Mechanistic descriptions of cAMP production have evolved

significantly since the 1960s when Sutherland and Rall

hypothesized the existence of a single polypeptide that would

both recognize hormone and synthesize cAMP. We now

appreciate that the hormone-activated synthesis of cAMP involves

multiple polypeptides, including a membrane-bound receptor; a

heterotrimeric, guanine nucleotide–binding protein (G protein);

and a membrane-bound adenylyl cyclase (AC). Biochemical and

structural biological studies have provided a firm understanding

for the regulation of AC by G proteins and elucidated the catalytic

mechanism. In addition, a number of small molecules have been

developed that modulate AC activity, introducing AC as a potential

therapeutic target. Many paradigms of multi-modal regulation of

AC have been investigated from a physiological perspective. This

review addresses the complexity of the direct modulators of AC

and summarizes the current biological models of their function.

168

Roger K. Sunahara1 and Ron Taussig2

1The Department of Pharmacology

University of Michigan Medical School

Ann Arbor, MI 48109-06322The Department of Pharmacology

and the Alliance for Cellular Signaling

University of Texas Southwestern Medical Center

Dallas TX 75390-9041

Isoforms of MammalianAdenylyl Cyclase:

Multiplicities of Signaling

With nine different isoforms ofmembrane-associated adenylylcyclases (ACs) and one isoform ofsoluble AC, there is much to learnand even more to understandregarding the expression of tissue-specific AC isoforms. However, onthe protein level, there are manyproteins and small molecules thataffect the catalytic activity of ACs.Knowing how to tailor AC activity,or how to exploit the activity of oneisoform over another in a giventissue, may give rise to therapeuticagents that can inhibit AC-dependent disease states or, atleast, lessen their severity.

INTRODUCTION

The activation of adenylyl cyclase (AC), resulting in theintracellular production of adenosine-3',5'-monophosphate (i.e.,cyclic AMP [cAMP]), is initiated by the binding of hormones tocell surface receptors (1). Epinephrine, dopamine, prostaglandinPGE2, adenosine, and glucagon are a few examples of the manyhormones that activate AC through membrane-bound receptors.Glucagon, for example, a hormone that regulates glycogenmetabolism in liver and skeletal muscle, recognizes membranereceptors in these tissues, markedly stimulates AC to produceintracellular cAMP. Glucagon-bound receptors communicate withan intracellular, membrane-associated heterotrimeric G protein (2)composed of a guanosine diphosphate (GDP)–bound �-subunitand an obligate �� heterodimer. Hormone-dependent activation ofreceptors leads to the exchange of GDP for guanosine triphosphate(GTP). Conformational changes due to GTP binding result in thedissociation of the heterotrimeric G protein into � and ��

subunits, which then interact with their respective effectors(Figure 1).

There are multiple classes of �-subunits that regulate AC,either in a stimulatory (G�s family), or inhibitory (G�i family)manner (2). The two G� families are normally coupled to distinctreceptor subtypes. The ��-subunits also regulate AC, but in an ACsubtype–specific manner (3–5). Additionally, calcium ions are verystrong modulators of some isoforms of AC (6–9); thus, G proteinsthat regulate calcium entry through voltage-dependent Ca2+

channels may also regulate AC activity (10–12).ACs have been extensively characterized, and great advances

have been made in our understanding of how they function. Theirmechanism of action can now be incorporated into model systems

to explain drug responses that occur in tissues or wholeorganisms. For example, learning and memory are associated withthe activation of protein kinase activity and proteinphosphorylation (13), two processes that are strongly regulated byAC activity. Genetically modified mouse strains that containaltered AC genes display considerable behavioral defects,particularly in learning and memory (14–16). The effects of Ca2+

and Ca2+–calmodulin (CaM) on AC activity are also stronglyimplicated in learning and memory (17–19). AC activity itself canbe modulated through phosphorylation of the enzyme, andalterations in the expression of AC isoforms also accompany drug-induced receptor effects that have been related to symptoms ofdrug dependence (20–23).

Much of the research surrounding cAMP signaling hasfocused on AC activity that is regulated by G proteins. However, abona fide soluble form of AC that is insensitive to G proteins andforskolin has been cloned (24). Soluble AC (sAC) activity wasidentified in testis during the early 1980s, but the activityremained an enigma until the corresponding cDNA was isolated.Elevated concentrations of cAMP in the testis are crucial for spermdevelopment and capacitation. The discovery of sAC, along withthe finding that it is activated by bicarbonate, has forced areexamination of how cAMP signals are propagated into the cellbeyond the cell membrane.

MULTIPLE AC ISOFORMS

Molecular cloning techniques have identified nine mammaliangenes that encode membrane-bound ACs (3–5, 25), and one geneencoding a soluble isoform (24). These genes do not tend tocluster within the genome, but rather are distributed among

Multiple Isoforms of Adenylyl Cyclase

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Figure 1. G proteins mediate the effects of hormone signals on adenylyl cyclase.A. Hormones that affect intracellular adenylyl cyclase activity bind to protein receptors that contain seven transmembrane domains (blue) and are thusanchored at the cell surface (brown double bar). The intracellular portion of these receptors interacts with GDP-bound heterotrimeric G proteins, resultingin the displacement of the bound GDP by GTP and concomitant dissociation of the GTP-bound G�-subunit from the G��-dimer. B. Two families of G� exist: GTP-bound G�s stimulates adenylyl cyclase, whereas GTP-bound G�i inhibits adenyly cyclase. G� subunits possess anintrinsic GTPase activity, so that their dissociation from G�� and their effect on AC activity are transitory. (Yellow triangle represents GDP; red trianglerepresents GTP.)

A Families of G�B

Epinepherine

Dopamine

PGE2

Adenosine

Glucagon

stimulates AC(e.g., �s-short, �s-long, �s XL)

regulatory role unclear

inhibits AC(e.g., �i1, �i2, �i3, �o, �z)

�

��

�

�s

�i

�q� �

different chromosomes, with the exception that the two genes thatencode AC7 and AC9 are located on chromosome 16, albeit atopposites arms. The ten AC isoforms can be divided into fivedistinct families based on their amino acid sequence similarity andfunctional attributes. The Ca2+–CaM-sensitive forms are typesAC1, AC3, and AC8 (Figure 2A). The G��-stimulatory forms arerepresented by AC2, AC4, and AC7 (Figure 2B). AC5 and AC6 aredistinguished by their sensitivity to inhibition by both Ca2+ andG�i isoforms (G�o, G�i1, G�i2, G�i3, and G�z) (Figure 2C).AC9 is the most divergent of the membrane-bound family and ishighly insensitive to the diterpene forskolin. The last isoform, sAC,is the most divergent of all the mammalian cyclases, and is similarto cyclases found in cyanobacteria (24).

The distribution of these AC isoforms, according to mRNAdetection, is summarized in Table 1. In general, all membrane-bound AC isoforms are found in, but not limited to, excitabletissues such as neurons and muscle (3, 26). Within the brain, ACisoforms localize to different, discrete brain regions (27–29).Although most isoforms are widely expressed, AC1 and AC3 areexpressed only in brain (29). Soluble cyclase is expressedpredominantly in the testis, although splice variants have beenidentified displaying a broader distribution pattern (30). The broaddistribution of AC isoforms suggests that any given cell containsmultiple isoforms.

REGULATION OF AC ACTIVITY

STIMULATION BY G�s

Hormonal activation of AC occurs primarily through receptorscoupled to the stimulatory G protein G�s. G�s is the most widelydistributed activator of all mammalian membrane-bound ACisoforms. Multiple splice variants of G�s have been identified:G�s-short, G�s-long and G�sXL. Although the former twoisoforms have been extensively characterized both physiologicallyand biochemically, G�sXL is a relatively new member and is lesswell characterized (31). The long and short splice forms arebiochemically indistinguishable in their capacity to directlyactivate AC (32); however, the behavior of the hormonereceptor–stimulated AC varies considerably (33). G�sXL canactivate AC directly, but no hormone receptor–mediated effectsthrough G�sXL have been demonstrated (34).

G�s in the GTP-bound form displays a tenfold greater affinityfor activating AC compared to the GDP-bound form (35).Crystallographic evidence suggests that the main contact betweenG�s and AC occurs through a short �-helix that is highly mobilethroughout the GTPase cycle of all G proteins (36, 37). Thedecreased affinity of the GDP-bound form for AC suggests that theGTPase activity of G�s serves as a timing mechanism to delimitcyclase activation. Following GTP hydrolysis, G�s dissociates fromcyclase, reassociates with G��, and thereby terminates both G�sand G�� signaling. The deactivation of G�s can be accelerated by

a specific Regulator of G protein Signaling (RGS) molecule, PX1-RGS, that serves as a GTPase-accelerating protein (GAP) for G�s(38). AC itself can weakly accelerate the GTPase activity of G�s(39), similar to the accelerating effect of PLC� isoforms on theiractivator G�q (40).

INHIBITION BY G�i

Members of the G�i family inhibit AC but can manifest selectivityfor given AC isoforms. G�i1, G�i2, G�i3, G�o, and G�z caninhibit AC5 and AC6 (Figure 2C) (41–43). Interestingly, theirmode of inhibition is not through direct competition with G�s,

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170

Figure 2. Multiple modes of regulation of adenylyl cyclaseisoforms. (A) The pattern of regulation of AC1 as illustrated isrepresentative also for AC3 and AC8. R1 represents a Gprotein–coupled receptor, such as the glucagon or �2-adrenergicreceptor, that couples to the stimulatory G protein G�s. R2represents a G protein–coupled receptor, such as the muscarinic M2or �1-adrenergic receptor, that couples to the inhibitory G proteinG�i. (B) The pattern of regulation of AC2 as illustrated isrepresentative of the regulation of AC4 and AC7. Note that G��

regulation of AC2 is dependent on G�s co-activation and does notactivate AC by itself. PKC can use AC as a substrate, resulting inelevation of basal activity and inhibition of the G�� superactivation.(C) The pattern of regulation of AC5 is representative also of AC6.(PKA, protein kinase A; PKC, protein kinase C; CaM, calmodulin;CaMK, calmodulin-dependent kinase; NO, nitric oxide; VDCC,voltage-dependent Ca2+ channel.)

A

B

C

��

�� ��

��

��

Ca2+ CaM

�s

�s

�s

�� �i

�i

�i

Ca2+

Ca2+

R1

R1

R1

AC1

CaMK

AC2

AC5

PKA

PKA NO

R2

R2

R2

VDCC

VDCC

PKC

PKC

because forskolin-stimulated activity is also inhibited. In addition,mutagenesis experiments and structural modeling suggest thatG�i exerts its effects at a site, symmetrical to the G�s bindingsite, located on the side opposite the AC molecule (44). Thehighly expressed brain-specific G�o can inhibit AC1 (andpossibly AC8), although it is not as potent as the other G�isubunits on AC5 and AC6. The G �i subunits areposttranslationally modified by long-chain acyl (myristoyl) andthioacyl (palmitoyl) moieties (45); myristoylation is required forG�i-mediated inhibition of AC.

REGULATION BY G��

The contributions of the G�� heterodimer to the modulation ofACs have been, at least until recently, largely unappreciated (46). G�-subunits had long been presumed to predominate in theregulation of AC; however, G�� subunits are strong modulators ofAC activity that can either be stimulatory, as in the case of AC2,AC4, and AC7, or inhibitory, as for AC1 and AC8 (Figure 2) (46,47). In fact, G�� subunits are among the most potent of allnegative regulators of AC1 and AC8, and can markedly inhibit theeffects of forskolin, G�s, and Ca2+–CaM on AC activities. Thesefindings are particularly relevant for brain physiology because theG�i family and their accompanying �� subunits are, along withAC1 and AC8, highly expressed in the brain (48).

In contrast, G�� subunits act to stimulate the cyclase activityof AC2, AC4 and AC7, albeit only when G�s is co-activated(Figure 2B). G�� and G�s could thus establish a synergisticrelationship, whereby the presence of G�� might dramaticallyenhance the ability of G�s to activate AC. Indeed, the activationof those hormone receptors coupled to G�i subunits could liberateG�� dimers that could synergistically potentiate AC activity thathad been stimulated by distinct, G�s-activated, hormonereceptors. It is important to note that the AC isoforms thatundergo stimulation by G�� (i.e., AC2, AC4 and AC7) are notdirectly modulated by the � subunits of the Gi family (3, 26). Lesswell understood is the relationship between G�� and the othercyclase isoforms such as AC5 and AC6. Transfection experimentssuggest that G�� can inhibit AC5 and AC6 activity, perhaps in anindirect manner (49).

The putative binding site for G�� on the G��-stimulatedfamily of ACs (i.e., AC2, AC4, and AC7) has been mapped on thebasis of peptide inhibition studies (50, 51). Peptidescorresponding to amino acid residues 956 to 982 of AC2, that is,derived from the middle of the second of the two catalyticdomains (i.e., C2), potently inhibit the ability of G�� to stimulatethe enzyme activity of intact AC2. Despite the high degree ofsequence conservation among AC catalytic domains, this sequence(i.e., corresponding to residues 956 to 982 of AC2) is not found inAC isoforms that are not modulated by G��. Indeed, thissequence also contains the short putative G��-binding motifQXXER, the consensus for which is based on GRK2, the �-

adrenergic receptor kinase that requires G�� for activation, as wellas G��-activated inwardly rectifying K+ channels, the G��-activated PLC� isoforms, and the G��-inhibited AC1. Disruptionof the consensus QXXER motif in any of these instances abrogatesall G�� effects. The C2 domain of AC2, possessing the QXXERmotif, is located near the plasma membrane face, but the precisestructure of this motif is unknown because the region isdisordered in the crystal structure. An additional region within theregulatory region of the C1 domain, juxtaposed to thetransmembrane domain, may also be important for G�� regulationof AC2, AC4, and AC7 (52).

A peptide generated from the catalytic region of the AC1isoform analogous to that containing the QXXER motif in the AC2sequence also displays dramatic effects on G�� regulation of ACactivity (52). The peptide could reverse both G��-dependentinhibition of AC1 activity and G��-dependent superactivation ofG�s-stimulated AC2, suggesting that the region of the AC1isoform also serves for binding of G��. For example, it has beenreported that this region contributes to G��-mediated inhibitionof AC1 activity (53).

The recent findings outlined above underscore theimportance of the G�� heterodimer in modulating AC activity andsuggest that it may be naïve to regard the importance of G�� assecondary to that of G�. In addition to its role in regulating AC,G�� subunits have been implicated as the primary component ofG proteins that directly regulate ion channels (e.g., K+-channelactivation and Ca2+- and Na+-channel inhibition) as well as othereffectors systems: GPCR kinases (activation), phospholipase C�

isoforms (activation), and the mitogen-activated protein (MAP)kinase pathway (activation) (10, 54). The MAP kinase-dependentmating response in yeast is solely dependent on G�� for signalingand only requires the G� subunit for its inactivation.

CALMODULIN AND CA2+ AS REGULATORS

Ca2+–CaM activates isoforms AC1, AC8, and possibly AC3(Figure 2A) (55–57). Some, but not all, agents that elevate localCa2+ levels in intact cells may thus dramatically enhance theactivity of these isoforms. Specifically, intracellular Ca2+ fromIP3-sensitive stores are unable to affect these Ca2+-sensitive ACisoforms, whereas activation of Ca2+ entry through voltage-gatedCa2+ channels or through capacitative entry is effective atactivating these AC isoforms (58, 59).

Although millimolar (i.e., non-physiological) concentrationsof Ca2+ inhibit all AC isoforms (Figure 3C), AC5 and AC6 areinhibited by concentrations of Ca2+ in the �M range, well withinthe dynamic range of intracellular levels (Figure 2C) (60); Ca2+

from capacitative entry are thought to be the sole physiologicalsource of Ca2+ to inhibit the AC5 and AC6 (6). The fact that theseAC isoforms are restricted mostly to brain-specific and excitablecell types, and are compartmentalized with voltage-gated Ca2+

channels, is consistent with this notion (Table 1).

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REGULATION BY OTHER PROTEINS

A number of other proteins have recently been identified thatinteract directly with ACs, but their biological significance has yetto be determined. Several of these proteins have been identifiedthrough yeast two-hybrid or copurification experiments using thecytosolic domains as baits. The protein associated with Myc (PAM)

potently inhibits AC1 and AC5, but not AC2 (61), whereas theEscherichia coli protein SlyD, a cis-trans peptidylprolyl isomerase(PPIase), copurifies with bacterially expressed AC7 so as to inhibitits activity (62). A conceptually more relevant interaction wasidentified between another RGS protein, RGS2, and AC3 (63). RGSmolecules are mostly noted for their ability to accelerate theGTPase activity of the heterotrimeric G proteins of the G�i, G�q,

Review

172

TABLE 1. REGULATORY PROPERTIES OF MAMMALIAN ADENYLYL CYCLASE ISOFORMS

AC Chromosomal Tissue Regulation by G Protein Calcium PutativeIsoforma,b Location Distribution Protein Subunits Kinases Effects FunctionAC1 7p12 (154)c Brain Stimulated by G�s PKC: weak Stimulated by Learning,

adrenal medulla Inhibited by G�� stimulation Ca2+-CaM memory,Inhibited by G�o CaMKIV: inhibition LTP

inhibition Synapticplasticity

AC2 5p15 (155, 156) Brain, skeletal Stimulated by G�s PKC:muscle, lung, heart Stimulated by G��d stimulation

AC3 2p22-p24 (157) Brain, olfactory Stimulation by G�s PKC: weak Stimulated by Olfactionepithelium stimulation Ca2+-CaM

CaMKII:inhibition

AC4 14q11.2 (156) Brain heart, kidney, Stimulation by G�s PKC: liver, lung, BAT, Stimulated by G��d inhibitionuterus

AC5 3q13.2-q21 (157) Heart, brain, Stimulation by G�s PKA: Inhibitedkidney, liver, lung, Inhibited by G��e inhibitionuterus, adrenal, Inhibited by G�i

f PKC�,�:BAT stimulation

AC6 12q12-q13 (157) Ubiquitous Stimulation by G�s PKA: InhibitedInhibited by G��e inhibitionInhibited by G�i

f PKC:inhibition

AC7 16q12-q13 (158) Ubiquitous, highly Stimulation by G�s PKC: Drugexpressed in brain Stimulated by G��d stimulation dependency

AC8 8q24 (155, 156) Brain, lung (testis, Stimulation by G�s Stimulated by Learning,adrenal, uterus, Ca2+-CaM memory,heart) LTP, Synaptic

plasticityAC9g 16p13.3 (159) Brain, skeletal Stimulation by G�s

muscleSACh 1q24 (24) Testis Not regulated by G Sperm

protein subunits capacitationBAT, brown adipose tissue; LTP, long-term potentiation.a All isoforms except sAC are inhibited by P-site inhibitors.b Forskolin stimulates human AC1-AC8, whereas AC9 is weakly stimulated by forskolin. sAC is not affected by forskolin.c Cited reference numbers.d G�� stimulation of AC isoforms is conditional upon G�s co-activation.e Inhibition determined by transfection only and could be an indirect G�� effect.f Denotes G�i family members G�i1, G�i2, G�i3, and G�z.g Inhibited by calcineurin.h Stimulated by bicarbonate.

and G�12 types. The RGS2 isoform can enhance the intrinsicGTPase rate of both the G�i and G�q (64, 65). A direct associationbetween G�i or G�q and AC3 has not been demonstrated;however, this interaction would provide an additional avenue ofcrosstalk.

REGULATION BY SMALL MOLECULES

Forskolin

The diterpene forskolin (from Coleus forskohlii) potently activatesall known isoforms of mammalian membrane-bound ACs with theexception of AC9 (66). The sensitivity difference may beaccounted for by as few as two residues, Ala1112 and Tyr1082,

corresponding to Leu912 and Ser942 of AC2 (67). In anunexpected divergence of evolution, however, the D. melanogasterortholog of AC9 is sensitive to forskolin (68). The forskolin-dependent activation of AC2, AC4, AC5, AC6, and AC7 issynergistic with G�s-mediated coactivation, whereas activation byforskolin and G�s is additive for isoforms AC1, AC3, and AC8 (3).

The binding site for forskolin is located within the catalyticcore of AC, at the interface between the intracellular catalytic (C1and C2) domains (Figure 3). G�s binds similarly between the twodomains, but at a location on the perimeter of the catalytic core.The relationship between the two binding sites and their proposedmechanism of action may explain the cooperativity of bindingobserved between forskolin and G�s. Why other isoforms displayadditive effects with forskolin and G�s is not obvious from thecrystal structure. Stimulation of activation by forskolin andCa2+–CaM is cooperative in the cases of AC1, AC8, andpresumably AC3 (56, 69).

Since the elucidation of the crystal structure of AC bound toeither forskolin or its water-soluble analog 7-deacetyl-7-(O-N-methylpiperazino)-�-butyryl forskolin (5, 43), researchers haveattempted to design isoform-selective forskolin analogs. Althoughthis methodology is still in its infancy, several compounds havebeen synthesized that contain subtle modifications of forskolinand display a two- to threefold preference for certain cyclaseisoforms (70).

Pyrophosphate

Adenylyl cyclase hydrolyses ATP to produce pyrophosphate andcAMP. In a steady-state AC assay, the rate-limiting step is normallythe release of pyrophosphate (71). Elevated concentrations ofpyrophosphate can thus be used to force AC into a product-boundconformation that prevents the binding of ATP. The antiviral agent,

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Figure 3. Structure of membrane-bound mammalian adenylyl cyclasebound to the activator G�s. (A) Illustration of the crystal structure of thecatalytic domain of adenylyl cyclase bound to G�s (36) and superimposedonto the membrane-spanning region of mammalian adenylyl cyclase.G�s•GTP�–S in its activated form is demarcated in gray. The cyclasedomains, C1 (tan) and C2 (mauve) interact and form the binding sites forforskolin and the substrate, ATP. (B) The same structure as in (A) butrotated around the x-axis of vision to give a perspective from the innersurface of the membrane. G�i (in red) is overlaid onto the G�s•C1•C2structure at the pseudosymmetrically related binding site to theG�s•GTP�–S site. (C) The active site of adenylyl cyclase bound to theATP analog ATP�–S(RP). Highlighted are residues that make contact withthe nucleotide and that are conserved in all mammalian adenylyl cyclases.Asp396 (D396), Asp440 (D440), and Arg484 (R484) are in the C1domain of AC5. Lys938 (K938), Asp1018 (D1018), Arg1029 (R1029),and Lys1065 (K1065) are in the C2 domain of AC2. Also indicated aretwo Mg2+ ions liganded by the phosphates of ATP��S(RP) and the twoaspartate residues. The 3D structure was visualized withSwissPDBViewer™(178) and rendered with POV-RAY™ using coordinatesfrom the G�s•C1•C2•forskolin•ATP�–S(RP) (PDB id:1CJK) and G�i(PDB id:1GIA) structures.

A

B

C

G�s

C2

C2G�s

ATP�–S

G�i

Forskolin

Forskolin

90º

K1065R1029

D1018

D440

K938D396

R484Mg2+

Mg2+

ATP�–S

ATP�–S

foscarnet, or phosphonoformic acid, mimics pyrophosphate andlikewise inhibits AC activity (72).

P-Site Inhibitors

One collection of adenosine analogs, classified as P-site inhibitors,inhibits AC activity in a manner without competing with ATPbinding (71, 73, 74). These compounds inhibit AC by binding to aconformation of the enzyme that closely resembles the product-bound state, or posttransition state (75). The capacity of P-siteinhibitors to inhibit AC activity is thus dramatically affected by thecatalytic activity of cyclase itself, which in turn is a function of theenzyme’s conformational state (73); most notably, P-site inhibitorsare dramatically potentiated by the presence of pyrophosphate.The majority of P-site inhibitors lack one or more hydroxyl groupsrelative to the ribose ring structure (75). Additionally, most ofthese inhibitors are mono- or polyphosphates and are structuralanalogs of cAMP. Thus, 2'-deoxy-3'-AMP (IC50 ~10 �M), and themore potent inhibitors 2',5'-dideoxy-3'-ADP and 2'-5'-dideoxy-3'ATP (IC50 ~ 40 nM), inhibit AC by stabilizing the quasi-product–bound state (76). P-site inhibitors are generally notspecific for individual AC isoforms. The only exceptions are 9-(cyclopentyl)-adenine and 9-(tetrahydro-2-furyl)-adenine; they areineffective on AC2, but equally inhibit AC1, AC3, AC5, AC6,AC7, and AC8 (70, 77).

Other Small-Molecule Modulators of AC Activity

Potent inhibitors of AC activity include the RP stereoisomer of �-thio-ATP (IC50 ~ 1 �M), although the SP isomer is actually aweak inhibitor (78). �,�-Methyleneadenosine-5'-triphosphates(AMP-CPP), which contains a methylene group between the �-and �-phosphates, is also an effective inhibitor of AC activity(IC50~300 �M) (79). The most potent inhibitor of AC activitycurrently available is �–L-2',3'-dideoxy-5'-ATP (IC50 ~ 24 nM)(80). As is the case with other inhibitors, such as 9-(2-phosphonylmethoxyethyl)-adenine and derivatives (80, 81), theability of any these compounds to specifically modulate ACisoforms is not established.

REGULATION BY POSTTRANSLATIONAL MODIFICATION

Several modes of posttranslational modification, includingphosphorylation, glycosylation, and S-nitrosylation, can alter theactivity of ACs. The phosphorylation of AC by protein kinasesgenerally has an inhibitory effect, not on basal activity, but onenhanced stimulation by various activators. These effects are partof a negative feedback mechanism; for example, PKA-mediatedphosphorylation is thought on negatively regulate AC5 and AC6activity (Figure 2C)(82, 83).

Much attention has focused on the role of phosphorylation byprotein kinase C (PKC) in regulating AC activity since the initial

report that AC purified from brain can be directly phosphorylatedby this kinase (84). The activities of AC1, AC2, AC3, and AC5 canbe stimulated following phorbol ester treatment, whereas those ofAC4 and AC6 are inhibited, suggesting that PKC can regulate ACsin an isoform-specific manner (85–90). For AC2, AC5, and AC6,this regulation is due to direct phosphorylation by PKC (87, 91).Interestingly, although PKC has opposite effects on the G�s-stimulated activities of AC2 (enhanced by PKC) and AC4(inhibited by PKC), PKC causes both AC2 and AC4 to loseresponsiveness to the (stimulatory) effect of G��. In this way, PKCbears the role, with regard to AC2-like cyclases, of modulating theintegration of G�s and G�� inputs (85).

It is perhaps counterintuitive that Ca2+–CaM, whichnormally activates AC1, AC3, and AC8, can also inhibit AC1 andAC3 indirectly through phosphorylation by CaM kinase II and IV,respectively (92, 93). This mode of regulation most likely reflects anegative feedback loop that controls Ca2+-mediated stimuli.

Both hormone- and forskolin-stimulated AC5 and AC6activity are inhibited by nitric oxide (NO) (94). In addition to itsprimary target—soluble guanylyl cyclase (GC) (95, 96)—NOaffects the ryanodine receptor and the NMDA receptor (97–98).These effects are largely inhibitory and involve S-nitrosylation.More recently, N-linked glycosylation was demonstrated to beimportant for AC responsiveness to hormones and forskolin (99,100). Although tunicamycin treatment, or substitution ofglutamine for Asn805 and Asn890 of AC6, has very little effect onthe targeting of AC6 to the plasma membrane and on G proteinactivation, G protein–mediated inhibition and responses toforskolin are impaired by as much as fifty percent (99). Incontrast, a variant of AC8 requires N-linked glycosylation forplasma membrane targeting and thus for activation by membrane-bound G protein–coupled receptors (100, 101).

STRUCTURE: PRIMARY, SECONDARY, ANDTERTIARY

AC is an integral membrane protein composed of twelvetransmembrane segments. The protein can be visualized as twotandemly repeated domains, each containing six transmembranesegments and a large cytoplasmic (catalytic) loop (Figure 4). Thetwelve-transmembrane domain topology is reminiscent of the ABCfamily of transporters such as the cystic fibrosis transmembranerectifier and the P-glycoprotein, which is responsible for multidrugresistance and is encoded by the MDR1 gene (102, 103). Thesequence similarity between the two cytosolic domains is striking:approximately forty percent, over a span of 250 residues,regardless of which membrane-bound isoform is considered (3).The catalytic cytosolic regions of mammalian ACs also sharesignificant sequence similarity to the corresponding regions of GCs(96) and ACs from prokaryotes (Figure 4).

The biochemical characterization of recombinant forms ofthe AC cytoplasmic domains has provided extremely useful

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174

insights into AC regulation and catalysis. The construction offused C1–C2 domains (104) and individual soluble domains (35,105–108) results in activities characteristic of the full-lengthmembrane-bound AC forms in terms of modulation by G protein�-subunits, forskolin, substrate inhibitors, and P-site inhibitors(35, 105–108). The utilization of the recombinant, solubledomains of AC has facilitated the biophysical characterization ofenzyme function.

STRUCTURAL BASIS FOR THE REGULATION OF AC

The C1 and C2 domains that form the catalytic core of AC arerelated by two-fold pseudosymmetry (Figure 3). Current modelsof the AC structure are based on the forskolin-bound form in thepresence (C1–C2 heterodimer) or absence (inactive C2homodimer) of G�s. The forskolin binding site is located in ahydrophobic pocket at the interface between the two domains (36,109). Like forskolin, G�s also contacts both domains, with most ofthe binding surface (approximately seventy-five per cent)contributed by the C2 domain. The binding of G�s induces a 7˚rotation of the C1 domain around the C2 domain, presumablypositioning the active site for catalysis. The C2 domain contactsG�s primarily in the switch II region, one of three segments of Gproteins that are highly mobile throughout the cycle of GTP

hydrolysis (37). The pseudosymmetrical structure of the catalyticcore makes apparent the likely binding site for other G proteins,such as G�il, that regulate catalysis (Figure 3B). G�i selectivelyinhibits AC5 and AC6, for example, presumably by binding to theC1 domain (and perhaps through an interaction with the C2domain) and stabilizing the two helices of C1, thus allostericallymodifying the proximally located active site (44, 108).

SUBSTRATE BINDING AND THE MECHANISM OF HYDROLYSIS

The active site, revealed by x-ray diffraction ofC1•C2•G�s•forskolin co-crystals, is located at the interfacebetween the C1 and C2 domains in at a site pseudosymmetricallyrelated to the forskolin binding site (36, 109, 110). Residues thatcontact the substrate (or substrate analog) are conserved in all ACisoforms (Figure 3C). Interestingly, the positions of Lys938 andAsp1018 in AC2, residues that contribute most of the bindingenergy to the adenine ring, are occupied in GCs by glutamate andcysteine residues, respectively. Indeed, the substrate specificity ofAC can be changed from ATP to GTP by making the appropriateamino acid substitutions (111). Similarly, the conversion of a GCto an AC has also been demonstrated (111, 112).

The residues that coordinate the binding of the ribose andtriphosphate portion of the nucleotide are conserved in all

isoforms AC and GC (Figure 3C).Non-conservative substitution ofany of these residues severelyimpairs cyclase activity. Arg484,Arg1029, and Lys1065 in AC2share coordination of the �-, �-,and �-phosphates of thenucleotide. Two highly conservedaspartate residues (Asp396 andAsp440 in AC5) also help tocoordinate the phosphates bycoupling to two Mg2+ cations thatstabilize the �-phosphate duringcatalysis. The overall structure isstrikingly similar to that found inother phosphoryl transferases,such as T7 DNA polymerase andHIV reverse transcriptase (110,113–115). A model of the catalyticmechanism for these enzymesinvolves the contribution of onethe Mg2+ ions acting as a generalbase that deprotonates the 3'-OHof the ribose ring (116). The newlyformed oxyanion is thus poisedfor nucleophilic attack of the �-phosphate with elimination ofpyrophosphate.

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Cyclase HomologyDomain

Cyclase HomologyDomain

MammalianMembrane-BoundAdenylyl Cyclase

Mammalian SolubleAdenylyl Cyclase

D. discoideumMembrane-BoundAdenylyl Cyclase

Membrane-BoundGuanylyl Cyclase

SolubleGuanylyl Cyclase

Membrane-BoundGuanylyl CyclaseTetrahymena,D. discoideum

Soluble GuanylylCyclaseD. discoideum

TM

TM1-6

C1

C1

C1

TM7-12

TM CHD

TM CHD

TM KHD CHD

TM KHD CHD

CHD

CHD

C2TM1-6

C1

TM7-12

C2

C2

C2

Nitric Oxide Binding

ANP Binding

TM

AC1

sAC

AGC

GCAHomodimer

GC�–GC�Heterodimer

GCA

sGC

Figure 4. Sequence alignment of the adenylyl and guanylyl cyclases. The catalytic domains (yellow) displayconsiderable similarity in amino acid sequence and have been coined the Cyclase Homology Domain (CHD).Illustrated in light blue are the membrane-spanning regions as predicted from amino acid sequence. (TM,transmembrane; ANP, atrial natriuretic factor; KHD, kinase homology domain.) Adapted from Wedel andGarbers (96).

PHYSIOLOGY AND FUNCTION OF MAMMALIAN ACS

The biochemical assessment of the ACs has revealed severalregulatory pathways that control AC activity. In contrast, anumber of factors have resulted in a relative paucity ofphysiological data describing these complex regulatory pathways.The most notable obstacle is the multiplicity of the isotypesexpressed within a given cell type, further complicated by thevarying effects of modulators such as Ca2+ and G��, as well asthe particular intracellular milieu. The majority of data come form:1) overexpression studies using cell transfection or transgenicanimals; 2) gene disruption studies utilizing genetic knockouts;and 3) the identification of natural gene mutations.

SENSITIZATION

The importance of AC sensitization has long been appreciated inmodel systems for drug abuse, withdrawal, and recovery (117).Cells chronically treated with opiates (which activate G�i-coupledreceptors) exhibit AC activity that is supersensitive to stimulationby either forskolin or G�s following withdrawal of the opiate.Similar sensitization is observed with chronic activation of otherhormone receptors that couple through G�i—such as the A3adenosine (118), D2 and D4 dopamine (23, 119), and M2muscarinic receptor subtypes (23, 120)—and is dependent on theexpression of particular ACs. In transfection studies, sensitization,in the form of superactivation, is observed for AC1, AC5, AC6,and AC8, but not for AC2, AC3, AC4, or AC7 (21, 119, 120), butthe mechanisms underlying this form of sensitization remainobscure. Interestingly, chronic opioid treament leads to relativedesensitization of the AC2, AC4 and AC7 isoforms, which appearsto be regulated through G�s, G�i, G��, and PKC (119, 120,121–126). PKC-mediated phosphorylation of AC5 increases ACactivity in vivo and in vitro (127). Chronic hormone stimulationleading to higher steady-state amounts of PKC-mediated phospho-AC5 may account for some of the apparent sensitization. G�� hasbeen implicated in the cannabinoid (CB1) receptor-mediatedsuperactivation of AC1, AC3, AC5, AC6, and AC8, but not theACs that are normally activated by G�� (128).

Supersensitization might result as an effect of increasedexpression of specific AC isoforms, PKA, and the cAMP-responsiveelement binding protein (CREB) (117, 129). In sharp contrast,chronic exposure to ethanol reduces such expression and leads toAC desensitization (130), suggesting that ethanol dependencegreatly relies on the cAMP signaling pathway (131).

GENETIC MANIPULATION OF AC ISOFORMS

AND THE MOUSE MODEL

Much information on the physiological role of specific AC

isoforms has come from studies on genetically altered animals. Therole of CaM-regulated ACs in learning and memory has beenhypothesized ever since the discovery that the basis for thelearning defects in the Drosophila mutant rutabaga is aninactivating mutation of a CaM-activated AC (132). Doubleknockout mice deficient in both of the CaM-stimulated ACs, AC1and AC8, exhibit neither long-term memory nor late long-termpotentiation (133). Each of the single knockouts is normal in thesefunctions; however, they display other neurological defects. Theseresults emphasize the involvement of cAMP signaling pathways inpattern formation of the brain and provide definitive evidence forroles of the CaM-regulated ACs in higher brain function.

Studies on AC3-deficient mice demonstrate a critical role forAC3 in olfaction. These mice fail several olfaction-based behavioraltests, and lack electro-olfactogram responses elicited by eithercAMP or IP3, despite the presence of other AC isoforms inolfactory cilia (134). These knockout mice also implicate AC3 asan important integrator of growth-inhibitory signals that stimulatecAMP formation and that inhibit the growth of arterial smoothmuscle cells (135).

Mice that overexpress ACs provide additional insight into thephysiological roles of specific isoforms. For example, theoverexpression of AC7 in the central nervous system enhancesacute responsiveness and tolerance to morphine (136). Indisagreement with cell transfection data, the transgenic AC7 miceare also supersensitive to G�s responses following morphinetreatment. The cause of this discrepancy is unknown. Studies ontransgenic mice overexpressing either AC5 or AC6 demonstrateimportant differences between these two prominent isoforms inthe heart (137–138). Among the major differences are the cardiac�-adrenergic–dependent regulation of heart rate and contractilityresponses, and the cardioprotective effects of AC6, but not AC5,observed in mouse models of heart failure (induced byoverexpression of G�q).

MUTATIONS OF THE AC SYSTEM IN HUMAN DISEASE

A number of studies have associated impairments of AC systemswith certain human diseases. Mutations causing constitutivelyactive receptors—resulting in elevated intracellular cAMPconcentrations—have been found in patients with: familial maleprecocious puberty/testitoxicosis (emanating from a constitutivelyactivate mutant luteinizing hormone receptor) (139); overactivethyroid adenomas and non-autoimmune autosomal dominanthyperthyroidism (arising from excessive activation of thyroid-stimulating hormone receptor) (140); and Jansen-type metaphysealchondrodysplasia (resulting from a constitutively active mutantparathyroid hormone receptor) (141). Similarly, diseases associatedwith mutations yielding constitutively active G proteins (G�s) arefound in patients with endocrine tumors, McCune-Albrightsyndrome, and testitoxicosis (142–144). Because elevated cAMPconcentrations in isolated endocrine tumors can arise

Review

176

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4. Patel, T.B., Du, Z., Pierre, S., Cartin,L., and Scholich, K. Molecularbiological approaches to unraveladenylyl cyclase signaling andfunction. Gene 269, 13–25 (2001).

5. Smit, M.J. and Iyengar, R.Mammalian adenylyl cyclases. Adv.Second Messenger Phosphoprotein Res.32, 1–21 (1998).

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independently of oncogenic mutations of the G protein �-subunit,moreover, activating mutations of AC might participate in thesedisorders (145). Alternatively, enhanced cyclase activity may resultfrom an increased expression of a particular AC isoform. Indeed,point mutations in the promoter region of the AC3-encoding genethat are associated with decreased insulin release are observed in arat model of type 2 diabetes (146). Conversely, reduced AC activitymay also contribute to pathophysiological states. For example,patients with an unusual form of pseudohypoparathyroidism havenormal G�s protein but have reduced AC activity, suggesting thepresence of inactivating mutations in ACs (147).

SOLUBLE AC

The last mammalian AC isoform to be identified was the solubleform, sAC (24). Although this unique testis-specific and solubleenzymatic activity was identified in the mid 1970s, isolation of thecorresponding protein and cDNA eluded investigators for twodecades (148). The enzymatic activity diverges significantly fromthe membrane-bound relatives in that it is unresponsive tohormones, G proteins, and forskolin. The sAC (24) is ubiquitouslyexpressed in low amounts, but is very highly expressed in spermcells, consistent with the role of AC activity in sperm maturation,motility, capacitation, and the acrosome reaction (149–151).Stimuli such as GTP, G proteins, and forskolin are incapable ofregulating these processes. In contrast, bicarbonate and Ca2+

strongly regulate these activities, as well as increase cAMP levelsand sAC activity (152). Furthermore, the concentration range ofbicarbonate at which recombinant sAC is activated (EC50 ~ 20–50mM) is well within the range found in epididymal fluid (30, 152).Analysis of the amino acid sequence of sAC indicates someresemblance to the membrane-bound isoforms, and tocyanobacterial isoforms of AC. The protein topology is predictedto be similar, and accordingly, most of the residues responsible forcatalysis are conserved. There are two splice forms resulting in187- and 48-kDa proteins. The catalytic domain of the enzyme islocated in the N-terminal region of the full-length 187-kDa form.The truncated form lacks exon 11 and results in prematuretermination. Messenger RNA from the truncated form is about

twenty-five percent as abundant as the full-length transcript;however, the maximal activity of the truncated form is at least ten-fold greater than the full-length form in response to bicarabonate.The precise role of the C-terminal domain of the 187-kD form isunknown. The important relationship of AC and cAMP withsperm maturation and function makes sAC a very attractivepotential pharmacological target. Moreover, sAC has beenpostulated to function as a ubiquitous metabolic sensor, similar toACs found in cyanobacteria (153).

SUMMARY

Our understanding of the hormonal control of intracellular cAMPconcentrations has come a long way since the discovery of AC,and has benefited greatly from the application of moleculargenetics and structural biology. The isolation and characterizationof a gene family encoding nine membrane-bound AC isoformsand one soluble isoform has increased our appreciation for theintricate complexity of the AC signaling system. Manyunanswered questions still remain. For example, why is thetwelve-transmembrane domain structure preserved in nine ACisoforms instead of a simpler structure like the soluble form?More directly, what is the function of the transmembranedomains? Is AC a transporter as suggested by the authors in thefirst paper describing the cloning of an AC (55)? Why do thesimilarly related nucleotide cyclases, the GCs, incorporate a morediverse structure? If cells express multiple AC isoforms, thenhow do they distinguish the stimulatory or inhibitory outcomesfollowing modulation by “on-off switch” regulators, such as G��

and Ca2+? It is clear from a large body of literature that Gprotein–mediated hormonal pathways impinge on the regulationof AC activity using distinct mechanisms, and each cyclaseisoform integrates this information in a specific manner. A majoravenue of research will be to continue to define the regulatoryrepertoires of AC isoforms and to couple this with informationconcerning tissue and subcellular localization. Genetic knockoutapproaches and further structural analyses will be necessary tounderstand the precise physiological and biochemical roles ofeach AC family member.

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Roger K. Sunahara, PhD, (left) is an Assistant Professor in the Department ofPharmacology at the University of Michigan Medical School.Ron Taussig, PhD, (right) is an Assisitant Professor in the Department of Pharmacology atthe University of Texas, Southwestern Medical Center, and is a member of the Alliance forCellular Signaling. Address correspondence to either RKS or RT. E-mailsunahara@umich.edu; fax 734-763-4450. E-mail ron.taussig@utsouthwestern.edu.