Molecular and Cellular Endocrinology, 16 (1979) 129-146 0 Elsevier/North-Holland Scientific Publishers, Ltd.

129

REVIEW GUANYL NUCLEOTIDE REGULATION ADENYLYL CYCLASES

Joel ABRAMOWITZ,

OF HORMONALLY-RESPONSIVE

Ravi IYENGAR and Lutz BIRNBAUMER

Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030 (U.S.A.) Received 21 September 1979

INTRODUCTION A large number of hormones and neurotransmitters activate adenylyl cyclase [ATP, pyrophosphate lyase (cyclizing; EC 4.6.1.1)] catalyzing the formation of CAMP and PPi from ATP in the presence of Mg*+. The CAMP formed is in turn responsible for eliciting the physiological responses of these hormones and neurotransmitters. In addition to hormones and neurotransmitters, fluoride ion, cholera toxin and guanyl nucleotides (GTP and GTP analogs such as GTPyS and GMPP(NH)P) also stimulate adenylyl cyclase activity (Perkins, 1974; Bimbaumer, 1977; Gill, 1977). It has become evident that hormonally-responsive adenylyl cyclase is a multi-component system consisting of at least 3 physically distinct units. The first is the hormone receptor containing a specific site for a given hormone. The second is the catalytic moiety (Ccomponent) of adenylyl cyclase bearing the site responsible for catalysis of the cyclizing reaction. The third is the guanyl nucleotide regulatory subunit (G component) which binds guanyl nucleotide. Recently, a CTPase activity has been found to be associated with the G component of adenylyl cyclase (Cassel and Selinger, 1976; Cassel et al., 1977a, b; Lambert et al., 1979). In this review we will present information on the regulation of hormonally-responsive adenylyl cyclases. This is not intended to be a comprehensive review of the literature. Rather, it represents our views on the current status of the regulation of CAMP formation.

HORMONE RECEPTORS Receptors are entities specific for hormone. They are molecularly separate from the rest of the adenylyl cyclase system (Birnbaumer and Rodbell, 1969; Limbird and Lefkowitz, 1977; Welton et al., 1977). Receptors “float” freely in the phospholipid bilayer of the plasma membrane and appear to do so independently of the adenylyl cyclase system. This was elegantly shown via cell-cell fusion experiments

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by Schramm and Orly (1976). These authors tested for complementation of hormonally-responsive cyclase by fusion of turkey erythrocytes, containing normal catecholamine-binding receptors but N-ethylmaleimide-inactivated adenylyl cyclase, with Friend erythroleukemia cells that had active cyclase but no detectable catecholamine receptors. After fusion, a catecholamine-responsive adenylyl cyclase system was obtained indicative of interaction of the Friend cell cyclase with turkey erythrocyte receptor. These studies have been extended to include cell-cell transfer of receptors for prostaglandins (Schramm et al., 1977), glucagon (Schramm, 1979) and LH (Dufau et al., 1978). More recently, Pike et al. (1979) showed that upon complementation, the receptor retains its hormone specificity but loses or exchanges its intrinsic activity characteristics. This was demonstrated fusing appropriately treated turkey and frog erythrocytes. In the turkey erythrocyte catecholamine agonists demonstrate a beta 1 order of potency for activating adenylyl cyclase, while in the frog erythrocyte a beta2 pattern is observed. Treatment of frog erythrocytes with dicyclohexylcarbodiimide which inactivates the beta-adrenergic receptor followed by fusion to N-ethylmaleimide-treated turkey erythrocytes resulted in a catecholamine-sensitive frog adenylyl cyclase with a beta1 affinity response pattern of the turkey erythrocyte, but with the “beta*” intrinsic activity pattern of response. Thus, even though in the hybrid cells turkey beta1 receptors are activating the frog cyclase, soterenol, a poor partial agonist in the frog and a good partial agonist in turkey, continues being a poor stimulator of cyclase.

GUANYL NUCLEOTIDE

REGULATORY

SUBUNIT

The original evidence for the existence of a role for guanyl nucleotides in the regulation of hormonally-responsive adenylyl cyclase came from studies on the effects of guanyl nucleotides on glucagon binding to liver membranes (Rodbell et al., 1971a, b, 1974). It is now clear that GTP exerts 3 actions on this and many other hormone-responsive adenylyl cyclase systems: (1) GTP stimulates basal activity in the absence of hormone; (2) GTP accelerates the rate of hormone binding to and release from its receptor; and (3) GTP promotes coupling of receptors to adenylyl cyclases. Physical separation followed by reconstitution, and isolation followed by identification of an S49 lymphoma cell variant containing an inoperative catalytic component and missing a guanyl nucleotide regulatory component, indicate the guanyl nucleotides interact with a component (G component) that is separate from the catalytic component. Physical separation was achieved by Pfeuffer (1977, 1979) by subjecting Lubrol-solubilized pigeon erythrocyte membranes to GTP-affinity chromatography. A resolved catalytic component was obtained that exhibits little or no activity under standard Mg2+ ion-containing assay conditions and that is unresponsive to guanyl nucleotide stimulation. It exhibits measurable activity, however, when Mn2+ is substituted for Mg2’ and re-acquires both Mg2+-dependent cyclizing

Guanyl nucleotide

regulation of adenylyl cyclases

131

activity and guanyl nucleotide sensitivity when placed in presence of a fraction eluted from the GTP-affinity matrix with guanyl nucleotide. Similarly, the above mentioned S49 cell variant isolated by Bourne et al. (1975) shows no cyclizing activity when assayed with Mg’+, is unresponsive to guanyl nucleotide, and is active when assayed with Mn*+ (Ross and Gilman, 1977; Ross et al., 1978; Howlett et al., 1978; Sternweiss and Gilman, 1979). As in the pigeon erythrocyte system, also the S49 system is easily recombined by mixing detergent extracts of the variant cell (called cyc- for its lack of activity under physiological and standard Mg*+ conditions) with extracts from “wild-type” S49 cells from which the catalytic activity has been removed by a short lo-min heating process at 37’C.

AN ADENYLYL CYCLASE-ASSOCIATED

GTPase

Studies by Cassel and Selinger (1976, 1977a, b) on the catecholamine-sensitive adenylyl cyclase system in turkey erythrocytes demonstrated the presence of a catecholamine-stimulated cholera toxin-inhibited GTPase activity associated with adenylyl cyclase. This GTPase has a low K, for GTP similar to the concentration of GTP needed to half-maximally activate adenylyl cyclase in a number of systems. These authors found that inhibition of GTPase activity and activation of adenylyl cyclase by cholera toxin are processes that occur concurrently and proposed adenylyl cyclase to be in constant turnover regulated by an intrinsic GTPase (Cassel and Selinger, 1977a; Cassel et al., 1977). The initial assumption was made that the active state of the enzyme is an enzyme-GTP complex and that under basal conditions the fraction of the enzyme in the GTP state is low because of constant breakdown to GDP, Pi and inactive (free) enzyme. Stimulation of adenylyl cyclase by hormones was then assumed to result from an increased rate of enzyme-GTP formation (with concomitant increased GTPase activity via increased substrate availability), and stimulation of adenylyl cyclase by cholera toxin was proposed to be due to accumulation of enzyme-GTP complex because of a decreased rate of hydrolysis of bound GTP. Work by Levinson and Blume (1977) on neuroblastoma adenylyl cyclase showed that high concentrations of Mg*+ can counteract GTP stimulation of adenylyl cyclase activity, that this effect of Mg*+ is reduced by cholera toxin and that GDP can competitively counteract GTP stimulation. These findings suggested the existence both of a Mg*+-dependent “GTPase-like” activity responsible for the “turn off’ of adenylyl cyclase activity and of an inactive enzyme-GDP complex. Levinson and Blume (1977) designated the offrate of GDP from this complex as a possible point of hormonal regulation. More recently, Cassel and Selinger (1978) showed that exposure of turkey erythrocyte membranes to [3H]GTP results in retention of [3H]GDP on the membrane and that catecholamine stimulation of membranes containing [jH]GDP results in release of this nucleotide. This suggests the existence of a stable enzyme-GDP complex. Further, this complex is inactive, for initial rates in the presence of GMP-P(NH)P are negli-

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gible. Thus, it would appear that adenylyl cyclases are regulated by a GTPase-like turnover mechanism and that in some systems hormonal stimulation is associated with a GTP : GDP exchange process that promotes dissociation of inhibitory GDP and allows association of stimulatory GTP. It is difficult to measure a hormonesensitive GTPase in mammalian membranes. Thus direct measurement of such an activity other than in turkey erythrocytes has thus far been reported only for a secretin- and CCK-stimulated system in isolated rat pancreatic membranes (Lambert et al., 1979) and been impossible to detect in membranes from other tissues such as liver, heart, brain and corpus luteum. Yet, in spite of this difficulty in determining hormone-stimulated GTPase, the kinetics of nucleotide regulation of mammalian hormonally-sensitive adenylyl cyclases are consistent with the existence of a GTPase associated with cyclase, and it is safe to assume that such a GTPase activity is associated with all adenylyl-cyclase systems. Not so clear, however, is the possible universality of the primary point of action of hormones being that of promoting release of GDP. Thus, while hormonal stimulation does indeed result in increased exchange rates of GDP as shown in the turkey erythrocyte system by Cassel and Selinger (1979) and more recently also in rat pancreatic membranes by Christophe and Svoboda (1979), there is evidence from our laboratory that in liver membranes, GDP exchange is not rate-limiting and that indeed hormone stimulation can occur in the presence of excess GDP. A detailed analysis of the situation leads us to propose that hormone action in both the rat liver and turkey erythrocytes can be accounted for if the primary effect is one of promoting a transition from an inactive to an active form of the cyclase system rather than one of modifying association and dissociation of nucleotides to and from the system (see below).

THE ACTION OF CHOLERA TOXIN Cholera toxin has proven to be a useful tool to explore the regulation of adenylyl cyclases (Gill, 1977). The active form of cholera toxin is the Al-subunit. To affect adenylyl cyclase the Al-subunit requires NAD’ and GTP (Gill, 1977; Moss and Vaughan, 1977; Enomoto and Gill, 1979). Studies with cholera toxin and [32P]NAD+ indicate that concomitant with adenylyl cyclase activation, a component with properties of the guanyl nucleotide binding component is ADP-ribosylated. This was shown by SDS-polyacrylamide gel electrophoresis in the pigeon erythrocyte system (Cassel and Pfeuffer, 1978; Gill and Meren, 1978) in the S49 lymphoma cell system (Johnson et al., 1978a) and in the turkey and human erythrocyte systems (Kaslow et al., 1979). An apparent molecular weight between 42 000 and 45 000 dalton has been ascribed to both the affinity chromatographyresolved guanyl nucleotide regulatory component (Pfeuffer and Helmreich, 1975; Pfeuffer, 1977) and the cholera toxin-mediated ADP-ribosylated component. That cholera toxin does indeed lead to covalent modification of the G component of the adenylyl cyclase system (which has not been purified to homogeneity) was demon-

Guanyl nucleotide

regulation of adenylyl cyclases

133

strated by Bourne and coworkers via reconstitution analyses of functional activity correlated with SDS-polyacrylamide gel electrophoretic analysis of [32P]ADPribosylation (Johnson et al., 1978a, b). In these studies it was shown that under conditions where cholera toxin-treatment of “wild-type” S49 cells resulted in incorporation of [32P] ADP-ribose into 42 000-45 000 dalton protein, it also resulted in a G component capable of conferring “toxin-treated” properties to the cyc- system. Toxin treatment of cyc- cell membranes, however, resulted in neither incormaterial of 42 OOOporation of [32P]ADP-ribose into a “G-component-like” 45 000 dalton nor in reconstitution of adenylyl cyclase activities that displayed characteristics of a toxin-treated enzyme. In addition, G component isolated by affinity chromatography from Lubrol-solubilized toxin-treated pigeon erythrocytes was shown to confer toxin-treated characteristics to an adenylyl cyclase catalytic moiety resolved from turkey erythrocytes (Cassel and Pfeuffer, 1978). Intracellular ADP-ribosylation may be a normal mechanism for regulating adenylyl cyclase. This is supported by the findings of Moss and Vaughan (1977) who isolated a soluble ADP-ribosylating factor from the cytosol of turkey erythrocytes that is capable of activating adenylyl cyclase in a NAD+-dependent manner similar to that seen with cholera toxin. Further evidence for a physiologically significant role of intracellular ADP-ribosylation in activation of adenylyl cyclase activity comes from the studies of Cassel and Pfeuffer (1978) and Gill and Meren (1978). Independently, these two groups have demonstrated that ADP-ribosylation of the G component from pigeon erythrocytes and activation of adenylyl cyclase by cholera toxin is reversible in the presence of cholera toxin and nicotinamide. The concept has emerged that adenylyl cyclase, in addition to being the transduction element for hormonal stimuli, is also a regulatory element modulated from within the cell by levels of guanyl nucleotides and by ADP-ribosylation of the G component (Moss and Vaughan, 1977; Iyengar and Birnbaumer, 1979).

GUANYL NUCLEOTIDE ADENYLYL CYCLASE

REGULATION

OF THE CATALYTIC

MOIETY

OF

As mentioned above, a role for GTP in the regulation of adenylyl cyclase was originally suggested based on its effects on glucagon action on the rat-liver plasmamembrane system (Rodbell et al., 1971a, b, 1974; Birnbaumer and Yang, 1974). introduced GMPRodbell and coworkers (Londos et al., 1974) subsequently P(NH)P, a GTP analog that exerted all the effects of GTP. Interestingly, it was found that this analog has a higher efficiency in stimulating cyclizing activity than GTP. These authors suggested that the larger effect of GMP-P(NH)P was due to the non-hydrolyzable nature of the NH bond between the beta and gamma phosphates and speculated that the poorer stimulating ability of GTP might be due to the hydrolysis of its gamma phosphate at a regulatory site on the enzyme system. Other analogs such as GTPyS, GMP-P(CH2)P and GMP-(CH2)PP act in a manner similar

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to GMP-P(NH)P (Pfeuffer and Helmreich, 1975; Londos et al., 1977). In the absence of hormone all of these guanyl nucleotide analogs stimulate adenylyl cyclase better than GTP; all are resistant to hydrolysis by beta-gamma nucleotidases. In fact, both GMP-P(NH)P and GTPyS inhibit the adenylyl cyclase associated GTPase in turkey erythrocyte membranes (Cassel and Selinger, 1977b). Analysis of the action of some of these analogs, especially GMP-P(NH)P, has been the center of attention of many studies. In addition to stimulating adenylyl cyclase more than GTP, GMP-P(NH)P activates adenylyl cyclases in a timedependent manner giving rise to lag periods in product accumulation. This slow activation of adenylyl cyclase by GMP-P(NH)P was proposed by Rodbell and coworkers (Salomon et al., 1975; Lin et al., 1975; Rendell et al., 1975,1977) to be the result of a two-step process involving 3 states of the enzyme system wherein the nucleotide binds rapidly to a resting state of the enzyme and yields an active form of the enzyme that is inhibited strongly by free, protonated ATP and which then slowly isomerizes to a second active form of the enzyme no longer inhibited by ATP. The studies by Levinson and Blume (1977), mentioned above, suggested that the lags observed after GMP-P(NH)P treatment may be due to a slow displacement of tightly bound GDP rather than slow isomerization. Studies from our laboratory on the same liver-membrane system studied by Rodbell’s group (Birnbaumer and Swartz, 1977; Birnbaumer et al., submitted) indicate that lags in time courses of product accumulation in the presence of GTP analogs may be accounted for by involving only two states of the enzyme with isomerization from inactive to active being slow and rate-limiting. In these studies transient and steady-state kinetics of the interaction of liver plasma-membrane adenylyl cyclase with GTP, GMP-P(NH)P, GMP-P(CH2)P and GTPyS were evaluated before and after treatment of membranes with cholera toxin. In control experiments, GTP stimulated the enzyme partially and without a noticeable lag. The GTP analogs stimulated to varying degrees [GTPyS 2 GMP-P(NH)P >> GMP-P(CH2)P > GTP] and with lag periods that varied with the nucleotide [GMP-P(NH)P> GMPP(CHa)P > GTPyS] indicating the unlikelihood that dissociation of GDP is the rate-limiting step in this system. In cholera-toxin-treated membranes transient kinetics and the degree of activation by analogs of GTP are unaltered; but activation by GTP, which remained rapid, became as effective as the most effective of the nucleotide analogs. Furthermore, cholera toxin treatment had no effect on the concentration-effect curves for analogs of GTP but resulted in a 5- to 9-fold lowering of the apparent K, with which GTP stimulates the system. These studies indicate that in this system the lags in progress curves are not due to slow dissociation of “resident” GDP molecules but rather to isomerization reactions of inactive to active forms of the enzyme. Time transients of the interaction of the rat-liver adenylyl cyclase with combinations of GTP and GMP-P(NH)P were also studied and found to be of a competitive type regardless of the time and sequence of addition of the two nucleotides (Birnbaumer and Swartz, 1977). These experimental results, which agree with results reported by Londos et al. (1977) are consistent with GMP-

Guanyl nucleotide regulation of adenylyl cyclases

,““cnvE-

- - .-

135

--ACTIVE

Fig. 1. Schematic representation of a two-state model of an enzyme regulated positively by GTP and negatively by GDP, and having a ligand-metabolizing activity (GTPase) capable of converting bound activating ligand to bound inactivating ligand. The directions towards which GTP and GDP displace the equilibrium between the f?’ and the E’ state are indicated by the arrows.

P(NH)P interacting with and dissociating from the system very slowly and not leading to the formation of an irreversibly activated state of the enzyme which had been suggested (Schramm and Rodbell, 1975; Lefkowitz and Caron, 1975; Cuatrecasas et al., 1975). We found that all data available on the liver system as well as many other systems such as a catecholamine-sensitive system from the heart and a prostaglandinsensitive system from corpora lutea can be interpreted in terms of a simple model of an enzyme that exists in only two preferred states: an inactive ‘-(zero) state and an active ‘-(prime) state, the equilibrium between these states being displaced towards the O-state in the absence of ligand or in the presence of an inactivating ligand, towards the ‘-state in the presence of an effectively stimulating ligand, and not being displaced predominately towards either state in the presence of partially effective ligands (Fig. 1). By assuming rapid binding of the nucleotide to the O-state and giving values to the transition rates from ligand-occupied inactive to the ligandoccupied active state, such a model can simulate nucleotide-specific lag periods in progress curves, and GTP “reversal” of a GMP-P(NH)P treated enzyme. By assuming that the active ligand-occupied ‘-state can decay to ligand-free ‘-state not only through dissociation of ligand but also through an active process that operates independently of all other equilibria in the system, a two-state view of the system can account for the presence of a GTPase-type turn-off mechanism in adenylyl cyclases. An interesting corollary to this type of model (Fig. 1, Scheme 1) is that because of thermodynamic considerations the intrinsic affinity of the inactive state of the enzyme for activating ligand is less than that of the active state of the enzyme. Activation of the enzyme by any means that increases isomerization toward the active form would therefore lock nucleotide in place. The same considerations also indicate that the intrinsic affinity of the inactive enzyme system for an inactivating ligand is greater than that of the active state of the enzyme and hence that activation of the enzyme via right shifts in the isomerization equilibria would result in a release of the inhibitory nucleotide. Some properties of the rat liver, of a GMP and isoproterenol-treated turkey erythrocyte and of the NS-20 neuroblastoma cell adenylyl cyclase systems are presented in Table 1.

Joel A bramowitz, Ravi Iyengar, Lutz Bimbaumer

136

Table 1 Adenylyl cyclase activities in rat liver, NS-20 neuroblastoma branes: effects of hormones and guanyl nucleotides Source of membranes a

Rat liver

Turkey erythrocyte

NS-20 neuroblastoma

cell

Nucleotide addition b

and turkey erythrocyte

mem-

Adenylyl cyclase activity ’ _

in the presence of hormone b

A. No regenerating system _ GDP d GMP-P(NH)P

11.2 11.6 122.3

35.2 145.6 194.1

B. Plus regenerating system GTP GMP-P(NH)P

12.2 30.4 110.2

51.6 193.1 220.3

A. No regenerating system _ GDP GMP-P(NH)P

87.2 0.8 141.2

69.5 0.9 52.7

B. Plus regenerating system _ GTP GMP-P(NH)P

138.0 13.7 213.8

07.8 42.3 239.0

A. No regenerating system _ GDP d GTP

17.3 6.7 26.7

17.5 38.1 77.3

a Liver membranes (1.1 pg protein per assay) were prepared and incubated as described (Iyengar, Swartz and Birnbaumer, 1979a) in 50 ~1 of medium containing 0.5 mM [oi-32P]ATP (50 X lo6 cpm; lo4 ct/5 min/pmol), 5.0 mM MgCla, 1.0 mM EDTA, 1.0 mM [3H]cAMP (ca. 10 000 cpm) and 25 mM Tris-HCl, pH 7.6 Incubations were for 5 min at 32.5”C. Turkeyerythrocyte membranes (5.3 pg/assay) were prepared by sucrose densitygradient centrifugation of a Polytron homogenate subjected to a 30-min treatment with lob3 M GMP and lo4 M isoproterenol to clear the adenylyl cyclase system from endogenous GDP, and then assayed in 50 nl of medium containing 0.75 mM AMP-P(NH)P, 10 CLM[ol-32P]ATP (15 X lo6 cpm), 5 mM MgCla, 1.0 mM EGTA, 10 mM KCI, 1.0 mM 13H]cAMP (ca. 10 000 cpm) and 10 mM Tris-HCl, pH 7.5. Incubations were for 2.5 min at 32.5”C. NS-20 neuroblastoma cell membranes (1.25 pg protein) were prepared as described by Foster and Blume (1975) and assayed in a final volume of 50 ~1 containing 0.5 mM AMP-P(NH)P, 10 dM ((r-32P]ATP (50 x lo6 cpm), 5.0 mM MgCla, 1.0 mM EDTA, 1.0 mM [3H]cAMP (ca. 10 000 cpm) and 25 mM TrisHCl, pH 7.5. Incubations were for 5 min at 32.5”C. Reactions were stopped and quantified by a modification (Bockaert et al., 1976) of the procedure of Salomon et al. (1974). b When present, GDP was lo4 M, GTP was 10T5 M, GMP-P(NH)P was 10e5 M and the regenerating system consisted of 2 mM creatine phosphate, 20 pg/ml creatine phosphokinase and 2 pg/ml myokinase. Hormones added were 10 pg/ml glucagon when assaying liver membranes, lO+ M isoproterenol when assaying turkeyerythrocyte membranes and 10 fig/ml prostaglandin Er when assaying NS-20 neuroblastoma-cell membranes.

Cuanyl nucleotide I. Rat Liver

regulation of adenylyl cyclases

137

II. Turkey Erythrocyte

k,

INACTIVE-ACUVE

INACUVE

-ACT/

VE

Scheme 1. Two-state model of adenylyl cyclase: assignment of equilibrium relationships for the enzyme system in rat liver and turkey erythrocyte. I values represent equilibrium constants between E’ and Ee states: lo = E’/@, IGTP = Eb~p/p~~p and IGDP = E~DP/E$DP; K values represent equilibrium dissociation constants of GTP and GDP binding to E’ (K’ values) or E” (K” values). Directions of arrows symbolize sense towards which equilibria are displaced; the thicker the arrows, the more displaced are the equilibria in the direction indicated. Doubleheaded hatched arrow symbolizes K$TP value; it is taken as point of reference for all the nucleotide-binding constants. Relative directionality of arrows is based on direct determinations (Table 1) coupled to thermodynamic analysis of the closed-loop arrangements that ariSe as a Consequence of a two-state assumption. Note: (1) While concerted right shifts of all I values (resulting in activation of cyclizing activity) leads to a system with increased aftinity for GTP in both rat liver and turkey erythrocytes, it clearly also leads to decreased affinity for GDP, especially in turkey membranes; (2) Although glucagon does not activate liver-membrane cyclizing activity to a large extent in the absence of nucleotide, suggesting IGDP > 10 and affinity of GDP for E” to be lower than that for E’ (a situation opposite to that in turkey erythrocyte membranes), this assertion is speculative since nucleotide activation of receptor rather than IGDP may be the limiting step in the receptor-mediated activation of cyclizing activity. Thus, it is quite possible that in liver IGDP < 10 (similar to the situation in turkeyerythrocyte membranes) and hence K&DP > K’GDP, i.e. affinity for GDP in the active “prime” state being less than that of the inactive “zero” state; and (3) the GTPase activity level of the turkey-erythrocyte system is represented as being notably higher than that of the rat-liver system, thus accounting for the otherwise unusual result that GTP addition is inhibitory rather than stimulatory in the turkey system (see Table 1). kG represent fist-order rate constant of GTPase activity.

’ Results are expressed in cpm X lo4 of CAMP formed from ATP in 5 mm per mg liver-membrane protein, in cpm X low3 of CAMP formed from AMP-P(NH)P and ATP in 2.5 mm per mg erythrocyte-membrane protein and in cpm X 10-4 of CAMP formed from AMP-P(NH)P and ATP in 5 mm per mg NS-20 neuroblastoma cell-membrane proteins. The numerical values are equivalent to pmoles/min/mg if it is assumed that AMP-P(NH)P and ATP are equally efficient as substrates and that reactions were linear with time. Values are means of triplicate determinations which agree within 5% of the means in all instances except in the determination of conversion rates by turkey membranes in the presence of GDP. These were not significantly different from zero. d In parallel experiments it was demonstrated that the incubation conditions shown here lead to less than 5% of added GDP transphosphorylated to GTP (Iyengar and Birnbaumer, 1979; Iyengar, Abramowitz, Riser, Blume and Birnbaumer, submitted).

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Studies of the subunit composition and on the kinetics of guanyl nucleotide regulation of adenylyl cyclase systems indicate that the activation process of the cyclizing activity must be accompanied not only by a state change in the catalytic unit, but also by a state change of the G component and possibly in profound alterations of the interaction of G and catalytic components. A recent report by Pfeuffer (1979) examined this question using separated and partially purified components from pigeonerythrocyte membranes. In this study pigeon erythrocytes were ADP-ribosylated by treatment with [32P]NAD+ plus cholera toxin. Membranes were solubilized with Lubrol PX and placed on a GTP-Sepharose affinity matrix to separate G component from catalytic unit. G component was then eluted with either GTPyS or GDP and subjected to sucrose-gradient centrifugation alone or in combination with resolved catalytic component. The ADP-ribosylated G component in the absence of catalytic component had an apparent S value of 3.4 when eluted with GTPyS and of 5.5 when eluted with GDP. The material eluted with GTPyS, but not that eluted with GDP, was capable of conferring adenylyl cyclase activity to resolved catalytic component. When the GTPyS-eluted material was applied to the gradient in combination with the resolved catalytic component, its S value changed from 3.4 to 7.6, and this complex had adenylyl cyclase activity. Neither the position of the material eluted with GDP (5.5 S) nor that of the catalytic moiety (6.0 S) were altered when the two were applied to the sucrose gradient simultaneously. These results indicate that in order for the G and catalytic components to combine and form an active enzyme, an effective ligand, GTP or a GTP analog such as GTP$S, must be bound to the G component and that lack of cyclizing activity in the presence of GDP may be due to physical dissociation of G component from the catalytic component.

HORMONE ACTION Regulation of coupling of homone-receptor to adenylyl cyclase Coupling comprises all of the events that intervene between hormone-receptor complex formation and modification of adenylyl cyclase activity. A wealth of data has been collected in recent years both on qualitative and quantitative aspects of coupling. Except for a few instances where hormones inhibit, and which will be discussed in an accompanying article by Jakobs (1979), the general effect of hormone is one of stimulation. At saturating concentrations of hormone the stimulation develops rapidly (Birnbaumer et al., 1974; Rodbell et al., 1974; Kaumann and Birnbaumer, 1974; Nakahara and Birnbaumer, 1974). Hormonal stimulation is also reversible as demonstrated by the fact that it can be washed off and blocked by post-addition of competitive blocker (Birnbaumer et al., 1972; Birnbaumer and Pohl, 1973; Kaumann and Birnbaumer, 1974). The first evidence for GTP being involved in coupling of hormone receptors to adenylyl cyclase was obtained by Rodbell and collaborators in 1971 using hepatic

Guanyl nucleotide

regulation of adenylyl cyclases

139

plasma membranes. Since then in most systems examined it has been found that under conditions where assay components are free of GTP or “GTP-like” contaminants there is an obligatory requirement for guanyl nucleotides for coupling of the hormone receptor to adenylyl cyclase. This has been observed of receptors for catecholamines (Ross et al., 1977; Lefkowitz and Pike, 1978) serotonin (Northup and Mansour, 1978), parathyroid hormone (Goltzman et al., 1977), prostaglandin (Krishna et al., 1972; Lefkowitz et al., 1977) and prostacyclin (Abramowitz and Birnbaumer, 1979). The only exception has been the LH receptor in the corpus luteum where ATP and not GTP is required for optimal coupling (Birnbaumer and Yang, 1974; Birnbaumer et al., 1976). In addition, evidence was presented for a role of intracellular GTP in the regulation of isoproterenol and prostaglandin El elevation of CAMP levels in intact cells (Johnson and Mukku, 1979; Mukku et al., 1979), clearly indicating that these in vitro effects have physiological bearing. Further evidence for a role of GTP in hormone-receptor coupling comes from the studies of Gilman and coworkers (Ross et al., 1978; Howlett et al., 1979). Using the S49 cyc- variant which contains beta-adrenergic receptors and the catalytic moiety of adenylyl cyclase but not the G component, these authors have demonstrated that functional reconstitution of hormone sensitivity requires both the G component and a guanyl nucleotide. Without added guanyl nucleotide during reconstitution of the G component with cyc- membranes, catecholamine-sensitive adenylyl cyclase activity was not restored even though adenylyl cyclase activity could be obtained under these conditions. Interestingly, not only is the G component necessary for hormonal stimulation of adenylyl cyclase, but the source of the G component can alter the kinetics of hormone activation of adenylyl cyclase. Kaslow et al. (1979) reported that transfer of the G component sulubilized from turkey erythrocytes into S49 cyc- membranes results in recombinant membranes that respond to nucleotide and hormone as if they were turkey erythrocyte membranes. Findings by Pike et al. (1979) showing turkey erythrocyte receptors activating frog erythrocyte cyclase (with frog G component) in a “frog fashion” (see above), underscore further the key role of the G component in expression of receptor coupling. Levitzki and Tolkovsky (1978a, b) explored coupling of receptor to adenylyl cyclases by studying the catecholamine-induced increase of the otherwise slow rate of activation of adenylyl cyclase by GMP-P(NH)P in turkey-erythrocyte membranes. Their studies indicate that a progressive decrease in membrane receptors results in a proportional slow down of the rate of activation of GMP-P(NH)P without a decrease in the final activity achieved. Based on these results and mathematical fitting experiments, Levitzki and Tolkovsky proposed catecholamine receptors to act through a so-called “collision-coupling” process in which the receptoractivated state of the enzyme is required to have a half-life that is longer than that of the receptor-cyclase complex, thus permitting one receptor to maintain continuously active more than one cyclase system. While providing interesting insight into one aspect of coupling, the experiments by Levitzki and Tolkovsky did not address

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Joel Abramowitz,

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the question as to what the actual kinetic structure of the basic nucIeotideregulated adenylyl cyclase affected by receptor might be. The rather simple and straight-forward two-state model for the regulation of the basic adenylyl cyclase system in the absence of hormone, presented above (Fig. 1 and Scheme 1) provides such a structure from the kinetic point of view (lyengar et al., 1979b). In view of the observation made in many laboratories that the liver adenylyl cyclase is activated by GMP-P(NH)P faster in the presence than in the absence of hormone, and since in this system isomerization of inactive to active form of the enzyme seems to be the rate-limiting step, it appears that one way for occupied hormone receptor to act is by promoting right shifts in the isomerization reaction. Thermodynamic considerations require that both isomerization in the presence of ligands (ICrP and ZGDp in Scheme 1) and that in the absence of ligand (I,, in Scheme 1) have to be affected concertedly. Simulations carried out in our laboratory according to analytical solutions of this model in which hormone causes a change in the isomerization reaction that is linearly dependent on receptor occupancy, give rise to a non-linear relationship with built-in hysteresis such as required by the “collision-coupling” hypothesis. Thus, if a given nucleotide ligand leads to a steady-state isomerization ratio of active/inactive forms of the enzyme of 0.1 (enzyme being consequently 10% active) and the coupling of hormone receptor to the system changes this by a factor of 1000 to a final isomerizat~on ratio of 100, it follows that after uncoupling the system can relax by as much as 90% to an isomerization ratio of 10 without detectable or significant decay in activity. Only after more than 90% of the system has relaxed will activity stop falling detectably, thus allowing “collision-coupling” to work. Since not only ZGTp but also Ze and ZGDP are affected by the hormone-receptor coupling, a two-state model such as presented in Fig. 1 and Scheme 1, can account as well for existence of hormonal stimulation even in the absence of a nucleotide or in the presence of a very ineffective nucleotide such as GDP (Table 1). At first thought, the two-state model affected by hormones as described above may appear to be in contradiction to the turnover proposals and experimental results of Levinson and Blume (1977) and Cassel and Selinger (1977, 1978a, b) in which coupling of hormone receptor to adenylyl cyclase would result primarily in release of tightly (and limitingly) bound GDP. However, as pointed out above, since GDP is an inhibitory nucleotide, feading to stabilization of the system in a more inactive state in its presence than in its absence (Scheme l)! a right shift in the isomerization equilibria by hormone receptor would not only lead to activation but also (and concurrently) to a loss in affinity of this system for inhibitory GDP as observed (Cassel and Selinger, 1979). The following proposals are therefore presented here: (1) that adenylyl cyclases behave as two-state enzyme systems giving rise to closed-loop arrangements such as shown in Scheme 1; (2) that hormonal activation resulting from coupling of occupied and active receptors to the cyclase system is due to a concurrent right shift of all isomerization equilibria between inactive and active forms (free, occupied by

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GTP and occupied by GDP); and (3) that both these features account for changes in lag times in activation by GTP analogs and in observed affinity changes of the system for inhibitory GDP upon hormone addition. Nucleotide effects on hormone binding to its receptor Rodbell and coworkers (Rodbell et al., 1971; Lin et al., 1977) found that guanyl nucleotides increased the rates of association and dissociation of labeled glucagon to and from its receptor. The overall effect is a decrease in the affinity of the hormone for its receptor. In S49 mouse-lymphoma cell membranes, guanyl nucleotides decrease the affinity of the beta-adrenergic receptor for agonists but not for antagonists (McGuire et al., 1976; Ross et al., 1977). Increased dissociation rates and decreased affinity of hormone for its receptor by guanyl nucleotides have also been observed for angiotensin binding to the adrenal cortex (Glossman et al., 1974); catecholamine (Williams and Lefkowitz, 1977) and prostaglandin (Lefkowitz, 1977) binding to frog erythrocytes; catecholamine binding to alpha-adrenergic receptors in calf-brain membranes (UPrichard and Snyder, 1978); vasoactive intestinal peptide binding to guinea-pig brain membranes (Robberecht et al., 1978); dopamine binding to rat corpus striatum membranes (Creese et al., 1979); and muscarinic ligand binding to rat myocardial membranes (Berrie et al., 1979). On the other hand, in the corpus luteum where ATP is required for coupling of LH receptors to adenylyl cyclase (Birnbaumer and Yang, 1974; Birnbaumer et al., 1976) no conclusive evidence is available on the effects of GTP on LH binding to its receptor. It is clear therefore that guanyl nucleotides affect not only the basic adenylyl cyclase system (in the absence of added hormone), but also and simultaneously receptor-hormone interaction. Since extensive removal of guanyl nucleotides by purification of reagent used and of the membranes onto which they act, leads either to loss or to drastically decreased hormonal stimulation (10% of maximum), the question arises as to which effect of the nucleotide is responsible for hormonal stimulation: that on the receptor or that on the cyclase. Further are both effects, as well as the resulting coupling, mediated by a single site or by more than one site? Experiments with GDP may give a clue as to the appropriate answers to these questions. Neuroblastoma cell adenylyl cyclase (Blume and Foster, 1975; Levinson and Blume, 1977) when assayed in the absence of hormone (prostaglandin El), is inhibited by about 50% upon addition of GDP. In terms of the two-state model, this indicates that I0 >IGDP, a situation qualitatively similar to that of the turkey erythrocyte adenylyl cyclase. Experiments carried out recently in our laboratory Abramowitz, Riser, Blume and Birnbaumer, unpublished; Table 1) (Iyengar, showed that prostaglandin does not stimulate this system in the absence of nucleotide addition, but does so both in the presence of GTP as well as in the presence of GDP. If receptor were active in the absence of nucleotide and nucleotide required at the cyclase level only, then prostaglandin should have stimulated either only in the presence of GTP, or in the presence or absence of GTP or under all 3 conditions: presence of GTP, presence of GDP and absence of nucleotide. The fact that

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stimulation was observed only in the presence of guanyl nucleotide and not in their absence indicates that nucleotide is required at the receptor level. Since this requirement leads to stimulation, it follows that the role of nucleotide at the receptor level is one of receptor activation. Since hormonal stimulation of cyclase does not occur in the absence of hormone the final conclusion can be drawn that also receptors exist in at least two states: a resting state and an active state, the latter being stabilized (or induced) by the simultaneous interaction of hormone and nucleotide with receptor. From the foregoing, it may be concluded: (1) that the active form of the receptor that couples to adenylyl cyclase is the one that exhibits a somewhat lower aftinity for stimulatory hormone than the respective inactive hormone, but that interacts more rapidly and hence in a more reversible manner with the hormone; (2) that the active form of receptor does not arise unless both hormone and nucleotide interact with it simultaneously and hence, that in the presence of hormone, it is the nucleotide-affected and not the nucleotide-free form of the receptor that couples to stimulate adenylyl cyclase. On the number of nucleotide sites involved in hormone action Based again on thermodynamic considerations of a closed-loop system such as arises from a two-state receptor with two ligands (hormone and guanyl nucleotide), it follows that if nucleotide interaction with receptor leads to decreased affinity of receptor for hormone, then hormone interaction with receptor has to lead also to a decrease in the affinity of the receptor for guanyl nucleotide. This argument is of importance in the consideration of the last major issue we wish to discuss in this article: How many guanyl nucleotide sites are involved in receptor activation, coupling and regulation of cyclizing activity? There are indications that there are two such sites: (1) Rodbell and co-workers prelabeled liver membranes with ‘251-glucagon and prepared a Lubrol PX extract which was subjected to gel filtration. They analyzed the column fractions for GMP-P(NH)P-stimulable adenylyl cyclase activity and for guanyl nucleotide stimulation of dissociation of ’ 2‘I-glucagon from macromolecular protein. They found that the pattern of elution of nucleotide-sensitive receptor differed from that of nucleotide-sensitive enzyme (Welton et al., 1977). This indicates that receptor and enzyme can interact independently with guanyl nucleotides. (2) Ross et al. (1977) found that treatment of S49 cell membranes with GMPP(NH)P followed by washing, resulted in complete reversal of the effects of the nucleotide on agonist binding but not of the effects on the enzyme which remained active. This suggests strongly that while GMP-P(NH)P dissociates very slowly from the site responsible for cyclase activation, it dissociates readily from the site responsible for receptor activation. (3) Preactivation of the liver-membrane adenylyl cyclase with GMP-P(NH)P, followed by washing, leads to an active enzyme system that does not respond further to addition of GMP-P(NH)P alone or of glucagon alone, but which is stimulated

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further if glucagon is added in combination with GMP-P(NH)P, GTP or GDP (Iyengar, Swartz and Birnbaumer, 1979a). These results suggest both that continued presence of free nucleotide is necessary for receptor activation and coupling, and that the characteristics of the nucleotide site associated with cyclase (leading to wash-resistant “permanent” activation by GMP-P(NH)P are different from those of the receptor-related nucleotide site. (4) Finally, it is difficult to reconcile how hormonal activation of cyclase in the presence of the activating nucleotide GMP-P(NH)P can lead to increased affinity at the cyclase level (Scheme 1) while at the same time leading to a decrease in affinity at the receptor level (Scheme 2) without invoking two sites. Arguments may be set forth to oppose the above conclusion that more than one nucleotide site is involved in regulation of hormone action on adenylyl cyclase: (a) Separation of G nucleotide-sensitive, bound 1251-glucagon from nucleotide-sensitive adenylyl cyclase was by no means total and excess G components over cyclase and receptors may exist; (b) The reason for which GMP-P(NH)P pretreated membranes “lose” their nucleotide effect on binding or coupling is that at the moment of addition of hormone the bound nucleotide dissociates giving rise to hormone-induced “clearing” and resulting in not enough free nucleotide to lead to significant occupancy of the nucleotide site and hence giving rise to loss of nucleotide effect. How-

Scheme 2. Two-state model of receptor: assignment of possible equilibrium relationships. Z’s represent equilibrium constants between R’ and R” states where IGD > 10 = IG = ID. K’s represent equilibrium-dissociation constants of guanyl nucleotide (subscript G) and hormone (subscript H) binding to R’ (K’ values) and R” (K” values) forms of the receptor. Directions of arrows symbolize sense towards which equilibria are displaced; the thicker the arrows, the more displaced are the equilibria in the directions indicated. Relative directionality is based on direct measurements of changes in receptor activity and hormone binding upon nucleotide addition. In addition to setting the above relationships between I constants, the relationship Kt < GKfI was set, i.e. affinity of free inactive receptor for hormone is greater than that of nucleotide occupied and active receptor. It should be noted that after setting these 6 constants (I’s and Kk and GKfI) assignment of any additional K value fixes all other K values. Double-headed hatched arrow symbolizes the KE value taken as point of reference for all ligand remaining (G and H) binding constants. Kh and GKfl represent the equilibrium dissociation constants of hormone H from R& and R~;H, respectively; K% represents the equilibrium dissociation constant of nucleotide G from R&

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ever, to overcome the thermodynamic considerations, it will be necessary to show that a true resting state of the liver-membrane system, as will as of most other mammalian systems is one where enzyme is more active in the absence of GMPP(NH)P than in its presence, i.e. where 1, >IGMP_P(NH)P. With the experimental evidence currently at hand, such speculation does not seem warranted. Thus, until such measurements are made accurately, we feel inclined to analyze the mode of action of nucleotides in terms of an interaction with two sites: one at the receptor level that decreases its affinity upon hormone binding and another at the cyclase level that increases its affinity for activating nucleotide upon coupling of active hormone receptor. It should be noted that the distinction is functional and that both functions could be mediated by the same G component. While further work is necessary in exploring the nucleotide regulation of hormone action at the levels of receptor and adenylyl cyclase, it is clear that major advances have been made in the understanding of these systems in the last 2-3 years. Further analysis wilI have to take into consideration the multicomponent arrangement of the system, the possibility of more than one guanyl nucleotide site being involved in modulation of receptor mediated activation of adenylyl cyclases and, importantly, the possibility of regulation at the level of the GTPase associated with these adenylyl cyclase systems.

ACKNOWLEDGEMENTS This work was supported in part by National Institutes of Health Grants AM-19318, HL-19423 and HD-09581. J.A. is the recipient of an Individual National Institutes of Health Postdoctoral Fellowship HD-05757. RI. is the recipient of an Individual National Institutes of Health Postdoctoral Fellowship AM-06066.

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