biochemical properties of hormone-sensitive adenylate cyclase

Ann. Rev. Biochcm. 1980. 49;533-64Copyright © 1980 by Annual Reviews Inc. All rights reserved

BIOCHEMICAL PROPERTIESOF HORMONE-SENSITIVEADENYLATE CYCLASE1

012051

Elliott M. Ross

Departments of Pharmacology and Biochemistry, University of Virginia Schoolof Medicine, Charlottesville, Virginia 22908

Alfred G. Gilman

Department of Pharmacology, University of Virginia School of Medicine,Charlottesville, Virginia 22908

CONTENTS

PERSPECTIVES AND SUMMARY .......................................................................... 534PROTEIN COMPONENTS OF HORMONE-SENSITIVEADENYLATE CYCLASE ............................................................................................ 535

Hormone Receptors and .4denylate Cy¢lase ............................................................ 535Hormone Receptors Linked to Adenylate Cyclase .................................................. 537Size and Shape of Adenylate Cy¢lase ...................................................................... 538Resolution of Catalytic and Regulatory Proteins .................................................... 540Properties of the Catalytic Protein (C) .................................................................... 542The Guanine Nucleotide-Binding Regulatory Protein (G/F) .................................. 543UNC Lesion: Coupling Factor or Covalent Modification ........................................ 545Calcium-Dependent Regulatory Protein .................................................................. 546Other Protein Factors ....................................................................’ ............................547Reconstitution of Hormone-Sensitive Activity .......................................................... 548

1Abbreviations used: Gpp(NH)p, guanyl-5’-yl-imidodiphosphate; GTP-~-S, guanosine-5’-O-(3-thiotriphosphate); CDR, calcium-dependent regulatory protein (calmodulin); C, cata-lytic protein of adenylate cyelase; G/F, guanine nucleotide-binding regulatory protein ofadenylate cyclase.

We use the word "hormone" to represent any hormone, autaeoid, neurotransmitter, or drugthat can activate adenylate cyclase in a receptor-mediated fashion.

533006-4154/80/0701-0533501.00

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534 ROSS & GILMAN

EFFECTS OF LIPIDS AND MEMBRANE STRUCTURE .................................... 549REGULATION OF ADENYLATE CYCLASE ACTIVITY .................................. 553

Regulation by Guanine Nucleotides ..........................................................................554Catecholaraine-Stiraulated GTPase ..........................................................................555A Regulatory GTPase Cycle ......................................................................................557

CONCLUSION AND QUESTIONS ............................................................................ 559

PERSPECTIVES AND SUMMARY

Adenosine-3’:5’-monophosphate (cyclic AMP) is now recognized as ubiquitous regulatory molecule, controlling diverse metabolic processes inboth prokaryotic and eukaryotic organisms. In animals, its principal roleis as an intracellular "second messenger" in the transduction of informationcarried by numerous hormones, and its synthesis is catalyzed almost exclu-sively by the hormone-sensitive adenylate cyelase system [ATP pyrophos-phate-lyase (cyclizing), E.C.4.6.1.1]. Hormone-sensitive adenylate cyclaseactivity is found in almost all animal cells (some erythrocytes and culturedcells are exceptions) and, depending upon the cell, can be stimulated by oneor more of a large number of hormones. These include various biogenicamines, proteins, polypeptides, and some prostaglandins. Recently, interesthas also focused on negative hormonal control of adenylate cyclase byopiates, c~-adrenergic amines, adenosine, and acetylcholine.

Because of the importance of cyclic AMP as a second messenger, interestin adenylate cyclase has centered on the regulation of its activity by hor-mones and other ligands. As assayed in plasma membrane preparations,adenylate cyclase displays a "basal" activity which varies enormously ac-cording to tissue and assay procedure. It is unclear whether this representsthe true activity of the unperturbed enzyme or slight stimulation of aninitially inactive enzyme by regulatory ligands (provided as impurities in themembrane preparation or in the ATP used as substrate). This basal activitycan be elevated by the addition of appropriate hormones or analoguesthereof to the assay mixture, but the extent of hormonal activation assayedin vitro is generally less than that observed when the synthesis of cyclicAMP is studied in intact cells or tissues. The concentration of hormonesrequired to stimulate the enzyme is also frequently increased after homo-genization of tissues.

Hormonal stimulation of adenylate cyclase also requires the presence ofa guanine (or related purine) nucleotide in addition to substrate. This is general requirement for all cells studied in detail and has led to the findingthat various analogues of GTP, such as Gpp(NH)p or GTP-T-S, can stimu-late adenylate cyclase activity in the absence of hormones, as can GTP itselfunder some conditions. Fluoride is another ubiquitously stimulatory ligandof eukaryotic adenylat¢ cyclase. Activation usually requires greater thanmillimolar concentrations of fluoride and is irreversible or only slowly


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HORMONE-SENSITIVE ADENYLATE CYCLASE 535

reversible. Cholera toxin and several other bacterial toxins also stimulateadenylatc cyclas¢, apparently by catalyzing the covalent modification of oneof the components of the enzyme.

This review discusses primarily the biochemical basis of these regulatoryphenomena and how the composition and structure of the system are mir-rored in its regulation. The progress of research in this area reflects threechallenging properties of the enzyme. First, adenylate cyclase appears to becomposed exclusively of intrinsic membrane proteins and depends upontheir proper integration in a membrane for hormonal regulation. Thus,while the catalytic, guanine nucleotide-binding, and hormone-binding pro-teins may be solubilized with detergents and assayed according to theirindividual activities, hormonal stimulation of adenylate cyclase is observedonly in intact membranes. Second, the proteins of adcnylatc cyclasc arcscarce, and probably none exists in a concentration greater than I0pmol/mg membrane protein; some arc lO0-fold less abundant. Third, sev-eral of the proteins arc quite labile in detergent solution, and further appar-ent lability is probably caused by their dissociation during manipulation.Nevertheless, the past several years have sccn a major increase in ourunderstanding of the individual components of the system and the mecha-nism of their interaction. It is now clear that hormone-sensitive adcnylatccyclasc is composed of at least three proteins: a catalytic protein that isrelatively inactive and displays none of the regulatory properties describedabove, a guanine nuclcotidc-binding protein that mediates the action of thevarious regulatory ligands, and one or more hormone receptors. Our under-standing of hormonal stimulation of adcnylatc cyclasc suggests that theregulatory protein is the proximal stimulator of the catalyst and that thereceptor-hormone complex acts by mediating the binding of pudne nucleo-tides to a regulatory site.

PROTEIN COMPONENTS OF HORMONE-SENSITIVEADENYLATE CYCLASE

Hormone Receptors and Adenylate CyclaseIt is now clear that receptors for hormones are indeed individual proteins,distinct from adenylate cyclase. This idea derived first from kinetic studiesof the activation of adenylate eyclase, particularly in membranes of adipo-cytes. In these cells multiple hormones, which bind to different receptorsites, seem to compete for a fixed number of molecules of adenylate cyclase(1, 2). Other arguments for the nonidentity of hormone receptors andenzyme have stemmed from observations of their independent ontogeneticregulation (3-6). More direct experimental approaches used chemical genetic manipulation to resolve receptor and enzyme. Schramm showed


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536 ROSS & GILMAN

that N-ethylmaleimide, which was known to inactivate adenylate cyclase (7,8), did not destroy the ligand-binding activity of/~-adrenergic receptors ofavian erythrocytes (8). Conversely, N,N-dicyclohexylearbodiimide inac-tivated binding sites for the fl-adrenergic ligand iodohydroxybenzylpindololat concentrations that did not inhibit the enzyme (9). Using a geneticapproach, Insel et al (10) demonstrated that two clones of cultured cells thatare phenotypically deficient in adenylate cyclase (HTC rat hepatoma andan $49 mouse lymphoma variant) both retain/3-adrenergie receptors (10).While each does in fact retain one of the two proteins necessary for adeny-late cyclase activity, the data clearly showed that B-adrenergic receptors aredistinct from assayable adenylate cyclase.

A more direct demonstration that adenylate cyclase and hormone recep-tors are distinct proteins came from the cell fusion experiments of Orly,Sehramm, and co-workers (11-14). Sendai virus was used to fuse Frienderythroleukemia cells to turkey erythrocytes that had been treated withN-ethylmaleimide to inactivate adenylate cyelase. The Friend cells haveadenylate cyclase but lack B-adrenergic receptors. Plasma membranes fromthe resultant erythrocyte-Friend cell heterokaryons displayed catechola-mine-stimulatable adenylate cyclase activity. Cell fusion and membranepreparation were performed in the presence of cycloheximide to prevent denovo protein synthesis, which indicates that the source of stimulation wasthe interaction of Friend cell enzyme with erythrocyte ~-adrenergic recep-tor. This receptor-enzyme interaction takes place rapidly (~ 5 min) in theheterokaryon membrane (13). Cell-to-membrane fusion rather than cell-to-cell fusion can yield similar reconstitution of hormonal stimulation (14, 15),and membranes from various cell types containing a variety of receptorshave now been used successfully in this procedure. Thus the central postu-late of the "floating receptor" model of the regulation of adenylate cyclaseappears to be essentially accurate: hormone receptors and adenylate cyclasemolecules are discrete proteins, relatively free to diffuse and interact in theplane of the bilayer.

Clear physical separation and molecular characterization of adenylatecyclase and its related hormone receptors were first achieved by Limbird& Lefkowitz (16) and by Haga et al (17). These groups solubilized adenylate cyelase activity and ~-adrenergic receptor sites with reasonablygood recoveries, and separated them both by gel exclusion chromatographyand sucrose density gradient eentrifugation in detergent solution. A similarstrategy was used by Vauquelin et al (18), who used a $-adrenergic a~nitymatrix to separate receptor from enzyme. Separation of other receptorsfrom adenylate cyclase has been less clear cut, presumably for technicalreasons. Welton et al (19) showed that the hepatic glucagon receptor didnot exactly cofractionate with adenylate cyclase during gel exclusionchromatography. When Dufau et al (20) chromatographed detergent ex-


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tracts of testis and ovary on agarose gel, they found several peaks of receptorbinding activity for LH/hCG, one of which coincided with adenylate cy-clase. Whether this was fortuitous or representative of real association ofproteins is unclear, as is the significance of the multiple peaks.

Hormone Receptors Linked to Adenylate Cyclase

The ligand-binding properties of a wide variety of hormone receptors thatact via adenylate cyclase have been studied in the membranes of target cellsby the use of appropriate radioactive ligands; a rather large number ofreceptors have also been characterized after detergent solubilization. The/~-adrenergic receptor has been partially purified (18, 21), and there is onebrief report on the possible purification of a receptor for LH (22). In severalcases [ADH (23), LH (24), FSH (25), PTH (26), and B-adrenergic 27, 28)] ligand binding characteristics of the soluble receptor are essentiallyunaltered from those of the membrane-bound protein. In the case of theglucagon receptor (19), it has not been possible to bind ligands to thesolubilized protein, although a receptoroligand complex has been solubilizedfrom hepatic membranes that were first incubated with lzSI-glueagon. Thismay reflect a stabilizing effect of ligand upon the receptor, and the relativeinstability of the unliganded, detergent-solubilized protein, as was noted toa lesser extent with the receptor for LH (24) and ADH (23). It is interestingthat ~-adrenergic receptors solubilized with digitonin (a saponin) can bindagonist or antagonist ligands in solution (16, 27, 28), whereas linear alkyl-polyethyleneoxide detergents (Lubrol, Brij) have permitted only the solubil-ization of a receptor-ligand complex (17).

Detergent-solubilized preparations of LH and B-adrenergic receptorshave been characterized with respect to their hydrodynamic properties, andboth appear to be rather asymmetric proteins that bind fairly large amountsof detergent in solution (Table 1). [The data of Abou-Issa & Reichert (25)on receptors for FSH from calf testis, in which no correction for detergentbinding was made, are not easily interpretable but are not grossly discrepantfrom those shown.] Detergent binding to a protein can be taken to indicatethe presence of significant hydrophobic surface area, which suggests thatthese molecules may interact with or span the hydrocarbon portion of theplasma membrane bilayer (30). However, the amount of detergent boundper receptor molecule is in each case about that found in one micelle [110molecules of Lubrol PX, 140 molecules of Triton X-100 (30, 31)]. This also consistent with the interaction of a small region of the protein with asingle micelle. It is thus possible that these receptors are localized on theouter face of the plasma membrane and interact with the bilayer via a smallhydrophobic region, as is the case for cytochrome b5 and cytochrome b5reductase (32).


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Table 1 Hydrodynamic properties of hormone receptors

Receptor fl-adrenergica Gonadotropinb

Stokes radius (~) 64 64S2o,w (S) 3.1 6.5

f/fo 1.8 1.6Mr 7.5 x 104 1.6 × 105

Detergent bound(g/g protein) 0.7 (Lubrol PX) 0.22c (Triton X-100)

aFrom (17). Source: $49 lymphoma cell.bFrom (24, 29). Sources: rat testis or ovary.CCalculated from (29). Partial specific volumes determined by CsCi gradient centrifuga-

tion in presence of Triton X-100.

Size and Shape of Adenylate CyclaseWhile adenylate eyelase was solubilized by nonionic detergents as early as1962 (33), it has been only recently that careful hydrodynamic studies the size and shape of the solubilized enzyme have been undertaken. In 1974Neer (34) used relatively standard methods to determine the sedimentationcoefficient and Stokes radius of rat renal adenylate eyelase (Table 2) andcalcdated a molecular weight of 159,000 for that enzyme. These studieswere performed in the presence of the nonionic detergent Triton X-100Osoocytylphenoxypolyethyleneoxide), which was used to solubilize the en-zyme. Adenylate cyclase displayed an apparent partial specific volume of0.74 in these experiments, suggesting that little detergent was bound to it.(This is a typical partial specific volume for a protein; that of Triton X-100is 0.94.) Neer argued, therefore, that the renal enzyme has a minimalhydrophobie surface area and probably does not penetrate the lipid bilayersignificantly. By similar experimental approaches, Neer (3 5), Haga et al (17),and Stengel & Hanoune (36) have shown that adenylate eyelase from brain,$49 lymphoma cells, and liver are larger proteins (Mr "~ 2-3 X 10~). Theseenzymes also have larger apparent partial specific volumes, which suggests

¯ significant detergent binding and, hence, a greater relative hydrophobicsurface area. In an alternative approach, Schlegd et al (37, 38) used targetsize theory to estimate the size of rat hepatic adenylate cyclase in intactmembranes. When the enzyme was assayed with MgATP as substrate, atarget size of 2.3 - 2.4 X 10~ was obtained, in surprisingly good agreementwith hydrodynamic measurements of solubilized enzyme. However, nonlin-ear decay curves suggested the presence of large aggregates within themembrane with sizes similar to those determined by Houslay et al (39).These large targets were hypothesized to represent catalytic protein inassociation with multimedc regulatory protein. Detailed studies of the sizeand shape of adenylate cyelase have not been undertaken with enzyme frommany different sources (el Table 2), so that the generality of these measure-


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8

x ~ ~~ z ~=~


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540 ROSS & GILMAN

ments is unknown. However, the interaction of the regulatory and catalyticproteins of the enzyme derived from diverse tissues and animals (see below)suggests that most tissues probably have structurally homologous enzymes.

Resolution of Catalytic and Regulatory Proteins

It had been hypothesized for several years, without much experimentalsupport, that hormone-sensitive adenylate cyclase is composed of a regula-tory protein in addition to a catalytic protein and hormone receptors. Whenthe requirement for a guanine nucleotide for hormonal activation wasnoted, it was natural that the nucleotide binding site was assumed to be onthis regulatory protein. The existence of these two distinct proteins wasrecently demonstrated by two groups. Pfeuffer made use of the atfinity ofthe regulatory protein for GTP to resolve it from the catalyst (42, 43) whileRoss & Gilman took advantage of somatic cell variants that were genet-ically deficient in one or the other protein (41, 44). Properties. of theseproteins are summarized in Table 3.

Pfeuffer (43) found that when a Lubrol PX extract of pigeon erythrocytemembranes was passed over a column of .GTP-substituted agarose, stimula-tion of adenylate cyclase activity in the extract was decreased when mea-sured in the presence of Mg2+ and either NaF or Gpp(NH)p. Elution of thecolumn with either GTP or Gpp(NH)p yielded a fraction that would com-bine with the unadsorbed fraction to restore activity, but that itself hadvirtually no adenylate cyclase activity. If elution was performed with GTP,stimulation of the recombined fractions by NaF could be demonstrated,while elution with Gpp(NH)p yielded a reconstituted mixture that wastypically activated by that nucleotide. While this separation was not com-plete (i.e. some basal and stimulatable activity remained in the unadsorbedfraction), this was the first concrete argument for the involvement of twoseparate proteins in fluoride- or guanine nucleotide-stimulatable adenylatecyclase activity. Since the affinity chromatography was based on binding ofa regulatory ligand (GTP), and because the residual activity in the unboundfraction had a differentially depleted response to fluoride and Gpp(NH)p,Pfeuffer assumed that it was the regulatory subunit of the enzyme that wasbound to the agarose and that the catalytic protein remained in the unboundfraction. In this same report, Pfeuffer also showed that solubilization ofmembranes under conditions that yielded the resolution described abovewould also solubilize a 42,000-dalton protein that had been labeled with thephotoattinity ligand GTP-~-azidoanilide. Circumstantial data arguedstrongly that this protein was responsible for the reconstitution of activityin the unbound fraction from the atfinity column (43, 45).

Ross & Gilman resolved the two proteins of adenylate eyclase by virtueof their differing thermal stabilities and their presence or absence in differentclonal cell lines. They had previously shown that a detergent extract of


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HORMONE-SENSITIVE ADENYLATE CYCLASE

Table 3 Protein components of hormone-sensitive adenylate cyclase

541

Hormone receptor

Contains hormone binding site on extracellular faceOne or more different receptors per target cell

Catalytic Protein (C)

Activity with Mg2+ " ATP less than 10% of activity with Mn2+ ̄ ATPNot stimulated by hormones, fluoride, or guanine nucleotidesMr = 190,000Sensitive to mild heating, low concentrations of sulfhydryl reagents

Guanine nucleotide-binding regulatory protein (G/F)

Confers upon C ability to use MgATP as substrateMediates regulation of C’s activity by fluoride and guanine nucleotidesBinds guanine nucleotides and fluorideProbable GTPaseContains 41,000-45,000 mol wt cholera toxin substrateAbsent in cyc- $49 lymphoma cellsMore stable to heat or sulfhydryl reagents than is C

plasma membranes that contained adenylate cyclase could, under appropri-ate conditions, recombine with membranes of a phenotypically adenylatecyclase-deficient $49 lymphoma cell (denoted cyc-) to yield hormone-sensi-tive activity (46).2 The eye- variant ceils retain fl-adrenergic.receptors (10)but lack adenylate cyclase activity assayable in the presence of MgATP,which suggests that the mechanism of the reconstitution may be the interac-tion of solubilized enzyme with B-adrenergic receptors in or on the eye-membranes. However, thermal denaturation of the soli~ble enzymatic activ-

ity at 30° led to only slightly decreased levels of activity in the reconstitutedmixture. Thus a heat-inactivated detergent extract of plasma membranescould combine with the inactive eye- $49 membranes to yield relatively highlevels of adenylate cyclase activity that could be stimulated by fluoride,Gpp(NH)p, or hormone. Similarly, the heated extract could reconstitutesoluble fluoride- or Gpp(NH)p-stimulatable activity upon combination with

a detergent extract of eye- membranes. These authors argued that the eye-membranes (or extracts therefrom) were supplying a heat-labile factor in-trinsic to adenylate cyclase which was destroyed by the heating of thecomplementary extract; and that the heated extract was hypothesized toprovide a second, more stable component that the eye- cells lacked. Sen-

2Clones of eye- variant S49 cells were selected from wild-type cells by Bourne et al (47). $49cells are killed by elevated intracellular concentrations of cyclic AMP (48). Therefore agentsthat activate adenylate cyelase in vivo, such as cholera toxin or fl-adrenergic agonists, maybe used for such selection.


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542 ROSS & GILMAN

sitivity to proteases, sulfhydryl reagents, and temperature suggested thatboth factors were proteins.

Ross et al (41) were led to propose that the more heat-labile protein,which is retained in cyc- $49 cells, is the catalytic protein of adenylatecyclase. This suggestion derived from the finding that cyc- membranes (orextracts thereof) contain a Mn2+-dependent adenylate cyclase activity. Theprotein that displays this Mn2+-dependent activity has hydrodynamic prop-erties identical to those of the protein that is active in the reconstitution.The two activities are also similarly thermolabile and are similarly stabilizedand labilized by a variety of mixtures of nuclcotides and divalent cations,which suggests that the rcconstitution factor is a Mn2+-dcpcndcnt adcnylatecyclase. It is therefore likely that the Mn2+-dependent activity is a non-physiological manifestation of thc catalytic protein of adcnylatc cyclasc.This protein is referred to as C.

From these and following studies it is apparent that a minimum of twoproteins arc necessary for the expression of physiological adcnylatc cyclascactivity and that a third protein, the receptor, is the site of hormone binding.If these proteins are not tightly bound to each other, the prospects ofpurifying "holoadcnylatc cyclasc" may bc bleak. Homey ct al (49, 50) havereported about 5000-fold purification of the cardiac enzyme after stabiliza-tion of the complex with sodium fluoride, but this success is nearly uniqueand has not yet bccn exploited further. Various claims of the purificationof adenylatc cyclasc to homogeneity (51, 52) arc not convincing, in partbecause of the very low specific-activities of the products.

Properties of the Catalytic Protein¯ Little is currently known about the physical properties of the catalytic

protein (C) of adenylatc cyclase. This paucity of information is duc to thelability and difficulty of preparation of resolved C. The best characterizedpreparation of C is in a crude Lubrol 12A9 extract of the cyc- $49 cellplasma membrane (41, 44). More promising preparations may result fromefforts by Pfeuffer to purify C from the unbound fraction in his affinitychromatographic procedure (53) and from Ross’s separation of C from G/Fby gel exclusion chromatography of a somewhat stabilized cholatc extractof hepatic plasma membranes (E. M. Ross, in preparation). Londos ct (54) have also reported the solubilization with deoxycholate of 2+-

dependent adcnylatc cyclase activity from liver, but the ability to reconsti-tute activity in the presence of MgATP upon addition of G/F was notexplored. Ross ct al (41) reported a molecular weight of C from cyc- $49cells of 1.9 X 105 (see Table 2), and Schlegcl et ai (38) found, by target analysis, that Mn2+-dependent activity from liver has a volume correspond-ing to a mass of 1.5 X 105 daltons. Nothing is known of a possible subunitcomposition of C. Determination of partial specific volume makes it seem


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likely that C has a relatively large hydrophobic surface area, as is consistentwith the ability of cholate-solubilized C to be reincorporated into monolam-ellar phospholipid vesicles upon the removal of detergent (E. M. Ross, inpreparation).

The MnATP-dependent adenylate cyclase activit~ of C is sensitive toseveral proteases and sulfhydryl reagents (44). Evidently a second, morereactive, cysteine residue is necessary for the interaction of C with theregulatory protein, since N-ethylmaleimide destroys the reconstitutive ac-tivity of C at a concentration more than tenfold below that which inhibitscatalysis (41).

C must have a substrate site for ATP, presumably as a divalent cation-ATP complex, but it is not known if this is the same site at which the effectsof nucleotides and metals on stability are exerted. There is at least oneregulatory divalent cation-binding site associated with adenylate cyclase(55), but it need not be located on C. There is some evidence for a divalentcation site on the regulatory protein (56).

It should be noted that there exist two other Mn2+-dependent adenylatecyclase activities in mammalian tissues, but neither appears to be related toC. Mittal & Murad (57) showed that guanylate cyclase, upon activation free radicals, can utilize ATP as an alternative substrate and catalyze theformation of cyclic AMP. However, guanylate cyelase from $49 cells ismore thermostable than is C (41), and Mn2+-dependent adenylate cyclaseactivity attributable to C is much higher than is guanylate cyclase in thesame preparation of eye- membranes. A crude preparation of the guanine-nucleotide-binding regulatory protein also failed to confer upon hepaticguanylate cyclase the ability to utilize MgATP as substrate. A solubleMn2+-dependent, hormone-insensitive adenylate cyclase has also beenfound in testis (40, 58). However, it does not interact productively with theregulatory protein (41) and is much smaller than C (40, 41).

Guanine Nucleotide-Binding Regulatory Protein (G/F)

Much more is known about the molecular characteristics of G/F than isknown about C because of the former’s greater stability and ease of prepara-tion and the ease with which it (or, at least, one of its subunits) can radioactively labeled. Several different preparations of G/F, resolved fromC, are now available in different states of purity. The G/F protein of pigeonery~hrocytes was initially separated from C by affinity chromatography onGTP-substituted agarose by Pfeuffer (43, 45) and subsequently by Spiegelet al (59). Purification by this procedure (about 60-fold) can be improvedupon by subsequent sucrose density gradient centrifugation (43). Mam-malian G/F was resolved from plasma membranes of various tissues andcultured cells by the thermal or chemical inactivation of C and was initiallycharacterized in such crude preparations (41, 44). t3/F from rabbit liver


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plasma membranes has now been partially purified (perhaps 1000-3000-fold over the activity in crude extracts) following solubilization by cholate(J. K. Northup, P. C. Sternweis, and A. G. Gilman, unpublished data).While these techniqu, es denature the activity of the catalytic protein, it islikely that C is physically removed during the early steps in the preparation.G/F is also found free of C in the plasma membranes of certain clones ofHTC hepatoma cells and in detergent extracts therefrom (41). These cellsare phenotypically very similar to the cyc- $49 cell variants, and the exis-tence of these two complementary clones helps to substantiate the in vivorequirement for both the G/F and C proteins for adenylate cyclase activity.

Initial data on the composition of G/F came from the work of Pfeuffer(43), who used [32p]GTP-~-azidoanilide to label pigeon erythrocyte mem-branes. Of four specifically labeled proteins, the fractionation of a 42,000-dalton band on dodecyl sulfate-polyacrylamide gels was consistent with itsinvolvement with adenylate cyclase. Further support for the involvementof a 42,000-dalton protein with G/F function derives from studies usingcholera toxin. Strong evidence, reviewed in the previous volume (60), sug-gests that the crucial step in the stimulation of adenylate cyclase activityby toxin is the ADP-ribosylation of the protein involved with regulation ofactivity by guanine nucleotides. Using [32p]NAD as the substrate for thetoxin, both Gill & Meren (61) and Cassel & Pfeuffer (45) were able to labelprimarily a 42,000-dalton protein in pigeon erythrocyte membranes underconditions where stimulation of adenylate cyclase is optimal. ADP-ribosy-lation of this protein has both time and temperature dependence and re-quirements for GTP and a cytosolic protein that are strikingly similar tothose observed for activation of adenylate cyclase (62, 63). The 42,000-dalton protein also binds to GTP-agarose and is eluted in parallel withreconstitutive G/F activity (45). Johnson et al (64) have similarly labeledwild-type $49 cell plasma membranes with cholera toxin and [32p]NAD.Johnson et al (65) and Howlett et al (67) demonstrated that G/F, ratherthan C, was the probable site of action of the toxin. It was therefore ofinterest that Kaslow et al (64, 66) could label a 45,000-dalton protein membranes of wild-type $49 cells (phenotype +, G/F+), o r HTC cells a ndhuman erythrocytes (C-, G/F+) but not in the cyc- $49 cell variant (C+,

G/F-), which supports the association of this protein with G/F function.A 45,000-dalton protein in hepatic plasma membranes is also ADP-ribosylated by cholera toxin, and coelectrophoreses with a major Coomasieblue-stained band in untreated, partially purified preparations of G/F. Thisprotein is also labeled when cholera toxin is used to label eye- membranesthat have been previously reconstituted with hepatic G/F (67a).

Initial hydrodynamic studies of native solubilized G/F are not consistentwith the 4.2 X 104 - 4.5 X 104 molecular weight obtained in dodecyl sulfate.Howlett et al (56) find varying molecular sizes for G/F from $49 lymphoma


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cells depending upon the presence or absence of regulatory ligands. Calcula-tions of Mr range from 9 X 104 - 1.3 X l05. Pfeuffer (53) has also determinedthat the S2o, w of G/F varies with ligand present (GTP-’z-S versus GDP) a range consistent with a monomer-dimer interconversion. Whether thesediscrepancies truly represent the formation of a dimer or trimer or theassociation of dissimilar proteins is uncertain. An intriguing and perhapsrelated finding by Johnson et al (64) is that cholera toxin catalyzes theADP-ribosylation of two other proteins in wild-type $49 cell plasma mem-branes (Mr ~" 5.2 - 5.5 X 104) which are also absent in the cyc- (i.e’.G/F-deficient) variant. Whether these proteins are related to G/F or notis unclear, since human erythrocytes display G/F activity but lack thelarger proteins as detected by cholera toxin-catalyzed ADP-ribosylation(66). The kinetics of the thermal denaturation of G/F activity also led speculation as to its possible subunit structure. It was found that, uponheating at 50° C, the ability of crude G/F to reconstitute Gpp(NH)p-stimulated activity decayed about twice as fast as its ability to reconstitutefluoride-stimulated activity. It was hypothesized that one protein, "F",might restore Mg2+-dependent, fluoride-stimulated activity, and that a sec-ond protein, "G", might mediate the guanine nucleotide responses of an"F.C" complex (41, 44). The copurification of these two activities nowmakes this idea less appealing, as does the finding that GTP or a mixtureof fluoride plus ATP stabilizes both activities.

It is likely that G/F represents the binding site for ligands that activateadenylate cyclase in the presence of MgATP. The affinity chromatographicprocedure of Pfeuffer and his affinity labelling of a 42,000-dalton proteinboth imply that G/F contains a binding site for GTP (43, 59). This is alsosupported by the findings that G/F activity is stabilized by GTP orGpp(NH)p (41), and that its sedimentation coefficient is decreased in presence of GTP-’/-S or Gpp(NH)p (53, 56). Similarly, fluoride plus divalent cation (Mn2+ or Mg2+) decreases the sedimentation coefficient ofG/F (56), which suggests that fluoride also binds to this protein. Effects guanine nucleotides and fluoride upon resolved G/F are generally revers-ible, in contrast to their irreversible or poorly reversible effects upon intactadenylate cyclase (67).

UNC Lesion: Coupling Factor or Covalent Modification

Selection of $49 lymphoma cells in medium containing/~-adrenergic ago-nists yields clones of a second resistant phenotype in addition to cyc-. Thesecells retain plasma membrane adenylate cyclase activity, which is stimu-lated by fluoride or GppfNH)p in the presence of Mg2+, and the intact cellsrespond to cholera toxin with increased production of cyclic AMP. How-ever, these cells have lost responsiveness both to/~-adrenergie agonists, the


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selecting agent, and also to prostaglandins E1 and F-,2. Since they retainfl-adrencrgic receptors, as assayed by the binding of [125I]iodohydroxybcn-zylpindolol, the lesion appears as an uncoupling of enzyme and receptor--hence the name UNC, for uncoupled phenotype (68). The UNC adenylatecyclase system is thus similar to the adenylate cyclase systems of tempo-rarily refractory astrocytoma cells (69, 70), adipocytes of hypothyroid rats(71), and those systems artificially uncoupled by treatment with phospholi-pases (72), the polyene antibiotic filipin (73-76), or certain other amphi-philic compounds (73). The lesion is not total, since a small response prostaglandins (10% as opposed to tenfold in wild type) is noted, andisoproterenol slightly stimulates the rate of activation of the enzyme byGpp(NH)p (68). The cyc- lesion (loss of the (3/F protein) and lesion are not complementary with regard to hormonal stimulation of theenzyme as assayed by reconstitution protocols (15, 46, 77) or in somatic cellhybrids (78). It can be inferred, therefore, that either cyc- is deficient bothin G/F and a putative "UNC factor" or that (3/F in UNC cells is somehowdefective. Sternweis & Gilman demonstrated that a crude preparation of(3/F from wild-type $49 cells or rabbit liver can restore responsiveness tohormone to UNC plasma membranes (77), and the ability to reconstitutehormone responses in UNC membranes cofractionates several thousandfold with (3/F (P. C. Sternweis and A. (3. (3ilman et al, unpublished). UNC plasma membranes are labelled with [32p]NAD and cholera toxin, itis also observed that the 45,000-dalton protein characteristic of G/F isshifted to a more acidic isoelectric point (67a). These results, taken together,provide a strong argument that the UNC lesion represents a modification(or lack of required modification) of the G/F protein such that it can longer fulfill its role as a coupling factor between receptor and C. The UNClesion also abolishes control by guanine nucieotides of the affinity of hor-mone-binding to receptor (68). This loss is also restored by rcconstitutionwith crude G/F (77). It is tempting to speculate that the molecular defectfound in the UNC variant is the site of physiological regulation of G/Ffunction in some of those physiologically uncoupled states mentioned above(69-71).

Calcium-Dependent Regulatory Protein

The calcium-dependent regulatory protein (CDR, calmodulin) is an acidic,low-molecular-weight, ubiquitous, Ca2÷-binding protein of mammaliancells. It is recognized as the mediator of Ca2+-dependent control of anincreasingly large number of enzymes (79, 80). In 1975, Brostrom et al (81)and Cheung et al (82) showed that adenylate cyclase in Lubrol extracts cerebral cortex particulate fractions displayed a requirement for CDR forthe stimulatory effects of Ca2+. The requirement in the extracts was demon-


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strated after the chromatographic removal of endogenous CDR. Elution ofendogenous CDR by EGTA can also allow the demonstration of regulationof membrane-bound enzyme by CDR (83-85). There is a wealth of kineticevidence that CDR. Ca2+ is the active species and that free CDR has littleeffect. Brostrom ct al (86) have also demonstrated an enhancement Ca2+ of the hormone-stimulated accumulation of cyclic AMP in C6 gliomacells, which supports the physiological relevance of the action of CDR.CDR has not, however, been shown to have any effect in preparations froma number of cells whose adenylate cyclase does not normally respond toCa2+ (86, 87).

The mechanism of the effect of CDR-Ca2+ is just beginning to be studiedin detail. Storm and co-workers have recently separated two fractions ofbrain adenylate cyclase by virtue of the affinity of one fraction for CDR-substituted agarose (88). They find that the species that does not bind unresponsive to CDR-Ca2+ and is also unresponsive to Gpp(NH)p andfluoride. Some response to these ligands can be restored to this fraction bythe addition of a diluted crude membrane extract, and the authors speculatethat t3/F is the protein that restores stimulation by t3pp(NH)p, fluoride,and CDR-Ca2+ (89). The interaction of CDR-Ca2+ with G/F is also consis-tent with the finding by Moss & Vaughan (90) that CDR-Ca2+ is requiredfor the activation of detergent-solubilized rat brain adenylate cyclase bycholera toxin. Why CDR-Ca2+ activates some adenylate cyclases and notothers is of great interest and is presumably subject to analysis by reconsti-tution protocols. It is unknown whether unresponsive systems lack themechanism for interaction with CDR-Ca2+ or whether CDR is so tightlybound to "unresponsive" cyclase as to be unremovable, as is the case withthe phosphorylase kinase-CDR complex (91). The lack of sensitivity unresponsive systems to EGTA or to a wide range of Ca2+ concentrationsargues for the former explanation (87).

Other Protein Factors

During preparation of mammalian plasma membranes, a variable andsometimes substantial amount of adenylate cyclase activity or responsive-ness to activators is lost. Many investigators have noticed amelioration ofthis loss by resuspension of the membranes in the cytosolic fraction (e.g.92-95). Most of these findings probably relate to loss of nucleotides or metalions. Pecker & Hanoune (92, 93) reported that the stimulatory activity rat liver cytosol was at least 80% sensitive to treatment with phosphataseand was less active than GTP. Katz et al (94) have described a cytosolicfraction of rat liver with similar properties, but have claimed that it wassensitive to proteases and not dialyzable. The absence of chromatographiccharacterization and the lack of a linear assay make the data difficult to


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evaluate. A number of investigators have noted alterations in the activityof adenylate cyclase upon extraction or treatment with detergents and haveinterpreted them in terms of the removal of protein factors. Bradham foundthat extraction of rat brain membranes with Lubrol PX decreased theresponsiveness to fluoride ion of activity that remained in the pellet, andreturn of the detergent extract yielded a responsive preparation (96). How-ever, the extract was itself stimulatory after treatment with trypsin or heatand data on .the recovery of total activity are not available. Similar experi-ments by Cuatrecasas’s group, who used multiply extracted brain mem-branes and solubilized liver adenylate cyclase, are also unclear (97, 98).Both groups may have removed some G/F, some CDR, or both. It cannotbe stressed too often, however, that adenylate cyclase activity and its re-sponsiveness to stimulators are exquisitely sensitive both to detergent con-centration and to the ratio of detergent to lipid and protein. This sensitivitycan be biphasic (stimulatory and inhibitory) and can be reflected differen-tially when different activators [e.g. fluoride, Gpp(NH)p] are used. almost arbitrary ratio of activities assayed in the presence of these twoligands can be achieved by manipulation of the concentration of detergentsand salts. Hence, it is absolutely essential to monitor total activity and touse appropriate detergent and detergent plus protein controls.

Reconstitution of Hormone-Sensitive Activity

Reconstitution of hormone-sensitive adenylate cyclase from purified com-ponents is an obvious prerequisite to detailed studies of the mechanism oftheir interaction. Progress toward this goal has so far been limited.Sehramm’s group and others have utilized membrane fusion to allow theinteraction of proteins present in the membranes of different cells (11-15,99) (see above). After fusion of complementary cells or of membranes fromdifferent cells, the relevant proteins apparently diffuse laterally throughoutthe hybrid membrane such that they can interact productively. While proto-cols of this sort have potential as a valuable assay for the presence ofreceptors, G/F and C, they are limited in that intact membranes must beused. An attractive development would of course be the insertion of solubil-ized, purified proteins into monolamellar liposomes prior to reconstitutionby fusion of different liposomal preparations or of liposomes with mem-branes.

Ross & Gilman (41, 46) have demonstrated that membranes of cyc- $49lymphoma cells, which are deficient in G/F protein, can be reconstitutedby the addition of detergent-solubilized G/F to yield hormone-stimulatableactivity. The physical mechanism by which the G/F reassociates with thecyc- membrane is obscure and is possibly dependent on the detergent usedto solubilize G/F. If G/F solubilized by Lubrol 12A9 is mixed with cyc-


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membranes, hormone-stimulated activity can be assayed in the mixture.However, if the mixture is centrifuged, G/F activity remains soluble andcan be separated from the apparently unaltered cyc- membranes (46). Stableattachment of Lubrol-solubilized G/F to the cyc- membranes is only ob-served if the mixture is first incubated with an irreversible activator of theenzyme [Gpp(NH)p or a mixture of fluoride plus ATP or ADP] and yieldsan irreversibly activated enzyme that cannot be stimulated further by hor-mones (67). If G/F is solubilized with cholate, however, mixing of G/F andcyc- membranes in the presence of MgATP (followed by dilution of cholateand incubation above 4° C) yields stable binding of G/F to the membranewithout permanent activation of the enzyme. Sternweis & Gilman (77) haveused this protocol to reconstitute what is in most functional respects awild-type membrane, using either eye- or UNC membranes as a startingpoint.

The reconstitution of solubilized catalytic protein or of hormone recep-tors into either depleted membranes or phospholipid vesicles has provenmore ditficult, and there is only one plausible report of the reconstitutionof hormone-stimulated activity using totally soluble proteins. Hoffmann(100, 101) has described the reconstitution of dopamine-stimulated adeny-late cyclase activity by a cholate-dilution protocol. The starting material isa cholate extract of membranes from bovine caudate nucleus mixed withasolectin. Other data suggest that the components of the system can alsobe partially resolved before reconstitution and that substitution of differentdetergents is feasible (101). This is quite a promising technical advance,since, if it is generally applicable, it will allow the use of increasingly purifiedproteins and lipids in reconstituted systems.

EFFECTS OF LIPIDS AND MEMBRANE STRUCTURE

It is clear that at least some of the protein components of the adenylatecyclase system must be bound to an appropriate membrane structure if theenzyme is to respond to hormones. However, the role of the structure andcomposition of the plasma membrane in the regulation of adenylate cyclaseactivity is one of the most confusing and least carefully documented areasin the study of this enzyme. The basic observation is that solubilization ofthe plasma membrane or the addition of any number of agents that disruptmembrane structure causes a loss of responsiveness to hormone. Whilesolubilizations of active Mg2+-dependent adenylate cyclase, C, G/F, andreceptors have been documented, there is to our knowledge no report ofsoluble, hormone-stimulatable activity in which both the specificity of theeffect of hormone and the true solubility of the preparation have beendemonstrated. The latter criterion has been conspicuously absent from the


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literature until recently; mere failure to pellet activity at 100,000 X g isinadequate.

The role of the membrane probably can be best considered in terms ofwhat a membrane provides: a permeability barrier and a topologicallyclosed asymmetric surface, a mechanism for the variable and regulatableassociation and segregation of molecules within a structured millieu, ahydrophobic environment and a stable hydrophobic-hydrophilic interface,and the opportunity to interact with a variety of specific lipid molecules.Each of these properties has been shown to be important for some enzymeor transport system.

There is little evidence to suggest that coupling between hormone recep-tors and adenylate cyclase is obligately involved with plasma membranetransport or with the existence of a membrane potential, 3 although cyclicAMP itself is involved in the regulation of numerous transport processes(105). While Moore & Wolff (73) have demonstrated that various iono-phores uncouple the TSH-stimulated adenylate cyclase system at lowerconcentrations than those needed to inhibit enzymatic activity, these con-centrations are higher than those needed for ionophoric activity in othermembrane systems and the effect possibly reflects disruption of bilayerstructure. We have also found that a number of proton and cation iono-photos do not prevent stimulation of adcnylatc cyclasc by catecholaminesin membranes of wild-type $49 cells at concentrations that arc sufficient touncouple oxidative phosphorylation (El M. Ross, unpublished). Since theionophores and such unrelated compounds as filipin [a polyene antibioticthat can grossly disrupt membranes by interaction with cholesterol (106)],nonionic detergents, cholatc, and phenothiazines all cause qualitativelysimilar multiphasic effects on adenylate cyclase as a function of increasingconcentration (stimulation, uncoupling of hormonal activation, and inhibi-tion), a primary action on the structure of the bilaycr seems more likely thandoes an increase in membrane permeability. Arguing for a direct relation-ship between ion gradients and adcnylatc cyclase, Grollman ct al (107)showed that thyrotropin causes hyperpolarization of cultured thyroid cellsand, in the presence of 50 mM chloride ion, of plasma membrane vesiclestherefrom. This effect preceded activation of adenylate cyclase by 4-5 min.Although the proton ionophore CCCP (2.5 ktM) abolished the membranepotential, its effect on TSH-stimulated adenylate cyclase was not reported;any causal relationship between effects of TSH on membrane potential andon adenylate cyclase is thus uncertain.

A number of membrane-bound enzymes have been shown to require aspecific lipid for activity or to be specifically influenced by one or more

3The situation is probably different among fungi and bacteria (102-104).


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lipids, and such suggestions have been advanced for adenylate cyclase.There exist numerous reports on the stimulatory effects of phospholipids,particularly acidic phospholipids, on preparations of adenylate cyclase thatwere previously perturbed by detergents, organic solvents, or phospholi-pases (108-111). Most of these early findings, including Levey’s provocativereports on hormone-specific requirements for phospholipids (112, 113),have been neither reproduced nor exploited further. In general, no particu-lar effort was made to promote the reassociation of the lipid (frequentlyadded in bulk form) with the membrane or with soluble adenylate cyclase,nor are there data to indicate whether added lipid interacted with themembrane or merely bound detergent (including endogenous fatty acids,lysophosphatides, etc). The best-documented study of this sort is probablythat of Rubalcava & Rodbell (72), who showed that treatment of rat liverplasma membranes with a phospholipase C with some specificity for acidicphospholipids abolished stimulation of adenylate cyclase by glucagon with-out inhibiting fluoride-stimulated activity. Concurrently, the Ka for bindingof glucagon was increased and the effect of GTP on glucagon binding waslost (72, 76). Similar results were obtained with a phospholiPase A2. neither ease was there a report of restoration of activity after treatment.

An alternative approach to the question of requirements for lipids hasbeen to alter metabolically the phospholipid composition of intact plasmamembranes. Engelhard et al (114, 115) grew LM fibroblasts in media sup-plemented with different ethanolamine/choline analogues. They found acorrelation between the degree of substitution of the ethanolamine nitrogenin membrane phospholipids and the prostaglandin El-stimulated adenylatecyelase activity. However, when GTP was added to the assay the effect wasmuch less striking, which suggests that variable contamination of mem-branes with endogenous GTP may have occurred. A variation of the Kmfor ATP as a function of phospholipid head group was also noted. The dataof Hirata et al (116) on effects of the methylation of phosphatidylethanola-mine to phosphatidylcholine on hormonal stimulation of the enzyme areintriguing but preliminary. A specific role of cholesterol is even less clearthan is that of phospholipids. Klein et al (117) noted a monotonic decreasein adenylate cyclase activity with increasing amounts of cholesterol in some(but not other) clones of cultured kidney cells, but Hanski & Levitzki (118)noted the opposite effect in turkey erythroeytes.

The lateral distribution and association of the protein components ofadenylate cyclase may be crucial to hormonal regulation of adenylate cy-clase. The notion of an enzyme (or perhaps free G/F, free C, and G/F plusC) and a receptor "floating" in a fluid mosaic bilayer is probably adequate,but their absolute freedom of motion is unclear. Physically, this motion hasnot been defined at all. The apparent ability of multiple receptors to corn-


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pete for a pool of adenylate cyclase molecules, as occurs in the multireceptoradipocyte~ system (2), and the ability of receptor from one cell to activateenzyme from another in the membrane of a heterokaryon within minutesof fusion (13) argue for at least relative freedom. On the other hand,Sahyoun et al (119) have found that pretreatment of frog erythrocytes withisoproterenol alters the distribution of fl-adrenergic receptors among subse-quently prepared membrane fractions, which suggests some restriction oftheir lateral distribution. Varga et al (120) were able to demonstrate cluster-ing of MSH receptors by labelling Cloudman melanoma cells with12~I-MSH, either at 0°C or after fixation with paraformaldehyde. Maguireet al (121) have also argued against totally random distribution of fl-adren-ergic receptors and enzyme based on their apparent stoichiometric relation-ship in small membrane vesicles prepared from $49 cells.

The effects of the physical state of the plasma membrane bilayer on theadenylate cyclase system are being studied in increasing detail. Severalgroups have tried to correlate adenylate cyclase activity and its regulationwith the physical state of endogenous membrane lipids by studying thetemperature dependence of catalysis in the presence of various activators(113, 122-i25). Numerous linear, upward and downward curved, and mul-tiphasic Arrhenius plots have been produced, but few general conclusionscan be drawn from these studies. More direct attempts to relate the fluidityof the bilayer to regulation of adenylate cyclase have involved enrichmentof isolated membranes with different exogenous phospholipids. Houslay ¢tal (126, 127) enriched hepatic plasma membranes with synthetic phos-phatidylcholines and were able to relate, qualitatively, changes in the tem-peratures of inflections of Arrhenius plots of glucagon-stimulated activitywith the thermotropic properties of the lipids used. No inflection was ob-served when fluoride-stimulated activity was measured. This study was thefirst to suggest strongly that the state of the bilayer might mediate a rate-limiting process in hormonal activation. Recently, Bakardjieva et al (128)enriched membranes of Chang liver cells with different phospholipids andcompared adenylate cyclase activity of membranes high in dimyristoylphos-phatidylcholine (DMPC) with membranes high in dipalmitoylphos-phatidylcholine (DPPC) or dioleoylphosphatidylcholine (DOPC). At 37°C,above the gel-liquid crystal transition temperature (T,~) of DMPC andDOPC, the membranes enriched in these lipids had lower adenylate cyclaseactivity than did control preparations or DPPC-enriched membranes. At17°C, below the Tm of DMPC but above that of DOPC, the membranesenriched in DMPC or DPPC had roughly equivalent activities that werehigher than those of the DOPC-endched membranes. It thus appeared asthough a relative decrease of activity occurred above Tm. Since the differ-


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ence appeared to be most marked when isoproterenol-stimulated activitywas measured, binding to B-adrenergie receptors was measured and foundto selectively de~rease at temperatures above the T,n of the added lipid. Itis not known whether the changes in activity and in the number of assayablefl-adrenergic receptors occur by the same or different mechanisms.

A third finding with relevance to the effects of membrane fluidity uponadenylate cyclase is that of Levitzki and co-workers, who demonstrated thatcis-vaccenic acid causes an increase in the rate of activation of turkeyerythrocyte adenylate cyclase by Gpp(NH)p plus epinephrine. These exper-iments were based on the finding of Orly & Schramm (129) that A9-10 Al1-12 cis-monounsaturated fatty acids stimulate adenylate cyclase inthese membranes. Levitzki and co-workers (130, 131) found that the in-crease in the rate constant for activation by Gpp(NH)p plus epinephrinewas inversely related to the "microviscosity" of the bilayer, as assayed bythe fluorescence anisotropy of 1,6-diphenyl-l,3,5-hexatriene (132, 133).Since Gpp(NH)p does not markedly stimulate activity by itself in thesemembranes, the authors interpreted their results as indicating that collisionof receptor and enzyme, controlled by their rates of lateral diffusion andhence by the viscosity of the membrane, was the rate-limiting factor inactivation by Gpp(NH)p plus hormone. These experiments do not of courseindicate which motions are limited, which proteins are involved, or even ifthe apparent effect of microviscosity is on the motion or conformation ofa protein. However, taken together with the studies of Bakardjieva et al(128) and Houslay et al 026, 127), they begin to suggest the major role thatthe structure of the bilayer plays in the regulation of the system.

REGULATION OF ADENYLATE CYCLASEACTIVITY

Most studies of the regulation of adenylate cyclase activity by hormones,nucleotides, and other ligands have been undertaken with a view towardunderstanding the physiological regulation of cyclic AMP metabolism in aparticular tissue. Consequently, there exists a great mass of data derivedfrom many different cells, frequently obtained using crude homogenates,and only rarely directed toward understanding the primary biochemicalmechanism of that regulation. In this section we draw upon only a smallnumber of studies in an attempt to organize what mechanistic informationis available and to correlate it with knowledge of the individual proteins.Regulation by divalent cations has been discussed elsewhere (55), and still know frustratingly little about the molecular actions of fluoride. Hence,


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554 ROSS & GILMAN

we will stress the interacting effects of hormones and guanine nucleotideson the activity and association of G/F and C.

Regulation by Guanine Nucleotides

Much of our information on the regulation of hormone-stimulatable adeny-late cyclase stems from observations on the effects of guanine nucleotides,first noted by Cryer et al (134) and studied more extensively by Rodbell andco-workers (135-138). GTP, GDP, or ITP by themselves display diverseinhibitory and sfimulatory effects on adenylate cyclase activity, and thesevary with cell type, pH, divalent cation concentration, ionic strength, pres-ence of detergent, and probably a dozen other variables. The key observa-tion, however, is that the presence of some guanine (or related purine)nucleotide is an absolute requirement for the stimulation of adenylate cy-clase activity by hormones (136). This requirement has been observed plasma membranes of a number of different cell types (121) but is oftendifficult to demonstrate due to the presence of contaminating guanine nu-cleotides in the ATP used as substrate (139) or in the membrane preparation(140), and to the possible ability of ATP itself to serve this function at highconcentrations. This effect of guanine nucleotides is also suggested to besignificant in vivo by the finding that depletion of intracellular GTP bygrowth of cells in the presence of mycophcnolic acid decreases the intracel-lular accumulation of cyclic AMP in response to hormones (141-143). addition to the requirement of guanine nucleotides for hormonal activationof adenylate cyclase, the concentration of hormone necessary to stimulateactivity is altered by the identity of the nucleotide used in the assay. Thus,isoprotcrenol causes half-maximal stimulation of adenylate cyclase in $49cell membranes at 100 nM in the presence of GTP, 50 nM in the presenceof ITP, 20 nM in the presence of XTF, and 15 nM in the presence ofGpp(NH)p (at t=0; see below) (140).

Rodbell and co-workers, using [12~I]iodoglucagon, were also first to ob-serve that those purine nucleotides that permit hormonal activation ofadenylate cyclase also frequently decrease the affinity of the receptor forhormone (144). This effect has now been described in some detail forreceptors for glucagon (19, 76, 145), prostaglandin l ( 146, 147), aB-adrenergic agonists (140, 148-150), and has also been observed withreceptors for TSH (73) and FSH (151). The effect is specific for agonist antagonist) ligands of the receptor (148, 149). It should be noted that thisinteraction of regulatory nucleotides and hormones is not competitive butrather is a negative heterotropic binding interaction. Thus GTP, ITP, andGpp(NH)p all decrease the affinity of the receptor for hormones to an equalmaximal extent, although with varying potency (140, 144, 147, 149), andact by increasing the dissociation rate constant for hormone (19, 144, 150).


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It has been inferred by us and others that the ability of nucleotides to alterbinding al~nity for hormone reflects association of receptor with adenylatecyclase [most probably with G/F (41)]. This inference is based on severalobservations: (a) the effect is lost when the enzyme is inactivated withsulfhydryl reagents (O/F must bc inactivated--inactivation of C is insufli-cient) (75, 150); (b) it is absent in UNC and cyc- variant S49 cells in G/F is either absent or altered (68, 140); (c) it is restored by reconstitutionof UNC or eye- membranes with crude preparations of G/F (77); (d) it lost when the fl-adrenergic receptor and adenylate cyclase are uncoupledby the polyene antibiotic filipin (74, 75); (e) it is lost upon solubilization(and hence, uncoupling) of/~-adrenergic receptors (150); and (f) it is only in those fl-adrenergic systems that display highly ctficient couplingbetween hormone binding and activation of adenylate cyclase (121).

More information on the mechanism of hormonal control came from theuse of poorly hydrolyzable analogues of GTP [Gpp(NH)p, Gpp(CH2)p,t3TP-T-S]. Although Gpp(NH)p was introduced as a substitute for GTPin assays where hydrolysis of nucleotide was likely, it quickly becameapparent that it was unique in that Gpp(NH)p almost invariably activatedadcnylatc cyclasc in the absence of hormone, the activation was frequentlygreater than that caused by fluoride or by GTP plus hormone, activationwas either irreversible or poorly reversible, and adenylate cyclase was muchmore stable after activation (42, 138, 152-154). While hormones andGpp(NH)p may appear to act synergistically, it was shown by Bennett,Jacobs & Cuatrecasas (155, 156) and others (140, 153) that the effect hormone is only to increase the rate of activation by Gpp(NH)p rather thanto increase the final activity attainable. The intensively studied turkey ery-throcyte enzyme, where activation by Gpp(NH)p appears to be totallydependent on hormone, is probably only an extreme case (129, 153). have already stressed that the irreversibility of activation by Gpp(NH)p andthe effect of hormones upon the rate of activation make the quantitativestudy of the kinetics of activation by this nucleotide somewhat treacherous(121). There is, in essence, no defined Kact for hormone under these condi-tions unless extrapolation to zero time is made (140), and few have tried quantitate accurately the effects of hormone on activation kinetics (131,140, 157-159). Furthermore, any calculation of an incremental activity dueto hormone is a function of time. Many misleading conclusions have beengenerated by the application of equilibrium assumptions to the analysis ofdata gathered using Gpp(NH)p.

Catecholamine-Stirnulated GTPase

In spite of experimental pitfalls, the data obtained with Gpp(NH)p,Gpp(CH2)p, and ITP led a number of investigators to speculate on the role


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556 ROSS & GILMAN

of either GTP hydrolysis (140, 160, 166) and/or phosphotransferase reac-tions (161) as a mechanism of regulation of adenylate cyclase. More impor-tantly, it led Cassel & Selinger (162) to discover a catecholamine-stimulatedGTPase activity in turkey erythrocyte membranes and to relate this activityto the regulation of adenylate cyclase. The specific activity of the catechola-mine-stimulated GTPase is low (less than 10 pmol min-1 mg-l) and isassayed above a somewhat larger basal activity. App(NH)p must be in-cluded in the assay to inhibit nonspecific nucleoside triphosphatases, andeven this strategy is insufficient to assay the enzyme in hepatic plasmamembranes, where background activity is 50-fold greater than that in tur-key erythrocyte membranes. Nevertheless, these authors showed that thepharmacological specificity, Km for GTP, and inhibition by either phos-pholipases, detergent, or GTP-T-S were all consistent with the involvementof this activity with the hormonal control of adenylate cyclase (162, 163).Perhaps their most provocative finding, however, was that treatment ofplasma membranes with cholera toxin markedly inhibited the catechola-mine-stimulated GTPase activity without decreasing the "basal" or back-ground level (164). The dependence of this inhibition on the concentrationsof toxin and NAD is similar to that observed for the activation of adenylatecyclase. The decrease in GTPase activity is also consistent with the observa-tions of others that cholera toxin alters the regulation of adenylate cyclaseby GTP so as to make GTP appear similar to the poorly hydrolyzablcGpp(NH)p; i.e. treatment with toxin causes activation by GTP to be greaterand longer lasting, increases the potency of hormone in the presence ofGTP, and decreases the incremental activity caused by hormones (140,165-168).

These data led Cassel & Selinger to propose an explicit model for theregulation of adenylate cyclase by hormones and guanine nucleotides basedupon the binding and hydrolysis of GTP (164, 165); the model was quitesimilar to the proposal of Levinson & Blume (166). Drawing heavily uponknowledge of the interaction of the prokaryotic translational elongationfactors Tu and Ts (169), Cassel & Selinger proposed a regulatory GTPasecycle in which GTP-liganded adenylate cyclas¢ was the active species andhydrolysis of GTP to GDP was the primary mechanism of inactivation.Fractional activation of adenylate cyclase would thus be proportional to thesteady-state concentration of the GTP-liganded state. According to themodel, inhibition of GTP hydrolysis by cholera toxin would sustain activa-tion, as would the binding of a nonhydrolyzable GTP analogue such asGpp(NH)p. Conversely, hormone was proposed to promote binding GTP, thereby stimulating both GTPase activity and adenylate cyclase ac-tivity. This facilitation of GTP binding was suggested to represent the"opening" of the binding site such that GDP could dissociate and GTPcould bind. Using this model, Cassel & Selinger have been able to relate


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activation and inactivation rate constants for adenylate cyclase to steady-state activities of the enzyme (165, 170). As expected from their model,cholera toxin decreases the inactivation rate constant without altering therate of activation by GTP plus hormone (165), and membranes labeled with[3H]GTP could be shown to release [3H]t3DP upon the addition of hor-mone (171). Simultaneous addition of Gpp(NH)p plus hormone to membranes led to the concomitant release of [3H]GDP and reactivation ofenzyme (172).

At this point, it should be mentioned that hormone-stimulated GTPaseactivity still has been assayed only in turkey erythrocytes and only with/~-adrenergic agonists. No rigorous proof of the coupling of GTP hydrolysisto the inactivation of adenylate cyclase exists. Such proof will probably beavailable only when the activities of purified G/F, C, and receptors can bestudied in properly reconstituted systems. The supporting evidence is strik-ing, however, and the concept of a regulatory GTPase cycle has provideda motivating and organizing hypothesis for the analysis of the regulation ofadenylate cyclase; such a paradigm has been absent for twenty years.

A Regulatory GTPase Cycle

The elegant studies by Cassel & Selinger and data from other laboratoriescan now form the basis of a much more detailed description of the regula-tory GTPase cycle. In this section we will try to relate the steady-state’ andkinetic data on the binding of regulatory ligands to their possible effects onthe activities and interactions of the proteins discussed in earlier sections.We will stress, as a unifying concept, the positive and negative effects of thebinding of one ligand to a component of the adenylate cyclase system uponthe binding of a second ligand or protein and the effects of such bindingupon enzymatic activity.

A schematic diagram of the proposed cycle is shown in Figure 1. Thecentral features are those suggested by Cassel & S elinger (165) and Levinson& Blume (166). Details are as follows:

1. It is assumed that G/F is the only site of binding of GTP and is thesite of GTP hydrolysis. Rodbell and co-workers (173) have proposed multi-ple binding sites for GTP, but their arguments do not seem to us compellingwhen it is realized that free G/F and G/F bound to receptors (R) or to can have strikingly different properties. It is assumed G/F-GTP is theactive species and is the proximal and sole stimulator of the adenylatecyclase activity of C. (G/F-GTP-R-H is presumably too short-lived to besignificant.) At present, there are few data to suggest whether GTP pro-motes the association of G/F and C (i.e. that the binding of C and guaninenucleotides to G/F is positively cooperative) or whether the nucleotidemerely activates a preexistent G/F-C complex. Pfeuffer’s demonstration ofan increased sedimentation coefficient of G/F in the presence of both active


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558 ROSS & GILMAN

_ ~/ ’,~ kIt; ~"--#GIF’GTP’- "- GIF’GDP

G/F-GTP.R’H G/F’GDP-R.H

G/F.N.N/k5

Figure ] A hypothetical regulatory GTPase cycle in which hormone-receptor complex(H.R) acts by catalizing the displacement of GDP from G/F by means of negative heterotropicbinding.

C and GTP-T-$ suggests that GTP-~-S promotes stable binding of 12 toO/F (53). Schlegel et al (38) found increases in the apparent size of adeny-late cyelase upon treatment with Gpp(NH)p, which also argues for a posi-tive interaction of the binding of (2 and GTP to ~/F. The data of Limbirdet al (1"/4) suggest, however, that the G/F-C complex may be relativelystable in the unliganded state.

2. Hydrolysis of GTP by G/F (k 1) causes the inactivation of adenylatecyclase, again as proposed by Cassel & Selinger. C may dissociate at thispoint. Normally kI is not rate-limiting, but the use of a nonhydrolyzableGTP analogue or treatment of G/F with cholera toxin decreases kl (165),which increases the concentration of G/F-GTP. It is unknown if free G/Fhas GTPase activity or whether the activity is expressed only when G/Fis associated with another protein (e.g. C). In an analogous system, freeelongation factor Tu is nearly inactive but becomes an active GTPase whenbound to the ribosome (169). However, Cassel & Selinger (162) were to inactivate adenylate cyclase with N-ethylmaleimide without inhibitingGTPase activity.

3, In the absence of hormone, k_2 is the rate-limiting step in the regenera-tion of G/F-GTP, and k2~ k_2. In turkey erythroeytes, k_2 "~ 0, so thatafter one cycle of GTP hydrolysis all G/F is in the G/F-GDP form andadenylate cyclase is inactive. Gpp(NH)p alone cannot activate adenylatecyclase in turkey erythrocytes because there is no free G/F. In mammaliancalls, where Gpp(NH)p alone does activate the enzyme, k_2 is assumed be finite but low. In $49 lymphoma cell membranes, k_z can be assumedto be at least 0.06 rain-I, the rate constant for activation of adenylate eyclaseby Gpp(NH)p (41).


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4. This model differs substantially from that of Cassel & Selinger onlyin the mechanism by which hormone is proposed to facilitate regenerationof G/F-GTP, and reflects the suggestion of Blume and co-workers (160,166) that dissociation of GDP rather than binding of GTP is the regulatedstep. It is generally compatible with the concept of "collision coupling"suggested by Levitzki and co-workers (158). We suggest that the onlyrelevant action of hormone is the negative heterotropic nature of its bindingto G/F-R with respect to guanine nucleotides. By this we mean that cre-ation of a G/F-R-H complex decreases the affinity of G/F-R for nucleotide.We base this suggestion on the negative heterotropic effect of guaninenucleotides upon hormone binding, where finite increases in both the Kd(144-150) and the dissociative rate constant (144, 147, 150) for hormonehave been documented. From thermodynamic considerations, such aneffect must be reciprocal; i.e. if nucleotide increases the Kd for hormone,then hormone must increase the Kd for nucleotide. This concept is consis-tent with the demonstration by Cassd & Selinger of the catecholamine-stimulated dissociation of [3H]GDP or [3H]Gpp(NH)p from turkeyerythrocyte membranes (171, 172); also consistent is the requirement forhormone noted by Pfeuffer (42, 43) and Spiegel et al (59) to allow exchange ofGDP (a tight ligand) with GMP (a loose ligand) prior to affinitychromatography of G/F. It should be noted that the reaction path k4" k_5¯ k6’k_7 is thermodynamically equivalent to the path k_2̄ k3. The receptor-catalyzed path is kinetically much faster. Formation of the unstable G/F-R-H-GDP complex promotes the rapid dissociation (k_5) of what wouldotherwise be a slowly dissociating (k_2) nueleotide ligand. [See (175) for detailed discussion of the general kinetic implications of negative hetero-tropic binding.]

It can be questioned why such diverse effects of purine nueleotides areobserved if only one binding protein is involved (173). It is dear that G/Fbehaves differently when free or when complexed with C. Thus activationof adenylate cyelase is generally irreversible when intact membranes orunfraetionated extracts are treated with Gpp(NH)p or fluoride but theeffects of these ligands upon free G/F are reversible (56, 67). Similarly, thereversibility of the effect of Gpp(NI-I)p upon hormone binding (140), opposed to its irreversible activation of enzyme, can be assumed to reflectaction of that nueleotide on C~/F-R as opposed to G/F-C. A number ofother discrepancies (173) are similarly explicable. One must always considerwhether free G/F, G/F-R, or G/F-C is the species under study in a particu-lar experiment.

CONCLUSIONS AND QUESTIONS

As we can describe it at this time, hormone-sensitive adenylate eyelaseappears to be composed of at least three interacting proteins. The catalyst


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560 ROSS & GILMAN

by itself is relatively inactive, but can be stimulated by a guanine nucleotide-binding protein, G/F, when G/F binds GTP. Upon hydrolysis of GTP,activation is terminated until G/F-GTP is regenerated. This regenerationis catalyzed by the receptor-hormone complex, in that the rate of dissocia-tion of GDP is stimulated by the binding of the hormone-receptor complexto G/F. There is as yet no evidence for the direct interaction of receptorwith the catalyst. Evidently, the interaction of G/F with receptor can occuronly in a relatively unperturbed membrane, since only then can hormonalstimulation of C be observed.

The study of the biochemistry of adenylate cyclase is just beginning.Details are missing and questions proliferate. The kinetics and thermody-namics of the multiple interactions of ligands and proteins are yet to bequantitated. The variability of hormonal responses among different tissuesis yet to be related to these basic parameters in any meaningful way. Thepermissive role of an intact plasma membrane for the interaction of G/Fand R has barry been approached. What is the mechanism of action offluoride on G/F?. What is the nature and significance of the hypotheticallymodified G/F in the UNC variant? Beyond this level, biochemical studiesof long-term regulation (refractoriness) of adenylate cyelase by hormoneshave been initiated in several laboratories. How are the synthesis and stoi-chiometry of G/F, C, and receptors coordinated? We are just beginning tolearn to phrase these questions in a meaningful way.

ACKNOWLED(3MENTS

We would like to thank many of our colleagues for sending us preprints of

publications. We thank Mrs. Wendy Deaner for help in the preparation ofthe manuscript. Our own studies have been supported by USPHS grantsGM26445, AM22125, and NS 10193, and by Amerlean Cancer Society

Grant BC240.

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biochemical properties of hormone-sensitive adenylate cyclase

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