192 Ejendal, Przybyla, and Watts

Table 10-1. Regulatory Properties of Adenylyl Cyclase Isoforms

Group Type Response to Regulators

Gαs Ca2+ βγ Gαi/o FSK PKA and PKC

AC1 ↑ ↑ (Cam) ↓ ↓ (αi) ↑ ↑ PKC (weak)

1 AC3 ↑ ↑ (Cam) ↓ ↓ (weak) ↑ ↑ PKC (weak)
AC8 ↑ ↑ (Cam) ↓ ↓ (weak) ↑
AC2 ↑ ↑* ↑ ↑ PKC
2 AC4 ↑ ↑* ↑ ↓ PKC
AC7 ↑ ↑* ↑ ↑ PKC
3 AC5 ↑ ↓ ↑* ↓ ↑ ↓ PKA, ↑ PKC
AC6 ↑ ↓;↑ (Cam) ↑* ↓ ↑ ↓ PKA; ↓ or ↑*
PKC
4 AC9 ↑ ↓ Calcineurin ↓ (weak) ↑(weak);↓* ↓ PKC
↑ (CamK)

↑ = stimulatory; ↓ = inhibitory; *, conditional; Cam, Calmodulin

Reprinted from Watts and Neve,59 with permission from Elsevier.

selective adenylyl cyclase antibodies have been too difficult to obtain by indi-
vidual investigators, and those available commercially have limited utility.4,18 In
general, the expression of the Group I adenylyl cyclases (AC1, AC3, and AC8)
appears to be primarily in neuronal tissue.17,19 In contrast, the expression pattern
of Group II adenylyl cyclases is generally broader, with the highest expression
levels seen in brain and muscle tissue.17 Group III adenylyl cyclases are highly
expressed in cardiac muscle and striatal neurons; however, AC6 is also highly
expressed in many other tissues.17 AC9 is highly expressed in brain, lung, and
skeletal muscle.17

GPCR regulation of adenylyl cyclases

Copyright © 2010. Cambridge University Press. All rights reserved.

Historical perspective of GPCR modulation of adenylyl cyclase

Early studies of adenylyl cyclase signal transduction were aimed at ­understanding

how hormones regulate carbohydrate metabolism. Through the course of more
than twenty-five years, a number of seminal experiments allowed scientists to
eventually determine that hormones were binding to a “receptor” that then
transduced its signal through a guanine nucleotide binding protein (G protein).
The G protein was capable of activating adenylyl cyclase, thereby increasing
cAMP accumulation. Thus, this G protein was designated Gαs for the stimula-
tory properties it exerts on all adenylyl cyclase isoforms.4 Throughout this pro-
cess, scientists discovered the receptor-G protein cycle. Agonist activation of the
GPCR activation causes a conformational change in heterotrimeric (Gαβγ) G
protein that promotes a GDP to GTP nucleotide exchange on the Gα subunit of
the interacting trimeric G protein. The GTP-bound Gα subunits then dissociate

Siehler, S., & Milligan, G. (Eds.). (2010). G protein-coupled receptors : Structure, signaling, and physiology. Cambridge University Press.
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 Adenylyl cyclase isoform-specific signaling of GPCRs 193

Inhibitor y AC 1,3,8 Stimulatory

I.

β γ β γ
Gαi/0 – + GαS
+/– +

2+
Ca
+
CaM CaMK

Inhibitor y AC 2,47 Stimulatory

II.

β γ + β γ
Gαi/0 + + GαS
PKC
Inhibitor y AC 5,6 Stimulatory

III.

β γ +
β γ
Gαi/0 – – + GαS
–
2+
+/–
Ca
PKA PKC

Inhibitor y Stimulatory
AC 9

IV .

β γ β γ
Gαi/0 – + GαS
– –
+
Calcineurin PKC CaMK
Copyright © 2010. Cambridge University Press. All rights reserved.

Figure 10-2: The nine membrane-bound isoforms of adenylyl cyclase are classified into four
categories/groups based on their regulatory properties. Group I (top) includes AC1, AC3, and AC8.
Group II is represented by AC2, 4, and 7. Group III is made up of AC5 and AC6. AC9 (bottom) is
the sole member of Group IV (see text for details on their regulation).

from (or change conformation relative to) the Gβγ subunits, allowing interac-
tions between Gα-GTP and Gβγ with effector proteins.5, 20 The intrinsic GTPase
activity of the Gα subunit returns Gα-GTP to Gα-GDP, allowing it to reassociate
with Gβγ and its receptor.
To date, there are four families of Gα subunits, five Gβ subunits, and twelve Gγ
subunits (Table 10.2). The Gαs family is comprised of a series of splice variants
(i.e., Gαsshort, Gαslong, and GαsXL) and Gαolf that is expressed in the olfactory
neuroepithelium and the striatum.4 All Gαs splice variants and Gαolf stimulate
adenylyl cyclase. In contrast, there are several inhibitory Gα subunits (i.e., Gαi,

Siehler, S., & Milligan, G. (Eds.). (2010). G protein-coupled receptors : Structure, signaling, and physiology. Cambridge University Press.
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 194 Ejendal, Przybyla, and Watts

Table 10-2. Subunits of the Heterotrimeric G proteins

Class Members Effect(s) on Adenylyl Comments

Cyclase

Gαs Gαs short Activation of AC Gαs activated by cholera toxin. Gαs

Gαs long stimulates AC by enhancing C1-C2
Gαs XL interactions
Gαolf
Gαi /o Gαi1–3 Inhibition of ACs in group Pertussis toxin prevents receptor
Gαo 1, 3, 4 activation by ADP-ribosylation of
Gαt Gαi/o/t subunits.
Gαg Gαi inhibition occurs via binding to
Gαz the C1 domain
Gαq Gαq Indirect, AC isoform Activates PLC, which generates
Gα11 dependent. the second messengers DAG and
Gα14 IP3 and subsequent downstream
Gα15 effectors (i.e. PKC and Ca2+).
Gα16
Gα12/13 Gα12 Selective activation of AC7
Gα13
Gβ/γ Gβ1–5 Inhibition or conditional AC dependent; may be dependent
Gγ1–5, 7, 8, 10–14 activation on Gα.

Gαo, and Gαz) that can inhibit select adenylyl cyclase isoforms (Tables 10.1
and 10.2). Very recently it was suggested that receptor activation of Gα12/13 sub-
units can selectively and directly stimulate the activity of AC7.21 In contrast,
GPCR activation of Gαq leads to an indirect modulation of adenylyl cyclases that
involves stimulation of phospholipase C (PLC) and subsequent modulation of
intracellular Ca2+ levels, as well as the activation of members of the PKC ­family
(see group discussions further in this chapter). The co-expression of stimulatory
and inhibitory Gα subunits and their respective receptors appears to provide
balance for regulation of adenylyl cyclase activity and consequently for cAMP
signaling in the cell. Many adenylyl cyclases are also regulated by G protein βγ
Copyright © 2010. Cambridge University Press. All rights reserved.

subunits following GPCR activation.4 Group II adenylyl cyclases are character-

ized by their ability to be conditionally activated by Gβγ. In contrast, several
other adenylyl cyclases are negatively modulated by Gβγ (see group discussions
further in this chapter). The ability of Gαs, Gαi/o, and Gβγ to regulate adenylyl
cyclases is thought to involve direct interactions with adenylyl cyclases.
G protein signaling and thus regulation of adenylyl cyclase can also be modu-
lated by RGS proteins.4 RGS proteins were originally identified as GTPase acti-
vators for Gαi/o and Gαq; however, they also have a variety of effects on other
signaling molecules, including adenylyl cyclase. Studies to date reveal that RGS2
inhibits several adenylyl cyclase isoforms (i.e., AC3, AC5, and AC6) through
direct interactions. However, there are more than twenty RGS proteins, and
much remains to be learned regarding their actions and specificity for regulating
adenylyl cyclase.4

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 Adenylyl cyclase isoform-specific signaling of GPCRs 195

GPCR regulation of group I adenylyl cyclases (AC1, AC3, and AC8)

Adenylyl cyclases in group I are all stimulated by Ca2+, and studies with AC1 and
AC8 have made it increasingly apparent that these two adenylyl cyclase isoforms
are stimulated primarily by elevated cytoplasmic Ca2+ through capacitative Ca2+
entry (CCE). The mechanism of CCE was first proposed by Putney.22 CCE is trig-
gered by depletion of intracellular Ca2+ stores, primarily the endoplasmic reticu-
lum (ER). Activation of subunits of the Gαq class stimulates membrane-bound
PLC generating IP3 and DAG from PIP2. The main role of the second messenger
IP3 is to mobilize Ca2+ from intracellular stores via activation of the IP3 receptor,
which then allows for CCE. Thus, depletion of intracellular Ca2+ stores can be
evoked experimentally by stimulating Gαq-coupled receptors with agonists, such
as carbachol-mediated stimulation of M1-like muscarinic receptors. Alternatively,
CCE can also be passively induced in a Gαq-independent manner by treatment
with the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitor, thapsi-
gargin. The CCE machinery is composed of the Orai proteins that form the pore
in the plasma membrane, and the STIM proteins that constitute the Ca2+ sensor
and are located in the ER. Although heterologous expression of Orai1 and STIM1
functionally reconstitutes CCE,23 the transient receptor potential (TRP) channels
may also play a role in CCE.10
A number of additional divergent regulatory properties relevant to GPCR
modulation are also present for group I adenylyl cyclases. For example, AC1 is
markedly inhibited by Gαi and Gαi-coupled receptors, whereas AC8 appears to
have reduced sensitivity to Gαi and regulation by Gαi/o-coupled receptors.24–26
Additional diversity for AC1 and AC8 activation reflect their ability to respond
to convergent activation by Ca2+/calmodulin and Gαs. Specifically, AC1 shows
robust synergistic responses to activation by the Ca2+ ionophore, A23187, and
Gαs activation, whereas the AC8 response is simply additive.24,25 In a recent report
directly comparing AC1 and AC8, it was also shown that AC1 is more sensitive to
Ca2+ as well as to stimulation by carbachol-mediated Ca2+ release when compared
to AC8.27 Such divergent regulation translates to modulation by Gαq-coupled
receptors, where Gαq-mediated activation of PLC precipitates the release of Ca2+
Copyright © 2010. Cambridge University Press. All rights reserved.

from intracellular stores and the activation of PKC as well as other Ca2+-dependent
pathways (e.g., calmodulin kinase activation). For example, in HEK293 cells
­stably expressing AC1, carbachol stimulation of endogenously expressed mus-
carinic M1-like receptors has been shown to increase cAMP levels and synergisti-
cally enhance Gαs activation of AC1 in a Ca2+-dependent manner.28,29 A similar
synergistic effect of Gαs activation and Gαq-modulated Ca2+ have been observed
on endogenous AC3 activity in cardiac fibroblasts.30,31 These observations suggest
that there are overlapping modes of AC1 and AC3 regulation in intact cellular
models. However, the overall mechanisms for Ca2+-stimulated adenylyl cyclase
regulation involving calmodulin appear to differ across group I adenylyl cycla-
ses. AC1 is inhibited by calmodulin kinase IV (CaMK IV) and AC3 is inhibited
by calmodulin kinase II (CaMK II) providing unique negative feedback mecha-
nisms.4 Also, both AC1 and AC8 are directly activated by calmodulin,27 whereas

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 196 Ejendal, Przybyla, and Watts

calmodulin activation of AC3 requires Gαs activation.4 Further diversity of group

I adenylyl cyclase regulation reflects their ability to be modulated by protein
kinases. For example, AC8 appears insensitive to PKC modulation, and both AC1
and AC3 show modest activation in response to phorbol esters (i.e., phorbol
12-myristate 13-acetate; PMA).32
A common feature of group I adenylyl cyclases is their ability to be directly
inhibited by Gβγ.4 The Gβγ inhibitory response blunts the activation following
stimulation of Gαs- and Gαq-coupled receptors, whereas the Gαi response of AC1
is enhanced. In summary, group I adenylyl cyclases are sensitive to regulation by
GPCRs linked to Gαs, Gαi/o, and Gαq as well as being inhibited by Gβγ.

GPCR regulation of group II adenylyl cyclases (AC2, AC4, and AC7)

Group II adenylyl cyclases appear to be unique in that they are directly or indi-
rectly sensitive to regulation by receptors linked to all families of G proteins (Gαs,
Gαi/o, Gαq, Gα12/13, and Gβγ). Gαs stimulates all group II adenylyl cyclases, and
the Gαs-stimulated activity is enhanced five- to tenfold by the Gβγ subunits.4
The Gβγ enhancement has been demonstrated in vitro with exogenous puri-
fied Gβγ and in intact cells via the release of Gβγ subunits from Gαi/o-coupled
receptors.33,34 The ability of Gβγ subunits to potentiate Gαs stimulation of AC2,
AC4, and AC7 is the result of direct interaction of both Gαs and Gβγ subunits
with these adenylyl cyclase isoforms, and may reflect the ability of Gβγ subunits
to enhance Gαs-adenylyl cyclase interactions.35 Extensive work with AC2 has
provided ­evidence that Gβγ binding may be shared across the enzyme involving
both a well-characterized motif on the C1b domain as well as the C2 domain.4
AC2 and AC7 also show conditional Gβγ activation in the presence of PKC
activation.36–38 This regulatory property appears to be a result of the divergent
regulation of the group II adenylyl cyclases to PKC. The PKC family of proteins
consists of nine genes that encode for serine/threonine kinases that share a com-
mon kinase domain but differ in their regulatory regions.39 The PKC isoforms can
be subdivided into three groups based on their structure and activating charac-
teristics. The classical/conventional (α, β, and γ) isoforms are Ca2+ dependent and
Copyright © 2010. Cambridge University Press. All rights reserved.

are activated by diacylglycerol (DAG), phosphatidylserine (PS), or phorbol esters.

Novel (δ, ε, η, ξ) isoforms are Ca2+-independent, but are activated by DAG, PS, or
phorbol esters. Atypical (ζ, λ) isoforms are Ca2+-independent and do not require
DAG for activation. Basal activity as well as forskolin-, Gβγ-, or Gαs-stimulated
AC2 activity can be enhanced by the activation of PKC.40, 41 Activation of PKC via
Gαq-coupled receptors (e.g., 5HT2A and M5) or following expression of a constitu-
tive Gαq also enhances Gαs-stimulated AC2 activity.11 These receptor-mediated
effects are blocked by the PKC inhibitor, bisindolylmaleimide I.11 Previous stud-
ies suggest that AC2 is regulated by both conventional and novel PKCs as PKCα
phosphorylates AC2 in vitro and PKCδ appears to stimulate AC2 in HEK293
cells.40,41 AC7 is also activated by PKC; however, the effects of PKC appear to dis-
sipate quickly.42,43 Since those initial studies, it has been determined that AC7 is
synergistically stimulated by Gαs and PKCδ.4 Together, these observations suggest

Siehler, S., & Milligan, G. (Eds.). (2010). G protein-coupled receptors : Structure, signaling, and physiology. Cambridge University Press.
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 Adenylyl cyclase isoform-specific signaling of GPCRs 197

potential overlap for PKCδ stimulation of AC2 and AC7. The basal activity of
AC2 is exquisitely sensitive to PKC activation, showing robust responses to phor-
bol esters and Gαq activation.12,32 In contrast, both basal and forskolin-stimulated
AC4 activity are resistant to PKC activation by phorbol esters or reconstituted
PKCα.32,40 It was also revealed that PKCα activation can inhibit both Gαs stimula-
tion and conditional Gβγ activation of AC4.40

GPCR regulation of group III adenylyl cyclases (AC5 and AC6)

AC5 and AC6 are inhibited by submicromolar concentrations of Ca2+ (0.2–0.6

μM) and make up the Ca2+-inhibited family of adenylyl cyclases.4,10 This Ca2+
sensitivity contrasts all other adenylyl cyclases that are inhibited by high con-
centrations of Ca2+ (10–25 μM), which competes with magnesium at the active
site.4 It is proposed that the source of Ca2+ to regulate AC5 and AC6 is CCE that
can be mediated by Gαq-coupled receptor activation.10,44 As discussed previously,
two membrane proteins, STIM1 and Orai1, can mediate CCE, thus understand-
ing their regulation by G proteins may have implications in GPCR-AC5/AC6
signaling.45,46
In many ways, AC5 and AC6 appear to be the prototypical adenylyl cyclases in
that they are robustly activated by Gαs and inhibited by the inhibitory G proteins,
Gαi and Gαz.4 Activated Gαs promotes the interaction between the C1 and C2
loops of adenylyl cyclase, whereas Gαi binds to the C1 domain and impairs the
C1-C2 interaction.4 AC5 and AC6 are the most sensitive isoforms to inhibition
by inhibitory G proteins (Gαi1,2,3/z).4 In vitro studies suggest that AC5 and AC6
are insensitive to inhibition to Gαo.26,35 However, several lines of evidence sug-
gest that Gαo can inhibit adenylyl cyclase activity in intact cells expressing group
III adenylyl cyclases.9 Studies with pertussis toxin-insensitive (PTXi) Gαo or Gαo
knockdown studies suggest that the inhibitory GPCRs (i.e., µ opioid and D2 dop-
amine) activate Gαo to inhibit AC6 activity in cultured cells. In addition, antibod-
ies against Gαo decrease opioid receptor-mediated inhibition of adenylyl cyclase
in striatal tissue where AC5 and AC6 are expressed at high levels.9 Although the
in vitro studies demonstrate that Gαo is not sufficient to inhibit AC5 or AC6,
Copyright © 2010. Cambridge University Press. All rights reserved.

­studies in cell lines and tissues suggest that receptor activation of Gαo signaling
can inhibit cAMP accumulation in cells expressing those isoforms.9
The effect of Gβγ subunits on AC5 and AC6 activity appears to differ based on
the experimental approach employed. Simple overexpression studies reveal that
Gβγ subunits reduce cAMP accumulation in cells expressing AC5 or AC6.47 In
contrast, a recent study demonstrated that Gβγ subunits enhance Gαs-stimulated
AC5 and AC6 activity.48 The mechanism for this stimulatory effect involved Gβγ
binding to the N-terminus of AC5 or AC6, and it was suggested that the source
of potentiating Gβγ is the Gαs-Gβγ heterotrimer.48 Previous truncation studies
using recombinant AC6 demonstrated that the removal of the N-terminal 86
residues of AC6 enhances the ability of Gαi to inhibit AC6 activity.49 This later
observation may suggest that removal of portions of the Gβγ binding region on
the N-terminus facilitates Gαi inhibition of AC6.

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 198 Ejendal, Przybyla, and Watts

Protein kinases can also significantly regulate AC5 and AC6 activity by phos-
phorylation of several serine and threonine residues. PKA is directly regulated
by cAMP, and both AC5 and AC6 are phosphorylated in vitro by PKA. This
PKA-dependent phosphorylation decreases the ability of Gαs and forskolin to
­stimulate cAMP production in cells expressing AC5 or AC6.9 Mutating a single
serine in the C1 domain (S674) to alanine prevents phosphorylation and PKA-
mediated inhibition of AC6.50,51 The precise PKA phosphorylation sites in AC5
have not been identified, although AC5 contains fourteen putative PKA phos-
phorylation consensus sequences, including serine 788 that corresponds to ser-
ine 674 in AC6.51,52
Similar to AC2, phosphorylation of recombinant AC5 by PKCα or PKCζ
enhances basal activity as well as forskolin- and Gαs-stimulated cAMP accumu-
lation.53 Evidence for PKC (e.g., α, δ, or ζ) enhancement of AC5 activity has
also been reported in gallbladder epithelium and MCF-7 cells expressing recom-
binant AC5.9 However, one study indicates that the phorbol ester, PMA, does
not stimulate AC5 activity.32 The effects of PKC activation on AC6 activity are
also inconsistent, revealing inhibition, stimulation, or no effect.9 For example,
in vitro studies with recombinant AC6 and a mixture of PKC isoforms isolated
from rat brain (PKCα, βI, βII, γ, δ, ε, and μ) suggest a PKC-mediated decrease in
forskolin-stimulated cAMP accumulation.9 In contrast, phorbol esters (i.e., PMA)
potentiate drug-stimulated cAMP accumulation in stably-transfected HEK-AC6
cells through a novel PKC isoform (i.e., δ, ε, η, or ξ) without altering basal cAMP
levels.50 PMA also potentiates drug-stimulated cAMP accumulation in other cell
lines that express endogenous levels of AC6, including Chinese hamster ovary
and Cath.a differentiated (CAD) cells.9 These studies contrast the initial reports
that that phorbol esters do not alter AC6 activity in transiently transfected
HEK293 cells.9,32
The regulation of AC5 and AC6 likely involves coincidence detection and may
reflect isoform specificity for the downstream signaling of Gαq-coupled ­receptors.
For example, in tissues that express AC5 and AC6, such as cardiac fibroblasts
and gastric smooth muscle, activation of Gαq-coupled receptors enhances adeny-
lyl cyclase signaling.9,30,31 Using stably transfected HEK-AC5 and HEK-AC6 cells,
Copyright © 2010. Cambridge University Press. All rights reserved.

it was demonstrated that Gαq-coupled receptor signaling enhances AC6 activa-

tion, but not AC5 activation.54 Specifically, the M1 muscarinic receptor agonist
carbachol significantly enhances the activity of forskolin-stimulated AC6, but
not AC5.54 Subsequent experiments reveal a similar potentiation of AC6 by the
Gαq-coupled 5HT2A receptor as well as by expression of constitutively active
Gαq (Q209L). These Gαq-mediated effects are not prevented by PKC inhibi-
tors, and subsequent biochemical studies suggest the involvement of a novel
Ca2+- calmodulin-dependent pathway.54

GPCR regulation of group IV adenylyl cyclases (AC9)

The least characterized and most divergent in sequence of the adenylyl

cyclase isoforms is AC9.12 Wild-type AC9 is insensitive to the small molecule

Siehler, S., & Milligan, G. (Eds.). (2010). G protein-coupled receptors : Structure, signaling, and physiology. Cambridge University Press.
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