Isoforms of Mammalian

Adenylyl Cyclase:
Multiplicities of Signaling
echanistic descriptions of cAMP production have evolved

M 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

With nine different isoforms of appreciate that the hormone-activated synthesis of cAMP involves
membrane-associated adenylyl
cyclases (ACs) and one isoform of multiple polypeptides, including a membrane-bound receptor; a
soluble AC, there is much to learn
heterotrimeric, guanine nucleotide–binding protein (G protein);
and even more to understand
regarding the expression of tissue- and a membrane-bound adenylyl cyclase (AC). Biochemical and
specific AC isoforms. However, on
the protein level, there are many
structural biological studies have provided a firm understanding
proteins and small molecules that for the regulation of AC by G proteins and elucidated the catalytic
affect the catalytic activity of ACs.
Knowing how to tailor AC activity, mechanism. In addition, a number of small molecules have been
or how to exploit the activity of one
developed that modulate AC activity, introducing AC as a potential
isoform over another in a given
tissue, may give rise to therapeutic therapeutic target. Many paradigms of multi-modal regulation of
agents that can inhibit AC-
dependent disease states or, at
AC have been investigated from a physiological perspective. This
least, lessen their severity. review addresses the complexity of the direct modulators of AC
and summarizes the current biological models of their function.

Roger K. Sunahara1 and Ron Taussig2

1
The Department of Pharmacology
University of Michigan Medical School
Ann Arbor, MI 48109-0632
2
The Department of Pharmacology
and the Alliance for Cellular Signaling
University of Texas Southwestern Medical Center
Dallas TX 75390-9041

168
 Multiple Isoforms of Adenylyl Cyclase

INTRODUCTION to explain drug responses that occur in tissues or whole

The activation of adenylyl cyclase (AC), resulting in the
intracellular production of adenosine-3',5'-monophosphate (i.e.,
cyclic AMP [cAMP]), is initiated by the binding of hormones to
cell surface receptors (1). Epinephrine, dopamine, prostaglandin
PGE2, adenosine, and glucagon are a few examples of the many
hormones that activate AC through membrane-bound receptors.
Glucagon, for example, a hormone that regulates glycogen
metabolism in liver and skeletal muscle, recognizes membrane
receptors in these tissues, markedly stimulates AC to produce
intracellular cAMP. Glucagon-bound receptors communicate with
an intracellular, membrane-associated heterotrimeric G protein (2)
composed of a guanosine diphosphate (GDP)–bound
-subunit
and an obligate  heterodimer. Hormone-dependent activation of
receptors leads to the exchange of GDP for guanosine triphosphate
(GTP). Conformational changes due to GTP binding result in the
dissociation 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 distinct
receptor subtypes. The -subunits also regulate AC, but in an AC
subtype–specific manner (3–5). Additionally, calcium ions are very
strong modulators of some isoforms of AC (6–9); thus, G proteins
that 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. Their
mechanism of action can now be incorporated into model systems
organisms. For example, learning and memory are associated with
the activation of protein kinase activity and protein
phosphorylation (13), two processes that are strongly regulated by
AC activity. Genetically modified mouse strains that contain
altered 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 strongly
implicated in learning and memory (17–19). AC activity itself can
be modulated through phosphorylation of the enzyme, and
alterations in the expression of AC isoforms also accompany drug-
induced receptor effects that have been related to symptoms of
drug dependence (20–23).
Much of the research surrounding cAMP signaling has
focused on AC activity that is regulated by G proteins. However, a
bona fide soluble form of AC that is insensitive to G proteins and
forskolin has been cloned (24). Soluble AC (sAC) activity was
identified in testis during the early 1980s, but the activity
remained an enigma until the corresponding cDNA was isolated.
Elevated concentrations of cAMP in the testis are crucial for sperm
development and capacitation. The discovery of sAC, along with
the finding that it is activated by bicarbonate, has forced a
reexamination of how cAMP signals are propagated into the cell
beyond the cell membrane.
MULTIPLE AC ISOFORMS
Molecular cloning techniques have identified nine mammalian
genes that encode membrane-bound ACs (3–5, 25), and one gene
encoding a soluble isoform (24). These genes do not tend to
cluster within the genome, but rather are distributed among

A B Families of G

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

PGE2

inhibits AC

i (e.g.,
i1,
i2,

Adenosine 
i3,
o,
z)

Glucagon   regulatory role

q unclear

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 thus
anchored at the cell surface (brown double bar). The intracellular portion of these receptors interacts with GDP-bound heterotrimeric G proteins, resulting
in 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 an
intrinsic GTPase activity, so that their dissociation from G and their effect on AC activity are transitory. (Yellow triangle represents GDP; red triangle
represents GTP.)

June 2002
Volume 2, Issue 3 169
 Review

A R1 AC1 R2 VDCC
different chromosomes, with the exception that the two genes that
encode AC7 and AC9 are located on chromosome 16, albeit at

s 
i
opposites arms. The ten AC isoforms can be divided into five
Ca2+
distinct families based on their amino acid sequence similarity and
CaMK
functional attributes. The Ca2+–CaM-sensitive forms are types Ca2+ CaM
AC1, AC3, and AC8 (Figure 2A). The G-stimulatory forms are
represented by AC2, AC4, and AC7 (Figure 2B). AC5 and AC6 are
distinguished by their sensitivity to inhibition by both Ca2+ and
G
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 is B R2 R1 AC2
highly insensitive to the diterpene forskolin. The last isoform, sAC,

i 
s
is the most divergent of all the mammalian cyclases, and is similar
to cyclases found in cyanobacteria (24).
PKA
The distribution of these AC isoforms, according to mRNA
detection, is summarized in Table 1. In general, all membrane-
PKC
bound AC isoforms are found in, but not limited to, excitable
tissues such as neurons and muscle (3, 26). Within the brain, AC
isoforms localize to different, discrete brain regions (27–29).
Although most isoforms are widely expressed, AC1 and AC3 are C VDCC R1 AC5 R2
expressed only in brain (29). Soluble cyclase is expressed
predominantly in the testis, although splice variants have been 
s
i 
identified displaying a broader distribution pattern (30). The broad
distribution of AC isoforms suggests that any given cell contains Ca2+
multiple isoforms.
PKC PKA
NO
REGULATION OF AC ACTIVITY Figure 2. Multiple modes of regulation of adenylyl cyclase
isoforms. (A) The pattern of regulation of AC1 as illustrated is
STIMULATION BY G
s representative also for AC3 and AC8. R1 represents a G
protein–coupled receptor, such as the glucagon or 2-adrenergic
Hormonal activation of AC occurs primarily through receptors receptor, that couples to the stimulatory G protein G
s. R2
coupled to the stimulatory G protein G
s. G
s is the most widely represents a G protein–coupled receptor, such as the muscarinic M2
or
1-adrenergic receptor, that couples to the inhibitory G protein
distributed activator of all mammalian membrane-bound AC
G
i. (B) The pattern of regulation of AC2 as illustrated is
isoforms. Multiple splice variants of G
s have been identified: representative of the regulation of AC4 and AC7. Note that G
G
s-short, G
s-long and G
sXL. Although the former two regulation of AC2 is dependent on G
s co-activation and does not
isoforms have been extensively characterized both physiologically activate AC by itself. PKC can use AC as a substrate, resulting in
and biochemically, G
sXL is a relatively new member and is less elevation of basal activity and inhibition of the G superactivation.
(C) The pattern of regulation of AC5 is representative also of AC6.
well characterized (31). The long and short splice forms are
(PKA, protein kinase A; PKC, protein kinase C; CaM, calmodulin;
biochemically indistinguishable in their capacity to directly CaMK, calmodulin-dependent kinase; NO, nitric oxide; VDCC,
activate AC (32); however, the behavior of the hormone voltage-dependent Ca2+ channel.)
receptor–stimulated AC varies considerably (33). G
sXL can
activate AC directly, but no hormone receptor–mediated effects a specific Regulator of G protein Signaling (RGS) molecule, PX1-
through G
sXL have been demonstrated (34). RGS, that serves as a GTPase-accelerating protein (GAP) for G
s
G
s in the GTP-bound form displays a tenfold greater affinity (38). AC itself can weakly accelerate the GTPase activity of G
s
for activating AC compared to the GDP-bound form (35). (39), similar to the accelerating effect of PLC isoforms on their
Crystallographic evidence suggests that the main contact between activator G
q (40).
G
s and AC occurs through a short
-helix that is highly mobile
throughout the GTPase cycle of all G proteins (36, 37). The INHIBITION BY G
i
decreased affinity of the GDP-bound form for AC suggests that the
GTPase activity of G
s serves as a timing mechanism to delimit Members of the G
i family inhibit AC but can manifest selectivity
cyclase activation. Following GTP hydrolysis, G
s dissociates from for given AC isoforms. G
i1, G
i2, G
i3, G
o, and G
z can
cyclase, reassociates with G, and thereby terminates both G
s inhibit AC5 and AC6 (Figure 2C) (41–43). Interestingly, their
and G signaling. The deactivation of G
s can be accelerated by mode of inhibition is not through direct competition with G
s,

170

because forskolin-stimulated activity is also inhibited. In addition,
mutagenesis experiments and structural modeling suggest that
G
i exerts its effects at a site, symmetrical to the G
s binding
site, located on the side opposite the AC molecule (44). The
highly expressed brain-specific G
o can inhibit AC1 (and
possibly AC8), although it is not as potent as the other G
i
subunits on AC5 and AC6. The G
i subunits are
posttranslationally modified by long-chain acyl (myristoyl) and
thioacyl (palmitoyl) moieties (45); myristoylation is required for
G
i-mediated inhibition of AC.
REGULATION BY G
The contributions of the G heterodimer to the modulation of
ACs have been, at least until recently, largely unappreciated (46). G

-subunits had long been presumed to predominate in the
regulation of AC; however, G subunits are strong modulators of
AC 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 all
negative regulators of AC1 and AC8, and can markedly inhibit the
effects of forskolin, G
s, and Ca2+–CaM on AC activities. These
findings are particularly relevant for brain physiology because the
G
i family and their accompanying  subunits are, along with
AC1 and AC8, highly expressed in the brain (48).
In contrast, G subunits act to stimulate the cyclase activity
of AC2, AC4 and AC7, albeit only when G
s is co-activated
(Figure 2B). G and G
s could thus establish a synergistic
relationship, whereby the presence of G might dramatically
enhance the ability of G
s to activate AC. Indeed, the activation
of those hormone receptors coupled to G
i subunits could liberate
G dimers that could synergistically potentiate AC activity that
had been stimulated by distinct, G
s-activated, hormone
receptors. It is important to note that the AC isoforms that
undergo stimulation by G (i.e., AC2, AC4 and AC7) are not
directly modulated by the
subunits of the Gi family (3, 26). Less
well understood is the relationship between G and the other
cyclase isoforms such as AC5 and AC6. Transfection experiments
suggest that G can inhibit AC5 and AC6 activity, perhaps in an
indirect manner (49).
The putative binding site for G on the G-stimulated
family of ACs (i.e., AC2, AC4, and AC7) has been mapped on the
basis of peptide inhibition studies (50, 51). Peptides
corresponding to amino acid residues 956 to 982 of AC2, that is,
derived from the middle of the second of the two catalytic
domains (i.e., C2), potently inhibit the ability of G to stimulate
the enzyme activity of intact AC2. Despite the high degree of
sequence conservation among AC catalytic domains, this sequence
(i.e., corresponding to residues 956 to 982 of AC2) is not found in
AC isoforms that are not modulated by G. Indeed, this
sequence also contains the short putative G-binding motif
QXXER, the consensus for which is based on GRK2, the -
Multiple Isoforms of Adenylyl Cyclase
adrenergic receptor kinase that requires G for activation, as well
as G-activated inwardly rectifying K+ channels, the G-
activated PLC isoforms, and the G-inhibited AC1. Disruption
of the consensus QXXER motif in any of these instances abrogates
all G effects. The C2 domain of AC2, possessing the QXXER
motif, is located near the plasma membrane face, but the precise
structure of this motif is unknown because the region is
disordered in the crystal structure. An additional region within the
regulatory region of the C1 domain, juxtaposed to the
transmembrane domain, may also be important for G regulation
of AC2, AC4, and AC7 (52).
A peptide generated from the catalytic region of the AC1
isoform analogous to that containing the QXXER motif in the AC2
sequence also displays dramatic effects on G regulation of AC
activity (52). The peptide could reverse both G-dependent
inhibition of AC1 activity and G-dependent superactivation of
G
s-stimulated AC2, suggesting that the region of the AC1
isoform also serves for binding of G. For example, it has been
reported that this region contributes to G-mediated inhibition
of AC1 activity (53).
The recent findings outlined above underscore the
importance of the G heterodimer in modulating AC activity and
suggest that it may be naïve to regard the importance of G as
secondary to that of G
. In addition to its role in regulating AC,
G subunits have been implicated as the primary component of
G proteins that directly regulate ion channels (e.g., K+-channel
activation and Ca2+- and Na+-channel inhibition) as well as other
effectors systems: GPCR kinases (activation), phospholipase C
isoforms (activation), and the mitogen-activated protein (MAP)
kinase pathway (activation) (10, 54). The MAP kinase-dependent
mating response in yeast is solely dependent on G for signaling
and 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 local
Ca2+ levels in intact cells may thus dramatically enhance the
activity of these isoforms. Specifically, intracellular Ca2+ from
IP3-sensitive stores are unable to affect these Ca2+-sensitive AC
isoforms, whereas activation of Ca2+ entry through voltage-gated
Ca2+ channels or through capacitative entry is effective at
activating these AC isoforms (58, 59).
Although millimolar (i.e., non-physiological) concentrations
of Ca2+ inhibit all AC isoforms (Figure 3C), AC5 and AC6 are
inhibited by concentrations of Ca2+ in the M range, well within
the dynamic range of intracellular levels (Figure 2C) (60); Ca2+
from capacitative entry are thought to be the sole physiological
source of Ca2+ to inhibit the AC5 and AC6 (6). The fact that these
AC isoforms are restricted mostly to brain-specific and excitable
cell types, and are compartmentalized with voltage-gated Ca2+
channels, is consistent with this notion (Table 1).
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 Review

TABLE 1. REGULATORY PROPERTIES OF MAMMALIAN ADENYLYL CYCLASE ISOFORMS

AC Chromosomal Tissue Regulation by G Protein Calcium Putative
Isoforma,b Location Distribution Protein Subunits Kinases Effects Function
AC1 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 Synaptic
plasticity
AC2 5p15 (155, 156) Brain, skeletal Stimulated by G
s PKC:
muscle, lung, heart Stimulated by Gd stimulation
AC3 2p22-p24 (157) Brain, olfactory Stimulation by G
s PKC: weak Stimulated by Olfaction
epithelium stimulation Ca2+-CaM
CaMKII:
inhibition
AC4 14q11.2 (156) Brain heart, kidney, Stimulation by G
s PKC:
liver, lung, BAT, Stimulated by Gd inhibition
uterus
AC5 3q13.2-q21 (157) Heart, brain, Stimulation by G
s PKA: Inhibited
kidney, liver, lung, Inhibited by Ge inhibition
uterus, adrenal, Inhibited by G
if PKC
,:
BAT stimulation
AC6 12q12-q13 (157) Ubiquitous Stimulation by G
s PKA: Inhibited
Inhibited by Ge inhibition
Inhibited by G
if PKC:
inhibition
AC7 16q12-q13 (158) Ubiquitous, highly Stimulation by G
s PKC: Drug
expressed in brain Stimulated by Gd stimulation dependency
AC8 8q24 (155, 156) Brain, lung (testis, Stimulation by G
s Stimulated by Learning,
adrenal, uterus, Ca2+-CaM memory,
heart) LTP, Synaptic
plasticity
AC9g 16p13.3 (159) Brain, skeletal Stimulation by G
s
muscle
SACh 1q24 (24) Testis Not regulated by G Sperm
protein subunits capacitation
BAT, 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.

REGULATION BY OTHER PROTEINS

A number of other proteins have recently been identified that
interact directly with ACs, but their biological significance has yet
to be determined. Several of these proteins have been identified
through yeast two-hybrid or copurification experiments using the
cytosolic domains as baits. The protein associated with Myc (PAM)
potently inhibits AC1 and AC5, but not AC2 (61), whereas the
Escherichia coli protein SlyD, a cis-trans peptidylprolyl isomerase
(PPIase), copurifies with bacterially expressed AC7 so as to inhibit
its activity (62). A conceptually more relevant interaction was
identified between another RGS protein, RGS2, and AC3 (63). RGS
molecules are mostly noted for their ability to accelerate the
GTPase activity of the heterotrimeric G proteins of the G
i, G
q,

172
 Multiple Isoforms of Adenylyl Cyclase

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

corresponding to Leu912 and Ser942 of AC2 (67). In an

unexpected divergence of evolution, however, the D. melanogaster
ortholog of AC9 is sensitive to forskolin (68). The forskolin-
C2 dependent activation of AC2, AC4, AC5, AC6, and AC7 is
G
s Forskolin G
i
synergistic with G
s-mediated coactivation, whereas activation by
forskolin and G
s is additive for isoforms AC1, AC3, and AC8 (3).
90º The binding site for forskolin is located within the catalytic
C core of AC, at the interface between the intracellular catalytic (C1
K1065 and C2) domains (Figure 3). G
s binds similarly between the two
R1029 domains, but at a location on the perimeter of the catalytic core.
The relationship between the two binding sites and their proposed
ATP
–S mechanism of action may explain the cooperativity of binding
Mg2+ observed between forskolin and G
s. Why other isoforms display
D440 R484 additive effects with forskolin and G
s is not obvious from the
Mg2+
crystal structure. Stimulation of activation by forskolin and
D1018 D396 Ca2+–CaM is cooperative in the cases of AC1, AC8, and
K938 presumably AC3 (56, 69).
Since the elucidation of the crystal structure of AC bound to
either forskolin or its water-soluble analog 7-deacetyl-7-(O-N-
and G
12 types. The RGS2 isoform can enhance the intrinsic methylpiperazino)--butyryl forskolin (5, 43), researchers have
GTPase rate of both the G
i and G
q (64, 65). A direct association attempted to design isoform-selective forskolin analogs. Although
between G
i or G
q and AC3 has not been demonstrated; this methodology is still in its infancy, several compounds have
however, this interaction would provide an additional avenue of been synthesized that contain subtle modifications of forskolin
crosstalk. and display a two- to threefold preference for certain cyclase
isoforms (70).
REGULATION BY SMALL MOLECULES
Pyrophosphate
Forskolin
Adenylyl cyclase hydrolyses ATP to produce pyrophosphate and
The diterpene forskolin (from Coleus forskohlii) potently activates cAMP. In a steady-state AC assay, the rate-limiting step is normally
all known isoforms of mammalian membrane-bound ACs with the the release of pyrophosphate (71). Elevated concentrations of
exception of AC9 (66). The sensitivity difference may be pyrophosphate can thus be used to force AC into a product-bound
accounted for by as few as two residues, Ala1112 and Tyr1082, conformation that prevents the binding of ATP. The antiviral agent,

June 2002
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 Review
foscarnet, or phosphonoformic acid, mimics pyrophosphate and
likewise 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 ATP
binding (71, 73, 74). These compounds inhibit AC by binding to a
conformation of the enzyme that closely resembles the product-
bound state, or posttransition state (75). The capacity of P-site
inhibitors to inhibit AC activity is thus dramatically affected by the
catalytic activity of cyclase itself, which in turn is a function of the
enzyme’s conformational state (73); most notably, P-site inhibitors
are dramatically potentiated by the presence of pyrophosphate.
The majority of P-site inhibitors lack one or more hydroxyl groups
relative to the ribose ring structure (75). Additionally, most of
these inhibitors are mono- or polyphosphates and are structural
analogs of cAMP. Thus, 2'-deoxy-3'-AMP (IC50 ~10 M), and the
more 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 not
specific for individual AC isoforms. The only exceptions are 9-
(cyclopentyl)-adenine and 9-(tetrahydro-2-furyl)-adenine; they are
ineffective 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 a
weak 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 activity
currently 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), the
ability of any these compounds to specifically modulate AC
isoforms is not established.
REGULATION BY POSTTRANSLATIONAL MODIFICATION
Several modes of posttranslational modification, including
phosphorylation, glycosylation, and S-nitrosylation, can alter the
activity of ACs. The phosphorylation of AC by protein kinases
generally has an inhibitory effect, not on basal activity, but on
enhanced stimulation by various activators. These effects are part
of a negative feedback mechanism; for example, PKA-mediated
phosphorylation is thought on negatively regulate AC5 and AC6
activity (Figure 2C)(82, 83).
Much attention has focused on the role of phosphorylation by
protein kinase C (PKC) in regulating AC activity since the initial
174
report that AC purified from brain can be directly phosphorylated
by this kinase (84). The activities of AC1, AC2, AC3, and AC5 can
be stimulated following phorbol ester treatment, whereas those of
AC4 and AC6 are inhibited, suggesting that PKC can regulate ACs
in 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 lose
responsiveness to the (stimulatory) effect of G. In this way, PKC
bears the role, with regard to AC2-like cyclases, of modulating the
integration of G
s and G inputs (85).
It is perhaps counterintuitive that Ca2+–CaM, which
normally activates AC1, AC3, and AC8, can also inhibit AC1 and
AC3 indirectly through phosphorylation by CaM kinase II and IV,
respectively (92, 93). This mode of regulation most likely reflects a
negative feedback loop that controls Ca2+-mediated stimuli.
Both hormone- and forskolin-stimulated AC5 and AC6
activity are inhibited by nitric oxide (NO) (94). In addition to its
primary target—soluble guanylyl cyclase (GC) (95, 96)—NO
affects 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 be
important for AC responsiveness to hormones and forskolin (99,
100). Although tunicamycin treatment, or substitution of
glutamine for Asn805 and Asn890 of AC6, has very little effect on
the targeting of AC6 to the plasma membrane and on G protein
activation, G protein–mediated inhibition and responses to
forskolin are impaired by as much as fifty percent (99). In
contrast, a variant of AC8 requires N-linked glycosylation for
plasma membrane targeting and thus for activation by membrane-
bound G protein–coupled receptors (100, 101).
STRUCTURE: PRIMARY, SECONDARY, AND
TERTIARY
AC is an integral membrane protein composed of twelve
transmembrane segments. The protein can be visualized as two
tandemly repeated domains, each containing six transmembrane
segments and a large cytoplasmic (catalytic) loop (Figure 4). The
twelve-transmembrane domain topology is reminiscent of the ABC
family of transporters such as the cystic fibrosis transmembrane
rectifier and the P-glycoprotein, which is responsible for multidrug
resistance and is encoded by the MDR1 gene (102, 103). The
sequence 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 share
significant sequence similarity to the corresponding regions of GCs
(96) and ACs from prokaryotes (Figure 4).
The biochemical characterization of recombinant forms of
the AC cytoplasmic domains has provided extremely useful
 Multiple Isoforms of Adenylyl Cyclase

insights into AC regulation and catalysis. The construction of hydrolysis (37). The pseudosymmetrical structure of the catalytic
fused C1–C2 domains (104) and individual soluble domains (35, core makes apparent the likely binding site for other G proteins,
105–108) results in activities characteristic of the full-length such as G
il, that regulate catalysis (Figure 3B). G
i selectively
membrane-bound AC forms in terms of modulation by G protein inhibits AC5 and AC6, for example, presumably by binding to the

-subunits, forskolin, substrate inhibitors, and P-site inhibitors C1 domain (and perhaps through an interaction with the C2
(35, 105–108). The utilization of the recombinant, soluble domain) and stabilizing the two helices of C1, thus allosterically
domains of AC has facilitated the biophysical characterization of modifying the proximally located active site (44, 108).
enzyme function.
SUBSTRATE BINDING AND THE MECHANISM OF HYDROLYSIS
STRUCTURAL BASIS FOR THE REGULATION OF AC
The active site, revealed by x-ray diffraction of
The C1 and C2 domains that form the catalytic core of AC are C1•C2•G
s•forskolin co-crystals, is located at the interface
related by two-fold pseudosymmetry (Figure 3). Current models between the C1 and C2 domains in at a site pseudosymmetrically
of the AC structure are based on the forskolin-bound form in the related to the forskolin binding site (36, 109, 110). Residues that
presence (C1–C2 heterodimer) or absence (inactive C2 contact the substrate (or substrate analog) are conserved in all AC
homodimer) of G
s. The forskolin binding site is located in a isoforms (Figure 3C). Interestingly, the positions of Lys938 and
hydrophobic pocket at the interface between the two domains (36, Asp1018 in AC2, residues that contribute most of the binding
109). Like forskolin, G
s also contacts both domains, with most of energy to the adenine ring, are occupied in GCs by glutamate and
the binding surface (approximately seventy-five per cent) cysteine residues, respectively. Indeed, the substrate specificity of
contributed by the C2 domain. The binding of G
s induces a 7˚ AC can be changed from ATP to GTP by making the appropriate
rotation of the C1 domain around the C2 domain, presumably amino acid substitutions (111). Similarly, the conversion of a GC
positioning the active site for catalysis. The C2 domain contacts to an AC has also been demonstrated (111, 112).
G
s primarily in the switch II region, one of three segments of G The residues that coordinate the binding of the ribose and
proteins that are highly mobile throughout the cycle of GTP triphosphate portion of the nucleotide are conserved in all
isoforms AC and GC (Figure 3C).
Cyclase Cyclase Non-conservative substitution of
Homology Homology any of these residues severely
TM Domain TM Domain
Mammalian impairs cyclase activity. Arg484,
Membrane-Bound TM1-6 C1 TM7-12 C2 AC1 Arg1029, and Lys1065 in AC2
Adenylyl Cyclase
share coordination of the
-, -,
Mammalian Soluble C1 C2 sAC and -phosphates of the
Adenylyl Cyclase nucleotide. Two highly conserved
D. discoideum TM CHD aspartate residues (Asp396 and
Membrane-Bound AGC
TM CHD Asp440 in AC5) also help to
Adenylyl Cyclase
ANP Binding coordinate the phosphates by
coupling to two Mg2+ cations that
Membrane-Bound TM KHD CHD GCA stabilize the
-phosphate during
Guanylyl Cyclase TM KHD CHD Homodimer catalysis. The overall structure is
Nitric Oxide Binding strikingly similar to that found in
Soluble CHD GC
–GC other phosphoryl transferases,
Guanylyl Cyclase Heterodimer such as T7 DNA polymerase and
CHD
Membrane-Bound HIV reverse transcriptase (110,
Guanylyl Cyclase 113–115). A model of the catalytic
Tetrahymena, TM1-6 C2 TM7-12 C1 GCA
mechanism for these enzymes
D. discoideum
involves the contribution of one
Soluble Guanylyl
Cyclase the Mg2+ ions acting as a general
C1 C2 sGC
D. discoideum base that deprotonates the 3'-OH
of the ribose ring (116). The newly
Figure 4. Sequence alignment of the adenylyl and guanylyl cyclases. The catalytic domains (yellow) display
considerable similarity in amino acid sequence and have been coined the Cyclase Homology Domain (CHD). formed oxyanion is thus poised
Illustrated in light blue are the membrane-spanning regions as predicted from amino acid sequence. (TM, for nucleophilic attack of the
-
transmembrane; ANP, atrial natriuretic factor; KHD, kinase homology domain.) Adapted from Wedel and phosphate with elimination of
Garbers (96). pyrophosphate.

June 2002
Volume 2, Issue 3 175
 Review
PHYSIOLOGY AND FUNCTION
OF MAMMALIAN ACS
The biochemical assessment of the ACs has revealed several
regulatory pathways that control AC activity. In contrast, a
number of factors have resulted in a relative paucity of
physiological data describing these complex regulatory pathways.
The most notable obstacle is the multiplicity of the isotypes
expressed within a given cell type, further complicated by the
varying effects of modulators such as Ca2+ and G, as well as
the particular intracellular milieu. The majority of data come form:
1) overexpression studies using cell transfection or transgenic
animals; 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 in
model systems for drug abuse, withdrawal, and recovery (117).
Cells chronically treated with opiates (which activate G
i-coupled
receptors) exhibit AC activity that is supersensitive to stimulation
by either forskolin or G
s following withdrawal of the opiate.
Similar sensitization is observed with chronic activation of other
hormone receptors that couple through G
i—such as the A3
adenosine (118), D2 and D4 dopamine (23, 119), and M2
muscarinic receptor subtypes (23, 120)—and is dependent on the
expression 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), but
the mechanisms underlying this form of sensitization remain
obscure. Interestingly, chronic opioid treament leads to relative
desensitization of the AC2, AC4 and AC7 isoforms, which appears
to be regulated through G
s, G
i, G, and PKC (119, 120,
121–126). PKC-mediated phosphorylation of AC5 increases AC
activity in vivo and in vitro (127). Chronic hormone stimulation
leading to higher steady-state amounts of PKC-mediated phospho-
AC5 may account for some of the apparent sensitization. G has
been implicated in the cannabinoid (CB1) receptor-mediated
superactivation of AC1, AC3, AC5, AC6, and AC8, but not the
ACs that are normally activated by G (128).
Supersensitization might result as an effect of increased
expression of specific AC isoforms, PKA, and the cAMP-responsive
element binding protein (CREB) (117, 129). In sharp contrast,
chronic exposure to ethanol reduces such expression and leads to
AC desensitization (130), suggesting that ethanol dependence
greatly 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
176
isoforms has come from studies on genetically altered animals. The
role of CaM-regulated ACs in learning and memory has been
hypothesized ever since the discovery that the basis for the
learning defects in the Drosophila mutant rutabaga is an
inactivating mutation of a CaM-activated AC (132). Double
knockout mice deficient in both of the CaM-stimulated ACs, AC1
and AC8, exhibit neither long-term memory nor late long-term
potentiation (133). Each of the single knockouts is normal in these
functions; however, they display other neurological defects. These
results emphasize the involvement of cAMP signaling pathways in
pattern formation of the brain and provide definitive evidence for
roles of the CaM-regulated ACs in higher brain function.
Studies on AC3-deficient mice demonstrate a critical role for
AC3 in olfaction. These mice fail several olfaction-based behavioral
tests, and lack electro-olfactogram responses elicited by either
cAMP or IP3, despite the presence of other AC isoforms in
olfactory cilia (134). These knockout mice also implicate AC3 as
an important integrator of growth-inhibitory signals that stimulate
cAMP formation and that inhibit the growth of arterial smooth
muscle cells (135).
Mice that overexpress ACs provide additional insight into the
physiological roles of specific isoforms. For example, the
overexpression of AC7 in the central nervous system enhances
acute responsiveness and tolerance to morphine (136). In
disagreement with cell transfection data, the transgenic AC7 mice
are also supersensitive to G
s responses following morphine
treatment. The cause of this discrepancy is unknown. Studies on
transgenic mice overexpressing either AC5 or AC6 demonstrate
important differences between these two prominent isoforms in
the heart (137–138). Among the major differences are the cardiac
-adrenergic–dependent regulation of heart rate and contractility
responses, and the cardioprotective effects of AC6, but not AC5,
observed in mouse models of heart failure (induced by
overexpression of G
q).
MUTATIONS OF THE AC SYSTEM IN HUMAN DISEASE
A number of studies have associated impairments of AC systems
with certain human diseases. Mutations causing constitutively
active receptors—resulting in elevated intracellular cAMP
concentrations—have been found in patients with: familial male
precocious puberty/testitoxicosis (emanating from a constitutively
activate mutant luteinizing hormone receptor) (139); overactive
thyroid adenomas and non-autoimmune autosomal dominant
hyperthyroidism (arising from excessive activation of thyroid-
stimulating hormone receptor) (140); and Jansen-type metaphyseal
chondrodysplasia (resulting from a constitutively active mutant
parathyroid hormone receptor) (141). Similarly, diseases associated
with mutations yielding constitutively active G proteins (G
s) are
found in patients with endocrine tumors, McCune-Albright
syndrome, and testitoxicosis (142–144). Because elevated cAMP
concentrations in isolated endocrine tumors can arise
 Multiple Isoforms of Adenylyl Cyclase

independently of oncogenic mutations of the G protein
-subunit,
moreover, activating mutations of AC might participate in these
disorders (145). Alternatively, enhanced cyclase activity may result
from an increased expression of a particular AC isoform. Indeed,
point mutations in the promoter region of the AC3-encoding gene
that are associated with decreased insulin release are observed in a
rat model of type 2 diabetes (146). Conversely, reduced AC activity
may also contribute to pathophysiological states. For example,
patients with an unusual form of pseudohypoparathyroidism have
normal G
s protein but have reduced AC activity, suggesting the
presence of inactivating mutations in ACs (147).
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 is
unknown. The important relationship of AC and cAMP with
sperm maturation and function makes sAC a very attractive
potential pharmacological target. Moreover, sAC has been
postulated to function as a ubiquitous metabolic sensor, similar to
ACs found in cyanobacteria (153).
SUMMARY

SOLUBLE AC Our understanding of the hormonal control of intracellular cAMP

The last mammalian AC isoform to be identified was the soluble
form, sAC (24). Although this unique testis-specific and soluble
enzymatic activity was identified in the mid 1970s, isolation of the
corresponding protein and cDNA eluded investigators for two
decades (148). The enzymatic activity diverges significantly from
the membrane-bound relatives in that it is unresponsive to
hormones, G proteins, and forskolin. The sAC (24) is ubiquitously
expressed in low amounts, but is very highly expressed in sperm
cells, 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 of
regulating these processes. In contrast, bicarbonate and Ca2+
strongly regulate these activities, as well as increase cAMP levels
and sAC activity (152). Furthermore, the concentration range of
bicarbonate at which recombinant sAC is activated (EC50 ~ 20–50
mM) is well within the range found in epididymal fluid (30, 152).
Analysis of the amino acid sequence of sAC indicates some
resemblance to the membrane-bound isoforms, and to
cyanobacterial isoforms of AC. The protein topology is predicted
to be similar, and accordingly, most of the residues responsible for
catalysis are conserved. There are two splice forms resulting in
187- and 48-kDa proteins. The catalytic domain of the enzyme is
located in the N-terminal region of the full-length 187-kDa form.
The truncated form lacks exon 11 and results in premature
termination. Messenger RNA from the truncated form is about
concentrations has come a long way since the discovery of AC,
and has benefited greatly from the application of molecular
genetics and structural biology. The isolation and characterization
of a gene family encoding nine membrane-bound AC isoforms
and one soluble isoform has increased our appreciation for the
intricate complexity of the AC signaling system. Many
unanswered questions still remain. For example, why is the
twelve-transmembrane domain structure preserved in nine AC
isoforms instead of a simpler structure like the soluble form?
More directly, what is the function of the transmembrane
domains? Is AC a transporter as suggested by the authors in the
first paper describing the cloning of an AC (55)? Why do the
similarly related nucleotide cyclases, the GCs, incorporate a more
diverse structure? If cells express multiple AC isoforms, then
how do they distinguish the stimulatory or inhibitory outcomes
following modulation by “on-off switch” regulators, such as G
and Ca2+? It is clear from a large body of literature that G
protein–mediated hormonal pathways impinge on the regulation
of AC activity using distinct mechanisms, and each cyclase
isoform integrates this information in a specific manner. A major
avenue of research will be to continue to define the regulatory
repertoires of AC isoforms and to couple this with information
concerning tissue and subcellular localization. Genetic knockout
approaches and further structural analyses will be necessary to
understand the precise physiological and biochemical roles of
each AC family member.

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