Physiol Rev 87: 965–1010, 2007;
doi:10.1152/physrev.00049.2006.
Organization and Ca2⫹ Regulation of Adenylyl Cyclases
in cAMP Microdomains
DEBBIE WILLOUGHBY AND DERMOT M. F. COOPER
Department of Pharmacology, University of Cambridge, Cambridge, United Kingdom
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I. Introduction
II. Adenylyl Cyclases
A. General structure of ACs
B. Are the ACs cAMP exporters?
C. Voltage sensitivity
D. Dimerization
E. Catalytic core
F. AC distribution
G. Soluble AC
III. Type-specific Regulation
A. G proteins
B. PKA
C. PKC
D. Calmodulin kinase
E. Receptor tyrosine kinase
F. Calcineurin
G. RGS proteins
IV. Regulation by Calcium In Vitro
A. Stimulation by Ca2⫹
B. Inhibition by Ca2⫹
V. Regulation by Calcium In Vivo
A. Ca2⫹ homeostasis in nonexcitable cells
B. Dependence of Ca2⫹-sensitive ACs on CCE over release in nonexcitable cells
C. Dependence of Ca2⫹-sensitive ACs on CCE rather than other forms of Ca2⫹ entry in
nonexcitable cells
D. Close apposition of ACs with CCE channels
E. Regulation by calcium in excitable cells
F. What is the nature of the Ca2⫹ signal to which the ACs respond in vivo?
G. Role of CaM recruitment in the regulation of AC8 by Ca2⫹
VI. Compartmentalization of the Adenylyl Cyclases
A. Lipid rafts
B. Lipid rafts and ACs
C. Components of the microdomain
VII. Dynamic and Local Changes in cAMP
A. Methodologies for single-cell cAMP measurements
B. Local dynamics of cAMP signals detected with real-time probes
C. Oscillations in intracellular cAMP
VIII. Organization of cAMP Signaling Via Direct Protein-Protein Interactions
A. AKAP-based signaling complexes
B. Other AC-associated proteins
IX. Physiological Systems
A. Specialized actions of Ca2⫹-inhibitable AC6
B. Physiological role of AC3 in olfaction
C. Functions of the Ca2⫹/CaM-stimulated ACs
X. Conclusions and Future Issues
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Willoughby D, Cooper DMF. Organization and Ca2⫹ Regulation of Adenylyl Cyclases in cAMP Microdomains.
Physiol Rev 87: 965–1010, 2007; doi:10.1152/physrev.00049.2006.—The adenylyl cyclases are variously regulated by
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0031-9333/07 $18.00 Copyright © 2007 the American Physiological Society
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DEBBIE WILLOUGHBY AND DERMOT M. F. COOPER
G protein subunits, a number of serine/threonine and tyrosine protein kinases, and Ca2⫹. In some physiological
situations, this regulation can be readily incorporated into a hormonal cascade, controlling processes such as
cardiac contractility or neurotransmitter release. However, the significance of some modes of regulation is obscure
and is likely only to be apparent in explicit cellular contexts (or stages of the cell cycle). The regulation of many of
the ACs by the ubiquitous second messenger Ca2⫹ provides an overarching mechanism for integrating the activities
of these two major signaling systems. Elaborate devices have been evolved to ensure that this interaction occurs, to
guarantee the fidelity of the interaction, and to insulate the microenvironment in which it occurs. Subcellular
targeting, as well as a variety of scaffolding devices, is used to promote interaction of the ACs with specific signaling
proteins and regulatory factors to generate privileged domains for cAMP signaling. A direct consequence of this
organization is that cAMP will exhibit distinct kinetics in discrete cellular domains. A variety of means are now
available to study cAMP in these domains and to dissect their components in real time in live cells. These topics are
explored within the present review.
cAMP is the prototypical second messenger, which
impacts on every aspect of the life of the cell, from
differentiation and development through to cell death.
The converse is probably also true, i.e., every major cellular event will have direct or indirect consequences for
cAMP signaling. Production of cAMP results from the
activity of the adenylyl cyclase (AC) family of enzymes.
Not so long ago, AC was viewed as a single entity that was
regulated by hormones in a stimulatory or inhibitory manner through the mediation of stimulatory and inhibitory G
proteins (159). The discovery of multiple AC isoforms (9
membrane-bound and 1 soluble) rapidly revealed that not
only was this regulation by G proteins diverse, but also an
array of regulatory properties were displayed that permitted numerous points of interaction with a range of major
signaling pathways (373). This potential for diverse interplay with other key regulatory molecules is accompanied
by an elegant spatiotemporal organization of cAMP signals to maintain tight control of cAMP-dependent events.
Details are now emerging of directed targeting of the ACs
to various microdomains of the cell and their close association with, or integration into, macromolecular complexes containing other essential signaling proteins and
their modulators. These proteins include cAMP effectors
such as protein kinase A (PKA) and specific cAMP phosphodiesterases (PDEs), which are themselves subject to
refined targeting and modulation. A further level of sophistication comes from the fact that many of the ACs are
regulated by intracellular Ca2⫹ changes, which can be highly
complex in space and time. A major consequence of such
elaborate microdomain organization, and the complexity
of the signals that influence enzyme activity, is the likelihood that cAMP signals are not simple, and this may be an
essential element of their regulatory influence.
To appreciate the questions and opportunities provided by current insights into cAMP signaling, this review
first focuses on recent advances in our understanding of
the structure and dynamic regulation of the ACs, with
particular emphasis on the molecular and cellular specializations developed for the regulation of the Ca2⫹-sensitive
Physiol Rev • VOL
ACs. We describe recently published data on the regulation of these ACs by Ca2⫹ in vitro and begin to assemble
a picture to detail how their regulation in vivo depends
upon the “context” in which the ACs are found. We will
expand on what is known of the compartmentalization of
ACs and cAMP signals to specific regions of the cell, and
their association with numerous regulatory and scaffold
proteins. The use of improved real-time biosensors to
monitor cAMP changes in single live cells is described in
some detail. We discuss how such methodological advances are beginning to open the door not only on the
consequences, but also the mechanisms underlying such
compartments of dynamic cAMP signals. We conclude
this review by looking at some examples of discrete regulation and compartmentalization of the ACs in specific
physiological situations and attempt to anticipate future
directions in unraveling the diversity and complexity of
cAMP signaling. The scope of this article touches on a
large number of areas related to cAMP signaling, some of
which have already been covered in recent reviews elsewhere. The reader will be directed to these sources when
extra background seems appropriate.
II. ADENYLYL CYCLASES
A. General Structure of ACs
Following the purification, partial sequencing, and
subsequent cloning of the first AC in 1989, a structure was
revealed that comprised 12 transmembrane-spanning
(TM) domains, two homologous apparent ATP-binding
regions and extensive NH2 and COOH termini (215). The
remarkable complexity of the structure for AC1 went
some way towards explaining previous difficulties associated with stabilizing and purifying the enzyme. Over
subsequent years, eight more species of AC were cloned
(21, 60, 144, 150, 205, 216, 309, 397, 422), all of which
displayed this broad structure first described for AC1
(Fig. 1).
This precise topology has not been proven for any
AC, but partial supporting evidence is available from a
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I. INTRODUCTION
ADENYLYL CYCLASES
number of approaches. For instance, fluorescently tagged
NH2 and COOH termini of AC8 are intracellular (174);
phosphorylation sites on AC6 are found intracellularly at
the NH2 terminus or within the cytosolic C1 and C2 domains (68, 218, 227); N-glycosylation sites (Asn-805 and
Asn-890) are located on the fifth and sixth extracellular
loops of AC6 (416). These glycosylation sites have been
substantiated in detail in the case of AC6, by mutagenesis
and deglycosylation experiments, to reveal a specific role
for N-glycosylation in the regulation and catalytic activity
of the AC independently of affecting its localization to the
plasma membrane (416). In particular, Chern and colleagues (416) found that mutation of the glycosylation
sites compromised catalytic activity of the enzyme in
response to specific activators (e.g., forskolin) and limited
its regulation by Gi␣ or protein kinase C (PKC). In the
case of AC8, transmembrane clusters fluorescently tagged
at the NH2 or COOH termini are sufficient to traffic the AC
to the plasma membrane independently from the cytoplasmic C1 and C2 domains (174). However, the C1 and
C2 domains are critical for the more specific targeting of
AC8 to lipid rafts (99). The significance of specific Nglycosylation sites, in terms of their intracellular processing of AC, on the activity and selective targeting of ACs to
specific cellular domains remains a topic worthy of further exploration.
B. Are the ACs cAMP Exporters?
In the early days of cAMP research, the issue of
exporting the cyclic nucleotide from the cell was considered by Davoren and Sutherland (108) as a possible
means of controlling intracellular [cAMP]. A sizable body
of studies provided evidence to confirm that cAMP extrusion did occur (122, 407) and that it was a unidirectional,
Physiol Rev • VOL
ATP-dependent process with activity that correlated with
intracellular [cAMP] (51). It was concluded that the energy-dependent cAMP extrusion, which was inhibited by
probenecid [subsequently identified as a nonspecific anion transport inhibitor (112)], was independent of the
activities of either ACs or PDEs (51, 122, 407). Indeed, in
many cell types, extrusion of cAMP is only detectable
after prolonged and high stimulation of AC activity. However, in the kidney cAMP extrusion is quite significant, to
the extent that urinary cAMP levels largely reflect the
activity of renal ACs. This situation is particularly notable
as a measure of hyperparathyroidism in which parathyroid hormone exerts its effects on renal tubule Ca2⫹
reabsorption by stimulating AC activity (15, 103).
The potential role of the ACs in cAMP extrusion was
revisited when the structure of the first AC was revealed.
Krupinski and colleagues (215) noted its remarkable similarity to classic ATP-driven transporters and speculated
that the 12 TM domains coupled with the two putative
ATP binding sites (see Fig. 1) might reflect an ability of
the AC to extrude cAMP. These studies were driven further by the knowledge that exported cAMP acted at extracellular receptors to mediate critical developmental
functions in the slime mould Dictyostelium discoidium
(164, 326, 327). However, deletion of a 12-TM domain AC
(ACA) from Dictyostelium, with the retention of a simple
AC enzyme (ACG) possessing only one TM span, did not
perturb cAMP secretion, which again strongly suggested
that the ACs themselves were not involved in exporting
cAMP (303). The possibility that ACs were involved in
cAMP extrusion was further dissipated by the lack of
selective inhibitor of cAMP export, coupled with the identification of a plethora of alternate transporters that were
capable of extruding cAMP (for example, the multidrug
resistance proteins, as recently reviewed by Refs. 71, 112,
178).
C. Voltage Sensitivity
The ACs could be considered superficially to resemble ion channels due to the complexity of their membrane-spanning organization. Although the mammalian
ACs do not possess “signature” S4 voltage sensors (116)
or a canonical potassium pore loop often associated with
ion channels, there have been some grounds to support
the possibility that the ACs could function as ion channels
from studies on the unicellular organism Paramecium.
Synthesis of cAMP increased upon membrane depolarization of Paramecium; in addition, a purified AC protein
from Paramecium placed in artificial lipid bilayers conducted K⫹ (342). Subsequently, an AC from Paramecium
was cloned that displayed a clear voltage-sensor pore
loop in tandem with a cytosolic AC homology domain
(399). This structure was also observed in a number of
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FIG. 1. Adenylyl cyclase (AC) structure. The ACs can be divided
into 5 major domains: the NH2 terminus, the first transmembrane cluster
(TM1, blue cylinders), the first cytoplasmic loop comprised of C1a (red)
and C1b (black), the second transmembrane cluster (TM2, blue cylinders) with extracellular N-glycosylation sites, and the second cytoplasmic loop comprising C2a (orange) and C2b (black). C1a and C2a are
highly conserved catalytic ATP-binding regions, which dimerize to form
the catalytic site. C1b and C2b domains are less conserved. Comparison
of this general AC structure with that of multidrug resistance transporters shows a remarkably similar topology in terms of the transmembrane
architecture and cytosolic organization (see Ref. 112).
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DEBBIE WILLOUGHBY AND DERMOT M. F. COOPER
D. Dimerization
Evidence has been reviewed elsewhere that ACs may
exist in higher-order states than simple monomers (91). In
brief, early sedimentation and target size analysis studies
indicated that dimerization or tetramerization of ACs
could occur (276, 325). In the case of AC8, dimerization
occurs as a result of interactions between the TM sections
(Fig. 2).
Live cell fluorescence resonance energy transfer
(FRET) studies using separately CFP- and YFP-tagged
transmembrane cassettes of AC8 molecules revealed that
this AC dimerizes via interactions between the second TM
domains (171). Similar studies using FRET/BRET methods to observe dimerization of other AC isoforms are yet
to be reported. Using a more biochemical approach, Ding
et al. (117) showed that a truncation mutant of AC6,
comprised of the first six transmembrane-spanning domains and half of the C1 catalytic domain, coimmunoprecipitated with full-length AC6. Biotinylation assays and
assessment of enzyme distribution using sucrose density
gradients showed that this truncated AC6 mutant impaired the ability of wild-type AC6 to traffic to the plasma
membrane and significantly reduced AC activity, suggesting dimerization of the AC molecules.
1
In the presence of Ca2⫹, Ca2⫹ rather than Na⫹ is carried by the
L-type channel and a far greater stimulation is observed.
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FIG. 2. Dimerization of AC8 molecules via their transmembrane
domains. AC8 associates internally by interactions between transmembrane clusters TM1 and TM2, or externally via interactions between two
TM2 domains. [From Cooper and Crossthwaite (91).]
Further support for AC dimerization comes from kinetic modeling studies which suggest that dimerization is
required for the activation of purified ACs by Gs␣ (72).
Although a growing body of evidence suggests that ACs
can form dimers and that these dimers may be of regulatory significance, we cannot exclude the possibility that
even higher-order associations may occur. Such propensity for oligomerization would provide further similarity
between the ACs and the ABC transporters. Indeed, oligomerization is a level of organization seen in several of
the ABC transporters, and it is considered to play an
important role in their ER processing and membrane
insertion (52). The similarity between ACs and ABC transporters might even be fancifully expanded to include
heterooligomerization with other regulatory elements.
For instance, an analogy with the sulfonylurea receptors
(SUR1 and SUR2A/B) from the ABC transporter family
can be considered. The SUR proteins, like the ACs, possess two ATP binding motifs (e.g., Refs. 52, 61, 255). In
this case, a heteromultimeric protein assembly comprised of four regulatory SUR subunits and four inwardly rectifying ATP-activated K⫹ channel (KATP) subunits has been identified (13, 256). The structure suggests that SUR1 directly interacts with Kir6.2 (431).
Both the SUR and KATP subunits possess ER retention
motifs, which are complementarily obscured to ensure
their coexpression at the plasma membrane. Adjacent
SUR1 proteins appear to interact and form a large
docking platform for cytosolic proteins, which can add
to the complexity and function of this complex. We are
inclined to speculate that the Ca2⫹-regulated ACs might
form similar, large heteromultimeric complexes to pro-
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other lower organisms, such as Plasmodium and Tetrahymena (399).
The precedent set by the Paramecium study may
have contributed to the enthusiasm for an apparent voltage-sensitive AC in rat cerebellar granule cells. A prolonged (30 min) depolarization of these cells, in the absence of extracellular Ca2⫹, led to an increase in cAMP
accumulation (316). A later study showed that this effect
was in fact due to Na⫹ entry through L-type Ca2⫹ channels, as a consequence of membrane depolarization;1 the
effect could be mimicked by Na⫹ entry through gramicidin-generated pores where the membrane potential had
been dissipated (96). There have since been a number of
cases where the depolarization of cells, in the absence of
extracellular Ca2⫹, can bring about unexpected effects
that are more typically associated with Ca2⫹ influx. For
example, in astrocytoma cells, modest increases in extracellular [K⫹] can stimulate inositol 1,4,5-trisphosphate
(IP3)-mediated Ca2⫹ release from the endoplasmic reticulum (ER), independently of Ca2⫹ influx (304). The effect
can be explained in part by depolarization-dependent activation of Gq-coupled purinergic receptors, but it is
largely independent of changes in membrane potential
(243, 244).
ADENYLYL CYCLASES
vide an intimate association with Ca2⫹ entry channel
elements.2
E. Catalytic Core
2
The relevance of the similarity of properties of ACs and transporters may become more apparent when the issue of their sensitivity to
2⫹
Ca entry is considered (see sect. V).
3
Even though only one ATP molecule is used per catalytic center
of mammalian ACs, bacterial and metazoan ACs, which also possess two
ATP binding domains, actually have two catalytic centers, which utilize
two ATP molecules (228).
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Two metal binding sites are identified: one, “site A,”
is a tetrahedral coordination sphere, which comprises an
oxygen from the ␣-phosphate of ATP, a carboxylate oxygen from each of the aspartates, and a water molecule; the
second metal binding site, “B,” is octahedrally coordinated by an oxygen from each of the aspartates, an oxygen from each of the ␣, ␤, and ␥, unesterified oxygens of
ATP, and a backbone carbonyl group of a nearby isoleucine (I397). In the crystallization study, by way of confirming the liganding atoms proposed above, Zn2⫹ bound
preferentially to site A, while Mn2⫹ bound to site B (377;
see Fig. 3). Although Mg2⫹ is expected to be the physiologically relevant cation in these sites, Mn2⫹and Zn2⫹ are
used in crystallization studies to provide a more stable
representation of these states. Both Mn2⫹and Zn2⫹ have
similar atomic radii (and identical charge) with differences in their binding properties presumably reflecting
their ability to accommodate four and six (or eight) ligands, respectively.
F. AC Distribution
The initial cloning of AC1 and AC2 led to a flurry of
AC cloning in the early 1990s, which resulted in the identification of nine mammalian species (21, 60, 144, 150, 192,
205, 215, 216, 309, 397, 422). They all showed the same
gross topological design with highly conserved catalytic
domains, displaying up to 65% amino acid sequence homology. However, significant differences in sequence
were observed between the noncatalytic cytosolic domains, which later turned out to accommodate different
regulatory features (Fig. 4). Cloning the same AC in different animal species also revealed differences in some
unconserved regions. For example, the NH2 terminus of
the canine AC5 is 19 amino acids longer than that of the
comparable murine or rabbit species.
Northern blotting and PCR analysis has allowed preliminary descriptions of the tissue distribution patterns of
these AC species. Unfortunately, the ACs are not expressed with sufficient abundance, nor are there adequately sensitive or specific antibodies to allow finer descriptions to be made of protein level abundance. However,
the following distributions can be presented (Table 1),
which has been verified in some cases by functional assays, based on the individual regulatory properties of the
AC species (reviewed in detail in Refs. 180, 262, 393).
A recent study by Visel et al. (388) using nonradioactive in situ hybridization techniques is worthy of specific
mention. This study carefully describes the expression
patterns of all nine membrane-associated ACs in mouse
brain at different stages of development (embryo, neonate, and adult). Interestingly, their findings suggest regional coexpression of ACs with different regulatory
properties but more exclusive expression of genes for
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A remarkable and puzzling feature of the ACs noted
upon their first structural characterization was their possession of two ATP binding domains.3 These domains are
highly homologous and complementary both within single
ACs and among different mammalian AC isoforms. When
the domains were expressed separately in Escherichia
coli and purified, they had to dimerize for full AC activity;
upon their recombination, AC activity was revealed,
which could be regulated by Gs␣ and forskolin (376).
Functional chimeric molecules can be formed between
separate AC1 C1a and AC2 C2a domains, as well as between AC5 C1a and AC2 C2a (368, 376). Even an AC2 C2a
dimer has been generated that displays some catalytic
activity (376, 432). A peptide-linked, but soluble, AC1 C1a
and AC2 C2a have also been created with full activity,
when purified from E. coli (115, 368). Indeed, even in
mammalian cells, two half AC molecules that comprise
the respective first or second TM cassettes with their
associated cytosolic domains, which lack activity when
expressed on their own, can be coexpressed to display
full activity (174). Such observations suggest great avidity
for the halves of AC molecules, both in their TM and
cytosolic domains. Seebacher et al. (345) had shown that
for retention of functional activity, TM domains could not
be swapped between ACs, even if the cognate catalytic
domains were retained in chimeric constructs.
The independence of the cytosolic domains from the
TM domains, at least in the neutral in vitro environment,
allowed the crystal structure of the catalytic domain to be
determined. The first structure was that of a C2a dimer
(432), which was shortly followed by the structure of an
AC2 C1a/AC5 C2a dimer (376). Although no linear sequence homology is observed, the overall structure is
remarkably similar to the catalytic site of DNA polymerases. Thus, like DNA polymerases, ACs possess a
␤␣␤␤␣␤-domain, which also possesses two aspartate residues at characteristic locations that coordinate two
Mg2⫹. These mediate the attack of the 3⬘-OH on the
␣-phosphate of ATP, promoting the cyclization of cAMP
and release of PPi. Both C1a and C2a domains possess
this palm motif, but only the C1a domain possesses the
catalytic aspartates (376, 377, 432).
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ACs with similar regulatory features. For example, expression of AC1 and AC8 mRNA in the hippocampus
reveals strong expression of AC1 in the dentate gyrus and
CA2 regions whilst AC8 expression is restricted to the
CA1 region. Similarly, in several cortical layers of the
olfactory bulb and striatum, these two Ca2⫹-stimulatable
AC isoforms are expressed in a mutually exclusive manner. In contrast, G␤␥-stimulated ACs (such as AC2) and
Gi␣-inhibited ACs (such as AC5 and AC6) tend to be
regionally coexpressed (388). Although such studies provide important information on the localization of specific
AC genes in particular groups of cells, they by no means
confirm the expression of functional AC proteins, or the
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coexpression of different, complementary ACs within the
same cell.
G. Soluble AC
A soluble AC (sAC) was originally identified in
seminiferous tubules and sperm from various animals
(42). This enzyme preferentially utilizes MnATP, rather
than MgATP, as substrate. Unlike membrane-bound
ACs, it is insensitive to G protein regulation and displays a lower affinity for ATP (Km ⬃1 mM, compared
with a Km ⬃100 ␮M for the membrane-bound ACs). The
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FIG. 3. Crystal structure showing complexes of ATP analog inhibitors with the catalytic core of AC. A: the C1a:C2a catalytic core of
AC. Forskolin (FSK) and ATP, which bind between the C1a (tan) and C2a (mauve) domains,
are shown as stick models. The switch II helix
of Gs␣, which forms much of the interface with
AC, is shown in red. B: model of ATP bound to
specific amino acids in the active site. Amino
acids are labeled according to their position
in canine type V AC for C1a and rat type II
AC for C2a. C, left: the active site of the
AC 䡠 ␤LddATP 䡠 Mn complex. Superimposed is
electron density from a 2.8-Å resolution
FoMg ⫺ Fc omit map (blue wire cage). Structural elements donated by the C1a and C2a
domains of AC are shown in tan and mauve,
respectively. In the right panel(shown close
up), the green and purple wire cages represent
electron density contoured at 5␴ for 3.0 Å
FoZn ⫺ FoMg and 2.8 Å FoMn ⫺ FoMg omit
maps, respectively, demonstrating that Zn2⫹
preferentially binds at site A and Mn2⫹ at site B.
D: the active site of AC 䡠 ATP␣S-Rp 䡠 Mn (left) and
a close up view of its thiotriphosphate (right).
Superimposed is electron density from a 3.0 Å
Fo ⫺ Fc omit map contoured at 2.5␴. A 7␴
peak marks the position of metal B and is modeled as Mn2⫹. A 2␴ peak marks the position of
metal A and is modeled as Mg2⫹, although it
could also be a weakly bound Mn2⫹. [From
Tesmer et al. (377).]
ADENYLYL CYCLASES
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III. TYPE-SPECIFIC REGULATION
The discovery of several AC isoforms to generate a
family of AC enzymes was rapidly succeeded by evidence
to reveal diversity in their regulation by G proteins, plus a
multiplicity of regulatory options for individual AC molecules (373). Although the ACs can be loosely subclassified
in terms of their responsiveness or not to Ca2⫹ (see Table 1
and sects. IV and V), an outline is first presented here of the
regulatory options available to specific AC isoforms, beginning with the complex options utilized by G proteins.4
1. Gs␣
FIG. 4. AC isoform sequences are presented in cylindrical schematic form and grouped according to their regulatory properties. ACs
1, 3, and 8 are stimulated by Ca2⫹ acting via calmodulin (see later);
ACs 2, 4, and 7 are not susceptible to Ca2⫹ regulation; ACs 5 and 6 are
directly inhibited by Ca2⫹; AC9 is unique in its calcineurin-mediated
inhibition by Ca2⫹. The relative positions of distinct domains have
been determined by alignment of all known ACs and the positions of
sequences predicted to form membrane-spanning helices. Basic domains are indicated by color: NH2 terminus (Nt), green; TM1 and
TM2, gray; C1a, yellow; C1b, red; C2a, light blue; C2b, dark blue. C1a
and C1b comprise the first, and C2a and C2b form the second, large
cytosolic domain. It can be clearly appreciated that although the
catalytic domains are conserved in terms of size, significant differences occur between the less-conserved domains. [Modified from
Hanoune et al. (181).]
enzyme was purified and cloned from rat testis, and its
catalytic domain was found to resemble that of ACs
expressed in various microorganisms, such as cyanobacteria (53). Uniquely among ACs, the sAC enzyme
is activated by bicarbonate ions in vivo and in vitro in
a pH-independent manner (69) and may mediate the
effect of bicarbonate ions on sperm motility. Indeed, if
the sAC is genetically deleted from mice, their sperm
mobility is inhibited and the animals are infertile (132).
Although the initial isolation of sAC was from sperm,
sAC is also expressed in other bicarbonate-responsive
tissues such as the kidney, which suggests that bicarbonate regulation of cAMP signaling may play a more
widespread role in biological systems (69), and perhaps, the modest formation of cAMP of which this AC
is capable may be of significance within discrete cellular domains (438). One elegant demonstration of this
possibility was recently published by Zippin et al. (439)
who showed that the perinuclear pool of sAC may
provide a pathway for CREB transcription following an
increase in intracellular bicarbonate levels, such as
might arise during metabolic activity.
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All of the cloned ACs can be stimulated by Gs␣
(reviewed in Ref. 365). Although no gross differences in
sensitivity between the different species are observed
during in vitro reconstitution assays, some apparent differences have emerged when the ACs are expressed in
cell lines and then stimulated by endogenous Gs-coupled
hormone receptors (e.g., Ref. 422). However, it has been
argued that selective expression of specific G proteincoupled receptors (GPCRs) and ACs in particular subcellular domains, or limitations in the amounts of AC relative
to the G protein, could give rise to apparent differences in
sensitivity to Gs␣ stimulation that might not be borne out
in extremely well-controlled experimental situations
(365). This argument underscores a central point of this
review, which is that it is the overall cellular context
rather than the absolute affinities of proteins for each
other that determine their regulation. Later in the review,
such cellular context issues are explored in more detail,
but for the moment our discussion centers on insights
from in vitro experiments.
2. Gi␣
In vitro assays reveal clear and absolute differences
in how, and whether, different AC species are regulated
by Gi␣. AC1, AC3, AC5, AC6, AC8, and AC9 are all inhibited by Gi␣, whereas AC2, AC4, and AC7 are not (90, 180,
365; see Table 1). It is perhaps no coincidence that inhibitory receptors that couple to Gi␣ inhibition of AC are
absent from tissues where AC2, AC4, and AC7 are prominent, e.g., lung and liver (406).
4
The reader is directed to reviews in References 30, 90, 180, and
365 for further detail, much of which is summarized in Table 1. It is
important to recognize that certain of these type-specific observations
have only been made in one or two sources, or with overexpressed
proteins. Nevertheless, these enzymes are remarkably understudied, and
the discovery of an appropriate context could render some of the
apparently “minor” regulatory properties of major significance.
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A. G Proteins
972
TABLE 1.
DEBBIE WILLOUGHBY AND DERMOT M. F. COOPER
Distribution and regulation of membrane-bound adenylyl cyclases
Response of AC Isoform to Other Signaling Molecules
AC Isoform
Major Source of Expression
Gi␣
G␤␥
Protein kinases
2
1
1
2
CaMK IV
RTK
PKC
CaMK II
1
1
2
1
2
2
1
1
3
2
PKC
PKC (␨⬎␣)
PKA
RTK
or 1 PKC
PKA
RTK
PKC (␦)
PKC
PKC
AC1
Brain
1
2
2
AC2
AC3
Lung, brain
Olfactory epithelium, pancreas
1
1
3
2
1
3
AC4
AC5
Widespread
Heart, striatum
1
1
3
2
1
3
AC6
Heart, kidney, widespread
1
2
3
AC7
AC8
AC9
Widespread
Brain, pancreas
Testis, widespread
1
1
1
3
2
2
3
2
3
RGS
Calcium
1 (Via CaM)
2
3
1 (?) (CaM in vitro)
2 (CaMK II)
3
2
2
2
2
3
1 (Via CaM)
2 (Via calcineurin)
Listed are the major sources of expression of each of the nine membrane-associated adenylyl cyclase (AC) isoforms (reviewed in detail in Refs.
180, 262, 393) and their regulatory properties. Stimulation, inhibition, or no effect of signaling molecule is represented by 1, 2, or 3, respectively.
The mode of Ca2⫹ regulation is given in parentheses unless Ca2⫹ effect is direct.
3. G␤␥
G␤␥ exerts type-specific effects on AC species. AC1
and AC8 are inhibited (361, 369, 370), whereas AC2, AC4,
and AC7 are stimulated (365, 369). AC3, AC5, AC6, and
AC9 are insensitive to direct regulation by G␤␥ (309, 369).
These effects are seen in vitro and in transfected cells
upon stimulation of appropriate receptors. Thus, in
HEK293 (human embryonic kidney) cells expressing AC7,
dopamine via a coexpressed D2 receptor stimulates
cAMP accumulation as a result of liberating ␤␥-subunits
from endogenous Gi proteins; however, when AC5 is expressed in these experiments, cAMP accumulation is inhibited by dopamine as a consequence of liberating Gi␣
(423).
4. Selectivity in G protein subunit requirements
Given the plethora of G protein subunits, i.e., 15␣, 5␤,
and 13␥, coupled with nine membrane-bound ACs, there
is potential for an extremely large variety of combinations
of G protein subunit and AC interactions, so that the issue
of selectivity among potential partners arises (406). This
subject again raises the conflict between in vitro and in
vivo assessments of affinity versus compatibility. Typical
of a rigorous in vitro approach was a study by Taussig
et al. (374) in which a variety of purified G protein subunits were reconstituted with recombinant AC1, AC2,
AC5, or AC6 that had been purified from Sf9 cell membranes expressing high levels of individual ACs. Concentration response curves were obtained and compared, and
based on the findings, the following selectivities were
observed: all four ACs were activated by Gs␣; AC2 was
not inhibited by Gi␣ or Go␣ but was stimulated by G␤␥;
AC1 was inhibited by G␤␥, Gi␣, and Go␣ with selectivity
Physiol Rev • VOL
of G␤␥ ⬎ Gi␣ ⬎ Go␣; whilst AC5 and AC6 were unaffected
by Go␣ but potently inhibited by Gi␣. Interestingly, there
were no differences in the abilities of Gi-1␣, Gi-2␣, and
Gi-3␣ to inhibit AC5, AC6, and AC1. The value of such
studies is the fact that affinities are obtained in a reasonably well-defined milieu; however, it is not known
whether such precise mixtures of components ever encounter each other in live cells. Deriving from that comment, an insightful, though still limited, approach would
be to determine the ACs and G protein subunits that are
expressed in a particular single cell. Although heroic, the
exercise might be beneficial in at least a few situations to
yield a sense of whether in vitro sensitivities were ever
exploited, or whether other factors in an in vivo context
changed the desirability of particular combinations. Such
a study was performed for olfactory neurons and led to
the discovery that the G protein Golf must stimulate AC3
(199). These two proteins colocalize in the distal segments of olfactory cilia where they play an essential role
in the early stages of olfactory transduction (252). Thus
certainly in that context, whatever other options might
have been preferable from a currently informed biochemical viewpoint, that mixture was selected during evolution. One early approach to assess the selectivity of ACs
for specific G proteins in an in situ context was by the use
of microinjected antisense oligonucleotides against individual G protein subunits in single-cell experiments,
where modulation by a GPCR linked to AC modified the
activity of L-type Ca2⫹ channels (238). Another approach
has been the use of tissue-targeted transgenic mice (195).
This study showed that Gi-2␣ mediated the effect of carbachol on isoprenaline stimulation of cardiac L-type Ca2⫹
currents. However, a number of variable factors, e.g.,
unexpected developmental abnormalities, the pluripo-
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Gs␣
ADENYLYL CYCLASES
B. PKA
PKA-mediated phosphorylation inhibits the activity
of AC5 and AC6 (30, 180). This effect was proposed to be
part of the hormone-induced desensitization of AC activity (308) and was confirmed by the Ishikawa lab, which
showed that PKA directly phosphorylates AC5, resulting
in reduced enzyme activity (194). It was later shown that
Physiol Rev • VOL
phosphorylation of AC6 by PKA, in vitro, mediated a
similar decrease in activity of the AC in response to Gs␣
or forskolin (68). Mutation of the serine at position 674 to
an alanine in AC6 prevented the phosphorylation-dependent inhibition both in vitro and in vivo (28, 68). This
amino acid sits within the C1b catalytic site of AC6 in a
region involved in Gs␣ stimulation. Other PKA phosphorylation sites are yet to be identified, although AC6
contains at least 14 putative sites for PKA interaction
(68, 194). In AC5, the targeting of serine at position 676
by PKA to reduce AC activity has been demonstrated in
a recent study revealing that PKA-dependent phosphorylation of the ACs is optimized by the localization of
PKA in a signaling complex incorporating both AKAP79
(an A-kinase anchoring protein) and AC5/6 (25; see
below).
C. PKC
PKC activation stimulates the Gi␣- and Ca2⫹-insensitive ACs (AC2, AC4, and AC7) (90, 338, 366) and inhibits
AC9 (101). Initial reports were established by activation
of PKC using phorbol esters. Thus all ACs are potentially
subject to regulation as a consequence of activation of the
phospholipase C (PLC) pathway, either via IP3-mediated
Ca2⫹ elevation (and subsequent regulation of Ca2⫹-sensitive ACs, see below) or via production of diacylglycerol
(and subsequent PKC activation of Ca2⫹-insensitive ACs).
However, AC5 and AC6 are potentially regulated by both
Ca2⫹ and PKC (30, 90, 180). Although several studies
provide support for PKC-regulated AC activity in vitro, the
significance of this effect in the intact cell is not well
established. The importance of similar regulation in vivo
is, however, suggested by the fact that individual ACs are
selective for specific PKC isoforms. For instance, work by
Kawabe and colleagues (208, 209) revealed preferential
stimulation of AC5 by PKC-␨ over PKC-␣. In contrast, AC6
exhibited reduced activity following activation of numerous PKC isoforms (␤, ␦, and ␨) in osteoblastic cells (76).
Along similar lines, in human erythroleukemia cells, ethanol was found to potentiate AC7 activity through a PKC␦-mediated phosphorylation (277, 366).
A study by Levin and Reed (223) has identified a
small unconserved region near the COOH terminus as the
site of action of PKC-mediated upregulation of AC2. In
contrast, Chern’s group (218) demonstrated that NH2terminal deletion of AC6 (amino acids 1– 86) or mutation
of S10A reduced the PKC-mediated inhibition and phosphorylation of the AC when expressed in Sf-21 insect
cells. Further studies showed that PKC-mediated inhibition of AC6 was ablated by mutating two of the three
potential phosphorylation sites in the Nt and C1 regions
(S10, S568, and S674) or a single mutation in the C2 region
(T931) (227). More recent studies using HEK293 cells
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tency of G proteins, and their involvement in processes
other than AC regulation, as well as compensatory
changes in other tissues render this approach difficult to
apply lightly. A more recent, direct approach to identify
protein interacting partners is the use of interference RNA
(RNAi) to knock-down specific proteins of interest. The
rationale is that if a particular response can be abrogated
by the selective elimination of a particular protein, and
ideally restored by expression of an ortholog of the
knocked out protein, then that target protein is most
likely involved in the original context.
Very recently, an extensive assessment of G protein
interaction has been made using RNAi techniques. The
authors, wishing to answer the apparently simple question of which G protein subunits were associated with
particular AC regulation, performed selective RNAi targeted knockdowns of various G protein ␣-, ␤-, and ␥-subunits. However, instead of merely checking that their
intended targets were knocked down, they also examined
the consequences of such knockdown on the expression
of other G protein subunits and ACs, in addition to the
effect on AC responsiveness (214). What they found was
a dramatic interdependence and a far-ranging series of
consequences for their knockdowns, not only in terms of
compensatory or apparently unrelated changes in other G
protein subunits, but also changes in cognate receptors
and effectors. A striking example from their study illustrates the problem; simultaneous knockdown of G␤1 and
G␤2 in HeLa cells resulted in a nontranscriptionally mediated increase in G␤4, which was subsequently able to
maintain Gs␣ activation of two AC isoforms whose expression levels had also unexpectedly increased as a consequence of the knockdown. Thus, although severe impairment was anticipated, an enhanced response to agonist was actually encountered. This perturbation also
resulted in a substantial decrease in Gi␣ protein (despite
a large increase in mRNA for Gi-1␣), with unknown effects
on G␥ subunits. Given the multiple roles of G proteins in
numerous signaling pathways, cautionary measures are
required to avoid naive application and interpretation of
such approaches (214). Thus awareness and addressing of
potential pitfalls by independent experimental strategies
may be required to establish with any confidence the
composition of specific signaling complexes, including
those involving the ACs.
973
974
DEBBIE WILLOUGHBY AND DERMOT M. F. COOPER
stably expressing AC6 demonstrate a potentiation of epidermal growth factor (EGF)-evoked AC activity when
cells are treated with phorbol esters, which suggests an
even more complex relationship, wherein a PKC-dependent upregulation of AC6 in the intact cell can be mediated via the direct interaction of AC6 with a protein
downstream of PKC, Raf1 (28).
D. Calmodulin Kinase
E. Receptor Tyrosine Kinase
A number of reports of modulation of ACs by receptor tyrosine kinases (RTK) are mentioned below.
However, it should be noted that in the life of a cell, the
responsiveness to an RTK may occur in a very narrow
time frame and/or a very discrete cellular domain, e.g.,
at a membrane ruffle, somewhere where movement or
cell cycle stage-dependent changes are occurring, and a
very local change in cAMP levels is of significance.
Consequently, it should not be surprising if the effects
that have been observed in the literature are rather
modest, which may merely reflect the lack of temporal
or contextual appropriateness for the proposed measurements, rather than the overall significance of the
effect. In many of the quoted examples that follow, ACs
have been expressed heterologously and are definitely
out of context.
One elegant example of a contextual effect of a
growth factor on AC is provided by the case of ephrin and
AC1. The expression of ephrin-A5 (a membrane-bound
guidance molecule) and its receptor (EphA) play a critical
Physiol Rev • VOL
F. Calcineurin
The role of calcineurin in AC9 function has been the
subject of some controversy, with suggestions of either
inhibition of AC9 activity (11, 295, 296) or no effect of the
Ca2⫹-dependent protein phosphatase on overexpressed
AC9 activity (309). Work by Antoni and colleagues (10, 11)
revealed inhibition of AC9 by the same range of intracellular Ca2⫹ changes that stimulated AC1. In the case of
AC9, however, the Ca2⫹-dependent inhibition of cAMP
production was clearly attenuated by calcineurin blockers. High AC9 mRNA levels in the brain and potential
regulation of this AC by Ca2⫹/calcineurin is proposed to
account for some of the diverse actions of the protein
phosphatase in brain function (11, 66).
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AC3 seems unique in its sensitivity to calmodulin
(CaM) kinase. Early work provided evidence that AC3
was inhibited by CaM kinase II in vivo (398),which yielded
an additional link between Ca2⫹ and the ACs, alongside
the well-described direct Ca2⫹- or Ca2⫹/CaM-mediated
regulation of several AC isoforms (see sects. IV and V). In
HEK293 cells stably expressing AC3, increases in intracellular Ca2⫹ concentration ([Ca2⫹]i) induced by ionophore or carbachol inhibited glucagon- and isoprenalinemediated AC activity. This effect was antagonized by an
inhibitor of CaM kinase, while coexpression of constitutively activated CaM kinase II completely inhibited Gsmediated AC3 activity (398). Mutation of a CaM kinase II
consensus site (S1076 to A1076) in AC3 suggested direct
phosphorylation of the AC (401). Later studies suggest
that CaM kinase II inhibition of AC3 may contribute to the
termination or adaptation of olfactory signaling (222,
402). This inhibition of AC3 by CaM kinase II is also
thought to underlie the long-lasting inhibition of AC activity observed following Ca2⫹ release from ER stores in
the A7r5 smooth muscle cell line (128).
role in defining the protrusion of axonal fibers in the
developing retina (63, 248). AC1 is necessary to enact a
retraction response of the retinal axons to ephrin-A5 during the refinement of the retinotopic map (278). This
requirement for AC1 in mediating the actions of ephrin-A5
was demonstrated in retinal axons from AC1 knockout
mice. These axons maintained their profusion but had lost
their selectivity in branching towards their targets and
inhibitory response to ephrin (278).
EGF increases AC activity and elevates cAMP accumulation in cells expressing AC5, while not affecting overexpressed AC1, AC2, or AC6 (70). The EGF stimulation of
AC5 required Gs␣, a finding that is consistent with previous data concerning EGF effects on cardiac AC activity
(70, 271). In studies using recombinant AC5, such activation of the enzyme was found to be due to the phosphorylation of Gs␣, by EGF, at one or more tyrosine residues
(306). The stimulation of AC5 by EGF is required for
activation of a KCa1.1 channel in rat vascular smooth
muscle and the subsequent upregulation of genes that are
critical for cell proliferation (193). RTKs also modulate
the activity of AC6. Insulin-like growth factor I (IGF-I)
activation of RTK and tyrosine phosphatase inhibition
(using sodium orthovanadate) has been shown to phosphorylate several serine residues within AC6 and increase
enzyme activity. Mutation of either S603 and S608 or S744,
S746, S750, and S754 (which occur in the C1 region)
attenuated both the RTK-mediated enhancement of enzyme phosphorylation, as well as the sensitization of function (367). Studies showing that AC6 phosphorylation is
similarly increased by Raf1 (a component of the ERK
signaling pathway), and that AC6 and Raf1 coimmunoprecipitate, suggest that pathways downstream of the RTK
may potentiate AC signaling (28, 30, 118, 367). This latter
effect also accounts for the PKC-dependent upregulation
of AC6 (28).
ADENYLYL CYCLASES
G. RGS Proteins
975
1. AC1
IV. REGULATION BY CALCIUM IN VITRO
A. Stimulation by Ca2ⴙ
AC1 and AC8 are clearly stimulated by Ca2⫹ in a
CaM-dependent manner. Almost coincident with the discovery of CaM as an activator of PDE (77, 201) came the
finding that Ca2⫹/CaM could stimulate AC from brain
membranes (48). Purification studies later showed that a
Ca2⫹-stimulable form of AC could be separated from a
Ca2⫹-insensitive form (404). Cloning and expression of
AC1 from brain (215) led to the demonstration that this
species was potently stimulated by Ca2⫹/CaM in membranes (370). AC8 was discovered a few years later, as
part of a PCR screening for the diversity of ACs that
existed in mammalian cDNA libraries (216). Because of
the abundance of AC8 mRNA in brain, Ca2⫹ simulation
was also explored. The expressed protein was Ca2⫹/CaM
stimulable, although with a lower sensitivity to Ca2⫹ than
AC1 (60). Interestingly, the identification of a second
Ca2⫹/CaM-stimulated AC in brain had been anticipated,
since the hypothalamus displayed high levels of Ca2⫹stimulated AC activity but no AC1 mRNA (263). AC8
turned out to be expressed well in the hypothalamus (60).
Physiol Rev • VOL
AC1 is stimulated in a CaM-dependent manner by
Ca
with an apparent Kd of ⬃0.1 ␮M (136, 418). An
examination of the linear amino acid sequence of AC1
revealed a number of potential CaM binding sites of the
“classical” amphipathic helix design. Indeed, dansylated
CaM bound to synthetic peptides corresponding to these
sequences (390). A further study showed that mutation of
AC1 in one putative CaM binding site (F503A) eliminated
the Ca2⫹ stimulation of the enzyme in vitro (418). This
binding site is located close to the catalytic domain of
AC1, within the C1b domain; however, information is not
yet available on how the activation occurs.
2⫹
2. AC8
AC8 is stimulated by Ca2⫹/CaM, with an apparent Kd
of ⬃0.5 ␮M (60, 136). Examination of the amino acid
sequence of AC8 suggested two types of candidate CaM
binding sites: an amphipathic helix at the NH2 terminus
and an apparent IQ motif at the COOH terminus. Deletion,
mutagenesis, and pull-down studies revealed that both
potential sites were utilized (173, 354).
An intriguing mechanism for Ca2⫹/CaM regulation of
AC8 has now been suggested, as depicted in Figure 5. A
common mode of enzyme activation by CaM is the relief
of autoinhibition (185); that is, under basal conditions, the
catalytic core of an enzyme is sterically hindered by a
regulatory domain within the enzyme, which contains a
pseudo-substrate sequence and a CaM binding domain
(CaMBD). CaM binding induces a conformational change
within the enzyme that subsequently removes the inhibition, exposing the catalytic site to the substrate. In many
cases, such as myosin light-chain kinase, CaM kinase II,
and the plasma membrane Ca2⫹-ATPase (PMCA), CaM
acts through a single CaMBD in the target enzyme, and
these enzymes are not associated with CaM when inactive (4, 62, 80, 179, 250). In contrast, AC8 presents a
variation on this theme, as it is apparently autoinhibited
at resting Ca2⫹, an effect that can be relieved upon CaM
interaction with the COOH terminus of AC8. However,
the CaM utilized is not derived directly from the cytosol. The CaMBD at the NH2 terminus of AC8 appears
to recruit CaM before activation. The preassociated
CaM can then relieve autoinhibition, following a stimulating elevation in [Ca2⫹]i and subsequent binding of a
fully liganded CaM to the COOH-terminal CaMBD. Although several proteins, particularly ion channels,
share this ability to preassociate with CaM, preassociation has not previously been linked to relief of autoinhibition by Ca2⫹. AC8, therefore, represents a novel
mode of activation, combining two established CaM
interactions via coordination of two CaMBDs (351).
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An example of the likely major gaps that still remain
in our knowledge of AC signaling complexes are provided
by the intracellular regulator of G protein signaling (RGS)
proteins. RGS proteins were first identified as the GTPaseactivating proteins (GAPs) for the G protein ␣-subunits,
Gi␣ and Gq␣, but not for Gs␣. This class of proteins has
since been found to regulate numerous GPCRs and their
effector molecules, including the ACs (reviewed in Ref.
2). In olfactory epithelial membranes, odorant-stimulated
cAMP production, which occurs via the Gs␣ family member Golf␣ was reduced by the addition of recombinant
RGS1, -2, and -3, with RGS2 having the largest inhibition
(RGS4 and -5 had no effect). Similarly, Sf9 cells expressing AC3 displayed reduced cAMP production when stimulated with either forskolin or the nonhydrolyzable GTP
analog guanosine 5⬘-O-(3-thiotriphosphate) (GTP␥S). Unexpectedly, RGS2 reduced odorant-elicited cAMP production, not by acting on Golf␣ but by directly inhibiting the
activity of AC3, the predominant AC isoform in olfactory
neurons (352). RGS2 also inhibited AC5 and AC6 when
expressed in HEK293 cells, apparently via a direct interaction with the C1 domain (330, 333). Recent imaging
studies in live cells reinforce the binding of RGS2 to a
receptor/G protein/effector signaling complex to regulate
AC activity (329). The full significance of RGS proteins in
AC signaling remains to be determined.
976
DEBBIE WILLOUGHBY AND DERMOT M. F. COOPER
parison between AC1, AC3, and AC8 expressed in
HEK293 cells, only AC3 was not stimulated by physiologically relevant [Ca2⫹] (136). No follow-up study since that
time has suggested any explanation for the initial reported
stimulatory Ca2⫹ effect. Unlike AC1 and AC8, there is no
experimental evidence, or likely amino acid sequence,
indicating that AC3 binds CaM. In terms of sequence, AC3
is an outlier from all of the other AC isoforms (216, 365),
and it may be appropriate to place it in a separate category, where its robust inhibition by CaM kinase may be
the major effect of Ca2⫹ (see sect. IIID).
B. Inhibition by Ca2ⴙ
1. AC5 and AC6
2⫹
FIG. 5. Model of Ca /calmodulin (CaM) activation of AC8. The
domains of AC8 are indicated as follows: transmembrane domains (light
blue), NH2 terminus (green), C1a (red), C1b (orange), C2a (purple), and
C2b (blue). The lobes of CaM are either Ca2⫹ free (white arc) or Ca2⫹
loaded (orange circle). Top panel: under resting Ca2⫹ conditions, CaM is
partially liganded, loaded at the C lobe, which binds to the NH2-terminal
amphipathic helix CaM binding domain (CaMBD) of AC8. Middle panel:
transition state following Ca2⫹ entry. Ca2⫹ binds to the N lobe of CaM,
which subsequently binds to the COOH-terminal IQ-like CaMBD. Bottom
panel: activated AC8. Both lobes of Ca2⫹/CaM bind to the COOHterminal CaMBD triggering relief of autoinhibition.
Although these ACs differ significantly from each
other in their NH2-terminal domains, they are strikingly
similar in their catalytic domains sharing 93% amino acid
identity (365). As mentioned above, although AC activity
from all tissues displays a low-affinity inhibition by Ca2⫹,
early studies revealed that tissues such as cardiac, renal,
anterior pituitary, and various cell lines also displayed a
high-affinity, submicromolar inhibition by Ca2⫹ (49, 58,
3. AC3
The situation with AC3 is less clear. AC3 is grouped
with the Ca2⫹/CaM-stimulated ACs because of one early
report stating that it could be stimulated by high (submillimolar) concentrations of Ca2⫹ in the presence of forskolin or GppNHP, a nonhydrolyzable GTP analog, in a
CaM-dependent manner (82). As AC3 had been cloned
from an olfactory cDNA library and was expressed in
olfactory neurons, and AC1 was found in central neurons,
the possibility was entertained that AC3 would be similarly stimulated by Ca2⫹/CaM. However, follow-up studies
suggested that AC3 was either inhibited directly by Ca2⫹
or inhibited via CaM kinase II (393). In one direct comPhysiol Rev • VOL
2⫹
FIG. 6. Response of the ACs to Ca . Representation of the range of
responses evoked by Ca2⫹ in tissues expressing Ca2⫹-stimulable, Ca2⫹insensitive, and Ca2⫹-inhibitable ACs. Comparisons are made in the
presence and absence of exogenous CaM. Note that regardless of the
response to Ca2⫹ at submicromolar concentrations, all classes of AC
exhibit inhibition at supramicromolar Ca2⫹ concentrations. [Modified
from Cooper (90).]
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All ACs are inhibited by supramicromolar concentrations of Ca2⫹(Fig. 6). This is most likely due to Ca2⫹
competing for what one presumes to be a low-affinity “site
A” metal binding site in the catalytic domain (see sect. IIE).
Although all ACs are inhibited by very high [Ca2⫹], only
two (AC5 and AC6) are inhibited by what are considered
more physiological Ca2⫹ changes.
ADENYLYL CYCLASES
5
The numbering of these cDNAs was a little confused initially;
AC6 was first described as AC5. Confusion as to which AC was in press
first resulted in the “AC5” reported in Reference 422 being renamed as
AC6.
Physiol Rev • VOL
ACs are highly conserved, sufficient differences exist between AC5 and AC2 (they are only 62% identical at the
amino acid level, Ref. 365) to allow Ca2⫹ to be implicated
in the catalytic cycle of AC5, but not accommodated by
AC2 at the same part of the reaction pathway. A crystal
structure utilizing C1a from AC2 would confirm this speculation.
V. REGULATION BY CALCIUM IN VIVO
Resting global cytosolic [Ca2⫹] is typically ⬃100 nM
and rises to 0.8 –1 ␮M, upon the triggering of Ca2⫹-elevating mechanisms. The sensitivity in vitro of the Ca2⫹stimulable AC1 and AC8 and Ca2⫹-inhibitable AC5 and
AC6 to submicromolar concentrations of Ca2⫹ has driven
interest in the regulation of these activities by Ca2⫹ in the
intact cell. One of the first detailed explorations of this
issue showed that in NCB-20 cells (the original source of
AC6), bradykinin-mediated [Ca2⫹]i increases were associated with an inhibition of cAMP accumulation, which
mirrored the temporal features of the [Ca2⫹]i rise (39).
The possibility that other mechanisms, such as Ca2⫹-stimulated PDE activity, G␤␥, and PKC effects, might account for
the effects of bradykinin was eliminated and pointed to a
direct effect of Ca2⫹ on the endogenous AC6 (154).
In subsequent studies, to eliminate the possibility of
(unknown) hormone receptor occupancy-dependent effects (such as G protein subunit effects, receptor associated signals such as kinases or arrestins, and unknown
contextual contributions) the cloned ACs have been heterologously expressed and their sensitivities to evoked
Ca2⫹ events assessed. Under these conditions, the expected effects of elevating [Ca2⫹]i on cAMP accumulation
were first shown with transfected AC1 and AC6 (92); this
was later extended to AC5 and AC8 (136, 264). The fact
that these Ca2⫹-sensitive ACs can all be regulated by
Ca2⫹, either directly or via CaM, renders this mode of
regulation particularly important as a device for harmonizing or integrating the activities of these two ubiquitous
signaling systems. Later studies showed that this regulation is more than a simple option, but actually reflective of
an intimate dependence on specific modes of [Ca2⫹]i rise.
A. Ca2ⴙ Homeostasis in Nonexcitable Cells
Since much of this discussion depends on awareness
of the context of physiological Ca2⫹ signaling, it is appropriate to briefly review Ca2⫹ homeostasis in nonexcitable
cells (as summarized in Fig. 8A).6 In brief, cytosolic free
calcium levels are remarkably well regulated, with resting
6
More detail can be found in reviews specific to this area (e.g.,
Refs. 129, 294).
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87). Due to specific interest in determining the nature of
such high-affinity inhibition by Ca2⫹, AC6 was first cloned
from NCB-20 cells, a cell line in which the inhibitory
process had been characterized in some detail (39, 422).
At the same time, the potential therapeutic interest of
cardiac AC triggered the cloning of AC5 from a cardiac
cDNA library (192).5 AC5 mRNA was expressed at high
levels in cardiac and striatal tissue. AC6 was also expressed in these tissues, although at lower mRNA levels,
but it was also more widely distributed in other cell types
including renal, endothelial, and blood cells (216). Expression of AC5 and AC6 in vitro revealed the expected
high-affinity inhibition by Ca2⫹ (176, 188, 422), displaying
a Kd for Ca2⫹ of ⬃0.2 ␮M.
Unlike AC1 and AC8, high-affinity inhibition by Ca2⫹
does not require readily dissociable CaM. In addition, no
readily identifiable motif such as an EF hand is displayed
anywhere within the sequence of these Ca2⫹-inhibitable
ACs. A kinetic characterization of high-affinity inhibition
indicated that it was a competition by Ca2⫹ for the activation of AC5 and AC6 by Mg2⫹, which was intrinsic to
the catalytic process (176). Thus inhibition was maximal
at low levels of activation, but antagonized by higher
levels of activation. A more detailed structural study used
mutants that showed a range of susceptibilities to activation by Mg2⫹; the efficacy of Ca2⫹ at causing inhibition
was a direct reflection of their susceptibility to be activated. Thus poorly activated mutant ACs were poorly
inhibited by Ca2⫹, even though Ca2⫹ still competed for
the activation. In addition, chimeras were constructed
between the Ca2⫹-inhibitable AC5 and the Ca2⫹-insensitive AC2. Fully active ACs were generated whose ability
to be inhibited by low [Ca2⫹] relied on them retaining the
C1a catalytic domain of AC5, rather than the analogous
domain of AC2 (188).
From the crystal structure of AC which is available,
and which fortuitously happens to utilize the catalytic C1a
domain of AC5, it is possible to speculate that the site of
high-affinity inhibition by Ca2⫹ is the “B” site described
earlier. Although no EF hand is shown in the catalytic
domain, the octahedral coordination capability of the “B”
site aspartates and the phosphoryl oxygen can readily
accommodate, and even select for, the eight liganding
form of Ca2⫹ (as it readily binds Mn2⫹) so it would not be
unexpected that a structure might be obtained containing
Ca2⫹ at that site (see Fig. 7).
In keeping with the selectivity for Ca2⫹ at that site, a
potency series of Ca2⫹ ⬎ Ba2⫹ ⬎ Sr2⫹ is displayed which
correlates with the cations’ atomic radii (1.06, 1.21, and
1.38 Å, respectively) (141). Although C1a domains of the
977
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DEBBIE WILLOUGHBY AND DERMOT M. F. COOPER
levels held at ⬃100 nM, creating a ⬃10,000-fold concentration gradient for Ca2⫹ across the plasma membrane.
This Ca2⫹ gradient, coupled with a negative resting membrane potential, generates a huge likelihood for Ca2⫹
entry when permeability of the cell membrane to Ca2⫹ is
increased, even in nonexcitable cells that lack voltagegated Ca2⫹ channels (VGCCs). Cytosolic Ca2⫹ rises are
typically initiated by the activation of PLC-linked GPCRs
or RTKs. This stimulates the production of IP3, which
then acts at specific receptors (IP3R) located on the ER to
promote release of Ca2⫹ into the cell cytosol. The ensuing
transient rise in [Ca2⫹]i is typically followed by a plateau
phase that depends on Ca2⫹ entry across the plasma
membrane. The initial transient Ca2⫹ rise is the consequence of a hormone-evoked propagating “wave” of Ca2⫹
seen globally that arises from the spatiotemporal recruitment of much smaller elementary Ca2⫹ release events
known as “blips” or “puffs” associated with the opening of
either one, or a group of, IP3R Ca2⫹ channels (32). The
Physiol Rev • VOL
sustained phase following the Ca2⫹ transient is referred to
as capacitative- or store operated-Ca2⫹ entry (CCE or
SOCE, respectively) as it is coupled to the depletion of the
ER Ca2⫹ stores, although the exact nature of this coupling
is still contentious.
Significant clarification of the molecular interactions
accompanying CCE has been provided by the recent identification of two proteins, STIM1 and Orai1 (298, 356).
Growing evidence suggests that STIM1 acts as a sensor of
Ca2⫹ store content, and Orai1 may be the ICRAC channel
itself, or at least an essential component of the channel
(reviewed in Ref. 355). The transient receptor potential
(TRPC) proteins (see sect. VIC) are also proposed as
essential Ca2⫹-entry components of CCE channels under
some physiological conditions. It is conceivable that the
TRPCs could even associate with Orai1 and STIM1 to
form Ca2⫹ entry channels (288).
Experimentally, CCE can be triggered using Gq-coupled receptor agonists such as carbachol to actively de-
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2⫹
FIG. 7. Structure of the catalytic domain of AC showing sites for high-affinity Ca
inhibition. Left panel: complex of AC5C1a and AC2C2a
showing forskolin binding site and active site of AC chimera. Forskolin, shown in red, binds in the cleft that contains the active site. The ATP analog
[2⬘,3⬘-dideoxy 5⬘-ATP (DAD), in yellow] and two Mg2⫹ (represented as orange spheres) are bound in the AC active site. Right panel: a ribbon
representation of the active site and location of the four active site mutations. The two Mg2⫹ are again displayed as orange spheres, whereas DAD
and the amino acids that are mutated are shown in stick representation. Each residue is colored by atom (carbon, yellow; nitrogen, blue; oxygen,
red; sulfur, pink). Each of the four mutations occurs proximate to the Mg2⫹ binding sites. Cys-441 and Tyr-442 are the two residues just after Asp-440,
which is of prime importance to Mg2⫹ binding and AC activity. The Arg-434 and Phe-423 are located more distally from the active site. Both residues
contact Tyr-442, suggesting that mutations of these amino acids might transmit their effects on Mg2⫹ activation through this residue. [From Hu et
al. (188).]
979
ADENYLYL CYCLASES
plete ER Ca2⫹ stores, or with sarco/endoplasmic reticulum Ca2⫹ ATPase (SERCA) inhibitors, such as thapsigargin (Tg), to passively deplete the stores.
Although CCE is the predominant mechanism regulating Ca2⫹ entry in nonexcitable cells, noncapacitative
pathways for Ca2⫹ entry also exist and include arachidonic acid-activated and diacylglycerol (DAG)- or 1-oleyl2-acetyl-sn-glycerol (OAG)-activated Ca2⫹ entry (242, 349,
375). In addition, mechanisms exist to terminate the signal of elevated [Ca2⫹]i; these include reuptake into ER
stores (via SERCA) and extrusion from the cell by a
PMCA (260, 442). Ca2⫹-buffering proteins, such as CaM
and calbindin, exert profound effects on the diffusion of
Ca2⫹ within cells limiting the amplitude and duration of
Ca2⫹ signals. Experimentally, nonspecific sustained increases in cytosolic Ca2⫹ may also be evoked by Ca2⫹
ionophores (such as ionomycin or A23187) that trigger
Physiol Rev • VOL
Ca2⫹ entry across the plasma membrane, but also release
of Ca2⫹ from intracellular sources, including the ER. As a
consequence, ionophores can trigger CCE along with
“nonspecific” Ca2⫹ entry. In the following sections we
discuss the ability of various modes of [Ca2⫹]i rise to
regulate the ACs in vivo.
B. Dependence of Ca2ⴙ-Sensitive ACs on CCE
Over Release in Nonexcitable Cells
Regulation of defined ACs by Ca2⫹ in the intact cell
was first demonstrated by evoking CCE, which was triggered following depletion of ER stores with Tg (94). The
ability of Ca2⫹-sensitive ACs to be regulated by CCE
prompted an investigation into whether other means of
elevating [Ca2⫹]i would be equally effective (81). The
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2⫹
FIG. 8. Mechanisms of Ca
regulation in nonexcitable cells and effects on AC activity. A: activation of a
Gq-coupled receptor generates the diffusible messenger
IP3 to stimulate Ca2⫹ release from the ER. Subsequent
emptying of this Ca2⫹ pool activates, via an unknown
mechanism, a store-operated Ca2⫹ channel (SOCC) triggering Ca2⫹ entry. Other Ca2⫹ entry pathways include
ionophore-, arachidonic acid-, and OAG-mediated Ca2⫹
entry. Basal cytosolic Ca2⫹ levels recover as a consequence of Ca2⫹ reuptake into the ER via SERCA, buffering,
or extrusion from the cell via the PMCA. B: of the four
Ca2⫹ entry pathways shown, only that occurring via
SOCCs (or CCE) can regulate AC activity in nonexcitable
cells. IP3-mediated Ca2⫹ release from the ER also has the
potential to act as a weak regulator of Ca2⫹-sensitive ACs.
AC, adenylyl cyclase; ARC, arachidonic acid-regulated
Ca2⫹ selective channel; DAG, 1,2-diacylglycerol; ER, endoplasmic reticulum; GPCR, G protein-coupled receptor; i,
inside the cell; IP3, inositol 1,4,5-trisphosphate; o, outside
the cell; OAG, 1-oleoyl-2-acetyl-sn-glycerol; PLC, phospholipase C; PMCA, plasma membrane Ca2⫹-ATPase; SERCA,
sarco/endoplasmic reticulum Ca2⫹-ATPase; SOCC, storeoperated Ca2⫹ channel.
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DEBBIE WILLOUGHBY AND DERMOT M. F. COOPER
C. Dependence of Ca2ⴙ-Sensitive ACs on CCE
Rather Than Other Forms of Ca2ⴙ Entry in
Nonexcitable Cells
1. Ionophore-mediated entry
The selectivity for CCE over Ca2⫹ release from the
ER, even when the magnitude of the release exceeded
that of the entry signal, prompted a comparison of AC
responsiveness to ionophore- versus CCE-mediated Ca2⫹
entry. Ionophores readily insert into all cellular membranes, triggering concentration-dependent shuttling of
Ca2⫹. Thus, in the absence of extracellular Ca2⫹, ionophore promotes intracellular store depletion and Ca2⫹
extrusion, priming the cells for CCE upon readdition of
extracellular Ca2⫹. This action is accompanied by non-
7
In this study, AC3 was also included; no effect of Ca2⫹ was
observed.
Physiol Rev • VOL
specific ionophore-mediated Ca2⫹ entry across the
plasma membrane. When the efficacy of ionophore-mediated entry was compared with that of CCE in promoting
inhibition of the endogenous AC6 of C6-glioma cells,
stores were first depleted with either Tg alone or Tg plus
ionomycin, and increasing amounts of extracellular
[Ca2⫹] were added to promote either CCE alone, or ionophore-mediated Ca2⫹ entry plus CCE, respectively (137).
At low extracellular [Ca2⫹] (e.g., 0.6 mM), very large
[Ca2⫹]i were achieved in ionophore-treated cells (⬃1,500
nM) due primarily to nonspecific Ca2⫹ entry and to a
lesser extent CCE. However, the degree of AC6 inhibition
seen under these conditions was no greater than produced by the relatively modest [Ca2⫹]i increase evoked by
CCE alone in Tg-treated cells (⬃400 nM) (137). Thus, of
the two components of the ionomycin-mediated Ca2⫹
entry, only the CCE element regulated AC6. Although not
conducted with similar awareness of the dual effects of
ionomycin, exposure of cells expressing AC1, AC6, and
AC8 to ionophore and high extracellular [Ca2⫹] yielded
the anticipated effects on cAMP accumulation, which
again probably reflects the underlying CCE triggered by
store depletion rather than the substantial and ineffective
direct ionophore-mediated trafficking of Ca2⫹ (59, 81,
396).
2. Arachidonic acid-mediated entry
In HEK293 cells, arachidonic acid activates a Ca2⫹
entry mechanism that is independent of CCE, although it
is of similar magnitude. Indeed, it may be an alternative to
CCE in some cells, since arachidonic acid-mediated entry
potently inhibits CCE and vice versa (235). Shuttleworth
and Thompson (349) compared the efficacy of arachidonic acid-mediated entry with CCE on transfected AC8
in HEK293 cells and found that AC8 was stimulated by
CCE as previously reported, but arachidonic acid-mediated entry was ineffective. These authors presented such
data as support for the independence of the two modes of
Ca2⫹ entry; however, the data also reinforce the discrimination of AC8 for CCE.
3. OAG-mediated entry
OAG, a synthetic analog of DAG (1,2-diacylglycerol),
triggers a Ca2⫹ entry in HEK293 cells that is distinct from
that mediated by arachidonic acid, since OAG does not
activate arachidonic acid-regulated Ca2⫹ (ARC) channels
(254). OAG activates a subset of the TRP (transient receptor potential) family of channels (TRPC3, TRPC6, and
TRPC7) which have received much attention as candidates for the elusive CCE channel. In HEK293 cells, OAG
and CCE evoked a Ca2⫹ rise of equivalent magnitude;
however, only the latter stimulated AC8 (242). The inability of OAG-activated Ca2⫹ entry to activate AC8 indicates
that this type of Ca2⫹ entry is mediated by channels that
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inhibition of the endogenous AC6 in C6 –2B glioma cells
was assessed by comparing the efficacy of Ca2⫹ when
elevated by release from stores or by CCE. Store release
was evoked by purinergic receptor stimulation, ionophore, or Tg in the absence of extracellular Ca2⫹. CCE
was triggered by active agonist, UTP, ER store depletion
or passive store depletion by Tg, and subsequent addition
of extracellular Ca2⫹ to promote store refilling. In all
cases, even though [Ca2⫹]i following release from intracellular stores (e.g., in response to ionomycin) was
greater than that evoked by CCE (in response to store
emptying), only CCE could regulate the endogenous AC6.
A recent hint of isoform-selective complexities in responses to Ca2⫹ release versus entry emerged in a study
that showed enhancement of AC6 activity by muscarinic
receptor-stimulated Ca2⫹ release in HEK293 cells (29).
Even though both AC5 and AC6 were inhibited by CCE
when stably expressed in HEK293 cells, Gq-coupled receptor potentiated AC6 (but not AC5) activity in a Ca2⫹dependent manner that was independent of PKC or Gi/o␣
modulation.
In a follow-up to the earlier findings by Chiono et al.
(81), heterologously expressed Ca2⫹-stimulated ACs
(AC1, AC8) were studied. Again, although ionomycinmediated Ca2⫹ release was substantial (⬃600 nM compared with just 250 nM evoked by Tg-mediated CCE), only
CCE could regulate AC activity.7 However, it was observed that release triggered by the muscarinic agonist
carbachol could elicit 15% of the maximal AC stimulation
that could be achieved by CCE (136). This is consistent
with more recent, single-cell experiments that demonstrate stimulation of transfected AC8 in a subpopulation
of HEK293 cells during carbachol-evoked Ca2⫹ release
(408).
ADENYLYL CYCLASES
are distinct from CCE channels. This finding is in keeping
with the recent discovery that Stim1 and Orai1 reconstitute CCE function (298, 356), even though the involvement of TRPC subunits is not precluded (430). Moreover,
the pathways mediating CCE and OAG-activated Ca2⫹
entry operate independently of one another, as they can
be triggered simultaneously in HEK293 cells with additive
effects (242). In this respect, the OAG-activated pathway
differs from the arachidonic acid-activated pathway (see
above). These observations further underline the dependence of AC8 on CCE.
D. Close Apposition of ACs With CCE Channels
The apposition of ACs and CCE channels was addressed by preloading cells with cell-permeable esters of
BAPTA and EGTA. These two Ca2⫹ chelators have almost
equal affinities for Ca2⫹, but BAPTA has a faster kon value;
thus equal BAPTA and EGTA concentrations produce
equivalent equilibrated Ca2⫹ solutions. However, at any
given point source of Ca2⫹, BAPTA is far more effective at
chelating Ca2⫹ than is EGTA. As a consequence, these
chelators have been used to estimate effective “distances”
between entry sites and regulatory sites of Ca2⫹; most
notably in the case of CCE channels by Zweifach and
Lewis (441) in characterizing Ca2⫹ regulation of ICRAC and
also by Naraghi and Neher (275) in studying the Ca2⫹
regulation of VGCCs. When BAPTA and EGTA were compared in their abilities to attenuate the effects of CCE on
AC8 activity, only BAPTA was effective (the cells having
been preloaded with BAPTA-AM or EGTA-AM) (137). Attempts were made to verify that the same concentrations
of EGTA and BAPTA were achieved inside the AC8-expressing cells, by demonstrating that equivalent loading
concentrations of the cell-permeable esters were equipotent at blunting the binding of Ca2⫹ by the fluorescent ion
sensor fura 2. Although it is relatively impossible to know
what the final concentrations of these chelators were at
their site of action, if we assume that equal concentrations were achieved, it is very difficult to interpret the
data otherwise than by concluding that the source of the
Ca2⫹ that regulates these ACs is very close to its site of
action. It is likely that they are not more than 50 nm apart,
since at greater distances diffusion allows EGTA and
BAPTA to be equally effective at chelating Ca2⫹ (275, 441).
2. Measurements with aequorin
An elegant strategy for assessing local Ca2⫹ was
developed by Rizzuto and colleagues (79, 321). The jellyfish Ca2⫹-binding protein aequorin can be heterologously
expressed in cells and directed, by the use of appropriate
tags, to specific domains within the cell such as the miPhysiol Rev • VOL
tochondria (322), nucleus (46), and ER (265). Upon binding Ca2⫹ in the presence of its cofactor, coelenterazine,
photons are emitted and can be detected using a photomultiplier. This strategy was adopted with AC6, where the
COOH terminus was tagged with aequorin and expressed
in HEK293 and HeLa cells. The [Ca2⫹] measured by the
AC6-aequorin construct was compared with those measured by cytosolic aequorin in response to Ca2⫹ release
from stores or CCE. The AC6-aequorin detected much
higher [Ca2⫹] in response to CCE than to release triggered
by Gq-coupled receptor stimulation; the converse was
true for the cytosolic aequorin (89, 273). However, calibration of the absolute Ca2⫹ values is difficult with aequorin in response to rapid phenomena, since the coelenterazine is consumed as a consequence of the light emission. As a result, these promising findings were never
formalized, even though they clearly supported the notion
of close apposition of CCE channels and Ca2⫹-sensitive
ACs (89, 273; K. Fagan, R. Rizzuto, and D. Cooper, unpublished observations).
3. Comparisons between the efficacy of Ca2⫹, Ba2⫹,
and Sr2⫹
The ability of Ca2⫹, Ba2⫹, and Sr2⫹ to stimulate heterologously expressed AC8 in vitro yields a potency series
of 1 Ca2⫹:40 Sr2⫹:1,600 Ba2⫹ (172), which is reflective of
the discrimination of the EF hands of CaM for this IIa
series of cations (141). However, in the intact cell, Sr2⫹
entry (via putative CCE channels) can stimulate AC8 activity ⬃50% as efficaciously as Ca2⫹, while Ba2⫹ is largely
ineffective (172). The efficacy of Sr2⫹ is hard to understand unless the intimacy of the CCE channels and the
ACs is considered.
A somewhat similar situation is seen when the inhibitory regulation of the endogenous AC6 of C6 –2B cells is
examined (172). In this case, only Ca2⫹ yields high-affinity
inhibition in vitro, although all three cations produce
low-affinity inhibition, with a relative potency series of 1
Ca2⫹:5 Sr2⫹:100 Ba2⫹. In the intact cell, in response to
CCE, again Sr2⫹ yields a response that appears equally
potent but only ⬃50% as efficacious as Ca2⫹, while Ba2⫹
is ineffective (172). One component of these observations
is the selectivity of the CCE channels for the cations,
since they display a similar selectivity order of Ca2⫹ ⬎
Sr2⫹ ⬎ Ba2⫹ and are very poor at carrying Ba2⫹ (134, 440).
Even though both of these cell types can carry large
amounts of Sr2⫹ via their CCE channels, it still seems
surprising, given the in vitro impotence of Sr2⫹, that any
in vivo effects can be seen. The only reasonable explanation would seem to be that the ACs and CCE channels are
very close and, as a consequence, the concentration of
Sr2⫹ is even higher than suggested by the global cation
measurement. A contributing factor may be that Ca2⫹ can
inhibit its own passage through CCE channels (440) and
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1. BAPTA versus EGTA
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DEBBIE WILLOUGHBY AND DERMOT M. F. COOPER
Sr2⫹ might be expected to be less efficacious at this effect
so that even more Sr2⫹ permeates the channels, or with
longer periodicity than Ca2⫹, to compensate for its in
vitro ineffectiveness. Again, intimacy of the CCE channels
and the ACs is strongly suggested.
4. Selective regulation of Ca2⫹-sensitive ACs by CCE
in endogenous sources
E. Regulation by Calcium in Excitable Cells
Notwithstanding the dependence of Ca2⫹-sensitive
ACs on CCE in nonexcitable cells, these enzymes can be
clearly regulated by alternate modes of Ca2⫹ entry in
excitable cells. Specifically, both Ca2⫹-inhibitable and
Ca2⫹-stimulable ACs are regulated as a consequence of
VGCC activity, which produces large, dynamic Ca2⫹ increases in cells from excitable tissue. Thus the AC5/6
activity of cardiac myocytes is inhibited by L-type Ca2⫹channel activity in response to ␤-adrenoceptor (␤-AR)
stimulation (425). Similarly, in hippocampal, cerebellar,
and hypothalamic neurons, AC1 and/or AC8 are stimulated by Ca2⫹ entry mediated through L-type channels (96,
145, 212). Further studies suggest that the robust Ca2⫹
signals associated with NMDA receptor activation are
also capable of stimulating AC1 activity in a CaM-dependent manner (74, 75).
The relative potential for regulation of ACs in excitable cells, by various means of [Ca2⫹]i increase, was
addressed directly in a study using the GH3 anterior piPhysiol Rev • VOL
F. What Is the Nature of the Ca2ⴙ Signal to Which
the ACs Respond In Vivo?
One of the enduring puzzles in the regulation of ACs
by Ca2⫹ is why apparently equivalent, or higher, [Ca2⫹]i
achieved by means other than CCE are incapable of regulating these enzymes, when in solution there is a simple
concentration effect relationship (cf. Fig. 6). One likely
explanation concerns the location of the ACs relative to
the global [Ca2⫹]i rise that is typically reported by cytosolically distributed Ca2⫹ indicators. Thus apparently
equivalent [Ca2⫹]i are not distributed uniformly across the
cell. Support for this comes from the targeted aequorin
studies alluded to in section VD, where an aequorin-tagged
AC reports large [Ca2⫹] changes in response to CCE but
much smaller changes during release from stores, even
though the amplitude of [Ca2⫹] increase under both conditions was comparable when measured globally (89,
273). Another possibility is that Ca2⫹-regulated ACs are
more sensitive to the nature of the Ca2⫹ rise achieved by
rapid opening and closing of Ca2⫹ entry channels. If, as
the data indicate, ACs are located very close to Ca2⫹
channels such that only BAPTA can attenuate their regulation, then it might be imagined that these enzymes are
exposed to an extremely rapid and substantial change in
local [Ca2⫹], a situation that bears little relationship to
more steady-state Ca2⫹ concentrations established during
in vitro assays. Thus, at least in the case of AC8, during
rapid channel opening and closing, binding and unbinding
of Ca2⫹ to CaM and the movement of CaM from the NH2
to the COOH terminus of AC8 (sect. IVA) are reactions
that must occur or alternatively an incremental accumulation of CaM must occur. The in vitro responsiveness of
the ACs to very fast Ca2⫹ transients may be approached
by rapid stopped-flow experiments, although such experiments might prove challenging with an enzyme of such a
low turnover number as AC (⬃20 molecules/s) (115). To
be predictive rather than experimental, another approach
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The majority of the foregoing studies were conducted with heterologously expressed ACs, but it is important to point out that selectivity for CCE over release
has also been seen with the endogenous ACs, where it has
been expressly examined. For instance, AC6 in pulmonary endothelial cells, C6 –2B glioma, and NCB-20 cells is
regulated by receptor-induced CCE (73, 111, 422). Similarly, AC6 in adrenal glomerulosa cells (56) and AC8 in
parotid cells (396) were both dependent on CCE for their
regulation. In contrast, very few examples of Ca2⫹ regulation mediated by release are apparent but include the
endogenous AC5/6 in ascending renal tubules that was
equally affected by release and CCE (65), and inhibition of
endogenous AC3 activity via CaM kinase as a consequence of IP3-evoked Ca2⫹ release in A7r5 cells (128). In
summary, in nonexcitable cells, both endogenous and
heterologously expressed Ca2⫹-sensitive ACs are typically very close to CCE sites and are highly discriminating
for this source, regardless of the apparent global [Ca2⫹]i
(Fig. 8B). A direct association between Ca2⫹-sensitive
ACs and specific components of CCE (e.g., STIM1 or
TRPC isoforms) could underlie the selectivity of ACs for
this specific form of Ca2⫹ entry, but no direct evidence for
any such interaction has yet been obtained.
tuitary cell line to compare the efficacy of Ca2⫹ entry
occurring via CCE or voltage-gated channels. Somewhat
surprisingly, it was revealed that the comparatively modest degree of CCE was equally effective at stimulating
transfected AC8 as the extremely robust Ca2⫹ entry arising from the opening of L-type Ca2⫹ channels (135). Despite this, the prevalence and specific role of CCE in
excitable cells is undefined, and it might be expected that
most Ca2⫹-regulated AC activity occurs as a consequence
of the activation of VGCC or glutamate receptor activation. Thus, notwithstanding the situation in nonexcitable
cells, and the potential for CCE to potently stimulate ACs
in excitable cells, there is no absolute exclusive dependence of the ACs on CCE, just independence from a
number of other mechanisms.
ADENYLYL CYCLASES
G. Role of CaM Recruitment in the Regulation of
AC8 by Ca2ⴙ
A consideration in the regulation of AC8 by Ca2⫹ is
the availability of CaM. CaM-regulated proteins compete
within cells for the limiting amount of this modulator,
Physiol Rev • VOL
whose free concentration is estimated to be ⬃100 nM,
while the total (available/bound) concentration is as high
as 8 ␮M (34). Persechini and Stemmer (299) point out that
the number of binding sites for CaM in the cell exceeds
the available [CaM]. Thus competition for CaM can determine whether a potential target is regulated by Ca2⫹
(299). The NH2 terminus of AC8 appears to garner CaM
avidly either during its passage to the plasma membrane
or within the domain where it resides. Whether this occurs upon or before the elevation of [Ca2⫹]i is not known;
however, it renders the enzyme potentially immune to
fluctuations in free [CaM] and only dependent on elevation of [Ca2⫹]i for activation. Furthermore, whereas mutated forms of CaM with impaired Ca2⫹ binding are ineffective at mediating Ca2⫹ stimulation of AC8 in vitro, they
cannot be expressed at sufficiently high levels to perturb
Ca2⫹ stimulation in the intact cell (351). It appears as
though either the expressed mutant CaM molecules simply cannot compete away the endogenous bound CaM, or
they cannot access the pool of CaM that is sampled by
AC8. The NH2 terminus of AC8 plays a key role in that
although an NH2-terminal deleted form of AC8 is fully
stimulated by Ca2⫹ in vitro, this truncated AC8 cannot be
activated by Ca2⫹ in vivo (173). Furthermore, the stimulation of the truncated form cannot be restored even
when high levels of CaM are heterologously expressed
along with the truncated AC8. These data underline both
the importance of the NH2 terminus in recruiting CaM and
the lack of access of the AC8 to global CaM pools. It
seems unlikely that a similar situation applies to AC1,
since this AC possesses apparently only one CaM binding
site; thus a mechanism which involves passing a recruited
CaM to a regulatory site seems unlikely. The mechanism
of AC8 activation by CaM is very reminiscent of the
situation with certain Ca2⫹/CaM modified ion channels
(246). The dynamics of the process, and its possible dependence on Ca2⫹ influx, may be further illuminated by
conducting live cell FRET analyses analogous to those
used to establish the Ca2⫹/CaM modulation of cyclic nucleotide-gated channels (CNGCs) (437) and L-type Ca2⫹
channels (130, 131, 266).
VI. COMPARTMENTALIZATION OF THE
ADENYLYL CYCLASES
The organization of ACs in “privileged domains”
within the cell is readily rationalized from a variety of
regulatory view-points. One obvious rationale is to arrange components that potentially interact in close proximity to each other. Other rationales for compartmentalizing the ACs include the following: the provision of a
microdomain that is insulated from metabolic fluctuations in cellular activity, concentration of substrate that is
not at the discretion of the second-to-second metabolic
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might be to simulate the behavior based on the known
properties of the ACs, along the lines used by Dupont et
al. (125) for the analysis of CaM kinase II by Ca2⫹ oscillation frequency, where the extremely high local [Ca2⫹]
and adjacency was taken into account in CaM recruitment
and activation, in an extension of the elegant work of De
Koninck and Schulman (109).
Another possibility is that CCE channels form clusters, which do not necessarily open and close synchronously, but which permit high local [Ca2⫹] to be achieved
in discrete domains where the ACs also reside, as mooted
by Luik et al. (234). Evidence for this possibility comes
from recent studies which show that candidate CCE channels, Orai1, and the probable store Ca2⫹ sensor, Stim1, are
recruited into channel clusters upon the triggering of CCE
(234, 307, 356).
A different situation occurs in the case of the Ca2⫹inhibitable AC5 and AC6, where the mechanism of action
of Ca2⫹ represents only a relatively simple competition
for Mg2⫹ binding sites. Again, the relationship between
steady-state in vitro concentrations and those arising at
the AC in vivo should be considered unknown. Given that
all ACs are capable of low-affinity inhibition by Ca2⫹ in
vitro, one could wonder why all ACs, even the presumed
Ca2⫹-insensitive ACs, do not display inhibitory responses
to Ca2⫹, if transient high [Ca2⫹] can be achieved within
the local domain of the ACs in the intact cell. In this case,
their distinct cellular location really may provide the answer (see sect. VI) or the precise conformation that the
catalytic domain adopts may render it more or less susceptible to Ca2⫹ occupancy (see sect. IVB).
A final possibility to consider is that it is not so much
the amount of Ca2⫹ moving through the channels that is
significant, but the conformational changes in the channels that are sensed by ACs in an intimate complex, i.e., a
form of vectorial or conformational sensing. This possibility was addressed in one study, where the ability of
Ba2⫹, which is ineffective at regulating AC activity in
vitro, but is transported by SOCCs (186, 441) was evaluated in a CCE-triggering experiment. Even though Ba2⫹
was effectively carried upon triggering CCE, it had no
effect on AC activity in in vivo experiments, which suggests that the conformational change associated with cation conductance through CCE channels is insufficient to
regulate the AC and that Ca2⫹ is essential (137). The
ability of BAPTA to attenuate regulation of the ACs (134)
also argues against this possibility.
983
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DEBBIE WILLOUGHBY AND DERMOT M. F. COOPER
activity of the cell, to provide a supply of regulatory
factors not in competition with other targets, or finally, to
protect the rest of the cell from the hyperactivity of cAMP
signaling, e.g., by arranging a barrier of PDEs to limit
cAMP diffusion. Physical barriers, both at the level of the
plasma membrane and the underlying (cytoskeleton) substratum, may also contribute to such compartmentalization. Our current knowledge of AC organization provides
examples of each of these options (with considerable
overlap). Some rationales are now well-established, while
others are more tenuous but still worthy of consideration.
A. Lipid Rafts
Physiol Rev • VOL
B. Lipid Rafts and ACs
Caveolae or rafts in their proposed role as devices for
concentrating interacting signaling molecules (347) are
obvious candidates to provide a means for concentrating
ACs with essential regulatory elements, such as G proteins, receptors, and Ca2⫹ entry channels (see Table 2). In
1999 AC5 was identified in lipid rafts in cardiomyocytes
via a proposed interaction with the raft scaffold protein
caveolin (344). Peptides derived from caveolin inhibited
AC5 activity, and the notion was advanced that AC5 resided in rafts where it was primed for activation upon
release from the caveolin (344, 379). The first indication
that ACs and elements of the Ca2⫹ regulatory apparatus of
cells coresided in rafts came from studies in C6 –2B glioma cells, where destruction of the rafts by extraction of
the cholesterol with methyl-␤-cyclodextrin (MBCD)
moved AC6 immunoreactivity and catalytic activity out of
the rafts and destroyed the AC regulation by CCE, without
affecting CCE itself (140). Elements of the CCE apparatus, namely, TRPC1 and TRPC3, have since been reported
in rafts (8, 9). There is also evidence for localization of
CNGCs in rafts (41), which may explain an earlier observation that the modest Ca2⫹ current carried by these
channels is capable of regulating a coexpressed AC8
(138). It has since been found that all of the Ca2⫹-sensitive
ACs (AC1/3/5/6/8), plus numerous associated proteins, are
found in rafts from various sources (see Table 2) whilst
the Ca2⫹-insensitive AC2/4/7 isoforms are excluded. Raft
localization of all of the Ca2⫹-regulated ACs has been
demonstrated using a combination of immunolocalization
and fractionation to reveal AC activity in detergent-resis-
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Lipid rafts are areas of plasma membrane heterogeneity, characterized by high concentrations of cholesterol
and sphingolipids, which are distinct from the glycerophospholipid-rich (nonraft) plasma membrane domains
(350). In many cell types raft domains also contain the
scaffold protein caveolin, which enables the rafts to adopt
invaginations, termed caveolae. Among other cellular
roles, caveolae have been proposed to traffic components
from the Golgi to the cell membrane (314, 347). The
notion that signaling elements can be concentrated in
such domains and so rendered more efficient in their
interaction has been enthusiastically embraced in many
areas of signaling (302), but it would be reasonable to
acknowledge that there is far from a universal consensus
on what may be occurring (16, 269). It seems undeniable
that certain proteins attract, or at least associate with, a
selective coterie of lipids. This may result in the production of a transient population of enriched domains centered on such proteins in the plasma membrane. It is then
conceivable that such microassociations may coalesce
with others to form aggregations of proteins with similar
physical lipid preferences. Such associations may well be
ephemeral unless 1) they are reinforced by stronger organizing forces, e.g., the cytoskeleton (217, 241) or caveolin, or 2) there is inherent attraction between partners
that find themselves in an appropriate microdomain.
For biochemical purposes, lipid rafts can be isolated
by two main types of procedure: either selective cold
detergent extraction or sonication. Extraction with nonionic detergents relies on the insolubility of lipid rafts,
due to close packing of the saturated acyl chains of
sphingolipids and stabilization via cholesterol interdigitation. The second method is independent of detergent and
relies on sonication at high pH to break the plasma membrane into small fragments. It is important to acknowledge that both of these preparations at best may be frozen
snapshots in the lives of what are dynamic assemblies
(217, 241) or at worst, preparation-induced assemblies
(16, 269). In a detailed comparison, proteomic analysis
was used to assess which proteins were found in rafts
when prepared by the two methods (147). It turned out
that the presence of some proteins in rafts was variable
and dependent on the method used, but other proteins
were always present in rafts regardless of the preparation
method. It is important to acknowledge that finding a
molecule by only one of these methods does not mean
that it has no lipid association, only that it may be more
tentative. Perhaps the most robust and credible data for
proteins associating with lipid rafts comes from single
molecule tracking in live cells (217, 241). It seems important to maintain a flexible position in assessing the significance of lipid associations of signaling molecules. For
instance, under some circumstances, passing associations
may form between proteins and lipid rafts, such as at
leading or trailing edges of cells (400). Alternatively, stable associations may form e.g., in neutrophils (312, 371),
during the control of barrier function in endothelial cells
(45, 84) or at postsynaptic densities (e.g., Ref. 142). In
addition, the packaging to rafts may provide a device for
protein delivery to the plasma membrane (312).
985
ADENYLYL CYCLASES
TABLE 2.
Components of lipid rafts associated with cAMP signaling
Caveolar/Raft Protein
Adenylyl cyclases
AC1
AC3
AC5/6
AC5
AC6
G i␣
RGS proteins
TRPC channels
TRPC1
TRPC3
CNGC
␣-Subunit (CNGA2)
pH regulators
NHE1
NHE3
PMCA
H⫹-ATPase
Miscellaneous
eNOS
PP2A
Arrestin
Reference Nos.
HEK293 cells
Cardiac fibroblasts
Vascular smooth muscle
Sf9 cells
PC12 cells
Cardiac myocytes
Cardiac fibroblasts
Vascular smooth muscle
HEK293 cells (AC5)
Sf9 cells
C6 glioma
PC12 cells
Cardiac myocytes
HEK293 cells
Masada et al., unpublished data
292
291
343
289
290, 332, 343
292, 332
291
99
343
140
289
293
99, 354
Neurons
Adipocytes
T lymphocytes
283
282
1, 12
Cardiac myocyte
Cardiac myocyte
PC12 cells
Cardiac myocyte
Vascular smooth muscle
290
359, 420
289
146
384
Cardiac myocyte
Vascular smooth muscle
Cardiac myocyte
HEK293 cells
Rat liver
COS-7 cells
332
291
332
184
184
184
Mouse sperm
Submandibular gland
MDCK cells
HEK293 cells
380
43
43
232
HEK293 cells
COS-1 cells
Rat olfactory tissue
41
41
41
HEK293, C6 glioma
AP-1 cells
Ileal brush border cells
Endothelial cells
Visceral smooth muscle cells
Hepatic stellate cells
Kidney
410
54
224, 270
148, 340
148
284
258
Cardiac myocytes
Mouse forebrain
Photoreceptors
T lymphocytes
146, 290
99
272
1
Listed are the ACs and some of their associated proteins that have been reported in lipid rafts of various cell types. It should be noted that
this is by no means an exhaustive list, and the presence of any given protein in this table does not preclude its localization to non-raft regions in
other cell types, under other experimental conditions.
tant membrane fractions. Postsynaptic densities appear
to provide variations of the lipid raft theme in neurons
and are similarly enriched with AC immunoreactivity
(142, 152, 189, 262).
Despite uncertainty surrounding the nature, precise
molecular composition, lifetime, and physiological role of
Physiol Rev • VOL
rafts (be it trafficking, endocytosis, cholesterol homeostasis, and/or organization of signaling complexes, Ref. 86),
there seems little doubt that some ACs and elements of
the CCE apparatus coassociate in these enriched regions
of the plasma membrane. As indicated earlier, the rafts
may be dynamic structures, whose significance increases
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AC8
Phosphodiesterases
PDE2
PDE3B
PDE4 (A4/B2/D1,2)
GPCRs
␤1-AR
␤2-AR
nAChR␣7
mAChR
AT1R
G proteins and regulators
Gs␣
Cell Type
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DEBBIE WILLOUGHBY AND DERMOT M. F. COOPER
or decreases at various times depending on the physiological requirements of the cell. It is also conceivable that
specific AC isoforms may be dynamically recruited to the
lipid rafts in response to certain stimuli. In any case, lipid
rafts are clearly an important contributing factor to the
organization of signaling complexes containing Ca2⫹-sensitive ACs.
C. Components of the Microdomain
In addition to the Ca2⫹-regulated ACs, a number of
associated proteins have been shown to reside in lipid
rafts, which may play an important part in the localized
regulation of cAMP synthesis (see Fig. 9). Below we detail
findings from a few of these studies.
1. NHE1
The Ca2⫹-regulated ACs are subject to in vitro regulation by modest alterations in pH (⫾0.3 pH units) with
acidification and alkalinization, respectively, decreasing
and increasing both the activity of the ACs (33, 198, 226)
and dramatically altering their sensitivity to Ca2⫹ (410).
The latter is clearly a consequence of the pH sensitivity of
the aspartates that bind Ca2⫹ in CaM and the AC catalytic
domains. Surprisingly, imposed changes in intracellular
pH in the intact cell were unable to produce the anticipated changes in the response of the ACs to [Ca2⫹]i rises.
This lack of in vivo pH effect turned out to be due to the
colocalization of Ca2⫹-stimulable AC8 and the Ca2⫹-inhibitable AC6, with the acid extruder, sodium-hydrogen exchanger 1 (NHE1), in lipid raft regions of HEK293 and
C6 –2B glioma cells, respectively (410). Such abundance
of NHE1 activity in lipid rafts appears to generate a
privileged microdomain that protects the Ca2⫹-sensitive
Physiol Rev • VOL
ACs from local acid shifts. Consequently, only when
NHE1 is selectively inhibited can the pH-dependent modulation of AC activity during Ca2⫹ entry be detected. This
localization of NHE1 to lipid raft regions of HEK293 and
C6 –2B cells is consistent with the distribution of transfected NHE1 to caveolin-containing fractions of AP-1 cells
(54). Similarly, studies in rabbit ileal brush-border cells
have revealed the localization and functional dependence
of the NHE3 isoform on its localization in lipid rafts (224,
270). The colocalization of specific NHE isoforms and
ACs in lipid rafts may in fact provide a site for bidirectional synergy between two key regulatory molecules as
local changes in cAMP have the potential to up- or downregulate NHE activity (203). In addition to acid extruders
such as NHE1 localizing to lipid rafts, there is also evidence for the localization of an acid loader to caveolincontaining membrane fractions. Concentration of the
PMCA within caveolae (148, 284, 340) suggests that these
subcellular domains may be exposed to substantial acid
loads as a consequence of Ca2⫹ extrusion in exchange for
protons (257), although such acidifications could be counterbalanced as a consequence of localized NHE activity. It
is not currently known if other pH regulators are localized
to lipid rafts. Conflicting evidence suggests the presence
or absence of the proton-translocating H⫹-ATPase in lipid
rafts in cells originating from the kidney (44, 258).
2. PDEs
PDEs, which provide the sole means for degrading
cAMP within the cell, are essential components of cAMP
signaling microdomains. Since the identification of the
Ca2⫹-stimulated PDE1 in 1970 (77, 201), 10 further members of this superfamily have been cloned and characterized (27, 88, 97, 113, 133, 149, 187, 240, 251, 427). The
family now consists of more than 20 different gene prod-
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2⫹
FIG. 9. Localization of Ca -sensitive
ACs and associated proteins in lipid rafts. All
Ca2⫹-sensitive ACs (AC1/3/5/6/8) preferentially locate in lipid rafts. TRPC1/3 and
NHE1/3 isoforms are also selectively located
in rafts where they may influence AC activity. Signaling modules that include AKAPs,
PKA, and anchored PDEs, along with nonanchored PDE, may provide a microdomain of
cAMP that differs from the broad cytosol
and yield local regulatory consequences.
GPCRs localize to raft or nonraft fractions
depending on receptor subtype and physiological context.
ADENYLYL CYCLASES
well be competed for, by proteins such as CaM kinase II,
Ca2⫹-activated ATPases, and CaM-modulated ion channels.
4. TRPC channels
The TRP family of ion channels, originally identified
in Drosophila, now encompass more than 30 cation channels that are divided into several subfamilies, including
the “canonical” TRPC family (reviewed in Ref. 297). The
TRPC channels consist of seven members (TRPC1–
TRPC7) that are candidate components of capacitative
Ca2⫹ entry channels (8, 311). Despite immense interest in
the TRPCs in recent years, their precise function and
mechanism of action remain unclear, as does the subunit
composition and assembly of their associated Ca2⫹ entry
channels that are activated by IP3-mediated Ca2⫹ release
from the ER. With the recent discovery of Orai1 as a likely
component of CCE channels, it is conceivable that the
TRPCs associate with this protein to form sites for Ca2⫹
entry. The selectivity of the Ca2⫹-regulated ACs for this
mode of Ca2⫹ entry and the proposed close association of
these ACs with sites of CCE (see sect. V) prompts specific
interest into the subcellular localization of the TRPC
channels. Both TRPC1 and TRPC3 have been shown to
form signaling complexes in caveolar domains (8, 9).
TRPC1 coimmunolocalizes with caveolin-1 (380) and
forms a multimeric complex with other Ca2⫹ signaling
proteins in lipid raft domains, coimmunoprecipitating
alongside caveolin-1, Gq␣ and IP3R3 to enable activation
of store-operated Ca2⫹ entry (233). A direct interaction
between caveolin-1 and the NH2 terminus of TRPC1 is
thought to be responsible for the plasma membrane localization of the TRPC1 (43). Other studies from Ambudkar’s group (232) have identified a multimeric caveolar
TRPC3 signaling complex that has associations with IP3R,
SERCA, Gq/11␣, PLC-␤, ezrin, and caveolin-1 (232).
Thus it is becoming clear that not only are ACs
organized in a microdomain with their effectors or regulators, they are also organized with contextual factors
that contribute to the efficient and “privileged” functioning of the domain. Further elements that could be considered to contribute to the domain are Ca2⫹-buffering proteins, and dynamic pools of ATP, subplasmalemmal scaffolding elements of the cytoskeleton and abutting
intracellular structures, which might contribute to transient isolation of these compartments.8 One final consideration for the composition and functioning of the microdomain has to do with the effect of macromolecular
3. Calmodulin
As alluded to in section VG, CaM recruitment and
availability are a determinant of AC responsiveness. Consequently, CaM availability should be considered an element of the cAMP signaling microdomain, which might
Physiol Rev • VOL
8
The recent flurry of literature suggesting that the ER is recruited
by the interaction of STIM1 and Orai1 at the plasma membrane (298,
356) as a consequence of depletion of intracellular Ca2⫹ stores is likely
to have implications for the physical organization of the AC microdomain.
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ucts, with a large number of splice variants (88). Given
this degree of heterogeneity, the homology (⬎50%) within
their catalytic domains is striking (88, 133). However,
slight structural differences in the catalytic domain determine whether the PDE is cAMP specific, cGMP specific,
or has dual substrate specificity (435). Further differential
regulation of the PDEs arises from divergent NH2 termini
which contain domains for CaM or cGMP-binding and
phosphorylation (88, 187, 357, 436) or intracellular targeting (187, 346). The COOH termini of PDEs may have a role
in kinase docking and dimerization (237).
Two PDE families of particular interest to this review
are PDE1 and PDE4. PDE1 has dual substrate specificity
and is the only PDE to be dependent on Ca2⫹/CaM. Consequently, it is in a prime position to facilitate cross-talk
between intracellular cAMP and Ca2⫹ signals (417, 436).
Studies in 1321N1 human astrocytoma cells suggest that
endogenous PDE1 remains inactive until Ca2⫹ entry is
triggered (166). Although it is yet to be seen if this is a
general property of all PDE1 isoforms, studies in insulinsecreting MIN6 cells propose that Ca2⫹-dependent activation of endogenous PDE1C isoforms plays some role in
the generation of dynamic cAMP changes (219). In the
case of the cAMP-dependent PDE4, intracellular targeting
of specific PDE4 isoforms plays a central role in generating spatially distinct cAMP pools (see sect. VII). Such
discrete targeting arises from the association of the NH2
terminus of specific PDE4 isoforms with accessory proteins such as ␤-arrestins (18, 20), SH3 domains (26, 249),
AKAPs (120, 372, 411), myomegalin (196), and the PKC
binding protein RACK1 (187, 421). Additional domains
within the NH2 terminus of the protein may also facilitate
its association with the phospholipid-rich environment of
intracellular membranes (19, 168).
A number of fairly recent studies have suggested the
specific targeting of PDEs to lipid rafts. Interestingly,
T-cell activation has been shown to recruit several PDE4
isoforms (namely, PDE4A4, PDE4B2, and PDE4D1/2) to
lipid rafts and increase PDE4 activity in these membrane
fractions (1). In the case of PDE4B2, localization to T-cell
lipid rafts is dependent on an intact NH2 terminus (12). In
primary adipocytes, PDE3B localizes to caveolae (282),
whilst a study by Noyama and Maekawa (283) using rat
cortical neurons suggested the enrichment of PDE2 isoforms in lipid raft fractions. The localization of PDE2 to
lipid raft fractions of neuronal preparations was sensitive
to depalmitoylation and was accompanied by other
cAMP-related proteins including PKA and AC5/6 (283).
987
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DEBBIE WILLOUGHBY AND DERMOT M. F. COOPER
crowding on the kinetic parameters of the proteins
present, which is likely to render affinities determined
from in vitro experiments to be underestimates (259).
VII. DYNAMIC AND LOCAL CHANGES IN cAMP
A. Methodologies for Single-Cell cAMP
Measurements
Over the past two decades, advances in the design of
fluorescent Ca2⫹ sensors and the improved resolution of
imaging systems have collectively allowed an awareness
of the spatiotemporal complexities of intracellular Ca2⫹
signaling (14, 36, 163, 300, 301). It is now accepted that
Ca2⫹ microdomains, oscillations, and propagating Ca2⫹
TABLE 3.
1. PKA-based probes
The earliest measurements of cAMP in single cells
used a fluorescent probe based on PKA. This ingeniously
designed probe was termed “FlCRhR” (“flicker”) due to its
Comparison of single-cell cAMP probes
PKA Based
Epac Based
Method of
measurement
FRET between
fluorescein⁄rhodamine or CFP⁄YFP
FRET between CFP⁄YFP
Dynamic range
⬃0.1–5 ␮M cAMP
⬃2–50 ␮M cAMP
Localization
Global, or localized to site of AKAPs
Advantages
First genetically encoded probe
Global, or selectively
targeted to plasma
membrane,
mitochondria, or nucleus
Better FRET efficiency and
temporal resolution than
PKA-based sensors
(largely due to
unimolecular nature of
sensors)
Buffering of cAMP
Potential catalytic activity
of full-length version
Good cAMP sensitivity
Disadvantages
Limited temporal resolution
Tetrameric, requiring binding of four
cAMP molecules before
dissociation
Potential saturation
Reassociation with endogenous
subunits complicates dynamics of
response
Microinjection of “FlCRhR” is
technically demanding
Buffering of cAMP
Potential catalytic activity
Likelihood of saturation due to
cooperative binding
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CNG or HCN Channel
Perforated or excised patch
clamp, or Ca2⫹ entry
measurements (fura 2)
⬃0.1–50 ␮M cAMP (varies
with mutant used)
Plasma membrane
Excellent temporal
resolution
Good cAMP sensitivity
Can be calibrated in vivo
Do not desensitize
Potential inhibition by Ca2⫹
Limited to subplasmalemmal
cAMP measurements
Potential saturation
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The case for compartmentalization of cAMP signaling that has been made up to this point begs the question
of whether local cAMP concentration changes can be
observed in the proposed microdomains. Below we compare a number of biosensors that can monitor real-time
changes in cAMP at the single-cell level to visualize the
spatiotemporal features of cAMP signaling, and we describe their use in a range of cell types to observe and
further characterize the dynamics and components of
specific cAMP microdomains.
waves are widespread in the control of numerous physiological events. In contrast, cAMP has been viewed traditionally to be a rather nondynamic, information-poor
second messenger. This view very much reflected the
limited technologies available. However, recent efforts to
develop sensors that monitor changes in [cAMP] with
improved sensitivity and spatiotemporal resolution (aided
by subcellular targeting) have enabled researchers to
make comparable insights into the detailed dynamics of
single-cell cAMP events. The biosensors used are based
on cAMP effector molecules including PKA (5, 127, 428,
434), CNGCs (139, 320), and most recently, exchangeprotein directly activated by cAMP (Epac) (119, 279, 305).
These real-time cAMP probes have enabled the complexities of cAMP signaling to be visualized, such as the
generation of cAMP oscillations and waves, and cAMP
microdomains, and thereby accommodated in the regulatory repertoire of the second messenger. In the following
section we discuss the development and characteristics
of the main classes of single-cell cAMP sensor (summarized in Table 3).
989
ADENYLYL CYCLASES
although this technique is subject to diffusion of cAMP
from the cytosol into the patch pipette (161, 387). After
some years, the FlCRhR method was modified to produce
a genetically encoded version of a PKA-based cAMP sensor that avoided the technical difficulties related to microinjection (428, 429). The FRET-based probe, using CFP
(donor) fused via a polypeptide linker to the RII subunit
of PKA, and YFP (acceptor) fused to the catalytic subunit,
has been successfully expressed in cultured nonexcitable
cells and neonatal cardiac myocytes to monitor changes
in cAMP (157, 429). In cardiac cells, the sensor localized
near the Z lines within the myocytes, a consequence of
anchoring to AKAPs, where it was able to detect local
cAMP signals following AC activation (429). This PKAbased sensor was recently introduced into an adenovirus
vector, enabling it to be expressed in adult ventricular
myocytes where it displayed a similar pattern of expression (395). Genetically encoded versions of the PKAbased cAMP sensor are a major improvement in their
application.
Other PKA-based sensors that are growing in popularity are the A-kinase activity reporters (AKAR), which
are composed of a phosphoamino acid binding domain
and PKA-specific substrate sandwiched between CFP and
FIG. 10. Comparison of the temporal characteristics of cAMP signals using the three main classes of
cAMP biosensor. A: cAMP signals measured in response to agonist stimulation of endogenous AC activity using PKA-based FRET probe, Epac-based
FRET probe, or CNGC in HEK293 cells. B: subplasmalemmal cAMP changes detected with the CNGC
method are clearly more transient than global cAMP
changes measured using either the PKA- or Epacbased probes. [Revised from Willoughby and Cooper
(409).]
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composition of fluorescein labeled catalytic (C) subunits
and rhodamine labeled regulatory (R) subunits of PKA
(5). The fluorescently tagged PKA subunits were microinjected into single cells where they associated to form a
PKA holoenzyme with FRET occurring between the fluorescein (donor) and rhodamine (acceptor) molecules
upon excitation of the donor fluorophore. The concept
behind the probe was that elevated cAMP would bind to
the regulatory subunits of PKA, causing the tagged complex to dissociate with a concurrent decrease in FRET
signal. This signal would increase again upon reassociation of the fluorescently tagged subunits when cAMP
levels declined. This pioneering technique has been successfully used to monitor cAMP changes in a number of
cell types including smooth muscle, fibroblasts, melanocytes, neurons, and cardiac myocytes (5, 17, 162, 167, 183,
334). However, this method has some drawbacks (see
Table 3) including its limited temporal resolution (Fig.
10). The FlCRhR method requires microinjection of the
fluorescent subunits into cells, thus limiting its use to
relatively large, robust cells such as invertebrate neurons.
The ability to introduce FlCRhR into cells via diffusion
from a patch-clamp electrode has extended the use of this
probe to vertebrate neurons in brain slice preparations,
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DEBBIE WILLOUGHBY AND DERMOT M. F. COOPER
YFP, which can be used to report functional endogenous PKA activity (7, 310, 335, 433, 434). PKA-dependent phosphorylation of the genetically encoded construct results in a conformational change that alters the
distance and/or orientation of the two GFP proteins
fused to the NH2 and COOH termini (detected as a
change in FRET). Other probes based around PKA
include a number of bioluminescent resonance energy
transfer (BRET)-based sensors that determine interactions of both RI and RII with PKA catalytic subunits to
monitor their dynamics in vivo (310).
2. Epac-based probes
Physiol Rev • VOL
CNGCs have been extensively studied in the visual
and olfactory system where they play an essential role in
signal transduction (206). The CNGCs form part of a large
family of ion channels that are directly activated by cyclic
nucleotides, and exhibit different selectivities for cGMP
and cAMP. Activation of both the CNGCs, and the related
hyperpolarization-activated cation channel (HCN), by cyclic nucleotides has been exploited to provide single-cell
sensors that monitor near-membrane cAMP or cGMP fluctuations with unmatched temporal resolution (182, 317,
318, 382). Native HCN channels are 10- to 100-fold more
sensitive to cAMP (206, 207) than the native CNG channel,
which is preferentially activated by cGMP. However, one
or two site-directed mutations of the rat olfactory CNG
␣-subunit alter its preference for cAMP rather than cGMP
(320).9
Although these channels are restricted to monitoring
cAMP changes close to the plasma/sarcolemmal membrane (317, 318, 324), they exhibit greater temporal resolution than fluorescent-based sensors (Fig. 10), provide
good sensitivity to cAMP changes in the physiological
range (0.1–5 ␮M), and unlike many ion channels, do not
desensitize (182, 206, 317, 320). Their activation results in
conduction of Na⫹, K⫹, and/or Ca2⫹, which can be measured using perforated whole cell, or excised, patchclamp methods, or by imaging with a Ca2⫹-sensitive dye,
such as fura 2, to assess Ca2⫹ entry into the cell via the
open channel. Both CNG and HCN channel types can be
calibrated to calculate their affinities for cAMP by exposing the expressed channels to known concentrations of
cyclic nucleotide and measuring either current amplitude
as a proportion of the peak ICNG (317) or channel activation time constant (182). The sensitivities of the mutant
CNGCs for cAMP described above (0.1–5 ␮M) are based
on measurements using the most sensitive C460W/E583M
mutant (320). However, other mutants useful for detecting higher [cAMP] within the cells include E583M and
⌬61–90/C460W/E583M that display ⬃10-fold lower cAMP
sensitivity (320, 324). This range of sensitivities to cAMP
is similar to that estimated for PKA- (17) and Epac-based
probes (279, 305). One potential drawback of using the
CNGCs to monitor cAMP is their possible inhibition by
Ca2⫹. Thus the presence of external Ca2⫹ can either cause
a permeation block of the channel pore (155) or upon
entering the cell, Ca2⫹ can bind to CaM and feedback on
the channel to limit its sensitivity to cAMP (40, 170, 230,
383). This potential inhibition of the CNGC by Ca2⫹ can
be avoided by performing electrophysiological experiments in Ca2⫹-free conditions or using mutant channels
9
Native CNG channels are heteromers of ␣- and ␤-subunits. However, homomeric assemblies of ␣-subunits form fully functional channels when expressed as sensors using an adenovirus system (320).
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Epac is a guanine nucleotide exchange factor for
Ras-like small GTPases that is directly activated by cAMP.
Epac1 has a single cAMP binding domain, while Epac2
possesses two cAMP binding domains (37). Clear physiological functions for cAMP-Epac signaling are beginning
to emerge and include roles in cell adhesion and insulin
secretion (35). This relatively recent discovery of Epac as
an important cAMP effector has led to the introduction of
a new family of FRET-based cAMP sensors (119, 279, 305)
that are rapidly becoming a popular alternative for imaging cAMP dynamics. Compared with PKA-based probes,
the Epac-based sensors exhibit an extended dynamic
range and have better signal-to-noise ratio. The improved
FRET efficiency is largely due to the unimolecular nature
of the Epac probes that have CFP and YFP attached to
opposite ends of the effector molecule, which undergoes
a conformational change in response to a local rise in
cAMP. The affinity of the single cAMP binding site in Epac
is at least an order of magnitude lower than that of PKA
(110), giving rise to cAMP sensors that are less likely to
saturate than PKA-based probes (305). Both full-length
catalytically active Epac and shortened inactive cytosolic
Epac variants have been successfully used to monitor
cAMP with a Kd ranging from 2.3 to 50 ␮M cAMP (279,
305). Nikolaev et al. (281) demonstrated faster activation
kinetics of their cytosolic Epac-based probes than the
slowly dissociating PKA-based sensor, which suggests
that it is more suitable than PKA-based probes for detecting rapid changes in cytosolic cAMP. A sensor based on
Epac-2 has since been encoded into an adenovirus, enhancing its potential usefulness in a wider range of cell
types (281). DiPilato et al. (119) have designed a number
of modified Epac-based probes that are selectively targeted to the plasma membrane, the mitochondria, or the
nucleus to monitor local cAMP changes in different compartments of the cell (119). As with all single-cell cAMP
sensors, these FRET-based probes are subject to some
limitations including potential buffering of cAMP signal
and catalytic activity (see Table 3).
3. CNGCs
ADENYLYL CYCLASES
(such as ⌬61–90/C460W/E583M) that lack the Ca2⫹/CaM
binding site (320). Indirect cyclic nucleotide-mediated activation of other ion channels, such as the cystic fibrosis
transmembrane conductance regulator (CFTR) chloride
channel, has also been used as an endogenous functional
readout of local cAMP changes in airway epithelia (23,
190).
B. Local Dynamics of cAMP Signals Detected With
Real-Time Probes
991
cAMP in intact cells are similar to the diffusion coefficient
for cAMP in aqueous solution (38), which suggests that
there are no gross diffusional barriers for cAMP in neurons. This does not, however, preclude the possibility of
compartmentalized cAMP signals, or cAMP-mediated
events, within specific subcellular domains, as supported
by the identification of macromolecular signaling complexes based around AKAP79 in postsynaptic regions of
hippocampal neurons (165).
2. Studies in cardiac myocytes
1. Neuronal studies
Elegant studies using the PKA-based FlCRhR sensor
in invertebrate neurons were the first to demonstrate
compartmentalization of cAMP at the single-cell level,
with gradients of cAMP being generated along neuronal
processes following either bath application of agonist
(17) or localized release of endogenous neurotransmitter
in response to electrical stimulation (183). This probe was
also used in isolated Xenopus neurons in the first study to
report dynamic interactions between Ca2⫹ transients and
cAMP (167; see below). The loading of FlCRhR via a
patch-pipette into vertebrate neurons in brain slices has
enabled researchers to correlate cAMP responses seen in
small dendrites, main dendritic trunks, and cell soma with
neuronal excitability (387). More recently, the Epac1based FRET sensor has been successfully expressed in rat
hippocampal neurons to beautifully illustrate propagating
cAMP waves following local agonist stimulation of AC
activity (279). These signals were used to estimate a
diffusion coefficient for cAMP of ⬃500 ␮m2/s, which correlates well with those determined from the diffusion of
microinjected cAMP in invertebrate neurons using the
FlCRhR method (780 ␮m2/s) (17) and from patch-clamp
recording of CNGC activation in frog olfactory cilia (270
␮m2/s) (67). These values are higher than estimated diffusion coefficients for Ca2⫹, which range from ⬃10 to 80
␮m2/s, reflecting greater Ca2⫹ buffering (6, 274, 378).
Interestingly, the estimated diffusion coefficients for
Physiol Rev • VOL
cAMP is an essential modulator of cardiac function,
and much interest centers on characterizing the mechanisms that produce localized pools of cAMP in distinct
subcellular locations. The first experiments to correlate
the time course of single-cell cAMP measurements with
the functional consequences of ␤-AR agonist application
were performed using the archetypal FlCRhR cAMP sensor (162). This study simultaneously monitored the
[cAMP] and PKA-mediated regulation of L-type Ca2⫹
channels in isolated frog ventricular myocytes to examine
the role of PDE activity on the time course of activation
(162). Because of the potential drawbacks of this PKAbased probe, more recent studies have employed mutant
CNGCs to directly examine some of the regulatory mechanisms that shape real-time ventricular cAMP signals. In a
study using adult rat ventricular myocytes expressing
mutant CNGCs to monitor subsarcolemmal changes in
cAMP, Fischmeister and colleagues (324) revealed that
both PDE4, and to a lesser extent PDE3, were stimulated
by PKA to rapidly hydrolyze subsarcolemmal cAMP levels. The latest use of CNGCs in adult ventricular myocytes, to examine the regulation of cAMP signals generated by different GPCR agonists, has revealed that different dynamics of cAMP signal may arise from the variable
contributions of individual PDE families to hydrolyze local cAMP signals (323, 385).
Other investigations into the organization of cAMP
microdomains in isolated cardiac cells have focused on
the use of the genetically encoded PKA sensor to reveal
discrete microdomains of cAMP produced as a consequence of ␤-AR stimulation (429). The role of different
PDE isoforms in the regulation of such localized cAMP
levels in neonatal cardiac myocytes suggests that PDE3
and PDE4 localize to distinct compartments within the
cell where they have differential effects on resting, and
stimulated, cAMP levels (261), in agreement with CNGCbased studies (324). By expressing dominant negative
versions of selective PDE4 isoforms, Mongillo et al. (261)
suggest that it is the PDE4B2 isoform that regulates the
duration of norepinephrine-induced cAMP increases.
How this relates to the essential role of PDE4D isoforms
in cardiac function is not yet clear. Lehnart et al. (221)
have suggested unchanged global cAMP production in
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The concept that distinct pools of cAMP could arise
within the cell was introduced on the basis of well-designed biochemical experiments performed more than 20
years ago (57) followed by some later creative electrophysiology (200). Nevertheless, our understanding of the
organization of such cAMP microdomains has only recently started to emerge with the ability to directly measure real-time changes in spatially and temporally discrete cAMP signals. Here we review the use of single-cell
cAMP sensors in various cell types (neurons, cardiac
myocytes, and nonexcitable cells) which have furthered
our understanding of the generation and regulation of
highly organized and tightly controlled cellular cAMP signals.
992
DEBBIE WILLOUGHBY AND DERMOT M. F. COOPER
Physiol Rev • VOL
3. Studies in HEK293 cells
Numerous studies have utilized the model cell line
HEK293 to characterize cAMP signals. The expression of
genetically modified CNGCs in HEK293 cells has provided
significant insight into the dynamics of cAMP events
within the vicinity of the plasma membrane where they
have provided compelling evidence for the existence of
cAMP microdomains just beneath the plasma membrane
where agonist-evoked changes in cAMP are larger and
more dynamic than those seen in the bulk cytosol as
detected by cell population radioassays (317, 318, 320) or
by single-cell cAMP measurements using a cytosolic
Epac-based sensor (411). These CNGC studies suggest
that enzymatic barriers restrict diffusion of the second
messenger away from the cell periphery (317, 318).
DiPilato et al. (119) using HEK293 cells described
cAMP measurements using several variants of Epacbased probes that are selectively targeted to different
regions of the cell. Consistent with work using mutant
CNGCs to monitor subplasmalemmal cAMP changes
(318), they found that Gs-coupled receptor activation in
HEK293 cells generated a faster rise in subplasmalemmal
cAMP, as detected by the plasma membrane targeted
Epac probe, than global cAMP rises detected using a
cytosolic version of the probe (t1/2 values of ⬃25 vs. ⬃40
s). The authors suggest that this difference is due to the
restricted release of cAMP from its site of synthesis in the
vicinity of the cell membrane to the bulk cytosol. However, in contrast to studies using CNGCs, a sustained
stimulation of near-membrane cAMP levels was recorded
with the plasma membrane-targeted Epac probe upon
stimulation of HEK293 cells with 10 ␮M prostaglandin E1.
Possible explanations for such discrepancy include differential localization of the two cAMP sensors at the level of
the plasma membrane, or possible saturation of the Epacbased sensor.
DiPilato et al. (119) also reveal that cAMP dynamics
monitored by FRET-based Epac probes targeted to the
mitochondria or to the nucleus responded to isoprenaline
stimulation with a rate equivalent to that seen globally
(t1/2 value of ⬃40 s), although the surprisingly fast rise in
nuclear cAMP levels is not sufficient to generate detectable phosphorylation of a genetically encoded PKA activity reported (AKAR) localized to the nucleus (119). This
observation is consistent with a slower translocation of
the catalytic subunit of PKA into the nucleus following its
dissociation from RII subunits in the cytoplasm in response to elevated cAMP levels.
C. Oscillations in Intracellular cAMP
Improved single-cell sensors for cAMP have given
rise to a number of recent studies providing evidence for
cAMP oscillations that are associated with dynamic intra-
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cardiac cells from PDE4D knockout mice but a significant
increase in the localized cAMP signal at the cardiac myocyte Z line, which corresponds to the location of type 2
ryanodine receptor (RyR2). It is yet to be established
whether such localized reduced PDE4D activity contributes to heart failure and arrhythmias in humans. Nevertheless, it is likely that any PDE4D and RyR interactions
may involve the muscle-selective PKA-anchoring protein
(mAKAP), which can associate with the RyRs (204, 331)
and with PDE4D3 (120, 121) (see sect. VIIIA).
Although these studies have provided major insight
into the dynamics of cAMP changes at the single-cell
level, PKA-based probes and CNGCs are restricted to
measuring cAMP changes where they are expressed either colocalized with AKAPs, or at the sarcolemmal membrane, respectively. Thus uncertainty remains as to
whether the localized signals that they report are due to
genuine compartmentalization of the cAMP signals to
subcellular microdomains or, to some extent, the localization of the cAMP sensor itself. Future experiments
using nontargeted, high-resolution cAMP sensors should
clarify this point. One such study by Lohse and colleagues
(280) describes a novel FRET-based cAMP biosensor that
uses the cAMP binding domain of a cyclic-nucleotide
gated channel (HCN2) to assess compartmentalization of
cAMP signals in cardiac myocytes during activation of
␤-ARs. Cardiomyocytes from transgenic mice expressing
the HCN2-based sensor, which is homogenously distributed throughout the cell cytosol, showed that ␤1-AR stimulation generates a cAMP signal that propagates throughout the cell cytosol and is controlled by PDE4, whilst
stimulation of ␤2-ARs produces a spatially restricted
cAMP signal that is controlled by both PDE4 and PDE3
activities. These data also implicate a role for PDE-independent mechanisms in restricting the ␤2-AR-mediated
cAMP signal to just beneath the plasma membrane. Indeed, the authors suggest that differential distribution of
the two receptors subtypes to caveolae and t-tubular
structures could contribute to such distinct cAMP signals.
In addition to investigating the spatiotemporal pattern of cAMP signals, an elegant report by Saucerman
et al. (335) investigates the dynamics of the cAMP effector
PKA in live neonatal cardiac myocytes using cytosolic and
plasma membrane-targeted variants of the FRET-based
PKA activity reporter AKAR2. Their findings provide evidence for gradients of PKA phosphorylation from the
plasma membrane to the bulk cytosol, which confirms
that characteristics of the cAMP signal may influence the
dynamics of downstream events. Restricted diffusion of
cAMP from the plasma membrane, PDE-mediated cAMP
degradation, and cAMP buffering by PKA were all thought
to contribute to the generation of these PKA gradients
(335).
993
ADENYLYL CYCLASES
(fluo 4) in embryonic spinal neurons, Gorbunova and
Spitzer (167) showed reciprocity between Ca2⫹ and
cAMP, with cAMP oscillations arising from specific patterns of Ca2⫹ transient, and conversely, spontaneous
cAMP transients modulating the frequency of Ca2⫹ spikes
in the neurons. Mathematical modeling of their data predicted synchronous changes in Ca2⫹ and cAMP, with
cAMP transients generated by Ca2⫹ increases and terminated by cAMP-dependent PDE activity (Fig. 11). The
frequency of these cAMP transients, when measured directly, was low with just four transients per hour, each
lasting for several minutes (167). However, mathematical
modeling predicts that much faster cAMP oscillations
could arise from physiological Ca2⫹ changes (95, 426). It
is possible that the slow response time of PKA-based
probes (such as FlCRhR) precludes, or at least limits, the
detection of rapid cAMP kinetics (319). Recently, significant steps in unraveling the complexities of dynamic Ca2⫹
and cAMP interplay have been made using newer sensors
with improved temporal resolution (127, 219, 408).
A recent study by Tengholm and colleagues (127)
using evanescent wave microscopy to measure FRET between a truncated form of the CFP-tagged PKA RII subunit (targeted to the plasma membrane) and YFP tagged
catalytic subunit of PKA demonstrated bidirectional interactions between Ca2⫹ and cAMP in insulin-secreting
␤-cells following hormone stimulation. A clear interdependence of near-membrane [cAMP] and cytosolic [Ca2⫹]
(monitored with fura 2 or fura red) was observed in
individual pancreatic cells, with an incidence frequency of
⬃0.2–1.5 transients/min. The authors predict that such
temporal coding of cAMP signals is necessary for the
differential regulation of downstream cellular targets regulating cell survival and proliferation. In contrast, Landa
et al. (219) have used an Epac-based cAMP probe to
report an inverse relationship between Ca2⫹ and cAMP
2⫹
FIG. 11. Modeling the dynamics of Ca -cAMP interactions in embryonic spinal neurons. Top and bottom panels
show simulated spontaneous Ca2⫹ and cAMP oscillations,
respectively. Baseline [Ca2⫹] and [cAMP] are ⬃20 and ⬃200
nM, respectively. [Data from Gorbunova and Spitzer (167).]
Physiol Rev • VOL
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cellular Ca2⫹ changes. Calcium is the classic example of a
dynamic second messenger, and the development of
probes that can detect changes in [Ca2⫹] with high sensitivity and spatial resolution has led to the discovery of
intracellular Ca2⫹ oscillations (102, 415) arising from very
rapid and highly localized Ca2⫹ events (14, 36). However,
the potential for interaction of these dynamic changes in
[Ca2⫹]i with Ca2⫹-regulated signaling pathways has been
difficult to address experimentally. A number of Ca2⫹sensitive proteins (e.g., Ras, PLC, and PKC) do show
changes in activities that mirror the frequency of Ca2⫹
transients (24, 100, 424). In addition, there is great potential for direct interaction between Ca2⫹ and cAMP regulation given the sensitivity of specific AC and PDE isoforms for submicromolar [Ca2⫹] (see sect. V) (81, 90, 135,
137, 140, 166). The ability of Ca2⫹ to regulate the production and degradation of cAMP, coupled with the potential
feedback of cAMP on Ca2⫹ entry, release, and extrusion
pathways (50, 202), has provided the basis for a number of
models predicting interdependent oscillations of Ca2⫹
and cAMP in excitable cells (95, 167, 313, 426). The regulation of the cAMP signaling pathway by dynamic
changes in Ca2⫹ to produce parallel changes in cAMP has
the potential to greatly enhance the signaling specificity of
both second messengers. Furthermore, the potential for
cAMP to signal specific cellular events by frequency coding, rather than amplitude and duration alone, provides a
more discerning mechanism of signaling. Here we discuss
some of the latest advances, made possible by the introduction of high-resolution cAMP probes, to specifically
examine the influence of dynamic Ca2⫹ changes on singlecell cAMP signals.
The first compelling evidence for cAMP oscillations
in a single cell was obtained in Xenopus spinal neurons
microinjected with the PKA-based cAMP probe FlCRhR
(167). By combining FlCRhR and a Ca2⫹-sensitive dye
994
DEBBIE WILLOUGHBY AND DERMOT M. F. COOPER
sients but generates more sustained cAMP rises in response to rapid Ca2⫹ oscillations. This latter point is
consistent, at least in part, with work by Gerbino et al.
(157) which also suggests that the cAMP machinery in
HEK293 cells acts as a dampening device for rapid Ca2⫹
events. In this study, using FRET-based PKA and Epac
probes to monitor changes in cAMP, Hofer’s group (157)
found that prolonged, low-frequency Ca2⫹ signals gave
rise to a stepped decrease in agonist-evoked cAMP levels,
whilst endogenous AC and/or PDE activity was unresponsive to relatively high frequency changes in Ca2⫹.
Recent demonstrations of synchronous cAMP and
Ca2⫹ changes brought about by the interaction of these
two discrete signaling pathways provide an important
mechanism by which cells can discriminate between various stimuli to mediate a diverse range of downstream
events critical to cell survival. Oscillations in cAMP have
been known to occur during the myocardial contraction
cycle for many years (47), although Ca2⫹-driven cAMP
oscillations in single cardiac myocytes are yet to be dem-
2⫹
2⫹
FIG. 12. Schematic illustration of events underlying Ca -dependent cAMP oscillations. A: cytosolic Ca
transients evoked by store-mediated
Ca2⫹ entry or activation of VGCCs stimulates Ca2⫹/CaM-dependent PDE1 activation and hydrolysis of cAMP for the duration of the Ca2⫹ rise. This
2⫹
2⫹
potentially generates antiphasic oscillations in Ca and cAMP. B: alternately, the rise in [Ca ]i stimulates AC activity in a CaM-dependent manner
to increase cAMP production. When [Ca2⫹]i returns to baseline, cAMP is rapidly hydrolyzed by PKA-dependent PDE4 activity. Subsequent increases
in Ca2⫹ repeat the cycle of events to generate phasic oscillations in Ca2⫹ and cAMP. AC, adenylyl cyclase; AKAP, A-kinase anchoring protein; GPCR,
G protein-coupled receptor; PDE, phosphodiesterase; PKA, protein kinase A; SOCC, store-operated Ca2⫹ channel; VGCC, voltage-gated Ca2⫹
channel.
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oscillations in ␤-cells during the induction of depolarization-dependent Ca2⫹ oscillations. Landa et al. (219) suggest that the periodic activation and inactivation of a
Ca2⫹/CaM-activated PDE1 is central to mediating such
events (Fig. 12A). We have recently used the same FRETbased Epac probe in nonexcitable HEK293 cells expressing the Ca2⫹/CaM-stimulated AC8 (408). Our experiments
revealed that regular Ca2⫹ rises were accompanied by
simultaneous increases in cAMP with the Ca2⫹-mobilizing
agonist carbachol, evoking cAMP events at a rate of up to
3 transients/min. These dynamic changes in cAMP depended on a combination of Ca2⫹-stimulated AC8 activity
(as a consequence of CCE) and cAMP-dependent PDE
(PDE4) activity (Fig. 12B). Although higher frequencies of
Ca2⫹ transient could be evoked, this was not paralleled by
higher frequency cAMP events, suggesting that AC8 and
the endogenous cAMP signaling system of HEK293 cells
acts as a low-pass filter under such conditions. Thus it is
capable of generating relatively low-frequency cAMP oscillations that are in phase with low-frequency Ca2⫹ tran-
ADENYLYL CYCLASES
onstrated experimentally. Oscillations in cAMP in neurons have been proposed to provide a biochemical basis
for the pulsatile release of hormones (389) or to influence
growth cone turning (268). One important concept to
consider is the role of ACs as coincidence detectors that
are dually regulated by both converging Gs stimulation
and Ca2⫹ signals (3, 191). Such dependence of AC activity
on two separate signaling pathways has also been demonstrated at the single-cell level (408), and the finely
tuned responsiveness of Ca2⫹/CaM regulated ACs (AC8
and AC1) to relatively high-frequency Ca2⫹ oscillations
may underlie the proposed role of these enzymes in learning and memory (3, 191).
The dynamic nature of cAMP signals goes some way
towards mediating specific and controlled actions for this
messenger. Nevertheless, cAMP can regulate a number of
target proteins either directly (e.g., PKA or Epac) or indirectly (e.g., downstream of PKA). This coupled with a
multitude of regulatory influences to which the ACs and
cAMP (via PDEs) are susceptible, highlights the need for
further refinement in the organization of cAMP signaling
so that regulatory mayhem does not arise. We know little
about whether more than one AC isoform can exist within
a single cell,10 and if so, whether they are distributed to
different subcellular regions (for example, preferentially
located in apical or basolateral surfaces, in postsynaptic
densities, or in raft versus nonraft regions of the cell
membrane). However, several recent studies have shown
that a further level of cAMP signaling refinement can be
achieved by the presence of macromolecular signaling
complexes that help to establish preferential activation of
downstream cAMP effector molecules, or discrete regulation of ACs from specific GPCRs or modulatory proteins
(such as protein kinases and phosphatases). We are now
beginning to understand some aspects of these apparently
widely used themes in detail; others remain more hazy
concepts. It seems reasonable to suggest that the organization of signaling complexes may depend on a specific
phase in the life of the cell, and about this we know
almost nothing. Thus regulatory susceptibility must be
contextual both in time and in space. Below are summarized some current examples that emphasize the importance of protein-protein interactions to generate tightly
organized macromolecular complexes to facilitate specificity in the actions of the ACs.
10
PCR suggests the presence of multiple ACs in tissue samples.
Physiol Rev • VOL
A. AKAP-Based Signaling Complexes
The A-kinase anchoring proteins, or AKAPs, are a
large family of scaffold proteins that tether inactive PKAs
to specific sites within the cell where they are readily
available to phosphorylate local proteins (85, 160, 210,
239, 247, 253, 331, 353, 371, 372, 414). Many studies have
now demonstrated protein-protein interactions between
specific AKAP isoforms and a vast array of signaling
molecules to control numerous physiological events (for
recent reviews, see Refs. 239, 247, 353, 371, 414). Here we
focus on some recent findings that have combined more
traditional biochemical techniques with real-time singlecell imaging of cAMP as a powerful means of characterizing the components and the functional consequences of
multiprotein AKAP-based signaling complexes and, in
some cases, the microdomain kinetics of cAMP.
1. Perinuclear mAKAP/PDE4D3 signaling complex in
cardiac myocytes
Previous studies using PKA- and CNGC-based cAMP
biosensors established the spatiotemporal compartmentalization of cAMP in cardiac tissue, emphasizing the
importance of PDE4 isoforms in shaping the localized
cAMP signal (221, 261, 324). The regulation of cAMP pools
in cardiac cells has been further characterized in biochemical studies using rat ventricular myocytes to provide the first evidence that specific isoforms of PDE4 can
complex an AKAP, in this case the muscle selective A-kinase anchoring protein (mAKAP) at perinuclear regions
of the cell, to facilitate the local degradation of cAMP and
limit the activation of downstream cAMP targets (120). A
further level of complexity for this mAKAP/PDE4D3 signaling complex was recently described by Dodge-Kafka
et al. (121) who revealed that the signaling complex can
exquisitely coordinate the activity of two integrated
cAMP effector pathways (PKA and Epac1) with tethered
PDE4D3 acting as the key regulatory enzyme. This study
combined more traditional biochemical methods with fluorescent reporters of PKA activity (based on AKAR2) to
demonstrate the bidirectional control of mAKAP associated PDE4D3 activity in real-time. At the functional level
this is thought to have important regulatory effects on the
degree of extracellular signal-regulated kinase (ERK) activity and ultimately the induction of cardiac hypertrophy.
2. Subplasmalemmal gravin/PDE4D signaling complex
in HEK293 cells
A recent study in HEK293 cells combined the use of
recombinant CNGCs as cAMP biosensors with coimmunoprecipitation and RNAi techniques to identify a PDE4/
PKA/AKAP signaling complex that facilitated the rapid
hydrolysis of cAMP close to its site of synthesis at the
plasma membrane (411). The endogenous membrane-as-
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VIII. ORGANIZATION OF cAMP SIGNALING VIA
DIRECT PROTEIN-PROTEIN
INTERACTIONS
995
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DEBBIE WILLOUGHBY AND DERMOT M. F. COOPER
regulated by an agonist recruited PDE4D5/␤-arrestin complex (236). Such coordinated regulation of subplasmalemmal cAMP levels via AKAP-associated, or ␤-arrestinrecruited, PDE4 activity has important implications for
the numerous cAMP-regulated ion channels or other
plasma membrane associated enzymes that are regulated
either directly, or indirectly, by cAMP. The cAMP-dependent regulation of many of these ion channels may itself
be regulated by the action of PKA targeted to specific
channel-associated AKAPs (104, 153, 197, 245, 328, 405).
3. AKAP79 and AC5/6 interactions in cortical tissue
and nonexcitable cells
Bauman et al. (25) combined coimmunoprecipitation, RNAi, and imaging techniques to identify and func-
FIG. 13. Schematic diagrams of two AC-dependent multimeric signaling complexes showing specific protein-protein interactions. A: an
AKAP250 (gravin)-based complex targets PKA-dependent PDE activity (PDE4D3/5) to the plasma membrane for the rapid hydrolysis of cAMP during
prostanoid receptor (EP-R) occupancy. AKAP250 also binds to PKC. Bottom panel: representation of the attenuated subplasmalemmal cAMP
recovery profile following AKAP250 knockdown (AKAP250 RNAi) or the inhibition of PDE4 (rolipram), PKA (H89), or PKA-AKAP interaction (Ht31).
B: proposed multiprotein signaling complex encompassing ␤2-AR, Gs protein, PKA and its anchoring protein, AKAP, the phosphatase PP2A, and
Cav1.2 L-type Ca2⫹ channel, thought to ensure localized activation of Cav1.2 by ␤2-AR activation. Bottom panel: comparison of L-type Ca2⫹ channel
current amplitudes in primary hippocampal neurons in response to the ␤2-AR specific agonist albuterol applied either via the bath (black line) or
via the patch pipette (red line). [Data modified from Davare et al. (105) and Willoughby et al. (411).]
Physiol Rev • VOL
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sociated AKAP involved was identified as gravin
(AKAP250), with the signaling complex generating discrete, dynamic cAMP signals during activation of ACcoupled prostanoid or ␤2-receptors, where local cAMP
hydrolysis by tethered cAMP-specific PDE4D isoforms
provided an efficient, tightly controlled feedback mechanism (Fig. 13A). Inhibition or knock-down of any of the
components disrupted the dynamics of the subplasmalemmal cAMP signal. In the case of ␤2-AR activation, it is
conceivable that PDE4D could associate with the gravin
complex via ␤-arrestin and be recruited to the receptor
during agonist stimulation (143, 225). AKAP79 has also
been linked with reducing cAMP levels in the vicinity of
␤2-ARs by tethering PKA that mediates receptor phosphorylation and switching of Gi activation. PKA activity is
determined by the local [cAMP] that is thought to be
ADENYLYL CYCLASES
tionally characterize a novel protein-protein interaction
between AKAP79 (or AKAP150) and AC5/6 in cortical
tissue, HeLa, and HEK293 cells. Both PKA activity and
cAMP production were assessed, using AKAR and CNGC
sensors, respectively, to provide evidence of a functional
role for this interaction. The AKAP79/PKA/AC5,6 complex
was shown to facilitate PKA-mediated inhibition of AC
activity, thereby providing a negative-feedback loop that,
in addition to PDE4 activity, could regulate local dynamics of cAMP production (25).
4. AC and L-type Ca2⫹ channel-associated complex in
rat forebrain and ventricular myocytes
B. Other AC-Associated Proteins
A number of proteins have been found associated
with ACs, whose full significance is not yet realized. For
instance, Crossthwaite et al. (98) using a yeast two-hybrid
(Y2H) screen and in vitro binding assays identified a novel
interaction between the catalytic subunit of PP2A
(PP2AC) and the NH2 terminus of AC8 that is sensitive to
Ca2⫹/CaM. The scaffolding subunit of PP2A (PP2AA) was
also pulled down with AC8, suggesting that AC8 interacts
with the PP2A core dimer. The physiological significance
of this recently identified AC8/PP2A interaction is yet to
be established, but it seems likely to function as part of a
larger signaling complex.
In a separate study, the SNAP25 binding protein
Snapin was found to interact with the NH2 terminus of
AC6, where it appears to specifically reverse the inhibitory effects of PKC on AC6, but has no effect on the
Physiol Rev • VOL
suppression of AC6 activity mediated via PKA or Ca2⫹
(83). Snapin is associated with cAMP-dependent neurotransmitter release due to its regulation by PKA (78).
Chou et al. (83) suggest that the Snapin/AC6/PKC interaction may play some part in either modulating channel
activities or the organization of synaptic proteins during
cAMP-mediated plasticity. Another protein associated
with synaptic function, PAM (protein associated with
Myc), has also recently been found to directly interact
with a Ca2⫹-inhibitable AC. With the use of a Y2H approach, PAM was found to interact with the C2 region of
AC5 and inhibit AC activity when stimulated by Gs␣. PAM
was also found to inhibit AC1 activity in the picomolar to
nanomolar range (in S49 cyc⫺ cells and HeLa cells) but
was ineffective at regulating stimulated AC2 activity
(341). Alternate AC interactions were proposed in previous coimmunoprecipitation studies that provided evidence for inwardly rectifying potassium channel (Kir3)
and AC association with ␤2-AR. Live cell studies confirmed a direct interaction between the K⫹ channel, ␤2AR, and AC2 (220). Later studies using BRET demonstrated that heterotrimeric G proteins and RGS2 also
preassociate with AC2 prior to targeting the multimeric
complex to the plasma membrane (126, 315, 329). It
seems certain from these recent findings that currently
recognized signaling complexes will be enlarged to incorporate other proteins, and many more new AC-associated
signaling complexes will be identified.
IX. PHYSIOLOGICAL SYSTEMS
The foregoing has summarized current awareness of
the regulation and compartmentalization of the ACs in
distinct regions of the cell. Much of the experimental
evidence for such organization is based on work using
continuous cell lines that are readily amenable to the
experimental manipulations necessary to establish the
factors that contribute to cellular cAMP signaling. Below
we summarize a few examples where specific AC isoforms are centrally involved in explicit physiological contexts in a manner that makes sense of their regulatory
capabilities.
A. Specialized Actions of Ca2ⴙ-Inhibitable AC6
1. Role of AC6 in cardiac tissue
It had originally been observed that cAMP levels
oscillated in cardiac tissue as part of the contractile cycle
(47). Since the description of AC5 and AC6 as the predominant AC isoforms in cardiac tissue (in adult and
neonate, respectively), it had been suggested that their
susceptibility to inhibition by Ca2⫹ could underlie such
oscillatory behavior, since cAMP-facilitated Ca2⫹ entry
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Recent reports from Hell’s group have provided compelling evidence for an L-type Ca2⫹ channel interacting
with numerous signaling proteins, including an AC, to
create a large signaling complex in neurons that provides
rapid and specific signaling of Cav1.2 during ␤2-AR activation (22, 105, 107). Coimmunoprecipitation revealed
interactions between an unidentified AC isoform, Gs␣,
Cav1.2, ␤2-AR, PKA, and the counterbalancing phosphatase PP2A (105, 107) (Fig. 13B). Colocalization and locally
restricted signaling between the AC-coupled ␤2-AR and
Cav1.2 were confirmed by immunofluorescence confocal
imaging and cell-attached patch-clamp recordings, respectively (105).
It was further demonstrated in ventricular myocytes
that the multimeric complex requires localization in
caveolar microdomains to function appropriately (22).
Although these studies did not demonstrate the presence
of a specific AKAP within the macromolecular complex,
the authors hypothesize that the microtubule-associated
protein MAP2B may assemble the proteins, as this AKAP
has been shown to directly bind, and recruit PKA, to
Cav1.2 in neurons (106).
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DEBBIE WILLOUGHBY AND DERMOT M. F. COOPER
2. Function of AC6 in endothelial cells
In endothelial cells, the Ca2⫹-inhibitable species,
AC6, is critical to maintaining barrier function as a bridge
between the opposing actions of Ca2⫹-mobilizing versus
cAMP-elevating hormones (55, 73, 362). Endothelial cell
studies using an adenovirus to overexpress a Ca2⫹-stimulable AC isoform, AC8, demonstrated disruption of the
processes underlying effective barrier function (84). The
significance of cAMP compartmentalization in protecting
AC6-regulated endothelial cell barrier function was demonstrated by expressing a cytosolic forskolin-activated
sAC chimaera (sAC/II) that generated gaps in monolayers
of pulmonary vascular endothelial cells in culture (336).
3. Differential expression of AC5 and AC6
in the kidney
In renal tissue a very discrete expression of ACs has
been described (31, 64, 348). By RT-PCR screening, AC5
was found to be restricted to the glomerulus and outer
medullary collecting duct, with AC6 also localized to the
outer medullary collecting duct (64). Despite this overlap
in expression patterns of AC5 and AC6, functional and
differential expression of the two isoforms in two different cell types was suggested due to different sensitivities
to specific agonists and imposed Ca2⫹ changes (64).
Physiol Rev • VOL
These very small differences in the expression of two
such closely related ACs suggest differences in their properties that are yet to be revealed, but which underline the
likely critical importance of context in establishing rationale for particular isoform expression.
B. Physiological Role of AC3 in Olfaction
The initial cloning of AC3 from olfactory epithelium
and its colocalization with Golf in olfactory cilia provides
evidence for the role of this AC isoform in the olfactory
sensory system (21, 199, 252). This role has been substantiated in more recent studies in which AC3 knockout mice
were shown to exhibit peripheral and behavioral anosmia
(413). Similarly, pheromone detection in male mice depends on signaling through AC3 in the main olfactory
epithelium (394). However, AC3 is not solely responsible
for transducing olfactory signals since some odorants can
be detected in an AC3-independent manner via the vomeronasal organ (381). The role of other AC isoforms in
controlling our sense of smell is yet to be established.
C. Functions of the Ca2ⴙ/CaM-Stimulated ACs
1. Role in synaptic function
The case for the importance of the Ca2⫹ stimulability
of AC1 and AC8 in contributing to long-term potentiation
(LTP) and its behavioral extension, conditioned learning,
has long been asserted (74, 75, 392, 403). This essential
role of the Ca2⫹/CaM-regulated ACs in learning and memory was first proposed following innovative studies in
Drosophila (123, 124, 231). This was followed by landmark studies in Aplysia neurons that revealed the requirement for dual regulation of the AC by both Gs activation
and Ca2⫹/CaM in a time-dependent manner, which suggested its function as a coincidence detector (3). Several
AC1/AC8 knock-out studies have alluded to essential and
differential roles of AC1 and AC8 in neuronal function
(267, 337, 364, 386, 412, 419), which may in part reside in
their different sensitivities to Ca2⫹ (60, 145). In contrast to
impairment of function as a consequence of gene knockout, one example is available where the acquisition of a
learned task demonstrated a positive feedback on Ca2⫹stimulated AC levels. Guillou et al. (177) showed that
Ca2⫹-stimulated AC activity was enhanced by spatial
learning in the hippocampus, whereas Ca2⫹-insensitive
AC activity was decreased. Similarly, overexpression of
AC1 in mouse forebrain has been shown to enhance LTP
(391).
2. Role in the pancreas
AC8 is upregulated in diabetic ␤- and ␣-islet cells,
which suggests an important physiological role for this
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through VGCCs could feed back on the Ca2⫹-inhibitable
ACs (93). Transgenic studies have gone some way to
establishing the role of the individual ACs in mediating
␤-AR responses in the heart; for instance, the specific
knockdown of AC6, or AC5, resulted in a decrease in the
development of heart failure, due to reduced sensitivity to
pressure overload and attenuated sympathetic responsiveness (285, 286). Overexpression of AC6 constitutively
increased ␤-AR signaling without upregulating ␤-AR number or G protein expression (151), causing an increase in
the amplitude of muscle contraction that was mediated by
the coupling of AC6 to ␤1-ARs rather than ␤2-ARs (358,
360). The specific role of AC5/6 isoforms in cardiac function was further explored in studies overexpressing the
Ca2⫹/CaM-stimulated AC8 to reveal no effect on global
cardiac function. It had been anticipated that expression
of AC8 might have disrupted the underlying oscillatory
interaction between Ca2⫹ and cAMP, based on the Ca2⫹
inhibitability of AC5/6, but enhanced cardiac function on
release of parasympathetic tone was observed (156, 229).
Although there is some doubt whether the overexpressed
human AC8 gene was a splice variant (AC8B) that lacks
sites for N-glycosylation, there are as yet undetermined
outcomes with respect to the precise subcellular localization and regulation of the AC. Further elaboration of the
specificity of actions of AC isoforms in the heart is to be
expected in cells from discrete regions of cardiac tissue,
e.g., sinoatrial and atrioventricular nodes.
ADENYLYL CYCLASES
X. CONCLUSIONS AND FUTURE ISSUES
Clearly, the family of ACs provides a rich opportunity
to modulate and respond to every stage in the life of a cell,
from ultraslow quiescent cell maintenance through to the
ultradynamic responses to ion channel activities in the central nervous system. What has been learned during the last
few years in the study of these enzymes allows more
questions to be raised and some predictions to be made
on progress in the near future. The physiological significance of individual ACs and their susceptibility to Ca2⫹
and other factors remains to be determined in the majority of tissues in which they occur. For instance, whereas
some rationale is available for Ca2⫹-stimulable ACs in
facilitating or even underpinning synaptic plasticity, no
immediate rationale is apparent for the unique abundance
of the Ca2⫹-inhibitable AC5 in the striatum. Furthermore,
hardly anything is known of the trafficking, assembly, and
targeting of ACs, even though this topic may provide vital
clues to the temporal roles and associations of ACs. It
should be safe to predict that as the mechanism responsible for coupling CCE to store depletion is now becoming
apparent, the mechanism entwining Ca2⫹-sensitive ACs
and CCE may begin to be revealed. In addition, it seems
extremely likely that associations of specific ACs with a
broad range of AKAPs and scaffolding elements will soon
be uncovered. Studies in live cells using FRET/BRETbased analyses should allow the study and appreciation of
the dynamics and role of these associations. It does not
seem too fanciful to anticipate that either specific inhibiPhysiol Rev • VOL
tors of individual ACs11 or AKAP/AC disruptors may be
developed that could provide powerful investigative tools
or therapeutic agents in the foreseeable future. The speed
with which tools have been developed to characterize the
behavior of cAMP in the microdomains from which the
cAMP signal emanates and in which it also operates
presages the likelihood that such dynamic changes in
cAMP may soon be understood in cellular contexts.
Whether cAMP targets can respond to the rapid changes
in cAMP that are being reported, in an analogous manner
to that proposed for Ca2⫹, is on the experimentally addressable horizon.
Oscillations in cAMP are now being observed in,
among other cell types, neurons and secretory cells where
their significance and precise roles will most likely differ.
Clearly both Ca2⫹ and cAMP are involved in secretion;
thus exocytosis and secretion may be rendered rhythmic
by this interaction. While individual secretory cells release hormones in a unique pulsatile manner that tracks
cellular Ca2⫹ oscillations (213, 339, 363), the intact secretory organ releases pulses of the product in a globally
coordinated manner (35). The dynamic instability provided by the interaction between cAMP and Ca2⫹ at the
single-cell level, coupled with some form of cell connectivity, may be an essential component of synchronizing
the pulsatility of hormone release from tissue. The observation of single-cell cAMP dynamics in, for instance, intact pituitary slice systems from transgenic animals expressing a cAMP reporter may not be a particularly distant objective with which to address interdependent
changes in signals and product. The evolution of the ACs,
with their multiple regulatory responsiveness and localization to specific microenvironments, coupled with the
organized compartmentalization and dynamism of cAMP
signals, represents a remarkably economic device for
yielding such a diverse and commanding role over the
many aspects in the life of the cell and the homeostasis of
the organism.
ACKNOWLEDGMENTS
We thank our colleagues in the lab for helpful comments on
the manuscript and with some of the illustrations.
Address for reprint requests and other correspondence:
D. M. F. Cooper, Dept. of Pharmacology, Univ. of Cambridge,
Tennis Court Road, Cambridge CB2 1PD, UK (e-mail: dmfc2@
cam.ac.uk).
11
Specific inhibitors of the AC reaction have been developed that
are based around the structure of forskolin, or ATP and related compounds (158, 287). Those currently available display a degree of selectivity for some AC species. Increasing their AC selectivity and their cell
permeability would enhance their usefulness.
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AC in the pancreas (175). It has been suggested that AC8
may function as a coincidence signal detector/generator
for glucose and GLP-1 in insulin-secreting ␤-cells (114).
Recent single-cell studies in pancreatic cell lines have
provided compelling evidence for cAMP oscillations that
may provide an essential component of hormone secretion. But the phase of the cAMP and Ca2⫹ oscillations, and
whether a Ca2⫹/CaM-stimulable AC8 or PDE1 is responsible for linking Ca2⫹ and cAMP dynamics, is still debatable (127, 219). Nevertheless, such oscillatory behavior is
commonly considered as an underpinning of pulsatile
hormone secretion (35, 169, 211, 339, 389).
These findings, which directly address the discrete
localization and role of specific AC isoforms in physiological contexts, emphasize the evolutionary importance of
cells having access to a range of individual ACs with
numerous regulatory features. Notwithstanding the limitations inherent in knock-out studies in terms of compensatory or unknown responses and mistargeting, the significance of specialized roles of specific AC isoforms in
different tissue types, and even discrete cell types within
a particular tissue, is beginning to emerge.
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DEBBIE WILLOUGHBY AND DERMOT M. F. COOPER
GRANTS
The work in the authors’ lab has been supported by the
National Institutes of Health and the Wellcome Trust. D. M. F.
Cooper is a Royal Society Wolfson Research fellow.
20.
21.
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