Dec 19, 1979 - Thomas H. Hudson and Gary L. Johnson. From the Section of Physiological Chemistry, Division of Biology and Medicine, Brown University, ...
THEJOURNAL OF BIOLOC~CAL CHEMISTRY Vol. 255, No. 15. Issue of August 10, pp. 7 M - 7 4 8 6 , 1980 Printed in U.S.A.
Peptide Mapping of Adenylate Cyclase Regulatory Proteins That Are Cholera Toxin Substrates* (Received for publication, December 19, 1979)
Thomas H. Hudson and Gary L. Johnson From the Section of Physiological Chemistry, Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912
Cholera toxin, in the presence of NAD, specifically of the M, = 52,000 t o 53,000 peptide that is absent with ADP-ribosylates a M, = 42,000 peptide which appears the M, = 42,000 peptide. The similarity in digestion t o be a subunit of a regulatory protein of hormonepatterns of the toxin-specific substrates indicates that sensitive adenylate cyclase in plasma membranes of they are closely related peptides. S49 mouse lymphoma,HTC4 rat hepatoma, and human and pigeon erythrocytes. Partial proteolytic peptide maps of the M, = 42,000 cholera toxin substrate from Hormonal stimulation of adenylate cyclase activity requires pigeon and human erythrocyte membranes radiolabeled using [32P]NAD suggest a significant degree of the interaction of at least three classes of proteins associated similarity but not identityin their primary structure. with the plasma membrane: hormone receptor, catalytic cyPrevious characterization of the regulatory protein of clase, and a component responsible for the coupling of their human erythrocyte membranes indicates that it is an functions. Considerable evidence from several different horelongated intrinsic membrane protein that binds little mone-responsiveadenylate cyclase systems indicates that the o r n o detergent after solubilization(Kaslow, H. R., coupling component includes regulatory proteins that mediate Johnson, G. L., Brothers, V.M.,and Bourne, H. R. (1980) guanine nucleotide effects on cyclase activity (1-5). RegulaJ. Biol. Chem. 255, 3736-3741). Partial proteolytic tory proteins apparently require a guanine triphosphate for digestion of the radiolabeled cholera toxin substrate activation of adenylate cyclase by the hormone. receptor comfrom pigeon erythrocyte membranes produces similar plex (3), and the activation of adenylate cyclasemaybe peptide maps with the native membrane-bound form terminated by the hydrolysis of GTP associated with this and after solubilization with Lubrol PX. These results regulatory site (6-8). indicate that the peptide is largely exposed on the Cholera toxin modifiesthe guanine nucleotide regulation of intracellular side of the membrane, supporting the hy- adenylate cyclase apparently by inhibiting GTP hydrolysis pothesis that it is a “stalked” intrinsic membrane pro- (6).This action of cholera toxin ismediated apparently by the tein. specific ADP-ribosylation of a M , = 42,000 membrane protein LubrolPXextracts of pigeonerythrocytememthat is a subunit of the guanine nucleotide regulatory combranes, containing the toxin-labeled peptide, are capable of reconstituting a hormone-sensitive adenylate cy- ponent (9-12). Several lines of evidence support this hypothesis. I) Reconstitution studies using variants of S49 lymphoma clase when mixed with membranes from regulatory cells indicate that cyc- variants,’ whichlack the coupling protein-deficient S49 cell variants (cyc-). -tic and chymotryptic digestion of Lubrol PX extracts destroys component (4, 13), also lack the factor required for cholera 50% of the reconstituting capability of the extract with toxin action (14).2) The proteins in wild type S49 membranes little or no decrease in the M, = 42,000 toxin-labeled specifically radiolabeled by cholera toxin using [”*PINADare peptide. These results suggest that either a second absent in cyc- membranes (11).3) The cholera toxin substrate subunit of the oligomericM, = 126,000 protein exhibits in human erythrocyte membranes co-migrates in sucrose dengreater sensitivity to digestion bytrypsin and chymo- sity gradients and gel filtration columns with the protein that trypsin than the M, = 42,000 toxin-labeled peptide o r reconstitutes adenylate cyclase when mixed with cyc- memthat a second protein exists independent of the M , = branes (15). 4) The M , = 42,000 toxin substrate of pigeon 126,000 regulatory protein which is required for recon- erythrocytes binds specifically to a GTP affinity column (9) stitution that demonstrates greater sensitivity to pro- and shifts to a higher sedimentation coefficient in sucrose teases. gradients when first mixed in the presence of guanine nucleothe M,= 42,000 cholera toxinsubstrate, tide with membrane extracts containing catalytic adenylate In addition to membranes from 549 lymphoma and HTC4 hepatoma cyclase, suggesting its association with cyclase under these cells have a second toxin-specific substrate of M , = conditions (16). 52,000 t o 53,000. Comparison of partial proteolytic diGuanine nucleotide regulatory proteins from various gests of the M, = 42,000 and M, = 52,000 t o 53,000 toxin sources are functionally distinguishable as measured by their substrates indicates nearly identical peptide maps, ex- properties in the in vitro reconstitution assay with plasma cept for a single unique fragment present in the digest membranes fromcyc-S49cells (12). The cyc- membrane contributes /?-adrenergic receptors and catalytic adenylate * This work wassupported by National Institutesof Health Grants GM 26776,AM 07187,GM 07546,and HL 06285, American Cancer Society Grant IN45-T, and Biomedical Research Support Grant funds from Brown University. The costs of publication of this article were defrayed in part by the payment of page charges. This article musttherefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.
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The abbreviations used are: cyc-, adenylate cyclase activity-defiGpp(NH)p, cient549 cell variant; SDS, sodiumdodecylsulfate; guanyl-5’yl imidodiphosphate;Hepes, 4-(2-hydroxyethyl)-l-piperazine-ethanesulfonic acid; EGTA,ethylene glycol bis(B-aminoethyl ether)N, N, N , N-tetraacetic acid.
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cyclase to the reconstituted system, while detergent extracts phoresis was performed by the method of Laemmli (19) as described by O'Farrell(20) using either 10% or 15% acrylamide.Two-dimenfrom donor cells contribute the guanine nucleotideregulatory proteins. These differences canbe accounted for by differencessional gels were performed by a modification of the technique described by Cleveland et al. (21). Briefly. detergent extracts containing in 1) accessibility of the GTP binding site to GTP a n d 2 ) the the labeled cholera toxin substrate are electrophoresed on 10%acrylactivity of the endogenous GTPase. amide-SDS gels. This first dimension SDS gel is then placed on top T h i s report is a s t u d y of the structure/function relationship of a discontinuous SDS gel system containing 15% acrylamide in the separating gel. A Irk agarose gel is used to keep the first dimension of the guanine nucleotide regulatory component from cells with distinguishable regulatorycharacteristics. We have used SIX gel in place and also contains the specific proteolvtic enzyme. proteases to examine the structure of the M , = 42,000 protein Electrophoresis was performed in the normal manner except that the current is turned off for 30 min when the bromphenol blue dye nears specifically radiolabeled by cholera toxin. This analysis has the bottom of the stacking gel. The power is then turned on at 20 been extended to examine the orientation of the toxin-labeled mA/gel and the peptide fragments generated are then separated in protein in the p l a s m a m e m b r a n e of pigeon erythrocytes. I n the 157 acrylamide-separatinggel. Gels were stained and fixed in 2 9 Coomassie blue, 50% acetic acid, and 10% ethanol and destained in addition, we show that a second cholera toxin-specific substrate of 52,000 to 53,000 daltons in S49 lymphoma and H T C 4 10% acetic acid and 10% ethanol. Driedgelswere used to expose rat hepatoma cells generates partial proteolytic peptidemaps Kodak X-Omat H film with Dupont lightening-plusintensifying screens at -70°C. that are very similar to the M , = 42,000 cholera toxin subMaterials-Staphylococcus aureus V8 protease was from Miles. strate, suggesting that t h e y are closely related proteins. Trypsin, chymotr.ypsin. and elastase were purchased from Worthington. Gpp(NH)p was from Boehringer Mannheim. [ "PINAD was MATERIALSANDMETHODS synthesized from a-[:"P]ATP as described (11) or purchased from New England Nuclear. Cholera toxin was obtained from Schwarz/ Cell Culture-S49 mouse lymphoma cells weregrown in Dulbecco's Sigma. All modified Eagle's medium containing 10% heat-inactivated horse se- Mann. Lubrol I'X andaprotinin werepurchasedfrom other materials were reagent grade. rum. HTC4 cellswere grown in suspension in S77 basal medium supplemented with 10%calf serum. RESULTS Isolation a n d Solubilization of Plasma Membranes-Membranes from S49 and HTC4cells were prepared by a modification (17) of the Fig. 1 shows the radiolabeled pattern of m e m b r a n e extracts method described by Ross et al. ( 3 ) .Pigeon erythrocytes were lysed in the presence of DNaseandmembranespreparedas described from pigeon and human er.ythroc.ytes, S49 mouse lymphoma, a n d H T C 4rat hepatoma cells after incubation with [."PINAD previously for turkeyerythrocytes ( 6 ) . Humanerythrocytemembranes were prepared by the same procedure with the omission of a n d cholera toxin. Previously, it was demonstrated that the DNase.Membranes were frozen in dryice/ethanolandstored a t M , = 42,000 protein in pigeon (9, 10) and human (12) eryth-6OOC. r o c y t e s a n d b o t h the M , = 42,000 a n d t h e M , = 52,000 to Detergent extracts of S49. HTC4. and turkey erythrocyte mem53,000 d o u b l e t i n S 4 9 l y m p h o m a a n d HTC Hepatoma membranes (10 mg of protein/ml) were prepared by adding Lubrol PX to (11) are specific cholera toxin substrates. With S49 branes a final concentration of 1.0% (w/v) and incubating overnight on ice. Extracts from human erythrocyte membranes (3 mg of protein/ml) membranes (Fig. 1, lane 3 ) , a n u m b e r of other proteins are were prepared similarly using 0.2% Lubrol PX (12). Cholera Toxin Treatment of Membranes-Membranes (3 to 5 mg of protein/ml) were washed by centrifugation in 500 mM potassium phosphate (pH 7.0) and resuspended in 250 nm potassium phosphate (pH 7.0). 20 mM thymidine, 5 mM ADP-ribose, 20 mM arginine/HCl, 100 p~ GTP, 400 p~ ATP, 10 pg/ml of cholera toxin (activated with 20 mM dithiothreitol (17). and 25 [:"PINAD' (1 to 10 Ci/mmol). The reaction mix was incubated for 30 min a t 30°C and terminated by diluting in 10 ml of ice-cold 20 mM Hepes (pH 8.0), 2 mMMgC12, 1 mM EDTA. and 1 mM 2-mercaptoethanol. The membranes were centrifuged and resuspended in the same buffer at 10 mg of protein/ ml with 1% Lubrol PX and incubated overnight on ice. The detergenttreated membranes were then centrifuged a t 100,OOO X g and used for experiments. In experiments in which cholera toxin-labeled proteins were examined by protease digestion of intact membranes, the membranes were centrifuged in an Eppendorf microfuge for 10 min, the supernatant wasremoved and S I X (1% final concentration) was added to the pellets. Protease Digestions-Digestions were performed in 20 mM Hepes (pH 8.0), 2 mMMgC12, 1 mM EDTA. and 1 mM 2-mercaptoethanol for 15 min a t 2 2 T . T h e reactionwas inhibited by theaddition of aprotinin followed by the addition of SDS toa final concentration of
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Reconstitution of Adenylate Cyclase-Components of adenylate cyclasewere reconstituted by adaptingthemethod of Ross and Gilman (4) as describedpreviously (12). Lubrol PX extracts were treated with various proteases for 15 min a t 22°C and the reaction stopped by the addition of aprotinin. Extracts were mixed with 25 to 30 pg ofcyc- membranesin a reaction mix containing 50 mM NaHepes (pH 8.0), 6 nm MgClz, 0.2 mM EGTA, 2 mM 2-mercaptoethanol, 0.1 mg/ml of bovine serum albumin, 10 mM creatine phosphate, 10 units/ ml of creatine phosphokinase, and 0.4 mM ATP. After 20 min a t 30°C. [,"'P]ATP (IOti cpm) was added to give a specific activity of 25 cpm/ pmol in a final volume of 100 pl. The reaction mixture was incubated for an additional 40 min a t 30°C and terminated by the addition of 1 ml of 1%SDS. ['"PICAMP was purifiedby sequential chromatography on Dowex and alumina (18). SDS-Polyacrylamide Gel Electrophoresis-Membranes of Lubrol PX extracts in 1% SDS were reduced with 5%2-mercaptoethanol and boiled for 1 min. One-dimensional SDS-polyacrylamide gel electro-
FIG. 1. Autoradiograph of SDS-polyacrylamide gel electrophoresis of Lubrol PX extractsof membranes incubated with ['*PINAD' and cholera toxin. Incubation and solubilization were performed as described under"Materialsand Methods." Specific activity of [:"PINAD' was 5 Ci/mmol. Approximately25 pg of protein wereloaded in each lane of a 10% acrylamide gel. The gels were exposed to x-ray film for 2 days. Lanes represent human erythrocyte membranes ( 1 ) . pigeon erythrocytemembranes ( 2 ) .wild t.ye S49 membranes ( 3 ) . andHTC4membranes ( 4 ) . Doublearrows, M. = 52,(KK)/53,000 doublet; single arrow,M. = 42,000 protein.
Adenylate Cyclase
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labeled with [32P]NAD in the absence of cholera toxin (11). and human erythrocyte M , = 42,000 protein. Elastase (lanes These proteins are also labeled incyc- membranes which lack 5 and 6) generates a large fragment that is common to both pigeon erythrocyte protein and several smaller functional guaninenucleotideregulatoryproteins (11) and the human and cholera toxin-specific substrates. The other cell types (Fig. 1, fragments with the pigeon protein that were not detectable human and pigeon erythrocytes and HTC4 hepatoma, lanes with the human protein. Both the pigeon and human eryth1, 2, and 4, respectively) show very little or no nonspecific rocyteproteins wererelatively resistantto proteolysis by trypsin (lanes 1 and 2). At high concentrations of trypsin (200 labeling when incubated with [:'2P]NAD alone. The highly specific cholera toxin radiolabeling of adenylate pg/ml), two prominentfragments were detected with the with cyclase regulatory proteins in pigeon and human erythrocyte pigeon erythrocyte protein and no detectable fragments differences membranes allowed partial proteolytic digestions to be done the human protein. These results indicate that with intact membranes and detergent extracts containing theexist in the structure of the M , = 42,000 toxin substrate from cholera toxin-specific substrates. Such analysisof proteolytic human and pigeon erythrocytes. These differencesmay in digests allows one to probe the structure and orientation of part explain some of the differences in properties between the these proteins in the membrane. Thiswas of particular inter- two guanine nucleotide regulatory proteins. If proteolytic digestion of the toxin-labeled regulatory proest in light of the recentfindings that the cholera toxin-specific substrate of human (15) and pigeon (16) erythrocytes andwild tein is performed directly in Lubrol PX extracts(Fig. 3) using t-ype S49 (22) appears to be a subunit of a protein of approxi- conditions where the protein is active, a single prominent differences in guanine fragment is generated by elastase (lanes 5 and 6 ) with the mately M , = 126,000 andthatthe nucleotide regulatory propertiesof these systems appear be to labeled protein from both human and pigeon erythrocytes. a property of this protein (1 1 ) . This appears tobe the samesize fragment which is also most Fig. 2 shows partial digests of the toxin-labeled regulatory prominent using the denatured protein (Fig. 2). The protein protein from both human and pigeon erythrocytes after reis relatively resistant to trypsindigestion, but at trypsin conduction and denaturation in SDS. The exposure time of this centrations of 200 pg/ml, a t least four fragments are detectable autoradiograph has been increased in order to detect as manywith the pigeon erythrocyte protein and two fragments with fragments as possible. After these relatively long exposure the human protein. Information concerning the orientationof the choleratoxintimes, two faint low molecular weight bands are observablein the pigeon erythrocytemembranecontrol whichwas not labeled protein in the membrane canbe obtained by comparexposed to protease (lane 8).These proteins are also seen in ing digests of solubilized and membrane-associated protein. control preparations which were treated in exactly the same Pigeon erythrocytemembranes, whoselabeling pattern is manner except cholera toxin was omitted from the labeling highly specific for the M , = 42,000 protein, demonstrate that elastase gives a single large digestion product that remains procedure. When control membranes are treated with proteases, these nonspecifically labeled bands arelost and do not bound to the membrane(Fig. 4, lane 4). This peptide appears generate any fragments observed in Fig. 2, lanes 2, 4, and 6 to have the same molecular weight as that generated in the (data not shown). Therefore, the presence of the two low Lubrol PX extracts. This size fragment is also the predomibackground nonspecifically labeled bands does not interfere nant sue peptide generated with the denatured and reduced with the analysis of cholera toxin-specific peptide fragments. protein (Fig. 2, lane 6). In preliminary experiments, the disIt can be seen that chymotrypsin (lanes 3 and 4 ) generates tribution of labeled fragments remaining in the membrane or numerous fragments that are similar in size for the protein from bothhumanand pigeon erythrocytes.Theseresults suggest a significant degree of homology between the pigeon
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FIG. 3. Autoradiograph of SDS-polyacrylamide gel electrophoresis of protease digestion products of the nondenatured FIG. 2. Autoradiograph of SDS-polyacrylamide gel electro- 42,000-dalton cholera toxin substrate of human and pigeon phoresis of proteasedigestionproducts erythrocytes. Membraneswerelabeled of thedenatured with [,"PINAD' (10 Ci/ 42,000-dalton cholera toxin substrate of human and pigeon mmol) in the presence of cholera toxin and solubilized overnight with erythrocytes. Membraneswere labeled with ['"PINAD (10 Ci/ Lubrol PX. The membrane extract was then incubated with various mmol) in the presence of cholera toxin and solubilized overnight with proteases for 15 min at 22°C. Digestion was stopped by the addition Lubrol PX. SDS and 2-mercaptoethanolwere added to the membrane of aprotinin and SDS and 2-mercaptoethanolwere added togive final extract to give final concentrations of 1% and 5%. respectively, and concentrations of 1% and 5%, respectively. Samples were boiledfor 1 the samples boiled for 1 min. Protease digestions were for 15 min at min and approximately 30 pg of membrane protein loaded in each 22°C. Lanes represent extracts of human erythrocyte membranes (1. lane. Lanes represent extracts of human erythrocyte membranes ( I , 3, 5, and 7) and extracts of pigeon erythroc-yte membranes (2. 4, 6, 3, 5, and and 7) extracts of pigeon erythrocyte membranes (2, 4, 6, and 8). Lanes 1 and 2, 200 pg/ml of tr.ypsin; 3 and 4, 50 pg/ml of and 8).Lanes 1 and 2, control without protease;3 and 4, 100 p g / d of chymotrypsin; 5 and 6.25 pg/ml of elastase; 7 and 8, controls without trypsin; 5 and 6,25 pg/ml of elastase; 7 and 8. 50 p g / d of S. aweus V8 protease. protease treatment.
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examine the digestion of the M , = 42,000 protein. Fig. 5 shows that with both trypsin and chymotrypsin 50%inhibition of the reconstitutionactivityoccurs at a proteaseconcentration where 5% or less of the M , = 42,000 toxin-labeled protein is digested. These results are consistent with there being at least one other peptide that is more sensitive to trypsin and chymotrypsin which is also required for guanine nucleotide regulation of hormone-activated adenylate cyclase. Since cholera toxin specifically radiolabeled proteins of M , = 42,000 and 52,000 to 53,000 in S49 and HTC4 cells, proteolysis could not be conducted directlyin membranes or detergentextracts. T o circumventthis problem, theprocedure described by Cleveland et al. (21) was modified as described under “Materials and Methods.” Peptide maps generated by elastase, chymotrypsin, and S. aureus VS protease are presented in Fig. 6. Three observations are readily apparent. 1) The peptides generated from the M , = 42,000 and 52,000 to 53,000 proteins are remarkably similar, suggestingthe proteins are closely related. A single fragment is present in the map of the M , = 52,000 to 53,000 protein which is absent in the M , = 42,000 toxin-labeled protein. This proteinis always slightly off-center, toward the M , = 53,000 component of the doublet, suggesting that it maybe unique to thispeptide. 2) The maps of both the M , = 42,000 toxin substrate and the M , = 52,000 100
FIG.4. Autoradiograph of SDS-polyacrylamide gel electrophoresis of protease digestion products of the 42,000-dalton cholera toxin substrate in intact pigeon erythrocyte membranes. Membranes were labeled with [.’?]NAD’ (10 Ci/mmol) in the presence of cholera toxin and washed by dilution and centrifugation. The membranes were resuspended in 20 mM NaHepes, pH 8.0 2.5 mM MgCI2, 1.0 m~ EDTA, and 1.0 mM 2-mercaptoethanol and incubated with various proteases for 15 min a t 22°C. Digestion was stopped by the addition of aprotinin and membranes were pelleted by centrifugation for 10 min in an Eppendorf microfuge. The supernatant wa.. removed and the pellet was resuspended in 1%SDS and 5% 2-mercaptoethanol and boiled for 1 min. Samples were run on 15% acrylamide-SDS gels. No label could be detected released into the supernatant under these conditions. Lanes represent 200 pg/ml of trypsin ( I ) , 100pg/ml of chymotrypsin (2),100pg/ml of S. aureus (3). 100 pg/ml of elastase (4). and control without protease(5).
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FIG. 5. Comparison of reconstitution capacity anddigestion of the M, = 42,000 cholera toxin substrate in Lubrol PX exreleased into the supernatant was examined (not shown); no tracts of pigeon erythrocyte membranes. Membranes were la-
fragments were detected in the supematant afterproteolysis, beled with [,‘”PINAD’ (10 Ci/mmol) in the presence of cholera toxin and solubilized as described under “Materials and Methods.” The even though conditions were used where fragments released membrane extract was then incubated with various concentrationsof from the membrane should havebeen detected. Digestion of trypsin (W, 0) or chymotrypsin (0.0) for 15 min a t 22°C. Digestion the toxin-labeled membranes with trypsin gave similar results was stopped by the additionof aprotinin. Controlsconsisted of trypsin to those with the Lubrol PX-solubilized protein (Fig. 3) in or chymotrypsin (100 pg/ml) treatment of membrane extract with of protease. An aliquot that fragments generatedwere similar to those obtained with aprotinin added immediately prior to addition trypsin treatment of the Lubrol PX-solubilized protein. The of each digestion was mixed with cyc- S49 membranes and assayed for their ability to reconstitute isoproterenol-stimulated adenylate membrane-associated labeled protein is insensitive to chy- cyclase activity as described under “Materials and Methods.” Ademotryptic digestion (Fig. 4, lune 2). Similarresults were nylate cyclase activity of cyc- membranesreconstituted withall obtained with the Lubrol-solubilized protein (data not shown).control extractswas 30 pmol/min/mg of cyc- membrane protein.The values obtained from extracts treated with trypsin (W) or chymotrypPrevious reports had indicated that the reconstituting factor in detergent extracts of wild type (4, 23) and unc (24) S49 sin (0)are plotted as per cent of control values. Another aliquot of membranes, referred to variously as G/F (23), G (16), and N each digestion was made 1% for SDS and 5% for 2-mercaptoethanol electrophoresed on 158 acrylamide-SDS gels as detailed under (14), is sensitive to trypsin and chymotrypsin at concentra- and “Materials and Methods.” Gelswere exposed to film fordifferent tions that weresignificantly lower than those required to time periods and the bandscorresponding to theM,= 42,000 cholera generate detectable fragments of the toxin-labeled protein. toxin substrate were quantified using an E-C scanning electrophoresis Because of this observation, we performed experiments vary- densitometer. Exposures which fell in the linear range of the densiing the concentration of protease used to treat a Lubrol PX tometer sensitivity were used to calculate, as a per cent of control extract of pigeon erythrocyte membranes previously labeled digests, the amountsof M,= 42,000 protein present in trypsin- (0)or chymotrypsin- (0) treated extracts. The inset shows representative with cholera toxin and [:’”PINAD+.Aliquots were then either autoradiographs of extracts treated with various concentrations of reconstitutedwith cyc- membranes and adenylate cyclase trypsin ( A ) or chymotr.ypsin ( B ) .Lane I , control extracts; lane2. 100 activity examinedin the presence of isoproterenoland pg/ml of protease; fane 3. 40 pg/ml of protease; lane 4, 10 pg/ml of Gpp(NH)p or reduced and denatured and run on SDS gels to protease; lane 5,4 pg/ml of protease; and lane 6, 1 pg/ml of protease.
7484
Cyclase WTS49 42
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to 53,000 doublet are nearly identical in S49 and HTC membranes, supporting the notion of their conserved structure and function. 3 ) The peptide maps generated by the three proteases are remarkablysimilar. This is striking because of the difference in substrate specificity between S. aureus V8 proteaseandelastaseorchymotrypsin.Thisresult is not an artifact of the toxin labeling because nonspecifically labeled proteins in other regions of the gel generate different peptide maps from the cholera toxin-specific substrates with the same protease. Inaddition,the nonspecifically labeled proteins demonstrated different maps withdifferent proteases(not shown). This result suggests that even in the denatured and reduced state specific domains of this protein are more susceptible than others to proteolysis, generating similar partial digest patterns with different proteases. DISCUSSION
FIG. 6. Autoradiographs of partial proteolytic digests of the 42,000-dalton and 52,000 to 53,000-dalton cholera toxin substrates of wild type S49 and HTC4 membranes. Membranes were labeled with [ "P]NAD+ (10 Ci/mmol) in the presence of cholera toxin and solubilized for 2 h with Lubrol I'X. SDS and 2-mercaptoethanol were added to the membrane extract to give a final concentration of 1% and 5%. respectively, and the samples boiled for 1 min. A two-
A M , = 42,000 protein that is specifically radiolabeled by cholera toxin using ["'PINAD as substrate has been examined by partial proteolytic peptide mapping. The structure of the protein appears to be conserved between two widely divergent species; however, structural differences existwhich may relate to the differences in guanine nucleotide regulation observed in the reconstitution procedure withcyc- S49 membranes. Digestion of the labeled protein in intact membranes yielded similar maps as those obtained with the active solubilized protein. Farfel et al. (25) reported that the cyclase regulatory protein is exposed on the cytoplasmic face of the human erythrocyte membrane. Proteolytic inactivation andcholera toxin activation occurred with sealed inside-out membrane vesicles but not with sealed right-side-out vesicles. Kaslow et al. (15) then characterized thehydrodynamicproperties of thehuman erythrocyte guanine nucleotide regulatory protein.Their findings support the hypothesis that a protein which behaves like a single molecular species on sucrose gradients andgel filtraof adenylate tioncolumnscanmediatethereconstitution cyclase activity when mixed with cyc- membranes. The cholera toxin substrate appears to be a subunit of the guanine nucleotide regulatory component which had a calculated M , = 126,000. Similar results havebeen reported for the guanine nucleotide regulatory protein from S49 cells (22) and pigeon erythrocytes (16). The findings of Kaslow et al. (15)indicate that the guanine nucleotide regulatory protein of adenylate cyclase is an intrinsic membrane protein by the criteriaof Steck andco-workers (26, 27); detergents such as Lubrol 12A9 and Triton X-100 selectively solubilize the protein and the radiolabeled toxin substrate; however, treatments which selectively and almost quantitatively release extrinsic proteins by interfering with protein-protein interactionsfail to release the regulatory protein or thetoxin substrate. The regulatory protein had a high frictional ratio ( f / f o = 1.85), suggesting it is an elongated molecule and bound little detergent aftersolubilization. From these findings, the regulatory protein has been postulated to be a "stalked intrinsicmembrane protein" (28) that protrudes into the interior of the cell with only a small portion of the protein embedded in the membrane bilayer. Our findings are consistent with this h-ypothesis becauseproteolysis of the membrane-associated andLubrol PX-solubilized protein generates essentially the same fragments. This is the expected result if the proteinis indeed embedded in the membranecore dimensional modification of the procedure of Cleveland et a f .(21) was used to generate partial proteolytic digests as described under "Materials and Methods." A, S.aureus V8 protease, 12.5 p g ; B, elastase, 8 pg; C. chymotr-ypsin, 50 p g .
Cyclase
Adenylate
by a stalk and the rest of the protein protrudes into the cytoplasm. Such an orientation of the guanine nucleotide regulatory protein within the membrane would allow accessibility to proteases similar to that of the solubilized protein. The failure to observe fragments released into the supernatant following protease digestion is probably a result of the Mr = 42,000 peptide being ADP-ribosylated at only one or a few sites so that not all of the fragments generated wouldbe detected. In addition to this, agents like NaOH, 6 M urea, and p-chloromercuriphenylsulfonatefail to release the M , = 42,000 peptide (15), suggesting that it is probably part of the stalk that protrudes into thehydrophobic domain of the membrane. If this is the case and the peptide is ADP-ribosylated at a site that is near or in a region of the peptide that is embedded in the membrane bilayer, then labeled fragments would not be released into the supernatant. The finding that theM , = 42,000 cholera toxin' substrate of pigeon erythrocyte membranes is relatively insensitive to trypsin and chymotrypsin, compared to inactivation of the detergent extract's ability to reconstitute a catecholaminestimulated adenylate cyclasewhenmixed with cyc- membranes, suggests that a second peptide is required for reconstitution. This hypothesis is appealing because of the findings that the M , = 42,000 peptide is a subunit of a larger M , = 126,000 protein (15, 16) and that careful inactivation experiments performed with Lubrol 12A9 extracts of wild type 549 membranes (13, 23) or cholate extracts ofwild type or unc S49 membranes (24) indicate that more than one peptide may be required for isoproterenol and guanine nucleotide-stimulated activity. Based on the apparent subunit composition of the M, = 126,000 protein required for reconstitution, the simplest explanation would be the second peptide is also a subunit of the larger protein. This explanation will await further characterization of the M , = 126,000 protein and does not exclude the possibility of a second protein separate from the M , = 126,000 protein that is required for reconstitution, or that theprotease could remove a small fragment from the M , = 42,000 peptide so that it was inactive, but not distinguishable from the native peptide on SDS-acrylamide gels. However, we favor the notion that a second subunit of the regulatory protein is more sensitive to proteases than the toxin substrate for three reasons. 1)The proteins from S49 cells and human and pigeon erythrocytes required for stimulation of adenylate cyclase by guanine nucleotides, fluoride ion, and cholera toxin behave like a single molecule on sucrose gradients and gel filtration columns (15, 16, 22). 2) The M , = 126,000 protein from pigeon erythrocyte membranes binds to a GTP affinity column, resulting in an approximately 100-fold purification of the protein (9, 16). Eluates from theGTP affinity column containing the M , = 126,000 protein reconstitutean isoproterenol-stimulated adenylate cyclasewhen mixed with cyc- S49 membranes2 3) The properties of the protein that appear to exert guanine nucleotide regulation on ,8-adrenergic receptors are indistinguishable from those of the M , = 126,000protein that imposes guanine nucleotide, fluoride ion, and cholera toxin regulation on adenylate cyclase (4, 24). These findings suggest that the M , = 126,000 protein is sufticient for regulation of hormone receptors and adenylate cyclase by these effectors. Guanine nucleotide regulatory components having only the M . = 42,000 toxin substrate can reconstitute isoproterenol, guanine nucleotide, and NaF sensitivity to cyc- membranes. However, wild type S49 and HTC4 membranes have a second cholera toxin substrate which is a doublet of M , = 52,000 to 53,000. NObiological function has as yet been assigned to this
* G. L. Johnson, unpublished observation.
7485 higher molecular weight toxin substrate. Exposure of intact wild type S49 cells to cholera toxin prior to the preparation of membranes prevents the specific labeling of the M , = 42,000 and 52,000 to 53,000 peptides, whereas nonspecific labeling remains unchanged or even enhanced (11). This result strongly suggests that both of these proteins are relevant toxin substrates in the intact cell. The partial proteolytic peptide maps of the cholera toxinspecific substrates in wild type S49 and HTC4 membranes indicate a striking similarity between the M , = 42,000 and 52,000 to 53,000 proteins. A single major peptide appears to be present in the higher molecular weight doublet that may be a fragment of the M , = 53,000 moiety. In addition, preliminary results indicate that thetwo toxin substrates from HTC4 and wild type S49 membranes co-migrate in sucrose gradients. This finding suggeststhat thetwo proteins may be associated with one another in membrane detergent extracts. Given the similarities in peptide maps, at least three possibilities exist for the relationship between the M, = 42,000 and 52,000 to 53,000 cholera toxin substrates. 1) The two peptides are separate gene products, although quite similar, and may possibly have different regulatory functions. 2) Either of the two peptides could be covalently modified forms of the othercausing the generation of a unique fragment in the higher molecular form. 3) The M , = 52,000 to 53,000 doublet couldbe a precursor of the M, = 42,000 protein. It is tempting to speculate about the possible functions of the M , = 52,000 to 53,000 toxin substrate based on the complexities and regulatory characteristics of the adenylate cyclase system of different cell types. Avian erythrocytes demonstrate asingle M, = 42,000 peptide labeled by cholera toxin. Lad et al. (29) have shown that if turkey erythrocytes are first incubated with isoproterenol and EDTA under conditions which have been shown to release ["HIGDP ( 5 ) ,the affinity of the /I-adrenergic receptor for agonist in the presence or absence of added guanine nucleotide behaves similar to the /3-adrenergic receptor in wild type S49 membranes. We have recently found that pigeon erythrocytes demonstrate the phenomenon of agonist-induced desensitization, measured by the specific decrease in the ability of catecholamines to stimulate adenylate cyclase in membranes prepared from cells exposed to isoproterenol relative to controls not incubated with agonists.* This is in spite of the finding that exposure of avian erythrocytes to isoproterenol did not cause a loss of /I-adrenergic binding sites (30). Thus, the presence of a M, = 52,000 to 53,000 peptide that is a substrate for cholera toxin is not required for these effects. It is possible, however, that a similar peptide is present that is not a cholera toxin substrate that is involved in these regulatory phenomenon. The possible functions of this peptide and its relationship to the M, = 42,000 peptide are presently under investigation. REFERENCES 1. Rodbell, M., Lin, M. C., and Salomon, Y. (1974) J. Biol. Chem. 249,5945 2. Rodbell, M., Lin, M. C., Salomon, Y., Londos, C., Harwot d, J. P., Martin, B. R., Rendell, M., and Berman, M.(1975) AdL. Cyclic Nucleotide Res. 5, 3-29 3. Ross, E. M., Maguire, M. E., Sturgdl,T. W., Biltonen, R. L., and Gilman, A. G. (1977) J. Biol. Chem. 252, 5761-5775 4. Ross, E. M., and Gilman, A. G. (1977) Proc. Natl. Acad. Sci. U. S. A . 74,3715-3719 5. Cassel, D., and Selinger, 2. (1978) Proc. Natl. Acad. Sci. U. S. A. 75,4155-4159 6. Cassel, D., and Selinger, 2. (1977) Proc. Natl. Acad.Sei. U. S. A . 84, 3307-3310 7. Cassel, D., and Selinger, Z. (1977) J. Cyclic Nucleotide Res. 3, 393-406 8. Cassel, D., and Selinger, 2. (1976) Biochim. Biophys. Acta 452, 538-551
7486
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9. Cassel, D., and Pfeuffer, T. (1978) Proc. Natl. Acad.Sci. U. S. A . 75,2669-2673 10. Gill, D. M., and Meren, R. (1978) Proc. Natl. Acad. Sci. U. S. A . 75,3050-3054 11. Johnson, G. L., Kaslow, H. R., and Bourne, H. R. (1978) J. Biol. Chem. 253,7120-7123 12. Kaslow, H. R., Farfel, Z., Johnson, G. L., and Bourne, H. R. (1978) Mol. Pharmacol. 15,472-483 13. Ross, E. M., and Gilman, A. G . (1977) J. Biol. Chem. 252,69666969 14. Johnson, G.L., Kaslow, H. R., and Bourne, H. R. (1978) Proc. Natl. Acad. Sci. U. S.A. 75,3113-3117 15. Kaslow, H. R., Johnson, G. L., Brothers, V., and Bourne, H. R. (1980) J. Biol. Chem. 255,3736-3741 16. Pfeuffer, T. (1979) FEBS Lett. 101,85-89 17. Johnson, G. L., and Bourne, H. R. (1977) Biochem. Biophys. Res. Commun. 78, 792-798 18. Salomon, Y.,Londos, C., and Rodbell, M. (1974) Anal. Biochem. 58, 541-548 19. Laemmli, U.K. (1970) Nature 227, 680-685
20. O’Farrell, P. H. (1975) J. Biol. Chem. 250,4007-4021 21. Cleveland, D. W., Fischer, S. G., Kirschner, M. W., and Laemmli, U. K. (1977) J. Biol. Chem. 252, 1102-1106 22. Howlett, A. C., Smigel, M., Crane, J. K., and G h a n , A. G. (1979) Fed. Proc. 38, 317 23. Ross, E. M., Howlett, A. C., Ferguson, K. M., and Gilman, A. G . (1978) J. Biol. Chem. 253,6401-6412 24. Sternweis, P. C., and Gilman, A. G. (1979) J. Biol. Chem. 254, 3333-3340 25. Farfel, Z., Kaslow, H. R., and Bourne, H. R. .(1979) Biochem. Biophys. Res. Commun. 90, 1237-1241 26. Steck, T., and Yu, J. (1973) J. Supramol. Struct. 1,220-232 27. Yu, J., Fischman, D. A,, and Steck, T. L. (1973) J. Supramol. Struct. 1,233-248 28. Brunner, J., Hauser, H., Braun, H., Wilson, K. J., Wacker, H., ONeill, B., and Semenza, G. (1979) J. Biol. Chem. 254, 18211828 29. Lad, P. M., Nielsen, T., Preston, M. S., and Rodbell, M. (1980) J . Biol. Chem. 255,988-995 30. Hanski, E., and Levitzki, A. (1978) Life Sci. 22,53-60
