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(Received for publication, November 2, 1994; and in revised form, January 20, 1995)
From the
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Adenylyl cyclase, the effector molecule of the cAMP signaling
pathway, is composed of a family of isoforms that differ in their modes
of regulation. Many of these modulatory interactions are dependent upon
well characterized molecules from various second messenger pathways;
however, very little is known about their mechanisms or sites of action
on adenylyl cyclase. Chimeras were produced by a novel in vivo mechanism between two differentially modulated adenylyl cyclases
to identify their regulatory domains. The basal activity of the type I
adenylyl cyclase (AC1) is activated by calcium/calmodulin, inhibited by
G protein 
subunits, and insensitive to protein kinase C
regulation. In contrast, type II adenylyl cyclase (AC2) is insensitive
to calcium/calmodulin regulation and is activated by G protein
subunits as well as by activated protein kinase C.
Expression and biochemical characterization of chimeras between AC1 and
AC2 identified a single specific domain of AC1 responsible for
calmodulin binding and a small, well defined region near the C terminus
of AC2 required for protein kinase C activation.
Adenylyl cyclase synthesizes the second messenger cAMP in
response to a variety of extracellular signals. Various hormones and
neurotransmitters modulate cAMP production by binding to members of the
seven-transmembrane receptor family coupled to stimulatory
(G
) and inhibitory (G
) G proteins.
Intracellular cAMP levels are also affected through mechanisms that
utilize other signaling pathways to modulate cyclase activity.
Ca
and protein kinase C, which function in discrete
second messenger pathways, can independently modulate the activity of
adenylyl cyclase. Ca activation synergizes with
G stimulation, allowing adenylyl cyclase to integrate
signals from different second messenger pathways. Current biochemical
models for associative learning in the sea slug Aplysia and
the fruit fly Drosophila suggest that dual activation of
adenylyl cyclase is responsible for the integration of independent
stimuli. The G protein subunits participate in the guanine
nucleotide exchange cycle and the transient activation of 
subunits, but they also regulate adenylyl cyclase directly. This
G
stimulation may account for the ability of
certain G-coupled neurotransmitters and hormones to
potentiate the effects of G-coupled agonists in the brain.
To date, seven mammalian isoforms of adenylyl cyclase have been
characterized and can be distinguished by their modes of regulation.
Three forms, types I, III, and VIII (AC1, (
)AC3, and AC8),
appear to be activated by Ca, through the modulatory
protein calmodulin (CaM), but AC1 and AC3 differ in their responses to
G(1, 2, 3, 4) .
Types V and VI (AC5 and AC6) are the most closely related cyclase
isoforms and appear to be similarly regulated; both are inhibited by
physiological levels of Ca and insensitive to G
protein subunit regulation (5, 6, 7, 8, 9) . The
activity of AC5 is also stimulated when the protein is phosphorylated
by protein kinase C(10) . Adenylyl cyclase types II (AC2) and
IV (AC4) are highly related in sequence and display similar biochemical
profiles(11, 12) . Both are insensitive to
Ca regulation, and their
G
-stimulated activities are potentiated by G protein
subunits(2, 11) . AC2 differs from AC4 by
being dramatically stimulated by protein kinase C in vivo(13, 14, 15) . The ability of adenylyl
cyclase to integrate various signals and to generate different
responses to identical inputs provides considerable versatility to this
second messenger cascade.
In each case, the mechanism of action of
regulation is not known; however, all have been shown to directly
modulate adenylyl cyclase activity in
vitro(1, 2, 10, 16, 17, 18) .
A specific region of AC1 is thought to bind Ca/CaM,
leading to activation, but the sites of action of the other known
cyclase regulators have not been identified. While protein kinase C has
been shown to stimulate AC5 in vitro, its phosphorylation
sites and mechanism of action have not yet been determined. Jacobowitz
and Iyengar (31) demonstrated that protein kinase C stimulation
of AC2 activity following phorbol ester treatment of cells correlated
with an increase in AC2 phosphorylation. Whether the protein kinase C
activation of AC2 arises from this direct phosphorylation or
alternatively through indirect processes remains to be determined.
Additionally, nothing is known about the structural requirements for
subunit regulation of adenylyl cyclases.
All known
metazoan adenylyl cyclase isozymes share a predicted topological
structure(19) . Two cytoplasmic domains (C
and
C
) display homology to each other as well as to the
catalytic portion of guanylyl cyclases(20) . These regions
exhibit the greatest degree of sequence conservation between isozymes
and are thought to be responsible for adenylyl cyclase catalytic
activity. Each catalytic domain is preceded by a set of six
transmembrane spans. These transmembrane domains share little sequence
homology, but are predicted to be structurally similar. Between the
first catalytic domain (C) and the second set of
transmembrane domains is a nonconserved cytoplasmic region
(C
) of unknown function.
Functional domains have been
identified within families of distinctly regulated, but related
proteins by investigating the properties of chimeric molecules. Such
studies have been particularly successful in determining the
ligand-binding and G protein-coupling domains of the seven
transmembrane receptors (21) as well as the
subunit-binding, receptor-interacting, and effector-activating regions
of G
subunits(22, 23) . In the case of
the G protein-coupled, seven transmembrane receptors, chimeras were
produced by swapping independently identified, putative structural
domains. Previous studies of adenylyl cyclase (1) indicated
that no functional protein could be formed when the cytoplasmic domains
of two different isoforms were expressed in the same cell, suggesting a
need for an alternative approach. We have utilized a novel method for
producing chimeras between two homologous genes that results in a
collection of molecules with crossovers scattered throughout their most
conserved regions.
We investigated the regulatory domains of two
adenylyl cyclase isoforms that differentially respond to each of the
known mechanisms of regulation. The basal activity of AC1 is activated
by Ca/CaM, unresponsive to protein kinase C, and
inhibited by G protein subunits. AC2 is unresponsive to
Ca/CaM, but is activated by either protein kinase C
or G protein subunits. Chimeric cyclases were produced
between these two isoforms by a variety of methods and have defined
domains essential for regulation by Ca/CaM and
protein kinase C.
-
P]ATP,
[
H]adenine, and 
I-calmodulin were
purchased from DuPont NEN. N-Ethylmaleimide was purchased from
Sigma. 
AR was kindly provided by Dr. Lee Limbird
(Vanderbilt University Medical Center). UK14304 was purchased from RBI.
precipitation.
After 3 days, cells were suspended in 200 µl of lysis buffer (50
mM Tris (pH 7.5), 1 mM EDTA, 1 mM dithiothreitol, 0.1 mg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride) and disrupted by sonication (cup
sonicator for 1 min on ice). Extracts were centrifuged at 100,000
g for 10 min, and pelleted membranes were resuspended
in 400 µl of lysis buffer by repeated passage through a 22-gauge
needle. Adenylyl cyclase assays were performed, in duplicate, on
approximately 20 µl (1/20 of transfected plate) of resuspended
membranes. Specific activity was determined and expressed as picomoles
of cAMP formed per minute/milligram of protein.
H]adenine. Labeled
cells were preincubated with 1 mM isobutylmethylxanthine in
Hepes-buffered Dulbecco's modified Eagle's medium for 30
min and then treated for 30 min with either 100 nM PMA or 1
µM UK14304 in the presence of isobutylmethylxanthine.
Cells were lysed with cold 5% trichloroacetic acid, and adenine
nucleotides were fractionated by the method of Salomon(26) .
Accumulation of cAMP is presented as a percentage of total adenine
nucleotides to normalize for labeling variability.
I-calmodulin as described(27) .
Membrane was exposed to film for approximately 3 days with an
intensifying screen.
transformation. Transformants, derived from plasmids
recircularized in vivo or not cleaved in the initial
restriction enzyme digestion, were recovered at a frequency of
approximately 10/µg of DNA. Recircularization likely
occurs by repair pathways at regions of nucleotide identity shared
between the two genes, resulting in chimeric cyclase coding regions. Chimeras were expressed from the cytomegalovirus promoter in HEK293
cells and assayed for adenylyl cyclase activity. The basal cyclase
activity of AC1-transfected cell membranes was further stimulated by
the addition of exogenous Ca/CaM and inhibited by the
Ca chelator EGTA, reflecting activation by endogenous
Ca/CaM (Fig. 1). In contrast, cells
transfected with calcium-insensitive AC2 (11) are unaffected by
Ca/CaM or EGTA addition. One particular AC1-2 chimera
(AC12x56) displayed an activity profile similar to that of AC1.
Activity was increased by exogenous Ca/CaM and
inhibited by EGTA, indicating that AC12x56 retains responsiveness to
Ca/CaM. This chimera defined the CaM-responsive
domain to within the N-terminal two-thirds of AC1 (Fig. 2). An
AC2-1 chimera (AC21x15) displayed an activity profile similar to that
of AC2 (Fig. 1), indicating that it is unresponsive to
Ca/CaM regulation.
Figure 1:
Ca/CaM-responsive
activity of adenylyl cyclase chimeras. Adenylyl cyclase activity is
shown normalized to vector-transfected HEK293 cells in the absence of
any additions (blackbars), in the presence of 30
µM CaCl and 50 µg/ml CaM (stripedbars), or in the presence of 1 mM EGTA (graybars). Bars represent the average of multiple
membrane preparations derived from independent transfections, each
assayed in duplicate (AC1, 10 transfections; AC2, nine transfections;
AC12x56, eight transfections; AC12x75, three transfections; and
AC21x15, six transfections).
Figure 2:
Summary of Ca/CaM
responsiveness and I-CaM binding of chimeric adenylyl
cyclases. Selected adenylyl cyclase chimeras are depicted
diagrammatically. Light-gray portions are derived from AC1,
and dark-gray portions are derived form AC2. Cyclases are
shown divided into putative structural domains as described in the
text. Ca/CaM-responsive chimeras (as determined from Fig. 1) are indicated by plus signs. Inactive chimeras
are indicated by emptyparentheses. Chimeras that
bind CaM (as determined from Fig. 3A) are indicated by plus signs; those that do not are indicated by minussigns. TM,
transmembrane.
Figure 3:
Calmodulin binding of adenylyl cyclase
chimeras. A, I-CaM overlay of whole cell
extracts from HEK293 cells transfected with the indicated cyclases or
chimeras or with vector alone (pGW1). Molecular weight markers are
indicated on the left. The dots indicate the position of AC1. B, I-CaM overlay of transfected HEK293 extracts
treated with N-ethylmaleimide (NEM; leftpanel) or untreated (rightpanel) prior
to denaturation in sample buffer.
Several in vivo chimeras with changeover points within the first catalytic domain
contained no detectable activity. Cyclase activity in extracts from
cells transfected with these chimeras was indistinguishable from vector
alone-transfected cells under basal and forskolin-stimulated conditions
(data not shown). Additional chimeras were produced by site-directed
mutagenesis and domain swapping. One of these targeted AC1-2 chimeras
(AC12x75) was responsive to Ca/CaM (Fig. 1);
however, tripartite chimeras made within the context of the
CaM-responsive AC12x56 chimera were inactive. Direct assessment of the
calmodulin binding using an overlay technique (28) delineated
the CaM-binding domain of the active as well as the enzymatically
inactive proteins. Whole cell extracts of transfected cells were
incubated with I-CaM in the presence of calcium. Cells
transfected with AC1 contained a CaM-binding protein at approximately
110 kDa that was absent in vector-transfected and AC2-transfected cells (Fig. 3A). The CaM-responsive chimeras (AC12x56 and
AC12x75) also displayed this specific I-CaM binding.
Among the inactive chimeras, only those containing the C domain of AC1 (Fig. 2) were able to bind I-CaM (Fig. 3A). Expression of adenylyl
cyclase protein by all the chimeric constructs was confirmed by Western
blotting with antibodies raised against the C terminus of either AC1 or
AC2 (data not shown). These results define C as the only
domain of AC1 responsible for CaM binding.
region has been shown to bind CaM with high
affinity(29) . This sequence encompasses two cysteine residues,
and treatment of extracts from cells expressing AC1 with
sulfhydryl-modifying reagents specifically attenuated the
Ca/CaM activation of AC1(30) . We
investigated whether cysteine modification directly inhibits CaM
binding by AC1. In an overlay experiment, N-ethylmaleimide
treatment prevented the I-CaM binding of AC1 and selected
chimeras while not altering CaM binding by endogenous proteins (Fig. 3B). These results directly demonstrate that at
least one cysteine residue is in or near the only CaM-binding site of
AC1 and suggest that the region defined by the CaM-binding peptide
within the C domain is the only binding site for CaM in
AC1.
/CaM-responsive AC1-2 chimera (AC12x56) and an
AC2-1 chimera (AC21x15), were also unresponsive to PMA stimulation (Fig. 4A). AC21x15 is identical to AC2 except for its
final 50 amino acids, which are replaced by the C terminus of AC1. This
chimera was enzymatically active in an in vitro cyclase assay (Fig. 1) and resembled AC2 in its responses to
AR (Fig. 4B). Agonist-dependent
activation of AR results in the stimulation of a
variety of G proteins, including G, G, and
G
(32) , each of which could stimulate AC2 via
different mechanisms. Stimulation of AC21x15 by AR is
sensitive to pertussis toxin (data not shown), implying that this
chimera, like AC2, is responsive to G protein
subunits.
Figure 4:
Phorbol ester response of chimeras. A, cAMP accumulation in HEK293 cells transfected with the
indicated cyclases or vector alone (0) either in the presence (stripedbars) or absence (solidbars) of 100 nM PMA. Shown is a representative
assay performed in duplicate. Errorbars indicate
standard deviation of the mean. B, HEK293 cells cotransfected
with the indicated cyclases and AR. cAMP accumulation
is measured in the presence (stripedbars) or absence (solidbars) of the AR-specific
agonist UK14304 (10 µM).
Targeted chimeras constructed by site-directed mutagenesis
and domain swapping (AC21x82 and AC212x(15/81)) confined the region
required for protein kinase C responsiveness to 39 amino acids, of
which 19 are identical between AC1 and AC2 (Fig. 5A).
There are no putative targets for protein kinase C phosphorylation
within this region of AC2. Additional chimeras were generated using a
PCR-based strategy to identify the specific residues within this region
required for PMA stimulation. Each resulting protein still responded to
AR stimulation (Fig. 4B), and as
summarized in Fig. 5B, a four-amino acid stretch of AC2
is required for responsiveness to phorbol ester treatment. The
corresponding region of AC1 exchanged for these residues does conform
to a consensus protein kinase C phosphorylation site (RRGSYR), but the
data presented here suggest that introduction of the putative protein
kinase C target sequence results in loss of phorbol ester
responsiveness. Models for this site's importance posit a
negative regulatory modification in AC1 that can be introduced into
AC2. This does not appear to be the case as mutation of serine 1035 to
alanine in AC21x15 did not render the enzyme phorbol ester-responsive
(data not shown).
Figure 5:
Summary of phorbol ester responsiveness of
chimeric adenylyl cyclases. A, adenylyl cyclase chimeras are
depicted diagrammatically as described in the legend to Fig. 2,
except this time, the C domain is drawn out of proportion
to depict the C-terminal chimeras. Phorbol ester response is indicated
to the right of each chimera. B, shown are the amino acid
sequences of the C terminus of AC2 (11) and the corresponding
regions of the PCR-generated chimeras (as outlined under
``Experimental Procedures'' and in Table 2):
AC1(20) , AC3(34) , AC4 (12) ,
AC5(6, 8, 9) ,
AC6(5, 6, 7) , and AC8(4) . Amino
acids differing from AC2 are shaded. The average -fold
stimulation in the presence of PMA is indicated to the right (averages
derived from at least 10 distinct cAMP accumulation
assays).
We have described a novel method for rapidly producing chimeric molecules with changeover points randomly scattered throughout the most conserved regions of homologous genes. The method is recA-independent and likely involves circularization of linear DNA molecules by natural bacterial repair systems. Presumably, one strand of the transformed linear DNA is preferentially digested by single-stranded exonucleases, revealing regions of nucleotide identity on opposite strands of the homologous genes. Pairing of the two single-stranded regions at small patches of sequence homology shared between related genes results in DNA polymerase-mediated repair using the two genes as templates for extension in opposing directions. This bacterial repair mechanism results in the conversion to one gene sequence on one side of the initial annealed region and to the other homologous gene on the other side, resulting in a single chimeric gene. The crossover point of the chimera is defined by the stretch of identical nucleotide sequence that initially annealed in vivo.
This method has many advantages over more traditional procedures for producing chimeras. Crossover points are generated at the most similar regions between genes, and the resulting linear sequence contains only naturally occurring amino acid neighbors. Additionally, there is no experimenter bias in selecting crossover sites based on predetermined, but potentially inappropriate structural domains. The changeover points are therefore less apt to alter important structural motifs or functional domains. Generating in vivo chimeras from two starting plasmids, with the homologous genes in the alternative head-to-tail positions, will produce reciprocal pairs of chimeras; however, it may require extensive screening to isolate every partner. This method efficiently produces a large number of chimeras randomly distributed throughout the conserved regions of two genes. A single transformation generates a large number of chimeras that can be rapidly screened and mapped by analysis of simple restriction digests. Finally, this method does not require the time and cost of oligoncleotide synthesis and site-directed mutagenesis.
We used this method to generate chimeras between AC1 and AC2, two genes that are <60% identical within their most conserved catalytic domains. Of 72 miniplasmid DNAs screened by restriction analysis, 29 contained an insert of the appropriate size for a single chimeric cyclase gene. Further restriction analysis determined that these were composed of elements from each of the starting cyclase genes, and nucleotide sequencing confirmed that all were precise chimeras. Chimeric genes were expressed in HEK293 cells, and each produced protein of the predicted molecular weight (data not shown).
Many of the in vivo chimeras had no detectable catalytic activity, and almost all of the directed chimeric cyclases, generated by swapping supposedly separate domains, were inactive. Additionally, every construct representing a reciprocal of an active chimera was enzymatically inactive (data not shown). Our difficulty in producing chimeras and the behavior of reciprocal molecules are consistent with previous attempts at producing chimeras between adenylyl cyclase isoforms. Coexpression of mixtures of structural domains from different cyclase isoforms in insect cells revealed a bias between the putative catalytic portions of AC1 and AC2(1) . The first catalytic domain of AC1 in combination with the second catalytic domain of AC2, similar to the active AC12x75 chimera described here, resulted in at least partial activity. In contrast, the reciprocal arrangement, the first catalytic domain of AC2 coexpressed with the second catalytic domain of AC1, resulted in no detectable activity. This arrangement is analogous to the inactive AC21x76 and AC21x78 chimeras we produced.
Both active
and inactive chimeras were useful in defining the CaM-binding domain of
AC1 to the nonconserved cytoplasmic portion between the first catalytic
domain and the second set of transmembrane domains (C).
CaM binding by chimeras 212x(78/56) and 12x79 demonstrates that the
C domain of AC1 is sufficient to confer CaM binding, while
the inability of chimeras 12x77 and 21x80 to bind indicates that it
encompasses the only detectable CaM-binding site in AC1. Computer
programs predicted that a specific sequence within this domain
comprises a CaM-binding site, and a synthetic peptide corresponding to
that sequence was able to bind CaM with high affinity(29) .
Additionally, amino acid substitutions within this sequence
specifically affected CaM activation of AC1(33) . These results
and the identification of the C domain as being both
necessary and sufficient for CaM binding confirm that this region (and
presumably the sequence defined by the peptide) mediates the
Ca/CaM activation of AC1.
A small region near the C terminus of AC2 was found to be required for PMA responsiveness; however, it does not contain any potential protein kinase C phosphorylation sites. Phorbol ester treatment has been shown to increase AC2 phosphorylation, which was correlated with stimulation of cyclase activity(31) . This suggests that the C terminus of AC2 plays a more general catalytic role and interacts with specific phosphorylation site(s) to increase cyclase activity. AC1-2 reciprocal constructs of the PMA-insensitive chimeras (AC21x15 and AC21x81) were inactive (data not shown), but the presence of this region in the CaM-responsive AC1-2 chimeras, which were insensitive to phorbol ester treatment (Fig. 4), demonstrated that this region was not sufficient to confer PMA responsiveness. The amino acid sequence of the required region is not conserved between AC2 and the only other adenylyl cyclase isoform (AC5) whose activity is known to be directly modulated by protein kinase C phosphorylation(10) . In fact, the only cyclase displaying any conservation in this region is AC4, but its activity is not affected by phorbol ester activation of protein kinase C(14) .
)AR,
-adrenergic receptor; PCR, polymerase chain reaction;
PMA, phorbol 12-myristate 13-acetate.AR. We also thank Dr. Bruce Conklin (University of
California, San Francisco) and Dr. Ravi Iyengar (Mt. Sinai School of
Medicine) for communicating results prior to publication.
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