate cyclase activity (Fig. 623). This is compared with a 327% stimulation in cyc- membranes (Fig. 6, inset), suggesting that in wild-type cells antibody inhibition ofGi is balanced by a concomitant inhibition ofG.. GTPyS exerts abimodal effect, causing inhibition of adenylate cyclase M (Fig. 6 B ) . The mAb at lo-' M andstimulation above 4A-induced stimulation of adenylate cyclase at low GTP+ concentration (caused by blockade of Gi) is followed at higher GTPyS concentration M) by a leveling of adenylate cyclase activity (caused by blockade ofG,) compared with control. A t M , nonhydrolyzable guanine nucleotide ana-

DISCUSSION

An antibody against ROS at which blocks light-activated phosphodiesterase activity in ROS membranes recognizes native homologous G proteins of the adenylate cyclase system and has effects on adenylate cyclase activity in ,949 lymphoma, C6 glioma, rat pineal, and cerebral cortex synaptic membranes. Additional at antibodies that bind to other regions of at but do not affect G,function do not affect adenylate cyclase activity. Since the antibody has no effect on resolved catalytic subunit, we conclude that itaffects adenylate cyclase activity via its interaction with G proteins. Since both G. and Gi are present in most membrane systems and mAb 4A recognizes both proteins, the effect of antibody blockade depends on the relative efficacy of the antibody 4A interaction with the particular G proteinsin each membrane. A resolution to thiscomplexity has been approached with the use of S49 cyc- membranes, which contain only Gi. In cyc-, mAb 4A causes an increase in forskolin-stimulated adenylate cyclase activity, which appears to be due to its blockade of Gi-mediated inhibition of adenylate cyclase. In wild-type S49 lymphoma membranes, mAb 4A also causes a stimulation of adenylate cyclase although to a lesser extent than in cyc-. This is due, we believe, to a concomitant blockade ofG.stimulated adenylate cyclase. In contrast, the predominant effect of mAb 4A in rat brain synaptic membranes as well as pineal and C6 glioma membranes is an inhibition of adenylate cyclase activity under a variety of conditions, suggesting an efficacious blockade of G, in this tissue. It thus appears that the effects ofmAb4A on adenylate cyclase depend on factors that vary from one membrane type to another. Such factors might include accessibility of the antibody to thecomponents of the adenylate cyclase system, the state of interaction between these components, and the physiological and ionic conditions. The analysis of these factors may give insights into theorganization of the components of the adenylate cyclase system in different membranes. The mAb 4A-induced blockade of adenylate cyclase was qualitatively different from that seen for ROS phosphodiesterase. 1) The blockade was only partial in synaptic membranes, with a maximal 63% inhibition of adenylate cyclase. In ROS membranes, however, mAb 4A completely blocked both the light activation of cGMP phosphodiesterase (Hamm and Bownds, 1984) and thelight-induced binding of G protein to rhodopsin (Hamm et al., 1987).2) A large excess of antibody was needed to observe the effect in synaptic membranes, whereas in the ROS, a stoichiometric molar ratio (1antibody/ G protein) was sufficient for complete blockade. Two different processes may be responsible for the lower potency and diminished efficacy in the homologous systems. One is a decreased affinity of the antibody for a different antigenic site on G. and Gi. A comparison of the deduced amino acid sequences of a,, aB,and ai in the region of the antigenic site (Deretic and Hamm, 1987; Hamm et al., 1988) is shown in Fig. 7. The homology with Gi is greater than with G, in this region as well as overall, suggesting that the antibody should recognize Gi moreeasily than G.. Dot blotting of native (Y subunits with mAb 4A reveals a preference of the antibody for the ai subtypes over as.Physiological evidence for more effective recognition ofGi than G, is given by the more dramatic mAb 4A blockade ofGi in cyc- membranes than G, in wild-type S49 lymphoma membranes. Nonhydrolyzable guanine nucleotide analogs do not effectively override mAb 4A blockade of Gi, but under certain conditions they can


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not block nucleotide binding to Gi or G, (data not shown). Inhibition of G Protein-G Protein Interaction-In cerebral cortex synaptic membranes, direct exchange of GTP between 31 1 329 Gi and G, appears to be one form ofG.-Gi interaction and at may be a constitutive process in the regulation of adenylate 80% ail cyclase (Hatta et al., 1986; Rasenick et al., 1987). It was 80% ai2 thought that if mAb 4A blocked only the interaction ofGi 80% ai3 with the catalytic moiety of adenylate cyclase, inhibition of G. activity could be observed asaresult of inhibition of 70% LYO nucleotide transfer from Gi to GB.Addition of mAb 4A to membranes that had been preincubated with AAGTP did not 50% as block Gpp(NH)p-mediated nucleotide exchange (datanot FIG. 7. Homology with other GTP-binding proteins aligned shown). as in Jones andReed (1989). Inhibition of Receptor-G Protein Interaction-In the ROS, mAb 4A appears to block rhodopsin-Gt interaction directly override antibody blockade of GB (see Figs.5B and6B). (Hamm et al., 1987, 1988). In pineal and C6 glioma memFurther evidence of effective interaction of mAb 4A with Gi branes, receptor-mediated activation of adenylate cyclase is is given by the recent report of a potent inhibition by mAb also partially blocked. In synaptic membranes, hormone 4A of the muscarinic K channel activationby the endogenous receptors are bypassed due to direct incorporation of hydrolheart GI (Codina et al., 1988), Gk (Yatani etal., 1988). Half- ysis-resistant GTP analogs by G. or Gi. Nonetheless, activamaximal inhibition of the carbachol- and GTP-activated K tion or inhibition of adenylate cyclase through these proteins channel was obtained at 65 nM mAb 4A. mAb 4A also blocked as well as basal and forskolin-stimulated adenylate cyclase K channel activationby exogenous purified Gi3or its activated activity were affected by mAb 4A.It is possible that G proteins ai3subunit (Yatani et al., 1988). are “precoupled” to receptors (Henis et al., 1982), and this Another possible reason for the diminished mAb 4A effec- persists throughout preparation of membranes. mAb 4A might tiveness is the different accessibility of the antigenic sites in block this inherent (“basal”) receptor activation of G proteins. the different G proteins. In the photoreceptor, Gt is periph- In reconstitution experiments, the presence of receptors in erally bound membrane protein, 90% of which can be removed phospholipid vesicles, even without agonist, is sufficient to from the membrane by low ionic strength buffers (Kuhn, stimulate GTP/GDP exchange and GTPase activities of G 1980), however, only 15% of a8 is removed from synaptic proteins (Cerione et al., 1985b, 1986). The effect of mAb 4A membranes by similar treatments (Rasenick et al., 1984). on basal adenylate cyclase levelsmight be explained by blockRecent data demonstrate that aimay have similar solubility ade of this interaction. (Neer et al., 1984; Sternweis, 1986), thus, it may also have More recentstudies indicate that G. displays distinctly similar partial accessibility to mAb 4A. Treatments that in- different behavior when uncoupled from receptors than under crease G protein mobility increase a, release to 30% of the conditions where receptors retain the possibility of coupling total, which corresponds to the loss of 50% of a. activity to G, (even in the absence of agonist). Specifically, GDPPS (Rasenick et al., 1984). This also suggests the existence of acts as a partial agonist for cerebral cortex G,, whereas this multiple populations of a,, some but not all of which might compound acts as an antagonist for Gi. Furthermore, in membe accessible to mAb 4A. Two further observations also sug- branes prepared from C6 cells (where a moderate coupling to gest that not all G proteins are blocked by antibody. 1) The the P-adrenergic receptor is retained), GDPpS antagonizes antibody dose-response curve does not saturate at the anti- the effects of GTP or its analogs in eliciting the activation of body concentrations used (Fig. 4). 2) GTPyS can effect only adenylate cyclase. GDPDS functions as an antagonist in the a partial inhibition of cyc- adenylate cyclase in the presence presence or absence of isoproterenol (Rasenick et al., 1989). of mAb 4A (Fig. 6A). This distinction between the G proteins of the two systems Manganese stimulates adenylate cyclase activity approxi- appears to be linked to receptors “making contact” with G., mately 6-fold both in the resolved catalytic subunit and in an event that mayoccur whether or not the receptor is S49 cyc- membranes. There is no effect of mAb 4A on Mn2+occupied by an agonist. Since the region of G, recognized by stimulated adenylate cyclase activity of the resolved catalytic mAb4A is that which interacts with the receptors, it is subunit andin pineal membranes and arelatively small effect possible that theregions of G, involved in receptor interaction in S49 cyc- membranes. This is consistent with the view that would cause an “uncoupled” G, protein to respond differently adenylate cyclase activity in the presence of Mn2+ reflects to mAb 4A than a receptor-coupled G,. catalytic subunit activity (Rasenick et al., 1981; Childers and G, Conformational Change Induced by ActiuaInhibition of LaRiviere, 1984). In the brain and C6 glioma cells, however, mAb 4A effectively inhibits Mn2+-stimulated adenylate cy- tion-Another possibility is that themAb 4A antigenic site is clase activity, suggesting that MnZ+might also potentiate G,- close to thesite of receptor interaction, but4A binding to this catalytic unit interaction in a manner similar to forskolin site canalso block conformational changes normally triggered (Green and Clark, 1982). Such an interaction has recently by interaction with receptor. In ROS, mAb 4A has no effect on aluminum fluoride-mediated activation ofGt (Maheras been reported (Peres-Reyes and Cooper, 1986), and mAb 4A and Hamm, 1989). In contrast to ROS phosphodiesterase, could have its effect by interfering with this interaction. The mechanism of antibody 4A blockade of adenylate cy- adenylate cyclase activity of synaptic membranes can be clase activity is not clear. Several possible mechanisms of activated or inhibited by hydrolysis-resistant GTP analogs without agonist occupancy of receptors. Also, unlike the ROS mAb 4A effects were considered. phosphodiesterase system (Hamm and Bownds, 1984), mAb Inhibition of GTP Binding to G Protein-mAb 4A does not directly block GTP binding to Gt.’ Similarly, studies with the 4A effects on adenylate cyclase Gproteins occur without photoaffinity GTP analog AAGTP indicate that mAb 4A does addition of agonists. There appears to be a “loose” coupling between receptors and G proteins elicited by the preparation * H. Hamm, unpublishedobservations. of membrane fractions (Rasenick and Kaplan,1986; Rasenick HOMOLOGY WITH at

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et al., 1987). Under such conditions, mAb4A might have access to a site on membrane-associated G proteins which blocks the ability of hydrolysis-resistant GTP analogs to promote inhibition or stimulation of adenylate cyclase. More work willbe needed to understand the mechanism of mAb 4A blockade of G proteins and the details of conformational changes involved in G proteinactivation. Comparative studies in homologous systems may be important for determination of the activated conformation of these G proteins blocked by mAb 4A. It is apparent that thefamily of GTP-binding signal-transducing proteins displays significant structural andfunctional homology. We have resolved one aspect of this homology in the use of mAb 4A to show that similar domains of G proteins interact with distinct receptor or effector molecules. We have also discovered variability between membranes in responses to antibody treatment, suggesting tissue-specific and physiological differences in the interactions between components of the adenylate cyclase system. Future work on this antigenic site may allow a more detailed description of these interactions and theirregulation. Acknowledgments-We thank Drs. Juan Codina and L. Birnbaumer for providing us with S49 wild type and cyc- membranes and purified ai3,Dr. Y. Kaziro for purified ail and (yi2, Drs. M. Graziano, M. Linder, and A. Gilman for purified cloned a., ail, and ai3,and M. M. Marcus for performing photoaffinity labeling experiments. REFERENCES Abood,M.E., Hurley, J. B., Pappone, M.-C., Bourne, H. R., and Stryer, L. (1982) J. Biol. Chem. 2 5 7 , 10540-10543 Bender, J. L., and Neer, E. J. (1983) J. Biol. Chem. 258, 2432-2439 Bitensky, M.W., Wheeler, M.A., Rasenick, M. M., Yamazaki, A., Stein, P. J., Halliday, K. R., and Wheeler, G. L. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 3408-3412 Bradford, M. M. (1976) Anal. Biochem. 7 2 , 248-254 Cerione, R. A., Codina, J., Kilpatrick, B. F., Staniszewski, C., Gierschik, P., Somers, R. L., Spiegel, A. M., Birnbaumer, L., Caron, M. G., and Lefkowitz, R. J. (1985a) Biochemistry 24,4499-4503 Cerione, R. A., Staniszewski, C., Benovic, J. L., Lefkowitz, R. J., Caron, M. G., Gierschik, P., Somers, R., Spiegel, A. M., Codina, J., and Birnbaumer, L. (1985b) J. Biol. Chem. 260,1493-1500 Cerione, R. A,, Regan, J. W., Nakata, H., Codina, J., Benovic, J. L., Gierschik, P., Somers, R. L., Spiegel, A.M., Birnbaumer, L., Lefkowitz, R. J., and Caron, M. G. (1986) J. Biol. Chem. 2 6 1 , 3901-3909 Childers, S. R., and La Riviere, G . (1984) J. Neurosci. 4, 2764-2771 Codina, J., Olate, J., Abramowitz, J., Mattera, R., Cook, R. G., and Birnbaumer, L. (1988) J. Biol. Chern. 263,6746-6750 Deretic, D., and Hamm, H. E. (1987) J. Biol. Chem. 2 6 2 , 1083910847 Dietzel, C., and Kurjan, J. (1987) Cell 50, 1001-1010 Fong, H. K., Yoshimoto, K. K., Eversole-Cire, P., and Simon, M. I. (1988) Proc. Natl. Acad. Sci. U. 5'. A . 85, 3066-3070 Gierschik, P., Codina, J., Simons, C., Birnbaumer, L., and Spiegel, A. (1985) Proc. Natl. Acad. Sci. U. S. A . 8 2 , 727-731 Gill, M., and Wollakis, M. (1985) CZBA Syrnp. 1 1 2 , 57-73 Gilman, A. (1987) Annu. Rev. Biochem. 56, 615-649 Graziano, M. P., Casey, P. J., and Gilman, A. G. (1987) J. Biol. Chern. 262,11375-11381 Green, D.A., and Clark, R. B. (1982) J. Cyclic Nucleotide Res. 8 , 337-346 Hamm, H. E., and Bownds, M. D. (1984) J . Gen. Physiol. 8 4 , 265280 Hamm, H. E., Deretic, D., Hofmann, K. P., Schleicher, A., and Kohl, B. (1987) J. Biol. Chem. 2 6 2 , 10831-10838 Hamm, H. E., Deretic, D., Arendt, A., Hargrave, P. A., Koenig, B., and Hofmann, K. P. (1988) Science 241,832-835 Harris, B. A., Robishaw, J. D., Mumby, S. M., and Gilman, A. G. (1985) Science 2 2 9 , 1274-1277 Hatta, S., Marcus, M., and Rasenick, M. M. (1986) Proc. Natl. Acad. Sci. U. S. A . 83, 5439-5443

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