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Control of catecholamine stimulated adenylate cyclase in pigeon erythrocyte membranes by guanylnucleotides
CONTROL OF CATECHOLAMINE STIMULATED ADENYLATE CYCLASE IN PIGEON ERYTHROCYTE MEMBRANES BY GUANYLNUCLEOTIDES ERNST J. M. HELMREICHand THOMASPFEUFFER Department of PhysiologicalChemistry,Universityof Wiirzburg, D-8700 Wiirzburg,Federal Republic of Germany INTRODUCTION We have recently called attention to the peculiar property of hormone receptor complexes which seem to be operationally irreversible (1,2). In some instances hormone receptor complexes, for example, insulin- glucagon- and vasopressinreceptor complexes, have long lifetimes. Assuming that dissociation of the hormone-receptor complex [n] • [R] ~ o f f [n] + [R] is first order, t/~ = In 2/koff may be calculated and is found to be/> 10 rain at 30 ° (1, 2). Moreover, exposure of frog erythrocyte membranes to f3-adrenergic hormones has recently been shown by Mukherjee and Lefkowitz to result in inactivation (desensitization) of receptor binding sites (3). The desensitized receptors no longer bind/3-adrenergic ligands and the latter fail to stimulate membrane bound adenylate cyclase.* We have therefore postulated regulatory steps modulating coupling between receptor and adenylate cyclase (1,4, 5). In this context, we have focussed on the role of guanylnucleotides in the control of adenylate cyclase activity (5-7). But we are aware that other factors, for example Ca2+ ions, may also play an important role in the regulation of hormonally stimulated adenylate cyclase (8). METHODS Pigeon erythrocyte membranes were prepared according to Oye and Sutherland (9) with the minor modifications described by Puchwein et al. (10). Adenylate cyclase was solubilized with Lubrol PX and measured in particulate and soluble preparations as described by Pfeuffer and Helmreich(6). [32 p] cAMP formed from [a a 2 p] ATP was determined according to (11). Binding of [all] Gpp(NH)p or [7a2 p] p3.(4_azidoanilido).pl guanosine triphosphate to particulate or solubilized membrane preparations was measured as described in (6). Analytical sodium dodeeyl sulfate polyacrylamide gel dec*Adenylatecyclase (EC 4.6.1.1) 209
210
ERNST J. M. HELMREICH et al.
trophoresis was carried out according to Neville and Glossmann (12). The synthesis of p3.(4.azidoanilido) p1-GTP, photoactivation and the preparation of the GTP-sepharose derivative are described by Pfeuffer (7). Colchicine Sepharose-4B derivatives are described by Zenner and Pfeuffer (13).
Activation by Guanylnucleotides Purine nucleotides, especially GTP, seem to play an ubiquitous role in optimizing the response of adenylate cyclase to hormones (2). In the nucleated avian erythrocytes which possess an adenylate cyclase system highly sensitive to catecholamines, guanylnucleotide analogs such as Gpp(CH2)p, Gpp(NH)p and GTP-7-S are much more effective activators than the natural effector GTP (6, 7) (See Fig. 1). We have made use of the high efficacy and the tight binding of the guanylnucleotide analogs in a search for GTP-binding proteins in pigeon erythrocyte membranes.
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FIG. 1. Activation of adenylate cyclase in pigeon ~rythrocyte membranes by guanylnucleotides. D,L-Isoproterenol (50/~M), the guanyinucleotides and ATP (0.1 raM) were added together at 37°C. cAMP formed was measured with 220 ~g of membrane protein after 10 rain at 37°C. (Reproduced by permission of the J. Biol. Chem. from T. Pfeuffer and E. Helmreieh (6).)
Guanylnucleotide Binding Sites Previous studies had shown that 120 pmoles of [all] Gpp(CH2)p per mg of protein were bound to pigeon erythrocyte membranes (6). Levitzki et al. (14) had, however, convincingly shown that only 1-2 pmoles of catecholamine per mg of protein were bound to turkey erythrocyte membranes. Subsequently, a
GUANYLNUCLEOTIDEACTIVATIONOF ADENYLATECYCLASE
211
comparably low value has also been found in our laboratory for pigeon erythrocyte membranes (cf:reference 7). Levitzki et al. (14) have made the interesting point that assuming a 1 : 1 relationship between receptor sites and catalytic sites, 1-2 pmoles are a realistic estimate for adenylate cyclase active sites because calculated on that basis the turnover number would be about 1,400 min-1 and thus approximate the turnover number of the crystalline soluble adenylate cyclase from Brevibacterium liquefaciens (15). Thus, following this argument, the guanylnucleotide binding sites are far in excess over the catalytic sites, suggesting that the greater part of them is unrelated to adenylate cyclase activation. This was shown to be correct in recent experirnents (of:reference 7) carried out to identify the GTP-binding site of catecholamine stimulated adenylate cyclase in pigeon erythrocyte membranes. This was achieved with the aid of a photoreactive [73ZP] labeled GTP derivative, pa.(4.azidoanilido) p l _ guanosine triphosphate, which is a potent activator of adenylate cyclase and which binds with high affinity (Kdiss = 3.3 X 10 -7 M). This GTP derivative competes with GTP or GTP-analogs for common binding sites in membranes. Only those binding sites for which the photoreactive GTP-azidoanilide and Gpp(NH)p effectively compete were considered specific binding sites. On photolysis by light of wavelengths > 350 nm nitrenes are generated which are I attacked by nucleophlles and also inserted into - C - H bonds of amino acid residues. Besides membrane proteins lipids also react with GTP azidoanilide but both materials can easily be separated. No covalent incorporation of the GTP-analog occurred in the dark or when the [Ta:P] GTP-azidoanilide was photolyzed before it was added. The proteins labeled in intact membranes and of Lubrol PX solubllized membranes containing adenylate cyclase activity were separated and analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis (see Figs. 2A and 2B). Four major proteins with MW 86,000, 52,000, 42,000 and 23,000 wete labeled (Fig. 2A). In addition a membrane protein of MW of about 250,000 which co-migrates with spectrin I was labeled but in this case an excess of Gpp(NH)p did not prevent incorporation of the radioactive photoaffinity reagent. In Lubrol solubilized membranes which lack the spectrin complex only the GTP-binding proteins with MW 42,000 and 23,000 were specifically labeled (Fig. 2B). Thus, because soluble adenylate cyclase is fully responsive to nucleotide stimulation only the binding proteins with MW 23,000 and 42,000 need to be considered for a role in adenylate cyclase activation. On centrifugation of the soluble preparation through a linear gradient of 5% to 30% sucrose more than 95% of the nucleotide binding proteins sediment slower than adenylate cyclase activity and only about 5% cosediment with it. The guanylnucleotide binding fractions separated by sucrose gradient centrifugation were labeled with [Ta2P] GTP-azidoanilide and analyzed by sodium dodecylsulfate acrylamide gel electrophoresis. The slow sedimenting nucleotide binding peak which could be separated from adenylate cyclase activity without loss of guanylnucleotide activation was exclusively the 23,000 MW protein. The binding
212
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FIGS. 2A and 2B. Photoaffinity-labeling of membranous and Lubrol PX solubilized proteins. A: Membranes, 2.5 mg per ml, were incubated with 3 ~M [~/32 p] -GTP azidoanilide (specific activity 5 Ci/mmole) and 50 izM D,L-isoproterenol for 30 min at 37°C. After removal of excess nucleotide by 3 washes with buffer each with 5 times the vol of the membrane suspension (see legend to Table 1), the membranes were incubated for another 25 rain with ( . o) and without (o o) 0.1 mM Gpp(NH)p and photolyzed for 60 min at 4°C. The irradiated samples (250-600 #g protein) were treated with 1% sodium dodecylsuifate and 10 mM 2-mercaptoethanol for 15 min at 37°C and electrophoresed on 12.5% polyacrylamide gels containing 0.1% SDS. Phospholipids have about the same mobility as the tracking dye (TD). B: Membranes were incubated with GTP-azidoanilide and D,L-isoproterenol as described in A. The washed membranes were solubilized with 20 mM Lubrol PX for 30 min at 4°C as descr~ed previously (6) and incubated for 25 min at 37°C in the presence (e , ) and absence (o o) of 0.1 mM Gpp(NH)p and then photolyzed and separated electrophoreticaUy on polyacrylamide gels as described in A and Methods.
GUANYLNUCLEOTIDE ACTIVATION OF ADENYLATE CYCLASE
213
protein which cosedimented with adenylate cyclase activity was mainly the 42,000 MW protein together with some of the 23,000 MW protein and traces of the 86,000 MW protein, b u t the latter was only unspecifically labeled since the attachment of the photoaffinity analog was not prevented by high concentrations of Gpp(NH)p (see reference 7).
Separation and Reconstitution of Adenylate Cyclase Activity With the Sepharose b o u n d GTP derivative, Seph4B-NH(CH2)3-CONH- Q -NH-pppG proteins including the guanylnucleotide binding protein with MW 42,000 could be detached from a detergent solubilized adenylate cyclase preparation with loss of nucleotide stimulation which could be partly restored b y recombining the guanylnucleotide binding protein with the other fractions not retained by the matrix (see Table 1). TABLE 1.
SEPARATION AND RECONSTITUTION OF ADENYLATE CYCLASE ACTIVITY*
Preparations
Adenylate Cyclase Activity tested with Gpp(NH)p Mg2+/F (nmoles/mg/min)
A. Control: Soluble adenylate cyclase after treatment with Sepharose B. Soluble adenylate cyelase after treatment with a GTP-Sepharose derivative C. Proteins detached from GTPSepharose with Gpp(NH)p D. B and C combined E. Proteins detached from GTPSepharose with GTP F. B and E combined
1.5 (100%)
0.81 (100%)
0.15-0.45t (10%-30%)
0.35 (43%)
0.03 (2%) 1.11
(74%) 0.015 (1.85%)$ 0.64 (79%)
*Adenylate cyclase was solubilized with Lubrol PX from membranes treated with 1 mM GMP and 50 ~M D,L-isoproterenol for 30 min at 37°C as described in references 6 and 7. One ml of the soluble protein (1.1 mg/ml) was added to 0.5 ml of a suspension of packed Sepharose 4B or of the GTP-Sepharose 4B derivative in 1.5 vol of a 250 mM NaCI solution. The mixtures were gently shaken at 22° C for 30 rain and centrifuged at 10,000 × g for 10 rain at 4°C and the supernatant solutions were tested for adenylate cyclase activity with 0.1 mM Gpp(NH)p or with 10 mM Mg2+/F-. Percentages of the activity of the complete system (100%) are given in parentheses. Sepharose with attached protein was separated from the supernatant solution by centIffugation washed 3 times with 83 mM NaCI, 0.66 mM Lubrol PX, 7 mM "Iris HC1 buffer pH 7.4, and finally brought to the original vol (1.5 mD with the same buffer. To onehalf of the suspension Gpp(NH)p at a final concentration of 0.1 mM was
214
ERNST J. M. HELMREICHet al. added to the other half GTP at a final concentration of 0.2 mM and the mixtures were gently shaken at 22°C for 120 rain and Sepharose separated by centrifugation. 50 ~tl aliquots of the detached proteins C and E were combined with fraction B and activity was measured. The supernatants in A and B used for fluoride activation received also final 0.05 mM GTP. In experiments with supernatant solutions (A and B) the vol was made equal by additions of 50 pl of the buffer without Lubrol PX. The relation of protein to detergent concentration is important for the expression of activity, presumably because of micelle formation. This is documented in detail in reference 7. tThere is some detachment of GTP and GDP from the matrix which binds to the proteins and interferes with Gpp(NH)p. This is mainly responsible for the variable results. 38GTP in contrast to Gpp(NH)p barely activates and hence does not interfere with fluoride activation.
Thomas Pfeuffer (7, 16) observed that the fluoride activation of adenylate cyclase was also lost concomitantly with guanylnucleotide activation (Table 1). That the loss of guanylnucleotide and fluoride stimulation was due to the removal of protein components of the adenylate cyclase system was shown by reconstitution experiments. Recombination with an adenylate cyclase preparation depleted of guanylnucleotide binding protein restored about ¾ of the original nucleotlde and fluoride sensitivity. No reconstitution occurs when matrix bound or soluble fractions are treated with trypsin or N-ethylmaleimide. The guanylnucleotide binding fraction had practically no adenylate cyclase activi~ when tested with Gpp(NH)p or NaF (Table 1). The guanylnucleotide binding proteins attached to the GTP-Sepharose affinity matrix are heterogeneous. They were removed from the matrix by addition of [3H] Gpp(NH)p and subjected to sucrose density gradient centrifugation. Only the heavier fractions containing the 42,000 MW protein did reactivate adenylate cyclase. The lighter fractions containing the 23,000 MW proteins were ineffective. While. this report was prepared it was found that GTP was hydrolyzed by the matrix bound fraction. The effect of fluoride on GTP hydrolysis is currently being studied (cf:reference 16). In this context it is of interest that Mittal e t al. (17) have recently separated a macromolecular nondialyzable and heat-labile fraction from guanylate cyclase of rat liver which was responsible for azide stimulation. In order to decide whether the GTPase activity actually resides in the guanylnucleotide binding protein or is due to a contaminating enzyme it will be necessary to purify the binding protein to homogeneity. Such experiments are currently underway. Experiments published elsewhere (7) show that one can reactivate adenylate cyclase in soluble fractions from rabbit myocard with guanylnucleotide binding proteins obtained from pigeon erythrocyte membranes. This finding agrees with the observation that activation by guanylnucleotides is a property shared by many adenylate cyclases from different cells which are responsive to different hormones. Thus, hormonally activated adenylate cyclases seem to have
GUANYLNUCLEOTIDE ACTIVATION OF ADENYLATE CYCLASE
215
structural features in common which endow them with similar regulatory properties and render them responsive to guanylnucleotide regulation.
Guanylnucleotides and Hormone Receptor An interesting, at present not yet understood relationship of the guanylnucleotide binding protein to the hormone receptor becomes apparent from the experiments in Table 2 (cf:reference 7). TABLE 2. THE EFFECT OF D,L-ISOPROTERENOLON THE REACTIVATION OF ADENYLATE CYCLASE BY THE GUANYLNUCLEOTIDE BINDING FRACTION* Activity on addition of Soluble Membrane Fractions
buffer
binding proteins from membranes treated with without Isoproterenol Isoproterenol
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0.45
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Adenylate cyelase from isoproterenol-treated membranes
1.6 (100%)
lII.
Fraction obtained from untreated membranes and deprived of guanyinueleotide binding proteins
0.I
1.5-1.1 (95%-69%)
0.3-0.2 (19%-12.5%)
IV.
Fraction obtained from isoproterenol-treated membranes and deprived of guanyinucleotide binding proteins
0.18
1.5 (94%)
0.47 (29%)
*Adenylate cyclase was solubilized with Lubrol PX from membranes preineubated with 0.1 mM GMP and with and without 50 t~M D,L-isoproterenol for 30 rain at 37°C. The solubilized material was treated with the G'I'P Sepbarose derivative as described in the legend to Table 1. The soluble fractions which passed through the GTP matrix were combined with 50 ;tl aliquots o f buffer (without Lubrol) or of the binding fractions which were retained by the matrix bound GTP-derivative, and adenylate cyelase activity was measured.
It is known that guanylnucleotides and hormones activate synergistically adenylate cyclase from a variety of sources (2, 6). Moreover, the activity elicited by both agents, for example catecholamine and Gpp(NH)p, could not be reversed by the /3-adrenergic antagonist propranolol (6, 18). We now found (Table 2)(cf:reference 7) that exposure of intact membranes to Isoprenaline (D,L-isoproterenol) makes the guanylnucleotide binding proteins isolated by affinity chromatography more effective in reactivating adenylate cyclase. Thus, the hormone acts v/a the matrix bound proteins, because recombination with guanylnucleotide binding fractions from inactive membranes yields little activity even when the soluble fractions which are not bound to the matrix come from hormonally activated membranes. Recent findings of Mukherjee and Lefkowitz (3) point likewise to a relationship between hormone receptor and guanyinucleotides. These authors found that desensitized/3-adrenergic receptors in frog erythrocyte membranes were rapidly and completely resensitized by
216
ERNST J. M. HELMREICHet al.
exposure of membranes to Gpp(NH)p and other guanine nucleotides. The efficacy of nucleotides for resensitization was Gpp(NH)p > GTP > GDP > GMP > ATP and hence the same as that of guanine nucleotide activation of adenylate cyclase.
Biological Significance o f Regulation o f Adenylate Cyclase by Guany lnuc leo tide s Although there exists an abundant literature (cf:reference 2) on guanylnucleotide activation of adenylate cyclases from a variety of broken cells and membrane preparations, the biological significance of naturally occurring guanylnucleotides such as GTP as regulators of hormonally activated adenylate cyclase in intact cells is still obscure. Purine nucleoside phosphates cannot enter intact cells. Although in most cells the concentration of GTP in the intracellular water is about 10-4 M, the concentration which usually gives maximal activation of adenylate cyclase in membranes, one has to consider that the intracellular concentrations of ATP are even higher (in the mM range). But high concentrations of ATP effectively compete and partially replace GTP as activator. GTP (lIT 4 M) for example, activated D,L-isoproterenol stimulated adenylate cyclases 2-5-fold in pigeon erythrocyte membranes only at 10-4 M--10-s M ATP concentrations. With App(NH)p, however, which compared with ATP was about 60% as effective as substrate, D,L-isoproterenol stimulated adenylate cyclase became nearly completely dependent on /aM concentrations of GTP for activity. App(NH)p did not compete with [a H] -Gpp(CH2 )p for binding in pigeon erythrocyte membranes (7). We have assumed that the much greater efficacy of the guanylnucleotide analogs compared with GTP is due to quasi irreversible binding of the nonhydrolyzable GTP analogs resulting in irreversibly activated enzyme complexes (6). As a consequence of that hypothesis one would have to postulate that the natural activator GTP is hydrolyzed by GTPase activity of the regulator protein. Further experiments will have to show whether the nucleotide binding protein fraction is itself a specific GTPase with a sufficiently high turnover. There is an interesting difference between GTP and GTP-~,-S. GTP-7-S is the most effective guanylnucleotide activator for pigeon erythrocyte membrane adenylate cyclase which we have tested (see Fig. 1 and reference (6)). GTP and GTP-~,-S seem to bind to the same sites in pigeon erythrocyte membranes (6) and are hydrolyzed at comparable rates at saturating concentrations. But an analysis of the membrane bound guanylnucleotides indicated that after 15 min of incubation at 37°C only about 10% was recovered unchanged in the case of GTP whereas 80% of the bound GTP-3,-S was intact. This suggests that GTP was hydrolyzed at additional sites in the membrane which are probably unrelated to adenylate cyclase activation and could explain why GTP-3,-S is a much better activator than GTP.
GUANYLNUCLEOTIDEACTIVATIONOF ADENYLATECYCLASE
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The majority of GTP binding proteins in erythrocyte membranes may be unrelated to adenylate cyclase regulation, although some of them may hydrolyze GTP and all of them share with the regulatory sites for adenylate cyclase the same high affinity and specificity for guanyl nucleotides and sensitivity towards SH-reagents. The function of these sites is unknown. To illustrate that point we refer to mammalian erythrocytes. The mature rat erythrocyte does not have adenylate cyclase activity but possesses about the same number of GTPmembrane-binding sites as does the reticulocyte which still has a highly active guanylnucleotide regulated adenylate cyclase (cf:reference 16). Although it has not been excluded that the catalytic part of adenylate cyclase becomes inactivated or is lost in the course of erythrocyte maturation, we still wonder what other functions in addition to that of regulating adenylate cyclase activity guanylnucleotide binding proteins may have in eucaryotic membranes. The fact that the major GTP binding proteins with MW 42,000 and 23,000 can be released from the membrane by low ionic strength - EDTA buffers, that is under conditions where contractile proteins in membranes, e.g.: components of the spectrin complex, are solubilized, hints at an as yet obscure involvement of the guanyl nucleotide binding proteins in other membranous processes in which contractile proteins take part (cf:reference 7).
Tubulin in l~geon Erythrocyte Membranes We have looked for a possible relationship to microtubular proteins which also bind GTP. As is shown in Figures 3A and 3B, Zenner and Pfeuffer (13) verified the presence of tubulin in membranes by binding intact pigeon erythrocytes to colchicine-Sepharose beads at 37°C. Addition of free colchicine (5 × 10-3 M) or incubation at 0°C prevented binding. Furthermore, lumicolchicine-Sepharose beads did not attach to erythrocytes at 37°C and binding of colchicine to pigeon erythrocytes decreased with time. These observations agree with the well-documented characteristics of colchicine binding to tubulin from various sources(19). Since colchicine was attached directly without a spacer to the Sepharose beads, it is unlikely that cytoplasmic microtubular proteins could have reached the immobilized colchicine. Zenner and Pfeuffer have also raised an antibody against bovine brain tubulin in rabbits and have demonstrated the presence 0f an immunologically crossreacting material in pigeon erythrocyte membranes solubilized with 2% sodium cholate (13). We therefore conclude that tubulin or an immunologically related protein with similar colchicine binding properties exists in pigeon erythrocyte membranes, but we have failed until now to demonstrate a link of the tubulin network to adenylate cyclase. The antitubulin antibody did not affect guanylnucleotide activation, hormonal stimulation or NaF activation of membranous or solubilized adenylate cyclase (13).
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ERNST J. M. HELMREICHet
al.
DISCUSSION Obviously, we have barely begun to probe into the molecular structure and function of the membrane bound hormonally stimulated adenylate cyclase complex. There are several models to explain the results presented here: all models visualize adenylate cyclase as a multicomponent system consisting of regulatory and catalytic components. In Model one removal of the regulatory GTP binding subunits causes inactivation. The loss of fluoride activation which accompanies the loss of nucleotide activation is thought to result from the dissociation of the holoenzyme complex which leaves the catalytic subunit in an inactive conformation. An alternative model suggests that the guanylnucleotide binding proteins are themselves involved in fluoride activation. Obviously, a decision for or against these and other plausible possibilities will only become feasible once the components of the adenylate cyclase system are isolated in pure form and characterized. Only then will it be possible to decide whether the effector binding sites and the catalytic sites are on separate or on the same polypeptide chains. Finally, we must consider the membranous location of hormonally responsive adenylate cyclase systems. Therefore the interaction of membrane lipids with the adenylate cyclase system needs to be studied. This is now made possible by manipulation of lipids and cholesterol (see also reference 10) in membranes from eucaryotic cells which have hormonally responsive adenylate cyclase activity and which can be propagated in tissue culture. Such experiments which have been initiated in our laboratory should tell us whether more general influences such as changes in micro-viscosity and fluidity, or more localized changes in the structural organization of the lipid halo surrounding adenylate cyclase affect the protein-protein interactions regulating its activity.
SUMMARY Hormone receptor complexes are tight complexes and rather long-lived. Thus several cycles of'activation and deactivation of adenylate cyclase might conceivably occur while the hormone is bound. Moreover,/~-adrenergic receptors can become desensitized. Regulatory steps are therefore postulated which control adenylate cyclase activity and in addition might also modulate the hormone receptor interaction. Isoprenaline (D,L-isoproterenol) stimulated adenylate cyclase from pigeon erythrocyte membranes is synergistically activated by analogs of GTP" Guanylylimidodiphosphate (Gpp(NH)p), Guanylylmethylenediphosphonate (Gpp(CH2)p) and Guanosine-5'-O-(3-thiotriphosphate) (GTP3,S). Among several
GUANYLNUCLEOTIDEACTIVATIONOF ADENYLATECYCLASE
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photoaffmity labels of GTP synthesized, GTP-?-azidoanilide (Gppp-NH- ( ~ N3) was best suited for the identification of GTP-binding proteins in pigeon erythrocyte membranes, because it binds with high affinity (Kd~-3 X 10-7 M) and activates adenylate cyclase about one-half as effectively as Gpp(NH)p. Four major protein fractions were labeled in membranes on photoactivation of GTP-7-azidoanilide. In solubilized membranes which contain adenylate cyclase fully responsive to guanylnucleotide activation, only two guanylnucleotide binding proteins were reactive with the photoaffinity derivative of GTP. These proteins had molecular weights of 42,000 and 23,000, respectively. Soluble guanylnucleotide binding proteins were removed by affmity chromatography with Sepharose-NH~CH2)3-CO-NH- Q - N H - p p p G with loss of guanylnucleotide and fluoride activation. Readdition of the protein fraction released from the affinity column restored guanylnucleotide and fluoride activation. This suggests the existence of separable protein moieties which are required for adenylate cyclase activation. ACKNOWLEDGEMENTS The work reported here was supported by grants from the Deutsche Forschungsgemeinschaft and by the Fonds der Chemischen Industrie. Some of the results reported here were submitted to the Journal of Biological Chemistry for publication (cf:reference 7). REFERENCES 1. E.J.M. HELMREICH,Hormone-receptorinteractions, FEBS Letters 61, 1-5 (1976). 2. E.J.M. HELMREICH,H. P. ZENNER, T. PFEUFFER and C. F. CORI, Signal transfer from hormone receptor to adenylate cyelase, pp. 41-87 in Current Topics in Cellular Regulation (B. L. HORECKER and E. R. STADTMAN, eds.), Academic Press, New York, San Francisco, London, Vol. 10, (1976). 3. C. MUKHERJEE and R. J. LEFKOWITZ,Desensitization of~-adrenergic agonists in a cell-free system: Resensitization by guanosine 5'-(~, ~-imino) triphosphate and other purine nucleotides, Proc. Natl. Acad. Sci. USA 73, 1494-1498 (1976). 4. E. J. M. HELMREICH, Die Obersetzung der Hormonrezeptorwechselwitkung in biologische Funktion, M~nch. med. Wschr. 117, 1215-1220 (1975). 5. T. PFEUFFER and E. J. M. HELMREICH, Signaloverdracht van de /3-adrenergische Receptor naar Adenylcyclase,Chemisch Weekblad, 30/31,370-372 (1976). 6. T. PFEUFFER and E. J. M. HELMREICH, Activation of pigeon erythrocyte membrane adenylate cyelase by guanylnucleotide analogues and separation of a nucleotide binding protein, J. Biol. C h e ~ 250, 867-876 (1975). 7. T. PFEUFFER, GTP-binding proteins in membranes and the control of adenylate cyclase activity, J. Biol. Cher~ submitted for publication (1976). 8. M.J. BERRIDGE, The interaction of cyclic nucleotides and calcium in the control of cellular activity, Advances in Cyclic Nucleotide R el. 6, 1-98 (1975). 9. J. ~YE and E. W. SUTHERLAND, The effect of epinephrine and other agents on adenyl cyclase in the cell membrane of avian erythrocytes, Biochim. Biophys. Acta 127, 347-354 (1966). 10. G. PUCHWEIN, T. PFEUFFER and E. J. M. HELMREICH, Uncoupling of catecholamine activation of pigeon erythroeyte membrane adenylate cyelase by t~dipin, J. Biol. Chem. 249, 3232-3240 (1974).
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11. J. RAMACHANDRAN, A new simple method for separation of adenosine 3',5'-cyclic monophosphate from other nucleotides and its use in the assay of adenyl cyclase, Anal. Biochem. 4 3 , 2 2 7 - 2 3 9 (1971). 12. D. M. NEVILLE, Jr. and H. GLOSSMANN, Plasma membrane protein subunit composition, A comparative study by discontinuous electrophoresis in sodium dodecyl sulfate, J. Biol. Chem. 246, 6335-6338 (1971). 13. H. P. ZENNER and T. PFEUFFER, Microtubular proteins in pigeon erythrocyte membranes, Europ. J. Biochem. 71,177-184 (1976). 14. A. LEVITZKI, N. SEVILLA, D. ATLAS and M. L. STEER, Ligand specificity and characteristics of the ~-adrenergic receptor in turkey erythrocyte plasma membranes, J. Mol. Biol. 97, 35-53 (1975). 15. K. TAKAI, Y. KUROSHIMA, C. SUZUKI-HORI, H. OKAMOTO and O. HAYAISHI, Adenylate cyclase from brevibacterium liquefaeiens. I. Purification, crystallization, and some properties, J. Biol. Chem. 249, 1965-1972 (1974). 16. T. PFEUFFER, unpublished experiments (1976). 17. C. K. MITTAL, H. KIMURA and F. MURAD, Requirement for a macromolecular factor for sodium azide activation of guanylate cyclase, J. o f Cyclic Nucleotide Research, 1 , 2 6 1 - 2 6 9 (1975). 18. M. SCHRAMM and M. RODBELL, A persistent active state of the adenylate cyclase system produced by the combined actions of isoproterenol and guanylyl imidodiphosphate in frog erythrocyte membranes, J. Biol. Chem. 250, 2232-2237 (1975). 19. L. WILSON, J. R. BAMBURG, S. B. MIZEL, L. M. GRISHAM and K. M. CRESWELL, Interaction of drugs with microtubule proteins, Fed. Proc. 33,158-166 (1974).
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ERNST J. M. HELMREICH et al.
trophoresis was carried out according to Neville and Glossmann (12). The synthesis of p3.(4.azidoanilido) p1-GTP, photoactivation and the preparation of the GTP-sepharose derivative are described by Pfeuffer (7). Colchicine Sepharose-4B derivatives are described by Zenner and Pfeuffer (13).
Activation by Guanylnucleotides Purine nucleotides, especially GTP, seem to play an ubiquitous role in optimizing the response of adenylate cyclase to hormones (2). In the nucleated avian erythrocytes which possess an adenylate cyclase system highly sensitive to catecholamines, guanylnucleotide analogs such as Gpp(CH2)p, Gpp(NH)p and GTP-7-S are much more effective activators than the natural effector GTP (6, 7) (See Fig. 1). We have made use of the high efficacy and the tight binding of the guanylnucleotide analogs in a search for GTP-binding proteins in pigeon erythrocyte membranes.
PARTICULATEADENYLATECYCLASE '.~-m400I x
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i0-6 GUANYLNUCLEOTIDES
GTP I
i0-4 [M]
FIG. 1. Activation of adenylate cyclase in pigeon ~rythrocyte membranes by guanylnucleotides. D,L-Isoproterenol (50/~M), the guanyinucleotides and ATP (0.1 raM) were added together at 37°C. cAMP formed was measured with 220 ~g of membrane protein after 10 rain at 37°C. (Reproduced by permission of the J. Biol. Chem. from T. Pfeuffer and E. Helmreieh (6).)
Guanylnucleotide Binding Sites Previous studies had shown that 120 pmoles of [all] Gpp(CH2)p per mg of protein were bound to pigeon erythrocyte membranes (6). Levitzki et al. (14) had, however, convincingly shown that only 1-2 pmoles of catecholamine per mg of protein were bound to turkey erythrocyte membranes. Subsequently, a
GUANYLNUCLEOTIDEACTIVATIONOF ADENYLATECYCLASE
211
comparably low value has also been found in our laboratory for pigeon erythrocyte membranes (cf:reference 7). Levitzki et al. (14) have made the interesting point that assuming a 1 : 1 relationship between receptor sites and catalytic sites, 1-2 pmoles are a realistic estimate for adenylate cyclase active sites because calculated on that basis the turnover number would be about 1,400 min-1 and thus approximate the turnover number of the crystalline soluble adenylate cyclase from Brevibacterium liquefaciens (15). Thus, following this argument, the guanylnucleotide binding sites are far in excess over the catalytic sites, suggesting that the greater part of them is unrelated to adenylate cyclase activation. This was shown to be correct in recent experirnents (of:reference 7) carried out to identify the GTP-binding site of catecholamine stimulated adenylate cyclase in pigeon erythrocyte membranes. This was achieved with the aid of a photoreactive [73ZP] labeled GTP derivative, pa.(4.azidoanilido) p l _ guanosine triphosphate, which is a potent activator of adenylate cyclase and which binds with high affinity (Kdiss = 3.3 X 10 -7 M). This GTP derivative competes with GTP or GTP-analogs for common binding sites in membranes. Only those binding sites for which the photoreactive GTP-azidoanilide and Gpp(NH)p effectively compete were considered specific binding sites. On photolysis by light of wavelengths > 350 nm nitrenes are generated which are I attacked by nucleophlles and also inserted into - C - H bonds of amino acid residues. Besides membrane proteins lipids also react with GTP azidoanilide but both materials can easily be separated. No covalent incorporation of the GTP-analog occurred in the dark or when the [Ta:P] GTP-azidoanilide was photolyzed before it was added. The proteins labeled in intact membranes and of Lubrol PX solubllized membranes containing adenylate cyclase activity were separated and analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis (see Figs. 2A and 2B). Four major proteins with MW 86,000, 52,000, 42,000 and 23,000 wete labeled (Fig. 2A). In addition a membrane protein of MW of about 250,000 which co-migrates with spectrin I was labeled but in this case an excess of Gpp(NH)p did not prevent incorporation of the radioactive photoaffinity reagent. In Lubrol solubilized membranes which lack the spectrin complex only the GTP-binding proteins with MW 42,000 and 23,000 were specifically labeled (Fig. 2B). Thus, because soluble adenylate cyclase is fully responsive to nucleotide stimulation only the binding proteins with MW 23,000 and 42,000 need to be considered for a role in adenylate cyclase activation. On centrifugation of the soluble preparation through a linear gradient of 5% to 30% sucrose more than 95% of the nucleotide binding proteins sediment slower than adenylate cyclase activity and only about 5% cosediment with it. The guanylnucleotide binding fractions separated by sucrose gradient centrifugation were labeled with [Ta2P] GTP-azidoanilide and analyzed by sodium dodecylsulfate acrylamide gel electrophoresis. The slow sedimenting nucleotide binding peak which could be separated from adenylate cyclase activity without loss of guanylnucleotide activation was exclusively the 23,000 MW protein. The binding
212
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FIGS. 2A and 2B. Photoaffinity-labeling of membranous and Lubrol PX solubilized proteins. A: Membranes, 2.5 mg per ml, were incubated with 3 ~M [~/32 p] -GTP azidoanilide (specific activity 5 Ci/mmole) and 50 izM D,L-isoproterenol for 30 min at 37°C. After removal of excess nucleotide by 3 washes with buffer each with 5 times the vol of the membrane suspension (see legend to Table 1), the membranes were incubated for another 25 rain with ( . o) and without (o o) 0.1 mM Gpp(NH)p and photolyzed for 60 min at 4°C. The irradiated samples (250-600 #g protein) were treated with 1% sodium dodecylsuifate and 10 mM 2-mercaptoethanol for 15 min at 37°C and electrophoresed on 12.5% polyacrylamide gels containing 0.1% SDS. Phospholipids have about the same mobility as the tracking dye (TD). B: Membranes were incubated with GTP-azidoanilide and D,L-isoproterenol as described in A. The washed membranes were solubilized with 20 mM Lubrol PX for 30 min at 4°C as descr~ed previously (6) and incubated for 25 min at 37°C in the presence (e , ) and absence (o o) of 0.1 mM Gpp(NH)p and then photolyzed and separated electrophoreticaUy on polyacrylamide gels as described in A and Methods.
GUANYLNUCLEOTIDE ACTIVATION OF ADENYLATE CYCLASE
213
protein which cosedimented with adenylate cyclase activity was mainly the 42,000 MW protein together with some of the 23,000 MW protein and traces of the 86,000 MW protein, b u t the latter was only unspecifically labeled since the attachment of the photoaffinity analog was not prevented by high concentrations of Gpp(NH)p (see reference 7).
Separation and Reconstitution of Adenylate Cyclase Activity With the Sepharose b o u n d GTP derivative, Seph4B-NH(CH2)3-CONH- Q -NH-pppG proteins including the guanylnucleotide binding protein with MW 42,000 could be detached from a detergent solubilized adenylate cyclase preparation with loss of nucleotide stimulation which could be partly restored b y recombining the guanylnucleotide binding protein with the other fractions not retained by the matrix (see Table 1). TABLE 1.
SEPARATION AND RECONSTITUTION OF ADENYLATE CYCLASE ACTIVITY*
Preparations
Adenylate Cyclase Activity tested with Gpp(NH)p Mg2+/F (nmoles/mg/min)
A. Control: Soluble adenylate cyclase after treatment with Sepharose B. Soluble adenylate cyelase after treatment with a GTP-Sepharose derivative C. Proteins detached from GTPSepharose with Gpp(NH)p D. B and C combined E. Proteins detached from GTPSepharose with GTP F. B and E combined
1.5 (100%)
0.81 (100%)
0.15-0.45t (10%-30%)
0.35 (43%)
0.03 (2%) 1.11
(74%) 0.015 (1.85%)$ 0.64 (79%)
*Adenylate cyclase was solubilized with Lubrol PX from membranes treated with 1 mM GMP and 50 ~M D,L-isoproterenol for 30 min at 37°C as described in references 6 and 7. One ml of the soluble protein (1.1 mg/ml) was added to 0.5 ml of a suspension of packed Sepharose 4B or of the GTP-Sepharose 4B derivative in 1.5 vol of a 250 mM NaCI solution. The mixtures were gently shaken at 22° C for 30 rain and centrifuged at 10,000 × g for 10 rain at 4°C and the supernatant solutions were tested for adenylate cyclase activity with 0.1 mM Gpp(NH)p or with 10 mM Mg2+/F-. Percentages of the activity of the complete system (100%) are given in parentheses. Sepharose with attached protein was separated from the supernatant solution by centIffugation washed 3 times with 83 mM NaCI, 0.66 mM Lubrol PX, 7 mM "Iris HC1 buffer pH 7.4, and finally brought to the original vol (1.5 mD with the same buffer. To onehalf of the suspension Gpp(NH)p at a final concentration of 0.1 mM was
214
ERNST J. M. HELMREICHet al. added to the other half GTP at a final concentration of 0.2 mM and the mixtures were gently shaken at 22°C for 120 rain and Sepharose separated by centrifugation. 50 ~tl aliquots of the detached proteins C and E were combined with fraction B and activity was measured. The supernatants in A and B used for fluoride activation received also final 0.05 mM GTP. In experiments with supernatant solutions (A and B) the vol was made equal by additions of 50 pl of the buffer without Lubrol PX. The relation of protein to detergent concentration is important for the expression of activity, presumably because of micelle formation. This is documented in detail in reference 7. tThere is some detachment of GTP and GDP from the matrix which binds to the proteins and interferes with Gpp(NH)p. This is mainly responsible for the variable results. 38GTP in contrast to Gpp(NH)p barely activates and hence does not interfere with fluoride activation.
Thomas Pfeuffer (7, 16) observed that the fluoride activation of adenylate cyclase was also lost concomitantly with guanylnucleotide activation (Table 1). That the loss of guanylnucleotide and fluoride stimulation was due to the removal of protein components of the adenylate cyclase system was shown by reconstitution experiments. Recombination with an adenylate cyclase preparation depleted of guanylnucleotide binding protein restored about ¾ of the original nucleotlde and fluoride sensitivity. No reconstitution occurs when matrix bound or soluble fractions are treated with trypsin or N-ethylmaleimide. The guanylnucleotide binding fraction had practically no adenylate cyclase activi~ when tested with Gpp(NH)p or NaF (Table 1). The guanylnucleotide binding proteins attached to the GTP-Sepharose affinity matrix are heterogeneous. They were removed from the matrix by addition of [3H] Gpp(NH)p and subjected to sucrose density gradient centrifugation. Only the heavier fractions containing the 42,000 MW protein did reactivate adenylate cyclase. The lighter fractions containing the 23,000 MW proteins were ineffective. While. this report was prepared it was found that GTP was hydrolyzed by the matrix bound fraction. The effect of fluoride on GTP hydrolysis is currently being studied (cf:reference 16). In this context it is of interest that Mittal e t al. (17) have recently separated a macromolecular nondialyzable and heat-labile fraction from guanylate cyclase of rat liver which was responsible for azide stimulation. In order to decide whether the GTPase activity actually resides in the guanylnucleotide binding protein or is due to a contaminating enzyme it will be necessary to purify the binding protein to homogeneity. Such experiments are currently underway. Experiments published elsewhere (7) show that one can reactivate adenylate cyclase in soluble fractions from rabbit myocard with guanylnucleotide binding proteins obtained from pigeon erythrocyte membranes. This finding agrees with the observation that activation by guanylnucleotides is a property shared by many adenylate cyclases from different cells which are responsive to different hormones. Thus, hormonally activated adenylate cyclases seem to have
GUANYLNUCLEOTIDE ACTIVATION OF ADENYLATE CYCLASE
215
structural features in common which endow them with similar regulatory properties and render them responsive to guanylnucleotide regulation.
Guanylnucleotides and Hormone Receptor An interesting, at present not yet understood relationship of the guanylnucleotide binding protein to the hormone receptor becomes apparent from the experiments in Table 2 (cf:reference 7). TABLE 2. THE EFFECT OF D,L-ISOPROTERENOLON THE REACTIVATION OF ADENYLATE CYCLASE BY THE GUANYLNUCLEOTIDE BINDING FRACTION* Activity on addition of Soluble Membrane Fractions
buffer
binding proteins from membranes treated with without Isoproterenol Isoproterenol
[nmoles,'mg/minwith 0.1 m M Gpp(NH)p] 1.
Adenylate cyclase from untreated membranes
0.45
II.
Adenylate cyelase from isoproterenol-treated membranes
1.6 (100%)
lII.
Fraction obtained from untreated membranes and deprived of guanyinueleotide binding proteins
0.I
1.5-1.1 (95%-69%)
0.3-0.2 (19%-12.5%)
IV.
Fraction obtained from isoproterenol-treated membranes and deprived of guanyinucleotide binding proteins
0.18
1.5 (94%)
0.47 (29%)
*Adenylate cyclase was solubilized with Lubrol PX from membranes preineubated with 0.1 mM GMP and with and without 50 t~M D,L-isoproterenol for 30 rain at 37°C. The solubilized material was treated with the G'I'P Sepbarose derivative as described in the legend to Table 1. The soluble fractions which passed through the GTP matrix were combined with 50 ;tl aliquots o f buffer (without Lubrol) or of the binding fractions which were retained by the matrix bound GTP-derivative, and adenylate cyelase activity was measured.
It is known that guanylnucleotides and hormones activate synergistically adenylate cyclase from a variety of sources (2, 6). Moreover, the activity elicited by both agents, for example catecholamine and Gpp(NH)p, could not be reversed by the /3-adrenergic antagonist propranolol (6, 18). We now found (Table 2)(cf:reference 7) that exposure of intact membranes to Isoprenaline (D,L-isoproterenol) makes the guanylnucleotide binding proteins isolated by affinity chromatography more effective in reactivating adenylate cyclase. Thus, the hormone acts v/a the matrix bound proteins, because recombination with guanylnucleotide binding fractions from inactive membranes yields little activity even when the soluble fractions which are not bound to the matrix come from hormonally activated membranes. Recent findings of Mukherjee and Lefkowitz (3) point likewise to a relationship between hormone receptor and guanyinucleotides. These authors found that desensitized/3-adrenergic receptors in frog erythrocyte membranes were rapidly and completely resensitized by
216
ERNST J. M. HELMREICHet al.
exposure of membranes to Gpp(NH)p and other guanine nucleotides. The efficacy of nucleotides for resensitization was Gpp(NH)p > GTP > GDP > GMP > ATP and hence the same as that of guanine nucleotide activation of adenylate cyclase.
Biological Significance o f Regulation o f Adenylate Cyclase by Guany lnuc leo tide s Although there exists an abundant literature (cf:reference 2) on guanylnucleotide activation of adenylate cyclases from a variety of broken cells and membrane preparations, the biological significance of naturally occurring guanylnucleotides such as GTP as regulators of hormonally activated adenylate cyclase in intact cells is still obscure. Purine nucleoside phosphates cannot enter intact cells. Although in most cells the concentration of GTP in the intracellular water is about 10-4 M, the concentration which usually gives maximal activation of adenylate cyclase in membranes, one has to consider that the intracellular concentrations of ATP are even higher (in the mM range). But high concentrations of ATP effectively compete and partially replace GTP as activator. GTP (lIT 4 M) for example, activated D,L-isoproterenol stimulated adenylate cyclases 2-5-fold in pigeon erythrocyte membranes only at 10-4 M--10-s M ATP concentrations. With App(NH)p, however, which compared with ATP was about 60% as effective as substrate, D,L-isoproterenol stimulated adenylate cyclase became nearly completely dependent on /aM concentrations of GTP for activity. App(NH)p did not compete with [a H] -Gpp(CH2 )p for binding in pigeon erythrocyte membranes (7). We have assumed that the much greater efficacy of the guanylnucleotide analogs compared with GTP is due to quasi irreversible binding of the nonhydrolyzable GTP analogs resulting in irreversibly activated enzyme complexes (6). As a consequence of that hypothesis one would have to postulate that the natural activator GTP is hydrolyzed by GTPase activity of the regulator protein. Further experiments will have to show whether the nucleotide binding protein fraction is itself a specific GTPase with a sufficiently high turnover. There is an interesting difference between GTP and GTP-~,-S. GTP-7-S is the most effective guanylnucleotide activator for pigeon erythrocyte membrane adenylate cyclase which we have tested (see Fig. 1 and reference (6)). GTP and GTP-~,-S seem to bind to the same sites in pigeon erythrocyte membranes (6) and are hydrolyzed at comparable rates at saturating concentrations. But an analysis of the membrane bound guanylnucleotides indicated that after 15 min of incubation at 37°C only about 10% was recovered unchanged in the case of GTP whereas 80% of the bound GTP-3,-S was intact. This suggests that GTP was hydrolyzed at additional sites in the membrane which are probably unrelated to adenylate cyclase activation and could explain why GTP-3,-S is a much better activator than GTP.
GUANYLNUCLEOTIDEACTIVATIONOF ADENYLATECYCLASE
217
The majority of GTP binding proteins in erythrocyte membranes may be unrelated to adenylate cyclase regulation, although some of them may hydrolyze GTP and all of them share with the regulatory sites for adenylate cyclase the same high affinity and specificity for guanyl nucleotides and sensitivity towards SH-reagents. The function of these sites is unknown. To illustrate that point we refer to mammalian erythrocytes. The mature rat erythrocyte does not have adenylate cyclase activity but possesses about the same number of GTPmembrane-binding sites as does the reticulocyte which still has a highly active guanylnucleotide regulated adenylate cyclase (cf:reference 16). Although it has not been excluded that the catalytic part of adenylate cyclase becomes inactivated or is lost in the course of erythrocyte maturation, we still wonder what other functions in addition to that of regulating adenylate cyclase activity guanylnucleotide binding proteins may have in eucaryotic membranes. The fact that the major GTP binding proteins with MW 42,000 and 23,000 can be released from the membrane by low ionic strength - EDTA buffers, that is under conditions where contractile proteins in membranes, e.g.: components of the spectrin complex, are solubilized, hints at an as yet obscure involvement of the guanyl nucleotide binding proteins in other membranous processes in which contractile proteins take part (cf:reference 7).
Tubulin in l~geon Erythrocyte Membranes We have looked for a possible relationship to microtubular proteins which also bind GTP. As is shown in Figures 3A and 3B, Zenner and Pfeuffer (13) verified the presence of tubulin in membranes by binding intact pigeon erythrocytes to colchicine-Sepharose beads at 37°C. Addition of free colchicine (5 × 10-3 M) or incubation at 0°C prevented binding. Furthermore, lumicolchicine-Sepharose beads did not attach to erythrocytes at 37°C and binding of colchicine to pigeon erythrocytes decreased with time. These observations agree with the well-documented characteristics of colchicine binding to tubulin from various sources(19). Since colchicine was attached directly without a spacer to the Sepharose beads, it is unlikely that cytoplasmic microtubular proteins could have reached the immobilized colchicine. Zenner and Pfeuffer have also raised an antibody against bovine brain tubulin in rabbits and have demonstrated the presence 0f an immunologically crossreacting material in pigeon erythrocyte membranes solubilized with 2% sodium cholate (13). We therefore conclude that tubulin or an immunologically related protein with similar colchicine binding properties exists in pigeon erythrocyte membranes, but we have failed until now to demonstrate a link of the tubulin network to adenylate cyclase. The antitubulin antibody did not affect guanylnucleotide activation, hormonal stimulation or NaF activation of membranous or solubilized adenylate cyclase (13).
218
ERNST J. M. HELMREICHet
al.
DISCUSSION Obviously, we have barely begun to probe into the molecular structure and function of the membrane bound hormonally stimulated adenylate cyclase complex. There are several models to explain the results presented here: all models visualize adenylate cyclase as a multicomponent system consisting of regulatory and catalytic components. In Model one removal of the regulatory GTP binding subunits causes inactivation. The loss of fluoride activation which accompanies the loss of nucleotide activation is thought to result from the dissociation of the holoenzyme complex which leaves the catalytic subunit in an inactive conformation. An alternative model suggests that the guanylnucleotide binding proteins are themselves involved in fluoride activation. Obviously, a decision for or against these and other plausible possibilities will only become feasible once the components of the adenylate cyclase system are isolated in pure form and characterized. Only then will it be possible to decide whether the effector binding sites and the catalytic sites are on separate or on the same polypeptide chains. Finally, we must consider the membranous location of hormonally responsive adenylate cyclase systems. Therefore the interaction of membrane lipids with the adenylate cyclase system needs to be studied. This is now made possible by manipulation of lipids and cholesterol (see also reference 10) in membranes from eucaryotic cells which have hormonally responsive adenylate cyclase activity and which can be propagated in tissue culture. Such experiments which have been initiated in our laboratory should tell us whether more general influences such as changes in micro-viscosity and fluidity, or more localized changes in the structural organization of the lipid halo surrounding adenylate cyclase affect the protein-protein interactions regulating its activity.
SUMMARY Hormone receptor complexes are tight complexes and rather long-lived. Thus several cycles of'activation and deactivation of adenylate cyclase might conceivably occur while the hormone is bound. Moreover,/~-adrenergic receptors can become desensitized. Regulatory steps are therefore postulated which control adenylate cyclase activity and in addition might also modulate the hormone receptor interaction. Isoprenaline (D,L-isoproterenol) stimulated adenylate cyclase from pigeon erythrocyte membranes is synergistically activated by analogs of GTP" Guanylylimidodiphosphate (Gpp(NH)p), Guanylylmethylenediphosphonate (Gpp(CH2)p) and Guanosine-5'-O-(3-thiotriphosphate) (GTP3,S). Among several
GUANYLNUCLEOTIDEACTIVATIONOF ADENYLATECYCLASE
219
photoaffmity labels of GTP synthesized, GTP-?-azidoanilide (Gppp-NH- ( ~ N3) was best suited for the identification of GTP-binding proteins in pigeon erythrocyte membranes, because it binds with high affinity (Kd~-3 X 10-7 M) and activates adenylate cyclase about one-half as effectively as Gpp(NH)p. Four major protein fractions were labeled in membranes on photoactivation of GTP-7-azidoanilide. In solubilized membranes which contain adenylate cyclase fully responsive to guanylnucleotide activation, only two guanylnucleotide binding proteins were reactive with the photoaffinity derivative of GTP. These proteins had molecular weights of 42,000 and 23,000, respectively. Soluble guanylnucleotide binding proteins were removed by affmity chromatography with Sepharose-NH~CH2)3-CO-NH- Q - N H - p p p G with loss of guanylnucleotide and fluoride activation. Readdition of the protein fraction released from the affinity column restored guanylnucleotide and fluoride activation. This suggests the existence of separable protein moieties which are required for adenylate cyclase activation. ACKNOWLEDGEMENTS The work reported here was supported by grants from the Deutsche Forschungsgemeinschaft and by the Fonds der Chemischen Industrie. Some of the results reported here were submitted to the Journal of Biological Chemistry for publication (cf:reference 7). REFERENCES 1. E.J.M. HELMREICH,Hormone-receptorinteractions, FEBS Letters 61, 1-5 (1976). 2. E.J.M. HELMREICH,H. P. ZENNER, T. PFEUFFER and C. F. CORI, Signal transfer from hormone receptor to adenylate cyelase, pp. 41-87 in Current Topics in Cellular Regulation (B. L. HORECKER and E. R. STADTMAN, eds.), Academic Press, New York, San Francisco, London, Vol. 10, (1976). 3. C. MUKHERJEE and R. J. LEFKOWITZ,Desensitization of~-adrenergic agonists in a cell-free system: Resensitization by guanosine 5'-(~, ~-imino) triphosphate and other purine nucleotides, Proc. Natl. Acad. Sci. USA 73, 1494-1498 (1976). 4. E. J. M. HELMREICH, Die Obersetzung der Hormonrezeptorwechselwitkung in biologische Funktion, M~nch. med. Wschr. 117, 1215-1220 (1975). 5. T. PFEUFFER and E. J. M. HELMREICH, Signaloverdracht van de /3-adrenergische Receptor naar Adenylcyclase,Chemisch Weekblad, 30/31,370-372 (1976). 6. T. PFEUFFER and E. J. M. HELMREICH, Activation of pigeon erythrocyte membrane adenylate cyelase by guanylnucleotide analogues and separation of a nucleotide binding protein, J. Biol. C h e ~ 250, 867-876 (1975). 7. T. PFEUFFER, GTP-binding proteins in membranes and the control of adenylate cyclase activity, J. Biol. Cher~ submitted for publication (1976). 8. M.J. BERRIDGE, The interaction of cyclic nucleotides and calcium in the control of cellular activity, Advances in Cyclic Nucleotide R el. 6, 1-98 (1975). 9. J. ~YE and E. W. SUTHERLAND, The effect of epinephrine and other agents on adenyl cyclase in the cell membrane of avian erythrocytes, Biochim. Biophys. Acta 127, 347-354 (1966). 10. G. PUCHWEIN, T. PFEUFFER and E. J. M. HELMREICH, Uncoupling of catecholamine activation of pigeon erythroeyte membrane adenylate cyelase by t~dipin, J. Biol. Chem. 249, 3232-3240 (1974).
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11. J. RAMACHANDRAN, A new simple method for separation of adenosine 3',5'-cyclic monophosphate from other nucleotides and its use in the assay of adenyl cyclase, Anal. Biochem. 4 3 , 2 2 7 - 2 3 9 (1971). 12. D. M. NEVILLE, Jr. and H. GLOSSMANN, Plasma membrane protein subunit composition, A comparative study by discontinuous electrophoresis in sodium dodecyl sulfate, J. Biol. Chem. 246, 6335-6338 (1971). 13. H. P. ZENNER and T. PFEUFFER, Microtubular proteins in pigeon erythrocyte membranes, Europ. J. Biochem. 71,177-184 (1976). 14. A. LEVITZKI, N. SEVILLA, D. ATLAS and M. L. STEER, Ligand specificity and characteristics of the ~-adrenergic receptor in turkey erythrocyte plasma membranes, J. Mol. Biol. 97, 35-53 (1975). 15. K. TAKAI, Y. KUROSHIMA, C. SUZUKI-HORI, H. OKAMOTO and O. HAYAISHI, Adenylate cyclase from brevibacterium liquefaeiens. I. Purification, crystallization, and some properties, J. Biol. Chem. 249, 1965-1972 (1974). 16. T. PFEUFFER, unpublished experiments (1976). 17. C. K. MITTAL, H. KIMURA and F. MURAD, Requirement for a macromolecular factor for sodium azide activation of guanylate cyclase, J. o f Cyclic Nucleotide Research, 1 , 2 6 1 - 2 6 9 (1975). 18. M. SCHRAMM and M. RODBELL, A persistent active state of the adenylate cyclase system produced by the combined actions of isoproterenol and guanylyl imidodiphosphate in frog erythrocyte membranes, J. Biol. Chem. 250, 2232-2237 (1975). 19. L. WILSON, J. R. BAMBURG, S. B. MIZEL, L. M. GRISHAM and K. M. CRESWELL, Interaction of drugs with microtubule proteins, Fed. Proc. 33,158-166 (1974).