Molecular biological approaches to unravel adenylyl cyclase signaling and function

Review

Molecular biological approaches to unravel adenylyl cyclasesignaling and function

Tarun B. Patel*, Ziyun Du, Sandra Pierre, Laura Cartin, Klaus Scholich

Department of Pharmacology and the Vascular Biology Center of Excellence, University of Tennessee, Memphis, 874 Union Avenue,

Memphis, TN 38163, USA

Received 29 December 2000; received in revised form 9 March 2001; accepted 19 March 2001

Received by A.J. van Wijnen

Abstract

Signal transduction through the cell membrane requires the participation of one or more plasma membrane proteins. For many transmem-

brane signaling events adenylyl cyclases (ACs) are the ®nal effector enzymes which integrate and interpret divergent signals from different

pathways. The enzymatic activity of adenylyl cyclases is stimulated or inhibited in response to the activation of a large number of receptors in

virtually all cells of the human body. To date, ten different mammalian isoforms of adenylyl cyclase (AC) have been cloned and character-

ized. Each isoform has its own distinct tissue distribution and regulatory properties, providing possibilities for different cells to respond

diversely to similar stimuli. The product of the enzymatic reaction catalyzed by ACs, cyclic AMP (cAMP) has been shown to play a crucial

role for a variety of fundamental physiological cell functions ranging from cell growth and differentiation, to transcriptional regulation and

apoptosis. In the past, investigations into the regulatory mechanisms of ACs were limited by dif®culties associated with their puri®cation and

the availability of the proteins in any signi®cant amount. Moreover, nearly every cell expresses several AC isoforms. Therefore, it was

dif®cult to perform biochemical characterization of the different AC isoforms and nearly impossible to assess the physiological roles of the

individual isoforms in intact cells, tissue or organisms. Recently, however, different molecular biological approaches have permitted several

breakthroughs in the study of ACs. Recombinant technologies have allowed biochemical analysis of adenylyl cyclases in-vitro and the

development of transgenic animals as well as knock-out mice have yielded new insights in the physiological role of some AC isoforms. In

this review, we will focus mainly on the most novel approaches and concepts, which have delineated the mechanisms regulating AC and

unravelled novel functions for this enzyme. q 2001 Elsevier Science B.V. All rights reserved.

1. Adenylyl cyclase family: topology, homology andchromosomal location

To date, nine membrane-bound isoforms and one soluble

form of mammalian AC have been cloned and characterized

(Table 1 (Krupinski et al., 1989; Bakalyar and Reed, 1990;

Feinstein et al., 1991; Gao and Gilman, 1991; Tang et al.,

1991; Ishikawa et al., 1992; Katsushika et al., 1992;

Krupinski et al., 1992; Premont et al., 1992; Yoshimura

and Cooper, 1992; Watson et al., 1994; Paterson et al.,

1995; Cali et al., 1996)). Examination of the amino acid

sequences of the membrane-bound ACs reveals 12 stretches

of hydrophobic residues in conserved positions which are

arranged in two sets of six, separated by a large hydrophilic

domain. Each of these hydrophobic stretches is presumed to

be a transmembrane region. Thus, the predicted topology of

the membrane-bound ACs is depicted in Fig. 1. The proposed

structure includes a short variable amino terminus, followed

by six transmembrane spans (M1), a large cytoplasmic

domain (C1), a second set of six transmembrane regions

(M2), and another large cytoplasmic domain (C2). The over-

all similarity among the different ACs is roughly 60%: the

most conserved sequences are located in the cytoplasmic

domains (C1 and C2) and range from 50±90%. Additionally,

there is considerable homology between the ACs and guany-

lyl cyclases. Indeed, by introducing three point mutations in

an engineered soluble form of AC it is possible to convert an

Gene 269 (2001) 13±25

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PII: S0378-1119(01)00448-6

www.elsevier.com/locate/gene

Abbreviations: AC, adenylyl cyclase; ACs, adenylyl cyclases; Roman

Numeral after AC designates AC type, e.g. ACV, type V AC; Gs, hetero-

trimeric stimulatory GTP binding protein of AC; Gi, heterotrimeric inhibi-

tory GTP binding protein of AC; Gsa, a subunit of Gs; Gia, subunit of Gi;

Gia1, type 1 isoform of Gia; Gbg, bg subunits of heterotrimeric G

proteins; PKA, cAMP dependent protein kinase; PKC, protein kinase C;

CaM, calmodulin; Fsk, forskolin; RGS, regulators of G protein signaling;

GAP, GTPase activating protein; GEF, guanine nucleotide exchange factor;

GTP[g-S], guanosine 5 0-[g-thio]triphosphate; CRE, cAMP response

element; CREB, CRE binding protein; For de®nitions of the C1, C1b,

C2, C2I subdomains of ACV please refer to Fig. 1

* Corresponding author. Tel.: 11-901-448-6006; fax: 11-901-448-4828.

E-mail address: [email protected] (T.B. Patel).

AC into a guanylyl cyclase (Sunahara et al., 1998; Beuve,

1999).

Although the various isoforms of AC share a considerable

degree of homology (Fig. 2), the genes for the different AC

isoforms are distributed independently from each other

throughout the genome (Table 2). While the chromosomal

locations of the genes for only some of the different

isoforms is known, so far no cluster of genes on one chro-

mosome has been found (Table 2). The tissue distribution of

adenylyl cyclases is not discussed here because this is very

well described in previous reviews (Smit and Iyengar, 1998;

Defer et al., 2000).

Presently, the amount of genome sequence data available

for the different isoforms of AC is limited. The mouse

ACVIII gene structure has been characterized (Muglia et

al., 1999) and this enzyme is encoded by 18 exons which

T.B. Patel et al. / Gene 269 (2001) 13±2514

Fig. 1. Schematic of the proposed structure for membrane-bound adenylyl

cyclases. Panel (A) represents the putative topology of AC isoforms. The

location of the major cytosolic regions C1 and C2 are shown in reference to

the whole molecule. M1 and M2 denotes the regions in the AC molecule

which span the membrane 6 times each. Panel (B) shows the boundaries of

the C1 and C2 domains as well as the C1b subdomain and two highly

homologous regions within the C2 designated C2I and C2II in ACV. The

amino acid numbers referred to are those for canine ACV. Note: The C2b

domain on the C terminus of the C2 region is not shown. This domain is

present only in ACI, ACII, ACIII and ACVIII isoforms.

Table 1

List of AC Isoforms cloned from different species. The cDNAs cloned for different isoforms of AC are shown. The Genbank accession numbers for the cDNAs

that have been cloned are provideda

Human Mouse Rat Bovine Dog Rabbit Chicken

ACI L05500 M25579

ACII X74210 M80550 U25635

AC III NM_004036.2 AF253540 M55075

AC IV M80633

AC V U65473 M96159 M88649 Z29371 AJ293817.1

AC VI NM_020983 M93422 L01115 M94968

AC VII D25538 U12919 AF184150 Z49806

AC VIII NM_001115.1 U85021 L26986

AC IX NM_001116.1 U30602 AJ401469

Soluble NM_018673 AF081941.1

a Human, Homo sapiens; Dog, Canis familiaris; Mouse, Mus musculus; Rabbit, Oryctolagus cuniculus; Rat, Rattus norvegicus; Chicken, Gallus gallus;

Bovine, Bos taurus.

Fig. 2. Phylogenetic tree of membrane bound adenylyl cyclase isoforms.

Amino acid sequence homologies among the various membrane bound

isoforms of mammalian ACs is shown. For clarity, the isoforms from

different species are not shown. As discussed under `Regulation of AC

Isoforms' in the text, the sequence homologies also permit the classi®cation

of the isoforms in four major groups. The ®fth group consisting of soluble

AC is not shown.

are distributed over approximately 200 kb of genomic DNA.

Comparison of the ACVIII gene with the information avail-

able about the ACIII gene suggests that the structures of the

genes encoding various membrane bound AC isoforms are

different. Thus, the ®rst intron of the ACIII gene is not

translated (Wang et al., 1993). Additionally, less than 1 kb

region of the promoter preceding the ®rst exon is suf®cient

for expression of the enzyme (Abdel-Halim et al., 1998).

However, in the case of ACVIII, the 10 kb region of the

DNA preceding the ®rst exon is necessary to obtain tissue

speci®c expression of this isoform (Muglia et al., 1999).

Interestingly, none of the two genes contain a canonical

TATA box and the promoters of both these genes contain

binding sites for transcriptional factors, which may be

physiologically relevant in their transcription. In this

respect, the ACIII promoter contains a binding site for the

transcriptional factor Olf-1 which is speci®c for olfactory

neurons (Wang and Reed, 1993; Wang et al., 1993). Since

ACIII was originally identi®ed as the predominant isoform

in olfactory cilia (Bakalyar and Reed, 1990; Menco et al.,

1992) the expression of this isoform in this tissue could be

regulated by Olf-1. Similarly, the ACVIII promoter contains

a cAMP response element (CRE) which would bind the

CRE binding protein (CREB). This could be important in

understanding the reasons underlying decreased ACVIII

expression in certain brain regions in response to drugs

such as morphine (Matsuoka et al., 1994; Lane-Ladd et

al., 1997) after injection of anti-sense oligonucleotides

directed toward CREB (Lane-Ladd et al., 1997).

2. Regulation of adenylyl cyclases

The different AC isoforms demonstrate signi®cant diver-

sity in their regulation. Therefore, the various family

members can be broadly divided into groups according to

the similarities in their sequences (Fig. 2) and regulatory

properties (Table 3). Group 1 consists of AC type I (ACI),

III (ACIII) and VIII (ACVIII), which are regulated by Ca21

and calmodulin (reviewed in (Bakalyar and Reed, 1990;

Tang et al., 1991; Choi et al., 1992; Krupinski et al., 1992;

Cali et al., 1996). While ACI and ACVIII are stimulated by

Ca21 and calmodulin (Tang et al., 1991; Krupinski et al.,

1992; Cali et al., 1996) ACIII is only activated by Ca21/

T.B. Patel et al. / Gene 269 (2001) 13±25 15

Table 2

Chromosomal localization of genes for adenylyl cyclases in human and mice are shown. References describing the chromosomal localization are provided

AC isoform Human chromosomes References Mouse chromosomes References

Soluble 1q24 (Buck et al., 1999;

Chen et al., 2000)

I 7p13-p12 (Villacres et al., 1993)

II 5p15.3 (Stengel et al., 1992)

III 2p24-p22 (Haber et al., 1994) 12 in the A-B region (Edelhoff et al., 1995)

IV 14q11.2 (Edelhoff et al., 1995) 14 in the D3 region (Edelhoff et al., 1995)

V 3q13.2-q21 (Haber et al., 1994) 16 in the B5 region (Edelhoff et al., 1995)

VI 12q12-q13 (Haber et al., 1994) 15 in the F region (Edelhoff et al., 1995)

VII 16q12-q13 (Hellevuo et al., 1995)

VIII 8q24.2 (Stengel et al., 1992)

IX 16p13.3 (Hacker et al., 1998) 16 band B1 (Hacker et al., 1998)

Table 3

Classi®cation of mammalian ACs according to their regulation by various modulators. The table summarizes the regulation of each isoform (see text for

details). The abbreviations used are: Fsk, forskolin; CaM, calmodulin; PKA, cAMP dependent protein kinase; PKC, protein kinase C

Group Isoforms Activators Inhibitors

Group1 ACI Gsa, Fsk, Ca21/CaM Giaa, Gbg, CaM Kinase IV, P-site analogs

ACIII Gsa, Fsk, Ca21/CaMb CaM Kinase II, P-site analogs

ACVIII Gsa, Fsk, Ca21/CaM P-site analogs

Group 2 ACII Gsa, Fsk, Gbg, PKC P-site analogs

ACIV Gsa, Fsk, Gbg P-site analogs

ACVII Gsa, Fsk, Gbg, PKC P-site analogs

Group 3 ACV Gsa, Fsk, PKC & z Gia, Ca21, PKA, P-site analogs, Gbgc

ACVI Gsa, Fsk Gia, Ca21, PKA, PKC, P-site analogs

Group 4 ACIX Gsa Calcineurin, P-site analogs

Group 5 Soluble AC HCO32 ?

a Gia inhibits Ca21/CaM stimulated activity of ACI; Gsa- and forskolin stimulated activity of ACI is either not or very weakly (,10±20%) inhibited by Gia.b Ca21/CaM stimulate ACIII activity in-vitro in the presence of Gpp(NH)p or forskolin. In intact cells Ca21/CaM inhibit ACIII activity (see text).c Direct inhibition of ACV by Gbg has not been observed. However, in cells over-expressing AC isoforms and Gbg, inhibition of ACV has been reported

(see text).

calmodulin if Gpp(NH)p or forskolin are present (Choi et al.,

1992). However, in intact cells the ACIII is inhibited by Ca21

via the actions of Ca21/calmodulin dependent protein kinase

II (Wayman et al., 1995; Wei et al., 1996; Wei et al., 1998).

ACI can also be inhibited by Gbg subunits of heterotrimeric

G proteins and by the a subunit (Gia) of the inhibitory GTP

binding protein of adenylyl cyclase Gi. This latter inhibition

of ACI is observed if the enzyme activity is stimulated by

calcium and calmodulin (Wittpoth et al., 1999; Taussig et al.,

1994). The second group comprises type II (ACII), IV

(ACIV) and VII (ACVII) isoforms. These isoforms are

stimulated by Gbg subunits of the heterotrimeric G proteins

provided that the active a subunit (Gsa) of the stimulatory

GTP binding protein Gs is also present (Feinstein et al., 1991;

Gao and Gilman, 1991; Tang and Gilman, 1991; Yoshimura

et al., 1996). Type V (ACV) and VI (ACVI) isoforms which

are the predominant ACs in the heart (Ishikawa et al., 1992;

Katsushika et al., 1992; Premont et al., 1992; Yoshimura and

Cooper, 1992) form the third group. Both these enzymes are

inhibited by Gia subunit and directly by calcium (Yoshi-

mura and Cooper, 1992; Ishikawa et al., 1992; Katsushika et

al., 1992; Premont et al., 1992; Yoshimura and Cooper,

1992). Although ACV and ACVI are not inhibited by Gbgsubunits in-vitro (Premont et al., 1992; Wittpoth et al., 1999),

in cells transfected to over-express these isoforms, it has been

reported that ACV and ACVI activity can be decreased by

Gbg subunits, especially b1g2 (Bayewitch et al., 1998).

Whether this is a direct or indirect effect of Gbg subunits,

remains to be determined. The fourth group consists of a

recently characterized AC isoform (type IX) which is regu-

lated by calcineurin (Paterson et al., 1995). The last group

comprises the only soluble mammalian AC described so far

(Chen et al., 2000). This enzyme is not stimulated by Gsa or

forskolin (Chen et al., 2000).

The membrane associated AC isoforms are also regulated

by phosphorylation events. Thus, ACII and ACVII are

stimulated by protein kinase C (Jacobowitz and Iyengar,

1994; Watson et al., 1994) but the Gsa-stimulated activity

of ACIV and ACVI is decreased by PKC (Zimmermann and

Taussig, 1996; Lai et al., 1999). Using puri®ed ACV as well

as PKC-a and z isoforms, Kawabe et al., (Kawabe et al.,

1994) demonstrated that the ACV activity could be

enhanced by 50-fold. However, whether or not ACV activ-

ity is altered by PKC in intact cells remains to be determined

since treatment of cells with phorbol esters did not alter

ACV activity to any signi®cant extent (Jacobowitz et al.,

1993). Likewise, although PKA has been shown to phos-

phorylate and inhibit the activities of ACV and ACVI

(Iwami et al., 1995; Chen et al., 1997), whether or not

PKA inhibits ACV in intact cells remains to be determined.

Interestingly, although calcium and calmodulin activate

ACI and ACIII these isoforms are phosphorylated and

inhibited by calmodulin kinase IV and II, respectively

(Wayman et al., 1996; Wei et al., 1996). The activity of

calcium and calmodulin stimulated ACVIII is not altered

by either calmodulin kinase II or IV (Wayman et al., 1996).

Despite the differences in the regulation of the different

ACs, the one common feature shared by all membrane-

bound isoforms of the enzyme is that they are all stimulated

by the GTP bound form of the a subunit of the stimulatory

GTP-binding protein Gs (reviewed in Sunahara et al., 1996;

Smit and Iyengar, 1998). Additionally, all membrane-bound

ACs, except for type IX isoform (Premont et al., 1996; Yan

et al., 1998) are stimulated by the diterpene, forskolin.

Moreover, all members of the membrane bound ACs are

inhibited by P-site analogs ((Desaubry et al., 1996) and

references therein). P-site inhibitors are essentially adenine

nucleoside 3 0 polyphosphates which inhibit AC activity by a

dead-end non-competitive mechanism (Johnson and

Shoshani, 1990; Dessauer and Gilman, 1997). A summary

of the regulation of various AC isoforms is provided in

Table 3.

3. Structure±function studies on adenylyl cyclases

Several studies have been performed to delineate the

regions of adenylyl cyclase which are essential for activity

and regulation by various regulators of the enzymes. In this

section we have described the regions of the enzymes which

are essential for activity and also discussed the interactions

of adenylyl cyclase with its regulators.

As mentioned before, all membrane-bound AC isoforms

share the characteristic structure depicted in Fig. 1. They all

consist of a short and variable amino-terminus, followed by

two repeats of a module predicted to be composed of six

trans-membrane spans (M1 and M2) and two cytoplasmic

domains (C1 and C2) of approximately 40 kDa each. The

C1 and C2 cytosolic domains can be subdivided into à' and

`b' subdomains (Fig. 1). The most highly conserved regions

among the ACs are within the amino-terminal halves of

each cytosolic domain (C1a and C2a). Notably, the C2b

region is only present in Type I, II, III and VIII isoforms

(Hurley, 1999). None of the two halves of the AC molecule

when expressed alone (i.e. M1C1 or M2C2) exhibit AC

activity (Katsushika et al., 1993; Tang et al., 1995).

However, the co-expression of the two halves of the AC

molecule (M1C1 and M2C2) (Katsushika et al., 1993;

Tang et al., 1995) or the expression of the C1a and C2a

domains joined by a linker (Tang and Gilman, 1995; Scho-

lich et al., 1997a), reconstitutes AC activity which can be

stimulated by forskolin and GTP bound Gsa. Individually

expressed C1a and C2 domains also reconstitute enzyme

activity when mixed together (Whisnant et al., 1996; Yan

et al., 1996; Wittpoth et al., 1999). These data suggested that

the two cytoplasmic domains interact with each other to

form a catalytic site for the enzyme. Indeed, this prediction

was borne out by the crystal structure of the C1a region of

ACV with the C2a domain of ACII (Tesmer et al., 1997).

Essentially, this structure demonstrated that the two cytoso-

lic domains of AC form an anti-parallel pseudosymmetrical

structure (Tesmer et al., 1997) and resembled the structure


of the dimeric C2 domain of ACII (Zhang et al., 1997).

However, whereas the C2 dimer demonstrated two forskolin

binding sites (Zhang et al., 1997), the C1a and C2a complex

showed only one forskolin binding site. This single site is

made up of residues F394, Y443, W507, and V511 in the

C1a region of ACV and K896, I940, G941, and S942 in the

C2a region of ACII (Tesmer et al., 1997). By interacting

with residues in both domains, forskolin stabilizes the inter-

actions between the two cytoplasmic domains and increases

the af®nity of the two domains for each other by $10-fold

(Sunahara et al., 1997). This increased interaction between

the two domains and their stabilization in the presence of

forskolin would then augment the activity of the enzyme.

Since both cytosolic domains are required for catalytic

activity, it would be predicted that the binding site for

ATP would comprise of both these domains. Indeed, the

crystal structure of C1a and C2a regions of ACV and

ACII, respectively, in the presence of forskolin and Gsapermitted the modelling of a hypothetical ATP binding

site within the molecule (Tesmer et al., 1997).

4. Interactions with the stimulatory and inhibitory GTPbinding proteins, Gsa and Gia

The crystal structure of soluble domains of adenylyl

cyclase in combination with forskolin and GTPgS bound

Gsa (Tesmer et al., 1997) revealed some interesting features

about the intermolecular interactions between these two

proteins. First, the switch II (aa 225±240) of Gsa molecule

inserts itself into a grove formed by thea2 0 helix anda3 0-b4 0

loop of AC (Tesmer et al., 1997). Second, the a3-b5 loop of

Gsa interacts with both the C1a domain of ACV (VC1a) and

C2a region of ACII (IIC2a) (Tesmer et al., 1997) (Fig. 3).

Interestingly, the complex of the Gsa and soluble AC in the

presence offorskolin demonstrated that residues in the switch

II region and a3-b5 loop of Gsa make contacts predomi-

nantly with the IIC2a. The GsaÍIC2a interface is a mixture

of hydrophobic and polar contacts, whereas Gsa´VC1a

contact is entirely hydrophobic (Tesmer et al., 1997). Only

one hydrophobic interaction was observed between W281 in

Gsa and F379 on the C1a subdomain of ACV (Tesmer et al.,

1997). The interactions of the switch II residues of Gsa with

AC were previously demonstrated by mutagenesis studies of

Berlot and Bourne (Berlot and Bourne, 1992). Likewise,

previous studies from the Gilman laboratory (Itoh and

Gilman, 1991) had identi®ed residues in the a3-b5 loop of

Gsa which interacted with AC. The structural studies also

con®rmed ®ndings of the mutagenesis approaches that had

determined several residues on the C2 domain of AC to be

important for interactions with Gsa (Yan et al., 1997).

Perhaps the most revealing aspect of the structural studies

has been the suggestion that Gsa activates the enzyme by

stabilizing the catalytically active transition form of the

protein formed by the interactions between the C1 and C2

domains. Essentially, the switch II of Gsa inserts into a cleft

formed by the a2 0 helix and a3 0-b4 0 loop (Tesmer et al.,

1997) (Fig. 3). This widens the cleft and the a1 0-a2 0 rotates

away from the C2 domain and because of its interactions with

the C1 region rotates the C1 region by 78. This rotation results

in a decrease in the pseudosymmetry of the C1Ć2 dimer and

brings D440 closer to the 3 0-hydroxyl group of ATP (Tesmer

et al., 1997). Moreover, this change may be necessary for

enhancing the interaction of R1029 with the pentavalent tran-

sition state of the substrate (see (Tesmer et al., 1997)).

From the pseudosymmetrical structure of the AC, it was

predicted that the inhibitory GTP binding protein of AC, Giabinds to a similar site on the C1a domain as the Gsa site on the

C2a region (Tesmer et al., 1997). Indeed, studies of Dessauer

et al. (1998) and from our laboratory (Wittpoth et al., 1999)

have shown that the C1a region on type V AC is suf®cient to

observe inhibition of enzyme activity. Moreover, mutagen-

esis approaches have demonstrated that mutation of certain

residues, especially F400 on the C1 region of AC reverses the

effects of Gia on ACV from inhibition to stimulation

(Zimmermann et al., 1999). The precise mechanisms

involved in Gia-elicited inhibition of AC is not known.

However, it has been postulated (Tesmer et al., 1997) that

just as Gsa stabilizes the transitional active state of the

enzyme, Gia by rotating the C1a domain in the opposite

direction may help decrease the stability of this form or

increase the stability of the inactive form. Such a structural

change would then diminish enzyme activity.

Although from the description of the structural and muta-

genesis studies discussed above, it would appear that the

C1b domain of various ACs is not essential for enzyme

activity, we and others have shown that this region is

involved in regulation of enzyme activity by a variety of

modulators. Thus, the C1b region in ACI is necessary for the

binding of Ca21/calmodulin and stimulation of enzyme

activity by these modulators (Wu et al., 1993; Levin and

Reed, 1995). In ACVIII, the splice variant in which portion

of the C1b region is deleted shows different sensitivity to

Ca21/calmodulin (Cali et al., 1996). The C1b region may

also be important in the regulation of ACIX by calcineurin

(Antoni et al., 1995). Notably, the Ca21/calmodulin depen-

dent protein kinase IV phosphorylates ACI on S545 and

S552 which are located in the C1b region of this enzyme

(Wayman et al., 1996).

With respect to activation of enzyme activity by Gsa,

several pieces of evidence suggest that the C1b region of

ACs is necessary for stimulation of enzyme activity by this

G protein. First, Chen et al., (Chen et al., 1997) demon-

strated that increasing concentrations of Gsa stimulated

ACVI in a biphasic manner, suggesting the existence of a

high and low af®nity sites for the G protein on ACVI. This

biphasic curve was converted into a monophasic curve in

which stimulation of ACVI by higher concentrations of Gsawas inhibited upon phosphorylation of S674 by cAMP

dependent protein kinase; S674 is located in the C1b region

of the ACVI. Second, using the yeast two hybrid assay,

experiments from our laboratory (Scholich et al., 1997b)


demonstrated that the C1b region of ACV interacts with a 64

amino acid long region C2I (Fig. 1) within the C2 domain of

ACV. Moreover, in the bacterially expressed soluble forms

of ACV, deletion of the C1b region altered the pro®le of

stimulation of enzyme activity by varying concentrations of

Gsa (Scholich et al., 1997b). We identi®ed two peptides,

corresponding to small regions (aa 1042±1058 and aa 1042±

1051) in the C-terminus of the C2I domain (Fig. 1) of ACV

which disrupt the C1b-C2 interaction (Scholich et al.,

1997b). Using these peptides, the pro®le of stimulation of

the full-length enzyme by different concentrations of Gsacould be converted to that observed with the soluble form in

which the C1b region was missing (see (Scholich et al.,

1997b) for details). Thus the C1b region of ACV is impor-

tant in stabilizing the interactions of AC with Gsa for stimu-

lation of activity by high concentrations of the G protein.

Further evidence that C1b region of ACV is important in

activation of enzyme activity by Gsa is provided by the

recent studies from Tang's group (Yan et al., 2000). These

authors demonstrated that the C1b region of ACVII interacts

with the C1a and C2a regions of this enzyme. Additionally,

the C1b region of ACVII was found to inhibit enzyme activ-

ity and interfere with the interactions of Gsa with C1a and

C2a regions of the enzyme (Yan et al., 2000).

The C1b region is also important in the regulation of

ACV activity by the inhibitory GTP binding protein Gia.

Thus, although the C1a region of ACV is suf®cient to

observe inhibition of activity by Gia, the presence of the

C1b region increases the sensitivity of inhibition by the G

protein (Dessauer et al., 1998; Wittpoth et al., 1999). Like-

wise, with respect to ACI, we have demonstrated that

although neither the C1b nor the C2 regions of ACI by

themselves are suf®cient to observe inhibition by G protein

bg (Gbg) subunits (Wittpoth et al., 1999). However, when

the C1b and C2 regions of ACI are present together, Gbgsubunits can inhibit enzyme activity (Wittpoth et al., 1999).

Interestingly, the C1a region of ACI is also suf®cient to

observe inhibition of enzyme activity by Gbg subunits

(Wittpoth et al., 1999). These ®ndings (Wittpoth et al.,

1999) suggest that on ACI there are two sites of Gbg inter-

action, one site is formed by the C1a region and the other by

the C1b and C2 regions in combination.

The signi®cance of the C1b region of AC in regulation of

enzyme activity discussed above was not made evident by the

structural studies of the C1a and C2a regions of AC (Tesmer

et al., 1997). Likewise, this limited structure of the enzyme

also leaves unexplained the signi®cance of the ®ndings that

mutations in the a4-b6 region of Gsa decreases or abolishes

the ability of the G protein to stimulate AC (Berlot and

Bourne, 1992). It is therefore, possible that the a4-b6 region

of Gsa is important in the modulation of AC activity invol-

ving the C1b region. Unfortunately, interactions of the C1b

region with the C2 region of AC and/or with Gsa (or Gbg)

cannot be resolved at the structural level because the expres-

sion of longer C1 domains in large amounts have proven to be

extremely dif®cult (Tesmer et al., 1997; Scholich and Patel,

unpublished observations).

5. Novel functions of adenylyl cyclases

Recently, we have discovered two novel functions for

membrane bound adenylyl cyclases. To facilitate their

discussion, ®rst the normal activation and inactivation

cycles of heterotrimeric G proteins is brie¯y described.

Essentially, following the binding of hormones to their


Fig. 3. Interactions between Gsa and the C2a domain of ACII in complex with the C1a region of ACV. This ®gure is derived from the structure of the complex

of GTPgS-Gsa and soluble AC formed by the C1a region of ACV and C2a region of ACII (Tesmer et al., 1997). Panel (A) The a3-b5 loop and switch II

regions of Gsa are shown in magenta and red, respectively. The C1a region of ACV is shown in dark gray. The a2 0 helix on the C2a region of ACII is depicted

in blue and the a3 0-b4 0 region including the loop are colored green. Panel (B) Enlargement of the regions between AC and Gsa that are involved in

intermolecular interactions.

respective receptors which couple with G proteins, the

receptors are activated and act as guanine nucleotide

exchange factors (GEF) for their respective G proteins. In

Fig. 4 the active receptor is designated as R*. The hormone

binding elicits a change on the receptor structure and

permits the receptor to augment the exchange of GTP for

GDP on the a subunit of the trimeric G protein. The GTP-

bound Ga subunit has a lower af®nity for the Gbg subunits

and dissociates from them (Fig. 4A). The GTP-bound, acti-

vated, a subunit and the Gbg subunits then interact with

their respective effectors to generate the appropriate signals.

In the case of ACs, Gsa and Gia activate and inhibit activity

of the enzyme, respectively. The Ga subunits of heterotri-

meric G proteins express an intrinsic GTPase activity,

which hydrolyzes the GTP and converts the GTP-bound

form to the GDP bound, inactive form (Fig. 4A). The

GDP-bound, inactive Ga then associates with the Gbgsubunits and returns to the resting state terminating the

transmission of signal (Fig. 4A).

In recent years, a large family of proteins (Regulators of

G-protein Signaling or RGS proteins) has been cloned and

characterized (see Siderovski et al., 1999; Burchett, 2000)

for reviews). These RGS proteins enhance the intrinsic

GTPase activity of a variety of Ga subunits and expedite

the termination of signals from these subunits (reviewed in

(Siderovski et al., 1999; Burchett, 2000). Although a large

number of RGS proteins which act as GTPase Activating

Proteins (GAPs) against a variety of Ga subunits have been

cloned and characterized, no RGS proteins or GAPs for Gsahave been found. Therefore, we proposed the hypothesis

that AC acts as a GAP for Gsa. Indeed, the C1±C2 form

of ACV and its C2 domain were found to act as GAPs for


Fig. 4. The activation and inactivation cycle of G proteins regulating adenylyl cyclase activity. Panel A depicts the classical activation and inactivation cycle of

G proteins by their receptors. Panel B has been revised to incorporate the two novel functions of AC. The hatched arrow denotes the GAP activity of AC against

Gsa only. AC does not act as a GAP against Gia (see text). However, AC enhances signal onset via both Gs and Gi coupled receptors (see text) and, therefore,

this arrow is common to both types of receptors and heterotrimeric G proteins.

Gsa (see (Scholich et al., 1999) for elaboration). Notably,

the soluble form of ACV does not act as a GAP for Gia(Scholich et al., 1999). Thus, AC is selective as a GAP for

the active form of Gsa. This action of AC is similar to that

of other effector enzymes such as phospholipase Cb(Berstein et al., 1992), and the g subunit of cGMP phospho-

diesterase (Arshavsky and Bownds, 1992) which can also

act as GAPs for Gqa and Gta, respectively.

Because the GAP activity of AC can expedite the termi-

nation of signaling via Gsa, we investigated the effects of

AC on signal onset. For these experiments, the Gs hetero-

trimer was reconstituted from puri®ed recombinant Gsa and

puri®ed bovine brain Gbg subunits ((Scholich et al., 1999),

also see (Patel et al., 2001) for experimental details). A

peptide bIII-2 corresponding to amino acids 259±272 of

the b2-adrenergic receptors was used as a constitutively

active receptor. This peptide has previously been shown to

mimic the actions of the b2-adrenergic receptor (Okamoto

et al., 1991; Sun et al., 1995). By monitoring activation of

Gs using three parameters, i.e. steady state GTPase activity,

GTPgS binding, and activation of AC in membranes of S49

cyc2 cells, we demonstrated that in the presence of the

soluble form of ACV (C1±C2 ACV), the concentration of

the bIII-2 required to activate the G protein was decreased

by 100-fold (Scholich et al., 1999). Overall, these experi-

ments showed that AC facilitates the onset of signaling via

receptors so that the amount of active receptor required to

activate the enzyme is decreased by 100-fold (Scholich et

al., 1999). In the case of Gs, AC and its C2 domain as well as

smaller regions within the C2 domain, C2I and C2II (Fig. 1)

were suf®cient to enhance the actions of the peptide bIII-2

(Scholich et al., 1999). This novel function of AC would

serve to amplify signaling in the presence of low concentra-

tions of ligand or active receptors, which signal via Gs. In

Fig. 4B, we have revised the classical activation/inactiva-

tion cycle to include the GAP and GEF enhancing actions of

AC.

Since the C2 domain of AC which interacts with Gsacould augment the GEF activity of Gs, it may be postulated

that the C1 domain of the enzyme in ACV which interacts

with the Gia would also enhance the GEF activity of Gi

coupled receptors. Indeed, using a number of different

approaches, including the inhibition of AC activity in

membranes of S49 cyc2 cells by somatostatin, we demon-

strated that indeed the C1 domain of ACV could increase

the onset of signaling via Gi coupled receptors (Wittpoth et

al., 2000). Since AC does not act as a GAP for Gi, in the new

activation/inactivation cycle for Gi, in Fig. 4B, the hatched

arrow from AC to the `signal off' portion of the cycle should

be omitted.

An interesting aspect of the novel functions of AC with

respect to Gsa and Gs, is that the enzyme both inactivates

Gsa (GAP activity of AC) and facilitates activation of Gs

(GEF enhancing activity of AC). This apparently paradox is

also applicable to other proteins with GAP activity against

Ga subunits. For example, besides acting as GAPs some

RGS proteins may also enhance the onset of signaling.

Thus, it has been demonstrated that RGS4 and RGS8 can

increase the activation kinetic of G protein coupled inward

rectifying K1 current (Doupnik et al., 1997; Saitoh et al.,

1997). More recently, Chen and Lambert (Chen and

Lambert, 2000) have shown that endogenous RGS proteins

in hippocampal neurons can increase the onset of presynap-

tic inhibition in response to adenosine and baclofen. There-

fore, the concept that GAPs, which expedite signal

termination, can also augment or facilitate signal onset is

more generally applicable than previously thought and AC

and RGS proteins could be interchangeably used in Fig. 4B.

While the membrane bound ACs may act as GAPs or

GEF enhancers, the soluble AC may act as a bicarbonate

sensor. The soluble enzyme is expressed in tissues such as

the testes, kidneys and the choroid plexus (Buck et al., 1999)

where changes in bicarbonate levels alter cAMP levels. It is

well recognized that spermatozoa undergo a number of

bicarbonate induced changes which increase motility, capa-

citation, and the acrosome reaction (Garty and Salomon,

1987; Okamura et al., 1991; Visconti et al., 1998). These

processes are also cAMP dependent. Interestingly, Chen et

al. (2000) found that the soluble AC is not modulated by the

regulators which alter the activity of membrane bound ACs,

but is activated by bicarbonate ions independently of

changes in pH. These ®ndings have provided the link

between changes in bicarbonate levels and alterations in

cAMP content and explain why increases in bicarbonate

and cAMP levels are associated with certain alterations in

spermatozoa function.

6. Knockout and transgenic models for adenylyl cyclases

Although the different isoforms of membrane bound ACs

are differentially regulated by a variety of modulators,

because some of these isoforms which are co-expressed in

some organs and cell types demonstrate similar modes of

regulation one wonders what physiological role each isoform

plays. To address this and other issues concerning the physio-

logical relevance of each isoform, several studies have

utilized knock-out and transgenic mice. Some of the clues

concerning the physiological role of AC isoforms were

derived from the drosophila mutants that were de®cient in

learning and memory. Four of these mutants, involved

defects in genes encoding one of the following: AC activat-

ing peptide (amnesiac) (Feany and Quinn, 1995), AC (ruta-

baga) (Livingstone et al., 1984), PKA (DCO) (Foster et al.,

1984), or cAMP phosphodiesterase (dunce) (Chen et al.,

1986). The drosophila AC is stimulated by Ca21 and calmo-

dulin and is similar to the mammalian ACI and ACVIII

isoforms. Furthermore, earlier studies had demonstrated

that in mammals PKA is important for synaptic plasticity

and some forms of long-term potentiation (LTP) (Frey et

al., 1993; Abrams et al., 1991). Therefore, to study the role

of ACI in synaptic plasticity and LTP, Storm and co-workers


knocked out this isoform in mice. Essentially, their ®ndings

demonstrated that the ACI is important in behaviour and LTP

in mice (Wu et al., 1995). In additional studies the Storm

laboratory demonstrated that in ACI knockout mice, AC

activity in cerebellar cortex was reduced by 65% and this

was accompanied by an almost complete blockade of cere-

bellar LTP (Storm et al., 1998). These ®ndings demonstrate

that ACI plays a critical role in regulating synaptic plasticity

and LTP. Interestingly, in mice lacking ACI or ACVIII late

phase LTP (L-LTP) and long-term memory are unaffected

(Storm et al., 1998). However, the double knock-out mice in

which both ACI and ACVIII genes have been disrupted do

not exhibit L-LTP or LTM (Storm et al., 1998). Injection of

the diterpene forskolin, which activates all isoforms of AC

except ACIX, in the Cornu Ammonis 1 (CA1) region of the

brain in double knockout mice, restored LTM (Storm et al.,

1998). These ®ndings clearly demonstrate that the Ca21 and

calmodulin stimulated ACs (I and VIII) are required for L-

LTP and LTM.

The ACVIII knockout mice have also unveiled at least

two other functions for this isoform. First, it has been

shown that capacitative Ca21 entry stimulates cAMP synth-

esis in the parotid acini. The parotid acini which contain a

variety of AC isoforms including ACI, ACIII, ACV, ACVI

and ACVIII, (Watson et al., 2000). Therefore, Watson et al

(Watson et al., 2000) used the ACI and ACVIII knockout

mice to determine the isoform which was activated by

agonists such as carbachol which increase Ca21 entry.

The disruption of ACVIII, but not ACI, gene obliterated

increase in cAMP levels in response to calcium (Watson et

al., 2000). In fact, in parotid acini from ACVIII de®cient

mice, increase in calcium entry decreased cAMP accumu-

lation (Watson et al., 2000). This latter effect can be attrib-

uted to the presence of the Ca21 inhibited ACV and ACVI

in parotid acini (Watson et al., 2000). Thus, the same

modulator may alter the activities of more than one isoform

in cells and the net of the positive and negative input from

different AC isoforms determines the ®nal level of cAMP

accumulation. A second physiological function of ACVIII

was reported by Schaefer et al., (Schaefer et al., 2000).

Essentially, mice de®cient in ACVIII do not show

increased anxiety in repeated stress tests. The ACVIII

knock-out mice also do not show CA1 region long-term

depression after low frequency stimulation and do not acti-

vate the CREB in the CA1 region after repeated stress tests

(Schaefer et al., 2000).

More recently, by disrupting the gene encoding ACIII,

the Storm laboratory has investigated the role of ACIII in

olfactory responses (Wong et al., 2000). Olfactory cilia

express ACII, III, and IV (Wong et al., 2000). However,

disruption of the ACIII gene abrogated electro-olfactogram

responses to a number of cAMP or inositol trisphosphate

elevating agents (Wong et al., 2000). Moreover, in olfac-

tion-based avoidance tests, the ACIII knockout mice

performed signi®cantly poorly as compared with their

wild type controls (Wong et al., 2000). These ®ndings

demonstrate that the ACIII is critical and of paramount

importance in olfaction and olfaction related responses.

Besides the knockout approach, several investigators

have used transgenic mice in which a given isoform of

AC is over-expressed in a tissue speci®c manner. These

studies have provided insights into the physiological role

of some of the AC isoforms. For instance, since the Ca21

inhibited ACV and ACVI are the predominant isoforms

expressed in the heart (Ishikawa et al., 1992; Katsushika

et al., 1992; Premont et al., 1992; Yoshimura and Cooper,

1992), it has been postulated that elevations in intracellular

Ca21 concentrations during the contractile cycle may inhibit

the activity of these isoforms in a feed-back regulatory

manner. Indeed, cyclical alterations in cAMP levels in

ventricular strips have been observed with each contractile

cycle (Brooker, 1973). Therefore, if the inhibition of ACV

or ACVI activity at the peak of a contraction is physiologi-

cally relevant, then it would be expected that the over-

expression of an AC isoform such as ACI or ACVIII

whose activity is augmented by Ca21 (and calmodulin),

would grossly alter cardiac function. Surprisingly, however,

cardiac speci®c over-expression of ACVIII did not alter

basal heart rate and contractility (Lipskaia et al., 2000).

However, when the parasympathetic tone was released,

cardiac contractility and heart rate of the ACVIII transgenic

mice increased markedly and was unresponsive to modula-

tion by b-adrenergic receptor agonists (Lipskaia et al.,

2000). These ®ndings differ from the study of Gao et al.,

(Gao et al., 1999) which demonstrated that over expression

of ACVI in the heart increased the responsiveness to b-

adrenergic receptor stimulation. One reason for this differ-

ence could be related to the cAMP levels in the hearts of

transgenic mice over-expressing ACVI and ACVIII. Since

Ca21 regulates these two enzymes in opposite ways, it

would be expected that in hearts over-expressing ACVIII,

the cAMP levels would be higher than in those over-expres-

sing ACVI. Such an increase in cAMP levels may desensi-

tize the b-adrenergic receptors in hearts of ACVIII

expressing animals but not in ACVI over-expressing

animals. Unfortunately, Lipskaia (Lipskaia et al., 2000)

did not measure cAMP levels in ACVIII transgenic animals.

However, the basal cAMP levels in myocytes derived from

ACVI transgenic animals were similar to those in controls

(Gao et al., 1999). Moreover, as expected from the func-

tional responsiveness to b-adrenergic receptor activation,

isoproterenol increased cAMP levels to a greater extent in

ACVI transgenic mice as compared with controls (Gao et

al., 1999).

One of the implications of the studies with cardiac speci-

®c expression of ACVI is that in certain pathological states

such as ischemia or hypertrophy in which responsiveness

of AC system to b-adrenoreceptor agonists is decreased

(Vatner et al., 1988; Strasser et al., 1990; D'Angelo et

al., 1997), the over-expression of ACVI or ACV, would

restore the responsiveness of the heart to b-receptor stimu-

lation. Indeed, the studies of Roth et al. (1999) demon-


strated that over-expression of ACVI in hearts of mice

over-expressing Gqa improved cardiac function and

restored the cAMP generating capacity in response to cate-

cholamines. Similarly, because over-expression of Gqaresults in approximately 45% decrease in ACV expression,

decreased b-adrenoreceptor mediated activation of AC,

and hypertrophy (D'Angelo et al., 1997; Roth et al.,

1999), Tepe and Liggett (1999) investigated whether or

not over-expression of ACV in Gqa over-expressing

animals would ameliorate the cardiac myopathy and restore

function. Essentially, these studies (Tepe and Liggett,

1999) demonstrated that cardiac speci®c over-expression

of ACV in the Gqa over-expressing mice improved cardiac

function and responsiveness to b-adrenoreceptor activa-

tion. However, cardiac hypertrophy and expression of

hypertrophy related genes was not altered (Tepe and

Liggett, 1999). These ®ndings demonstrate that neither

the decrease in ACV nor the loss of responsiveness to b-

receptor activation was the underlying cause of cardiac

hypertrophy. Nevertheless, the studies with ACV and

ACVI over-expression in the heart provide important infor-

mation concerning how the cardiac responsiveness to b-

adrenergic receptors can be restored. In this respect, it

should be noted that over-expression of ACV or ACVI

may improve cardiac function since it is generally accepted

that in the receptor ± Gs ± AC complex, AC is in limiting

amounts (Post et al., 1995). Thus, over-expression of the

limiting signaling element (AC) may amplify the actions of

activated receptors. On the other hand, it should be noted

that we have demonstrated that AC and its domains which

interact with G protein a subunits, facilitate the onset of

signaling via receptors which couple to Gs and Gi (Scho-

lich et al., 1999; Wittpoth et al., 2000). Therefore, by this

mechanism, it is equally possible that the over-expression

of adenylyl cyclase augments the onset of signaling from

the receptor to the effector.

7. Concluding remarks and future directions

Since the discovery of cAMP as a second messenger

some forty years ago by Dr. Earl Sutherland, the amount

of research that has been performed in understanding of the

regulation and role of the diverse family of ACs is beyond

the scope of any single review. Therefore, we have focused

on the aspects of ACs not previously represented in other

reviews. However, the readers are strongly encouraged to

read other comprehensive reviews on the subject which

address the ®ndings and inferences derived from studies

in different tissues (Defer et al., 2000), the distribution of

Ca21 modulated AC isoforms in the brain (Mons et al.,

1998) and the role of ACs as signal integrators (Ishikawa

and Homcy, 1997). The underlying message from the

numerous reviews and research articles published in the

area is that the family of diverse ACs offers a fertile ground

for future investigations. Thus, while the knock-out and

transgenic mice experiments have provided some informa-

tion on the physiological roles that some of the AC

isoforms may play in the context of the nervous system

and the heart, the roles of other members of the family

remain to be clearly de®ned. This task is made even

more daunting since most tissues and organs express a

variety of AC isoforms. Moreover the structures of the

genes encoding the majority of AC isoforms remain to

be determined. Additionally, the role of ACs in disease

conditions involving mutant Gsa forms also remains to

be determined. For instance, certain pituitary tumors are

associated with the expression of constitutively active

mutant forms of Gsa (Lyons et al., 1990). Recently, Gu

et al. (2000) have shown that Gsa also activates the tyro-

sine kinase Lck. Thus, it could be argued that the pituitary

tumors associated with mutant, constitutively active, Gsaare due to activation of Lck rather than AC. To investigate

this and other possibilities it is essential that selective inhi-

bitors of AC be developed. Presently, the only inhibitors

which selectively inhibit ACs are the `P site' inhibitors

developed by Johnson and colleagues (see e.g. Doronin

et al., 1999; Shoshani et al., 1999a). Most of these,

however, do not permeate the cell membrane. The few

inhibitors of AC, which are taken up by cells, exert other

non-speci®c effects (Shoshani et al., 1999b; Kudlacek et

al., 2000). Moreover, no inhibitors such as dominant nega-

tive forms of AC are presently available. Therefore, efforts

in this area would probably present another breakthrough

in the ®eld. Likewise, our recent studies (Scholich et al.,

1999; Wittpoth et al., 2000) have shown that AC clearly

has a function in regulation Gsa activity and signaling via

receptors coupled to Gs and Gi. Therefore, with the intent

to increase or decrease the signaling via receptors it is

essential to determine the molecular mechanisms underly-

ing this mode of regulation of G proteins by AC.

Acknowledgements

We are greatly indebted to numerous investigators in the

®eld who provided their most recent publications and

preprints of their publications to ensure that our review

would be as up to date as possible. Due to the numerous

publications in this ®eld and because of space limitations,

we had to refer to some previous reviews in our citations,

and, therefore, we apologize to those authors whose original

®ndings were not cited in this review. This work was

supported by grants from the NIH (HL59679 and HL

07641).

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Molecular biological approaches to unravel adenylyl cyclase signaling and function