Cell Calcium 6: 213-226, 1985

CALMODULIN REGULATION OF ADENYLATE CYCLASE ACTIVITY

S Mac Neil, T Lakey and S Tomlinson

Department of Medicine, Clinical Sciences Centre, Northern General Hospital, Sheffield, S5 7AU, UK. (reprint requests to SMN)

Calmodulin activation of adenylate cyclase, originally described for brain, (1,2) has now been demonstrated in a variety of cell and tissue types as indicated in Table 7. In this review we discuss the evidence that calmodulin activation of adenylate cyclase is a generalised phenomenon and we examine current knowledge about the mechanisms of interaction of calmodulin with this enzyme.

Discovery of calmodulin-dependent adenylate cyclase It has been known for some time that many adenylate cyclase

systems are calcium-dependent, low concentrations of calcium stimulating enzyme activity, high concentrations inhibiting enzyme activity - see Bradham and Cheung 1980 (27) for a review of this area. Calmodulin activation of adenylate cyclase is a more recent discovery, being first shown for brain in 1975 (1,2), 5 years after the initial discovery of calmodulin as a regulator of cyclic nucleotide phosphodiesterase in the brain (25,261. For both enzymes the accidental removal of endogenous calmodulin was the key to the discovery of their activation by calmodulin. The relevance of calmodulin to non-neural adenylate cyclase systems is only now becoming apparent, (Table 1). This delay appears largely attributable to two factors: difficulties in the removal of endogenous calmodulin from the particulate enzyme and the need to investigate enzyme activity over an appropriate range of calcium concentrations.

Removal of endogenous calmodulin Approximately 40-50X of the cell calmodulin is present in a

crude membrane preparation (11,281. With only one exception (ll), the investigators listed in Table 1 have found it necessary to remove at least some of this endogenous calmodulin in order to subsequently demonstrate calmodulin activation of the enzyme. (Several authors who did not measure membrane calmodulin nevertheless used EDTA and EGTA, the most common approach to the removal of calmodulin, in their membrane preparation). Also, in at least some of the tissues where calmodulin was reported not to activate the adenylate cyclase enzyme,

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endogenous calmodulin was either incompletely removed - cardiac muscle (29) or not removed at all - Chinese hamster ovary cells (30).

Removal of calmodulin from brain adenylate cyclase was first achieved in 1975 (1,2) by chromatography of solubilised enzyme preparations. These preparations lose agonist-responsiveness during solubilisation, however, and in 1977 (31) removal of calmodulin from particulate adenylate cyclase was also demonstrated in brain using the chelators EDTA and EGTA. These chelators will sequester calcium, (and other ions to varying degrees) without which calmodulin cannot bind to its acceptor proteins. However the effectiveness of this technique for removal of membrane calmodulin varies from tissue to tissue. For example, over 95% removal was found for rat brain (31) and 70% for bovine adrenal medulla (7) but in other tissues little or no reduction in calmodulin was found: guinea pig sperm (321, rat pituitary GH, cells (1 I), human thyroid membranes (23) and mouse B16 melanoma (24). These variations in the ease with which calmodulin is removed suggest that membrane binding sites can differ appreciably in their affinity for calmodulin.

Recently lanthanum (La'+) has been reported to be more effective than EGTA in the depletion of calmodulin from the ciliary membrane of Paramecium (33) and our own results confirm LaCl, to be effective in removing calmodulin from human thyroid (23) and mouse B16 melanoma membranes (24). This technique may prove valuable for the demonstration of calmodulin-dependent adenylate cyclase activity in other tissues also.

Calcium sensitivity of the adenylate cyclase enzyme, with and without calmodulin present

Essentially4 removal of endogenous calmodulin (whether by EGTA/EDTA or La3 ) is associated with a loss of both basal (eg 8) and hormone-stimulated (eg 9) adenylate cyclase activation normally observed at submicromolar Ca2+ concentrations.

Thus, it has been known for some time that many adenylate cyclase systems have a biphasic response to calcium, requiring comparatively low concentrations of calcium for maximal enzyme activity and being inhibited at higher concentrations of calcium (see 27). Marked inhibition of enzyme activity was often found around 1 IJ-M calcium, complete inhibition usually occurring above 10 @I calcium eg (23,241. Because of difficulties of removing endogenous calcium from the particulate enzyme and in accurately measuring free calcium in such studies, investigators have of necessity used EGTA and calcium buffers, calculating the actual concentration of free calcium present in the assay. Using this approach several groups of workers including ourselves (7,11,14,16,19,20,23,24) calculate that the free calcium requirement for maximal activation of the native enzyme (with calmodulin present) is submicromolar (see Table I). Many other authors who did not calculate the theoretical free calcium in the assay, eg (3-6,8,9) nevertheless report the common finding that maximum enzyme activity occurred in the presence of roughly equimolar concentrations of EGTA and calcium under standard assay conditions (Table I).

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Table 1 Calmodulin-stimulated adenylate cyclase activity

Species Tissue Authors Calcium Requirement

1975 Pig Bovine

1976 Rat

1979 Rat 1980 Rat Bordatella pertussis

Bovine

1981 Rat Pig Rat

1982 Rat Rat Rat

1983 Rat Crayfish Insect Rat Guinea pig

1984 Rabbit Bovine Abalone Neurospora

crassa Human Mouse

Brain a Brain a

Glial tumour cells

Islets of Langerhans

Islets of Langerhans

Adrenal medulla

Lung Ofulue & Nijjar(8 Renal cell line Ansiello & Hall(9 Testicular germ cells Gordeladze et al(

Brostrom et al (I) Cheung et al(2)

Brostrom et al(31

Valverde et al(4)

Sharp et al(5)

Wolff et al (6) Le Donne & Coffee

Pituitary tumour cells Brostrom et al( 11) Islets of Langerhans Platelet

Smooth muscle Abdominal muscle Brain Intestinal epithelium Intestinal epithelium

Intestinal epithelium Retina Spermatozoa

Thyroid B16 melanoma

Thams et al(12) Adnot et al(13)

Piascik et al(14)

7)

0)

b

b

b

P0-G"l

b b

4x1 _e-RH 10 M b

I-8x10-7M Sedlmeir & Dieberg(l5) b Bodnaryk(l6) ~xIO-~M Amiranoff et al(17) 1-30x10-% Pinkus et al(18)

Lazo et al (I 9) ~xIO-~M Gnegy et al(20) 1 .2xl0_7M Kopf et al(21) b

Reig et al(22) b Lakey et al(23) ZXIO-~M Mac Neil et al(24) 7xlO_9M

a For many other references on calmodulin regulation of brain adenylate cyclase see Brostrom & Cheung, 1980.

b Optimal enzyme activity was observed at roughly equimolar concentrations of EGTA and calcium in the assay.

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Most authors, rat'0 -3

(with some exceptions [eg I?]) agree that-&his of EGTA to Ca"+ generates submicromolar free calcium (10 to

IO M, see Table 1). It is possible that calculated free Ca2+ concentrations may be somewhat different from actual free Ca'+ concentrations. However, it is possible to confirm that adenylate cyclase requires a lower calcium concentration for activation than enzymes with a known requirement for micromolar calcium. The approach used by Potter et al (29) and confirmed by us (unpublished observations) was to use the same calcium/EGTA buffers to assay both adenylate cyclase and a calmodulin-dependent cyclic AMP phosphodiesterase. for activation of

The calculated free calciu8 concen$ration required the phosphodiesterase (10 to lo- M) was found to

be significanbly high r than that required for activation of adenylate cyclase ilO- to 10 -7 M). We shall discuss the possible physiological relevance of this difference in calcium optima in a later section. On a purely practical level the fact that calmodulin stimulation of the adenylate cyclase occurs only.over a relatively low and narrow range of calcium concentrations may have contributed to this calmodulin activation of the enzyme being missed in some investigations.

The calcium sensitivity of many adenylate cyclase enzymes seems largely attributable to the presence of particulate calmodulin. Thus, removal of endogenous calmodulin results in a loss of the enzymes' ability to be stimulated by submicromolar concentrations of calcium; furthermore addition of exogenous calmodulin to the enzyme restores this calcium sensitivity (eg 8,9,14,29). Interestingly, there is no direct evidence for a role for calmodulin in the inhibitory effects of calcium - indeed the loss of endogenous calmodulin does not generally prevent the inhibition of enzyme activity observed at higher calcium concentrations, (eg 9,291. This implies that this inhibitory effect is direct or mediated by some factor(s) other than calmodulin.

The use of calmodulin antagonists in detecting calmodulin-dependent adenylate cyclase

In addition to the known calcium-dependency of many adenylate cyclase systems indirect evidence of calmodulin-involvement in the regulation of these enzymes has also been obtained using calmodulin antagonists. Many of the authors who subsequently demonstrated direct activation of the enzyme by calmodulin initially demonstrated that calmodulin antagonists inhibit adenylate cyclase activity in the native calmodulin-containing membrane preparation (see Table 1, refs 6,7,9-15,24). However a wide range of structurally dissimilar drugs will inhibit calmodulin activation of calmodulin-dependent enzymes in vitro (34) but none of these is truly specific for calmodulin alone. %?ivo the drugs can inhibit other aspects of cell metabolism, eg inhibition of ATP substrate availability (35) and even in vitro their actions may not be confined to calmodulin. Thus trifluoperazine, the most common anti-calmodulin drug used, was found to inhibit the activation of a calmodulin-dependent enzyme (erythrocyte Ca'+ ATPase) when the enzyme was being activated by another agent, the fatty acid, oleic acid (36). This latter example strongly suggests that the drug was binding directly to the enzyme in such a way as to prevent its activation by calmodulin or oleate. Even the naphthalenesulfonamides, preferable to many drugs in their specificity against calmodulin, can

216

inhibit calmodulin-independent enzymes such as protein kinase C (37). Accordingly, most authors now use anti-calmodulin drugs simply

as a first indicator that calmodulin may be involved in the process under investigation. In order to discuss how calmodulin interacts with the adenylate cyclase enzyme we shall first summarise the current concepts of the structure of this enzyme.

Structure of the adenylate cyclase enzyme The structure of the adenylate cyclase enzyme is depicted in

Figure 1. The enzyme is commonly thought of as three discrete functional units - a hormone-specific receptor (R) located in the outer plasma membrane; a guanyl nucleotide dependent protein regulator complex (NJ, mainly located within the plasma membrane and a catalytic unit (Cl, located on the inner plasma membrane. Occupation of receptors by the appropriate agonist is coupled to activation or inhibition of the enzyme catalytic units by the GTP dependent-activation of the N unit. The stimulatory and inhibitory forms of N, Ns and Ni, each comprise an CL subunit (45K for Ns and 41K for Ni) and an identical g subunit (35K) (52,531. Subunits bind GTP, required for the activated state of both Ns and Ni. These subunits are also the specific substrates for ADP-ribosylation when acted on by cholera-toxin (which permanently activates the enzyme via Ns) and pertussis toxin (which prevents the response of the enzyme to inhibitory agonists via Nij(54). It is unlikely that the ful.1 structure of the N protein complex is yet known.

- GTP GDP

ATPACAMP - GDP GTP

Figure 1 Model of functional components of adenylate cyclase enzyme.

Mechanism of calmodulin regulation of adenylate cyclase The molecular mechanism by which calmodulin activates adenvlate

cyclase is not yet understood for either neural or non-neural tissues. From the available evidence it is not clear whether calmodulin interacts with adenylate cyclase at the level of the guanine nucleotide regulatory subunit (N-unit) or directly with the catalytic unit.

Most of the investigations to-date into the interaction of calmodulin with adenylate cyclase have used brain adenylate cyclase,

217

presumably because this was the first calmodulin-responsive enzyme to be characterised. However, even within this one system, there is no clear agreement on how calmodulin affects this enzyme. Accordingly, we shall discuss what is known for both neural and non-neural adenylate cyclase systems together.

Calmodulin can affect both basal (eg brain (I), pancreatic islets (4) and adrenal gland(7)) and hormonally-stimulated enzyme activity (eg norepinephine-stimulated brain (3), vasopressin-stimulated renal cell (9) and melanotrophin-stimulated B16 melanoma (24) 1. These effects of calmodulin are not mimicked by structurally related proteins such as troponin C (7).

Several studies support the interpretation that calmodulin acts directly with the catalytic unit and does not interact with the N unit. Thus calmodulin has been found to directly activate the isolated catalytic unit of the brain enzyme (39) and in both neural and non-neural tissue several authors have shown the action of calmodulin to be independent of GTP (eg 7,18,40,41). In addition, differences in the thermal inactivation properties of calmodulin-sensitive brain adenylate cyclase and of the enzyme sensitive to Gpp (NH)p (the non-hydrolysable analogue of GTP) have been reported (421, suggesting that calmodulin and guanyl nucleotides are acting at different sites of the enzyme complex. Futhermore, in brain, loss of Gpp (NH)p stimulation following detergent treatment (which apparently caused selective release of the N-unit) did not change the sensitivity of the enzyme to calmodulin (43).

However, in contrast, several studies suggest that calmodulin can interact at the level of the N unit. Thus, hormonal stimulation of the enzyme is not only increased by calcium plus calmodulin (eg 9,241 but in at least some studies the sensitivity to hormone is also increased (eg in striatum, (44) and retina (20)). In a partially purified calmodulin-insensitive and Gpp (NH)p-insensitive adenylate cyclase preparation of brain, the sensitivity to both calmodulin and Gpp (NH)p was restored by reconstitution of the enzyme with a detergent-solubilised membrane preparation (containing N-unit activity). In addition such calmodulin sensitivity was only seen in the presence of Gpp (NH)p (45). Finally, there is kinetic evidence that Gpp (NH)p and calmodulin compete for a single component of brain adenylate cyclase (46).

Although the concept which is emerging of calmodulin interacting in some way with both the catalytic unit and with N is not simple, it is perhaps comparable to what we now know concerning the interactions of fluoride and forskolin with this enzyme. Again, what at first seemed to be direct interaction of these non-specific agents with the catalytic unit of the enzyme turned out to be much more complicated: both, on close examination, also involving interactions with the N unit, (47,48). For calmodulin it is certainly premature to conclude that it acts on either C or N or both. The recent finding of calmodulin-activated adenylate cyclase in so many non-neural tissues should help clarify this area within the next few years.

Prevalance of calmodulin-dependent adenylate cyclase Two other questions which remain to be answered are whether both

calmodulin-dependent and calmodulin-independent forms of adenylate

218

cyclase exist within any one tissue and whether some tissues lack a calmodulin-dependent enzyme entirely. There is evidence from brain that only some proportion of the total adenylate cyclase activity is calmodulin-dependent (49) but this remains to be investigated in other tissues. Some tissues have been reported to lack any calmodulin sensitive adenylate cyclase activity. In some cases (eg Chinese hamster ovary cells, 30) this may be because the tissues have not been subjected to the experimental conditions (ie calmodulin depletion and calcium dependency investigation) that the majority of investigators (see Table I) have found necessary for the demonstration of calmodulin-dependent adenylate cyclase. However, in cardiac muscle, workers who have used these experimental criteria find no evidence for calmodulin activation of the enzyme (29,381. It is apparent that this point is not yet resolved since a report of calmodulin activation of cardiac adenylate cyclase has appeared during the preparation of this manuscript (55). Current evidence suggests that the presence in cell membranes of a calmodulin-dependent adenylate cyclase is a general phenomenon rather than a unique feature of neural tissues.

Physiological relevance of calcium/calmodulin regulation of adenylate cyclase

Brostrom et al (3) Ca'+/calmodulin-dependent

first suggested in 1976 that the phosphodiesterase and the

Ca'+/calmodulin-dependent adenylate cyclase, both present in the C-6 glioma cell type, might be coordinately intracellular free CaZ+

regulated by changes in concentration acting through calmodulin. In

the presence of calmodulin, the adenylate cyclase present was activated at low concentrations of Ca'+ and inhibited at concentrations of Ca'+ which activated the phosphodiesterase. (In many other tissues also calmodulin-dependent activation of phosphodiesterase is known to require micromolar concentrations of free calcium (50) 1. Since then, many authors have noted that activation calmodulin-dependent adenylate cyclase occurs at low calcium 27 (10 M or lower, see Table I), inhibit'on of th's enzyme occurring at higher calcium concentrations (IO -3 -5 to 10 M). The former calcium concentration is comparable to that now thought to be present as free calcium in the resting cell, the latter concentrations would occur only transiently in the hormone-stimulated cell (51). In Figure 2 we depict schematically these very different calcium optima for the calmodulin-dependent adenylate cyclase and phosphodiesterase enzymes based on our current knowledge of the of these enzymes in vitro in both neural and non-neural tissue. investigations

Furthermore, direct into the difference in calcium optima of these two

enzymes (such as the work of Potter et al (291, described in a previous section) confirm that the optimum calcium concentration required by calmodulin-dependent phosphodiesterase is 10 to 100 times higher than that required by calmodulin-dependent adenylate cyclase.

Such a model may have some important physiological implications. We would speculate that little or no elevation in intracellular free calcium would be required for the initial activation of the calmodulin

219

CaPf/CsM-dependent Ca2’/CaM-dependent

adenylate cyclase phosphod,esterase

Free calcium concentration

Figure 2 Schematic representation of in vitro calcium requirements of calmodulin-dependent adenylate cyclase and calmodulin-dependent phosphodiesterase activity.

dependent adenylate cyclase by hormone, any subsequent increase in intracellular calcium would tend rather to inhibit the activity of this enzyme whilst increasing the activity of the calmodulin-dependent cyclic nucleotide phosphodiesterase. Thus more recent knowledge of calmodulin activation of adenylate cyclase in non-neural tissues supports the proposal of calcium-coordinated regulation of cyclic AMP synthesis and degradation originally proposed by Brostrom et al (3). We cannot at the present time explain how the nanomolar calcium which we and others find optimum for activation of the calmodulin-dependent adenylate cyclase may in vivo activate calmodulin which has an affinity for calcium in the micromolar range.

Summary and conclusions Calmodulin-dependent stimulation of adenylate cyclase was

initially thought to be a unique feature of neural tissues. In,recent years evidence to the contrary has accumulated, calmodulin-dependent stimulation of adenylate cyclase now being demonstrated in a wide range of structurally unrelated tissues and species. Demonstration of the existance of calmodulin-dependent adenylate cyclase has in nearly all instances required the removal of endogenous calmodulin. It is not yet clear whether calmodulin-dependent and calmodulin-independent forms of the enzyme exist and whether some tissues (such as heart) lack a calmodulin-dependent adenylate cyclase. The presence of calmodulin appears largely responsible for the ability of the adenylate cyclase enzyme to be stimulated by submicromolar concentrations of calcium; it may not be relevant to the inhibition of the enzyme which occurs at higher concentrations of calcium. The physical relationship of calmodulin to the plasma membrane bound enzyme (or to the soluble forms of the enzyme) is not known nor is the

220

mechanism of adenylate cyclase activation by calmodulin clear; current data suggest some involvement with both the N and C units of the enzyme. Finally, it is possible that in vivo calcium contributes to the duration of the hormone stimulated cyclic AMP signal. Thus current in vitro data suggest that optimal hormonal activation of calmodulin-dependent adenylate cyclase occurs at very low intracellular calcium concentrations, comparable to those found in the resting cell; conversely the enzyme is inhibited as intracellular calcium increases, following for example agonist stimulation of the cell. These higher calcium concentrations would then activate calmodulin-dependent phosphodiesterase. Such differential effects of calcium on adenylate cyclase and phosphodiesterase would ultimately restrict the duration of the hormone-induced cyclic AMP signal.

Acknowledgements We are grateful to the Medical Research Council for financial

support for S Mac Neil. T Lakey is a Medical Research Council postgraduate student. S Tomlinson is a Wellcome Trust Senior Lecturer. We thank MS L Richardson for the typing of this manuscript.

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Received 23.10.84. Revised version received 21.12.84.

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