From the
Demonstration that the principal control of cellular cyclic AMP
concentrations lies at the level of its synthesis has focused attention
on adenylyl cyclase, the enzyme that catalyzes the conversion of
intracellular ATP to cyclic AMP. The prototypical hormone-sensitive
adenylyl cyclase system is comprised of three types of plasma
membrane-associated components: heptahelical, G protein-coupled
receptors for a variety of hormones, neurotransmitters, and autacoids;
stimulatory and inhibitory heterotrimeric G proteins; and the catalytic
entity itself. The G proteins regulate the activity of the enzyme in
response to interaction of ligands with appropriate receptors. Each G
protein contains a guanine nucleotide-binding subunit and a
complex of tightly associated
and
subunits. Upon activation
of a G protein by an agonist-bound receptor, GDP is released from
in exchange for GTP. Binding of GTP causes conformational changes that
result in dissociation of GTP-
from
, liberating two
macromolecular complexes capable of regulation of downstream effectors.
Mammalian adenylyl cyclases are activated by the diterpene forskolin, and the development of a forskolin affinity matrix by Pfeuffer and Metzger (1) greatly facilitated purification of the enzyme. The scarcity of the protein and its considerable lability in detergent-containing solutions had impeded progress for years. However, it was apparent that there were at least two distinct forms of adenylyl cyclase that differed in their capacity to be stimulated by calmodulin (2, 3, 4, 5, 6) . Using a forskolin affinity resin, Krupinski and co-workers (7) were able to purify a sufficient amount of a calmodulin-sensitive adenylyl cyclase from bovine brain to obtain partial amino acid sequence and thereby isolate cDNA clones encoding the full-length protein; this was termed the type I isoform. Application of low stringency hybridization and polymerase chain reaction techniques has now permitted isolation of six additional full-length clones (types II-VI and VIII)(8, 9, 10, 11, 12, 13, 14) . However, definition of the extent of molecular diversity in this family is not yet complete. Both alternatively spliced transcripts of adenylyl cyclases (15) and partial sequences of novel isoforms (e.g. type VII) (16) are in evidence.
Adenylyl cyclases have
molecular weights of roughly 120,000 (range of 1064-1248 amino
acid residues) and a complex (but as yet only deduced) topology within
the membrane (Fig. 1). A short cytoplasmic amino terminus is
followed by six transmembrane spans (designated M) and a
large (roughly 40 kDa) cytoplasmic domain (C
). The motif is
then repeated: a second set of six transmembrane spans (M
)
is followed by a second cytoplasmic domain (C
). This
structure is (to date) unique for a ``simple'' enzyme. It is,
however, highly reminiscent of the structures of certain channels and
ATP-dependent transporters, particularly the P glycoprotein and the
cystic fibrosis transmembrane conductance regulator. This relationship
prompted speculation that the adenylyl cyclases might also serve as
channels or transporters, but there is as yet no evidence to support
this conjecture. Of interest, an adenylyl cyclase from Paramecium does appear to be a K
channel(17) ;
however, its structure is not yet known.
Figure 1:
Structure of adenylyl
cyclase. The predicted topology of membrane-bound adenylyl cyclases is
shown (see text for discussion). Cylinders represent
membrane-spanning regions, while boldfacelines indicate regions of high amino acid similarity among all members
of the family. Nomenclature is as follows: N, amino-terminal
domain: M, first set of membrane-spanning
regions; C
and C
, the first large intracellular
cytoplasmic domain; M
, second set of
transmembrane spanning regions; and C
and C
, second large
intracellular domain.
The overall amino acid
sequence similarity among the different isoforms of adenylyl cyclase is
roughly 50%. However, two regions highlighted in Fig. 1(designated C and C
) are more highly conserved (up to 93%
sequence identity), and this strong relationship extends to
corresponding domains of topographically similar adenylyl cyclases from Drosophila(18) and Dictyostelium(19) . Of importance, the C
and C
domains are also highly homologous to each
other as well as to the catalytic domains of membrane-bound guanylyl
cyclases and domains that are found in each of the subunits of
cytosolic heterodimeric guanylyl cyclases. Based on these
relationships, it is predicted that one or both of these domains of
mammalian adenylyl cyclases is the site for catalysis of cyclic AMP
synthesis.
Analysis of sequence relationships among the adenylyl cyclases permits some grouping. The type II and IV isoforms are clearly more related to each other than to the others; a similar relationship exists between type V and type VI. The regulatory properties of these isoforms appear to reflect their evolutionary relationship (see below). Although type I, III, and VIII adenylyl cyclases all are activated by calmodulin, they do not form a closely related group.
Thorough documentation of the cellular and subcellular localization of the different isoforms of adenylyl cyclase in mammalian tissues has been hampered by low levels of expression (generally 0.01-0.001% of membrane protein) and limited availability of high affinity, isoform-specific antibodies. Most information thus comes from analysis of patterns of mRNA expression. All isoforms of adenylyl cyclase appear to be expressed in the brain, apparently in region-specific patterns. The type I enzyme is largely restricted to the nervous system, while type III is found predominantly in olfactory neuroepithelium. There are substantial differences in patterns of expression in peripheral tissues.
Structural Correlates of Activity
Despite the similarities discussed above that lead to the
conclusion that the C and C
domains of the
adenylyl cyclases represent catalytic domains, it has not been possible
to detect significant enzymatic activity after expression of either of
these domains as discrete proteins. The same is true of an entire
C
or C
domain or of M
C
or M
C
(halves of the molecule). However,
concurrent expression of constructs encoding M
C
and M
C
in Sf9 cells using recombinant
baculoviruses permits detection of the encoded proteins by
immunoblotting and a substantial amount of enzymatic activity that can
be regulated in characteristic type-specific fashions (see below) by G
protein subunits or calmodulin(20) . It is thus assumed that
interaction between the C
and C
domains is
essential for catalysis. This is consistent with the fact that neither
subunit of the heterodimeric cytosolic guanylyl cyclases is sufficient
for catalysis (21) and that the membrane-bound guanylyl
cyclases are oligomers(22, 23, 24) . It is
also notable that point mutations in either the C
or
C
domains of the adenylyl cyclases can impair enzymatic
activity severely (with retention of G
binding
activity) and that mutations in either domain can elevate the K
for substrate. (
)It is thus
speculated that both domains can bind ATP. It is unknown if both
domains can catalyze cyclic AMP synthesis or if one is the dominant
catalyst while the other is regulatory.
The front half of one
isoform of adenylyl cyclase can be coexpressed with the back half of
another, creating so-called noncovalent chimeras. Concurrent expression
of a ``front half'' construct of type I adenylyl cyclase,
truncated to remove domain C, with the back half of type
II adenylyl cyclase, which largely lacks C
, permits
assembly of a functional adenylyl cyclase that responds very well to
both forskolin and activated G
. The variable C
and C
domains are thus not necessary for responses
to these regulators.
We assume that G
interacts with C
and/or C
and perhaps
regulates interactions between these domains. A peptide corresponding
to residues 495-522 of the C
domain of type I
adenylyl cyclase binds calmodulin and inhibits calmodulin-stimulated
enzymatic activity(26) . Point mutations in this region also
interfere with activation of adenylyl cyclase by
calmodulin(27) . The C
domain is thus likely to be
a (or the) site of interaction of calmodulin with adenylyl cyclases
that are sensitive to the protein.
Regulation of Enzymatic Activity
Studies of the regulation of the isoforms of mammalian
adenylyl cyclases reveal a wealth of common and disparate features. All
isoforms are activated by both forskolin and the GTP-bound
subunit of the stimulatory G protein G
. All are inhibited
by certain adenosine analogs termed P-site inhibitors; 2`-deoxy-3`-AMP
is particularly potent. However, all of the isoforms of adenylyl
cyclase are further regulated in type-specific patterns by other
inputs, particularly including those that are dependent on
Ca
or that arise from other (non-G
)
G protein subunits (Fig. 2).
Figure 2: Patterns of regulation of adenylyl cyclase activity. PKC, protein kinase C; CAM, calmodulin; AC, adenylyl cyclase. See text for discussion.
Smigel (4) and Katada et al.(28) first noted inhibition of type I adenylyl cyclase
activity by the G protein subunit complex, but in neither
case was it clear that the effect was exerted directly. In fact, Katada et al.(28) attributed the inhibition to sequestration
of calmodulin by
. Interest in this phenomenon was rekindled
when Tang et al.(20) noted prominent inhibition by
of type I adenylyl cyclase expressed in Sf9 cells, and
purification of the expressed protein permitted demonstration of its
direct interaction with this subunit complex(29) .
Inhibition of adenylyl cyclases by is confined for now to
the type I enzyme. Notably, however, when the effects of
on
other isoforms were tested, surprising stimulatory effects were
observed with type II (25) and type IV (10) adenylyl
cyclases. Of particular interest, stimulation of these enzymes by
is highly conditional. Effects of
alone are
barely detectable, but the magnitude of stimulation of activity is
substantial (5-10-fold) in the presence of G
.
Stimulation of type II adenylyl cyclase by
requires
significantly higher concentrations of
than of
G
, and the source of the
is presumed to be
the G
or G
oligomers, which are present in high
concentrations in brain. The potential significance of this synergistic
interaction is discussed below.
The G proteins designated as
Gs (G
, G
, and
G
) were discovered as substrates for pertussis toxin
and as the G protein oligomers responsible for inhibitory regulation of
adenylyl cyclase activity. However, when the effects of the resolved
subunits of G
were tested, it was difficult to observe
substantial inhibition of the enzyme by the
subunits of these
proteins. Nevertheless, Wong and co-workers (30) showed that
transfection of cells with cDNAs encoding constitutively activated
G
subunits lowered intracellular concentrations of
cyclic AMP, suggesting that these proteins could inhibit adenylyl
cyclase activity. We know, retrospectively, that the failures to
reconstitute the response in vitro were due to the use of
inadequate concentrations of G
purified from natural
sources (or to the counteracting stimulatory effects of contaminating
G
when higher concentrations of G
were used), to the use of nonmyristoylated G
expressed in Escherichia coli, and/or to tests performed
with isoforms of adenylyl cyclase not susceptible to inhibition by
G
. When adequate concentrations of E.
coli-derived myristoylated G
were tested on type
V and type VI adenylyl cyclase, prominent inhibition of
G
- and forskolin-stimulated activity was
observed(31, 32) . The three isoforms of G
are equally potent and efficacious. The three G
proteins can also inhibit type I adenylyl cyclase, but the effect
is not as prominent as that observed with
. Furthermore,
inhibition of type I adenylyl cyclase activity by G
is
largely absent when the G
-stimulated activity is
examined; inhibition is largely confined to activity observed in the
presence of calmodulin or forskolin. Type I adenylyl cyclase can also
be inhibited by G
, while this subunit has no effect on
the type V enzyme. Both type I and V adenylyl cyclases are also
inhibited by G
, (
)the only member of the
G
subfamily of
subunits that is not a substrate for
pertussis toxin.
The subject of inhibitory regulation of type II
adenylyl cyclase is problematic. We have demonstrated that this isoform
(when expressed in Sf9 cell membranes) is not inhibited by G (either native or myristoylated recombinant protein). However,
Chen and Iyengar (33) noted that expression of a constitutively
activated mutant of G
inhibited type II adenylyl
cyclase activity when coexpressed in COS-7 cells. In view of the
observation of Tang and Gilman(25) , discussed above, that
activates type II adenylyl cyclase, we view it as unlikely
that activation of heterotrimeric G
would release two
regulators (G
and
) with opposing effects on
the same enzyme. In support of this hypothesis, Federman et al.(34) noted that activation of G
resulted in
conditional activation (and not inhibition) of type II adenylyl cyclase
expressed in HEK-293 cells.
All
adenylyl cyclases are inhibited by high (100-1000
µM) concentrations of Ca as a result of
competition for Mg
, which is required for catalysis.
By contrast, types V and VI adenylyl cyclase are inhibited by low
micromolar concentrations of
Ca
(11, 13, 35) . This
effect is independent of calmodulin and is presumably mediated
directly. Inhibition of cyclic AMP accumulation in some intact cells
has been shown to follow elevation of Ca
concentrations(36, 37, 38) . In some
cases this has been correlated with expression of either type V or VI
adenylyl cyclase(11, 13, 35, 39) .
The possibility of feedback inhibition of adenylyl cyclase activity in response to phosphorylation by cyclic AMP-dependent protein kinase is obvious, but evidence for this mechanism remains sparse. Its existence is suggested by studies of chick hepatocytes and variants of the S49 lymphoma cell line(43) . These cells have several isoforms of adenylyl cyclase but share the type VI enzyme. It contains two consensus sites for phosphorylation by cyclic AMP-dependent protein kinase, one of which is conserved in the closely related type V isoform.
Potential regulation of adenylyl cyclase by protein kinase C has been explored more extensively, prompted by a wealth of confusing literature demonstrating almost every imaginable effect of phorbol esters on cellular cyclic AMP concentrations. Three reports indicate that the activity of type II adenylyl cyclase, expressed by transfection, can be augmented substantially by stimulation of protein kinase C(44, 45, 46) . Nevertheless, we (and presumably others) have failed to detect phosphorylation of type II adenylyl cyclase (expressed in Sf9 cell membranes, for example) by addition of activated protein kinase C. The effect may be indirect or mediated by a specific isoform of the kinase.
Kawabe and associates (47) have phosphorylated type V adenylyl cyclase in vitro using protein kinase C and demonstrated a marked increase in
enzymatic activity. The effect was specific for the and
isoforms of protein kinase C, suggesting cross-talk between this
adenylyl cyclase and both G
-mediated pathways (for protein
kinase C-
) and growth factor tyrosine kinase pathways (for protein
kinase C-
). Despite the robust nature of the response observed in vitro, only modest effects of activation of protein kinase
C in vivo have been observed with type V adenylyl
cyclase(44) .
There is less consensus with regard to effects
of protein kinase C on type I adenylyl cyclase. Again, the
Ca connection would seem to make this isoform (and
type VIII) logical candidates for feedback regulation by a
Ca
-activated kinase. Although some have observed
enhanced forskolin- (48) or calmodulin- (44) stimulated
type I adenylyl cyclase activity in response to activation of protein
kinase C, others have not(45) .
Patterns of Regulation and Functional
Consequences
Three distinct patterns of regulation of mammalian adenylyl cyclases are evident for the type I, II, V, and VI isoforms (Fig. 2). Types III, IV, and VIII have been omitted from Fig. 2 because they have been studied less extensively. However, type IV appears to resemble type II closely, and the features of type VIII noted to date resemble those of type I.
As noted above, the
only constant feature (with regard to G protein-mediated regulation) is
that all membrane-bound mammalian adenylyl cyclases discovered to date
are activated by G. One can make reasonable hypotheses
for regulation of each of the well studied isoforms of adenylyl cyclase
(directly or indirectly) by both of the other two major subclasses of G
proteins whose signaling mechanisms are (at least partially)
understood: the G
subfamily and the G
subfamily. Accepting this premise, there are then four
possibilities: G
and G
both stimulate adenylyl
cyclase activity; G
and G
both inhibit; G
inhibits/G
stimulates; G
stimulates/G
inhibits. Remarkably, three of these
four patterns appear to be represented by the first four adenylyl
cyclases to be studied in detail. Thus, even at what will ultimately be
considered to be an extremely superficial level of analysis, the system
has surely evolved to permit extensive integration and cross-talk, and
adenylyl cyclases can be considered as focal points or final common
paths for convergence of the activities of a very large number of
regulatory elements (particularly if receptors are included in the
counting game).
Five years ago the prevailing view was of two basic
types of adenylyl cyclase: calmodulin stimulated or not, both of these
likely inhibited in a G-mediated fashion. These basic
patterns are represented by type I (VIII) on the one hand and by types
V and VI on the other. However, each scenario has offered surprises.
For type I, Ca
-mediated stimulatory signals may arise
from either calmodulin or protein kinase C. Inhibition (at least in
vitro) may be mediated more by
than by
G
's. This brain-specific isoform can also be
inhibited by G
, which is expressed predominantly in
brain. We speculate that the
-mediated inhibitory effects
arise predominantly from activation of G
or G
because these G proteins are expressed at the highest
concentrations (particularly in brain), and the effects of
require higher protein concentrations than do those of G
or G
.
Studies of learning and memory in a
number of animal models demonstrate a likely role for
calmodulin-activated adenylyl cyclases. A
Ca-stimulated adenylyl cyclase is postulated to be
the locus of integration of inputs underlying the development of
classical conditioning in the marine mollusk Aplysia(49) . Impaired learning is evident in the Drosophila mutant rutabaga. These flies lack
calmodulin-stimulated adenylyl cyclase activity(50) , and the
defect has been mapped to a gene encoding a calmodulin-activated
adenylyl cyclase that is homologous to the mammalian type I and VIII
isoforms(18) . Activation of glutamate-gated Ca
channels within the CA1 field of the mammalian hippocampus
activates a calmodulin-stimulated adenylyl cyclase(51) . This
has been proposed to trigger cyclic AMP-dependent protein kinases and
transcription factors that contribute to the development of long term
potentiation(52) . Types I and VIII adenylyl cyclase are both
present in this region of the hippocampus.
For type V and type VI
adenylyl cyclase, inhibition can be mediated directly by
Ca, which can arise from G
-regulated
pathways and, of course, others. The observation that
Ca
-dependent isoforms of protein kinase C can
phosphorylate and activate type V adenylyl cyclase in vitro suggests that Ca
may under some circumstances
have a stimulatory effect on this enzyme; however, this situation has
not been observed in vivo. Regulation of types V and VI
adenylyl cyclase by
has not been detected. Although these
isoforms are also found in brain, types V and VI may be the dominant
forms of adenylyl cyclase in peripheral tissues, where concentrations
of
are low compared with values in brain. Regulation of
types V and VI by G
has also dropped from view.
Type II adenylyl cyclase has provided the biggest surprise. This
enzyme is activated by G,
, and
(indirectly?) by protein kinase C. The effects of
are
largely dependent on concurrent activation of the enzyme by
G
. This adenylyl cyclase is thus designed to detect
coincidental activation of regulatory inputs. The capacity of
to serve as an appropriate regulator of such a coincidence
detector is dependent on its affinity for the enzyme (rather than
release of distinct isoforms of
following activation of
different pathways). G
activates adenylyl cyclase at
picomolar concentrations, while
is effective at nanomolar
concentrations. Liberation of both G
and
by
activation of the G
oligomer is presumed to provide
insufficient
to stimulate adenylyl cyclase. Activation by
is thus likely dependent on liberation of the subunit from a
G protein present at significantly higher concentration, such as
G
or G
. The biochemical properties of type II
adenylyl cyclase provide a gratifying explanation for a phenomenon
described extensively in the 1970s, highly synergistic stimulation of
cyclic AMP accumulation in brain slices following stimulation by pairs
of neurotransmitters and/or neuromodulators, now known to work through
G
- and G
-regulated pathways. These responses
have often been observed in regions of the brain where expression of
type II adenylyl cyclase is particularly abundant (e.g. cerebellum and hippocampus)(53) . Electrophysiological
studies of hippocampal pyramidal cells appear to provide a specific
physiological example of the significance of conditional regulation of
type II adenylyl cyclase activity(54) .
This discussion of
patterns of regulation of adenylyl cyclase activity and a few of their
physiological consequences is necessarily simplistic. We have ignored a
number of other regulatory phenomena, either because they have been
incompletely defined and/or because there is little meaningful to say,
other than to note their existence. Included among these are
synergistic interactions between a variety of regulators (e.g. G and calmodulin, G
and
forskolin), complex kinetic patterns of inhibition, activator-dependent
patterns of inhibition, and the likelihood that regulation by
mechanisms such as phosphorylation will also be highly dependent on the
context of concurrently acting activators or inhibitors. Also ignored
have been the effects of agents such as forskolin or P-site inhibitors,
whose physiological correlates are unknown. Compilation of the
catalogue of adenylyl cyclase phenomenology is a necessary task for the
immediate future. This will require more labor than intellect and may
at times be frankly boring. However, appreciation of the chemical and
structural basis of this catalogue and its interpretation, particularly
with regard to the physiology of the central nervous system, will be
major challenges and accomplishments.